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WO2018162439A1 - New predictive biomarker for the sensitivity to a treatment of cancer with a dbait molecule - Google Patents

New predictive biomarker for the sensitivity to a treatment of cancer with a dbait molecule Download PDF

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Publication number
WO2018162439A1
WO2018162439A1 PCT/EP2018/055396 EP2018055396W WO2018162439A1 WO 2018162439 A1 WO2018162439 A1 WO 2018162439A1 EP 2018055396 W EP2018055396 W EP 2018055396W WO 2018162439 A1 WO2018162439 A1 WO 2018162439A1
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Prior art keywords
cancer
nucleic acid
cells
acid molecule
treatment
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PCT/EP2018/055396
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French (fr)
Inventor
Marie Dutreix
Wael Jdey
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Onxeo
Institut Curie
INSERM (Institut National de la Santé et de la Recherche Médicale)
Centre National De La Recherche Scientifique
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Publication of WO2018162439A1 publication Critical patent/WO2018162439A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the field of the medicine, more particularly of the oncology. It relates to a biomarker of the sensitivity to treatment with a Dbait molecule.
  • Dbait molecules are a new class of DNA repair inhibitors triggering false DNA damage signaling in cancer cells. These molecules are short double-stranded DNA with a free double strand blunt end, which target key damage signal transducers such as DNA dependent protein kinase (DNA-PK) and Poly-ADP- Ribo-Polymerase (PARP), triggering their activation and amplifying false damage signaling (WO2005/040378, WO2008/034866 and WO2008/084087). In order to increase the efficiency of cellular uptake, the Dbait molecule was modified by covalently linking a cholesterol moiety to the 5'- end (DT01 or AsiDNA) (WO2011/161075; Berthault et al, 2011, Cancer gene therapy, 18, 695-706).
  • DT01 or AsiDNA 5'- end
  • the inventors characterized potential predictive biomarkers for treatment with Dbait molecules. Sensitivity to Dbait molecules was associated with a high spontaneous frequency of cells with micronuclei (MN) and the number of large-scale chromosomal rearrangements (LSTs). A high basal level of MN as a predictive biomarker for treatment with Dbait molecules, especially with AsiDNA, was validated in 43 solid tumor cell lines from various tissues and 16 models of cell- and patient-derived xenografts. MN quantification was also possible in patient biopsies. Overall, this study identified genetic instability as a predictive biomarker for sensitivity to treatment with Dbait molecules, especially with AsiDNA. The inventors have also shown on several hematologic cell lines that MN quantification is also useful for predicting sensitivity to treatment with Dbait molecules, especially with AsiDNA.
  • the present invention relates to the use of genetic instability as measured by a frequency of cells with micronuclei and/or a number of large-scale chromosomal rearrangements (LSTs) as a predictive biomarker of a sensitivity or resistance to a treatment of cancer with a nucleic acid molecule, said nucleic acid molecule having at least one free end and comprising a hairpin with a DNA double stranded portion of 24-200 bp with less than 60% sequence identity to any gene in a human genome.
  • LSTs large-scale chromosomal rearrangements
  • the present invention also relates to a method for determining the sensitivity or resistance of a subject having a cancer to a treatment with a nucleic acid molecule, said nucleic acid molecule having at least one free end and comprising a hairpin with a DNA double stranded portion of 24-200 bp with less than 60% sequence identity to any gene in a human genome, wherein the method comprises determining a frequency of cells with micronuclei and/or a number of large-scale chromosomal rearrangements (LSTs) in a biological sample from the subject, the frequency of cells with micronuclei and/or the number of large-scale chromosomal rearrangements (LSTs) being positively correlating to the sensitivity of the subject to a treatment of cancer with the nucleic acid molecule.
  • LSTs large-scale chromosomal rearrangements
  • the present invention further relates to a nucleic acid molecule having at least one free end and comprising a hairpin with a DNA double stranded portion of 24-200 bp with less than 60% sequence identity to any gene in a human genome for use for treating a cancer in a subject having a high genetic instability as measured by a frequency of cells with micronuclei and/or a number of large-scale chromosomal rearrangements (LSTs).
  • the nucleic acid molecule has one of the following formulae:
  • N is a deoxynucleotide
  • n is an integer from 19 to 195
  • the underlined N refers to a nucleotide having or not a modified phosphodiester backbone
  • L' is a linker
  • C is the molecule facilitating endocytosis selected from a lipophilic molecule or a ligand which targets cell receptor enabling receptor mediated endocytosis
  • L is a linker
  • m and p independently, are an integer being 0 or 1.
  • nucleic acid molecule of formula (I), (II) or (III) has one or several of the following features:
  • - n is an integer from 23 to 195 or from 27 to 95, and/or
  • - N is a deoxynucleotide selected from the group consisting of A (adenine), C (cytosine), T (thymine) and G (guanine) and selected so as to avoid occurrence of a CpG dinucleotide and to have less than 80% sequence identity to any gene in a human genome.; and/or,
  • the linked L' is selected from the group consisting of hexaethyleneglycol, tetradeoxythymidylate (T4), l,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane and 2,19-bis(phosphor)-8-hydraza- l-hydroxy-4-oxa-9-oxo-nonadecane; and/or,
  • L is a carboxamido polyethylene glycol, more preferably carboxamido triethylene glycol or carboxamido tetraethylene glycol; and/or,
  • - C is selected from the group consisting of a cholesterol, single or double chain fatty acids such as octadecyl, oleic acid, dioleoyl or stearic acid, or ligand (including peptide, protein, aptamer) which targets cell receptor such as folic acid, tocopherol, sugar such as galactose and mannose and their oligosaccharide, peptide such as RGD and bombesin, and protein such transferring and integrin, preferably is a cholesterol or a tocopherol, still more preferably a cholesterol.
  • ligand including peptide, protein, aptamer which targets cell receptor such as folic acid, tocopherol, sugar such as galactose and mannose and their oligosaccharide, peptide such as RGD and bombesin, and protein such transferring and integrin, preferably is a cholesterol or a tocopherol, still more preferably a cholesterol.
  • the biological sample of said subject is a cancer sample, in particular a tumor biopsy or a biological fluid comprising cancer cells.
  • the frequency of cells with micronuclei can be determined by counting the number of cells having micronuclei in a cancer sample.
  • a frequency higher than 1%, preferably higher than 2%, more preferably higher than 2.5 or 3% is indicative of a sensitivity to the treatment with said nucleic acid molecule.
  • the number of large-scale chromosomal rearrangements can be determined with a SNP-array.
  • the number of large-scale chromosomal rearrangements (LSTs) may correspond to the number, per genome, of breakpoints resulting in segments of at least 3, 4, 5, 6, 7, 8 9, 10 megabases, preferably at least 10 megabases.
  • a number of large-scale chromosomal rearrangements (LSTs) of at least 10 large-scale chromosomal rearrangements (LSTs), preferably at least 15, is indicative of a sensitivity to the treatment with said nucleic molecule.
  • the method may further comprise selecting the subject as for determining the sensitivity or resistance of a subject having a cancer to a treatment with said nucleic acid molecule.
  • the present invention relates to the use of a kit comprising means for determining the frequency of micronuclei in a cell population or for determining the number of large-scale chromosomal rearrangements (LSTs) for determining the sensitivity or resistance of a subject having a cancer to a treatment with a nucleic acid molecule, for selecting a subject affected with a cancer or tumor for a treatment with a nucleic acid molecule or for determining whether a subject affected with a cancer or tumor is susceptible to benefit from a treatment with said nucleic acid molecule.
  • LSTs large-scale chromosomal rearrangements
  • the present invention relates to the use of genetic instability as a biomarker for the sensitivity of a subject to a treatment with DBait molecules. It also relates to a method for determining the sensitivity of a subject having a cancer to a treatment with DBait molecules comprising determination the level of genetic instability in a sample from the subject, the genetic instability being positively correlated with the sensitivity of the subject having a cancer to a treatment with DBait molecules.
  • the method may further comprise administering DBait molecules to the selected subject, alone or in combination with other treatments.
  • a DBait molecule, alone or in combination with other treatments for use in the treatment of a cancer or tumor in a subject having a high level of genetic instability.
  • a method for treating a cancer in a subject comprising administering a therapeutic effective amount of a DBait molecule, alone or in combination with other treatments, to a subject having a high level of genetic instability. It also relates to the use of a DBait molecule for the manufacture of a drug for treating a cancer in a subject having a high level of genetic instability, optionally in combination with other treatments.
  • the term "marker” and “biomarker” are interchangeable and refer to biological parameters that aid the selection of patients who will benefit from a specific treatment. This term refers particularly to "tumor biomarkers”. It is a measurable indicator for predicting the responsiveness or sensitivity of a patient to a specific treatment, in particular a treatment with DBait molecules.
  • a biomarker can be detected in the blood, urine, stool, tumor tissue, or other tissues or bodily fluids of some patients with cancer, in particular in a tumor tissue.
  • the terms “subject”, “individual” or “patient” are interchangeable and refer to an animal, preferably to a mammal, even more preferably to a human.
  • the term “patient” can also refer to non-human animals, in particular mammals such as dogs, cats, horses, cows, pigs, sheep and non- human primates, among others, that are in need of a treatment.
  • the genetic instability is measured in a sample from the subject having a cancer.
  • the sample is a cancer sample.
  • cancer sample refers to any biological sample containing tumoral cells derived from the patient.
  • tumoral cells may be obtained from fluid sample such as blood, plasma, urine, cerebrospinal fluid and seminal fluid samples as well as from biopsies, organs, tissues or cell samples.
  • tumoral cells are obtained from tumor biopsy or resection sample from the patient.
  • the sample contains only tumoral cells.
  • the method may comprise an initial step of providing a sample from the subject, preferably a tumor or cancer sample.
  • a sample from the subject preferably a tumor or cancer sample.
  • two samples may be provided from the same patient, a normal and a tumor samples.
  • the tumor and normal samples are histologically matched tissues.
  • the genetic instability can be measured by the frequency of cells with micronuclei and/or by the number of large-scale chromosomal rearrangements (LSTs). Accordingly, in a first embodiment, the genetic instability is measured by the frequency of cells with micronuclei. Criteria for identifying micronuclei are well-established, for instance as described in Countryman & Heddle (1976, Mutation Research, 41, 321-332). In particular, the criteria are the followings:
  • the present invention relates to the use of a frequency of cells with micronuclei as a biomarker for the sensitivity of a subject to a treatment with DBait molecules. It also relates to a method for determining the sensitivity of a subject having a cancer to a treatment with DBait molecules comprising determination the frequency of cells with micronuclei in a sample from the subject, the frequency of cells with micronuclei being positively correlated with the sensitivity of the subject having a cancer to a treatment with DBait molecules.
  • the method may further comprise administering DBait molecules to the selected subject, alone or in combination with other treatments.
  • a DBait molecule, alone or in combination with other treatments for use in the treatment of a cancer or tumor in a subject having a high frequency of cells with micronuclei.
  • a method for treating a cancer in a subject comprising administering a therapeutic effective amount of a DBait molecule, alone or in combination with other treatments, to a subject having a high frequency of cells with micronuclei. It also relates to the use of a DBait molecule for the manufacture of a drug for treating a cancer in a subject having a high frequency of cells with micronuclei, optionally in combination with other treatments.
  • the determination of the frequency of cells with micronuclei can be measured by the proportion of cells that contain micronuclei in a sample, in particular a tissue sample, preferably a tumor or cancer sample.
  • the determination of the frequency of cells with micronuclei is carried out by the cytokinesis-block micronucleus (CBMN) assay.
  • CBMN cytokinesis-block micronucleus
  • This assay has been described in Fenech and Morley (1985, Mutation Res., 148, 29-36) and been improved as disclosed in WO02/14859 (see also, Fenech, 2007, Nature Protocols, 2, 1084-1104), the disclosure thereof being incorporated herein by reference.
  • the frequency of cells with micronuclei is determined on cells having undergone one cellular division, more particular only one division.
  • the cells of the sample are incubated with a cytokinesis blocking agent such as cytochalasin-B.
  • a cytokinesis blocking agent such as cytochalasin-B.
  • This cytokinesis blocking agent prevents cells from separating into daughter nuclei, thereby allowing unambiguous identification of cells having undergone one or more cellular divisions.
  • the method may comprise a step of either determining the proportion of cells having undergone one cellular division or of sorting the cells for obtaining a population of cells having undergone one cellular division.
  • the counting of the number of cells having micronuclei can be determined under microscope after appropriate staining.
  • the micronuclei are clearly smaller than the cell nuclei and are physically separate from them.
  • the counting can be carried out by visual scoring or by automated scoring procedures. Prototypes of software for automated image analysis have been described in the literature since the early 1990s and developed by Bee ton-Dickinson and Loats Associates, Inc (LAI Automated Micronucleus Assay System;. 201 East Main St. Riverside, MD 21157 410-876-8055). Alternative methods are disclosed in WO02/14859 and WO2010/068799.
  • the method for determining the frequency of cells with micronuclei comprises lysing cells of the sample to release nuclei and micronuclei, counting the nuclei and micronuclei, and determining the frequency of cells with micronuclei.
  • the lysis can be achieved by any known procedures, such as those described in Nusse and Marx (1997, Mutat Res. 392(1-2): 109-15), the disclosure of which being incorporated by reference.
  • the nuclei and micronuclei can be analyzed by flow cytometry. When nuclei and micronuclei are analyzed by flow cytometry, the nuclei and micronuclei are stained with a dye prior to flow cytometry.
  • nuclei and micronuclei can be performed by flow cytometry as described in Nusse and Marx (1997, Mutat Res. 392(1-2): 109-15), the disclosure of which being incorporated by reference.
  • nuclei may be distinguished from micronuclei by their greater light scatter and greater DNA content.
  • the frequency of cells with micronuclei is calculated as the micronuclei frequency per nuclei.
  • the micronuclei and nuclei can be stained by any appropriate dye.
  • the dye is a fluorescent dye.
  • the fiorescent dye can be selected from the group consisting of DAPI (4',6-diamidino-2-phenylindole), propidium iodide, Hoechst 33342, carboxyfluorescein diacetate succinimidyl ester (CFSE) and dyes of the PKH series.
  • the dye can be selected from the group consisting of DAPI (4',6-diamidino-2- phenylindole), propidium iodide and Hoechst 33342. It will be appreciated by those skilled in the art that a number of alternative nucleic acid dyes may also be used.
  • a method for determining the frequency of micronuclei can comprise: incubating cells from the sample for a sufficient time to allow a substantial population of cells to complete at least one cellular division;
  • the threshold between high frequency and low frequency can be determined by the person skilled in the art, especially as detailed in the examples. Accordingly, the status of high or low frequency is determined by comparing the frequency to the threshold and the frequency is high if it is greater than the threshold and is low if it is lower than the threshold.
  • a threshold can be defined for each kind of cancer. For instance, a frequency higher than 1%, preferably higher than 2%, more preferably higher than 2.5%, still more preferably higher than 3%, is indicative of a sensitivity to the treatment with Dbait molecules.
  • a frequency lower than 3%, preferably lower than 2.5%, more preferably lower than 2%, still more preferably lower than 1, 0.75 or 0.5%, is indicative of a resistance to the treatment with Dbait molecules.
  • 1 % is intended to refer to a sample with 1 % of cells having micronuclei.
  • the frequency is determined by analyzing at least 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 cells.
  • a frequency of cells with micronuclei higher than 1%, preferably higher than 2%, more preferably higher than 2.5%, still more preferably higher than 3% can be considered as a high frequency of cells with micronuclei for selecting a subject as suitable for being treated with DBait molecules or for determining whether a subject affected with a cancer or tumor is susceptible to benefit from such a treatment.
  • the genetic instability is measured by the number of large-scale chromosomal rearrangements (LSTs).
  • the present invention relates to the use of a number of large-scale chromosomal rearrangements (LSTs) as a biomarker for the sensitivity of a subject to a treatment with DBait molecules. It also relates to a method for determining the sensitivity of a subject having a cancer to a treatment with DBait molecules comprising determination the number of large-scale chromosomal rearrangements (LSTs) in a sample from the subject, the number of large-scale chromosomal rearrangements (LSTs) being positively correlated with the sensitivity of the subject having a cancer to a treatment with DBait molecules.
  • LSTs large-scale chromosomal rearrangements
  • the method may further comprise administering DBait molecules to the selected subject, alone or in combination with other treatments.
  • a DBait molecule for use in the treatment of a cancer or tumor in a subject having a high number of large-scale chromosomal rearrangements (LSTs). Accordingly, it relates to a method for treating a cancer in a subject, comprising administering a therapeutic effective amount of a DBait molecule, alone or in combination with other treatments, to a subject having a high number of large-scale chromosomal rearrangements (LSTs). It also relates to the use of a DBait molecule for the manufacture of a drug for treating a cancer in a subject having a high number of large-scale chromosomal rearrangements (LSTs), optionally in combination with other treatments.
  • LSTs large-scale chromosomal rearrangements
  • the method of determining the number of large-scale chromosomal rearrangements (LSTs) in a sample comprises the step of quantifying the number of rearrangements in the genomic DNA of the tumor or cancer sample obtained from the subject, wherein the number of rearrangements corresponds to the number, per genome, of breakpoints resulting in segments of at least 3 megabases, preferably at least 4 megabases, more preferably at least 5, 6, 7, 8 9, 10, 11 12, 13, 14, 15, 16, 17, 18, 19 or 20 megabases, still more preferably from at least 3 to 11 megabases, even more preferably at least 10 megabases.
  • the number of rearrangements corresponds to the number, per genome, of breakpoints resulting in segments of at least 10 megabases.
  • the step of quantifying the number of rearrangements per genome in the genomic DNA of the sample can be performed by any suitable method in the art.
  • the step of quantifying rearrangements is carried out by sequencing techniques, such as next- generation sequencing using mate paired libraries, or longer reads (e.g., Stephens et al, 2009, Nature, 462, 1005-1010).
  • the step of quantifying rearrangements is performed by quantifying the number of copy number variations per genome. Typically, this can be done by hybridization techniques such as comparative genomic hybridization (CGH) array and Single Nucleotide Polymorphism (SNP) array.
  • CGH comparative genomic hybridization
  • SNP Single Nucleotide Polymorphism
  • Suitable methods for quantifying rearrangements include, but are not limited to, those described in Le Scouarnec and Gribble, Heredity, 2012, 108, 75-85.
  • the number of rearrangements per genome is defined with a SNP (Single Nucleotide Polymorphism) array by determining absolute copy number (CN) and allelic content profiles, for instance by GAP methodology (Popova et al. Genome Biol. 2009; 10(11):R128; Popova et al. Cancer Res. 2012 Nov l ;72(21):5454-62). More preferably, the number of breakpoints in each genomic profile is estimated based on the resulting copy number profile and after filtering smaller tha 50 SNPs variation.
  • LSTs large-scale chromosomal rearrangements
  • the threshold between high number and low number of LSTs can be determined by the person skilled in the art, especially as detailed in the examples. Accordingly, the status of high or low number is determined by comparing the number of LSTs to the threshold and the number is high if it is greater than the threshold and is low if it is lower than the threshold. For instance, a threshold can be defined for each kind of cancer.
  • a number higher than 9, preferably higher than 10, for instance higher than 11, 12, 13, 14 or 15, is indicative of a sensitivity to the treatment with Dbait molecules.
  • a number of large-scale chromosomal rearrangements (LSTs) higher than 9, preferably higher than 10, more preferably higher than 11, 12, 13, 14 or 15, can be considered as a high number of large-scale chromosomal rearrangements (LSTs) for selecting a subject as suitable for being treated with DBait molecules or for determining whether a subject affected with a cancer or tumor is susceptible to benefit from such a treatment.
  • the threshold of the number of LSTs will depend on the size of the considered segment for determining the number of rearrangements (e.g., longer or shorter than 10 megabases). Therefore, the threshold will increase if the size of the considered segment for determining the number of rearrangements is smaller than 10 megabases and will decrease if the size of the considered segment for determining the number of rearrangements is greater than 10 megabases.
  • the genetic instability is measured by the combination of the frequency of cells with micronuclei and the number of large-scale chromosomal rearrangements (LSTs) as detailed above.
  • LSTs large-scale chromosomal rearrangements
  • the nucleic acid molecules have at least one free end and comprises a hairpin with a DNA double stranded portion of 24-200 bp with less than 60% sequence identity to any gene in a human genome.
  • the nucleic acid molecules, conjugated or not, can be described by the following formulae:
  • N is a nucleotide
  • n is an integer from 15 to 195
  • the underlined N refers to a nucleotide having or not a modified phosphodiester backbone
  • L' is a linker
  • C is a molecule facilitating endocytosis
  • L is a linker
  • m and p independently, are an integer being 0 or 1.
  • C-L m is respectively linked to the 5' end or the 3' end of the nucleotide.
  • C-L m is preferably linked to L' through a disulfide bond (S-S).
  • S-S disulfide bond
  • p is 1.
  • the underlined N refers to a nucleotide having a modified phosphodiester backbone.
  • the molecule of formula (I), (II) or (III) has one or several of the following features:
  • - N is a deoxynucleotide, preferably selected from the group consisting of A (adenine), C (cytosine), T (thymine) and G (guanine) and selected so as to avoid occurrence of a CpG dinucleotide and to have less than 80% or 70%, even less than 60% or 50% sequence identity to any gene in a human genome.; and/or,
  • n is an integer from 15 to 195, preferably from 23 to 195, or from 25 to 195, optionally from 15 to 195, from 19-95, from 21 to 95, from 23 to 95, from 25 to 95, from 27 to 95, from 15 to 45, from 19 to 45, from 21 to 45, or from 27 to 45.
  • n is 27; and/or,
  • the underlined N refers to a nucleotide having or not a phosphorothioate or methylphosphonate backbone, more preferably a phosphorothioate backbone; preferably, the underlined N refers to a nucleotide having a modified phosphodiester backbone; and/or,
  • the linked L' is selected from the group consisting of hexaethyleneglycol, tetradeoxythymidylate (T4), l,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane and 2,19-bis(phosphor)-8-hydraza- 1 -hydroxy-4-oxa-9-oxo-nonadecane; and/or,
  • - m is 1 and L is a carboxamido polyethylene glycol, more preferably carboxamido triethylene glycol or carboxamido tetraethylene glycol; and/or, - C is selected from the group consisting of a cholesterol, single or double chain fatty acids such as octadecyl, oleic acid, dioleoyl or stearic acid, or ligand (including peptide, protein, aptamer) which targets cell receptor such as folic acid, tocopherol, sugar such as galactose and mannose and their oligosaccharide, peptide such as RGD and bombesin, and protein such transferring and integrin, preferably is a cholesterol or a tocopherol, still more preferably a cholesterol;
  • C-Lm is a triethyleneglycol linker (10-O-[l-propyl-3-N-carbamoylcholesteryl]- triethyleneglycol radical.
  • C-Lm is a tetraethyleneglycol linker (10-O-[l-propyl-3-N- carbamoylcholesteryl] -tetraethyleneglycol radical.
  • conjugated Dbait molecule or hairpin nucleic acid molecule has the following formula:
  • the nucleic acid molecules can be Dbait molecules such as those extensively described in PCT patent applications WO2005/040378, WO2008/034866 and WO2008/084087, the disclosure of which is incorporated herein by reference.
  • Dbait molecules may be defined by a number of characteristics necessary for their therapeutic activity, such as their minimal length, the presence of at least one free end, and the presence of a double stranded portion, preferably a DNA double stranded portion. As will be discussed below, it is important to note that the precise nucleotide sequence of Dbait molecules does not impact on their activity. Furthermore, Dbait molecules may contain a modified and/or non-natural backbone.
  • Dbait molecules are of non-human origin (i.e., their nucleotide sequence and/or conformation (e.g., hairpin) does not exist as such in a human cell), most preferably of synthetic origin.
  • sequence of the Dbait molecules plays little, if any, role, Dbait molecules have preferably no significant degree of sequence homology or identity to known genes, promoters, enhancers, 5'- or 3'- upstream sequences, exons, introns, and the like.
  • Dbait molecules have less than 80% or 70%, even less than 60% or 50% sequence identity to any gene in a human genome. Methods of determining sequence identity are well known in the art and include, e.g., Blast.
  • Dbait molecules do not hybridize, under stringent conditions, with human genomic DNA. Typical stringent conditions are such that they allow the discrimination of fully complementary nucleic acids from partially complementary nucleic acids.
  • sequence of the Dbait molecules is preferably devoid of CpG in order to avoid the well- known toll-like receptor-mediated immunological reactions.
  • the length of Dbait molecules may be variable, as long as it is sufficient to allow appropriate binding of Ku protein complex comprising Ku and DNA-PKcs proteins. It has been showed that the length of Dbait molecules must be greater than 20 bp, preferably about 32 bp, to ensure binding to such a Ku complex and allowing DNA-PKcs activation.
  • Dbait molecules comprise between 20-200 bp, more preferably 24-100 bp, still more preferably 26-100, and most preferably between 24-200, 25-200, 26-200, 27-200, 28-200, 30-200, 32-200, 24-100, 25-100, 26-100, 27-100, 28-100, 30-100, 32-200 or 32-100 bp.
  • Dbait molecules comprise between 24-160, 26-150, 28-140, 28-200, 30-120, 32-200 or 32-100 bp.
  • bp is intended that the molecule comprise a double stranded portion of the indicated length.
  • the Dbait molecules having a double stranded portion of at least 32 pb, or of about 32 bp comprise the same nucleotide sequence than Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5).
  • the Dbait molecules have the same nucleotide composition than Dbait32, Dbait32Ha, Dbait32Hb, Dbait32Hc or Dbait32Hd but their nucleotide sequence is different.
  • the Dbait molecules comprise one strand of the double stranded portion with 3 A, 6 C, 12 G and 11 T.
  • the sequence of the Dbait molecules does not contain any CpG dinucleotide.
  • the double stranded portion comprises at least 16, 18, 20, 22, 24, 26, 28, 30 or 32 consecutive nucleotides of Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5).
  • the double stranded portion consists in 20, 22, 24, 26, 28, 30 or 32 consecutive nucleotides of Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5).
  • the nucleic acid as disclosed herein must have at least one free end, as a mimic of DSB. Said free end may be either a free blunt end or a 5'-/3'- protruding end.
  • the "free end” refers herein to a nucleic acid molecule, in particular a double-stranded nucleic acid portion, having both a 5' end and a 3' end or having either a 3 'end or a 5' end.
  • one of the 5' and 3' end can be used to conjugate the nucleic acid molecule or can be linked to a blocking group, for instance a or 3'-3'nucleotide linkage.
  • the nucleic acid molecules contain two free ends and can be linear. Accordingly, Dbait molecules may also be a double stranded molecule with two free ends and having the nucleotide sequence of Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 1)
  • Dbait molecules are made of hairpin nucleic acids with a double-stranded DNA stem and a loop.
  • the loop can be a nucleic acid, or other chemical groups known by skilled person or a mixture thereof.
  • a nucleotide linker may include from 2 to 10 nucleotides, preferably, 3, 4 or 5 nucleotides.
  • Non-nucleotide linkers non exhaustively include abasic nucleotide, polyether, polyamine, poly amide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.
  • oligoethylene glycols such as those having between 2 and 10 ethylene glycol units, preferably 4, 5, 6, 7 or 8 ethylene glycol units).
  • a preferred linker is selected from the group consisting of hexaethyleneglycol, tetradeoxythymidylate (T4), 1,19- bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane and other linkers such as 2,19- bis(phosphor)-8-hydraza-l-hydroxy-4-oxa-9-oxo-nonadecane.
  • the Dbait molecules can be a hairpin molecule having a double stranded portion or stem comprising at least 16, 18, 20, 22, 24, 26, 28, 30 or 32 consecutive nucleotides of Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5) and a loop being a hexaethyleneglycol linker, a tetradeoxythymidylate linker (T4), 1,19- bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane or 2,19-bis(phosphor)-8-hydraza-l- hydroxy-4-oxa-9-oxo-nonadecane.
  • Dbait32 SEQ ID No 1
  • Dbait32Ha SEQ ID No 2
  • Dbait32Hb SEQ ID No 3
  • those Dbait molecules can have a double stranded portion consisting in 20, 22, 24, 26, 28, 30 or 32 consecutive nucleotides of Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5).
  • Dbait molecules preferably comprise a 2'-deoxynucleotide backbone, and optionally comprise one or several (2, 3, 4, 5 or 6) modified nucleotides and/or nucleobases other than adenine, cytosine, guanine and thymine. Accordingly, the Dbait molecules are essentially a DNA structure. In particular, the double-strand portion or stem of the Dbait molecules is made of deoxyribonucleotides.
  • Preferred Dbait molecules comprise one or several chemically modified nucleotide(s) or group(s) at the end of one or of each strand, in particular in order to protect them from degradation.
  • the free end(s) of the Dbait molecules is(are) protected by one, two or three modified phosphodiester backbones at the end of one or of each strand.
  • Preferred chemical groups, in particular the modified phosphodiester backbone comprise phosphorothioates.
  • preferred Dbait have 3'- 3' nucleotide linkage, or nucleotides with methylphosphonate backbone.
  • modified backbones are well known in the art and comprise phosphoramidates, morpholino nucleic acid, 2'-0,4'- C methylene/ethylene bridged locked nucleic acid, peptide nucleic acid (PNA), and short chain alkyl, or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intrasugar linkages of variable length, or any modified nucleotides known by skilled person.
  • the Dbait molecules have the free end(s) protected by one, two or three modified phosphodiester backbones at the end of one or of each strand, more preferably by three modified phosphodiester backbones (in particular phosphorothioate or methylphosphonate) at least at the 3 'end, but still more preferably at both 5' and 3' ends.
  • modified phosphodiester backbones in particular phosphorothioate or methylphosphonate
  • the Dbait molecule is a hairpin nucleic acid molecule comprising a DNA double-stranded portion or stem of 32 bp (e.g., with a sequence selected from the group consisting of SEQ ID Nos 1-5, in particular SEQ ID No 4) and a loop linking the two strands of the DNA double- stranded portion or stem comprising or consisting of a linker selected from the group consisting of hexaethyleneglycol, tetradeoxythymidylate (T4), l,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9- oxo-nonadecane and 2,19-bis(phosphor)-8-hydraza-l-hydroxy-4-oxa-9-oxo-nonadecane, the free ends of the DNA double-stranded portion or stem (i.e. at the opposite of the loop) having three modified phosphodiester backbones (in particular phosphorothioate internucle
  • nucleic acid molecules are made by chemical synthesis, semi-biosynthesis or biosynthesis, any method of amplification, followed by any extraction and preparation methods and any chemical modification.
  • Linkers are provided so as to be incorporable by standard nucleic acid chemical synthesis. More preferably, nucleic acid molecules are manufactured by specially designed convergent synthesis: two complementary strands are prepared by standard nucleic acid chemical synthesis with the incorporation of appropriate linker precursor, after their purification, they are covalently coupled together.
  • the nucleic acid molecules may be conjugated to molecules facilitating endocytosis or cellular uptake.
  • the molecules facilitating endocytosis or cellular uptake may be lipophilic molecules such as cholesterol, single or double chain fatty acids, or ligands which target cell receptor enabling receptor mediated endocytosis, such as folic acid and folate derivatives or transferrin (Goldstein et al. Ann. Rev. Cell Biol. 1985 1 : 1-39; Leamon & Lowe, Proc Natl Acad Sci USA. 1991, 88: 5572-5576.).
  • the molecule may also be tocopherol, sugar such as galactose and mannose and their oligosaccharide, peptide such as RGD and bombesin and protein such as integrin.
  • Fatty acids may be saturated or unsaturated and be in C4-C28, preferably in C14-C22, still more preferably being in Ci8 such as oleic acid or stearic acid.
  • fatty acids may be octadecyl or dioleoyl.
  • Fatty acids may be found as double chain form linked with in appropriate linker such as a glycerol, a phosphatidylcholine or ethanolamine and the like or linked together by the linkers used to attach on the Dbait molecule.
  • folate is meant to refer to folate and folate derivatives, including pteroic acid derivatives and analogs.
  • the analogs and derivatives of folic acid suitable for use in the present invention include, but are not limited to, antifolates, dihydrofolates, tetrahydrofolates, folinic acid, pteropolyglutamic acid, 1- deza, 3-deaza, 5-deaza, 8-deaza, 10-deaza, 1,5-deaza, 5,10 dideaza, 8,10-dideaza, and 5,8-dideaza folates, antifolates, and pteroic acid derivatives. Additional folate analogs are described in US2004/242582.
  • the molecule facilitating endocytosis may be selected from the group consisting of single or double chain fatty acids, folates and cholesterol. More preferably, the molecule facilitating endocytosis is selected from the group consisting of dioleoyl, octadecyl, folic acid, and cholesterol. In a most preferred embodiment, the nucleic acid molecule is conjugated to a cholesterol.
  • the molecules facilitating endocytosis are conjugated to Dbait molecules, preferably through a linker. Any linker known in the art may be used to covalently attach the molecule facilitating endocytosis to Dbait molecules For instance, WO09/126933 provides a broad review of convenient linkers pages 38- 45.
  • the linker can be non-exhaustively, aliphatic chain, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon , or other polymeric compounds (e. g. oligoethylene glycols such as those having between 2 and 10 ethylene glycol units, preferably 3, 4, 5, 6, 7 or 8 ethylene glycol units, still more preferably 6 ethylene glycol units), as well as incorporating any bonds that may be break down by chemical or enzymatical way, such as a disulfide linkage, a protected disulfide linkage, an acid labile linkage (e.g., hydrazone linkage), an ester linkage, an ortho ester linkage, a phosphonamide linkage, a biocleavable peptide linkage, an azo linkage or an aldehyde linkage.
  • cleavable linkers are detailed in WO2007/040469 pages 12-14, in WO2008/022309 pages 22-28.
  • the nucleic acid molecule can be linked to one molecule facilitating endocytosis.
  • several molecules facilitating endocytosis e.g., two, three or four
  • the linker between the molecule facilitating endocytosis, in particular cholesterol, and nucleic acid molecule is wherein n is an integer from 1 to 10,
  • the linker is (carboxamido tetraethylene glycol). In another very particular embodiment, the linker is (carboxamido triethylene glycol).
  • the linker can be
  • the linker can be linked at the 5' end, at the 3' end or in the loop when the nucleic acid molecule is a hairpin. Therefore, in a preferred embodiment, the contemplated conjugated Dbait molecule is a Dbait molecule having a hairpin structure and being conjugated to the molecule facilitating endocytosis, preferably through a linker, at its 5' end.
  • the linker between the molecule facilitating endocytosis, in particular cholesterol, and nucleic acid molecule is dialkyl -disulfide ⁇ e.g., with r and s being
  • the conjugated Dbait molecule is a hairpin nucleic acid molecule comprising a DNA double-stranded portion or stem of 32 bp (e.g., with a sequence selected from the group consisting of SEQ ID Nos 1-5, in particular SEQ ID No 4) and a loop linking the two strands of the DNA double-stranded portion or stem comprising or consisting of a linker selected from the group consisting of hexaethyleneglycol, tetradeoxythymidylate (T4), l,19-bis(phospho)-8-hydraza-2- hydroxy-4-oxa-9-oxo-nonadecane and 2,19-bis(phosphor)-8-hydraza-l-hydroxy-4-oxa-9-oxo- nonadecane, the free ends of the DNA double-stranded portion or stem (i.e.
  • a linker e.g. carboxamido oligoethylene glycol, preferably carboxamido triethylene glycol or carboxamido tetraethylene glycol.
  • NNNN-(N) disregard-N comprises at least 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 consecutive nucleotides of Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5) or consists in 20, 22, 24, 26, 28, 30 or 32 consecutive nucleotides of Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5).
  • NNNN-(N)n-N comprises or consists in Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5), more preferably Dbait32Hc (SEQ ID No 4).
  • conjugated Dbait molecule or hairpin nucleic acid molecule may be selected from the group consisting of:
  • the underlined nucleotide refers to a nucleotide having or not a phosphorothioate or methylphosphonate backbone, more preferably a phosphorothioate backbone; preferably, the underlined nucleotide refers to a nucleotide having a phosphorothioate or methylphosphonate backbone, more preferably a phosphorothioate backbone and/or,
  • the linked L' is selected from the group consisting of hexaethyleneglycol, tetradeoxythymidylate (T4), l,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane and 2,19-bis(phosphor)-8-hydraza- l-hydroxy-4-oxa-9-oxo-nonadecane; and/or,
  • L is a carboxamido polyethylene glycol, more preferably carboxamido triethylene glycol or carboxamido tetraethylene glycol; and/or,
  • C-Lm is selected from the group consisting of a cholesterol, single or double chain fatty acids such as octadecyl, oleic acid, dioleoyl or stearic acid, or ligand (including peptide, protein, aptamer) which targets cell receptor such as folic acid, tocopherol, sugar such as galactose and mannose and their oligosaccharide, peptide such as RGD and bombesin, and protein such transferring and integrin, preferably is a cholesterol.
  • C-Lm is a triethyleneglycol linker (10-O-[l-propyl-3-N-carbamoylcholesteryl]- triethyleneglycol radical.
  • C-Lm is a tetraethyleneglycol linker (10-O-[l-propyl-3-N- carbamoylcholesteryl] -tetraethyleneglycol radical.
  • T4 tetradeoxythymidylate
  • the conjugated Dbait molecule or hairpin nucleic acid molecule is selected from the roup consisting of (II), (IF), (Ila), (lib), (lie), (lid), and (He), wherein C-L m being the radical
  • L' is preferably selected from the group consisting of hexaethyleneglycol, tetradeoxythymidylate (T4), l,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane and 2,19-bis(phosphor)-8-hydraza-l-hydroxy-4-oxa-9-oxo-nonadecane, more preferably 2,19- bis(phosphor)-8-hydraza-l-hydroxy-4-oxa-9-oxo-nonadecane.
  • T4 tetradeoxythymidylate
  • T4 l,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane
  • 2,19-bis(phosphor)-8-hydraza-l-hydroxy-4-oxa-9-oxo-nonadecane more preferably 2,19- bis(phosphor)-8-hydraza-l-hydroxy-4-oxa-9-ox
  • the Dbait molecule or hairpin nucleic acid molecule has the following formula
  • L' is 2,19-bis(phosphor)-8-hydraza-l-hydroxy-4-oxa-9-oxo-nonadecane and wherein the underlined nucleotides have a phosphorothioate backbone. Accordingly, the molecule has the following structure and it is referred thereto in the Example section as "coDbait".
  • the conjugated Dbait molecule or hairpin nucleic acid molecule is selected from the group consisting of (II), (IF), (Ila), (lib), (lie), (lid), and (He), wherein C-L m being the tetraethyleneglycol linker (10-O-[l-propyl-3-N-carbamoylcholesteryl]-tetraethyleneglycol radical and wherein L' is preferably selected from the group consisting of hexaethyleneglycol, tetradeoxythymidylate (T4), l,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane and 2,19-bis(phosphor)-8-hydraza-l-hydroxy-4-oxa-9-oxo-nonadecane, more preferably 1,19- bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane.
  • C-L m being the te
  • C-L m is the tetraethyleneglycol linker (10-O-[l-propyl-3-N-carbamoylcholesteryl]- tetraethyleneglycol radical, and L' is l,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo- nonadecane.
  • SEQ ID No 21 with the underlined nucleotide having phosphorothioate internucleotide linkages.
  • the nucleic acid molecule has one of the following formulae
  • N is a deoxy nucleotide
  • n is an integer from 1 to 15
  • the underlined N refers to a nucleotide having or not a modified phosphodiester backbone
  • L' is a linker
  • C is a cholesterol
  • L is a linker
  • m is an integer being 0 or 1
  • p is 1.
  • the underlined N refers to a nucleotide having a modified phosphodiester backbone.
  • the nucleic acid molecule as the formula (II).
  • the present invention also relates the use of a Dbait molecule or a nucleic acid molecule as disclosed above, a pharmaceutical composition comprising it and optionally a pharmaceutically acceptable carrier, for use in the treatment of cancer in combination with a PARP inhibitor, and/or with radiotherapy and/or radioisotope therapy and/or an antitumor chemotherapy, preferably with a DNA damaging antitumoral agent, as detailed below.
  • the treatment with a nucleic acid molecule as disclosed herein and a PARP inhibitor can be used in combination with a radiotherapy, a radioisotope therapy and/or another antitumor chemotherapy, immunotherapy, or hormonal therapy.
  • the antitumor chemotherapy is a treatment by a DNA damaging antitumor agent, either directly or indirectly.
  • the term "antitumor chemotherapy” or “chemotherapy” refers to a cancer therapeutic treatment using chemical or biochemical substances, in particular using one or several antineoplastic agents. In particular, it also includes hormonal therapy and immunotherapy.
  • hormonal therapy refers to a cancer treatment having for purpose to block, add or remove hormones. For instance, in breast cancer, the female hormones estrogen and progesterone can promote the growth of some breast cancer cells. So in these patients, hormone therapy is given to block estrogen and a non-exhaustive list commonly used drugs includes: Tamoxifen, Fareston, Arimidex, Aromasin, Femara, Zoladex/Lupron, Megace, and Halotestin.
  • immunotherapy refers to a cancer therapeutic treatment using the immune system to reject cancer. The therapeutic treatment stimulates the patient's immune system to attack the malignant tumor cells.
  • the nucleic acid molecule as disclosed herein and PARP inhibitor are used in combination with a DNA-damaging treatment.
  • the DNA-damaging treatment can be radiotherapy, or chemotherapy with a DNA-damaging antitumoral agent, or a combination thereof.
  • DNA-damaging treatment refers to a treatment inducing DNA strand breakage, preferably relatively specifically in cancer cells. DNA strand breakage can be achieved by ionized radiation (radiotherapy). Radiotherapy includes, but is not limited to, ⁇ -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other radiotherapies include microwaves and UV -irradiation. Other approaches to radiation therapy are also contemplated in the present invention.
  • DNA strand breakage can be achieved by radioisotope therapy, in particular by administration of a radioisotope, preferably a targeted radioisotope.
  • a radioisotope preferably a targeted radioisotope.
  • Targeting can be due to the chemical properties of the isotope such as radioiodine which is specifically absorbed by the thyroid gland a thousand fold better than other organs.
  • the targeting can be achieved by attaching to the radioisotope another molecule having targeting properties such hapten or antibody.
  • radioactive isotopes can be used, including, but not limited to, Indium-111, Lutetium-171, Bismuth-212, Bismuth- 213, Astatine-211, Copper-62, Copper-64, Copper-67, Yttrium-90, Iodine-125, Iodine-131, Phosphorus-32, Phosphorus-33, Scandium-47, Silver-I l l, Gallium-67, Praseodymium- 142, Samarium- 153, Terbium-161, Dysprosium-166, Holmium-166, Rhenium-186, Rhenium-188, Rhenium-189, Lead- 212, Radium-223, Actinium-225, Iron-59, Selenium-75, Arsenic-77, Strontium-89, Molybdenum-99, Rhodium-105, Palladium- 109, Praseodymium- 143, Promethium-149, Erbium-169
  • the DNA-damaging antitumor agent is preferably selected from the group consisting of an inhibitor of topoisomerases I or II, a DNA crosslinker, a DNA alkylating agent, an anti-metabolic agent and inhibitors of the mitotic spindles.
  • Inhibitors of topoisomerases I and/or II include, but are not limited to, etoposide, topotecan, camptothecin, irinotecan, amsacrine, intoplicine, anthracyclines such as doxorubicine, epirubicine, daunorubicine, idanrubicine and mitoxantrone.
  • Inhibitors of Topoisomerase I and II include, but are not limited to, intoplecin.
  • DNA crosslinkers include, but are not limited to, cisplatin, carboplatin and oxaliplatin.
  • Anti-metabolic agents block the enzymes responsible for nucleic acid synthesis or become incorporated into DNA, which produces an incorrect genetic code and leads to apoptosis.
  • Non-exhaustive examples thereof include, without limitation, folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors, and more particularly Methotrexate, Floxuridine, Cytarabine, 6- Mercaptopurine, 6- Thioguanine, Fludarabine phosphate, Pentostatine, 5-fiuorouracil, gemcitabine and capecitabine.
  • the DNA-damaging anti-tumoral agent can be alkylating agents including, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, metal salts and triazenes.
  • Non- exhaustive examples thereof include Uracil mustard, Chlormethine, Cyclophosphamide (CYTOXAN(R)), Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine, Triethylenethiophosphor amine, Busulfan, Carmustine, Lomustine, Fotemustine, cisplatin, carboplatin, oxaliplatin, thiotepa, Streptozocin, dacarbazine, and Temozolomide.
  • Inhibitors of the mitotic spindles include, but are not limited to, paclitaxel, docetaxel, vinorelbine, larotaxel (also called XRP9881 ; Sanofi-Aventis), XRP6258 (Sanofi-Aventis), BMS-184476 (Bristol- Meyer-Squibb), BMS-188797 (Bristol-Meyer-Squibb), BMS-275183 (Bristol-Meyer-Squibb), ortataxel (also called IDN 5109, BAY 59-8862 or SB-T-101131 ; Bristol-Meyer-Squibb), RPR 109881 A (Bristol- Meyer-Squibb), RPR 116258 (Bristol-Meyer-Squibb), NBT-287 (TAPESTRY), PG-paclitaxel (also called CT-2103, PPX, paclitaxel poliglumex, paclitaxel polyglutamate or
  • PARP inhibitor refers to any compound which has the ability to decrease the activity of a poly (ADP-ribose) polymerase (PARP).
  • PARP inhibition relies mainly on two different mechanisms: (i) catalytic inhibition that act mainly by inhibiting PARP enzyme activity and (ii) bound inhibition that block PARP enzyme activity and prevent its release from the damage site. Bound inhibitors are more toxic to cells than catalytic inhibitors.
  • PARP inhibitors according to the inventions are preferably catalytic and/or bound inhibitors.
  • the PARP inhibitor is an inhibitor of any enzyme of the PARP family, preferentially PARP1 and/or PARP2.
  • the PARP activity can be determined by a variety of techniques well known by the skilled person. Usually, these techniques comprise measuring the incorporation of a labeled poly(ADP-ribose) onto histone proteins. Commercial kits for such techniques are available (see for example, Tervigen's kits with biotinylated poly(ADP-ribose)). Other methods may also be used such as the one developed by Putt KS et al (Anal Biochem, 326(l):78-86, 2004), the disclosure of which is hereby incorporated by reference in his entirety. These methods are ideal for the determination of IC50 values of known or suspected PARP inhibitors.
  • PARP inhibitors are known and, thus, can be synthesized by known methods from starting materials that are known, may be available commercially, or may be prepared by methods used to prepare corresponding compounds in the literature.
  • Suitable PARP inhibitors include, but are not limited to, olaparib (AZD-2281 , 4-[(3-[(4-cyclopropylcarbonyl)piperazin-4-yl]carbonyl)-4-fiuorophenyl]methyl(2H)- phthalazin-l-one), veliparib (ABT-888, CAS 912444-00-9, 2-((fi)-2-methylpyrrolidin-2-yl)-lW- benzimidazole-4-carboxamide), CEP-8983 (ll-methoxy-4,5,6,7-tetrahydro-lH- cyclopenta[a]pyrrolo[3,4-c]carbazole-l,3(2H)-dione) or a prodrug thereof (e.g.
  • the PARP inhibitor compound is selected from the group consisting of rucaparib (AG014699, PF-01367338), olaparib (AZD2281), veliparib (ABT888), iniparib (BSI 201), niraparib (MK 4827), talazoparib (BMN673), AZD 2461, CEP 9722, E7016, INO-1001, LT-673, MP- 124, NMS-P118, XAV939, analogs, derivatives or a mixture thereof.
  • the PARP inhibitor is selected from the group consisting of rucaparib (AG014699, PF-01367338), olaparib (AZD2281), veliparib (ABT888), iniparib (BSI 201), niraparib (MK 4827), talazoparib (BMN673), AZD 2461, analogs, derivatives or a mixture thereof.
  • the term treatment denotes curative, symptomatic, and preventive treatment.
  • Pharmaceutical compositions, kits, products and combined preparations of the invention can be used in humans with existing cancer or tumor, including at early or late stages of progression of the cancer.
  • the pharmaceutical compositions, kits, products and combined preparations of the invention will not necessarily cure the patient who has the cancer but will delay or slow the progression or prevent further progression of the disease, ameliorating thereby the patients' condition.
  • the pharmaceutical compositions, kits, products and combined preparations of the invention reduce the development of tumors, reduce tumor burden, produce tumor regression in a mammalian host and/or prevent metastasis occurrence and cancer relapse.
  • the pharmaceutical composition of the invention is administered in a therapeutically effective amount.
  • treatment of a cancer or the like is mentioned with reference to the pharmaceutical composition of the invention, there is meant: a) a method for treating a cancer, said method comprising administering a pharmaceutical composition of the invention to a subject in need of such treatment; b) the use of a pharmaceutical composition of the invention for the treatment of a cancer; c) the use of a pharmaceutical composition of the invention for the manufacture of a medicament for the treatment of a cancer; and/or d) a pharmaceutical composition of the invention for use in the treatment a cancer.
  • cancer refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, and/or immortality, and/or metastatic potential, and/or rapid growth and/or proliferation rate, and/or certain characteristic morphological features. This term refers to any type of malignancy (primary or metastases) in any type of subject. In particular, the term encompasses renal cancer at any stage of progression. Examples of cancer include, for example, leukemia, lymphoma, blastoma, carcinoma and sarcoma.
  • cancers include chronic myeloid leukemia, acute lymphoblastic leukemia, Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL), squamous cell carcinoma, small-cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatocarcinoma, breast cancer, colon carcinoma, and head and neck cancer, gastric cancer, germ cell tumor, pediatric sarcoma, multiple myeloma, acute myelogenous leukemia (AML), chronic lymphocytic leukemia, mastocytosis and any symptom associated with mastocytosis.
  • AML acute myelogenous leukemia
  • Leukemia refers to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease— acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number of abnormal cells in the blood— leukemic or aleukemic (subleukemic).
  • Leukemia includes, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell le
  • carcinoma including that of the bladder (including accelerated and metastatic bladder cancer), breast, colon (including colorectal cancer), kidney, liver, lung (including small and non-small cell lung cancer and lung adenocarcinoma), ovary, prostate, testis, genitourinary tract, lymphatic system, rectum, larynx, pancreas (including exocrine pancreatic carcinoma), esophagus, stomach, gall bladder, cervix, thyroid, and skin (including squamous cell carcinoma); hematopoietic tumors of lymphoid lineage including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T- cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, histiocytic lymphoma, and Burketts
  • the cancer is a hematopoietic cancer, especially a leukemia or lymphoma.
  • the cancer is a solid tumor.
  • the cancer may be sarcoma and oestosarcoma such as Kaposi sarcome, AIDS-related Kaposi sarcoma, melanoma, in particular ulveal melanoma, and cancers of the head and neck, kidney, ovary, pancreas, prostate, thyroid, lung, esophagus, breast, bladder, colorectum, liver and biliary tract, uterine, appendix, and cervix, testicular cancer, gastrointestinal cancers and endometrial and peritoneal cancers.
  • the cancer may be sarcoma, melanoma, in particular ulveal melanoma, and cancers of the head and neck, kidney, ovary, pancreas, prostate, thyroid, lung, esophagus, breast, bladder, colorectum, liver, cervix, and endometrial and peritoneal cancers.
  • the cancer may be selected from the group consisting of breast cancer, hepatocellular carcinoma, colorectal cancer, glioblastoma, melanoma, and head and neck cancer.
  • the cancer is a breast cancer. Kits and uses thereof
  • the present invention finally relates to the use of a kit comprising means for determining the frequency of micronuclei in a cell population or for determining the number of large-scale chromosomal rearrangements (LSTs) for determining the sensitivity or resistance of a subject having a cancer to a treatment with a DBait molecule, for selecting a subject affected with a cancer or tumor for a treatment with a DBait molecule or for determining whether a subject affected with a cancer or tumor is susceptible to benefit from a treatment with a DBait molecule.
  • LSTs large-scale chromosomal rearrangements
  • FIG. 1 Spontaneous DNA damages and sensitivity to AsiDNA in breast cancer (BC) cell lines.
  • A. Spontaneous DNA damages were monitored by alkaline comet assay.
  • Figure 3 Lack of a correlation between sensitivity of BC cells to AsiDNA and their proliferation rate. Survival (ordinate) was monitored by trypan blue staining and manual counting (10 days after treatment). Survival is expressed as the percentage of living cells relative to non-treated cells (NT). The doubling time (abscissa) was calculated by counting the cells during the 10 days of exponential growth. The correlation analysis between survival in the presence of AsiDNA and doubling time was performed using GraphPadPrism5 software and indicates that there is no significant correlation.
  • Figure 4. DNA repair and cell cycle pathway profiles of BC cells with a MN High or MN Low status.
  • Hierarchical clustering of the BC cell lines based on gene expression values of 13 DNA repair and cell cycle genes. Clustering was done using Euclidean distance and Ward linkage. The color intensity indicates the level of change relative to the mean value of the BC cell line group.
  • FIG. 1 Micronuclei are predictive biomarkers of AsiDNA treatment.
  • mice bearing patient-derived xenografts (PDX) or cell-derived xenografts (CDX) were treated (black) or not (grey) with AsiDNA for 3 weeks.
  • Vt tumor volume at a given time;
  • Vi initial tumor volume before treatment.
  • TGD values are the mean of at least 6 xenografted mice per cancer type: black circles, glioblastoma; squares, uveal melanoma; diamonds, breast cancer; grey circle, colon cancer; black triangles, skin melanoma. *p ⁇ 0.05.
  • FIG. 7 Assessment of micronuclei in breast cancer patient biopsies.
  • A. Micronuclei (MN) quantification in 12 breast cancer (BC) patient' biopsies. Biopsies showing a % of cells with MN ⁇ 1% are considered MN Low and biopsies containing a % of cells with MN > 1% are considered MN Hlgh .
  • Micronuclei frequency is predictive biomarker for sensitivity to AsiDNA in hematopoietic cancer cell lines. Spearman correlation between AsiDNA efficiency (AsiDNA EC50, ⁇ ), and Micronuclei frequency of the different cells lines (expressed as the percentage of cells with micronuclei). P values are shown.
  • EXAMPLE 1 Genetic instability (MN and LSTs) as predictive biomarkers for treatment with AsiDNA in solid tumors.
  • MN and LSTs Genetic instability
  • the inventors characterized potential predictive biomarkers for treatment with AsiDNA, a first-in- class DNA repair inhibitor. Tumors showing spontaneous genetic instability are dependent on DNA repair to survive. Thus the inventors determined whether this intrinsic characteristic is a prerequisite for sensitivity to AsiDNA. They evaluated genetic instability and DNA repair defects by direct and indirect assays in 12 breast cancer cell lines to estimate the spontaneous occurrence of single-strand and double- strand breaks (DSBs).
  • DSBs single-strand and double- strand breaks
  • PAR Poly(ADP-Ribose)
  • DSBs are the most harmful type of DNA damage. Dbait inhibits both HR and NHEJ repair pathways, and should thus be toxic to cells with a high basal content of DSBs.
  • the inventors tested the persistence of broken chromosomes and abnormal chromosome segregation in BC cell cultures by investigating the presence of MN after DAPI staining. MN were identified as small nuclear bodies containing DNA and chromatin located in the vicinity of the nucleus (Fenech et al. Mutat Res. 1993 Jan;285(l):35-44). MN assays have been developed in human lymphocytes to measure both whole chromosome loss and chromosome breaks upon genotoxic exposure.
  • MN are also frequently found in some solid tumors (Bhatia et al. APMIS. 2013 Jul; 121(7):569-81).
  • the inventors analyzed the frequency of cells with MN in growing cultures. BC cell lines showed MN frequencies from 1 to 12% ( Figure 2B).
  • MN appear after cell division.
  • sensitivity of BC cell lines to AsiDNA or MN frequency and their rate of proliferation (Figure 3), indicating that sensitivity is due to defects in DSB repair (revealed by MN) and not a high rate of cell proliferation.
  • the inventors confirmed the MN data by performing genome analysis to estimate the number of chromosomal breakpoints within a tumor cell population genome by measuring the level of LSTs, corresponding mainly to copy number alterations.
  • the BC cell lines showed different levels of LSTs (Figure 2B), reflecting large differences in genetic instability.
  • the frequency of cells with MN highly correlated with the level of LSTs ( Figure 2E; Spearman r: 0.88, P value ⁇ 0.0001).
  • This result shows that the genetic instability in most of the cell lines, as measured by LSTs, which reflect the history of the accumulated events in the tumor cell genome during tumor development, correlates with their recent genomic instability and defects in DSB repair, revealed by the presence of MN.
  • the inventors investigated the mechanisms at the origin of MN formation, a prerequisite for sensitivity to AsiDNA, by evaluating the performance of the DNA repair pathways and cell cycle checkpoints of the BC cell lines. For these analyses, they integrated mRNA expression and copy number variations, of DNA repair pathways and cell cycle checkpoints of the BC cell lines. Gene expression analysis showed that cells which were MN Low and resistant to AsiDNA up-regulated all DNA repair pathways and cell cycle checkpoints, whereas these pathways were down-regulated in the MN Hlgh cells ( Figure 4A). Moreover, they observed many genetic alterations in the DNA repair pathways and cell cycle checkpoints of the MN Hlgh group, where copy number losses were abundant (48% of genes with copy losses).
  • the MN levels were generally lower in the xenografts than in the corresponding cell cultures (Table 1).
  • the three MN Hlgh cell lines formed tumors with detectable MN, whereas the MN Low MDA-MB-231 cell line, gave rise to tumors with no detectable MN.
  • Tumor sensitivity to AsiDNA correlated with the MN frequency in the xenografts.
  • the inventors observed complete tumor growth arrest for up to 60 days after treatment in the MN Hlgh xenografts, whereas the MN Low MDA-MB-231 tumor escaped treatment immediately after completion (three weeks) (Figure 6A). They observed no partial (> 20% decrease in the initial size of the tumor) or complete responses in the MN Low model.
  • MN Hlgh tumors showed partial or complete responses (Table 2). They confirmed the general predictive value of MN by evaluating the presence of MN in histological samples of all tumor models used in previous studies and comparing them to their response to AsiDNA monotherapy. They included models for BC, colorectal cancer, skin melanoma, uveal melanoma, and glioblastoma. As observed for BC, the frequency of MN did not depend on the cancer type, but rather the tumor model studied. The xenografts were classified MN Low if they presented ⁇ 2.8% cells with MN and MN High if they showed > 2.8% cells with MN, similar to the threshold used for the BC model.
  • Xenografts with a MN Hlgh status showed a greater tumor growth delay (TGD) induced by AsiDNA treatment than xenografts with MN Low status (p ⁇ 0.05; Figure 6B and Table 2).
  • All MN ⁇ W models were poor responders and did not show a significant difference in growth after AsiDNA treatment, with a maximal TGD of approximately 160%, meaning that it took only 60% more time for the tumors to achieve a four-fold increase in size relative to vehicle-treated tumors.
  • most tumor types (5/8) with a MN Hlgh status showed an at least three-fold increase (300%) in TGD after AsiDNA treatment.
  • the three remaining MNHigh tumors behaved like the MNLow models ( Figure 6B).
  • the inventors analyzed whether MN can be observed in patient biopsies. They quantified the MN in hematoxylin/eosin stained tumor sections of biopsies from unselected BC samples from the Curie Hospital Pathology Department (Figure 7). Half of the 12 analyzed BC biopsies displayed a MN Hlgh status (% cells with MN > 3%), whereas only few or no MN were detectable in the others (MN 1 TM; % cells with MN ⁇ 1%). There was no evident link between MN status and the type or grade of the tumors (Table 3). The lack of correlation between genetic instability and tumor grade or type has already been reported in a large study of 5,371 tumors using three different methods to estimate genetic instability.
  • the inventors determined the sensitivity of a set of tumor cell lines to AsiDNA and estimated the presence of DNA damage by various direct and indirect methods. Sensitivity to AsiDNA was associated with the presence of unrepaired or misrepaired DSBs, revealed by MN formation, and large genome rearrangements (measured by LST analysis). SSBs and spontaneous PARP activation were not essential, confirming that DSBs are probably the main factor for cell survival. Transcriptomic and genomic analysis revealed down regulation of DNA repair pathways and several alterations, especially in DSB repair pathways of sensitive BC cells. These DSB repair defects may explain the high frequency of MN and LST in these cells, and therefore their sensitivity to AsiDNA treatment.
  • the inventors analyzed the sensitivity of BC cell lines to Olaparib (a PARP inhibitor) in a previous study (Jdey et al. Clin Cancer Res. 2017 Feb 15;23(4): 1001- 1011). They did not observe a correlation between the frequency of MN and LST and sensitivity to Olaparib in these cells. Thus, these predictive biomarkers appear to be specific to AsiDNA and are not applicable to other DNA repair inhibitors.
  • MN could be used as a predictive biomarker for response to AsiDNA. Detection of basal MN frequencies in biopsies of different tumor types, before AsiDNA treatment, could help to predict good and poor responders. This tool would allow to stratify patients for tailored treatment and appropriate dosing. In general, the present finding that low basal levels of LSTs and MN could be biomarkers of resistance to AsiDNA suggests that aggressive tumors with high genetic instability (frequently with a poor prognosis) may be the preferential indication for AsiDNA treatment.
  • MN Micronuclei
  • BC Breast Cancer
  • C canalar
  • L lobular. Materials and Methods Cell culture
  • BRCA deficient BC cell lines BC227, BRCA2 deficient, from the Institut Curie, and HCC1937, HCC38, and MDAMB436, BRCA1 deficient, from the ATCC
  • eight BRCA proficient BC cell lines BC173 from Institut Curie, and BT20, HCC1143, HCC1187, HCC70, MCF7, MDAMB231 , and MDAMB468 from the ATCC
  • three non-tumor mammary cell lines (184B5, MCF10A, and MCF12A from the ATCC)
  • three human cervical cancer HeLa cell lines silenced for BRCA1 HelaBRCAlSX, Tebu-Bio, reference 00301-00041
  • BRCA2 HelaBRCA2SX, Tebu-Bio, reference 00301-00028
  • control HeLaCTLSX, Tebu-Bio 01-00001
  • seven human glioblastoma cell lines M059K, M059J, SF767, SF763,
  • Cell lines were authenticated by short tandem repeat profiling (Geneprint 10, Promega) at 10 different loci (TH01, D21S11, D5S818, D13S317, D7S820, D16S539, CSF1PO, AMEL, vWA, TPOX). Cell lines were verified to be negative for Mycoplasma contamination using the VenorGeM Avance Kit (Biovalley). Cells were grown according to the supplier's instructions. Cell lines were maintained at 37°C in a humidified atmosphere at 5% C02. AsiDNA molecule
  • AsiDNA is a New Chemical Entity, a 64-nucleotide (nt) oligodeoxyribonucleotide consisting of two 32- nt strands of complementary sequence connected through a l,19-bis(phospho)-8-hydraza-2-hydroxy-4- oxa-9-oxo-nonadecane linker with a cholesterol at the 5'-end and three phosphorothioate internucleotide linkages at each of the 5' and the ⁇ ends (Agilent, USA).
  • the sequence is: 5'- X GsCsTs GTG CCC ACA ACC CAG CAA ACA AGC CTA GA - L - CL - TCT AGG CTT GTT TGC TGG GTT GTG GGC AC sAsGsC -3' (SEQ ID NO 23), where L is an amino linker, X a Cholesteryl tetraethyleneglycol, CL a Carboxylic (Hydroxyundecanoic) Acid Linker, and s a Phosphorothioate linkage.
  • AsiDNA cytotoxicity was measured by quantification of relative survival and cell death.
  • Adherent cells were seeded in 24-well culture plates at appropriate densities and incubated for 24 h at 37°C before AsiDNA addition. Cells and supernatant were harvested on day 10 of treatment, stained with 0.4% trypan blue (Sigma Aldrich, Saint-Louis, USA), and counted with a Burker chamber. Cell survival was calculated as the ratio of living treated cells to living mock-treated cells. Cell death was calculated as the number of dead cells over the total number of counted cells.
  • MN result from chromosomal breakage or spindle damage. They arise in the nuclei of daughter cells following cell division and form single or multiple MN in the cytoplasm.
  • cells were grown on cover slips in a Petri dish. Cells were then fixed with PFA (4%), permeabilized with Triton (0.5%), and stained with DAPI The frequency of MN was estimated as the percentage of cells with MN over the total number of cells. At least 1000 cells were analyzed for each cell line.
  • MN assessment in tumors was performed as follows: tumors were fixed in formalin and then embedded in paraffin. Sections were cut and stained with hematoxylin, eosin, and saffron. The percentage of MN was estimated in the non-necrotic and proliferative area by quantifying the cells presenting MN in the cytoplasm, according to their described characteristics in at least 1000 cells.
  • LSTs were defined as a chromosomal breakpoint (change in copy number or allelic content) between adjacent regions, each of at least 10 megabases (Mb). The number of LSTs, representing the number of breakpoints between large chromosome fragments, was calculated as previously described (Popova et al. Cancer Res. 2012 Nov l ;72(21):5454-62). Alkaline Single-cell electrophoresis "COMET Assay"
  • the variables of the "comets” were quantified using Comet Assay 2 software (Perceptive Instrument). Triplicate slides were processed for each experimental point.
  • the tail moment is defined as the product of the percentage of DNA in the tail and the displacement between the head and the tail of the comet.
  • a sandwich ELISA was used to detect Poly(ADP-Ribose) (PAR) polymers.
  • Cells were boiled in PathScan Sandwich ELISA Lysis Buffer (Cell Signaling Technology) supplemented with ImM PMSF (Phenylmethanesulfonyl Fluoride, Sigma). Cell extracts were then diluted in Superblock buffer (Thermo Scientific) prior to the ELISA Assay. Briefly, 100 carbonate buffer (1.5 g/L sodium carbonate Na2C03, 3 g/L NaHC03) containing the capture antibody (mouse anti-PAR at 4 ⁇ g/ml, Trevigen) was added to 96 well plates and incubated overnight at 4°C.
  • the plates were then washed and blocked with Superblock at 37°C for 1 h, and 10 ⁇ L. of cell extract added to 65 ⁇ L. of Superblock in each well and the plate incubated overnight at 4°C. After washing, 75 ⁇ L. buffer (PBS/2% milk/1% mouse serum) containing detection antibody (Rabbit anti-PAR, diluted 1/1000, Trevigen) was added and the plate incubated for 1 h at RT (Room Temperature). The plates were then washed and the wells incubated with 75 ⁇ L. buffer (PBS/2% milk/1% mouse serum) containing an HRP-conjugated anti-rabbit antibody (diluted 1/5000, Abeam) for 1 h at RT.
  • 75 ⁇ L. buffer PBS/2% milk/1% mouse serum
  • HRP-conjugated anti-rabbit antibody diluted 1/5000, Abeam
  • mRNA expression analysis mRNA expression data for the BC cell lines were produced using Human Exon 1.0 ST Affymetrix microarrays. Raw data were RMA normalized and summarized with FAST DB annotation (version 2013_1) (Irizarry et al. Biostatistics. 2003 Apr;4(2):249-64; de la Grange et al. Nucleic Acids Res. 2005 Jan;33(13):4276-84). Gene expression data were log2 transformed and the mean centered over all the cell line samples which were then assigned into the two groups (MN Hlgh , MN Low ). Genes associated with Cell cycle and DNA repair pathways were retrieved from Atlas of Cancer Signalling Networks (https://acsn.curie.fr). The mean expression of these genes in MN Hlgh and MN Low samples was represented in heatmaps showing the distance to mean expression for each gene. The clustering has been performed using euclidean distance and Ward agglomeration method.
  • Copy number data analysis The copy number (CN) values for each gene for the cell lines was assessed by GAP analysis of the data generated on the Affymetrix Genome Wide SNP Array 6.0 (Popova et al. Genome Biol. 2009 Jan; 10(l l):R128) and corrected for ploidy. Two CN were considered to be 'normal', less than two CN as a loss and more than two CN as a gain. Each gene was then given a score using the average copy number across samples of the cell lines in the same group (MN Hlgh , MN Low ).
  • MDA-MB-231 and MDA-MB-468 Cell-Derived-Xenografts were obtained by injecting 107 Breast Cancer (BC) tumor cells into the mammary fat pad of six to eight-week-old adult female nude NMRI-nu Rj : NMRI-Foxn 1 nu/ Foxnlnu mice (Janvier).
  • BC227 and BC173 BC tumor models are Patient-Derived Xenografts (PDXs) and were established at the Curie Institute (France) as described in (Marangoni et al. Clin Cancer Res. 2007 Jul 1 ; 13(13):3989— 98).
  • the uveal melanoma models were also PDXs (MM26, MP34, MP41 and MP55).
  • Glioblastoma models were either PDXs (GBM14-RAV, TGI -HAM, ODA4_GEN) or CDXs (CB 193, T98G, SF763 and SF767).
  • the colorectal cancer (HT29) and skin melanoma models (SK28) were CDXs.
  • fragments of 30 to 60 mm3 were grafted into the mammary fat pad (BC227 and BC173) or right flank of six to eight- week-old female nude mice (Janvier).
  • mice were housed at least one week before tumor engraftment, under controlled conditions of light and dark (12h-12h), relative humidity (55%), and temperature (21°C). When engrafted tumors reached 80-250 mm3, mice were individually randomized into groups of 8-12 to different treatment groups. AsiDNA was injected locally (intratumoral and peritumoral subcutaneous administration). Tumor growth was evaluated three times a week using a caliper and calculated using the following formula: (length x width x width)/2. Mice were followed for up to six months, and ethically killed when the tumor volume reached 2,000 mm3. The Local Animal Experimentation Ethics Committee approved all experiments.
  • TGD tumor growth delay
  • EXAMPLE 2 Genetic instability (MN and LSTs) as predictive biomarkers for treatment with AsiDNA in hematologic cancers (leukemia and lymphoma).
  • THP-1 and U-937 myeloid leukemia cells including THP-1 and U-937 myeloid leukemia cells; Jurkat-E6.1, MOLT-4, 174xCEM.T2m and MT4 acute CD4 + T-cell leukemia; Raji and IM-9 B-cell Burkitt lymphoma, and HuT-78 CD4 + T-cell cutaneous lymphoma and SupT-1 CD4 + T-cell pleural lymphoma were purchased from the ATCC (Raji, CCL-86; IM-9, CCL-159; HuT-78, CRMB-TIB-161 ; Sup-Tl, CRL-1942; Jurkat-E6.1, TIB-152; MOLT-4, CRL-1582; 174xCEM.T2, CRL-1992; U-937, CRL- 1593.2, and THP-1, TIB- 202 respectively).
  • Cell lines were authenticated at the beginning and at the end of the study by short tandem repeat profiling (Geneprint 10, Promega) at 9 different loci (TH01, D5S818, D13S317, D7S820, D16S539, CSF1PO, AMEL, vWA, and TPOX). Cell lines were verified to be negative for mycoplasma contamination using the VenorGeM Advance Kit (Biovalley). Cells were grown according to the supplier's instructions in RPMI1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere at 5% CO 2 . Reagents for cell culture were purchased from Gibco Invitrogen.
  • cells were cultured in 96-well plates: the MT-4, MOLT-4, 174xCEM.T2, Jurket-E6.1, Sup-Tl, U-937, IM-9, and Raji cell lines were seeded at 2xl0 5 cells/ml , HuT-78 at 4xl0 5 cells/ml, and THP-1 at 10 6 cells/ml.
  • Cell proliferation and cell death were monitored after staining with 0.4% trypan blue (Sigma Aldrich, Saint-Louis, USA) by visual 166 counting using a Burker chamber.
  • Cell survival was calculated as the ratio of living treated cells to living mock-treated cells.
  • Cell death was calculated as the number of dead cells divided by the total number of cells.
  • Relative cell survival was measured by the mitochondrial MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-154 diphenyltetrazolium bromide) colorimetric assay or live-cell monitoring.
  • MTT assay was modified for cells in suspension. Briefly, one-tenth of the culture volume of MTT solution (5 mg/ml in PBS was added to the cultures and the cells incubated at 37°C in 5% C02 for 30 min to 4 h (depending on the cells).
  • AsiDNA is toxic to malignant hematologic cells and MN frequency is a predictive biomarker.
  • the toxicity of AsiDNA monotherapy was tested on a wide range of hematologic cancer cells, including two myeloid leukemias, four acute T-cell leukemias, two Burkitt's B-cell lymphomas, and two T-cell lymphomas.
  • EC50 half maximal effective concentration
  • S U- 937, IM-9, MOLT-4, and Sup-Tl
  • I MT4
  • 174xCEM.T2 HuT-78

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Abstract

The present invention relates to a biomarker which is predictive of the sensitivity to a treatment of cancer with a Dbait molecule.

Description

NEW PREDICTIVE BIOMARKER FOR THE SENSITIVITY TO A TREATMENT OF CANCER WITH A DBAIT MOLECULE
FIELD OF THE INVENTION
The present invention relates to the field of the medicine, more particularly of the oncology. It relates to a biomarker of the sensitivity to treatment with a Dbait molecule.
BACKGROUND OF THE INVENTION
Dbait molecules are a new class of DNA repair inhibitors triggering false DNA damage signaling in cancer cells. These molecules are short double-stranded DNA with a free double strand blunt end, which target key damage signal transducers such as DNA dependent protein kinase (DNA-PK) and Poly-ADP- Ribo-Polymerase (PARP), triggering their activation and amplifying false damage signaling (WO2005/040378, WO2008/034866 and WO2008/084087). In order to increase the efficiency of cellular uptake, the Dbait molecule was modified by covalently linking a cholesterol moiety to the 5'- end (DT01 or AsiDNA) (WO2011/161075; Berthault et al, 2011, Cancer gene therapy, 18, 695-706). These molecules act on enzymes involved in the HR, NHEJ, BER, and SSBR pathways (Quanz et al. Clin Cancer Res. 2009 Feb 15; 15(4): 1308-16; Croset et al. Nucleic Acids Res. 2013 Aug;41(15):7344- 55; Jdey et al. Clin Cancer Res. 2017 Feb 15;23(4): 1001-1011) and thus broadly inhibits DNA repair. Their antitumor activity has been demonstrated in preclinical studies in association with DNA damaging treatments, such as radiotherapy and chemotherapy in many tumor models, and in the clinic in association with radiotherapy.
However, its activity as a monotherapy is more selective as tumors must be dependent on DNA repair for their survival to be sensitive to its inhibition. In particular, monotherapy with Dbait molecules, especially with AsiDNA, has been recently demonstrated to show efficacy in cells and tumors. All cell lines deficient in HR via inactivation of BRCA function were highly sensitive to AsiDNA, but this sensitivity did not appear to be restricted to HR defects, as some of the BRCA+/+ cell lines were also highly sensitive. Dbait molecules such as AsiDNA are indeed effective against any cancer cells independently of their BRCA status contrary to PARP inhibitors (PARPi) which, for their part, are 100- to 1,000-fold more effective against Bi?CA-deficient cancer cells (Jdey et al. Clin Cancer Res. 2017 Feb 15;23(4): 1001-1011). It has also been shown that cancer cells such as breast cancer cell lines display different profiles of sensitivity to Dbait molecules and PARPi. Therefore, it could be useful identify predictive biomarkers for the sensitivity to treatment with Dbait molecules, especially with AsiDNA. SUMMARY OF THE INVENTION
Here, the inventors characterized potential predictive biomarkers for treatment with Dbait molecules. Sensitivity to Dbait molecules was associated with a high spontaneous frequency of cells with micronuclei (MN) and the number of large-scale chromosomal rearrangements (LSTs). A high basal level of MN as a predictive biomarker for treatment with Dbait molecules, especially with AsiDNA, was validated in 43 solid tumor cell lines from various tissues and 16 models of cell- and patient-derived xenografts. MN quantification was also possible in patient biopsies. Overall, this study identified genetic instability as a predictive biomarker for sensitivity to treatment with Dbait molecules, especially with AsiDNA. The inventors have also shown on several hematologic cell lines that MN quantification is also useful for predicting sensitivity to treatment with Dbait molecules, especially with AsiDNA.
Accordingly, the present invention relates to the use of genetic instability as measured by a frequency of cells with micronuclei and/or a number of large-scale chromosomal rearrangements (LSTs) as a predictive biomarker of a sensitivity or resistance to a treatment of cancer with a nucleic acid molecule, said nucleic acid molecule having at least one free end and comprising a hairpin with a DNA double stranded portion of 24-200 bp with less than 60% sequence identity to any gene in a human genome. The present invention also relates to a method for determining the sensitivity or resistance of a subject having a cancer to a treatment with a nucleic acid molecule, said nucleic acid molecule having at least one free end and comprising a hairpin with a DNA double stranded portion of 24-200 bp with less than 60% sequence identity to any gene in a human genome, wherein the method comprises determining a frequency of cells with micronuclei and/or a number of large-scale chromosomal rearrangements (LSTs) in a biological sample from the subject, the frequency of cells with micronuclei and/or the number of large-scale chromosomal rearrangements (LSTs) being positively correlating to the sensitivity of the subject to a treatment of cancer with the nucleic acid molecule.
The present invention further relates to a nucleic acid molecule having at least one free end and comprising a hairpin with a DNA double stranded portion of 24-200 bp with less than 60% sequence identity to any gene in a human genome for use for treating a cancer in a subject having a high genetic instability as measured by a frequency of cells with micronuclei and/or a number of large-scale chromosomal rearrangements (LSTs). Preferably, the nucleic acid molecule has one of the following formulae:
Figure imgf000003_0001
Figure imgf000004_0002
wherein N is a deoxynucleotide, n is an integer from 19 to 195, the underlined N refers to a nucleotide having or not a modified phosphodiester backbone, L' is a linker, C is the molecule facilitating endocytosis selected from a lipophilic molecule or a ligand which targets cell receptor enabling receptor mediated endocytosis, L is a linker, m and p, independently, are an integer being 0 or 1.
More preferably, the nucleic acid molecule of formula (I), (II) or (III) has one or several of the following features:
- n is an integer from 23 to 195 or from 27 to 95, and/or
- N is a deoxynucleotide selected from the group consisting of A (adenine), C (cytosine), T (thymine) and G (guanine) and selected so as to avoid occurrence of a CpG dinucleotide and to have less than 80% sequence identity to any gene in a human genome.; and/or,
- the linked L' is selected from the group consisting of hexaethyleneglycol, tetradeoxythymidylate (T4), l,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane and 2,19-bis(phosphor)-8-hydraza- l-hydroxy-4-oxa-9-oxo-nonadecane; and/or,
- m is 1 and L is a carboxamido polyethylene glycol, more preferably carboxamido triethylene glycol or carboxamido tetraethylene glycol; and/or,
- C is selected from the group consisting of a cholesterol, single or double chain fatty acids such as octadecyl, oleic acid, dioleoyl or stearic acid, or ligand (including peptide, protein, aptamer) which targets cell receptor such as folic acid, tocopherol, sugar such as galactose and mannose and their oligosaccharide, peptide such as RGD and bombesin, and protein such transferring and integrin, preferably is a cholesterol or a tocopherol, still more preferably a cholesterol.
Figure imgf000004_0001
with the underlined nucleotide having phosphorothioate internucleotide linkages.
Preferably, the biological sample of said subject is a cancer sample, in particular a tumor biopsy or a biological fluid comprising cancer cells. The frequency of cells with micronuclei can be determined by counting the number of cells having micronuclei in a cancer sample. Optionally, a frequency higher than 1%, preferably higher than 2%, more preferably higher than 2.5 or 3% is indicative of a sensitivity to the treatment with said nucleic acid molecule.
The number of large-scale chromosomal rearrangements (LSTs) can be determined with a SNP-array. The number of large-scale chromosomal rearrangements (LSTs) may correspond to the number, per genome, of breakpoints resulting in segments of at least 3, 4, 5, 6, 7, 8 9, 10 megabases, preferably at least 10 megabases. Optionally, a number of large-scale chromosomal rearrangements (LSTs) of at least 10 large-scale chromosomal rearrangements (LSTs), preferably at least 15, is indicative of a sensitivity to the treatment with said nucleic molecule.
In one embodiment, the method may further comprise selecting the subject as for determining the sensitivity or resistance of a subject having a cancer to a treatment with said nucleic acid molecule.
The present invention relates to the use of a kit comprising means for determining the frequency of micronuclei in a cell population or for determining the number of large-scale chromosomal rearrangements (LSTs) for determining the sensitivity or resistance of a subject having a cancer to a treatment with a nucleic acid molecule, for selecting a subject affected with a cancer or tumor for a treatment with a nucleic acid molecule or for determining whether a subject affected with a cancer or tumor is susceptible to benefit from a treatment with said nucleic acid molecule.
DETAILED DESCRIPTION OF THE INVENTION
Biomarker predictive of the sensitivity to a treatment with the nucleic acid molecules
The inventors surprisingly established that the genetic instability is positively correlated with the sensitivity of tumors to the treatment with DBait molecules.
Therefore, the present invention relates to the use of genetic instability as a biomarker for the sensitivity of a subject to a treatment with DBait molecules. It also relates to a method for determining the sensitivity of a subject having a cancer to a treatment with DBait molecules comprising determination the level of genetic instability in a sample from the subject, the genetic instability being positively correlated with the sensitivity of the subject having a cancer to a treatment with DBait molecules. It further relates to a method for selecting a subject affected with a cancer or tumor for a treatment with DBait molecules or for determining whether a subject affected with a cancer or tumor is susceptible to benefit from a treatment with DBait molecules, comprising determination the level of genetic instability in a sample from the subject and selecting the subject having a high level of genetic instability. The method may further comprise administering DBait molecules to the selected subject, alone or in combination with other treatments. Finally, it relates to a DBait molecule, alone or in combination with other treatments, for use in the treatment of a cancer or tumor in a subject having a high level of genetic instability. Accordingly, it relates to a method for treating a cancer in a subject, comprising administering a therapeutic effective amount of a DBait molecule, alone or in combination with other treatments, to a subject having a high level of genetic instability. It also relates to the use of a DBait molecule for the manufacture of a drug for treating a cancer in a subject having a high level of genetic instability, optionally in combination with other treatments.
As used herein, the term "marker" and "biomarker" are interchangeable and refer to biological parameters that aid the selection of patients who will benefit from a specific treatment. This term refers particularly to "tumor biomarkers". It is a measurable indicator for predicting the responsiveness or sensitivity of a patient to a specific treatment, in particular a treatment with DBait molecules. A biomarker can be detected in the blood, urine, stool, tumor tissue, or other tissues or bodily fluids of some patients with cancer, in particular in a tumor tissue.
As used herein, the terms "subject", "individual" or "patient" are interchangeable and refer to an animal, preferably to a mammal, even more preferably to a human. However, the term "patient" can also refer to non-human animals, in particular mammals such as dogs, cats, horses, cows, pigs, sheep and non- human primates, among others, that are in need of a treatment.
The genetic instability is measured in a sample from the subject having a cancer. Preferably, the sample is a cancer sample. As used herein, the term "cancer sample" refers to any biological sample containing tumoral cells derived from the patient. In particular, tumoral cells may be obtained from fluid sample such as blood, plasma, urine, cerebrospinal fluid and seminal fluid samples as well as from biopsies, organs, tissues or cell samples. In a preferred embodiment, tumoral cells are obtained from tumor biopsy or resection sample from the patient. Preferably, the sample contains only tumoral cells.
According, the method may comprise an initial step of providing a sample from the subject, preferably a tumor or cancer sample. Optionally, two samples may be provided from the same patient, a normal and a tumor samples. Preferably, the tumor and normal samples are histologically matched tissues.
The genetic instability can be measured by the frequency of cells with micronuclei and/or by the number of large-scale chromosomal rearrangements (LSTs). Accordingly, in a first embodiment, the genetic instability is measured by the frequency of cells with micronuclei. Criteria for identifying micronuclei are well-established, for instance as described in Countryman & Heddle (1976, Mutation Research, 41, 321-332). In particular, the criteria are the followings:
diameter less than l/3rd the main nucleus;
non-re fractility (to exclude small stain particles);
- colour same as or lighter than the nucleus (to exclude large stain particles);
location within 3 or 4 nuclear diameters of of a nucleus; and not touching the nucleus (to make frequency measurements meaningful); and
no more than 2 micronuclei associated with one nucleus. Therefore, the present invention relates to the use of a frequency of cells with micronuclei as a biomarker for the sensitivity of a subject to a treatment with DBait molecules. It also relates to a method for determining the sensitivity of a subject having a cancer to a treatment with DBait molecules comprising determination the frequency of cells with micronuclei in a sample from the subject, the frequency of cells with micronuclei being positively correlated with the sensitivity of the subject having a cancer to a treatment with DBait molecules. It further relates to a method for selecting a subject affected with a cancer or tumor for a treatment with DBait molecules or for determining whether a subject affected with a cancer or tumor is susceptible to benefit from a treatment with DBait molecules, comprising determination the frequency of cells with micronuclei in a sample from the subject and selecting the subject having a high frequency of cells with micronuclei. The method may further comprise administering DBait molecules to the selected subject, alone or in combination with other treatments. Finally, it relates to a DBait molecule, alone or in combination with other treatments, for use in the treatment of a cancer or tumor in a subject having a high frequency of cells with micronuclei. Accordingly, it relates to a method for treating a cancer in a subject, comprising administering a therapeutic effective amount of a DBait molecule, alone or in combination with other treatments, to a subject having a high frequency of cells with micronuclei. It also relates to the use of a DBait molecule for the manufacture of a drug for treating a cancer in a subject having a high frequency of cells with micronuclei, optionally in combination with other treatments.
The determination of the frequency of cells with micronuclei can be measured by the proportion of cells that contain micronuclei in a sample, in particular a tissue sample, preferably a tumor or cancer sample. In one particular embodiment, the determination of the frequency of cells with micronuclei is carried out by the cytokinesis-block micronucleus (CBMN) assay. This assay has been described in Fenech and Morley (1985, Mutation Res., 148, 29-36) and been improved as disclosed in WO02/14859 (see also, Fenech, 2007, Nature Protocols, 2, 1084-1104), the disclosure thereof being incorporated herein by reference. The frequency of cells with micronuclei is determined on cells having undergone one cellular division, more particular only one division. Accordingly, the cells of the sample are incubated with a cytokinesis blocking agent such as cytochalasin-B. This cytokinesis blocking agent prevents cells from separating into daughter nuclei, thereby allowing unambiguous identification of cells having undergone one or more cellular divisions. Optionally, the method may comprise a step of either determining the proportion of cells having undergone one cellular division or of sorting the cells for obtaining a population of cells having undergone one cellular division.
The counting of the number of cells having micronuclei can be determined under microscope after appropriate staining. The micronuclei are clearly smaller than the cell nuclei and are physically separate from them. The counting can be carried out by visual scoring or by automated scoring procedures. Prototypes of software for automated image analysis have been described in the literature since the early 1990s and developed by Bee ton-Dickinson and Loats Associates, Inc (LAI Automated Micronucleus Assay System;. 201 East Main St. Westminster, MD 21157 410-876-8055). Alternative methods are disclosed in WO02/14859 and WO2010/068799.
In one embodiment, the method for determining the frequency of cells with micronuclei comprises lysing cells of the sample to release nuclei and micronuclei, counting the nuclei and micronuclei, and determining the frequency of cells with micronuclei. The lysis can be achieved by any known procedures, such as those described in Nusse and Marx (1997, Mutat Res. 392(1-2): 109-15), the disclosure of which being incorporated by reference. The nuclei and micronuclei can be analyzed by flow cytometry. When nuclei and micronuclei are analyzed by flow cytometry, the nuclei and micronuclei are stained with a dye prior to flow cytometry.
Analysis of nuclei and micronuclei can be performed by flow cytometry as described in Nusse and Marx (1997, Mutat Res. 392(1-2): 109-15), the disclosure of which being incorporated by reference. In this embodiment, nuclei may be distinguished from micronuclei by their greater light scatter and greater DNA content. The frequency of cells with micronuclei is calculated as the micronuclei frequency per nuclei.
The micronuclei and nuclei can be stained by any appropriate dye. In a preferred embodiment, the dye is a fluorescent dye. For instance, the fiorescent dye can be selected from the group consisting of DAPI (4',6-diamidino-2-phenylindole), propidium iodide, Hoechst 33342, carboxyfluorescein diacetate succinimidyl ester (CFSE) and dyes of the PKH series. When nuclei and micronuclei are analyzed by flow cytometry, the dye can be selected from the group consisting of DAPI (4',6-diamidino-2- phenylindole), propidium iodide and Hoechst 33342. It will be appreciated by those skilled in the art that a number of alternative nucleic acid dyes may also be used.
For instance, a method for determining the frequency of micronuclei can comprise: incubating cells from the sample for a sufficient time to allow a substantial population of cells to complete at least one cellular division;
optionally, sorting the cells to obtain a substantially pure population of cells which have undergone one cellular division;
- lysing the cells to release nuclei and micronuclei; and
determining the nuclei and micronuclei in order to determine the frequency of micronuclei.
Based on the frequency of cells with micronuclei, it is possible to predict the sensitivity or resistance of the tumor to a treatment with Dbait molecules. The threshold between high frequency and low frequency can be determined by the person skilled in the art, especially as detailed in the examples. Accordingly, the status of high or low frequency is determined by comparing the frequency to the threshold and the frequency is high if it is greater than the threshold and is low if it is lower than the threshold. For instance, a threshold can be defined for each kind of cancer. For instance, a frequency higher than 1%, preferably higher than 2%, more preferably higher than 2.5%, still more preferably higher than 3%, is indicative of a sensitivity to the treatment with Dbait molecules. On the opposite, a frequency lower than 3%, preferably lower than 2.5%, more preferably lower than 2%, still more preferably lower than 1, 0.75 or 0.5%, is indicative of a resistance to the treatment with Dbait molecules. By 1 % is intended to refer to a sample with 1 % of cells having micronuclei. In a preferred embodiment, the frequency is determined by analyzing at least 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 cells.
Accordingly, a frequency of cells with micronuclei higher than 1%, preferably higher than 2%, more preferably higher than 2.5%, still more preferably higher than 3%, can be considered as a high frequency of cells with micronuclei for selecting a subject as suitable for being treated with DBait molecules or for determining whether a subject affected with a cancer or tumor is susceptible to benefit from such a treatment.
Accordingly, in a second embodiment, the genetic instability is measured by the number of large-scale chromosomal rearrangements (LSTs).
Therefore, the present invention relates to the use of a number of large-scale chromosomal rearrangements (LSTs) as a biomarker for the sensitivity of a subject to a treatment with DBait molecules. It also relates to a method for determining the sensitivity of a subject having a cancer to a treatment with DBait molecules comprising determination the number of large-scale chromosomal rearrangements (LSTs) in a sample from the subject, the number of large-scale chromosomal rearrangements (LSTs) being positively correlated with the sensitivity of the subject having a cancer to a treatment with DBait molecules. It further relates to a method for selecting a subject affected with a cancer or tumor for a treatment with DBait molecules or for determining whether a subject affected with a cancer or tumor is susceptible to benefit from a treatment with DBait molecules, comprising determination the number of large-scale chromosomal rearrangements (LSTs) in a sample from the subject and selecting the subject having a high number of large-scale chromosomal rearrangements (LSTs). The method may further comprise administering DBait molecules to the selected subject, alone or in combination with other treatments. Finally, it relates to a DBait molecule, alone or in combination with other treatments, for use in the treatment of a cancer or tumor in a subject having a high number of large-scale chromosomal rearrangements (LSTs). Accordingly, it relates to a method for treating a cancer in a subject, comprising administering a therapeutic effective amount of a DBait molecule, alone or in combination with other treatments, to a subject having a high number of large-scale chromosomal rearrangements (LSTs). It also relates to the use of a DBait molecule for the manufacture of a drug for treating a cancer in a subject having a high number of large-scale chromosomal rearrangements (LSTs), optionally in combination with other treatments.
The method of determining the number of large-scale chromosomal rearrangements (LSTs) in a sample comprises the step of quantifying the number of rearrangements in the genomic DNA of the tumor or cancer sample obtained from the subject, wherein the number of rearrangements corresponds to the number, per genome, of breakpoints resulting in segments of at least 3 megabases, preferably at least 4 megabases, more preferably at least 5, 6, 7, 8 9, 10, 11 12, 13, 14, 15, 16, 17, 18, 19 or 20 megabases, still more preferably from at least 3 to 11 megabases, even more preferably at least 10 megabases. In a specific embodiment, the number of rearrangements corresponds to the number, per genome, of breakpoints resulting in segments of at least 10 megabases. The number of rearrangements is determined as detailed in WO2013/182645, in Popova et al. Cancer Res. 2012 Nov l;72(21):5454-62 and/or Popova et al, Genome Biol. 2009;10(11):R128, the disclosure thereof being incorporated herein by reference.
The step of quantifying the number of rearrangements per genome in the genomic DNA of the sample can be performed by any suitable method in the art. In one embodiment, the step of quantifying rearrangements is carried out by sequencing techniques, such as next- generation sequencing using mate paired libraries, or longer reads (e.g., Stephens et al, 2009, Nature, 462, 1005-1010). In another embodiment, the step of quantifying rearrangements is performed by quantifying the number of copy number variations per genome. Typically, this can be done by hybridization techniques such as comparative genomic hybridization (CGH) array and Single Nucleotide Polymorphism (SNP) array. Suitable methods for quantifying rearrangements include, but are not limited to, those described in Le Scouarnec and Gribble, Heredity, 2012, 108, 75-85. In a preferred embodiment, the number of rearrangements per genome is defined with a SNP (Single Nucleotide Polymorphism) array by determining absolute copy number (CN) and allelic content profiles, for instance by GAP methodology (Popova et al. Genome Biol. 2009; 10(11):R128; Popova et al. Cancer Res. 2012 Nov l ;72(21):5454-62). More preferably, the number of breakpoints in each genomic profile is estimated based on the resulting copy number profile and after filtering smaller tha 50 SNPs variation.
Based on the number of large-scale chromosomal rearrangements (LSTs), it is possible to predict the sensitivity or resistance of the tumor to a treatment with Dbait molecules. The threshold between high number and low number of LSTs can be determined by the person skilled in the art, especially as detailed in the examples. Accordingly, the status of high or low number is determined by comparing the number of LSTs to the threshold and the number is high if it is greater than the threshold and is low if it is lower than the threshold. For instance, a threshold can be defined for each kind of cancer.
For instance, based on the number of large-scale chromosomal rearrangements (LSTs) with breakpoints resulting in segments of at least 10 megabases and for tumor being diploid or near-diploid, a number higher than 9, preferably higher than 10, for instance higher than 11, 12, 13, 14 or 15, is indicative of a sensitivity to the treatment with Dbait molecules.
Accordingly, based on the number of large-scale chromosomal rearrangements (LSTs) with breakpoints resulting in segments of at least 10 megabases and for tumor being diploid or near-diploid, a number of large-scale chromosomal rearrangements (LSTs) higher than 9, preferably higher than 10, more preferably higher than 11, 12, 13, 14 or 15, can be considered as a high number of large-scale chromosomal rearrangements (LSTs) for selecting a subject as suitable for being treated with DBait molecules or for determining whether a subject affected with a cancer or tumor is susceptible to benefit from such a treatment.
However, the threshold of the number of LSTs will depend on the size of the considered segment for determining the number of rearrangements (e.g., longer or shorter than 10 megabases). Therefore, the threshold will increase if the size of the considered segment for determining the number of rearrangements is smaller than 10 megabases and will decrease if the size of the considered segment for determining the number of rearrangements is greater than 10 megabases.
In a third embodiment, the genetic instability is measured by the combination of the frequency of cells with micronuclei and the number of large-scale chromosomal rearrangements (LSTs) as detailed above. Nucleic acid molecules
The nucleic acid molecules have at least one free end and comprises a hairpin with a DNA double stranded portion of 24-200 bp with less than 60% sequence identity to any gene in a human genome. In particular, the nucleic acid molecules, conjugated or not, can be described by the following formulae:
Figure imgf000012_0001
wherein N is a nucleotide, n is an integer from 15 to 195, the underlined N refers to a nucleotide having or not a modified phosphodiester backbone, L' is a linker, C is a molecule facilitating endocytosis, L is a linker, m and p, independently, are an integer being 0 or 1. In Formulae (II) and (III), C-Lm is respectively linked to the 5' end or the 3' end of the nucleotide. In formula (I-III), C-Lm is preferably linked to L' through a disulfide bond (S-S). When the molecule is conjugated, p is 1. Preferably, the underlined N refers to a nucleotide having a modified phosphodiester backbone. In preferred embodiments, the molecule of formula (I), (II) or (III) has one or several of the following features:
- N is a deoxynucleotide, preferably selected from the group consisting of A (adenine), C (cytosine), T (thymine) and G (guanine) and selected so as to avoid occurrence of a CpG dinucleotide and to have less than 80% or 70%, even less than 60% or 50% sequence identity to any gene in a human genome.; and/or,
- n is an integer from 15 to 195, preferably from 23 to 195, or from 25 to 195, optionally from 15 to 195, from 19-95, from 21 to 95, from 23 to 95, from 25 to 95, from 27 to 95, from 15 to 45, from 19 to 45, from 21 to 45, or from 27 to 45. In a particularly preferred embodiment, n is 27; and/or,
- the underlined N refers to a nucleotide having or not a phosphorothioate or methylphosphonate backbone, more preferably a phosphorothioate backbone; preferably, the underlined N refers to a nucleotide having a modified phosphodiester backbone; and/or,
- the linked L' is selected from the group consisting of hexaethyleneglycol, tetradeoxythymidylate (T4), l,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane and 2,19-bis(phosphor)-8-hydraza- 1 -hydroxy-4-oxa-9-oxo-nonadecane; and/or,
- m is 1 and L is a carboxamido polyethylene glycol, more preferably carboxamido triethylene glycol or carboxamido tetraethylene glycol; and/or, - C is selected from the group consisting of a cholesterol, single or double chain fatty acids such as octadecyl, oleic acid, dioleoyl or stearic acid, or ligand (including peptide, protein, aptamer) which targets cell receptor such as folic acid, tocopherol, sugar such as galactose and mannose and their oligosaccharide, peptide such as RGD and bombesin, and protein such transferring and integrin, preferably is a cholesterol or a tocopherol, still more preferably a cholesterol;
Preferably, C-Lm is a triethyleneglycol linker (10-O-[l-propyl-3-N-carbamoylcholesteryl]- triethyleneglycol radical. Alternatively, C-Lm is a tetraethyleneglycol linker (10-O-[l-propyl-3-N- carbamoylcholesteryl] -tetraethyleneglycol radical.
In a preferred embodiment, the conjugated Dbait molecule or hairpin nucleic acid molecule has the following formula:
Figure imgf000013_0001
with the same definition than formulae (I), (II), (IF) and (III) for and m.
Figure imgf000013_0002
In a particular embodiment, the nucleic acid molecules can be Dbait molecules such as those extensively described in PCT patent applications WO2005/040378, WO2008/034866 and WO2008/084087, the disclosure of which is incorporated herein by reference.
Dbait molecules may be defined by a number of characteristics necessary for their therapeutic activity, such as their minimal length, the presence of at least one free end, and the presence of a double stranded portion, preferably a DNA double stranded portion. As will be discussed below, it is important to note that the precise nucleotide sequence of Dbait molecules does not impact on their activity. Furthermore, Dbait molecules may contain a modified and/or non-natural backbone.
Preferably, Dbait molecules are of non-human origin (i.e., their nucleotide sequence and/or conformation (e.g., hairpin) does not exist as such in a human cell), most preferably of synthetic origin. As the sequence of the Dbait molecules plays little, if any, role, Dbait molecules have preferably no significant degree of sequence homology or identity to known genes, promoters, enhancers, 5'- or 3'- upstream sequences, exons, introns, and the like. In other words, Dbait molecules have less than 80% or 70%, even less than 60% or 50% sequence identity to any gene in a human genome. Methods of determining sequence identity are well known in the art and include, e.g., Blast. Dbait molecules do not hybridize, under stringent conditions, with human genomic DNA. Typical stringent conditions are such that they allow the discrimination of fully complementary nucleic acids from partially complementary nucleic acids. In addition, the sequence of the Dbait molecules is preferably devoid of CpG in order to avoid the well- known toll-like receptor-mediated immunological reactions.
The length of Dbait molecules may be variable, as long as it is sufficient to allow appropriate binding of Ku protein complex comprising Ku and DNA-PKcs proteins. It has been showed that the length of Dbait molecules must be greater than 20 bp, preferably about 32 bp, to ensure binding to such a Ku complex and allowing DNA-PKcs activation. Preferably, Dbait molecules comprise between 20-200 bp, more preferably 24-100 bp, still more preferably 26-100, and most preferably between 24-200, 25-200, 26-200, 27-200, 28-200, 30-200, 32-200, 24-100, 25-100, 26-100, 27-100, 28-100, 30-100, 32-200 or 32-100 bp. For instance, Dbait molecules comprise between 24-160, 26-150, 28-140, 28-200, 30-120, 32-200 or 32-100 bp. By "bp" is intended that the molecule comprise a double stranded portion of the indicated length.
In a particular embodiment, the Dbait molecules having a double stranded portion of at least 32 pb, or of about 32 bp, comprise the same nucleotide sequence than Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5). Optionally, the Dbait molecules have the same nucleotide composition than Dbait32, Dbait32Ha, Dbait32Hb, Dbait32Hc or Dbait32Hd but their nucleotide sequence is different. Then, the Dbait molecules comprise one strand of the double stranded portion with 3 A, 6 C, 12 G and 11 T. Preferably, the sequence of the Dbait molecules does not contain any CpG dinucleotide.
Alternatively, the double stranded portion comprises at least 16, 18, 20, 22, 24, 26, 28, 30 or 32 consecutive nucleotides of Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5). In a more particular embodiment, the double stranded portion consists in 20, 22, 24, 26, 28, 30 or 32 consecutive nucleotides of Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5).
The nucleic acid as disclosed herein must have at least one free end, as a mimic of DSB. Said free end may be either a free blunt end or a 5'-/3'- protruding end. The "free end" refers herein to a nucleic acid molecule, in particular a double-stranded nucleic acid portion, having both a 5' end and a 3' end or having either a 3 'end or a 5' end. Optionally, one of the 5' and 3' end can be used to conjugate the nucleic acid molecule or can be linked to a blocking group, for instance a or 3'-3'nucleotide linkage.
In an alternative embodiment, the nucleic acid molecules contain two free ends and can be linear. Accordingly, Dbait molecules may also be a double stranded molecule with two free ends and having the nucleotide sequence of Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID
No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5). In another particular embodiment, they contain only one free end. Preferably, Dbait molecules are made of hairpin nucleic acids with a double-stranded DNA stem and a loop. The loop can be a nucleic acid, or other chemical groups known by skilled person or a mixture thereof. A nucleotide linker may include from 2 to 10 nucleotides, preferably, 3, 4 or 5 nucleotides. Non-nucleotide linkers non exhaustively include abasic nucleotide, polyether, polyamine, poly amide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e. g. oligoethylene glycols such as those having between 2 and 10 ethylene glycol units, preferably 4, 5, 6, 7 or 8 ethylene glycol units). A preferred linker is selected from the group consisting of hexaethyleneglycol, tetradeoxythymidylate (T4), 1,19- bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane and other linkers such as 2,19- bis(phosphor)-8-hydraza-l-hydroxy-4-oxa-9-oxo-nonadecane. Accordingly, in a particular embodiment, the Dbait molecules can be a hairpin molecule having a double stranded portion or stem comprising at least 16, 18, 20, 22, 24, 26, 28, 30 or 32 consecutive nucleotides of Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5) and a loop being a hexaethyleneglycol linker, a tetradeoxythymidylate linker (T4), 1,19- bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane or 2,19-bis(phosphor)-8-hydraza-l- hydroxy-4-oxa-9-oxo-nonadecane. In a more particular embodiment, those Dbait molecules can have a double stranded portion consisting in 20, 22, 24, 26, 28, 30 or 32 consecutive nucleotides of Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5).
Dbait molecules preferably comprise a 2'-deoxynucleotide backbone, and optionally comprise one or several (2, 3, 4, 5 or 6) modified nucleotides and/or nucleobases other than adenine, cytosine, guanine and thymine. Accordingly, the Dbait molecules are essentially a DNA structure. In particular, the double-strand portion or stem of the Dbait molecules is made of deoxyribonucleotides.
Preferred Dbait molecules comprise one or several chemically modified nucleotide(s) or group(s) at the end of one or of each strand, in particular in order to protect them from degradation. In a particular preferred embodiment, the free end(s) of the Dbait molecules is(are) protected by one, two or three modified phosphodiester backbones at the end of one or of each strand. Preferred chemical groups, in particular the modified phosphodiester backbone, comprise phosphorothioates. Alternatively, preferred Dbait have 3'- 3' nucleotide linkage, or nucleotides with methylphosphonate backbone. Other modified backbones are well known in the art and comprise phosphoramidates, morpholino nucleic acid, 2'-0,4'- C methylene/ethylene bridged locked nucleic acid, peptide nucleic acid (PNA), and short chain alkyl, or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intrasugar linkages of variable length, or any modified nucleotides known by skilled person. In a first preferred embodiment, the Dbait molecules have the free end(s) protected by one, two or three modified phosphodiester backbones at the end of one or of each strand, more preferably by three modified phosphodiester backbones (in particular phosphorothioate or methylphosphonate) at least at the 3 'end, but still more preferably at both 5' and 3' ends. In a most preferred embodiment, the Dbait molecule is a hairpin nucleic acid molecule comprising a DNA double-stranded portion or stem of 32 bp (e.g., with a sequence selected from the group consisting of SEQ ID Nos 1-5, in particular SEQ ID No 4) and a loop linking the two strands of the DNA double- stranded portion or stem comprising or consisting of a linker selected from the group consisting of hexaethyleneglycol, tetradeoxythymidylate (T4), l,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9- oxo-nonadecane and 2,19-bis(phosphor)-8-hydraza-l-hydroxy-4-oxa-9-oxo-nonadecane, the free ends of the DNA double-stranded portion or stem (i.e. at the opposite of the loop) having three modified phosphodiester backbones (in particular phosphorothioate internucleotidic links).
Said nucleic acid molecules are made by chemical synthesis, semi-biosynthesis or biosynthesis, any method of amplification, followed by any extraction and preparation methods and any chemical modification. Linkers are provided so as to be incorporable by standard nucleic acid chemical synthesis. More preferably, nucleic acid molecules are manufactured by specially designed convergent synthesis: two complementary strands are prepared by standard nucleic acid chemical synthesis with the incorporation of appropriate linker precursor, after their purification, they are covalently coupled together.
Optionally, the nucleic acid molecules may be conjugated to molecules facilitating endocytosis or cellular uptake. In particular, the molecules facilitating endocytosis or cellular uptake may be lipophilic molecules such as cholesterol, single or double chain fatty acids, or ligands which target cell receptor enabling receptor mediated endocytosis, such as folic acid and folate derivatives or transferrin (Goldstein et al. Ann. Rev. Cell Biol. 1985 1 : 1-39; Leamon & Lowe, Proc Natl Acad Sci USA. 1991, 88: 5572-5576.). The molecule may also be tocopherol, sugar such as galactose and mannose and their oligosaccharide, peptide such as RGD and bombesin and protein such as integrin. Fatty acids may be saturated or unsaturated and be in C4-C28, preferably in C14-C22, still more preferably being in Ci8 such as oleic acid or stearic acid. In particular, fatty acids may be octadecyl or dioleoyl. Fatty acids may be found as double chain form linked with in appropriate linker such as a glycerol, a phosphatidylcholine or ethanolamine and the like or linked together by the linkers used to attach on the Dbait molecule. As used herein, the term "folate" is meant to refer to folate and folate derivatives, including pteroic acid derivatives and analogs. The analogs and derivatives of folic acid suitable for use in the present invention include, but are not limited to, antifolates, dihydrofolates, tetrahydrofolates, folinic acid, pteropolyglutamic acid, 1- deza, 3-deaza, 5-deaza, 8-deaza, 10-deaza, 1,5-deaza, 5,10 dideaza, 8,10-dideaza, and 5,8-dideaza folates, antifolates, and pteroic acid derivatives. Additional folate analogs are described in US2004/242582. Accordingly, the molecule facilitating endocytosis may be selected from the group consisting of single or double chain fatty acids, folates and cholesterol. More preferably, the molecule facilitating endocytosis is selected from the group consisting of dioleoyl, octadecyl, folic acid, and cholesterol. In a most preferred embodiment, the nucleic acid molecule is conjugated to a cholesterol. The molecules facilitating endocytosis are conjugated to Dbait molecules, preferably through a linker. Any linker known in the art may be used to covalently attach the molecule facilitating endocytosis to Dbait molecules For instance, WO09/126933 provides a broad review of convenient linkers pages 38- 45. The linker can be non-exhaustively, aliphatic chain, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon , or other polymeric compounds (e. g. oligoethylene glycols such as those having between 2 and 10 ethylene glycol units, preferably 3, 4, 5, 6, 7 or 8 ethylene glycol units, still more preferably 6 ethylene glycol units), as well as incorporating any bonds that may be break down by chemical or enzymatical way, such as a disulfide linkage, a protected disulfide linkage, an acid labile linkage (e.g., hydrazone linkage), an ester linkage, an ortho ester linkage, a phosphonamide linkage, a biocleavable peptide linkage, an azo linkage or an aldehyde linkage. Such cleavable linkers are detailed in WO2007/040469 pages 12-14, in WO2008/022309 pages 22-28.
In a particular embodiment, the nucleic acid molecule can be linked to one molecule facilitating endocytosis. Alternatively, several molecules facilitating endocytosis (e.g., two, three or four) can be attached to one nucleic acid molecule.
In a specific embodiment, the linker between the molecule facilitating endocytosis, in particular cholesterol, and nucleic acid molecule is wherein n is an integer from 1 to 10,
Figure imgf000017_0001
preferably n being selected from the group consisting of 3, 4, 5 and 6. In a very particular embodiment, the linker is
Figure imgf000017_0003
(carboxamido tetraethylene glycol). In another very particular embodiment, the linker is (carboxamido triethylene glycol). The linker can be
Figure imgf000017_0002
linked to nucleic acid molecules at any convenient position which does not modify the activity of the nucleic acid molecules. In particular, the linker can be linked at the 5' end, at the 3' end or in the loop when the nucleic acid molecule is a hairpin. Therefore, in a preferred embodiment, the contemplated conjugated Dbait molecule is a Dbait molecule having a hairpin structure and being conjugated to the molecule facilitating endocytosis, preferably through a linker, at its 5' end.
In another specific embodiment, the linker between the molecule facilitating endocytosis, in particular cholesterol, and nucleic acid molecule is dialkyl -disulfide {e.g., with r and s being
Figure imgf000017_0004
integer from 1 to 10, preferably from 3 to 8, for instance 6}. In a most preferred embodiment, the conjugated Dbait molecule is a hairpin nucleic acid molecule comprising a DNA double-stranded portion or stem of 32 bp (e.g., with a sequence selected from the group consisting of SEQ ID Nos 1-5, in particular SEQ ID No 4) and a loop linking the two strands of the DNA double-stranded portion or stem comprising or consisting of a linker selected from the group consisting of hexaethyleneglycol, tetradeoxythymidylate (T4), l,19-bis(phospho)-8-hydraza-2- hydroxy-4-oxa-9-oxo-nonadecane and 2,19-bis(phosphor)-8-hydraza-l-hydroxy-4-oxa-9-oxo- nonadecane, the free ends of the DNA double-stranded portion or stem (i.e. at the opposite of the loop) having three modified phosphodiester backbones (in particular phosphorothioate internucleotidic links) and said Dbait molecule being conjugated to a cholesterol at its 5' end, preferably through a linker (e.g. carboxamido oligoethylene glycol, preferably carboxamido triethylene glycol or carboxamido tetraethylene glycol).
In a preferred embodiment, NNNN-(N)„-N comprises at least 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 consecutive nucleotides of Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5) or consists in 20, 22, 24, 26, 28, 30 or 32 consecutive nucleotides of Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5). In a particular embodiment, NNNN-(N)n-N comprises or consists in Dbait32 (SEQ ID No 1), Dbait32Ha (SEQ ID No 2), Dbait32Hb (SEQ ID No 3), Dbait32Hc (SEQ ID No 4) or Dbait32Hd (SEQ ID No 5), more preferably Dbait32Hc (SEQ ID No 4).
According, the conjugated Dbait molecule or hairpin nucleic acid molecule may be selected from the group consisting of:
with NNNN-(N)n-N being SEQ ID No 1
Figure imgf000018_0001
Figure imgf000019_0001
In preferred embodiments, the molecule of formulae (la), (Ila), (Ilia), (lb), (lib), (Illb), (Ic), (lie), (IIIc), (Id), (lid), (Hid), (Ie), (He) and (Hie), preferably of formulae (II), (Ila), (lib), (lie), (lid) and (He), has one or several of the following features:
- the underlined nucleotide refers to a nucleotide having or not a phosphorothioate or methylphosphonate backbone, more preferably a phosphorothioate backbone; preferably, the underlined nucleotide refers to a nucleotide having a phosphorothioate or methylphosphonate backbone, more preferably a phosphorothioate backbone and/or,
- the linked L' is selected from the group consisting of hexaethyleneglycol, tetradeoxythymidylate (T4), l,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane and 2,19-bis(phosphor)-8-hydraza- l-hydroxy-4-oxa-9-oxo-nonadecane; and/or,
- m is 1 and L is a carboxamido polyethylene glycol, more preferably carboxamido triethylene glycol or carboxamido tetraethylene glycol; and/or,
- p is 1 ; and/or,
- C is selected from the group consisting of a cholesterol, single or double chain fatty acids such as octadecyl, oleic acid, dioleoyl or stearic acid, or ligand (including peptide, protein, aptamer) which targets cell receptor such as folic acid, tocopherol, sugar such as galactose and mannose and their oligosaccharide, peptide such as RGD and bombesin, and protein such transferring and integrin, preferably is a cholesterol. Preferably, C-Lm is a triethyleneglycol linker (10-O-[l-propyl-3-N-carbamoylcholesteryl]- triethyleneglycol radical. Alternatively, C-Lm is a tetraethyleneglycol linker (10-O-[l-propyl-3-N- carbamoylcholesteryl] -tetraethyleneglycol radical.
In a specific embodiment of the Dbait molecules or hairpin nucleic acid molecules of formulae (I), (II), (IF), (HI), (la), (Ila), (Ilia), (lb), (lib), (nib), (Ic), (lie), (IIIc), (Id), (lid), (Hid), (Ie), (He) and (Hie), preferably of formulae (II), (IF), (Ila), (lib), (lie), (lid) and (He), L' is preferably selected from the group consisting of hexaethyleneglycol, tetradeoxythymidylate (T4), l,19-bis(phospho)-8-hydraza-2- hydroxy-4-oxa-9-oxo-nonadecane and 2,19-bis(phosphor)-8-hydraza-l-hydroxy-4-oxa-9-oxo- nonadecane.
In a specific embodiment of the Dbait molecules or hairpin nucleic acid molecules of formulae (I), (II), (IF), (HI), (la), (Ila), (Ilia), (lb), (lib), (Illb), (Ic), (lie), (IIIc), (Id), (lid), (Hid), (Ie), (He) and (Hie), preferably of formulae (II), (IF), (Ila), (lib), (lie), (lid) and (He), with C being cholesterol, C-Lm is the radical
Figure imgf000021_0001
In a preferred embodiment, the conjugated Dbait molecule or hairpin nucleic acid molecule is selected from the roup consisting of (II), (IF), (Ila), (lib), (lie), (lid), and (He), wherein C-Lm being the radical
Figure imgf000021_0002
and wherein L' is preferably selected from the group consisting of hexaethyleneglycol, tetradeoxythymidylate (T4), l,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane and 2,19-bis(phosphor)-8-hydraza-l-hydroxy-4-oxa-9-oxo-nonadecane, more preferably 2,19- bis(phosphor)-8-hydraza-l-hydroxy-4-oxa-9-oxo-nonadecane.
In a very specific embodiment, the Dbait molecule or hairpin nucleic acid molecule has the following formula
Figure imgf000021_0003
wherein is the radical
Figure imgf000021_0004
wherein L' is 2,19-bis(phosphor)-8-hydraza-l-hydroxy-4-oxa-9-oxo-nonadecane and wherein the underlined nucleotides have a phosphorothioate backbone. Accordingly, the molecule has the following structure and it is referred thereto in the Example section as "coDbait".
Figure imgf000022_0001
SEQ ID No 22
In a specific embodiment of the Dbait molecules or hairpin nucleic acid molecules of formulae (I), (II), (II ), (HI), (la), (Ha), (Ilia), (lb), (lib), (Illb), (Ic), (lie), (IIIc), (Id), (lid), (Hid), (Ie), (He) and (Hie), preferably of formulae (II), (IF), (Ila), (lib), (lie), (lid) and (He), with C being cholesterol, C-Lm is a tetraethyleneglycol linker ( 10-O-[ 1 -propyl-3 -N-carbamoylcholesteryl] -tetraethyleneglycol radical. In a preferred embodiment, the conjugated Dbait molecule or hairpin nucleic acid molecule is selected from the group consisting of (II), (IF), (Ila), (lib), (lie), (lid), and (He), wherein C-Lm being the tetraethyleneglycol linker (10-O-[l-propyl-3-N-carbamoylcholesteryl]-tetraethyleneglycol radical and wherein L' is preferably selected from the group consisting of hexaethyleneglycol, tetradeoxythymidylate (T4), l,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane and 2,19-bis(phosphor)-8-hydraza-l-hydroxy-4-oxa-9-oxo-nonadecane, more preferably 1,19- bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane. In a very specific embodiment, the Dbait molecule or hairpin nucleic acid molecule (AsiDNA or DTOl) has the following formula
Figure imgf000022_0002
wherein C-Lm is the tetraethyleneglycol linker (10-O-[l-propyl-3-N-carbamoylcholesteryl]- tetraethyleneglycol radical, and L' is l,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo- nonadecane. SEQ ID No 21
Figure imgf000023_0001
with the underlined nucleotide having phosphorothioate internucleotide linkages.
In another preferred embodiment, the nucleic acid molecule has one of the following formulae
Figure imgf000023_0002
wherein N is a deoxy nucleotide, n is an integer from 1 to 15, the underlined N refers to a nucleotide having or not a modified phosphodiester backbone, L' is a linker, C is a cholesterol, L is a linker, m is an integer being 0 or 1, and p is 1. Preferably, the underlined N refers to a nucleotide having a modified phosphodiester backbone. In a preferred embodiment, the nucleic acid molecule as the formula (II).
Further combinations
Accordingly, the present invention also relates the use of a Dbait molecule or a nucleic acid molecule as disclosed above, a pharmaceutical composition comprising it and optionally a pharmaceutically acceptable carrier, for use in the treatment of cancer in combination with a PARP inhibitor, and/or with radiotherapy and/or radioisotope therapy and/or an antitumor chemotherapy, preferably with a DNA damaging antitumoral agent, as detailed below.
Optionally, the treatment with a nucleic acid molecule as disclosed herein and a PARP inhibitor can be used in combination with a radiotherapy, a radioisotope therapy and/or another antitumor chemotherapy, immunotherapy, or hormonal therapy. Preferably, the antitumor chemotherapy is a treatment by a DNA damaging antitumor agent, either directly or indirectly.
As used herein, the term "antitumor chemotherapy" or "chemotherapy" refers to a cancer therapeutic treatment using chemical or biochemical substances, in particular using one or several antineoplastic agents. In particular, it also includes hormonal therapy and immunotherapy. The term "hormonal therapy" refers to a cancer treatment having for purpose to block, add or remove hormones. For instance, in breast cancer, the female hormones estrogen and progesterone can promote the growth of some breast cancer cells. So in these patients, hormone therapy is given to block estrogen and a non-exhaustive list commonly used drugs includes: Tamoxifen, Fareston, Arimidex, Aromasin, Femara, Zoladex/Lupron, Megace, and Halotestin. The term "immunotherapy" refers to a cancer therapeutic treatment using the immune system to reject cancer. The therapeutic treatment stimulates the patient's immune system to attack the malignant tumor cells.
In a particular aspect, the nucleic acid molecule as disclosed herein and PARP inhibitor are used in combination with a DNA-damaging treatment. The DNA-damaging treatment can be radiotherapy, or chemotherapy with a DNA-damaging antitumoral agent, or a combination thereof. DNA-damaging treatment refers to a treatment inducing DNA strand breakage, preferably relatively specifically in cancer cells. DNA strand breakage can be achieved by ionized radiation (radiotherapy). Radiotherapy includes, but is not limited to, γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other radiotherapies include microwaves and UV -irradiation. Other approaches to radiation therapy are also contemplated in the present invention. DNA strand breakage can be achieved by radioisotope therapy, in particular by administration of a radioisotope, preferably a targeted radioisotope. Targeting can be due to the chemical properties of the isotope such as radioiodine which is specifically absorbed by the thyroid gland a thousand fold better than other organs. Alternatively, the targeting can be achieved by attaching to the radioisotope another molecule having targeting properties such hapten or antibody. Any of a number of suitable radioactive isotopes can be used, including, but not limited to, Indium-111, Lutetium-171, Bismuth-212, Bismuth- 213, Astatine-211, Copper-62, Copper-64, Copper-67, Yttrium-90, Iodine-125, Iodine-131, Phosphorus-32, Phosphorus-33, Scandium-47, Silver-I l l, Gallium-67, Praseodymium- 142, Samarium- 153, Terbium-161, Dysprosium-166, Holmium-166, Rhenium-186, Rhenium-188, Rhenium-189, Lead- 212, Radium-223, Actinium-225, Iron-59, Selenium-75, Arsenic-77, Strontium-89, Molybdenum-99, Rhodium-105, Palladium- 109, Praseodymium- 143, Promethium-149, Erbium-169, Iridium-194, Gold- 198, Gold-199, and Lead-211.
The DNA-damaging antitumor agent is preferably selected from the group consisting of an inhibitor of topoisomerases I or II, a DNA crosslinker, a DNA alkylating agent, an anti-metabolic agent and inhibitors of the mitotic spindles. Inhibitors of topoisomerases I and/or II include, but are not limited to, etoposide, topotecan, camptothecin, irinotecan, amsacrine, intoplicine, anthracyclines such as doxorubicine, epirubicine, daunorubicine, idanrubicine and mitoxantrone. Inhibitors of Topoisomerase I and II include, but are not limited to, intoplecin.
DNA crosslinkers include, but are not limited to, cisplatin, carboplatin and oxaliplatin.
Anti-metabolic agents block the enzymes responsible for nucleic acid synthesis or become incorporated into DNA, which produces an incorrect genetic code and leads to apoptosis. Non-exhaustive examples thereof include, without limitation, folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors, and more particularly Methotrexate, Floxuridine, Cytarabine, 6- Mercaptopurine, 6- Thioguanine, Fludarabine phosphate, Pentostatine, 5-fiuorouracil, gemcitabine and capecitabine.
The DNA-damaging anti-tumoral agent can be alkylating agents including, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, metal salts and triazenes. Non- exhaustive examples thereof include Uracil mustard, Chlormethine, Cyclophosphamide (CYTOXAN(R)), Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine, Triethylenethiophosphor amine, Busulfan, Carmustine, Lomustine, Fotemustine, cisplatin, carboplatin, oxaliplatin, thiotepa, Streptozocin, Dacarbazine, and Temozolomide. Inhibitors of the mitotic spindles include, but are not limited to, paclitaxel, docetaxel, vinorelbine, larotaxel (also called XRP9881 ; Sanofi-Aventis), XRP6258 (Sanofi-Aventis), BMS-184476 (Bristol- Meyer-Squibb), BMS-188797 (Bristol-Meyer-Squibb), BMS-275183 (Bristol-Meyer-Squibb), ortataxel (also called IDN 5109, BAY 59-8862 or SB-T-101131 ; Bristol-Meyer-Squibb), RPR 109881 A (Bristol- Meyer-Squibb), RPR 116258 (Bristol-Meyer-Squibb), NBT-287 (TAPESTRY), PG-paclitaxel (also called CT-2103, PPX, paclitaxel poliglumex, paclitaxel polyglutamate or XyotaxTM), ABRAXANE® (also called Nab-Paclitaxel ; ABRAXIS BIOSCIENCE), Tesetaxel (also called DJ-927), IDN 5390 (INDENA), Taxoprexin (also called docosahexanoic acid-paclitaxel ; PROTARGA), DHA-paclitaxel (also called Taxoprexin®), and MAC-321 (WYETH). Also see the review of Hennenfent & Govindan (2006, Annals of Oncology, 17, 735-749).
As used herein, the term "PARP inhibitor" refers to any compound which has the ability to decrease the activity of a poly (ADP-ribose) polymerase (PARP). PARP inhibition relies mainly on two different mechanisms: (i) catalytic inhibition that act mainly by inhibiting PARP enzyme activity and (ii) bound inhibition that block PARP enzyme activity and prevent its release from the damage site. Bound inhibitors are more toxic to cells than catalytic inhibitors. PARP inhibitors according to the inventions are preferably catalytic and/or bound inhibitors. In a preferred embodiment, the PARP inhibitor is an inhibitor of any enzyme of the PARP family, preferentially PARP1 and/or PARP2.
The PARP activity can be determined by a variety of techniques well known by the skilled person. Usually, these techniques comprise measuring the incorporation of a labeled poly(ADP-ribose) onto histone proteins. Commercial kits for such techniques are available (see for example, Tervigen's kits with biotinylated poly(ADP-ribose)). Other methods may also be used such as the one developed by Putt KS et al (Anal Biochem, 326(l):78-86, 2004), the disclosure of which is hereby incorporated by reference in his entirety. These methods are ideal for the determination of IC50 values of known or suspected PARP inhibitors.
Many PARP inhibitors are known and, thus, can be synthesized by known methods from starting materials that are known, may be available commercially, or may be prepared by methods used to prepare corresponding compounds in the literature.
Examples of suitable PARP inhibitors according to the invention include, but are not limited to, olaparib (AZD-2281 , 4-[(3-[(4-cyclopropylcarbonyl)piperazin-4-yl]carbonyl)-4-fiuorophenyl]methyl(2H)- phthalazin-l-one), veliparib (ABT-888, CAS 912444-00-9, 2-((fi)-2-methylpyrrolidin-2-yl)-lW- benzimidazole-4-carboxamide), CEP-8983 (ll-methoxy-4,5,6,7-tetrahydro-lH- cyclopenta[a]pyrrolo[3,4-c]carbazole-l,3(2H)-dione) or a prodrug thereof (e.g. CEP- 9722), rucaparib (AG014699, PF-01367338, 8-Fluoro-2-{4-[(methylamino)methyl]phenyl}-l,3,4,5-tetrahydro-6H- azepino[5,4,3-cd]indol-6-one), E7016 (GPI-21016, 10-((4-Hydroxypiperidin-l-yl)methyl)chromeno- [4,3,2-de]phthalazin-3(2H)-one), talazoparib (BMN-673, (8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(l- methyl-lH-l,2,4-triazol-5-yl)-8,9-dihydro-2H-pyrido[4,3,2de]phthalazin-3(7H)-one), INO-1001 (4- phenoxy-3-pyrrolidin-l-yl-5-sulfamoyl-benzoic acid), KU0058684 (CAS 623578-11-0), niraparib (MK 4827, Merck & Co Inc), iniparib (BSI 201), iniparib-met (C-nitroso metabolite of Iniparib), CEP 9722 (Cephalon Inc), LT-673, MP-124, NMS-P118, XAV939, AZD 2461, nicotinamides, 5-methyl nicotinamide, 4-Amino-l,8-naphthalimide, picolinamide, benzamides, 3-substituted benzamides, 3- methoxybenzamide, 3-hydroxybenzamide, 3-aminobenzamide, 3-chloroprocainamide, 3- nitrosobenzamide, 4-aminobenzamide, 2-aminobenzamide, methyl 3,5-diiodo-4-(4'-methoxyphenoxy) benzoate, methyl-3,5-diiodo-4-(4'-methoxy-3',5'-diiodo-phenoxy) benzoate, cyclic benzamides, 1,5- di[(3-carbamoylphenyl)aminocarbonyloxy] pentane, indoles, benzimidazoles, benzoxazole -4- carboxamides, benzimidazole-4-carboxamides, 2-substituted benzoxazole 4-carboxamides, 2- substituted benzimidazole 4-carboxamides, 2-aryl benzimidazole 4-carboxamides, 2- cycloalkylbenzimidazole -4-carboxamides, 2-(4-hydroxphenyl) benzimidazole A-carboxamide, quinoxalinecarboxamides, imidazopyridinecarboxamides, 2-phenylindoles, 2-substituted benzoxazoles, 2-phenyl benzoxazole, 2-(3-methoxyphenyl) benzoxazole, 2-substituted benzimidazoles, 2-phenyl benzimidazole, 2-(3-methoxyphenyl) benzimidazole, 1 ,3,4,5-tetrahydro-azepino[5,4,3-cd]indol-6-one, azepinoindoles, azepinoindolones, 1 ,5-dihydro-azepino[4,5,6-cd]indolin-6-one, dihydrodiazapinoindolinone, 3-substituted dihydrodiazapinoindolinones, 3-(4-trifluoromethylphenyl)- dihydrodiazapinoindolinone, tetrahydrodiazapinoindolinone, 5,6-dihydroimidazo[4,5, 1-j, k] [l,4]benzodiazopin-7(4H)-one, 2-phenyl-5,6-dihydro-imidazo[4,5,l-jk] [l,4]benzodiazepin-7(4H)-one, 2,3-dihydro-isoindol-l-one, benzimidazole -2 -piperazine, benzimidazole-2-piperazine heterocyclic derivatives, 4-iodo-3-nitrobenzamide, benzopyrones, 1 ,2-benzopyrone 6-nitrosobenzopyrone, 6-nitroso 1,2-benzopyrone, 5-iodo-6-aminobenzopyrone, benzoylurea, quinolone, isoquinolone, isoquinolinones, dihydroisoquinolinones, 2H-isoquinolin-l-ones, 3H-quinazolin-4-ones, 5-substituted dihydroisoquinolinones, 5-hydroxy dihydroisoquinolinone, 5-methyl dihydroisoquinolinone, 5-hydroxy isoquinolinone, 5-amino isoquinolin-l-one, 5-dihydroxyisoquinolinone, 1,5-dihydroxyisoquinoline, 1,5-isoquinolinediol, 4-hydroxyquinazoline, substituted thiazolyl-isoquinolinones, substituted oxazoyl- isoquinolinones, tetrahydro-2H-isoquinolin-l-one, 3,4-dihydroisoquinolin-l(2H)-ones, 3,4-dihydro-5- methoxy-isoquinolin-l(2H)-one, 3,4-dihydro-5-methyl-l(2H)isoquinolinone, 3H-quinazolin-4-one, isoquinolin-l(2H)-ones, 3,4 dihydroisoquinolin-l(2H)-one, 4-carboxamido-benzimidazole, substituted 6-cyclohexylalkyl substituted 2-quinolinones, substituted 6-cyclohexylalkyl substituted 2- quinoxalinones, 7-phenylalkyl substituted 2-quinolinones, 7-phenylalkyl substituted 2-quinoxalinones, 6-substituted 2-quinolinones, 6-substituted 2-quinoxalinones, l-(arylmethyl)quinazoline-2,4(lH,3H)- dione, 4,5-dihydro-imidazo[4,5,l-ij]quinolin-6-ones, l,6-naphthyridine-5(6H)-ones, 1,8-naphthalimides, 4-amino-l,8-naphthalimides, 3,4-dihydro-5-[4-l(l-piperidinyl)butoxy]-l(2H)-isoquinolinone, 2,3- dihydrobenzo[de]isoquinolin-l-one, 1-1 lb-dihydro-[2H]benzopyrano[4,3,2-de]isoquinolin-3-one, tetracyclic lactams, benzpyranoisoquinolinones, benzopyrano[4,3,2-de]isoquinolinone, quinazolines, quinazolinones, quinazolinediones, A-hydroxyquinazoline, 2-substituted quinazolines, 8-hydroxy-2- methylquinazolin-4-(3H)one, phthalazines, phthalazinones, phthalazin-l(2H)-ones, 5-methoxy-4- methyl-l(2) phthalazinones, 4-substituted phthalazinones, 4-(l-piperazinyl)-l(2H)-phthalazinone, tetracyclic benzopyrano[4,3,2-de]phthalazinones and tetracyclic indeno [l,2,3-de]phthalazinones, tricyclic phthalazinones, 2-aminophthalhydrazide, phthalazinone ketone, dihydropyridophthalazinone, 6-substituted 5-arylamino-lh-pyidine-2-ones, pyridazinones, tetrahydropyridopyridazinone, tetraaza phenalen-3-one, thieno[2,3-c]isoquinolin-5-one (TIQ-A), 2,5-diazabicyclo[2.2.1]heptane, pyrimidoimidazole, isoindolinones, phenanthridines, phenanthridinones, 5[H]phenanthridin-6-one, substituted 5[H] phenanthridin-6-ones, 2,3-substituted 5[H]phenanthridin-6-ones, sulfonamide/carbamide derivatives of 6(5H)phenanthridinones, thieno[2, 3-c]isoquinolones, 9-amino thieno[2,3-c]isoquinolone, 9- hydroxythieno[2,3-c]isoquinolone, 9-methoxythieno[2,3-c]isoquinolone, N-(6-oxo-5,6-dihydrophenanthridin-2-yl] -2-(N,N-dimethylamino } acetamide, substituted 4,9- dihydrocyclopenta[imn]phenanthridine-5-ones, unsaturated hydroximic acid derivatives, 0-(3- piperidino-2-hydroxy-l-propyl)nicotinic amidoxime, 0-(2-hydroxy-3-piperidino-propyl)-3-carboxylic acid amidoxime, pyridazines, pyrazinamide, BGB-290, PF-1367338 (Pfizer Inc), AG014699 (Pfizer, Inc.), KU-59436 (KuDOS/AstraZeneca PJ34, 4-amino-l,8-naphfhalirnide (Trevigen), 6(5H)- phenanthridinone (Trevigen), NU1025, 4-HQN, BGP -15, A-966492, GPI21016, 6(5H)- phenanthridinone (Phen), theobromine, theophylline, caffeine, methylxanthines, thymidine, 3- aminophtalhydrazide, analogs, derivatives or a mixture thereof.
Additional PARP inhibitors are described for example in WO14201972, WO14201972, WO12141990, WO10091140, W09524379, WO09155402, WO09046205, WO08146035, WO08015429, WO0191796, WO0042040, US2006004028, EP2604610, EP1802578, CN104140426, CN104003979, US060229351, US7041675, WO07041357, WO2003057699, US06444676, US20060229289, US20060063926, WO2006033006, WO2006033007, WO03051879, WO2004108723, WO2006066172, WO2006078503, US20070032489, WO2005023246, WO2005097750, WO2005123687, WO2005097750, US7087637, US6903101, WO20070011962, US20070015814, WO2006135873, UA20070072912, WO2006065392, WO2005012305, WO2005012305, EP412848, EP453210, EP454831, EP879820, EP879820, WO030805, WO03007959, US6989388, US20060094746, EP1212328, WO2006078711, US06426415, US06514983, EP1212328, US20040254372, US20050148575, US20060003987, US06635642, WO200116137, WO2004105700, WO03057145A2, WO2006078711, WO2002044157, US20056924284, WO2005112935, US20046828319, WO2005054201, WO2005054209, WO2005054210, WO2005058843, WO2006003146, WO2006003147, WO2006003148, WO2006003150, WO2006003146, WO2006003147, UA20070072842, US05587384, US20060094743, WO2002094790, WO2004048339, EP1582520, US20060004028, WO2005108400, US6964960, WO20050080096, WO2006137510, UA20070072841, WO2004087713, WO2006046035, WO2006008119, WO06008118, WO2006042638, US20060229289, US20060229351, WO2005023800, WO1991007404, WO2000042025, WO2004096779, US06426415, WO02068407, US06476048, WO2001090077, WO2001085687, WO2001085686, WO2001079184, WO2001057038, WO2001023390, WO01021615A1, WO2001016136, WO2001012199, WO95024379, WO200236576, WO2004080976, WO2007149451, WO2006110816, WO2007113596, WO2007138351, WO2007144652, WO2007144639, WO2007138351, WO2007144637, Banasik et al. (J. Biol. Chem., 267:3, 1569-75, 1992), Banasik et al. (Molec. Cell. Biochem, 138: 185-97, 1994), Cosi et al. (Expert Opin. Ther. Patents 12 (7), 2002), Southan and Szabo (Curr Med Chem, 10 321-340, 2003), Underhill C. et al. (Annals of Oncology, doi: 10.1093/annonc/mdq322, pp 1-12, 2010), Murai J. et al. (J. Pharmacol. Exp. Ther., 349:408-416, 2014), all these patents and publications being hereby incorporated by reference in their entirety. In a preferred embodiment, the PARP inhibitor compound is selected from the group consisting of rucaparib (AG014699, PF-01367338), olaparib (AZD2281), veliparib (ABT888), iniparib (BSI 201), niraparib (MK 4827), talazoparib (BMN673), AZD 2461, CEP 9722, E7016, INO-1001, LT-673, MP- 124, NMS-P118, XAV939, analogs, derivatives or a mixture thereof.
In an even more preferred embodiment, the PARP inhibitor is selected from the group consisting of rucaparib (AG014699, PF-01367338), olaparib (AZD2281), veliparib (ABT888), iniparib (BSI 201), niraparib (MK 4827), talazoparib (BMN673), AZD 2461, analogs, derivatives or a mixture thereof.
Cancer or tumor treatment
Within the context of the invention, the term treatment denotes curative, symptomatic, and preventive treatment. Pharmaceutical compositions, kits, products and combined preparations of the invention can be used in humans with existing cancer or tumor, including at early or late stages of progression of the cancer. The pharmaceutical compositions, kits, products and combined preparations of the invention will not necessarily cure the patient who has the cancer but will delay or slow the progression or prevent further progression of the disease, ameliorating thereby the patients' condition. In particular, the pharmaceutical compositions, kits, products and combined preparations of the invention reduce the development of tumors, reduce tumor burden, produce tumor regression in a mammalian host and/or prevent metastasis occurrence and cancer relapse. In treating the cancer, the pharmaceutical composition of the invention is administered in a therapeutically effective amount. Whenever within this whole specification "treatment of a cancer" or the like is mentioned with reference to the pharmaceutical composition of the invention, there is meant: a) a method for treating a cancer, said method comprising administering a pharmaceutical composition of the invention to a subject in need of such treatment; b) the use of a pharmaceutical composition of the invention for the treatment of a cancer; c) the use of a pharmaceutical composition of the invention for the manufacture of a medicament for the treatment of a cancer; and/or d) a pharmaceutical composition of the invention for use in the treatment a cancer.
The term "cancer" or "tumor", as used herein, refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, and/or immortality, and/or metastatic potential, and/or rapid growth and/or proliferation rate, and/or certain characteristic morphological features. This term refers to any type of malignancy (primary or metastases) in any type of subject. In particular, the term encompasses renal cancer at any stage of progression. Examples of cancer include, for example, leukemia, lymphoma, blastoma, carcinoma and sarcoma. More particular examples of such cancers include chronic myeloid leukemia, acute lymphoblastic leukemia, Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL), squamous cell carcinoma, small-cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatocarcinoma, breast cancer, colon carcinoma, and head and neck cancer, gastric cancer, germ cell tumor, pediatric sarcoma, multiple myeloma, acute myelogenous leukemia (AML), chronic lymphocytic leukemia, mastocytosis and any symptom associated with mastocytosis.
"Leukemia" refers to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease— acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number of abnormal cells in the blood— leukemic or aleukemic (subleukemic). Leukemia includes, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocyte leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblasts leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, and undifferentiated cell leukemia. In certain aspects, the present invention provides treatment for chronic myeloid leukemia, acute lymphoblastic leukemia, and/or Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL).
Various cancers are also encompassed by the scope of the invention, including, but not limited to, the following: carcinoma including that of the bladder (including accelerated and metastatic bladder cancer), breast, colon (including colorectal cancer), kidney, liver, lung (including small and non-small cell lung cancer and lung adenocarcinoma), ovary, prostate, testis, genitourinary tract, lymphatic system, rectum, larynx, pancreas (including exocrine pancreatic carcinoma), esophagus, stomach, gall bladder, cervix, thyroid, and skin (including squamous cell carcinoma); hematopoietic tumors of lymphoid lineage including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T- cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, histiocytic lymphoma, and Burketts lymphoma; hematopoietic tumors of myeloid lineage including acute and chronic myelogenous leukemias, myelodysplastic syndrome, myeloid leukemia, and promyelocytic leukemia; tumors of the central and peripheral nervous system including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; other tumors including melanoma, xenoderma pigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer, and teratocarcinoma; melanoma, unresectable stage III or IV malignant melanoma, squamous cell carcinoma, small-cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatocarcinoma, breast cancer, colon carcinoma, and head and neck cancer, retinoblastoma, gastric cancer, germ cell tumor, bone cancer, bone tumors, adult malignant fibrous histiocytoma of bone; childhood malignant fibrous histiocytoma of bone, sarcoma, pediatric sarcoma; myelodysplastic syndromes; neuroblastoma; testicular germ cell tumor, intraocular melanoma, myelodysplastic syndromes; myelodysplastic/myeloproliferative diseases, synovial sarcoma.
In a preferred embodiment, the cancer is a hematopoietic cancer, especially a leukemia or lymphoma. In another preferred embodiment of the present invention, the cancer is a solid tumor. For instance, the cancer may be sarcoma and oestosarcoma such as Kaposi sarcome, AIDS-related Kaposi sarcoma, melanoma, in particular ulveal melanoma, and cancers of the head and neck, kidney, ovary, pancreas, prostate, thyroid, lung, esophagus, breast, bladder, colorectum, liver and biliary tract, uterine, appendix, and cervix, testicular cancer, gastrointestinal cancers and endometrial and peritoneal cancers. Preferably, the cancer may be sarcoma, melanoma, in particular ulveal melanoma, and cancers of the head and neck, kidney, ovary, pancreas, prostate, thyroid, lung, esophagus, breast, bladder, colorectum, liver, cervix, and endometrial and peritoneal cancers. For instance, the cancer may be selected from the group consisting of breast cancer, hepatocellular carcinoma, colorectal cancer, glioblastoma, melanoma, and head and neck cancer.
In a preferred embodiment, the cancer is a breast cancer. Kits and uses thereof
The present invention finally relates to the use of a kit comprising means for determining the frequency of micronuclei in a cell population or for determining the number of large-scale chromosomal rearrangements (LSTs) for determining the sensitivity or resistance of a subject having a cancer to a treatment with a DBait molecule, for selecting a subject affected with a cancer or tumor for a treatment with a DBait molecule or for determining whether a subject affected with a cancer or tumor is susceptible to benefit from a treatment with a DBait molecule. Further aspects and advantages of the present invention will be disclosed in the following experimental section, which should be regarded as illustrative and not limiting the scope of the present application. A number of references are cited in the present specification; each of these cited references is incorporated herein by reference.
DESCRIPTION OF THE FIGURES
Figure 1. Spontaneous DNA damages and sensitivity to AsiDNA in breast cancer (BC) cell lines. A. Spontaneous DNA damages were monitored by alkaline comet assay. B. Basal PARP proteins activation was measured by Sandwich ELISA anti-Poly(ADP-Ribose) polymers. C, D, E. Spearman correlations performed using GraphPadPrism5 software show no correlation. C. Spearman r: -0.05; P value: 0.85; D. Spearman r: 0.19; P value: 0.46; E. Spearman r: -0.12; P value: 0.65.
Figure 2. Effects of endogenous double-strand break repair defects on sensitivity to AsiDNA.
A. Effect of AsiDNA (4.8μΜ) on 15 malignant (6 BRCA deficient, grey; 9 BRCA proficient, black) and 3 non-malignant (white) cell lines. Survival is defined as the percentage of living cells relative to non-treated cells (NT). B. Analysis of cell lines for LST or MN: LSTs, which indicate large genomic rearrangements, were assessed for BC cells by SNP-array profiling (ND: Not Determined). MN were quantified in 1000 cells cultured on cover slips after DAPI staining. Results are given as the percentage of cells harboring one or two MN. C, D, E. Spearman correlation analysis between: sensitivity to AsiDNA and basal levels of MN (C), sensitivity to AsiDNA and number of LSTs (D), basal levels of MN and number of LSTs (E).
Figure 3. Lack of a correlation between sensitivity of BC cells to AsiDNA and their proliferation rate. Survival (ordinate) was monitored by trypan blue staining and manual counting (10 days after treatment). Survival is expressed as the percentage of living cells relative to non-treated cells (NT). The doubling time (abscissa) was calculated by counting the cells during the 10 days of exponential growth. The correlation analysis between survival in the presence of AsiDNA and doubling time was performed using GraphPadPrism5 software and indicates that there is no significant correlation. Figure 4. DNA repair and cell cycle pathway profiles of BC cells with a MNHigh or MNLow status. A. Pathway-based heatmap showing the distance to mean expression for each pathway in MNHlgh and MNLow BC cell line samples. B. Hierarchical clustering of the BC cell lines based on gene expression values of 13 DNA repair and cell cycle genes. Clustering was done using Euclidean distance and Ward linkage. The color intensity indicates the level of change relative to the mean value of the BC cell line group. Abbreviations: CP, Checkpoint; NER, nucleotide excision repair; BER, base excision repair; MMR, mismatch repair; SSA, single-strand annealing; A_NHEJ, Alternative non-homologous end- joining; C_NHEJ, Classical non-homologous end-joining; MMEJ, microhomology-mediated end- joining; HR, homologous recombination.
Figure 5. Micronuclei are predictive biomarkers of AsiDNA treatment. A. Forty-three tumor cell lines were characterized for the frequency of MN (black) and survival (grey) in the presence of AsiDNA (4.8μΜ) as described in Material and Methods. B. Statistical analysis of the correlation between survival to AsiDNA and the percentage of cells with MN. Cells were grouped in two groups: MNLow (% cells with MN <3.6%) and MNHigh (% cells with MN >3.6%). T test significance ****p < 0.0001. Figure 6. Effect of AsiDNA monotherapy on xenografts. A. Relative tumor growth compared to the initial tumor volume. Mice bearing patient-derived xenografts (PDX) or cell-derived xenografts (CDX) were treated (black) or not (grey) with AsiDNA for 3 weeks. Vt, tumor volume at a given time; Vi, initial tumor volume before treatment. B. Effect of AsiDNA in xenografts showing a low (MNLow) or high (MNHlgh) basal level of MN, as measured by tumor growth delay (TGD). TGD values are the mean of at least 6 xenografted mice per cancer type: black circles, glioblastoma; squares, uveal melanoma; diamonds, breast cancer; grey circle, colon cancer; black triangles, skin melanoma. *p < 0.05.
Figure 7. Assessment of micronuclei in breast cancer patient biopsies. A. Micronuclei (MN) quantification in 12 breast cancer (BC) patient' biopsies. Biopsies showing a % of cells with MN < 1% are considered MNLow and biopsies containing a % of cells with MN > 1% are considered MNHlgh. B. Representative images of tumor sections after hematoxylin/Eosin staining and MN (arrows) assessment in BC patient biopsies. Scale: 100 μιη.
Figure 8. Micronuclei frequency is predictive biomarker for sensitivity to AsiDNA in hematopoietic cancer cell lines. Spearman correlation between AsiDNA efficiency (AsiDNA EC50, μΜ), and Micronuclei frequency of the different cells lines (expressed as the percentage of cells with micronuclei). P values are shown.
Further aspects and advantages of the present invention will be described in the following examples, which should be regarded as illustrative and not limiting. EXAMPLES
EXAMPLE 1: Genetic instability (MN and LSTs) as predictive biomarkers for treatment with AsiDNA in solid tumors. Here, the inventors characterized potential predictive biomarkers for treatment with AsiDNA, a first-in- class DNA repair inhibitor. Tumors showing spontaneous genetic instability are dependent on DNA repair to survive. Thus the inventors determined whether this intrinsic characteristic is a prerequisite for sensitivity to AsiDNA. They evaluated genetic instability and DNA repair defects by direct and indirect assays in 12 breast cancer cell lines to estimate the spontaneous occurrence of single-strand and double- strand breaks (DSBs). For each cell line, they monitored constitutive poly(ADP-ribose) polymerase (PARP) activation, spontaneous DNA damage by alkaline comet assay, basal Micronuclei (MN) levels, and the number of large-scale chromosomal rearrangements (LSTs) in a SNP-array tumor profile, and the status of several DNA repair pathways by transcriptome and genome analysis. Sensitivity to AsiDNA was associated with a high spontaneous frequency of cells with MN, LSTs and specific alterations in DNA repair pathways that essentially monitor DSB repair defects. A high basal level of MN as a predictive biomarker for AsiDNA treatment was validated in 43 tumor cell lines from various tissues and 16 models of cell- and patient-derived xenografts. MN quantification was also possible in patient biopsies. Overall, this study identified genetic instability as a predictive biomarker for sensitivity to AsiDNA treatment.
Results
Genetic instability is a predictive biomarker of response to AsiDNA in breast cancer cell lines
The inventors assessed whether the level of spontaneous DNA damage could determine sensitivity to AsiDNA, as it is a DNA repair pathway inhibitor. They measured endogenous SSBs and base damage to reveal differences between cell lines using the alkaline COMET assay (which essentially measures SSBs) and ELISA to detect Poly(ADP-Ribose) (PAR) levels (which measures the spontaneous activation of the Poly(ADP-ribose) polymerase (PARP) enzyme) (Figure 1 A, B). There was no correlation between comet tail moments and PAR content of the cells, although PARP activation, detected by PAR polymer formation, is thought to be mainly triggered by single-strand breaks. This indicates that other factors, such as PARP and Poly-ADP-ribose Glycohydrolase (PARG) levels or other signaling enzymes, may affect the PARP-dependent signal (Figure 1 E). Statistical analyses showed no correlation between survival in the presence of AsiDNA with either comet tail moments or PAR levels (Figure 1 C, D), suggesting that the level of SSBs and oxidative damage to chromosomes is not predictive of sensitivity to AsiDNA.
DSBs are the most harmful type of DNA damage. Dbait inhibits both HR and NHEJ repair pathways, and should thus be toxic to cells with a high basal content of DSBs. The inventors tested the persistence of broken chromosomes and abnormal chromosome segregation in BC cell cultures by investigating the presence of MN after DAPI staining. MN were identified as small nuclear bodies containing DNA and chromatin located in the vicinity of the nucleus (Fenech et al. Mutat Res. 1993 Jan;285(l):35-44). MN assays have been developed in human lymphocytes to measure both whole chromosome loss and chromosome breaks upon genotoxic exposure. MN are also frequently found in some solid tumors (Bhatia et al. APMIS. 2013 Jul; 121(7):569-81). The inventors analyzed the frequency of cells with MN in growing cultures. BC cell lines showed MN frequencies from 1 to 12% (Figure 2B). The frequency of MN in cell cultures highly correlated with sensitivity to AsiDNA (Figure 2C; Spearman r: -0.77, P value = 0.0008). MN appear after cell division. Importantly there was no correlation between sensitivity of BC cell lines to AsiDNA or MN frequency and their rate of proliferation (Figure 3), indicating that sensitivity is due to defects in DSB repair (revealed by MN) and not a high rate of cell proliferation.
The inventors confirmed the MN data by performing genome analysis to estimate the number of chromosomal breakpoints within a tumor cell population genome by measuring the level of LSTs, corresponding mainly to copy number alterations. The BC cell lines showed different levels of LSTs (Figure 2B), reflecting large differences in genetic instability. The number of LSTs correlated with sensitivity to AsiDNA (Figure 2D; Spearman r: -0.7, P value = 0.0078). The frequency of cells with MN highly correlated with the level of LSTs (Figure 2E; Spearman r: 0.88, P value < 0.0001). This result shows that the genetic instability in most of the cell lines, as measured by LSTs, which reflect the history of the accumulated events in the tumor cell genome during tumor development, correlates with their recent genomic instability and defects in DSB repair, revealed by the presence of MN.
Mechanisms underlying MN formation and sensitivity to AsiDNA
The inventors investigated the mechanisms at the origin of MN formation, a prerequisite for sensitivity to AsiDNA, by evaluating the performance of the DNA repair pathways and cell cycle checkpoints of the BC cell lines. For these analyses, they integrated mRNA expression and copy number variations, of DNA repair pathways and cell cycle checkpoints of the BC cell lines. Gene expression analysis showed that cells which were MNLow and resistant to AsiDNA up-regulated all DNA repair pathways and cell cycle checkpoints, whereas these pathways were down-regulated in the MNHlgh cells (Figure 4A). Moreover, they observed many genetic alterations in the DNA repair pathways and cell cycle checkpoints of the MNHlgh group, where copy number losses were abundant (48% of genes with copy losses). There were very few alterations in the MNLow group, but they included copy number gains, which could increase the performance of these pathways, and only 9% of genes with copy losses. Interestingly, 13 genes among these pathways allowed the BC cell lines to cluster according to their MN status (Figure 4B). These genes were significantly differentially expressed between the MNHlgh and the MNLow groups (fold change > 1.5; p<0.05), and belong essentially to the DSB repair pathways HR and NHEJ (BRCA1, POLD3, MRE11, DCLRE) and to the BER pathway (SMUG, MBD4). Defects in the DSB repair pathways may explain the high frequency of MN and LSTs, and ultimately the sensitivity of these cell lines to AsiDNA. MN are predictive biomarkers of AsiDNA sensitivity in all tumor type cell lines
Analyses of the genomes of multiple tumor types have shown that genomic alterations vary between tumors from different tissues of origin. The inventors tested whether a high level of MN could also predict the response to AsiDNA in cancer cell models other than BC. They assessed 43 different cell lines of various cancer type (hepatocellular carcinoma, colorectal cancer, glioblastoma, melanoma, and head and neck cancer) for their sensitivity to AsiDNA and their basal level of MN (Figure 5A). All cell lines with a high level of MN (> 3.6% of cells with MN - MNHigh) showed higher sensitivy to AsiDNA treatment than those with low levels of MN (< 3.6% of cells with MN - MNkw), confirming the predictive value of MN levels (P value < 0.0001) in all the tested models (Figure 5B). MN are detected in xenografted tumors and predict response to AsiDNA
The inventors validated the predictive value of MN levels in tumors using four BC-xenografted models derived from BC cell lines already studied in vitro. Two models were patient-derived xenografts BC227 and BC173, which were used to generate the BC227 and BC173 cell lines, studied in vitro. The two other models were obtained by grafting the MDA-MB-231 and the MDA-MB-468 cells. The cell-line analysis classified MDA-MB-231 as MNLow and the three others as MNHigh (Figure 4A). Histological samples of the tumors were stained with Hematoxylin/eosin and analyzed by microscopy for the frequency of tumor cells with MN. The MN levels were generally lower in the xenografts than in the corresponding cell cultures (Table 1). The three MNHlgh cell lines formed tumors with detectable MN, whereas the MNLow MDA-MB-231 cell line, gave rise to tumors with no detectable MN. Tumor sensitivity to AsiDNA correlated with the MN frequency in the xenografts. The inventors observed complete tumor growth arrest for up to 60 days after treatment in the MNHlgh xenografts, whereas the MNLow MDA-MB-231 tumor escaped treatment immediately after completion (three weeks) (Figure 6A). They observed no partial (> 20% decrease in the initial size of the tumor) or complete responses in the MNLow model. In contrast, all MNHlgh tumors showed partial or complete responses (Table 2). They confirmed the general predictive value of MN by evaluating the presence of MN in histological samples of all tumor models used in previous studies and comparing them to their response to AsiDNA monotherapy. They included models for BC, colorectal cancer, skin melanoma, uveal melanoma, and glioblastoma. As observed for BC, the frequency of MN did not depend on the cancer type, but rather the tumor model studied. The xenografts were classified MNLow if they presented < 2.8% cells with MN and MNHigh if they showed > 2.8% cells with MN, similar to the threshold used for the BC model. Xenografts with a MNHlgh status showed a greater tumor growth delay (TGD) induced by AsiDNA treatment than xenografts with MNLow status (p < 0.05; Figure 6B and Table 2). All MN^W models were poor responders and did not show a significant difference in growth after AsiDNA treatment, with a maximal TGD of approximately 160%, meaning that it took only 60% more time for the tumors to achieve a four-fold increase in size relative to vehicle-treated tumors. In contrast, most tumor types (5/8) with a MNHlgh status showed an at least three-fold increase (300%) in TGD after AsiDNA treatment. The three remaining MNHigh tumors behaved like the MNLow models (Figure 6B). Taken together with the in vitro analysis, our results suggest that the absence of MN could be a predictive biomarker for resistance to AsiDNA monotherapy, whereas the presence of MN is necessary, but not sufficient, to provide sensitivity. MN frequency in patient biopsies
The inventors analyzed whether MN can be observed in patient biopsies. They quantified the MN in hematoxylin/eosin stained tumor sections of biopsies from unselected BC samples from the Curie Hospital Pathology Department (Figure 7). Half of the 12 analyzed BC biopsies displayed a MNHlgh status (% cells with MN > 3%), whereas only few or no MN were detectable in the others (MN1™; % cells with MN < 1%). There was no evident link between MN status and the type or grade of the tumors (Table 3). The lack of correlation between genetic instability and tumor grade or type has already been reported in a large study of 5,371 tumors using three different methods to estimate genetic instability.
Discussion
In this study, the inventors determined the sensitivity of a set of tumor cell lines to AsiDNA and estimated the presence of DNA damage by various direct and indirect methods. Sensitivity to AsiDNA was associated with the presence of unrepaired or misrepaired DSBs, revealed by MN formation, and large genome rearrangements (measured by LST analysis). SSBs and spontaneous PARP activation were not essential, confirming that DSBs are probably the main factor for cell survival. Transcriptomic and genomic analysis revealed down regulation of DNA repair pathways and several alterations, especially in DSB repair pathways of sensitive BC cells. These DSB repair defects may explain the high frequency of MN and LST in these cells, and therefore their sensitivity to AsiDNA treatment. The inventors analyzed the sensitivity of BC cell lines to Olaparib (a PARP inhibitor) in a previous study (Jdey et al. Clin Cancer Res. 2017 Feb 15;23(4): 1001- 1011). They did not observe a correlation between the frequency of MN and LST and sensitivity to Olaparib in these cells. Thus, these predictive biomarkers appear to be specific to AsiDNA and are not applicable to other DNA repair inhibitors.
The occurrence of spontaneous DSBs is difficult to quantify as cells do not survive, except if they are repaired in a faithful (HR) or unfaithful (NHEJ) manner. Unfaithful repair leads to an accumulation of breaks and ligations that can be detected in the tumor cell genomes as discontinuous or amplified/deleted large sequences (LSTs). However, these scars accumulate during tumor development and do not represent the temporal status of the genetic events at the time of treatment. Therefore, the detection of MN is an additional piece of information that reflects the real-time occurrence of spontaneous damage as they do persist for more than one or two divisions. Moreover, they are specific to the proliferating population, as they are produced at mitosis. Here, MN and LST frequencies correlated in cell line cultures.
The present study suggests, for the first time, that MN could be used as a predictive biomarker for response to AsiDNA. Detection of basal MN frequencies in biopsies of different tumor types, before AsiDNA treatment, could help to predict good and poor responders. This tool would allow to stratify patients for tailored treatment and appropriate dosing. In general, the present finding that low basal levels of LSTs and MN could be biomarkers of resistance to AsiDNA suggests that aggressive tumors with high genetic instability (frequently with a poor prognosis) may be the preferential indication for AsiDNA treatment.
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
MN, Micronuclei; BC, Breast Cancer; C: canalar; L: lobular. Materials and Methods Cell culture
Cell cultures were maintained for four BRCA deficient BC cell lines (BC227, BRCA2 deficient, from the Institut Curie, and HCC1937, HCC38, and MDAMB436, BRCA1 deficient, from the ATCC), eight BRCA proficient BC cell lines (BC173 from Institut Curie, and BT20, HCC1143, HCC1187, HCC70, MCF7, MDAMB231 , and MDAMB468 from the ATCC), three non-tumor mammary cell lines (184B5, MCF10A, and MCF12A from the ATCC), three human cervical cancer HeLa cell lines silenced for BRCA1 (HelaBRCAlSX, Tebu-Bio, reference 00301-00041), BRCA2 (HelaBRCA2SX, Tebu-Bio, reference 00301-00028), and control (HeLaCTLSX, Tebu-Bio 01-00001), seven human glioblastoma cell lines (M059K, M059J, SF767, SF763, FOG, U87, and T98G), three human melanoma cell lines (SK28, SK28LshCTL, and SK28 LshDNA-PKcs), seven human colorectal cancer cell lines (HCT116, HT29, CAC02, SW480, SW620, CT329X12, and CRLRB018), seven human hepatocellular carcinoma cell lines (HepG2, SNU423, HLE, PLP-PRF5, SNU449, Huh7, and Huh6) and the human head and neck cancer cell line Hep2 (from ATCC). Cell lines were authenticated by short tandem repeat profiling (Geneprint 10, Promega) at 10 different loci (TH01, D21S11, D5S818, D13S317, D7S820, D16S539, CSF1PO, AMEL, vWA, TPOX). Cell lines were verified to be negative for Mycoplasma contamination using the VenorGeM Avance Kit (Biovalley). Cells were grown according to the supplier's instructions. Cell lines were maintained at 37°C in a humidified atmosphere at 5% C02. AsiDNA molecule
AsiDNA is a New Chemical Entity, a 64-nucleotide (nt) oligodeoxyribonucleotide consisting of two 32- nt strands of complementary sequence connected through a l,19-bis(phospho)-8-hydraza-2-hydroxy-4- oxa-9-oxo-nonadecane linker with a cholesterol at the 5'-end and three phosphorothioate internucleotide linkages at each of the 5' and the Ύ ends (Agilent, USA). The sequence is: 5'- X GsCsTs GTG CCC ACA ACC CAG CAA ACA AGC CTA GA - L - CL - TCT AGG CTT GTT TGC TGG GTT GTG GGC AC sAsGsC -3' (SEQ ID NO 23), where L is an amino linker, X a Cholesteryl tetraethyleneglycol, CL a Carboxylic (Hydroxyundecanoic) Acid Linker, and s a Phosphorothioate linkage.
Figure imgf000041_0001
Measurement of cellular sensitivity to AsiDNA
AsiDNA cytotoxicity was measured by quantification of relative survival and cell death. Adherent cells were seeded in 24-well culture plates at appropriate densities and incubated for 24 h at 37°C before AsiDNA addition. Cells and supernatant were harvested on day 10 of treatment, stained with 0.4% trypan blue (Sigma Aldrich, Saint-Louis, USA), and counted with a Burker chamber. Cell survival was calculated as the ratio of living treated cells to living mock-treated cells. Cell death was calculated as the number of dead cells over the total number of counted cells.
Micronuclei detection
MN result from chromosomal breakage or spindle damage. They arise in the nuclei of daughter cells following cell division and form single or multiple MN in the cytoplasm. For in vitro analysis, cells were grown on cover slips in a Petri dish. Cells were then fixed with PFA (4%), permeabilized with Triton (0.5%), and stained with DAPI
Figure imgf000041_0002
The frequency of MN was estimated as the percentage of cells with MN over the total number of cells. At least 1000 cells were analyzed for each cell line. MN assessment in tumors was performed as follows: tumors were fixed in formalin and then embedded in paraffin. Sections were cut and stained with hematoxylin, eosin, and saffron. The percentage of MN was estimated in the non-necrotic and proliferative area by quantifying the cells presenting MN in the cytoplasm, according to their described characteristics in at least 1000 cells.
SNP array processing and evaluation of the number of LSTs
SNP-arrays were processed using GAP methodology to obtain absolute copy number (CN) and allelic content profiles (Popova et al. Genome Biol. 2009; 10(11):R128). LSTs were defined as a chromosomal breakpoint (change in copy number or allelic content) between adjacent regions, each of at least 10 megabases (Mb). The number of LSTs, representing the number of breakpoints between large chromosome fragments, was calculated as previously described (Popova et al. Cancer Res. 2012 Nov l ;72(21):5454-62). Alkaline Single-cell electrophoresis "COMET Assay"
Cells (2-3x105 cells/ml) were suspended in 0.5% low melting point agarose in culture medium and transferred onto a frosted glass microscope slide pre-coated with a layer of 0.5% normal melting point agarose. Slides were immersed in lysis solution [2.5 mol/L NaCl, 100 mmol/L EDTA,10 mmol/L Tris, 1% sodium lauryl sarcosinate, 10% DMSO, and 1% Triton X-100 (pH 10)] at 4°C for 1 h, placed in an electrophoresis tank containing 0.3 mol/L NaOH (pH 13) and 1 mmol/L EDTA for 40 min, subjected to electrophoresis for 25 min at 25 V (300 mA), washed with neutral buffer [400 mmol/L Tris-HCl (pH 7.5)], and stained with 20 μg/mL ethidium bromide. The variables of the "comets" were quantified using Comet Assay 2 software (Perceptive Instrument). Triplicate slides were processed for each experimental point. The tail moment is defined as the product of the percentage of DNA in the tail and the displacement between the head and the tail of the comet.
ELISA
A sandwich ELISA was used to detect Poly(ADP-Ribose) (PAR) polymers. Cells were boiled in PathScan Sandwich ELISA Lysis Buffer (Cell Signaling Technology) supplemented with ImM PMSF (Phenylmethanesulfonyl Fluoride, Sigma). Cell extracts were then diluted in Superblock buffer (Thermo Scientific) prior to the ELISA Assay. Briefly, 100 carbonate buffer (1.5 g/L sodium carbonate Na2C03, 3 g/L NaHC03) containing the capture antibody (mouse anti-PAR at 4 μg/ml, Trevigen) was added to 96 well plates and incubated overnight at 4°C. The plates were then washed and blocked with Superblock at 37°C for 1 h, and 10 μL. of cell extract added to 65 μL. of Superblock in each well and the plate incubated overnight at 4°C. After washing, 75 μL. buffer (PBS/2% milk/1% mouse serum) containing detection antibody (Rabbit anti-PAR, diluted 1/1000, Trevigen) was added and the plate incubated for 1 h at RT (Room Temperature). The plates were then washed and the wells incubated with 75 μL. buffer (PBS/2% milk/1% mouse serum) containing an HRP-conjugated anti-rabbit antibody (diluted 1/5000, Abeam) for 1 h at RT. Following multiple washes, 75μL. Supersignal Pico (Pierce) was added to the wells. The optical absorbance (OD 425 nm) of each well was determined at various time points (1, 5, and 15 min) to optimize the signal to noise ratio. In Each condition was performed in duplicate wells for all experiments.
High-throughput data analysis
mRNA expression analysis: mRNA expression data for the BC cell lines were produced using Human Exon 1.0 ST Affymetrix microarrays. Raw data were RMA normalized and summarized with FAST DB annotation (version 2013_1) (Irizarry et al. Biostatistics. 2003 Apr;4(2):249-64; de la Grange et al. Nucleic Acids Res. 2005 Jan;33(13):4276-84). Gene expression data were log2 transformed and the mean centered over all the cell line samples which were then assigned into the two groups (MNHlgh, MNLow). Genes associated with Cell cycle and DNA repair pathways were retrieved from Atlas of Cancer Signalling Networks (https://acsn.curie.fr). The mean expression of these genes in MNHlgh and MNLow samples was represented in heatmaps showing the distance to mean expression for each gene. The clustering has been performed using euclidean distance and Ward agglomeration method.
Copy number data analysis: The copy number (CN) values for each gene for the cell lines was assessed by GAP analysis of the data generated on the Affymetrix Genome Wide SNP Array 6.0 (Popova et al. Genome Biol. 2009 Jan; 10(l l):R128) and corrected for ploidy. Two CN were considered to be 'normal', less than two CN as a loss and more than two CN as a gain. Each gene was then given a score using the average copy number across samples of the cell lines in the same group (MNHlgh, MNLow).
In vivo experiments
MDA-MB-231 and MDA-MB-468 Cell-Derived-Xenografts (CDXs) were obtained by injecting 107 Breast Cancer (BC) tumor cells into the mammary fat pad of six to eight-week-old adult female nude NMRI-nu Rj : NMRI-Foxn 1 nu/ Foxnlnu mice (Janvier). BC227 and BC173 BC tumor models are Patient-Derived Xenografts (PDXs) and were established at the Curie Institute (France) as described in (Marangoni et al. Clin Cancer Res. 2007 Jul 1 ; 13(13):3989— 98). The uveal melanoma models were also PDXs (MM26, MP34, MP41 and MP55). Glioblastoma models were either PDXs (GBM14-RAV, TGI -HAM, ODA4_GEN) or CDXs (CB 193, T98G, SF763 and SF767). The colorectal cancer (HT29) and skin melanoma models (SK28) were CDXs. For PDX engraftment, fragments of 30 to 60 mm3 were grafted into the mammary fat pad (BC227 and BC173) or right flank of six to eight- week-old female nude mice (Janvier). The animals were housed at least one week before tumor engraftment, under controlled conditions of light and dark (12h-12h), relative humidity (55%), and temperature (21°C). When engrafted tumors reached 80-250 mm3, mice were individually randomized into groups of 8-12 to different treatment groups. AsiDNA was injected locally (intratumoral and peritumoral subcutaneous administration). Tumor growth was evaluated three times a week using a caliper and calculated using the following formula: (length x width x width)/2. Mice were followed for up to six months, and ethically killed when the tumor volume reached 2,000 mm3. The Local Animal Experimentation Ethics Committee approved all experiments.
Statistical analysis
Two-sided unpaired t tests were used for comparisons between MN formation and tumor growth delay (TGD). TGD was calculated by subtracting the mean tumor volume quadrupling time of the control group from the tumor volume quadrupling times of individual mice in each treatment group. The mean TGD was calculated for each treatment group from the individual measurements. Statistical analyses were performed using GraphPadPrism (GraphPad Software, Inc., San Diego, USA) and statEL software (Ad Science, Paris, France).
EXAMPLE 2: Genetic instability (MN and LSTs) as predictive biomarkers for treatment with AsiDNA in hematologic cancers (leukemia and lymphoma).
Materials and Methods
Culture of malignant hematologic cell lines.
Ten hematologic malignant cell lines including THP-1 and U-937 myeloid leukemia cells; Jurkat-E6.1, MOLT-4, 174xCEM.T2m and MT4 acute CD4+ T-cell leukemia; Raji and IM-9 B-cell Burkitt lymphoma, and HuT-78 CD4+ T-cell cutaneous lymphoma and SupT-1 CD4+ T-cell pleural lymphoma were purchased from the ATCC (Raji, CCL-86; IM-9, CCL-159; HuT-78, CRMB-TIB-161 ; Sup-Tl, CRL-1942; Jurkat-E6.1, TIB-152; MOLT-4, CRL-1582; 174xCEM.T2, CRL-1992; U-937, CRL- 1593.2, and THP-1, TIB- 202 respectively). Cell lines were authenticated at the beginning and at the end of the study by short tandem repeat profiling (Geneprint 10, Promega) at 9 different loci (TH01, D5S818, D13S317, D7S820, D16S539, CSF1PO, AMEL, vWA, and TPOX). Cell lines were verified to be negative for mycoplasma contamination using the VenorGeM Advance Kit (Biovalley). Cells were grown according to the supplier's instructions in RPMI1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere at 5% CO2. Reagents for cell culture were purchased from Gibco Invitrogen.
Micronuclei detection
MN assessment in hematologic malignant cell lines was performed as described in Example 1. Measurement of cellular sensitivity to AsiDNA
For proliferation studies, cells were cultured in 96-well plates: the MT-4, MOLT-4, 174xCEM.T2, Jurket-E6.1, Sup-Tl, U-937, IM-9, and Raji cell lines were seeded at 2xl05 cells/ml , HuT-78 at 4xl05 cells/ml, and THP-1 at 106 cells/ml. Cell proliferation and cell death were monitored after staining with 0.4% trypan blue (Sigma Aldrich, Saint-Louis, USA) by visual 166 counting using a Burker chamber. Cell survival was calculated as the ratio of living treated cells to living mock-treated cells. Cell death was calculated as the number of dead cells divided by the total number of cells. Relative cell survival was measured by the mitochondrial MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-154 diphenyltetrazolium bromide) colorimetric assay or live-cell monitoring. MTT assay was modified for cells in suspension. Briefly, one-tenth of the culture volume of MTT solution (5 mg/ml in PBS was added to the cultures and the cells incubated at 37°C in 5% C02 for 30 min to 4 h (depending on the cells). Cells were lysed by adding one culture volume of lysis buffer (50% dimethylformamide, 20% SDS, 0.22% acetic acid) directly to the medium and incubated for 2 h at 37°C before reading at 550 nm (Victor-X3, PerkinElmer). Statistical analysis
Correlations were evaluated using the Spearman test (nonparametric test). P values < 0.05 were considered to be significant for all statistical tests applied.
Results
AsiDNA is toxic to malignant hematologic cells and MN frequency is a predictive biomarker.
The toxicity of AsiDNA monotherapy was tested on a wide range of hematologic cancer cells, including two myeloid leukemias, four acute T-cell leukemias, two Burkitt's B-cell lymphomas, and two T-cell lymphomas. We evaluated the half maximal effective concentration (EC50) of AsiDNA for all cell lines by measuring the survival four days after treatment with various doses, ranging from 0.16 to 48 μΜ the maximum concentration close to the level observed in Monkeys after IV injection. AsiDNA was toxic for eight of the 10 tested cell lines, with EC50s inferior to 16 μΜ for the most sensitive (class "S": U- 937, IM-9, MOLT-4, and Sup-Tl) and ranging from 16 to 48 μΜ for the cells with intermediate sensitivity (class "I": MT4, Jurkat-E6.1, 174xCEM.T2, and HuT-78). Two cell 226 lines were resistant up to the maximum tested dose of 48 μΜ (class "R": Raji and THP1). Similar results were obtained with the colorimetric MTT assay, or direct counting of viable cells by trypan blue enumeration.
Sensitivity to AsiDNA was independent of the cellular subset (myeloid, B cells, T cells) and the type of disease, either leukemia or lymphoma. Unexpectedly, the efficacy of AsiDNA in the various cell lines did not appear to depend on the level of p53 activity, as the most sensitive (U-232 937 and Sup-Tl) and resistant cells (Raji and THP-1) were all p53 deficient (p value= 0.3387, ns).
Interestingly, the results indicate that genetic instability is a biomarker of AsiDNA sensitivity in hematologic malignancies. Indeed, micronuclei frequency was found to correlate with the sensitivity of the hematologic cell lines to AsiDNA (Figure 8). Moreover, neither AsiDNA cell uptake nor activation of H2AX phosphorylation are predictive biomarker for sensitivity to AsiDNA.

Claims

1- Use of genetic instability as measured by a frequency of cells with micronuclei and/or a number of large-scale chromosomal rearrangements (LSTs) as a predictive biomarker of a sensitivity or resistance to a treatment of cancer with a nucleic acid molecule, said nucleic acid molecule having at least one free end and comprising a hairpin with a DNA double stranded portion of 24-200 bp with less than 60% sequence identity to any gene in a human genome.
2- A method for determining the sensitivity or resistance of a subject having a cancer to a treatment with a nucleic acid molecule, said nucleic acid molecule having at least one free end and comprising a hairpin with a DNA double stranded portion of 24-200 bp with less than 60% sequence identity to any gene in a human genome, wherein the method comprises determining a frequency of cells with micronuclei and/or a number of large-scale chromosomal rearrangements (LSTs) in a biological sample from the subject, the frequency of cells with micronuclei and/or the number of large-scale chromosomal rearrangements (LSTs) being positively correlating to the sensitivity of the subject to a treatment of cancer with the nucleic acid molecule.
3- A nucleic acid molecule having at least one free end and comprising a hairpin with a DNA double stranded portion of 24-200 bp with less than 60% sequence identity to any gene in a human genome for use for treating a cancer in a subject having a high genetic instability as measured by a frequency of cells with micronuclei and/or a number of large-scale chromosomal rearrangements (LSTs).
4- The use of claim 1, the method of claim 2 or the nucleic acid molecule for use of claim 3, wherein the nucleic acid molecule has one of the following formulae:
Figure imgf000046_0001
wherein N is a deoxynucleotide, n is an integer from 19 to 195, the underlined N refers to a nucleotide having or not a modified phosphodiester backbone, L' is a linker, C is the molecule facilitating endocytosis selected from a lipophilic molecule or a ligand which targets cell receptor enabling receptor mediated endocytosis, L is a linker, m and p, independently, are an integer being 0 or 1. 5- The use, method or nucleic acid molecule for use of claim 4, wherein the nucleic acid molecule of formula (I), (II) or (III) has one or several of the following features:
- n is an integer from 23 to 195 or from 27 to 95, and/or
- N is a deoxynucleotide selected from the group consisting of A (adenine), C (cytosine), T (thymine) and G (guanine) and selected so as to avoid occurrence of a CpG dinucleotide and to have less than 80% sequence identity to any gene in a human genome.; and/or,
- the linked L' is selected from the group consisting of hexaethyleneglycol, tetradeoxythymidylate (T4), l,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane and 2,19-bis(phosphor)-8-hydraza- 1 -hydroxy-4-oxa-9-oxo-nonadecane; and/or,
- m is 1 and L is a carboxamido polyethylene glycol, more preferably carboxamido triethylene glycol or carboxamido tetraethylene glycol; and/or,
- C is selected from the group consisting of a cholesterol, single or double chain fatty acids such as octadecyl, oleic acid, dioleoyl or stearic acid, or ligand (including peptide, protein, aptamer) which targets cell receptor such as folic acid, tocopherol, sugar such as galactose and mannose and their oligosaccharide, peptide such as RGD and bombesin, and protein such transferring and integrin, preferably is a cholesterol or a tocopherol, still more preferably a cholesterol.
6- The use, method or nucleic acid molecule for use of claim 5, wherein the nucleic acid molecule is
Figure imgf000047_0001
with the underlined nucleotide having phosphorothioate internucleotide linkages.
7- The method of any one of claims 2 and 4-6, wherein the biological sample of said subject is a cancer sample, in particular a tumor biopsy or a biological fluid comprising cancer cells.
8- The method of any one of claims 2 and 4-7, wherein the frequency of cells with micronuclei is determined by counting the number of cells having micronuclei in a cancer sample.
9- The method of claim 8, wherein a frequency higher than 1%, preferably higher than 2%, more preferably higher than 2.5 or 3% is indicative of a sensitivity to the treatment with said nucleic acid molecule. 10- The method of any one of claims 2 and 4-7, wherein the number of large-scale chromosomal rearrangements (LSTs) is determined with a SNP-array.
11- The method of claim 10, wherein the number of large-scale chromosomal rearrangements (LSTs) corresponds to the number, per genome, of breakpoints resulting in segments of at least 3, 4, 5, 6, 7, 8
9, 10 megabases, preferably at least 10 megabases.
12- The method of claim 11, wherein a number of large-scale chromosomal rearrangements (LSTs) of at least 10 large-scale chromosomal rearrangements (LSTs), preferably at least 15, is indicative of a sensitivity to the treatment with said nucleic molecule.
13- The method of any one of claims 2 and 4-12, wherein the method further comprises selecting the subject as for determining the sensitivity or resistance of a subject having a cancer to a treatment with a nucleic acid molecule as defined in claims 2 and 4-6.
14- Use of a kit comprising means for determining the frequency of micronuclei in a cell population or for determining the number of large-scale chromosomal rearrangements (LSTs) for determining the sensitivity or resistance of a subject having a cancer to a treatment with a nucleic acid molecule, for selecting a subject affected with a cancer or tumor for a treatment with a nucleic acid molecule or for determining whether a subject affected with a cancer or tumor is susceptible to benefit from a treatment with a nucleic acid molecule, said nucleic acid molecule being as defined in any one of claims 1 and 4- 6.
15- Nucleic acid molecule for use of any one of claim 3-6, wherein a sample of the cancer of said subject has at least 1 % of cells with micronuclei, preferably at least 2%, or presents at least 10 large-scale chromosomal rearrangements (LSTs), preferably at least 15.
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WO2023111203A1 (en) 2021-12-16 2023-06-22 Onxeo New conjugated nucleic acid molecules and their uses

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