HK1182016B - Use of rnai inhibiting parp activtiy for the manufacture of a medicament for the treatment of cancer - Google Patents

Description

Use of RNAi inhibiting PARP activity for the manufacture of a medicament for the treatment of cancer

The present application is a divisional application of chinese patent application 200480027294.1 (international application number PCT/GB2004/003235) entitled "use of RNAi that inhibits PARP activity for the manufacture of a medicament for the treatment of cancer", filed on 7/23/2004.

Technical Field

The present invention relates to the use of agents that inhibit the activity of enzymes that mediate DNA strand break repair for the treatment of certain forms of cancer, particularly breast cancer.

Background

It has been shown that in mammalian cells, Homologous Recombination (HR) plays an important role in the repair of damage that occurs at the DNA replication fork (2). Thus, HR-deficient cells appear to be slow growing and exhibit a higher level of genetic instability. It is thought that genetic instability in human cancers due to loss of HR repair contributes significantly to the development of cancer in these cells (1).

Post-transcriptional modification of nuclear proteins by poly (ADP-ribosyl) ation (PARP) plays an important role in DNA repair, regulation of programmed cell death, and maintenance of genomic stability in response to DNA strand breaks.

Poly (ADP-ribose) polymerase (PAPR-1), a nuclear protein abundantly present in mammalian cells, utilizes NAD⁺As a substrate, to catalyze the formation of poly (ADP-ribose) (PAR) multimers. Upon DNA damage, PAPR-1 rapidly binds to DNA strand breaks (single or double stranded) and catalyzes the addition of negatively charged PAR strands to itself (self-modification) and other proteins (reviewed in [3, 4]]). Binding of PAPR-1 to DNA strand breaks is thought to protect DNA damage from further processing until PAPR-1 is dissociated from the break by the accumulated negative charge caused by the PAR multimer (5, 6).

PAPR-1 knockout mice develop normally, although PAPR-1 is involved in some nuclear processes, such as regulation of chromatin structure, DNA replication, DNA repair and transcription (7). Cells isolated from these mice display an excessively recombinant phenotype and elevated levels of SCE, genetic instability of micronuclei and tetraploidy (8-10). Genetic instability can also occur in these PAPR-1 knockout mice through telomere shortening, increased frequency of chromosome fusion and aneuploidy (11), although all of these results are not reproducible in another group of PARP-1 knockout mice (12). In the previous mouse knockdown, PAPR-1 null mutations rescued impaired V (D) J recombination in SCID mice (13). These results support the view proposed by Lindahl and coworkers that PARP-1 has protection against recombination (5). They suggest that PARP-1 in combination with DNA strand breaks prevents the recombination machinery from recognizing and handling DNA damage, or alternatively that negative charges accumulated after poly ADP-ribosylation repel adjacent recombinant DNA sequences. Only the latter model is consistent with the self-inhibition of PARP-1 and the expression, induction of SCE, gene amplification and homologous recombination of the dominant negative mutant PARP-1 (HR [14-18 ]).

Studies based on PARP inhibitor treated cells or cells derived from PARP-1 or PARP-2 knockout mice show that inhibition of PARP-1 activity increases the sensitivity of cells to DNA damaging agents and inhibits strand break reconnection (3,4,8-11,19,20, 47).

Inhibitors of PARP-1 activity have been used in combination with conventional anti-cancer agents such as radiation therapy and chemotherapy (21). The use of inhibitors in combination with methylating agents, topoisomerase poisons and ionising radiation has been found to improve the effectiveness of these forms of treatment. However, such treatments are known to cause injury and death to non-cancerous or "healthy" cells and are associated with some undesirable side effects.

There is therefore a need for a cancer treatment that is both effective and selective in killing cancer cells and that does not have to be used in conjunction with radiation therapy and chemotherapy.

The present inventors have surprisingly found that cells deficient in Homologous Recombination (HR) are hypersensitive to PARP inhibitors compared to wild type cells. This is surprising because PARP-1 knockout mice live normally, suggesting that PARP-1 is not essential for life. Thus, it cannot be expected that cells will be sensitive to PARP inhibition.

According to a first aspect of the present invention there is provided the use of an agent that inhibits the activity of an enzyme that mediates repair of a DNA strand break for the manufacture of a medicament for the treatment of a disease caused by a genetic defect in a gene that mediates homologous recombination.

In another aspect, the invention provides a method of treating a disease or condition in a mammal (including a human being) which disease or condition is caused by a genetic defect in a gene which mediates homologous recombination, the method comprising administering to the mammal a therapeutically effective amount of an agent which inhibits the activity of an enzyme which mediates repair of DNA strand breaks or other damage present in the replication fork.

In a preferred aspect, the enzyme is PARP. In a more preferred aspect, the agent is a PARP inhibitor or an RNAi molecule specific to the PARP gene.

In a further preferred aspect, the use is the treatment of cancer.

Preferably, the medicament is a pharmaceutical composition consisting of a PARP inhibitor and a pharmaceutically acceptable carrier or diluent.

The specific sensitivity of homologous recombination-deficient tumors to PARP-1 inhibitors means that normally dividing cells in a patient will not be affected by the treatment. A further benefit of treating homologous recombination-deficient cancer cells with PARP-1 inhibitors is that it need not be administered as a combination therapy with conventional radiation therapy or chemotherapy, thereby avoiding the side effects associated with these conventional forms of treatment.

The genetic defect in the gene mediating homologous recombination may be due to a mutation, a deletion or defective expression in the gene encoding the protein involved in homologous recombination.

In another aspect, the present invention also provides the use of a PARP-1 inhibitor for the manufacture of a medicament for inducing apoptosis in a homologous recombination defective cell.

In another aspect, the present invention provides a method of inducing apoptosis in homologous recombination defective cells in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a PARP inhibitor.

By causing programmed cell death in cells deficient in homologous recombination, it is possible to reduce or stop tumor growth in mammals.

Preferably, the homologous recombination-defective cell is a cancer cell.

Cancer cells deficient in homologous recombination can be partially or completely deficient in HR. Preferably, the cancer cells are completely deficient in HR.

The term "cancer" or "tumor" includes lung, colon, pancreatic, gastric, ovarian, cervical, breast or prostate cancer. The cancer may also include skin cancer, kidney cancer, liver cancer, bladder cancer, or brain cancer.

The cancer to be treated may be a hereditary form of cancer, where the patient to be treated has a familial predisposition to develop cancer. Preferably, the cancer to be treated is a genetically linked hereditary cancer. In a preferred embodiment of the invention, the cancer is a genetically linked hereditary breast cancer.

In a preferred aspect, PARP-1 inhibitors are useful in the treatment of cancer cells deficient in the expression of genes involved in HR. Genes having proposed functions in homologous recombination include XRCC1, ADPRT (PARP-1), ADPRTL2(PARP-2), CTPS, RPA1, RPA2, RPA3, XPD, ERCC1, XPF, MMS19, RAD51, RAD51B, RAD51C, DMC C, XRCC C, BRCA C, RAD C, MRE C, NBS C, WRN, BLM, Ku C, ATM, ATR, chkl, chk C, FANCA, FANCB, FANCC, FANCD C, FANCE, FANCF, FANCG C, EMK C, FENCA C, FANCB C, FANCD C, FANCF, FANCG C, MUDDS C, DDS 363, DDS C, and DDS.

The gene involved in homologous recombination may be a tumor suppressor gene. The invention thus provides for the treatment of cancer cells that are deficient in expression of tumor suppressor genes. Preferably, the tumor suppressor gene is BRCA1 or BRCA 2.

Breast cancer is now the most common cancer in women in western countries. Some families have strong breast cancer causes, often due to genetic mutations in one allele of BRCA1 or BRCA 2. However, these patients still maintain one functional allele. Thus, these patients develop normally and do not have the phenotypic consequences of the mutation. However, in a cell, the functional allele may be lost, making this cell cancerous, and defective in homologous recombination. This step is critical for tumorigenesis (1).

The present inventors have surprisingly found that BRCA2 deficient cells are 100 times more sensitive to cytotoxicity of the PARP inhibitor NU1025 than wild-type cells.

Thus, in a preferred aspect, the present invention provides the use of a PARP inhibitor for the manufacture of a medicament for the treatment of cancer cells deficient in HR (e.g. due to loss of expression of BRCA1 and/or BRCA 2).

The cancer cells to be treated may be partially or completely deficient in BRCA1 or BRCA2 expression. BRCA1 and BRCA2 mutations can be identified using multiplex PCR techniques, array techniques (29, 30), or other screens known to the skilled artisan.

PARP inhibitors useful in the present invention may be selected from PARP-1, PARP-2, PARP-3, PARP-4, tankyrase 1 or tankyrase 2. (for review see 31). In a preferred embodiment, PARP inhibitors useful in the present invention are inhibitors of PARP-1 activity.

PARP-1 inhibitors useful in the present invention include benzimidazole-carboxamides, quinazolin-4- [3H ] -one, and isoquinoline derivatives (e.g., 2- (4-hydroxyphenyl) benzimidazole-4-carboxamide (NU1085), 8-hydroxy-2-methyl quinazolin-4- [3H ] one (NU1025), 6(5H) phenanthridinone, 3 aminobenzamide, benzimidazole-4-carboxamide (BZ1-6), and tricycloacylamide indole (TI1-5) [32] other inhibitors of PARP may be identified by design [33] or novel FlashPlalate assay [34 ].

PARP inhibitors formulated as pharmaceutical compositions can be administered in a manner effective and convenient for targeting cancer cells, e.g., oral, intravenous, intramuscular, intradermal, intranasal, topical routes, and the like. Carriers or diluents useful in the pharmaceutical composition can include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

In therapy or as a prophylactic agent, the active agent may be administered to the subject as an injectable composition, for example as a sterile aqueous dispersion. The inhibitor may be administered directly to the tumor, or may be targeted to the tumor by systemic administration.

A therapeutically effective amount of an inhibitor is an amount sufficient to achieve the desired effect and may vary depending on the nature and severity of the disease condition and the potency of the inhibitor. It will be appreciated that in addition to treating active disease, different concentrations may be used prophylactically.

For administration to mammals, particularly humans, it is expected that the daily dose of the active agent will be up to 100mg/kg, for example from 0.01mg/kg to 50mg/kg body weight, usually up to 0.1, 0.5, 1.0, 2.0, 5.0, 10, 15, 20 or 30mg/kg body weight. Ultimately, however, the amount of inhibitor administered and the frequency of administration are determined by the physician.

The therapeutic advantage of using PARP inhibitors to treat cancer cells is that therapeutic efficacy in treating cancer can be achieved with only small doses, thereby reducing the systemic accumulation of inhibitors and associated toxic effects.

In a preferred aspect of the invention, an agent is provided which is an inhibitory RNA (RNAi) molecule.

One technique for specifically abolishing gene function is by introducing double-stranded RNA, also known as inhibitory (RNAi), into a cell, resulting in the destruction of mRNA that is complementary to the sequence included in the RNAi molecule. RNAi molecules comprise two complementary strands of RNA (the sense and antisense strands) that anneal to each other to form a double-stranded RNA molecule. RNAi molecules are typically derived from an exon sequence or a coding sequence of a gene to be abolished.

Preferably, the RNAi molecule is derived from a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:

a) a nucleic acid sequence represented by the sequence in figures 9, 10, 11, 12, 13 or 14 or a fragment thereof;

b) a nucleic acid sequence which hybridizes to the nucleic acid sequence of figure 9, 10, 11, 12, 13 or 14 or a gene encoding PARP;

c) nucleic acid sequences comprising sequences which result from the degeneracy of the genetic code of the nucleic acid sequences defined in a) and b).

Recent studies suggest that RNAi molecules of 100-1000bp derived from the coding sequence are effective inhibitors of gene expression. Surprisingly, only a few RNAi molecules are required to block gene expression, suggesting that its mechanism is catalytic. The site of action appears to be in the nucleus, since little, if any, RNAi is detectable in the cytoplasm of the cell, indicating that the RNAi molecule exerts its effect during mRNA synthesis or processing.

More preferably, the RNAi molecules are 10 nucleotide bases (nb) to 1000nb in length. Even more preferably, the RNAi molecules are 10nb, 20nb, 30nb, 40nb, 50nb, 60nb, 70nb, 80nb, 90nb, or 100nb in length. Even more preferably, the RNAi molecule is 21nb in length.

Even more preferably, the RNAi molecule comprises nucleic acid sequence aaa agc cau ggu gga gua uga (PARP-1).

Even more preferably, the RNAi molecule consists of nucleic acid sequence aag acc aau cuc ucc agu uca ac (PARP-2).

Even more preferably, the RNAi molecule consists of nucleic acid sequence aag acc aac auc gag aac aac (PARP-3).

The RNAi molecule can comprise modified nucleotide bases.

Preferred features of each aspect of the invention are applicable to each of the other aspects mutatis mutandis.

The invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

FIG. 1 is a graph showing that HR deficient cells are hypersensitive to cytotoxicity resulting from the inhibition of PARP-1. Colony growth halo when Chinese hamster cell line AA8 (wild type), irsISF (HR deficient [4]), CXR3 (irsISF [2] complementary to XRCC 3), V79 (wild type), irsI (HR deficient [5]), or irsIX2.2 (irsI [1] complementary to XRCC 2) is exposed to 3-AB (A), ISQ (B), or NU1025 (C). The mean (symbols) and standard deviation (bars) of at least three experiments are shown. Using colony growth halo assay;

FIG. 2 is a graph showing cell viability in the presence of the PARP inhibitor NU1025 in wt V79 cells, BRCA2 deficient VC-8 cells, and VC-8 cells complementary to the functional BRCA2 gene (VC-8#13, VC-8+ B2);

FIG. 3 is a bar graph showing the percentage of apoptotic cells after 72 hours incubation with NU 1025;

FIG. 4(a) transfection of 48 hours with siRNA from MCF-7(p 53)^wt) Or MDA-MB-231(p 53)^mut) Western blot analysis of protein lysates isolated from breast cancer cells. (b) Halo of colony growth of siRNA treated MCF-7 cells or (c) MDA-MB-231 cells after exposure to the PARP inhibitor NU 1025. The mean (symbols) and standard deviation (bars) of at least three experiments are shown.

FIG. 5.BRCA2 deficient cells are unable to repair the recombination lesions formed by PARP inhibitors at the replication fork. Cells with BRCA2 intact or defective after 24 hours of treatment with NU1025(0.1mM) were visualized by pulsed gel electrophoresis. 2mM hydroxyurea was used as a positive control. (b) Display of γ H2Ax foci in untreated V-C8+ B2 cells and V-C8 cells. (c) Number of cells containing γ H2Ax foci or (C) RAD51 foci (d) shown in V-C8+ B2 and V-C8 cells 24 hours after treatment with NU1025(10 μ M). The mean (symbols) and standard deviation (bars) of at least three experiments are shown. (e) Proposed model of cell death induced in BRCA2 deficient cells.

FIG. 6 PARP-1, but not PARP-2, is important in preventing the formation of recombinant lesions that cause cell death in the absence of BRCA 2. (a) RT-PCR of RNA isolated from SW480SN.3 cells treated with BRCA2, PARP-1, and PARP-2 in the indicated combinations for 48 hours. (b) Clonal producing cells survive 48 hours of exhaustion of BRCA2, PARP-1 and PARP-2. The mean (symbols) and standard deviation (bars) of at least three experiments are shown. Two asterisks and three asterisks refer to statistical significance of p <0.01 and p <0.001, respectively, in the student's t-test. (c) Western blot of PARP-1 in SW480SN.3 cells treated with different siRNAs.

FIG. 7 shows PAR multimers of (a) untreated and (b) V79 cells treated with thymidine (5mM for 24 hours). (c) Hydroxyurea (0.2mM) and thymidine (5mM) after treatment>Percentage of cells of 10 PARP active sites. At least 300 nuclei were counted for each treatment and experiment. (d) Survival of V-C8+ B2 cells after co-treatment with hydroxyurea (e) or thymidine with NU1025(10 μ M). (f) After treatment with MMS, hydroxyurea (0.5mM) or thymidine (10mM), by free NAD (P) H¹¹Measure the activity of PARP. The mean (symbols) and standard deviation (error bars) from at least 3 experiments are depicted.

FIG. 8 shows PAR multimers in (a) untreated V-C8 cells and (B) V-C8+ B2 cells. (c) Quantification of the percentage of cells containing >10 PARP active sites in untreated V-C8 cells and V-C8+ B2 cells (d) nad (p) H levels measured in untreated V-C8 and V-C8+ B2 cells. 3 asterisks indicate p <0.001 in student's t-test. (e) Display of RAD51 and PARP active sites in V79 cells after 24 hours thymidine (5mM) treatment. (f) Models of the role of PARP and HR in stopped replication forks.

FIG. 9 cDNA sequence of human PARP-1;

FIG. 10 cDNA sequence of human PARP-2;

FIG. 11. cDNA sequence of human PARP-3;

FIG. 12 is the cDNA sequence of human terminal Anchor polymerase 1;

FIG. 13 mRNA sequence of human terminal Anchor polymerase 2;

FIG. 14 mRNA sequence of human VPARP.

Materials and methods

PARP inhibitors on HR-deficient cells: XRCC2, XRCC3 or BRCA2 cells

Toxicity

Cell culture

irsI, irsIX2.1 and V79-4 cell lines were donated by John Thacker [40], AA8, irsISF and CXR3 cell lines were supplied by Larry Thompson [41 ].

VC-8, VC-8+ B2, VC-8#13 are given by Malgorzarata ZDzienicka [42 ]]. All cell lines in this study contained 5% CO at 37C₂In Dulbecco's Modified Eagle's Medium (DMEM) Medium to which 10% fetal bovine serum and penicillin (100U/mL) and streptomycin sulfate (100. mu.g/mL) were added.

Toxicity assay-colony outgrowth assay

The plates were plated with 500 cells suspended in medium and after 4 hours 3-AB, ISQ or NU1025 was added. ISQ and NU1025 were dissolved in DMSO at a final concentration of 0.2% in the treatment medium. After 7-12 days, when cell colonies could be observed, these colonies were fixed and stained with methylene blue (4g/L) in methanol. Colonies consisting of more than 50 cells were then counted.

Programmed cell death assay

0.25x10⁶The individual cells were plated on a petri dish and cultured for 4 hours before treatment with NU 1025. After 72 hours, the cells were trypsinized and resuspended in media containing any suspended cells from that sample. Cells were pelleted by centrifugation and resuspended and then analyzed for programmed cell death using FITC-conjugated annexin V and Propidium Iodide (PI) (ApoTarget, biosource international) according to the manufacturer's protocol. Samples were analyzed by flow cytometry (Becton-Dickenson facport, 488nm laser) and the percentage of programmed cell dead cells was determined by the fraction of viable cells (PI negative) that bound to FITC-conjugated annexin V.

Immunofluorescence

Cells were plated on cover slips, 4 hrsAfter aging, the treatment was carried out for 24 hours as described above. After treatment, the medium was removed, the coverslips washed with PBS at 37 ℃ and fixed as described elsewhere [2]. The primary antibodies and dilutions used in this study were: rabbit polyclonal anti-PAR (Trevigen;1: 500); sheep polyclonal anti-Rad 51(C-20, Santa Cruz;1: 200); rabbit polyclonal anti-Rad 51(H-92, Santa Cruz;1: 1000). The secondary antibody was Cy-3 conjugated goat anti-rabbit IgG antibody (Zymed;1:500), Alexa555 goat anti-rabbit F (ab')₂IgG antibodies (Molecular Probes;1:500), Alexa546 donkey anti-goat IgG antibodies (Molecular Probes;1:500) and Alexa488 donkey anti-goat IgG antibodies (Molecular Probes;1: 500). The antibody was diluted with PBS containing 3% bovine serum albumin. The DNA was stained with 1. mu.g/ml of To Pro (Molecular Probes). Images were acquired with a Zeiss LSM510 inverted confocal microscope using a 63X/NA1.4 oil immersion objective and 488, 546 and 630 nm. Images were obtained from optical slices separated by 0.50 μm and 1.0 μm slice thickness by focus maximum projection. The images were processed using Adobe Photoshop (Abacus Corp.). At least 300 nuclei were counted on each slide and those containing more than 10 RAD51 foci or PARP active sites were classified as positive.

PARP Activity assay

Reduction to yellow formazan with water-soluble tetrazolium salt (5mM WST-8) by itDyes to monitor the amount of NAD (P) H [43]. 5000 cells were plated in wells of a 96-well plate in at least triplicate and cultured in 100. mu.l of normal medium at 37 ℃ for 4 hours. CK8 buffer (Dojindo Molecular Technology, Gaithersburg, USA) containing WST-8 was then added, with or without treatment with the indicated concentrations of DNA damaging agent. By measuring visible absorption (OD) every 30 minutes₄₅₀) Determination of the reduction of WST-8 in the presence of NAD (P) H. A media blank containing only media and CK8 buffer was also prepared. Changes in nad (p) H levels can be calculated by comparing the uptake of wells containing cells treated with DNA damaging agents to the uptake of wells containing cells treated with DMSO alone. Alternatively, the relative levels of nad (p) H in different cell lines were calculated after 4 hours of incubation in CK8 buffer.

By Halldorsson et al [44]Improved methods of (1) (details elsewhere [45 ]]) The ability of NU1025 to inhibit PARP-1 activity in permeabilized cells was determined. Briefly, 300. mu.L of NU 1025-treated permeabilized cells were incubated with oligonucleotide (final concentration 2.5. mu.g/mL) and 75. mu.M NAD +, [2]³²P]NAD (Amersham Pharmacia, Amersham, UK) was incubated in a total volume of 400. mu.L. After 5 minutes, the reaction was stopped by adding ice-chilled 10% TCA, 10% Na Ppi for 60 minutes, followed by filtration through a Whatman GF/C filter (LabSales, Maidstone, UK), washing 6 times with 1% TCA1% NaPPi, allowing to dry and measuring the incorporated radioactivity to determine PARP-1 activity. Reference [2]³²P]NAD standards, data expressed as pmol NAD/10 incorporated⁶And (4) cells.

Pulsed gel electrophoresis

Mix 1.5x10⁶The individual cells were plated in 100mm dishes and attached for 4 hours. Exposure to the drug was 18 hours, after which the cells were digested with pancreatin. Melting 10 in each 1% agarose well (insert)⁶And (4) cells. These wells were incubated as described elsewhere (8) and separated by pulsed gel electrophoresis for 24 hours (BioRad;120 ℃ angle, 60 to 240 transition time, 4V/cm). The gel was then stained with ethidium bromide for analysis.

SiRNA treatment

The pre-designed BRCA2SMART library and scrambled siRNAs (Dharmacon, Lafayette, CO) were purchased. 10000 cells were seeded in 6-well plates and left overnight before transfection with 100nM siRNA using Oligofectamine Reagent (Invitrogen) according to the manufacturer's instruction manual. After culturing the cells in normal growth medium for 48 hours, they were digested with trypsin, replated and subjected to toxicity assay. Inhibition of BRCA2 was demonstrated by western blotting (as described previously [46]) of the protein extract treated with siRNA using an antibody against BRCA2 (Oncogene, Nottingham, UK).

Examples

Homologous recombination defective cellHypersensitiveness to PARP-1 inhibition

To investigate the involvement of HR in the cellular response to PARP-1 inhibition, the effect of PARP-1 inhibitors on the survival of HR repair deficient cell lines was investigated. We found that cells deficient in HR (i.e. irslSF deficient in XRCC3 or irsl deficient in XRCC2 [ see table 1]) were very sensitive to the toxic effects of 3-aminobenzamide (3-AB) and were sensitive to two more potent PARP-1 inhibitors: 1, 5-dihydroxyisoquinoline (ISQ; [37]) or 8-hydroxy-2-methyl quinazoline (NU1025[38,39]) was also very sensitive (FIG. 1). Sensitivity of irslSF cells to 3-AB, ISQ or NU1025 can be corrected by introducing cosmids containing a functional XRCC3 gene (CXR 3). Similarly, the sensitivity of irsl cells to 3-AB, ISQ or NU1025 can be corrected by introducing cosmids containing functional XRCC2 genes (irslX2.2).

BRCA2 deficient cells are hypersensitive to PARP-1 inhibition

The survival of BRCA 2-deficient cells (VC8) and wild-type cells (V79Z) in the presence of inhibitors of PARP-1 was studied. VC8 cells were found to be very sensitive to the toxic effects of NU1025 (fig. 2). The sensitivity of VC8 cells can be corrected by introducing a functional BRCA2 gene on chromosome 13 (VC 8# 13) or on an overexpression vector (VC8+ B2). The results indicate that sensitivity to PARP-1 inhibitors is a direct result of loss of function of BRCA 2.

To investigate whether inhibition of PARP-1 triggered programmed cell death in BRCA2 deficiency, the level of programmed cell death after NU102572 hour exposure was investigated. NU1025 was found to trigger programmed cell death only in VC8 cells, indicating that loss of PARP-1 activity in BRCA2 deficient cells triggers this death (fig. 3).

BRCA2 deficient breast cancer cells hypersensitive to PARP-1 inhibition

It was examined whether MCF7 (wild-type p53) and MDA-MB-231 (mutant p53) breast cancer cell lines show similar sensitivity to NU1025 when BRCA2 was depleted. It was found that PARP-1 inhibitors could greatly reduce the survival of MCF7 and MDA-MB-231 cells only when BRCA2 was depleted with the BRCA2siRNA cocktail (fig. 4). This suggests that BRCA2 depleted breast cancer cells are sensitive to PARP inhibitors, which are not associated with p53 status.

BRCA2 deficient cells die due to PARP-1 inhibition in the absence of DNA Double Strand Break (DSB) but in the presence of gamma H2Ax

HR is known to be involved in the repair of DSB and other lesions in DNA replication [2 ]. To determine whether sensitivity of BRCA 2-deficient cells was the result of inability to repair DSM following NU1025 treatment, DSB accumulation in V79 and V-C8 cells was measured following treatment with high toxicity levels of NU 1025. It was found that no DSB was detected by pulse gel electrophoresis analysis of DNA obtained from treated cells (fig. 5A), indicating that low levels of DSB or other recombination substrates accumulate after PARP inhibition in HR-deficient cells, which elicits γ H2Ax (fig. 5B). The reason why BRCA 2-deficient cells die after induction of these recombinant lesions may be due to the inability to repair these lesions. To test this, BRCA2 deficient V-C8 cells and BRCA2 complementing cells were tested for their ability to form RAD51 foci in response to NU 1025. RAD51 foci were indeed induced in V-C8+ B2 cells after NU1025 treatment (statistically significant when p <0.05 in t-test; fig. 5D). This indicates that the damage caused by recombination causes recombination repair in these cells, allowing these cells to survive. In contrast, V-C8 cells deficient in BRCA2 failed to form RAD51 foci in response to NU1025 (fig. 5D), suggesting the absence of HR, which would leave the recombination lesions unrepaired and thereby lead to cell death.

PARP-1, but not PARP-2, is important in preventing the formation of recombinant lesions

There are two major species of PARP in mammalian cell nuclei: PARP-1 and PARP-2. All reported PARP inhibitors are able to inhibit both PARPs. To distinguish which PARP caused the effect, we tested whether none of PARP-1 and/or PARP-2 caused accumulation of toxic lesions by depleting PARP-1 and/or PARP-2 and BRCA2 in human cells with siRNA (fig. 6 a). We found that the survival of clonogenic cells was significantly reduced when PARP-1 and BRCA2 were simultaneously depleted from the cells (fig. 6 b). Depletion of PARP-2 and BRCA2 had no effect on clonogenic cell survival and depletion of PARP-2, PARP-1 and BRCA2 did not result in additional toxicity. These results suggest that PARP-1, but not PARP-2, is responsible for mitigating toxic recombinant damage in human cells. The efficiency of cloning in cells co-depleted in PARP-1 and BRCA2 decreased only to 60% of controls, whereas cells without HR deficiency survived treatment with PARP inhibitor. This may be associated with incomplete depletion of large amounts of PARP-1 protein by siRNA (fig. 6c), which may be sufficient to maintain PARP-1 function in some cells.

Activation of PARP-1 by replication inhibitors

HR is also associated with damage repair occurring on stopped replication forks, which may not include detectable DSBs. To test whether PARP has a role in the replication fork, PARP activation in cells treated with agents (thymidine or hydroxyurea) that retard or prevent DNA replication forks was examined. Thymidine depletes the dCTP of the cell and slows the rate of replication forks but does not cause DSBs. Hydroxyurea depletes several dNTPs and blocks the replication fork, with the formation of DSB 2 at the replication fork. Both agents are effective in inducing HR 2. PAR multimers were stained with V79 hamster cells treated with thymidine or hydroxyurea for 24 hours. This revealed a significant increase in the number of cells containing the PARP active site (fig. 7C). This result suggests the function of PARP at the stopped replication fork. It was also shown that inhibition of PARP with NU1025 enhanced sensitivity to thymidine or hydroxyurea in V-C8+ B2 cells (fig. 7D, E). This result suggests that PARP activity is important in the repair of stopped replication forks or alternatively, it prevents the induction of death in cells with stopped replication forks.

PARP is rapidly activated at DNA Single Strand Breaks (SSBs) and attracts DNA repair enzymes [3-6 ]. Methyl methane sulfonic acid (MMS) results in the alkylation of DNA, which is repaired by base excision repair. PARP is rapidly activated by SSB intermediates formed during this repair process, which deplete nad (p) H levels (fig. 7F). We found that PARP activation and nad (p) H levels decrease much more slowly after thymidine or hydroxyurea treatment. This slowing of PARP activation can be explained by the indirect effects of thymidine and hydroxyurea and the time required for replication forks to stop after the cell enters the S-phase of the cell cycle.

PARP-1 and HR have separate roles in the stopped replication fork

The number of PARP active sites in untreated BRCA2 deficient V-C8 cells was determined. More V-C8 cells were found to contain PARP active sites compared to V-C8+ B2 cells (FIG. 8A, B, C). Also the levels of free nad (p) H in V-C8 cells were lower than those of corrected cells (fig. 8D), probably due to increased PARP activity. Importantly, these PARP active sites did not overlap with RAD51 foci (fig. 8E).

The results here suggest that PARP and HR have separate roles in protecting or rescuing the stopped replication fork (fig. 8F). Loss of PARP activity can be compensated by increased HR, which can be compensated by increased PARP activity. However, the simultaneous loss of both pathways leads to the accumulation of halted replication forks to cell death, just as does the PARP-inhibited BRCA 2-deficient cells.

As shown in the model outlined in fig. 8F, PARP and HR have complementary effects at the stopped replication fork. (i) The replication fork may stop when an obstacle to the DNA template is encountered. In addition, replication forks are also temporarily halted due to the absence of dNTPs or other DNA replication cofactors. (ii) PARP binds to a stopped replication fork or other replication-related lesions, triggering PAR multimerization. The resulting negatively charged PAR multimers can protect the stopped replication fork by normal handling of the protein (e.g., resolvase) of the replication fork until the replication fork can be spontaneously restored when dNTPs or other cofactors are available. Alternatively, PAR multimers or PARPs can attract proteins to break down replication forks by other means. (iii) In the absence of PARP activity, HR may serve as an alternative pathway to replication forks that stop repair. This compensatory model accounts for the elevated levels of HR and RAD51 foci found in PARP deficient cells (i.e., V-C8)^3-5. In the absence of both PARP and HR, spontaneous replication block/damage is rather fatal

TABLE 1 genotype and source of cell lines used in the study

Reference to the literature

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Claims (17)

1. Use of a poly ADP-ribose polymerase-1 inhibitor for the manufacture of a medicament for treating a cancer cell deficient in homologous recombination, wherein the cancer cell is deficient in a gene selected from the group consisting of RAD51, XRCC2, XRCC3, BRCA1, and BRCA 2.

2. Use according to claim 1, wherein the poly ADP-ribose polymerase-1 inhibitor is selected from benzimidazole-carboxamides, quinazolin-4- [3H ] -one and isoquinoline derivatives.

3. Use according to claim 2, wherein the poly ADP-ribose polymerase-1 inhibitor is selected from the group consisting of 2- (4-hydroxyphenyl) benzimidazole-4-carboxamide, 8-hydroxy-2-methyl quinazolin-4- [3H ] one, 6(5H) phenanthridinone, 3-aminobenzamide, benzimidazole-4-carboxamides and tricycloacylaminoindole.

4. Use according to any of the preceding claims, wherein the cancer cell deficient in homologous recombination is partially deficient in homologous recombination.

5. Use according to any one of claims 1 to 3, wherein the cancer cell deficient in homologous recombination is completely deficient in homologous recombination.

6. The use as claimed in any one of claims 1 to 3, wherein the defect is a mutation in a gene encoding a protein involved in homologous recombination.

7. The use as claimed in any one of claims 1 to 3, wherein the defect is a deletion of a gene encoding a protein involved in homologous recombination.

8. The use as claimed in any one of claims 1 to 3, wherein the defect is in the expression of a gene encoding a protein involved in homologous recombination.

9. The use as claimed in any one of claims 1 to 3, wherein the cancer cell deficient in homologous recombination is selected from the group consisting of cells of lung cancer, colon cancer, pancreatic cancer, gastric cancer, ovarian cancer, cervical cancer, breast cancer and prostate cancer.

10. The use as claimed in any one of claims 1 to 3 wherein the cancer cell is in a human.

11. The use as claimed in claim 10, wherein the cancer cell deficient in homologous recombination is a genetically linked hereditary cancer cell.

12. The use as claimed in claim 11, wherein the cancer cell deficient in homologous recombination is a breast cancer cell.

13. The use as claimed in any one of claims 1 to 3 wherein the cancer cell deficient in homologous recombination is deficient in expression of BRCA 1.

14. The use as claimed in claim 13 wherein the cancer cell fraction lacks BRCA1 expression.

15. The use as claimed in claim 13 wherein the cancer cells are completely devoid of BRCA1 expression.

16. The use as claimed in claim 6, wherein the gene mediating homologous recombination is a tumor suppressor gene.

17. The use as claimed in claim 16, wherein the tumor suppressor gene is BRCA 1.