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WO2018167780A1 - Méthodes de diagnostic et de traitement du cancer - Google Patents

Méthodes de diagnostic et de traitement du cancer Download PDF

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Publication number
WO2018167780A1
WO2018167780A1 PCT/IL2018/050289 IL2018050289W WO2018167780A1 WO 2018167780 A1 WO2018167780 A1 WO 2018167780A1 IL 2018050289 W IL2018050289 W IL 2018050289W WO 2018167780 A1 WO2018167780 A1 WO 2018167780A1
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Prior art keywords
cancer
subject
level
activity
otc
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PCT/IL2018/050289
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English (en)
Inventor
Ayelet Erez
Eytan Ruppin
Joo Sang Lee
Hiren KARATHIA
Sridhar Hannenhalli
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Yeda Research And Development Co. Ltd.
University Of Maryland
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Publication of WO2018167780A1 publication Critical patent/WO2018167780A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
    • 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
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention in some embodiments thereof, relates to methods of prognosing and treating cancer.
  • Cancer diagnosis at early stage is essential when it comes to treatment outcome and survival, especially when it comes to highly malignant tumors.
  • Clinically practiced methods for cancer diagnosis include general well-being of the patient, screening tests and medical imaging.
  • Cancer cells typically undergo metabolic transformations leading to synthesis of biological molecules that are essential for cell division and growth.
  • the urea cycle is a metabolic process which converts excess nitrogen derived from the breakdown of nitrogen-containing molecules to the excretable nitrogenous compound - urea.
  • Urea a colorless, odorless solid which is highly soluble in water and practically non-toxic is the main nitrogen-containing substance in the urine of mammals.
  • Several studies have reported altered expression of specific UC components in several types of cancer and also indicated an association between the pattern of these UC components and poor survival or increased metastasis [see e.g. Chaerkady, R. et al. (2008) J Proteome Res 7, 4289-4298; Lee, Y. Y. et al. (2014) Tumour Biol 35: 11097-11105; Syed, N. et al.
  • a method of treating cancer in a subject in need thereof comprising: (a) determining a shift from the urea cycle to pyrimidine synthesis in a cancerous cell of the subject as compared to a control sample; and
  • a method of treating cancer in a subject in need thereof comprising:
  • the shift is determined by level of purine to pyrimidine transversion mutations.
  • the shift is determined by a level and/or activity of a urea cycle enzyme and/or a CAD enzyme.
  • the urea cycle enzyme is selected from the group consisting of ASL, ASS1, OTC, SLC25A15, CPS1 and SLC25A13.
  • a method of prognosing cancer in a subject diagnosed with cancer comprising determining a level of purine to pyrimidine transversion mutations in a cancerous cell of the subject as compared to a control sample, wherein the level of the purine to pyrimidine transversion mutations above a predetermined threshold is indicative of poor prognosis, thereby prognosing cancer in the subject.
  • a method of monitoring efficacy of cancer therapy in a subject comprising determining a level of purine to pyrimidine transversion mutations in a cancerous cell of the subject undergoing or following the cancer therapy, wherein a decrease in the level of the purine to pyrimidine transversion mutations from a predetermined threshold or in comparison to the level in the subject prior to the cancer therapy, indicates efficacious cancer therapy.
  • a method of prognosing cancer in a subject diagnosed with cancer comprising determining a level and/or activity of a urea cycle enzyme SLC25A15 in a cancerous cell of the subject as compared to a control sample, wherein the level and/or activity of the SLC2SA1S below a predetermined threshold is indicative of poor prognosis, thereby prognosing cancer in the subject.
  • a method of monitoring efficacy of cancer therapy in a subject comprising determining a level and/or activity of a urea cycle enzyme SLC25A15 in a cancerous cell of the subject undergoing or following the cancer therapy, wherein an increase in the level and/or activity of the a urea cycle enzyme SLC2SA1S from a predetermined threshold or in comparison to the level and/or activity in the subject prior to the cancer therapy, indicates efficacious cancer therapy.
  • the cancer is selected from the group consisting of thyroid cancer, hepatic cancer, bile duct cancer and kidney cancer.
  • a method of prognosing cancer in a subject diagnosed with cancer comprising determining a level and/or activity of at least two urea cycle enzymes selected from the group consisting of ASL, ASS1, OTC, SLC25A15, CPS1 and SLC25A13 in a cancerous cell of the subject as compared to a control sample, wherein the urea cycle enzymes comprise ASS1 and SLC25A13 the at least two is at least three; wherein the level and/or activity of the ASL, the ASS1, the OTC and/or the SLC25A15 below a predetermined threshold and/or the level and/or activity of the CPS1 and/or the SLC2SA13 above a predetermined threshold is indicative of poor prognosis, thereby prognosing cancer in the subject.
  • a method of monitoring efficacy of cancer therapy in a subject comprising determining a level and/or activity of at least two urea cycle enzymes selected from the group consisting of ASL, ASS1, OTC, SLC25A15, CPS1 and SLC25A13 in a cancerous cell of the subject undergoing or following the cancer therapy, wherein the urea cycle enzymes comprise ASS1 and SLC25A13 the at least two is at least three; wherein an increase in the level and/or activity of the ASL, the ASS1, the OTC and/or the SLC25A15 and/or a decrease in the level and/or activity of the CPS1 and/or the SLC2SA13 from a predetermined threshold or in comparison to the level and/or activity in the subject prior to the cancer therapy indicates efficacious cancer therapy.
  • the cancer is thyroid, stomach and/or bladder cancer and the at least two urea cycle enzymes are selected from the group consisting of OTC, SLC25A15 and SLC25A13.
  • the cancer is prostate cancer and the at least two urea cycle enzymes comprise ASS1 and CSP1.
  • the cancer is lung and/or head and neck cancer and the at least two urea cycle enzymes are selected from the group consisting of ASL, OTC, CPS1 and SLC25A13.
  • the cancer is hepatic, bile duct and/or kidney cancer and the at least two urea cycle enzymes are selected from the group consisting of ASL, ASS1, OTC and SLC25A15.
  • the cancer is breast cancer and the at least three urea cycle enzymes comprise ASS 1 , OTC and SLC25 A 13.
  • a method of prognosing breast cancer in a subject diagnosed with breast cancer comprising determining a level and/or activity of at least two urea cycle enzymes selected from the group consisting of ASS1, OTC and SLC25A13 in a cancerous cell of the subject as compared to a control sample,
  • the level and/or activity of the ASS1 and/or the OTC below a predetermined threshold and/or the level and/or activity of the SLC2SA13 above a predetermined threshold is indicative of poor prognosis, thereby prognosing the breast cancer in the subject.
  • a method of monitoring efficacy of cancer therapy in a subject diagnosed with breast cancer comprising determining a level and/or activity of at least two urea cycle enzymes selected from the group consisting of ASS1, OTC and SLC2SA13 in a cancerous cell of the subject undergoing or following the cancer therapy, wherein an increase in the level and/or activity of the ASS1 and/or the OTC and/or a decrease in the level and/or activity of the SLC2SA13 from a predetermined threshold or in comparison to the level and/or activity in the subject prior to the cancer therapy, indicates efficacious cancer therapy.
  • the method further comprising determining a level and/or activity of a CAD enzyme, wherein the level and/or activity of the CAD enzyme above a predetermined threshold is indicative of poor prognosis.
  • the method further comprising determining a level and/or activity of a CAD enzyme, wherein a decrease in the level and/or activity of the CAD enzyme from a predetermined threshold or in comparison to the level in the subject prior to the cancer therapy, indicates efficacious cancer therapy.
  • the CAD is activated CAD.
  • the method comprising corroborating the prognosis using a state of the art technique.
  • a method of treating cancer in a subject in need thereof comprising:
  • a method of treating cancer in a subject in need thereof comprising:
  • a method of treating cancer in a subject in need thereof comprising:
  • a method of treating cancer in a subject in need thereof comprising:
  • the predetermined threshold is at least 1.1 fold.
  • control sample is a non-cancerous cell of the subject.
  • control sample is a cancerous cell with a level and/or activity of the urea cycle enzyme, the purine to pyrimidine transversion mutations and/or the CAD enzyme similar to levels and/or activity of same in a healthy cell of the same type.
  • the cancer is selected from the group consisting of hepatic cancer, osteosarcoma, breast cancer, colon cancer, thyroid cancer, stomach cancer, lung cancer, kidney cancer, prostate cancer, head and neck cancer, bile duct cancer and bladder cancer.
  • the cancer is selected from the group consisting of hepatic cancer, osteosarcoma, breast cancer and colon cancer.
  • the cancer therapy comprises a therapy selected from the group consisting of L-arginine depletion, glutamine depletion, pyrimidine analogs, thymidylate synthase inhibitor and mammalian target of Rapamycin (mTOR) inhibitor.
  • the cancer therapy comprises an immune modulation agent
  • the cancer therapy further comprises an agent which induces a pyrimidines to purines nucleotide imbalance.
  • the method further comprising treating the subject with an agent which induces a pyrimidines to purines nucleotide imbalance when the shift is indicated.
  • the method further comprising treating the subject with the immune modulation agent.
  • the immune modulation agent comprises anti-PDl.
  • the immune modulation agent comprises anti-CTLA4.
  • the agent which induces a pyrimidines to purines nucleotide imbalance comprises an anti-folate agent.
  • the anti-folate agent comprises methotrexate.
  • the purine to pyrimidine transversion mutations are non-synonymous purine to pyrimidine transversion mutations.
  • the determining the level of purine to pyrimidine transversion mutations is effected at the genomic level.
  • the determining the level of the enzyme is effected at the transcript level.
  • the determining the level of the enzyme is effected at the protein level.
  • Figures 1A-E demonstrate the association between the urea cycle (UC) enzymes and CAD.
  • Figure 1A is a schematic representation demonstrating that the UC enzymes alternate substrates with CAD.
  • Figure IB shows a representative photograph and a bar plot summarizing the crystal violet staining which indicates increased proliferation of cultured fibroblasts extracted from ORNT1 deficient (ORNT1D) or OTC deficient (OTCD) patients as compared to fibroblasts extracted from healthy controls.
  • Figure 1C is a western blot photograph demonstrating increased levels of CAD and phosphorylated CAD in fibroblasts extracted from ORNT1D and OTCD patients as compared to fibroblasts extracted from healthy patient (NF).
  • Figure ID is a plot showing decreased expression of ASS1 and increase expression of SLC2SA13 and CAD in fibroblasts extracted from healthy patients following human Cytomegalovirus (CMV) infection as measured by ribosome profiling.
  • Y-axis represents expression normalized to non-infected control.
  • Figure IE demonstrates high homology and identity between the UC enzymes and CAD. Protein domain structures were annotated using the NCBI BLAST
  • Results show high homology between the proximal UC enzymes proteins CPSI and OTC, and two CAD domains CPS2 and ATC, respectively.
  • Figures 2A-E demonstrate that downregulation of UC enzymes increases cancer proliferation and pyrimidine synthesis.
  • Figure 2A is a western blow photograph demonstrating the extent of OTC downregulation using several shRNAs in HepG2 hepatic cancer cell line.
  • Figure 2B shows a representative photograph and a bar plot summarizing the crystal violet staining which indicates increased proliferation of HepG2 hepatic cancer cells transduced with OTC shRNA, as compared to HepG2 hepatic cancer cell transduced with an empty vector (EV).
  • Figures 2F-H demonstrate that specific dysregulation of UC enzymes facilitates cancer proliferation.
  • Figure 2F shows western blot photographs demonstrating the specific UC perturbations induced in different cancer cells [i.e. downregulation of OTC (shOTC) or ORNT1 (shORNTl) or overexpression of citrin (OE-Citrin)] and the resultant effect on CAD activation compared to control cells transfected with empty vector (EV).
  • Figure 2G upper left bar plot is a quantification of crystal violet staining showing increased proliferation of different cancer cells following the indicated UC perturbations.
  • Figure 2G lower left bar plot shows that rescue experiments for the specific UC perturbation reverses the proliferative phenotype.
  • Figure 2G right bar plots show RT-PCR quantification for the changes in UC genes RNA expression levels following transfection with the specific rescue plasmid versus control plasmids.
  • Figure 2H left bar plots show enhanced synthesis of labelled M+l uracil from ISN-a-glutamine in HepG2 cancer cells transduced with OTC shRNA and SKOV cancer cells transduced with ORNT1 shRNA as compared to controls transduced with empty vector.
  • Figure 2H right bar plots show in vivo growth of HepG2 transduced with OTC shRNA and SKOV transduced with ORNTl shRNA xenografts compared to xenografts transduced with an empty vector.
  • Figures 3A-E demonstrate that dysregulation of the UC genes (denoted herein as UCD) in cancer activates CAD and correlates with worse prognosis.
  • Figure 3A shows relative expression of 6 UC genes in tumors from the cancer genome atlas (TCGA) with respect to their expression in healthy control tissues. Most tumors have aberrant expression of at least 2 UC components in the direction that metabolically supplies the required substrates for CAD activity [that is, decreased expression of ASL, ASS1, OTC and/or ONRT1D (SLC23A15) and/or increased expression of CPS1 and/or SLC25A13, P ⁇ 2.67E-3].
  • Tumor type's abbreviations are as follows: THCA - Thyroid cancer, STAD - Stomach adenocarcinoma, PRAD - Prostate cancer, LUSC - Lung squamous carcinoma, LHHC - Liver hepatocellular carcinoma, KIRP - Kidney renal papillary cell carcinoma, KIRC - Kidney renal Clear Cell Ca, KICH - Kidney chromophobe, HNSC- Head Neck Squamous Cell Carcinoma, CHOL - cholangiocarcinoma, BRCA - breast cancer, BLCA - Bladder cancer.
  • Figure 3B shows immunohistochemistry images of cancer tissues with their respective healthy tissue controls stained with the indicated UC components or PCNA as a marker for proliferation, showing inverse correlation between the expression of UC genes and the proliferation marker.
  • Figure 3C shows bar plots summarizing staining intensity of the PCNA positive cell count and UC proteins. Each staining was calibrated and repeated in two technical repetitions per patient sample in each slide (intensity OD level was compared in a matched T-student test).
  • Figure 3D is a graph demonstrating that UCD-scores (X-axis, equally divided into 5 bins) are positively correlated with CAD expression. Each paired consecutive bins were compared using the Wilcoxon rank sum test.
  • Figure 3E is a Kaplan-Meier survival curve showing that UCD is associated with worse survival of patients computed across all TCGA samples (i.e. pan cancer analysis).
  • Figures 4A-E demonstrate that UCD in cancer correlates with tumor grade.
  • Figure 4A is a schematic representation demonstrating the direction of UC enzymes expression that supports CAD activation (represented in blue arrows). The resulting changes in metabolites' levels following these expression alterations are represented by red arrows.
  • Figure 4B shows immunohistochemistry images of cancer tissues with their respective healthy tissue controls stained with OTC Magnification X10; and a bar plot summarizing OTC staining intensity. Each staining was calibrated and repeated in 2 technical repetitions per patient sample in each slide (intensity OD level was compared in a matched T-student test, ****P ⁇ 0.0001).
  • Figure 4C shows immunohistochemistry images of thyroid cancer tissues stained with ORNT1 Magnification X10; and a bar plot summarizing ORNT1 staining intensity; demonstrating that low levels of ORNT1 are associated with more advanced thyroid tumor grades. Each staining was calibrated and repeated in 2 technical repetitions per patient sample in each slide (intensity OD level was compared in a matched T-student test, ***P ⁇ 0.001).
  • Figure 4D is a Kaplan- Meier survival curve showing that CAD is associated with worse survival of patients computed across all TCGA samples (i.e. pan cancer analysis).
  • Figure 4E shows a Cox regression analysis of the UCD-score and CAD expression, demonstrating that both variables are independently significant.
  • Figures 5A-G demonstrate that UCD in cancer increases nitrogen utilization.
  • Figure SA shows metabolic modelling which predicts decreased urea excretion (left panel) and increased nitrogen utilization (right panel) with increased CAD activity, at high biomass production (that is, higher cell proliferation) conditions.
  • Figure SC shows plots demonstrating the distribution of the ratio of pyrimidine to purine metabolites for samples with low and high UCD-scores (top and bottom IS %).
  • Figure SD is a plot showing urea plasma levels in children with different cancers. The dashed red line demonstrates the normal age matched mean urea value.
  • Figure SF shows metabolic modelling which predicts a significant increase in metabolic flux reactions involving pyrimidine metabolites following UCD.
  • Figure SG shows western blot photographs and their quantification bar plots demonstrating that the increased pyrimidine pathway metabolites' in urine of colon tumors bearing mice shown in Figure SB correlates with UCD in the tumors compared to control healthy colon.
  • Figures 6A-D demonstrate that tumors with UCD have increased transverse coding mutations.
  • Figure 6A is a bar plot demonstrating mat downregulation of ASS1 in osteosarcoma cancer cells using shRNA increases pyrimidine to purines ratio as compared to osteosarcoma cancer cells transduced with an empty vector (EV), (****P-value ⁇ 0.0001, two way ANOVA with Dunnett's correction).
  • Figure 6B is a plot demonstrating that UCD (UC-dys) increases DNA purine to pyrimidine transversion mutations at a pan-cancer scale and across different tumor types compared to tumors with intact UC (UC-WT).
  • Figure 6C is a plot demonstrating that UCD samples show a higher fraction of nonsynonymous purine to pyrimidine transversion mutations as compared to UC-WT across all TCGA data (P ⁇ 4.93E-3). Such a significant bias is not present for any of the other transversion mutation types (Y ->Y, R->R, and Y->R).
  • Figure 6D shows a Cox regression analysis demonstrating that only R->Y mutation levels are significantly associated with survival (while overall mutation levels and Y->R mutation levels are not).
  • Figures 7A-F demonstrate that UCD increases transversion mutations in tumors.
  • Figure 7A is a bar plot demonstrating that downregulation of OTC in hepatic cancer cells using shRNA increases pyrimidine to purines ratio as compared to hepatic cancer cells transduced with an empty vector (EV), as measured by LCMS Bars represent the mean of >2 biological repeats, *P ⁇ 0.05, one way anova with dunnet correction.
  • Figure 7B is a plot demonstrating that tumors with UCD (UC-dys) have significantly higher number of transversion mutations from purines to pyrimidines on the coding (sense) DNA strand versus tumors with intact UC (UC-WT), Wilcoxon rank sum P ⁇ 2.3SE-3), while such a significance is not observed for transition mutations.
  • Figure 7D is a plot demonstrating that tumors with UCD have significantly greater fractions of transversion mutations from purines to pyrimidines at the mRNA level, based on 18 breast cancer samples (Wilcoxon rank sum, **P ⁇ 0.001). Only those variants that were detected as a somatic mutation in the exome sequence and were mapped in the corresponding RNA sequence were considered.
  • Figure 7E is a plot representing genome wide proteomic analysis of 42 breast cancers demonstrating a significantly increased R->Y mutation rates in UCD tumors as compared to tumors with intact UC (Wilcoxon rank sum PO.02).
  • Figure 7F is a plot demonstrating that CAD, SLC25A13 and SLC25A 15 genes' expression are among the top 10 % of genes that correlate most strongly with DNA purines to pyrimidines transversion mutations.
  • Figure 8 is a bar plot demonstrating that specific UC perturbations induced in different cancer cells [i.e. downregulation of OTC (shOTC), ORNT1 (shSLC25A15) or ASS1 (shASSl) or overexpression of citrin (Citrin OE)] increases pyrimidine to purines ratio as compared to control cancer cells transduced with an empty vector (EV), as measured by LCMS. Shown is a representative of the mean of more than two biological repeats. (*P ⁇ 0.05, ** P ⁇ 0.01, one way ANOVA with Dunnet's correction).
  • Figure 9 is a bar graph demonstrating mat specific UC perturbations induced in different cancer cells [i.e. downregulation of OTC (shOTC), ORNT1 (shSLC25A15) or ASS1 (shASSl) or overexpression of citrin (Citrin OE)] increases purines to pyrimidines (R->Y) mutations using a Fisher's exact test.
  • FIGS 10A-F demonstrate that UCD score correlates with response to immune modulation therapy (ICT).
  • Figure 10A demonstrates that UCD-scores are significantly higher in human patients responding to anti-PDl (left panel) and anti-CTLA4 (right panel) therapies (orange) compared to non-responders (grey) (Wilcoxon ranksum PO.05).
  • Figure 10B shows ROC curves demonstrating higher predictive power of pyrimidine-rich transversion mutational bias (PTMB, AUO0.77, blue) compared to mutational load (AUO0.34, red) in predicting the response to anti-PDl therapy (Roh et al., 2017).
  • PTMB pyrimidine-rich transversion mutational bias
  • AUO0.34, red mutational load
  • Figures 10C-E demonstrates that anti-PDl therapy is more efficient in UCD tumors, as determined in an in-vivo syngeneic mouse model of colon cancer.
  • Figure IOC demonstrates tumor volume 22 days following inoculation (Wilcoxon ranksum P ⁇ 0.007).
  • Figure 10E demonstrates tumor growth over time in the shASSl group with or without anti-PDl (PO.01, ANOVA with Dunnett's correction).
  • Figure 10F is a schematic representation summary the "UCD effect': while in normal tissues excess nitrogen is disposed as urea, in cancer cells most nitrogen is utilized for synthesis of macromolecules, with pyrimidine synthesis playing a major role in carcinogenesis and effecting patients' prognosis and response to ICT.
  • Figures 11 A-D demonstrate the impact of CAD and PTMB on ICT response and HLA- peptide presentation.
  • Figure 11B shows peptidomics analysis which demonstrates that UCD cell lines have higher MS/MS intensity than control cell lines (Wilclxon ranksum PO.001).
  • Figure 11C demonstrates that UCD cell lines have more hydrophobic peptides than control cell lines (Wilcoxon ranksum P ⁇ 0.0002).
  • Figures 12A-E demonstrates that UCD perturbed mouse colon cancers respond better to ICT.
  • Figure 12A shows western blot photograph and a quantification bar graph demonstrating that MC-38 mouse colon cancer cells infected with different shASSl clones demonstrate downregulation of ASS1 at the protein level as compared to control cells infected with an empty vector (EV).
  • Figure 12B is a RT PCR quantification bar graph demonstrating decreased ASS1 levels in MC38 infected with different shASSl clones as compared to MC38 infected with EV.
  • Figure 12C is a bar graph demonstrating that in vivo tumor growth was enhanced in MC38 transduced with shASSl as compared to the growth of MC38-EV tumors 22 days following inoculation.
  • Figure 12E demonstrates tumor growth over time in the control group (EV) with (red) or without (blue) anti-PDl (ANOVA P>0.12).
  • the present invention in some embodiments thereof, relates to methods of prognosing and treating cancer.
  • UC urea cycle
  • the present inventors show that fibroblasts from patients with deficiency in the UC enzymes OTC or SLC25A15 are more proliferative and exhibit elevated levels of activated CAD (Example 1, Figures 1A-E). Following, the present inventors have uncovered that downregulation of the UC enzymes ASS1 or OTC in cancer cells using shRNA resulted in increased proliferation and pyrimidine synthesis (Example 2, Figures 2A-D).
  • the inventors then developed a computational method to quantify the extent of the metabolic redirection from the UC towards CAD and pyrimidine synthesis (denoted herein as UCD) by calculating a UCD-score which sums up expression of 6 UC genes - ASL, ASS1, CPS1, OTC, SLC25A13 and SLC25A15.
  • UCD a computational method to quantify the extent of the metabolic redirection from the UC towards CAD and pyrimidine synthesis (denoted herein as UCD) by calculating a UCD-score which sums up expression of 6 UC genes - ASL, ASS1, CPS1, OTC, SLC25A13 and SLC25A15.
  • the inventors show that a majority of tumors harbour expression alterations in at least two UC components in the direction that enhances CAD activity (Example 2, Figures 3A-D and 4A-4B).
  • up-regulation of CPS1 and/or SLC25A13; and/or down-regulation of ASL, ASS1, OTC and/or SLC25A15 were detected in different kinds of cancer and were associated with increased CAD expression and activity and pyrimidine synthesis.
  • UCD and the UCD-score
  • activated CAD expression were associated with higher tumor grade and decreased cancer survival (Example Figures 3E, 4B-E).
  • the inventors further demonstrate mat UCD in cancer is also associated with increased pyrimidine to purine ratio (Example 3, Figure SC) and with increased purine to pyrimidine transversion mutations at the DNA, RNA and protein levels (Example 4, Figures 6A-B, 7A-F).
  • the inventors present several computational modeling and experimental studies of urine and plasma samples, which show increased levels of pyrimidine synthesis metabolites (Uracil, Thymidine, Orotic acid and Orotidine) and decreased levels of urea in urine and plasma samples of tumor bearing mice and cancer patients, respectively, compared to cancer-free mice and patients (Example 3, Figures SA-B, SD-E). Consequently, according to some embodiments, the presence of a shift in the metabolic urea cycle (UC) to CAD enzyme associated with a mutation bias toward pyrimidines, can be used as a marker for prognosing and treating cancer.
  • UC metabolic urea cycle
  • purine to pyrimidine transversion mutations and particularly non- synonymous purine to pyrimidine transversion mutations result in presentation of new tumor- associated antigens that can activate the host immune response toward the cancer
  • the present teachings suggest that cancers prognosed and/or monitored according to some embodiments of the present invention are more susceptible to treatment by immune modulation which can deprive the tumor of its protective immune suppression that enables the cancer to evade the host immune response toward the new antigens.
  • a method of treating cancer in a subject in need thereof comprising:
  • a method of treating cancer in a subject in need thereof comprising:
  • treating refers to inhibiting, preventing or arresting the development of a pathology (e.g. cancer) and/or causing the reduction, remission, or regression of a pathology.
  • pathology e.g. cancer
  • Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.
  • subject refers to a mammal (e.g., human being) at any age or of any gender.
  • the subject is a human subject.
  • the subject is diagnosed with a disease (i.e., cancer) or is at risk of to develop a disease (i.e. cancer).
  • the subject is not afflicted with an ongoing inflammatory disease (other than cancer).
  • the subject is not a pregnant female.
  • Cancers which may be prognosed, monitored and/or treated by some embodiments of the invention can be any solid or non-solid cancer and/or cancer metastasis. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastema, sarcoma, and leukemia.
  • cancers include, but not limited to, tumors of the gastrointestinal tract (colon carcinoma, rectal carcinoma, colorectal carcinoma, colorectal cancer, colorectal adenoma, hereditary nonpolyposis type 1, hereditary nonpolyposis type 2, hereditary nonpolyposis type 3, hereditary nonpolyposis type 6; colorectal cancer, hereditary nonpolyposis type 7, small and/or large bowel carcinoma, esophageal carcinoma, tylosis with esophageal cancer, stomach carcinoma, pancreatic carcinoma, pancreatic endocrine tumors), endometrial carcinoma, dermatofibrosarcoma protuberans, gallbladder carcinoma, Biliary tract tumors, prostate cancer, prostate adenocarcinoma, renal cancer (e.g., Wilms' tumor type 2 or type 1), liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma, hepatocellular cancer), bladder cancer
  • colon carcinoma
  • the cancer is carcinoma.
  • the cancer is selected from the list of cancers presented in Figure 3A, each possibility represents a separate embodiment of the present invention.
  • the cancer is selected from the group consisting of hepatic cancer, osteosarcoma, breast cancer, colon cancer, thyroid cancer, stomach cancer, lung cancer, kidney cancer, prostate cancer, head and neck cancer, bile duct cancer and bladder cancer, each possibility represents a separate embodiment of the present invention.
  • the cancer is selected from the group consisting of hepatic cancer, osteosarcoma, breast cancer and colon cancer, each possibility represents a separate embodiment of the present invention.
  • the cancer is selected from the group consisting of thyroid cancer, hepatic cancer, bile duct cancer and kidney cancer, each possibility represents a separate embodiment of the present invention.
  • the cancer is lung cancer.
  • the lung cancer is lung squamous carcinoma.
  • the cancer is a hepatic cancer.
  • the hepatic cancer is hepatocellular carcinoma.
  • the cancer is not hepatocellular carcinoma.
  • the cancer is kidney cancer.
  • the kidney cancer is kidney renal papillary cell carcinoma.
  • the kidney cancer is kidney renal clear cell carcinoma.
  • the kidney cancer is Kidney chromophobe.
  • the cancer is a head and neck cancer.
  • the head and neck cancer is Head Neck Squamous Cell Carcinoma.
  • the cancer is bile duct cancer.
  • the bile duct cancer is cholangiocarcinoma.
  • the cancer is thyroid cancer.
  • the cancer is not thyroid cancer.
  • the cancer is breast cancer.
  • the methods of some embodiments of the present invention comprise determining a shift from the urea cycle to pyrimidine synthesis in a cancerous cell of the subject.
  • a cell of the subject refers to at least one cell (e.g., an isolated cell), cell culture, cell content and/or cell secreted content which contains RNA and/or proteins of the subject.
  • the cell is comprised in a tissue or an organ.
  • the cell is a cancerous cell, which can be primary or metastatic.
  • the cell may be isolated from the subject (e.g., for in-vitro detection) or may optionally comprise a cell that has not been physically removed from the subject (e.g., in-vivo detection).
  • the determining is effected in-vivo using detection methods suitable for the subject (e.g. human) body (e.g. using antibodies, such as the antibodies e.g. conjugated to a label or to a detectable moiety).
  • the determining is effected in-vitro or ex-vivo.
  • the method comprising obtaining the cell prior to the determining.
  • the cell is comprised in a biological sample.
  • biological sample refers to any cellular biological sample which may express a UC enzyme and CAD enzyme. Examples include but are not limited to, a cell obtained from any tissue biopsy, a tissue, an organ, a blood cell, a bone marrow cell, body fluids such as blood, saliva, spinal fluid, lymph fluid, rinse fluid that may have been in contact with the tumor, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, urine and feces. According to specific embodiments, the biological sample is an in-situ sample (i.e. of the cancer).
  • the method of the present invention comprises obtaining the biological sample prior to the determining,
  • the biological sample can be obtained using methods known in the art such as using a syringe with a needle, a scalpel, fine needle biopsy, needle biopsy, core needle biopsy, fine needle aspiration (FN A), surgical biopsy, buccal smear, lavage and the like. According to specific embodiments, the biological sample is obtained by biopsy.
  • a specific cell type may be further isolated from the biological sample obtained from the subject.
  • Methods of isolating specific cell types are well known in the art including, but not limited to, density gradient centrifugation, flow cytometry and magnetic beads separation.
  • a cancerous cell can be isolated from the biological sample by e.g. tumor specific markers.
  • a shift from the urea cycle to pyrimidine synthesis refers to a change in the metabolic balance characterized by a decrease in the urea cycle (UC) activity and an increase in pyrimidine synthesis in a cell as compared to a control sample, which may be manifested in e.g. alterations in level and/or activity of a UC enzyme, increased level and/or activity of CAD enzyme, decreased levels of UC metabolites, increased levels of pyrimidines and/or an increased level of purine to pyrimidine transversion mutations.
  • UC urea cycle
  • a shift from the urea cycle to pyrimidine synthesis is indicated when the change (e.g. alterations in level and/or activity of a UC enzyme gene, increased level and/or activity of CAD enzyme, increased level of purine to pyrimidine transversion mutations) is above or below a predetermined threshold (depending on the component analyzed).
  • the change e.g. alterations in level and/or activity of a UC enzyme gene, increased level and/or activity of CAD enzyme, increased level of purine to pyrimidine transversion mutations
  • predetermined threshold refers to a level and/or activity
  • a component e.g. a UC enzyme gene, CAD enzyme, purine to pyrimidine transversion mutations
  • a level can be experimentally determined by comparing samples with normal levels of the component (e.g., samples obtained from healthy subjects e.g., not having cancer) to samples derived from subjects diagnosed with cancer. Alternatively, such a level can be obtained from the scientific literature and from databases.
  • the increase/decrease above or below a predetermined threshold is statistically significant.
  • the predetermined threshold is derived from a control sample.
  • control samples can be used with specific embodiments of the present invention.
  • the control sample has a balance of UC to pyrimidine synthesis representative of a healthy biological sample.
  • control sample contains a level and/or activity of a UC enzyme comparable to a healthy biological sample.
  • control sample contains a level and/or activity of a CAD enzyme comparable to a healthy biological sample.
  • control sample contains a level of purine to pyrimidine transversion mutations comparable to a healthy biological sample.
  • control sample is obtained from a subject of the same species, age, gender and from the same sub-population (e.g. smoker/nonsmoker).
  • control sample comprises a cell of the same type as the cell of the subject.
  • control sample is from the same type as the biological sample obtained from the subject.
  • control sample is a healthy control sample.
  • control sample is a non-cancerous tissue of said subject.
  • control sample is a cancerous cell with a level and/or activity of said urea cycle enzyme, said purine to pyrimidine transversion mutations and/or said CAD enzyme similar to levels and/or activity of same in a healthy cell of the same type.
  • control sample is obtained from the scientific literature or from a database, such as the known age matched mean value in a non-cancerous population.
  • the predetermined threshold is at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least l.S fold, at least 2 fold, at least 3 fold, at least 5 fold, at least 10 fold, or at least 20 fold as compared the level and/or activity of the component (e.g. a UC enzyme gene, CAD enzyme, purine to pyrimidine transversion mutations) in a control sample as measured using the same assay such as any DNA (e.g. genome sequencing) RNA (e.g. RNA sequencing, PCR, Northern blot), protein (e.g. western blot, immunocytochemi stry, flow cytometry), chromatography and mass spectrometry (e.g. LC-MS), enzymatic and/or chemical assay suitable for measuring level and/or activity of a compound, as further disclosed herein.
  • the predetermined threshold is at least 1.1 fold compared to a control sample.
  • the predetermined threshold is at least 2 %, at least 5 %, at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least SO %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, e.g., 100 %, at least 200 %, at least 300 %, at least 400 %, at least 500 %, at least 600 % as compared the level and/or activity of the component in a control sample.
  • the shift is determined by a level of purine to pyrimidine transversion mutations.
  • Determining the level of purine to pyrimidine transversion mutation can be effected by any method known in the art, such as but not limited to, whole genome sequencing, DNA sequencing and/or RNA sequencing as further described in the Examples section which follows.
  • determining the level of purine to pyrimidine transversion mutations is effected at the genomic level.
  • determining the level of purine to pyrimidine transversion mutations is effected at the transcript level.
  • the present inventors have shown that the shift from the UC to pyrimidine synthesis in cancer is associated with increased purine to pyrimidine transversion mutations at the DNA, RNA and at times at the protein levels, also referred to as non-synonymous, i.e. they change the encoded amino acid.
  • the purine to pyrimidine transversion mutations are non-synonymous purine to pyrimidine transversion mutations.
  • the shift from the UC to pyrimidine synthesis can be determined by analyzing at the proteomics of newly formed peptide antigens (peptidomics).
  • Determining non-synonymous transversion mutations are well known in the art and can be effected by direct analysis of the encoded protein(s) or using in-silico proteomics. However, such a determination can also be performed at the RNA or DNA level and using in-silico translational tools to test the effect on the translated sequence.
  • DNA or RNA is first extracted from a biological sample of the tested subject, by methods well known in the art. According to specific embodiments, the DNA or RNA sample is amplified prior to determining sequence alterations, since many genotyping methods require amplification of the region carrying the sequence alteration of interest.
  • the purine to pyrimidine transversion mutations of some embodiments of the invention can be identified using a variety of methods. For example, one option is to determine the entire gene sequence of a PCR reaction product. Alternatively, a given segment of nucleic acid may be characterized by the size of the molecule as may be determined by e.g. electrophoresis by comparison to a known standard run on the same gel.
  • a more detailed picture of the molecule may be achieved by cleavage with combinations of restriction enzymes prior to electrophoresis, to allow construction of an ordered map.
  • the presence of specific sequences within the fragment can be detected by hybridization of a labeled probe, or the precise nucleotide sequence can be determined by partial chemical degradation or by primer extension in the presence of chain- terminating nucleotide analogs.
  • detecting purine to pyrimidine transversion mutations involves directly determining the identity of the nucleotide at the alteration site by a sequencing assay, an enzyme-based mismatch detection assay, or a hybridization assay.
  • Sequencing analysis an isolated DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-terminator (unlabeled primer and labeled di-deoxy nucleotides) or a dye-primer (labeled primers and unlabeled di-deoxy nucleotides) cycle sequencing protocols.
  • a dye-terminator reaction a PCR reaction is performed using unlabeled PCR primers followed by a sequencing reaction in the presence of one of the primers, deoxynucleotides and labeled di-deoxy nucleotide mix.
  • a PCR reaction is performed using PCR primers conjugated to a universal or reverse primers (one at each direction) followed by a sequencing reaction in the presence of four separate mixes (correspond to the A, G, C, T nucleotides) each containing a labeled primer specific the universal or reverse sequence and the corresponding unlabeled di-deoxy nucleotides.
  • Hybridization Assay Methods - Hybridization based assays which allow the detection of single base alterations rely on the use of oligonucleotide which can be 10, IS, 20, or 30 to 100 nucleotides long preferably from 10 to SO, more preferably from 40 to SO nucleotides.
  • the oligonucleotide includes a central nucleotide complementary to a specific site of a gene and flanking nucleotide sequences spanning on each side of the central nucleotide and substantially complementary to the nucleotide sequences of the gene. Sequence alteration can be detected by hybridization of the oligonucleotide of some embodiments of the invention to the template sequence under stringent hybridization reactions.
  • the hybridization assay can be effected with oligonucleotide arrays.
  • the chip/array technology has already been applied with success in numerous cases. For example, the screening of mutations has been undertaken in the BRCA1 gene, in S. cerevisiae mutant strains, and in the protease gene of HIV-1 virus [see Hacia et al., (1996) Nat Genet 1996;14(4):441-447; Shoemaker et al., (1996) Nat Genet 1996;14(4):450-456; Kozal et al., (1996) Nat Med 1996;2(7):753-759].
  • sets of four oligonucleotide probes are generally designed that span each position of a portion of the candidate region found in the nucleic acid sample, differing only in the identity of the mutation base.
  • the relative intensity of hybridization to each series of probes at a particular location allows the identification of the base corresponding to the mutation base of the probe.
  • SSCP Single-Strand Conformation Polymorphism
  • the SSCP process involves denaturing a DNA segment (e.g., a PCR product) that is labeled on both strands, followed by slow electrophoretic separation on a non-denaturing polyacrylamide gel, so that intra-molecular interactions can form and not be disturbed during the run.
  • a DNA segment e.g., a PCR product
  • This technique is extremely sensitive to variations in gel composition and temperature.
  • a serious limitation of this method is the relative difficulty encountered in comparing data generated in different laboratories, under apparently similar conditions.
  • Dideoxy fingerprinting (ddF) -
  • the ddF technique combines components of Sanger dideoxy sequencing with SSCP (see Liu and Sommer, PCR Methods Appli., 4:97, 1994).
  • a dideoxy sequencing reaction is performed using one dideoxy terminator and then the reaction products are electrophoresed on nondenaturing polyacrylamide gels to detect alterations in mobility of the termination segments as in SSCP analysis.
  • Restriction fragment length polymorphism This method uses a change in a single nucleotide which modifies a recognition site for a restriction enzyme resulting in the creation or destruction of an RFLP.
  • Single nucleotide mismatches in DNA heteroduplexes are also recognized and cleaved by some chemicals, providing an alternative strategy to detect single base substitutions, generically named the "Mismatch Chemical Cleavage” (MCC) (Gogos et al., Nucl. Acids Res., 18:6807-6817, 1990).
  • MCC Mismatch Chemical Cleavage
  • this method requires the use of osmium tetroxide and piperidine, two highly noxious chemicals which are not suited for use in a clinical laboratory.
  • elevation of at least 2 %, at least 5 %, at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least SO %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, e.g., 100 %, at least 200 %, at least 300 %, at least 400 %, at least 500 %, at least 600 % in the level of non-synonymous purine to pyrimidine transversion mutations in a genome sequence, a RNA sequence or in a protein sequence as compared to a control sample is indicative of a shift from the UC to pyrimidine synthesis.
  • the present inventors have also shown that the shift from the UC to pyrimidine synthesis in cancer is associated with alterations in the expression levels of UC and CAD enzymes.
  • the shift is determined by a level and/or activity of a urea cycle enzyme and/or a CAD enzyme.
  • the urea cycle is a metabolic process which converts excess nitrogen derived from the breakdown of nitrogen-containing molecules to the excretable nitrogenous compound - urea.
  • the UC enzymes encompassed by specific embodiments of the present invention are selected from the group consisting of ASL, ASS1, OTC, SLC25A15, CPS1 and SLC25A13.
  • ASL argininosuccinate lyase
  • ASAL argininosuccinase
  • ASL catalyzes the hydrolytic cleavage of argininosuccinate (ASA) into arginine and fumarate.
  • the ASL refers to the human ASL, such as provided in the following Accession Numbers: NM 000048, NM 001024943, NM 001024944, NM_001024946, NP_000039, NP OO 1020114, NP_001020115 and NP_001020117 (SEQ ID NO: 8).
  • ASS1 Argininosuccinate synthase 1
  • E.C. No. 6.3.4.5 also known as or
  • Argininosuccinate synthase refers to the polynucleotide or polypeptide expression product of the ASS1 gene (Gene ID 445).
  • ASS1 catalyzes the synthesis of argininosuccinate from citrulline and aspartate.
  • the ASS1 refers to the human ASS1, such as provided in the following Accession Numbers: NM_000050, NM_054012, XM_005272200, XM_011518705, XM 017014729, NP 000041, NP_446464, XP_005272257, XP_011517007 and XP_016870218.
  • OTC Ornithine transcarbamylase
  • E.C. No. 2.1.3.3 also known as ornithine carbamoyltransferase and OTCase, refers to the polynucleotide or polypeptide expression product of the OTC gene (Gene ID 5009).
  • OTC catalyzes the reaction between carbamoyl phosphate and ornithine to form citrulline and phosphate.
  • the OTC refers to the human OTC, such as provided in the following Accession Numbers: NM 000531, XM 017029556, NP_000522 and XP_016885045.
  • SLC25A15 Solute Carrier Family 25 Member 15
  • ORNT1 Ornithine Transporter 1
  • SLC25A15 a member of the mitochondrial carrier family, transports ornithine from the cytosol into the mitochondria.
  • the SLC25A15 refers to the human SLC25A15, such as provided in the following Accession Numbers: NM_014252 and NP_055067.
  • CPS1 Carbamoyl Phosphate Synthetase I
  • CPS1 Carbamoyl Phosphate Synthetase I
  • CPS1 catalyzes synthesis of carbamoyl phosphate from ammonia and bicarbonate.
  • the overall reaction that occurs in CPSI is:
  • the CPSI refers to the human CPSI, such as provided in the following Accession Numbers: NM_001122633, NM_001122634, NM_001875, XM_011510640, XM_011510641, NP_001116105, NP_001116106, NP_001866, XP_011508942, XP_011508943, XP_011508944, XP_011508945 and XP_011508946.
  • SLC25A13 Solute Carrier Family 25 Member 13
  • citrin Mitochondrial Aspartate Glutamate Carrier 2, ARALAR2, CTLN2 and Calcium-Binding Mitochondrial Carrier Protein Aralar2
  • SLC25A13 a protein member of the mitochondrial carrier family, catalyzes the exchange of aspartate for glutamate and a proton across the inner mitochondrial membrane, and is stimulated by calcium on the external side of the inner mitochondrial membrane.
  • the SLC25A13 protein contains four EF-hand Ca(2+) binding motifs in the N-terminal domain, and is localized in the mitochondria.
  • the SLC25A13 refers to the human SLC25A13, such as provided in the following Accession Numbers: NM_001160210, NM_014251, XM 006715831, XM_011515727, XM_017011663, NP_001153682, NP_055066, XP_006715894, XP_011514029, XP_016867152, XP_016867153 and XP_016867154.
  • CAD carbamoyl-pho sphate synthetase 2, aspartate transcarbamylase, and dihydroorotase
  • CAD is a trifunctional protein which is associated with the enzymatic activities of the first 3 enzymes in the de-novo pyrimidine synthesis pathway:
  • the CAD refers to the human CAD, such as provided in the following Accession Numbers: NM_001306079, NM 004341, XM 005264555, XM_006712101, NP 001293008, NP_004332, XP_005264612, and XP_006712164.
  • the trifunctional protein encoded by CAD is regulated by the mitogen-activated protein kinase (MAPK) cascade.
  • MAPK mitogen-activated protein kinase
  • CAD is CAD is activated CAD.
  • An activated CAD is phosphorylated. Phosphorylation of CAD can be determined by any method known in the art, such as but not limited to, Western blot, Elisa, flow cytometry and mass spectrometry.
  • ASL, ASS1, OTC, SLC25A15, CPS1, SLC25A13 and CAD also encompass functional homologues (naturally occurring or synthetically/recombinantly produced), orthologs (from other species) which exhibit the desired enzymatic activity described hereinabove.
  • the functional homologs also refer to functional portions of ASL, ASS1, OTC, SLC25A15, CPS1, SLC2SA13 and CAD which maintain the enzymatic activity of the full length proteins, as described hereinabove.
  • activity refers to activity of an enzyme per cell. Methods of determining activity of the enzymes disclosed herein are well known in the art and include both enzymatic and chemical assays.
  • the phrase "level" when relating to an enzyme refers to the degree of gene expression (e.g. mRNA or protein).
  • the expression level can be determined in arbitrary absolute units, or in normalized units (relative to known expression levels of a control sample). For example, when using DNA chips, the expression levels are normalized according to internal controls or by using quantile normalization such as RMA. Expression level can be determined in the cell using any structural, biological or biochemical method which is known in the art for detecting the expression level at the transcript or the protein level.
  • the RNA or the protein molecules are extracted from the cell of the subject
  • the method further comprises extracting RNA or a protein from the cell prior to the determining.
  • RNA, cDNA or protein molecules can be characterized for the level of various RNA, cDNA and/or protein molecules using methods known in the arts.
  • determining the level of the enzyme is effected at the transcript level using RNA or DNA detection methods
  • detection of the level of the UC enzyme and/or CAD enzyme is performed by contacting the biological sample, the tissue, the cell, or fractions or extracts thereof with a probe (e.g. oligonucleotide probe or primer) which specifically hybridizes to a polynucleotide expressed from the UC gene (e.g. ASL, ASS1, OTC, SLC25A15, CPS1, SLC2SA13) and/or CAD gene.
  • a probe can be at any size, such as a short polynucleotide (e.g., of 15-200 bases), an intermediate polynucleotide of 100-2000 bases and a long polynucleotide of more than 2000 bases.
  • the probe used by the present invention can be any directly or indirectly labeled RNA molecule [e.g., RNA oligonucleotide (e.g., of 17-50 bases), an in-vitro transcribed RNA molecule], DNA molecule (e.g., oligonucleotide, e.g., 15-50 bases, cDNA molecule, genomic molecule) and/or an analogue thereof [e.g., peptide nucleic acid (PNA)] which is specific to the RNA transcript of the UC and/or CAD gene.
  • the probe is bound to a detectable moiety.
  • Oligonucleotides designed according to the teachings of the present invention can be generated according to any oligonucleotide synthesis method known in the art such as enzymatic synthesis or solid phase synthesis.
  • the contacting is effected under conditions which allow the formation of a complex comprising mRNA or cDNA of a UC (e.g. ASL, ASS1, OTC, SLC25A15, CPS1, SLC25A13) and/or CAD gene present in the cell and the probe.
  • a complex comprising mRNA or cDNA of a UC (e.g. ASL, ASS1, OTC, SLC25A15, CPS1, SLC25A13) and/or CAD gene present in the cell and the probe.
  • the complex can be formed at a variety of temperatures, salt concentration and pH values which may vary depending on the method and the biological sample used and those of skills in the art are capable of adjusting the conditions suitable for the formation of each nucleotide/probe complex.
  • composition of matter comprising RNA of a cancerous cell of a subject and a probe capable of detecting a polynucleotide expressed from a UC (e.g. ASL, ASS1, OTC, SLC25A15, CPS1, SLC25A13) and/or a CAD gene.
  • a UC e.g. ASL, ASS1, OTC, SLC25A15, CPS1, SLC25A13
  • the composition further comprises an RNase inhibitor.
  • Non-limiting examples of methods of detecting RNA and/or cDNA molecules in a cell sample include Northern blot analysis, RT-PCR [e.g., a semi-quantitative RT-PCR, quantitative RT-PCR using e.g., the Light CyclerTM (Roche)], RNA in-situ hybridization (using e.g., DNA or RNA probes to hybridize RNA molecules present in the cells or tissue sections), in-situ RT-PCR (e.g., as described in Nuovo GJ, et al. Am J Surg Pathol. 1993, 17: 683-90; Karlinoth P, et al. Pathol Res Pract.
  • RT-PCR e.g., a semi-quantitative RT-PCR, quantitative RT-PCR using e.g., the Light CyclerTM (Roche)
  • RNA in-situ hybridization using e.g., DNA or RNA probes to hybridize RNA molecules present in the cells or tissue
  • oligonucleotide microarray e.g., by hybridization of polynucleotide sequences derived from a sample to oligonucleotides attached to a solid surface [e.g., a glass wafer) with addressable location, such as Affymetrix microarray (Affymetrix®, Santa Clara, CA)].
  • Affymetrix microarray Affymetrix®, Santa Clara, CA
  • determining the level of the enzyme is effected at the protein level using protein detection methods.
  • detection of the level of the protein of the UC and/or CAD is performed by contacting the biological sample, the tissue, the cell, or fractions or extracts thereof with an antibody which specifically binds to a UC enzyme (e.g. ASL, ASS1, OTC, SLC25A15, CPS1, SLC25A13) and/or a CAD enzyme.
  • a UC enzyme e.g. ASL, ASS1, OTC, SLC25A15, CPS1, SLC25A13
  • CAD enzyme e.g. ASL, ASS1, OTC, SLC25A15, CPS1, SLC25A13
  • the contacting is effected under conditions which allow the formation of a complex comprising a UC enzyme (e.g. ASL, ASS1, OTC, SLC25A15, CPS1, SLC25A13) and/or a CAD enzyme present in the cell and the antibody (i.e. immunocomplex).
  • the immunocomplex can be formed at a variety of temperatures, salt concentration and pH values which may vary depending on the method and the biological sample used and those of skills in the art are capable of adjusting the conditions suitable for the formation of each immunocomplex.
  • composition of matter comprising a lysate of a cancerous cell of a subject, and an antibody capable of detecting a UC enzyme (e.g. ASL, ASS1, OTC, SLC25A15, CPS1, SLC25A13) and/or a CAD enzyme.
  • a UC enzyme e.g. ASL, ASS1, OTC, SLC25A15, CPS1, SLC25A13
  • CAD enzyme e.g. ASL, ASS1, OTC, SLC25A15, CPS1, SLC25A13
  • the composition further comprises a secondary antibody capable of binding the antibody.
  • the composition further comprises a protease inhibitor.
  • Non-limiting examples of methods of detecting the level of specific protein molecules in a cell sample include Enzyme linked immunosorbent assay (ELISA), Western blot analysis, immunoprecipitation (IP), radio-immunoassay (RIA), Fluorescence activated cell sorting (FACS), immunohistochemical analysis, in-situ activity assay (using e.g., a chromogenic substrate applied on the cells containing an active enzyme), in-vitro activity assays (in which the activity of a particular enzyme is measured in a protein mixture extracted from the cells) and molecular weight-based approach.
  • ELISA Enzyme linked immunosorbent assay
  • IP immunoprecipitation
  • RIA radio-immunoassay
  • FACS Fluorescence activated cell sorting
  • immunohistochemical analysis using e.g., a chromogenic substrate applied on the cells containing an active enzyme
  • in-vitro activity assays in which the activity of a particular enzyme is measured in a protein
  • the antibody or probe used by the present invention can be any directly or indirectly labeled antibody or probe. According to specific embodiments, the antibody or probe is bound to a detectable moiety.
  • the detectable moiety used by some embodiments of the invention can be, but is not limited to a fluorescent chemical (fluorophore), a phosphorescent chemical, a chemiluminescent chemical, a radioactive isotope (such as [12s] iodine), an enzyme, a fluorescent polypeptide, an affinity tag, and molecules (contrast agents) detectable by Positron Emission Tomagraphy (PET) or Magnetic Resonance Imaging (MRI).
  • the methods of the present invention can be used for prognosing and treating cancer and for monitoring efficacy of cancer therapy.
  • a method of prognosing cancer in a subject diagnosed with cancer comprising determining a level of purine to pyrimidine transversion mutations in a cancerous cell of the subject as compared to a control sample, wherein said level of said purine to pyrimidine transversion mutations above a predetermined threshold is indicative of poor prognosis, thereby prognosing cancer in the subject.
  • an increased level of purine to pyrimidine transversion mutations is indicative of poor prognosis.
  • no change in the purine to pyrimidine transversion mutations levels, or a decreased level of the purine to pyrimidine transversion mutations indicates better prognosis.
  • prognosing refers to determining the outcome of the disease (cancer).
  • poor prognosis refers to increased risk of death due to the disease, increased risk of progression of the disease (e.g. cancer grade), and/or increased risk of recurrence of the disease.
  • a method of prognosing cancer in a subject diagnosed with cancer comprising determining a level and/or activity of a urea cycle enzyme SLC2SA1S in a cancerous cell of the subject as compared to a control sample, wherein said level and/or activity of said SLC25A15 below a predetermined threshold is indicative of poor prognosis, thereby prognosing cancer in the subject.
  • a method of prognosing cancer in a subject diagnosed with cancer comprising determining a level and/or activity of at least two urea cycle enzymes selected from the group consisting of ASL, ASS1, OTC, SLC25A15, CPS1 and SLC25A13 in a cancerous cell of the subject as compared to a control sample, wherein said urea cycle enzymes comprise ASS1 and SLC2SA13 said at least two is at least three; wherein said level and/or activity of said ASL, said ASS1, said OTC and/or said SLC25A15 below a predetermined threshold and/or said level and/or activity of said CPS1 and/or said SLC25A13 above a predetermined threshold is indicative of poor prognosis, thereby prognosing cancer in the subject.
  • a method of prognosing breast cancer in a subject diagnosed with breast cancer comprising determining a level and/or activity of at least two urea cycle enzymes selected from the group consisting of ASS1, OTC and SLC25A13 in a cancerous cell of the subject as compared to a control sample, wherein said level and/or activity of said ASS1 and/or said OTC below a predetermined threshold and/or said level and/or activity of said SLC25A13 above a predetermined threshold is indicative of poor prognosis, thereby prognosing the breast cancer in the subject.
  • a decreased level and/or activity of a UC enzyme selected from the group consisting of ASL, ASS1, OTC and SLC25A15 and/or an increased level a UC enzyme selected from the group consisting of CPS1 and SLC25A13 is indicative of poor prognosis.
  • no change in the UC enzyme selected from the group consisting of ASL, ASS1, OTC, SLC2SA1S, CPS1 and SLC25A13 or an increased level and/or activity of a UC enzyme selected from the group consisting of ASL, ASS1, OTC and SLC25A15 and/or a decreased level and/or activity of the UC enzyme selected from the group consisting of CPS1 and SLC2SA13 indicates better prognosis.
  • At least 2, at least 3, at least 4, at least 5 or all 6 UC enzymes are detennined, each possibility represent a separate embodiment of the present invention.
  • the at least two UC enzymes determined are ASL+ASSI, ASL-tOTC, ASL+SLC25A15, ASL+CPS1, ASL+SCL25A13, ASSl+OTC, ASS1+SLC25A15, ASS1+CPS1, ASS1+SLC25A13, OTC+SLC25A15, OTC+CPS1, OTC+SLC25A13, SLC25A15+CPS1, SLC25A15+SLC25A13 or CPS1+SLC25A13, each possibility represent a separate embodiment of the present invention.
  • the at least three UC enzymes determined are ASL+ASS l+OTC, ASL+ASS 1+SLC25A15, ASL+ASS 1+CPS 1 , ASL+ASS1+SLC25A13, AS L+OTC+SLC25 A 15, ASL+OTC+CPS1, ASL+OTC+SLC25A13, ASL+SLC25A15+CPS 1, ASL+SLC25A15+SLC25A13, ASL+CPS1+SLC25A13, ASS1+OTC+SLC25A15,
  • ASS1+OTC+CPS1 ASS1+OTC+SLC25A13, ASS1+SLC25A15+CPS1,
  • the at least four UC enzymes determined are ASL+ASS 1-K)TC+SLC25A15, ASL+ASS l+OTC+CPSl, ASL+ASS 1+OTC+SLC25 A 13, ASL+OTC+SLC25A15+CPS 1 , ASL+OTC+CLC25A15+SLC25A13, ASL+SLC25 A 15+CPS 1+SLC25 A 13, ASS 1+OTC+SLC25 A 15+CPS 1 , ASS1+OTC+SLC25A15+SLC25A13, ASS1+SLC25A15+CPS1+SLC25A13 or
  • the at least five UC enzymes determined are ASL+ASS 1+OTC+SLC25 A 15+CPS 1 , ASL+ASS1+OTC+SLC25A15+SLC25A13 or ASS 1+OTC+SLC25A15+CPS 1+ SLC25A13, each possibility represent a separate embodiment of the present invention.
  • the cancer is thyroid, stomach and/or bladder cancer and the at least two UC enzymes are selected from the group consisting of OTC, SLC25A15 and SLC25A13.
  • the cancer is prostate cancer and the at least two UC enzymes comprise ASS1 and CSP1.
  • the cancer is lung and/or head and neck cancer and the at least two UC enzymes are selected from the group consisting of ASL, OTC, CPS1 and SLC25A13.
  • the cancer is hepatic, bile duct and/or kidney cancer and the at least two UC enzymes are selected from the group consisting of ASL, ASS1, OTC and SLC25A15.
  • the cancer is breast cancer and the at least three UC enzymes comprise ASS1, OTC and SLC25A13.
  • alteration in level and/or activity in the specified direction of at least 1, at least 2, at least 3, at least 4, at least 5 or all 6 UC enzymes is indicative of poor prognosis, each possibility represent a separate embodiment of the present invention
  • alteration in level and/or activity in the specified direction of the at least two UC enzymes ASL+ASSL ASL+OTC, ASL+SLC25A15, ASL-fCPSl, ASL+SCL25A13, ASS1+OTC, ASS1+SLC25A15, ASS1+CPS1, ASS1+SLC25A13, OTC+SLC25A15, OTC+CPS1, OTC+SLC25A13, SLC25A15+CPS 1, SLC25A15+SLC25A13 or CPS1+SLC2SA13 is indicative of poor prognosis, each possibility represent a separate embodiment of the present invention
  • alteration in level and/or activity in the specified direction of the least three UC enzymes ASL+ASS1+OTC, ASL+ASS1+SLC25A15, ASL+ASS1+CPS1, ASL+ASS1+SLC25A13, ASL+OTC+SLC25A15, ASL+OTC+CPS 1 , ASL+OTC+SLC25A13, ASL+SLC25 Al 5+CPS 1 , ASL+SLC25 A 15+SLC25 A 13, ASL+CPS 1+SLC25 Al 3, ASS1+OTC+SLC25A15, ASS1+OTC+CPS1,
  • ASS 1+OTC+SLC25 A13, ASS 1+SLC25 A 15+CPS 1 , ASS1+SLC25A15+SLC25A14, ASS1+CPS1+SLC25A13, OTC+SLC25A15+CPS 1, OTC+CPS 1+SLC25 Al 3 or SLC25A15+CPS1+SLC25A13, is indicative of poor prognosis, each possibility represent a separate embodiment of the present invention.
  • alteration in level and/or activity in the specified direction of the at least four UC enzymes ASL+ASS 1+OTC+SLC25 A 15, ASL+ASSl+OTC+CPSl, ASL+ASS1+OTC+SLC25A13, ASL+OTC+SLC25A15+CPS 1, ASL+OTC+CLC25A15+SLC25A13, ASL+SLC25A15+CPS 1+SLC25A13,
  • ASS 1+OTC+SLC25A15+CPS 1 , ASS1+OTC+SLC25A15+SLC25A13, ASS1+SLC25A15+CPS1+SLC25A13 or OTC+SLC25A15+CPS1+SLC25A13, is indicative of poor prognosis, each possibility represent a separate embodiment of the present invention.
  • alteration in level and/or activity in the specified direction of the at least five UC enzymes is indicative of poor prognosis, each possibility represent a separate embodiment of the present invention.
  • the methods disclosed herein further comprising determining a level and/or activity of a CAD enzyme, wherein said level and/or activity of said CAD enzyme above a predetermined threshold is indicative of poor prognosis.
  • the methods disclosed herein comprise corroborating the prognosis using a state of the art technique.
  • CBC complete blood count
  • tumor marked tests also known as biomarkers
  • imaging such as MRI, CT scan, PET-CT, ultrasound, mammography and bone scan
  • endoscopy colonoscopy
  • biopsy and bone marrow aspiration.
  • a method of monitoring efficacy of cancer therapy in a subject comprising determining a level of purine to pyrimidine transversion mutations in a cancerous cell of the subject undergoing or following the cancer therapy, wherein a decrease in said level of said purine to pyrimidine transversion mutations from a predetermined threshold or in comparison to said level in said subject prior to said cancer therapy, indicates efficacious cancer therapy.
  • a decrease in the level of purine to pyrimidine transversion mutations is indicative of the cancer therapy being efficient.
  • the cancer therapy is not efficient in eliminating (e.g., killing, depleting) the cancerous cells from the treated subject and additional and/or alternative therapies (e.g., treatment regimens) may be used.
  • a method of monitoring efficacy of cancer therapy in a subject comprising determining a level and/or activity of a urea cycle enzyme SLC25A15 in a cancerous cell of the subject undergoing or following the cancer therapy, wherein an increase in said level and/or activity of said a urea cycle enzyme SLC2SA1S from a predetermined threshold or in comparison to said level and/or activity in said subject prior to said cancer therapy, indicates efficacious cancer therapy.
  • a method of monitoring efficacy of cancer therapy in a subject comprising determining a level and/or activity of at least two urea cycle enzymes selected from the group consisting of ASL, ASS1, OTC, SLC25A15, CPS1 and SLC25A13 in a cancerous cell of the subject undergoing or following the cancer therapy, wherein said urea cycle enzymes comprise ASS1 and SLC2SA13 said at least two is at least three; wherein an increase in said level and/or activity of said ASL, said ASS1, said OTC and/or said SLC2SA1S and/or a decrease in said level and/or activity of said CPS1 and/or said SLC2SA13 from a predetermined threshold or in comparison to said level and/or activity in said subject prior to said cancer therapy indicates efficacious cancer therapy.
  • a method of monitoring efficacy of cancer therapy in a subject diagnosed with breast cancer comprising determining a level and/or activity of at least two urea cycle enzymes selected from the group consisting of ASS1, OTC and SLC2SA13 in a cancerous cell of the subject undergoing or following the cancer therapy, wherein an increase in said level and/or activity of said ASS1 and/or said OTC and/or a decrease in said level and/or activity of said SLC2SA13 from a predetermined threshold or in comparison to said level and/or activity in said subject prior to said cancer therapy, indicates efficacious cancer therapy.
  • an increase in the level and/or activity of a UC enzyme selected from the group consisting of ASL, ASS1, OTC and SLC2SA1S and/or a decrease in the level and/or activity of a UC enzyme selected from the group consisting of CPS1 and SLC2SA13 is indicative of the cancer therapy being efficient.
  • the cancer therapy is not efficient in eliminating (e.g., killing, depleting) the cancerous cells from the treated subject and additional and/or alternative therapies (e.g., treatment regimens) may be used.
  • alteration in level and/or activity in the specified direction of at least 1, at least 2, at least 3, at least 4, at least 5 or all 6 UC enzymes is indicative of efficacious cancer therapy, each possibility represent a separate embodiment of the present invention.
  • alteration in level and/or activity in the specified direction of the at least two UC enzymes ASL+ASSI, ASL+OTC, ASL+SLC25A15, ASL+CPS1, ASL+SCL25A13, ASSl+OTC, ASS1+SLC25A15, ASS1+CPS1, ASS1+SLC25A13, OTC+SLC25A15, OTC+CPS1, OTC+SLC25A13, SLC25A15+CPS1, SLC25A15+SLC25A13 or CPS1+SLC25A13 is of efficacious cancer therapy, each possibility represent a separate embodiment of the present invention.
  • alteration in level and/or activity in the specified direction of the least three UC enzymes ASL+ASSI +OTC, ASL+ASS 1 +SLC25 Al 5, ASL+ASS1+CPS1, ASL+ASS1+SLC25A13, ASL+OTC+SLC25A15, ASL+OTC+CPS1, ASL+OTC+SLC25A13, ASL+SLC25 Al 5+CPS 1 , ASL+SLC25A15+SLC25A13,
  • ASS1+OTC+SLC25A13, ASS1+SLC25A15+CPS1, ASS1+SLC25A15+SLC25A14, ASS1+CPS1+SLC25A13, OTC+SLC25A15+CPS 1, OTC+CPS1+SLC25A13 or SLC25A15+CPS1+SLC25A13, is indicative of efficacious cancer therapy, each possibility represent a separate embodiment of the present invention.
  • alteration in level and/or activity in the specified direction of the at least four UC enzymes ASL+ASS 1+OTC+SLC25 Al 5, ASL+ASSl+OTC+CPSl, ASL+ASS 1+OTC+SLC25A13, ASL+OTC+SLC25A15+CPS1, ASL+OTC+CLC25A15+SLC25A13, ASL+SLC25 Al 5+CPS 1+SLC25 Al 3, ASS1+OTC+SLC25A15+CPS1, ASS1-K)TC+SLC25A15+SLC25A13, ASS 1+SLC25 Al 5+CPS 1+SLC25 Al 3 or OTC+SLC25A15+CPS1+SLC25A13, is indicative of efficacious cancer therapy, each possibility represent a separate embodiment of the present invention.
  • alteration in level and/or activity in the specified direction of the at least five UC enzymes is indicative of efficacious cancer therapy, each possibility represent a separate embodiment of the present invention.
  • the methods disclosed herein further comprising determining a level and/or activity of a CAD enzyme, wherein a decrease in the level and/or activity of said CAD enzyme from a predetermined threshold or in comparison to said level in said subject prior to said cancer therapy, indicates efficacious cancer therapy.
  • the predetermined threshold is in comparison to the level in the subject prior to cancer therapy.
  • the predetermined threshold is at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 2 fold, at least 3 fold, at least 5 fold, at least 10 fold, or at least 20 fold as compared the level and/or activity of the component (e.g. a UC enzyme, CAD enzyme, purine to pyrimidine transversion mutations) in a control sample or in the subject prior to the cancer therapy as measured using the same assay such as any DNA (e.g. genome sequencing) RNA (e.g. RNA sequencing, PGR, Northern blot), protein (e.g. western blot, immunocytochemistry, flow cytometry), chromatography and mass spectrometry (e.g. LC-MS), enzymatic and/or chemical assay suitable for measuring level and/or activity of a compound, as further disclosed herein.
  • the component e.g. a UC enzyme, CAD enzyme, purine to pyrimidine transversion mutations
  • RNA e
  • the predetermined threshold is at least 1.1 fold as compared the level and/or activity of the component in a control sample or in the subject prior to the cancer therapy.
  • the predetermined threshold is at least 2 %, at least 5 %, at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, e.g., 100 %, at least 200 %, at least 300 %, at least 400 %, at least 500 %, at least 600 % as compared the level and/or activity of the component in a control sample or in the subject prior to the cancer therapy.
  • the predetermined threshold can be determined in a subset of subjects with known outcome of cancer therapy.
  • a method of treating cancer in a subject in need thereof comprising:
  • a method of treating cancer in a subject in need thereof comprising:
  • a method of treating cancer in a subject in need thereof comprising:
  • a method of treating cancer in a subject in need thereof comprising:
  • prognosis of the cancer is indicated by the levels of purine to pyrimidine transversion mutations and/or levels and/or activity of a UC enzyme and/or CAD enzyme; according to specific embodiments, the cancer therapy is selected based on the levels and/or activity of the determined component (e.g. a UC enzyme, CAD enzyme, purine to pyrimidine transversion mutations).
  • the phrase "cancer therapy” refers to any therapy that has an anti-tumor effect including, but not limited to, anti-cancer drugs, radiation therapy, cell transplantation and surgery.
  • anti-cancer drugs used with specific embodiments of the present invention include chemotherapy, small molecules, biological drugs, hormonal therapy, antibodies and targeted therapy.
  • the cancer therapy is selected from the group consisting of radiation therapy, chemotherapy and immunotherapy.
  • Anti-cancer drugs that can be used with specific embodiments of the invention include, but are not limited to: Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adriamycin;
  • Adozelesin Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide;
  • Amsacrine Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa;
  • Docetaxel Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride;
  • Esorubicin Hydrochloride Estramustine; Estramustine Phosphate Sodium; Etanidazole;
  • Etoposide Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide;
  • Floxuridine Fhidarabine Phosphate; Fluorouracil; Fhirocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride;
  • Mitocarcin Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper, Mitotane; Mitoxantrone
  • Paclitaxel Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofiirin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogennariium Hydrochloride; Spiromustine; Spiroplatin; Streptomgrin; Streptozocin; Sulofenur; Talisomycin; Taxol; Tecogalan Sodium; Tegafur, Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone
  • Additional antineoplastic agents include those disclosed in Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner), and the introduction thereto, 1202-1263, of Goodman and Gilman's "The Pharmacological Basis of Therapeutics", Eighth Edition, 1990, McGraw-Hill, Inc. (Health Professions Division).
  • Non-limiting examples for anti-cancer approved drugs include: abarelix, aldesleukin, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, asparaginase, azacitidine, AZD9291, AZD4S47, AZD2281, bevacuzimab, bexarotene, bleomycin, bortezomib, busulfan, calusterone, capecitabine, carboplatin, carmustine, celecoxib, cetuximab, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dabrafenib, dacarbazine, dactinomycin, actinomycin D, Darbepoetin alfa, Darbepoetin alfa, daunor
  • the anti-cancer drug is selected from the group consisting of Gefitinib, Lapatinib, Afatinib, BGJ398, CH5183284, Linsitinib, PHA665752, Crizotinib, Sunitinib, Pazopanib, Imatinib, Ruxolitinib, Dasatinib, BEZ23S, Pictilisib, Everolimus, MK-2206, Trametinib / AZD6244, Vemurafinib / Dabrafenib, CCT196969 / CCT241161, Barasertib, VX-680, Nutlin3, Palbociclib, BI 2536, Bardoxolone, Vorinostat, Navitoclax (ABT263), Bortezomib, Vismodegib, Olaparib (AZD2281), Simvastatin, 5- Fluorouricil, Irinotecan, Epirubicin, Ci
  • cancer is associated with a shift from the UC to pyrimidine synthesis in the cancerous cells
  • the present inventors contemplate that cancers prognosed and/or monitored according to some embodiments of the present invention are more susceptible to treatment with agents targeting components associated with these pathways.
  • the cancer therapy is selected from the group consisting of L-arginine depletion, glutamine depletion, pyrimidine analogs, mymidylate synthase inhibitor and mammalian target of Rapamycin (mTOR) inhibitor.
  • Non-limiting examples of Lrarginine depletion agents which can be used with specific embodiments of the present invention include arginine deiminase (ADI) polypeptide, arginase I polypeptide, arginase II polypeptide, arginine decarboxylase polypeptide and arginine kinase polypeptide.
  • ADI arginine deiminase
  • arginase I polypeptide arginase II polypeptide
  • arginine decarboxylase polypeptide arginine decarboxylase polypeptide
  • arginine kinase polypeptide arginine kinase
  • a pegylated form of the indicated enzymes can also be used, according to specific embodiments, such as ADI-PEG 20 is a formulation of ADI with polyethylene glycol (PEG) having an average molecular weight of 20 kilodaltons (PEG 20) and a pegylated form of the catabolic enzyme arginase I (peg-Argl, such as disclosed in Fletcher M et al., (2015) Cancer Res. 75(2):275-83).
  • PEG polyethylene glycol
  • arginase I peg-Argl, such as disclosed in Fletcher M et al., (2015) Cancer Res. 75(2):275-83
  • a cobalt-containing arginase polypeptide such as described in WO2010/051533 can be used.
  • Glutamine depletion agents that can be used with specific embodiments of the invention can act on intracellular and/or extracellular glutamine, e.g., on the glutamine present in the cytosol and/or the mitochondria, and/or on the glutamine present in the peripheral blood.
  • glutamine depleting agents include, inhibitors of glutamate-oxaloacetate- transaminase (GOT), carbamoyl-phosphate synthase, glutamine-pyruvate transaminase, glutamine-tRNA ligase, glutaminase, D-glutaminase, glutamine N-acyltransferase, glutaminase- asparaginase Aminooxyacetate (AOA, an inhibitor of glutamate-dependent transaminase), phenylbutyrate and phenylacetate.
  • GAT glutamate-oxaloacetate- transaminase
  • AOA an inhibitor of glutamate-dependent transaminase
  • phenylbutyrate an inhibitor of glutamate-dependent transaminase
  • Non-limiting examples of pyrimidine analogs which can be used with specific embodiments of the invention include arabinosylcytosine, gemcitabine and decitabine.
  • Non-limiting examples of thymidilate synthase inhibitor that can be used according to specific embodiments of the present invention include fluorouracil (5-FU), capecitabine (an oral 5-FU pro-drug) and pemetrexed.
  • mTOR inhibitors include Rapamycin and rapalogs [rapamycin derivatives e.g. temsirolimus (CCI-779), everolimus (RADOOl), and ridaforolimus (AP-23573), deforolimus (AP23573), everolimus (RADOOl), and temsirolimus (CCI-779)].
  • Rapamycin and rapalogs rapamycin derivatives e.g. temsirolimus (CCI-779), everolimus (RADOOl), and ridaforolimus (AP-23573), deforolimus (AP23573), everolimus (RADOOl), and temsirolimus (CCI-779)].
  • the cancer therapy comprises any agent which downregulates expression and/or activity of the upregulated enzyme (e.g. CPS1, SLC25A13, CAD).
  • the upregulated enzyme e.g. CPS1, SLC25A13, CAD.
  • Downregulation can be at the genomic (e.g., homologous recombination, genome editing and/or site specific endonucleases), the transcript level using a variety of molecules which interfere with transcription and/or translation [e.g., RNA silencing agents (e.g., antisense, siRNA, shRNA, micro-RNA), Ribozyme, DNAzyme, TFO] or at the protein level (e.g., aptamers, small molecules and inhibitory peptides, antagonists, enzymes that cleave the polypeptide, antibodies and the like).
  • RNA silencing agents e.g., antisense, siRNA, shRNA, micro-RNA
  • Ribozyme e.g., Ribozyme, DNAzyme, TFO
  • protein level e.g., aptamers, small molecules and inhibitory peptides, antagonists, enzymes that cleave the polypeptide, antibodies and the like.
  • the cancer therapy comprises over-expressing within tumor cells of the subject the downregulated (e.g. ASL, ASS1, OTC, SLC25A15).
  • the downregulated e.g. ASL, ASS1, OTC, SLC25A15.
  • Over-expression can be at the genomic level [i.e., activation of transcription via promoters, enhancers, regulatory elements, genome editing e.g., using homology directed repair (HDR), and/or by small molecules which can activate expression], at the transcript level (i.e., correct splicing, polyadenylation, activation of translation) or at the protein level (i.e., post- trans lational modifications, interaction with substrates and the like, and/or delivery of the protein itself or of a functional portion thereof into the cells).
  • HDR homology directed repair
  • Upregulation can be also achieved using vectors (nucleic acid constructs) comprising an exogenous polynucleotide encoding the desired expression product or a functional portion thereof, which are designed and constructed to express the desired expression product in the mammalian cells (preferably in tumor cells).
  • cancers prognosed and/or monitored according to some embodiments of the present invention are more susceptible to treatment with immune modulating agent
  • the cancer therapy comprises an immune modulation agent.
  • Immune modulating agents are typically targeting an immune-check point protein.
  • immuno-check point protein refers to an antigen independent protein that modulates an immune cell response (i.e. activation or function).
  • Immune-check point proteins can be either co-stimulatory proteins [i.e. positively regulating an immune cell activation or function by transmitting a co-stimulatory secondary signal resulting in activation of an immune cell] or inhibitory proteins (i.e. negatively regulating an immune cell activation or function by transmitting an inhibitory signal resulting in suppressing activity of an immune cell).
  • check-point proteins include, but not limited to, PD1, PDL-1, B7H2, B7H3, B7H4, BTLA-4, HVEM, CTLA-4, CD80, CD86, LAG-3, TIM-3, KIR, ⁇ , CD19, OX40, OX40L, 4-1BB (CD137), 4-1BBL, CD27, CD70, CD40, CD40L, GITR, CD28, ICOS (CD278), ICOSL, VISTA and adenosine A2a receptor.
  • the immune modulating agent is a PD1 antagonist, such as, but not limited to an anti-PDl antibody.
  • PD1 Programmed Death 1
  • gene symbol PDCDl is also known as CD279.
  • the PD1 protein refers to the human protein, such as provided in the following GenBank Number NP 005009.
  • Anti-PDl antibodies suitable for use in the invention can be generated using methods well known in the art. Alternatively, art recognized anti-PDl antibodies can be used. Examples of anti-PDl antibodies are disclosed for example in Topalian, et al. NEJM 2012, US Patent Nos. US 7,488,802; US 8,008,449; US 8,609,089; US 6,808,710; US 7,521,051; and US 8168757, US Patent Application Publication Nos. US20140227262; US20100151492; US20060210567; and US20060034826 and International Patent Application Publication Nos.
  • Specific anti-PDl antibodies that can be used according to some embodiments of the present invention include, but are not limited to, Nivolumab (also known as MDX1106, BMS- 936558, ONO-4538, marketed by BMY as Opdivo); Pembrolizumab (also known as MK-3475, Keytruda, SCH 900475, produced by Merck); Pidilizumab (also known as CT-011, hBAT, hBAT-1, produced by CureTech); AMP-514 (also known as MEDI-0680, produced by AZY and Medlmmune); and Humanized antibodies h409Al 1, h409A16 and h409A17, which are described in PCT Patent Application No. WO2008/156712.
  • Nivolumab also known as MDX1106, BMS- 936558, ONO-4538, marketed by BMY as Opdivo
  • Pembrolizumab also known as MK-3475, Keytruda,
  • the immune modulating agent is a CTLA4 antagonist, such as, but not limited to an anti-CTLA4 antibody.
  • CTLA4 cytotoxic T-lymphocyte-associated protein 4
  • CD 152 is also known as CD 152.
  • the CTLA-4 protein refers to the human protein, such as provided in the following GenBank Number NP_001032720.
  • Anti-CTLA4 antibodies suitable for use in the invention can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA4 antibodies can be used. Examples of anti-CTLA4 antibodies are disclosed for example in Hurwitz et al. (1998) Proc. Natl. Acad. Sci. USA 95(17): 10067-10071; Camacho et al. (2004) J. Clin. Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res. 58:5301-5304; US Patent Nos.
  • Specific anti-CTLA4 antibodies that can be used according to some embodiments of the present invention include, but are not limited to Ipilimumab (also known as 10D1, MDX-D010), marketed by BMS as YervoyTM; and Tremelimumab, (ticilimumab, CP-675,206, produced by Medlmmune and Pfizer).
  • the present invention discloses that the a shift from the UC to pyrimidine synthesis and the pyrimidine-rich transversion mutational bias enhance the response to immune- modulation therapy independently of mutational load both in mouse models and in patient correlative studies, the present inventors contemplate that cancers diagnosed, prognosed and/or monitored according to some embodiments of the present invention are more susceptible to treatment with immune-modulation therapy in combination with agents that specifically promote pyrimidines to purines nucleotide imbalance.
  • the cancer therapy comprises an agent which induces a pyrimidines to purines nucleotide imbalance.
  • the cancer therapy comprises an immune modulation agent and an agent which induces a pyrimidines to purines nucleotide imbalance.
  • a method of potentiating cancer treatment with an immune modulating agent in a subject in need thereof comprising:
  • the term "induces a pyrimidines to purines nucleotide imbalance” refers to an increase in the ratio of pyrimidines to purines in a cell in the presence of the agent as compared to same in the absence of the agent, which may be manifested in e.g. increased levels of pyrimidines, decreased levels of purines and/or increased level of purine to pyrimidine transversion mutations.
  • the increase is at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least l.S fold, at least 2 fold, at least 3 fold, at least 5 fold, at least 10 fold, or at least 20 fold in the ratio of pyrimidines to purines in a cell in the presence of the agent as compared to same in the absence of the agent, which may be determined by e.g. chromatography and mass spectrometry (e.g. LC-MS), whole genome sequencing, DNA sequencing and/or RNA sequencing.
  • chromatography and mass spectrometry e.g. LC-MS
  • the predetermined threshold is at least 2 %, at least S %, at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, e.g., 100 %, at least 200 %, at least 300 %, at least 400 %, at least 500 %, at least 600 % in the ratio of pyrimidines to purines in a cell in the presence of the agent as compared to same in the absence of the agent.
  • the agent which induces a pyrimidines to purines nucleotide imbalance comprises an anti-folate agent
  • Anti-folate agents which can be used with specific embodiments of the invention are known in the art and include, but not limited to, methotrexate, pemetrexed, proguanil, pyrimethamine, trimethoprim, aminopterin, trimetrexate, edatrexate, piritrexim, ZD 1694, lometrexol, AG337, LY231514 and 1843U89.
  • the anti-folate agent comprises methotrexate.
  • abouf ' refers to ⁇ 10 %
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.
  • the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the f equency of such variations is less than 1 in SO nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.
  • UCD-score is a weighted sum of rank-normalized expression of the 6 urea cycle (UC) genes - ASL, ASS1, CPS1, OTC, SLC25A13 and SLC25A15; wherein:
  • TCGA DNA mutation analysis - TCGA mutation profiles of 7,462 tumor samples encompassing 18 cancer types were downloaded from cbioportal 18 on Feb 1, 2017.
  • the data from cBioPortal does not include healthy control samples but integrates the mutation analysis from different TCGA centers to avoid center specific bias in mutation calls. Samples with less than 5 mutation events were excluded from further analysis.
  • the 13 cancer types that had sufficient sample size N>150, which results in 983,404 single point mutation events (including 745,712 non-synonymous mutations) in 4963 samples, were used.
  • the transversion rates were quantified based on the coding (sense) strand (i.e. the TCGA mutation data was converted to its complementary sequences in genes transcribed from the (-)-strand of the genomic DNA).
  • the fraction of transversions from pyrimidines to purines, denoted herein as f(Y->R) was determined and defined in an analogous manner. Following, the association between UC deregulation and R->Y transverse mutations was analyzed using four different approaches:
  • SR.>Y denotes synonymous mutation level of purine to pyrimidine transversions
  • Naii denotes nonsynonymous mutation level of all mutation events
  • Sail denotes synonymous mutation level of all mutation events.
  • s is an indicator variable over all possible combinations of patients' stratifications based on race, sex and cancer type
  • ft is the hazard function (defined as the risk of death of patients per time unit); and is the baseline-hazard function at time t of the s ft stratification.
  • the model contains two covariates: (i) UCD: UCD-score based on the urea cycle deregulation signatures, and (ii) age: age of the patient.
  • the #s are the regression coefficients of the covariates, which quantify the effect of covariates survival, determined by standard likelihood maximization of the model 19 .
  • the results of this analysis are presented in (Figure 3E).
  • exome-seq data of 18 individual cancer and matched normal cohorts was downloaded from TCGA portal.
  • BAM file of normal and cancer variants were called using the GATK (V. 3.6) 'HaplotypeCaller' 20 * 1 utility with same hg38 assembly that the TCGA used for exome-seq mapping and applying '-ERC GVCF mode to produce a comprehensive record of genotype likelihoods for every position in the genome regardless of whether a variant was detected at that site or not.
  • the purpose of using the GVCF mode was to capture confidence score for every site represented in a paired normal and cancer cohort for detecting somatic mutation in cancer.
  • RNA-Seq The final somatic mutations were mapped on an exonic site of a transcript by 'bcftools' tool (V. 1.3) 21 using BED file of coding region in hg38 assembly.
  • BAM files of RNA-Seq data was downloaded for the same normal and cancer cohorts as described above.
  • GATK's 'SplitNCigar Reads' utility was used to split the reads into exon segments and hard-clipped to any sequence overhanging into the intronic regions.
  • GATK's 'HaplotypeCaller' utility was used with the same hg38 assembly that the TCGA used for RNA-Seq mapping.
  • the 'dontUseSofiClippedBases' argument with the 'HaplotypeCaller' with minimum phred-scaled confidence threshold was used for calling variants set to be 20.
  • the variants were filtered using 'VariantFiltratiori utility based on Fisher Strand values (FS > 30) and Qual By Depth values (QD ⁇ 2.0).
  • Each of the output VCF files was used for annotation of coding regions on the transcripts to which the variants were mapped by using bcftoob' with BED file of coding region in hg38 assembly. Based on this data, the overall R->Y mutation bias, f(R- >Y)-f(Y->R) was compared between UC dysregulated vs. UC intact samples using Wilcoxon rank sum test.
  • Genome-scale metabolic network modeling was used to study the stoichiometric balance of nitrogen metabolism between urea production and pyrimidine synthesis.
  • the stoichiometric constraints can be represented by a stoichiometric matrix S, as follows: where the entry Sy represents the stoichiometric coefficients of metabolite i in reaction j, and v j stands for the metabolic flux vector for all reactions in the model.
  • the model assumes steady metabolic state, as represented in equation (3) above, constraining the production rate of each metabolite to be equal to its consumption rate.
  • a constraint-based model limits the space of possible fluxes in the metabolic network's reactions through a set of (inequalities imposed by thermodynamic constraints, substrate availability and the maximum reaction rates supported by the catalyzing enzymes and transporting proteins, as follows: (4) where oj and fij defines the lower and upper bounds of the metabolic fluxes for different types of metabolic fluxes, (i) The exchange fluxes model the metabolite exchange of a cell with the surrounding environment via transport reactions, enabling a pre-defined set of metabolites to be either taken up or secreted from the growth media, (ii) Enzymatic directionality and flux capacity constraints define lower and upper bounds on the fluxes as represented in equation (4) above.
  • the human metabolic network model 24 was used with biomass function introduced in Folger et al. 25 under the Roswell Park Memorial Institute Medium (RPMI)-1640.
  • fibroblast studies were performed anonymously on cells devoid of all patient identifiers. Punch biopsies were taken from UC deficient patients to generate fibroblast cell line. HepG2 cell line was purchased from ATTC. OTC and CPS1 deficient cell lines as well as control fibroblasts were purchased from Coriell Institute for Medical Research (GM06902; GM 12604). Cells were cultured using standard procedures in a 37 °C humidified incubator with 5 % CO 2 in Dulbecco's Modified Eagle's Medium (DMEM, sigma-aldrich) supplemented with 10-20 % heat-inactivated fetal bovine serum, 10 % pen-strep and 2 mM glutamine. All cells were tested routinely for Mycoplasma using Mycoplasma EZ- PCR test kit (#20-700-20, Biological Industries, Kibbutz Beit Ha'emek).
  • DMEM Dulbecco's Modified Eagle's Medium
  • Crystal Violet Staining - Cells were seeded in 12- wells plates at 75,000-150,000 cells / well in triplicates. Time 0 was determined as the time the cells adhered to the culture plate, which was about 10 hours following seeding. For each time point, cells were washed with PBS XI and fixed in 4 % PFA (in PBS). Following, cells were stained with 0.5 % Crystal Violet (Catalog number C0775, Sigma- Aldrich) for 20 minutes (1 ml per well) and washed with water. The cells were then incubated with 10 % acetic acid for 20 minutes with shaking. The extract was diluted 1 : 1 - 1 : 4 in water and absorbance was measured for each time point at 595 ran every 24 hours.
  • Immunohtstochemtstry Four micrometer paraffin embedded tissue sections were deparaffinized and rehydrated. Endogenous peroxidase was blocked with three percent H 2 0 2 in methanol. For ASL, ASS1 and ORNT1 (SLC25A15) staining, antigen retrieval was performed in citric acid (pH 6), for 10 minutes, using a low boiling program in the microwave to break protein cross-links and unmask antigens. Following, the sections were pre-incubated with 20 % normal horse serum and 0.2 % Triton X-100 for 1 hour at RT, biotin block via Avidin/Biotin Blocking Kit (SP-2001, Vector Laboratories, Ca, USA).
  • the blocked sections were incubated overnight at room temperature followed by 48 hours at 4 °C with the following primary antibodies: ASL (1 : 50, Abeam, ab97370, CA, USA); ASS1 (1 : 50, Abeam, abl 24465, CA, USA), ORNTl (1 : 200, ⁇ 2-20387, novus biologicals, CO, USA), OTC (1 : 3-1 : 200, HPA000570, Sigma-aldrich). All antibodies were diluted in PBS containing 2 % normal horse serum and 0.2 % Triton.
  • HFF human foreskin fibroblasts
  • Metabolomics analysis - HepG2 cell lines were seeded at 3 - 5 x 10 6 cells per 10 cm plate and incubated with 4 mM L-glutamine (a-lSN, 98 %, Cambridge Isotope Laboratories) for 24 hours. Subsequently, cells were washed with ice-cold saline, lysed with a mixture of 50 % methanol in water added with 2 ⁇ g / ml ribitol as an internal standard and quickly scraped followed by three freeze-thaw cycles in liquid nitrogen. Following, the sample was centrifuged in a 4 °C cooled centrifuge and the supernatant was collected for consequent GC-MS analysis.
  • the pellets were dried under air flow at 42 °C using a Techne Dry-Block Heater with sample concentrator (Bibby Scientific) and the dried samples were treated with 40 ⁇ of a methoxyamine hydrochloride solution (20 mg ml-1 in pyridine) for 90 minutes while shaking at 37 °C followed by incubation with 70 ⁇ ⁇ , ⁇ -bis (trimethylsilyl) trifluoroacetamide (Sigma) for additional 30 minutes at 37 °C.
  • a methoxyamine hydrochloride solution (20 mg ml-1 in pyridine) for 90 minutes while shaking at 37 °C followed by incubation with 70 ⁇ ⁇ , ⁇ -bis (trimethylsilyl) trifluoroacetamide (Sigma) for additional 30 minutes at 37 °C.
  • Isotopic labeling - Hepatocellular and ovarian carcinoma cells were seeded in 10 cm plates and once cell confluency reached 80 % cells were incubated with 4 mM L- GLUTAMINE, (ALPHA-15N, 98 %, Cambridge Isotope Laboratories, Inc.) for 24 hours.
  • GC-MS analysis - GC-MS analysis used a gas chromato graph (7820AN, Agilent Technologies) interfaced with a mass spectrometer (597S Agilent Technologies).
  • Helium carrier gas was maintained at a constant flow r°C via a ramp of 4 °C min -1 , 250-215 °C via a ramp of 9 °C min -1 , 215-300 °C via a ramp of 25 °C min -1 and maintained at 300 °C for additional 5 minutes.
  • the inlet and MS transfer line temperatures were maintained at 280 °C, and the ion source temperature was 250 °C.
  • Sample injection (1 - 3 ⁇ ) was in split less mode.
  • Nucleotide analysis - Materials Ammonium acetate (Fisher Scientific) and ammonium bicarbonate (Fluka) of LC-MS grade; Sodium salts of AMP, CMP, GMP, TMP and UMP (Sigma- Aldrich); Acetonitrile of LC grade (Merck); water with resistivity 18.2 ⁇ obtained using Direct 3-Q UV system (Millipore).
  • Extract preparation Samples were concentrated in speedvac to eliminate methanol, and then lyophilized to dryness, re-suspended in 200 ⁇ of water and purified on polymeric weak anion columns [Strata-XL-AW 100 ⁇ (30 mg ml -1 , Phenomenex)] as follows: each column was conditioned by passing 1 ml of methanol followed by 1 ml of formic acid/methanol/water (2/25/73) and equilibrated with 1 ml of water. The samples were loaded, and each column was washed with 1 ml of water and 1 ml of 50 % methanol.
  • the purified samples were eluted with l ml of ammonia/methanol/water (2/25/73) followed by 1 ml of ammonia/methanol/water (2/50/50) and then collected, concentrated in speedvac to remove methanol and lyophilized. Following, the obtained residues were re-dissolved in 100 ⁇ of water and centrifuged for 5 minutes at 21 ,000 g to remove insoluble material.
  • LC-MS analysis The LC-MS/MS instrument used for analysis of nucleoside monophosphates was an Acquity I-class UPLC system (Waters) and Xevo TQ-S triple quadrupole mass spectrometer (Waters) equipped with an electrospray ion source and operated in positive ion mode. MassLynx and TargetLynx software (version 4.1, Waters) were applied for data acquisition and analysis.
  • Chromatographic separation was done on a 100 mm x 2.1 mm internal diameter, 1.8 ⁇ UPLC HSS T3 column equipped with 50 mm x 2.1 mm internal diameter, 1.8 ⁇ UPLC HSS T3 pre-column (both Waters Acquity) with mobile phases A (10 mM ammonium acetate and 5 mM ammonium hydrocarbonate buffer, pH 7.0 adjusted with 10 % acetic acid) and B (acetonitrile) at a flow rate of 0.3 ml min -1 and column temperature 35 °C.
  • mobile phases A (10 mM ammonium acetate and 5 mM ammonium hydrocarbonate buffer, pH 7.0 adjusted with 10 % acetic acid
  • B acetonitrile
  • a gradient was used as follows: for 0-3 min the column was held at 0 % B, 3-3.5 min a linear increase to 100 % B, 3.5-4.0 min held at 100 % B, 4.0-4.5 min back to 0 % B and equilibration at 0 % B for 2.5 min. Samples kept at 8 °C were automatically injected in a volume of 3 ⁇ . For mass spectrometry, argon was used as the collision gas with a flow of 0.15 ml min -1 . The capillary voltage was set to 2.90 kV, source temperature 150 °C, desolvation temperature 350 °C, cone gas flow 150 1 hr -1 , desolvation gas flow 6501 hr -1 .
  • OTC - HEPG2 Cells were infected with pLKO-based lenti viral vector with or without the human OTC short hairpin RNA (shRNA) encoding one or two separate sequences combined (RHS4533-EG5009, GE Healthcare, Dharmacon). Transduced cells were selected with 4 ⁇ g ml -1 puromycin.
  • shRNA human OTC short hairpin RNA
  • Virus infection -_Primary fibroblasts were infected with HCMV and harvested at different time points following infection for ribosome footprints (deep sequencing of ribo some- protected mRNA fragments) as previously described (Tirosh et al., 2015). Briefly human foreskin fibroblasts (HFF) were infected with the Merlin HCMV strain and harvested cells at S, 12, 24 and 72 hours post infection. Cells were pre-treated with Cylcoheximide and ribosome protected fragments were then generated and sequenced. Bowtie vO.12.7 (allowing up to 2 mismatches) was used to perform the alignments. Reads with unique alignments were used to compute footprints densities in units of reads per kilobase per million (RPKM).
  • HFF human foreskin fibroblasts
  • Cancer cells were infected with pLKO-based lenti viral vector with or without the human OTC and SLC25A15, ASS1 short hairpin RNA (shRNA) (Dharmacon). Transduced cells were selected with 2-4 ug ml -1 puromycin.
  • shRNA short hairpin RNA
  • Transient transfection - LOX-IMVI melanoma cells were seeded in 6-well plates at 70,000cells/ well, or in 12-well plates at lOO.OOOcells/ plate. At the following day, cells were transfected with either 700 pmol or 350 pmol siRNA siGenome SMARTpool targeted to human SLC25A13 mRNA (#M-007472-01, Dharmacon), respectively. Hepatocellular and ovarian carcinoma cells were seeded in 6-well plate at 10 6 or 70,000 cells/ well respectively, transfected with 2-3 ⁇ g of the OTC (EXa3688-LV207 GENECOPOEIA) or ORNT1 (EXu0560-LV207 GENECOPOEIA) plasmids.
  • OTC EXa3688-LV207 GENECOPOEIA
  • ORNT1 EXu0560-LV207 GENECOPOEIA
  • Transfection was effected with Lipofectamine® 2000 Reagent (#11668027, ThennoFisher Scientific), in the presence of Opti-MEM® I Reduced Serum Medium (#11058021, ThennoFisher Scientific). Four hours following transfection, medium was replaced and the experiments were performed 48-108 hours post transfection.
  • Over expression - LOX-IMVI melanoma cells were transduced with pLEX307-based lenti-viral vector with or without the human SLC25A13 transcript, encoding for Citrin. Transduced cells were selected with 2 ⁇ g / ml Puromycin.
  • In-vivo experiments 8 weeks old Balb/c or C57BL mice were injected with 4T1 breast cancer cells (in the mammary fat fad) or with CT26 colon cancer cells (sub-cutaneous). 3 weeks following injection an advanced tumor was observed and palpated. Urine was collected from mice presenting adverse tumors. Pyrimidine pathway related metabolites were assessed by LC- MS at Baylor College of medicine. Control urine was obtained from Balb/c or C57BL mice similar in age which were not injected. Samples below 100 ⁇ were excluded from the analysis. All animal experiments were approved by the Weizmann Institute Animal Care and Use Committee Following US National Institute of Health, European Commission and the Israeli guidelines (IACUC 21131015-4).
  • mice Syngeneic mouse models - 8 weeks old CS7BL/6 male mice were injected sub-cutaneous in the right flank with MC38 mouse colon cancer cells infected with either an empty vector (EV) or with shASSl. For each injection, 5x10 s tumor cells were suspended in 200 ⁇ DMEM containing 5 % matrigel. Following injection, on days 8, 13, 17, 20, mice were treated with 250 ⁇ g of anti PD-1 antibody (Clones 29F.1A12, RPM114, Bio Cell) or PBS (control) as control.
  • PD-1 antibody Clones 29F.1A12, RPM114, Bio Cell
  • mice were euthanized and tumors were removed and incubated in 1 ml of PBS containing Ca2+, Mg2+ (Sigma D8662) with 2.5 mg / ml Collagenase D (Roche) and 1 mg / ml DNase I (Roche). Following 20 minutes incubation at 37 °c, the tumors were processed into a single cell suspension by mechanically grinding on top of wire mesh and repeated washing and filtering onto 70 ⁇ filter (Falcon). Single cell suspensions from tumors were stained for flow cytometry analysis with CD3-FITC (clone 17A2), CD4-PE (clone GK15) and CD8a-APC (clone 53-6.7) all from Biolegend.
  • CD3-FITC clone 17A2
  • CD4-PE clone GK15
  • CD8a-APC clone 53-6.7
  • the tumor volume was quantified by the formula, (/ and normalized by their volume on day 11 when the mean tumor volume reached around 100 mm 3 .
  • the response to anti-PDl therapy (and empty vector) was quantified by the tumor volume change at time where V t denotes the normalized tumor volume at a given time t, and VQ denotes the tumor volume on day 11.
  • the overall response of treated and control groups was compared by Wilcoxon ranksum test of AV, on day 21, and the sequential tumor growth was compared using ANOVA over the whole period (where the internal tumor volume was measured on day 9, 13,17, and 19).
  • Nonspecific binding was blocked by incubation with TBST [ 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1 % Tween 20] containing 5 % skim milk or BSA 3 % (Sigma catalog no: A7906) for 1 hour at room temperature.
  • Membranes were subsequently incubated with primary antibodies against: p97 (1 : 10,000, PA5-22257, Thermo Scientific), GAPDH (1 : 1000, 14C10, #2118, Cell Signaling), CAD (1 : 1000, ab40800, abeam), phospho-CAD (Serl859) (1 : 1000, #12662, Cell Signaling), ASL (1 : 1000, ab97370, Abeam ), MAP2K1 (1 : 10000, MFCD00239713, Sigma- Aldrich), OTC (1 : 1000, ab2038S9, Abeam).
  • the membranes were incubated with the secondary antibodies used were: using peroxidase-conjugated AffiniPure goat anti-rabbit IgG or goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) and detected by enhanced chemiluminescence western blotting detection reagents (EZ-Gel, Biological Industries).
  • the bands were quantified by Gel DocTM XR+ (BioRad) and analyzed by ImageLab 5.1 software (BioRad).
  • the lysates were cleared by centrifugation at 48,000g for 60 minutes at 4 °C, and then were passed through a pre- clearing column containing Protein-A Sepharose beads.
  • HLA- 1 molecules were immunoaffinity purified from cleared lysate with the pan-HLA-I antibody (W6/32 antibody purified from HB95 hybridoma cells) covalently bound to Protein-A Sepharose beads.
  • Affinity column was washed first with 10 column volumes of 400 mM NaCl, 20 mM Tris-HCl followed by 10 volumes of 20 mM Tris-HCl, pH 8.0.
  • HLA peptides and HLA molecules were then eluted with 1 % trifluoracetic acid followed by separation of the peptides from the proteins by binding the eluted fraction to disposable reversed-phase C18 columns (Harvard Apparatus). Elution of the peptides was effected with 30 % acetonitrile in 0.1 % trifluoracetic acid (Milner et al., 2013). The eluted peptides were cleaned using C18 stage tips as described previously (Rappsilber et al., 2003).
  • HLA peptides were dried by vacuum centrifugation, solubilized with 0.1 % formic acid, and resolved on capillary reversed phase chromatography on 0.075x300 mm laser-pulled capillaries, self-packed with CI 8 reversed-phase 3.5um beads (Reprosil-C 18-Aqua, Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) (Ishihama et al., 2002).
  • the peptide match option was set to Preferred. Normalized collision energy was set to 25 % and MS/MS resolution was 17,500 at 200 m/z. Fragmented m/z values were dynamically excluded from further selection for 20 seconds.
  • the MS data were analyzed using MaxQuant (Cox and Mann, 2008) version 1.5.3.8, with 5 % false discovery rate (FDR). Peptides were searched against the UniProt human database (July 2015) and customized reference databases that contained the mutated sequences identified in the sample by WES. N- terminal acetylation (42.010565 Da) and methionine oxidation (15.994915 Da) were set as variable modifications. Enzyme specificity was set as unspecific and peptides FDR was set to 0.05. The match between runs option was enabled to allow matching of identifications across the samples belonging the same patient.
  • HLA typing was determined from the WES data by POLYSOLVER version 1.0 (Shukla et al., 2015); and the HLA allele to which the identified peptides match to was determined using the NetMHCpan version 4.0 (Hoof et al., 2009; Nielsen and Andreatta, 2016).
  • the abundance of the peptides was quantified by the MS/MS intensity values, following normalization with the summed intensity of both UC -perturbed and control cell lines.
  • the hydrophobicity of a peptide was determined by the fraction of hydrophobic amino acid in the peptide, which we termed hydrophobic score.
  • the abundance of the peptides of top 20 % hydrophobic score vs bottom 20 % of hydrophobic score was compared using Wilcoxon ranksum test in UCD cell lines and control cell lines.
  • terminal group molecular weight (Da) the default 1.0078 and 17.0027 were chosen respectively for N-terminal and C -terminal attached chemical group, which accounts for the Hydrogen signal and -COOH group respectively.
  • the default mass tolerance (Da) of 1.0 in precursor ion and 0.2 in product ion parameters were used.
  • the reference protein sequence database from NCBI (Refseq release 82) was used to map the peptides to protein IDs. In identifying single amino acid polymorphisms (SAPs) all amino acids were allowed for.
  • the RAId DbS outputs were used to map the amino- acid change to non-synonymous mutations on genes, separately for R->Y and Y->R cases, reported in VCF files, using in-house python script.
  • fibroblasts from OTC deficient (OTCD) and ORNTl deficient (ORNTID) patients were studied. As shown in Figures 1B-C, these fibroblasts were significantly more proliferative (as evident by the crystal violet stain) and exhibited elevated levels of activated CAD protein as compared to fibroblasts from healthy controls. On the contrary, fibroblasts from CPS1 deficient patients proliferated to the same extent and exhibited the same levels of activated CAD protein as fibroblasts from healthy controls (data not shown).
  • Metabolic redirection from the UC towards CAD is expected from down-regulation of ASS1, ASL, OTC, or SLC25A15 (ORNT1), or from up-regulation of CPS1 or SLC25A13 (citrin).
  • UCD Metabolic redirection from the UC towards CAD
  • UC dysregulation and the consequent flux of nitrogen towards CAD can be achieved through specific alterations in expression of different enzymes in the cycle ( Figure 1A).
  • Figure 1A To quantify the total extent of expression deregulation in the above described 6 UC enzymes [i.e. ASS1, ASL, OTC, SLC25A15 (ORNT1), CPS1, SLC25A13 (citrin)] a UCD-score was computed.
  • the UCD-score takes the aggregate expression of the 6 enzymes in the direction that supports metabolic redirection toward CAD.
  • UCD in cancer is a result of coordinated alterations in UC enzyme activities, where CPS1 and SLC25A13 tend to be up-regulated, while ASL, ASS1, OTC and SLC2SA1S tend to be down-regulated to increase substrate supply to CAD and enhance pyrimidine synthesis (see Figure 4A); and most importantly UCD correlates with cancer prognosis and patient's survival.
  • Table 1 Fraction of the samples of UC dysregulated and PTMB in different cancer types.
  • the data shows deregulation of UC enzyme(s) in cancer resulting in increased CAD activity that leads to increased pyrimidine levels.
  • perturbed UC enzyme activity increased pyrimidine levels and significantly altered the ratio between purines and pyrimidines ( Figures 6A and 7A).
  • a cellular increase in the ratio of pyrimidine to purine metabolites was also found in the other UCD induced cancer cells generated ( Figure 2F and 8).
  • UCD-elicited pyrimidine-rich transversion mutational bias could result in the presentation of neo- antigens in tumor cells. Due to the outstanding relevance of this phenomenon for immunotherapy (Topalian et al., 2016), UCD and PTMB effects on the efficacy of immune checkpoint therapy (ICT) was evaluated. To this end, the trans criptomics of published data of melanoma patients treated with ICT (Van Allen et al., 201S),(Hugo et al., 2016) was analyzed and the UCD scores of the tumors were computed (where the gene expression of the 6 UC genes were available).
  • ICT immune checkpoint therapy
  • the data reveals an oncogenic metabolic rewiring that maximizes the use of nitrogen by cancer cells and has diagnostic and prognostic values.
  • UCD was shown to be a common event in cancer which enhances nitrogen anabolism to pyrimidines by supplementing CAD with the three substrates needed for its function, supporting cell proliferation and mutagenesis, and correlating with survival risk.
  • the data reveals the hitherto unknown direct link between metabolic alterations in cancer, changes in nitrogen composition in biofluids and a genome-wide shift in mutational bias toward pyrimidines, generating metabolic and mutational signatures which encompass a persistent disruption in purine to pyrimidine nucleotide balance.
  • the pyrimidine-rich transversion mutational bias propagates from the DNA to RNA and protein levels, leading to the generation of peptides with increased predicted immunogenicity, enhancing the response to immune-modulation therapy independently of mutational load both in mouse models and in patient correlative studies (Figure lOF).

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Abstract

L'invention concerne des méthodes de traitement du cancer. L'invention concerne ainsi une méthode de traitement du cancer chez un sujet en ayant besoin, la méthode consistant à déterminer un passage du cycle d'urée à la synthèse de pyrimidine dans une cellule cancéreuse du sujet par comparaison avec un échantillon témoin; et à traiter le sujet avec un agent de modulation immunitaire lorsqu'est indiqué un tel passage. L'invention concerne également des méthodes de diagnostic du cancer et de traitement du cancer en fonction du diagnostic.
PCT/IL2018/050289 2017-03-12 2018-03-12 Méthodes de diagnostic et de traitement du cancer WO2018167780A1 (fr)

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WO2021260675A1 (fr) 2020-06-24 2021-12-30 Yeda Research And Development Co. Ltd. Agents pour sensibiliser des tumeurs solides à un traitement
WO2023214405A1 (fr) * 2022-05-01 2023-11-09 Yeda Research And Development Co. Ltd. Réexpression de hnf4a pour atténuer la cachexie associée au cancer

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