Use of RNAI Inhibiting PARP Activity for the Manufacture of a Medicament for the Treatment of Cancer

Abstract

The present invention relates to the use of an agent that inhibits the activity of an enzyme that mediates repair of a DNA strand break in the manufacture of a medicament for the treatment of diseases caused by a defect in a gene that mediates homologous recombination.

Claims

1-32. (canceled)

33. A method of treatment of cancer cells defective in homologous recombination (HR) in a human patient, the method comprising; administering to the patient a therapeutically effective amount of a compound which inhibits PARP-1.

34. The method of claim 33 wherein the PARP inhibitor is selected from the group consisting of benzimidazole-carboxamides, quinazolin-4-[3H]-ones and isoquinolone derivatives.

35. The method of claim 34 wherein the PARP inhibitor is selected from the group consisting of 2-(4-hydroxyphenyl)benzimidazole-4-carboxamide, 8-hydroxy-2-methylquinazolin-4-[3H]one, 6(5H)phenanthridinone, 3-aminobenzamide, benzimidazole-4-carboxamides and tricyclic lactam indoles.

36. The method of claim 33 wherein the cancer cells have defect in a gene encoding a protein involved in HR.

37. The method of claim 36 wherein the human patient has one functional allele of said gene, said functional allele being lost in the cancer cells.

38. The method of claim 33 wherein the gene encoding a protein involved in HR is selected from the group consisting of XRCC1, CTPS, RPA, RPA1, RPA2, RPA3, XPD, ERCC1, XPF, MMS19, RAD51, RAD51B, RAD51C, RAD51D, DMC1, XRCC2, XRCC3, BRCA1, BRCA2, RAD52, RAD54, RAD50, MRE11, NBS1, WRN, BLM, Ku70, Ku80, ATM, ATR, chkl, chk2, FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, RAD1, RAD9, FEN-1, Mus81, Emel, DDS1 and BARD.

39. The method of claim 33 wherein the cancer cells are selected from the group consisting of lung, colon, pancreatic, gastric, ovarian, cervical, breast and prostate cancer.

40. The method of claim 33 wherein the cancer cells are selected from the group consisting of renal, liver, and bladder cancer.

41. The method of claim 33 wherein the cancer is gene-linked hereditary cancer.

42. The method of claim 41 wherein the cancer is breast cancer.

43. The method of claim 33 wherein the cancer cells to be treated are defective in BRCA1 expression.

44. The method of claim 33 wherein the cancer cells to be treated are defective in BRCA2 expression.

45. The method of claim 33 comprising determining the inhibition of PARP-1 in said individual following said administration.

46. The method of claim 33 comprising identifying a patient with a familial predisposition to said cancer and administering said compound to said patient.

47. The method of claim 33 wherein the daily dosage of said compound is sufficient to induce apoptosis in the cancer cells without affecting normally dividing cells in the human patient.

48. The method of claim 33 wherein the daily dosage of said compound is up to 20 mg/Kg body weight.

49. The method of claim 48 wherein the daily dosage of said compound is more than 2 mg/Kg body weight.

50. The method of claim 33 wherein the compound which inhibits PARP-1 is not administered in combination with radio- or chemo-therapy.

51. The method of claim 33 wherein the compound which inhibits PARP-1 is administered in a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent.

Description

EXAMPLES

[0082] Homologous Recombination Deficient Cells are Hypersensitive to PARP-1 Inhibition

[0083] To investigate the involvement of HR in cellular responses to inhibition of PARP-1, the effects of PARP-1 inhibitors on the survival of HR repair deficient cell lines were studied. It was found that cells deficient in HR (i.e., irs1SF which is defective in XRCC3 or irs1 which is defective in XRCC2 [see Table 1] were very sensitive to the toxic effect of 3-aminobenzamide (3-AB) and to two more potent inhibitors of PARP-1: 1,5-dihydroxyisoquinoline (ISQ; [37]) or 8-hydroxy-2-methylquinazolinone (NU1025 [38, 39]) (FIG. 1). The sensitivity in irs1SF cells to 3-AB, ISQ or NU1025 was corrected by the introduction of a cosmid containing a functional XRCC3 gene (CXR3). Similarly, the sensitivity in irs1 cells to 3-AB, ISQ or NU1025 was corrected by the introduction of a cosmid containing a functional XRCC2 gene (irs1X2.2).

[0084] BRCA2 Deficient Cells are Hypersensitive to PARP-1 Inhibition

[0085] The survival of BRCA2 deficient cells (VC8) and wild type cells (V79Z) in the presence of inhibitors of PARP-1 was investigated. It was found that VC8 cells are very sensitive to the toxic effect of NU1025 (FIG. 2). The sensitivity in VC8 cells was corrected by the introduction of a functional BRCA2 gene either on chromosome 13 (VC8#13) or on an overexpression vector (VC8+B2). This result demonstrates that the sensitivity to PARP-1 inhibitors is a direct consequence of loss of the BRCA2 function.

[0086] To investigate if inhibition of PARP-1 triggers apoptosis in BRCA2 deficient cells, the level of apoptosis 72 hours following exposure to NU1025 was investigated. It was found that NU1025 triggered apoptosis only in VC8 cells, showing that loss of PARP-1 activity in BRCA2 deficient cells triggers this means of death (FIG. 3).

[0087] BRCA2 Deficient Breast Cancer Cells are Hypersensitive to PARP-1 Inhibition

[0088] It was examined whether the MCF7 (wild-type p53) and MDA-MB-231 (mutated p53) breast cancer cell lines displayed a similar sensitivity to NU1025 upon depletion of BRCA2. It was found that PARP inhibitors profoundly reduced the survival of MCF7 and MDA-MB-231 cells only when BRCA2 was depleted with a mixture of BRCA2 siRNA (FIG. 4). This shows that BRCA2 depleted breast cancer cells are sensitive to PARP inhibitors regardless of p53 status.

[0089] BRCA2 Deficient Cells Die from PARP-1 Inhibition in Absence of DNA Double-Strand Breaks (DSBs) but in Presence of γH2Ax

[0090] HR is known to be involved in the repair of DSBs and other lesions that occur during DNA replication [2]. To determine whether the sensitivity of BRCA2 deficient cells is the result of an inability to repair DSBs following NU1025 treatment, the accumulation of DSBs in V79 and V-C8 cells was measured following treatments with highly toxic levels of NU1025. It was found that no DSBs were detectable by pulsed field gel electrophoretic analysis of DNA obtained from the treated cells (FIG. 5A), suggesting that low levels of DSBs or other recombinogenic substrates accumulated following PARP inhibition in HR deficient cells, which trigger .gamma.H2Ax FIG. 5B). The reason why BRCA2 deficient cells die following induction of these recombinogenic lesions is likely to be due to an inability to repair such lesions. To test this, the ability of BRCA2 deficient V-C8 cells and BRCA2 complimented cells to form RAD51 foci in response to NU1025 was determined. It was found that RAD51 foci were indeed induced in V-C8+B2 cells following treatment with NU1025 (statistically significant in t-test p<0.05; FIG. 5D). This indicates that the recombinogenic lesions trigger HR repair in these cells allowing them to survive. In contrast, the BRCA2 deficient V-C8 cells were unable to form RAD51 foci in response to NU1025 treatment (FIG. 5D) indicating no BR, which would leave the recombinogenic lesions unrepaired and thus cause cell death.

[0091] PARP-1 and not PARP-2 is Important in Preventing Formation of a Recombinogenic Lesion

[0092] There are two major PARPs present in the nucleus in mammalian cells, PARP-1 and PARP-2 and all reported PARP inhibitors inhibit both. In order to distinguish which PARP was responsible for the effect, we tested if the absence of PARP-1 and/or PARP-2 results in accumulation of toxic lesions, by depleting these and BRCA2 with siRNA in human cells (FIG. 6a). We found that the clonogenic survival was significantly reduced when both PARP-1 and BRCA2 proteins were co-depleted from human cells (FIG. 6b). Depletion of PARP-2 with BRCA2 had no effect on the clonogenic survival and depletion of PARP-2 in PARP-1 and BRCA2 depleted cells did not result in additional toxicity. These results suggest that PARP-1 and not PARP-2 is responsible for reducing toxic recombinogenic lesions in human cells. The cloning efficiency was only reduced to 60% of control in PARP-1 and BRCA2 co-depleted cells, while no HR deficient cells survived treatments with PARP inhibitors. This is likely to do with incomplete depletion of the abundant PARP-1 protein by siRNA (FIG. 6c), which might be sufficient to maintain PARP-1 function in some of the cells.

[0093] PARP-1 is Activated by Replication Inhibitors

[0094] HR is also involved in repair of lesions occurring at stalled replication forks, which may not involve detectable DSBs [2]. To test if PARP has a role at replication forks, PARP activation in cells treated cells with agents (thymidine or hydroxyurea) that retard or arrest the progression of DNA replication forks was examined. Thymidine depletes cells of dCTP and slows replication forks without causing DSBs. Hydroxyurea depletes several dNTP and block the replication fork, which is associated with the formation of DSBs at replication forks [2]. Both of these agents potently induce HR [2]. V79 hamster cells treated for 24 hours with thymidine or hydroxyurea were stained for PAR polymers. This revealed a substantial increase in the number of cells containing sites of PARP activity (FIG. 7C). This result suggests a function for PARP at stalled replication forks. It was also shown that inhibition of PARP with NU1025 enhances the sensitivity to thymidine or hydroxyurea in V-C8+B2 cells (FIG. 7D,E). This result suggests that PARP activity is important in repair of stalled replication forks or alternatively that it prevents the induction of death in cells with stalled replication forks.

[0095] PARP is rapidly activated at DNA single-strand breaks (SSB) and attracts DNA repair enzymes [3-6]. Methylmethane sulphonate (MMS) causes alkylation of DNA, which is repaired by base excision repair. PARP is rapidly activated by the SSB-intermediate formed during this repair, which depletes the NAD(P)H levels (FIG. 7F). We found that the activation of PARP and reduction of NAD(P)H levels is much slower following thymidine or hydroxyurea treatments. This slow PARP activation can be explained by the indirect action of thymidine and hydroxyurea and the time required to accumulate stalled replication forks as cells enter the S phase of the cell cycle.

[0096] PARP-1 and HR have Separate Roles at Stalled Replication Forks

[0097] The number sites of PARP activity in untreated BRCA2 deficient V-C8 cells was determined. It was found that more V-C8 cells contain sites of PARP activity compared to V-C8+B2 cells (FIG. 8A,B,C). Also, the V-C8 cells have lower free NAD(P)H levels than the corrected cells (FIG. 8D), as a likely result of the increased PARP activity. Importantly these sites of PARP activity do not overlap with RAD51 foci (FIG. 8E).

[0098] The results herein suggest that PARP and HR have separate roles in the protection or rescue of stalled replication forks (FIG. 8F). A loss of PARP activity can be compensated by increased HR while a loss of HR can be compensated by increased PARP activity. However, loss of both these pathways leads to accumulation of stalled replication forks and to death, as in the case of PARP inhibited BRCA2 deficient cells.

[0099] As shown in the model outlined in FIG. 8F PARP and HR have complementary roles at stalled replication forks. (i) Replication forks may stall when encountering a roadblock on the DNA template. In addition, they may also stall temporarily, due to lack of dNTPs or other replication co-factors. (ii) PARP binds stalled replication forks or other replication-associated damage, triggering PAR polymerization. Resulting negatively charged PAR polymers may protect stalled replication forks, by repelling proteins that normally would process replication forks (e.g., resolvases), until the replication fork can be restored spontaneously when dNTPs or other co-factors become available. Alternatively, PAR polymers or PARP may attract proteins to resolve the replication block by other means. (iii) In absence of PARP activity, HR may be used as an alternative pathway to repair stalled replication forks. This compensatory model explains the increased level of HR and RAD51 foci found in PARP deficient cells.sup.3-5 and higher PARP activity found in HR deficient cells (i.e. V-C8). Spontaneous replication blocks/lesions are only lethal in the absence of both PARP and HR.

TABLE-US-00001 TABLE 1 Genotype and origin of cell lines used in this study. Cell line Genotype Defect Origin Reference AA8 Wt Wt CHO [41] irs1SF XRCC3.sup.− XRCC3.sup.−, deficient in HR AA8 [41] CXR3 XRCC3.sup.− + Wt irs1SF [41] hXRCC3 V79-4 Wt Wt V79 [40] irs1 XRCC2.sup.− XRCC2.sup.−, deficient in HR V79-4 [40] irs1X2.2 XRCC2.sup.− + Wt irs1 [40] hXRCC2 V79-Z Wt Wt V79 [42] VC8 BRCA2.sup.− BRCA2.sup.−, deficient in HR V79-Z [42] VC8#13 BRCA2.sup.− + Wt VC8 [42] hBRCA2 VC8 + B2 BRCA2.sup.− + Wt VC8 [42] hBRCA2

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