METHODS FOR THE TREATMENT OF HRD CANCER AND BRCA-ASSOCIATED CANCER

Abstract

The invention relates to methods and pharmaceutical compositions for the treatment of cancer, particularly BRCA-associated cancer and chemo-resistance BRCA-associated cancer. The inventors investigated the role of NMNAT1 in cancer, particularly in BRCA-associated cancer. The inventors demonstrated that NMNAT1 inhibition kills BRCA1 and BRCA2-mutated tumor cells but does not affect the survival of non-BRCA-mutated cells. The inventors also demonstrated that NMNAT1, a nuclear enzyme other than PARP1, is crucial for the survival of HRD cells and indicate that NMNAT1 is a key factor which activities are necessary for the survival of HRD cells. The inventors also demonstrated that inhibition of NMNAT1 kills PARP-inhibitor and cisplatin-resistant BRCA1 and BRCA2-mutated tumors and show that targeting NMNAT1 kills chemo-resistance HRD cells, particularly PARPi-resistant HRD cells. Altogether, the present invention highlights the role of NMNAT1 inhibitors in cancer and the use of NMNAT1 inhibitors in the treatment of cancer, particularly BRCA-associated cancer including BRCA-associated cancer with acquired drug resistance in mono- or combination therapy with PARPi. In the present invention, the inventors provide in vitro evidences towards a direct role of NMNAT1 in BRCA-associated cancer. Thus, the present invention relates to NMNAT1 inhibitor for use in the treatment of cancer, particularly HRD cancer. BRCA-associated cancer and chemo-resistance BRCA-associated cancer.

Claims

1.-11. (canceled)

12. A method for treating HRD cancer and BRCA-associated cancer comprising administering to a subject in need thereof, a therapeutically effective amount of a NMNAT1 inhibitor.

13. The method according to claim 12, wherein the cancer is chemo-resistance BRCA-associated cancer.

14. The method according to claim 12, wherein the cancer is PARPi resistant BRCA-associated cancer or cisplatin resistant BRCA-associated cancer, including those with somatic reversion of the BRCA mutation and HR restoration.

15. The method according to claim 12, wherein said NMNAT1 inhibitor is a small organic molecule, a polypeptide, an aptamer, an oligonucleotide or an antibody.

16. The method according to claim 12, wherein said NMNAT1 inhibitor is an antisense oligonucleotide, a siRNA, a shRNA, a DNA aptamer or a RNA aptamer.

17. The method according to claim 12, wherein said NMNAT1 inhibitor is administered in combination with a PARP inhibitor, optionally said PARP inhibitor is selected from the group consisting of olaparib, rucaparib, niraparib, talazoparib, iniparib, veliparib, Pamiparib (BGB-290), CEP 9722, E7016, E7449 and 3-Aminobenzamide.

18. The method according to claim 12, wherein said NMNAT1 inhibitor is administered in combination with cisplatin or a Pole inhibitor such as novobiocin.

19. The method according to claim 13, wherein said NMNAT1 inhibitor is a small organic molecule, a polypeptide, an aptamer, an oligonucleotide or an antibody.

20. The method according to claim 13, wherein said NMNAT1 inhibitor is an antisense oligonucleotide, a siRNA, a shRNA, a DNA aptamer or a RNA aptamer.

21. The method according to claim 13, wherein said NMNAT1 inhibitor is administered in combination with a PARP inhibitor, optionally said PARP inhibitor is selected from the group consisting of olaparib, rucaparib, niraparib, talazoparib, iniparib, veliparib, Pamiparib (BGB-290), CEP 9722, E7016, E7449 and 3-Aminobenzamide.

22. The method according to claim 13, wherein said NMNAT1 inhibitor is administered in combination with cisplatin or a Pole inhibitor such as novobiocin.

23. The method according to claim 14, wherein said NMNAT1 inhibitor is a small organic molecule, a polypeptide, an aptamer, an oligonucleotide or an antibody.

24. The method according to claim 14, wherein said NMNAT1 inhibitor is an antisense oligonucleotide, a siRNA, a shRNA, a DNA aptamer or a RNA aptamer.

25. The method according to claim 14, wherein said NMNAT1 inhibitor is administered in combination with a PARP inhibitor, optionally said PARP inhibitor is selected from the group consisting of olaparib, rucaparib, niraparib, talazoparib, iniparib, veliparib, Pamiparib (BGB-290), CEP 9722, E7016, E7449 and 3-Aminobenzamide.

26. The method according to claim 14, wherein said NMNAT1 inhibitor is administered in combination with cisplatin or a Pole inhibitor such as novobiocin.

27. A method for treating HRD cancer and BRCA-associated cancer comprising administering to a subject in need thereof, a pharmaceutical composition comprising a NMNAT1 inhibitor and a PARP inhibitor.

28. The method according to claim 27, wherein said NMNAT1 inhibitor is a small organic molecule, a polypeptide, an aptamer, an oligonucleotide or an antibody.

29. The method according to claim 27, wherein the PARP inhibitor selected from the group consisting of olaparib, rucaparib, niraparib, talazoparib, iniparib, veliparib, Pamiparib (BGB-290), CEP 9722, E7016, E7449 and 3-Aminobenzamide.

30. A method for treating HRD cancer and BRCA-associated cancer comprising administering to a subject in need thereof, a pharmaceutical composition comprising a NMNAT1 inhibitor and cisplatin or a Pole inhibitor such as novobiocin.

Description

FIGURES

[0095] FIG. 1: a, Quantification (left) and representative images (right) of clonogenic formation of wild type (WT) and BRCA2.sup./ (clones C1 and C2) RPE-1 cells with and without BRCA2 cDNA complementation, after transfection with the indicated NMNAT1 siRNAs. b, Clonogenic formation (left) and WB-analysis (right) of BRCA2 mAID HeLa cells in the presence or absence of auxin (IAA) after transduction with viral particles carrying NMNAT1 shRNA or control. c, Clonogenic formation of WT and BRCA1.sup./ RPE-1 cells after transfection with siNMNAT1 #2. d, Clonogenic formation of WT and NMNAT1.sup./ RPE-1 cells after transfection with the indicated BRCA1/2 siRNAs. e, Quantification (left) and representative images (right) of clonogenic formation of the indicated HR-proficient and HR-deficient cells upon siNMNAT1. f, DNA damage quantification by alkaline COMET assay of P53/ RPE-1 cells in the presence or absence of NMNAT1. g, Survival assay of WT and NMNAT1.sup./ RPE-1 cells exposed to the indicated doses of the PARP inhibitor rucaparib (PARPi).

[0096] FIG. 2: a,b Proliferation curve of WT and NMNAT1.sup./ cells in P53.sup./ (a) and P53++ (b) RPE-1. c, DNA damage quantification by alkaline COMET assay of the same cells as in a.b.

[0097] FIG. 3: a, WB analysis showing PARP1 activation in WT and NMNAT1.sup./ RPE-1 cells (G9) complemented with wild type (WT), catalytically dead (MUT, W169A) NMNAT1 cDNA or empty vector (EV) following exposure to PARG inhibitor and MMS. b, DNA damage quantification by alkaline COMET assay of WT and NMNAT1.sup./ (clone G9) RPE-1 cells complemented or not (EV) with wild type (WT) or catalytically dead (MUT, W169A) NMNAT1 cDNA. c,d, Clonogenic formation of cells as in b in response to PARPi (c) or following transfection with cither BRCA2 or control (siCTRL) siRNAs (d left panel). Representative images are shown in d right panel.

[0098] FIG. 4: a, Schematic of the drug-resistant cell lines used in this work. b, Clonogenic formation of wild type (WT), BRCA2.sup./ (clones C1 and C2) and BRCA2.sup./- derived PARPi-resistant RPE-1 cells after control or NMNAT1 (siNMNAT1 #2) siRNAs transfection. c, Clonogenic formation of parental and derived PARPi-resistant CAPAN-1 cells after transduction with shNMNAT1 lentiviral particles. Representative images are shown in the right panel. d, Clonogenic formation of PEO-1 and PEO-4 after transduction with shNMNAT1 lentiviral particles. Lentiviral particles carrying scramble shRNA (shSCR) were used as control for all the experiments shown in c,d.

[0099] FIG. 5: NMNAT1 inhibition kills HRD tumors in vivo. A, Growth (relative tumor volume, RTV) of indicated OVCAR8 xenografts in vivo. The OVCAR8 is a BRCA1 deficient (hypermethylated promoter) human ovarian cell line, xenotransplanted subcutaneously in athymic nude mice. Doxycycline was supplemented in the food (625 p.p.m.). B, Growth (relative tumor volume, RTV) of indicated KURAMOCHI xenografts in vivo. The KURAMOCHI is a BRCA2 deficient (homozygous deletion) human ovarian cell line, xenotransplanted intraperitonealy in NSG mice. Doxycycline was given IP to the mice (indicated by arrow), at the concentration of 10 mg/Kg.

[0100] FIG. 6: NMNAT1 inhibition kills PARPi-resistant HRD tumors in vivo. A. Growth (relative tumor volume, RTV) of parental and PARPi-resistant OVCAR8 xenografts in vivo upon PARPi (rucaparib) or vehicle treatment. B, Growth (relative tumor volume, RTV) of PARPi-resistant OVCAR8 xenografts in vivo. C, Relative tumour volumes (RTV) for individual mice treated in B after five weeks of treatment. D, Overall survival for mice bearing PARPi-resistant OVCAR8 xenografts treated with vehicle or doxycycline. For data shown in FIG. 6, the PARPi-resistant OVCAR8 cells were xenotransplanted subcutaneously in athymic nude mice. Doxycycline was supplemented in the food (625 p.p.m.).

EXAMPLE

Results

NMNAT1 Inhibition Kills BRCA1 and BRCA2-Mutated Tumor Cells but does not Affect the Survival of Non-BRCA-Mutated Cells

[0101] To study the role of NMNAT1-produced nuclear NAD+ in homologous recombination-deficient (HRD) cells, we generated several BRCA1/2 isogenic cell systems, namely BRCA1.sup./ and BRCA2.sup./ knockout clones in P53.sup./ RPE-1 cells and BRCA2 mini auxin-inducible degron (mAID) in Hela cells. In P53.sup./ RPE-1 cells, NMNAT1 knockdown by two different short interphering RNA sequences (siRNA) impaired the clonogenic ability of BRCA2.sup./ clones, while having no effect on the survival of parental HRP cells (FIG. 1a). Complementation of BRCA2.sup./ cells with a full length BRCA2 cDNA (C2+BRCA2) rescued the survival of BRCA2.sup./ cells upon NMNAT1 depletion (FIG. 1a). Likewise, in the mAID BRCA2 HeLa isogenic model NMNAT1 knockdown by short hairpin RNA (shRNA) reduced cell growth only in the presence of auxin, i.e. upon BRCA2 protein degradation (FIG. 1b). Furthermore, NMNAT1 silencing also impaired the clonogenic ability of BRCA1.sup./ P53.sup./ RPE-1 cells, while sparing parental HRP cells (FIG. 1c). These data suggest that NMNAT1 is synthetically lethal with BRCA1/2.

[0102] To corroborate these findings, we used a complementary approach by silencing BRCA1/2 in NMNAT1.sup./ RPE-1 cells that were generated by CRISP-Cas9 genome editing. While having only a mild effect on P53.sup./ RPE-1 cells, knockdown of BRCA1/2 by siRNA completely impaired the clonogenic ability of NMNAT1.sup./ P53.sup./ RPE-1 cells (FIG. 1d).

[0103] Next, we compared siNMNAT1 cytotoxicity between HRP and HR-deficient (HRD) cancer cells. siNMNAT1 had a strong impact on the clonogenic ability of HRD cells, whereas it induced only mild effects in HRP cells, including the immortalized normal breast epithelial MCF-10A cells (FIG. 1e). Altogether, we collected data showing that NMNAT1 is synthetically lethal with BRCA1/2.

[0104] Considering that HRD cells have higher level of replication stress than HRP cells, we reasoned that NMNAT1 might play a key role in maintenance of genome stability, which would explain why its loss is detrimental only in BRCA-deficient cells. To test this, we quantified DNA breaks in RPE-1 cells with and without NMNAT1 by alkaline COMET assay. P53.sup./ NMNAT1.sup./ clones showed higher amount of DNA lesions if compared with parental cells (FIG. 1f), indicating that NMNAT1 contributes to maintain genome stability.

[0105] Accumulation of DNA lesions can result in increased sensitivity to genotoxic drugs. Given that NMNAT1 functions upstream of PARP1 by providing NAD.sup.+, the reduced PARP1 activity upon NMNAT1 loss might account for the observed synthetic lethality between NMNAT1 and BRCA. Interestingly, NMNAT1.sup./ cells were sensitized to the PARP inhibitor rucaparib (PARPi) (FIG. 1g), suggesting that the role of NMNAT1 is not completely epistatic with PARP1, but rather nuclear NAD.sup.+ could be used by other downstream enzymes to sustain HRD cell survival.

NMNAT1 Loss is not Detrimental in Normal Immortalized P53+/+ Cells

[0106] For the successful translation of synthetic lethal interactions into cancer treatments, one important consideration is the toxic impact of the inhibition of the gene under investigation in normal tissue. To rule out any possible detrimental effect of NMNAT1 loss in HRP cells, we monitored the proliferation rate of P53.sup./ NMNAT1.sup./ RPE-1 and parental cells for one week. All the three NMNAT1.sup./ clones showed a mild reduction in terms of proliferation when compared to parental P53.sup./ RPE-1 cells (FIG. 2a). To better evaluate the effect of NMNAT1 loss in normal tissues, we generated NMNAT1 knockout clones in P53.sup.+/+ RPE-1, as normal cells are P53-proficient. Contrarily, when P53.sup.+/+ NMNAT1.sup./ RPE-1 clones were compared to parental cells, we observed that NMNAT1 loss did not have any impact in terms of cell proliferation, as shown by the similar growth of P53.sup.+/+ NMNAT1-clones (C12, C14, C17 C19 and C22) and parental P53.sup.+/+ cells (FIG. 2b). These data indicate that whereas NMNAT1 loss slightly reduces the proliferation of P53.sup./ cells, it does not have any detrimental effect in P53.sup.+/+ cells, thus suggesting that targeting NMNAT1 could have no toxic impact in normal tissue.

[0107] The observed accumulation of DNA lesions in NMNAT1.sup./ P53.sup./ cells (FIG. 1g) might account for the reduced proliferation upon NMNAT1 loss in P53.sup./ RPE-1 cells (FIG. 2a). However, when we measured DNA breaks by alkaline COMET assay in NMNAT1.sup./ P53.sup.+/+ cells, the increase of DNA breaks was less considerable than in NMNAT1.sup./ P53.sup./ RPE-1 if compared to the respective parental cells (FIG. 2c). These results are consistent with the observation that NMNAT1 loss had no impact on the proliferation of NMNAT1.sup./ P53.sup.+/+ cells and thus does not have any detrimental effect on normal cells.

[0108] To evaluate whether the catalytic activity of NMNAT1, i.e. nuclear NAD.sup.+, is essential for the survival of HRD cells, we complemented NMNAT1.sup./ RPE-1 cells with either wild type (WT) or a catalytically-dead version of the enzyme (W169A, MUT) and tested cell survival upon BRCA2 knockdown. While complementation with WT NMNAT1 increased PARP1 activation, and thus was able to rescue the amount of DNA lesions in NMNAT1.sup./ cells to the same extent than in parental cells, the W169A mutant did not (FIG. 3a,b). In accordance with these results, both PARPi sensitivity and synthetic lethality with BRCA2 were rescued by WT NMNAT1 complementation, but not by W169A NMNAT1 (FIG. 3c,d). Altogether, these datas demonstrate that NMNAT1, a nuclear enzyme other than PARP1, is crucial for the survival of HRD cells. These results indicate that NMNAT1 is a key factor which activities are necessary for the survival of HRD cells.

Inhibition of NMNAT1 Kills PARP-Inhibitor and Cisplatin-Resistant BRCA1/2-Mutated Tumors, Including Those with Somatic Reversion of the BRCA1/2 Mutations

[0109] Despite the striking cytotoxic effect of PARPi in BRCA-mutated cells, insurgence of resistance is ubiquitous in clinic and calls for the design of alternative therapies for the treatment of advanced diseases. Our findings that inhibition of NMNAT1 kills HRD cells in a PARP1-independent manner suggest that targeting this axis might also tackle BRCA-mutated cells that developed resistance to PARPi. To test this, the inventors used several cellular models, which recapitulated the major known mechanisms of resistance, including fork stabilization and HR restoration (FIG. 4a).

[0110] First, the inventors generated resistant cells by continuous exposure of BRCA2.sup./ RPE-1 to rucaparib. The inventors did not observe HR restoration in any of the derived clones, but resistance arose through fork stabilization. In those clones, NMNAT1 knockdown by siRNA impaired the clonogenic ability to a similar extent than that of the drug-nave BRCA2.sup./ cells, while sparing the HRP RPE-1 (FIG. 4b).

[0111] Then using a similar approach, several clones were derived from the BRCA2-mutated pancreatic cancer cell line CAPAN-1. After becoming resistant to rucaparib, those clones did not restore HR but rather developed resistance through other mechanisms that need to be further investigated. Knockdown of NMNAT1 impaired the clonogenic ability of all the CAPAN-1-derived resistant clones (FIG. 4c). Together, these data show that targeting NMNAT1 also kills PARPi-resistant HRD cells.

[0112] Nonetheless, HR restoration by secondary mutations in the BRCA genes is the only mechanism of resistance to PARPi validated so far in clinic. For this reason, the inventors evaluated the effect of NMNAT1 inhibition in HRD cells that developed chemo-resistance through BRCA2 secondary mutations restoring the open reading frame of the gene and thus HR. In particular, the inventors tested the chemo-resistant HR-restored ovarian cancer cell line PEO4together with its BRCA2-mutated paired parental PEO1 cells- and five clones derived from prolonged in vitro cisplatin exposure of CAPAN-1 cells, each bearing different secondary mutation in BRCA2 gene. Surprisingly, shRNA-mediated knockdown of NMNAT1 impaired the colony formation ability of both PEO4 cells (FIG. 4d) and resistant CAPAN-1 clones, suggesting that targeting NMNAT1 kills chemo-resistant cells regardless the mechanism of drug resistance.

NMNAT1 Inhibition Kills Homologous Recombination (HR)-Deficient (HRD) Tumor Cells In Vivo

[0113] The inventors performed in vivo xenotransplantation studies in mice, using HRD human ovarian tumor lines expressing either doxycycline-inducible NMNAT1 or scrambled (Scr) shRNA. NMNAT1 depletion significantly impaired BRCA1-deficient (OVCAR8) xenograft tumor growth, in athymic nude mice (FIG. 5A). Next, the inventors xenotransplanted the luciferase-tagged Kuramochi (BRCA2-deficient) line intraperitoneally in NOD scid gamma (NSG) mice and waited 3 months so the tumors burden reached high radiance signal (i.e. confluency in the peritoneum). Strikingly, NMNAT1 depletion in these conditions achieved rapid and significant Kuramochi (BRCA2-deficient) tumor killing (FIG. 5B).

[0114] These data demonstrate that HRD cells are highly sensitive to NMAT1 inhibition in vivo.

NMNAT1 Inhibition Kills PARPi-Resistant HRD Tumor Cells In Vivo

[0115] The inventors collected data in vitro showing that targeting NMNAT1 kills chemoresistant HRD cells regardless the mechanism of acquired resistance.

[0116] To translate these findings into a translational setting, the inventors derived a PARPi-resistant clone from the PARPi-naive BRCA1-deficient OVCAR8 lines, expressed either doxycycline-inducible NMNAT1 or scrambled (Scr) shRNA in these cells, and xenograft them in athymic nude mice.

[0117] As expected the OVCAR8 PARPi-resistant cells were resistant to the PARPi rucaparib in vivo (FIG. 6A). It was found that NMNAT1 depletion significantly impaired tumor growth (FIG. 6B, C). Moreover, mice bearing NMNAT1-depleted tumors had a survival advantage compared to control mice (FIG. 6D).

REFERENCES

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