The Combination Of Parp Inhibitor Talazoparib With Low-Dose Temozolomide Results In Increased Cell Lethality In Brca1/2 Wild-Type Melanoma, Small Cell Lung Cancer, Ovarian, And Colon Cancer Cell Lines And Mouse Xenografts Via The Formation Of Dna Double-Strand Breaks During S-Phase

Background: Previous studies of PARP inhibitors have shown efficacy in tumors with defects in homologous recombination repair, particularly in persons with ovarian and breast cancers with germline deleterious BRCA1/2 mutations (gBRCA1/2+). There is much interest in expanding the use of PARP inhibitors beyond these populations, with one strategy being the creation of DNA damage with cytotoxic chemotherapy with concomitant inhibition of its repair with a PARP inhibitor. Murai, et al, have also shown that PARP is trapped on the DNA at the site of damage in the presence of a PARP inhibitor. In April 2016, my colleague Dr Zev Wainberg presented Phase I safety and efficacy data at AACR (abstract CT011) with PARP inhibitor talazoparib in combination with alkylating agent temozolomide in cancer patients without gBRCA1/2+, demonstrating this to be a promising combination. Methods: We evaluated the molecular mechanisms underlying cancer cell lethality with talazoparib plus temozolomide in melanoma, small cell lung, ovarian, and colorectal cancer cell lines and mouse xenografts. We determined growth-adjusted IC50s for talazoparib in multiple histologies to choose cell lines of low and high sensitivity to the PARP inhibitor as monotherapy. We studied percent growth inhibition of talazoparib, temozolomide, and combination therapy at increasing doses. We investigated cell cycle dynamics at 24 hours and 48 hours; PARP inhibitors have been shown to activate the G2/M cell cycle checkpoint, which could confound growth arrest and DNA repair studies. We performed western blot analysis to identify phosphorylated histone H2AX as a marker of DNA double-strand breaks. We used FACS to determine apoptotic fractions and to correlate phospho-H2AX formation with cell cycle phase. We used confocal microscopy to correlate phospho-H2AX with nuclear PARP. We used mouse xenografts to demonstrate sensitivity of tumors to temozolomide, talazoparib, and combination therapy. Results: The combination of temozolomide with talazoparib results in the formation of DNA double-strand breaks during S-phase, which results in increased cell lethality in BRCA1/2 wild-type cell lines and mouse xenografts of melanoma, colorectal, ovarian, and small cell lung histologies even when those cell lines are not sensitive to either talazoparib or temozolomide alone. We were unable to demonstrate colocalization of PARP and phospho-H2AX due to PARP9s high expression in the nucleus during S-phase. We were also not able to demonstrate PARP-trapping by western blot at doses utilized in human patients, but this is likely due to lack of assay sensitivity. Conclusions: Taking into consideration both the molecular data demonstrating efficacy of talazoparib plus temozolomide therapy in BRCA1/2 wild-type cell lines and mouse xenografts and the Phase I trial data showing safety and efficacy with this combination, PARP inhibitors likely have utility beyond treatment of gBRCA1/2+ patients. Use of the alkylating agent might be particularly important, as it is recognized during S-phase and trapping of PARP on the DNA could result in collapse of a stalled replication fork into a DNA double-strand break. Citation Format: Kelly Elizabeth McCann, Erika von Euw, Neil O9Brien, Dennis Slamon. The combination of PARP inhibitor talazoparib with low-dose temozolomide results in increased cell lethality in BRCA1/2 wild-type melanoma, small cell lung cancer, ovarian, and colon cancer cell lines and mouse xenografts via the formation of DNA double-strand breaks during S-phase [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4124.