Abstract B30: Talazoparib efficacy is enhanced by noncytotoxic doses of temozolomide-mediated DNA damage in prostate cancer cell lines

Abstract

Abstract Purpose: To investigate the effects of talazoparib alone and in combination with temozolomide (TMZ) on prostate cancer cells Introduction: Talazoparib is a potent, orally bioavailable, small molecule inhibitor of PARP (poly-ADP ribose polymerase) enzyme activity that also traps PARP protein onto DNA at sites of DNA damage; both activities result in cancer cell death (Murai et al. Mol Cancer Ther. 2014;13:433-43). Talazoparib is currently under investigation for clinical activity in patients who have locally advanced and/or metastatic breast cancer with germline BRCA mutations (EMBRACA; Clinical Trials.gov; NCT01945775). In the current nonclinical study, talazoparib demonstrated potent antitumor effects on androgen receptor (AR)-positive prostate cancer cells expressing both full length AR (LNCaP cells) as well as the androgen-independent splice variant, AR-V7 (22RV1 cells), when used either as a single agent or in combination with low, noncytotoxic concentrations of TMZ, a DNA alkylating therapeutic. Methods: Viability of prostate cancer cells (LNCaP and 22RV1 cells) after 7 days of treatment was measured by Cell Titer Glo (Promega) after treatment with vehicle, talazoparib, TMZ or a combination of the two. DNA damage was determined by immunofluorescence against gH2AX (EMD Millipore), and the amount of PARP trapped on DNA, determined by Western Blot analysis of chromatin probed with antibodies specific for PARP. Cellular homeostasis was determined by flow cytometry analysis for cell cycle stage, and apoptosis indicated by Annexin V staining (BD Pharmingen) or caspase activation with Caspase Glo (Promega) assays. Nucleotide adducts, N7 methyl-2'-deoxyguanosine (N7-MedG) and O6-Methyl-2'-deoxyguanosine (O6-MedG), from 1-, 2- and 4-hour TMZ-treated calf thymus DNA and genomic DNA from LNCaP cells treated with TMZ were detected after DNA hydrolysis and quantitated by LC-mass spectrometry in multiple reaction monitoring mode. Results: Talazoparib treatment resulted in a concentration-dependent inhibition of cell viability in LNCaP (AR wildtype) and 22RV1 (AR-V7) cells. Talazoparib's antitumor effects were greatly enhanced when combined with noncytotoxic TMZ concentrations. In a cell-free study using calf thymus DNA, TMZ generated both N7-MedG and O6-MedG DNA adducts, with the N7-MedG being more abundant than the O6-MedG. Analysis of DNA from LNCaP cells treated with TMZ generated detectable N7-MedG DNA adducts. Low, noncytotoxic concentrations of TMZ potentiated the effects of talazoparib on prostate cancer cell viability independent of AR variant status. Treatment with talazoparib alone and in combination with TMZ was associated with increased gH2AX staining (DNA damage) and enhanced PARP-DNA trapping complexes. The combination treatment also induced an increase in apoptotic cells at early time points. Conclusion: Talazoparib is cytotoxic to prostate cancer cells as a single agent in nonclinical cell based models. Herein, we demonstrate that combining noncytotoxic doses of TMZ with talazoparib improves efficacy in killing of LNCaP and 22RV1 cells. The increased efficacy corresponds with increased DNA-damage and increased trapped PARP-DNA complexes. Low, noncytotoxic doses of TMZ are shown to generate DNA damage sites in vitro and in treated LNCaP cells, providing a mechanistic basis for the combination effect of talazoparib and TMZ. Together, these nonclinical data provide scientific rationale for a clinical trial strategy of combining talazoparib with low, noncytotoxic doses of TMZ to enhance efficacy in prostate cancer. Citation Format: Jeffrey N. Lindquist, Vernon T. Phan, Eduardo Riquelme, Kakoli Mukherjee, Olivia Farías, Ashu Gupta, Mohd Raja, Roopa Rai, Hirdesh Uppal, Andrew A. Protter. Talazoparib efficacy is enhanced by noncytotoxic doses of temozolomide-mediated DNA damage in prostate cancer cell lines [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B30.