Abstract B12: Co-targeting PTEN-defined TNBC with p110beta-isoform specific inhibitor and PARP inhibitor BMN 673: A predictive context to sensitize PARP inhibitors in TNBC
Authors: Dey Nandini , Carlson Jennifer H. , De Pradip , Leyland-Jones Brian 
Published: May 2016

Abstract
Background: TNBC is a highly aggressive form of BRCA-associated BC subtype for which chemotherapy still remains a major form of treatment to which patients initially respond yet a majority of them inevitably relapse. PARP1 has been identified as a target in BRCA-defined cancers and inhibitors of PARP1 has been recently approved by FDA as targeted agents in BRCA-defined cancers. The loss of PTEN is the most common “first event” associated with basal-like subtype (Martins et al., 2012) and this PI3K-pathway activating event (deletion/mutation/loss of PTEN) occurs more frequently (35%) than PIK3CA mutations in this subtype of BC (Ellis & Perou, 2013). In the cytoplasm, PTEN-loss mediated upregulation of PI3K/AKT signal is known to depend on the PI3Kbeta-isoform. In contrast nuclear PTEN controls DNA repair (Bassi et al., 2013). A failure to repair the damaged DNA upon inhibition of PARP causes accumulation of DNA double-strand breaks and leads to apoptosis of cancer cells. Thus we hypothesized that a combination of p110beta-isoform specific inhibitor and PARP inhibitor will sensitize PARP inhibitors in PTEN-null TNBC model. Methods: Five PARP inhibitors, Talazoparib (BMN673, B), Niraparib (N), Olaparib (O), Rucaparib (R) and Veliparib (V) were tested in four PTEN-null TNBC cell lines, MDA-MB468, HCC70 (p.F90fs*9), BT549 (p.V275fs*1) and SUM149 cell lines in combination with p110beta specific inhibitor, AZD6482. Proliferative, apoptotic and PARylation signals following drug combinations were demonstrated by WB in a dose and time dependent manner. Pro-apoptotic and anti-proliferative effects were verified using complementary 3D ON-TOP assay, real time proliferation (Incucyte), AnnexinV and cl-caspase3 analyses. In order to understand the initial effects of isoform-specific inhibitors of PI3K we also compared anti-proliferative signals of GDC-0032 (p110 beta sparing inhibitor) and GDC-0941 (pan PI3K inhibitor) with that of AZD6482 at 3/6 hours in MDA-MB468 and SUM149 cells. Results: In PTEN null TNBC cells 500 nM BMN673 alone was effective in slowing cell proliferation and inducing apoptosis while AZD6482 (both 5-10 uM) did not have anti-proliferative or pro-apoptotic effects at 48 and 72 hours. Combinations of different PARPi (100nM of B, 1uM of N, 10uM of O, 10uM of R and 10uM of V) with AZD6482 blocked 3D growth of PTEN null BT549 and MDA-MB468 TNBT cells in a time dependent manner (96 hours & 7 days). PARP inhibitors abrogated PAR signals either alone or in combination with carboplatin in both BRCA1/2 WT and mutated cells. The most pronounced anti-tumor effect was observed with the combination of B and AZD6482 which was mechanistically explained by the robust increase of AnnexinV positive cells following a single 500nM dose of B at both 48 and 72 hours. In both MDA-MB468 and SUM149 cells although panPI3K inhibitor, GDC-0941 downregulated pAKT (S473, T308), pP70S6K (T389), pS6RP and p4EBP1 (T37/46) levels, p110 alpha-specific inhibitor GDC-0032 failed to perturb these signals while AZD6482 blocked them all. In summary, we demonstrate a remarkable sensitivity of tumor cells to PARP inhibition in PTEN-defined TNBC models and identify that PTEN-nullness as a potential predictive context for a possible co-targeting of PI3K pathway to further sensitize TNBC to PARP inhibitors. Significance: Our findings squarely place PTEN expression at the nexus of oncogenic signals in TNBC and indicate that TNBC patients with no PTEN activity in their tumors might benefit from combined p110beta-isoform specific inhibitor and PARP inhibitor.
Citation Format: Nandini Dey, Jennifer H. Carlson, Pradip De, Brian Leyland-Jones. Co-targeting PTEN-defined TNBC with p110beta-isoform specific inhibitor and PARP inhibitor BMN 673: A predictive context to sensitize PARP inhibitors in TNBC. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B12.