Abstract P2-03-06: PTEN expression at the nexus of oncogenic signals in TNBC: Testing combination of p110beta-isoform-specific inhibitor with five PARP inhibitors

Abstract

Abstract Background: TNBC is the most aggressive form of BRCA-associated BC subtype. The loss of PTEN is a common “first event” associated with basal-like subtype (Martins et al., 2012) and this mode of PI3K-pathway activation (deletion/mutation/loss of PTEN) occurs more frequently (35%) than PIK3CA (5-10%) mutations in TNBC (Ellis & Perou, 2013). Several reports suggest upregulation of PI3K/AKT mediated by PTEN loss depends on the PI3Kbeta-isoform. PARP1 is identified as a target of BRCA-defined cancers. Inhibitors of PARP1 have been recently approved by the FDA as targeted agents in BRCA-defined breast cancers. Failure to repair damaged DNA upon PARP inhibition causes accumulation of DNA double-strand breaks and leads to apoptosis of cancer cells. Also, nuclear PTEN controls DNA repair (Bassi et al., 2013). Here we hypothesized that a combination of PARP inhibitor with p110beta-isoform-specific inhibitor would sensitize the effect of PARP inhibitor(s) in PTEN-deprived TNBC model. Methods: We tested five PARP inhibitors, Talazoparib (BMN673, B), Niraparib (N), Olaparib (O), Rucaparib (R) and Veliparib (V) in combination with p110beta specific inhibitor, AZD6482. Four PTEN-null TNBC cell lines (BRCA WT/null), MDA-MB468, HCC70 (p.F90fs*9), BT549 (p.V275fs*1) and SUM149 cell lines were used for the study. 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. As an internal control, 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. Mode of apoptosis was tested using triple fluorescence staining in live cells. Results: A dose of 500 nM BMN673 alone was effective in slowing cell proliferation and induced apoptosis. AZD6482 (5-10 uM) did not have anti-proliferative or pro-apoptotic effects at 48 and 72 hours. When compared, combinations of different PARPi (100nM of B, 1uM of N, 10uM of O, 10uM of R and 10uM of V) with AZD6482 abrogated 3D growth in BT549 and MDA-MB468 TNBC cells in a time-dependent manner. The effect of PARP inhibitor was tested by PAR signals which were abrogated 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. Pan PI3K inhibitor, GDC-0941, and AZD6482 blocked the activation of PI3K and its downstream effectors in contrast to p110 alpha-specific inhibitor, GDC-0032 in MDA-MB468 and SUM149 cells. Summary: We demonstrated a remarkable sensitivity of tumor cells to PARP inhibition in PTEN-defined TNBC models and identified PTEN-nullness as a potential predictive biomarker for a possible co-targeting of the PI3K pathway to further sensitize TNBC to PARP inhibitors. Citation Format: Dey N, Carlson JH, De P, Leyland-Jones B. PTEN expression at the nexus of oncogenic signals in TNBC: Testing combination of p110beta-isoform-specific inhibitor with five PARP inhibitors [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-03-06.