Abstract P1-06-04: Small-molecule screening nominates diverse combination therapies that sensitize BRCA mutant and wild-type triple negative breast cancer to PARP inhibition
Authors: Sammons S , Yip C , Anderson G , Force J , Marcom K , Westbrook K , Anders CK , Blackwell K , Wood K 
Published: May 2019

Abstract
Background: Triple negative breast cancer (TNBC) remains a heterogeneous clinical phenotype with few, known therapeutic targets. PARP inhibitors (PARPi) are the first approved, targeted therapy in TNBC, limited to germline BRCA mutant (BRCAm) cancers that lack homologous recombination repair capacity. Even in this context, resistance quickly emerges via secondary mutations that restore DNA repair ability. While DNA damage repair is an intriguing target in BRCA wild type (BRCAwt) TNBC due to inherent, genomic instability, PARPi alone have been ineffective in unselected populations. Systematic approaches to define novel drugs that sensitize BRCAwt and BRCAm TNBC to PARPi would greatly improve therapeutic efficacy and durability.
Methods: BRCAwt (HCC1806) and BRCAm (SUM149PT) cell lines were screened in duplicate using a 2,100-compound small molecule library. Cell lines were plated in media containing DMSO or sub-lethal doses of the PARPi, olaparib, onto Selleck Bioactive drug plates. Cell viability was assessed after 72 hours, then normalized to vehicle control. Hit cut-offs were predefined as log2 drug/DMSO of ≤ -0.7 with a viability difference greater than 20% -where stringent scoring thresholds were chosen to exceed the full range of scores observed in 816 empty control wells. Hits were sorted by target and pathway to provide mechanistic insight into the synergy of combinations. Drug combinations with the highest potential for near term translation were validated using GI50 viability assays in 9 BRCAwt and BRCAm TNBC cell lines. The most promising combination was further validated via immunoblotting, colony formation, and apoptosis assays.
Results: Several drug classes affecting well-known oncogenic signaling pathways conferred sensitivity to PARPi, with more hits in the BRCAm cell line. Relevant druggable targets sensitizing cells to olaparib in BRCAm TNBC that met the predefined cut-point were inhibitors of PI3K (pan-PI3K, PI3Kα and PI3Kβ specific), VEGFR, MEK, EGFR, NF-kB, aurora kinase and several DNA damaging agents. Aurora kinase, EGFR, and NF-kB inhibition sensitized cells to olaparib, yet upon further validation, synergy was mild. The screen identified ATM inhibitors, KU-55933 and KU-60019, as sensitizers of BRCAm cells to olaparib. The potent ATM inhibitor, AZD0156, and olaparib were a highly synergistic combination validated in all 9 BRCAm and BRCAwt TNBC cell lines via cell viability, annexin V, and colony formation assays. Immunoblotting of relevant DNA damage repair proteins showed that olaparib caused upregulation of p-ATM in BRCAm and BRCAwt cells. p-ATM expression decreased in response to combination ATM and PARP inhibition. Attenuated levels of p-ATM resulted in increased levels of p- and T-γH2AX, indicating an accumulation of double stranded DNA breaks.
Conclusion: In vitro, inhibition of several relevant, oncogenic pathways yielded sensitivity to PARPi in TNBC. We identified the ATM inhibitor, AZD0156, and olaparib as a potent combination regardless of BRCA status, a finding currently being evaluated in patient-derived in vivo models. Combination ATM plus PARP inhibitor therapy is a promising and feasible approach for near term translation in metastatic TNBC.
Citation Format: Sammons S, Yip C, Anderson G, Force J, Marcom K, Westbrook K, Anders CK, Blackwell K, Wood K. Small-molecule screening nominates diverse combination therapies that sensitize BRCA mutant and wild-type triple negative breast cancer to PARP inhibition [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 P1-06-04.