Abstract 626: Mechanistic differentiation of targeted DDR agent combinations
Authors: Forment Josep V. , Wijnhoven Paul , Ramos-Montoya Antonio , Fok Jacqueline , Follia Valeria , Vazquez-Chantada Mercedes , Oderda Rajesh , Wilson Zena , Lau Alan , Leo Elisabetta , Durant Stephen , Cadogan Elaine , O'Connor Mark J. 
Published: September 2020

Abstract
The DNA damage response (DDR) safeguards genome stability and promotes cellular survival by counteracting deleterious consequences of DNA damage. Deficiencies in DDR pathways contribute to genome instability, a hallmark of cancer development, and targeting the DDR is a promising anti-cancer therapy strategy. We describe here in vitro and in vivo studies allowing mechanistic understanding of the benefit of combining our DDR kinase inhibitors (DDRi) targeting ATM (AZD0156), ATR (AZD6738) and DNA-PK (AZD7648) with the PARP inhibitor, olaparib, in homologous recombination-deficient backgrounds. Using the break-induced replication (BIR) assay, which measures homologous-recombination repair (HRR) of DNA double-strand breaks resembling broken replication forks, we observed that ATRi decreases this type of homologous recombination repair by up to 50%, while inhibiting DNA-PK or ATM showed no effect. This provides a mechanistic rationale for combining ATRi with compounds that cause replication-fork collapse, such as PARPi. Indeed, combination of olaparib with the ATRi in BRCA1 mutant UWB1.289 cells enhanced olaparib sensitivity a thousand-fold (GI50 from 590 nM to 0.6 nM). This correlates with the complete tumour growth inhibitions (TGI) observed for the ATRi + olaparib combination in the BRCA2 mutant HBCx-10 triple negative breast cancer PDX model and is consistent with a synergistic increase of genome instability measured by metaphase spread analysis (total aberrations: olaparib; 11, ATRi; 8, combination; 35). DNA-PKi, however, did not synergise with olaparib, showing little enhancement of olaparib single-agent treatment (GI50 from 0.59 μM to 0.33 μM) and no significant impact on the total amount of chromosomal aberrations (total aberrations: olaparib; 11, DNA-PKi ; 3, combination; 14), suggesting that DNA-PKi alone or in combination with olaparib have little impact on BRCA1 mutated tumours. In ATM KO FaDu xenografts, ATRi or DNA-PKi single-agent treatment resulted in TGI, with complete tumour regressions when combined. Focusing on the olaparib combination, metaphase spread analysis of ATRi-treated FaDu ATM KO cells showed an enhancement of DNA replication-dependent chromatid breaks (1.3 vs 0.2 breaks/spread in ATM WT), while DNA-PKi treatment predominantly caused chromosome breaks (1.7 vs 0.2 breaks/spread in ATM WT) that are not dependent on replication. When combined with olaparib, ATRi acted synergistically and in a manner similar to that observed in the BRCA1 mutant cells, while the DNA-PKi + olaparib combination was additive. These data suggest that enhanced olaparib sensitisation of ATM KO cells by ATRi or DNA-PKi combinations occurs through different mechanisms. Together, these findings provide mechanistic differentiation of our DDRi in specific genetic backgrounds. This work informs how to clinically position these agents to benefit the right patients not only as single-agent treatments, but also in combination.
Citation Format: Josep V. Forment, Paul Wijnhoven, Antonio Ramos-Montoya, Jacqueline Fok, Valeria Follia, Mercedes Vazquez-Chantada, Rajesh Oderda, Zena Wilson, Alan Lau, Elisabetta Leo, Stephen Durant, Elaine Cadogan, Mark J. O'Connor. Mechanistic differentiation of targeted DDR agent combinations [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 626.