Identifying circulating biomarkers of acute response and resistance to clinical ATR and Chk1 inhibitors

Objectives: Clinical ATR kinase inhibitors (ATRi) are an emerging class of therapeutics being used to treat diverse solid tumor malignancies, including ovarian cancers. The development of circulating biomarkers indicating response or resistance to clinical ATRi will support the ongoing development of this emerging class of therapeutics. This study is focused on establishing isogenic models of ATRi-resistance in ovarian cancer cell lines and identifying conserved secreted protein alterations correlating with acute response or resistance to ATRi treatment in these models. Methods: We generated isogenic cell line models of ATRi-resistant, CCNE1-amplified (OVCAR3) and non-CCNE1 amplified (OV90) ovarian cancer cell by metronomic treatment of cell lines with the ATR inhibitor AZD6738 (Astra Zeneca). We characterized sensitivity of models to AZD6738 as well as inhibitors of checkpoint kinase 1 (Chk1, LY2606368), ataxia-telangiectasia mutated (ATM, KU55933) poly (ADP-ribose) polymerase (PARP, BMN-673) and cisplatin combination by dose response assay and further assessed impact of ATRi treatment on cell cycle. Lastly, we analyzed conditioned media by LC-MS/MS-based proteomic analyses from ATRi-resistant OVCAR3 cells or following acute treatment with ATRi. Results: Metronomic treatment of ovarian cancer cells with ATRi induced resistance to ATRi irrespective of CCNE1 status. ATRi-resistant cells were also resistant to Chk1i, but not to ATM, PARP inhibitors or combinations of ATRi and cisplatin. Cell cycle analyses revealed that ATRi-sensitive cells arrest in S-phase and continue active DNA replication, whereas ATRi-resistant cells arrest in G1/S phase and cease DNA replication following ATRi treatment. Comparison of proteins identified in conditioned media revealed u003e 30 proteins bearing signal peptide sequences potentially mediating cellular secretion as significantly elevated in the cellular secretomes of ATRi-resistant or ATRi sensitive cells treated with ATRi, with only a single protein co-altered between these sample, i.e. alpha-N-acetylgalactosaminidase (NAGA). Conclusions: Our findings show that metronomic treatment of ovarian cancer cells with ATR inhibitors induces resistance to ATRi as well as an inhibitor of Chk1, an immediate downstream effector of ATR. Our analyses show that ATRi-resistant cells remain sensitive to inhibitors of orthogonal DNA damage response signaling pathways, i.e. ATM and PARP, as well as to cisplatin sensitization induced by ATRi co-treatment. ATRi-resistant cells further exhibit a conserved G1/S-phase cell cycle arrest response to ATRi treatment. Lastly, our preliminary secretome analyses provide proof of concept data that identification of secreted biomarkers specific for acute response or resistance to ATRi (Chk1i)-treatment is feasible. Citation Format: Nicholas Bateman, Wei Ao, Domenic Tommarello, Kelly Conrads, Pang-ning Teng, Kathleen Darcy, Chad Hamilton, G. Larry Maxwell, Christopher Bakkenist, Thomas Conrads. Identifying circulating biomarkers of acute response and resistance to clinical ATR and Chk1 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2852.