Abstract A116: Temozolomide-resistant MGMT/MSI double-negative cancer cells rely on ATR signaling and homologous recombination for DNA repair and survival
Authors: El Touny Lara H. , Harris Erik , Hose Curtis , Connelly John , Monks Anne , Newton Dianne , Stockwin Luke , Hollingshead Melinda , Parchment Ralph , Doroshow James H. , Teicher Beverly A. , Rapisarda Annamaria 
Published: August 2018

Abstract
Although loss of MGMT expression is used as a predictive biomarker for response to temozolomide (TMZ), some patients do not benefit from treatment, suggesting that additional criteria for patient selection remain to be identified. We used a panel of newly developed patient derived models and well-characterized cancer lines to evaluate potential determinants of TMZ response and identify strategies to overcome TMZ resistance in MGMT negative cancers. There was 100% association between microsatellite instability (MSI) and TMZ resistance in the lines tested. Moreover, only a fraction of MGMT/MSI double-negative lines were sensitive to TMZ at clinically relevant concentrations (37.6µM). TMZ-induced DNA damage and repair (DDR) were compared in a bladder line sensitive to TMZ (BLX1, IC50 11.4µM) and a resistant large neuroendocrine cancer line (LNX1, IC50&gt;80 µM). We observed DDR activation in both models, but only BLX1 had measurable DNA damage (PARP cleavage and γH2AX accumulation, starting 24 h post treatment). An extended G2/M arrest occurred in BLX1 but not in LNX1, suggesting that LNX1 efficiently repaired TMZ-induced damage. ATR inhibition (VE821, VX970 or AZD6738), but not ATM inhibition, in LNX1 cells increased response to TMZ (IC50 27.5µM), PARP cleavage, γH2AX accumulation, and protracted G2/M arrest. BLX1 had minimal homologous recombination (HR) capacity (measured by pDR-GFP/SceI assay) compared to LNX1; however, ATR inhibition abrogated HR activity in LNX1 cells, suggesting HR importance in repair of TMZ-induced damage. To assess whether HR defects sensitize cells to TMZ, we used MGMT positive BRCA1 null cells (UWB1.289) and their BRCA1 reconstituted counterpart line. BRCA1 status did not affect TMZ response in the presence of an MGMT inhibitor (TMZ IC50&gt;80µM); however, BRCA1 null cells were sensitive to TMZ in combination with an ATR inhibitor (VE821 0.3µM, TMZ IC50 10µM). BRCA1 reconstituted cells were sensitized to TMZ+VE821 using a Rad51 inhibitor (B02 2.5µM, TMZ IC50 22.5µM), suggesting that when HR is compromised, cells rely on ATR signaling for repair of TMZ-induced damage. Our data suggest that MGMT/MSI double-negative cells rely on ATR-dependent signaling for repair of TMZ-induced DSBs and that HR defects could be used as additional selection criteria for TMZ+ATRi treatment. Funded by NCI Contract No. HHSN261200800001E. This research was supported, in part, by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute.
Citation Format: Lara H. El Touny, Erik Harris, Curtis Hose, John Connelly, Anne Monks, Dianne Newton, Luke Stockwin, Melinda Hollingshead, Ralph Parchment, James H. Doroshow, Beverly A. Teicher, Annamaria Rapisarda. Temozolomide-resistant MGMT/MSI double-negative cancer cells rely on ATR signaling and homologous recombination for DNA repair and survival [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A116.