Abstract B19: The ATR inhibitor, AZD6738, synergizes with other DNA damage response inhibitors and genotoxic drugs in pancreatic ductal adenocarcinoma cell lines: Opportunities for new therapeutic combinations
Authors: Wallez Yann , Koh Siang-Boon , Bhogadi Venkata Sai Vivek , Lau Alan , Richards Frances M. , Jodrell Duncan I. 
Published: April 2017

Mutations in oncogenes, tumor suppressor and DNA damage response (DDR) mediator genes drive or permit malignant transformation but also increase endogenous replication stress. The serine/threonine kinase ATR plays a critical role in safeguarding genome integrity from such replication stress and several studies have demonstrated the increased reliance of cancer cells on ATR function. We investigated the therapeutic opportunities for the ATR inhibitor, AZD6738, in combination with DNA damaging or DDR-targeted agents, in the context of pancreatic ductal adenocarcinoma (PDAC).

We evaluated four DNA-damaging agents (gemcitabine, 5-fluorouracil, oxaliplatin, SN38 (the active metabolite of irinotecan)) and three DDR-targeted agents (Wee1 inhibitor (AZD1775), Chk1 inhibitor (MK8776), PARP inhibitor (AZD2281)), each in combination with AZD6738 at multiple concentrations. Efficacy of these combinations was tested in growth inhibition assays in vitro, using a panel of cell lines in order to capture some of the genetic heterogeneity observed in PDAC: two human cell lines and four lines from the KrasG12D; Trp53R172H; Pdx-Cre (KPC) mouse. Synergistic growth inhibition was identified applying both Bliss Independence and Loewe models, using Combenefit software. All the KPC mouse cell lines were sensitive to AZD6738 as a single agent, with GI50 ranging from 346 to 566 nM. MIA PaCa-2 were sensitive to AZD6738, achieving &gt;90% growth inhibition, with GI50 of 2.2 μM. PANC-1 cells were less sensitive, with GI50 21 μM and achieving only ~60% GI, at the highest concentration tested. PANC-1 cells are also less sensitive to gemcitabine than the other cell lines. Synergy was detected in most of the cell lines, with each of the seven drug combinations tested. The combinations of AZD6738 with gemcitabine and with oxaliplatin showed synergy in all cell lines tested.

We next investigated scheduling of the gemcitabine/ATRi combination, at the specific GI50 concentrations for each cell line, using kinetic live-cell imaging assays. Concurrent treatment of gemcitabine/ATRi for 16h proved to be most effective, almost completely inhibiting cell growth for more than three days after washout. Sequential treatment (irrespective of the order) or shorter pulses (8h) were less effective. Maintaining ATRi after gemcitabine washout further enhanced growth inhibition for most cell lines. Mechanistically, ATRi impaired Chk1 activation (p-Ser345) and, in combination with gemcitabine, strongly potentiated DNA damage (gamma H2AX). Maintaining ATRi after gemcitabine washout helped to sustain the level of DNA damage. In vivo studies are underway to determine whether the gemcitabine/ATRi combination enhances efficacy compared to gemcitabine alone. The ATRi/oxaliplatin combination is also being investigated in vitro and in vivo using similar methods.

Several genes have been described in the literature to increase the reliance on ATR functions when altered. Mining two published datasets (TCGA, 186 samples and UTSW, Nat. Commun. 2015, 109 samples) we have investigated the frequencies at which 21 of these genes were altered in human PDAC. Overall ~95% of PDAC samples exhibit at least one (9% only one, 28% two and 57% three or more) genetic alteration likely to sensitize to ATRi, potentially improving the therapeutic index of combination approaches. Thus, combinations including ATRi merit further evaluation as they have the potential to be effective in the treatment of patients with PDAC.

Citation Format: Yann Wallez, Siang-Boon Koh, Venkata Sai Vivek Bhogadi, Alan Lau, Frances M. Richards, Duncan I. Jodrell. The ATR inhibitor, AZD6738, synergizes with other DNA damage response inhibitors and genotoxic drugs in pancreatic ductal adenocarcinoma cell lines: Opportunities for new therapeutic combinations [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B19.