Abstract 3497: Mechanism-based scheduling of triple therapy gemcitabine/CHK1i/WEE1i in pancreatic cancer at submaximal yet synergistic concentrations

Abstract

Abstract Sensitization of cancer cells to gemcitabine has been shown with checkpoint kinase CHK1 and WEE1 inhibitors. To rationalize and optimize the concomitant use of these three agents, we first performed growth inhibition assays on MIA PaCa-2 pancreatic cancer cells using gemcitabine in combination with either CHIR-124 (CHK1 inhibitor) or MK-1775 (WEE1 inhibitor). In silico analysis with three mathematical models (Bliss Independence, Loewe and Highest Single Agent) identified synergistic growth inhibition at submaximal (i.e. <GI50) concentrations of the single agents (10nM gemcitabine, 20nM CHIR-124, 300nM MK-1775) in MIA PaCa-2 cells. At these concentrations of gemcitabine+CHIR-124, quantitative image-based cytometry demonstrated S-phase redistribution, with 31% of >8000 individual cells undergoing extensive fork collapse (compared to 0-1% with single agents) as evidenced by co-staining of yH2AX and hyper-phosphorylated RPA32. Conversely, submaximal gemcitabine+MK-1775 induced clear reduction of inhibitory CDK1 Y15 level compared to single agents. Consistent with this G2/M abrogation, in a Fucci-based CDT1/geminin-expressing MIA PaCa-2 stable cell line, gemcitabine+MK-1775 partially reversed the accumulation of the geminin-expressing (S/G2) population induced by gemcitabine alone. This was accompanied by an increase in the CDT1-expressing (G1) population, alongside aberrant mitotic cells with features of perturbed kinetochore-microtubule integrity. Based on these findings, we performed a series of long-term, kinetic live-cell imaging assays to determine the optimal scheduling for gemcitabine/CHK1i/WEE1i as a triplet at synergistic concentrations. In contrast to the widely proposed scheduling regimen, we found that delayed administration of CHK1i (at 24 hours), relative to gemcitabine, did not lead to synergy. However, concurrent administration yielded durable growth inhibition, even when the two agents were removed after 24 hours. This inhibition was further enhanced with the subsequent addition of WEE1i at 24 hours, but not continuation of a CHK1i. Together, our results illustrate differential mechanistic effects of CHK1 and WEE1 inhibitors with gemcitabine, when combined at sub-GI50 concentrations, and propose a novel triple agent schedule that will be evaluated in vivo. Citation Format: Siang-Boon Koh, Frances M. Richards, Esther Rodriguez, Scott K. Lyons, Duncan I. Jodrell. Mechanism-based scheduling of triple therapy gemcitabine/CHK1i/WEE1i in pancreatic cancer at submaximal yet synergistic concentrations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3497. doi:10.1158/1538-7445.AM2015-3497