Abstract B018: RNR inhibitors enhance chemotherapeutic activity in pancreatic cancer cells through altering replication stress

Abstract

Abstract Despite extensive study, pancreatic ductal adenocarcinoma (PDAC) remains highly lethal resulting in a need for new effective therapies and, in particular, combination therapies. Gemcitabine that targets ribonucleotide reductase (RNR) is active in PDAC, albeit with modest impacts on long-term outcomes. Inhibition of RNR leads to depletion of dNTP pools, provoking replication stress (RS). RS activates the ATR/CHK1/WEE1 S phase DNA damage checkpoint cascade. Our study provides insights into the dynamic interplay between inhibitors of RRM1 (gemcitabine, clofarabine, fludarabine) and RRM2 (triapine) and targeting of the ATR/WEE1 DNA damage checkpoint cascade. Understanding the mechanisms underlying the combination therapy activity will enable development of optimal dosing approaches that optimize efficacy while limiting toxicity. RNR inhibitors and DNA damage checkpoint inhibitors were assessed on viability and cell growth as mono and combination therapy in human PDAC cell lines and in mouse transgenic PDAC cell lines (KMC cell lines). We detected RS using DNA combing and pRPA32 foci/H2AX staining and correlated these with response to single and combination therapy. Gemcitabine caused greater S phase to G2 cell cycle delay in sensitive cell lines (CFPAC1, HPAFII) compared to resistant cell lines (Miapaca2, PANC1). Delayed S phase progression induced by gemcitabine appears to be due to impairment of the replication machinery leading to a DNA damage response (γH2AX S139) and fork stalling. During fork stalling, RPA32 protects single-strand DNA (ssDNA). Gemcitabine increases γH2AX, as well as double positive staining for γH2AX and pRPA32 foci, and decreases replication fork speed indicative of RS. These changes correlated with the sensitivity to gemcitabine monotherapy. KMC cell lines exhibited lower sensitivity to the ATR inhibitor, AZD6738, and the WEE1 inhibitor, MK1775, than human PDAC cell lines. MK1775 and AZD6738 IC50s in PDAC cell lines were highly correlated (Pearson r=0.888, p value=0.0006), although the correlation was weaker in KMC PDAC cell lines (Pearson r=0.353, p value=0.350). Combined treatment with gemcitabine and ATR/WEE1 inhibitors was effective in gemcitabine-resistant human and KMC PDAC cell lines both in terms of decreased viability and increased RS. Other inhibitors of RRM1 (clofarabine) and also RRM2 (triapine) also effectively decreased cell viability in combination with ATR/WEE1 inhibitors. The viability was recovered with exogenous dNTPs in cells treated with the RRM1 inhibitors gemcitabine and clofarabine, but not the RRM2 inhibitor triapine, consistent with the RS induced by RRM1 inhibitors being due to decreased endogenous dNTP levels. RRM1 and RRM2 inhibitors cause replication stress and are synergistic with inhibition of the ATR/WEE1 pathway that contributes to ability of cells to survive RS-driven cell death. These findings support the design and implementation of in vivo studies that will subsequently drive clinical trials with potential benefit for PDAC patients. Citation Format: Soon Young Park, Kang Jing Jeong, Alfonso Poire, Isabel English, Jonathan R. Brody, Rosalie C. Sears, Gordon B. Mills. RNR inhibitors enhance chemotherapeutic activity in pancreatic cancer cells through altering replication stress [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B018.