Abstract 4851: Improving the efficacy of dual ERK-MAPK and autophagy inhibition as a therapeutic strategy for pancreatic ductal adenocarcinoma

Abstract

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent growth. We and others recently demonstrated that inhibition of the RAF-MEK-ERK pathway resulted in upregulated autophagy, and that dual treatment with the autophagy inhibitor hydroxychloroquine (HCQ)/chloroquine (CQ) and MEK or ERK inhibitors (MEKi, ERKi) synergistically blocked PDAC growth. These findings provided rationale for our initiation of Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT04132505) or ERKi (LY3214996; NCT04386057) with HCQ in PDAC. However, HCQ/CQ are not specific or potent autophagy inhibitors. Thus, we sought to identify a more efficacious autophagy inhibition strategy. To this end, we performed a CRISPR-Cas9 mediated genetic loss-of-function screen with a library encompassing cancer signaling pathways in the presence or absence of CQ. In the absence of CQ, we identified PIKfyve, a lipid kinase critical for the recycling dynamics of lysosomes, as an essential autophagy-related gene in PDAC cells. PIKfyve inhibition, by the clinical candidate inhibitor apilimod, resulted in potent reduction of autophagic flux and growth. Additionally, PIKfyve inhibition resulted in robust intracellular vacuolization. Vacuoles stained positive for the lysosomal marker, LAMP1, and exhibited reduced acidity and impaired cargo degradation. Importantly, when we inhibited MEK, with the clinical stage MEKi mirdametinib, and PIKfyve (with apilimod) together, we observed decreased MEKi-induced autophagic flux and synergistic impairment of PDAC cell proliferation. Growth inhibition was due in part to an induction of apoptosis unique to combination treatment. Synergistic growth suppression was maintained in a panel of patient derived PDAC organoids, suggesting this may be an efficacious therapeutic strategy for PDAC treatment. In the presence of CQ, our CRISPR-Cas9 screen showed that loss of upstream autophagy-related genes sensitized PDAC cells to CQ treatment. Validation indicated that vertical inhibition (dual inhibition of distinct nodes of a linear pathway) of the autophagy pathway further reduced autophagic flux relative to inhibition of any single node and synergistically reduced PDAC cell growth. Furthermore, combining ERK-MAPK inhibition with vertical inhibition of autophagy improves the in vitro efficacy of this treatment strategy. Ongoing studies are aimed at delineating the mechanism underlying the synergy observed with anti-autophagy inhibitor combinations and further validation in more advanced preclinical models of PDAC. Citation Format: Jonathan M. DeLiberty, Clint A. Stalnecker, Kristina Drizyte-Miller, Elyse G. Schechter, Noah L. Pieper, Runying Yang, Channing J. Der, Adrienne D. Cox, Kirsten L. Bryant. Improving the efficacy of dual ERK-MAPK and autophagy inhibition as a therapeutic strategy for pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4851.