Abstract B034: Determination of KRAS- and ERK-regulated phosphoproteomes in KRAS-mutant cancers (2023)

Abstract Direct inhibitors of mutationally activated KRAS are currently under intense preclinical and clinical development, with one KRASG12C mutant-selective inhibitor approved. However, treatment-associated resistance to KRASG12C inhibitors has been reported, with ~60% of relapsed patients acquiring mutations in signaling components both upstream and downstream, and at the level of RAS itself, that led to reactivation of RAF-MEK-ERK and PI3K-AKT effector pathways. Unexpectedly, we found that ectopic expression of constitutively activated MEK1, ERK1 or ERK2, but not AKT1, drove near-complete resistance to direct inhibitors of KRASG12C or KRASG12D in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). Despite comprehensive studies, how ERK supports KRAS-dependent cancer growth remains poorly understood. Therefore, we focused on delineating the ERK-dependent phosphoproteome. We treated a panel of six KRAS-mutant PDAC cell lines acutely (1 h) and long-term (24 h) with the highly selective ERK1/2 inhibitor SCH772984. Our proteomics analyses identified 5,117 phosphosites on 2,252 proteins significantly dysregulated by ERK inhibition. We first compared our PDAC ERK phosphoproteome with a recent compendium of published direct/indirect ERK substrates identified in non-PDAC cells and found, surprisingly, only 12% overlap. Thus, our dataset substaintially extends the complexity of ERK-regulated phosphoproteins, in part, reflecting a pancreatic cancer selective profile. To elucidate the kinase network that drives our PDAC ERK-dependent phosphoproteome, we utilized our newly described global atlas of kinase-substrate specificities and reported kinase-substrate interaction datasets. This revealed a highly dynamic kinome dominated by ERK at 1 h, then dominated by ERK-regulation of CDK1-6 cell cycle components and RHO GTPase signaling. Next, we established the subcellular distribution of the PDAC ERK phosphoproteome, then evaluated data from DepMap, and found enrichment of nuclear ERK substrates that displayed strong dependencies in PDAC. We also determined the KRASG12C-regulated phosphoproteome in pancreatic, lung, and colorectal cancer cells using the KRASG12C selective inhibitor MRTX1257. We used our global atlas of kinase-motif specificities and ERK-regulated phosphoproteome to determine KRAS-regulated signaling pathways in KRASG12C mutant cancers. We found a high correlation between KRAS- and ERK-regulated phosphorylations, primarily driven by CDK substrates. However, we also noted key differences, principally in DNA damage response pathways not identified in our ERK-regulated phosphoproteome. Finally, we performed a high-throughput drug sensitivity and resistance screen of (DSRT) comprised of &amp;gt;500 clinically actionable drugs in combination with a KRASG12C inhibitor, and found ERK inhibitors as top hits. In summary, our studies establish a comprehensive profile of KRAS-ERK signaling output that may define new therapeutic targets for targeting KRAS- and ERK-dependent cancer growth. Citation Format: Jennifer E. Klomp, Jeff A. Klomp, Nathaniel J. Diehl, Cole A. Edwards, Kristina Drizyte-Miller, Priya S. Hibshman, Runying Yang, Alexis J. Morales, Khalilah E. Taylor, Mariaelena Pierobon, Emanuel F. Petricoin III, Johnson L. Jared, Emily M. Huntsman, Tomer M. Yaron, Markus Vähä-Koskela, Laura E. Herring, Alex W. Prevatte, Natalie K. Barker, Lee M. Graves, Wennerberg Krister, Lewis C. Cantley, James G. Christensen, Adrienne D. Cox, Channing J. Der, Clint A. Stalnecker. Determination of KRAS- and ERK-regulated phosphoproteomes in KRAS-mutant cancers [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B034.