The COVID-19 pandemic has had a significant impact on communities across the United States (US). Three vaccines have now been granted Emergency Use Authorization by the Food and Drug Administration (FDA) and the Centers for Disease Control and Prevention (CDC) for use in the US. However, barriers to vaccination exist, some of which are well documented in the literature, including lack of knowledge, fear, accessibility, mistrust in the healthcare system, and systemic and operational obstacles. Vaccine hesitancy in the US could potentially hinder all the efforts and resources being used to beat COVID-19, which has resulted in more than 594 000 deaths in the US per the CDC as of early June 2021. In order to overcome this pandemic, vaccine distribution and uptake is crucial. Pharmacists play a crucial role as healthcare providers as they can dismantle vaccine hesitancy and make an outstanding impact on the efforts to overcome this pandemic.
KRAS mutations occur in 95% of pancreatic ductal adenocarcinomas (PDAC) and are a well-validated driver of PDAC growth. Therefore, anti-KRAS therapies are expected to make a significant impact on the treatment of this deadly cancer, where there are currently no effective targeted therapies. Supporting this premise, early clinical trial results with KRASG12C inhibitors have shown promising disease control rates (84-100%) in KRASG12C-mutant PDAC. Despite these observations, two key issues limit the impact of KRASG12C inhibitors in PDAC. First, KRASG12C(OFF) mutations comprise less than 2% of KRAS mutations in PDAC. Second, patients initially responsive to KRASG12C inhibitors invariably relapse due to treatment-induced resistance. To begin qualifying KRAS inhibitors that target KRAS mutations more frequently found in PDAC, we characterized a RAS inhibitor that targets the multiple KRAS mutations as well as wild-type RAS proteins. We evaluated the impact of this inhibitor on RAS signaling and anti-proliferative activity in KRAS-mutant pancreatic cancer human cell lines and in a panel of RASless MEFs (ras-null mouse embryo fibroblasts) stably expressing exogenous RAS mutant proteins that are commonly found in PDAC. To identify genetic mechanisms of resistance to KRAS inhibition in pancreatic cancer, we applied CRISPR-Cas9 loss-of-function screens to KRASG12C-, KRASG12D-, KRASG12R-, and KRASQ61H-mutant PDAC cells treated with KRAS inhibitors to identify genes that modulate KRAS inhibitor anti-proliferative activity. We identified expected and novel mechanisms of resistance, including those that have been observed in patients treated with KRASG12C inhibitors. Citation Format: Andrew Michael Waters, Wen-Hsuan Chang, Clint Stalnecker, Cole Edwards, Runying Yang, Craig M. Goodwin, Adrienne D. Cox, Channing J. Der. Identification of resistance mechanisms to direct KRAS inhibition in pancreatic cancer [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 1075.
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 >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.
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