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Figures showing on-target effects of Kras knockdown in murine and human PDAC cells in vitro and in vivo, Gpr56 knockdown, SILAC and iTRAQ workflow and additional proteomics data, pharmacological inhibition of Src and FAK, and genetic inhibition of FAK.

Activating mutations in KRAS are the hallmark genetic alterations in pancreatic ductal adenocarcinoma (PDAC) and the key drivers of its initiation and progression. Longstanding efforts to develop novel KRAS inhibitors have been based on the assumption that PDAC cells are addicted to activated KRAS, but this assumption remains controversial. In this study, we analyzed the requirement of endogenous Kras to maintain survival of murine PDAC cells, using an inducible shRNA-based system that enables temporal control of Kras expression. We found that the majority of murine PDAC cells analyzed tolerated acute and sustained Kras silencing by adapting to a reversible cell state characterized by differences in cell morphology, proliferative kinetics, and tumor-initiating capacity. While we observed no significant mutational or transcriptional changes in the Kras-inhibited state, global phosphoproteomic profiling revealed significant alterations in cell signaling, including increased phosphorylation of focal adhesion pathway components. Accordingly, Kras-inhibited cells displayed prominent focal adhesion plaque structures, enhanced adherence properties, and increased dependency on adhesion for viability in vitro. Overall, our results call into question the degree to which PDAC cells are addicted to activated KRAS, by illustrating adaptive nongenetic and nontranscriptional mechanisms of resistance to Kras blockade. However, by identifying these mechanisms, our work also provides mechanistic directions to develop combination strategies that can help enforce the efficacy of KRAS inhibitors.Significance: These results call into question the degree to which pancreatic cancers are addicted to KRAS by illustrating adaptive nongenetic and nontranscriptional mechanisms of resistance to Kras blockade, with implications for the development of KRAS inhibitors for PDAC treatment. Cancer Res; 78(4); 985–1002. ©2017 AACR.

Detailed information on shRNA and primer sequences, tumor initiating frequency of Kras-inhibited cells, RNA-Seq data, and phosphoproteomic data.

Activating mutations in KRAS are the hallmark genetic alterations in pancreatic ductal adenocarcinoma (PDAC) and the key drivers of its initiation and progression. Longstanding efforts to develop novel KRAS inhibitors have been based on the assumption that PDAC cells are addicted to activated KRAS, but this assumption remains controversial. In this study, we analyzed the requirement of endogenous Kras to maintain survival of murine PDAC cells, using an inducible shRNA-based system that enables temporal control of Kras expression. We found that the majority of murine PDAC cells analyzed tolerated acute and sustained Kras silencing by adapting to a reversible cell state characterized by differences in cell morphology, proliferative kinetics, and tumor-initiating capacity. While we observed no significant mutational or transcriptional changes in the Kras-inhibited state, global phosphoproteomic profiling revealed significant alterations in cell signaling, including increased phosphorylation of focal adhesion pathway components. Accordingly, Kras-inhibited cells displayed prominent focal adhesion plaque structures, enhanced adherence properties, and increased dependency on adhesion for viability in vitro. Overall, our results call into question the degree to which PDAC cells are addicted to activated KRAS, by illustrating adaptive nongenetic and nontranscriptional mechanisms of resistance to Kras blockade. However, by identifying these mechanisms, our work also provides mechanistic directions to develop combination strategies that can help enforce the efficacy of KRAS inhibitors. Keywords: Pancreatic cancer; cell adhesion; cell signaling; protein tyrosine kinases; animal models of cancer; gene expression profiling; oncogenes; tumor suppressor genes; gene products as targets for therapy; novel mechanisms, NCIK08 Award, KL2/Catalyst MeRIT award, CCF/ASCO Young Investigator Award, Lustgarten Foundation Consortium grant, Blum-Kovler Pancreatic Cancer Action Network-AACR Innovative grant, Department of Defense Congressionally-Directed Medical Research Program (Grant W81XWH-12-043), National Cancer Institute. Cancer Center Support (Grant P30-CA14051)

Obesity is a major modifiable risk factor for pancreatic ductal adenocarcinoma (PDAC), yet how obesity contributes to pancreatic cancer development is not well understood. Moreover, whether and at what point weight loss or other obesity-related interventions may impact PDAC progression remains largely unexplored, with significant implications for prevention and treatment. Leveraging an autochthonous genetically engineered model of PDAC, we demonstrate that obesity plays a stage-specific causal and reversible role in early PDAC progression. Molecular and histologic analyses reveal prominent microenvironmental alterations in tumors from obese mice, including increased inflammation and fibrosis and evidence for marked pancreatic islet cell adaptation. In particular, we identify aberrant expression of the peptide hormone cholecystokinin (CCK) in pancreatic islets in tumors as an adaptive response to obesity and show that islet CCK expression promotes oncogenic Kras-driven pancreatic ductal tumorigenesis. Together, these studies link obesity, changes in the local microenvironment, and tumorigenesis and implicate islet-derived hormones beyond insulin in PDAC development. Furthermore, the reversible nature of these adaptations supports the potential of antiobesity strategies to intercept PDAC early during progression. Citation Format: Katherine Minjee Chung, Jaffarguribal Singh, Lauren Lawres, Kimberly Judith Dorans, Rebecca Robbins, Arjun Bhutkar, Ana Babic, Sara A. Vayrynen, Andressa D. Costa, Jonathan A. Nowak, Daniel T. Chang, Richard F. Dunne, Aram F. Hezel, Albert C. Koong, Joshua J. Wilhelm, Melena D. Bellin, Vibe Nylander, Anna L. Gloyn, Mark I. McCarthy, Brian M. Wolpin, Tyler Jacks, Charles S. Fuchs, Mandar Deepak Muzumdar. Microenvironmental adaptations drive obesity-associated pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A35.