Cox, Adrienne D.

Of the thousands of genes and substrates identified in KRAS-mutant signaling networks in pancreatic ductal adenocarcinoma (PDAC), more than 200 are transcription factors, implying extensive and complex transcriptional regulation. However, we observed that genetic suppression of the transcription factor MYC alone was sufficient to phenocopy the effect of KRAS suppression in signaling, growth, and metabolic processes in PDAC cells. We determined the gene transcription changes caused by acute suppression of MYC function in KRAS -mutant PDAC cell lines and performed dependency map and pathway analyses on the affected gene sets. The expression of 1685 genes was increased upon suppression of MYC, and this gene set may comprise the bulk of the MYC-regulated genes essential for PDAC growth. In contrast, the 1325 genes whose expression was inhibited may comprise a compensatory response to oncogenic stress, mediated in part by the GTPase RHO. MYC-dependent transcriptional activity was largely ERK dependent, and almost one-third of ERK-regulated genes were also regulated by MYC in PDAC cells. Furthermore, chemical proteomic profiling revealed MYC-regulated protein kinases that can be targeted therapeutically. Together, these data provide a molecular portrait of MYC-dependent signaling that encompasses potentially exploitable mechanisms for treating PDAC.

How the KRAS oncogene drives cancer growth remains poorly understood. Therefore, we established a systemwide portrait of KRAS- and extracellular signal–regulated kinase (ERK)–dependent gene transcription in KRAS-mutant cancer to delineate the molecular mechanisms of growth and of inhibitor resistance. Unexpectedly, our KRAS-dependent gene signature diverges substantially from the frequently cited Hallmark KRAS signaling gene signature, is driven predominantly through the ERK mitogen-activated protein kinase (MAPK) cascade, and accurately reflects KRAS- and ERK-regulated gene transcription in KRAS-mutant cancer patients. Integration with our ERK-regulated phospho- and total proteome highlights ERK deregulation of the anaphase promoting complex/cyclosome (APC/C) and other components of the cell cycle machinery as key processes that drive pancreatic ductal adenocarcinoma (PDAC) growth. Our findings elucidate mechanistically the critical role of ERK in driving KRAS-mutant tumor growth and in resistance to KRAS-ERK MAPK targeted therapies.

Abstract ECT2 is an activator of RHO GTPases that is essential for cytokinesis. In addition, ECT2 was identified as an oncoprotein when expressed ectopically in NIH/3T3 fibroblasts. However, oncogenic activation of ECT2 resulted from N-terminal truncation, and such truncated ECT2 proteins have not been found in patients with cancer. In this study, we observed elevated expression of full-length ECT2 protein in preneoplastic colon adenomas, driven by increased ECT2 mRNA abundance and associated with APC tumor-suppressor loss. Elevated ECT2 levels were detected in the cytoplasm and nucleus of colorectal cancer tissue, suggesting cytoplasmic mislocalization as one mechanism of early oncogenic ECT2 activation. Importantly, elevated nuclear ECT2 correlated with poorly differentiated tumors, and a low cytoplasmic:nuclear ratio of ECT2 protein correlated with poor patient survival, suggesting that nuclear and cytoplasmic ECT2 play distinct roles in colorectal cancer. Depletion of ECT2 reduced anchorage-independent cancer cell growth and invasion independent of its function in cytokinesis, and loss of Ect2 extended survival in a KrasG12D Apc-null colon cancer mouse model. Expression of ECT2 variants with impaired nuclear localization or guanine nucleotide exchange catalytic activity failed to restore cancer cell growth or invasion, indicating that active, nuclear ECT2 is required to support tumor progression. Nuclear ECT2 promoted ribosomal DNA transcription and ribosome biogenesis in colorectal cancer. These results support a driver role for both cytoplasmic and nuclear ECT2 overexpression in colorectal cancer and emphasize the critical role of precise subcellular localization in dictating ECT2 function in neoplastic cells. Significance: ECT2 overexpression and mislocalization support its role as a driver in colon cancer that is independent from its function in normal cell cytokinesis.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent tumorigenic growth. We determined the role of mutationally activated KRAS, found in ~95% of PDAC, in supporting autophagy. Surprisingly, acute KRAS suppression, which blocks proliferation, was associated with increased rather than decreased autophagic flux. Pharmacologic inhibition of ERK MAPK phenocopied the genetic silencing of KRAS and also increased autophagic flux. We speculated that the loss of ERK-driven metabolic processes may induce compensatory mechanisms to increase autophagy. Addressing a mechanism for ERK suppression-increased autophagy, we describe three mechanisms: 1) ERK inhibition-mediated increased transcription of autophagy and lysosomal genes, 2) ERK inhibitor-induced AMPK activation and suppression of mTOR signaling, and 3) ERK inhibition-facilitated decreased glycolytic flux. We then addressed whether ERK inhibition increased PDAC dependence on autophagy. Supporting this possibility, we found that cotreatment with the autophagy inhibitor chloroquine (CQ) synergistically enhanced ERK inhibitor-mediated antiproliferative activity. Similarly, genetic or pharmacologic inhibition of specific regulators of autophagy also enhanced ERK inhibitor activity. Encouraged by the synergistic relationship between ERK and autophagy inhibition, we performed a CRISPR/Cas-9 mediated genetic loss of function screen in the presence of CQ to determine additional sensitizers as well as mediators of resistance to autophagy inhibition. Top sensitizers included multiple mediators of the DNA damage response. One such sensitizing gene was CHEK1, which encodes the CHK1 serine/threonine kinase, required for checkpoint-mediated cell cycle arrest and activation of DNA repair in response to DNA damage. Accordingly, we showed that treatment with prexasertib, a clinical candidate CHK1 inhibitor, increased autophagic flux in PDAC cells and synergized with CQ to decrease PDAC cell proliferation and increase apoptosis. We conclude that concurrent suppression of multiple metabolic processes, to block compensatory rebound activities, will be needed for effective PDAC treatment. This abstract is also being presented as Poster B07. Citation Format: Kirsten L. Bryant, Jennifer E. Klomp, Ye S. Lee, Clint A. Stalnecker, Kajal R. Grover, A. Cole Edwards, Sen Peng, Mariaelena Pierobon, Emanuel F. Petricoin III, Nhan Tran, Alec C. Kimmelman, Adrienne D. Cox, Channing J. Der. Enhancing the effect of autophagy inhibition for pancreatic cancer treatment [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 PR10.