How theKRASoncogene 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.

Primary/intrinsic and treatment-induced acquired resistance limit the initial response rate to and long-term efficacy of direct inhibitors of the KRASG12C mutant in cancer. To identify potential mechanisms of resistance, we applied a CRISPR/Cas9 loss-of-function screen and observed loss of multiple components of the Hippo tumor suppressor pathway, which acts to suppress YAP1/TAZ-regulated gene transcription. YAP1/TAZ activation impaired the antiproliferative and proapoptotic effects of KRASG12C inhibitor (G12Ci) treatment in KRASG12C-mutant cancer cell lines. Conversely, genetic suppression of YAP1/WWTR1 (TAZ) enhanced G12Ci sensitivity. YAP1/TAZ activity overcame KRAS dependency through two distinct TEAD transcription factor–dependent mechanisms, which phenocopy KRAS effector signaling. First, TEAD stimulated ERK-independent transcription of genes normally regulated by ERK (BIRC5, CDC20, ECT2, FOSL1, and MYC) to promote progression through the cell cycle. Second, TEAD caused activation of PI3K–AKT–mTOR signaling to overcome apoptosis. G12Ci treatment-induced acquired resistance was also caused by YAP1/TAZ-TEAD activation. Accordingly, concurrent treatment with pharmacologic inhibitors of TEAD synergistically enhanced KRASG12C inhibitor antitumor activity in vitro and prolonged tumor suppression in vivo. In summary, these observations reveal YAP1/TAZ-TEAD signaling as a crucial driver of primary and acquired resistance to KRAS inhibition and support the use of TEAD inhibitors to enhance the antitumor efficacy of KRAS-targeted therapies. Significance: YAP1/TAZ-TEAD activation compensates for loss of KRAS effector signaling, establishing a mechanistic basis for concurrent inhibition of TEAD to enhance the efficacy of KRASG12C-selective inhibitor treatment of KRASG12C-mutant cancers. See related commentary by Johnson and Haigis, p. 4005

To delineate the mechanisms by which the ERK1 and ERK2 mitogen-activated protein kinases support mutant KRAS–driven cancer growth, we determined the ERK-dependent phosphoproteome in KRAS-mutant pancreatic cancer. We determined that ERK1 and ERK2 share near-identical signaling and transforming outputs and that the KRAS-regulated phosphoproteome is driven nearly completely by ERK. We identified 4666 ERK-dependent phosphosites on 2123 proteins, of which 79 and 66%, respectively, were not previously associated with ERK, substantially expanding the depth and breadth of ERK-dependent phosphorylation events and revealing a considerably more complex function for ERK in cancer. We established that ERK controls a highly dynamic and complex phosphoproteome that converges on cyclin-dependent kinase regulation and RAS homolog guanosine triphosphatase function (RHO GTPase). Our findings establish the most comprehensive molecular portrait and mechanisms by which ERK drives KRAS-dependent pancreatic cancer growth.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent growth. Inhibition of the KRAS–RAF–MEK–ERK pathway enhances autophagic flux and dependency, and concurrent treatment with the nonspecific autophagy inhibitor chloroquine (CQ) and ERK–MAPK pathway inhibitors can synergistically block PDAC growth. However, CQ is limited in terms of specificity and potency. To find alternative anti-autophagy strategies, in this study, we performed a CRISPR-Cas9 loss-of-function screen in PDAC cell lines that identified the lipid kinase phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) as a growth-promoting gene. PIKfyve inhibition by the small molecule apilimod resulted in durable growth suppression, with much greater potency than CQ treatment. PIKfyve inhibition caused lysosomal dysfunction, reduced autophagic flux, and led to the accumulation of autophagy-related proteins. Furthermore, PIKfyve inhibition blocked the compensatory increases in autophagic flux associated both with MEK inhibition and with direct RAS inhibition. Accordingly, combined inhibition of PIKfyve and the RAS–MAPK pathway showed robust growth suppression across a panel of KRAS-mutant PDAC models. Growth suppression was due, in part, to potentiated cell-cycle arrest and induction of apoptosis following loss of inhibitor of apoptosis proteins. These findings indicate that concurrent inhibition of RAS and PIKfyve is a synergistic, cytotoxic combination that may represent a therapeutic strategy for PDAC. Significance: PIKfyve inhibition effectively blocks autophagy in multiple models of KRAS-mutant pancreatic cancer and can synergize with inhibitors of members of the RAS–MAPK pathway, providing an effective combination strategy for pancreatic cancer.

Protein arginine methyltransferase 5 (PRMT5) is a synthetic lethal target in MTAP-deleted (MTAP-del) cancers. The MTA-cooperative PRMT5 inhibitor BMS-986504 exhibited potent and selective anti-tumor activity in MTAP-del preclinical models and demonstrated activity in MTAP-del patients without the toxicity associated with previous PRMT5 inhibitors. Here, we focused on pancreatic ductal adenocarcinoma (PDAC), ~22% of which are MTAP-del, and demonstrated that BMS-986504 suppressed PRMT5 function and cell growth in MTAP-del cells and xenograft models. CRISPR/Cas9 loss-of-function screens implicated co-targeting KRAS as a combination strategy. Concurrent inhibition of PRMT5 and KRASG12C/D enhanced and prolonged suppression of PDAC growth. RNA-sequencing analysis revealed that PRMT5 inhibition disrupted RNA splicing of genes essential for PDAC growth. While PRMT5 and KRAS regulated distinct transcriptomes, they converged on pathways governing cancer cell growth and expression of PDAC-essential genes. These findings provide rationale for combined inhibition of PRMT5 and KRAS in MTAP-deleted/KRAS-mutant PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is a highly KRAS-addicted cancer. Although KRAS-mutant PDAC is susceptible to treatment with direct RAS inhibitors, drug resistance is an ongoing challenge. KRAS-driven PDAC is dependent on nutrient scavenging pathways such as macropinocytosis to fuel increased metabolic demands. Here, we report on the dynamics of macropinocytosis in response to genetic suppression and pharmacologic inhibition of KRAS and the RAS–ERK mitogen-activated protein kinase pathway. We show that macropinocytosis is transiently suppressed following acute genetic suppression of KRAS but is rapidly restored and even enhanced under prolonged RAS pathway inhibition. Furthermore, we demonstrate that human PDAC cell lines that have acquired resistance to RAS inhibitors, as well as cell lines derived from KPC tumors with acquired resistance to RAS inhibitors display markedly elevated macropinocytosis. RAS-inhibitor resistant cell lines also display increased abundance of macropinocytosis-related proteins and enhanced albumin uptake and sensitivity to albumin-bound paclitaxel. Mechanistically, this adaptive phenotype is mediated through both phosphoinositide 3-kinase (PI3K)-dependent and -independent pathways that converge on the activation of RAC1. Our results demonstrate a previously unrecognized link between RAS pathway inhibitor resistance and upregulation of macropinocytosis, underscoring the importance of further elucidating metabolic reprogramming in RASi-R PDAC. Significance: Macropinocytosis is upregulated following prolonged RAS-inhibitor treatment and in RASi-R models of PDAC via several heterogeneous mechanisms that converge on RAC1 activation and render RASi-R PDAC cell lines more sensitive to nab-paclitaxel.