Alexis Jean Morales

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.

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.

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.

<div>Abstract<p>Primary/intrinsic and treatment-induced acquired resistance limit the initial response rate to and long-term efficacy of direct inhibitors of the KRAS<sup>G12C</sup> 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 KRAS<sup>G12C</sup> inhibitor (G12Ci) treatment in <i>KRAS<sup>G12C</sup></i>-mutant cancer cell lines. Conversely, genetic suppression of <i>YAP1/WWTR1</i> (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 (<i>BIRC5</i>, <i>CDC20, ECT2, FOSL1</i>, and <i>MYC</i>) 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 KRAS<sup>G12C</sup> inhibitor antitumor activity <i>in vitro</i> and prolonged tumor suppression <i>in vivo</i>. 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.</p>Significance:<p>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 KRAS<sup>G12C</sup>-selective inhibitor treatment of <i>KRAS<sup>G12C</sup></i>-mutant cancers.</p><p><i><a href="https://aacrjournals.org/cancerres/article/doi/10.1158/0008-5472.CAN-23-3547" target="_blank">See related commentary by Johnson and Haigis, p. 4005</a></i></p></div>

Abstract Activated mutants of KRAS comprise the major oncogenic drivers in lung (LAC), colorectal (CRC), and pancreatic ductal (PDAC) adenocarcinoma. Recent success in covalently targeting one KRAS mutant (KRASG12C) led to FDA approval of the first anti-KRAS therapy (G12Ci). However, both primary and treatment-induced acquired resistance to G12Ci have been observed. While analyses of relapsed patients have identified reactivation of the key KRAS effector signaling network as a driver of resistance, the mechanisms in ~50% of patients are not known. To identify potential resistance mechanisms, we applied a CRISPR-Cas9 loss-of-function screen targeting the druggable genome. In addition to genes discovered recently in relapsed patients (e.g., PTEN, NF1), we also identified loss of multiple components of the Hippo tumor suppressor pathway as drivers of G12Ci resistance (NF2, LATS1/2, TAOK1/2 and STK3/4). We therefore determined if activation of the functionally related transcriptional co-activators, YAP1 and TAZ, normally inhibited by Hippo signaling, can drive resistance to G12Ci. We first determined that ectopic expression of constitutively activated YAP1/TAZ was sufficient to impair the anti-proliferative and pro-apoptotic effects of G12Ci treatment in KRASG12C-mutant LAC, CRC, and PDAC cell lines. Conversely, genetic suppression of YAP1/TAZ enhanced G12Ci sensitivity. YAP1/TAZ requires association with TEAD and other transcription factors to regulate transcription. We determined that YAP1/TAZ mutants deficient in TEAD binding failed to drive resistance to G12Ci treatment. Further supporting a role for TEAD, both overexpression of a TEAD dominant negative mutant and treatment with pan-TEAD pharmacological inhibitors phenocopied the effects of YAP1/TAZ genetic suppression and sensitized KRASG12C mutant cancer cells to G12Ci. Finally, transcriptional analyses support a model where YAP1/TAZ-TEAD overcomes KRASG12C addiction by restoring a subset of KRAS-dependent gene transcription. In summary, our observations support YAP1/TAZ-TEAD signaling as a novel driver of resistance to KRAS inhibition and support the use of TEAD inhibitors to enhance the anti-tumor efficacy of KRAS-targeted therapies. Citation Format: Alexander C. Edwards, Clint A. Stalnecker, Alexis J. Morales, Khalilah E. Taylor, Jill Hallin, Peter Olson, Tracy T. Tang, Lars Engstrom, Leonard Post, James G. Christensen, Adrienne D. Cox, Channing J. Der. TEAD inhibition overcomes YAP1/TAZ-driven resistance to RAS inhibitors in KRASG12C-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 B009.