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Galpha and MEK1/2 inhibition in combination with Baf A1 and CQ in MP41 cells

Response of uveal melanoma cell lines to MEK1/2 inhibition

Trametinib in combination with macropinocytosis or mitophagy inhibition

Bioluminescent images of hepatic tumors

YAP requirement in MP41 cells

Supplementary Materials and Methods

Purpose:Mutational activation of GNAQ or GNA11 (GNAQ/11), detected in >90% of uveal melanomas, leads to constitutive activation of oncogenic pathways, including MAPK and YAP. To date, chemo- or pathway-targeted therapies, either alone or in combination, have proven ineffective in the treatment of patients with metastatic uveal melanoma.Experimental Design:We tested the efficacy of chloroquine or hydroxychloroquine, in combination with MAPK pathway inhibition in GNAQ/11-mutated cells in vitro and in vivo and identified mechanisms of MEK1/2 inhibitor plus chloroquine-induced cytotoxicity.Results:Inhibition of GNAQ/11-mediated activation of MAPK signaling resulted in the induction of autophagy. Combined inhibition of Gα and autophagy or lysosome function resulted in enhanced cell death. Moreover, the combination of MEK1/2 inhibition, using trametinib, with the lysosome inhibitor, chloroquine, also increased cytotoxicity. Treatment of mice bearing GNAQ/11-driven melanomas with trametinib plus hydroxychloroquine resulted in inhibition of tumor growth and significantly prolonged survival. Interestingly, lysosomal- and autophagy-specific inhibition with bafilomycin A1 was not sufficient to promote cytotoxicity in combination with trametinib. However, the addition of YAP inhibition with trametinib plus bafilomycin A1 resulted in cell death at comparable levels to trametinib plus chloroquine (T/CQ) treatment. Furthermore, T/CQ-treated cells displayed decreased YAP nuclear localization and decreased YAP transcriptional activity. Expression of a constitutively active YAP5SA mutant conferred resistance to T/CQ-induced cell death.Conclusions:These results suggest that YAP, MEK1/2, and lysosome function are necessary and critical targets for the therapy of GNAQ/11-driven melanoma, and identify trametinib plus hydroxychloroquine as a potential treatment strategy for metastatic uveal melanoma.

YAP activation in OMM1 cells

Immunofluorescence analysis of autophagy in OMM2.5 cells with or without trametinib

Mutationally activated BRAF is detected in approximately 7% of human lung adenocarcinomas, with BRAFT1799A serving as a predictive biomarker for treatment of patients with FDA-approved inhibitors of BRAFV600E oncoprotein signaling. In genetically engineered mouse (GEM) models, expression of BRAFV600E in the lung epithelium initiates growth of benign lung tumors that, without additional genetic alterations, rarely progress to malignant lung adenocarcinoma. To identify genes that cooperate with BRAFV600E for malignant progression, we used Sleeping Beauty–mediated transposon mutagenesis, which dramatically accelerated the emergence of lethal lung cancers. Among the genes identified was Rbms3, which encodes an RNA-binding protein previously implicated as a putative tumor suppressor. Silencing of RBMS3 via CRISPR/Cas9 gene editing promoted growth of BRAFV600E lung organoids and promoted development of malignant lung cancers with a distinct micropapillary architecture in BRAFV600E and EGFRL858R GEM models. BRAFV600E/RBMS3Null lung tumors displayed elevated expression of Ctnnb1, Ccnd1, Axin2, Lgr5, and c-Myc mRNAs, suggesting that RBMS3 silencing elevates signaling through the WNT/β-catenin signaling axis. Although RBMS3 silencing rendered BRAFV600E-driven lung tumors resistant to the effects of dabrafenib plus trametinib, the tumors were sensitive to inhibition of porcupine, an acyltransferase of WNT ligands necessary for their secretion. Analysis of The Cancer Genome Atlas patient samples revealed that chromosome 3p24, which encompasses RBMS3, is frequently lost in non–small cell lung cancer and correlates with poor prognosis. Collectively, these data reveal the role of RBMS3 as a lung cancer suppressor and suggest that RBMS3 silencing may contribute to malignant NSCLC progression.Significance:Loss of RBMS3 cooperates with BRAFV600E to induce lung tumorigenesis, providing a deeper understanding of the molecular mechanisms underlying mutant BRAF-driven lung cancer and potential strategies to more effectively target this disease.

Validation of hepatic tumor model

Supplementary Data from Transposon Mutagenesis Reveals RBMS3 Silencing as a Promoter of Malignant Progression of BRAFV600E-Driven Lung Tumorigenesis

Purpose:Mutational activation of GNAQ or GNA11 (GNAQ/11), detected in >90% of uveal melanomas, leads to constitutive activation of oncogenic pathways, including MAPK and YAP. To date, chemo- or pathway-targeted therapies, either alone or in combination, have proven ineffective in the treatment of patients with metastatic uveal melanoma.Experimental Design:We tested the efficacy of chloroquine or hydroxychloroquine, in combination with MAPK pathway inhibition in GNAQ/11-mutated cells in vitro and in vivo and identified mechanisms of MEK1/2 inhibitor plus chloroquine-induced cytotoxicity.Results:Inhibition of GNAQ/11-mediated activation of MAPK signaling resulted in the induction of autophagy. Combined inhibition of Gα and autophagy or lysosome function resulted in enhanced cell death. Moreover, the combination of MEK1/2 inhibition, using trametinib, with the lysosome inhibitor, chloroquine, also increased cytotoxicity. Treatment of mice bearing GNAQ/11-driven melanomas with trametinib plus hydroxychloroquine resulted in inhibition of tumor growth and significantly prolonged survival. Interestingly, lysosomal- and autophagy-specific inhibition with bafilomycin A1 was not sufficient to promote cytotoxicity in combination with trametinib. However, the addition of YAP inhibition with trametinib plus bafilomycin A1 resulted in cell death at comparable levels to trametinib plus chloroquine (T/CQ) treatment. Furthermore, T/CQ-treated cells displayed decreased YAP nuclear localization and decreased YAP transcriptional activity. Expression of a constitutively active YAP5SA mutant conferred resistance to T/CQ-induced cell death.Conclusions:These results suggest that YAP, MEK1/2, and lysosome function are necessary and critical targets for the therapy of GNAQ/11-driven melanoma, and identify trametinib plus hydroxychloroquine as a potential treatment strategy for metastatic uveal melanoma.

NTRK1 gene fusions encode oncoprotein (TRKA) kinases that are actionable drivers of a number of human malignancies including a subset of pancreatic cancers. Here, we describe a new preclinical model of pancreatic cancer in which a TPR-NTRK1 fusion gene is expressed in conditionally immortalized mouse pancreatic ductal epithelial (IMPE) cells. Indeed, expression of TPR-NTRK1 in IMPE cells is sufficient to transform these cells without any additional engineered genetic alterations. Other oncogenes tested in this model include KRAS and EGFR, but neither were unable to replicate this one-hit model of transformation. This striking result may be explained by the differential levels of MAPK pathway activation produced by different oncogenes, and a “goldilocks zone” required to drive pancreatic cellular transformation. These data suggest NTRK1 is a unique and powerful driver of transformation in pancreatic epithelial cells. NTRK1-driven IMPE cells form fast growing tumors in immunocompromised mice both subcutaneously, and orthotopically in the mouse pancreas. Importantly, TPR-NTRK1-driven IMPE cell derived tumors are exquisitely sensitive to targeted inhibition of TRKA kinase activity. However, as also observed in pancreatic cancer patients treated with entrectinib, this therapeutic response is transient and tumors eventually develop resistance to single-agent TRKA inhibition. This model revealed that short drug treatments (2 hours) are sufficient to block autophosphorylation of the oncoprotein target, as well as blockade of the downstream MAPK signaling pathway. Critically, we observed that long-term drug treatments (24 hours) result in downstream MAPK pathway reactivation despite maintained drug activity against the target, TRKA. Consistent with this, the targeted inhibition of TRKA, which transiently blocks MAPK signaling results in a subsequent elevation of BIM protein expression, a pro-apoptotic member of the BCL2 family. Indeed, CRISPR/CAS9-mediated abrogation of BIM expression significantly diminishes the response of TPR-NTRK1-driven IMPE tumors to inhibition of TRKA kinase activity. In an effort to delay acquired resistance to single-agent TRKA inhibitors, we reasoned that combined vertical inhibition of both TRKA and MAPK signaling with the combination of an TRKA plus a MEK1/2 inhibitor might increase the durability of response in this model and counteract the observed pathway reactivation. To that end, we treated mice bearing TPR-NTRK-driven IMPE tumors with either a TRKA inhibitor, a MEK1/2 inhibitor or the combination of both therapeutic agents. Mice treated with the entrectinib/cobimetinib combination revealed the durability of the tumor regression was significantly increased compared to single-agent entrectinib. Importantly, no toxicity or adverse side effects were observed in mice receiving the combination. Additionally, the combined therapies also induced more stable expression of BIM than the single-agent, by delaying degradation of BIM by the proteasome following BIM phosphorylation. Collectively, these data provide a compelling rationale for the clinical deployment of the targeted combination of TRKA inhibition plus MEK1/2 inhibition first-line in patients whose cancers are driven by NTRK1 gene fusions. Citation Format: Aria Vaishnavi, Michael T. Scherzer, Conan Kinsey, Ignacio Garrido-Laguna, Martin McMahon. Combined TRKA and MEK1/2 Inhibition Forestalls the Onset of Acquired Resistance in a New Preclinical Model of NTRK1+ Pancreatic Cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3769.

GNAQ and GNA11 (GNAQ/11) mutations are found in less than 2% of all melanoma, but more than 80% of uveal melanoma. Mutations in these Gα proteins lead to constitutive activation of multiple oncogenic pathways, including MAPK (RAF->MEK1/2->ERK1/2) and YAP signaling. Metastatic uveal melanoma is refractory to all forms of pharmacologic treatment, such as FDA-approved targeted therapies inhibiting MEK1/2 (i.e. trametinib and binimetinib). We show that combining MEK1/2 inhibitors with 4-aminoquinoline antimalarials, chloroquine or hydroxychloroquine, resulted in synergistic and apoptosis-mediated cytotoxicity in GNAQ/11 mutant uveal melanoma cell lines. Interestingly, in contrast to our previous work in pancreatic and other RAS-driven cancers, the lysosomotropic role of chloroquine was not sufficient to promote cytotoxicity with MEK1/2 inhibitors, as neither lysosome inhibition with Bafilomycin A1 nor autophagy-specific and macropinocytosis-specific inhibition yielded enhanced cell death in combination with MEK1/2 inhibition. We then found that chloroquine prevented nuclear localization of the transcriptional coactivator, YAP, suggesting a novel mechanism of chloroquine. YAP inhibition combined with MEK1/2 inhibition enhanced cell death only in the presence of Bafilomycin A1. Gα-specific inhibition (inhibiting YAP and MAPK) combined with Bafilomycin A1 yielded similar results. This implies that the ability of chloroquine to inhibit both YAP signaling and lysosome function is required for promoting cell death in the presence of MEK1/2 inhibition. For in vivostudies, we utilized a hepatic colonization model using luciferized human metastatic uveal melanoma cell lines, OMM2.5 and OMM1. Daily treatment of trametinib with hydroxychloroquine in combination resulted in delayed tumor growth and increased overall survival compared to either treatment as monotherapy or chemotherapy. These findings were also recapitulated in an immunocompetent mouse model in which immortalized mouse melanocytes (Melan-A) with either a GNAQ or GNA11 activating mutation were implanted into syngeneic C57BL/6 mice. Our findings identify a novel mechanism of chloroquine and suggest a potentially effective strategy combining two FDA-approved drugs for the treatment of metastatic uveal melanoma. Citation Format: Amanda Truong, Michael Scherzer, Conan Kinsey, John Michael Sanchez, Jae Hyuk Yoo, Jackson Richards, Donghan Shin, Phaedra Ghazi, Michael Onken, Kendall Blumer, Shannon Odelberg, Martin McMahon. Chloroquine synergizes with MEK1/2 targeted therapy through dual YAP and lysosomal inhibition in GNAQ/11 mutant uveal melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1890.