HDAC Inhibition Enhances the in Vivo Efficacy of MEK Inhibitor Therapy in Uveal Melanoma

Author Manuscript Published OnlineFirst on June 21, 2019; DOI: 10.1158/1078-0432.CCR-18-3382 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. HDAC inhibition enhances the in vivo efficacy of MEK inhibitor therapy in uveal melanoma Fernanda Faião-Flores1, Michael F. Emmons1, Michael A. Durante2, Fumi Kinose3, Biswarup Saha1, Bin Fang4, John M. Koomen4, Srikumar Chellappan1, Silvya Stuchi Maria-Engler5, Uwe Rix3, Jonathan D. Licht6, J. William Harbour2, Keiran S.M. Smalley1* 1The Department of Tumor Biology, The Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL, USA. 2Bascom Palmer Eye Institute, Sylvester Comprehensive Cancer Center and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL, USA. 3Department of Drug Discovery, The Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL, USA 4Department of Molecular Oncology, The Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL, USA. 5Department of Clinical Chemistry and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil. 6Division of Hematology & Oncology, Department of Medicine, University of Florida Health Cancer Center, University of Florida, Gainesville, FL, USA. *To whom correspondence should be addressed Tel: 813-745-8725 Fax: 813-449-8260 e-mail: [email protected] Keywords: uveal melanoma, MEK, HDAC, endothelin, adaptation, resistance. Running title: HDAC-MEK inhibition in uveal melanoma Conflict of interest: Dr. Harbour is a paid consultant for Castle Biosciences, licensee of intellectual property related to uveal melanoma, and he receives royalties from its commercialization. All other authors declare no conflict of interest 1 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 21, 2019; DOI: 10.1158/1078-0432.CCR-18-3382 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ABSTRACT Purpose: The clinical use of MEK inhibitors in uveal melanoma is limited by the rapid acquisition of resistance. The current study has used multi-omics approaches and drug screens to identify the pan-HDAC inhibitor panobinostat as an effective strategy to limit MEK inhibitor resistance. Experimental Design: Mass spectrometry-based proteomics and RNA-Seq was used to identify the signaling pathways involved in the escape of uveal melanoma cells from MEK inhibitor therapy. Mechanistic studies were performed to evaluate the escape pathways identified and the efficacy of the MEK-HDAC inhibitor combination was demonstrated in multiple in vivo models of uveal melanoma. Results: We identified a number of putative escape pathways that were upregulated following MEK inhibition including the PI3K/AKT pathway, ROR1/2 and IGF-1R signaling. MEK inhibition was also associated with increased GPCR expression, particularly the Endothelin B receptor and this contributed to therapeutic escape through ET-3-mediated YAP signaling. A screen of 289 clinical grade compounds identified HDAC inhibitors as potential candidates that suppressed the adaptive YAP and AKT signaling that followed MEK inhibition. In vivo, the MEK-HDAC inhibitor combination outperformed either agent alone, leading to a long-term decrease of tumor growth in both subcutaneous and liver metastasis models and the suppression of adaptive PI3K/AKT and YAP signaling. Conclusions Together our studies have identified GPCR-mediated YAP activation and RTK-driven AKT signaling as key pathways involved in the escape of uveal melanoma cells from MEK inhibition. We further demonstrate that HDAC inhibition is a promising combination partner for MEK inhibitors in advanced uveal melanoma. 2 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 21, 2019; DOI: 10.1158/1078-0432.CCR-18-3382 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Statement of translational relevance: At this time there are no effective therapies for advanced uveal melanoma. One of the most thoroughly explored targeted therapies for uveal melanoma are small molecule inhibitors of MEK. Despite initial clinical responses to MEK inhibition, levels of progression-free survival are very short and the majority of patients fail within 3 months. Here, we used three unbiased platforms (proteomics, RNA-Seq, drug screens) to define the mechanisms by which uveal melanoma cells escaped MEK inhibitor therapy. Our studies identified a complex adaptive response involving G-protein coupled receptor (GPCR)-driven YAP activation and increased receptor tyrosine kinase (RTK)-driven AKT signaling, both of which were suppressed by the pan-HDAC inhibitor panobinostat. The combination of the MEK and HDAC inhibitor was highly effective at limiting therapeutic escape and led to durable anti-tumor responses in both subcutaneous xenograft and liver metastasis models of uveal melanoma. Together our results provide the rationale for the clinical co-targeting MEK and HDACs in advanced uveal melanoma. 3 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 21, 2019; DOI: 10.1158/1078-0432.CCR-18-3382 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Uveal melanoma is a highly aggressive tumor derived from the melanocytes of the eye, with a tendency to metastasize to the liver. Although few patients show signs of disseminated disease at diagnosis (~4%), up to half will eventually succumb to metastatic disease despite successful treatment of the primary tumor (1). The majority of uveal melanomas harbor activating mutations in the small G-proteins GNAQ and GNA11. These mutations (most commonly at Q209L/P) disable the intrinsic GTPase activity, leading to constitutive activation (2, 3). The major downstream signaling target of GNAQ and GNA11 is phospholipase-C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate to the second messengers: inositol triphosphate (IP3) and diacyl glycerol. Protein kinase C (PKC) is activated by these second messengers in GNAQ/GNA11 mutant melanomas (4). Recent work has shown that PKC and the small G-protein RasGRP3 are required for the GNAQ/GNA11-driven activation of the mitogen activated protein kinase (MAPK) pathway and that the majority of uveal melanomas have constitutive MAPK signaling that contributes to cell growth (5, 6). As a single agent, MEK inhibition has some activity against uveal melanoma cell lines, and is associated with reduced cell proliferation in vitro (7, 8). In light of this promising data, and the FDA-approval of MEK inhibitors for BRAF-mutant cutaneous melanoma, a number of clinical trials were undertaken to evaluate MEK inhibitors in uveal melanoma. In an open-label phase II clinical trial of uveal melanoma patients with no history of prior dacabarzine treatment, use of the MEK inhibitor selumetinib was associated with an increase in PFS from 7 to 16 weeks (9). These initially promising findings led to the initiation of a phase III double-blind clinical trial of selumetinib plus dacarbazine, which unfortunately failed to show any increase in PFS compared to dacarbazine alone (10). Despite these disappointing results, current strategies continue to focus upon combination therapies that include MEK inhibition as the backbone. There is promising preclinical data that indicates the combination of a MEK and a PKC inhibitor potently induces apoptosis and suppresses tumor growth in mouse xenograft models (5). Multiple other signal transduction cascades are also activated in uveal melanoma including the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling pathway, which has been implicated in survival and cell migration (11, 12) and the Hippo tumor suppressor pathway, which plays key roles in tissue homeostasis and organ size (13). Under normal physiological conditions, the MST1/2 and LATS1/2 kinases phosphorylate and inactivate 4 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 21, 2019; DOI: 10.1158/1078-0432.CCR-18-3382 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. YAP and TAZ, two transcriptional co-activators implicated in oncogenic transformation (13, 14). In uveal melanoma, GNAQ stimulates YAP through a Hippo-independent mechanism that is initiated through actin polymerization (15). Silencing of GNAQ/GNA11 in uveal melanoma cells led to decreased nuclear accumulation of YAP, with further studies showing that the YAP inhibitor verteporfin abrogates GNAQ/GNA11 driven tumor growth in an orthotopic uveal melanoma ocular xenograft model (15, 16). At this time, little is known about the systems level signaling adaptations of uveal melanoma cells to MEK inhibition. In the present study we used affinity-based protein profiling (ABPP) and RNA-Seq to identify key proteins involved in the adaptation of uveal melanoma cells to MEK inhibition, and identified novel drug combinations to overcome this adaptation. METHODS Reagents RPMI culture medium was purchased from Corning

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