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

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HDAC Inhibition Enhances the in Vivo Efficacy of MEK Inhibitor Therapy in Uveal Melanoma Published OnlineFirst June 21, 2019; DOI: 10.1158/1078-0432.CCR-18-3382 Translational Cancer Mechanisms and Therapy Clinical Cancer Research HDAC Inhibition Enhances the In Vivo Efficacy of MEK Inhibitor Therapy in Uveal Melanoma Fernanda Faiao-Flores~ 1, Michael F. Emmons1, Michael A. Durante2, Fumi Kinose3, Biswarup Saha1, Bin Fang4, John M. Koomen4, Srikumar P. Chellappan1, Silvya Stuchi Maria-Engler5, Uwe Rix3, Jonathan D. Licht6, J. William Harbour2, and Keiran S.M. Smalley1 Abstract Purpose: The clinical use of MEK inhibitors in uveal mel- expression, particularly the endothelin B receptor, and this anoma is limited by the rapid acquisition of resistance. This contributed to therapeutic escape through ET-3–mediated study has used multiomics approaches and drug screens to YAP signaling. A screen of 289 clinical grade compounds identify the pan-HDAC inhibitor panobinostat as an effective identified HDAC inhibitors as potential candidates that sup- strategy to limit MEK inhibitor resistance. pressed the adaptive YAP and AKT signaling that followed Experimental Design: Mass spectrometry–based proteo- MEK inhibition. In vivo, the MEK-HDAC inhibitor combina- mics and RNA-Seq were used to identify the signaling path- tion outperformed either agent alone, leading to a long-term ways involved in the escape of uveal melanoma cells from decrease of tumor growth in both subcutaneous and liver MEK inhibitor therapy. Mechanistic studies were performed to metastasis models and the suppression of adaptive PI3K/AKT evaluate the escape pathways identified, and the efficacy of the and YAP signaling. MEK-HDAC inhibitor combination was demonstrated in mul- Conclusions: Together, our studies have identified GPCR- tiple in vivo models of uveal melanoma. mediated YAP activation and RTK-driven AKT signaling as key Results: We identified a number of putative escape path- pathways involved in the escape of uveal melanoma cells from ways that were upregulated following MEK inhibition, includ- MEK inhibition. We further demonstrate that HDAC inhibi- ing the PI3K/AKT pathway, ROR1/2, and IGF-1R signaling. tion is a promising combination partner for MEK inhibitors in MEK inhibition was also associated with increased GPCR advanced uveal melanoma. Introduction commonly at Q209L/P) disable the intrinsic GTPase activity, leading to constitutive activation (2, 3). The major downstream Uveal melanoma is a highly aggressive tumor derived from the signaling target of GNAQ and GNA11 is phospholipase-C (PLC), melanocytes of the eye, with a tendency to metastasize to the liver. which hydrolyzes phosphatidylinositol 4,5-bisphosphate to the Although few patients show signs of disseminated disease at second messengers: inositol triphosphate (IP3) and diacyl glyc- diagnosis (4%), up to half will eventually succumb to metastatic erol. Protein kinase C (PKC) is activated by these second mes- disease despite successful treatment of the primary tumor (1). The sengers in GNAQ/GNA11–mutant melanomas (4). majority of uveal melanomas harbor activating mutations in the Recent work has shown that PKC and the small G-protein small G-proteins GNAQ and GNA11. These mutations (most RasGRP3 are required for the GNAQ/GNA11–driven activation of the MAPK pathway and that the majority of uveal melanomas have constitutive MAPK signaling that contributes to cell 1The Department of Tumor Biology, The Moffitt Cancer Center & Research Institute, Tampa, Florida. 2Bascom Palmer Eye Institute, Sylvester Comprehen- growth (5, 6). As a single agent, MEK inhibition has some activity sive Cancer Center and Interdisciplinary Stem Cell Institute, University of Miami against uveal melanoma cell lines, and is associated with reduced Miller School of Medicine, Miami, Florida. 3Department of Drug Discovery, The cell proliferation in vitro (7, 8). In light of this promising data, and Moffitt Cancer Center & Research Institute, Tampa, Florida. 4Department of the FDA approval of MEK inhibitors for BRAF-mutant cutaneous Molecular Oncology, The Moffitt Cancer Center & Research Institute, Tampa, melanoma, a number of clinical trials were undertaken to evaluate 5 Florida. Department of Clinical Chemistry and Toxicological Analysis, School of MEK inhibitors in uveal melanoma. In an open-label phase II Pharmaceutical Sciences, University of Sao~ Paulo, Sao~ Paulo, Brazil. 6Division of Hematology & Oncology, Department of Medicine, University of Florida Health clinical trial of patients with uveal melanoma with no history of Cancer Center, University of Florida, Gainesville, Florida. prior dacabarzine treatment, use of the MEK inhibitor selumetinib was associated with an increase in PFS from 7 to 16 weeks (9). Note: Supplementary data for this article are available at Clinical Cancer fi Research Online (http://clincancerres.aacrjournals.org/). These initially promising ndings led to the initiation of a phase III double-blind clinical trial of selumetinib plus dacarbazine, Corresponding Author: Keiran S.M. Smalley, Moffitt Cancer Center, 12902 which unfortunately failed to show any increase in PFS compared Magnolia Drive, Tampa, FL 33612. Phone: 813-745-8725; Fax: 813-449-8260; E-mail: keiran.smalley@moffitt.org with dacarbazine alone (10). Despite these disappointing results, current strategies continue Clin Cancer Res 2019;XX:XX–XX to focus upon combination therapies that include MEK inhibition doi: 10.1158/1078-0432.CCR-18-3382 as the backbone. There is promising preclinical data that indicates Ó2019 American Association for Cancer Research. the combination of a MEK and a PKC inhibitor potently induces www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst June 21, 2019; DOI: 10.1158/1078-0432.CCR-18-3382 Faiao-Flores~ et al. from Cell Signaling Technology, Sigma Chemical Co., Millipore, Translational Relevance and Abcam. The phospho-receptor tyrosine kinase and phospho- At this time, there are no effective therapies for advanced kinase array were purchased from R&D Systems. OptiMEM medi- uveal melanoma. One of the most thoroughly explored tar- um, Lipofectamine 2000, and live/dead viability stain were pur- geted therapies for uveal melanoma is small-molecule inhi- chased from Invitrogen/Life Technologies Corp). siRNA for bitors of MEK. Despite initial clinical responses to MEK ROR1/2, IGF-1R, and YAP were purchased from Dharmacon RNA inhibition, levels of progression-free survival are very short Technologies. Nontargeting siRNA was purchased from Santa and the majority of patients fail within 3 months. Here, we Cruz Biotechnology. The Endothelin-3 Assay Kit was purchased used three unbiased platforms (proteomics, RNA-Seq, drug from IBL. screens) to define the mechanisms by which uveal melanoma cells escaped MEK inhibitor therapy. Our studies identified a Uveal melanoma cell lines complex adaptive response involving G-protein coupled The uveal melanoma cell lines 92.1, Mel270, OMM1, MP41, receptor (GPCR)-driven YAP activation and increased receptor and MM28 were used as described previously (17). All uveal tyrosine kinase (RTK)-driven AKT signaling, both of which melanoma cell lines were cultured in RPMI1640 supplemented were suppressed by the pan-HDAC inhibitor panobinostat. with 10% FBS, L-glutamine, and antibiotics at 5% CO2. All cells The combination of the MEK and HDAC inhibitor was highly were tested for Mycoplasma contamination every month using the effective at limiting therapeutic escape and led to durable Plasmotest-Mycoplasma Detection Test (Invivogen). Last test antitumor responses in both subcutaneous xenograft and liver date: April 18, 2019. Each cell line was authenticated using the metastasis models of uveal melanoma. Together, our results Human short-tandem repeat human cell line authentication provide the rationale for the clinical cotargeting MEK and service (ATCC) and frozen stocks of cells were discarded after HDACs in advanced uveal melanoma. 10 passages. Cell viability assay (MTT assay) Uveal melanoma cells were plated in triplicate wells apoptosis and suppresses tumor growth in mouse xenograft (1 Â 103 cells/well) and treated with increasing concentrations models (5). Multiple other signal transduction cascades are also of MEK inhibitor (trametinib) for 72 hours. Cell viability was activated in uveal melanoma including the PI3K/AKT/mTOR determined using the MTT assay as described previously (18). signaling pathway, which has been implicated in survival and cell migration (11, 12) and the Hippo tumor suppressor pathway, Colony formation assay which plays key roles in tissue homeostasis and organ size (13). A total of 1 Â 103 cells were plated and allowed to attach Under normal physiologic conditions, the MST1/2 and LATS1/2 overnight. The medium and drug/vehicle was replaced every kinases phosphorylate and inactivate YAP and TAZ, two tran- 2 days for 4 weeks. After the specific treatments for each exper- scriptional coactivators implicated in oncogenic transforma- iment, colonies were stained with crystal violet dye, as described tion (13, 14). In uveal melanoma, GNAQ stimulates YAP through previously (18). a Hippo-independent mechanism that is initiated through actin polymerization (15). Silencing of GNAQ/GNA11 in uveal mel- Flow cytometry for apoptosis analysis anoma cells led to decreased nuclear accumulation of YAP, with A total of 1 Â 105 cells were plated and allowed to attach further studies showing that the YAP inhibitor verteporfin abro- overnight. After the specific treatments
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