Co‐targeting BET and MEK as salvage therapy for MAPK and checkpoint inhibitor‐resistant melanoma
2018; Springer Nature; Volume: 10; Issue: 5 Linguagem: Inglês
10.15252/emmm.201708446
ISSN1757-4684
AutoresIleabett M. Echevarría-Vargas, Patricia Reyes-Uribe, Adam N. Guterres, Xiangfan Yin, Andrew V. Kossenkov, Qin Liu, Gao Zhang, Clemens Krepler, Chaoran Cheng, Zhi Wei, Rajasekharan Somasundaram, Giorgos C. Karakousis, Wei Xu, Jennifer J.D. Morrissette, Yiling Lu, Gordon B. Mills, Ryan J. Sullivan, Benchun Miao, Dennie T. Frederick, Genevieve M. Boland, Keith T. Flaherty, Ashani T. Weeraratna, Meenhard Herlyn, Ravi K. Amaravadi, Lynn M. Schuchter, Christin E. Burd, Andrew E. Aplin, Xiaowei Xu, Jessie Villanueva,
Tópico(s)CAR-T cell therapy research
ResumoResearch Article11 April 2018Open Access Source DataTransparent process Co-targeting BET and MEK as salvage therapy for MAPK and checkpoint inhibitor-resistant melanoma Ileabett M Echevarría-Vargas Ileabett M Echevarría-Vargas Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Patricia I Reyes-Uribe Patricia I Reyes-Uribe Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Adam N Guterres Adam N Guterres Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Xiangfan Yin Xiangfan Yin Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Andrew V Kossenkov Andrew V Kossenkov Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Qin Liu Qin Liu Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Gao Zhang Gao Zhang Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Clemens Krepler Clemens Krepler Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Chaoran Cheng Chaoran Cheng College of Computing Sciences, New Jersey Institute of Technology, Newark, NJ, USA Search for more papers by this author Zhi Wei Zhi Wei College of Computing Sciences, New Jersey Institute of Technology, Newark, NJ, USA Search for more papers by this author Rajasekharan Somasundaram Rajasekharan Somasundaram Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Giorgos Karakousis Giorgos Karakousis Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA Department of Surgery, Hospital of the University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Wei Xu Wei Xu Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Jennifer JD Morrissette Jennifer JD Morrissette Center for Personalized Diagnostics, Hospital of the University of Pennsylvania, University of Pennsylvania, Philadelphia, PA, USA Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Yiling Lu Yiling Lu Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Gordon B Mills Gordon B Mills Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Ryan J Sullivan Ryan J Sullivan Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA Search for more papers by this author Miao Benchun Miao Benchun Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA Search for more papers by this author Dennie T Frederick Dennie T Frederick Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA Search for more papers by this author Genevieve Boland Genevieve Boland Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Keith T Flaherty Keith T Flaherty Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA Search for more papers by this author Ashani T Weeraratna Ashani T Weeraratna Melanoma Research Center, The Wistar Institute, Philadelphia, PA, USA Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Meenhard Herlyn Meenhard Herlyn Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Melanoma Research Center, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Ravi Amaravadi Ravi Amaravadi Department of Surgery, Hospital of the University of Pennsylvania, Philadelphia, PA, USA Department of Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Lynn M Schuchter Lynn M Schuchter Department of Surgery, Hospital of the University of Pennsylvania, Philadelphia, PA, USA Department of Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Christin E Burd Christin E Burd Departments of Molecular Genetics and Cancer Biology and Genetics, Ohio State University, Columbus, OH, USA Search for more papers by this author Andrew E Aplin Andrew E Aplin Department of Cancer Biology and Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Xiaowei Xu Xiaowei Xu Department of Surgery, Hospital of the University of Pennsylvania, Philadelphia, PA, USA Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Jessie Villanueva Corresponding Author Jessie Villanueva [email protected] orcid.org/0000-0001-5701-2609 Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Melanoma Research Center, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Ileabett M Echevarría-Vargas Ileabett M Echevarría-Vargas Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Patricia I Reyes-Uribe Patricia I Reyes-Uribe Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Adam N Guterres Adam N Guterres Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Xiangfan Yin Xiangfan Yin Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Andrew V Kossenkov Andrew V Kossenkov Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Qin Liu Qin Liu Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Gao Zhang Gao Zhang Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Clemens Krepler Clemens Krepler Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Chaoran Cheng Chaoran Cheng College of Computing Sciences, New Jersey Institute of Technology, Newark, NJ, USA Search for more papers by this author Zhi Wei Zhi Wei College of Computing Sciences, New Jersey Institute of Technology, Newark, NJ, USA Search for more papers by this author Rajasekharan Somasundaram Rajasekharan Somasundaram Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Giorgos Karakousis Giorgos Karakousis Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA Department of Surgery, Hospital of the University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Wei Xu Wei Xu Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Jennifer JD Morrissette Jennifer JD Morrissette Center for Personalized Diagnostics, Hospital of the University of Pennsylvania, University of Pennsylvania, Philadelphia, PA, USA Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Yiling Lu Yiling Lu Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Gordon B Mills Gordon B Mills Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Ryan J Sullivan Ryan J Sullivan Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA Search for more papers by this author Miao Benchun Miao Benchun Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA Search for more papers by this author Dennie T Frederick Dennie T Frederick Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA Search for more papers by this author Genevieve Boland Genevieve Boland Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Keith T Flaherty Keith T Flaherty Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA Search for more papers by this author Ashani T Weeraratna Ashani T Weeraratna Melanoma Research Center, The Wistar Institute, Philadelphia, PA, USA Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Meenhard Herlyn Meenhard Herlyn Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Melanoma Research Center, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Ravi Amaravadi Ravi Amaravadi Department of Surgery, Hospital of the University of Pennsylvania, Philadelphia, PA, USA Department of Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Lynn M Schuchter Lynn M Schuchter Department of Surgery, Hospital of the University of Pennsylvania, Philadelphia, PA, USA Department of Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Christin E Burd Christin E Burd Departments of Molecular Genetics and Cancer Biology and Genetics, Ohio State University, Columbus, OH, USA Search for more papers by this author Andrew E Aplin Andrew E Aplin Department of Cancer Biology and Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Xiaowei Xu Xiaowei Xu Department of Surgery, Hospital of the University of Pennsylvania, Philadelphia, PA, USA Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Jessie Villanueva Corresponding Author Jessie Villanueva [email protected] orcid.org/0000-0001-5701-2609 Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA Melanoma Research Center, The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Author Information Ileabett M Echevarría-Vargas1, Patricia I Reyes-Uribe1, Adam N Guterres1, Xiangfan Yin1, Andrew V Kossenkov1, Qin Liu1, Gao Zhang1, Clemens Krepler1, Chaoran Cheng2, Zhi Wei2, Rajasekharan Somasundaram1, Giorgos Karakousis3,4, Wei Xu3, Jennifer JD Morrissette5,6, Yiling Lu7, Gordon B Mills7, Ryan J Sullivan8, Miao Benchun8, Dennie T Frederick8, Genevieve Boland9, Keith T Flaherty8, Ashani T Weeraratna10,11, Meenhard Herlyn1,10, Ravi Amaravadi4,12, Lynn M Schuchter4,12, Christin E Burd13, Andrew E Aplin14, Xiaowei Xu4,7 and Jessie Villanueva *,1,10 1Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA 2College of Computing Sciences, New Jersey Institute of Technology, Newark, NJ, USA 3Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA 4Department of Surgery, Hospital of the University of Pennsylvania, Philadelphia, PA, USA 5Center for Personalized Diagnostics, Hospital of the University of Pennsylvania, University of Pennsylvania, Philadelphia, PA, USA 6Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA, USA 7Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 8Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA 9Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA 10Melanoma Research Center, The Wistar Institute, Philadelphia, PA, USA 11Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, PA, USA 12Department of Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA, USA 13Departments of Molecular Genetics and Cancer Biology and Genetics, Ohio State University, Columbus, OH, USA 14Department of Cancer Biology and Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA *Corresponding author. Tel: +1 215 495 6818; E-mail: [email protected] EMBO Mol Med (2018)10:e8446https://doi.org/10.15252/emmm.201708446 See also: Y Yu (May 2018) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Despite novel therapies for melanoma, drug resistance remains a significant hurdle to achieving optimal responses. NRAS-mutant melanoma is an archetype of therapeutic challenges in the field, which we used to test drug combinations to avert drug resistance. We show that BET proteins are overexpressed in NRAS-mutant melanoma and that high levels of the BET family member BRD4 are associated with poor patient survival. Combining BET and MEK inhibitors synergistically curbed the growth of NRAS-mutant melanoma and prolonged the survival of mice bearing tumors refractory to MAPK inhibitors and immunotherapy. Transcriptomic and proteomic analysis revealed that combining BET and MEK inhibitors mitigates a MAPK and checkpoint inhibitor resistance transcriptional signature, downregulates the transcription factor TCF19, and induces apoptosis. Our studies demonstrate that co-targeting MEK and BET can offset therapy resistance, offering a salvage strategy for melanomas with no other therapeutic options, and possibly other treatment-resistant tumor types. Synopsis Oncogenic NRAS has been deemed undrugabble; an alternative approach is to target NRAS effectors and non-oncogene addictions. Co-targeting MEK and BET synergistically downregulated TCF19 and restrained the growth of NRASMut melanoma tumors including tumors resistant to targeted and immunotherapies. High BRD4 levels are associated with poor outcome in NRASMut melanoma patients, suggesting that BRD4 plays a key role and hence, constitutes a vulnerability that can be therapeutically exploited. Combining BET and MEK inhibitors restrained the growth of NRASMut melanoma and prolonged the survival of mice bearing tumors refractory to MAPK and checkpoint inhibitors with no overt toxicity. Co-targeting BET and MEK mitigates a MAPK- and checkpoint-inhibitor resistance transcriptional signature (IPRES) and downregulates the transcription factor TCF19. TCF19 blockade triggers apoptosis of NRASMut melanoma cells. Downregulation of TCF19 is associated with response to targeted or immunotherapies. Introduction Promising new therapies have emerged for BRAF-mutant melanoma patients, but NRAS-mutant (NRASMut) melanoma, like other RAS-driven tumors, continues to have poor prognosis and limited therapeutic options (Sullivan & Flaherty, 2013; Johnson et al, 2014; Posch et al, 2016; Vu & Aplin, 2016; Wong & Ribas, 2016). Somatic mutations in NRAS account for approximately 26% of all malignant melanoma (Hodis et al, 2012). Additionally, development of secondary NRAS mutations is a frequent mechanism for acquired resistance to BRAF inhibitors (Nazarian et al, 2010; Van Allen et al, 2014). Currently, there are few effective therapeutic options for NRAS-driven melanoma (Johnson & Puzanov, 2015). Clinical studies have evaluated compounds that target RAS effectors, mainly inhibitors of the mitogen-activated protein kinase (MAPK, e.g., MEK inhibitors) and phosphatidylinositol-3 kinase (PI3K) signaling pathways (Kwong & Davies, 2014). However, the therapeutic efficacy of these drugs as single agents is modest (Johnson & Puzanov, 2015). Here, we focused on identifying novel approaches targeting "non-oncogene addictions", which have the potential to induce cell death of NRASMut melanoma when combined with inhibitors of RAS effectors, and explored the efficacy of this strategy in targeted and immune checkpoint inhibitor-resistant melanoma. Melanoma, like other cancers, is driven by genetic and epigenetic alterations. Epigenetic mechanisms implicated in melanomagenesis include altered gene expression via promoter hypo- or hypermethylation, histone modification, chromatin remodeling, and expression of non-coding RNAs (Sarkar et al, 2015). For example, hypermethylation of the CDKN2A tumor suppressor promoter occurs in ~ 20% of primary melanomas and is associated with reduced patient survival (Straume et al, 2002). Oncogenic pathways, like mutant NRAS, can modulate and interact with the cancer epigenome; hence, epigenetic factors could constitute therapeutic targets for NRASMut tumors (Besaratinia & Tommasi, 2014). For example, RAS/MAPK signaling promotes expression of the chromatin remodeler EZH2, which mediates chromatin compaction via histone H3K27 methylation, thereby repressing expression of its target genes (Fujii et al, 2011; Hou et al, 2012). Primary and metastatic melanomas express aberrantly high levels of EZH2, which is associated with poor survival (Zingg et al, 2015). Other epigenetic regulators that are commonly deregulated in melanoma include ATP-dependent chromatin remodelers belonging to the SWI/SNF family, mediators of DNA de/methylation such as TET2 and IDH1/2, and covalent modifiers of histones, including histone deacetylases (HDAC9) and methyltransferases (SETD2) (Hayward et al, 2017). One group of epigenetic regulators that has emerged as promising therapeutic targets for cancer is the Bromodomain and Extra-terminal Domain (BET) family of proteins (Filippakopoulos & Knapp, 2014; Ugurel et al, 2016). Bromodomains are known to bind acetylated lysine residues in the N-terminal tail of histones and non-histone proteins, serving as scaffolds facilitating gene transcription and regulating many cellular processes including DNA replication and cell cycle progression (Shi & Vakoc, 2014). Several small molecule inhibitors of BET proteins (BETi) have been developed as potential anti-cancer agents, and some are currently undergoing clinical investigation in various tumor types including melanoma (NCT02259114, NCT02369029, NCT01987362, NCT02683395, NCT01587703; Brand et al, 2015). In this study, we evaluated the efficacy of BETi when combined with MEKi in restraining NRASMut melanoma and offsetting drug resistance. Our data support the premise that there is a unique synergistic vulnerability exposed by combining BET and MEK inhibitors, and that this combination could be used as a salvage strategy for targeted- and immune checkpoint inhibitor-resistant melanoma. Results BRD4 as a molecular target for NRAS-mutant melanoma To identify therapeutic vulnerabilities in NRAS-mutant melanoma, we explored different potential targets for expression in the TCGA skin cutaneous melanoma dataset (SKCM, Provisional 2017; www.cbioportal.org) (Cerami et al, 2012; Gao et al, 2013). This analysis revealed that high BRD4 mRNA expression was associated with poor patient survival (P = 0.001; Fig 1A) and disease-free survival (P = 0.0008; Fig EV1) in NRAS-mutant melanoma patients, but not in other genetic cohorts (Appendix Fig S1). We next performed immunohistochemical analysis of biopsies from 54 patients with genetically diverse metastatic melanoma and confirmed high expression of BRD4 in NRASMut tumors; BRD4 levels were markedly higher than in tumors harboring mutant BRAF or wild-type for BRAF and NRAS (WT) (Fig 1B and C). To determine the effect of BRD4 blockade, we silenced BRD4 in NRAS-mutant melanoma cells (Appendix Fig S2). Depletion of BRD4 decreased the viability of NRAS-mutant melanoma cells (Fig 1D), but induced only modest apoptosis (Fig 1E). These data suggest that BRD4 plays an important role in NRAS-mutant melanoma and it is necessary for proliferation of these cells. Figure 1. BRD4 is associated with poor patient survival and constitutes a promising target for NRASMut melanoma A. NRAS-mutant melanoma samples (n = 98) were analyzed from the skin cutaneous melanoma TCGA database. Samples were classified into high or low BRD4, BRD3, and BRD2 expressing groups according to the median tissue mRNA expression levels. Overall survival Kaplan–Meier curves for BRD4, BRD3, BRD2 in the NRAS-mutant group are shown; P-values were calculated by long-rank tests comparing the two Kaplan–Meier curves. B, C. Fifty-four patient samples were categorized in subgroups based on mutation status: NRAS (n = 18), BRAF (n = 19), or BRAF-WT/NRAS-WT (WT/WT; n = 17). (B) BRD4 expression was assessed by immunohistochemistry (IHC) in biopsies from patients with metastatic melanoma. BRD4 staining is localized in the nucleus. High magnification (40×) representative images are shown; the scale bar represents 20 μm. (C) BRD4 expression by IHC was scored blindly as low (1), medium (2), or high (3) for each sample. Scatter plot showing BRD4 expression in each subgroup ranging from total absence of BRD4 in the tumor (H-score = 0) to high BRD4 expression (H-score = 300). Each point represents the H-score from a single tumor sample. The horizontal line represents the mean H-score ± SEM. D. BRD4 was silenced using two different hairpins (#4 and #7) in NRAS-mutant melanoma cells (FS13, M93-047, and WM3000). Cell viability was determined by MTT assays 7 or 10 days post-transduction (dpi) and calculated relative to empty vector (EV) control. E. Cell death (Annexin V+/PI+) was analyzed by flow cytometry 7 dpi. Data information: For (D, E), data represent the mean of three independent experiments ± SEM. P-values when comparing each condition with its corresponding control (empty/non-targeting vector) was calculated using Student's t-test are indicated in each figure. Source data are available online for this figure. Source Data for Figure 1 [emmm201708446-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. BRD4 expression levels correlate with disease-free survival in NRAS-mutant melanoma patientsFor all associations of gene expression with survival, patients were split into two groups of high and low BET/BRD expression based on the gene's median expression level. Disease-free survival Kaplan–Meier curves for BRD4, BRD3, BRD2 in the NRAS-mutant group are shown; P-values were calculated by long-rank test comparing the two Kaplan–Meier curves. Download figure Download PowerPoint We next evaluated the effect of JQ-1 (a prototype BET inhibitor) on cell viability and determined the half-maximal inhibitory concentration (IC50) of JQ-1 in NRAS-mutant melanoma cells intrinsically resistant to MAPK inhibitors as well as non-transformed cells (Fig 2A and Appendix Table S1). JQ-1 decreased the viability of NRAS-mutant melanoma cells; moreover, sensitivity to JQ-1 inversely correlated with BRD4 protein levels (Pearson's correlation coefficient = −0.759, P = 0.018; Figs 2A–C and EV2) but not with BRD2 or BRD3 levels (Fig 2D–H). Figure 2. BRD4 expression levels are associated with sensitivity to BET inhibition in NRAS-mutant melanoma A. Cells were treated with increasing doses of JQ-1 for 3 days, and the number of viable cells was determined by MTT assays. Concentrations inhibiting 50% of cell growth (IC50) were calculated at day 3 using GraphPad Prism V5.0a. Bottom panel: Expression of BRD4 was assessed by immunoblotting; β-actin was used as loading control. Representative Western blot is shown. B. Quantification of BRD4 protein levels from immunoblots is shown. Membranes were scanned and quantified using the LI-COR Odyssey system; average protein levels from three independent experiments ± SEM are depicted in the bar graphs. C. Linear correlation between BRD4 protein levels and JQ-1 IC50 was assessed using Pearson's correlation coefficient. Data analysis was performed using Stata version 13. D. Expression of BRD2 and BRD3 was evaluated by Western blot in a panel of NRAS-mutant melanoma and non-transformed cell lines. E, F. Quantification of BRD2 (E) and BRD3 (F) protein levels from immunoblots is shown. Membranes were scanned and quantified using the LI-COR Odyssey system; average protein levels from three independent experiments ± SEM are depicted in the bar graphs. G, H. Linear correlation between BRD2 (G) and BRD3 (H) protein levels and JQ-1 IC50 was assessed using Pearson's correlation coefficient. Data analysis was performed using Stata version 13. Source data are available online for this figure. Source Data for Figure 2 [emmm201708446-sup-0005-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Sensitivity to combinations of JQ-1 with inhibitors of RAS effector pathways in NRAS-mutant melanomaA panel of NRAS-mutant melanoma cells was treated with the indicated doses of JQ1 alone or in combination with the MEKi PD0325901, Cdk4/6i PD0332991, or PI3Ki BKM120 for 5 days. Cell viability was determined by Alamar Blue assay after 5 days of treatment. Relative cell viability (normalized to vehicle-treated cells) is shown for each combination at the indicated doses. Download figure Download PowerPoint To identify more effective therapies for NRAS-mutant melanoma, we evaluated combinations of JQ-1 with inhibitors of RAS effectors that are undergoing clinical evaluation for NRAS-mutant melanoma patients. We selected inhibitors of MEK (PD0325901; PD901), CDK4/6 (PD0332991; PD991), and PI3K (BKM120) (Fig EV2). We found that the MEK inhibitor (MEKi) PD901 potently synergized with JQ-1 (Figs EV2 and EV3A). Treatment of NRAS-mutant melanoma cells with a single dose of JQ-1 (0.5 μM) in combination with PD901 (0.1 μM) substantially impaired colony formation compared to single agent treatment (Fig EV3B). Single doses of either compound as a single agent transiently restrained cell proliferation, but this effect was not sustained at 14 days (Fig EV3C, top panels). In contrast, a single dose of the combination treatment led to sustained inhibition of melanoma cell proliferation (Fig EV3C, bottom panels), whereas it only transiently inhibited the growth of non-transformed cells (Fig EV3C). While BET or MEK inhibitors predominantly induced cytostatic effects as single agents, the combination of both compounds triggered significant apoptosis selectively in NRAS-mutant melanoma cells without affecting non-transformed cells (Fig EV3D). We further explored the efficacy of this combination using the structurally-related BET family inhibitor OTX-015 (MK-8628), which is currently in phase II clinical trials for solid tumors (NCT02698176; NCT02296476; Odore et al, 2016; Amorim et al, 2016) and the FDA-approved MEKi trametinib. Combining trametinib with the clinically relevant BETi OTX-015 more potently induced cell death compared to single agents (Appendix Fig S3), further supporting the notion that the combination of BET and MEK inhibitors elicit cytotoxic effects in NRAS-mutant melanoma. Click here to expand this figure. Figure EV3. The BET inhibitor JQ-1 in combination with the MEK inhibitor PD0325901 synergistically impairs cell proliferation and induces apoptosis of NRAS-mutant melanoma cells NRAS-mutant melanoma cells were treated with JQ-1 alone or in combination with PD901. Cell viability was determined by Alamar Blue assay after 5 days of treatment and calculated relative to DMSO-treated controls. Interaction index and 95% confidence interval (CI) were assessed for each cell line. The upper limit of its 95% CI < 1 was considered significant synergy. Cells were cultured in the presence of DMSO, 0.5 μM JQ-1, 0.1 μM PD901, or combo for 14 days followed by crystal violet staining. Colonies were imaged using a digital camera and quantitated by ImageJ. Representative photographs of crystal violet stained colonies are shown. Cells were treated with DMSO or a single dose of 0.5 μM JQ-1, 0.1 μM PD901, or combo. At day 6, cells were washed to remove the drugs and refed fresh (drug-free) medium. Cells were fixed after 7 or 14 days, stained with crystal violet, and relative number of cells quantified. NRAS-mutant melanoma and non-transformed cells were treated as in (C) for 7 days. Cells were stained with propidium iodide and Annexin V-FITC and analyzed by FACS; % Annexin V+/PI+ cells are shown. Data information: Data represent the mean of three independent experiments ± SEM. Statistically significant differences were determined by Student's t-test; P-values are shown. Download figure Download PowerPoint In similar experiments, we found that although combining JQ-1 with the CDK4/6 inhibitor, PD991, appeared to be effective in NRAS-mutant melanoma (Fig EV2, and Appendix Fig S4A and B), this combination significantly inhibited the proliferation of non-transformed cells (Appendix Fig S4B). Additionally, for the PI3K inhibitor, BKM120, concentrations above 1
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