Published OnlineFirst March 25, 2019; DOI: 10.1158/0008-5472.CAN-18-2331 Cancer Tumor Biology and Immunology Research MITF Expression Predicts Therapeutic Vulnerability to p300 Inhibition in Human Melanoma Edward Kim1, Beth E. Zucconi2,3, Muzhou Wu1, Sarah E. Nocco1, David J. Meyers4, Jean S. McGee1, Samantha Venkatesh1, Daniel L. Cohen1, Estela C. Gonzalez1, Byungwoo Ryu1, Philip A. Cole2,3, and Rhoda M. Alani1 Abstract Histone modifications, largely regulated by histone acetyl- p300–MITF axis driving cell growth that is selectively activated transferases (HAT) and histone deacetylases, have been rec- in human melanomas. Targeted chemical inhibition of p300 ognized as major regulatory mechanisms governing human acetyltransferase activity using a potent and selective catalytic diseases, including cancer. Despite significant effort and recent p300/CBP inhibitor demonstrated significant growth inhibi- advances, the mechanism by which the HAT and transcrip- tory effects in melanoma cells expressing high levels of tional coactivator p300 mediates tumorigenesis remains MITF. Collectively, these data confirm the critical role of the unclear. Here, we use a genetic and chemical approach to p300–MITF–FOXM1 axis in melanoma and support p300 as a identify the microphthalmia-associated transcription factor promising novel epigenetic therapeutic target in human (MITF) as a critical downstream target of p300 driving human melanoma. melanoma growth. Direct transcriptional control of MITF by p300-dependent histone acetylation within proximal gene Significance: These results show that MITF is a major regulatory regions was coupled to cellular proliferation, sug- downstream target of p300 in human melanoma whose gesting a significant growth regulatory axis. Further analysis expression is predictive of melanoma response to small- revealed forkhead box M1 (FOXM1) as a key effector of the molecule inhibition of p300 HAT activity. Introduction the transcriptional activity of target genes, and have demonstrated complex roles in determining cell fate in both normal and Dynamic changes in histone acetylation are major regulatory diseased tissues (3). p300 has notably been found to be amplified mechanisms governing gene transcription in human diseases in subsets of human melanomas and has been implicated as an including cancers (1, 2). Large-scale analyses of chromatin mod- oncogene in this and other malignancies (4). Our group previ- ifications in human cancers have prompted the development of ously reported the development of a small-molecule inhibitor of new epigenetic therapies including histone deacetylase (HDAC) p300/CBP HAT, C646, (5–7) and its potential therapeutic efficacy and DNA-methyltransferase inhibitors (DNMTi). Among newly in cancer, including myeloid leukemia and melanoma (5); how- identified epigenetic targets, histone acetyltransferases (HAT) and ever, its modest potency and electrophilic functionality have their therapeutic efficacies remain unclear. The transcriptional limited its pharmacologic applications, necessitating the search coactivator p300 possesses both lysine acetyltransferase (KAT) for more potent and specific reagents targeting p300. More enzymatic activity, as well as scaffolding abilities, which regulate recently, investigators have used a virtual ligand screen to identify A-485, a potent, selective, and drug-like catalytic p300/CBP inhibitor that targets lineage-specific tumors including hemato- 1Department of Dermatology, Boston University School of Medicine, Boston, logic malignancies and prostate cancers (8). Massachusetts. 2Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts. 3Department of Biological Chemistry Previously, p300 was found to serve as a coactivator for the and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts. Microphthalmia-associated transcription factor (MITF; refs. 9, 10), 4Department of Pharmacology & Molecular Sciences, Johns Hopkins School of regulating the expression of a subset of downstream target genes Medicine, Baltimore, Maryland. through consensus DNA binding E-box and M-box motifs (11). In Note: Supplementary data for this article are available at Cancer Research addition, varying levels of MITF expression have been associated Online (http://cancerres.aacrjournals.org/). with melanoma development and progression, and have Corresponding Authors: Rhoda M. Alani, Boston University School of Medicine, been found to contribute to BRAF-inhibitor therapeutic resis- 609 Albany Street, Suite J-507, Boston, MA 02118. Phone: 617-358-9770; tance (12–15). Transcriptional regulators such as SOX10, PAX3, Fax: 617-358-9769; E-mail: [email protected]; and Philip A. Cole, Division of Genet- CREB, LEF-1, and ATF2 have been shown to control MITF expres- ics, Department of Medicine, Brigham and Women's Hospital; Department of sion, although the precise mechanisms remain to be elucidat- Biological Chemistry and Molecular Pharmacology, Harvard Medical School, ed (10, 16–23). Given the importance of MITF in melanoma Boston, MA 02115. Phone: 617-525-5208; E-mail: [email protected] biology, and the significance of p300 acetyltransferase activity in doi: 10.1158/0008-5472.CAN-18-2331 regulating melanoma cell growth, we sought to determine the role Ó2019 American Association for Cancer Research. of p300 in melanoma development and progression and its www.aacrjournals.org 2649 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst March 25, 2019; DOI: 10.1158/0008-5472.CAN-18-2331 Kim et al. potential relevance to the master melanocyte differentiation gene, medium to the desired concentration. An equal amount of DMSO MITF. Here, we explore the functional role of p300 in human was used as the vehicle control. melanoma using both a genetic and chemical approach using the fi à potent and speci c inhibitor of p300/CBP HAT, A-485 , to further Cell-cycle analysis fi dissect the speci c functional contributions of p300 HAT activity Cells were stained with propidium iodide according to a to melanoma development and progression. These studies have published protocol (24). Data acquisition and analysis were allowed us to identify MITF as a critical downstream effector of performed on a FACSCalibur Flow Cytometer with the CellQuest p300 HAT activity and important stimulus for melanoma growth. Software (BD Biosciences). In addition, bioinformatic analysis of MITF target genes allowed us to identify forkhead box M1 (FOXM1) as a specific target of the p300–MITF signaling axis. Analysis of primary human melanoma Microarray studies genetic data from The Cancer Genome Atlas (TCGA) database RNA from melanoma cells transduced with either shp300 or identified specific and exclusive alterations in this signaling axis in scrambled lentiviruses was purified using the Qiagen RNeasy Plus primary human melanomas, suggesting a critical growth regula- Kit. Samples were submitted to Boston University Microarray and tory pathway. Moreover, chemical inhibition of p300/CBP HAT Sequencing Resource Core Facility for analysis on the Affymetrix activity by A-485à was found to significantly inhibit proliferation GeneChip Human Gene 2.0 ST. The initial data processing and of multiple melanoma lines in an MITF-dependent fashion, normalizations were performed by the core facility. The gene supporting the role of p300 as a promising therapeutic target in ontology analysis was performed with Ingenuity Pathway Anal- human melanoma and promoting a therapeutic strategy for p300 ysis (Qiagen). HAT inhibitor therapies in tumors expressing high levels of MITF. qRT-PCR Materials and Methods cDNA was synthesized using the Superscript III First Strand Synthesis System (Invitrogen). qRT-PCR was performed using the Cell culture SYBR Green PCR Master Mix (Applied Biosystems/Invitrogen) as Melanoma cell lines were kindly provided by Dr. Meenhard described previously (5). The primer pairs were designed using the Herlyn at the Wistar Institute (Philadelphia, PA) and Dr. Levi NCBI PrimerBlast tool and individually optimized. Gene expres- Garraway at the Broad Institute (Cambridge, MA). Melanoma sion values were determined with the DDCt method. GAPDH was cells were maintained in DMEM. The medium was supplemented used as an internal control. The absolute copy number of MITF with 10% FBS, 1% penicillin–streptomycin, and 1% L-glutamine. was determined by using a standard curve generated from a 500 bp The media, penicillin-streptomycin, and L-glutamine, FBS were MITF amplicon with varying concentrations. The list of primers purchased from Invitrogen. All cell lines were grown at 37 Cinan used in this study is provided in the Supplementary Information. atmosphere containing 5% CO2. Plasmids and transfection Chromatin immunoprecipitation assay followed by qPCR Lentiviral expression vectors pCW45-GFP and pCW45-MITF Chromatin immunoprecipitation (ChIP) was performed on were kindly provided by Dr. David Fisher (Massachusetts General the basis of a previously described protocol (5). The antibodies Hospital, Boston, MA). pGL2-MITF and control vector were used in the study include normal rabbit and mouse IgG (sc-2027x generous gifts from Dr. Hans Widlund (Brigham and Women's and sc-2025, Santa Cruz Biotechnology), or antibodies against Hospital, Boston, MA). The Lentiviral pLKO1-based short hairpin p300 (sc-585, Santa Cruz Biotechnology), histone H3K18 RNA (shRNA) vectors targeting p300 and MITF were purchased (13998, Cell Signaling Technology),