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Targeting Lineage-Specific MITF Pathway in Human Melanoma Cell Lines by A-485, the Selective Small Molecule Inhibitor of P300/CBP

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Targeting Lineage-Specific MITF Pathway in Human Melanoma Cell Lines by A-485, the Selective Small Molecule Inhibitor of P300/CBP Author Manuscript Published OnlineFirst on September 28, 2018; DOI: 10.1158/1535-7163.MCT-18-0511 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Targeting lineage-specific MITF pathway in human melanoma cell lines by A-485, the selective small molecule inhibitor of p300/CBP Rui Wang1,2, Yupeng He1, Valerie Robinson1, Ziping Yang1, Paul Hessler1, Loren M. Lasko1, Xin Lu1, Anahita Bhathena1, Albert Lai1, Tamar Uziel1, Lloyd T. Lam1,3 1Oncology Discovery, AbbVie, 1 N Waukegan Road, North Chicago, IL 60064-6101, USA. 2Former AbbVie employee Running title: p300/CBP inhibition targets MITF in melanoma cell lines Keywords: p300/CBP inhibitor, melanoma, MITF, A-485, senescence Word count: 3,040 (excluding the abstract, references and legends) References: 29 3Correspondence: Lloyd T. Lam, Oncology Discovery, AP10-214, Dept. R4CD 1 North Waukegan Road North Chicago, IL 60064-6098, USA. Phone: 847-937-5585 Fax: 847-935-4994 E-mail: [email protected] Financial information Y.H., V.R., Z.Y., P.H., L.M.L., X.L, A.B., A.L., T.U., L.T.L. are employees of AbbVie. R.W. was an employee of AbbVie at the time of the study. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the manuscript. R.W., Y.H., V.R., Z.Y., P.H., L.M.L., X.L, A.B., A.L., T.U., L.T.L. have nothing to disclose. Downloaded from mct.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 28, 2018; DOI: 10.1158/1535-7163.MCT-18-0511 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Metastatic melanoma is responsible for approximately 80 percent of deaths from skin cancer. Microphthalmia-associated transcription factor (MITF) is a melanocyte- specific transcription factor that plays an important role in the differentiation, proliferation and survival of melanocytes as well as in melanoma oncogenesis. MITF is amplified in approximately 15% of metastatic melanoma patients. However, no small molecule inhibitors of MITF currently exist. MITF was shown to associate with p300/CBP, members of the KAT3 family of histone acetyltransferase (HAT). p300/CBP regulate a wide range of cellular events such as senescence, apoptosis, cell cycle, DNA damage response and cellular differentiation. p300/CBP act as transcriptional co-activators for multiple proteins in cancers, including oncogenic transcription factors such as MITF. In this study, we showed that our novel p300/CBP catalytic inhibitor, A-485, induces senescence in multiple melanoma cell lines, similar to silencing expression of EP300 (encodes p300) or MITF. We did not observe apoptosis and increase invasiveness upon A-485 treatment. A-485 regulates the expression of MITF and its downstream signature genes in melanoma cell lines undergoing senescence. In addition, expression and copy number of MITF is significantly higher in melanoma cell lines that undergo A-485-induced senescence than resistant cell lines. Finally, we showed that A-485 inhibits histone-H3 acetylation but did not displace p300 at promoters of MITF and its putative downstream genes. Taken together, we provide evidence that p300/CBP inhibition suppressed the melanoma driven transcription factor, MITF and could be further exploited as a potential therapy for treating melanoma. Introduction Melanoma is one of the most frequent cancers with increased incidence in the Western societies. Melanoma is responsible for 80 percent of deaths from skin cancer. The median overall survival of patients with advanced-stage melanoma has increased from approximately 9 months 2 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 28, 2018; DOI: 10.1158/1535-7163.MCT-18-0511 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. before 2011 to at least 2 years (1). Melanoma has been studied extensively and is found to be related to the uncontrolled proliferation of melanocytes (2). Although there have been recent successes in developing effective treatments for melanoma such as targeted therapies against BRAF and MEK kinases, and immunotherapy, advanced melanoma still has a high mortality rate (3,4). The identification of a constitutively active mitogen-activated protein kinase (MAPK) pathway due to BRAF V600E mutation in about 40% melanoma has led to the development of selective BRAF inhibitors such as vemurafenib (5). Although great initial clinical benefits were observed in patients with BRAF V600E mutation, the long-term benefit of vemurafenib has been compromised due to the development of resistance to the therapy. Subsequent study indicated that acquired resistance is caused by additional mutations which can re-activate the MAPK pathway directly or indirectly (6,7). Interestingly, ~15-20% patients with BRAF mutation do not respond to vemurafenib and the mechanism underling the intrinsic resistance remains an intense area of investigation (7). Recently, it was shown that microphthalmia-associated transcription factor (MITF)-low melanoma is associated with intrinsic resistance to multiple targeted agents including BRAF inhibitor (8,9). MITF is a melanocyte-specific basic helix-loop-helix leucine zipper transcription factor that plays an important role in the differentiation, proliferation and survival of melanocytes (10). MITF has been reported as a lineage-specific oncogene in melanoma as primary cultures of human melanocytes can be transformed by enhanced expression of MITF (11). The oncogenic role of MITF is also reflected by the occurrence of its gene amplification in approximately 15% of metastatic melanomas as well as the association with resistance to conventional chemotherapy (11). However, it has been challenging to target MITF since it is a transcription factor. Since it was shown that the transcriptional co-activators p300/CBP interact with MITF and regulate the downstream target genes (12,13), we hypothesize that targeting p300/CBP may indirectly target the MITF pathway in melanoma. 3 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 28, 2018; DOI: 10.1158/1535-7163.MCT-18-0511 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. p300 and CREB-binding protein (CBP) (paralogs called p300/CBP thereafter) are members of the KAT3 family of histone acetyltransferase (HAT) that have a number of biological substrates (14). p300/CBP regulate a wide range of cellular events such as senescence, apoptosis, cell cycle, DNA damage response and cellular differentiation (15). p300/CBP act as transcriptional co-activators for multiple proteins, including oncogenes, suggesting the potential involvement of p300/CBP in cancers (16). Several p300/CBP mutations in melanoma patients have been identified, but these mutations have not been studied extensively (17). In addition, EP300 expression is upregulated and frequently amplified in melanoma cell lines (18). Together, the evidence indicates the potential therapeutic value of targeting p300 in melanoma cells. We recently identified a first-in-class selective catalytic inhibitor of p300/CBP A-485 (19) (Figure 1a). A-485 competes with acetyl coenzyme A, and robustly inhibited the HAT activity of p300 bromodomain-HAT-CH3 (BHC) as well as the BHC domain of CBP. A-485 also showed minimal activity against other HATs family members and negligible binding to BET bromodomain proteins and other protein targets such as G protein-coupled receptors, ion channels, transporters and kinases (19). In this study, we evaluated the activity of this specific p300/CBP inhibitor A-485 in a panel of melanoma cell lines. We demonstrated the modulation of MITF expression and pathway in response to A-485. Our results support a rationale for testing p300/CBP inhibitors in melanoma patients with MITF copy number gain and upregulated expression. Material and Methods Chemicals p300/CBP inhibitor A-485 and inactive compound A-486 were synthesized at AbbVie, Inc. (North Chicago, IL). Cells, cell culture, transfection, and cellular assays 4 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 28, 2018; DOI: 10.1158/1535-7163.MCT-18-0511 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Melanoma cell lines were cultured as recommended by the supplier (ATCC, Manassas, VA). The cells were tested for mycoplasma using MycoAlert Detection Kit (Lonza), authenticated using GenePrint 10 STR Authentication Kit (Promega). IC50 of acetylation marker H3K27 and cell viability were determined as previously described (19). Silencing of EP300 and MITF was performed by transfecting SKMEL5 cells with EP300 or MITF siRNAs or non-targeting control siRNAs synthesized by Dharmacon (Lafayette, CO) (On- Target-Plus SMARTpool siRNA for each gene). Reverse transfections were performed with Lipofectamine 2000 according to the manufacturer’s protocol (InvitrogenTM, Carlsbad, CA). Senescence was determined using a Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, Danvers, MA). Cells were cultured in six-well
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