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

Published OnlineFirst September 28, 2018; DOI: 10.1158/1535-7163.MCT-18-0511

Small Molecule Therapeutics Molecular Cancer Therapeutics Targeting Lineage-speciﬁc MITF Pathway in Human Melanoma Cell Lines by A-485, the Selective Small-molecule Inhibitor of p300/CBP Rui Wang, Yupeng He, Valerie Robinson, Ziping Yang, Paul Hessler, Loren M. Lasko, Xin Lu, Anahita Bhathena, Albert Lai, Tamar Uziel, and Lloyd T. Lam

Abstract

Metastatic melanoma is responsible for approximately catalytic inhibitor, A-485, induces senescence in multiple 80% of deaths from skin cancer. Microphthalmia-associated melanoma cell lines, similar to silencing expression of transcription factor (MITF) is a melanocyte-specifictran- EP300 (encodes p300) or MITF.Wedidnotobserveapo- scription factor that plays an important role in the differ- ptosis and increase invasiveness upon A-485 treatment. entiation, proliferation, and survival of melanocytes as well A-485 regulates the expression of MITF and its downstream as in melanoma oncogenesis. MITF is amplified in approx- signature genes in melanoma cell lines undergoing senes- imately 15% of patients with metastatic melanoma. How- cence. In addition, expression and copy number of MITF ever, no small-molecule inhibitors of MITF currently exist. is significantly higher in melanoma cell lines that undergo MITF was shown to associate with p300/CBP, members of A-485–induced senescence than resistant cell lines. Finally, the KAT3 family of histone acetyltransferase. p300 and we showed that A-485 inhibits histone-H3 acetylation but CREB-binding protein (p300/CBP) regulate a wide range did not displace p300 at promoters of MITF and its putative of cellular events such as senescence, apoptosis, cell cycle, downstream genes. Taken together, we provide evidence DNA damage response, and cellular differentiation. p300/ that p300/CBP inhibition suppressed the melanoma-driven CBP act as transcriptional coactivators for multiple proteins transcription factor, MITF, and could be further exploited in cancers, including oncogenic transcription factors such as as a potential therapy for treating melanoma. Mol Cancer Ther; MITF. In this study, we showed that our novel p300/CBP 17(12); 2543–50. 2018 AACR.

Introduction observed in patients with BRAF V600E mutation, the long-term benefit of vemurafenib has been compromised because of the Melanoma is one of the most frequent cancers with increased development of resistance to the therapy. Subsequent study incidence in the Western societies. Melanoma is responsible for indicated that acquired resistance is caused by additional 80% of deaths from skin cancer. The median overall survival of mutations, which can reactivate the MAPK pathway directly patients with advanced-stage melanoma has increased from or indirectly (6, 7). approximately 9 months before 2011 to at least 2 years (1). Interestingly, approximately 15%–20% patients with BRAF Melanoma has been studied extensively and is found to be mutation do not respond to vemurafenib and the mechanism related to the uncontrolled proliferation of melanocytes (2). underlying the intrinsic resistance remains an intense area of Although there have been recent successes in developing effec- investigation (7). Recently, it was shown that microphthalmia- tive treatments for melanoma such as targeted therapies against associated transcription factor (MITF)-low melanoma is asso- BRAF and MEK kinases, and immunotherapy, advanced mel- ciated with intrinsic resistance to multiple targeted agents anoma still has a high mortality rate (3, 4). including BRAF inhibitor (8, 9). MITF is a melanocyte-specific The identification of a constitutively active MAPK pathway basic helix-loop-helix leucine zipper transcription factor that due to BRAF V600E mutation in about 40% melanoma has led plays an important role in the differentiation, proliferation, to the development of selective BRAF inhibitors such as and survival of melanocytes (10). MITF has been reported as a vemurafenib (5). Although great initial clinical benefits were lineage-specific oncogene in melanoma as primary cultures of human melanocytes can be transformed by enhanced expres- fl Oncology Discovery, AbbVie, North Chicago, Illinois. sion of MITF (11). The oncogenic role of MITF is also re ected by the occurrence of its gene amplification in approximately Note: Supplementary data for this article are available at Molecular Cancer 15% of metastatic melanomas, as well as the association with Therapeutics Online (http://mct.aacrjournals.org/). resistance to conventional chemotherapy (11). However, it has Current address for R. Wang: Precision Oncology, Bristol-Myers Squibb, been challenging to target MITF because it is a transcription Princeton, New Jersey. factor. Because it was shown that the transcriptional coactiva- Corresponding Author: Lloyd T. Lam, AbbVie, Inc., 1 North Waukegan Road, tors p300/CBP interact with MITF and regulate the downstream AP10 Room 214, North Chicago, IL 60064-6098. Phone: 847-937-5585; Fax: target genes (12, 13), we hypothesize that targeting p300/CBP 847-935-4994; E-mail: [email protected] mayindirectlytargettheMITFpathwayinmelanoma. doi: 10.1158/1535-7163.MCT-18-0511 p300 and CREB-binding protein (CBP; paralogs called 2018 American Association for Cancer Research. p300/CBP thereafter) are members of the KAT3 family of

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histone acetyltransferase (HAT) that have a number of bio- RNA expression assays logical substrates (14). p300/CBP regulates a wide range of RNA was purified using the RNeasy Mini Kit (Qiagen) and cellular events such as senescence, apoptosis, cell cycle, DNA quantitative PCR (qPCR) reaction was performed using the damage response, and cellular differentiation (15). p300/CBP SuperScript III Platinum One-Step qPCR Kit (Thermo Fisher act as transcriptional coactivators for multiple proteins, Scientific) following the manufacturer's instructions. MITF and including oncogenes, suggesting the potential involvement of EP300 primers were designed and synthesized by Integrated DNA p300/CBP in cancers (16). Several p300/CBP mutations in Technologies. qPCR was performed with CFX96 Touch real-time patients with melanoma have been identified, but these muta- PCR Detection System (Bio-Rad), using GAPDH as an internal tions have not been studied extensively (17). In addition, control. The siRNA-induced reduction of gene expression was EP300 expression is upregulated and frequently amplified in calculated using the comparative Ct (DDCt) method following melanoma cell lines (18). Together,theevidenceindicatesthe the manufacturer's protocol (Thermo Fisher Scientific). The potential therapeutic value of targeting p300 in melanoma results were normalized to GAPDH and to the nontargeting cells. We recently identified a first-in-class selective catalytic siRNA control. inhibitor of p300/CBP A-485 (ref. 19; Fig. 1A). A-485 competes with acetyl-coenzyme A, and robustly inhibited the HAT activ- Microarray analysis ity of p300 bromodomain-HAT-CH3 (BHC) as well as the BHC Cells were treated with DMSO or 3 mmol/L of A-485 for 6 or domain of CBP. A-485 also showed minimal activity against 24 hours. Purified RNA was subjected to microarray analysis on other HATs family members and negligible binding to BET the GeneChip Human Genome U133 Plus 2.0 Array following bromodomain proteins and other protein targets such as G manufacturer's protocol (Affymetrix). The data normalization protein–coupled receptors, ion channels, transporters, and and processing were performed according to the previously kinases (19). In this study, we evaluated the activity of this described approach (20). The upstream regulator analysis was specific p300/CBP inhibitor A-485 in a panel of melanoma performed with Ingenuity Pathway Analysis (IPA; Qiagen). cell lines. We demonstrated the modulation of MITF expres- Z-scores of >2or<2 are considered significant. Microarray data sion and pathway in response to A-485. Our results support is deposited in GEO (accession no. GSE116459). a rationale for testing p300/CBP inhibitors in patients with melanoma with MITF copy number gain and upregulated Western blot analysis expression. Cell lysates were prepared in RIPA buffer (Sigma) plus pro- tease inhibitor cocktail (Roche Life Science). Thirty micrograms of total protein was resolved on a 12% SDS polyacrylamide gel. Materials and Methods Antibodies against MITF (Abcam) and control GAPDH (Cell Chemicals Signaling Technology) were used to detect protein level. After p300/CBP inhibitor A-485 and inactive compound A-486 incubation with secondary antibodies (LI-COR), blots were were synthesized at AbbVie, Inc. developed using Odyssey infrared imaging system (LI-COR).

Chromatin immunoprecipitation Cells, cell culture, transfection, and cellular assays Native chromatin immunoprecipitation (ChIP) was per- Melanoma cell lines were cultured as recommended by the formed as described previously (21). Briefly, nuclei were ini- Mycoplasma supplier (ATCC). The cells were tested for using tially released to generate the soluble chromatin. The immu- MycoAlert Detection Kit (Lonza), authenticated using GenePrint noprecipitation was then carried out overnight at 4Cwith 10 STR Authentication Kit (Promega). ChIP-grade anti-acetylated H3 antibody or anti-p300 antibody IC50 of acetylation marker H3K27 and cell viability were coupled with protein A/G magnetic beads (all from Millipore- determined as described previously (19). Sigma). The bound chromatin was eluted followed with Pro- EP300 MITF Silencing of and was performed by transfecting teinase K (Millipore-Sigma) digestion at 55C for 2 hours. EP300 MITF SKMEL5 cells with or siRNAs or nontargeting control The eluted DNA was purified by QIAquick PCR Purification siRNAs synthesized by Dharmacon (On-Target-Plus SMARTpool columns (Qiagen), and analyzed by qPCR using SYBR Green siRNA for each gene). Reverse transfections were performed with PCR Master Mix (Thermo Fisher Scientific) following the man- Lipofectamine 2000 according to the manufacturer's protocol ufacturers' instructions with CFX96 Touch Real-Time PCR (Invitrogen). Detection System (Bio-Rad). qPCR was performed using primer Senescence was determined by using a Senescence pairs near the transcription start sites of MITF and its putative b -Galactosidase Staining Kit (Cell Signaling Technology). Cells target genes. qPCR primers are: MITF primer,forward:50-CA- were cultured in 6-well plate and treated with DMSO or A-485 TTGTTATGCTGG AAATGCTAGAA-30, reverse: 50- GGCTTGCT- for 5 days before staining. GTATGTGGTACTTGG-30; MLANA primer, forward: 50-GGATA- GAG CACTGGGACTGG-30, reverse: 50- CTGACGGG GTCGT- Flow cytometry analysis for apoptosis CTGTAAT-30; TRPM1 primer, forward: 50- AAAGCTCATG- The percentages of apoptotic and early apoptotic cells were GAAAGCTG GAA-30,reverse:50- GCAT CCACAGTCACCTG- detected using Annexin V FITC and propidium iodide (PI) AAA-30; SEMA6A,forward:50-GCCTAAA CCTGTGGCTGGACA- staining (Thermo Fisher Scientific).Afterstaining,cellswere CAA-30, reverse: 50-CCCTGGAGGGTGGGATTCTCTAAA-30;and analyzed by BD LSR II Flow Cytometer (BD Biosciences). The TDRD7,forward:50- AGAGGGAGT GCTTCCGTTTTCA-30, percentage of early apoptotic (high Annexin V-FITC/low PI) reverse: 50- GCCATTAAAGGC TGCTCACAAC-30). Relative and late apoptotic (high Annexin V-FITC/high PI) cells was binding values were calculated using the DDCt method follow- determined. ing the manufacturer's protocol (Thermo Fisher Scientific) by

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comparing with DMSO enrichment relative to negative control a high-content imaging assay (23). We selected two melanoma (IgG) for ChIP is indicated; GAPDH promoter sequence is used cell lines that are vulnerable to MITF silencing (WM2664 and as endogenous control for qPCR. SKMEL5) and one cell line resistant to MITF silencing (A375) based on the Project DRIVE data (Supplementary Fig. S1). The results indicated that A-485 inhibited the H3K27Ac in Results all three melanoma cells lines with IC50 ¼ 0.59 mmol/L, p300/CBP inhibitor A-485 inhibits cell growth and induces 0.93 mmol/L, and 0.96 mmol/L for WM2664, SKMEL5, and cellular senescence in MITF-dependent melanoma cells A375 cells, respectively (Fig. 1A). In contrast, there was no effect MITF is a melanocyte-specific transcription factor that plays on H3K27 acetylation mark in all three cell lines using the in- an important role in the differentiation, proliferation, and active control compound A-486 (ref. 19; IC50 > 10 mmol/L). survival of melanocytes (10). Data from Project DRIVE (a Interestingly, even though A-485 could inhibit the H3K27 acet- large-scale RNAi screen in cancer cell lines that reveals vulner- ylation mark in all three cells lines, the cellular response to A-485 abilities to specific genes in cancer subtypes; ref. 22) shows that was different. In particular, proliferation of WM2664 and 18 of 34 melanoma cell lines have a dependency on MITF SKMEL5 cells was inhibited by A-485 (IC50 1 mmol/L) while (Supplementary Fig. S1). Previous publications showed that A375 cell line was resistant to the inhibitor (IC50 > 10 mmol/L; MITF recruits p300/CBP in activating transcription (12, 13). We Fig. 1A). In addition, we treated primary melanocytes (HEM reasoned that a small-molecule inhibitor of p300/CBP could cells) with A-485 and showed that HEM cells were also sensitive target the MITF pathway in melanoma. Because p300/CBP are to A-485 with IC50 < 1 mmol/L (Supplementary Fig. S2). HATs, we first assessed the effect of A-485 in modulating It was previously suggested that downregulation of MITF or histone acetylation of Histone H3 at Lys27 (H3K27Ac) using p300/CBP histone acetyltransferases activates a senescence

Figure 1. p300/CBP inhibition leads to senescence in sensitive melanoma cells. A, Structure and cellular activity of A-485 in melanoma cell lines. IC50 of acetylation marker H3K27 and cell viability were determined. B, Silencing EP300 or MITF induced senescence in SKMEL5 cells. Silencing of EP300 and MITF was performed by transfecting SKMEL5 cells with EP300 or MITF siRNAs or nontargeting control siRNAs. The siRNA-induced reduction of gene expression was calculated and results were normalized to GAPDH and to the nontargeting siRNA control (mean SD, n ¼ 3). C, A-485 treatment leads to senescence in sensitive melanoma cell lines. WM2664, SKMEL5, and A375 melanoma cells were treated with DMSO or A-485 for 5 days and levels of senescence were determined. D, A-485 treatment does not induce apoptosis in sensitive melanoma cells. WM2664 and SKMEL5 were treated with A-485 for 5 days. The percentages of apoptotic and early apoptotic cells were detected using Annexin V ﬂuorescein isothiocyanate (FITC) and propidium iodide (PI) staining.

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checkpoint in human melanocytes (24, 25). We first demon- (Fig. 2E) and low protein expression of MITF was observed in strated that silencing EP300 induced senescence in SKMEL5, the resistant cell line A375. Taken together, our data suggested similar to silencing MITF (Fig.1B).WefurthershowedthatA- that MITF gene expression and downstream pathway is selec- 485 induced pronounced cellular senescence in sensitive mel- tively regulated by p300/CBP inhibitor in melanoma. anoma cell lines WM2664 and SKMEL5 but not in the resistant line A375 (Fig. 1C). Our data suggested that p300/CBP inhib- MITF gene expression and DNA copy number predicts itor phenocopied the effect of silencing EP300 or MITF.In sensitivity to p300/CBP inhibitor A-485 in human melanoma contrast, we found no significant change in the percentages of cell lines early apoptotic and apoptotic cells in all three melanoma cell Because we observed that p300/CBP inhibitor–sensitive cell lines with A-485 treatment as shown by annexin V/PI staining lines express higher MITF mRNA and protein than resistant (Fig. 1D) and terminal deoxynucleotidyl transferase–mediated- cell line, we hypothesize that MITF level may play a role in dUTP nick end labeling assay (Supplementary Fig. S3). Taken predicting sensitivity to p300/CBP inhibitor in melanoma cell together, the major activity of p300/CBP inhibitor in the lines. We first evaluated the effect of A-485 in a panel of 17 sensitive melanoma cell lines is induction of cellular senes- melanoma cell lines using a 5-day senescence assay. On the cence but not apoptosis, similar to silencing the expression of basis of the staining results, we divided melanoma cell lines EP300 or MITF. into the senescence (>þ) and no senescence () groups. Melanomas are known to undergo phenotype switching. In Representative images of the staining results were shown in particular, tumors with high MITF are highly proliferative and Supplementary Fig. S5A. We found that eight cell lines under- tumors with low MITF are highly invasive (26, 27). We deter- went senescence and six cell lines did not with A-485 treatment mined whether A-485 treatment increases invasiveness in cells while three cell lines showed low levels of senescence (þ) with high MITF. We treated WM2664 cells with A-485 for 6 days (Supplementary Fig. S5B). To further dissect the differences and showed that A-485 treatment greatly reduced the cell number between the melanoma cell lines in response to p300/CBP but did not increase invasiveness (Supplementary Fig. S4). inhibitor, we utilized data from the Cancer Cell Line Encyclo- pedia (CCLE; http://cbioportal.org) to determine whether p300/CBP inhibition potently suppresses MITF signature genes there is differential gene expression of MITF between these To better understand the potential mechanisms of A-485 in two groups (29). We found that senescence cell lines were melanoma cell lines, we performed global gene expression associated with higher MITF expression than the no senescence analysis using microarrays on the two sensitive melanoma cell cell lines (P ¼ 0.02;Fig.3A).Incontrast,SOX10 was not lines, WM2664 and SKMEL5, as well as the resistant cell line differentially expressed between senescence and no senescence A375. To distinguish early and late transcriptional changes, we cell lines (P ¼ 0.55). EP300 and CREBBP (encoding CBP) were treated these cells with A-485 for 6 and 24 hours. Overall, also not differentially expressed between the two groups of global gene expression analysis showed strong inhibition of melanoma cell lines (Fig. 3A). Furthermore, we identified 430 gene expression in sensitive lines WM2664 and SKMEL5. genes that are differentially expressed with at least 2-fold Stronger modulation in gene expression was observed after change and P < 0.05 between the senescence and no senescence 24-hour exposure to A-485 as compared with 6-hour exposure groups. Twenty-six out of these 430 genes, including MITF, (Fig. 2A). Gene expression changes in the resistant cell line were identified by IPA to belong to the MITF gene signature A375 were different from the sensitive cell lines and to a lesser (Supplementary Fig. S5C). Next, we compared the copy num- extent. Minimal transcriptional effect was observed with the ber of MITF in the senescence and no senescence groups. We inactive compound, A-486 (data now shown). Upon perform- observed that cell lines that underwent senescence have higher ing Ingenuity upstream regulator analysis (IPA) across the MITF copy number than nonsenescence cell lines (P ¼ different cell lines, we showed that there was an enrichment 0.002; Fig. 3B). Finally, we determined the protein expression of transcriptional network regulated by MITF with p300/CBP of MITF in senescence and no senescence cell lines upon A-485 inhibitor treatment in the two sensitive lines (Fig. 2B). These treatment. We showed that cell lines that undergo senescence changes were much weaker in the resistant cell line A375. In upon A-485 treatment have higher basal expression of MITF contrast, there was no enrichment of transcriptional network than cell lines that do not (Fig. 3C). In addition, we showed regulated by Sry-related HMG-box-10 (SOX10), a neural crest that there was a good correlation between MITF protein expres- stem cell transcription factor that contributes to melanomagen- sion and viability/proliferation (IC50) to A-485 in melanoma esis (28). Figure 2C shows the gene expression changes within cell lines (Fig. 3D). Taken together, these findings indicate the MITF signature in the two sensitive melanoma cell lines that the expression and amplification of MITF mayplaya WM2664 and SKMEL5, as well as the resistant cell line A375. role in determining sensitivity to p300/CBP inhibitor in Quantification showed a 7- to 8-fold downregulation of MITF melanoma cell lines. This also suggests that MITF gain/ at 6 hours and 4- to 5-fold downregulation after 24 hours post amplification could be used to stratify patients for p300/CBP A-485 treatment in the two sensitive cell lines. An independent inhibitor treatment because previous study showed that up to qPCR analysis on gene expression indicated that the transcrip- 15% of patients with metastatic melanoma exhibited amplifica- tional levels of MITF was indeed downregulated in a dose- tion of MITF (11). dependent manner following 6 and 24 hours of A-485 treatment in the sensitive cells lines WM2664 and SKMEL5, but not Inhibition of p300/CBP HAT activity affects histone in the resistant cell line A375 (Fig. 2D). It should be noted that acetylation but does not displace p300 at promoters of the resistant line A375 had low expression of MITF. Similarly, MITF target genes MITF protein expression was reduced in sensitive cell lines Histone acetyltransferases have been suggested to function WM2664 and SKMEL5 following 24-hour treatment of A-485 as transcriptional coactivators and associate with activated

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Figure 2. A-485 induces a significant decrease in MITF expression and MITF signature genes in sensitive melanoma cells. A, A-485 induces different gene expression changes in sensitive and resistant melanoma cell lines. WM2664, SKMEL5, and A375 cells were treated with DMSO or 3 mmol/L of A-485 for 6 or 24 hours. Heatmap of statistically significant up- or downregulated genes is depicted (P < 0.01 with at least 2-fold change compared with DMSO in at least one experimental condition). Genes did not meet P < 0.01 with at least 2-fold change are indicated by the color black. B, A-485 decreases MITF but not SOX10 transcriptional network. The upstream regulator analysis was performed with IPA. Z-scores of >2or<2are considered significant. C, A-485 induces decrease in MITF-regulated genes. MITF-regulated genes (as determined by IPA analysis, P < 0.01 with at least 2-fold change) in A-485–treated WM2664, SKMEL5, and A375 cells at 6 or 24 hours. The color scale (red/blue) represents fold change of expression compared with the untreated control (up/down, respectively). D, A-485 decreases gene expression of MITF upon A-485 treatment (0.3 and 3 mmol/L) in human melanoma cells. qPCR was performed using GAPDH as an internal control and the results were normalized to GAPDH. Student t test was performed (, P < 0.05). E, A-485 decreases MITF protein expression. Immunoblot analysis of MITF protein levels after A-485 treatment (0.3 and 3 mmol/L) for 24 hours in melanoma cell lines. gene expression. We sought to understand whether p300 inhi- exposure to A-485 (Fig 4B), consistent with our microarray bition regulates transcription by affecting the p300-mediated data. However, we did not observe p300 displacement at the acetylation and/or p300 binding near the gene promoter promoter regions of these genes (Fig. 4C), suggesting that the regions. We used ChIP followed by qPCR (ChIP-qPCR) to p300/CBP inhibition affects its histone acetylation activity at detect histone H3 acetylation and p300 levels at the promoter the MITF gene promoter regions without interfering with p300 region of MITF and a few strongly inhibited MITF target genes chromatin binding (Fig. 4D). (MLANA, SEMA6A, TRPM1,andTDRD7)identified from our microarray results in WM2664 and SKMEL5 melanoma cells treated with A-485 (Fig. 4A). Notably, histone H3 acetylation Discussion level at the promoter regions of MITF and its signature genes The role of MITF in melanoma has been described but were reduced in A-485–sensitive cell lines after a 24-hour targeting MITF has been challenging because it is a transcription

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Figure 3. MITF expression and DNA copy number predicts sensitivity to p300/CBP inhibitor in human melanoma cell lines. A, Basal gene expression of MITF, EP300, CREBBP,andSOX10 in senescence and no senescence melanoma cell lines upon A-485 treatment. B, DNA copy number of MITF in senescence versus no senescence melanoma cell lines upon A-485 treatment. Data were obtained from the CCLE (http://cbioportal.org). Student t test was performed and P < 0.05 was considered signiﬁcant. C, HigherMITFproteinexpressioninsenescencethannonsenescence melanoma cell lines upon A-485 treatment. MITF and GAPDH protein expression was determined on the basis of immunoblot analysis in melanoma cell lines. D, Correlation between MITF protein

expression and viability/proliferation (IC50) to A-485 in melanoma cell lines.

factor. In this study, we show that a selective p300/CBP inhib- Finally, p300/CBP inhibitor decreased Histone-H3 acety- itor A-485 induced senescence in more than half the melanoma lation without displacing p300 at promoters of MITF and cell lines tested. The majority of these cell lines have high MITF its putative downstream genes. In addition, our current find- expression. In addition, MITF pathway was the most signifi- ing is consistent with our recent report that A-485 inhibited cantly inhibited pathway in p300/CBP inhibitor-sensitive cell proliferation in lineage-specific tumor types, such as andro- lines as compared with an insensitive cell line suggesting that gen receptor–positive prostate cancer (19). On the basis of MITF pathway could be targeted with a p300/CBP inhibitor. these observations, p300/CBP inhibitor could be further MITF expression and DNA copy number also differs between exploited as a potential therapy for treating MITF-amplified the senescence versus no senescence cell lines, suggesting melanoma. patients could be selected for this treatment. Interestingly, a MITF melanoma cell state with high expression dictates sensi- Disclosure of Potential Conflicts of Interest tivity to RAF inhibitor suggesting that a combination of p300/ A. Lai has ownership interest (including stock, patents, etc.) at Abbvie CBP inhibitor and RAF inhibitor in these patients could be Stock. No potential conflicts of interest were disclosed by the other more efficacious (8, 9). authors.

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Figure 4. A-485 decreases histone H3 acetylation but does not displace p300 at promoter of MITF target genes. A, Decrease in gene expression of downstream target genes of MITF (MLANA, SEMA6A, TRPM1,andTDRD7) upon A-485 treatment in WM2664, SKMEL5, and A375 cells based on microarray analysis as shown in Fig. 2C. Acetylated histone H3 (B)andp300(C) ChIP were performed in WM2664, SKMEL5, and A375 cells treated with 3 mmol/L A-485 for 24 hours. Relative binding value comparing with DMSO enrichment relative to negative control (IgG) for ChIP is indicated; GAPDH promoter sequence is used as endogenous control for qPCR. Student t test was performed (, P < 0.05; , P < 0.1). D, Model depicting inhibition of lineage-speciﬁc MITF pathway by A-485. A-485, a catalytic p300/CBP inhibitor, inhibits histone acetylation at promoters of MITF and its target genes, and subsequently decreases their expression. A-485 does not displace p300 at these loci.

Authors' Contributions Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Conception and design: R. Wang, Y. He, V. Robinson, Z. Yang, X. Lu, A. Lai, computational analysis): R. Wang, Y. He, V. Robinson, Z. Yang, P. Hessler, T. Uziel, L.T. Lam L.M. Lasko, X. Lu, A. Lai, T. Uziel, L.T. Lam Development of methodology: R.Wang,Y.He,Z.Yang,A.Lai, Writing, review, and/or revision of the manuscript: R. Wang, Y. He, Z. Yang, L.T. Lam P. Hessler, X. Lu, A. Bhathena, A. Lai, T. Uziel, L.T. Lam Acquisition of data (provided animals, acquired and managed patients, Administrative, technical, or material support (i.e., reporting or organizing provided facilities, etc.): R. Wang, Y. He, V. Robinson, Z. Yang, P. Hessler, data, constructing databases): R. Wang, Z. Yang, L.T. Lam L.M. Lasko, L.T. Lam Study supervision: A. Bhathena, A. Lai, T. Uziel, L.T. Lam

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Wang et al.

Acknowledgments study conduct, and ﬁnancial support for this research were provided by We thank the AbbVie Oncology Biomarkers group and epigenetic AbbVie. group for discussion and critical review of the manuscript. We thank Sujatha Jagadeeswaran for technical support; Saul Rosenberg, Ken Brom- Received May 11, 2018; revised July 11, 2018; accepted September 24, 2018; berg, and Josh Plotnik for critical review of the manuscript. The design, published ﬁrst September 28, 2018.

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2550 Mol Cancer Ther; 17(12) December 2018 Molecular Cancer Therapeutics

Targeting Lineage-specific MITF Pathway in Human Melanoma Cell Lines by A-485, the Selective Small-molecule Inhibitor of p300/CBP

Rui Wang, Yupeng He, Valerie Robinson, et al.

Mol Cancer Ther 2018;17:2543-2550. Published OnlineFirst September 28, 2018.

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