MITF Expression Predicts Therapeutic Vulnerability to p300 Inhibition in Human Melanoma Free

Dynamic changes in histone acetylation are major regulatory mechanisms governing gene transcription in human diseases including cancers (1, 2). Large-scale analyses of chromatin modifications in human cancers have prompted the development of new epigenetic therapies including histone deacetylase (HDAC) and DNA-methyltransferase inhibitors (DNMTi). Among newly identified epigenetic targets, histone acetyltransferases (HAT) and their therapeutic efficacies remain unclear. The transcriptional coactivator p300 possesses both lysine acetyltransferase (KAT) enzymatic activity, as well as scaffolding abilities, which regulate the transcriptional activity of target genes, and have demonstrated complex roles in determining cell fate in both normal and diseased tissues (3). p300 has notably been found to be amplified in subsets of human melanomas and has been implicated as an oncogene in this and other malignancies (4). Our group previously reported the development of a small-molecule inhibitor of p300/CBP HAT, C646, (5–7) and its potential therapeutic efficacy in cancer, including myeloid leukemia and melanoma (5); however, its modest potency and electrophilic functionality have limited its pharmacologic applications, necessitating the search for more potent and specific reagents targeting p300. More 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 hematologic malignancies and prostate cancers (8).

Previously, p300 was found to serve as a coactivator for the Microphthalmia-associated transcription factor (MITF; refs. 9, 10), regulating the expression of a subset of downstream target genes through consensus DNA binding E-box and M-box motifs (11). In addition, varying levels of MITF expression have been associated with melanoma development and progression, and have been found to contribute to BRAF-inhibitor therapeutic resistance (12–15). Transcriptional regulators such as SOX10, PAX3, CREB, LEF-1, and ATF2 have been shown to control MITF expression, although the precise mechanisms remain to be elucidated (10, 16–23). Given the importance of MITF in melanoma biology, and the significance of p300 acetyltransferase activity in regulating melanoma cell growth, we sought to determine the role of p300 in melanoma development and progression and its potential relevance to the master melanocyte differentiation gene, MITF. Here, we explore the functional role of p300 in human melanoma using both a genetic and chemical approach using the potent and specific inhibitor of p300/CBP HAT, A-485*, to further dissect the specific functional contributions of p300 HAT activity to melanoma development and progression. These studies have allowed us to identify MITF as a critical downstream effector of p300 HAT activity and important stimulus for melanoma growth. 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 genetic data from The Cancer Genome Atlas (TCGA) database identified specific and exclusive alterations in this signaling axis in primary human melanomas, suggesting a critical growth regulatory pathway. Moreover, chemical inhibition of p300/CBP HAT activity by A-485* was found to significantly inhibit proliferation of multiple melanoma lines in an MITF-dependent fashion, supporting the role of p300 as a promising therapeutic target in human melanoma and promoting a therapeutic strategy for p300 HAT inhibitor therapies in tumors expressing high levels of MITF.

Melanoma cell lines were kindly provided by Dr. Meenhard Herlyn at the Wistar Institute (Philadelphia, PA) and Dr. Levi Garraway at the Broad Institute (Cambridge, MA). Melanoma cells were maintained in DMEM. The medium was supplemented with 10% FBS, 1% penicillin–streptomycin, and 1% l-glutamine. The media, penicillin-streptomycin, and l-glutamine, FBS were purchased from Invitrogen. All cell lines were grown at 37 °C in an atmosphere containing 5% CO₂.

Lentiviral expression vectors pCW45-GFP and pCW45-MITF were kindly provided by Dr. David Fisher (Massachusetts General Hospital, Boston, MA). pGL2-MITF and control vector were generous gifts from Dr. Hans Widlund (Brigham and Women's Hospital, Boston, MA). The Lentiviral pLKO1-based short hairpin RNA (shRNA) vectors targeting p300 and MITF were purchased from Sigma Aldrich. Packaging and envelope-expressing plasmids (psPAX2 and pMD2.G) were obtained from the Addgene plasmid repository (https://www.addgene.org/Didier_Trono/; Addgene plasmid catalog no. 12260).

Selected p300 and MITF shRNA plasmids were cotransfected into HEK293T cells along with expression vectors containing lentiviral envelope and packaging plasmids via Lipofectamine 2000 according to the manufacturer's protocol. Lentiviruses were harvested 48 hours following transfection. A total of 25 × 10⁴ human melanoma cells were transduced with each harvested lentivirus (500 μL) in the presence of 8 μg/mL of polybrene. Subsequently, melanoma cells transduced with pLKO1-based shRNAs were selected in 1.5 μg/mL of puromycin after 48 hour following transduction in their respective culture medium. Information regarding p300 shRNAs used in this study is described in a previous publication (7).

To treat cells, compound A-485* was dissolved in anhydrous DMSO to make a 10 mmol/L solution and added to culture medium to the desired concentration. An equal amount of DMSO was used as the vehicle control.

Cells were stained with propidium iodide according to a published protocol (24). Data acquisition and analysis were performed on a FACSCalibur Flow Cytometer with the CellQuest Software (BD Biosciences).

RNA from melanoma cells transduced with either shp300 or scrambled lentiviruses was purified using the Qiagen RNeasy Plus Kit. Samples were submitted to Boston University Microarray and Sequencing Resource Core Facility for analysis on the Affymetrix GeneChip Human Gene 2.0 ST. The initial data processing and normalizations were performed by the core facility. The gene ontology analysis was performed with Ingenuity Pathway Analysis (Qiagen).

cDNA was synthesized using the Superscript III First Strand Synthesis System (Invitrogen). qRT-PCR was performed using the SYBR Green PCR Master Mix (Applied Biosystems/Invitrogen) as described previously (5). The primer pairs were designed using the NCBI PrimerBlast tool and individually optimized. Gene expression values were determined with the ΔΔC_t method. GAPDH was used as an internal control. The absolute copy number of MITF was determined by using a standard curve generated from a 500 bp MITF amplicon with varying concentrations. The list of primers used in this study is provided in the Supplementary Information.

Chromatin immunoprecipitation (ChIP) was performed on the basis of a previously described protocol (5). The antibodies used in the study include normal rabbit and mouse IgG (sc-2027x and sc-2025, Santa Cruz Biotechnology), or antibodies against p300 (sc-585, Santa Cruz Biotechnology), histone H3K18 (13998, Cell Signaling Technology), histone H3K27 (8173, Cell Signaling Technology), or RNA polymerase II (2629, Cell Signaling Technology). Primers for qPCR were designed to amplify a region near transcription start (Supplementary Table S1). qPCR was performed using SYBR Green PCR Master Mix (Applied Biosystems/Invitrogen) on StepOnePlus Real Time PCR System (Applied Biosystems).

HAT assays were performed as described previously (25). Briefly, reactions measured the p300 catalyzed incorporation of ¹⁴C from the acetyl-CoA substrate (60 mCi/mmol) into purified histone H3. A-485* was dissolved in 100% DMSO and diluted in 10% DMSO for a final reaction concentration of 1%. Reactions were performed in a buffer composed of 50 mmol/L HEPES (pH 7.9), 50 mmol/L NaCl, 1 mmol/L TCEP, and 25 μg/mL BSA at 30°C and initiated by the addition of ¹⁴C-acetyl-CoA to a final concentration of 200 nmol/L. After 5 minutes, the reaction was quenched and acetylated histone product was separated on a 16% tris-tricine gel and visualized by autoradiography. A ¹⁴C-BSA standard was run in parallel and used to quantify product formation.

Whole-cell lysates were prepared as described in the Supplementary Materials and Methods. Western blots were performed as described previously (26). A complete list of primary and secondary antibodies used in this study is included in the Supplementary Information.

Senescent cells were detected by staining for lysosomal senescence–activated β-galactosidase activity with a commercial kit from Cell Signaling Technology (catalog no. 9860) and by immunofluorescent staining of promyelocytic leukemia protein nuclear bodies using an established protocol (27).

For reporter assays, cells were plated in a 6-well culture plate (Corning) to a 5 × 10⁵ cells/well density and treated with either DMSO (control) or A485 (10 μmol/L) for 2 days. The following day, cells were transfected with 5 μg of pCW45-MITF vector (kind gift from Dr. Widlund and Dr. Fisher), 0.5 μg of pRL.null (Promega) using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific). Cells were allowed to incubate with the transfection mixture for 24 hours. Cells were washed once with PBS and lysed with 200 mL of 1 lysis buffer/well (Promega). The assay samples were then analyzed on a luminometer using the Dual-Luciferase Kit (Promega). Luciferase signals were normalized to corresponding Renilla signals. Results are expressed as fold activation over DMSO-treated control and are plotted as the means from at least three independent experimental data points with error bars representing SD.

Structural data that support the findings of this study have been deposited in PDB with the accession code 5KJ2, and microarray data have been deposited in Gene Expression Omnibus with the accession code GSE128737.

To analyze connections between p300 function and melanoma development, we evaluated the frequency of EP300 gene alterations present in human cancers using publicly available datasets from TCGA (28, 29). Fifty-four different types of human cancers were evaluated through TCGA where melanomas displayed an approximately 10% incidence of EP300 genetic alterations, half of which were classified as amplifications (Fig. 1A). In addition, the EP300 genetic locus was noted to be the site of frequently gained copy numbers in melanomas identified via a comprehensive genomic analysis of 101 melanoma short-term cultures and cell lines (4). Together, these findings suggest that p300 may play an important role in melanomagenesis. To investigate the functional role of p300 in human melanoma, we first evaluated basal levels of EP300 gene expression in a panel of melanoma cell lines and found that EP300 expressed uniformly throughout all cell lines examined (Supplementary Fig. S1A). We subsequently used targeted shRNA to knockdown p300 expression in four human metastatic melanoma cell lines (WM893B, 451Lu, SK-Mel5, and 1205Lu; Fig. 1B). Forty-eight hours following the depletion of p300, melanoma cells were evaluated phenotypically and found to display appreciable changes in morphology including an enlarged and flattened appearance (Supplementary Fig. S1B). Melanoma cell growth was also found to be significantly inhibited in all melanoma cell lines evaluated (Fig. 1B) in association with a G₀–G₁ growth arrest (Supplementary Fig. S1C). In addition, p300 knockdown was found to induce increased expression of senescence-associated β-galactosidase (Fig. 1C) and associated cellular senescence, which was similar to previously reported changes seen following p300 knockdown in primary human melanocytes (30). There was no appreciable increase in apoptosis following p300 knockdown as measured by annexin V staining and FACS analysis (Supplementary Fig. S1D); however, melanoma cells were found to exhibit increased expression of the DNA damage response (DDR) and cellular senescence marker, γH2AX. (Fig. 1D).

To obtain a comprehensive assessment of p300 downstream effector genes in human melanoma, we performed genome-wide expression profiling analysis of WM983B and SK-Mel5 melanoma cells following p300 depletion (Fig. 2). We identified 666 and 348 downregulated genes (>2-fold) in SK-Mel5 and WM983B, respectively, following silencing of p300. A total of 250 genes were found to be significantly downregulated in both melanoma cell lines following silencing of p300 and were considered as potential bona fide p300 downstream effectors, which were selected for further evaluation (Fig. 2A). Ingenuity Pathway Analysis (IPA) of the common p300 effector genes found them to be statistically (z-score) enriched for downregulation of cell-cycle control genes and upregulation of genes associated with the DDR (Fig. 2B). The two highest ranked genes identified in gene set enrichment analysis were associated with cell-cycle control and DNA replication and showed similar negative enrichment scores (Supplementary Fig. S2A). Expression profiling data were confirmed via qRT-PCR analysis, which was notable for significantly upregulated expression of the p21 cyclin–dependent kinase inhibitor CDKN1A following p300 knockdown, while expression of Cyclin A2 (CCNA2) was significantly reduced (Supplementary Fig. S2B), consistent with a cellular growth arrest phenotype.

Upstream regulator analysis by IPA was used to further identify global regulatory mechanisms associated with p300 knockdown in human melanoma cells. These studies allowed us to identify the MITF transcription factor as a significant upstream regulator affecting p300 downstream target gene expression (Fig. 2C). Among the 250 overlapping p300 target genes identified, 33 genes were listed as MITF transcriptional target genes (Fig. 2D; Supplementary Fig. S2C), which was confirmed through qRT-PCR analysis (Fig. 2D) and previously published ChIP-seq analysis (31). As p300 is a known transcriptional cofactor for MITF (10), we were not surprised to discover that loss of p300 was associated with downregulation of MITF target gene expression; however, our microarray analysis further demonstrated that depletion of p300 was also associated with reduced expression of MITF itself, suggesting a direct effect of p300 on MITF transcription (Fig. 2D). In addition, several known upstream regulators of MITF, such as Pax3 and Sox10, were found to be downregulated in the setting of p300 knockdown (Fig. 2D). These data were confirmed by qRT-PCR and Western blot analysis of knockdown cell lysates in melanoma WM983B and SK-Mel 5 cells; however, downregulation of Pax3 appeared to be cell line dependent (Fig. 2E).

To further confirm the functional impact of p300 and MITF expression in human melanoma cells, we used shRNA to knockdown MITF expression in four melanoma cell lines (WM983B, 451Lu, 1205Lu, and A375; Fig. 3A). Morphologically, all melanoma cell lines evaluated showed an enlarged and flattened cell phenotype following MITF depletion, which was similar to the morphologic phenotype of p300 knockdown (Fig. 3B; Supplementary Fig. S1B) and suggestive of cellular senescence as has been previously observed (31, 32). Interestingly, knockdown of MITF was associated with inhibition of cell growth in all melanoma cells tested, similar to that seen in p300-depleted melanoma cells (Fig. 3C–F; Supplementary Fig. S3A). To further define the significance of MITF as a mediator of p300-controlled cell growth, we ectopically expressed MITF in p300-knockdown melanoma cell lines (Fig. 3G). Strikingly, ectopic expression of MITF in p300-silenced WM983B and 1205Lu human melanoma cells was found to rescue the p300 knockdown growth inhibitory phenotype (Fig. 3H; Supplementary Fig. S3B), suggesting that MITF is an important mediator of p300-associated cell growth in human melanoma.

We evaluated MITF promoter accessibility in the setting of p300 knockdown using ChIP analysis spanning 2,000 bp of the promoter region proximal to the transcription start site (TSS) of MITF (Fig. 4A). p300 pulldown ChIP analysis identified significant occupancy of p300 within the promoter region proximal to the TSS of the MITF gene in human melanoma cells (Fig. 4B). ChIP analysis of the signature p300 histone marks, acetylated H3K18 and H3K27, has previously identified “active” regions of the MITF promoter (33), which demonstrate significant overlap with p300-associated regulatory regions. Acetylated H3K18 and H3K27 were readily detected within the promoter region of MITF, which was completely abolished following p300 depletion (Fig. 4C). In addition, recruitment of RNA polymerase II (RNAPII) to the proximal TSS was also eliminated following gene silencing of p300. Further evaluation of p300 regulation of MITF transcription was performed using an MITF promoter reporter assay (17), where MITF promoter activity was found to be significantly reduced following p300 depletion in human melanoma cells (Fig. 4D). Together, these data indicate that p300 mediates core histone acetylation within the promoter region of the MITF gene and critically regulates MITF expression in human melanoma cells.

Further evaluation of our microarray data was pursued to identify potential downstream effectors of the p300–MITF regulatory axis. Notably, expression of FOXM1, a pro-proliferative and prosurvival MEK-target gene that has been shown to be an important regulator of cell cycle in many human cancers (34–36), was found to be markedly suppressed following p300 depletion (Fig. 5A and B). In addition, target genes of FOXM1 were consistently downregulated in both SK Mel-5 and WM983B cells following EP300 gene silencing (Figs. 2C and 5A and C), whereas FOXM1 protein levels were also found to be reduced following p300 depletion (Fig. 5B). This is particularly noteworthy because human melanomas have recently been found to express elevated levels of FOXM1, which has been shown to be a potential therapeutic target for this disease (37, 38).

Because our work identified the p300–MITF signaling axis as a critical pathway for melanoma survival and proliferation, we hypothesized that a key function of MITF in melanoma may be to regulate the expression of FOXM1 in these tumors. To test this hypothesis, we performed MITF knockdown in three melanoma cell lines and investigated changes in FOXM1 expression. Notably, we found that both transcript and protein levels of FOXM1 were sharply reduced following MITF depletion in human melanoma cells, suggesting an important regulatory role in these cells (Fig. 5D and E). Because Strub and colleagues (31) had previously used ChIP-seq to explore the MITF transcriptome, we reviewed their dataset to evaluate MITF occupancy at the FOXM1 promoter, and remarkably identified a peak within the proximal promoter region of FOXM1. Of note, reverse phase protein array (RPPA) from the TCGA cutaneous melanoma dataset (28, 29) revealed that tumors from patients with elevated expression of p300 or MITF (denoted by gene amplifications in this dataset) also displayed significantly higher levels of FOXM1 protein expression (Fig. 5F), further suggesting that the p300–MITF–FOXM1 signaling axis is critical for the proliferative phenotype of human melanomas.

While direct transcriptional regulation of FOXM1 by MITF may be a principal mechanism governing FOXM1 expression and melanoma cell growth, alternative regulation of FOXM1 protein expression and function may be achieved through direct phosphorylation of FOXM1, which contributes to its protein stability (39). Because FOXM1 phosphorylation has been shown to be catalyzed by cyclin-dependent kinases (38), and CDK2 expression is transcriptionally regulated by MITF (40), we hypothesized that downregulated expression of CDK2/CyclinA complexes following MITF depletion would result in reduced phosphorylation of FOXM1, leading to decreased FOXM1 stability and subsequent degradation (41). Indeed, we found that both mRNA and protein expression levels of CDK2 and cyclin A2 were significantly reduced following MITF depletion, which could contribute to loss of FOXM1 stability and function (Fig. 5C and D).

Following the observation that the p300–MITF axis controls the expression of FOXM1, we explored the significance of these findings in human melanoma specimens by mining genetic data from 471 patient samples available through the TCGA database, using cBioPortal (28, 29). Our analysis of gene alteration patterns for p300, MITF, cyclin A2, CDK2, and FOXM1 demonstrated a strong pattern of mutual exclusivity, suggesting that genes within this set contribute to a common downstream pathway (Fig. 5F; ref. 42). Furthermore, patients with gene alterations within the p300/MITF/cyclinA2/CDK2/FOXM1 pathway showed significantly reduced overall survival compared with patients without such gene alterations (Fig. 5G and H). We therefore conclude that the p300–MITF–FOXM1 axis is important for tumor cell survival and may be predictive of patient outcomes.

Because p300-mediated histone acetylation appears to play a major role in regulating MITF transcription (Fig. 4), we sought to evaluate whether targeted inhibition of p300-mediated lysine acetylation would be sufficient to block MITF transcription and inhibit proliferation in human melanomas. The small-molecule inhibitor of p300 HAT activity, A-485*, (43), was synthesized as a mixture of diastereomers following the methods described in the Supplementary Materials (Fig. 6A). To confirm that A-485* is a potent inhibitor of p300 acetyltransferase activity, we performed in vitro enzymatic assays using purified recombinant full-length p300 with purified histone H3 protein as substrate. Acetyltransferase reactions were performed with ¹⁴C-acetyl-CoA and ¹⁴C incorporation into target histone H3 was evaluated using phosphorimage analysis of SDS-PAGE (Supplementary Fig. S4). Compound A-485* was found to inhibit p300 acetyltransferase activity on purified H3 with an IC₅₀ of approximately 0.5μmol/L (Supplementary Fig. S5) under the conditions of our assays, which was significantly more potent than that reported for C646 (IC₅₀ ∼20 μmol/L) determined under similar conditions (7). Upon treatment with A-485*, melanoma cells displayed altered morphologic features with an elongated, refractile, and spindled appearance reminiscent of that seen in primary human melanocytes (Fig. 6B). We therefore sought to determine whether inhibition of p300 HAT activity by A-485* might directly promote melanocyte differentiation. We found that the melanocyte differentiation genes, PAX3 and SOX10 were variably altered in expression following A-485* treatment of WM983B, 451Lu, and SK-Mel5 cells, whereas expression of MITF, and its target differentiation genes TYRP1, and PMEL was markedly reduced in all cell lines (Fig. 6C). Compound A-485* treatment of WM983B melanoma cells demonstrated effective inhibition of histone H3 acetylation at lysine 18 while sparing lysine 9, a histone site that is not preferentially acetylated by p300/CBP (Fig. 6D). Moreover, MITF protein expression was dramatically suppressed following A-485* treatment whereas protein levels of p300 and CBP remained unchanged (Fig. 6D).

Because we determined that FOXM1 gene expression is regulated by the p300–MITF signaling axis in human melanoma cells, we sought to evaluate whether A-485* treatment of melanoma cells would also suppress FOXM1 expression in a similar fashion to p300 knockdown. Consistent with our previous results, A-485* treatment of WM983B and 451Lu melanoma cells led to dramatically reduced transcription of MITF, CDK2, cyclin A2, and FOXM1 (Figs. 5B–D and 6E). In addition, protein levels of FOXM1 were reduced following A-485* treatment of three metastatic melanoma cell lines along with reduced expression of MITF, CDK2, and cyclin A (Fig. 6F). These results suggest that p300 HAT inhibition potently inhibits expression of the pro-proliferative FOXM1 gene in human melanomas, which may be mediated by downregulated MITF expression. Because FOXM1 serves as a major regulator of cell proliferation and survival, we sought to determine whether A-485* treatment of human melanoma cells would induce a cellular senescence phenotype. We found that both WM983B and 451Lu melanoma cells demonstrated marked increases in senescence-associated β-galactosidase staining following A-485* treatment, consistent with induction of cellular senescence (Fig. 6G). In addition, melanoma cells treated with A-485* displayed increased intensity of senescence-associated promyelocytic leukemia (PML) and a rise in PML nuclear bodies following treatment, supporting the induction of cellular senescence by this compound (Fig. 6H and I).

Melanoma cells have been noted to express varying levels of MITF, with MITF-high–expressing tumors exhibiting a more proliferative phenotype and MITF-low–expressing tumors demonstrating a more invasive/metastatic phenotype (44). Our library of human melanoma cell lines consists of melanoma cells possessing both high and low levels of MITF baseline expression (Fig. 7A; Supplementary Fig. S1A). We therefore sought to evaluate the effect of p300 inhibitor A-485* in human melanoma cells expressing varying levels of MITF, to determine whether the specific inhibition of MITF expression through p300 HAT allowed for targeted cell growth inhibition. Remarkably, we found that melanoma cells possessing high-MITF expression levels displayed significantly greater growth inhibition by A-485* (10 μmol/L) compared with low MITF–expressing melanoma cells (Fig. 7B and C). MITF promoter reporter assay was performed to evaluate the regulation of MITF transcription by A-485*, where MITF promoter activity was found to be significantly reduced following A-485* treatment in human melanoma cells (Fig. 7D). Together, these data suggest a model for p300 as a critical regulator of MITF expression in human melanoma cells and the specific therapeutic efficacy of targeting p300 HAT activity in human melanomas possessing increased expression of MITF (Fig. 7E).

Although genetic mutations in human melanomas have been explored extensively over the past decade, the role of epigenetic alterations in melanoma development and progression has been less clearly defined. We have undertaken a series of experiments to determine the functional significance of p300-mediated chromatin remodeling in human melanomas using both a genetic and chemical approach. Our studies have allowed us to identify a highly conserved molecular pathway driving melanoma cell growth, which is mediated by p300-associated epigenetic modifications of core histones at the MITF promoter. We have further determined that restoration of MITF expression in p300 knockdown melanoma cells reverses the growth arrest phenotype seen with depletion of p300, suggesting that MITF is a key regulator of p300-associated proliferation in these cells. MITF has been identified as a critical mediator of “phenotype switching” in melanoma, whereby cells are able to convert from a proliferative (high MITF) phenotype to an invasive/migratory (low MITF) phenotype (45); however, the precise mechanism governing this switch remains to be determined. In addition, single-cell RNA-seq of metastatic melanoma tissues identified subsets of cells within a tumor expressing high and low levels of MITF, supporting the potential interchangeability of this phenotype switch (46). Furthermore, MITF levels have been shown to be critically regulated in the context of tumor development, with up to 20% of advanced melanomas demonstrating MITF gene amplifications (Cancer Genome Atlas Network, 2015, http://cancergenome.nih.gov/; ref. 12), supporting its role as a bona fide oncogene in melanoma. Moreover, MITF has been identified as a key driver of acquired resistance to MAPK pathway inhibition through a variety of prosurvival mechanisms, as well as innate resistance to such treatments (47–50). Taken together, these data implicate MITF as a central mediator of melanoma cell fate and an important therapeutic target for melanoma; however, previous attempts to target MITF in human melanoma cells have been largely unsuccessful (51). Recently, Smith and colleagues undertook a unique approach to understanding acquired MAPK-targeted therapeutic resistance by exploring mediators of nonmutational drug tolerance in human melanoma cell lines, which allowed them to identify MITF as a key mediator of early drug tolerance (50). A large-scale drug repurposing screen allowed for identification of the HIV1 protease inhibitor, nelfinavir, as an inhibitor of MITF expression, which promoted resensitization of tolerant cell lines to inhibitors of BRAF and MEK through indirect transcriptional repression of MITF (50). While this work was intriguing, the lack of understanding of nelfinavir's molecular mechanism(s) and its narrow application to cases of nonmutational drug tolerance may limit nelfinavir's clinical development. In contrast, we have identified direct activation of the MITF promoter through p300-mediated acetylation of core histones at critical promoter sites, and identified a potent and specific inhibitor, A-485*, of this process and melanoma cell growth. This clear mechanism of A-485* effects on MITF, and the potent growth inhibitory effects in tumors possessing increased expression of MITF, raise the possibility of development of this and other novel epigenetic therapies for melanoma. In addition, as A-485* treatment of WM983B melanoma cells demonstrated effective inhibition of histone H3 acetylation at lysine 18 while sparing lysine 9, we suggest that H3K18 acetylation may be used as a surrogate marker for therapeutic targeting of p300 HAT activity in vivo. Indeed, as effective agents targeting the MAPK pathway have been developed for tumors with activating BRAF gene mutations, their sustained therapeutic utility has been stymied by the near-universal acquired resistance to these drugs over time, necessitating the need for a multipronged approach (52–54) and targeting of the numerous and varied resistance mechanisms (14, 15, 49, 55–57). Development of novel strategies targeting epigenetic pathways may promote more durable therapeutic responses in melanoma, as bypass mechanisms of resistance would be less probable.

The recent investigation of A-485, the pure stereoisomer form of A-485* across >100 cancer cell lines showed a wide range of sensitivities of the antiproliferative effects mediated by A-485 across these lines, including in human melanomas. In the case of prostate cancer, expression of the androgen receptor or a splice variant conferred sensitivity to A-485 and AML1-ETO appeared to predict sensitivity to p300 inhibition in acute leukemia. For the other cancer types, no clear biomarker that correlated with A-485 sensitivity had been established; however, the studies here now add MITF in melanoma as such a biomarker.

Our studies of downstream effectors of p300 in human melanomas allowed us to identify a specific growth regulatory axis involving MITF, cyclin A, and CDK2, and the prosurvival transcription factor, FOXM1. Notably, these genes were found to be altered in a mutually exclusive way in primary human tumors (Fig. 5G) and associated with a poor prognosis (Fig. 5H). Indeed, increased FOXM1 expression in melanoma has previously been associated with accelerated tumor progression and poor prognosis (58), as well as suppression of the senescence phenotype (38). Our studies also support a cell survival phenotype associated with the p300–MITF axis and FOXM1 expression in human melanoma cells, which is abrogated following treatment with the p300 HAT inhibitor, A-485*, and associated with subsequent cellular senescence (Fig. 6). Interestingly, recent data suggests that the novel anticancer agents Honokiol (51) and Imipramine Blue (52) exhibit antitumor effects through inhibition of FOXM1. Thus, combination treatment of melanomas with these agents and A-485 may demonstrate synergistic activity and should be further investigated.

Recent significant advances in melanoma immunotherapies have been the cause for great excitement in the cancer treatment field, but thus far have proven highly effective for only subsets of patients with advanced disease (59), suggesting a significant need for the development of additional therapeutic strategies for this disease. Moreover, while epigenetic modifying agents such as DNMTis have been demonstrated to enhance the host response to immunotherapies in mouse models for melanoma (60), such studies have had limited impact with regard to disease treatment to date. In addition, while several epigenetic modifying therapies have been approved by the FDA for use in patients, including DNMTis, and histone deacetylase inhibitors, such agents have not demonstrated widespread benefits in human cancers. Our data suggest that the time may be right to pursue new therapeutic strategies targeting epigenetic alterations associated with HAT activity in human melanoma. Our data further suggest that such therapies may prove beneficial in directly targeting tumors expressing high levels of MITF, but perhaps also by augmenting immune responsiveness (61). Finally, we expect that A-485* should prove useful, not only as a chemical probe to specifically dissect out the functional significance of MITF in human melanomas, but more broadly in understanding the role of protein acetylation in tumor biology and immunomodulation.

D.J. Meyers has ownership interest (including stock, patents, etc.) in Acylin Therapeutics. P.A. Cole has ownership interest (including stock, patents, etc.) in and is a consultant/advisory board member for Acylin Therapeutics. R.M. Alani has ownership interest (including stock, patents, etc.) in Acylin Therapeutics. No potential conflicts of interest were disclosed by the other authors.

Conception and design: E. Kim, B.E. Zucconi, M. Wu, B. Ryu, P.A. Cole, R.M. Alani

Development of methodology: E. Kim, J.S. McGee, B. Ryu, P.A. Cole, R.M. Alani

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): E. Kim, B.E. Zucconi, M. Wu, S.E. Nocco, S. Venkatesh, D.L. Cohen, B. Ryu, R.M. Alani

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): E. Kim, M. Wu, S.E. Nocco, B. Ryu, P.A. Cole, R.M. Alani

Writing, review, and/or revision of the manuscript: E. Kim, B.E. Zucconi, M. Wu, S. Venkatesh, D.L. Cohen, B. Ryu, P.A. Cole, R.M. Alani

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): E.C. Gonzalez, R.M. Alani

Study supervision: B. Ryu, P.A. Cole, R.M. Alani

Other (synthesis of A-485*): D.J. Meyers

We thank members of the Alani, Ryu, and Cole laboratories for helpful discussions. D.J. Meyers is supported by CTSA grant UL1TR001079 and FAMRI (Flight Attendant Medical Research Institute). B.E. Zucconi is supported by a Jane Coffin Childs Fund postdoctoral fellowship. E. Kim is supported by an ASA Medical Student Grant. P.A. Cole is supported by NIH grant GM62437. The results shown here are in whole or part based upon data generated by the TCGA Research Network (http://cancergenome.nih.gov/).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.