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

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.

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

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 plate and treated with DMSO or A-485

for five days before staining.

Flow cytometry analysis for apoptosis

The percentages of apoptotic and early apoptotic cells were detected using Annexin V

fluorescein isothiocyanate (FITC) and Propidium Iodide (PI) staining (Thermo Fisher Scientific).

After staining, cells were analyzed by BD LSR II flow cytometer (BD Biosciences, San Jose, CA).

The percentage of early apoptotic cells (high Annexin V-FITC/low PI) and late apoptotic (high

Annexin V-FITC/high PI) was determined.

RNA expression assays

RNA was purified using the RNeasy Mini kit (Qiagen Germantown, MD) and quantitative

PCR (qPCR) reaction was performed using the SuperScript III Platinum One-Step qPCR Kit

(ThermoFisher) following the manufacturers' instructions. MITF and EP300 primers were designed

and synthesized by Integrated DNA Technologies (Coralville, IA). qPCR was performed with CFX96

Touch real-time PCR Detection System (BioRad, Hercules, CA), using GAPDH as an internal

control. The siRNA-induced reduction of gene expression was calculated using the Comparative Ct

(ΔΔCt) method following the manufacturer's protocol (Thermo Fisher Scientific, Carlsbad, CA). The

results were normalized to GAPDH and to the non-targeting siRNA control.

Microarray analysis

Cells were treated with DMSO or 3µM of A-485 for 6 or 24 hours. Purified RNA was

subjected to microarray analysis on the GeneChip Human Genome U133 Plus 2.0 Array following

manufacturer’s protocol (Affymetrix, Santa Clara, CA). The data normalization and processing were

performed according to the previously described approach (20). The upstream regulator analysis was

performed with Ingenuity Pathway Analysis (IPA®) (Qiagen, Germantown, MD). Z-scores of > 2 or

< -2 are considered significant. Microarray data is deposited in GEO (accession no. GSE116459).

Western blot analysis

Cell lysates were prepared in RIPA buffer (Sigma, St. Louis, MO) plus protease inhibitor

cocktail (Roche Life Science, Indianapolis, IN). 30 μg of total protein was resolved on a 12% SDS

polyacrylamide gel. Antibody against MITF (Abcam, Cambridge, UK) and control GAPDH (Cell

Signaling Technology) were used to detect protein level. After incubation with secondary antibodies

(LI-COR, Lincoln, NE), blots were developed using Odyssey infrared imaging system (LI-COR).

Chromatin immunoprecipitation (ChIP)

Native ChIP was performed according to a previously described (21). Briefly, nuclei

were initially released to generate the soluble chromatin. The immunoprecipitation was then

carried out overnight at 4oC with ChIP grade anti-acetylated H3 antibody or anti-p300 antibody

coupled with protein A/G magnetic beads (all from Millipore-Sigma). The bound chromatin was

eluted followed with Proteinase K (MilliporeSigma) digestion at 55 °C for 2 h. The eluted DNA

was purified by QIAquick PCR Purification columns (Qiagen), and analyzed by quantitative PCR

(qPCR) using SYBR Green PCR Master Mix (ThermoFisher) following the manufacturers'

instructions with CFX96 Touch real-time PCR Detection System (BioRad, Hercules, CA). qPCR

was performed using primer pairs near the transcription start sites of MITF and its putative target

genes. qPCR primers are: MITF primer, forward: 5’- CATTGTTATGCTGG AAATGCTAGAA-

3’, reverse: 5’- GGCTTGCTGTATGTGGTACTTGG-3’; MLANA primer, forward: 5’-

GGATAGAG CACTGGGACTGG-3’, reverse: 5’- CTGACGGG GTCGTCTGTAAT-3’;

TRPM1 primer, forward: 5’- AAAGCTCATGGAAAGCTG GAA-3’, reverse: 5’- GCAT

CCACAGTCACCTGAAA-3’; SEMA6A: 5’-GCCTAAA CCTGTGGCTGGACACAA-3’,

reverse: 5’-CCCTGGAGGGTGGGATTCTCTAAA-3’; TDRD7: 5’- AGAGGGAGT

GCTTCCGTTTTCA-3’, reverse: 5’- GCCATTAAAGGC TGCTCACAAC-3’). Relative binding

values were calculated using the Comparative Ct (ΔΔCt) method following the manufacturer's

protocol (ThermoFisher) by comparing to DMSO enrichment relative to negative control (IgG)

for ChIP is indicated; GAPDH promoter sequence is used as endogenous control for qPCR.

Results

p300/CBP inhibitor A-485 inhibits cell growth and induces cellular senescence in MITF-

dependent melanoma cells

MITF is a melanocyte-specific transcription factor that plays an important role in the

differentiation, proliferation and survival of melanocytes(10). Data from Project DRIVE (a large-

scale RNAi screen in cancer cell lines that reveals vulnerabilities to specific genes in cancer

subtypes (22)) shows that 18 out of 34 melanoma cell lines have a dependency on MITF (Suppl.

Figure 1). Previous publications showed that MITF recruits p300/CBP in activating transcription

(12,13). We reasoned that a small molecule inhibitor of p300/CBP could target the MITF

pathway in melanoma. Since p300/CBP are histone acetyltransferases (HATs), we first assessed

the effect of A-485 in modulating histone acetylation of Histone H3 at Lys27 (H3K27Ac) using a

high-content imaging assay (23). We selected two melanoma cell lines that are vulnerable to

MITF silencing (WM2664 and SKMEL5) and one cell line resistant to MITF silencing (A375)

based on the Project DRIVE data (Suppl. Figure1). The results indicated that A-485 inhibited the

H3K27Ac in all three melanoma cells lines with IC50 = 0.59 M, 0.93 M, and 0.96 M for

WM2664, SKMEL5, and A375 cells, respectively (Figure 1a). By contrast, there was no effect on

H3K27 acetylation mark in all three cell lines using the inactive control compound A-486 

(IC50>10 . Interestingly, even though A-485 could inhibit the H3K27 acetylation mark in all

three cells lines, the cellular response to A-485 was different. In particular, proliferation of

WM2664 and SKMEL5 cells was inhibited by A-485 (IC50 ≤ 1 ) while A375 cell line was

resistant to the inhibitor (IC50 > 10  (Figure 1a). In addition, we treated primary melanocytes

(HEM cells) with A-485 and showed that HEM cells were also sensitive to A-485 with IC50 < 1

(Suppl. Figure 2).

It was previously suggested that down-regulation of MITF or p300/CBP histone

acetyltransferases activate a senescence checkpoint in human melanocytes (24,25). We first

demonstrated that silencing EP300 induced senescence in SKMEL5, similar to silencing MITF

(Figure 1b). We further showed that A-485 induced pronounced cellular senescence in sensitive

melanoma cell lines WM2664 and SKMEL5 but not in the resistant line A375 (Figure 1c). Our

data suggested that p300/CBP inhibitor phenocopied the effect of silencing EP300 or MITF. In

contrast, we found no significant change in the percentages of early apoptotic and apoptotic cells

in all three melanoma cell lines with A-485 treatment as shown by annexin V/propidium iodide

staining (Figure 1d) and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling

(TUNEL) assay (Suppl. Figure 3). Taken together, the major activity of p300/CBP inhibitor in the

sensitive melanoma cell lines is induction of cellular senescence but not apoptosis, similar to

silencing the expression of EP300 or MITF.

Melanomas are known to undergo phenotype switching. In particular, tumors with high

MITF are highly proliferative and tumors with low MITF are highly invasive (26,27). We

determined whether A-485 treatment increases invasiveness in cells with high MITF. We treated

WM2664 cells with A-485 for 6 days and showed that A-485 treatment greatly reduced the cell

number but did not increase invasiveness (Suppl. Fig.4).

p300/CBP inhibition potently suppresses MITF signature genes

To better understand the potential mechanisms of A-485 in melanoma cell lines, we

performed global gene expression analysis using microarrays on the two sensitive melanoma cell

lines, WM2664 and SKMEL5, as well as the resistant cell line A375. To distinguish early and

late transcriptional changes, we treated these cells with A-485 for 6 and 24 hours. Overall, global

gene expression analysis showed strong inhibition of gene expression in sensitive lines WM2664

and SKMEL5. Stronger modulation in gene expression was observed after 24-hour exposure to

A-485 as compared to 6-hour exposure (Figure 2a). Gene expression changes in the resistant cell

line A375 were different from the sensitive cell lines and to a lesser extent. Minimal

transcriptional effect was observed with the inactive compound, A-486 (data now shown). Upon

performing Ingenuity® upstream regulator analysis (IPA®) across the different cell lines, we

showed that there was an enrichment of transcriptional network regulated by MITF with

p300/CBP-inhibitor treatment in the two sensitive lines (Figure 2b). These changes were much

weaker in the resistant cell line A375. By contrast, there was no enrichment of transcriptional

network regulated by Sry-related HMG-box-10 (SOX10), a neural crest stem cell transcription

factor that contributes to melanomagenesis (28). Fig 2c shows the gene expression changes within

the MITF signature in the two sensitive melanoma cell lines WM2664 and SKMEL5 as well as

the resistant cell line A375. Quantification showed a 7-8-fold down-regulation of MITF at 6 hours

and 4-5-fold down-regulation after 24 hours post-A-485 treatment in the two sensitive cell lines.

An independent QPCR analysis on gene expression indicated that the transcriptional levels of

MITF was indeed down-regulated in a dose-dependent manner following 6 and 24 hours of A-485

treatment in the sensitive cells lines WM2664 and SKMEL5 but not in the resistant cell line A375

(Figure 2d). It should be noted that the resistant line A375 had low expression of MITF.

Similarly, MITF protein expression was reduced in sensitive cell lines WM2664 and SKMEL5

following 24-hour treatment of A-485 (Figure 2e) and low protein expression of MITF was

observed in the resistant cell line A375. Taken together, our data suggested that MITF gene

expression and downstream pathway is selectively regulated by p300/CBP inhibitor in melanoma.

MITF gene expression and DNA copy number predicts sensitivity to p300/CBP inhibitor A-

485 in human melanoma cell lines

Since we observed that p300/CBP inhibitor-sensitive cell lines express higher MITF

mRNA and protein than resistant cell line, we hypothesize that MITF level may play a role in

predicting sensitivity to p300/CBP inhibitor in melanoma cell lines. We first evaluated the effect

of A-485 in a panel of seventeen melanoma cell lines using a 5-day senescence assay. Based on

the staining results, we divided melanoma cell lines into the senescence (> +) and no senescence

(-) groups. Representative images of the staining results were shown in Suppl. Figure 5a. We

found that eight cell lines underwent senescence and six cell lines did not with A-485 treatment

while three cell lines showed low levels of senescence (+) (Suppl. Figure 5b). To further dissect

the differences between the melanoma cell lines in response to p300/CBP inhibitor, we utilized

data from the Cancer Cell Line Encyclopedia (CCLE) (http://cbioportal.org) to determine if there

is differential gene expression of MITF between these two groups (29). We found that senescence

cell lines were associated with higher MITF expression than the no senescence cell lines (p=0.02)

(Figure 3a). By contrast, SOX10 was not differentially expressed between senescence and no

senescence cell lines (p=0.55). EP300 and CREBBP (encoding CBP) were also not differentially

expressed between the two groups of melanoma cell lines (Figure 3a). Furthermore, we identified

430 genes that are differentially expressed with at least 2-fold change and p value < 0.05 between

the senescence and no senescence groups. Twenty-six out of these 430 genes, including MITF,

were identified by IPA® to belong to the MITF gene signature (Suppl. Figure 5c). Next, we

compared the copy number of MITF in the senescence and no senescence groups. We observed

that cell lines that underwent senescence have higher MITF copy number than non-senescence

cell lines (p=0.002) (Figure 3b). Finally, we determined the protein expression of MITF in

senescence and no senescence cell lines upon A-485 treatment. We showed that cell lines that

undergo senescence upon A-485 treatment have higher basal expression of MITF than cell lines

that do not (Figure 3c). In addition, we showed that there was a good correlation between MITF

protein expression and viability/proliferation (IC50) to A-485 in melanoma cell lines (Figure 3d).

Taken together, these findings indicate that the expression and amplification of MITF may play a

role in determining sensitivity to p300/CBP inhibitor in melanoma cell lines. This also suggests

that MITF gain/amplification could be used to stratify patients for p300/CBP inhibitor treatment

since previous study showed that up to 15% of metastatic melanoma patients exhibited

amplification of MITF (11).

Inhibition of p300/CBP HAT activity affects histone acetylation but does not displace p300

at promoters of MITF target genes

Histone acetyltransferases have been suggested to function as transcriptional co-

activators and associate with activated gene expression. We sought to understand if p300

inhibition regulates transcription by affecting the p300-mediated acetylation and/or p300 binding

near the gene promoter regions. We used chromatin immunoprecipitation (ChIP) followed by

qPCR (ChIP-qPCR) to detect histone H3 acetylation and p300 levels at the promoter region of

MITF and a few strongly inhibited MITF target genes (MLANA, SEMA6A, TRPM1, and TDRD7)

identified from our microarray results in WM2664 and SKMEL5 melanoma cells treated with A-

485 (Figure 4a). Notably, histone H3 acetylation level at the promoter regions of MITF and its

signature genes were reduced in A-485 sensitive cell lines after a 24-hour exposure to A-485 (Fig

4b), consistent with our microarray data. However, we did not observe p300 displacement at the

promoter regions of these genes (Figure 4c), suggesting that the p300/CBP inhibition affects its

histone acetylation activity at the MITF gene promoter regions without interfering with p300

chromatin binding (Figure 4d).

Discussion

The role of MITF in melanoma has been described but targeting MITF has been challenging

since it is a transcription factor. In this study, we show that a selective p300/CBP inhibitor A-485

induced senescence in more than half the melanoma cell lines tested. The majority of these cell lines

have high MITF expression. In addition, MITF pathway was the most significantly inhibited pathway

in p300/CBP inhibitor-sensitive cell lines as compared to an insensitive cell line suggesting that

MITF pathway could be targeted with a p300/CBP inhibitor. MITF expression and DNA copy

number also differs between the senescence vs. no senescence cell lines, suggesting patients could be

selected for this treatment. Interestingly, a melanoma cell state with high MITF expression dictates

sensitivity to RAF inhibitor suggesting that a combination of p300/CBP inhibitor and RAF inhibitor

in these patients could be more efficacious (8,9).

Finally, p300/CBP inhibitor decreased Histone-H3 acetylation without displacing p300 at

promoters of MITF and its putative downstream genes. In addition, our current finding is

consistent with our recent report that A-485 inhibited proliferation in lineage-specific tumor

types, such as androgen-receptor positive prostate cancer (19). On the basis of these observations,

p300/CBP inhibitor could be further exploited as a potential therapy for treating MITF amplified

melanoma.

Acknowledgements

We thank the AbbVie Oncology Biomarkers group and epigenetic group for discussion and critical

review of the manuscript. We thank Sujatha Jagadeeswaran for technical support; Saul Rosenberg,

Ken Bromberg, and Josh Plotnik for critical review of the manuscript.

References

1. Luke JJ, Flaherty KT, Ribas A, Long GV. Targeted agents and immunotherapies: optimizing outcomes in melanoma. Nat Rev Clin Oncol 2017;14(8):463-82. 2. Bandarchi B, Ma L, Navab R, Seth A, Rasty G. From melanocyte to metastatic malignant melanoma. Dermatology research and practice 2010;2010:583748. 3. Callahan MK, Flaherty CR, Postow MA. Checkpoint Blockade for the Treatment of Advanced Melanoma. Cancer treatment and research 2016;167:231-50. 4. Wong DJ, Ribas A. Targeted Therapy for Melanoma. Cancer treatment and research 2016;167:251-62. 5. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 2011;364(26):2507- 16. 6. Hertzman Johansson C, Egyhazi Brage S. BRAF inhibitors in cancer therapy. Pharmacol Ther 2014;142(2):176-82. 7. Manzano JL, Layos L, Buges C, de Los Llanos Gil M, Vila L, Martinez-Balibrea E, et al. Resistant mechanisms to BRAF inhibitors in melanoma. Annals of translational medicine 2016;4(12):237. 8. Konieczkowski DJ, Johannessen CM, Abudayyeh O, Kim JW, Cooper ZA, Piris A, et al. A melanoma cell state distinction influences sensitivity to MAPK pathway inhibitors. Cancer Discov 2014;4(7):816-27. 9. Muller J, Krijgsman O, Tsoi J, Robert L, Hugo W, Song C, et al. Low MITF/AXL ratio predicts early resistance to multiple targeted drugs in melanoma. Nature communications 2014;5:5712. 10. Goding CR. Commentary. A picture of Mitf in melanoma immortality. Oncogene 2011;30(20):2304-6. 11. Garraway LA, Widlund HR, Rubin MA, Getz G, Berger AJ, Ramaswamy S, et al. Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma. Nature 2005;436(7047):117-22. 12. Price ER, Ding HF, Badalian T, Bhattacharya S, Takemoto C, Yao TP, et al. Lineage-specific signaling in melanocytes. C-kit stimulation recruits p300/CBP to microphthalmia. J Biol Chem 1998;273(29):17983-6. 13. Sato S, Roberts K, Gambino G, Cook A, Kouzarides T, Goding CR. CBP/p300 as a co-factor for the Microphthalmia transcription factor. Oncogene 1997;14(25):3083-92. 14. Liu X, Wang L, Zhao K, Thompson PR, Hwang Y, Marmorstein R, et al. The structural basis of protein acetylation by the p300/CBP transcriptional coactivator. Nature 2008;451(7180):846-50. 15. Chan HM, La Thangue NB. p300/CBP proteins: HATs for transcriptional bridges and scaffolds. Journal of cell science 2001;114:2363-73. 16. Iyer NG, Ozdag H, Caldas C. p300/CBP and cancer. Oncogene 2004;23(24):4225-31. 17. Hodis E, Watson IR, Kryukov GV, Arold ST, Imielinski M, Theurillat JP, et al. A landscape of driver mutations in melanoma. Cell 2012;150(2):251-63.

18. Lin WM, Baker AC, Beroukhim R, Winckler W, Feng W, Marmion JM, et al. Modeling genomic diversity and tumor dependency in malignant melanoma. Cancer Res 2008;68(3):664-73. 19. Lasko LM, Jakob CG, Edalji RP, Qiu W, Montgomery D, Digiammarino EL, et al. Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours. Nature 2017;550(7674):128-32. . 20. Storey JD, Tibshirani R. Statistical significance for genomewide studies. Proc Natl Acad Sci U S A 2003;100(16):9440-5. 21. Wang R, Islam K, Liu Y, Zheng W, Tang H, Lailler N, et al. Profiling genome-wide chromatin methylation with engineered posttranslation apparatus within living cells. Journal of the American Chemical Society 2013;135(3):1048-56. 22. McDonald ER, 3rd, de Weck A, Schlabach MR, Billy E, Mavrakis KJ, Hoffman GR, et al. Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening. Cell 2017;170(3):577-92. 23. Bromberg KD, Mitchell TR, Upadhyay AK, Jakob CG, Jhala MA, Comess KM, et al. The SUV4- 20 inhibitor A-196 verifies a role for epigenetics in genomic integrity. Nat Chem Biol 2017;13(3):317-24. 24. Bandyopadhyay D, Okan NA, Bales E, Nascimento L, Cole PA, Medrano EE. Down-regulation of p300/CBP histone acetyltransferase activates a senescence checkpoint in human melanocytes. Cancer Res 2002;62(21):6231-9. 25. Giuliano S, Cheli Y, Ohanna M, Bonet C, Beuret L, Bille K, et al. Microphthalmia-associated transcription factor controls the DNA damage response and a lineage-specific senescence program in melanomas. Cancer Res 2010;70(9):3813-22. 26. Carreira S, Goodall J, Denat L, Rodriguez M, Nuciforo P, Hoek KS, et al. Mitf regulation of Dia1 controls melanoma proliferation and invasiveness. Genes Dev 2006;20(24):3426-39. doi: 10.1101/gad.406406. 27. Cheli Y, Giuliano S, Botton T, Rocchi S, Hofman V, Hofman P, et al. Mitf is the key molecular switch between mouse or human melanoma initiating cells and their differentiated progeny. Oncogene 2011;30(20):2307-18. doi: 10.1038/onc.2010.598. Epub 1 Jan 31. 28. Cronin JC, Watkins-Chow DE, Incao A, Hasskamp JH, Schonewolf N, Aoude LG, et al. SOX10 ablation arrests cell cycle, induces senescence, and suppresses melanomagenesis. Cancer Res 2013;73(18):5709-18. 29. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov 2012;2(5):401-4.

Figure legends

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

non-targeting control siRNAs. The siRNA-induced reduction of gene expression was calculated and

results were normalized to GAPDH and to the non-targeting siRNA control (mean +/- s.d., n = 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 five days and levels of senescence was

determined. (D) A-485 treatment does not induce apoptosis in sensitive melanoma cells. WM2664

and SKMEL5 were treated with A-485 for five days. The percentages of apoptotic and early apoptotic

cells were detected using Annexin V fluorescein isothiocyanate (FITC) and Propidium Iodide (PI)

staining.

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 3µM

of A-485 for 6 or 24 hours. Heat map of statistically significant up- or down-regulated genes is

depicted (p < 0.01 with at least two-fold change compared to DMSO in at least one experimental

condition). Genes did not meet p < 0.01 with at least two-fold change are indicted by the color black.

(B) A-485 decreases MITF but not SOX10 transcriptional network. The upstream regulator analysis

was performed with Ingenuity Pathway Analysis (IPA®). Z-scores of > 2 or < -2 are 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 two-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 μM) in human melanoma cells. qPCR was

performed using GAPDH as an internal control and the results were normalized to GAPDH. Student’s

t-test was performed and p-values of < 0.05 were labeled with *. (E) A-485 decreases MITF protein

expression. Immunoblot analysis of MITF protein levels after A-485 treatment (0.3 and 3µM) for 24

hours in melanoma cell lines.

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, and SOX10 in

senescence and no senescence melanoma cell lines upon A-485 treatment. (B) DNA copy number of

MITF in senescence vs. no senescence melanoma cell lines upon A-485 treatment. Data was obtained

from the Cancer Cell Line Encyclopedia (CCLE) (http://cbioportal.org). Student’s t-test was

performed and p-values of < 0.05 were considered significant. (C) Higher MITF protein expression in

senescence than non-senescence melanoma cell lines upon A-485 treatment. MITF and GAPDH

protein expression was determined based on immunoblot analysis in melanoma cell lines. (D)

Correlation between MITF protein expression and viability/proliferation (IC50) to A-485 in

melanoma cell lines.

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, and TDRD7) upon A-485 treatment in WM2664, SKMEL5 and

A375 cells based on microarray analysis as shown in Fig. 2c. Acetylated histone H3 (B) and p300

cells treated with 3μM A-485 for 24 hours. Relative binding value comparing to DMSO

enrichment relative to negative control (IgG) for ChIP is indicated; GAPDH promoter sequence is

used as endogenous control for qPCR. Student’s t-test was performed and p-values of < 0.05 were

labeled with * while p-values of < 0.1 was labeled with **. (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.

Figure 1. p300/CBP inhibition leads to senescence in sensitive melanoma cells

A Oxazolidinedione B Lead compound siCtrl siEP300 EP300 level A-485

siCtrl siEP300 siCtrl siMITF MITF level Cell lines H3K27ac IC50 (M) Proliferation IC50 (M) A-485 A-486 A-485 A-486

WM2664 0.6 > 10 1.0 > 10

SKMEL5 0.9 > 10 0.9 > 10 siCtrl siMITF A375 1.0 > 10 > 10 > 10 D C DMSO 0.3 µM A-485 3 µM A-485

WM2664

SKMEL5

A375

Figure 2. A-485 induces a significant decrease in MITF expression and MITF signature genes in sensitive melanoma cells

A D WM2664 * SKMEL5 2.5 * 6 * * * DMSO * DMSO 2.0 0.3uM A-1619485* 0.3uM A-1619485 43uM A-1619485 3uM A-1619485 1.5 *

1.0 2

0.5

Normalized Expression Normalized Expression Normalized 0.0 0 6 hrs. 24 hrs. 6 hrs. 24 hrs.

B A375 Z-scores WM2664 1.5 Upstream 2.5 DMSO DMSODMSO 0.3uM A-1619485 regulators WM2664 SKMEL5 A375 2.0 0.30.3uM mM A-1619485A-485 3uM A-1619485 MITF -6.69 -5.61 -2.15 1.0 33uM mM A-1619485A-485 N.S.1.5 SOX10 -2.15 -2.15 -2.56 N.S. 1.0 0.5 N.S. N.S. 0.5

6 hour 24 hour Normalized Expression Normalized C Sensitive Resistant Sensitive Resistant Expression Normalized 0.0 0.0 6 hour 24 hour Sensitive Resistant Sensitive Resistant 6 hrs. 24 hrs. WM2664 SKMEL5 A375 WM2664 SKMEL5 A375 6 hrs. 24 hrs. MITF E

A-485 (mM) MITF

GAPDH

Figure 3. MITF expression and DNA copy number predicts sensitivity to p300/CBP inhibitor in human melanoma cell lines

Figure 3. MITF expression and DNA copy number predicts sensitivity to p300/CBP inhibitor in human melanoma cell lines A B

p=0.02 N.S.

N.S. N.S.

C D

Figure 4 A

B ** ** * ** **

485 485

485 485 485

485

485 485

485 485 485

- -

- - -

- -

- - -

A A

A A A

A A

A A A

DMSO DMSO

DMSO DMSO DMSO

DMSO

DMSO DMSO

DMSO DMSO DMSO

485 485

485 485 485

485

485 485

485 485 485

- -

- - -

- -

- - -

A A

A A A

A A

A A A

DMSO DMSO

DMSO DMSO DMSO

DMSO

DMSO DMSO

DMSO DMSO DMSO D P300/CBP P300/CBP Ac Ac MLANA SEMA6A p300i TRPM1 TDTD7

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 Published OnlineFirst September 28, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-18-0511

Supplementary Access the most recent supplemental material at: Material http://mct.aacrjournals.org/content/suppl/2018/09/28/1535-7163.MCT-18-0511.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mct.aacrjournals.org/content/early/2018/09/28/1535-7163.MCT-18-0511. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.