Journal: bioRxiv

Article Title: HDAC8-mediated inhibition of EP300 drives a neural crest-like transcriptional state that increases melanoma brain metastasis

doi: 10.1101/2022.10.12.511971

Figure Lengend Snippet: A: HDAC8 expression revealed enriched chromatin accessibility/initiation of transcription for genes involved in migration and invasion. A GREAT analysis was performed on genes with enhanced accessibility/H3K27 acetylation around their transcription start sites. B-C: HDAC8 expression increases accessibility in NCSC genes while EV increases accessibility in melanocyte genes. There is increased accessibility in the promoter region of the (B) NCSC gene SOX2 while there is decreased accessibility in the promoter region of the (C) melanocyte gene MLANA upon HDAC8 expression. D-E: The HAT co-activators EP300 and CREBBP are deacetylated upon HDAC8 expression. A mass spectrometry-based acetylomics assay was performed on HDAC8 and EV expressing cells. D: Proteins deacetylated by HDAC8 were organized into signaling hubs using STRING. E: Significantly changed lysine acetylation sites in the CH3 domain of CREBBP and EP300 are shown. F: HDAC8 deacetylates EP300. Immunoprecipitation assays were performed with EP300 in the cell lines indicated. Lysates were probed for acetyl-lysine levels by Western Blot. Levels of acetyl-EP300 were normalized to EP300 input controls and quantified by ImageJ. G: HDAC8 reduces EP300 histone activity. Immunoprecipitation assays were performed with EP300 in two independent melanoma cell lines. Collected lysates were analyzed for EP300 mediated H4 histone acetylation. H: HDAC8 increases binding of EP300 to Jun bound promoter regions and decreases binding of EP300 to MITF bound promoter regions. A ChIP assay was performed on EP300 binding to the promoter region of Axl and MLANA . Significance in G was determined by a student’s t-test with *=p<0.005. Significance in (H) was determined by a one-way ANOVA followed by a post hoc t-test with *=p<0.05. Experiments were run 3 independent times with an n of 4 in each cohort.

Article Snippet: Antibodies against SMC3 (# 5696, D47B5), p-EGFR (Y1068, # 3777, D7A5), EGFR (#4267, D38B1), p-c-Jun (S73, #3270, D47G9), c-Jun (# 9165, 60A8), HDAC1 (#34589, D5C6U), HDAC2 (#57156, D6S5P), HDAC3 (#85057, D2O1K), acetyl-histone 3 (Lys27, #8173, D5E4), histone3 (#4499, D1H2), EP300 (# 86377, D8Z4E), and CREBBP (# 7389, D6C5) were purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Expressing, Migration, Mass Spectrometry, Immunoprecipitation, Western Blot, Activity Assay, Binding Assay

Journal: bioRxiv

Article Title: HDAC8-mediated inhibition of EP300 drives a neural crest-like transcriptional state that increases melanoma brain metastasis

doi: 10.1101/2022.10.12.511971

Figure Lengend Snippet: A: EP300 is downregulated in BRAFi resistant cells. Cell lines were probed for EP300, CREBBP and HDAC8 levels by western blot. B: Alterations in HDAC8/EP300 expression conferred a decrease in patient survival. Patient samples with decreased EP300 and increased HDAC8 RNA levels (change in 2-fold expression over all samples) were compared to unaltered patient samples using the TGCA melanoma database. C-D: Inhibition of EP300/CREBBP increases resistance to BRAFi. C: Cells were treated with vemurafenib (1 μmol/L, BRAFi), I-CBP112 (100 nmol/L, EP300i) or combination for 21 days. Colony formation was measured by Crystal Violet staining. D: Colonies were quantified using acetic acid permeabilization followed by ImageJ analysis. E: Silencing EP300 decreases sensitivity to BRAFi-MEKi. WM164 cells were transfected with a non-silencing siRNA (siNS) or an EP300 siRNA (siEP300_1 or siEP300_2). Cells were probed for EP300 and CREBBP after 72 hours transfection. Levels of CREBBP and EP300 were quantified by ImageJ. Cells were also treated with dabrafenib and trametinib (100 nmol/L dabrafenib, 10 nmol/L trametinib, BRAFi-MEKi) for 72 hours. Apoptosis was measured by Annexin V/PI staining using flow cytometry. F: Silencing of EP300 increases c-Jun phosphorylation. Cell lines were treated with I-CBP112 (100 nmol/L, EP300i) for 24 hours. Lysates were collected and probed for p-c-Jun and c-Jun by western blot. Levels of p-c-Jun were quantified by ImageJ. G: Forced expression of EP300 increases sensitivity to BRAFi-MEKi, whereas CREBBP does not. Cells with intrinsic resistance to BRAF inhibition were stably transfected with an empty vector, EP300 or CREBBP. Protein levels of EP300 and CREBBP were quantified for overexpression by ImageJ. Cells were treated with dabrafenib and trametinib (100 nmol/L dabrafenib, 10 nmol/L trametinib, BRAFi-MEKi) for 72 hours. Apoptosis was measured by Annexin V/PI staining using flow cytometry. H-I: Inhibition of EP300 increases migration in BRAFi sensitive cell lines. H: Melanoma spheroids were treated with ICBP-112 (100 nmol/L, EP300i) or VC and plated in collagen for indicated time points. I: Spheroid invasion area was quantified using ImageJ software. J-K: Introduction of EP300 decreases migration in BRAFi resistant cell lines. J: EP300- or EV-expressing spheroids were plated on collagen for indicated time points. K: Spheroid invasion area was quantified using ImageJ software. L: A schematic of the proposed mechanism of action for the HDAC8/EP300 modulated phenotype switch is shown. Significance in (D), (E) , and (G) was determined by a one-way ANOVA followed by a post hoc t-test with *=p<0.05, **=p<0.01, and #=p>0.05. Significance in (I) and (K) was determined by a student’s t-test with *=p<0.05. Experiments were run 3 independent times with an n of 3 in (D) , an n of 4 in (E) and (G) , and an n of 5 in (I) and (K) .

Article Snippet: Antibodies against SMC3 (# 5696, D47B5), p-EGFR (Y1068, # 3777, D7A5), EGFR (#4267, D38B1), p-c-Jun (S73, #3270, D47G9), c-Jun (# 9165, 60A8), HDAC1 (#34589, D5C6U), HDAC2 (#57156, D6S5P), HDAC3 (#85057, D2O1K), acetyl-histone 3 (Lys27, #8173, D5E4), histone3 (#4499, D1H2), EP300 (# 86377, D8Z4E), and CREBBP (# 7389, D6C5) were purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Western Blot, Expressing, Inhibition, Staining, Transfection, Flow Cytometry, Phospho-proteomics, Stable Transfection, Plasmid Preparation, Over Expression, Migration, Software