Background: Melanoma cells show great plasticity and can switch from a proliferative, drug-sensitive state to an invasive, drug-resistant phenotype through mechanisms that remain poorly defined.
Hypothesis: Transcriptional and chromatin regulatory factors alter the gene expression states of cell in response to targeted therapy treatment and allow melanoma cells to persist and grow in the face of initially effective therapy. These factors/pathways may represent additional targets for anti-melanoma therapy.
Methods: BRAF mutant melanoma cells were exposed to the BRAF inhibitor vemurafenib to generate cells that persisted and grew in the face of continued drug treatment. RNA-Seq, CHIP seq, ATAC-Seq and mass spectroscopy analyses were performed on persistent/resistant cells in comparison to parental cells. Additionally, histone deacetylase (HDAC) 8 and EP300 expression was enforced in drug naïve melanoma cell lines and the effects determined.
Results: HDAC8 (but not other HDACs) was consistently up-regulated in BRAFi resistant melanoma cells. Increased expression of HDAC8 reduced cell growth, increased p53 activation and suppressed microphthalmia-associated transcription factor MITF, suppressing the melanocyte lineage program driven, leading to increased drug resistance. Introduction of HDAC8 or its active mutant (S39A) increased the tolerance of melanoma cells to multiples stresses (UV-irradiation, hypoxia, and BRAF-MEK inhibitor therapy), reduced their proliferation and increased their invasive potential in in vitro and in vivo models. Gene expression analyses indicate genes associated with a proliferative, drug-sensitive phenotype (e.g. DCT, MITF, MLANA, SOX10) are downregulated in HDAC8 active cells while genes correlative with an invasive, drug-resistant phenotype are upregulated (e.g. AXL, EGFR, TGFB1, WNT5A). ATAC-Seq analysis showed that HDAC8 regulates chromatin structure and decreased accessibility in the DNA binding sites of MITF and Sox10 while increasing accessibility in the DNA binding sites of c-Jun and TEADs (e.g. YAP1). Additionally, ChIP-Seq showed an increase in HDAC8 activity decreased H3K27 acetylation in the promoter region of MITF and MITF regulated genes (e.g. DCT, MLANA, PMEL, TYR). Mass spectrometry-based acetylomics and co-immunoprecipitation experiments demonstrated that active HDAC8 associated with and deacetylated the histone acetyltransferase EP300, a known coactivator of MITF in melanocytes. Increased expression of EP300 increased the sensitivity of melanoma cells to UV-irradiation, hypoxia and BRAF-MEK inhibitor therapy.
Conclusions: HDAC8 associated with EP300 and mediated the switch to an invasive, stress resistant state in melanoma cells by suppressing MITF and increasing c-Jun driven transcriptional programs. Targeting HDAC8 could be a strategy to limit plasticity in advanced melanoma, increasing the efficacy of current therapies.
Citation Format: Michael Emmons, Richard Bennett, Alberto Riva, Bin Fang, Edward Seto, john koomen, Jonathan Licht, Keiran Smalley. HDAC8 activity regulates stress induced phenotype switching in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1116.