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posted on 2023-03-31, 02:27 authored by Michael F. Emmons, Fernanda Faião-Flores, Ritin Sharma, Ram Thapa, Jane L. Messina, Jurgen C. Becker, Dirk Schadendorf, Edward Seto, Vernon K. Sondak, John M. Koomen, Yian A. Chen, Eric K. Lau, Lixin Wan, Jonathan D. Licht, Keiran S.M. Smalley Supplementary Table 1: Clinical data on samples stained for HDAC8 expression. S1) HDAC expression levels in melanoma cells S2) Cell lines that are resistant to BRAF inhibitors have upregulated c-Jun protein expression S3) Inhibiting HDAC6 does not resensitize BRAF/MEK resistant cells to BRAF inhibitors S4) BRAF/MEK inhibitor withdrawal decreases HDAC8 expression over time S5) Knocking down or elevating HDAC8 levels does not change cell proliferation S6) Expression of HDAC8 increased ERK activity and resistance upon treatment with BRAF-MEK inhibitors S7) Knocking down HDAC8 in BRAFi naïve cells increased apoptosis and BIM expression while decreasing ERK activity after BRAFi activity. S8) HDAC8 expression increases baseline RTK signaling S9) Inhibiting MET or FGFR3 partially resensitizes HDAC8 expressing cells to BRAF inhibitors S10) HDAC8 expression increases baseline c-Jun, p53, AKT and HSP60 signaling S11) Inhibiting c-Jun resensitizes HDAC8 expressing cells to a BRAF inhibitor S12) Protein sequences of c-Jun acetyl mutants S13) Acetylation-deficient c-JUN at residue 273 increases melanoma cell survival following BRAF inhibition S14) Acetylation-deficient c-Jun at residue 273 increases p53, Akt 1/2/3, STAT3, WNK1 and HSP60 S15) Acetylation-deficient c-JUN at residue 273 increases baseline EGFR signaling
Funding
SPORE
NCI
Florida Department of Health
Forma Therapeutics
Proteomics and Metabolomics Core
Biostatistics and Bioinformatics Core
NCI-designated Comprehensive Cancer Center
History
ARTICLE ABSTRACT
Melanoma cells have the ability to switch to a dedifferentiated, invasive phenotype in response to multiple stimuli. Here, we show that exposure of melanomas to multiple stresses including BRAF–MEK inhibitor therapy, hypoxia, and UV irradiation leads to an increase in histone deacetylase 8 (HDAC8) activity and the adoption of a drug-resistant phenotype. Mass spectrometry–based phosphoproteomics implicated HDAC8 in the regulation of MAPK and AP-1 signaling. Introduction of HDAC8 into drug-naïve melanoma cells conveyed resistance both in vitro and in vivo. HDAC8-mediated BRAF inhibitor resistance was mediated via receptor tyrosine kinase activation, leading to MAPK signaling. Although HDACs function at the histone level, they also regulate nonhistone substrates, and introduction of HDAC8 decreased the acetylation of c-Jun, increasing its transcriptional activity and enriching for an AP-1 gene signature. Mutation of the putative c-Jun acetylation site at lysine 273 increased transcriptional activation of c-Jun in melanoma cells and conveyed resistance to BRAF inhibition. In vivo xenograft studies confirmed the key role of HDAC8 in therapeutic adaptation, with both nonselective and HDAC8-specific inhibitors enhancing the durability of BRAF inhibitor therapy. Our studies demonstrate that HDAC8-specific inhibitors limit the adaptation of melanoma cells to multiple stresses including BRAF–MEK inhibition.
This study provides evidence that HDAC8 drives transcriptional plasticity in melanoma cells in response to a range of stresses through direct deacetylation of c-Jun.
