ETV1-Driven Drug Resistance in BRAFV600E Melanoma
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Dang, Michelle
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Dang, Michelle. 2019. ETV1-Driven Drug Resistance in BRAFV600E Melanoma. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.Abstract
Melanoma is the most aggressive form of skin cancer and unresectable melanoma is treated with immunomodulators and targeted therapies. The emergence of drug resistance is a major clinical challenge and the reactivation of the MAPK and PI3K pathways are the most common mechanisms. The identification of novel resistance mechanisms will impact the development of better therapeutic strategies. Here we provide evidence that ETV1, an ETS family transcription factor that is amplified or upregulated in approximately 12% of human melanomas, is a transcriptional driver of drug resistance in BRAFV600E melanoma. Using the adult zebrafish melanoma model, I developed an oral gavage drug treatment platform and identified 10 transcription factors that conferred resistance against Vemurafenib, a BRAFV600E inhibitor. Of the candidates, overexpression of ETV1 and OCT4 did not accelerate median tumor onset and did not metastasize in a tumor transplantation assay. Characterization of the transcriptional and epigenetic signatures of ETV1 and OCT4 in a human melanoma line (A375) identified ETV1 signaling to be a driver of drug resistance. In chromatin immunoprecipitation sequencing (ChIP-seq) studies using DMSO-treated A375 cells overexpressing ETV1, ETV1 and endogenous OCT4 were globally co-bound. Upon Vemurafenib treatment, ETV1 binding significantly increased and OCT4 lost chromatin occupancy. In ChIP-seq studies performed in A375s overexpressing OCT4, OCT4 occupancy is lost upon Vemurafenib treatment and a subset of those binding sites become newly occupied by endogenous ETV1. Analysis of the new ETV1 peaks identified an enrichment for AP-1 and ETS binding motifs and gene set enrichment analysis (GSEA) using RNA-seq data revealed a negative enrichment for apoptosis. Key members of the BCL-2 family, including anti-apoptotic factors MCL1 and BCL2A1 were differentially upregulated via RNA-seq and decreased apoptotic activity in ETV1 and OCT4 overexpressing lines was measured via TUNEL. Finally, shRNAs were used to knockdown ETV1 in OCT4 overexpressing A375s and resulted in a restored sensitivity to Vemurafenib, suggesting the requirement of ETV1 in the OCT4 resistance. Taken together, our studies illustrate that Vemurafenib treatment increases ETV1 chromatin binding and transcriptionally activates an anti-apoptosis program to confer drug resistance.Terms of Use
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http://nrs.harvard.edu/urn-3:HUL.InstRepos:42029596
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