Abstract 5833: Escape form adaptive drug tolerance through OGT and TET1 mediated H3K4me3 remodeling in MAPKi resistant melanoma
Authors: Menon Dinoop Ravindran , Hammerlindl Heinz , Emran Abdullah Al , Torrano Joachim , Hammerlindl Sabrina , Zhang Gao , Somasundaram Rajasekharan , Sturm Richard A. , Haass Nikolas K. , Flaherty Keith , Herlyn Meenhard , Schaider Helmut 
Published: August 2018

Abstract
Background Acquired drug resistance in BRAF mutant melanoma is the main cause for disease relapse. We previously described a slow cycling induced drug tolerant state upon continuous BRAF/MEK treatment preceding permanent resistance with generic changes in histone methylation. Rationale Genetic alterations linked to acquired BRAF inhibitor resistance are absent in about 40% of relapsed melanoma patients suggesting the involvement of epigenetic alterations in the development of acquired drug resistance. We investigated epigenetic remodeling in BRAF mutant melanoma upon BRAF/MEK inhibition. Methods An in-vitro model of time lapse dependent transition to acquired drug resistance using mutant BRAF melanoma was used to investigate epigenetic changes following chronic drug exposure. Histone methylation patters were investigated using ChIP-seq, followed by target gene promoter ChIP-PCR and functional verification. Findings were confirmed by gene silencing, combined treatment regimes in vivo, in PDX tumors and clinical data sets. Results A state dependent response to chronic drug treatment was observed. Long term treatment of more than 45 days enables the cells to escape the slow cycling state which results in proliferating cellular clusters (drug-tolerant persister colonies) with stem-like characteristics that regain global H3K4me3. Persister colonies are then giving rise to fast proliferating BRAF/MEK inhibitor resistant cells. H3K4me3 ChIP-seq of colonies compared to parental cells revealed differential marking at promotor regions of several target genes involved in MAPKi resistance, including ARAF, BRAF, and CRAF. H3K4me3 remodeling corresponded to increased gene expression and susceptibility to pan-RAF inhibitors, suggesting an H3K4me3 mediated increase of ARAF and CRAF as a mechanism of BRAF/MEK inhibitor resistance. Two enzymes, OGT and TET1 that are both linked to H3K4me3 regulation in embryonic stem cells are highly upregulated in persister colonies and tumor tissue of PDXs from BRAF mutant melanoma patients under MEK1/2 inhibition. A shift in OGT nuclear localization and O-linked glycosylation patterns was observed in colonies compared to parental cells suggestive of altered transcriptional and protein activity. OGT ChIP-PCR of colonies compared to parental cells confirmed a set of genes with exclusively H3K4me3 marking in colonies. shRNA mediated knockdown of OGT and TET1 blocked H3K4me3 increase in IDTC colonies, prevented colony formation and delayed tumor relapse in a BRAF mutant xenograft mouse model. High TET1 mRNA expression is linked to significantly shorter survival in TGCA data. Conclusion OGT and TET1 mediated epigenetic remodeling through H3K4me3 with upregulation of MAPKi resistant genes is responsible for the emergence of permanent resistance. Both enzymes are promising targets to combat treatment failure and prolong overall survival.
Citation Format: Dinoop Ravindran Menon, Heinz Hammerlindl, Abdullah Al Emran, Joachim Torrano, Sabrina Hammerlindl, Gao Zhang, Rajasekharan Somasundaram, Richard A. Sturm, Nikolas K. Haass, Keith Flaherty, Meenhard Herlyn, Helmut Schaider. Escape form adaptive drug tolerance through OGT and TET1 mediated H3K4me3 remodeling in MAPKi resistant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5833.