Abstract LB-134: The identification and characterization of MLL2, an epigenetic enzyme, as a novel tumor suppressor in melanoma

Abstract

Abstract Introduction: Much has been learned about the molecular basis of melanoma in recent years. However, the role of epigenetic alterations in melanoma genesis is poorly understood. Here we report the identification and characterization of MLL2, which encodes a histone lysine methyltransferase, as a novel tumor suppressor in melanoma. Methods: We performed an in vivo shRNA screen using 475 shRNAs targeting 95 epigenetic enzymes to identify novel tumor suppressors of melanoma genesis. Immortalized human melanocytic cell lines expressing BrafV600E (HMEL/PMEL) were transduced with pooled shRNAs and injected subcutaneously in nude mice. Candidate genes were identified from orthotopic tumors formed using PCR and Sanger sequencing and independently validated using multiple independent shRNAs and human cell lines. Results: Among 11 candidate genes identified from tumors formed in the in vivo shRNA screen, MLL2 knockdown was associated with the shortest time to tumor appearance (6 weeks) and highest penetrance (tumor formation at 80% of sites injected). In validation experiments, loss of MLL2 in human melanocytic and melanoma cell lines resulted in worse tumor-free survival when injected orthotopically in mice. Analysis of human melanoma data from The Cancer Genome Atlas (TCGA) project revealed that MLL2 is mutated in 15% of TCGA cases and is not associated with either BRAF or NRAS mutations. We found that MLL2 knockdown in human cell lines was associated with decreased expression of transcription factor TCF3 by qRT-PCR as well as decreased expression of TCF3 gene targets by gene set enrichment analysis (GSEA) of microarray data. Furthermore, GSEA analysis of data from TCGA samples with MLL2 mutations across tumor types revealed decreased expression of genes with TCF3 binding sites. Finally, we found that MLL2 knockdown in a human melanocytic cell line resulted in decreased H3K4 di- and tri-methylation at the promoter region of the TCF3 gene as well as at transcriptional start sites of TCF3 gene targets in ChIP-Seq experiments, suggesting a possible mechanism by which MLL2 loss may contribute to melanoma genesis and progression through dysregulation of the transcription factor TCF3. Conclusions: MLL2 is a novel tumor suppressor in melanoma identified through an in vivo shRNA tumorigenesis screen targeting 95 epigenetic enzymes. MLL2, which encodes for an epigenetic enzyme (a histone methyltransferase), is significantly mutated in human melanoma as represented in the TCGA and loss of MLL2 results in enhanced tumorigenesis in vivo. Studies to elucidate the mechanisms by which MLL2 loss contributes to melanoma genesis and progression and which focus on the transcription factor TCF3 are ongoing. Citation Format: Emily Z. Keung, Kunal Rai, Kadir C. Akdemir, Liren Li, Sneha Sharma, Bryce Axelrad, Lynda Chin. The identification and characterization of MLL2, an epigenetic enzyme, as a novel tumor suppressor in melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-134. doi:10.1158/1538-7445.AM2014-LB-134