Abstract P1-05-10: MLL3 mutation in ER+ breast cancers leads to dramatic shift in ER binding locations and consequentially alters transcriptional programs

Abstract

Abstract Hypothesis: Endocrine therapy (ET) is an effective treatment for estrogen-receptor positive (ER+) breast cancer; however, more than 80% of women will develop endocrine resistance. ER ChIP-seq studies in clinical samples show tumors likely to relapse have a unique set of ER binding sites that correlate with gene signatures predicting clinical outcome. Understanding how several factors, including transcriptional co-activator-complexes, influence ER binding location is crucial to improving treatment. MLL3, which is recurrently mutated in ~7% of ER+ cases, is a member of one such complex that interacts with ER. It is a histone methyltransferase that marks active enhancers. We hypothesized that mutation of MLL3 may change the genomic landscape of enhancers and thus change binding patterns of ER, altering response to ET. Methods: To elucidate transcriptional consequences of MLL3 mutation in ER+ breast cancers, we leveraged TCGA ER+ breast cancer RNA-seq data. We used a gene-by-gene multivariate linear model to identify differentially expressed genes (DEGs) between MLL3-mutant and wild-type (WT) samples. This data was compared to DEG found with in-house RNA-seq data for MLL3-mutant MCF7 cells, MLL3-WT ZR751 cells, and MLL3-knockdown (KD) ZR751 cells to identify a testable MLL3-mutant signature. ChIP-seq data for ER and H3K4me1, the histone mark made by MLL3, was then produced using these cell lines in order to pinpoint genes with changes in both H3K4me1 and ER-binding upon MLL3 mutation/KD. This gene list was compared to DEG found through RNA-Seq. To illuminate inherent differences between MLL3-WT and mutant/KD cells in response to ET, Cell-Titer Blue, Cell-Titer Glo, and Caspase-Glo assays were performed using DMSO, 4-OHT, and Fulvestrant. Results: TCGA RNA-seq data revealed MLL3-mutant ER+ breast cancers have differentially expressed transcriptional regulators, including ER itself. iRegulon analysis showed enrichment for DEG-regulated by ER-tethering factor SP1, a phenomenon suggested to be associated with more aggressive behavior. ChIP-seq data revealed substantial shifts in both ER-binding locations and H3K4me1 marks upon MLL3 mutation/KD. Interestingly, annotated peaks for MLL3-KD ZR751 showed overlap with those of MLL3-mutant MCF7, and GREAT analysis of new ER binding sites in MLL3-KD ZR751 showed an up-regulation of signatures associated with endocrine resistance. Indeed, MLL3-KD ZR751 cells were more resistant to 4-OHT/Fulvestrant than MLL3-WT ZR751. Conclusions: Loss of MLL3 function leads to a massive shift in H3K4me1, indicating a shift in the genomic landscape of enhancers. This shift is associated with a shift in ER binding as well as alterations in gene expression. Pathway analysis of these genes suggest that loss of MLL3 function may increase endocrine resistance, and indeed, cell lines with loss of MLL3 function are more resistant to ER inhibition than MLL3-WT cell lines. This work demonstrates that loss of MLL3 function leads to shifts in the enhancer landscape, alterations in ER binding and regulation of gene expression, and contributes to more aggressive tumor behavior. Citation Format: Stauffer K, Stricker T. MLL3 mutation in ER+ breast cancers leads to dramatic shift in ER binding locations and consequentially alters transcriptional programs [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-10.