Background. Amplification of the 8p11-p12 region, occurring in 10-30% of luminal tumors, is associated with poor prognosis and resistance to endocrine therapy. The 8p11-p12 amplicon is heterogeneous and contains several validated oncogenes, including NSD3, ZNF703, and FGFR1. ASH2L, an epigenetic regulator of chromatin, is a member of the trithorax group of proteins that promote transcription, primarily through tri-methylation of lysine 4 on histone H3 (H3K4me3) in the promoter region of target genes. The 8p11-p12 amplicon has been well-characterized previously in the SUM44 cell line and the role of overexpressed oncogenes such as NSD3 has been demonstrated. We therefore selected this cell line for further investigation of ASH2L function in the context of amplification of this oncogene, and its relationship with response to CDK inhibitors.
Methods. We assessed cell growth following knockdown of ASH2L with shRNA constructs compared to LacZ control in MCF7 and in the amplicon-bearing SUM44, CAMA1, and SUM52 cell lines. To assess the influence of ASH2L expression on promoter H3K4me3, we performed chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) utilizing an H3K4me3 antibody in control SUM44 cells and following shRNA-mediated knockdown of ASH2L. Three biological replicates were prepared and the samples sequenced simultaneously. We treated LacZ control and ASH2L knockdown SUM44 and MCF7 cells with increasing doses of palbociclib and assessed cell growth over 7 days. From the set of genes that lost promoter H3K4me3 upon ASH2L knockdown and were downregulated in response to knockdown of ASH2L, NSD3, and ESR1, we selected 5 genes (FBXO5, EZH2, TTK, CCNE2, and BUB1) that were downregulated in response to treatment with palbociclib according to comparative toxicogenomics database data.
Results. Knockdown of ASH2L reduced cell proliferation, validating that ASH2L influences cell growth regardless of amplification status. From ChIP-seq data, we found ASH2L robustly regulates H3K4me3 at the promoter region of itself and other genes of the amplicon, and genes pertaining to cell cycle and proliferation, such as EZH2 and WDR5. To determine the influence of ASH2L-regulated promoter H3K4me3 on transcription, we compared the gene set that lost promoter H3K4me3 upon ASH2L knockdown to the genes that were downregulated at the mRNA level upon knockdown of ASH2L by shRNA. This analysis identified 438 genes that were directly transcriptionally regulated by ASH2L, including cell cycle processes, palbociclib response, EZH2-regulated genes, estradiol-upregulated genes, and ESR1 interactions. SUM44 cells were more resistant to palbociclib than MCF7, and knockdown of ASH2L rendered these cells more resistant yet. Expression of FBXO5, EZH2, TTK, CCNE2, and BUB1, was reduced in the palbociclib-sensitive MCF7 cell line, however SUM44 cells did not demonstrate a reduction in transcript level for these genes after palbociclib treatment.
Conclusion. We identified a suite of genes with ASH2L-dependent promoter H3K4me3 that are transcriptionally downregulated upon ASH2L knockdown and discovered that these genes are predicted to be implicated in many cell cycle processes as well as response to palbociclib.
Citation Format: Giordano A, Mills JN, Hazard SE, Liu Y, Britten CD, Wilson RC, Chung D, Hardiman G, Ethier SP. The 8p11-p12 amplicon oncogene ASH2L regulates expression of genes involved in tumorigenic processes and response to palbociclib via promoter H3K4me3 [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-04-11.
