To develop novel and improved cancer therapies, we aim to target the epigenome, which is often deregulated in cancer. We identified the splicing factor SF3B1 as a potential epigenetic regulator in a whole genome siRNA screen for reactivation of aberrantly silenced genes alone or in synergy with the DNMT inhibitor Decitabine (DAC). The screen was done in YB5 colon cancer cells which contain a methylated and silenced CMV promoter driving GFP expression, and confirmed in a similar model in HCT116, another colon cancer cell line. SF3B1 has been studied in splicing, but little is known about its effects on gene regulation outside of this function, prompting us to further investigate this. We validated the screen findings by siRNA (siSF3B1) and inhibition of SF3B1 with Pladienolide B (PB). We then performed RNA-Seq and Reduced Representation Bisulfite Sequencing (RRBS) in cells treated with siSF3B1 and DAC, and we performed ChIP-Seq to assess SF3B1 binding. By flow cytometry, we measured a ~7% and ~5% induction of GFP in YB5 cells after siSF3B1 and DAC, respectively. The combination treatment caused a synergistic increase to ~15% GFP positive cells (p < 0.001). siSF3B1 led to 423 up and 338 downregulated genes, while DAC induced 430 up and 135 downregulated genes. With the combination, there was activation of 1190 genes and downregulation of 904 genes (fold change > 2, FDR < 0.1). There were 695, 119, 1584 genes alternatively spliced following siSF3B1, DAC, and the combination treatment, respectively, but there was no significant overlap with the regulated genes (p < 0.05), suggesting a distinct mechanism for gene expression regulation. There were two major subsets of siSF3B1 upregulated genes. A set of genes had low promoter methylation (0-20% methylation) and were p53 activation targets such as CDKN1A and GADD45A. The other set of genes had high promoter methylation (80-100% methylation) and promoters containing TATA-box motifs. RRBS showed global DNA hypomethylation after DAC treatment as expected, with an average methylation of 76.2% compared to 84.7% methylation in siControl cells. Further demethylation occurred in the combination treated cells, which had an average methylation of 72.7% (p<0.001). ChIP-Seq showed differential binding of SF3B1 at the transcription start sites (TSSs) of genes based on their expression. SF3B1 was depleted at TSSs of expressed genes and enriched at nonexpressed genes, resembling the binding pattern of histone H3. Finally, SF3B1 inhibition with PB alone and with DAC also induced reactivation of gene expression and altered DNA methylation. Together, these findings suggest that SF3B1 may be playing a role in gene regulation outside of its role in splicing. In cancer, SF3B1 transcriptional target genes are potentially tumor suppressor genes and upon knockdown or inhibition of SF3B1, their expression is reactivated leading to antitumor effects.

Citation Format: Sandra Deliard, Yasuyuki Okamoto, Jozef Madzo, Somnath Pandey, Jaroslav Jelinek, Jean-Pierre Issa. Potential role of the splicing factor SF3B1 in epigenetic regulation and activation of p53 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4331.