Abstract 4404: EZH2 modulates mRNA splicing in chronic myeloid leukemia through the repression of splicing factors and exerts part of its oncogenic function through the regulation of CELF2
Authors: Brunmeir Reinhard , Li Ying , Lin Baohong , Zheng Leong Qiao , Yang Henry , Chng Wee Joo 
Published: March 2024

The polycomb protein EZH2 is a commonly deregulated or mutated transcriptional regulator in various cancers. In Chronic Myeloid Leukemia (CML) it has been reported to be upregulated and associated with transcriptional reprograming, rendering CML leukemic stem cells (LSCs) sensitive to EZH2 inhibitor treatment. Mechanistically this seems to be linked to the silencing of apoptotic target genes by EZH2-mediated histone H3 lysine 27 tri-methylation (H3K27me3). We hypothesized that the regulation of apoptotic genes is not the only pathway responsible for its oncogenic function, and that EZH2 might also act as a modulator of the mRNA splicing landscape. To test this, we treated the CML cell line K562 with the EZH2 inhibitors GSK126 and EPZ-6438 and performed RNA-seq followed by splicing analysis. Using two different bioinformatics tools, we detected differential splicing of several hundreds of splicing events. However, differentially spliced genes were not associated with increased EZH2 or H3K27me3 signals, suggesting an indirect mechanism of splicing regulation. Indeed, we found 11 splicing factors transcriptionally upregulated upon EZH2 inhibitor treatment. CELF2 was identified as top candidate to mediate part of the EZH2 inhibitor induced phenotype. Upon overexpression, we observed 1) reduced cell growth, viability, and colony formation of K562 cells, 2) a change in the splicing landscape, significantly overlapping with EZH2 inhibitor induced changes, 3) the downregulation of cMYC signaling, also found upon EZH2 inhibitor treatment. To test if CELF2 is indeed acting as EZH2-regulated tumor suppressor in CML, we extended our study to CML patient samples: we performed RNA-seq followed by splicing and gene expression analysis in seven CML patient-derived CD34+ samples and eight healthy donor-derived CD34+ samples, as well as three CML patient samples transduced with CELF2-containing expression constructs. While we found Celf2 mRNA downregulated in CML derived samples, overexpression led to decreased colony formation and cMYC signaling, validating our findings from K562 cells. We also performed an analysis of CML associated splicing changes and found hundreds of deregulated splicing events, of which a significant portion overlapped with CELF2 overexpression mediated changes, suggesting direct regulation by CELF2. Based on this data we propose a novel role of EZH2, where it exerts part of its oncogenic function in CML through the transcriptional repression of splicing factors, in particular CELF2, leading to splicing deregulation, increased cMYC signaling, and enhanced cell growth and viability. Finally, data from other publicly available EZH2 inhibitor treated cancer models suggests that splicing modulation by EZH2 might not be restricted to CML.

Citation Format: Reinhard Brunmeir, Ying Li, Baohong Lin, Leong Qiao Zheng, Henry Yang, Wee Joo Chng. EZH2 modulates mRNA splicing in chronic myeloid leukemia through the repression of splicing factors and exerts part of its oncogenic function through the regulation of CELF2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4404.