S161: ZRSR2 AND TET2 MUTATIONS PROMOTE MDS BY DYSREGULATING GENE EXPRESSION AND ABERRANT ALTERNATIVE SPLICING IN MICE

Abstract

Background: Mutations in splicing factors and epigenetic regulators are the most frequent genetic alterations in patients with myelodysplastic syndromes (MDS). The minor spliceosome factor ZRSR2 and the epigenetic regulator TET2 appear significantly associated in MDS patients. However, the functional impact of such mutations in the hematopoietic system and MDS have been scarcely studied. To address this question, we established a murine model (Zrsr2m/mTet2−/−) carrying mutations in both genes, which exhibited signs compatible with MDS disease in mice. However, the molecular disease mechanism has not yet been elucidated. Aims: To interrogate the impact of ZRSR2 and TET2 mutations in gene expression and alternative splicing in the context of hematopoiesis and MDS. Methods: Whole transcriptome sequencing was performed to investigate changes in gene expression and alternative splicing patterns. Lin-Sca-1+c-kit+ (LSK) cells (50,000 per sample) were sorted from pools of 3 mice, and mRNA was sequenced in an Illumina NovaSeq 6000 platform. Differential gene expression was determined using DESeq2 with adjusted P-value ≤ 0.05, log2FoldChange ≥ 0. Functional enrichment from differentially spliced genes was identified with KEGG, and the enrichment cut-off p-value was set as adjusted P-value ≤ 0.05. Relevant alternative splicing events were validated by RT-PCR. Results: RNA-seq analysis of Zrsr2m/mTet2−/−cells revealed 2952 differentially expressed genes (DEG), compared to 571 in Zrsr2m/m and 1203 in Tet2−/−. From 2952, 1327 were up-regulated and 1625 were downregulated. Interestingly, relevant genes for hematopoietic lineage specification were significantly dysregulated. In particular, genes related to lymphoid lineage (Flt3, Notch1, and Tlr7) were downregulated, while those related to myeloid lineage (Ccl9, Mpo, Ms4a6b, and Prtn3) and megakaryocytic-erythroid lineage (Pdfgrb, Itgb3, Optn, and Tgfbr3) were upregulated. This suggests an early myeloid and megakaryocyte-erythroid priming of LSK towards the production of cells from these lineages in Zrsr2m/mTet2−/− mice. Enrichment analysis of DEG using Go enrichment analysis identified ribosome function, inflammation, and migration/motility processes as the most significantly altered. Further, KEGG pathway enrichment pointed ribosome, the MAPK family, and pro-inflammatory pathways as significantly enriched in Zrsr2m/mTet2−/− cells. Finally, KEGG pathway enrichment of alternative splicing targets identified the MAPK family and the Fanconi anemia pathway as the most altered targets in Zrsr2m/mTet2−/− cells. Importantly, a total of 9 genes related to the MAPK pathway were identified as mis-spliced, from which Dusp1, Tgfbr2, and Fgf11 were validated in this study. Summary/Conclusion: In this study, we broaden our previous report and show that concurrent mutations in Zrsr2 and Tet2 dysregulate normal gene expression and cause aberrant mRNA splicing. Gene expression analysis identified ribosome function, inflammation, and migration/motility as the most altered processes in Zrsr2m/mTet2−/− cells. Alternative splicing analysis identified the MAPK and the Fanconi Anemia pathway as key targets of aberrant splicing. All in all, gene expression dysregulation and aberrant mRNA splicing disturb important biological pathways and drive the molecular pathomechanism in Zrsr2m/mTet2−/− mice.