1011 – UNCOVERING CONVERGENT ABERRANT SPLICING EVENTS DRIVING MYELODYSPLASTIC SYNDROME DEFECTS

Splicing defects are a characteristic feature of myelodysplastic syndromes (MDS) and typically associate with recurrent splicing factor mutations. However, a subset of transcripts exhibit convergent abnormal splicing, occurring even in the absence of splicing-related mutations. These shared splicing events likely include common drivers of MDS hematopoietic defects, yet the functions of the resulting transcripts remain unknown. We identified a long isoform of the heterochromatin enforcer Methyl-CpG-Binding Domain 1 (MBD1), as the product of one such mutation-independent splicing event. In cord blood CD34+ cells overexpression of the MDS-associated full-length isoform (MBD1-L), containing MBD1′s 3rd CXXC domain, impaired erythroid differentiation, stalled cell cycling and promoted apoptosis while the MBD1-ΔCXXC3 isoform (MBD1-S), preferentially produced in healthy cells, did not induce these defects. Similarly, only MBD1-L impaired reconstitution capacity in vivo, particularly in the erythroid and myeloid lineages, and produced an enrichment of the MDS transcriptomic signature. We show that inclusion of the exon containing CXXC3, unique in specifically binding non-methylated CpGs, disrupts MBD1′s co-localization with heterochromatin. This triggers a striking redistribution of MBD1 from gene bodies and intergenic regions to hypomethylated promoter CpGs, resulting in widespread repression of promoter chromatin accessibility and downregulation of cell-cycle-related transcripts through its recruitment of the SETDB1:ATF7IP H3K9 methylase complex. Through knockdown or delivery of splice-switching antisense oligonucleotides targeting the CXXC3 exon into MDS cells, we confirm that targeted MBD1-L reduction inverts the quiescent, differentiation-impaired phenotype imposed by its overexpression. These findings provide evidence that mutation-independent splicing changes can drive hematopoietic dysfunction and serve as therapeutic targets in MDS.