Targeting <i>EZH2</i> Oncogenic Splicing: Decoding the Regulatory Network and Antisense Correction

Recurrent mutations in splicing factors (SFs) have been established as crucial drivers of tumorigenesis in several types of blood cancer, and also common in a variety of solid tumors. Mutations change the RNA-binding preferences of SFs, promote global splicing alterations, and often generate erroneous mRNAs that are then degraded by nonsense-mediated mRNA decay (NMD). Consequently, several critical genes linked to hematopoiesis are dysregulated, leading to blood cancer. Although the field has progressed considerably in identifying aberrant genes and affected pathways, effective therapies have not yet emerged in SF-mutated cancers. To address this key gap, we instigated a gene-specific targeted strategy by unlocking the regulatory network. As a proof-of-concept, we scrutinized a tumor suppressor gene <i>EZH2</i> , which is a bona fide target in SRSF2-mutated cancer. We precisely defined splicing cis-elements in <i>EZH2</i> transcripts and illustrated the dynamic choreography of regulatory proteins in the entire splicing and NMD catalytic pathways. We uncovered a highly coordinated cross-regulation between splicing and NMD promoted by mutant SRSF2 by enhancing the deposition of critical spliceosome- and NMD-associated factors, augmenting mRNA decay to ablate tumor suppression. We then designed antisense oligonucleotides (ASOs) targeting important regulatory sites. Our lead ASO successfully corrects aberrant splicing and NMD, restores the expression and function of EZH2, and partially rescues hematopoietic defects and cellular properties. Our study demonstrates that ASO pharmacology is an actionable strategy for clinical development, challenging the existing paradigms in SF-mutated cancers.