CO36 | TGF-β pathway deregulation in SF3B1-mutated low risk myelodysplastic syndrome patients

Introduction. Myelodysplastic syndromes (MDS) associated with mutations in genes belonging to the splicing machinery, such as SF3B1, SRSF2, U2AF1 and ZRSR2, are characterized by inefficient hematopoiesis and variable risk of evolving into AML. Mutations affecting SF3B1 are among the most common alterations, correlating with low-risk phenotype (LR-MDS) and response to Luspatercept treatment. We were interested in the characterization of the expression profiles and the spliceosome of LR-MDS mutated for splicing factors (SF). Methods: We selected a study cohort of 50 LR-MDS (30 M and 20 F, med. age 75 yrs, range 40-91) characterized by mutations in SF genes (SF3B1=15, SRSF2=9, U2AF1=11 and SF-wt=15).Results. RNA-Seq on 50 LR-MDS vs 64 non-haematologic pts revealed 4221 differentially expressed genes (DEGs), 3921 down- and 300 up-regulated. When subgrouping patients according to specific SF mutations, SF3B1-mutated MDS (n=15) showed the highest number of DEGs (622: 10 down- and 612 up-regulated) when compared to SF-wt pts (n=7). Gene Ontology analysis identified 37 deregulated pathways in SF3B1-mut vs SF-wt pts. We found 6 DEGs in TGF-β pathway (CHRD, DCN, SMAD9, FST, ID4, PITX2) and 10 in TGF-β modulators (BDNF, BMPER, COL1A1, COL1A2, FN1, GLI2, IGF1, MMP2, NFIB and PLAU). DEGs were confirmed using Q-rt-PCR assays: DCN (p=0,004), FST (p=0,017), SMAD9 (p=0,030), FN1 (p=0,004), IGF1 (p=0,030) and MMP2 (p= 0,004). We then investigated the presence of alternative splicing events (ASE) within the TGF-β pathway, identifying an intron 15 retention on the USP15 gene, a ubiquitin specific protease. This yields a truncated protein isoform (USP15-TR), which was preferentially expressed in SF3B1-K700E vs wt MDS, without changes in total mRNA. USP15-TR was overexpressed in SF3B1-K700E mutated K562 cells (p