Abstract IA36: SRSF2 mutations impair hematopoietic differentiation by altering exonic splicing enhancer preference

• Published in: Clinical cancer research 2015-09, Vol.21 (17_Supplement), p.IA36-IA36
• Main Authors: Kim, Eunhee, Ilagan, Janine O., Lee, Stanley, Ramakrishnan, Aravind, Chung, Young Rock, Micol, Jean-Baptiste, Murphy, Michele E., Kim, Min-Kyung, Zebari, Ahmad S., Buonamici, Silvia, Smith, Peter, Deeg, H. Joachim, Lobry, Camille, Aifantis, Iannis, Bradley, Robert K., Abdel-Wahab, Omar
• Format: Article
• Language: English
• ISSN: 1078-0432; 1557-3265
• DOI: 10.1158/1557-3265.HEMMAL14-IA36

Spliceosomal mutations account for the most frequent class of mutations in patients with myelodysplastic syndromes, yet the mechanism by which these mutations perform their driver function is not well understood. Given the genetic heterogeneity of primary patient samples, we generated a model for conditional expression of the commonly occurring SRSF2P95H mutation from the endogenous murine locus of Srsf2 and compared expression of the Srsf2P95H mutation with genetic inactivation of 0, 1 or 2 copies of Srsf2. Mx1-cre Srsf2P95H/wildtype mice exhibited significant morphologic dysplasia, leukopenia, macrocytic anemia, and preserved bone marrow (BM) cellularity as early as 2 weeks after mutation expression. Moreover, Mx1-cre Srsf2P95H/wildtype mice exhibited an increase in hematopoietic stem/progenitor cells (HSPCs) with an increase in lineage-negative Sca1+ c-Kit+ cells (LSK cells) in S-phase and early apoptosis. In competitive transplantation, Srsf2P95H mice HPSCs were expanded in the BM at 16 weeks post-transplantation despite having a reduced contribution to peripheral blood chimerism. In contrast, mice with homozygous deletion of Srsf2 exhibited anemia and leukopenia due to BM aplasia with striking loss of HSPCs. Collectively, these data show that Srsf2 is required for hematopoiesis, while mutations in Srsf2 provide a competitive advantage at the level of HSPCs but impair differentiation into mature circulating blood elements. Next, to identify transcriptional alterations caused by SRSF2 mutations, we performed deep RNA-seq on sorted HSPC populations from wildtype and Srsf2P95H mice, stable K562 cell lines ectopically expressing an empty vector or a single allele of SRSF2 (WT, P95H, P95L, P95R), as well as primary CMML and AML patient samples. We quantified global changes in splicing of ~125,000 alternative splicing events and ~160,000 constitutive splice junctions associated with SRSF2 mutations in these datasets. Intersection of differentially spliced genes in primary murine HSPC, CMML, and AML samples identified 75 genes that were differentially spliced in association with SRSF2 mutations in murine HSPCs and at least one primary patient cohort. Many of the genes that were differentially spliced in SRSF2 mutant cells participate in biological processes of known importance in myeloid malignancies. For example, a cassette exon of EZH2 that alters the reading frame inducing nonsense-mediated decay was promoted by SRSF2 mutations. We next sought to determine