Myelodysplastic syndromes (MDS) are genetically complex diseases that require multiple mutations during disease development. Acquisition of somatic mutations results in clonal diversity and different responses to therapy. Genes encoding the spliceosomal proteins SRSF2, SF3B1, U2AF1, and ZRSR2 are frequently mutated in MDS, together affecting around 60% of patients. SRSF2 mutations are found in 10-13% of MDS patients and almost always present as heterozygous missense mutations. Several patient studies have shown that SRSF2 mutations are closely associated with RUNX1, ASXL1, IDH1, and IDH2 mutations. RUNX1 is an important transcription factor for hematopoiesis and mutations of RUNX1 are detected in 10-15% of MDS. Although RUNX1 is one of the most frequently co-mutated genes with SRSF2 in MDS patients, it remains unknown how this combination of genetic alterations affect hematopoiesis. Here we hypothesize that RUNX1 and SRSF2 mutations cooperate in MDS development and explored this biologically in vivo.
To investigate whether the coexistence of SRSF2 mutation and RUNX1 deficiency could impair hematopoiesis in vivo, we utilized previously reported Mx1-Cre based conditional knock-in Srsf2-P95H mutation (P95H/+) mice (which confers an MDS phenotype), and Mx1-Cre based Runx1 conditional knockout mice (Runx1 f/f) (which demonstrates a mild myeloproliferative phenotype). We crossed these two strains to establish a new mouse model (Srsf2 P95H/+ Runx1 f/f Mx1-Cre). To determine whether the phenotypic effects from the mutations are hematopoietic cell-intrinsic manner, we performed non-competitive bone marrow transplantation experiments by transplanting mouse bone marrow mononuclear cells collected from Mx1-Cre, Runx1 f/f Mx1-Cre, Srsf2 P95H/+ Mx1-Cre, and Srsf2 P95H/+ Runx1 f/f Mx1-Cre mice into lethally irradiated recipients. Four weeks after transplantation, we induced Cre expression by pIpC injection. RUNX1 loss and SRSF2 P95H together altered multi-lineage hematopoiesis in mice. Double mutant mice showed MDS features including severe leukopenia in multiple lineages, mild anemia, mild thrombocytopenia, and dysplastic morphology, such as hyposegmented/hypersegmented neutrophils and red blood cells with Howell-Jolly bodies in peripheral blood. Double mutant mice also displayed more dramatic skewing to the myeloid lineage at expense of the B cell lineage when compared to single mutant mice. Flow cytometric analysis of the peripheral blood cell lineages revealed reduced B cell percentages (Mean of B220+ percentage Mx1-Cre: 51.6%, Runx1 f/f Mx1-Cre: 40.5%, Srsf2 P95H/+ Mx1-Cre: 38.6%, Srsf2 P95H/+ Runx1 f/f Mx1-Cre:20%, N = 7-12 each genotype, 28 weeks after bone marrow transplantation) and increased myeloid percentages (Mean of CD11b+ percentage: 20.1%, 24.3%, 18.9%, 35.5% respectively). At 20-weeks post transplantation, we analyzed the spleen and bone marrow compartments of the mice. In the spleen, we found the same enhanced skewing from B-cells to myeloid lineage as in the peripheral blood compartment. However, in the bone marrow, single and double deficiency mice both demonstrated similar decreases in B cell percentage. To evaluate stem and progenitor function in vivo, we performed competitive bone marrow transplantation. We mixed CD45.1 bone marrow and CD45.2 (Mx1-Cre, Runx1 f/f Mx1-Cre, Srsf2 P95H/+ Mx1-Cre, Srsf2 P95H/+ Runx1 f/f Mx1-Cre) bone marrow in a 1:1 ratio, transplanted to lethally irradiated recipients, and injected pIpC 4 weeks after transplantation. The repopulation assays showed that SRSF2 P95H with RUNX1 deficiency confer a competitive disadvantage. Our findings from these mouse models indicate that RUNX1 deficiency and SRSF2 P95H mutation together impair multi-lineage hematopoiesis and exacerbate disease phenotypes caused by SRSF2 P95H mutation.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.