Impact of different Sf3b1 mutations on hematopoietic function and erythropoiesis

Abstract Myelodysplastic syndrome (MDS) is a group of hematological disorders defined by clonal expansion of hematopoietic stem cells (HSCs) with acquired somatic mutations that cause peripheral blood cytopenias and bone marrow dysplasia. Adults with MDS typically present with anemia and have a high risk of progression to acute myeloid leukemia (AML). Splicing factor 3B subunit 1 (SF3B1) is a crucial spliceosome complex component and is mutated in ~25% of MDS cases. SF3B1 mutations are point mutations within the HEAT repeat domain, most commonly at positions K700, K666, and R625. While these hotspots are in close spatial proximity, they are typically mutually exclusive in patients and have varying effects on disease progression and survival. The K700E mutation is the most common and is associated with lower risk disease whereas mutations at K666 and R625 are linked to more rapid progression to AML. In this study, we aim to compare the impact of different Sf3b1 point mutations on MDS pathogenesis. We used three conditional knock-in mouse models driven by the Mx1-Cre promoter to evaluate the impact of each Sf3b1 mutation: Sf3b1K700E/+, Sf3b1K666N/+, and Sf3b1R625H/+. Peripheral blood counts were followed for 28 - 36 weeks post mutant allele induction. Sf3b1R625H/+mice were more anemic hemoglobin (Hb) 6.3 – 12.8 g/dL than Sf3b1K700E/+and Sf3b1K666N/+mice (Hb 12.0 – 13.6 g/dL) and had significant red cell macrocytosis mean corpuscular volume (MCV) 50 – 57 fL vs 41 – 47 fL (Sf3b1K700E/+) and 41 – 45 fL (Sf3b1K666N/+and WT). No significant differences in total white blood cell counts or platelets were observed between the genotypes. However, mutant allele expression in the peripheral blood as measured by next generation sequencing was significantly lower in Sf3b1K700E/+mice (9.9 ± 3.7%) compared to Sf3b1K666N/+(18.7 ± 3.2%) and Sf3b1R625H/+(19.7 ± 5.1%) mice (p = 0.002). At this relatively early timepoint, Sf3b1R625H/+mice demonstrated a significant block in terminal erythroid maturation measured by CD71 and Ter119 staining with a significant increase in the less mature CD71+ Ter119+ R2 cells (p .001) and a significant decrease in the more mature Ter119+ R4 cells (p .05) in the spleen. Immunophenotypic analysis of hematopoietic stem and progenitor populations in the bone marrow 36 weeks post-mutant allele induction was notable for a decreased frequency of short-term HSCs (ST-HSCs, p0.0001) and increased multipotent progenitor 3 (MPP3) p=0.02 cells in Sf3b1R625H/+ mice. Common myeloid progenitors (CMPs) were decreased in Sf3b1R625H/+ (p=0.03), and megakaryocyte-erythroid progenitors (MEPs) were increased in Sf3b1K666N/+ and Sf3b1R625H/+ (p=0.005) mice. Histologic evaluation of bone marrow biopsies was notable for trilineage dysplasia and micromegakaryocytes with all mutations, however Sf3b1R625H/+ mice displayed more myeloid dysplasia while Sf3b1K700E/+ mice had the most significant erythroid dysplasia. Increased iron deposition was noted in the spleens of all Sf3b1 mutant mice, consistent with increased extramedullary erythropoiesis. Previously, we have shown that Sf3b1K700E/+ HSCs have a significant competitive disadvantage when transplanted 1:1 with WT HSCs into WT recipients with peripheral blood chimerism decreasing from 50% to 20% as early as 4 weeks post- bone marrow transplant (BMT). We found that Sf3b1R625H/+ and Sf3b1K666N/+possess a similar disadvantage. Sf3b1R625H/+displayed the greatest disadvantage, with peripheral blood chimerism less than 2% at 12, 16, and 20 weeks post-BMT. Notably, while Sf3b1K700E/+ and Sf3b1K666N/+recipients had higher bone marrow than peripheral blood chimerism 20 weeks post-BMT (38.4 vs 12%; 6 vs 11.1%, respectively), this was not observed in Sf3b1R625H/+ recipients. We have shown the R625H mutation in SF3B1 appears to be associated with severe red cell maturation and HSC fitness defects that may progress to bone marrow failure without the acquisition of secondary mutations while the K666N mutation is associated with less severe anemia and HSC fitness findings. Overall, these findings highlight the differences in the functional and physiological impact of different Sf3b1 mutations, underscoring their potential distinct roles in impairing hematopoietic fitness and altering erythropoiesis.