Sf3b1+/K700E hematopoietic stem cells create their own expansion niche

Abstract Myelodysplastic syndromes (MDS) is an heterogenous group of clonal hematopoietic disorders that affect older individuals. It is characterized by ineffective hematopoiesis and a variable risk of progression to acute myeloid leukemia (AML). About half of MDS patients carry somatic mutations in spliceosome genes, with SF3B1 being the most frequently mutated. It has long been considered that MDS is solely driven by genetic and epigenetic events in hematopoietic stem cells (HSCs) and our group has previously shown that hematopoietic-specific expression of Sf3b1+/K700E is sufficient to cause features of MDS including macrocytic anemia and significant HSC expansion within the bone marrow (BM) as the mice age. However, HSCs from Sf3b1+/K700E mice have a paradoxical competitive disadvantage when transplanted with an equal number of wild type (WT) HSCs into lethally irradiated WT recipients. These findings suggest that MDS progression in Sf3b1-mutant mice is related to the ability of Sf3b1+/K700EHSCs to alter their BM microenvironment to create a supportive milieu that promotes the expansion of mutant HSCs over WT HSCs. To test this hypothesis, we first evaluated the cytokines present in Sf3b1+/K700E and WT BM fluid using Luminex assays. Sf3b1+/K700E mice, aged 33 to 90 weeks after mutant allele induction with pIpC, had significantly increased levels of inflammatory cytokines including MCP-1 and IL1α (p 0.001) in addition to marked TGF-β1 elevation (p 0.05) in the BM compared to littermate controls. To functionally evaluate the effect of Sf3b1+/K700E HSC exposure on MSCs, we isolated BM MSCs from aged Sf3b1+/K700E and WT mice. After confirming the absence of Sf3b1 mutant allele expression within the MSCs by targeted deep next generation sequencing, we cultured MSCs from Sf3b1+/K700E and WT mice in adipogenic and osteogenic differentiation media. MSCs from Sf3b1+/K700E mice exhibit decreased capacity to differentiate towards adipogenic and osteogenic lineages in vitro. The osteogenic differences were further validated with micro-CT analysis of aged Sf3b1+/K700Eand WT femurs which revealed significantly increased trabecular spacing in Sf3b1+/K700Efemurs. Moreover, MSCs from Sf3b1+/K700Emice showed poor expansion in vitro and morphological changes suggesting increased cellular senescence. Indeed, senescence-associated β-galactosidase staining was markedly increased in MSCs isolated from Sf3b1+/K700Emice compared to WT. To determine whether MSCs are the cellular component of the BM niche that contributes to mutant HSCs expansion, we performed co-cultures of MSCs isolated from Sf3b1+/K700E and WT mice with HSCs sorted from mutant or WT animals in all possible combinations. While MSCs from Sf3b1+/K700E mice promoted a 5-fold expansion of WT HSCs compared to co-culture with MSCs from WT mice (44.4 ± 8.1 vs 214 ± 33.6 HSCs isolated after 10 days of co-culture, p 0.01), MSCs from Sf3b1+/K700E mice supported an even greater expansion of Sf3b1+/K700E HSCs (816 ± 24.7, p 0.001). To directly test whether Sf3b1+/K700E mutant HSCs alter healthy BM MSCs, we transplanted HSCs from aged CD45.2+WT and Sf3b1+/K700E mice into lethally irradiated young CD45.1+ WT recipients. As expected, WT mice transplanted with Sf3b1+/K700E HSCs developed a progressive macrocytic anemia. At 20 weeks post-transplant, we isolated BM MSCs from the recipients of Sf3b1+/K700E and WT HSCs and performed bulk RNA sequencing. We identified 92 differentially expressed genes in MSCs exposed to Sf3b1+/K700E compared to WT HSCs including an upregulation of genes associated with cell communication and cell interactions/migration. We further evaluated the function of these MSCs ex vivo with the in vitro co-culture assay. MSCs isolated from WT mice transplanted with Sf3b1+/K700E or WT HSCs were expanded and co-cultured with HSCs sorted from young WT mice. Importantly, we found that MSCs exposed to Sf3b1+/K700E HSCs compared to MSCs exposed to WT HSCs promoted a 3.3-fold expansion of WT HSCs (4480 ± 426.1 vs 14584 ± 1946 HSCs, p 0.05).In sum, our data demonstrate that HSCs with somatic Sf3b1+/K700Emutation can alter the gene expression and function of MSCs to promote the preferential expansion of mutantover WT HSCs. Identification of the mediators of these effects may lead to novel therapeutic targets to prevent MDS progression.