Functional and molecular consequences of SF3B1 mutations in human hematopoietic stem cells

ABSTRACT The surprising discovery that RNA splicing alterations induced by recurrent and common change-of-function mutations in the spliceosome can perturb hematopoiesis and lead to disease has raised a number of fundamental questions. SF3B1 mutations are found ~30% of patients with myelodysplastic syndromes (MDS) and are common disease-initiating events that first arise in hematopoietic stem cells (HSCs) in premalignant clonal hematopoiesis, or CHIP. The finding of SF3B1 mutations in CHIP suggests that these mutations confer a clonal fitness advantage to HSCs. However, how SF3B1 mutations dysregulate HSC function to promote clonal expansion and subsequent MDS development has not been established. We lack an understanding of how these mutations “re-wire” RNA splicing in HSCs, which mis-spliced genes are critical for promoting clonal expansion and impaired differentiation of mutant stem cells, and how to target pathogenic mis-splicing while sparing alternative splicing that is essential for normal HSC function. Here, we propose to gain a fundamental understanding of how SF3B1 mutations promote CHIP and MDS and use this knowledge to develop new therapeutic approaches. Our team consists of a stem cell biologist with expertise in hematologic disease modeling (Doulatov), a basic scientist with expertise in RNA splicing and functional genomics (Bradley), and a physician-scientist with expertise in MDS pathophysiology (Abkowitz). In preliminary studies, we leveraged precise gene editing in human HSCs to define the molecular and functional RNA splicing alterations induced by mutant SF3B1 in the HSC context. These experiments uncovered novel HSC-specific mis-spliced isoforms, implicating an altered response to inflammatory signaling in pathophysiology; showed that SF3B1-mutant human HSCs display robust in vivo reconstitution in humanized mice, including a competitive advantage over their wild-type counterparts; and identified checkpoint kinase 1 (CHK1) inhibition as a promising therapeutic strategy for selectively targeting SF3B1-mutant HSCs. We propose to build on these preliminary studies as follows: Aim 1, Define the molecular consequences of SF3B1 mutations on RNA splicing in human HSCs and progenitors; Aim 2, Define the functional basis of clonal expansion and aberrant differentiation of SF3B1-mutated HSCs; Aim 3, Identify therapeutic vulnerabilities and preclinical efficacy created by coordinated mis-splicing of mitotic regulators. The significance of these studies is that they will directly connect RNA mis-splicing to HSC dysfunction and pathology in MDS. The health relevance is that the proposed work will directly test novel treatments for MDS with SF3B1 mutations.