P713: exploring clonal competition through 12-year follow-up of an mds-rs patient with dual sf3b1 mutations

Background: Hematopoietic SF3B1 mutations are well-established as a driver event of clonal establishment and expansion in myelodysplastic syndromes with ring sideroblasts (MDS-RS). However, the mechanisms underlying the fitness advantage that allows SF3B1-mutant (SF3B1mt) hematopoietic stem and progenitor cells (HSPCs) to thrive remain poorly understood. Aims: Here we aim to unravel the molecular mechanisms of hematopoietic clonal competition through long-term longitudinal analysis of a dual SF3B1mt MDS-RS patient. This patient displayed active competition of two clones propagated by SF3B1N626D and SF3B1K666N mutations and was followed in the clinic over the course of 12 years, providing a unique opportunity to investigate clonal dynamics in MDS-RS (Figure 1). Methods: We performed whole-genome sequencing (WGS) of HSPC-generated colony-forming units (CFU), high-throughput 10X single-cell RNA sequencing (RNAseq) of CD34+ HSPCs and combined single-cell DNA + RNAseq (TargetSeq) of FACS-purified HSPC compartments to molecularly characterize the two distinct SF3B1mt clones in bone marrow samples acquired before, during and after the points of clonal inversion. Results: Genotype tracking displayed an initial rapid expansion of the N626D clone over approximately 2 years, followed by spontaneous expansion of the K666N clone. SF3B1K666N eventually dominated the bone marrow, driving a complete inversion of clonal prevalence. This active competition was underlined only by stable subclinical anemia (median 12.5 g/L, range 12.0 - 13.2 g/L) and a continued decrease in ring sideroblast frequency over time (from 42% to 8% RS), both in the absence of any treatment. The acquisition of either SF3B1 mutation was found to precede the diagnosis of MDS-RS by at least 20 years, and either mutation was associated with an increased mutational rate. Mirroring previous studies, we identified a deleterious alternative splicing profile common to both SF3B1 genotypes in HSPC subsets; however, this was accompanied by unique mis-splicing profiles specific to each mutation which potentially drive their differential expansion ability. Summary/Conclusion: We identified mutation-specific splicing profiles and intricate patterns of tumor suppressor gene expression as potential compound factors to the varied clonal fitness of SF3B1 mutations. This unique patient enabled the identification and tracking of mechanisms underlying long-term clonal expansion and competition, which we aim to verify in further work.Keywords: Hematopoiesis, Myelodysplastic syndrome, RNA-seq, Myelodysplasia