SKI Controls MDS-Associated Chronic TGFb Signaling, Aberrant Splicing, and Stem Cell Fitness

The transforming growth factor-beta (TGFβ) signaling pathway controls hematopoietic stem cell (HSC) behavior in the marrow niche; however, TGFb signaling becomes chronic in early-stage myelodysplasia (MDS) where it may select MDS-genic HSC clones. In MDS, the level of TGFb signaling may have prognostic value while TGFβ-receptor inhibitors improve hematopoiesis in MDS samples. Thus, to better understand MDS pathobiology, it is vital to understand the mechanisms underlying chronic TGFb signaling. Normally, TGFβ signaling is tightly controlled by antagonists (e.g. SMAD7, SKI) which block promiscuous activity. Upon ligand-receptor engagement, these antagonists are transiently eliminated to amplify the signal; however, they are re-induced by TGFβ signaling and subsequently terminate the signal (a negative feedback loop). Early-stage MDS marrow cells have significantly diminished expression of SMAD7 and elevated levels of microRNA-21 (miR-21), which targets SMAD7. Thus, miR-21 is one factor that interferes with the TGFβ negative feedback loop to generate a chronic TGFβ signal in MDS.

We bioinformatically reanalyzed a recently published RNA-Seq dataset of MDS patient samples and find that a TGFb-signal-correlated gene signature is sufficient to identify a population of MDS patients with abnormal RNA splicing (e.g. CSF3R) independent of splicing factor mutations, and coincident with low HNRNPK activity. Elevated levels of SKI mRNA, encoding a transcriptional corepressor and TGFb-antagonist, are sufficient to identify these patients. We questioned why elevated SKI mRNA (encoding a TGFβ-antagonist) would be associated with chronic activation of TGFβ. We show that miR-21 targets SKI to block translation, and that event is associated with slight elevation in SKI mRNA abundance. Moreover, SKI protein is reduced in primary bone marrow samples from early-stage MDS patients with elevated miR-21 and chronic TGFβ signaling. To determine the impact of SKI loss, we examined murine Ski^-/- HSC. Ski^-/- HSC are initially specified and rescue transplant recipients in the absence of competitors, where they participate in multilineage hematopoiesis. However, when challenged with wild-type competitors, Ski^-/- HSC display a profound defect in HSC fitness that can be rescued by increasing the number of Ski^-/- HSC transplanted. Ski wild type and null embryonic stem cell - blastocyst complementation assays confirm an intrinsic Ski^-/- HSC defect in the absence of transplantation. Using single-cell RNA-Seq, Ski^-/- HSC exhibited striking upregulation of TGFb signaling, including Tgfb1 itself. Novel bioinformatics single-cell-splicing analyses revealed splicing alterations in Ski^-/- HSC concordant with low HNRNPK activity. Strikingly, a large fraction of the differentially expressed genes and alternative splicing events in Ski^-/- HSC are found in SKI-high MDS patients. We conclude that miR-21-mediated loss of SKI contributes to early stage MDS pathogenesis by activating TGFβ signaling and alternative splicing while hindering HSC fitness.