SF3B1-Mutant Myelodysplastic Syndrome with Ringed Sideroblasts (MDS-RS) at the Single-Cell Level

The biological mechanisms of abnormal terminal erythroid differentiation (TED) in MDS-RS and SF3B1 mutations (SF3B1^MT) are largely unknown. This gap in understanding, which has dramatically delayed the design of second-line approaches for SF3B1^MT patients whose disease has failed hypomethylating agent (HMA) therapy, is primarily due to a lack of studies molecularly characterizing how SF3B1^MTaffect distinct stages of erythropoiesis.

Here, we dissected at the single-cell level the cellular and transcriptomic changes induced by SF3B1^MT in cells undergoing erythroid differentiation and elucidated how HMA therapy can overcome SF3B1^MT-defective erythropoiesis.

We first analyzed the expression profile of the lineage-negative CD34+ stem and progenitor (HSPC) compartment. Single-cell RNA sequencing (scRNA-seq) analysis of HSPCs isolated from 2 healthy donors (HDs) and 5 untreated MDS-RS patients with SF3B1^MT revealed cell clusters driven by the cells' differentiation potential that we defined based on the differential expression of validated lineage-specific transcriptional factors and cellular markers. Whereas the HD HSPCs had equally distributed erythroid/megakaryocyte and myeloid/lymphoid differentiation trajectories, the SF3B1^MT HSPCs had a predominant erythroid differentiation route (Fig. 1a). Differential expression analysis revealed that the SF3B1^MT HSPCs undergoing erythroid differentiation were characterized by the expression of genes involved in translation, oxidative phosphorylation, and cell cycle progression, which underlines these cells' metabolic and proliferative activation. Consistent with these findings, scRNA-seq of bone marrow (BM) mononuclear cells (MNCs) from the same samples showed that SF3B1^MT MNCs had a predominant population of erythroid cells at the expense of B lymphocytes and myeloid cells (Fig. 1b). An analysis of the erythroid cluster distribution inside the erythroid compartment showed that the frequency of early-stage maturation (BFU-E [cluster #18], CFU-E [#9]) was lower in the SF3B1^MT erythroblasts than in the HD erythroblasts, owing to a significantly increased frequency of SF3B1^MT cells in the latest stages of TED (proerythroblasts [#4]), basophilic normoblasts [#12], polychromatophilic normoblasts [#7], orthochromatic normoblasts [ONs; #1/11], pre-reticulocytes [#13]). These data suggest that SF3B1^MT enhance HSPC differentiation towards the erythroid lineage but arrest erythroblasts at the last step of their maturation by inhibiting the transition of ONs to pre-reticulocytes, resulting in the accumulation of TED cells in the BM. Transcriptomic analysis of SF3B1^MT ONs revealed a significant upregulation of genes regulating heme metabolism, including those involved in EIF2AK1's response to impaired heme production, and of major effectors of cell cycle checkpoint activation (e.g., CHK1, CDKN1A, GADD45). These data are consistent with previous studies showing that the SF3B1^MT-induced defective accumulation of iron in erythroblasts' mitochondria leads to activated cell stress response, cell cycle arrest, and apoptosis. Of note, the growth differentiation factor 11 receptors ACVR1B, TGFBR1, and ACVR1C were not expressed at any step of erythroid differentiation, which challenges this factor's role as a target of luspatercept-induced differentiation of late-stage erythroblasts.

To evaluate how HMA therapy can overcome inefficient erythropoiesis, we performed scRNA-seq of HSPCs and BM MNCs isolated from SF3B1^MT MDS-RS patients before any therapy and at the time of hematological response to HMA therapy. HMA therapy did not reduce the aberrant differentiation of SF3B1^MT HSPCs towards the erythroid lineage but temporarily induced the TED of SF3B1^MT ONs into reticulocytes and these cells' release into peripheral blood. Accordingly, blast progression following the initial HMA therapy-induced hematological response coincided with the further expansion of the erythroid-primed cells arising from the earliest stem cell population (Fig. 1c), which suggests that failure to eradicate or genetically correct SF3B1^MT stem cells leads to disease relapse.

In conclusion, our results elucidate how SF3B1^MT molecularly affect distinct stages of erythropoiesis and have implications for developing approaches that achieve lasting hematological remission in patients with MDS-RS.

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