Mitochondrial Transcriptional-Translational Conflict Contributes to Defective Erythropoiesis and Disease Progression in Splicing Factor Mutant Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are myeloid malignancies derived from hematopoietic stem progenitor cells (HSPCs), characterized by cytopenia, dysplastic and ineffective hematopoiesis and a high risk of developing acute myeloid leukemia. SF3B1 is the most frequently mutated splicing factor in MDS, especially in MDS with ring sideroblasts. Although SF3B1 mutation generally indicates good prognosis, some patients with excess blasts still harbor this mutation. Previous studies have reported SF3B1 mutation induce R-loop and DNA damage with therapeutic implications, however, the role of SF3B1 mutation in disease progression and ineffective erythropoiesis remains poorly understood.

To characterize the properties of SF3B1 mutated HSPCs in MDS, we performed single-cell RNA sequencing combined with single-cell genotyping of bone marrow-derived CD34+ HSPCs from 3 lower-risk MDS patients and 3 higher-risk MDS patients according to the Revised International Prognostic Scoring System for MDS. Although mutated cells in two groups were both originated from HSCs and were found across almost all CD34+ progenitor cells, we observed an accumulation of mutated cells along the erythroid trajectory in lower-risk MDS and more mutated immature myeloid populations in higher-risk MDS. This suggests SF3B1 mutant cell frequency varies as a function of the progenitor subtype in different disease stage. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of differentially expressed genes in SF3B1 K700E mutant cells showed mitochondrial gene expression and mitochondrial translation-related signaling pathways were abnormal.

To deeply explore the roles of SF3B1 mutation in MDS HSC and erythroid differentiation, we established an MDS patient-derived inducible pluripotent stem cell (iPSC) line with SF3B1 K700E mutation and SF3B1 wild type. To gain a comprehensive understanding of transcription and translation, we performed RNA sequencing (RNA-Seq) and ribosome sequencing (Ribo-Seq) of HSPCs derived from iPSCs. The translation efficiency obtained by RNA-seq and Ribo-seq of HSPCs revealed a higher score in nuclear events: kinase and transcription factor activation but a lower score of ribosome biogenesis and mitochondrial translation of SF3B1 K700E cells, indicating activated transcription and imbalanced translation in SF3B1 mutated cells. Additionally, Gene set enrichment analysis showed activation of the PI3K-Akt (the upstream of mTOR) signaling pathway in SF3B1 mutated HSPCs. During HSPC differentiation, SF3B1 K700E group showed a higher mitochondrial membrane potential and mitochondrial mass, elevated mitochondrial superoxide level, cytoplasmic and total ROS levels, and extremely decreased mitochondrial DNA copy number, demonstrating dysfunctional mitochondria in mutated cells. During erythroid differentiation, SF3B1 K700E group showed a significant decrease in the production of erythroblasts compared to the wild-type line. Increased protein synthesis, especially in unfolded protein, and activated ATF4-mediated endoplasmic reticulum stress (ESR) were also found in mutated erythroblasts. We screened ROS scavengers (N-acetylcysteine), NAD⁺ precursor (Nicotinamide Riboside), integrated stress response inhibitor or ESR inhibitor (ISRIB, 4-Phenylbutyric acid), NADPH oxidase inhibitor (Setanaxib), tropomyosin receptor kinase (TRK) inhibitor (Larotrectinib), and translation-associated drugs (A-484954, 4EGI-1) to test whether they can rescue anemia, but unfortunately all of these drugs failed to improve erythropoiesis of mutated cells. Interestingly, rapamycin, an mTOR inhibitor, improved erythroid differentiation in SF3B1 K700E iPSCs even to a normal level, but this effect was not observed in WT iPSCs, implying that rapamycin is specific to SF3B1 mutation.

In summary, our findings provide insights into the critical role of mitochondrial transcription and translation in governing erythropoiesis via the mTOR signaling pathway. We propose that SF3B1 mutation cause dysfunctional mitochondria and elevated ESR, leading to aberrant mitochondrial translation and impaired protein synthesis, and suggest rapamycin as a potential therapeutic strategy for MDS patients with SF3B1 mutation to alleviate anemia by inhibiting stress-induced mTOR activation.