Targeting the EIF2AK1 signaling pathway rescues red blood cell production in SF3B1-mutant myelodysplastic syndromes with ringed sideroblasts

SF3B1 mutations, which occur in 20% of patients with myelodysplastic syndromes (MDS), are the hallmarks of a specific MDS subtype, MDS with ringed sideroblasts (MDS-RS), which is characterized by the accumulation of erythroid precursors in the bone marrow and affects the elderly population. Here, using single-cell technologies and functional validation studies of primary SF3B1-mutant MDS-RS samples (MDS-RS-3-bis, MDS-RS-13, and MDS-RS-14), we show that SF3B1 mutations lead to the activation of the EIF2AK1 pathway in response to heme deficiency and that targeting this pathway rescues aberrant erythroid differentiation and enables the red blood cell maturation of MDS-RS erythroblasts. These data support the development of EIF2AK1 inhibitors to overcome transfusion dependency in patients with SF3B1-mutant MDS-RS with impaired red blood cell production. MACS-purified CD34+ cells were subjected to a 3-phase in vitro culture as described previously (Huang et al., 2020a). Cells were maintained in phase 1 from the day of collection until day 8, at which time the cells underwent ribonucleoprotein-based gene editing and were transitioned to phase 2 medium. On day 13 of culture, the cells were transitioned to phase 3 medium. Samples for Western blot and cytospin analyses, and sample for flow cytometry were harvested on day 13 or 15 of culture, respectively. RNA sequencing of NT vs. EIF2AK1 [KO] samples on day 13 was used to determine the VAF % of SF3B1 and to perform comparative gene expression profiling analysis of NT vs. EIF2AK1 [KO] samples. Three primary SF3B1-mutant MDS-RS samples were used (MDS-RS-3-bis, MDS-RS-13, and MDS-RS-14). The number 3, 13 and 14 in each sample name is a consecutive number of the sample in the study.