Erythropoiesis In SF3B1 Mutated RARS Is Disrupted During Terminal Erythroid Maturation

Refractory anemia with ring sideroblasts (RARS) is characterized by severe anemia, erythroid apoptosis and aberrant mitochondrial ferritin accumulation in erythroblasts. Dominant mutations in SF3B1, a core component of the splicing machinery are detected in more than 75% of patients. Of the recurrent mutations, SF3B1 alone is associated with the RARS phenotype. We recently showed that SF3B1 suppresses the expression of the mitochondrial transporter protein ABCB7, which in turn mediates erythroid failure in RARS. The exact mechanisms remain unclear, hence potential targets for treatment have not been identified. Fifty-six patients with RARS or RCMD-RS were subjected to targeted sequencing of which 52 (93%) had SF3B1 mutations and 43 also other recurrent mutations mostly involving epigenetic regulators. To explore the mechanisms behind the disrupted RARS erythropoiesis molecular and biological features of erythroid progenitors from SF3B1 mutated RARS and normal bone marrow (NBM) were monitored in vitro throughout a well-established 14-days liquid culture system. Normal and RARS transcriptomes (RNAseq) were analyzed at two time points (CD34+ cells at day 0 and early erythroblasts at day 4) during differentiation, followed by validation in an extended cohort of 11 RARS and 4 NBM using Taqman Low Density Array (TLDA). CD34+ RARS progenitors were characterized by activation of genes involved in the oxidative stress pathway and in particular genes involved in defense against oxidative stress, such as SEPP-1. This activation dropped dramatically at day 4. By contrast, early differentiation in RARS displayed a marked failure to up-regulate genes in the autophagy pathway, which is essential for removal of organelles and terminal maturation to erythrocytes.

We then followed the clone size of SF3B1 in 8 erythroid cultures, two of which also underwent myeloid differentiation. 4 cultures used RARS patients with stable untreated anemia (Hb 107 g/L ± 15), a high percentage of ring sideroblasts and a relatively normal reticulocyte count. SF3B1 allelic burden was assessed by pyrosequencing at day 0, 4, 7 and 14. To exclude the possibility that the culture system itself promoted survival of mutated progenitors we also analyzed isolated GPA+ progenitors from uncultured mononuclear BM, and finally we analyzed blood reticulocytes from the 4 untreated patients. We observed a normal expansion of both erythroid and myeloid progenitors during culture, with a stable allele burden (± 10%) in all patients. In fact, cultured and non-cultured erythroid progenitors showed a higher allelic burden than the corresponding myeloid cells. However, the allele burden in reticulocytes was only 60% of that found in the freshly isolated GPA+ fraction, indicating that reticulocytes to a greater extent derived from unmutated cells (Figure 1).

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In summary, we show that genes involved in autophagy are markedly down-regulated during early erythroid differentiation in RARS. While SF3B1 mutations do not confer a growth disadvantage to differentiating erythroid bone marrow progenitors, the final step of erythroid maturation to reticulocytes is clearly affected. Hence, we demonstrate a novel interference of the SF3B1 mutation with terminal erythroid maturation.