Hierarchical Analysis Of Recurrent Point Mutations In SF3B1 and TET2 In RARS Stem Cells

The MDS subgroup refractory anemia with ring sideroblasts (RARS) is characterised by aberrant mitochondrial ferritin accumulation in erythroblasts that fail to mature into erythrocytes. Recently, dominant mutations in SF3B1, a core component of the spliceosome were demonstrated in >75% of RARS, but only in a minority of other MDS subtypes. Many RARS patients also carry other driver mutations, such as epigenetic mutations in DNMT3A and TET2, but the order of occurrence and cooperation between these mutations have not been established. We recently showed that SF3B1 suppresses the expression of the mitochondrial transporter protein ABCB7, which in turn mediates erythroid failure in RARS, but the link to clonal advantage of RARS hemopoietic stem cells (HSC) remains unclear. To explore this link, as well as the impact of additional mutations, we studied RARS with normal karyotype. Screening for 111 recurrently mutated genes in myeloid malignancies revealed SF3B1 in 12 out of 13 patients, TET2 mutations in 3 of these patients (Q916*, H1881Y, Q690*, and R1404*), and DNMT3A mutations in 3 patients(E240fs*8, F414L, W305*, E285*). Other mutations occurred only once.

The frequencies of phenotypically defined RARS stem and myeloid-erythroid progenitor cells in the bone marrow (BM) did not differ from that of normal BM controls, whereas pro-B cells were significantly reduced in the RARS samples (p<0.005). However, functional in vitro analysis of sorted lineage-restricted RARS populations showed a 3-fold decrease in the number of granulocyte-macrophage progenitors (GMP) colonies (p<0.05) and a 5-fold decrease of megakaryocyte-erythroid progenitor (MEP) (p<0.001) compared to normal. Colony forming-units picked from these sorted linage-restricted RARS populations and analysed by pyrosequencing revealed remarkable differences; TET2 mutated RARS samples showed 90% and 87% SF3B1 mutated GMP and MEP subpopulations, respectively, while TET2 wild-type samples had much lower SF3B1 mutational frequencies (26% and 45%) in these subpopulations. Long-term culture initiating cell assays showed that only CD34⁺CD38^-CD90⁺CD45RA^- RARS stem cells could sustain long-term (6-week) generation of myeloid progenitors. Pyrosequencing of the different RARS subpopulations colonies helped us to determine the hierarchy of mutations, suggesting that TET2 mutations precede SF3B1 mutations at the HSC level (n=15). Interestingly, patients that were not TET2 but SF3B1 mutated showed a heterogeneous patterns. In some cases the SF3B1 mutation appeared at the HSC level and in others at the differentiated progenitor level. These results, together with an increased 22-week engraftment of TET2 mutated RARS HSC in NOD/SCID mice compared to HSC carrying SF3B1 mutation only constitute the basis for future investigation involving DNA and RNA sequencing of the sorted stem and lineage restricted RARS populations, in order to further explore the mutational hierarchy, as well as studies of the potential for clonal expansion and functional differentiation into progenitor cells.