TET2 Deficiency Led to Expansion of Dysfunctional Erythroid Progenitors

Erythrocyte development is a complex process involving multiple developmental states that include erythroid progenitors, burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) cells and erythroid precursors, proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts and orthochromatic erythroblasts in the bone marrow. Orthochromatic erythroblasts expel nucleus to become reticulocytes which mature into erythrocytes in blood stream. Defects at any of these developmental stages will lead to anemia. Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by cytopenia and ineffective hematopoiesis. Anemia is the defining cytopenia in the majority of patients with MDS, yet the molecular mechanisms for dyserythropoiesis in MDS remain largely unclear. Recent studies have revealed that heterozygous loss-of-function mutation of DNA dioxygenase TET2 is the most common mutation in MDS.

In the present study, we explored shRNA-mediated knockdown approach in human CD34⁺ cell to study the role of TET2 in human erythropoiesis. We show that TET2 is downregulated starting at the CFU-E stage. Growth curves of purified CFU-E cells show no difference between control and TET2 knockdown cells until day 6. An increased proliferation of TET2 knockdown-CFU-E cells is noted starting at day 7. Interestingly, while the growth of control CFU-E cells plateaued on day 11 with completion of terminal erythroid differentiation and enucleation, TET2 knockdown CFU-E cells continued their growth for additional 13 days. Flow cytometry and cytospin morphologic analyses revealed that by day 11 all control cells differentiated into GPA⁺ late stage erythroblasts or reticulocytes. In marked contrast, about 30 % of TET2 knockdown cells are still GPA^- but CD71⁺, IL-3R^-, CD34^-, CD36⁺, phenotypically resembling erythroid progenitor CFU-E cells. Surprisingly, in contrast to freshly purified control and TET2 knockdown CFU-E cells that generate colonies in methylcellulose colony forming assays, these cells fail to give rise to CFU-E colonies. We thus termed these late proliferating cells as "TET2 knockdown marker CFU-E". Further characterization of the "TET2 knockdown marker CFU-E" cells revealed that in contrast to control CFU-E, the "TET2 knockdown marker CFU-E" exhibit following distinct features: 1) they had lower proliferative potential; 2) their proliferation required both EPO and SCF (while control CFU-E cells only require EPO); 3) they underwent extensive apoptosis in the absence of SCF; 4) they failed to undergo terminal erythroid differentiation. These findings demonstrate that TET2 knockdown led to expansion of dysfunctional erythroid progenitors.

To explore the underlying molecular mechanisms, we performed RNA-seq analysis on control CFU-E, TET2 knockdown CFU-E and "TET2 knockdown marker CFU-E" cells. Bioinformatics analysis revealed that while only about 50 genes were differentially expressed between control CFU-E and TET2 knockdown CFU-E, about 400 genes were differentially expressed between control CFU-E and "TET2 knockdown marker CFU-E". Gene set enrichment analysis revealed upregulation of pathways associated with apoptosis/cell death. Notably, FAS (CD95), the cell surface death receptor previously shown to be upregulated in MDS bone marrow cells, was among the top five upregulated apoptosis associated genes. The increased surface expression of FAS was also noted in TET2-knockdown cells as assessed by flow cytometry. Furthermore, the increased surface expression of FAS was detected in primary bone marrow erythroblasts of MDS patients with TET2 mutation. Our findings uncovered previously unknown roles of TET2 and its potential underlying molecular mechanisms. As the phenotypic changes upon TET2 knockdown resemble the characteristic pathological changes of MDS, our findings suggest the involvement of TET2 deficiency in the pathophysiology of MDS.