ASXL1 Is a Key Regulator for Erythroid Development and Asxl1 Loss Impairs Erythropoiesis In Vivo

Objective:

Recently, ASXL1 mutations are found in a spectrum of numerous of myeloid malignancies, including myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML). ASXL1 mutation is a poor prognostic markers. Recently, we reported that Asxl1^+/- mice developed myelodysplastic syndrome (MDS)-like disease and MDS/MPN. Regardless of the existence of defective erythroid lineage maturation and anima in patients with MDS or MPN, the role of ASXL1 in erythropoiesis remains unveiled.

Methods:

Asxl1^+/- mice were utilized for hematopoietic analysis, including peripheral blood counts, blood smear, morphology, flow cytometry and histology. To assess the erythroid phenotypes in vivo, blood smears were subjected to acridine staining for reticulocyte counts. The percentage of reticulocytes and erythroid cell differentiation in peripheral blood and bone marrow were evaluated flow cytometrically following thiazole orange and CD71/Ter119 staining. Colony-forming cells (CFU) and in vitro erythroid differentiation assays were performed with methylcellulose (EPO, SCF) and liquid (EPO, transferrin) cultures of Lin-Kit+ cells of BM, spleen and fetal liver cells. In addition, the nuclear fractions were prepared from cultured erythoblasts of WT and Asxl1^+/- mice and subjected to western blotting analyses to determine the histone levels of H3K27me3. RNA-Seq was performed to survey the differentially expressed genes in LK cells of WT and Asxl1^+/- mice. Real time PCR was performed to confirm the alteration of erythroid development related gene.

Results:

Adult Asxl1^+/- mice were anemic. High frequencies of immature and dysplastic (tear drop) red blood cells were observed in the peripheral blood of Asxl1^+/- mice. Flow cytometric analysis revealed an increased percentage of pro-erythroblasts and basophilic erythroblasts populations in peripheral blood and bone marrow cells compared to that of WT controls, suggesting a blockage of erythroid maturation in Asxl1^+/- mice. In vitro methylcellulose cultures showed a dramatic decrease of BFU-E and CFU-GEMM in the bone marrow of Asxl1^+/- mice compared to WT controls. Western blot analysis showed that Asxl1^+/- erythroblast cells exhibited decreased global levels of H3K27me3 and qPCR of Asxl1^+/- erythroblast cells revealed altered expression of genes involved in development and homeostasis of erythroid lineage, including Gata1, Ldb1, Cdc42, and Zfpm1.

Conclusion:

Our study demonstrates that loss of Asxl1 results in blockage of erythroid cell maturation, indicating that ASXL1 is a key regulator for erythroid development and Asxl1 loss impairs erythropoiesis in vivo.