Myeloid Disease Mutations of Splicing Factor SRSF2 Cause G2-M Arrest and Skewed Differentiation of Human Hematopoietic Stem and Progenitor Cells

Abstract

Myeloid malignancies, including myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia, are characterized by abnormal proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). Reports on analysis of bone marrow samples from patients have revealed a high incidence of mutations in splicing factors in early stem and progenitor cell clones, but the mechanisms underlying transformation of HSPCs harboring these mutations remain unknown. Using ex vivo cultures of primary human CD34+ cells as a model, we find that mutations in splicing factors SRSF2 and U2AF1 exert distinct effects on proliferation and differentiation of HSPCs. SRSF2 mutations cause a dramatic inhibition of proliferation via a G2‐M phase arrest and induction of apoptosis. U2AF1 mutations, conversely, do not significantly affect proliferation. Mutations in both SRSF2 and U2AF1 cause abnormal differentiation by skewing granulo‐monocytic differentiation toward monocytes but elicit diverse effects on megakaryo‐erythroid differentiation. The SRSF2 mutations skew differentiation toward megakaryocytes whereas U2AF1 mutations cause an increase in the erythroid cell populations. These distinct functional consequences indicate that SRSF2 and U2AF1 mutations have cell context‐specific effects and that the generation of myeloid disease phenotype by mutations in the genes coding these two proteins likely involves different intracellular mechanisms. stem cells 2018;36:1663–1675

Myelomonocytic Skewing In Vitro Discriminates Subgroups of Patients with Myelofibrosis with A Different Phenotype, A Different Mutational Profile and Different Prognosis

“…Furthermore, the splicing factors SRSF2 and U2AF1 seem to impact skewing. Mutations in both SRSF2 and U2AF1 cause abnormal differentiation by skewing granulo-monocytic differentiation towards monocytes [4]. On the other hand, nonmalignant conditions may also contribute to in vitro myelomonocytic skewing.…”

Knockdown of spliceosome U2AF1 significantly inhibits the development of human erythroid cells

“…21 During human HSPCs megakaryo-erythroid differentiation, expression of U2AF1-S34F and U2AF1-Q157R resulted in a decrease in the megakaryocyte with a compensatory increase in the erythroid precursors populations. 27 However, it has remained unclear whether depletion of U2AF1 plays any vital role in human erythropoiesis. We analysed the expression of three isoforms of U2AF1 on the sorted cells during human erythropoiesis.…”

“…24,25 Recently study revealed some spliceosome components, such as SF3B1 and SRSF2 deficiency impairs human erythropoiesis. 26,27 But the functions of depletion U2AF1 in erythropoiesis remain unknown.…”

Pan-myeloid Differentiation of Human Cord Blood Derived CD34⁺ Hematopoietic Stem and Progenitor Cells

“…An advantage of this protocol is that it allows differentiation along all four myeloid populations, namely monocytes, granulocytes, megakaryocytes, and erythrocytes. Thus, it can be employed for studying normal myeloid differentiation and for investigating the impact of myeloid disease (myelodysplastic syndromes, acute myeloid leukemia, myeloproliferative neoplasms, and others) associated point mutations and chromosomal translocations on molecular and cellular phenotype during proliferation and differentiation of CD34 + cells 11,12,[23][24][25] . This protocol can also be employed for examining the impact of anti-and pro-inflammatory cytokines, and of potential therapeutic drugs on myeloid differentiation [26][27][28] .…”

“…Ex vivo differentiation of umbilical cord blood derived CD34 + HSPCs is a widely applied model for investigating normal hematopoiesis and hematopoietic disease mechanisms. When cultured with the appropriate cytokines, the UCB CD34 + HSPCs can be induced to differentiate along the myeloid or lymphoid lineages [11][12][13][14][15][16] . Here, we describe protocols for isolation and immunophenotypic characterization of the CD34 + HSPCs from human UCB, and for their differentiation to myeloid lineage cells.…”