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Erythroblast enucleation is a unique process during mammalian erythropoiesis, yet its regulatory mechanisms remain largely elusive. Here, we demonstrate the specific regulatory role of the oncogene PIM1, the most highly expressed protein kinase in orthochromatic erythroblasts, in enucleation. Unlike its well-established roles in cancer cell proliferation and survival, knockdown of PIM1 in human erythroid cells does not affect cell growth or apoptosis, but specifically inhibits erythroblast enucleation without altering differentiation. To elucidate the functional conservation of PIM1 in mammalian erythropoiesis, we generate Pim1fl/flEpoRCre mice in which Pim1 is deleted in erythroid cells. Consistent with human erythropoiesis, deletion of Pim1 in mice has no detectable effect on apoptosis or differentiation of erythroid cells, but specifically inhibits erythroblast enucleation. Phosphoproteomic analysis reveals that PIM1 deficiency causes a pronounced decrease in phosphorylation of GTPase-associated proteins involved in actin assembly and vesicle trafficking. Functionally, this perturbation results in an aberrant distribution of F-actin and endocytic vesicles within enucleating cells. These findings reveal the unexpected role of PIM1 in normal erythropoiesis and enhance our understanding of mammalian erythroblast enucleation. The oncogene PIM1 regulates erythroblast enucleation via GTPase-dependent cytoskeletal remodeling and vesicle trafficking, independent of its canonical roles in cell proliferation and apoptosis.

We examined the role of SLFN12, a member of the Schlafen (SLFN) family of interferon-regulated genes and proteins in leukemogenesis, and its potential as a therapeutic target in acute myeloid leukemia (AML). We explored the effects of velcrins, a class of small molecules able to modulate SLFN12 biological activity, on AML cells. Velcrin treatment of AML cells stabilized SLFN12 and promoted SLFN12 complex formation with phosphodiesterase 3A or phosphodiesterase 3B. Such effects were associated with growth-inhibitory and proapoptotic responses, as well as potent suppressive effects on leukemic cell growth. In addition, velcrin treatment suppressed clonogenic capacity of primitive leukemic progenitors and significantly extended survival in a mouse AML xenograft model. Taken together, these findings establish an important role of SLFN12 in leukemogenesis and raise the potential for the use of velcrins as a therapeutic strategy for AML. Significance: Our studies identify SLFN12 as a potential target in AML with important clinical–translational implications.

BackgroundAt present, healthcare facilities often face blood shortages because of the low supply of donated blood relative to the high demand. Therefore, efforts to develop red blood cell (RBC) production methods have gained traction. In this work, Lin−CD45−CD133+ cells were isolated from human umbilical cord blood (UCB) and subsequently differentiated into erythrocytes in vitro in serum-free culture medium.MethodsLin−CD45−CD133+ cells were prepared from mononuclear cells (MNCs) using magnetic-activated cell sorting (MACS). The characteristics of Lin−CD45−CD133+ cells were confirmed using flow cytometry analysis, colony-forming unit (CFU) assays, morphological analysis, immunocytochemistry (ICC) analysis, and real-time fluorescent quantitative polymerase chain reaction (RT–PCR). Erythrocytes were differentiated in serum-free medium supplemented with stem cell factor (SCF), interleukin-3 (IL-3), erythropoietin (EPO), and FK506 for 13 days, after which autoplasma derived from UCB was added at a concentration of 5% beginning on day 14. Erythroid differentiation and maturation were examined using electron microscopy and flow cytometric analysis.ResultsLin−CD45−CD133+ cells were successfully obtained from UCB. These cells were slightly smaller than normal RBCs and had a high nucleus-to-cytoplasm ratio. Oct-4 and Nanog were expressed at both the mRNA and protein levels in Lin−CD45−CD133+ cells. Most of the colonies were burst-forming unit-erythroid (BFU-E). After 7 days of in vitro culture, the Lin−CD45−CD133+ cells were negative for CD133 expression and positive for CD45 expression. The percentage of CD71+ cells gradually increased, peaked on day 10, and then started decreasing on day 13. The percentage of CD235a+ cells increased gradually after day 7 and peaked on day 13. CD240 expression was detected on day 18, with the highest level detected on day 20. The number of erythroid cells increased persistently during differentiation, and their morphology was consistent with that of normal erythrocytes.ConclusionAn ex vivo culture system was developed that can generate human erythrocytes from Lin−CD45−CD133+ cells isolated from human UCB.