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The myelodysplastic syndromes (MDSs) include a spectrum of stem cell malignancies characterized by an increased risk of developing acute myeloid leukemia. Heterozygous loss of chromosome 5q (del[5q]) is the most common cytogenetic abnormality in MDS. DIAPH1 is localized to 5q31 and encodes one of the formin proteins, mDia1, which is involved in linear actin polymerization. Mice with mDia1 deficiency develop hematologic features with age mimicking human myeloid neoplasm, but its role in the pathogenesis of MDS is unclear. Here we report that mDia1 heterozygous and knockout mice develop MDS phenotypes with age. In these mice, CD14 was aberrantly overexpressed on granulocytes in a cell-autonomous manner, leading to a hypersensitive innate immune response to lipopolysaccharide (LPS) stimuli through CD14/Toll-like receptor 4 signaling. Chronic stimulation with LPS accelerated the development of MDS in mDia1 heterozygous and knockout mice that can be rescued by lenalidomide. Similar findings of CD14 overexpression were observed on the bone marrow granulocytes of del(5q) MDS patients. Mechanistically, mDia1 deficiency led to a downregulation of membrane-associated genes and a specific upregulation of CD14 messenger RNA in granulocytes, but not in other lineages. These results underscore the significance of mDia1 heterozygosity in deregulated innate immune responses in del(5q) MDS., (© 2014 by The American Society of Hematology.)

Relapse of drug-resistant acute lymphoblastic leukemia (ALL) has been associated with increased expression of survivin/BIRC5, an inhibitor of apoptosis protein, suggesting a survival advantage for ALL cells. In the present study, we report that inhibition of survivin in patient-derived ALL can eradicate leukemia. Targeting survivin with shRNA in combination with chemotherapy resulted in no detectable minimal residual disease in a xenograft model of primary ALL. Similarly, pharmacologic knock-down of survivin using EZN-3042, a novel locked nucleic acid antisense oligonucleotide, in combination with chemotherapy eliminated drug-resistant ALL cells. These findings show the importance of survivin expression in drug resistance and demonstrate that survivin inhibition may represent a powerful approach to overcoming drug resistance and preventing relapse in patients with ALL.

Even though the production of orthochromatic erythroblasts can be scaled up to fulfill clinical requirements, enucleation remains one of the critical rate-limiting steps in the production of transfusable red blood cells. Mammalian erythrocytes extrude their nucleus prior to entering circulation, likely to impart flexibility and improve the ability to traverse through capillaries that are half the size of erythrocytes. Recently, there have been many advances in our understanding of the mechanisms underlying mammalian erythrocyte enucleation. This review summarizes these advances, discusses the possible future directions in the field, and evaluates the prospects for improved ex vivo production of red blood cells.

Enucleation of mammalian erythroblasts is a process whose mechanism is largely undefined. The prevailing model suggests that nuclear extrusion occurs via asymmetric cytokinesis. To test this hypothesis, we treated primary erythroblasts with inhibitors of cytokinesis, including blebbistatin, hesperadin, and nocodazole, and then assayed for enucleation. Although these agents inhibited cell-cycle progression and subsequent enucleation when added early in culture, they failed to block enucleation proper when added to postmitotic cells. These results suggest that contraction of the actomyosin ring is not essential for nuclear expulsion. Next, by ultrastructural examination of primary erythroblasts, we observed an accumulation of vacuoles in the cytoplasm proximal to the extruding nucleus. This finding led us to hypothesize that vesicle trafficking contributes to erythroblast enucleation. Here, we show that chemical inhibitors of vesicle trafficking block enucleation of primary erythroblasts without affecting differentiation, cell division, or apoptosis. Moreover, knock-down of clathrin inhibited the enucleation of late erythroblasts. In contrast, vacuolin-1, a small molecule that induces vacuole formation, increased the percentage of enucleated cells. Together, these results illustrate that vesicle trafficking, specifically the formation, movement, and subsequent coalescence of vacuoles at the junction of the nucleus and the cytoplasm, is a critical component of mammalian erythroblast enucleation.

Although erythroid cells and megakaryocytes arise from a common progenitor, their terminal maturation follows very different paths; erythroid cells undergo cell-cycle exit and enucleation, whereas megakaryocytes continue to progress through the cell cycle but skip late stages of mitosis to become polyploid cells. In our efforts to identify genes that participate in this process, we discovered that survivin, a member of the inhibitor of apoptosis family that also has an essential role in cytokinesis, is differentially expressed during erythroid versus megakaryocyte development. Erythroid cells express survivin throughout their maturation, whereas megakaryocytes express approximately 4-fold lower levels of survivin mRNA and no detectable protein. To investigate the role of survivin in these lineages, we overexpressed or knocked down survivin from mouse bone marrow cells and then examined erythroid and megakaryocyte development. These studies revealed that overexpression of survivin antagonized megakaryocyte growth, maturation, and polyploidization but had no effect on erythroid development. This block in polyploidization was accompanied by increased expression of p21 and decreased expression of megakaryocyte genes such as von Willebrand factor and beta(1)-tubulin. In contrast, a reduction in survivin expression interfered with the formation of erythroid cells but not megakaryocytes. Last, consistent with the requirement for survivin in the survival of proliferating cells, survivin-deficient hematopoietic progenitors failed to give rise to either erythroid or megakaryocytic colonies. Together, these studies show that whereas survivin expression is essential for megakaryocyte and erythroid progenitors, its down-regulation is required for terminal differentiation of megakaryocytes.