CBFβ-SMMHC Impairs Erythroid Differentiation and Induces Expansion of Aberrant Megakaryocytic/Erythroid Progenitors Capable of Leukemia Initiation

Abstract Approximately 12% of human acute myeloid leukemia (AMLs) harbor a recurrent chromosomal rearrangement inv(16)(p13q22). Inv(16) creates a fusion gene Cbfb-MYH11, encoding the fusion protein CBFß-SMMHC. Expressing CBFß-SMMHC in hematopoietic cells using a constitutive knock-in mouse model (Cbfb+/Cbfb-MYH11) or a conditional knock-in mouse model (Cbfb56M/+/Mx1-Cre; 129SvEv strain) causes defects in lymphoid and myeloid differentiation, and predisposes mice to AML. Previous studies with the constitutive knock-in mouse model showed impaired primitive erythropoiesis, however, Cbfb-MYH11 knocked-in cells were able to contribute to erythropoiesis in chimeric mice. To further delineate the effect of CBFß-SMMHC in adult erythropoiesis in the conditional knock-in mouse, we backcrossed Cbfb56M/+/Mx1-Cre into C57BL/6 and a Rosa26mT/mG Cre reporter strain. Induced expression of CBFß-SMMHC in adult mice leads to cell number dependent development of AML, consistent with previous studies in 129SvEv strain. Analysis of pre-leukemic bone marrow 2 weeks after induction revealed a 5.7-fold expansion of immunophenotypic pre-megakaryocyte/erythrocyte (Pre-Meg/E; Lin-cKit+Sca1-CD16-/loCD150+CD105-), and a 4.7 fold decrease of the erythroid progenitor (EP; Lin-cKit+Sca1-CD16-/loCD105hi) subset compared to similarly treated control mice. Both methylcellulose-based erythroid colony forming assay and in vitro erythroid differentiation culture showed that pre-leukemic Pre-Meg/Es expressing CBFß-SMMHC had an impaired differentiation potential for erythroid lineage. Using the Rosa26mT/mG Cre reporter allele, we tracked the proportions of CBFß-SMMHC- expressing cells (GFP+) in the Pre-Meg/E and EP subsets. We observed that the contribution of GFP+ cells sharply decreased in EPs but not in Pre-Meg/Es from primary pre-leukemic mice. Similar results were seen in transplant recipients engrafted with sorted GFP+ pre-leukemic Lin-cKit+Sca1+ cells. These results further confirmed that CBFß-SMMHC impairs cell-autonomous erythroid differentiation in vivo. Consistent with the impaired differentiation of Pre-Meg/Es, we observed altered expression pattern of erythroid regulatory genes, including Fog1, Gata2, and Gfi1b. The pre-leukemic Pre-Meg/Es exhibited increased colony forming and replating capacity in vitro and enhanced proliferation and survival in vivo. To determine whether these phenotypic Pre-Meg/E cells could be the cellular origin for leukemic transformation, we expressed a known cooperative onco-protein Mpl by retroviral transduction followed by transplantation. The majority of mice (83%) receiving 100,000 Pre-Meg/E cells developed leukemia with a medium onset of 92 days, suggesting that Pre-Meg/Es indeed are capable of leukemia initiation. In conclusion, the expression of CBFß-SMMHC impairs adult erythropoiesis at the transition of Pre-Meg/E to EPs, causing an expansion of Pre-Meg/E cells. These pre-leukemic Pre-Meg/Es could be the target cell of additional mutations contributing to leukemia transformation. Disclosures No relevant conflicts of interest to declare.

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Aberrant Megakaryocytic/Erythroid Progenitors Contributes To Transformation Of Cbfb-SMMHC Induced Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.1652.1652 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1652-1652

Author(s):

Qi Cai ◽

Robin Jeannet ◽

Hongjun Liu ◽

Ya-Huei Kuo

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Cell Of Origin ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Differentiation Potential ◽

Acute Myeloid

Abstract Inv(16)(p13q22) is a recurrent chromosomal rearrangement found in approximately 12% of human acute myeloid leukemia (AML) cases and creates a fusion gene between CBFb and MYH11. The fusion gene encodes a fusion protein CBFß-SMMHC which causes defects in lymphoid and myeloid differentiation. Previous studies also showed that primitive erythropoiesis is impaired by CBFß-SMMHC, however, CBFß-SMMHC knocked-in cells was able to contribute to adult erythropoiesis in chimeric mice. Expressing CBFß-SMMHC in the hematopoietic cells using a conditional knock-in mouse model (Cbfb56M/+/Mx1-Cre; 129SvEv strain) recapitulates inv(16)-associated AML. Previous studies in this model showed that CBFß-SMMHC expression leads to pre-leukemic hematopoietic alterations, and together with additional cooperative mutations, result in spontaneous myeloid leukemia in mice with a 3-6 month latency. We hypothesized that an expanded cell population at the pre-leukemic stage could be the target of additional mutations, and hence the cell of origin of leukemia initiating cells. To further delineate the pre-leukemic progenitors and leukemia initiating cells, we backcrossed Cbfb56M/+/Mx1-Cre into C57BL/6 for more than 10 generations. Similar to previous studies in the129SvEv strain, expressing CBFß-SMMHC in adult C57BL/6 mice leads to cell number dependent development of AML. Analysis of pre-leukemic bone marrow as early as 2 weeks after induction revealed a 5.7-fold expansion of Pre-Meg/E cells (Pre-Megakaryocyte/Erythrocyte: Lin-cKit+Sca1-CD16-/loCD150+CD105-) compared to similarly treated control mice. While there was a significant increase in Pre-Meg/E population, we did not find significant increase in their proliferation but observed a 4.7 fold decrease of the erythroid progenitor (EP; Lin-cKit+Sca1-CD16-/loCD105hi) subset. Methylcellulose-based colony forming assay showed that pre-leukemic Pre-Meg/E had an impaired differentiation potential for erythroid lineage. In vitro erythroid differentiation assay also showed a partial block of differentiation from pre-leukemic Pre-Meg/E progenitors. These Pre-Meg/E like progenitors were able to induce leukemia in the presence of a known cooperative oncoprotein MPL when transplanted in lethally irradiated congenic recipient mice. In summary, our Results suggest that expression of CBFß-SMMHC impairs adult erythropoiesis at the transition of Pre-Meg/E to EPs, causing an expansion of Pre-Meg/E cells, which can be the target cell of additional mutations contributing to leukemia transformation. Disclosures: No relevant conflicts of interest to declare.

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Probing Aberrant Splicing in a Novel Model of SF3B1-Mutant Myelodysplastic Syndromes

Blood ◽

10.1182/blood-2019-129176 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1706-1706

Author(s):

Courtnee Clough ◽

Stephanie Busch ◽

Joey Pangallo ◽

Robert K. Bradley ◽

Sergei Doulatov

Keyword(s):

Conflicts Of Interest ◽

Erythroid Differentiation ◽

Aberrant Splicing ◽

Erythroid Progenitor ◽

Differentiation Potential ◽

Normal Cells ◽

Major Mechanism ◽

Ring Sideroblasts ◽

Vitro Model

Splicing is a fundamental process by which introns are removed from primary RNA transcripts. Alternative splicing is widely appreciated to be a major mechanism of gene regulation by which eukaryotic cells expand their transcriptional repertoire. By contrast, aberrant splicing generates novel transcripts not found in normal cells. Heterozygous mutations in a core U2 spliceosome factor SF3B1 are strongly associated with MDS with ring sideroblasts (MDS-RS). MDS-RS is characterized by iron retention in mitochondria and ineffective erythropoiesis. How aberrant splicing induced by mutant SF3B1 causes ring sideroblasts and erythroid defects remains poorly understood. There are no genetically accurate models of MDS-RS that recapitulate ring sideroblast formation. Our lab has established an iPSC reprogramming approach to generate an in vitro model of MDS-RS. This method captures genetically distinct MDS subclones as well as normal cells in individual patients enabling internal normalization to the patient's isogenic normal clone. By differentiating iPSCs we generated inducibly immortalized MDS-RS CD34+ progenitor cell lines with robust expansion and erythroid differentiation potential enabling extensive analysis of SF3B1-mutant aberrant splicing throughout erythroid differentiation. We show that SF3B1-mutant lines form abundant ring sideroblasts compared to normal isogenic controls. We next performed RNA-sequencing and splicing analysis at different stages of erythroid differentiation. We show that global splicing patterns are maintained in SF3B1-mutant cells, while revealing distinct subsets of aberrantly spliced transcripts in CD34+ progenitors and erythroblast populations. This suggests that different aberrant splicing events contribute to erythroid progenitor expansion and inefficient erythropoiesis and iron dysregulation in MDS-RS. Taken together, this novel iPSC model of MDS-RS is a robust tool for studying the role of aberrant splicing in SF3B1-mutant erythroid progenitor expansion, inefficient erythropoiesis and ring sideroblast formation. Disclosures No relevant conflicts of interest to declare.

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Erythroid-Specific Variant of the Nuclear Exportin Xpo7 Conserved Only in Mammals May Explain Functional Differences Between Mammalian Definitive and Lower Vertebrate (or Primitive) Erythropoiesis

Blood ◽

10.1182/blood.v128.22.2440.2440 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2440-2440

Author(s):

Sandra Martinez-Morilla ◽

Srividhya Venkatesan ◽

Seibel Katharina ◽

Jeffrey D. Cooney ◽

Barry H. Paw ◽

...

Keyword(s):

Conflicts Of Interest ◽

Fetal Liver ◽

Chromatin Condensation ◽

Erythroid Differentiation ◽

Erythroid Progenitor ◽

Lower Vertebrate ◽

Primary Mouse ◽

Lower Vertebrates

Abstract Exportin 7 (Xpo7) was previously found to be crucial for late murine erythroid nuclear maturation when its knockdown significantly inhibited chromatin condensation and ultimate enucleation in a primary mouse erythroblast culture [1]. Additionally, Xpo7 was found to have an erythroid-specific isoform, transcribed from an erythroid-specific transcription start site (TSS) in the first intron of Xpo7 (named Xpo7-1B in contrast to the ubiquitously expressed variant -1A) [1]. Xpo7-1B differs from Xpo7-1B in 8 amino acid residues in the first exon and 9 residues in the 5th exon, and these differences are not conserved either in lower vertebrates or in primitive mouse erythropoiesis. It is intriguing that both lower vertebrate and primitive mammalian erythroblasts do not enucleate. In order to understand the in vivo function of Xpo7 and determine if these differences in the two isoforms confer differential functions to the proteins or are simply regulatory, we examined the definitive fetal liver erythropoiesis (considered adult "stress erythropoiesis") and adult steady state erythropoiesis of transgenic mouse models lacking either Xpo7-1A, Xpo7-1B, or both. We had previously knocked down Xpo7 in zebrafish using morpholinos (zebrafish only express the Xpo7-1A homologue) and found no effect on adult erythroid differentiation or proliferation. Adult Xpo7-1A knockout (KO) mice are born in normal Mendelian ratios, are not anemic, and express normal Xpo7-1B only in the blood. Closer examination reveals that fetal liver erythroid proliferation is actually higher in Xpo7-1A KO mice than either heterozygous (HET) or wild type (WT) while differentiation is normal. The double KO (Xpo7-1AB KO) appears to be embryonic lethal as only 4 live knockout pups were born out of 71 live births and even these 4 pups appear to be partial chimeras. We characterized the Xpo7-1AB HET mice in more detail and fetal liver erythroblasts of HET mice showed normal proliferation and differentiation compared to WT, and adults showed no differences in red blood cell (RBC) counts, hematocrit or hemoglobin. No complete live Xpo7-1AB KO has been born so we are now evaluating embryos at E3, E9.5, and E14.5. Given the possibility of embryonic lethality, in order to evaluate the erythroid-specific isoform Xpo7-1B, we have crossed a mouse floxed for the Xpo7-1B TSS/1st exon with a tamoxifen-inducible Cre-recombinase mouse. These mice have been given tamoxifen after reaching adulthood and we are currently evaluating their hematopoietic parameters. We have also set up HET-HET timed matings and administered tamoxifen to pregnant females to evaluate fetal liver erythropoiesis in Xpo7-1B HET, WT, and KO embryos. Not only will this work further our understanding of the process of global chromatin condensation during erythropoiesis, data from this in vivo study may reveal differences in erythropoiesis between mammals and lower vertebrates, which may also reflect intrinsic differences in mammalian primitive versus definitive erythropoiesis. Based on our in vitro knockdown studies, we anticipate that the phenotype of the Xpo7-1B KO mice may be similar to congenital dyserythropoietic anemia (CDA), a human disorder involving abnormalities in erythroid progenitor chromatin condensation and nuclear membrane substructure, so examination of these mice may also uncover a novel mechanism for this form of ineffective erythropoiesis. [1] Hattangadi, SM, et al. Blood (2014) 124, 1931-40. Disclosures No relevant conflicts of interest to declare.

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Monoclonal antibodies detecting antigenic determinants with restricted expression on erythroid cells: from the erythroid committed progenitor level to the mature erythroblast

Blood ◽

10.1182/blood.v63.6.1376.bloodjournal6361376 ◽

1984 ◽

Vol 63 (6) ◽

pp. 1376-1384 ◽

Cited By ~ 1

Author(s):

T Yokochi ◽

M Brice ◽

PS Rabinovitch ◽

T Papayannopoulou ◽

G Stamatoyannopoulos

Keyword(s):

Monoclonal Antibodies ◽

K562 Cells ◽

Erythroid Differentiation ◽

Antigenic Determinants ◽

Erythroid Progenitors ◽

Cell Surface Antigens ◽

Cytotoxicity Assays ◽

Complement Dependent Cytotoxicity

Two new cell surface antigens specific for the erythroid lineage were defined with cytotoxic IgM monoclonal antibodies (McAb) (EP-1; EP-2) that were produced using BFU-E-derived colonies as immunogens. These two antigens are expressed on in vivo and in vitro derived adult and fetal erythroblasts, but not on erythrocytes. They are not detectable on resting lymphocytes, concanavalin-A (Con-A) activated lymphoblasts, granulocytes, and monocytes or granulocytic cells or macrophages present in peripheral blood or harvested from CFU-GM cultures. Cell line and tissue distributions distinguish McAb EP-1 and EP-2 from all previously described monoclonal antibodies. McAb EP-1 (for erythropoietic antigen-1) inhibits the formation of BFU-E and CFU-E, but not CFU-GM, colonies in complement-dependent cytotoxicity assays. By cell sorting analysis, about 90% of erythroid progenitors (CFU-E, BFU-E) were recovered in the antigen-positive fraction. Seven percent of the cells in this fraction were progenitors (versus 0.1% in the negative fraction). The expression of EP-1 antigen is greatly enhanced in K562 cells, using inducers of hemoglobin synthesis. McAb EP-2 fails to inhibit BFU-E and CFU-E colony formation in complement-dependent cytotoxicity assays. EP-2 antigen is predominantly expressed on in vitro derived immature erythroblasts, and it is weakly expressed on mature erythroblasts. The findings with McAb EP-1 provide evidence that erythroid progenitors (BFU-E and CFU-E) express determinants that fail to be expressed on other progenitor cells and hence appear to be unique to the erythroid lineage. McAb EP-1 and EP-2 are potentially useful for studies of erythroid differentiation and progenitor cell isolation.

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Monoclonal antibodies detecting antigenic determinants with restricted expression on erythroid cells: from the erythroid committed progenitor level to the mature erythroblast

Blood ◽

10.1182/blood.v63.6.1376.1376 ◽

1984 ◽

Vol 63 (6) ◽

pp. 1376-1384 ◽

Cited By ~ 20

Author(s):

T Yokochi ◽

M Brice ◽

PS Rabinovitch ◽

T Papayannopoulou ◽

G Stamatoyannopoulos

Keyword(s):

Monoclonal Antibodies ◽

K562 Cells ◽

Erythroid Differentiation ◽

Antigenic Determinants ◽

Erythroid Progenitors ◽

Cell Surface Antigens ◽

Cytotoxicity Assays ◽

Complement Dependent Cytotoxicity

Abstract Two new cell surface antigens specific for the erythroid lineage were defined with cytotoxic IgM monoclonal antibodies (McAb) (EP-1; EP-2) that were produced using BFU-E-derived colonies as immunogens. These two antigens are expressed on in vivo and in vitro derived adult and fetal erythroblasts, but not on erythrocytes. They are not detectable on resting lymphocytes, concanavalin-A (Con-A) activated lymphoblasts, granulocytes, and monocytes or granulocytic cells or macrophages present in peripheral blood or harvested from CFU-GM cultures. Cell line and tissue distributions distinguish McAb EP-1 and EP-2 from all previously described monoclonal antibodies. McAb EP-1 (for erythropoietic antigen-1) inhibits the formation of BFU-E and CFU-E, but not CFU-GM, colonies in complement-dependent cytotoxicity assays. By cell sorting analysis, about 90% of erythroid progenitors (CFU-E, BFU-E) were recovered in the antigen-positive fraction. Seven percent of the cells in this fraction were progenitors (versus 0.1% in the negative fraction). The expression of EP-1 antigen is greatly enhanced in K562 cells, using inducers of hemoglobin synthesis. McAb EP-2 fails to inhibit BFU-E and CFU-E colony formation in complement-dependent cytotoxicity assays. EP-2 antigen is predominantly expressed on in vitro derived immature erythroblasts, and it is weakly expressed on mature erythroblasts. The findings with McAb EP-1 provide evidence that erythroid progenitors (BFU-E and CFU-E) express determinants that fail to be expressed on other progenitor cells and hence appear to be unique to the erythroid lineage. McAb EP-1 and EP-2 are potentially useful for studies of erythroid differentiation and progenitor cell isolation.

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Abnormal Distribution of Erythroid Progenitors Populations in Mice Lacking p45 NF-E2.

Blood ◽

10.1182/blood.v114.22.1988.1988 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1988-1988

Author(s):

Jadwiga Gasiorek ◽

Gregory Chevillard ◽

Zaynab Nouhi ◽

Volker Blank

Keyword(s):

Bone Marrow ◽

Red Blood Cells ◽

Blood Cells ◽

Conflicts Of Interest ◽

Globin Genes ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Adult Mice ◽

Deficient Mice

Abstract Abstract 1988 Poster Board I-1010 The NF-E2 transcription factor is a heterodimer composed of a large hematopoietic-specific subunit called p45 and widely expressed 18 to 20-kDa small Maf subunits. In MEL (mouse erythroleukemia) cells, a model of erythroid differentiatin, the absence of p45 is inhibiting chemically induced differentiation, including induction of globin genes. In vivo, p45 knockout mice were reported to show splenomegaly, severe thrompocytopenia and mild erythroid abnormalities. Most of the mice die shortly after birth due to haemorrhages. The animals that survive display increased bone, especially in bony sites of hematopoiesis. We confirmed that femurs of p45 deficient mice are filled with bone, thus limiting the space for cells. Hence, we observed a decrease in the number of hematopoietic cells in the bone marrow of 3 months old mice. In order to analyze erythroid progenitor populations we performed flow cytometry using the markers Ter119 and CD71. We found that p45 deficient mice have an increased proportion of early erythroid progenitors (proerythroblasts) and a decreased proportion of late stage differentiated red blood cells (orthochromatic erythroblasts and reticulocytes) in the spleen, when compared to wild-type mice. We showed that the liver of p45 knockout adult mice is also becoming a site of red blood cell production. The use of secondary sites, such as the spleen and liver, suggests stress erythropoiesis, likely compensating for the decreased production of red blood cells in bone marrow. In accordance with those observations, we observed about 2 fold increased levels of erythropoietin in the serum of p45 knockout mice.Overall, our data suggest that p45 NF-E2 is required for proper functioning of the erythroid compartment in vivo. Disclosures: No relevant conflicts of interest to declare.

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TIF1γ Knockdown Enhances Hematopoietic Stem Cell Self Renewal with Preferential Myeloid Differentiation and Delayed Erythropoiesis

Blood ◽

10.1182/blood.v118.21.4829.4829 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4829-4829

Author(s):

David C Dorn ◽

Wei He ◽

Joan Massague ◽

Malcolm A.S. Moore

Keyword(s):

Transforming Growth Factor ◽

Conflicts Of Interest ◽

Erythroid Differentiation ◽

Transforming Growth Factor Β ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 4829 The role of TIF1γ in hematopoiesis is still incompletely understood. We previously identified TIF1γ as a novel binding factor for Smad2/3 in the Transforming Growth Factor-β (TFGβ)-inducible signaling pathway implicated in the enhancement of erythropoiesis. To investigate the function of TIF1γ in regulation of hematopoietic stem cells we abrogated TIF1γ signaling by shRNA gamma-retroviral gene transfer in human umbilical cord blood-derived CD34+ hematopoietic stem/ progenitor cells (HCS/ HPCs). Upon blocking TIF1γ the self-renewal capacity of HSCs was enhanced two-fold in vitro as measured by week 5 CAFC assay and three-fold in vivo as measured by competitive engraftment in NOD/ SCID mice over controls. This was associated with a delay in erythroid differentiation and enhanced myelopoiesis. These changes were predominantly observed after TIF1γ knockdown and only mildly after Smad2 depletion but not after Smad3 or 4 reduction. Our data reveal a role for TIF1γ-mediated signaling in the regulation of HSC self-renewal and differentiation. Disclosures: No relevant conflicts of interest to declare.

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Identification of Non-Cardiotoxic hERG1 Blockers to Overcome Chemoresistance in Acute Lymphoblastic Leukemias

Blood ◽

10.1182/blood.v120.21.1506.1506 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1506-1506

Author(s):

Marika Masselli ◽

Serena Pillozzi ◽

Massimo D'Amico ◽

Luca Gasparoli ◽

Olivia Crociani ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Map Kinases ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Significant Proportion ◽

Leukemic Cells ◽

K Channel

Abstract Abstract 1506 Although cure rates for children with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have markedly improved over the last two decades, chemotherapy resistance remains a major obstacle to successful treatment in a significant proportion of patients (Pui CH et al. N Engl J Med., 360:2730–2741, 2009). Increasing evidence indicates that bone marrow mesenchymal cells (MSCs) contribute to generate drug resistance in leukemic cells (Konopleva M et al., Leukemia, 16:1713–1724, 2002). We contributed to this topic, describing a novel mechanism through which MSCs protect leukemic cells from chemotherapy (Pillozzi S. et al., Blood, 117:902–914, 2011.). This protection depends on the formation of a macromolecular membrane complex, on the plasma membrane of leukemic cells, the major players being i) the human ether-a-gò-gò-related gene 1 (hERG1) K+ channel, ii) the β1integrin subunit and iii) the SDF-1α receptor CXCR4. In leukemic blasts, the formation of this protein complex activates both the ERK 1/2 MAP kinases and the PI3K/Akt signalling pathways triggering antiapoptotic effects. hERG1 exerts a pivotal role in the complex, as clearly indicated by the effect of hERG1 inhibitors to abrogate MSCs protection against chemotherapeutic drugs. Indeed, E4031, a class III antiarrhythmic that specifically blocks hERG1, enhances the cytotoxicity of drugs commonly used to treat leukemia, both in vitro and in vivo. The latter was tested in a human ALL mouse model, consisting of NOD/SCID mice injected with REH cells, which are relatively resistant to corticosteroids. Mice were treated for 2 weeks with dexamethasone, E4031, or both. Treatment with dexamethasone and E4031 in combination nearly abolished bone marrow engraftment while producing marked apoptosis, and strongly reducing the proportion of leukemic cells in peripheral blood and leukemia infiltration of extramedullary sites. These effects were significantly superior to those obtained by treatment with either dexamethasone alone or E4031 alone. This model corroborated the idea that hERG1 blockers significantly increase the rate of leukemic cell apoptosis in bone marrow and reduced leukemic infiltration of peripheral organs. From a therapeutic viewpoint, to develop a pharmacological strategy based on hERG1 targeting we must consider to circumvent the side effects exerted by hERG1 blockers. Indeed, hERG1 blockers are known to retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, an effect that in some cases leads to life threatening ventricular arrhythmias (torsades de points). On the whole, it is mandatory to design and test non-cardiotoxic hERG1 blockers as a new strategy to overcome chemoresistance in ALL. On these bases, we tested compounds with potent anti-hERG1 effects, besides E4031, but devoid of cardiotoxicity (e.g. non-torsadogenic hERG1 blockers). Such compounds comprise erythromycin, sertindole and CD160130 (a newly developed drug by BlackSwanPharma GmbH, Leipzig, Germany). We found that such compounds exert a strong anti-leukemic activity both in vitro and in vivo, in the ALL mouse model described above. This is the first study describing the chemotherapeutic effects of non-torsadogenic hERG1 blockers in mouse models of human ALL. This work was supported by grants from the Associazione Genitori contro le Leucemie e Tumori Infantili Noi per Voi, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Istituto Toscano Tumori. Disclosures: No relevant conflicts of interest to declare.

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A NUP98-HOXD13 Leukemic Fusion Gene Leads To Aberrant Actin Localization In Dysplastic Megakaryocytes

Blood ◽

10.1182/blood.v122.21.2486.2486 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2486-2486

Author(s):

David L. Caudell ◽

Benjamin Okyere ◽

Jacob Cawley ◽

Abdul Gafoor A. Puthiyaveetil ◽

Bettina Heid

Keyword(s):

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Hematopoietic Malignancy ◽

Glass Slides ◽

Transmission Electron ◽

Platelet Release ◽

Actin Localization

Abstract Myelodysplastic syndrome (MDS) is a hematopoietic malignancy characterized by peripheral cytopenias due to bone marrow (BM) failure. Megakarypoiesis, megakaryocyte (MK) production, and platelet release are impaired in in some cases of MDS. Patients often have fewer, but larger circulating platelets, which have abnormal demarcation membrane systems (DMS); the DMS, which determines the number and size of platelets released, is dependent on actin formation. However, the precise role of actin during megakaryopoiesis is poorly understood. Transgenic mice that express the fusion gene NUP98-HOXD13 (NHD13) is a model for MDS and have dysplastic MKs in BM, and macro platelets in circulation. We hypothesized that expression of NHD13 disrupts actin localization during megakaryopoiesis resulting in reduced platelet release and macro platelet formation. To test the hypothesis, BM from wild type (WT) and NHD13 mice were flushed and cultured in media supplemented with Thrombopoietin for 5 days. Following in vitro propagation, MKs were harvested over a discontinuous gradient for downstream experiments. Sternums were also fixed in paraformaldehyde, stained with hematoxylin and eosin, and evaluated by light microscopy to analyze MK morphology in vivo. NHD13 BM contained many dysplastic MKs. Harvested MKs and BM cores from one femur were processed and analyzed by transmission electron microscopy (TEM) and the ultrastructural properties of the DMS detailed. TEM of MKs showed NHD13 leads to formation of an irregular DMS along with abnormal distribution of unusually large granules in MK cytoplasm. Cultured MKs were also cytospun onto glass slides, labeled with fluorescent-tagged F-actin and Myosin IIa and the cytoskeleton visualized by confocal microscopy. WT MKs in vitro had two phenotypes: (1) MKs with myosin and actin evenly dispersed in the cytoplasm and (2) MK with actin predominantly in the periphery of the cytoplasm. In contrast, transgenic MKs displayed only the former phenotype suggesting that actin localization is impaired in NHD13 MKs. Finally, MKs were stimulated with estrogen and adhered to fibrinogen matrices to determine their proplatelet formation functionality. Our results showed impaired proplatelets formation in NHD13 MKs. These data suggest that expression of NHD13 leads to aberrant actin localization leading to dysplastic MK differentiation and macro platelet release. Understanding molecular mechanisms of abnormal megakaryopoiesis in MDS is important as many MDS patients die of hemorrhagic complications. Further studies using this model system will provide a platform for translational research and should reveal potential therapeutic targets in MDS, leading to improved patient care/survival. Disclosures: No relevant conflicts of interest to declare.

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DMT1-Mutant Erythrocytes Have Shortened Life Span, Accelerated Glycolysis and Increased Oxidative Stress

Blood ◽

10.1182/blood.v122.21.957.957 ◽

2013 ◽

Vol 122 (21) ◽

pp. 957-957

Author(s):

Zuzana Zidova ◽

Pavla Pospisilova ◽

Renata Mojzikova ◽

Katarina Kapralova ◽

Dalibor Dolezal ◽

...

Keyword(s):

Oxidative Stress ◽

Life Span ◽

Conflicts Of Interest ◽

Transmembrane Protein ◽

Erythroid Differentiation ◽

Compound Heterozygous ◽

Divalent Metal Transporter 1 ◽

Divalent Metal Transporter

Abstract Divalent metal transporter 1 (DMT1, also known as NRAMP2 and SLC11A2) is a transmembrane protein important for intestinal iron (Fe2+) absorption and erythroid iron utilization. Homozygous or compound heterozygous mutations in DMT1 are associated with moderate to severe hypochromic microcytic anemia in human patients and a mouse model - mk/mk mice. We have previously reported that DMT1 deficiency leads to an impaired erythroid differentiation hallmarked by accumulation of immature forms of erythroblast which also showed increased rate of apoptosis. For human samples we observed suppression of colony-forming capacity of erythroid progenitors that can be corrected by the addition of iron saturated chelate Fe-SIH. Later we proved this result also for mk/mk progenitors and showed reduced number of mk/mk CFU-E (164±25 vs. 283±50) and BFU-E (9±4 vs. 22±5) colonies in comparison to the colonies of wild-type (wt) mice and improvement of the colony growth with Fe-SIH. In our following studies we focused on mature erythrocytes, the last stage of erythroid differentiation that has not been analyzed yet. We first determined the in vivo half-life of red blood cells (RBC). Isolated RBCs from mk/mk mice and wt controls were in vitro labeled with CFSE fluorescent dye and injected into the wt mice. The intensity of RBCs fluorescence was measured on the 1st, 7th, 10th, 14th, 19th, 26th and 30th day following the injection. We observed an accelerated clearance of CFSE-labeled mk/mk RBCs from circulating blood when compared to wt RBCs, which indicates increased destruction of DMT1-mutant erythrocytes in vivo. It is known, that mature RBCs retain the ability to undergo stress-induced death (eryptosis), characterized by their shrinkage, membrane blebbing and phosphatidylserine surface exposure. This process may be triggered by iron deficiency. To determine the involvement of eryptosis in mk/mk RBCs clearance, RBCs were exposed to different stress conditions in vitro. A significantly increased number of Annexin V-positive RBCs was detected for mk/mk RBCs when compared to wt RBCs after 5 and 7 hour exposure to hyperosmotic shock (400mM sucrose) and glucose depletion, respectively. These results indicate shortened life span of DMT1-mutant erythrocytes and their reduced ability to cope with stress. To unravel the possible underlying mechanisms we focus on two processes important for RBC survival; anti-oxidative defense and anaerobic glycolysis. We observed 1.5 to 2-fold higher activity of glutathione peroxidase, catalase and methemoglobin reductase and elevated levels of methemoglobin in mk/mk RBCs in comparison to wt RBCs, indicating increased oxidative stress in mk/mk RBCs. Increased activity of hexokinase (2.5 times) and pyruvatkinase (2.4 times) together with reduced ratio of ATP/ADP in mk/mk mice compared with wt mice (from 2.89±0.56 μmol/L to 1.71±0.49 μmol/L) shows enhanced demand for glycolytically derived ATP to maintain the stability of RBC membrane in mk/mk mice. Our analyzes suggest that DMT1 deficiency negatively affects metabolism and life span of mature erythrocytes; two other aspects of defective erythropoiesis contributing to the pathophysiology of the disease. Grant support Czech Grant Agency, grant No. P305/11/1745; Ministry of Health Czech Republic Grant No. NT11208 and Internal Grant of Palacky University Olomouc (LF_2013_010). Disclosures: No relevant conflicts of interest to declare.

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