Functional Impact of MacroH2A Histone Variants in Acute Myeloid Leukemia

Histone variants are emerging as key regulatory molecules in cancer. Recent data provided first evidence of how transcriptional deregulation and changes in the deposition of histone variants may affect malignant transformation. H2A variants are highly conserved and consist of 8 members. Among all histone variants, macroH2A variants hold a unique C-terminal macro domain, which is approximately twice the size of its H2A-like histone domain. In mammals, there are 3 macroH2A variants encoded by H2AFY (macroH2A1.1, macroH2A1.2) and H2AFY2 (macroH2A2). MacroH2A variants occupy large repressive domains throughout the genome, exerting a repressive role on transcription. So far, the function of macroH2A proteins in hematopoiesis and leukemic transformation is incompletely understood. Here, we report on a functional role of macroH2A histone variants that may influence cell competition in leukemia and normal hematopoietic stem and progenitor cell (HSPC) function. In published gene expression datasets, macroH2A variants H2AFY and H2AFY2 are highly expressed in hematopoietic stem cells (HSCs) compared to mononuclear cells (both p<0.0001****). Moreover, expression of H2AFY and H2AFY2 appeared even higher in acute myeloid leukemia (AML) (p=0.0750 and 0.0037**, respectively) when compared to the expression level in HSCs. This high expression is independent of different genetic subtypes and AML risk groups. To assess for the functional dependency of leukemic cells on the expression of macroH2A variants, we performed an in vitro CRISPR-Cas9 dropout screen focusing on macroH2A variants in MOLM-13 cells. Genetic inactivation of H2AFY but not H2AFY2 resulted in outcompetition of infected cells against non-infected competitors. This finding could be recapitulated in 3 additional AML cell lines (OCI-AML3, THP-1, HL-60), indicating a broader dependency on H2AFY irrespective of the oncogenic background or underlying driver mutation. To validate the functional impact of H2AFY on leukemia development in vivo, we generated a novel conditional knockout mouse model for H2afy and induced leukemia through retroviral infection of sorted Lineage-Sca1+cKit+ (LSK) cells with the oncogene MLL-AF9. Results of these functional studies will be presented. To understand the functional impact of macroH2A variants on normal HSPC function in vivo, we performed competitive repopulation studies. We used conventional knockout mouse models for the 2 H2afy isoforms: macroH2A1.1 and macroH2A1.2 In addition, for inactivation of both isoforms, we used the conditional macroH2A knockout mouse model. H2afy2 was either inactivated by RNAi or conditionally knockout through Mx1-Cre recombinase activation. In brief, whole bone marrow cells (WBMCs) were transplanted into primary recipient mice in a 1:1 competitive manner and compared to the respective wildtype littermate controls. Peripheral blood (PB) chimerism was monitored on a monthly basis 4 weeks post-transplantation for a total of 16 weeks. We observed no significant difference in the recipients of macroH2A1.1 (n=5/5) or macroH2A1.2 (n=10/9), suggesting both macroH2A isoforms are dispensable for intrinsic functions of HSCs. Remarkably, we found macroH2A2-deficient cells (n=6) competed significantly worse than macroH2A2+/+ (n=5) controls in PB (p=0.0043**) and in WBM (p=0.0043**), indicating a requirement for macroH2A2 in maintenance of HSPCs. These findings could be recapitulated by RNA-mediated inactivation of H2afy2 in sorted HSCs. We observed significant reduction of macroH2A2-depleted hematopoiesis after 16 weeks in WBM (p=0.0286*) and PB (p=0.0079**). Importantly, this functional impairment was less pronounced when investigating multipotent progenitor and committed progenitor cell function in spleen colony-forming unit (CFU) assay in vivo or CFU assay in vitro. Taken together, our data indicate distinct requirements for macrohistone variants in leukemic and normal hematopoietic cells. Dependency of leukemic cells but not normal HSPCs on macroH2A may indicate a potential therapeutic index. Disclosures No relevant conflicts of interest to declare.

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Evidence for a positive role of SHIP in the BCR-ABL–mediated transformation of primitive murine hematopoietic cells and in human chronic myeloid leukemia

Blood ◽

10.1182/blood-2003-05-1550 ◽

2003 ◽

Vol 102 (8) ◽

pp. 2976-2984 ◽

Cited By ~ 29

Author(s):

Xiaoyan Jiang ◽

Matthew Stuible ◽

Yves Chalandon ◽

Andra Li ◽

Wing Yiu Chan ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Fluorescent Protein ◽

Chronic Phase ◽

Leukemic Cells ◽

Mouse Bone Marrow ◽

Positive Role ◽

Hematopoietic Stem

Abstract Previous studies suggested that the SH2-containing inositol-5-phosphatase (SHIP) may play a tumor suppressor-like function in BCR-ABL–mediated leukemogenesis. To investigate this possibility, we first developed a new assay for quantitating transplantable multilineage leukemia-initiating cells (L-ICs) in hematopoietic stem cell (HSC)–enriched mouse bone marrow (BM) cells transduced with a BCR-ABL–GFP (green fluorescent protein) retrovirus. The frequency of L-ICs (1 of 430 Sca-1+lin– cells) was 7-fold lower than the frequency of HSCs in the Sca-1+lin– subset transduced with a control virus (1 of 65 cells). Forced BCRABL expression was also accompanied by a loss of regular HSC activity consistent with the acquisition of an increased probability of differentiation. Interestingly, the frequency and in vivo behavior of wild-type (+/+) and SHIP–/– L-ICs were indistinguishable, and in vitro, Sca-1+lin– BCR-ABL–transduced SHIP–/– cells showed a modestly reduced factor independence. Comparison of different populations of cells from patients with chronic myeloid leukemia (CML) in chronic phase and normal human BM showed that the reduced expression of full-length SHIP proteins seen in the more mature (CD34–lin+) leukemic cells is not mirrored in the more primitive (CD34+lin–) leukemic cells. Thus, SHIP expression appears to be differently altered in the early and late stages of differentiation of BCR-ABL–transformed cells, underscoring the importance of the cellular context in which its mechanistic effects are analyzed.

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Anti-MY9-blocked-ricin: an immunotoxin for selective targeting of acute myeloid leukemia cells

Blood ◽

10.1182/blood.v77.11.2404.2404 ◽

1991 ◽

Vol 77 (11) ◽

pp. 2404-2412 ◽

Cited By ~ 41

Author(s):

DC Roy ◽

JD Griffin ◽

M Belvin ◽

WA Blattler ◽

JM Lambert ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Leukemic Cells ◽

Short Exposure ◽

Continuous Exposure ◽

Dependent Manner ◽

Acute Myeloid

Abstract The use of immunotoxins (IT) to selectively destroy acute myeloid leukemia (AML) cells in vivo or in vitro is complicated by both the antigenic similarity of AML cells to normal progenitor cells and the difficulty of producing a sufficiently toxic conjugate. The monoclonal antibody (MoAb) anti-MY9 is potentially ideal for selective recognition of AML cells because it reacts with an antigen (CD33) found on clonogenic AML cells from greater than 80% of cases and does not react with normal pluripotent stem cells. In this study, we describe an immunotoxin that is selectively active against CD33+ AML cells: Anti- MY9-blocked-Ricin (Anti-MY9-bR), comprised of anti-MY9 conjugated to a modified whole ricin that has its nonspecific binding eliminated by chemical blockage of the galactose binding domains of the B-chain. A limiting dilution assay was used to measure elimination of HL-60 leukemic cells from a 20-fold excess of normal bone marrow cells. Depletion of CD33+ HL-60 cells was found to be dependent on the concentration of Anti-MY9-bR and on the duration of incubation with IT at 37 degrees C. More than 4 logs of these leukemic cells were specifically depleted following short exposure to high concentrations (10(-8) mol/L) of Anti-MY9-bR. Incubation with much lower concentrations of Anti-MY9-bR (10(-10) mol/L), as compatible with in vivo administration, resulted in 2 logs of depletion of HL-60 cells, but 48 to 72 hours of continuous exposure were required. Anti-MY9-bR was also shown to be toxic to primary AML cells, with depletion of greater than 2 logs of clonogenic cells following incubation with Anti- MY9-bR 10(-8) mol/L at 37 degrees C for 5 hours. Activity of Anti-MY9- bR could be blocked by unconjugated Anti-MY9 but not by galactose. As expected, Anti-MY9-bR was toxic to normal colony-forming unit granulocyte-monocyte (CFU-GM), which expresses CD33, in a concentration- and time-dependent manner, and also to burst-forming unit-erythroid and CFU-granulocyte, erythroid, monocyte, megakaryocyte, although to a lesser extent. When compared with anti-MY9 and complement (C′), Anti- MY9-bR could be used in conditions that provided more effective depletion of AML cells with substantially less depletion of normal CFU- GM. Therefore, Anti-MY9-bR may have clinical utility for in vitro purging of AML cells from autologous marrow when used at high IT concentrations for short incubation periods. Much lower concentrations of Anti-MY9-bR that can be maintained for longer periods may be useful for elimination of AML cells in vivo.

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Cooperation of Antiporter LAT2/CD98hc with Uniporter TAT1 for Renal Reabsorption of Neutral Amino Acids

Journal of the American Society of Nephrology ◽

10.1681/asn.2017111205 ◽

2018 ◽

Vol 29 (6) ◽

pp. 1624-1635 ◽

Cited By ~ 13

Author(s):

Clara Vilches ◽

Emilia Boiadjieva-Knöpfel ◽

Susanna Bodoy ◽

Simone Camargo ◽

Miguel López de Heredia ◽

...

Keyword(s):

Amino Acids ◽

Knockout Mouse ◽

Functional Relationship ◽

Tubular Reabsorption ◽

Compensatory Mechanisms ◽

Double Knockout ◽

Knockout Mouse Model ◽

Relationship Of

Background Reabsorption of amino acids (AAs) across the renal proximal tubule is crucial for intracellular and whole organism AA homeostasis. Although the luminal transport step is well understood, with several diseases caused by dysregulation of this process, the basolateral transport step is not understood. In humans, only cationic aminoaciduria due to malfunction of the basolateral transporter y+LAT1/CD98hc (SLC7A7/SLC3A2), which mediates the export of cationic AAs, has been described. Thus, the physiologic roles of basolateral transporters of neutral AAs, such as the antiporter LAT2/CD98hc (SLC7A8/SLC3A2), a heterodimer that exports most neutral AAs, and the uniporter TAT1 (SLC16A10), which exports only aromatic AAs, remain unclear. Functional cooperation between TAT1 and LAT2/CD98hc has been suggested by in vitro studies but has not been evaluated in vivo.Methods To study the functional relationship of TAT1 and LAT2/CD98hc in vivo, we generated a double-knockout mouse model lacking TAT1 and LAT2, the catalytic subunit of LAT2/CD98hc (dKO LAT2-TAT1 mice).Results Compared with mice lacking only TAT1 or LAT2, dKO LAT2-TAT1 mice lost larger amounts of aromatic and other neutral AAs in their urine due to a tubular reabsorption defect. Notably, dKO mice also displayed decreased tubular reabsorption of cationic AAs and increased expression of y+LAT1/CD98hc.Conclusions The LAT2/CD98hc and TAT1 transporters functionally cooperate in vivo, and y+LAT1/CD98hc may compensate for the loss of LAT2/CD98hc and TAT1, functioning as a neutral AA exporter at the expense of some urinary loss of cationic AAs. Cooperative and compensatory mechanisms of AA transporters may explain the lack of basolateral neutral aminoacidurias in humans.

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Selective Ablation of Acute Myeloid Leukemia Using Antibody-Targeted Chemotherapy: A Phase I Study of an Anti-CD33 Calicheamicin Immunoconjugate

Blood ◽

10.1182/blood.v93.11.3678 ◽

1999 ◽

Vol 93 (11) ◽

pp. 3678-3684 ◽

Cited By ~ 351

Author(s):

E.L. Sievers ◽

F.R. Appelbaum ◽

R.T. Spielberger ◽

S.J. Forman ◽

D. Flowers ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

High Rate ◽

Leukemic Cells ◽

Antitumor Antibiotic ◽

Dose Escalation Study ◽

Hematopoietic Stem ◽

Selective Ablation ◽

Acute Myeloid

Abstract Leukemic blast cells express the CD33 antigen in most patients with acute myeloid leukemia (AML), but this antigen is not expressed by hematopoietic stem cells. We conducted a study to determine whether normal hematopoiesis could be restored in patients with AML by selective ablation of cells expressing the CD33 antigen. In a dose escalation study, 40 patients with relapsed or refractory CD33+ AML were treated with an immunoconjugate (CMA-676) consisting of humanized anti-CD33 antibody linked to the potent antitumor antibiotic calicheamicin. The capacity of leukemic cells to efflux 3,3’-diethyloxacarbocyanine iodide (DiOC2) was used to estimate pretreatment functional drug resistance. Leukemia was eliminated from the blood and marrow of 8 (20%) of the 40 patients; blood counts returned to normal in three (8%) patients. A high rate of clinical response was observed in leukemias characterized by low dye efflux in vitro. Infusions of CMA-676 were generally well tolerated, and a postinfusion syndrome of fever and chills was the most common toxic effect. Two patients who were treated at the highest dose level (9 mg/m2) were neutropenic >5 weeks after the last dose of CMA-676. These results show that an immunoconjugate targeted to CD33 can selectively ablate malignant hematopoiesis in some patients with AML.

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HDAC3 Activity is Essential for Human Leukemic Cell Growth and the Expression of β-catenin, MYC, and WT1

Cancers ◽

10.3390/cancers11101436 ◽

2019 ◽

Vol 11 (10) ◽

pp. 1436 ◽

Cited By ~ 3

Author(s):

Mandy Beyer ◽

Annette Romanski ◽

Al-Hassan M. Mustafa ◽

Miriam Pons ◽

Iris Büchler ◽

...

Keyword(s):

Dna Damage ◽

Myeloid Leukemia ◽

Histone Deacetylase Inhibitors ◽

Wilms Tumor ◽

Leukemic Cell ◽

Replication Stress ◽

Leukemic Cells ◽

Hdac6 Inhibitor

Therapy of acute myeloid leukemia (AML) is unsatisfactory. Histone deacetylase inhibitors (HDACi) are active against leukemic cells in vitro and in vivo. Clinical data suggest further testing of such epigenetic drugs and to identify mechanisms and markers for their efficacy. Primary and permanent AML cells were screened for viability, replication stress/DNA damage, and regrowth capacities after single exposures to the clinically used pan-HDACi panobinostat (LBH589), the class I HDACi entinostat/romidepsin (MS-275/FK228), the HDAC3 inhibitor RGFP966, the HDAC6 inhibitor marbostat-100, the non-steroidal anti-inflammatory drug (NSAID) indomethacin, and the replication stress inducer hydroxyurea (HU). Immunoblotting was used to test if HDACi modulate the leukemia-associated transcription factors β-catenin, Wilms tumor (WT1), and myelocytomatosis oncogene (MYC). RNAi was used to delineate how these factors interact. We show that LBH589, MS-275, FK228, RGFP966, and HU induce apoptosis, replication stress/DNA damage, and apoptotic fragmentation of β-catenin. Indomethacin destabilizes β-catenin and potentiates anti-proliferative effects of HDACi. HDACi attenuate WT1 and MYC caspase-dependently and -independently. Genetic experiments reveal a cross-regulation between MYC and WT1 and a regulation of β-catenin by WT1. In conclusion, reduced levels of β-catenin, MYC, and WT1 are molecular markers for the efficacy of HDACi. HDAC3 inhibition induces apoptosis and disrupts tumor-associated protein expression.

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Involvement of the Integrin Alpha 6 Receptor in the Homing and Engraftment of Hematopoietic Stem and Progenitor Cells to Bone Marrow.

Blood ◽

10.1182/blood.v104.11.1293.1293 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1293-1293

Author(s):

Hong Qian ◽

Sten Eirik W. Jacobsen ◽

Marja Ekblom

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Progenitor Cell ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Functional Blocking

Abstract Within the bone marrow environment, adhesive interactions between stromal cells and extracellular matrix molecules are required for stem and progenitor cell survival, proliferation and differentiation as well as their transmigration between bone marrow (BM) and the circulation. This regulation is mediated by cell surface adhesion receptors. In experimental mouse stem cell transplantation models, several classes of cell adhesion receptors have been shown to be involved in the homing and engraftment of stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Using FACS analysis, the integrin a6 chain was now found to be ubiquitously (>95%) expressed in mouse hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, lin−Sca-1+c-Kit+CD34+) both in adult bone marrow and in fetal liver. In vitro, about 70% of mouse BM lin−Sca-1+c-Kit+ cells adhered to laminin-10/11 and 40% adhered to laminin-8. This adhesion was mediated by integrin a6b1 receptor, as shown by functional blocking monoclonal antibodies. We also used a functional blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of hematopoietic stem and progenitor cells. We found that the integrin a6 antibody inhibited the homing of bone marrow progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C was reduced by about 40% as compared to cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells (LTR), antibody treated bone marrow cells were first injected intravenously into lethally irradiated primary recipients. After three hours, bone marrow cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis 16 weeks after transplantation revealed an 80% reduction of stem cell activity of integrin a6 antibody treated cells as compared to cells treated with control antibody. These results suggest that integrin a6 plays an important role for hematopoietic stem and progenitor cell homing in vivo.

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Integrin alpha 6 Is Essential for Fetal Hematopoietic Progenitor, but Not Fetal Stem Cell Homing to Bone Marrow.

Blood ◽

10.1182/blood.v106.11.1387.1387 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1387-1387

Author(s):

Hong Qian ◽

Sten Eirik W. Jacobsen ◽

Marja Ekblom

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Progenitor Cell ◽

Fetal Liver ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Integrin Alpha 6

Abstract Homing of transplanted hematopoietic stem cells (HSC) in the bone marrow (BM) is a prerequisite for establishment of hematopoiesis following transplantation. However, although multiple adhesive interactions of HSCs with BM microenviroment are thought to critically influence their homing and subsequently their engraftment, the molecular pathways that control the homing of transplanted HSCs, in particular, of fetal HSCs are still not well understood. In experimental mouse stem cell transplantation models, several integrins have been shown to be involved in the homing and engraftment of both adult and fetal stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Furthermore, integrin a6 is required for adult mouse HSC homing to BM in vivo (Qian et al., Abstract American Society of Hematology, Blood 2004 ). We have now found that the integrin a6 chain like in adult HSC is ubiquitously (>99%) expressed also in fetal liver hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, LSK ). In vitro, fetal liver LSK cells adhere to laminin-10/11 and laminin-8 in an integrin a6b1 receptor-dependent manner, as shown by function blocking monoclonal antibodies. We have now used a function blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of fetal liver hematopoietic stem and progenitor cells to BM. The integrin a6 antibody inhibited homing of fetal liver progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C in BM was reduced by about 40% as compared to the cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells, BM cells were first incubated with anti-integrin alpha 6 or anti-integrin alpha 4 or control antibody, and then injected intravenously into lethally irradiated primary recipients. After three hours, BM cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis up to 16 weeks after transplantation showed that no reduction of stem cell reconstitution from integrin a6 antibody treated cells as compared to cells treated with control antibody. In accordance with this, fetal liver HSC from integrin a6 gene deleted embryos did not show any impairment of homing and engraftment in BM as compared to normal littermates. These results suggest that integrin a6 plays an important developmentally regulated role for homing of distinct hematopoietic stem and progenitor cell populations in vivo.

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The Role of STAT5 in HOXA9-Associated Leukemia

Blood ◽

10.1182/blood.v128.22.3919.3919 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3919-3919

Author(s):

Peilin Ma ◽

Yuqing Sun ◽

Jingya Wang ◽

Weihua Song ◽

Tao Xu ◽

...

Keyword(s):

Binding Sites ◽

Myeloid Leukemia ◽

Tandem Duplication ◽

Conflicts Of Interest ◽

Hematopoietic Stem ◽

Constitutive Activation ◽

Oncogenic Mutation

Abstract Homeobox A9 (HOXA9) is a homeodomain-containing transcription factor that is essential for hematopoietic stem cell expansion and differentiation. Deregulation of HOXA9 is commonly observed in human acute myeloid leukemia (AML). About half of AML patients overexpress HOXA9 as a result of MLL rearrangements, NUP98 translocations, NPM1 mutations or CDX2/CDX4 overexpression. Despite its central importance in leukemia, the mechanism of transcriptional regulation by HOXA9 and its downstream effectors are poorly understood. HOXA9 physically interacts with MEIS1, a cofactor that greatly accelerates leukemia development in transplanted animals. Our group recently identified a number of transcription factors as HOXA9 potential collaborators by genomic profiling of HOXA9 binding sites and mass spectroscopy. One of these putative collaborators is signal transducer and activator of transcription 5 (STAT5), which coimmunoprecipitates with HOXA9. Furthermore STAT motifs extensively overlap with HOXA9 binding sites. STAT5 is important for survival, proliferation and differentiation of hematopoietic cells and constitutive activation of STAT5 has also been observed in human leukemias bearing oncogenic mutation of Jak2, Bcr-Abl, c-Kit and Flt3. FLT3 internal tandem duplication (FLT3-ITD) is observed in 25% of patients with MLL-partial tandem duplication (MLL-PTD) and is associated with HOXA9 upregulation and unfavorable prognosis. Therefore, we hypothesized that the interaction of HOXA9 and STAT5 may play a role in HOXA9-associated leukemogenesis. Treatment of human cell lines bearing MLL-AF9 and FLT3-ITD with specific FLT3 and STAT5 inhibitors showed that suppression of the constitutive activation of STAT5 significantly inhibits the hyper-proliferation of these cells. We then overexpressed FLT3-ITD or active mutation of STAT5 (STAT5 1*6) in mouse hematopoietic stem cells /progenitor cells (HSC/PCs) transduced with MLL-AF9 or HOXA9 and found that constitutively active STAT5 enhances cell proliferation in vitro. We next transduced HOXA9 into HSC/Pcs from wild type (WT) or FLT3-ITD transgenic mice and transplanted these cells into sublethally irradiated WT mice. All of these recipients developed myeloid leukemia, with recipients transplanted with FLT3-ITD (n=4) developing leukemia significantly earlier than WT controls (n=5, p<0.05), suggesting that FLT3-ITD mediated STAT5 activation enhanced HOXA9-induced leukemogenesis in vivo. To further assess the role of STAT5 in HOXA9-mediated transformation, we performed ChIP-Seq assay with HOXA9-transformed cells and identified nearly half of STAT5 binding sites (228 out of 596) colocalized with HOXA9. Most of these cobound sites are located in distal intergenic (61.0%) and intron (35.1%) regions. Five cobound regions (Il2rα, Fgf1, Pdlim5, Pim1, Fabp5) were selected and confirmed by ChIP-qPCR. To further characterize the interaction between HOXA9 and STAT5, GST pull-down assays were performed that showed that the c-terminal of HOXA9 is critical for interaction with STAT5. Overall, the findings suggest that STAT5 promotes HOXA9-induced transformation by functionally interacting with HOXA9 at HOXA9-regulated enhancers. Disclosures No relevant conflicts of interest to declare.

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Co-inhibition of NF-κB and JNK is synergistic in TNF-expressing human AML

Journal of Experimental Medicine ◽

10.1084/jem.20130990 ◽

2014 ◽

Vol 211 (6) ◽

pp. 1093-1108 ◽

Cited By ~ 56

Author(s):

Andrew Volk ◽

Jing Li ◽

Junping Xin ◽

Dewen You ◽

Jun Zhang ◽

...

Keyword(s):

Nuclear Factor Κb ◽

Leukemic Cells ◽

Comprehensive Treatment ◽

Hematopoietic Stem ◽

Similar Treatment ◽

Stem And Progenitor Cells ◽

Treatment Paradigm ◽

Factor Α

Leukemic stem cells (LSCs) isolated from acute myeloid leukemia (AML) patients are more sensitive to nuclear factor κB (NF-κB) inhibition-induced cell death when compared with hematopoietic stem and progenitor cells (HSPCs) in in vitro culture. However, inadequate anti-leukemic activity of NF-κB inhibition in vivo suggests the presence of additional survival/proliferative signals that can compensate for NF-κB inhibition. AML subtypes M3, M4, and M5 cells produce endogenous tumor necrosis factor α (TNF). Although stimulating HSPC with TNF promotes necroptosis and apoptosis, similar treatment with AML cells (leukemic cells, LCs) results in an increase in survival and proliferation. We determined that TNF stimulation drives the JNK–AP1 pathway in a manner parallel to NF-κB, leading to the up-regulation of anti-apoptotic genes in LC. We found that we can significantly sensitize LC to NF-κB inhibitor treatment by blocking the TNF–JNK–AP1 signaling pathway. Our data suggest that co-inhibition of both TNF–JNK–AP1 and NF-κB signals may provide a more comprehensive treatment paradigm for AML patients with TNF-expressing LC.

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Ezh2 augments leukemogenicity by reinforcing differentiation blockage in acute myeloid leukemia

Blood ◽

10.1182/blood-2011-11-394932 ◽

2012 ◽

Vol 120 (5) ◽

pp. 1107-1117 ◽

Cited By ~ 122

Author(s):

Satomi Tanaka ◽

Satoru Miyagi ◽

Goro Sashida ◽

Tetsuhiro Chiba ◽

Jin Yuan ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Target Genes ◽

Fusion Gene ◽

Hematologic Malignancies ◽

Leukemic Cells ◽

Polycomb Repressive Complex 2 ◽

Acute Myeloid

Abstract EZH2, a catalytic component of the polycomb repressive complex 2, trimethylates histone H3 at lysine 27 (H3K27) to repress the transcription of target genes. Although EZH2 is overexpressed in various cancers, including some hematologic malignancies, the role of EZH2 in acute myeloid leukemia (AML) has yet to be examined in vivo. In the present study, we transformed granulocyte macrophage progenitors from Cre-ERT;Ezh2flox/flox mice with the MLL-AF9 leukemic fusion gene to analyze the function of Ezh2 in AML. Deletion of Ezh2 in transformed granulocyte macrophage progenitors compromised growth severely in vitro and attenuated the progression of AML significantly in vivo. Ezh2-deficient leukemic cells developed into a chronic myelomonocytic leukemia–like disease with a lower frequency of leukemia-initiating cells compared with the control. Chromatin immunoprecipitation followed by sequencing revealed a significant reduction in the levels of trimethylation at H3K27 in Ezh2-deficient leukemic cells, not only at Cdkn2a, a known major target of Ezh2, but also at a cohort of genes relevant to the developmental and differentiation processes. Overexpression of Egr1, one of the derepressed genes in Ezh2-deficient leukemic cells, promoted the differentiation of AML cells profoundly. Our findings suggest that Ezh2 inhibits differentiation programs in leukemic stem cells, thereby augmenting their leukemogenic activity.

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