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1.
Bull Cancer ; 110(3): 331-335, 2023 Mar.
Article in English | MEDLINE | ID: mdl-36775700

ABSTRACT

This article highlights the presentations from the 2021 scientific meeting of the Club Hematopoiesis and Oncogenesis. This annual meeting focuses on hematopoiesis and oncogenic mechanisms. Various topics were presented: expansion of hematopoietic stem cells with in vivo and ex vivo strategies, the role of the hematopoietic stem cell niches in aging and leukemic resistance, the crossroad between hematology and immunology, the importance of the metabolism in normal hematopoiesis and hematopoietic defects, solid tumors and oncogenesis, the noncoding genome, inflammation in monocyte differentiation and leukemia, and importantly, the recent advances in myeloid malignancies, lymphoid leukemia and lymphoma.


Subject(s)
Leukemia , Lymphoma , Humans , Hematopoiesis/genetics , Hematopoietic Stem Cells , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology
2.
Haematologica ; 104(6): 1244-1255, 2019 06.
Article in English | MEDLINE | ID: mdl-30545930

ABSTRACT

Germline RUNX1 mutations lead to thrombocytopenia and platelet dysfunction in familial platelet disorder with predisposition to acute myelogenous leukemia (AML). Multiple aspects of platelet function are impaired in these patients, associated with altered expression of genes regulated by RUNX1 We aimed to identify RUNX1-targets involved in platelet function by combining transcriptome analysis of patient and shRUNX1-transduced megakaryocytes (MK). Down-regulated genes included TREM-like transcript (TLT)-1 (TREML1) and the integrin subunit alpha (α)-2 (ITGA2) of collagen receptor α2-beta (ß)-1, which are involved in platelet aggregation and adhesion, respectively. RUNX1 binding to regions enriched for H3K27Ac marks was demonstrated for both genes using chromatin immunoprecipitation. Cloning of these regions upstream of the respective promoters in lentivirus allowing mCherry reporter expression showed that RUNX1 positively regulates TREML1 and ITGA2, and this regulation was abrogated after deletion of RUNX1 sites. TLT-1 content was reduced in patient MK and platelets. A blocking anti-TLT-1 antibody was able to block aggregation of normal but not patient platelets, whereas recombinant soluble TLT-1 potentiated fibrinogen binding to patient platelets, pointing to a role for TLT-1 deficiency in the platelet function defect. Low levels of α2 integrin subunit were demonstrated in patient platelets and MK, coupled with reduced platelet and MK adhesion to collagen, both under static and flow conditions. In conclusion, we show that gene expression profiling of RUNX1 knock-down or mutated MK provides a suitable approach to identify novel RUNX1 targets, among which downregulation of TREML1 and ITGA2 clearly contribute to the platelet phenotype of familial platelet disorder with predisposition to AML.


Subject(s)
Blood Platelet Disorders/genetics , Blood Platelet Disorders/metabolism , Core Binding Factor Alpha 2 Subunit/metabolism , Gene Expression Regulation , Integrin alpha2/genetics , Leukemia, Myeloid, Acute/etiology , Receptors, Immunologic/genetics , Blood Platelet Disorders/blood , Blood Platelets/metabolism , Disease Susceptibility , Gene Expression Profiling , Humans , Leukemia, Myeloid, Acute/diagnosis , Megakaryocytes/metabolism , Mutation , Platelet Aggregation , Platelet Function Tests , Protein Binding
3.
Genet Med ; 20(4): 458-463, 2018 04.
Article in English | MEDLINE | ID: mdl-28837157

ABSTRACT

PurposeMutations in genes involved in Fanconi anemia (FA)/BRCA DNA repair pathway cause cancer susceptibility diseases including familial breast cancer and Fanconi anemia (FA). A single FA patient with biallelic FANCM mutations was reported in 2005 but concurrent FANCA pathogenic mutations precluded assignment of FANCM as an FA gene. Here we report three individuals with biallelic FANCM truncating mutations who developed early-onset cancer and toxicity to chemotherapy but did not present congenital malformations or any hematological phenotype suggestive of FA.MethodsChromosomal breakages, interstrand crosslink sensitivity, and FANCD2 monoubiquitination were assessed in primary fibroblasts. Mutation analysis was achieved through Sanger sequencing. Genetic complementation of patient-derived cells was performed by lentiviral mediated transduction of wild-type FANCM complementary DNA followed by functional studies.ResultsPatient-derived cells exhibited chromosomal fragility, hypersensitivity to interstrand crosslinks, and impaired FANCD2 monoubiquitination. We identified two homozygous mutations (c.2586_2589del4; p.Lys863Ilefs*12 and c.1506_1507insTA; p.Ile503*) in FANCM as the cause of the cellular phenotype. Patient-derived cells were genetically complemented upon wild-type FANCM complementary DNA expression.ConclusionLoss-of-function mutations in FANCM cause a cancer predisposition syndrome clinically distinct from bona fide FA. Care should be taken with chemotherapy and radiation treatments in these patients due to expected acute toxicity.


Subject(s)
Alleles , DNA Helicases/genetics , Fanconi Anemia/genetics , Genetic Predisposition to Disease , Neoplasms/diagnosis , Neoplasms/genetics , Sequence Deletion , Adolescent , Cell Line , Chromosome Fragility/drug effects , DNA Helicases/metabolism , Female , Genetic Association Studies , Genetic Complementation Test , Homozygote , Humans , Male , Middle Aged , Pedigree , Phenotype
5.
J Clin Invest ; 126(9): 3580-4, 2016 09 01.
Article in English | MEDLINE | ID: mdl-27500492

ABSTRACT

Fanconi anemia (FA) is a recessive genetic disease characterized by congenital abnormalities, chromosome instability, progressive bone marrow failure (BMF), and a strong predisposition to cancer. Twenty FA genes have been identified, and the FANC proteins they encode cooperate in a common pathway that regulates DNA crosslink repair and replication fork stability. We identified a child with severe BMF who harbored biallelic inactivating mutations of the translesion DNA synthesis (TLS) gene REV7 (also known as MAD2L2), which encodes the mutant REV7 protein REV7-V85E. Patient-derived cells demonstrated an extended FA phenotype, which included increased chromosome breaks and G2/M accumulation upon exposure to DNA crosslinking agents, γH2AX and 53BP1 foci accumulation, and enhanced p53/p21 activation relative to cells derived from healthy patients. Expression of WT REV7 restored normal cellular and functional phenotypes in the patient's cells, and CRISPR/Cas9 inactivation of REV7 in a non-FA human cell line produced an FA phenotype. Finally, silencing Rev7 in primary hematopoietic cells impaired progenitor function, suggesting that the DNA repair defect underlies the development of BMF in FA. Taken together, our genetic and functional analyses identified REV7 as a previously undescribed FA gene, which we term FANCV.


Subject(s)
Fanconi Anemia/genetics , Mad2 Proteins/genetics , Mutation , Alleles , Animals , Cell Cycle , Cell Line, Tumor , Child , Chromosomal Instability , Chromosome Breakage , Cohort Studies , Cross-Linking Reagents/chemistry , DNA Damage , DNA Repair , Female , Fibroblasts/metabolism , Gene Silencing , Genetic Complementation Test , Genetic Predisposition to Disease , Genetic Variation , Hematopoietic Stem Cells/cytology , Humans , Lentivirus , Mad2 Proteins/metabolism , Male , Mice , Mice, Knockout , Mitosis , Phenotype
6.
Blood ; 125(6): 930-40, 2015 Feb 05.
Article in English | MEDLINE | ID: mdl-25490895

ABSTRACT

To explore how RUNX1 mutations predispose to leukemia, we generated induced pluripotent stem cells (iPSCs) from 2 pedigrees with germline RUNX1 mutations. The first, carrying a missense R174Q mutation, which acts as a dominant-negative mutant, is associated with thrombocytopenia and leukemia, and the second, carrying a monoallelic gene deletion inducing a haploinsufficiency, presents only as thrombocytopenia. Hematopoietic differentiation of these iPSC clones demonstrated profound defects in erythropoiesis and megakaryopoiesis and deregulated expression of RUNX1 targets. iPSC clones from patients with the R174Q mutation specifically generated an increased amount of granulomonocytes, a phenotype reproduced by an 80% RUNX1 knockdown in the H9 human embryonic stem cell line, and a genomic instability. This phenotype, found only with a lower dosage of RUNX1, may account for development of leukemia in patients. Altogether, RUNX1 dosage could explain the differential phenotype according to RUNX1 mutations, with a haploinsufficiency leading to thrombocytopenia alone in a majority of cases whereas a more complete gene deletion predisposes to leukemia.


Subject(s)
Core Binding Factor Alpha 2 Subunit/genetics , Hematopoiesis , Leukemia/genetics , Mutation , Thrombocytopenia/genetics , Cell Line , Cells, Cultured , Core Binding Factor Alpha 2 Subunit/metabolism , Gene Deletion , Genetic Predisposition to Disease , Genomic Instability , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Leukemia/metabolism , Leukemia/pathology , Molecular Sequence Data , Mutation, Missense , Thrombocytopenia/metabolism , Thrombocytopenia/pathology
7.
Stem Cell Reports ; 3(6): 1085-102, 2014 Dec 09.
Article in English | MEDLINE | ID: mdl-25458892

ABSTRACT

Hematopoietic stem cells (HSCs) are characterized by the capacity for self-renewal and the ability to reconstitute the entire hematopoietic compartment. Thrombopoietin maintains adult HSCs in a quiescent state through the induction of cell cycle inhibitors p57(Kip2) and p19(INK4d). Using the p19(INK4d-/-) mouse model, we investigated the role of p19(INK4d) in basal and stress-induced hematopoiesis. We demonstrate that p19(INK4d) is involved in the regulation of HSC quiescence by inhibition of the G0/G1 cell cycle transition. Under genotoxic stress conditions, the absence of p19(INK4d) in HSCs leads to accelerated cell cycle exit, accumulation of DNA double-strand breaks, and apoptosis when cells progress to the S/G2-M stages of the cell cycle. Moreover, p19(INK4d) controls the HSC microenvironment through negative regulation of megakaryopoiesis. Deletion of p19(INK4d) results in megakaryocyte hyperproliferation and increased transforming growth factor ß1 secretion. This leads to fibrosis in the bone marrow and spleen, followed by loss of HSCs during aging.


Subject(s)
Cellular Senescence/genetics , Cyclin-Dependent Kinase Inhibitor p19/genetics , DNA Damage , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Stem Cell Niche/genetics , Animals , Bone Marrow/metabolism , Bone Marrow/pathology , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Cell Count , Cell Differentiation/genetics , Cell Lineage/genetics , Cyclin-Dependent Kinase Inhibitor p19/deficiency , Cyclin-Dependent Kinase Inhibitor p19/metabolism , Hematopoiesis , Mice , Mice, Knockout , Osteosclerosis/genetics , Osteosclerosis/pathology , Primary Myelofibrosis/genetics , Primary Myelofibrosis/pathology , Resting Phase, Cell Cycle/genetics , Stromal Cells/metabolism
8.
J Clin Invest ; 124(2): 580-91, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24430186

ABSTRACT

Point mutations in the 5' UTR of ankyrin repeat domain 26 (ANKRD26) are associated with familial thrombocytopenia 2 (THC2) and a predisposition to leukemia. Here, we identified underlying mechanisms of ANKRD26-associated thrombocytopenia. Using megakaryocytes (MK) isolated from THC2 patients and healthy subjects, we demonstrated that THC2-associated mutations in the 5' UTR of ANKRD26 resulted in loss of runt-related transcription factor 1 (RUNX1) and friend leukemia integration 1 transcription factor (FLI1) binding. RUNX1 and FLI1 binding at the 5' UTR from healthy subjects led to ANKRD26 silencing during the late stages of megakaryopoiesis and blood platelet development. We showed that persistent ANKRD26 expression in isolated MKs increased signaling via the thrombopoietin/myeloproliferative leukemia virus oncogene (MPL) pathway and impaired proplatelet formation by MKs. Importantly, we demonstrated that ERK inhibition completely rescued the in vitro proplatelet formation defect. Our data identify a mechanism for development of the familial thrombocytopenia THC2 that is related to abnormal MAPK signaling.


Subject(s)
Chromosome Disorders/genetics , MAP Kinase Signaling System , Mutation , Nuclear Proteins/genetics , Thrombocytopenia/congenital , 5' Untranslated Regions , Adolescent , Adult , Base Sequence , Binding Sites , Cell Differentiation , Chromosome Breakage , Core Binding Factor Alpha 2 Subunit/metabolism , Enzyme Activation , Female , Gene Expression Regulation , Gene Silencing , Humans , Infant , Intercellular Signaling Peptides and Proteins , Male , Megakaryocytes/cytology , Middle Aged , Molecular Sequence Data , Pedigree , Proto-Oncogene Protein c-fli-1/metabolism , Receptors, Thrombopoietin/metabolism , Signal Transduction , Thrombocytopenia/genetics , Young Adult
9.
J Immunol ; 192(5): 2471-9, 2014 Mar 01.
Article in English | MEDLINE | ID: mdl-24477908

ABSTRACT

The interaction of integrin αE(CD103)ß7, often expressed on tumor-infiltrating T lymphocytes, with its cognate ligand, the epithelial cell marker E-cadherin on tumor cells, plays a major role in antitumor CTL responses. CD103 is induced on CD8 T cells upon TCR engagement and exposure to TGF-ß1, abundant within the tumor microenvironment. However, the transcriptional mechanisms underlying the cooperative role of these two signaling pathways in inducing CD103 expression in CD8 T lymphocytes remain unknown. Using a human CTL system model based on a CD8(+)/CD103(-) T cell clone specific of a lung tumor-associated Ag, we demonstrated that the transcription factors Smad2/3 and NFAT-1 are two critical regulators of this process. We also identified promoter and enhancer elements of the human ITGAE gene, encoding CD103, involved in its induction by these transcriptional regulators. Overall, our results explain how TGF-ß1 can participate in CD103 expression on locally TCR-engaged Ag-specific CD8 T cells, thus contributing to antitumor CTL responses and cancer cell destruction.


Subject(s)
Antigens, CD/immunology , Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/immunology , Integrin alpha Chains/immunology , Lung Neoplasms/immunology , NFATC Transcription Factors/immunology , Smad2 Protein/immunology , Smad3 Protein/immunology , Antigens, CD/biosynthesis , Antigens, CD/genetics , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/pathology , HEK293 Cells , Humans , Integrin alpha Chains/biosynthesis , Integrin alpha Chains/genetics , Jurkat Cells , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , Response Elements/genetics , Response Elements/immunology , Signal Transduction/genetics , Signal Transduction/immunology , Smad2 Protein/genetics , Smad2 Protein/metabolism , Smad3 Protein/genetics , Smad3 Protein/metabolism , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/immunology , Transforming Growth Factor beta1/metabolism
10.
Blood ; 120(13): 2708-18, 2012 Sep 27.
Article in English | MEDLINE | ID: mdl-22898599

ABSTRACT

FPD/AML is a familial platelet disorder characterized by platelet defects, predisposition to acute myelogenous leukemia (AML) and germ-line heterozygous RUNX1 alterations. Here we studied the in vitro megakaryopoiesis of 3 FPD/AML pedigrees. A 60% to 80% decrease in the output of megakaryocytes (MKs) from CD34(+) was observed. MK ploidy level was low and mature MKs displayed a major defect in proplatelet formation. To explain these defects, we focused on myosin II expression as RUNX1 has been shown to regulate MYL9 and MYH10 in an inverse way. In FPD/AML MKs, expression of MYL9 and MYH9 was decreased, whereas MYH10 expression was increased and the MYH10 protein was still present in the cytoplasm of mature MKs. Myosin II activity inhibition by blebbistatin rescued the ploidy defect of FPD/AML MKs. Finally, we demonstrate that MYH9 is a direct target of RUNX1 by chromatin immunoprecipitation and luciferase assays and we identified new RUNX1 binding sites in the MYL9 promoter region. Together, these results demonstrate that the defects in megakaryopoiesis observed in FPD/AML are, in part, related to a deregulation of myosin IIA and IIB expression leading to both a defect in ploidization and proplatelet formation.


Subject(s)
Blood Platelet Disorders/pathology , Core Binding Factor Alpha 2 Subunit/genetics , Gene Expression Regulation, Neoplastic , Genetic Predisposition to Disease , Leukemia, Myeloid, Acute/pathology , Megakaryocytes/pathology , Mutation/genetics , Nonmuscle Myosin Type IIA/metabolism , Nonmuscle Myosin Type IIB/metabolism , Blood Platelet Disorders/genetics , Blood Platelet Disorders/metabolism , Blotting, Western , Chromatin Immunoprecipitation , Female , Heterocyclic Compounds, 4 or More Rings/pharmacology , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Luciferases/metabolism , Male , Nonmuscle Myosin Type IIA/genetics , Nonmuscle Myosin Type IIB/genetics , Pedigree , Ploidies , Prognosis , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction
11.
Blood ; 120(13): 2719-22, 2012 Sep 27.
Article in English | MEDLINE | ID: mdl-22677128

ABSTRACT

RUNX1 gene alterations are associated with acquired and inherited hematologic malignancies that include familial platelet disorder/acute myeloid leukemia, primary or secondary acute myeloid leukemia, and chronic myelomonocytic leukemia. Recently, we reported that RUNX1-mediated silencing of nonmuscle myosin heavy chain IIB (MYH10) was required for megakaryocyte ploidization and maturation. Here we demonstrate that runx1 deletion in mice induces the persistence of MYH10 in platelets, and a similar persistence was observed in platelets of patients with constitutional (familial platelet disorder/acute myeloid leukemia) or acquired (chronic myelomonocytic leukemia) RUNX1 mutations. MYH10 was also detected in platelets of patients with the Paris-Trousseau syndrome, a thrombocytopenia related to the deletion of the transcription factor FLI1 that forms a complex with RUNX1 to regulate megakaryopoiesis, whereas MYH10 persistence was not observed in other inherited forms of thrombocytopenia. We propose MYH10 detection as a new and simple tool to identify inherited platelet disorders and myeloid neoplasms with abnormalities in RUNX1 and its associated proteins.


Subject(s)
Biomarkers/metabolism , Blood Platelet Disorders/diagnosis , Blood Platelets/metabolism , Core Binding Factor Alpha 2 Subunit/physiology , Myosin Heavy Chains/genetics , Nonmuscle Myosin Type IIB/genetics , Proto-Oncogene Protein c-fli-1/genetics , Animals , Blood Platelet Disorders/genetics , Blood Platelet Disorders/metabolism , Blood Platelets/pathology , Case-Control Studies , Comparative Genomic Hybridization , Female , Genetic Predisposition to Disease , Humans , Immunoblotting , Jacobsen Distal 11q Deletion Syndrome/diagnosis , Jacobsen Distal 11q Deletion Syndrome/genetics , Jacobsen Distal 11q Deletion Syndrome/metabolism , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myelomonocytic, Chronic/diagnosis , Leukemia, Myelomonocytic, Chronic/genetics , Leukemia, Myelomonocytic, Chronic/metabolism , Male , Megakaryocytes/pathology , Mice , Myosin Heavy Chains/metabolism , Nonmuscle Myosin Type IIB/metabolism , Pedigree , Ploidies , Prognosis , Proto-Oncogene Protein c-fli-1/metabolism , Thrombocytopenia/diagnosis , Thrombocytopenia/genetics , Thrombocytopenia/metabolism
12.
Nat Commun ; 3: 717, 2012 Mar 06.
Article in English | MEDLINE | ID: mdl-22395608

ABSTRACT

Megakaryocytes are unique mammalian cells that undergo polyploidization (endomitosis) during differentiation, leading to an increase in cell size and protein production that precedes platelet production. Recent evidence demonstrates that endomitosis is a consequence of a late failure in cytokinesis associated with a contractile ring defect. Here we show that the non-muscle myosin IIB heavy chain (MYH10) is expressed in immature megakaryocytes and specifically localizes in the contractile ring. MYH10 downmodulation by short hairpin RNA increases polyploidization by inhibiting the return of 4N cells to 2N, but other regulators, such as of the G1/S transition, might regulate further polyploidization of the 4N cells. Conversely, re-expression of MYH10 in the megakaryocytes prevents polyploidization and the transition of 2N to 4N cells. During polyploidization, MYH10 expression is repressed by the major megakaryocyte transcription factor RUNX1. Thus, RUNX1-mediated silencing of MYH10 is required for the switch from mitosis to endomitosis, linking polyploidization with megakaryocyte differentiation.


Subject(s)
Core Binding Factor Alpha 2 Subunit/metabolism , Megakaryocytes/cytology , Myosin Heavy Chains/genetics , Nonmuscle Myosin Type IIB/genetics , Polyploidy , Animals , Antigens, CD34/biosynthesis , Cell Line , Core Binding Factor Alpha 2 Subunit/genetics , Cytokinesis , Heterocyclic Compounds, 4 or More Rings/pharmacology , Humans , Megakaryocytes/metabolism , Mice , Mice, Knockout , Mitosis , Myosin Heavy Chains/biosynthesis , Myosin Heavy Chains/metabolism , Nonmuscle Myosin Type IIB/biosynthesis , Nonmuscle Myosin Type IIB/metabolism , RNA Interference , RNA, Small Interfering
13.
Blood ; 118(24): 6310-20, 2011 Dec 08.
Article in English | MEDLINE | ID: mdl-21725049

ABSTRACT

RUNX1 encodes a DNA-binding α subunit of the core-binding factor, a heterodimeric transcription factor. RUNX1 is a master regulatory gene in hematopoiesis and its disruption is one of the most common aberrations in acute leukemia. Inactivating or dominant-negative mutations in the RUNX1 gene have been also identified in pedigrees of familial platelet disorders with a variable propensity to develop acute myeloid leukemia (FPD/AML). We performed analysis of hematopoiesis from 2 FPD/AML pedigrees with 2 distinct RUNX1 germline mutations, that is, the R139X in a pedigree without AML and the R174Q mutation in a pedigree with AML. Both mutations induced a marked increase in the clonogenic potential of immature CD34(+)CD38(-) progenitors, with some self-renewal capacities observed only for R174Q mutation. This increased proliferation correlated with reduction in the expression of NR4A3, a gene previously implicated in leukemia development. We demonstrated that NR4A3 was a direct target of RUNX1 and that restoration of NR4A3 expression partially reduced the clonogenic potential of patient progenitors. We propose that the down-regulation of NR4A3 in RUNX1-mutated hematopoietic progenitors leads to an increase in the pool of cells susceptible to be hit by secondary leukemic genetic events.


Subject(s)
Core Binding Factor Alpha 2 Subunit/genetics , DNA-Binding Proteins/metabolism , Down-Regulation , Hematopoiesis , Leukemia, Myeloid, Acute/genetics , Platelet Storage Pool Deficiency/genetics , Receptors, Steroid/metabolism , Receptors, Thyroid Hormone/metabolism , Adolescent , Adult , Animals , Cell Proliferation , Cells, Cultured , Clone Cells/metabolism , Core Binding Factor Alpha 2 Subunit/metabolism , Female , HEK293 Cells , Hematopoietic Stem Cells/metabolism , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/physiopathology , Male , Mice , Middle Aged , Mutation , Pedigree , Platelet Storage Pool Deficiency/metabolism , Platelet Storage Pool Deficiency/physiopathology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , T-Lymphocytes/transplantation , Young Adult
14.
Blood ; 114(19): 4221-32, 2009 Nov 05.
Article in English | MEDLINE | ID: mdl-19724058

ABSTRACT

Megakaryoblastic leukemia 1 (MAL) is a transcriptional coactivator of serum response factor (SRF). In acute megakaryoblastic leukemia, the MAL gene is translocated and fused with the gene encoding one twenty-two (OTT). Herein, we show that MAL expression increases during the late differentiation steps of neonate and adult human megakaryopoiesis and localized into the nucleus after Rho GTPase activation by adhesion on collagen I or convulxin. MAL knockdown in megakaryocyte progenitors reduced the percentage of cells forming filopodia, lamellipodia, and stress fibers after adhesion on the same substrates, and reduced proplatelet formation. MAL repression led to dysmorphic megakaryocytes with disorganized demarcation membranes and alpha granules heterogeneously scattered in the cytoplasm. Gene expression profiling revealed a marked decrease in metalloproteinase 9 (MMP-9) and MYL9 expression after MAL inhibition. Luciferase assays in HEK293T cells and chromatin immunoprecipitation in primary megakaryocytes showed that the MAL/SRF complex directly regulates MYL9 and MMP9 in vitro. Megakaryocyte migration in response to stromal cell-derived factor 1, through Matrigel was considerably decreased after MAL knockdown, implicating MMP9 in migration. Finally, the use of a shRNA to decrease MYL9 expression showed that MYL9 was involved in proplatelet formation. MAL/SRF complex is thus involved in platelet formation and megakaryocyte migration by regulating MYL9 and MMP9.


Subject(s)
Blood Platelets/cytology , Blood Platelets/metabolism , Cardiac Myosins/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Matrix Metalloproteinase 9/genetics , Megakaryocytes/physiology , Myosin Light Chains/genetics , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Serum Response Factor/genetics , Serum Response Factor/metabolism , Adult , Cell Differentiation , Cell Line , Cell Movement/genetics , Cell Movement/physiology , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/chemistry , Fetal Blood/cytology , Focal Adhesions , Gene Expression Profiling , Humans , In Vitro Techniques , Infant, Newborn , Megakaryocytes/cytology , Multiprotein Complexes , Oligonucleotide Array Sequence Analysis , Oncogene Proteins, Fusion/antagonists & inhibitors , Oncogene Proteins, Fusion/chemistry , RNA, Small Interfering/genetics , Serum Response Factor/chemistry , Thrombopoiesis , Trans-Activators , rho GTP-Binding Proteins/metabolism
15.
J Clin Invest ; 119(4): 852-64, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19287095

ABSTRACT

Acute megakaryoblastic leukemia (AMKL) is a form of acute myeloid leukemia (AML) associated with a poor prognosis. The genetics and pathophysiology of AMKL are not well understood. We generated a knockin mouse model of the one twenty-two-megakaryocytic acute leukemia (OTT-MAL) fusion oncogene that results from the t(1;22)(p13;q13) translocation specifically associated with a subtype of pediatric AMKL. We report here that OTT-MAL expression deregulated transcriptional activity of the canonical Notch signaling pathway transcription factor recombination signal binding protein for immunoglobulin kappa J region (RBPJ) and caused abnormal fetal megakaryopoiesis. Furthermore, cooperation between OTT-MAL and an activating mutation of the thrombopoietin receptor myeloproliferative leukemia virus oncogene (MPL) efficiently induced a short-latency AMKL that recapitulated all the features of human AMKL, including megakaryoblast hyperproliferation and maturation block, thrombocytopenia, organomegaly, and extensive fibrosis. Our results establish that concomitant activation of RBPJ (Notch signaling) and MPL (cytokine signaling) transforms cells of the megakaryocytic lineage and suggest that specific targeting of these pathways could be of therapeutic value for human AMKL.


Subject(s)
Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Leukemia, Megakaryoblastic, Acute/genetics , Oncogene Fusion , Oncogene Proteins, Fusion/genetics , Animals , Disease Models, Animal , Hematopoiesis , Humans , Leukemia, Megakaryoblastic, Acute/etiology , Leukemia, Megakaryoblastic, Acute/metabolism , Leukemia, Megakaryoblastic, Acute/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation , Receptors, Notch/metabolism , Receptors, Thrombopoietin/genetics , Signal Transduction , Transcription, Genetic
16.
Mol Cell Biol ; 28(20): 6171-81, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18710951

ABSTRACT

The OTT-MAL/RBM15-MKL1 fusion protein is the result of the recurrent translocation t(1;22) in acute megakaryocytic leukemia in infants. How it contributes to the malignancy is unknown. The 3' fusion partner, MAL/MKL1/MRTF-A, is a transcriptional coactivator of serum response factor (SRF). MAL plays a key role in regulated gene expression depending on Rho family GTPases and G-actin. Here we demonstrate that OTT-MAL is a constitutive activator of SRF and target gene expression. This requires the SRF-binding motif and the MAL-derived transactivation domain. OTT-MAL localizes to the nucleus and is not regulated by upstream signaling. OTT-MAL deregulation reflects its independence from control by G-actin, which fails to interact with OTT-MAL in coimmunoprecipitation experiments. Regulation cannot be restored by reintroduction of the entire MAL N terminus into the fusion protein. OTT-MAL also caused a delayed induction of the MAL-independent, ternary complex factor-dependent target genes c-fos and egr-1 and the mitogen-activated protein kinase/Erk pathway. With testing in heterologous tissue culture systems, however, we observed considerable antiproliferative effects of OTT-MAL. Our data suggest that the deregulated activation of MAL-dependent and -independent promoters results in tissue-specific functions of OTT-MAL.


Subject(s)
Gene Expression Regulation , Oncogene Proteins, Fusion/metabolism , Serum Response Factor/metabolism , Actins/metabolism , Animals , Early Growth Response Protein 1/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , HeLa Cells , Humans , Intracellular Space/metabolism , Mice , NIH 3T3 Cells , Oncogene Proteins, Fusion/chemistry , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Protein Transport , Proto-Oncogene Proteins c-fos/metabolism , Signal Transduction , Ternary Complex Factors/metabolism , rhoA GTP-Binding Protein/metabolism
17.
Blood ; 111(8): 4081-91, 2008 Apr 15.
Article in English | MEDLINE | ID: mdl-18276842

ABSTRACT

The molecular mechanisms that regulate megakaryocyte (MK) ploidization are poorly understood. Using MK differentiation from primary human CD34(+) cells, we observed that p19(INK4D) expression was increased both at the mRNA and protein levels during ploidization. p19(INK4D) knockdown led to a moderate increase (31.7% +/- 5%) in the mean ploidy of MKs suggesting a role of p19(INK4D) in the endomitotic arrest. This increase in ploidy was associated with a decrease in the more mature MK population (CD41(high)CD42(high)) at day 9 of culture, which was related to a delay in differentiation. Inversely, p19(INK4D) overexpression in CD34(+) cells resulted in a decrease in mean ploidy level associated with an increase in CD41 and CD42 expression in each ploidy class. Confirming these in vitro results, bone marrow MKs from p19(INK4D) KO mice exhibited an increase in mean ploidy level from 18.7N (+/- 0.58N) to 52.7N (+/- 12.3N). Chromatin immunoprecipitation assays performed in human MKs revealed that AML-1 binds in vivo the p19(INK4D) promoter. Moreover, AML-1 inhibition led to the p19(INK4D) down-regulation in human MKs. These results may explain the molecular link at the transcriptional level between the arrest of endomitosis and the acceleration of MK differentiation.


Subject(s)
Cell Differentiation , Core Binding Factor Alpha 2 Subunit/metabolism , Cyclin-Dependent Kinase Inhibitor p19/metabolism , Megakaryocytes/cytology , Mitosis , Animals , Bone Marrow Cells/cytology , Cyclin-Dependent Kinase Inhibitor p19/antagonists & inhibitors , Cyclin-Dependent Kinase Inhibitor p19/deficiency , Cyclin-Dependent Kinase Inhibitor p19/genetics , Gene Expression Regulation , Humans , Mice , Platelet Glycoprotein GPIb-IX Complex/metabolism , Platelet Membrane Glycoprotein IIb/metabolism , Ploidies , Promoter Regions, Genetic/genetics
18.
Blood ; 108(1): 278-85, 2006 Jul 01.
Article in English | MEDLINE | ID: mdl-16410450

ABSTRACT

The incidence of follicular lymphoma (FL) in industrialized countries has been increasing since the 1950s. Polymorphisms in genes encoding key enzymes controlling folate-methionine metabolism, including methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MS or MTR), serine hydroxymethyltransferase (SHMT), and thymidylate synthase (TS or TYMS), modify the risk of various cancers and possibly FL. This study specifically looks for an association between MTHFR, MTR, TYMS, and SHMT polymorphisms and the risk of FL. We carried out a case-control study with 172 patients diagnosed with FL and 206 control subjects. We report that the risk of FL was doubled by the association of one mutant allele at both MTHFR polymorphisms. Individuals with MTR 2756AA had 2-fold higher risk of FL, and subjects not having at least one TYMS 2R allele showed a 2-fold higher risk of FL. The MTR 2756AA genotype conferred a greater multivariate-adjusted relative risk of FL, and the risk was multiplied by almost 5 in the TYMS2R(-)/MTR 2756AA combination. In conclusion, common polymorphisms in key enzymes of the folate-methionine metabolism pathway result in an increased risk of FL and suggest that inadequate intake of dietary folate and other methyl donor nutrients may contribute to the development of this malignancy.


Subject(s)
Enzymes/genetics , Folic Acid/metabolism , Genetic Predisposition to Disease , Lymphoma, Follicular/enzymology , Lymphoma, Follicular/genetics , Methionine/metabolism , 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase/genetics , 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase/metabolism , Adult , Alleles , DNA/genetics , Female , Folic Acid/blood , Genotype , Glycine Hydroxymethyltransferase/genetics , Glycine Hydroxymethyltransferase/metabolism , Humans , Lymphoma, Follicular/diagnosis , Male , Methionine/blood , Methylenetetrahydrofolate Reductase (NADPH2)/genetics , Methylenetetrahydrofolate Reductase (NADPH2)/metabolism , Middle Aged , Mutation , Polymorphism, Genetic/genetics , Risk Factors , Thymidylate Synthase/genetics , Thymidylate Synthase/metabolism
19.
Genes Chromosomes Cancer ; 41(3): 243-9, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15334547

ABSTRACT

Most chromosomal translocations observed in T-cell acute lymphoblastic leukemia (T-ALL) often produce transcriptional activation of transcription factor oncogenes. Ectopic expression of the TLX3 (also known as HOX11L2) gene has been shown to be associated with a cryptic t(5;14)(q35;q32) translocation specific for a subtype of T-ALL. Here we report several examples of variant and alternative translocations resulting in expression of TLX3 in T-ALL, and we describe three of these translocations in detail. In particular, the CDK6 gene was rearranged in two t(5;7)(q35;q21) translocations. In two additional instances, fusion of the BCL11B (also known as CTIP2) and RANBP17/TLX3 loci were shown to result from subtle genomic insertion/deletion within these loci. This study further underscores that TLX3 expression in T-ALL is strongly associated with the presence of genomic rearrangements.


Subject(s)
Homeodomain Proteins/genetics , Oncogene Proteins/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Adolescent , Blotting, Western , Cell Line, Tumor , Child , Child, Preschool , Chromosome Banding , Chromosomes/ultrastructure , Cyclin-Dependent Kinase 6 , Cyclin-Dependent Kinases/genetics , Female , Humans , Immunophenotyping , In Situ Hybridization, Fluorescence , Karyotyping , Male , Models, Genetic , Polymerase Chain Reaction , Proto-Oncogene Proteins , Translocation, Genetic , ran GTP-Binding Protein/genetics , ras Proteins/genetics
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