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1.
Leukemia ; 32(3): 820-827, 2018 03.
Article in English | MEDLINE | ID: mdl-28819285

ABSTRACT

Son of Sevenless 1 (SOS1) is a dual guanine nucleotide exchange factor (GEF) that activates the small GTPases RAC and RAS. Although the molecular mechanisms of RAS GEF catalysis have been unveiled, how SOS1 acquires RAC GEF activity and what is the physio-pathological relevance of this activity is much less understood. Here we show that SOS1 is tyrosine phosphorylated on Y1196 by ABL. Phosphorylation of Y1196 controls SOS1 inter-molecular interaction, is required to promote the exchange of nucleotides on RAC in vitro and for platelet-derived growth factor (PDGF) activation of RAC- and RAC-dependent actin remodeling and cell migration. SOS1 is also phosphorylated on Y1196 by BCR-ABL in chronic myelogenous leukemic cells. Importantly, in these cells, SOS1 is required for BCR-ABL-mediated activation of RAC, cell proliferation and transformation in vitro and in a xenograft mouse model. Finally, genetic removal of Sos1 in the bone marrow-derived cells (BMDCs) from Sos1fl/fl mice and infected with BCR-ABL causes a significant delay in the onset of leukemogenesis once BMDCs are injected into recipient, lethally irradiated mice. Thus, SOS1 is required for full transformation and critically contribute to the leukemogenic potential of BCR-ABL.


Subject(s)
Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Fusion Proteins, bcr-abl/genetics , Guanine Nucleotide Exchange Factors/metabolism , SOS1 Protein/metabolism , Animals , Cell Line, Tumor , Cell Movement , Cell Proliferation , Disease Models, Animal , Fusion Proteins, bcr-abl/metabolism , Humans , Leukemia/genetics , Leukemia/metabolism , Mice , Phosphorylation , Platelet-Derived Growth Factor/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , Tyrosine/metabolism , rac GTP-Binding Proteins , rac1 GTP-Binding Protein/metabolism
3.
Leukemia ; 31(11): 2315-2325, 2017 11.
Article in English | MEDLINE | ID: mdl-28216661

ABSTRACT

Deregulation of epigenetic mechanisms, including microRNA, contributes to leukemogenesis and drug resistance by interfering with cancer-specific molecular pathways. Here, we show that the balance between miR-194-5p and its newly discovered target BCL2-associated transcription factor 1 (BCLAF1) regulates differentiation and survival of normal hematopoietic progenitors. In acute myeloid leukemias this balance is perturbed, locking cells into an immature, potentially 'immortal' state. Enhanced expression of miR-194-5p by treatment with the histone deacetylase inhibitor SAHA or by exogenous miR-194-5p expression re-sensitizes cells to differentiation and apoptosis by inducing BCLAF1 to shuttle between nucleus and cytosol. miR-194-5p/BCLAF1 balance was found commonly deregulated in 60 primary acute myeloid leukemia patients and was largely restored by ex vivo SAHA treatment. Our findings link treatment responsiveness to re-instatement of miR-194-5p/BCLAF1 balance.


Subject(s)
Gene Expression Regulation , Leukemia, Myeloid, Acute/pathology , MicroRNAs/genetics , Repressor Proteins/genetics , Tumor Suppressor Proteins/genetics , Apoptosis , Cell Cycle , Cell Differentiation , Cell Line, Tumor , Down-Regulation , Humans , Leukemia, Myeloid, Acute/genetics
4.
Cell Death Dis ; 7: e2308, 2016 07 28.
Article in English | MEDLINE | ID: mdl-27468685

ABSTRACT

Proteins involved in DNA double-strand break (DSB) repair localize within the promyelocytic leukemia nuclear bodies (PML-NBs), whose disruption is at the root of the acute promyelocytic leukemia (APL) pathogenesis. All-trans-retinoic acid (RA) treatment induces PML-RARα degradation, restores PML-NB functions, and causes terminal cell differentiation of APL blasts. However, the precise role of the APL-associated PML-RARα oncoprotein and PML-NB integrity in the DSB response in APL leukemogenesis and tumor suppression is still lacking. Primary leukemia blasts isolated from APL patients showed high phosphorylation levels of H2AX (γ-H2AX), an initial DSBs sensor. By addressing the consequences of ionizing radiation (IR)-induced DSB response in primary APL blasts and RA-responsive and -resistant myeloid cell lines carrying endogenous or ectopically expressed PML-RARα, before and after treatment with RA, we found that the disruption of PML-NBs is associated with delayed DSB response, as revealed by the impaired kinetic of disappearance of γ-H2AX and 53BP1 foci and activation of ATM and of its substrates H2AX, NBN, and CHK2. The disruption of PML-NB integrity by PML-RARα also affects the IR-induced DSB response in a preleukemic mouse model of APL in vivo. We propose the oncoprotein-dependent PML-NB disruption and DDR impairment as relevant early events in APL tumorigenesis.


Subject(s)
Cell Nucleus/metabolism , DNA/metabolism , Gene Expression Regulation, Leukemic , Granulocyte Precursor Cells/metabolism , Leukemia, Promyelocytic, Acute/genetics , Oncogene Proteins, Fusion/genetics , Animals , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Nucleus/drug effects , Cell Nucleus/radiation effects , Cell Nucleus/ultrastructure , Checkpoint Kinase 2/genetics , Checkpoint Kinase 2/metabolism , DNA/genetics , DNA Breaks, Double-Stranded/radiation effects , Disease Models, Animal , Gamma Rays , Granulocyte Precursor Cells/drug effects , Granulocyte Precursor Cells/pathology , Granulocyte Precursor Cells/radiation effects , Histones/genetics , Histones/metabolism , Humans , Leukemia, Promyelocytic, Acute/metabolism , Leukemia, Promyelocytic, Acute/pathology , Mice , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oncogene Proteins, Fusion/metabolism , Signal Transduction , Tretinoin/pharmacology , Tumor Suppressor p53-Binding Protein 1/genetics , Tumor Suppressor p53-Binding Protein 1/metabolism
5.
Leukemia ; 26(7): 1630-7, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22333881

ABSTRACT

Aberrant histone acetylation was physiopathologically associated with the development of acute myeloid leukemias (AMLs). Reversal of histone deacetylation by histone deacetylase inhibitor (HDACis) activates a cell death program that allows tumor regression in mouse models of AMLs. We have used several models of PML-RARA-driven acute promyelocytic leukemias (APLs) to analyze the in vivo effects of valproic acid, a well-characterized HDACis. Valproic acid (VPA)-induced rapid tumor regression and sharply prolonged survival. However, discontinuation of treatment was associated to an immediate relapse. In vivo, as well as ex vivo, VPA-induced terminal granulocytic differentiation. Yet, despite full differentiation, leukemia-initiating cell (LIC) activity was actually enhanced by VPA treatment. In contrast to all-trans retinoic acid (ATRA) or arsenic, VPA did not degrade PML-RARA. However, in combination with ATRA, VPA synergized for PML-RARA degradation and LIC eradication in vivo. Our studies indicate that VPA triggers differentiation, but spares LIC activity, further uncouple differentiation from APL clearance and stress the importance of PML-RARA degradation in APL cure.


Subject(s)
Anticonvulsants/pharmacology , Cell Differentiation/drug effects , Cell Transformation, Neoplastic/drug effects , Leukemia, Promyelocytic, Acute/drug therapy , Leukemia, Promyelocytic, Acute/pathology , Valproic Acid/pharmacology , Acetylation/drug effects , Animals , Antineoplastic Agents/pharmacology , Flow Cytometry , Mice , Mice, Inbred C57BL , Mice, Nude , Neoplasm Recurrence, Local , Oncogene Proteins, Fusion/metabolism , Signal Transduction , Tretinoin/pharmacology , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
6.
Leukemia ; 25(5): 814-20, 2011 May.
Article in English | MEDLINE | ID: mdl-21331069

ABSTRACT

In acute promyelocytic leukemia (APL) the retinoic acid receptor alpha (RARα) becomes an oncogene through the fusion with several partners, mostly with promyelocytic leukemia protein (PML), all of which have in common the presence of a self-association domain. The new fusion proteins, therefore, differently from the wild-type RARα, which forms only heterodimers with retinoic X receptor alpha, are also able to homo-oligomerize. The presence of such a domain has been suggested to be crucial for the leukemogenic potential of the chimeric proteins found in APL blasts. Whether or not any self-association domain is sufficient to bestow a leukemogenic activity on RARα is still under investigation. In this work, we address this question using two different X-RARα chimeras, where X represents the coiled-coil domain of PML (CC-RARα) or the oligomerization portion of the yeast transcription factor GCN4 (GCN4-RARα). We demonstrate that in vitro both proteins have transforming potential, and recapitulate the main PML-RARα biological properties, but CC-RARα is uniquely able to disrupt PML nuclear bodies. Indeed, in vivo only the CC-RARα chimera induces efficiently APL in a murine transplantation model. Thus, the PML CC domain represents the minimal structural determinant indispensable to transform RARα into an oncogenic protein.


Subject(s)
Cell Transformation, Neoplastic , Leukemia, Promyelocytic, Acute/genetics , Nuclear Proteins/genetics , Receptors, Retinoic Acid/genetics , Recombinant Fusion Proteins/physiology , Transcription Factors/genetics , Tumor Suppressor Proteins/genetics , Animals , Blotting, Western , Chromatography, Gel , Fluorescent Antibody Technique , Hematopoietic Stem Cells/metabolism , Immunophenotyping , Immunoprecipitation , Leukemia, Promyelocytic, Acute/metabolism , Leukemia, Promyelocytic, Acute/pathology , Mice , Promyelocytic Leukemia Protein , Protein Multimerization , RNA, Messenger/genetics , Retinoic Acid Receptor alpha , Reverse Transcriptase Polymerase Chain Reaction
7.
Proc Natl Acad Sci U S A ; 99(3): 1359-64, 2002 Feb 05.
Article in English | MEDLINE | ID: mdl-11818541

ABSTRACT

The function of urokinase and its receptor is essential for cell migration in pathological conditions, as shown by the analysis of knockout mice phenotypes. How a protease of a fibrinolytic pathway can induce migration is not understood and no link between this protease and migration-promoting G protein-coupled receptors has been described. We now show that FPRL1/LXA4R, a G protein-coupled receptor for a number of polypeptides and for the endogenous lipoxin A4 (LXA4), is the link between urokinase-type plasminogen activator (uPA) and migration as it directly interacts with an activated, soluble, cleaved form of uPA receptor (uPAR) (D2D3(88-274)) to induce chemotaxis. In this article we show that (i) both uPAR and FPRL1/LXA4R are necessary for the chemotactic activity of uPA whereas FPRL1/LXA4R is sufficient to mediate D2D3(88-274)-induced cell migration. (ii) Inhibition or desensitization of FPRL1/LXA4R by antibodies or specific ligands specifically prevents chemotaxis induced by D2D3(88-274) in THP-1 cells and human peripheral blood monocytes. (iii) Desensitization of FPRL1/LXA4R prevents the activation of tyrosine kinase Hck induced by D2D3(88-274). (iv) D2D3(88-274) directly binds to FPRL1/LXA4R and is competed by two specific FPRL1/LXA4R agonists, the synthetic MMK-1 peptide and a stable analog of LXA4. Thus, a naturally produced cleaved form of uPAR is a unique endogenous chemotactic agonist for FPRL1/LXA4R receptor and its activity can be antagonized by specific ligands. These results provide the first direct link, to our knowledge, between the fibrinolytic machinery and the inflammatory response, demonstrating that uPA-derived peptide fragments can activate a specific chemotactic receptor.


Subject(s)
Lipoxins , Receptors, Cell Surface/metabolism , Receptors, Immunologic/physiology , Receptors, Lipoxin , Receptors, Peptide/physiology , Animals , Cell Line , Chemotaxis, Leukocyte/drug effects , Chemotaxis, Leukocyte/physiology , GTP-Binding Proteins/metabolism , Humans , Hydroxyeicosatetraenoic Acids/metabolism , Leukemia, Basophilic, Acute , Monocytes/physiology , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Peptides/pharmacology , Plasminogen Activators/metabolism , Protein-Tyrosine Kinases/metabolism , Rats , Receptors, Formyl Peptide , Receptors, Urokinase Plasminogen Activator , Tumor Cells, Cultured , Urokinase-Type Plasminogen Activator/metabolism
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