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PLoS One ; 11(1): e0146100, 2016.
Article in English | MEDLINE | ID: mdl-26745281

ABSTRACT

The four members of the epidermal growth factor receptor (EGFR/ERBB) family form homo- and heterodimers which mediate ligand-specific regulation of many key cellular processes in normal and cancer tissues. While signaling through the EGFR has been extensively studied on the molecular level, signal transduction through ERBB3/ERBB4 heterodimers is less well understood. Here, we generated isogenic mouse Ba/F3 cells that express full-length and functional membrane-integrated ERBB3 and ERBB4 or ERBB4 alone, to serve as a defined cellular model for biological and phosphoproteomics analysis of ERBB3/ERBB4 signaling. ERBB3 co-expression significantly enhanced Ba/F3 cell proliferation upon neuregulin-1 (NRG1) treatment. For comprehensive signaling studies we performed quantitative mass spectrometry (MS) experiments to compare the basal ERBB3/ERBB4 cell phosphoproteome to NRG1 treatment of ERBB3/ERBB4 and ERBB4 cells. We employed a workflow comprising differential isotope labeling with mTRAQ reagents followed by chromatographic peptide separation and final phosphopeptide enrichment prior to MS analysis. Overall, we identified 9686 phosphorylation sites which could be confidently localized to specific residues. Statistical analysis of three replicate experiments revealed 492 phosphorylation sites which were significantly changed in NRG1-treated ERBB3/ERBB4 cells. Bioinformatics data analysis recapitulated regulation of mitogen-activated protein kinase and Akt pathways, but also indicated signaling links to cytoskeletal functions and nuclear biology. Comparative assessment of NRG1-stimulated ERBB4 Ba/F3 cells revealed that ERBB3 did not trigger defined signaling pathways but more broadly enhanced phosphoproteome regulation in cells expressing both receptors. In conclusion, our data provide the first global picture of ERBB3/ERBB4 signaling and provide numerous potential starting points for further mechanistic studies.


Subject(s)
B-Lymphocytes/metabolism , Phosphoproteins/genetics , Proteome/genetics , Receptor, ErbB-3/genetics , Receptor, ErbB-4/genetics , Signal Transduction , Amino Acid Sequence , Animals , B-Lymphocytes/cytology , B-Lymphocytes/drug effects , Cell Line , Gene Expression Regulation , Gene Regulatory Networks , Genetic Engineering , Humans , Mice , Molecular Sequence Data , Neuregulin-1/metabolism , Neuregulin-1/pharmacology , Phosphoproteins/metabolism , Phosphorylation , Protein Binding , Protein Interaction Mapping , Proteome/metabolism , Receptor, ErbB-3/metabolism , Receptor, ErbB-4/metabolism
3.
Haematologica ; 100(10): 1301-10, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26206799

ABSTRACT

T-cell acute lymphoblastic leukemia is caused by the accumulation of multiple oncogenic lesions, including chromosomal rearrangements and mutations. To determine the frequency and co-occurrence of mutations in T-cell acute lymphoblastic leukemia, we performed targeted re-sequencing of 115 genes across 155 diagnostic samples (44 adult and 111 childhood cases). NOTCH1 and CDKN2A/B were mutated/deleted in more than half of the cases, while an additional 37 genes were mutated/deleted in 4% to 20% of cases. We found that IL7R-JAK pathway genes were mutated in 27.7% of cases, with JAK3 mutations being the most frequent event in this group. Copy number variations were also detected, including deletions of CREBBP or CTCF and duplication of MYB. FLT3 mutations were rare, but a novel extracellular mutation in FLT3 was detected and confirmed to be transforming. Furthermore, we identified complex patterns of pairwise associations, including a significant association between mutations in IL7R-JAK genes and epigenetic regulators (WT1, PRC2, PHF6). Our analyses showed that IL7R-JAK genetic lesions did not confer adverse prognosis in T-cell acute lymphoblastic leukemia cases enrolled in the UK ALL2003 trial. Overall, these results identify interconnections between the T-cell acute lymphoblastic leukemia genome and disease biology, and suggest a potential clinical application for JAK inhibitors in a significant proportion of patients with T-cell acute lymphoblastic leukemia.


Subject(s)
Epigenesis, Genetic , Janus Kinases/genetics , Mutation , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Receptors, Interleukin-7/genetics , Adult , Child , Clonal Evolution/genetics , DNA Copy Number Variations , Female , Gene Expression Profiling , Gene Expression Regulation, Leukemic , Genetic Association Studies , Genetic Variation , High-Throughput Nucleotide Sequencing , Humans , Janus Kinases/metabolism , Male , Polymorphism, Single Nucleotide , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Prognosis , Receptors, Interleukin-7/metabolism , Reproducibility of Results , Signal Transduction
5.
Cancer Discov ; 4(4): 415-22, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24469108

ABSTRACT

UNLABELLED: We discovered a novel somatic gene fusion, CD74-NRG1, by transcriptome sequencing of 25 lung adenocarcinomas of never smokers. By screening 102 lung adenocarcinomas negative for known oncogenic alterations, we found four additional fusion-positive tumors, all of which were of the invasive mucinous subtype. Mechanistically, CD74-NRG1 leads to extracellular expression of the EGF-like domain of NRG1 III-ß3, thereby providing the ligand for ERBB2-ERBB3 receptor complexes. Accordingly, ERBB2 and ERBB3 expression was high in the index case, and expression of phospho-ERBB3 was specifically found in tumors bearing the fusion (P < 0.0001). Ectopic expression of CD74-NRG1 in lung cancer cell lines expressing ERBB2 and ERBB3 activated ERBB3 and the PI3K-AKT pathway, and led to increased colony formation in soft agar. Thus, CD74-NRG1 gene fusions are activating genomic alterations in invasive mucinous adenocarcinomas and may offer a therapeutic opportunity for a lung tumor subtype with, so far, no effective treatment. SIGNIFICANCE: CD74­NRG1 fusions may represent a therapeutic opportunity for invasive mucinous lung adenocarcinomas, a tumor with no effective treatment that frequently presents with multifocal unresectable disease.


Subject(s)
Adenocarcinoma, Mucinous/genetics , Adenocarcinoma/genetics , Antigens, Differentiation, B-Lymphocyte/genetics , Histocompatibility Antigens Class II/genetics , Lung Neoplasms/genetics , Neuregulin-1/genetics , Oncogene Proteins, Fusion/genetics , Adenocarcinoma/pathology , Adenocarcinoma of Lung , Adenocarcinoma, Mucinous/pathology , Adult , Aged , Aged, 80 and over , Animals , Base Sequence , Cell Line, Tumor , Female , Gene Expression Profiling , Humans , Lung Neoplasms/pathology , Male , Mice , Middle Aged , Molecular Sequence Data , NIH 3T3 Cells , Oncogene Proteins, Fusion/metabolism , Sequence Analysis, DNA , Signal Transduction/genetics
6.
Eur J Med Chem ; 63: 713-21, 2013 May.
Article in English | MEDLINE | ID: mdl-23567961

ABSTRACT

FLT3 and PDGFR tyrosine kinases are important targets for therapy of different types of leukemia. Several FLT3/PDGFR inhibitors are currently under clinical investigation for combination with standard therapy for treatment of acute myeloid leukemia (AML), however these agents only induce partial remission and development of resistance has been reported. In this work we describe the identification of potent and novel dual FLT3/PDGFR inhibitors that resulted from our efforts to screen a library of 25,607 small molecules against the FLT3 dependent cell line MOLM-13 and the PDGFR dependent cell line EOL-1. This effort led to the identification of five compounds that were confirmed to be active on additional FLT3 dependent cell lines (cellular EC50 values between 35 and 700 nM), while having no significant effect on 24 other tyrosine kinases.


Subject(s)
Protein Kinase Inhibitors/pharmacology , Receptor, Platelet-Derived Growth Factor alpha/antagonists & inhibitors , Receptor, Platelet-Derived Growth Factor beta/antagonists & inhibitors , Small Molecule Libraries/pharmacology , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , Blotting, Western , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Hypereosinophilic Syndrome/metabolism , Hypereosinophilic Syndrome/pathology , Leukemia, Monocytic, Acute/metabolism , Leukemia, Monocytic, Acute/pathology , Molecular Structure , Phosphorylation/drug effects , Protein Kinase Inhibitors/chemistry , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Receptor, Platelet-Derived Growth Factor beta/metabolism , Small Molecule Libraries/chemistry , Structure-Activity Relationship , fms-Like Tyrosine Kinase 3/metabolism
7.
Nat Genet ; 45(2): 186-90, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23263491

ABSTRACT

T-cell acute lymphoblastic leukemia (T-ALL) is caused by the cooperation of multiple oncogenic lesions. We used exome sequencing on 67 T-ALLs to gain insight into the mutational spectrum in these leukemias. We detected protein-altering mutations in 508 genes, with an average of 8.2 mutations in pediatric and 21.0 mutations in adult T-ALL. Using stringent filtering, we predict seven new oncogenic driver genes in T-ALL. We identify CNOT3 as a tumor suppressor mutated in 7 of 89 (7.9%) adult T-ALLs, and its knockdown causes tumors in a sensitized Drosophila melanogaster model. In addition, we identify mutations affecting the ribosomal proteins RPL5 and RPL10 in 12 of 122 (9.8%) pediatric T-ALLs, with recurrent alterations of Arg98 in RPL10. Yeast and lymphoid cells expressing the RPL10 Arg98Ser mutant showed a ribosome biogenesis defect. Our data provide insights into the mutational landscape of pediatric versus adult T-ALL and identify the ribosome as a potential oncogenic factor.


Subject(s)
Exome/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Ribosomal Proteins/genetics , Transcription Factors/genetics , Animals , Base Sequence , Drosophila melanogaster , High-Throughput Nucleotide Sequencing , Humans , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation/genetics , Polyribosomes/genetics , RNA Interference , Ribosomal Protein L10 , Saccharomyces cerevisiae , Sequence Alignment
8.
Expert Rev Hematol ; 5(4): 381-93, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22992233

ABSTRACT

Since the discovery of the BCR-ABL1 fusion gene in chronic myeloid leukemia, many more fusion genes resulting from chromosomal rearrangements have been identified and characterized. The study of these fusion genes has been extremely important for our understanding of the role of chromosomal rearrangements in leukemogenesis and in oncology in general. In chronic myeloid leukemia, or related myeloproliferative malignancies caused by the expression of oncogenic fusion kinases, tyrosine kinase inhibitors are now successfully used to treat these diseases. In acute myeloid leukemias, the presence of chromosomal rearrangements, oncogenic fusion genes and point mutations in key oncogenic drivers has important prognostic value and determines the choice of therapy. In this review, the authors provide an overview of the important fusion genes present in various myeloid malignancies and their importance for clinical practice.


Subject(s)
Chromosome Aberrations , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Oncogene Proteins, Fusion/genetics , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Oncogene Proteins, Fusion/metabolism , Protein Kinase Inhibitors/therapeutic use
10.
PLoS One ; 7(6): e38463, 2012.
Article in English | MEDLINE | ID: mdl-22675565

ABSTRACT

With the advent of whole-genome and whole-exome sequencing, high-quality catalogs of recurrently mutated cancer genes are becoming available for many cancer types. Increasing access to sequencing technology, including bench-top sequencers, provide the opportunity to re-sequence a limited set of cancer genes across a patient cohort with limited processing time. Here, we re-sequenced a set of cancer genes in T-cell acute lymphoblastic leukemia (T-ALL) using Nimblegen sequence capture coupled with Roche/454 technology. First, we investigated how a maximal sensitivity and specificity of mutation detection can be achieved through a benchmark study. We tested nine combinations of different mapping and variant-calling methods, varied the variant calling parameters, and compared the predicted mutations with a large independent validation set obtained by capillary re-sequencing. We found that the combination of two mapping algorithms, namely BWA-SW and SSAHA2, coupled with the variant calling algorithm Atlas-SNP2 yields the highest sensitivity (95%) and the highest specificity (93%). Next, we applied this analysis pipeline to identify mutations in a set of 58 cancer genes, in a panel of 18 T-ALL cell lines and 15 T-ALL patient samples. We confirmed mutations in known T-ALL drivers, including PHF6, NF1, FBXW7, NOTCH1, KRAS, NRAS, PIK3CA, and PTEN. Interestingly, we also found mutations in several cancer genes that had not been linked to T-ALL before, including JAK3. Finally, we re-sequenced a small set of 39 candidate genes and identified recurrent mutations in TET1, SPRY3 and SPRY4. In conclusion, we established an optimized analysis pipeline for Roche/454 data that can be applied to accurately detect gene mutations in cancer, which led to the identification of several new candidate T-ALL driver mutations.


Subject(s)
DNA Mutational Analysis/methods , Genes, Neoplasm/genetics , Mutation/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Base Sequence , Cell Line, Tumor , Clone Cells , Humans , Molecular Sequence Data , Neoplasm Proteins/genetics , Time Factors , Tumor Suppressor Proteins/genetics
11.
Haematologica ; 96(10): 1448-56, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21750091

ABSTRACT

BACKGROUND: The EVI1 gene (3q26) codes for a zinc finger transcription factor with important roles in both mammalian development and leukemogenesis. Over-expression of EVI1 through either 3q26 rearrangements, MLL fusions, or other unknown mechanisms confers a poor prognosis in acute myeloid leukemia. DESIGN AND METHODS: We analyzed the prevalence and prognostic impact of EVI1 over-expression in a series of 476 patients with acute myeloid leukemia, and investigated the epigenetic modifications of the EVI1 locus which could be involved in the transcriptional regulation of this gene. RESULTS: Our data provide further evidence that EVI1 over-expression is a poor prognostic marker in acute myeloid leukemia patients less than 65 years old. Moreover, we found that patients with no basal expression of EVI1 had a better prognosis than patients with expression/over-expression (P=0.036). We also showed that cell lines with over-expression of EVI1 had no DNA methylation in the promoter region of the EVI1 locus, and had marks of active histone modifications: H3 and H4 acetylation, and trimethylation of histone H3 lysine 4. Conversely, cell lines with no expression of EVI1 have DNA hypermethylation and are marked by repressive trimethylation of histone H3 lysine 27 at the EVI1 promoter. CONCLUSIONS: Our results identify EVI1 over-expression as a poor prognostic marker in a large, independent cohort of acute myeloid leukemia patients less than 65 years old, and show that the total absence of EVI1 expression has a prognostic impact on the outcome of such patients. Furthermore, we demonstrated for the first time that an aberrant epigenetic pattern involving DNA methylation, H3 and H4 acetylation, and trimethylation of histone H3 lysine 4 and histone H3 lysine 27 might play a role in the transcriptional regulation of EVI1 in acute myeloid leukemia. This study opens new avenues for a better understanding of the regulation of EVI1 expression at a transcriptional level.


Subject(s)
DNA-Binding Proteins/genetics , Down-Regulation/genetics , Epigenesis, Genetic , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/genetics , Proto-Oncogenes/genetics , Transcription Factors/genetics , Aged , Aged, 80 and over , Alternative Splicing , Cell Line, Tumor , Chromosomes, Human, Pair 3 , Female , Gene Expression Profiling , Gene Expression Regulation, Leukemic , Gene Rearrangement , Humans , Kaplan-Meier Estimate , Leukemia, Myeloid, Acute/mortality , MDS1 and EVI1 Complex Locus Protein , Male , Middle Aged , Prognosis
12.
Blood ; 117(15): 4056-64, 2011 Apr 14.
Article in English | MEDLINE | ID: mdl-21325169

ABSTRACT

The genetics of classical Hodgkin lymphoma (cHL) is poorly understood. The finding of a JAK2-involving t(4;9)(q21;p24) in 1 case of cHL prompted us to characterize this translocation on a molecular level and to determine the prevalence of JAK2 rearrangements in cHL. We showed that the t(4;9)(q21;p24) leads to a novel SEC31A-JAK2 fusion. Screening of 131 cHL cases identified 1 additional case with SEC31A-JAK2 and 2 additional cases with rearrangements involving JAK2. We demonstrated that SEC31A-JAK2 is oncogenic in vitro and acts as a constitutively activated tyrosine kinase that is sensitive to JAK inhibitors. In vivo, SEC31A-JAK2 was found to induce a T-lymphoblastic lymphoma or myeloid phenotype in a murine bone marrow transplantation model. Altogether, we identified SEC31A-JAK2 as a chromosomal aberration characteristic for cHL and provide evidence that JAK2 rearrangements occur in a minority of cHL cases. Given the proven oncogenic potential of this novel fusion, our studies provide new insights into the pathogenesis of cHL and indicate that in at least some cases, constitutive activation of the JAK/STAT pathway is caused by JAK2 rearrangements. The finding that SEC31A-JAK2 responds to JAK inhibitors indicates that patients with cHL and JAK2 rearrangements may benefit from targeted therapies.


Subject(s)
Gene Rearrangement/genetics , Hodgkin Disease/genetics , Janus Kinase 2/genetics , Oncogene Proteins, Fusion/genetics , Vesicular Transport Proteins/genetics , Adult , Aged, 80 and over , Animals , Bone Marrow Transplantation , Chromosomes, Human, Pair 4 , Chromosomes, Human, Pair 9 , Disease Models, Animal , Female , Genetic Predisposition to Disease/epidemiology , HEK293 Cells , Hodgkin Disease/epidemiology , Humans , Male , Mice , Middle Aged , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Prevalence , Protein-Tyrosine Kinases/metabolism , Translocation, Genetic , Young Adult
13.
Nat Genet ; 42(6): 530-5, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20473312

ABSTRACT

PTPN2 (protein tyrosine phosphatase non-receptor type 2, also known as TC-PTP) is a cytosolic tyrosine phosphatase that functions as a negative regulator of a variety of tyrosine kinases and other signaling proteins. In agreement with its role in the regulation of the immune system, PTPN2 was identified as a susceptibility locus for autoimmune diseases. In this work, we describe the identification of focal deletions of PTPN2 in human T-cell acute lymphoblastic leukemia (T-ALL). Deletion of PTPN2 was specifically found in T-ALLs with aberrant expression of the TLX1 transcription factor oncogene, including four cases also expressing the NUP214-ABL1 tyrosine kinase. Knockdown of PTPN2 increased the proliferation and cytokine sensitivity of T-ALL cells. In addition, PTPN2 was identified as a negative regulator of NUP214-ABL1 kinase activity. Our study provides genetic and functional evidence for a tumor suppressor role of PTPN2 and suggests that expression of PTPN2 may modulate response to treatment.


Subject(s)
Gene Deletion , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 2/genetics , Animals , Benzamides , Cell Line , Cell Line, Tumor , Chemokine CCL1/metabolism , Gene Knockdown Techniques , Homeodomain Proteins/genetics , Humans , Imatinib Mesylate , Interleukin-2/metabolism , Interleukin-7/metabolism , Leukemia, Experimental/genetics , Mice , Piperazines/therapeutic use , Proto-Oncogene Proteins/genetics , Pyrimidines/therapeutic use
14.
Haematologica ; 93(4): 533-42, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18322257

ABSTRACT

BACKGROUND: Activating NOTCH1 mutations are common in T-cell acute lymphoblastic leukemia. Inhibition of NOTCH1 signaling with gamma-secretase inhibitors causes cell cycle block, but only after treatment for several days. We further documented the effects of gamma-secretase inhibitor treatment on T-cell acute lymphoblastic leukemia cell lines and tested whether combining gamma-secretase inhibitors with other anti-cancer drugs offers a therapeutic advantage. DESIGN AND METHODS: The effect of gamma-secretase inhibitor treatment and combinations of gamma-secretase inhibitors with chemotherapy or glucocorticoids was assessed on T-cell acute lymphoblastic leukemia cell lines. We sequenced NOTCH1 in T-cell acute lymphoblastic leukemia cases with ABL1 fusions and tested combinations of gamma-secretase inhibitors and the ABL1 inhibitor imatinib in a T-cell acute lymphoblastic leukemia cell line. RESULTS: gamma-secretase inhibitor treatment for 5-7 days reversibly inhibited cell proliferation, caused cell cycle block in sensitive T-cell acute lymphoblastic leukemia cell lines, and caused differentiation of some T-cell acute lymphoblastic leukemia cell lines. Treatment for 14 days or longer was required to induce significant apoptosis. The cytotoxic effects of the chemotherapeutic agent vincristine were not significantly enhanced by addition of gamma-secretase inhibitors to T-cell acute lymphoblastic leukemia cell lines, but gamma-secretase inhibitor treatment sensitized cells to the effect of dexamethasone. NOTCH1 mutations were identified in all T-cell acute lymphoblastic leukemia patients with ABL1 fusions and in a T-cell acute lymphoblastic leukemia cell line expressing NUP214-ABL1. In this cell line, the anti-proliferative effect of imatinib was increased by pre-treatment with gamma-secretase inhibitors. CONCLUSIONS: Short-term treatment of T-cell acute lymphoblastic leukemia cell lines with gamma-secretase inhibitors had limited effects on cell proliferation and survival. Combinations of gamma-secretase inhibitors with other drugs may be required to obtain efficient therapeutic effects in T-cell acute lymphoblastic leukemia, and not all combinations may be useful.


Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Benzodiazepinones/pharmacology , Carbamates/pharmacology , Dipeptides/pharmacology , Enzyme Inhibitors/pharmacology , Leukemia-Lymphoma, Adult T-Cell/drug therapy , Neoplasm Proteins/antagonists & inhibitors , Receptor, Notch1/antagonists & inhibitors , Antineoplastic Agents/administration & dosage , Apoptosis/drug effects , Benzamides , Carbamates/administration & dosage , Cell Cycle/drug effects , Cell Division/drug effects , Cell Line, Tumor/drug effects , Cell Line, Tumor/enzymology , DNA, Neoplasm/genetics , Daunorubicin/administration & dosage , Daunorubicin/pharmacology , Dexamethasone/administration & dosage , Dexamethasone/pharmacology , Dipeptides/administration & dosage , Drug Screening Assays, Antitumor , Drug Synergism , Enzyme Inhibitors/administration & dosage , Humans , Imatinib Mesylate , In Vitro Techniques , Leukemia-Lymphoma, Adult T-Cell/genetics , Leukemia-Lymphoma, Adult T-Cell/pathology , Mutation , Neoplasm Proteins/genetics , Oncogene Proteins, Fusion/antagonists & inhibitors , Oncogene Proteins, Fusion/genetics , Piperazines/administration & dosage , Piperazines/pharmacology , Pyrimidines/administration & dosage , Pyrimidines/pharmacology , Receptor, Notch1/genetics , Sequence Analysis, DNA , Vincristine/administration & dosage , Vincristine/pharmacology
15.
Haematologica ; 93(1): 49-56, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18166785

ABSTRACT

BACKGROUND: Translocations involving region 5q31-32 (PDGFRB) have been reported in a variety of myeloproliferative diseases and are often associated with significant peripheral eosinophilia. We report an unusual case of a patient presenting with peripheral basophilia and systemic mastocytosis in whom cytogenetic analysis revealed a t(4;5)(q21.1;q31.3). DESIGN AND METHODS: We used molecular analyses to determine the role of PDGFRB in this case. The patient was treated with imatinib. RESULTS: Fluorescence in situ hybridization (FISH) documented a breakpoint in PDGFRB. In agreement with this, the patient responded very well to imatinib with resolution of clinical symptoms, basophilia, and mast cell disease. Molecular analyses revealed that PDGFRB, encoding an imatinib-sensitive tyrosine kinase, was fused to PRKG2. The fusion gene incorporates the first two exons of PRKG2 fused to the truncated exon 12 of PDGFRB, resulting in the disruption of its juxtamembrane domain. Functional studies confirmed that the activity and transforming properties of PRKG2-PDGFRbeta were dependent on the disruption of the auto-inhibitory juxtamembrane domain. CONCLUSIONS: Our results identify a second case of the PRKG2-PDGFRB fusion and confirm the unusual PDGFRB breakpoint associated with this fusion. This work also illustrates the use of imatinib for the treatment of specific cases of systemic mastocytosis.


Subject(s)
Gene Expression Regulation, Leukemic , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Mastocytosis, Systemic/drug therapy , Mastocytosis, Systemic/genetics , Oncogene Proteins, Fusion/genetics , Piperazines/pharmacology , Pyrimidines/pharmacology , Receptor, Platelet-Derived Growth Factor beta/chemistry , Animals , Benzamides , Bone Marrow/pathology , Cytogenetics , Humans , Imatinib Mesylate , Immunophenotyping , In Situ Hybridization, Fluorescence , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/complications , Male , Mastocytosis, Systemic/complications , Mice , Middle Aged
16.
J Clin Invest ; 117(12): 3696-707, 2007 Dec.
Article in English | MEDLINE | ID: mdl-18037989

ABSTRACT

Transgenic expression of the abnormal products of acute myeloid leukemia-associated (AML-associated) primary chromosomal translocations in hematopoietic stem/progenitor cells initiates leukemogenesis in mice, yet additional mutations are needed for leukemia development. We report here aberrant expression of PR domain containing 16 (PRDM16) in AML cells with either translocations of 1p36 or normal karyotype. These carried, respectively, relatively high prevalence of mutations in the TP53 tumor suppressor gene and in the nucleophosmin (NPM) gene, which regulates p53. Two protein isoforms are expressed from PRDM16, which differ in the presence or absence of the PR domain. Overexpression of the short isoform, sPRDM16, in mouse bone marrow induced AML with full penetrance, but only in the absence of p53. The mouse leukemias were characterized by multilineage cellular abnormalities and megakaryocyte dysplasia, a common feature of human AMLs with 1p36 translocations or NPM mutations. Overexpression of sPRDM16 increased the pool of HSCs in vivo, and in vitro blocked myeloid differentiation and prolonged progenitor life span. Loss of p53 augmented the effects of sPRDM16 on stem cell number and induced immortalization of progenitors. Thus, overexpression of sPRDM16 induces abnormal growth of stem cells and progenitors and cooperates with disruption of the p53 pathway in the induction of myeloid leukemia.


Subject(s)
Cell Transformation, Neoplastic/metabolism , DNA-Binding Proteins/biosynthesis , Hematopoietic Stem Cells/metabolism , Leukemia, Myeloid, Acute/metabolism , Neoplastic Stem Cells/metabolism , Transcription Factors/biosynthesis , Tumor Suppressor Protein p53 , Animals , Bone Marrow/metabolism , Bone Marrow/pathology , Cell Differentiation/genetics , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Cellular Senescence/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation, Leukemic , Hematopoietic Stem Cells/pathology , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Knockout , Neoplastic Stem Cells/pathology , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nucleophosmin , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Protein Structure, Tertiary/genetics , Transcription Factors/genetics , Translocation, Genetic , Tumor Suppressor Protein p53/genetics
17.
Cancer Genet Cytogenet ; 175(1): 65-8, 2007 May.
Article in English | MEDLINE | ID: mdl-17498561

ABSTRACT

The simultaneous occurrence of two different translocations affecting both alleles of the IGH gene has rarely been reported in multiple myeloma. In such a case, two different oncogenes might become transcriptionally deregulated. To investigate this hypothesis, we have characterized the plasma cell leukemia cell line SK-MM2 and a primary myeloma both carrying simultaneous IGH-FGFR3/MMSET and IGH-CCND1 fusions as shown by multicolor fluorescence in situ hybridization. Remarkably, quantitative real-time polymerase chain reaction demonstrated that only one of the oncogene loci was transcriptionally upregulated in both instances. Moreover, the upregulated oncogenes differed between both samples. Thus, biallelic IGH translocations might exert different pathogenetic effects in plasma cell disorders.


Subject(s)
Cyclins/genetics , Histone-Lysine N-Methyltransferase/genetics , Immunoglobulin Heavy Chains/genetics , Multiple Myeloma/genetics , Receptor, Fibroblast Growth Factor, Type 3/genetics , Repressor Proteins/genetics , Translocation, Genetic , Alleles , Cell Line , Chromosome Banding , Chromosome Painting , Cyclin D , Gene Expression Regulation, Neoplastic , Humans , Karyotyping , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction
18.
Nat Genet ; 39(5): 593-5, 2007 May.
Article in English | MEDLINE | ID: mdl-17435759

ABSTRACT

We identified a duplication of the MYB oncogene in 8.4% of individuals with T cell acute lymphoblastic leukemia (T-ALL) and in five T-ALL cell lines. The duplication is associated with a threefold increase in MYB expression, and knockdown of MYB expression initiates T cell differentiation. Our results identify duplication of MYB as an oncogenic event and suggest that MYB could be a therapeutic target in human T-ALL.


Subject(s)
Cell Differentiation/genetics , Gene Duplication , Genes, myb/genetics , Leukemia-Lymphoma, Adult T-Cell/genetics , T-Lymphocytes/pathology , Cell Differentiation/immunology , Cell Line, Tumor , Chromosomes, Artificial/genetics , Flow Cytometry , Gene Dosage , Gene Expression Regulation, Neoplastic/genetics , Genetic Testing , Humans , In Situ Hybridization, Fluorescence , Karyotyping , Mutation/genetics , Nucleic Acid Hybridization/genetics , RNA, Small Interfering/genetics , Statistics, Nonparametric
19.
Haematologica ; 92(1): 27-34, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17229632

ABSTRACT

BACKGROUND AND OBJECTIVES: Activated tyrosine kinases are implicated in the pathogenesis of chronic and acute leukemia, and represent attractive targets for therapy. Sorafenib (BAY43-9006, Nexavar) is a small molecule B-RAF inhibitor that is used for the treatment of renal cell carcinoma, and has been shown to have activity against receptor tyrosine kinases from the platelet-derived growth factor receptor (PDGFR) and vascular endothelial growth factor receptor (VEGFR) families. We investigated the efficacy of sorafenib at inhibiting mutants of the receptor tyrosine kinases PDGFRbeta, KIT, and FLT3, which are implicated in the pathogenesis of myeloid malignancies. DESIGN AND METHODS: We tested the effect of sorafenib on the proliferation of hematopoietic cells transformed by ETV6-PDGFRbeta, FLT3 with an internal tandem duplication or D835Y point mutation, and the KIT(D816V) mutant. The direct effect of sorafenib on the activity of these kinases and their downstream signaling was tested using phospho-specific antibodies. RESULTS: We show that sorafenib is a potent inhibitor of ETV6-PDGFRbeta and FLT3 mutants, including some of the mutants that confer resistance to PKC412 and other FLT3 inhibitors. Sorafenib induced a cell cycle block and apoptosis in the acute myeloid leukemia cell lines MV4-11 and MOLM-13, both expressing FLT3 with an internal tandem duplication, whereas no effect was observed on four other acute myeloid leukemia cell lines. The imatinib-resistant KIT(D816V) mutant, associated with systemic mastocytosis, was found to be resistant to sorafenib. INTERPRETATION AND CONCLUSIONS: These results warrant further clinical studies of sorafenib for the treatment of myeloid malignancies expressing activated forms of PDGFRbeta and FLT3.


Subject(s)
Antineoplastic Agents/pharmacology , Benzenesulfonates/pharmacology , Drug Resistance, Neoplasm , Pyridines/pharmacology , Receptor, Platelet-Derived Growth Factor beta/biosynthesis , Receptor, Platelet-Derived Growth Factor beta/genetics , fms-Like Tyrosine Kinase 3/biosynthesis , fms-Like Tyrosine Kinase 3/genetics , Apoptosis , Cell Cycle , Cell Line , Cell Line, Transformed , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Hematopoietic Stem Cells/metabolism , Humans , Mutation , Niacinamide/analogs & derivatives , Phenylurea Compounds , Sorafenib
20.
Cancer Genet Cytogenet ; 172(1): 70-3, 2007 Jan 01.
Article in English | MEDLINE | ID: mdl-17175383

ABSTRACT

Rearrangement of the BCL6 gene is found in follicular lymphomas and in diffuse large B cell lymphomas of follicular center cell origin. The breakpoints cluster mainly in a region spanning the first noncoding exon of the gene (the major breakpoint region). A second breakpoint cluster has also been identified upstream of the first BCL6 noncoding exon (the alternative breakpoint region [ABR]). To date, eight different rearrangements involving the ABR have been reported. Here, we describe a novel rearrangement involving a t(2;3)(p11;q27) translocation that affects the ABR in an unusual combination with the IGK locus.


Subject(s)
Chromosomes, Human, Pair 2/genetics , Chromosomes, Human, Pair 3/genetics , Lymphoma, Follicular/genetics , Translocation, Genetic/genetics , Aged , Gene Rearrangement , Humans , Immunoglobulins/genetics , Lymphoma, Follicular/classification , Male , Polymerase Chain Reaction , Proto-Oncogene Proteins c-bcl-6/genetics
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