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1.
PLoS Genet ; 11(4): e1005144, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25919613

RESUMO

The hallmark of Philadelphia chromosome positive (Ph(+)) leukemia is the BCR/ABL kinase, which is successfully targeted by selective ATP competitors. However, inhibition of BCR/ABL alone is unable to eradicate Ph(+) leukemia. The t(9;22) is a reciprocal translocation which encodes not only for the der22 (Philadelphia chromosome) related BCR/ABL, but also for der9 related ABL/BCR fusion proteins, which can be detected in 65% of patients with chronic myeloid leukemia (CML) and 100% of patients with Ph+ acute lymphatic leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96(ABL/BCR) for the pathogenesis of Ph(+) ALL. The co-expression of p96(ABL/BCR) enhanced the kinase activity and as a consequence, the transformation potential of p185(BCR/ABL). Targeting p96(ABL/BCR) by RNAi inhibited growth of Ph(+) ALL cell lines and Ph(+) ALL patient-derived long-term cultures (PD-LTCs). Our in vitro and in vivo stem cell studies further revealed a functional hierarchy of p96(ABL/BCR) and p185(BCR/AB)L in hematopoietic stem cells. Co-expression of p96(ABL/BCR) abolished the capacity of p185(BCR/ABL) to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96(ABL/BCR) for the pathogenesis of Ph(+) ALL.


Assuntos
Proteínas de Fusão bcr-abl/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação Genética/genética , Linhagem Celular Tumoral , Cromossomos Humanos Par 22/genética , Cromossomos Humanos Par 9/genética , Proteínas de Fusão bcr-abl/biossíntese , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/patologia , Humanos , Cromossomo Filadélfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia
2.
Int J Oncol ; 34(6): 1521-31, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19424569

RESUMO

Acute myeloid leukemia (AML) is caused by the cooperation between class I, mostly mutated receptor tyrosine kinases (RTK), and class II oncoproteins, chimeric transcription factors derived from chromosomal translocations. The blasts of 80-90% of AML-patients are positive for the RTK c-Kit. In about 50% of the 'core binding factor' (CBF)-AMLs, c-Kit harbors additional gain-of-function mutations, whereas the t(15;17)-positive AML-M3 (100% c-Kit positive) presents virtually no c-Kit mutations. In all c-Kit-positive AMLs, c-Kit signaling is activated. Here, we investigated the role of c-Kit in the determination of the leukemic phenotype in a model of CBF-AML and AML-M3. We studied the role of aberrant c-Kit signaling on normal and leukemic murine stem cells by RNA interference, the c-Kit-inhibitor Imatinib and a constitutively-activated c-Kit mutant in well-established stem cell assays. Effects of the AML-M3-associated PML/RARalpha and the AML-1/ETO as a model for CBF-AML on c-Kit signaling were investigated in trans-activation assays on the Kit promoter. The contribution of activated c-Kit signaling to PML/RARalpha- and AML-1/ETO-induced leukemogenesis was investigated in a murine transduction/transplantation leukemia model. We report that: i) the inhibition of c-Kit impaired the stem cell capacity of PML/RARalpha- and AML-1/ETO-positive HSC; ii) PML/RARalpha was able to activate the c-Kit promoter; iii) constitutively-activated c-Kit increased the stem cell capacity of HSC; and iv) constitutively-activated c-Kit increased the leukemogenic potential of PML/RARalpha- and AML-1/ETO-positive HSC. Our data provide evidence that c-Kit does not have to be mutated to contribute to the determination of the leukemic phenotype in AML.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Proteínas de Fusão Oncogênica/fisiologia , Proteínas Proto-Oncogênicas c-kit/fisiologia , Transdução de Sinais/fisiologia , Animais , Western Blotting , Ciclo Celular , Cromossomos Humanos Par 15/genética , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 8/genética , Feminino , Humanos , Leucemia Promielocítica Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína 1 Parceira de Translocação de RUNX1 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Translocação Genética
3.
Genes Cancer ; 7(1-2): 36-46, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27014420

RESUMO

Targeting BCR/ABL with Tyrosine kinase inhibitors (TKIs) is a proven concept for the treatment of Philadelphia chromosome-positive (Ph+) leukemias but the "gatekeeper" mutation T315I confers resistance against all approved TKIs, with the only exception of ponatinib, a multi-targeted kinase inhibitor. Besides resistance to TKIs, T315I also confers additional features to the leukemogenic potential of BCR/ABL, involving endogenous BCR. Therefore we studied the role of BCR on BCR/ABL mutants lacking functional domains indispensable for the oncogenic activity of BCR/ABL. We used the factor independent growth of murine myeloid progenitor 32D cells and the transformation of Rat-1 fibroblasts both mediated by BCR/ABL. Here we report that T315I restores the capacity to mediate factor-independent growth and transformation potential of loss-of-function mutants of BCR/ABL. Targeting endogenous Bcr abrogated the capacity of oligomerization deficient mutant of BCR/ABL-T315I to mediate factor independent growth of 32D cells and strongly reduced their transformation potential in Rat-1 cells, as well as led to the up-regulation of mitogen activated protein kinase (MAPK) pathway. Our data show that the T315I restores the capacity of loss-of-function mutants to transform cells which is dependent on the transphosphorylation of endogenous Bcr, which becomes a putative therapeutic target to overcome resistance by T315I.

4.
Genes Cancer ; 5(11-12): 378-92, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25568664

RESUMO

Acute myeloid leukemia (AML) is characterized by an aberrant self-renewal of hematopoietic stem cells (HSC) and a block in differentiation. The major therapeutic challenge is the characterization of the leukemic stem cell as a target for the eradication of the disease. Until now the biology of AML-associated fusion proteins (AAFPs), such as the t(15;17)-PML/RARα, t(8;21)-RUNX1/RUNX1T1 and t(6;9)-DEK/NUP214, all able to induce AML in mice, was investigated in different models and genetic backgrounds, not directly comparable to each other. To avoid the bias of different techniques and models we expressed these three AML-inducing oncogenes in an identical genetic background and compared their influence on the HSC compartment in vitro and in vivo. These AAFPs exerted differential effects on HSCs and PML/RARα, similar to DEK/NUP214, induced a leukemic phenotype from a small subpopulation of HSCs with a surface marker pattern of long-term HSC and characterized by activated STAT3 and 5. In contrast the established AML occurred from mature populations in the bone marrow. The activation of STAT5 by PML/RARα and DEK/NUP214 was confirmed in t(15;17)(PML/RARα) and t(6;9)(DEK/NUP214)-positive patients as compared to normal CD34+ cells. The activation of STAT5 was reduced upon the exposure to Arsenic which was accompanied by apoptosis in both PML/RARα- and DEK/NUP214-positive leukemic cells. These findings indicate that in AML the activation of STATs plays a decisive role in the biology of the leukemic stem cell. Furthermore we establish exposure to arsenic as a novel concept for the treatment of this high risk t(6;9)-positive AML.

5.
Cancer Res ; 74(18): 5244-55, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25082812

RESUMO

Nonsteroidal anti-inflammatory drugs such as sulindac inhibit Wnt signaling, which is critical to maintain cancer stem cell-like cells (CSC), but they also suppress the activity of 5-lipoxygenase (5-LO) at clinically feasible concentrations. Recently, 5-LO was shown to be critical to maintain CSC in a model of chronic myeloid leukemia. For these reasons, we hypothesized that 5-LO may offer a therapeutic target to improve the management of acute myeloid leukemia (AML), an aggressive disease driven by CSCs. Pharmacologic and genetic approaches were used to evaluate the effects of 5-LO blockade in a PML/RARα-positive model of AML. As CSC models, we used Sca-1(+)/lin(-) murine hematopoietic stem and progenitor cells (HSPC), which were retrovirally transduced with PML/RARα. We found that pharmacologic inhibition of 5-LO interfered strongly with the aberrant stem cell capacity of PML/RARα-expressing HSPCs. Through small-molecule inhibitor studies and genetic disruption of 5-LO, we also found that Wnt and CSC inhibition is mediated by the enzymatically inactive form of 5-LO, which hinders nuclear translocation of ß-catenin. Overall, our findings revealed that 5-LO inhibitors also inhibit Wnt signaling, not due to the interruption of 5-LO-mediated lipid signaling but rather due to the generation of a catalytically inactive form of 5-LO, which assumes a new function. Given the evidence that CSCs mediate AML relapse after remission, eradication of CSCs in this setting by 5-LO inhibition may offer a new clinical approach for immediate evaluation in patients with AML. Cancer Res; 74(18); 5244-55. ©2014 AACR.


Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Inibidores de Lipoxigenase/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Leucemia Mieloide Aguda/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos , Transdução de Sinais , Transfecção
6.
Cell Cycle ; 11(17): 3219-26, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22895185

RESUMO

Acute myeloid leukemia (AML) is a highly malignant disease that is not curable in the majority of patients. Numerous non-random genetic abnormalities are known, among which several translocations such as PLZF/RARα or AML1/ETO are known to aberrantly recruit histone deacetylases. Deacetylase inhibitors (DACi) are promising drugs leading to growth inhibition, cell cycle arrest, premature senescence and apoptosis in malignant cells. It is believed that DACi may have clinical efficacy by eradicating the most primitive population of leukemic stem and progenitor cells, possibly by interfering with self-renewal. The aim of the study was to investigate the effects of DACi on leukemic stem and progenitor cells using murine transduction-transplantation models of hematopoietic cells harboring the leukemia-associated fusion proteins (LAFP) PLZF/RARα or a truncated AML1/ETO protein (AML1/ETO exon 9). We show that the self-renewal and short-term repopulation capacity of AML1/ETO- or PLZF/RARα-expressing Sca1+/lin- stem and progenitor cells are profoundly inhibited by clinically applicable concentrations of the DACi dacinostat and vorinostat. To further investigate the mechanisms underlying these effects, we examined the impact of DACi on the transcription factor c-MYC and the Polycomb group protein BMI1, which are induced by LAFP and involved in leukemic transformation. In AML1/ETO or PLZF/RARα-positive 32D cells, DACi-mediated antiproliferative effects were associated with downregulation of BMI1 and c-MYC protein levels. Similar effects were demonstrated in primary samples of cytogenetically defined high-risk AML patients. In conclusion, DACi may be effective as maintenance therapy by negatively interfering with signaling pathways that control survival and proliferation of leukemic stem and progenitor cells.


Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Animais , Western Blotting , Ensaio de Unidades Formadoras de Colônias , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Primers do DNA/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Camundongos , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vorinostat
7.
PLoS One ; 6(7): e22540, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21811629

RESUMO

Chromosomal translocations can lead to the formation of chimeric genes encoding fusion proteins such as PML/RARα, PLZF/RARα, and AML-1/ETO, which are able to induce and maintain acute myeloid leukemia (AML). One key mechanism in leukemogenesis is increased self renewal of leukemic stem cells via aberrant activation of the Wnt signaling pathway. Either X-RAR, PML/RARα and PLZF/RARα or AML-1/ETO activate Wnt signaling by upregulating γ-catenin and ß-catenin. In a prospective study, a lower risk of leukemia was observed with aspirin use, which is consistent with numerous studies reporting an inverse association of aspirin with other cancers. Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID), where the effects on AML risk was FAB subtype-specific. To better investigate whether NSAID treatment is effective, we used Sulindac Sulfide in X-RARα-positive progenitor cell models. Sulindac Sulfide (SSi) is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling. We found that SSi downregulated both ß-catenin and γ-catenin in X-RARα-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARα-positive HSCs. The data presented herein show that SSi inhibits the leukemic cell growth as well as hematopoietic progenitors cells (HPCs) expressing PML/RARα, and it indicates that Sulindac is a valid molecular therapeutic approach that should be further validated using in vivo leukemia models and in clinical settings.


Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas de Fusão Oncogênica/metabolismo , Sulindaco/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Camundongos , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Sulindaco/farmacologia , Fatores de Tempo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , gama Catenina/metabolismo
8.
PLoS One ; 4(10): e7661, 2009 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-19876398

RESUMO

BACKGROUND: t(9;22) is a balanced translocation, and the chromosome 22 breakpoints (Philadelphia chromosome--Ph+) determine formation of different fusion genes that are associated with either Ph+ acute lymphatic leukemia (Ph+ ALL) or chronic myeloid leukemia (CML). The "minor" breakpoint in Ph+ ALL encodes p185(BCR/ABL) from der22 and p96(ABL/BCR) from der9. The "major" breakpoint in CML encodes p210(BCR/ABL) and p40(ABL/BCR). Herein, we investigated the leukemogenic potential of the der9-associated p96(ABL/BCR) and p40(ABL/BCR) fusion proteins and their roles in the lineage commitment of hematopoietic stem cells in comparison to BCR/ABL. METHODOLOGY: All t(9;22) derived proteins were retrovirally expressed in murine hematopoietic stem cells (SL cells) and human umbilical cord blood cells (UCBC). Stem cell potential was determined by replating efficiency, colony forming--spleen and competitive repopulating assays. The leukemic potential of the ABL/BCR fusion proteins was assessed by in a transduction/transplantation model. Effects on the lineage commitment and differentiation were investigated by culturing the cells under conditions driving either myeloid or lymphoid commitment. Expression of key factors of the B-cell differentiation and components of the preB-cell receptor were determined by qRT-PCR. PRINCIPAL FINDINGS: Both p96(ABL/BCR) and p40(ABL/BCR) increased proliferation of early progenitors and the short term stem cell capacity of SL-cells and exhibited own leukemogenic potential. Interestingly, BCR/ABL gave origin exclusively to a myeloid phenotype independently from the culture conditions whereas p96(ABL/BCR) and to a minor extent p40(ABL/BCR) forced the B-cell commitment of SL-cells and UCBC. CONCLUSIONS/SIGNIFICANCE: Our here presented data establish the reciprocal ABL/BCR fusion proteins as second oncogenes encoded by the t(9;22) in addition to BCR/ABL and suggest that ABL/BCR contribute to the determination of the leukemic phenotype through their influence on the lineage commitment.


Assuntos
Linfócitos B/citologia , Cromossomos Humanos Par 22 , Cromossomos Humanos Par 9 , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Translocação Genética , Animais , Diferenciação Celular , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Retroviridae/genética , Células-Tronco/citologia , Células U937
9.
J Antimicrob Chemother ; 54(1): 232-5, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15192049

RESUMO

OBJECTIVES: The enterococcal surface protein gene, esp, is a major putative pathogenicity marker in clinical isolates of Enterococcus faecium and Enterococcus faecalis. This study demonstrates in vitro conjugative transfer of the esp gene among E. faecium and E. faecalis. MATERIALS AND METHODS: Enterococcal isolates from clinical samples, positive for esp, were mated on filters with enterococcal recipients. Transconjugants were checked for transfer of antibiotic resistance determinants and co-mobilization of the esp gene. They were also characterized by PCR and plasmid profiling/PFGE typing including Southern hybridizations with labelled esp probes. Transfer as triggered by excision was tested using Taqman PCR. RESULTS: Two of five E. faecalis and five of nine E. faecium transferred antibiotic resistance determinants into a recipient. Of the transconjugants analysed by PCR for acquisition of esp, only isolates from two E. faecalis and a single E. faecium mating were positive. In the donor strains, the esp gene was located on the chromosome. Molecular analysis revealed a plasmid localization of esp in the E. faecium transconjugant and chromosome-to-chromosome transfer in E. faecalis. CONCLUSION: The esp gene is transferable by conjugation among enterococcal isolates.


Assuntos
Conjugação Genética/genética , Enterococcus faecalis/genética , Enterococcus faecium/genética , Fatores de Virulência/genética , Primers do DNA , DNA Bacteriano/genética , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecium/efeitos dos fármacos , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Plasmídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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