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1.
Blood ; 132(1): 67-77, 2018 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-29784639

RESUMO

Mutations in FMS-like tyrosine kinase 3 (FLT3), such as internal tandem duplications (ITDs), can be found in up to 23% of patients with acute myeloid leukemia (AML) and confer a poor prognosis. Current treatment options for FLT3(ITD)-positive AMLs include genotoxic therapy and FLT3 inhibitors (FLT3i's), which are rarely curative. PARP1 inhibitors (PARP1i's) have been successfully applied to induce synthetic lethality in tumors harboring BRCA1/2 mutations and displaying homologous recombination (HR) deficiency. We show here that inhibition of FLT3(ITD) activity by the FLT3i AC220 caused downregulation of DNA repair proteins BRCA1, BRCA2, PALB2, RAD51, and LIG4, resulting in inhibition of 2 major DNA double-strand break (DSB) repair pathways, HR, and nonhomologous end-joining. PARP1i, olaparib, and BMN673 caused accumulation of lethal DSBs and cell death in AC220-treated FLT3(ITD)-positive leukemia cells, thus mimicking synthetic lethality. Moreover, the combination of FLT3i and PARP1i eliminated FLT3(ITD)-positive quiescent and proliferating leukemia stem cells, as well as leukemic progenitors, from human and mouse leukemia samples. Notably, the combination of AC220 and BMN673 significantly delayed disease onset and effectively reduced leukemia-initiating cells in an FLT3(ITD)-positive primary AML xenograft mouse model. In conclusion, we postulate that FLT3i-induced deficiencies in DSB repair pathways sensitize FLT3(ITD)-positive AML cells to synthetic lethality triggered by PARP1i's. Therefore, FLT3(ITD) could be used as a precision medicine marker for identifying AML patients that may benefit from a therapeutic regimen combining FLT3 and PARP1i's.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Reparo do DNA/efeitos dos fármacos , Leucemia Mieloide Aguda , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/metabolismo , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Benzotiazóis/farmacologia , Linhagem Celular Tumoral , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Mutação , Compostos de Fenilureia/farmacologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Tirosina Quinase 3 Semelhante a fms/genética
2.
Blood ; 130(26): 2848-2859, 2017 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-29042365

RESUMO

Myeloproliferative neoplasms (MPNs) often carry JAK2(V617F), MPL(W515L), or CALR(del52) mutations. Current treatment options for MPNs include cytoreduction by hydroxyurea and JAK1/2 inhibition by ruxolitinib, both of which are not curative. We show here that cell lines expressing JAK2(V617F), MPL(W515L), or CALR(del52) accumulated reactive oxygen species-induced DNA double-strand breaks (DSBs) and were modestly sensitive to poly-ADP-ribose polymerase (PARP) inhibitors olaparib and BMN673. At the same time, primary MPN cell samples from individual patients displayed a high degree of variability in sensitivity to these drugs. Ruxolitinib inhibited 2 major DSB repair mechanisms, BRCA-mediated homologous recombination and DNA-dependent protein kinase-mediated nonhomologous end-joining, and, when combined with olaparib, caused abundant accumulation of toxic DSBs resulting in enhanced elimination of MPN primary cells, including the disease-initiating cells from the majority of patients. Moreover, the combination of BMN673, ruxolitinib, and hydroxyurea was highly effective in vivo against JAK2(V617F)+ murine MPN-like disease and also against JAK2(V617F)+, CALR(del52)+, and MPL(W515L)+ primary MPN xenografts. In conclusion, we postulate that ruxolitinib-induced deficiencies in DSB repair pathways sensitized MPN cells to synthetic lethality triggered by PARP inhibitors.


Assuntos
Reparo do DNA/efeitos dos fármacos , Transtornos Mieloproliferativos/tratamento farmacológico , Neoplasias/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Pirazóis/farmacologia , Animais , Calreticulina/genética , Linhagem Celular , Sinergismo Farmacológico , Xenoenxertos , Humanos , Janus Quinase 2/genética , Camundongos , Transtornos Mieloproliferativos/genética , Neoplasias/genética , Nitrilas , Ftalazinas/farmacologia , Piperazinas/farmacologia , Pirimidinas , Receptores de Trombopoetina/genética , Células Tumorais Cultivadas
3.
Blood ; 127(17): 2131-43, 2016 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-26864341

RESUMO

Leukemias expressing constitutively activated mutants of ABL1 tyrosine kinase (BCR-ABL1, TEL-ABL1, NUP214-ABL1) usually contain at least 1 normal ABL1 allele. Because oncogenic and normal ABL1 kinases may exert opposite effects on cell behavior, we examined the role of normal ABL1 in leukemias induced by oncogenic ABL1 kinases. BCR-ABL1-Abl1(-/-) cells generated highly aggressive chronic myeloid leukemia (CML)-blast phase-like disease in mice compared with less malignant CML-chronic phase-like disease from BCR-ABL1-Abl1(+/+) cells. Additionally, loss of ABL1 stimulated proliferation and expansion of BCR-ABL1 murine leukemia stem cells, arrested myeloid differentiation, inhibited genotoxic stress-induced apoptosis, and facilitated accumulation of chromosomal aberrations. Conversely, allosteric stimulation of ABL1 kinase activity enhanced the antileukemia effect of ABL1 tyrosine kinase inhibitors (imatinib and ponatinib) in human and murine leukemias expressing BCR-ABL1, TEL-ABL1, and NUP214-ABL1. Therefore, we postulate that normal ABL1 kinase behaves like a tumor suppressor and therapeutic target in leukemias expressing oncogenic forms of the kinase.


Assuntos
Crise Blástica/genética , Genes Supressores de Tumor , Genes abl , Leucemia Experimental/genética , Leucemia Mieloide de Fase Crônica/genética , Proteínas Oncogênicas v-abl/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Proteínas Proto-Oncogênicas c-abl/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Crise Blástica/tratamento farmacológico , Crise Blástica/enzimologia , Crise Blástica/patologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citostáticos/farmacologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Instabilidade Genômica , Humanos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Leucemia Experimental/tratamento farmacológico , Leucemia Experimental/enzimologia , Leucemia Experimental/patologia , Leucemia Mieloide de Fase Crônica/tratamento farmacológico , Leucemia Mieloide de Fase Crônica/enzimologia , Leucemia Mieloide de Fase Crônica/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/enzimologia , Proteínas Oncogênicas v-abl/antagonistas & inibidores , Proteínas Oncogênicas v-abl/genética , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/genética , Estresse Oxidativo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-abl/genética , Piridazinas/farmacologia , Piridazinas/uso terapêutico , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética
4.
Blood ; 118(4): 1062-8, 2011 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-21653319

RESUMO

Chronic myeloid leukemia chronic phase (CML-CP) CD34(+) cells contain numerous DNA double-strand breaks whose unfaithful repair may contribute to chromosomal instability and disease progression to blast phase (CML-BP). These phenomena are often associated with the appearance of imatinib-resistant BCR-ABL1 kinase mutants (eg, T315I) and overexpression of BCR-ABL1. Here we show that BCR-ABL1 (nonmutated and T315I mutant) promoted RAD51 recombinase-mediated unfaithful homeologous recombination repair (HomeoRR) in a dosage-dependent manner. BCR-ABL1 SH3 domain interacts with RAD51 proline-rich regions, resulting in direct phosphorylation of RAD51 on Y315 (pY315). RAD51(pY315) facilitates dissociation from the complex with BCR-ABL1 kinase, migrates to the nucleus, and enhances formation of the nuclear foci indicative of recombination sites. HomeoRR and RAD51 nuclear foci were strongly reduced by RAD51(Y315F) phosphorylation-less mutant. In addition, peptide aptamer mimicking RAD51(pY315) fragment, but not that with Y315F phosphorylation-less substitution, diminished RAD51 foci formation and inhibited HomeoRR in leukemia cells. In conclusion, we postulate that BCR-ABL1 kinase-mediated RAD51(pY315) promotes unfaithful HomeoRR in leukemia cells, which may contribute to accumulation of secondary chromosomal aberrations responsible for CML relapse and progression.


Assuntos
Reparo do DNA/fisiologia , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Rad51 Recombinase/genética , Animais , Western Blotting , Linhagem Celular Tumoral , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Camundongos , Reação em Cadeia da Polimerase , Rad51 Recombinase/metabolismo , Transfecção , Tirosina/metabolismo
5.
Cell Rep ; 23(11): 3127-3136, 2018 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-29898385

RESUMO

PARP inhibitors (PARPis) have been used to induce synthetic lethality in BRCA-deficient tumors in clinical trials with limited success. We hypothesized that RAD52-mediated DNA repair remains active in PARPi-treated BRCA-deficient tumor cells and that targeting RAD52 should enhance the synthetic lethal effect of PARPi. We show that RAD52 inhibitors (RAD52is) attenuated single-strand annealing (SSA) and residual homologous recombination (HR) in BRCA-deficient cells. Simultaneous targeting of PARP1 and RAD52 with inhibitors or dominant-negative mutants caused synergistic accumulation of DSBs and eradication of BRCA-deficient but not BRCA-proficient tumor cells. Remarkably, Parp1-/-;Rad52-/- mice are normal and display prolonged latency of BRCA1-deficient leukemia compared with Parp1-/- and Rad52-/- counterparts. Finally, PARPi+RAD52i exerted synergistic activity against BRCA1-deficient tumors in immunodeficient mice with minimal toxicity to normal cells and tissues. In conclusion, our data indicate that addition of RAD52i will improve therapeutic outcome of BRCA-deficient malignancies treated with PARPi.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Poli(ADP-Ribose) Polimerase-1/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Animais , Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Reparo do DNA/efeitos dos fármacos , Feminino , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Recombinação Homóloga/efeitos dos fármacos , Humanos , Mesilato de Imatinib/farmacologia , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/deficiência , Proteína Rad52 de Recombinação e Reparo de DNA/antagonistas & inibidores , Proteína Rad52 de Recombinação e Reparo de DNA/deficiência , Mutações Sintéticas Letais , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
6.
Mol Cancer Res ; 15(8): 967-972, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28634224

RESUMO

Burkitt lymphoma/leukemia cells carry t(8;14)(q24;q32) chromosomal translocation encoding IGH/MYC, which results in the constitutive expression of the MYC oncogene. Here, it is demonstrated that untreated and cytarabine (AraC)-treated IGH/MYC-positive Burkitt lymphoma cells accumulate a high number of potentially lethal DNA double-strand breaks (DSB) and display low levels of the BRCA2 tumor suppressor protein, which is a key element of homologous recombination (HR)-mediated DSB repair. BRCA2 deficiency in IGH/MYC-positive cells was associated with diminished HR activity and hypersensitivity to PARP1 inhibitors (olaparib, talazoparib) used alone or in combination with cytarabine in vitro Moreover, talazoparib exerted a therapeutic effect in NGS mice bearing primary Burkitt lymphoma xenografts. In conclusion, IGH/MYC-positive Burkitt lymphoma/leukemia cells have decreased BRCA2 and are sensitive to PARP1 inhibition alone or in combination with other chemotherapies.Implications: This study postulates that IGH/MYC-induced BRCA2 deficiency may predispose Burkitt lymphoma cells to synthetic lethality triggered by PARP1 inhibitors.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/15/8/967/F1.large.jpgMol Cancer Res; 15(8); 967-72. ©2017 AACR.


Assuntos
Proteína BRCA2/genética , Linfoma de Burkitt/tratamento farmacológico , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Animais , Proteína BRCA2/deficiência , Linfoma de Burkitt/genética , Citarabina/administração & dosagem , Reparo do DNA/efeitos dos fármacos , Genes myc/genética , Recombinação Homóloga/efeitos dos fármacos , Humanos , Camundongos , Ftalazinas/administração & dosagem , Piperazinas/administração & dosagem , Poli(ADP-Ribose) Polimerase-1/genética , Mutações Sintéticas Letais/genética , Translocação Genética/genética , Ensaios Antitumorais Modelo de Xenoenxerto
7.
J Clin Invest ; 127(6): 2392-2406, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28481221

RESUMO

Quiescent and proliferating leukemia cells accumulate highly lethal DNA double-strand breaks that are repaired by 2 major mechanisms: BRCA-dependent homologous recombination and DNA-dependent protein kinase-mediated (DNA-PK-mediated) nonhomologous end-joining, whereas DNA repair pathways mediated by poly(ADP)ribose polymerase 1 (PARP1) serve as backups. Here we have designed a personalized medicine approach called gene expression and mutation analysis (GEMA) to identify BRCA- and DNA-PK-deficient leukemias either directly, using reverse transcription-quantitative PCR, microarrays, and flow cytometry, or indirectly, by the presence of oncogenes such as BCR-ABL1. DNA-PK-deficient quiescent leukemia cells and BRCA/DNA-PK-deficient proliferating leukemia cells were sensitive to PARP1 inhibitors that were administered alone or in combination with current antileukemic drugs. In conclusion, GEMA-guided targeting of PARP1 resulted in dual cellular synthetic lethality in quiescent and proliferating immature leukemia cells, and is thus a potential approach to eradicate leukemia stem and progenitor cells that are responsible for initiation and manifestation of the disease. Further, an analysis of The Cancer Genome Atlas database indicated that this personalized medicine approach could also be applied to treat numerous solid tumors from individual patients.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células , Leucemia/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Cricetinae , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Genes BRCA1 , Genes BRCA2 , Genes Letais , Genes abl , Humanos , Leucemia/tratamento farmacológico , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células-Tronco Embrionárias Murinas/fisiologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Transcriptoma , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Cell Cycle ; 13(23): 3727-41, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25483082

RESUMO

BRCA1 tumor suppressor regulates crucial cellular processes involved in DNA damage repair and cell cycle control. We showed that expression of BCR-ABL1 correlates with decreased level of BRCA1 protein, which promoted aberrant mitoses and aneuploidy as well as altered DNA damage response. Using polysome profiling and luciferase-BRCA1 3'UTR reporter system here we demonstrate that downregulation of BRCA1 protein in CML is caused by inhibition of BRCA1 mRNA translation, but not by increased protein degradation or reduction of mRNA level and half-life. We investigated 2 mRNA-binding proteins - HuR and TIAR showing specificity to AU-Rich Element (ARE) sites in 3'UTR of mRNA. BCR-ABL1 promoted cytosolic localization of TIAR and HuR, their binding to BRCA1 mRNA and formation of the TIAR-HuR complex. HuR protein positively regulated BRCA1 mRNA stability and translation, conversely TIAR negatively regulated BRCA1 translation and was found localized predominantly in the cytosolic stress granules in CML cells. TIAR-dependent downregulation of BRCA1 protein level was a result of ER stress, which is activated in BCR-ABL1 expressing cells, as we previously shown. Silencing of TIAR in CML cells strongly elevated BRCA1 level. Altogether, we determined that TIAR-mediated repression of BRCA1 mRNA translation is responsible for downregulation of BRCA1 protein level in BCR-ABL1 -positive leukemia cells. This mechanism may contribute to genomic instability and provide justification for targeting PARP1 and/or RAD52 to induce synthetic lethality in "BRCAness" CML and BCR-ABL1 -positive ALL cells.


Assuntos
Proteína BRCA1/metabolismo , Regulação para Baixo/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteína BRCA1/genética , Linhagem Celular Tumoral , Proteínas de Fusão bcr-abl/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Camundongos , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética
10.
Cancer Res ; 71(16): 5381-6, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21693657

RESUMO

Chronic myeloid leukemia in chronic phase (CML-CP) cells that harbor oncogenic BCR-ABL1 and normal ABL1 allele often become resistant to the ABL1 kinase inhibitor imatinib. Here, we report that loss of the remaining normal ABL1 allele in these tumors, which results from cryptic interstitial deletion in 9q34 in patients who did not achieve a complete cytogenetic remission (CCyR) during treatment, engenders a novel unexpected mechanism of imatinib resistance. BCR-ABL1-positive Abl1(-/-) leukemia cells were refractory to imatinib as indicated by persistent BCR-ABL1-mediated tyrosine phosphorylation, lack of BCR-ABL1 protein degradation, increased cell survival, and clonogenic activity. Expression of ABL1 kinase, but not a kinase-dead mutant, restored the antileukemic effects of imatinib in ABL1-negative chronic myelogenous leukemia (CML) cells and in BCR-ABL1-positive Abl1(-/-) murine leukemia cells. The intracellular concentration of imatinib and expression of its transporters were not affected, although proteins involved in BCR-ABL1 degradation were downregulated in Abl1(-/-) cells. Furthermore, 12 genes associated with imatinib resistance were favorably deregulated in Abl1(-/-) leukemia. Taken together, our results indicate that loss of the normal ABL1 kinase may serve as a key prognostic factor that exerts major impact on CML treatment outcomes.


Assuntos
Alelos , Antineoplásicos/farmacologia , Genes abl , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Piperazinas/farmacologia , Pirimidinas/farmacologia , Animais , Benzamidas , Linhagem Celular Tumoral , Bandeamento Cromossômico , Hibridização Genômica Comparativa , Humanos , Mesilato de Imatinib , Hibridização in Situ Fluorescente , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Camundongos
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