RESUMO
Mitochondrial DNA encodes 13 proteins that comprise components of the respiratory chain that maintain oxidative phosphorylation. The replication of mitochondrial DNA is performed by the sole mitochondrial DNA polymerase γ. As acute myeloid leukemia (AML) cells and stem cells have an increased reliance on oxidative phosphorylation, we sought to evaluate polymerase γ inhibitors in AML. The thymidine dideoxynucleoside analog, alovudine, is an inhibitor of polymerase γ. In AML cells, alovudine depleted mitochondrial DNA, reduced mitochondrial encoded proteins, decreased basal oxygen consumption, and decreased cell proliferation and viability. To evaluate the effects of polymerase γ inhibition with alovudine in vivo, mice were xenografted with OCI-AML2 cells and then treated with alovudine. Systemic administration of alovudine reduced leukemic growth without evidence of toxicity and decreased levels of mitochondrial DNA in the leukemic cells. We also showed that alovudine increased the monocytic differentiation of AML cells. Genetic knockdown and other chemical inhibitors of polymerase γ also promoted AML differentiation, but the effects on AML differentiation were independent of reductions in oxidative phosphorylation or respiratory chain proteins. Thus, we have identified a novel mechanism by which mitochondria regulate AML fate and differentiation independent of oxidative phosphorylation. Moreover, we highlight polymerase γ inhibitors, such as alovudine, as novel therapeutic agents for AML.
Assuntos
Diferenciação Celular/efeitos dos fármacos , DNA Polimerase gama/antagonistas & inibidores , Didesoxinucleosídeos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Mitocôndrias/patologia , Monócitos/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Animais , Antivirais/farmacologia , Apoptose , Proliferação de Células , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Timidina/química , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We evaluated outcomes of 100 patients with high risk AML treated with Ida-FLAG induction as first-line therapy. 72 achieved remission with one cycle; 19 did not. High risk cytogenetics and TP53 mutations were associated with failure to achieve remission. In those reaching remission, allogeneic bone marrow transplantation was associated with better relapse-free and overall survival. Those not achieving remission with induction therapy were extremely unlikely to reach remission with further therapy and had a dismal prognosis. Exploratory molecular analysis confirmed persistence of the dominant genetic mutations identified at diagnosis. Ex vivo chemosensitivity did not demonstrate significant differences between responders and non-responders. Thus, Ida-FLAG induction has a high chance of inducing remission in patients with high risk AML. Those achieving remission require allogeneic transplantation to achieve cure; those not achieving remission rarely respond to salvage chemotherapy and have a dismal outcome. Alternatives to conventional chemotherapy must be considered in this group.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Quimioterapia de Indução , Leucemia Mieloide Aguda/tratamento farmacológico , Vidarabina/análogos & derivados , Adolescente , Adulto , Idoso , Citarabina/uso terapêutico , Feminino , Genes p53 , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Humanos , Idarubicina/uso terapêutico , Leucemia Mieloide Aguda/genética , Masculino , Pessoa de Meia-Idade , Mutação , Prognóstico , Indução de Remissão , Estudos Retrospectivos , Resultado do Tratamento , Vidarabina/uso terapêutico , Adulto JovemRESUMO
Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acute myeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2'3'-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2'3'-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase γ. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML.
Assuntos
DNA Mitocondrial/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Fosforilação Oxidativa , Fosfotransferases/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Replicação do DNA , Humanos , Camundongos SCID , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Núcleosídeo-Fosfato Quinase/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Zalcitabina/metabolismoRESUMO
Human mitochondrial DNA (mtDNA) is replicated by the mitochondrial DNA polymerase gamma (POLG). Using proximity dependent biotin labelling (BioID), we characterized the POLG interactome and identified new interaction partners involved in mtDNA maintenance, transcription, translation and protein quality control. We also identified interaction with the nuclear AAA+ ATPase Ruvbl2, suggesting mitochondrial localization for this protein. Ruvbl2 was detected in mitochondria-enriched fractions in leukemic cells. Additionally, transgenic overexpression of Ruvbl2 from an alternative translation initiation site resulted in mitochondrial co-localization. Overall, POLG interactome mapping identifies novel proteins which support mitochondrial biogenesis and a potential novel mitochondrial isoform of Ruvbl2.
Assuntos
Proteínas de Transporte/análise , DNA Helicases/análise , DNA Polimerase Dirigida por DNA/metabolismo , Mitocôndrias/química , Mapeamento de Interação de Proteínas , ATPases Associadas a Diversas Atividades Celulares , DNA Polimerase gama , HumanosRESUMO
Acute myeloid leukemia (AML) cells have high oxidative phosphorylation and mitochondrial mass and low respiratory chain spare reserve capacity. We reasoned that targeting the mitochondrial RNA polymerase (POLRMT), which indirectly controls oxidative phosphorylation, represents a therapeutic strategy for AML. POLRMT-knockdown OCI-AML2 cells exhibited decreased mitochondrial gene expression, decreased levels of assembled complex I, decreased levels of mitochondrially-encoded Cox-II and decreased oxidative phosphorylation. POLRMT-knockdown cells exhibited an increase in complex II of the electron transport chain, a complex comprised entirely of subunits encoded by nuclear genes, and POLRMT-knockdown cells were resistant to a complex II inhibitor theonyltrifluoroacetone. POLRMT-knockdown cells showed a prominent increase in cell death. Treatment of OCI-AML2 cells with 10-50 µM 2-C-methyladenosine (2-CM), a chain terminator of mitochondrial transcription, reduced mitochondrial gene expression and oxidative phosphorylation, and increased cell death in a concentration-dependent manner. Treatment of normal human hematopoietic cells with 2-CM at concentrations of up to 100 µMdid not alter clonogenic growth, suggesting a therapeutic window. In an OCI-AML2 xenograft model, treatment with 2-CM (70 mg/kg, i.p., daily) decreased the volume and mass of tumours to half that of vehicle controls. 2-CM did not cause toxicity to major organs. Overall, our results in a preclinical model contribute to the functional validation of the utility of targeting the mitochondrial RNA polymerase as a therapeutic strategy for AML.
Assuntos
Adenosina/análogos & derivados , Antineoplásicos/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Adenosina/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Relação Dose-Resposta a Droga , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Células K562 , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos SCID , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Terapia de Alvo Molecular , Fosforilação Oxidativa , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Carga Tumoral/efeitos dos fármacos , Células U937 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The highly effective anticancer agent doxorubicin (Dox) is a frontline drug used to treat a number of cancers. While Dox has a high level of activity against cancer cells, its clinical use is often complicated by dose-limiting cardiotoxicity. While this side effect has been linked to the drug's direct activity in the mitochondria of cardiac cells, recent studies have shown that these result primarily from downstream effects of nuclear DNA damage. Our lab has developed a mitochondrially targeted derivative of Dox that enables the selective study of toxicity generated by the presence of Dox in the mitochondria of human cells. We demonstrate that mitochondria-targeted doxorubicin (mtDox) lacks any direct nuclear effects in H9c2 rat cardiomyocytes, and that these cells are able to undergo mitochondrial biogenesis. This recovery response compensates for the mitotoxic effects of Dox and prevents cell death in cardiomyocytes. Furthermore, cardiac toxicity was only observed in Dox but not mtDox treated mice. This study supports the hypothesis that mitochondrial damage is not the main source of the cardiotoxic effects of Dox.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/toxicidade , Núcleo Celular/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Doxorrubicina/toxicidade , Mitocôndrias/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Animais , Antibióticos Antineoplásicos/química , Cardiotoxicidade/genética , Cardiotoxicidade/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/genética , Dano ao DNA/efeitos dos fármacos , Doxorrubicina/análogos & derivados , Sistemas de Liberação de Medicamentos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , RatosRESUMO
Mitochondria contain multiple copies of their own 16.6 kb circular genome. To explore the impact of mitochondrial DNA (mtDNA) damage on mitochondrial (mt) function and viability of AML cells, we screened a panel of DNA damaging chemotherapeutic agents to identify drugs that could damage mtDNA. We identified bleomycin as an agent that damaged mtDNA in AML cells at concentrations that induced cell death. Bleomycin also induced mtDNA damage in primary AML samples. Consistent with the observed mtDNA damage, bleomycin reduced mt mass and basal oxygen consumption in AML cells. We also demonstrated that the observed mtDNA damage was functionally important for bleomycin-induced cell death. Finally, bleomycin delayed tumor growth in xenograft mouse models of AML and anti-leukemic concentrations of the drug induced mtDNA damage in AML cells preferentially over normal lung tissue. Taken together, mtDNA-targeted therapy may be an effective strategy to target AML cells and bleomycin could be useful in the treatment of this disease.