RESUMO
Azacitidine/venetoclax is an active regimen in patients with newly diagnosed AML. However, primary or secondary resistance to azacitidine/venetoclax is an area of unmet need and overexpression of MCL-1 is suggested to be a potential resistance mechanism. Pevonedistat inhibits MCL-1 through activation of NOXA, and pevonedistat/azacitidine has previously shown activity in AML. To assess the tolerability and efficacy of adding pevonedistat to azacitidine/venetoclax in relapsed/refractory AML, we conducted a phase I multicenter openlabel study in 16 adults with relapsed/refractory AML. Patients were treated with azacitidine, venetoclax along with pevonedistat intravenously on days 1, 3 and 5 of each 28-day cycle at 10, 15 or 20 mg/m2 in successive cohorts in the dose escalation phase. The impact of treatment on protein neddylation as well as expression of pro-apoptotic BCL2 family members was assessed. The recommended phase II dose of pevonedistat was 20 mg/m2. Grade 3 or higher adverse events included neutropenia (31%), thrombocytopenia (13%), febrile neutropenia (19%), anemia (19%), hypertension (19%) and sepsis (19%). The overall response rate was 46.7% for the whole cohort including complete remission (CR) in 5 of 7 (71.4%) patients who were naïve to the hypomethylating agent/venetoclax. No measurable residual disease (MRD) was detected in 80.0% of the patients who achieved CR. The median time to best response was 50 (range: 23 - 77) days. Four patients were bridged to allogeneic stem cell transplantation. The combination of azacitidine, venetoclax and pevonedistat is safe and shows encouraging preliminary activity in patients with relapsed/refractory AML. (NCT04172844).
RESUMO
OBJECTIVE: Poly(ADP-ribose) polymerase (PARP) inhibitors have shown substantial activity in homologous recombination- (HR-) deficient ovarian cancer and are undergoing testing in other HR-deficient tumors. For reasons that are incompletely understood, not all patients with HR-deficient cancers respond to these agents. Preclinical studies have demonstrated that changes in alternative DNA repair pathways affect PARP inhibitor (PARPi) sensitivity in ovarian cancer models. This has not previously been assessed in the clinical setting. METHODS: Clonogenic and plasmid-based HR repair assays were performed to compare BRCA1-mutant COV362 ovarian cancer cells with or without 53BP1 gene deletion. Archival biopsies from ovarian cancer patients in the phase I, open-label clinical trial of PARPi ABT-767 were stained for PARP1, RAD51, 53BP1 and multiple components of the nonhomologous end-joining (NHEJ) DNA repair pathway. Modified histochemistry- (H-) scores were determined for each repair protein in each sample. HRD score was determined from tumor DNA. RESULTS: 53BP1 deletion increased HR in BRCA1-mutant COV362 cells and decreased PARPi sensitivity in vitro. In 36 women with relapsed ovarian cancer, responses to the PARPi ABT-767 were observed exclusively in cancers with HR deficiency. In this subset, 7 of 18 patients (39%) had objective responses. The actual HRD score did not further correlate with change from baseline tumor volume (râ¯=â¯0.050; pâ¯=â¯0.87). However, in the HR-deficient subset, decreased 53BP1 H-score was associated with decreased antitumor efficacy of ABT-767 (râ¯=â¯-0.69, pâ¯=â¯0.004). CONCLUSION: Differences in complementary repair pathways, particularly 53BP1, correlate with PARPi response of HR-deficient ovarian cancers.
Assuntos
Benzamidas/administração & dosagem , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Sulfonamidas/administração & dosagem , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Reparo do DNA , Resistencia a Medicamentos Antineoplásicos , Feminino , Genes BRCA1 , Genes BRCA2 , Recombinação Homóloga , Humanos , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/biossíntese , Poli(ADP-Ribose) Polimerase-1/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/biossíntese , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiênciaRESUMO
Germline mutations in SPRTN cause Ruijs-Aalfs syndrome (RJALS), a disorder characterized by genome instability, progeria and early onset hepatocellular carcinoma. Spartan, the protein encoded by SPRTN, is a nuclear metalloprotease that is involved in the repair of DNA-protein crosslinks (DPCs). Although Sprtn hypomorphic mice recapitulate key progeroid phenotypes of RJALS, whether this model expressing low amounts of Spartan is prone to DPC repair defects and spontaneous tumors is unknown. Here, we showed that the livers of Sprtn hypomorphic mice accumulate DPCs containing Topoisomerase 1 covalently linked to DNA. Furthermore, these mice exhibited DNA damage, aneuploidy and spontaneous tumorigenesis in the liver. Collectively, these findings provide evidence that partial loss of Spartan impairs DPC repair and tumor suppression.
Assuntos
Carcinogênese/genética , Carcinoma Hepatocelular/genética , Proteínas Cromossômicas não Histona/deficiência , DNA Topoisomerases Tipo I/genética , Neoplasias Hepáticas/genética , Progéria/genética , Aneuploidia , Animais , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proteínas Cromossômicas não Histona/genética , Adutos de DNA/genética , Adutos de DNA/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Knockout , Progéria/metabolismo , Progéria/patologia , Proteólise , SíndromeRESUMO
The mammalian target of rapamycin (mTOR), a kinase that regulates proliferation and apoptosis, has been extensively evaluated as a therapeutic target in multiple malignancies. Rapamycin analogs, which partially inhibit mTOR complex 1 (mTORC1), exhibit immunosuppressive and limited antitumor activity, but sometimes activate survival pathways through feedback mechanisms involving mTORC2. Thus, attention has turned to agents targeting both mTOR complexes by binding the mTOR active site. Here we show that disruption of either mTOR-containing complex is toxic to acute lymphocytic leukemia (ALL) cells and identify 2 previously unrecognized pathways leading to this cell death. Inhibition of mTORC1-mediated 4EBP1 phosphorylation leads to decreased expression of c-MYC and subsequent upregulation of the proapoptotic BCL2 family member PUMA, whereas inhibition of mTORC2 results in nuclear factor-κB-mediated expression of the Early Growth Response 1 (EGR1) gene, which encodes a transcription factor that binds and transactivates the proapoptotic BCL2L11 locus encoding BIM. Importantly, 1 or both pathways contribute to death of malignant lymphoid cells after treatment with dual mTORC1/mTORC2 inhibitors. Collectively, these observations not only provide new insight into the survival roles of mTOR in lymphoid malignancies, but also identify alterations that potentially modulate the action of mTOR dual inhibitors in ALL.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Inibidores Enzimáticos/farmacologia , NF-kappa B/metabolismo , Fosfoproteínas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
A number of established and investigational anticancer drugs slow the religation step of DNA topoisomerase I (topo I). These agents induce cytotoxicity by stabilizing topo I-DNA covalent complexes, which in turn interact with advancing replication forks or transcription complexes to generate lethal lesions. Despite the importance of topo I-DNA covalent complexes, it has been difficult to detect these lesions within intact cells and tumors. Here, we report development of a monoclonal antibody that specifically recognizes covalent topo I-DNA complexes, but not free topo I or DNA, by immunoblotting, immunofluorescence or flow cytometry. Utilizing this antibody, we demonstrate readily detectable topo I-DNA covalent complexes after treatment with camptothecins, indenoisoquinolines and cisplatin but not nucleoside analogues. Topotecan-induced topo I-DNA complexes peak at 15-30 min after drug addition and then decrease, whereas indotecan-induced complexes persist for at least 4 h. Interestingly, simultaneous staining for covalent topo I-DNA complexes, phospho-H2AX and Rad51 suggests that topotecan-induced DNA double-strand breaks occur at sites distinct from stabilized topo I-DNA covalent complexes. These studies not only provide new insight into the action of topo I-directed agents, but also illustrate a strategy that can be applied to study additional topoisomerases and their inhibitors in vitro and in vivo.
Assuntos
Anticorpos Monoclonais/biossíntese , Antineoplásicos Fitogênicos/farmacologia , DNA Topoisomerases Tipo I/genética , DNA/genética , Regulação Neoplásica da Expressão Gênica , Inibidores da Topoisomerase I/farmacologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Apoptose/efeitos dos fármacos , Benzodioxóis/farmacologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/metabolismo , Células HCT116 , Histonas/genética , Histonas/metabolismo , Humanos , Isoquinolinas/farmacologia , Células K562 , Camundongos , Dados de Sequência Molecular , Ligação Proteica/efeitos dos fármacos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Alinhamento de Sequência , Relação Estrutura-Atividade , Topotecan/farmacologiaRESUMO
OBJECTIVE: Poly(ADP-ribose) polymerase (PARP) inhibitors have yielded encouraging responses in high-grade serous ovarian carcinomas (HGSOCs), but the optimal treatment setting remains unknown. We assessed the effect of niraparib on HGSOC patient-derived xenograft (PDX) models as well as the relationship between certain markers of homologous recombination (HR) status, including BRCA1/2 mutations and formation of RAD51 foci after DNA damage, and response of these PDXs to niraparib in vivo. METHODS: Massively parallel sequencing was performed on HGSOCs to identify mutations contributing to HR deficiency. HR pathway integrity was assessed using fluorescence microscopy-based RAD51 focus formation assays. Effects of niraparib (MK-4827) on treatment-naïve PDX tumor growth as monotherapy, in combination with carboplatin/paclitaxel, and as maintenance therapy were assessed by transabdominal ultrasound. Niraparib responses were correlated with changes in levels of poly(ADP-ribose), PARP1, and repair proteins by western blotting. RESULTS: Five PDX models were evaluated in vivo. Tumor regressions were induced by single-agent niraparib in one of two PDX models with deleterious BRCA2 mutations and in a PDX with RAD51C promoter methylation. Diminished formation of RAD51 foci failed to predict response, but Artemis loss was associated with resistance. Niraparib generally failed to enhance responses to carboplatin/paclitaxel chemotherapy, but maintenance niraparib therapy delayed progression in a BRCA2-deficient PDX. CONCLUSIONS: Mutations in HR genes are neither necessary nor sufficient to predict response to niraparib. Assessment of repair status through multiple complementary assays is needed to guide PARP inhibitor therapy, design future clinical trials and identify ovarian cancer patients most likely to benefit from PARP inhibition.
Assuntos
Recombinação Homóloga , Indazóis/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Piperidinas/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Feminino , Genes BRCA2 , Humanos , Neoplasias Ovarianas/genética , Regiões Promotoras GenéticasRESUMO
The topoisomerase (topo) I-DNA covalent complex represents an attractive target for developing diagnostic antibodies to measure responsiveness to drugs. We report a new antigen, peptide , and four murine monoclonal antibodies raised against that exhibit excellent specificity for recognition of in comparison to structurally similar peptides by enzyme-linked immunosorbent assays. Although topo I-DNA complex detection was not achieved in cellular samples by these new antibodies, a new strategy for antigen design is reported.
Assuntos
Anticorpos Monoclonais/química , Antígenos/química , DNA Topoisomerases Tipo I/química , DNA/química , Nucleotídeos/química , Peptídeos/química , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Antígenos/imunologia , Linhagem Celular Tumoral , DNA Topoisomerases Tipo I/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Estrutura Molecular , Nucleotídeos/síntese química , Peptídeos/síntese químicaRESUMO
Recombinant human tumor necrosis factor-α-related apoptosis inducing ligand (TRAIL), agonistic monoclonal antibodies to TRAIL receptors, and small molecule TRAIL receptor agonists are in various stages of preclinical and early phase clinical testing as potential anticancer drugs. Accordingly, there is substantial interest in understanding factors that affect sensitivity to these agents. In the present study we observed that the poly(ADP-ribose) polymerase (PARP) inhibitors olaparib and veliparib sensitize the myeloid leukemia cell lines ML-1 and K562, the ovarian cancer line PEO1, non-small cell lung cancer line A549, and a majority of clinical AML isolates, but not normal marrow, to TRAIL. Further analysis demonstrated that PARP inhibitor treatment results in activation of the FAS and TNFRSF10B (death receptor 5 (DR5)) promoters, increased Fas and DR5 mRNA, and elevated cell surface expression of these receptors in sensitized cells. Chromatin immunoprecipitation demonstrated enhanced binding of the transcription factor Sp1 to the TNFRSF10B promoter in the presence of PARP inhibitor. Knockdown of PARP1 or PARP2 (but not PARP3 and PARP4) not only increased expression of Fas and DR5 at the mRNA and protein level, but also recapitulated the sensitizing effects of the PARP inhibition. Conversely, Sp1 knockdown diminished the PARP inhibitor effects. In view of the fact that TRAIL is part of the armamentarium of natural killer cells, these observations identify a new facet of PARP inhibitor action while simultaneously providing the mechanistic underpinnings of a novel therapeutic combination that warrants further investigation.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Receptor fas/biossíntese , Humanos , Células K562 , Neoplasias/genética , Neoplasias/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Elementos de Resposta , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Receptor fas/genéticaRESUMO
Signaling through the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, which is aberrantly activated in >50% of carcinomas, inhibits apoptosis and contributes to drug resistance. Accordingly, several Akt inhibitors are currently undergoing preclinical or early clinical testing. To examine the effect of Akt inhibition on the activity of multiple widely used classes of antineoplastic agents, human cancer cell lines were treated with the Akt inhibitor A-443654 [(2S)-1-(1H-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan-2-amine; ATP-competitive] or MK-2206 (8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3-one;dihydrochloride; allosteric inhibitor) or with small interfering RNA (siRNA) targeting phosphoinositide-dependent kinase 1 (PDK1) along with cisplatin, melphalan, camptothecin, or etoposide and assayed for colony formation. Surprisingly different results were observed when Akt inhibitors were combined with different drugs. Synergistic effects were observed in multiple cell lines independent of PI3K pathway status when A-443654 or MK-2206 was combined with the DNA cross-linking agents cisplatin or melphalan. In contrast, effects of the Akt inhibitors in combination with camptothecin or etoposide were more complicated. In HCT116 and DLD1 cells, which harbor activating PI3KCA mutations, A-443654 over a broad concentration range enhanced the effects of camptothecin or etoposide. In contrast, in cell lines lacking activating PI3KCA mutations, partial inhibition of Akt signaling synergized with camptothecin or etoposide, but higher A-443654 or MK-2206 concentrations (>80% inhibition of Akt signaling) or PDK1 siRNA antagonized the topoisomerase poisons by diminishing DNA synthesis, a process that contributes to effective DNA damage and killing by these agents. These results indicate that the effects of combining inhibitors of the PI3K/Akt pathway with certain classes of chemotherapeutic agents might be more complicated than previously recognized.
Assuntos
Antineoplásicos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Indazóis/farmacologia , Indóis/farmacologia , Venenos/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Inibidores da Topoisomerase II/farmacologia , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Sinergismo Farmacológico , Células HCT116 , Compostos Heterocíclicos com 3 Anéis/metabolismo , Humanos , Indazóis/metabolismo , Indóis/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Venenos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Inibidores da Topoisomerase I/metabolismo , Inibidores da Topoisomerase I/farmacologia , Inibidores da Topoisomerase II/metabolismoRESUMO
The mammalian target of rapamycin (mTOR) plays crucial roles in proliferative and antiapoptotic signaling in lymphoid malignancies. Rapamycin analogs, which are allosteric mTOR complex 1 (mTORC1) inhibitors, are active in mantle cell lymphoma and other lymphoid neoplasms, but responses are usually partial and short-lived. In the present study we compared the effects of rapamycin with the dual mTORC1/mTORC2 inhibitor OSI-027 in cell lines and clinical samples representing divers lymphoid malignancies. In contrast to rapamycin, OSI-027 markedly diminished proliferation and induced apoptosis in a variety of lymphoid cell lines and clinical samples, including specimens of B-cell acute lymphocytic leukemia (ALL), mantle cell lymphoma, marginal zone lymphoma and Sezary syndrome. Additional analysis demonstrated that OSI-027-induced apoptosis depended on transcriptional activation of the PUMA and BIM genes. Overexpression of Bcl-2, which neutralizes Puma and Bim, or loss of procaspase 9 diminished OSI-027-induced apoptosis in vitro. Moreover, OSI-027 inhibited phosphorylation of mTORC1 and mTORC2 substrates, up-regulated Puma, and induced regressions in Jeko xenografts. Collectively, these results not only identify a pathway that is critical for the cytotoxicity of dual mTORC1/mTORC2 inhibitors, but also suggest that simultaneously targeting mTORC1 and mTORC2 might be an effective anti-lymphoma strategy in vivo.
Assuntos
Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Imidazóis/farmacologia , Linfoma/patologia , Proteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Triazinas/farmacologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Western Blotting , Proliferação de Células/efeitos dos fármacos , Humanos , Imunoprecipitação , Imunossupressores/farmacologia , Linfoma/tratamento farmacológico , Linfoma/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos , Fosforilação/efeitos dos fármacos , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
Novel combinations targeting new molecular vulnerabilities are needed to improve the outcome of patients with acute myeloid leukemia. We recently identified WEE1 kinase as a novel target in leukemias. To identify genes that are synthetically lethal with WEE1 inhibition, we performed a short interfering RNA screen directed against cell cycle and DNA repair genes during concurrent treatment with the WEE1 inhibitor MK1775. CHK1 and ATR, genes encoding two replication checkpoint kinases, were among the genes whose silencing enhanced the effects of WEE1 inhibition most, whereas CDK2 short interfering RNA antagonized MK1775 effects. Building on this observation, we examined the impact of combining MK1775 with selective small molecule inhibitors of CHK1, ATR and cyclin-dependent kinases. The CHK1 inhibitor MK8776 sensitized acute myeloid leukemia cell lines and primary leukemia specimens to MK1775 ex vivo, whereas smaller effects were observed with the MK1775/MK8776 combination in normal myeloid progenitors. The ATR inhibitor VE-821 likewise enhanced the antiproliferative effects of MK1775, whereas the cyclin-dependent kinase inhibitor roscovitine antagonized MK1775. Further studies showed that MK8776 enhanced MK1775-mediated activation of the ATR/CHK1 pathway in acute leukemia cell lines and ex vivo. These results indicate that combined cell cycle checkpoint interference with MK1775/MK8776 warrants further investigation as a potential treatment for acute myeloid leukemia.
Assuntos
Proteínas de Ciclo Celular/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/genética , Proteínas Tirosina Quinases/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Pirimidinonas , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais , Ensaio Tumoral de Célula-TroncoRESUMO
Although farnesyltransferase inhibitors have shown promising activity in relapsed lymphoma and sporadic activity in acute myelogenous leukemia, their mechanism of cytotoxicity is incompletely understood, making development of predictive biomarkers difficult. In the present study, we examined the action of tipifarnib in human acute myelogenous leukemia cell lines and clinical samples. In contrast to the Ras/MEK/ERK pathway-mediated Bim upregulation that is responsible for tipifarnib-induced killing of malignant lymphoid cells, inhibition of Rheb-induced mTOR signaling followed by dose-dependent upregulation of Bax and Puma occurred in acute myelogenous leukemia cell lines undergoing tipifarnib-induced apoptosis. Similar Bax and Puma upregulation occurred in serial bone marrow samples harvested from a subset of acute myelogenous leukemia patients during tipifarnib treatment. Expression of FTI-resistant Rheb M184L, like knockdown of Bax or Puma, diminished tipifarnib-induced killing. Further analysis demonstrated that increased Bax and Puma levels reflect protein stabilization rather than increased gene expression. In U937 cells selected for tipifarnib resistance, neither inhibition of signaling downstream of Rheb nor Bax and Puma stabilization occurred. Collectively, these results not only identify a pathway downstream from Rheb that contributes to tipifarnib cytotoxicity in human acute myelogenous leukemia cells, but also demonstrate that FTI-induced killing of lymphoid versus myeloid cells reflects distinct biochemical mechanisms downstream of different farnesylated substrates. (ClinicalTrials.gov identifier NCT00602771).
Assuntos
Antineoplásicos/farmacologia , Farnesiltranstransferase/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neuropeptídeos/metabolismo , Quinolonas/farmacologia , Proteína X Associada a bcl-2/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Farnesiltranstransferase/metabolismo , Humanos , Prenilação/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Células U937RESUMO
BH3 mimetics, including the BCL2/BCLXL/BCLw inhibitor navitoclax and MCL1 inhibitors S64315 and tapotoclax, have undergone clinical testing for a variety of neoplasms. Because of toxicities, including thrombocytopenia after BCLXL inhibition as well as hematopoietic, hepatic and possible cardiac toxicities after MCL1 inhibition, there is substantial interest in finding agents that can safely sensitize neoplastic cells to these BH3 mimetics. Building on the observation that BH3 mimetic monotherapy induces AMP kinase (AMPK) activation in multiple acute leukemia cell lines, we report that the AMPK inhibitors (AMPKis) dorsomorphin and BAY-3827 sensitize these cells to navitoclax or MCL1 inhibitors. Cell fractionation and phosphoproteomic analyses suggest that sensitization by dorsomorphin involves dephosphorylation of the proapoptotic BCL2 family member BAD at Ser75 and Ser99, leading BAD to translocate to mitochondria and inhibit BCLXL. Consistent with these results, BAD knockout or mutation to BAD S75E/S99E abolishes the sensitizing effects of dorsomorphin. Conversely, dorsomorphin synergizes with navitoclax or the MCL1 inhibitor S63845 to induce cell death in primary acute leukemia samples ex vivo and increases the antitumor effects of navitoclax or S63845 in several xenograft models in vivo with little or no increase in toxicity in normal tissues. These results suggest that AMPK inhibition can sensitize acute leukemia to multiple BH3 mimetics, potentially allowing administration of lower doses while inducing similar antineoplastic effects.
Assuntos
Proteínas Quinases Ativadas por AMP , Compostos de Anilina , Proteína de Sequência 1 de Leucemia de Células Mieloides , Pirimidinas , Sulfonamidas , Proteína bcl-X , Humanos , Animais , Compostos de Anilina/farmacologia , Sulfonamidas/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Camundongos , Proteína bcl-X/metabolismo , Proteína bcl-X/antagonistas & inibidores , Linhagem Celular Tumoral , Pirimidinas/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Pirazóis/farmacologia , Proteína de Morte Celular Associada a bcl/metabolismo , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Leucemia/tratamento farmacológico , Leucemia/patologia , Leucemia/metabolismo , Fosforilação/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Sinergismo FarmacológicoRESUMO
Poly(ADP-ribose) polymerase-1 (PARP1) plays critical roles in the regulation of DNA repair. Accordingly, small molecule inhibitors of PARP are being developed as agents that could modulate the activity of genotoxic chemotherapy, such as topoisomerase I poisons. In this study we evaluated the ability of the PARP inhibitor veliparib to enhance the cytotoxicity of the topoisomerase I poisons topotecan and camptothecin (CPT). Veliparib increased the cell cycle and cytotoxic effects of topotecan in multiple cell line models. Importantly, this sensitization occurred at veliparib concentrations far below those required to substantially inhibit poly(ADP-ribose) polymer synthesis and at least an order of magnitude lower than those involved in selective killing of homologous recombination-deficient cells. Further studies demonstrated that veliparib enhanced the effects of CPT in wild-type mouse embryonic fibroblasts (MEFs) but not Parp1(-/-) MEFs, confirming that PARP1 is the critical target for this sensitization. Importantly, parental and Parp1(-/-) MEFs had indistinguishable CPT sensitivities, ruling out models in which PARP1 catalytic activity plays a role in protecting cells from topoisomerase I poisons. To the contrary, cells were sensitized to CPT in a veliparib-independent manner upon transfection with PARP1 E988K, which lacks catalytic activity, or the isolated PARP1 DNA binding domain. These results are consistent with a model in which small molecule inhibitors convert PARP1 into a protein that potentiates the effects of topoisomerase I poisons by binding to damaged DNA and preventing its normal repair.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , DNA Topoisomerases Tipo I , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Embrião de Mamíferos/enzimologia , Embrião de Mamíferos/patologia , Fibroblastos/enzimologia , Fibroblastos/patologia , Humanos , Camundongos , Camundongos Knockout , Neoplasias/genética , Neoplasias/patologia , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Inibidores da Topoisomerase I/farmacologia , Topotecan/farmacologiaRESUMO
How BAK and BAX induce mitochondrial outer membrane (MOM) permeabilization (MOMP) during apoptosis is incompletely understood. Here we have used molecular dynamics simulations, surface plasmon resonance, and assays for membrane permeabilization in vitro and in vivo to assess the structure and function of selected BAK subdomains and their derivatives. Results of these studies demonstrate that BAK helical regions α5 and α6 bind the MOM lipid cardiolipin. While individual peptides corresponding to these helical regions lack the full biological activity of BAK, tandem peptides corresponding to α4-α5, α5-α6, or α6-α7/8 can localize exogenous proteins to mitochondria, permeabilize liposomes composed of MOM lipids, and cause MOMP in the absence of the remainder of the BAK protein. Importantly, the ability of these tandem helices to induce MOMP under cell-free conditions is diminished by mutations that disrupt the U-shaped helix-turn-helix structure of the tandem peptides or decrease their lipid binding. Likewise, BAK-induced apoptosis in intact cells is diminished by CLS1 gene interruption, which decreases mitochondrial cardiolipin content, or by BAK mutations that disrupt the U-shaped tandem peptide structure or diminish lipid binding. Collectively, these results suggest that BAK structural rearrangements during apoptosis might mobilize helices involved in specific protein-lipid interactions that are critical for MOMP.
Assuntos
Cardiolipinas , Citocromos c , Citocromos c/metabolismo , Cardiolipinas/metabolismo , Proteína X Associada a bcl-2/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Apoptose , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismoRESUMO
BACKGROUND: Despite recent approval of several new agents, relapsed acute lymphoblastic leukemia (ALL) remains challenging to treat. Sapanisertib (MLN0128/TAK-228) is an oral TORC1/2 inhibitor that exhibited preclinical activity against ALL. METHODS: We conducted a single-arm multi-center Phase II study of sapanisertib monotherapy (3 mg orally daily of the milled formulation for 21 days every 28 days) in patients with ALL through the Experimental Therapeutics Clinical Trials Network (NCI-9775). RESULTS: Sixteen patients, 15 of whom were previously treated (median 3 prior lines of therapy), were enrolled. Major grade 3-4 non-hematologic toxicities included mucositis (3 patients) and hyperglycemia (2 patients) as well as hepatic failure, seizures, confusion, pneumonitis, and anorexia (1 patient each). Grade >2 hematological toxicity included leukopenia (3), lymphopenia (2), thrombocytopenia, and neutropenia (1). The best response was stable disease in 2 patients (12.5%), while only 3 patients (19%) were able to proceed to Cycle 2. Pharmacokinetic analysis demonstrated drug exposures similar to those observed in solid tumor patients. Immunoblotting in serially collected samples indicated limited impact of treatment on phosphorylation of mTOR pathway substrates such as 4EBP1, S6, and AKT. CONCLUSION: In summary, single-agent sapanisertib had a good safety profile but limited target inhibition or efficacy in ALL as a single agent. This trial was registered at ClinicalTrials.gov as NCT02484430.
Assuntos
Benzoxazóis , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológicoRESUMO
Although treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) is known to protect a subset of cells from induction of apoptosis by death ligands such as Fas ligand and tumor necrosis factor-alpha-related apoptosis-inducing ligand, the mechanism of this protection is unknown. This study demonstrated that protection in short term apoptosis assays and long term proliferation assays was maximal when Jurkat or HL-60 human leukemia cells were treated with 2-5 nm PMA. Immunoblotting demonstrated that multiple PKC isoforms, including PKCalpha, PKCbeta, PKCepsilon, and PKC, translocated from the cytosol to a membrane-bound fraction at these PMA concentrations. When the ability of short hairpin RNA (shRNA) constructs that specifically down-regulated each of these isoforms was examined, PKCbeta shRNA uniquely reversed PMA-induced protection against cell death. The PKCbeta-selective small molecule inhibitor enzastaurin had a similar effect. Although mass spectrometry suggested that Fas is phosphorylated on a number of serines and threonines, mutation of these sites individually or collectively had no effect on Fas-mediated death signaling or PMA protection. Further experiments demonstrated that PMA diminished ligand-induced cell surface accumulation of Fas and DR5, and PKCbeta shRNA or enzastaurin reversed this effect. Moreover, enzastaurin sensitized a variety of human tumor cell lines and clinical acute myelogenous leukemia isolates, which express abundant PKCbeta, to tumor necrosis factor-alpha related apoptosis-inducing ligand-induced death in the absence of PMA. Collectively, these results identify a specific PKC isoform that modulates death receptor-mediated cytotoxicity as well as a small molecule inhibitor that mitigates the inhibitory effects of PKC activation on ligand-induced death receptor trafficking and cell death.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Indóis/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptor fas/metabolismo , Carcinógenos/farmacologia , Ativadores de Enzimas/farmacologia , Proteína Ligante Fas/farmacologia , Células HL-60 , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Células Jurkat , Fosforilação/efeitos dos fármacos , Proteína Quinase C beta , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND: In preclinical studies the heat shock protein 90 (Hsp90) inhibitor tanespimycin induced down-regulation of checkpoint kinase 1 (Chk1) and other client proteins as well as increased sensitivity of acute leukemia cells to cytarabine. We report here the results of a phase I and pharmacological study of the cytarabine + tanespimycin combination in adults with recurrent or refractory acute leukemia. DESIGN AND METHODS: Patients received cytarabine 400 mg/m(2)/day continuously for 5 days and tanespimycin infusions at escalating doses on days 3 and 6. Marrow mononuclear cells harvested before therapy, immediately prior to tanespimycin, and 24 hours later were examined by immunoblotting for Hsp70 and multiple Hsp90 clients. RESULTS: Twenty-six patients were treated at five dose levels. The maximum tolerated dose was cytarabine 400 mg/m(2)/day for 5 days along with tanespimycin 300 mg/m(2) on days 3 and 6. Treatment-related adverse events included disseminated intravascular coagulation (grades 3 and 5), acute respiratory distress syndrome (grade 4), and myocardial infarction associated with prolonged exposure to tanespimycin and its active metabolite 17-aminogeldanamycin. Among 21 evaluable patients, there were two complete and four partial remissions. Elevations of Hsp70, a marker used to assess Hsp90 inhibition in other studies, were observed in more than 80% of samples harvested 24 hours after tanespimycin, but down-regulation of Chk1 and other Hsp90 client proteins was modest. CONCLUSIONS: Because exposure to potentially effective concentrations occurs only for a brief time in vivo, at clinically tolerable doses tanespimycin has little effect on resistance-mediating client proteins in relapsed leukemia and exhibits limited activity in combination with cytarabine. (Clinicaltrials.gov identifier: NCT00098423).
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Leucemia/tratamento farmacológico , Doença Aguda , Adulto , Idoso , Benzoquinonas/administração & dosagem , Benzoquinonas/efeitos adversos , Quinase 1 do Ponto de Checagem , Citarabina/administração & dosagem , Citarabina/efeitos adversos , Feminino , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactamas Macrocíclicas/administração & dosagem , Lactamas Macrocíclicas/efeitos adversos , Leucemia/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Quinases/metabolismo , Recidiva , Fatores de TempoRESUMO
Inhibitors of the lipogenic enzyme fatty acid synthase (FASN) have attracted much attention in the last decade as potential targeted cancer therapies. However, little is known about the molecular determinants of cancer cell sensitivity to FASN inhibitors (FASNis), which is a major roadblock to their therapeutic application. Here, we find that pharmacological starvation of endogenously produced FAs is a previously unrecognized metabolic stress that heightens mitochondrial apoptotic priming and favors cell death induction by BH3 mimetic inhibitors. Evaluation of the death decision circuits controlled by the BCL-2 family of proteins revealed that FASN inhibition is accompanied by the upregulation of the pro-death BH3-only proteins BIM, PUMA, and NOXA. Cell death triggered by FASN inhibition, which causally involves a palmitate/NADPH-related redox imbalance, is markedly diminished by concurrent loss of BIM or PUMA, suggesting that FASN activity controls cancer cell survival by fine-tuning the BH3 only proteins-dependent mitochondrial threshold for apoptosis. FASN inhibition results in a heightened mitochondrial apoptosis priming, shifting cells toward a primed-for-death state "addicted" to the anti-apoptotic protein BCL-2. Accordingly, co-administration of a FASNi synergistically augments the apoptosis-inducing activity of the dual BCL-XL/BCL-2 inhibitor ABT-263 (navitoclax) and the BCL-2 specific BH3-mimetic ABT-199 (venetoclax). FASN inhibition, however, fails to sensitize breast cancer cells to MCL-1- and BCL-XL-selective inhibitors such as S63845 and A1331852. A human breast cancer xenograft model evidenced that oral administration of the only clinically available FASNi drastically sensitizes FASN-addicted breast tumors to ineffective single-agents navitoclax and venetoclax in vivo. In summary, a novel FASN-driven facet of the mitochondrial priming mechanistically links the redox-buffering mechanism of FASN activity to the intrinsic apoptotic threshold in breast cancer cells. Combining next-generation FASNis with BCL-2-specific BH3 mimetics that directly activate the apoptotic machinery might generate more potent and longer-lasting antitumor responses in a clinical setting.
Assuntos
Ácido Graxo Sintases/metabolismo , Mitocôndrias/metabolismo , Neoplasias/genética , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Nus , TransfecçãoRESUMO
The poly(ADP-ribose) binding protein CHFR regulates cellular responses to mitotic stress. The deubiquitinase UBC13, which regulates CHFR levels, has been associated with better overall survival in paclitaxel-treated ovarian cancer. Despite the extensive use of taxanes in the treatment of ovarian cancer, little is known about expression of CHFR itself in this disease. In the present study, tissue microarrays containing ovarian carcinoma samples from 417 women who underwent initial surgical debulking were stained with anti-CHFR antibody and scored in a blinded fashion. CHFR levels, expressed as a modified H-score, were examined for association with histology, grade, time to progression (TTP) and overall survival (OS). In addition, patient-derived xenografts from 69 ovarian carcinoma patients were examined for CHFR expression and sensitivity to paclitaxel monotherapy. In clinical ovarian cancer specimens, CHFR expression was positively associated with serous histology (p = 0.0048), higher grade (p = 0.000014) and higher stage (p = 0.016). After correction for stage and debulking, there was no significant association between CHFR staining and overall survival (p = 0.62) or time to progression (p = 0.91) in patients with high grade serous cancers treated with platinum/taxane chemotherapy (N = 249). Likewise, no association between CHFR expression and paclitaxel sensitivity was observed in ovarian cancer PDXs treated with paclitaxel monotherapy. Accordingly, differences in CHFR expression are unlikely to play a major role in paclitaxel sensitivity of high grade serous ovarian cancer.