RESUMO
Polymerase ß (POLB), with dual functionality as a lyase and polymerase, plays a critical role in the base excision repair (BER) pathway to maintain genomic stability. POLB knockout and rescue studies in BRCA1/2-mutant cancer cell lines revealed that inhibition of lyase and polymerase activity is required for the synthetic lethal interaction observed with PARP inhibitors, highlighting POLB as a valuable therapeutic target. Traditional biochemical assays to screen for enzyme inhibitors focus on a single substrate to product relationship and limit the comprehensive analysis of enzymes such as POLB that utilize multiple substrates or catalyze a multi-step reaction. This report describes the first high-throughput mass spectrometry-based screen to measure the two distinct biochemical activities of POLB in a single assay using a duplexed self-assembled monolayer desorption ionization (SAMDI) mass spectrometry methodology. A multiplexed assay for POLB dual enzymatic activities was developed optimizing for kinetically balanced conditions and a collection of 200,000 diverse small molecules was screened in the duplexed format. Small molecule modulators identified in the screen were confirmed in a traditional fluorescence-based polymerase strand-displacement assay and an orthogonal label-free binding assay using SAMDI affinity selection mass spectrometry (ASMS). This work demonstrates the flexibility of high-throughput mass spectrometry approaches in drug discovery and highlights a novel application of SAMDI technology that opens new avenues for multiplexed high-throughput screening.
RESUMO
CRISPR Cas9-based screening is a powerful approach for identifying and characterizing novel drug targets. Here, we elucidate the synthetic lethal mechanism of deubiquitinating enzyme USP1 in cancers with underlying DNA damage vulnerabilities, specifically BRCA1/2 mutant tumors and a subset of BRCA1/2 wild-type (WT) tumors. In sensitive cells, pharmacologic inhibition of USP1 leads to decreased DNA synthesis concomitant with S-phase-specific DNA damage. Genome-wide CRISPR-Cas9 screens identify RAD18 and UBE2K, which promote PCNA mono- and polyubiquitination respectively, as mediators of USP1 dependency. The accumulation of mono- and polyubiquitinated PCNA following USP1 inhibition is associated with reduced PCNA protein levels. Ectopic expression of WT or ubiquitin-dead K164R PCNA reverses USP1 inhibitor sensitivity. Our results show, for the first time, that USP1 dependency hinges on the aberrant processing of mono- and polyubiquitinated PCNA. Moreover, this mechanism of USP1 dependency extends beyond BRCA1/2 mutant tumors to selected BRCA1/2 WT cancer cell lines enriched in ovarian and lung lineages. We further show PARP and USP1 inhibition are strongly synergistic in BRCA1/2 mutant tumors. We postulate USP1 dependency unveils a previously uncharacterized vulnerability linked to posttranslational modifications of PCNA. Taken together, USP1 inhibition may represent a novel therapeutic strategy for BRCA1/2 mutant tumors and a subset of BRCA1/2 WT tumors.
Assuntos
Neoplasias , Mutações Sintéticas Letais , Humanos , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitina/genética , Ubiquitinação , Dano ao DNA , Neoplasias/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismoRESUMO
Synthetic lethality is a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone, which can be co-opted for cancer therapeutics. Pairs of paralog genes are among the most straightforward potential synthetic-lethal interactions by virtue of their redundant functions. Here, we demonstrate a paralog-based synthetic lethality by targeting vaccinia-related kinase 1 (VRK1) in glioblastoma (GBM) deficient of VRK2, which is silenced by promoter methylation in approximately two thirds of GBM. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells resulted in decreased activity of the downstream substrate barrier to autointegration factor (BAF), a regulator of post-mitotic nuclear envelope formation. Reduced BAF activity following VRK1 knockdown caused nuclear lobulation, blebbing, and micronucleation, which subsequently resulted in G2-M arrest and DNA damage. The VRK1-VRK2 synthetic-lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic expression of VRK2. In VRK2-methylated GBM cell line-derived xenograft and patient-derived xenograft models, knockdown of VRK1 led to robust tumor growth inhibition. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM. SIGNIFICANCE: A paralog synthetic-lethal interaction between VRK1 and VRK2 sensitizes VRK2-methylated glioblastoma to perturbation of VRK1 kinase activity, supporting VRK1 as a drug discovery target in this disease.
Assuntos
Glioblastoma , Humanos , Apoptose , Linhagem Celular Tumoral , Pontos de Checagem da Fase G2 do Ciclo Celular , Vaccinia virus , Fosforilação , Proteínas Serina-Treonina QuinasesRESUMO
Tumors lack a well-regulated vascular supply of O2 and often fail to balance O2 supply and demand. Net O2 tension within many tumors may not only depend on O2 delivery but also depend strongly on O2 demand. Thus, tumor O2 consumption rates may influence tumor hypoxia up to true anoxia. Recent reports have shown that many human tumors in vivo depend primarily on oxidative phosphorylation (OxPhos), not glycolysis, for energy generation, providing a driver for consumptive hypoxia and an exploitable vulnerability. In this regard, IACS-010759 is a novel high affinity inhibitor of OxPhos targeting mitochondrial complex-I that has recently completed a Phase-I clinical trial in leukemia. However, in solid tumors, the effective translation of OxPhos inhibitors requires methods to monitor pharmacodynamics in vivo. Herein, 18F-fluoroazomycin arabinoside ([18F]FAZA), a 2-nitroimidazole-based hypoxia PET imaging agent, was combined with a rigorous test-retest imaging method for non-invasive quantification of the reversal of consumptive hypoxia in vivo as a mechanism-specific pharmacodynamic (PD) biomarker of target engagement for IACS-010759. Neither cell death nor loss of perfusion could account for the IACS-010759-induced decrease in [18F]FAZA retention. Notably, in an OxPhos-reliant melanoma tumor, a titration curve using [18F]FAZA PET retention in vivo yielded an IC50 for IACS-010759 (1.4 mg/kg) equivalent to analysis ex vivo. Pilot [18F]FAZA PET scans of a patient with grade IV glioblastoma yielded highly reproducible, high-contrast images of hypoxia in vivo as validated by CA-IX and GLUT-1 IHC ex vivo. Thus, [18F]FAZA PET imaging provided direct evidence for the presence of consumptive hypoxia in vivo, the capacity for targeted reversal of consumptive hypoxia through the inhibition of OxPhos, and a highly-coupled mechanism-specific PD biomarker ready for translation.
Assuntos
Complexo I de Transporte de Elétrons/antagonistas & inibidores , Oxidiazóis/farmacologia , Piperidinas/farmacologia , Hipóxia Tumoral/efeitos dos fármacos , Animais , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Complexo I de Transporte de Elétrons/metabolismo , Feminino , Glioblastoma/diagnóstico por imagem , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Nus , Nitroimidazóis , Fosforilação Oxidativa/efeitos dos fármacos , Oxigênio/metabolismo , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Compostos RadiofarmacêuticosRESUMO
Bromodomain and extraterminal domain (BET) proteins help direct the differentiation of helper T cell subsets, but their role in activated T cell function has not been described in detail. In this study, we investigate various consequences of epigenetic perturbation in human T lymphocytes using MK-8628, a potent and highly selective inhibitor of BET proteins. MK-8628 reduces the expression of canonical transcripts directing the proliferation, activation, and effector function of T lymphocytes. Treatment with MK-8628 abolishes the expression of key cyclins required for cell cycle progression and induces G1 cell cycle arrest in TCR-activated lymphocytes. This antiproliferative phenotype partially results from T lymphocyte apoptosis, which is exacerbated by MK-8628. In naive and memory T cell subsets, MK-8628 antagonizes T cell activation and suppresses polyfunctional cytokine production. Collectively, our results describe potent immunosuppressive effects of BET inhibition on human T cell biology. These results have important implications for immune modulatory targeting of BET proteins in the settings of T cell-driven autoimmune inflammation.
Assuntos
Acetanilidas/farmacologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Compostos Heterocíclicos com 3 Anéis/farmacologia , Imunossupressores/farmacologia , Proteínas/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Voluntários Saudáveis , Humanos , Ativação Linfocitária/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
The plasticity of a preexisting regulatory circuit compromises the effectiveness of targeted therapies, and leveraging genetic vulnerabilities in cancer cells may overcome such adaptations. Hereditary leiomyomatosis renal cell carcinoma (HLRCC) is characterized by oxidative phosphorylation (OXPHOS) deficiency caused by fumarate hydratase (FH) nullizyogosity. To identify metabolic genes that are synthetically lethal with OXPHOS deficiency, we conducted a genetic loss-of-function screen and found that phosphogluconate dehydrogenase (PGD) inhibition robustly blocks the proliferation of FH mutant cancer cells both in vitro and in vivo. Mechanistically, PGD inhibition blocks glycolysis, suppresses reductive carboxylation of glutamine, and increases the NADP+/NADPH ratio to disrupt redox homeostasis. Furthermore, in the OXPHOS-proficient context, blocking OXPHOS using the small-molecule inhibitor IACS-010759 enhances sensitivity to PGD inhibition in vitro and in vivo. Together, our study reveals a dependency on PGD in OXPHOS-deficient tumors that might inform therapeutic intervention in specific patient populations.
Assuntos
Fosforilação Oxidativa , Fosfogluconato Desidrogenase/genética , Mutações Sintéticas Letais , Animais , Linhagem Celular Tumoral , Feminino , Fumarato Hidratase/genética , Genômica/métodos , Glicólise , Humanos , Mutação com Perda de Função , Camundongos , Camundongos NusRESUMO
Metabolic reprograming is an emerging hallmark of tumor biology and an actively pursued opportunity in discovery of oncology drugs. Extensive efforts have focused on therapeutic targeting of glycolysis, whereas drugging mitochondrial oxidative phosphorylation (OXPHOS) has remained largely unexplored, partly owing to an incomplete understanding of tumor contexts in which OXPHOS is essential. Here, we report the discovery of IACS-010759, a clinical-grade small-molecule inhibitor of complex I of the mitochondrial electron transport chain. Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in models of brain cancer and acute myeloid leukemia (AML) reliant on OXPHOS, likely owing to a combination of energy depletion and reduced aspartate production that leads to impaired nucleotide biosynthesis. In models of brain cancer and AML, tumor growth was potently inhibited in vivo following IACS-010759 treatment at well-tolerated doses. IACS-010759 is currently being evaluated in phase 1 clinical trials in relapsed/refractory AML and solid tumors.
Assuntos
Neoplasias/patologia , Fosforilação Oxidativa , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Metabolismo Energético , Glicólise/efeitos dos fármacos , Células HEK293 , Humanos , Ácido Láctico/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Mitocôndrias/metabolismo , Nucleotídeos/biossíntese , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
SIRT1 belongs to the silent information regulator 2 (Sir2) protein family of enzymes and functions as a NAD(+) -dependent class III histone deacetylase. Here, we examined the anti-multiple myeloma (MM) activity of a novel oral agent, SRT1720, which targets SIRT1. Treatment of MM cells with SRT1720 inhibited growth and induced apoptosis in MM cells resistant to conventional and bortezomib therapies without significantly affecting the viability of normal cells. Mechanistic studies showed that anti-MM activity of SRT1720 is associated with: (i) activation of caspase-8, caspase-9, caspase-3, poly(ADP) ribose polymerase; (ii) increase in reactive oxygen species; (iii) induction of phosphorylated ataxia telangiectasia mutated/checkpoint kinase 2 signalling; (iv) decrease in vascular endothelial growth factor-induced migration of MM cells and associated angiogenesis; and (v) inhibition of nuclear factor-κB. Blockade of ATM attenuated SRT1720-induced MM cell death. In animal tumour model studies, SRT1720 inhibited MM tumour growth. Finally, SRT1720 enhanced the cytotoxic activity of bortezomib or dexamethasone. Our preclinical studies provide the rationale for novel therapeutics targeting SIRT1 in MM.
Assuntos
Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Ácidos Borônicos/administração & dosagem , Bortezomib , Linhagem Celular Tumoral , Dexametasona/administração & dosagem , Feminino , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Compostos Heterocíclicos de 4 ou mais Anéis/química , Humanos , Imuno-Histoquímica , Camundongos , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Pirazinas/administração & dosagem , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Previous studies have established a role of vascular-disrupting agents as anti- cancer agents. Plinabulin is a novel vascular-disrupting agent that exhibits potent interruption of tumor blood flow because of the disruption of tumor vascular endothelial cells, resulting in tumor necrosis. In addition, plinabulin exerts a direct action on tumor cells, resulting in apoptosis. In the present study, we examined the anti-multiple myeloma (MM) activity of plinabulin. We show that low concentrations of plinabulin exhibit a potent antiangiogenic action on vascular endothelial cells. Importantly, plinabulin also induces apoptotic cell death in MM cell lines and tumor cells from patients with MM, associated with mitotic growth arrest. Plinabulin-induced apoptosis is mediated through activation of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase cleavage. Moreover, plinabulin triggered phosphorylation of stress response protein JNK, as a primary target, whereas blockade of JNK with a biochemical inhibitor or small interfering RNA strategy abrogated plinabulin-induced mitotic block or MM cell death. Finally, in vivo studies show that plinabulin was well tolerated and significantly inhibited tumor growth and prolonged survival in a human MM.1S plasmacytoma murine xenograft model. Our study therefore provides the rationale for clinical evaluation of plinabulin to improve patient outcome in MM.
Assuntos
Inibidores da Angiogênese/farmacologia , Apoptose/efeitos dos fármacos , Imidazóis/farmacologia , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Mieloma Múltiplo/irrigação sanguínea , Mieloma Múltiplo/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Piperazinas/farmacologia , Animais , Western Blotting , Proliferação de Células/efeitos dos fármacos , Dicetopiperazinas , Imunofluorescência , Humanos , Masculino , Camundongos , Camundongos SCID , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/genética , RNA Interferente Pequeno/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Aurora kinases, whose expression is linked to genetic instability and cellular proliferation, are being investigated as novel therapeutic targets in multiple myeloma (MM). In this study, we investigated the preclinical activity of a small-molecule multitargeted kinase inhibitor, AT9283, with potent activity against Aurora kinase A, Aurora kinase B, and Janus kinase 2/3. EXPERIMENTAL DESIGN: We evaluated the in vitro antimyeloma activity of AT9283 alone and in combination with lenalidomide and the in vivo efficacy by using a xenograft mouse model of human MM. RESULTS: Our data showed that AT9283 induced cell-growth inhibition and apoptosis in MM. Studying the apoptosis mechanism of AT9283 in MM, we observed features consistent with both Aurora kinase A and Aurora kinase B inhibition, such as increase of cells with polyploid DNA content, decrease in phospho-histone H3, and decrease in phospho-Aurora A. Importantly, AT9283 also inhibited STAT3 tyrosine phosphorylation in MM cells. Genetic depletion of STAT3, Aurora kinase A, or Aurora kinase B showed growth inhibition of MM cells, suggesting a role of AT9283-induced inhibition of these molecules in the underlying mechanism of MM cell death. In vivo studies showed decreased MM cell growth and prolonged survival in AT9283-treated mice compared with controls. Importantly, combination studies of AT9283 with lenalidomide showed significant synergistic cytotoxicity in MM cells, even in the presence of bone marrow stromal cells. Enhanced cytotoxicity was associated with increased inhibition of phosphorylated STAT3 and phosphorylated extracellular signal-regulated kinase. CONCLUSIONS: Demonstration of in vitro and in vivo anti-MM activity of AT9283 provides the rationale for the clinical evaluation of AT9283 as monotherapy and in combination therapy for treating patients with MM.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Benzimidazóis/administração & dosagem , Benzimidazóis/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Talidomida/análogos & derivados , Ureia/análogos & derivados , Animais , Aurora Quinase A , Aurora Quinase B , Aurora Quinases , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Lenalidomida , Masculino , Camundongos , Camundongos SCID , Modelos Biológicos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Fator de Transcrição STAT3/antagonistas & inibidores , Talidomida/administração & dosagem , Resultado do Tratamento , Ureia/administração & dosagem , Ureia/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PR-924 is an LMP-7-selective tripeptide epoxyketone proteasome inhibitor that covalently modifies proteasomal N-terminal threonine active sites. In the present study, we show that PR-924 inhibits growth and triggers apoptosis in multiple myeloma (MM) cell lines and primary patient MM cells, without significantly affecting normal peripheral blood mononuclear cells. PR-924-induced apoptosis in MM cells is associated with activation of caspase-3, caspase-8, caspase-9, BID, PARP and cytochrome-c release. In vivo administration of PR-924 inhibits tumour growth in human plasmacytoma xenografts. Results from SCID-hu model show a significant reduction in the shIL-6R levels in mice treated with PR-924 versus vehicle-control. PR-924 treatment was well tolerated as evidenced by the lack of weight loss. Importantly, treatment of tumour-bearing mice with PR-924, but not vehicle alone, prolonged survival. Our preclinical findings therefore validate immunoproteasome LMP-7 subunit as a novel therapeutic target in MM.
Assuntos
Antineoplásicos/farmacologia , Mieloma Múltiplo/patologia , Oligopeptídeos/farmacologia , Inibidores de Proteassoma , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Camundongos , Camundongos SCID , Mieloma Múltiplo/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Oligopeptídeos/uso terapêutico , Plasmocitoma/tratamento farmacológico , Plasmocitoma/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Análise de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Bortezomib therapy has proven successful for the treatment of relapsed, relapsed/refractory, and newly diagnosed multiple myeloma (MM). At present, bortezomib is available as an intravenous injection, and its prolonged treatment is associated with toxicity and development of drug resistance. Here we show that the novel proteasome inhibitor ONX 0912, a tripeptide epoxyketone, inhibits growth and induces apoptosis in MM cells resistant to conventional and bortezomib therapies. The anti-MM activity of ONX-0912 is associated with activation of caspase-8, caspase-9, caspase-3, and poly(ADP) ribose polymerase, as well as inhibition of migration of MM cells and angiogenesis. ONX 0912, like bortezomib, predominantly inhibits chymotrypsin-like activity of the proteasome and is distinct from bortezomib in its chemical structure. Importantly, ONX 0912 is orally bioactive. In animal tumor model studies, ONX 0912 significantly reduced tumor progression and prolonged survival. Immununostaining of MM tumors from ONX 0912-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Finally, ONX 0912 enhances anti-MM activity of bortezomib, lenalidomide dexamethasone, or pan-histone deacetylase inhibitor. Taken together, our study provides the rationale for clinical protocols evaluating ONX 0912, either alone or in combination, to improve patient outcome in MM.
Assuntos
Antineoplásicos/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Western Blotting , Caspases/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Camundongos SCID , Oligopeptídeos/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The bone marrow (BM) microenvironment consists of extracellular-matrix and the cellular compartment including immune cells. Multiple myeloma (MM) cell and BM accessory cell interaction promotes MM survival via both cell-cell contact and cytokines. Immunomodulatory agents (IMiDs) target not only MM cells, but also MM cell-immune cell interactions and cytokine signaling. Here we examined the in vitro effects of IMiDs on cytokine signaling triggered by interaction of effector cells with MM cells and BM stroma cells. IMiDs diminished interleukin-2, interferonγ, and IL-6 regulator suppressor of cytokine signaling (SOCS)1 expression in immune (CD4T, CD8T, natural-killer T, natural-killer) cells from both BM and PB of MM patients. In addition, coculture of MM cells with healthy PBMCs induced SOCS1 expression in effector cells; conversely, treatment with IMiDs down-regulated the SOCS1 expression. SOCS1 negatively regulates IL-6 signaling and is silenced by hypermethylation in MM cells. To define the mechanism of inhibitory-cytokine signaling in effector cells and MM cells, we next analyzed the interaction of immune cells with MM cells that were epigenetically modified to re-express SOCS1; IMiDs induced more potent CTL responses against SOCS1 re-expressing-MM cells than unmodified MM cells. These data therefore demonstrate that modulation of SOCS1 may enhance immune response and efficacy of IMiDs in MM.
Assuntos
Antineoplásicos/imunologia , Células da Medula Óssea/efeitos dos fármacos , Fatores Imunológicos/imunologia , Mieloma Múltiplo/imunologia , Linfócitos T/efeitos dos fármacos , Talidomida/análogos & derivados , Células da Medula Óssea/imunologia , Linhagem Celular Tumoral , Citocinas/imunologia , Epigênese Genética , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Lenalidomida , Mieloma Múltiplo/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Células Estromais/imunologia , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/imunologia , Linfócitos T/imunologia , Talidomida/imunologiaRESUMO
Aurora-A is a mitotic kinase that regulates mitotic spindle formation and segregation. In multiple myeloma (MM), high Aurora-A gene expression has been correlated with centrosome amplification and proliferation; thus, inhibition of Aurora-A in MM may prove to be therapeutically beneficial. Here we assess the in vitro and in vivo anti-MM activity of MLN8237, a small-molecule Aurora-A kinase inhibitor. Treatment of cultured MM cells with MLN8237 results in mitotic spindle abnormalities, mitotic accumulation, as well as inhibition of cell proliferation through apoptosis and senescence. In addition, MLN8237 up-regulates p53 and tumor suppressor genes p21 and p27. Combining MLN8237 with dexamethasone, doxorubicin, or bortezomib induces synergistic/additive anti-MM activity in vitro. In vivo anti-MM activity of MLN8237 was confirmed using a xenograft-murine model of human-MM. Tumor burden was significantly reduced (P = .007) and overall survival was significantly increased (P < .005) in animals treated with 30 mg/kg MLN8237 for 21 days. Induction of apoptosis and cell death by MLN8237 were confirmed in tumor cells excised from treated animals by TdT-mediated dUTP nick end labeling assay. MLN8237 is currently in phase 1 and phase 2 clinical trials in patients with advanced malignancies, and our preclinical results suggest that MLN8237 may be a promising novel targeted therapy in MM.
Assuntos
Apoptose/efeitos dos fármacos , Azepinas/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Aurora Quinase A , Aurora Quinases , Azepinas/uso terapêutico , Ácidos Borônicos/farmacologia , Bortezomib , Ciclo Celular , Linhagem Celular Tumoral , Senescência Celular/efeitos dos fármacos , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Dexametasona/farmacologia , Doxorrubicina/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos SCID , Mieloma Múltiplo/enzimologia , Transplante de Neoplasias , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/biossíntese , Pirazinas/farmacologia , Pirimidinas/uso terapêutico , Fuso Acromático/metabolismo , Fatores de Tempo , Transplante Heterólogo , Proteínas Supressoras de Tumor/biossíntese , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Multiple myeloma (MM) remains incurable despite novel therapies, suggesting the need for further identification of factors mediating tumorigenesis and drug resistance. Using both in vitro and in vivo MM xenograft models, we show that plasmacytoid dendritic cells (pDCs) in the bone marrow (BM) microenvironment both mediate immune deficiency characteristic of MM and promote MM cell growth, survival, and drug resistance. Microarray, cell signaling, cytokine profile, and immunohistochemical analysis delineate the mechanisms mediating these sequelae. Although pDCs are resistant to novel therapies, targeting toll-like receptors with CpG oligodeoxynucleotides both restores pDC immune function and abrogates pDC-induced MM cell growth. Our study therefore validates targeting pDC-MM interactions as a therapeutic strategy to overcome drug resistance in MM.
Assuntos
Células da Medula Óssea/imunologia , Comunicação Celular , Células Dendríticas/imunologia , Resistencia a Medicamentos Antineoplásicos , Mieloma Múltiplo/imunologia , Transdução de Sinais , Animais , Antineoplásicos/farmacologia , Apoptose , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Ácidos Borônicos/farmacologia , Bortezomib , Estudos de Casos e Controles , Comunicação Celular/efeitos dos fármacos , Proliferação de Células , Sobrevivência Celular , Quimiotaxia , Técnicas de Cocultura , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Imunofenotipagem , Camundongos , Camundongos SCID , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Oligodesoxirribonucleotídeos/farmacologia , Inibidores de Proteases/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirazinas/farmacologia , Receptores Imunológicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/imunologia , Fatores de Tempo , Receptores Toll-Like/efeitos dos fármacos , Receptores Toll-Like/metabolismo , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
A long-standing hypothesis on tumorigenesis is that cell division failure, generating genetically unstable tetraploid cells, facilitates the development of aneuploid malignancies. Here we test this idea by transiently blocking cytokinesis in p53-null (p53-/-) mouse mammary epithelial cells (MMECs), enabling the isolation of diploid and tetraploid cultures. The tetraploid cells had an increase in the frequency of whole-chromosome mis-segregation and chromosomal rearrangements. Only the tetraploid cells were transformed in vitro after exposure to a carcinogen. Furthermore, in the absence of carcinogen, only the tetraploid cells gave rise to malignant mammary epithelial cancers when transplanted subcutaneously into nude mice. These tumours all contained numerous non-reciprocal translocations and an 8-30-fold amplification of a chromosomal region containing a cluster of matrix metalloproteinase (MMP) genes. MMP overexpression is linked to mammary tumours in humans and animal models. Thus, tetraploidy enhances the frequency of chromosomal alterations and promotes tumour development in p53-/- MMECs.