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1.
Mol Cancer Ther ; 21(2): 322-335, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34789563

RESUMO

MET-targeted therapies are clinically effective in MET-amplified and MET exon 14 deletion mutant (METex14) non-small cell lung cancers (NSCLCs), but their efficacy is limited by the development of drug resistance. Structurally distinct MET tyrosine kinase inhibitors (TKIs) (type I/II) have been developed or are under clinical evaluation, which may overcome MET-mediated drug resistance mechanisms. In this study, we assess secondary MET mutations likely to emerge in response to treatment with single-agent or combinations of type I/type II MET TKIs using TPR-MET transformed Ba/F3 cell mutagenesis assays. We found that these inhibitors gave rise to distinct secondary MET mutant profiles. However, a combination of type I/II TKI inhibitors (capmatinib and merestinib) yielded no resistant clones in vitro The combination of capmatinib/merestinib was evaluated in vivo and led to a significant reduction in tumor outgrowth compared with either MET inhibitor alone. Our findings demonstrate in vitro and in vivo that a simultaneous treatment with a type I and type II MET TKI may be a clinically viable approach to delay and/or diminish the emergence of on target MET-mediated drug-resistance mutations.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Simulação de Acoplamento Molecular/métodos , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Feminino , Humanos , Camundongos , Inibidores de Proteínas Quinases/farmacologia
2.
Cancer Res ; 82(1): 130-141, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34548332

RESUMO

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) are the standard-of-care treatment for EGFR-mutant non-small cell lung cancers (NSCLC). However, most patients develop acquired drug resistance to EGFR TKIs. HER3 is a unique pseudokinase member of the ERBB family that functions by dimerizing with other ERBB family members (EGFR and HER2) and is frequently overexpressed in EGFR-mutant NSCLC. Although EGFR TKI resistance mechanisms do not lead to alterations in HER3, we hypothesized that targeting HER3 might improve efficacy of EGFR TKI. HER3-DXd is an antibody-drug conjugate (ADC) comprised of HER3-targeting antibody linked to a topoisomerase I inhibitor currently in clinical development. In this study, we evaluated the efficacy of HER3-DXd across a series of EGFR inhibitor-resistant, patient-derived xenografts and observed it to be broadly effective in HER3-expressing cancers. We further developed a preclinical strategy to enhance the efficacy of HER3-DXd through osimertinib pretreatment, which increased membrane expression of HER3 and led to enhanced internalization and efficacy of HER3-DXd. The combination of osimertinib and HER3-DXd may be an effective treatment approach and should be evaluated in future clinical trials in EGFR-mutant NSCLC patients. SIGNIFICANCE: EGFR inhibition leads to increased HER3 membrane expression and promotes HER3-DXd ADC internalization and efficacy, supporting the clinical development of the EGFR inhibitor/HER3-DXd combination in EGFR-mutant lung cancer.See related commentary by Lim et al., p. 18.


Assuntos
Antineoplásicos/uso terapêutico , Receptores ErbB/antagonistas & inibidores , Imunoconjugados/metabolismo , Receptor ErbB-3/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Humanos , Camundongos
3.
J Clin Invest ; 131(2)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33151910

RESUMO

Resistance to oncogene-targeted therapies involves discrete drug-tolerant persister cells, originally discovered through in vitro assays. Whether a similar phenomenon limits efficacy of programmed cell death 1 (PD-1) blockade is poorly understood. Here, we performed dynamic single-cell RNA-Seq of murine organotypic tumor spheroids undergoing PD-1 blockade, identifying a discrete subpopulation of immunotherapy persister cells (IPCs) that resisted CD8+ T cell-mediated killing. These cells expressed Snai1 and stem cell antigen 1 (Sca-1) and exhibited hybrid epithelial-mesenchymal features characteristic of a stem cell-like state. IPCs were expanded by IL-6 but were vulnerable to TNF-α-induced cytotoxicity, relying on baculoviral IAP repeat-containing protein 2 (Birc2) and Birc3 as survival factors. Combining PD-1 blockade with Birc2/3 antagonism in mice reduced IPCs and enhanced tumor cell killing in vivo, resulting in durable responsiveness that matched TNF cytotoxicity thresholds in vitro. Together, these data demonstrate the power of high-resolution functional ex vivo profiling to uncover fundamental mechanisms of immune escape from durable anti-PD-1 responses, while identifying IPCs as a cancer cell subpopulation targetable by specific therapeutic combinations.


Assuntos
Imunoterapia , Proteínas de Neoplasias , Neoplasias Experimentais , Receptor de Morte Celular Programada 1 , RNA-Seq , Análise de Célula Única , Esferoides Celulares , Animais , Linhagem Celular Tumoral , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Esferoides Celulares/imunologia , Esferoides Celulares/patologia
4.
Clin Cancer Res ; 27(1): 276-287, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33239433

RESUMO

PURPOSE: Dexamethasone, a uniquely potent corticosteroid, is frequently administered to patients with brain tumors to decrease tumor-associated edema, but limited data exist describing how dexamethasone affects the immune system systemically and intratumorally in patients with glioblastoma (GBM), particularly in the context of immunotherapy. EXPERIMENTAL DESIGN: We evaluated the dose-dependent effects of dexamethasone when administered with programmed cell death 1 (PD-1) blockade and/or radiotherapy in immunocompetent C57BL/6 mice with syngeneic GL261 and CT-2A GBM tumors. Clinically, the effect of dexamethasone on survival was evaluated in 181 patients with isocitrate dehydrogenase (IDH) wild-type GBM treated with PD-(L)1 blockade, with adjustment for relevant prognostic factors. RESULTS: Despite the inherent responsiveness of GL261 to immune checkpoint blockade, concurrent dexamethasone administration with anti-PD-1 therapy reduced survival in a dose-dependent manner. Concurrent dexamethasone also abrogated survival following anti-PD-1 therapy with or without radiotherapy in immune-resistant CT-2A models. Dexamethasone decreased T-lymphocyte numbers by increasing apoptosis, in addition to decreasing lymphocyte functional capacity. Myeloid and natural killer cell populations were also generally reduced by dexamethasone. Thus, dexamethasone appears to negatively affect both adaptive and innate immune responses. As a clinical correlate, a retrospective analysis of 181 consecutive patients with IDH wild-type GBM treated with PD-(L)1 blockade revealed poorer survival among those on baseline dexamethasone. Upon multivariable adjustment with relevant prognostic factors, baseline dexamethasone administration was the strongest predictor of poor survival [reference, no dexamethasone; <2 mg HR, 2.16; 95% confidence interval (CI), 1.30-3.68; P = 0.003 and ≥2 mg HR, 1.97; 95% CI, 1.23-3.16; P = 0.005]. CONCLUSIONS: Our preclinical and clinical data indicate that concurrent dexamethasone therapy may be detrimental to immunotherapeutic approaches for patients with GBM.


Assuntos
Edema Encefálico/tratamento farmacológico , Neoplasias Encefálicas/terapia , Dexametasona/farmacologia , Glioblastoma/terapia , Inibidores de Checkpoint Imunológico/farmacologia , Animais , Antígeno B7-H1/antagonistas & inibidores , Edema Encefálico/etiologia , Neoplasias Encefálicas/complicações , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Linhagem Celular Tumoral/transplante , Quimiorradioterapia/métodos , Dexametasona/uso terapêutico , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Interações Medicamentosas , Feminino , Seguimentos , Glioblastoma/complicações , Glioblastoma/genética , Glioblastoma/mortalidade , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Isocitrato Desidrogenase/genética , Estimativa de Kaplan-Meier , Camundongos , Prognóstico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Estudos Retrospectivos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia
5.
Cancer Cell ; 37(1): 104-122.e12, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31935369

RESUMO

Eradicating tumor dormancy that develops following epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment of EGFR-mutant non-small cell lung cancer, is an attractive therapeutic strategy but the mechanisms governing this process are poorly understood. Blockade of ERK1/2 reactivation following EGFR TKI treatment by combined EGFR/MEK inhibition uncovers cells that survive by entering a senescence-like dormant state characterized by high YAP/TEAD activity. YAP/TEAD engage the epithelial-to-mesenchymal transition transcription factor SLUG to directly repress pro-apoptotic BMF, limiting drug-induced apoptosis. Pharmacological co-inhibition of YAP and TEAD, or genetic deletion of YAP1, all deplete dormant cells by enhancing EGFR/MEK inhibition-induced apoptosis. Enhancing the initial efficacy of targeted therapies could ultimately lead to prolonged treatment responses in cancer patients.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Senescência Celular , Receptores ErbB/metabolismo , Feminino , Deleção de Genes , Humanos , Neoplasias Pulmonares/patologia , MAP Quinase Quinase 1/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mutação , Transdução de Sinais , Transcrição Gênica , Proteínas de Sinalização YAP
6.
Cancer Discov ; 9(1): 34-45, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30297358

RESUMO

KRAS-driven lung cancers frequently inactivate TP53 and/or STK11/LKB1, defining tumor subclasses with emerging clinical relevance. Specifically, KRAS-LKB1 (KL)-mutant lung cancers are particularly aggressive, lack PD-L1, and respond poorly to immune checkpoint blockade (ICB). The mechanistic basis for this impaired immunogenicity, despite the overall high mutational load of KRAS-mutant lung cancers, remains obscure. Here, we report that LKB1 loss results in marked silencing of stimulator of interferon genes (STING) expression and insensitivity to cytoplasmic double-strand DNA (dsDNA) sensing. This effect is mediated at least in part by hyperactivation of DNMT1 and EZH2 activity related to elevated S-adenylmethionine levels and reinforced by DNMT1 upregulation. Ectopic expression of STING in KL cells engages IRF3 and STAT1 signaling downstream of TBK1 and impairs cellular fitness, due to the pathologic accumulation of cytoplasmic mitochondrial dsDNA associated with mitochondrial dysfunction. Thus, silencing of STING avoids these negative consequences of LKB1 inactivation, while facilitating immune escape. SIGNIFICANCE: Oncogenic KRAS-mutant lung cancers remain treatment-refractory and are resistant to ICB in the setting of LKB1 loss. These results begin to uncover the key underlying mechanism and identify strategies to restore STING expression, with important therapeutic implications because mitochondrial dysfunction is an obligate component of this tumor subtype.See related commentary by Corte and Byers, p. 16.This article is highlighted in the In This Issue feature, p. 1.


Assuntos
Adenocarcinoma/genética , Deleção de Genes , Neoplasias Pulmonares/genética , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Quinases Proteína-Quinases Ativadas por AMP , Adenocarcinoma/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Fator Regulador 3 de Interferon/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/genética , Mutação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Fator de Transcrição STAT1/metabolismo
7.
Clin Cancer Res ; 25(20): 6127-6140, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31409614

RESUMO

PURPOSE: PARP inhibitors are approved for the treatment of high-grade serous ovarian cancers (HGSOC). Therapeutic resistance, resulting from restoration of homologous recombination (HR) repair or replication fork stabilization, is a pressing clinical problem. We assessed the activity of prexasertib, a checkpoint kinase 1 (CHK1) inhibitor known to cause replication catastrophe, as monotherapy and in combination with the PARP inhibitor olaparib in preclinical models of HGSOC, including those with acquired PARP inhibitor resistance. EXPERIMENTAL DESIGN: Prexasertib was tested as a single agent or in combination with olaparib in 14 clinically annotated and molecularly characterized luciferized HGSOC patient-derived xenograft (PDX) models and in a panel of ovarian cancer cell lines. The ability of prexasertib to impair HR repair and replication fork stability was also assessed. RESULTS: Prexasertib monotherapy demonstrated antitumor activity across the 14 PDX models. Thirteen models were resistant to olaparib monotherapy, including 4 carrying BRCA1 mutation. The combination of olaparib with prexasertib was synergistic and produced significant tumor growth inhibition in an olaparib-resistant model and further augmented the degree and durability of response in the olaparib-sensitive model. HGSOC cell lines, including those with acquired PARP inhibitor resistance, were also sensitive to prexasertib, associated with induction of DNA damage and replication stress. Prexasertib also sensitized these cell lines to PARP inhibition and compromised both HR repair and replication fork stability. CONCLUSIONS: Prexasertib exhibits monotherapy activity in PARP inhibitor-resistant HGSOC PDX and cell line models, reverses restored HR and replication fork stability, and synergizes with PARP inhibition.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Cistadenocarcinoma Seroso/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Pirazinas/farmacologia , Pirazóis/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteína BRCA1/genética , Linhagem Celular Tumoral , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patologia , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Humanos , Gradação de Tumores , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Ftalazinas/farmacologia , Ftalazinas/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirazinas/uso terapêutico , Pirazóis/uso terapêutico , Reparo de DNA por Recombinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Cancer Immunol Res ; 7(9): 1457-1471, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31331945

RESUMO

The success of targeted or immune therapies is often hampered by the emergence of resistance and/or clinical benefit in only a subset of patients. We hypothesized that combining targeted therapy with immune modulation would show enhanced antitumor responses. Here, we explored the combination potential of erdafitinib, a fibroblast growth factor receptor (FGFR) inhibitor under clinical development, with PD-1 blockade in an autochthonous FGFR2K660N/p53mut lung cancer mouse model. Erdafitinib monotherapy treatment resulted in substantial tumor control but no significant survival benefit. Although anti-PD-1 alone was ineffective, the erdafitinib and anti-PD-1 combination induced significant tumor regression and improved survival. For both erdafitinib monotherapy and combination treatments, tumor control was accompanied by tumor-intrinsic, FGFR pathway inhibition, increased T-cell infiltration, decreased regulatory T cells, and downregulation of PD-L1 expression on tumor cells. These effects were not observed in a KRASG12C-mutant genetically engineered mouse model, which is insensitive to FGFR inhibition, indicating that the immune changes mediated by erdafitinib may be initiated as a consequence of tumor cell killing. A decreased fraction of tumor-associated macrophages also occurred but only in combination-treated tumors. Treatment with erdafitinib decreased T-cell receptor (TCR) clonality, reflecting a broadening of the TCR repertoire induced by tumor cell death, whereas combination with anti-PD-1 led to increased TCR clonality, suggesting a more focused antitumor T-cell response. Our results showed that the combination of erdafitinib and anti-PD-1 drives expansion of T-cell clones and immunologic changes in the tumor microenvironment to support enhanced antitumor immunity and survival.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Imunidade/efeitos dos fármacos , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Biomarcadores , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sinergismo Farmacológico , Humanos , Imunofenotipagem , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Prognóstico , Receptor de Morte Celular Programada 1/genética , Pirazóis/farmacologia , Quinoxalinas/farmacologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Resultado do Tratamento , Microambiente Tumoral
9.
Elife ; 72018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30422115

RESUMO

High-grade serous ovarian cancer is characterized by extensive copy number alterations, among which the amplification of MYC oncogene occurs in nearly half of tumors. We demonstrate that ovarian cancer cells highly depend on MYC for maintaining their oncogenic growth, indicating MYC as a therapeutic target for this difficult-to-treat malignancy. However, targeting MYC directly has proven difficult. We screen small molecules targeting transcriptional and epigenetic regulation, and find that THZ1 - a chemical inhibiting CDK7, CDK12, and CDK13 - markedly downregulates MYC. Notably, abolishing MYC expression cannot be achieved by targeting CDK7 alone, but requires the combined inhibition of CDK7, CDK12, and CDK13. In 11 patient-derived xenografts models derived from heavily pre-treated ovarian cancer patients, administration of THZ1 induces significant tumor growth inhibition with concurrent abrogation of MYC expression. Our study indicates that targeting these transcriptional CDKs with agents such as THZ1 may be an effective approach for MYC-dependent ovarian malignancies.


Assuntos
Antineoplásicos/metabolismo , Proteína Quinase CDC2/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Neoplasias Ovarianas/patologia , Fenilenodiaminas/metabolismo , Proteínas Proto-Oncogênicas c-myc/biossíntese , Pirimidinas/metabolismo , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação para Baixo , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/metabolismo , Feminino , Xenoenxertos , Humanos , Camundongos SCID , Transplante de Neoplasias , Neoplasias Ovarianas/tratamento farmacológico , Fenilenodiaminas/administração & dosagem , Pirimidinas/administração & dosagem , Resultado do Tratamento , Quinase Ativadora de Quinase Dependente de Ciclina
10.
Cancer Immunol Res ; 6(12): 1511-1523, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30242021

RESUMO

We developed a screening assay in which luciferized ID8 expressing OVA was cocultured with transgenic CD8+ T cells specifically recognizing the model antigen in an H-2b-restricted manner. The assay was screened with a small-molecule library to identify compounds that inhibit or enhance T cell-mediated killing of tumor cells. Erlotinib, an EGFR inhibitor, was the top compound that enhanced T-cell killing of tumor cells. Subsequent experiments with erlotinib and additional EGFR inhibitors validated the screen results. EGFR inhibitors increased both basal and IFNγ-induced MHC class-I presentation, which enhanced recognition and lysis of tumor cell targets by CD8+ cytotoxic T lymphocytes. The ID8 cell line was also transduced to constitutively express Cas9, and a pooled CRISPR screen, utilizing the same target tumor cell/T-cell assay, identified single-guide (sg)RNAs targeting EGFR that sensitized tumor cells to T cell-mediated killing. Combination of PD-1 blockade with EGFR inhibition showed significant synergistic efficacy in a syngeneic model, further validating EGFR inhibitors as immunomodulatory agents that enhance checkpoint blockade. This assay can be screened in high-throughput with small-molecule libraries and genome-wide CRISPR/Cas9 libraries to identify both compounds and target genes, respectively, that enhance or inhibit T-cell recognition and killing of tumor cells. Retrospective analyses of squamous-cell head and neck cancer (SCCHN) patients treated with the combination of afatinib and pembrolizumab demonstrated a rate of clinical activity exceeding that of each single agent. Prospective clinical trials evaluating the combination of an EGFR inhibitor and PD-1 blockade should be conducted.


Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Receptores ErbB/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Inibidores de Proteínas Quinases/farmacologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Afatinib/administração & dosagem , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfócitos T CD8-Positivos , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Técnicas de Cocultura , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Luciferases de Vaga-Lume/genética , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Linfócitos T Citotóxicos/imunologia
11.
Clin Cancer Res ; 24(23): 5963-5976, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30072474

RESUMO

PURPOSE: MET inhibitors can be effective therapies in patients with MET exon 14 (METex14) mutant non-small cell lung cancer (NSCLC). However, long-term efficacy is limited by the development of drug resistance. In this study, we characterize acquired amplification of wild-type (WT) KRAS as a molecular mechanism behind crizotinib resistance in three cases of METex14-mutant NSCLC and propose a combination therapy to target it. EXPERIMENTAL DESIGN: The patient-derived cell line and xenograft (PDX) DFCI358 were established from a crizotinib-resistant METex14-mutant patient tumor with massive focal amplification of WT KRAS. To characterize the mechanism of KRAS-mediated resistance, molecular signaling was analyzed in the parental cell line and its KRAS siRNA-transfected derivative. Sensitivity of the cell line to ligand stimulation was assessed and KRAS-dependent expression of EGFR ligands was quantified. Drug combinations were screened for efficacy in vivo and in vitro using viability and apoptotic assays. RESULTS: KRAS amplification is a recurrent genetic event in crizotinib-resistant METex14-mutant NSCLC. The key characteristics of this genetic signature include uncoupling MET from downstream effectors, relative insensitivity to dual MET/MEK inhibition due to compensatory induction of PI3K signaling, KRAS-induced expression of EGFR ligands and hypersensitivity to ligand-dependent and independent activation, and reliance on PI3K signaling upon MET inhibition. CONCLUSIONS: Using patient-derived cell line and xenografts, we characterize the mechanism of crizotinib resistance mediated by KRAS amplification in METex14-mutant NSCLC and demonstrate the superior efficacy of the dual MET/PI3K inhibition as a therapeutic strategy addressing this resistance mechanism.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Éxons , Amplificação de Genes , Neoplasias Pulmonares/genética , Mutação , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Crizotinibe/farmacologia , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Modelos Biológicos , Fosfatidilinositol 3-Quinases/genética , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Cancer Cell ; 34(3): 439-452.e6, 2018 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30205046

RESUMO

Despite extensive efforts, oncogenic KRAS remains resistant to targeted therapy. Combined downstream RAL-TBK1 and MEK inhibition induces only transient lung tumor shrinkage in KRAS-driven genetically engineered mouse models (GEMMs). Using the sensitive KRAS;LKB1 (KL) mutant background, we identify YAP1 upregulation and a therapy-induced secretome as mediators of acquired resistance. This program is reversible, associated with H3K27 promoter acetylation, and suppressed by BET inhibition, resensitizing resistant KL cells to TBK1/MEK inhibition. Constitutive YAP1 signaling promotes intrinsic resistance in KRAS;TP53 (KP) mutant lung cancer. Intermittent treatment with the BET inhibitor JQ1 thus overcomes resistance to combined pathway inhibition in KL and KP GEMMs. Using potent and selective TBK1 and BET inhibitors we further develop an effective therapeutic strategy with potential translatability to the clinic.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antineoplásicos Imunológicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/imunologia , Células HEK293 , Humanos , Imunidade Inata/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/imunologia , Fator de Crescimento Insulin-Like I/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Transgênicos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfoproteínas/imunologia , Fosfoproteínas/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fatores de Transcrição , Proteínas de Sinalização YAP
13.
J Clin Invest ; 127(12): 4554-4568, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29130934

RESUMO

Transcriptional repression of ubiquitin B (UBB) is a cancer-subtype-specific alteration that occurs in a substantial population of patients with cancers of the female reproductive tract. UBB is 1 of 2 genes encoding for ubiquitin as a polyprotein consisting of multiple copies of ubiquitin monomers. Silencing of UBB reduces cellular UBB levels and results in an exquisite dependence on ubiquitin C (UBC), the second polyubiquitin gene. UBB is repressed in approximately 30% of high-grade serous ovarian cancer (HGSOC) patients and is a recurrent lesion in uterine carcinosarcoma and endometrial carcinoma. We identified ovarian tumor cell lines that retain UBB in a repressed state, used these cell lines to establish orthotopic ovarian tumors, and found that inducible expression of a UBC-targeting shRNA led to tumor regression, and substantial long-term survival benefit. Thus, we describe a recurrent cancer-specific lesion at the level of ubiquitin production. Moreover, these observations reveal the prognostic value of UBB repression and establish UBC as a promising therapeutic target for ovarian cancer patients with recurrent UBB silencing.


Assuntos
Inativação Gênica , Proteínas de Neoplasias/biossíntese , Neoplasias Ovarianas/metabolismo , Ubiquitina C/biossíntese , Ubiquitina/biossíntese , Linhagem Celular Tumoral , Feminino , Humanos , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Ubiquitina/genética , Ubiquitina C/genética
14.
Clin Cancer Res ; 23(5): 1263-1273, 2017 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-27573169

RESUMO

Purpose: Ovarian cancer is the leading cause of death from gynecologic malignancy in the United States, with high rates of recurrence and eventual resistance to cytotoxic chemotherapy. Model systems that allow for accurate and reproducible target discovery and validation are needed to support further drug development in this disease.Experimental Design: Clinically annotated patient-derived xenograft (PDX) models were generated from tumor cells isolated from the ascites or pleural fluid of patients undergoing clinical procedures. Models were characterized by IHC and by molecular analyses. Each PDX was luciferized to allow for reproducible in vivo assessment of intraperitoneal tumor burden by bioluminescence imaging (BLI). Plasma assays for CA125 and human LINE-1 were developed as secondary tests of in vivo disease burden.Results: Fourteen clinically annotated and molecularly characterized luciferized ovarian PDX models were generated. Luciferized PDX models retain fidelity to both the nonluciferized PDX and the original patient tumor, as demonstrated by IHC, array CGH, and targeted and whole-exome sequencing analyses. Models demonstrated diversity in specific genetic alterations and activation of PI3K signaling pathway members. Response of luciferized PDX models to standard-of-care therapy could be reproducibly monitored by BLI or plasma markers.Conclusions: We describe the establishment of a collection of 14 clinically annotated and molecularly characterized luciferized ovarian PDX models in which orthotopic tumor burden in the intraperitoneal space can be followed by standard and reproducible methods. This collection is well suited as a platform for proof-of-concept efficacy and biomarker studies and for validation of novel therapeutic strategies in ovarian cancer. Clin Cancer Res; 23(5); 1263-73. ©2016 AACR.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Ascite/patologia , Antígeno Ca-125/sangue , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Elementos Nucleotídeos Longos e Dispersos/genética , Proteínas de Membrana/sangue , Camundongos , Neoplasias Epiteliais e Glandulares/sangue , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/patologia , Transdução de Sinais/efeitos dos fármacos
15.
Leukemia ; 29(7): 1555-1563, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25703587

RESUMO

The rapid proliferation of myeloid leukemia cells is highly dependent on increased glucose metabolism. Through an unbiased metabolomics analysis of leukemia cells, we found that the glycogenic precursor UDP-D-glucose is pervasively upregulated, despite low glycogen levels. Targeting the rate-limiting glycogen synthase 1 (GYS1) not only decreased glycolytic flux but also increased activation of the glycogen-responsive AMP kinase (AMPK), leading to significant growth suppression. Further, genetic and pharmacological hyper-activation of AMPK was sufficient to induce the changes observed with GYS1 targeting. Cancer genomics data also indicate that elevated levels of the glycogenic enzymes GYS1/2 or GBE1 (glycogen branching enzyme 1) are associated with poor survival in AML. These results suggest a novel mechanism whereby leukemic cells sustain aberrant proliferation by suppressing excess AMPK activity through elevated glycogenic flux and provide a therapeutic entry point for targeting leukemia cell metabolism.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Glicogênio Sintase/metabolismo , Glicogênio/biossíntese , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Metabolômica , Animais , Apoptose , Estudos de Casos e Controles , Proliferação de Células , Citometria de Fluxo , Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase/genética , Glicólise , Células HEK293 , Humanos , Leucemia Mieloide/mortalidade , Camundongos , Fosforilação , Prognóstico , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Células Tumorais Cultivadas
16.
Mol Cancer Res ; 12(7): 1055-66, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24752990

RESUMO

UNLABELLED: TBK1 (TANK-binding kinase 1) is a noncanonical IκB protein kinase that phosphorylates and activates downstream targets such as IRF3 and c-Rel and, mediates NF-κB activation in cancer. Previous reports demonstrated synthetic lethality of TBK1 with mutant KRAS in non-small cell lung cancer (NSCLC); thus, TBK1 could be a novel target for treatment of KRAS-mutant NSCLC. Here, the effect of TBK1 on proliferation in a panel of cancer cells by both genetic and pharmacologic approaches was evaluated. In KRAS-mutant cancer cells, reduction of TBK1 activity by knockdown or treatment with TBK1 inhibitors did not correlate with reduced proliferation in a two-dimensional viability assay. Verification of target engagement via reduced phosphorylation of S386 of IRF3 (pIRF3(S386)) was difficult to assess in NSCLC cells due to low protein expression. However, several cell lines were identified with high pIRF3(S386) levels after screening a large panel of cell lines, many of which also harbor KRAS mutations. Specifically, a large subset of KRAS-mutant pancreatic cancer cell lines was uncovered with high constitutive pIRF3(S386) levels, which correlated with high levels of phosphorylated S172 of TBK1 (pTBK1(S172)). Finally, TBK1 inhibitors dose-dependently inhibited pIRF3(S386) in these cell lines, but this did not correlate with inhibition of cell growth. Taken together, these data demonstrate that the regulation of pathways important for cell proliferation in some NSCLC, pancreatic, and colorectal cell lines is not solely dependent on TBK1 activity. IMPLICATIONS: TBK1 has therapeutic potential under certain contexts and phosphorylation of its downstream target IRF3 is a biomarker of TBK1 activity.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/terapia , Fator Regulador 3 de Interferon/antagonistas & inibidores , Neoplasias Pulmonares/terapia , Neoplasias/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Terapia de Alvo Molecular , Neoplasias/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais
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