RESUMO
The composition of the tumor immune microenvironment (TIME) is considered a key determinant of patients' response to immunotherapy. The mechanisms underlying TIME formation and development over time are poorly understood. Glioblastoma (GBM) is a lethal primary brain cancer for which there are no curative treatments. GBMs are immunologically heterogeneous and impervious to checkpoint blockade immunotherapies. Utilizing clinically relevant genetic mouse models of GBM, we identified distinct immune landscapes associated with expression of EGFR wild-type and mutant EGFRvIII cancer driver mutations. Over time, accumulation of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) was more pronounced in EGFRvIII-driven GBMs and was correlated with resistance to PD-1 and CTLA-4 combination checkpoint blockade immunotherapy. We determined that GBM-secreted CXCL1/2/3 and PMN-MDSC-expressed CXCR2 formed an axis regulating output of PMN-MDSCs from the bone marrow leading to systemic increase in these cells in the spleen and GBM tumor-draining lymph nodes. Pharmacologic targeting of this axis induced a systemic decrease in the numbers of PMN-MDSC, facilitated responses to PD-1 and CTLA-4 combination checkpoint blocking immunotherapy, and prolonged survival in mice bearing EGFRvIII-driven GBM. Our results uncover a relationship between cancer driver mutations, TIME composition, and sensitivity to checkpoint blockade in GBM and support the stratification of patients with GBM for checkpoint blockade therapy based on integrated genotypic and immunologic profiles.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Células Supressoras Mieloides , Animais , Camundongos , Glioblastoma/terapia , Glioblastoma/tratamento farmacológico , Antígeno CTLA-4/genética , Antígeno CTLA-4/metabolismo , Receptor de Morte Celular Programada 1 , Linhagem Celular Tumoral , Imunoterapia , Mutação , Microambiente Tumoral/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapiaRESUMO
Long recognized as an evolutionarily ancient cell type involved in tissue homeostasis and immune defense against pathogens, macrophages are being re-discovered as regulators of several diseases, including cancer. Tumor-associated macrophages (TAMs) represent the most abundant innate immune population in the tumor microenvironment (TME). Macrophages are professional phagocytic cells of the hematopoietic system specializing in the detection, phagocytosis and destruction of bacteria and other harmful micro-organisms, apoptotic cells and metabolic byproducts. In contrast to these healthy macrophage functions, TAMs support cancer cell growth and metastasis and mediate immunosuppressive effects on the adaptive immune cells of the TME. Cancer is one of the most potent insults on macrophage physiology, inducing changes that are intimately linked with disease progression. In this Review, we outline hallmarks of TAMs and discuss the emerging mechanisms that contribute to their pathophysiological adaptations and the vulnerabilities that provide attractive targets for therapeutic exploitation in cancer.
Assuntos
Neoplasias , Microambiente Tumoral , Progressão da Doença , Humanos , Macrófagos , FagocitoseRESUMO
Glioblastoma (GBM) is an incurable primary malignant brain cancer hallmarked with a substantial protumorigenic immune component. Knowledge of the GBM immune microenvironment during tumor evolution and standard of care treatments is limited. Using single-cell transcriptomics and flow cytometry, we unveiled large-scale comprehensive longitudinal changes in immune cell composition throughout tumor progression in an epidermal growth factor receptor-driven genetic mouse GBM model. We identified subsets of proinflammatory microglia in developing GBMs and anti-inflammatory macrophages and protumorigenic myeloid-derived suppressors cells in end-stage tumors, an evolution that parallels breakdown of the blood-brain barrier and extensive growth of epidermal growth factor receptor+ GBM cells. A similar relationship was found between microglia and macrophages in patient biopsies of low-grade glioma and GBM. Temozolomide decreased the accumulation of myeloid-derived suppressor cells, whereas concomitant temozolomide irradiation increased intratumoral GranzymeB+ CD8+T cells but also increased CD4+ regulatory T cells. These results provide a comprehensive and unbiased immune cellular landscape and its evolutionary changes during GBM progression.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Animais , Neoplasias Encefálicas/metabolismo , Receptores ErbB , Glioblastoma/metabolismo , Humanos , Camundongos , Análise de Sequência de RNA , Análise de Célula Única , Temozolomida/uso terapêutico , Microambiente Tumoral/genéticaRESUMO
Focal amplification of epidermal growth factor receptor (EGFR) and its ligand-independent, constitutively active EGFRvIII mutant form are prominent oncogenic drivers in glioblastoma (GBM). The EGFRvIII gene rearrangement is considered to be an initiating event in the etiology of GBM, however, the mechanistic details of how EGFRvIII drives cellular transformation and tumor maintenance remain unclear. Here, we report that EGFRvIII demonstrates a reliance on PDGFRA co-stimulatory signaling during the tumorigenic process in a genetically engineered autochthonous GBM model. This dependency exposes liabilities that were leveraged using kinase inhibitors treatments in EGFRvIII-expressing GBM patient-derived xenografts (PDXs), where simultaneous pharmacological inhibition of EGFRvIII and PDGFRA kinase activities is necessary for anti-tumor efficacy. Our work establishes that EGFRvIII-positive tumors have unexplored vulnerabilities to targeted agents concomitant to the EGFR kinase inhibitor repertoire.
Assuntos
Neoplasias Encefálicas/metabolismo , Receptores ErbB/metabolismo , Glioblastoma/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Células HEK293 , Xenoenxertos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidoresRESUMO
The molecular alterations underlying progression of low-grade glial/glioneuronal tumors remain to be elucidated. We present a case of a 56-year-old male with an enhancing left temporal lobe tumor. Histology revealed a high-grade glioma adjacent to a low-grade glioneuronal component with abundant Rosenthal fibers, focal eosinophilic granular bodies, and CD34-positive neurons. The tumor was negative for IDH1 (R132H), BRAF-V600E, and the KIAA1549-BRAF fusion. Comparative genomic hybridization detected a large amplification (> 15 copies) of the Son of Sevenless 1 (SOS1) gene, a component of the MAPK pathway. Although activating mutations in the MAPK pathway occur frequently in gliomas and glioneuronal tumors, SOS1 gene amplification has not been reported previously. This case indicates another potential mechanism for MAPK activation in glial tumors.
Assuntos
Astrocitoma/genética , Glioma/patologia , Mutação/genética , Proteína SOS1/genética , Astrocitoma/diagnóstico , Astrocitoma/patologia , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/patologia , Hibridização Genômica Comparativa/métodos , Glioma/genética , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter D) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support activation of canonical NF-κB signaling in response to treatment with tumor necrosis factor. Nevertheless, non-canonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic wild-type NEMO, but not certain human NEMO disease mutants, in the edited cells restores downstream NF-κB signaling in response to tumor necrosis factor. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter D) in the human SNU-423 liver cancer cell line. Thus, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed.
Assuntos
Edição de Genes/métodos , Técnicas de Silenciamento de Genes/métodos , Quinase I-kappa B/genética , Elementos Reguladores de Transcrição/genética , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Células HEK293 , Humanos , NF-kappa B/metabolismo , Transdução de SinaisRESUMO
Glioblastoma multiforme (GBM) is an aggressive primary brain cancer that includes focal amplification of PDGFRα and for which there are no effective therapies. Herein, we report the development of a genetically engineered mouse model of GBM based on autocrine, chronic stimulation of overexpressed PDGFRα, and the analysis of GBM signaling pathways using proteomics. We discover the tubulin-binding protein Stathmin1 (STMN1) as a PDGFRα phospho-regulated target, and that this mis-regulation confers sensitivity to vinblastine (VB) cytotoxicity. Treatment of PDGFRα-positive mouse and a patient-derived xenograft (PDX) GBMs with VB in mice prolongs survival and is dependent on STMN1. Our work reveals a previously unconsidered link between PDGFRα activity and STMN1, and highlight an STMN1-dependent cytotoxic effect of VB in GBM.
Assuntos
Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Estatmina/metabolismo , Vimblastina/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose , Ciclo Celular , Sobrevivência Celular , Células Cultivadas , Biologia Computacional , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imageamento por Ressonância Magnética , Masculino , Camundongos , Transplante de Neoplasias , Fosforilação , Proteômica , Transdução de SinaisRESUMO
Glioblastoma Multiforme (GBM) is a highly malignant primary brain cancer that is associated with abysmal prognosis. The median survival of GBM patients is â¼15 months and there have not been any significant advance in therapies in over a decade, leaving treatment options limited. There is clearly an unmet need for GBM treatment. Immunotherapies are treatments based on usurping the power of the host's immune system to recognize and eliminate cancer cells. They have recently proven to be a successful strategy for combating a variety of cancers. Of the various types of immunotherapies, checkpoint blockade approaches have thus far produced significant clinical responses in several cancers including melanoma, non small-cell lung cancer, renal cancer, and prostate cancer. This review focuses on the biological rationale for using checkpoint blockade immunotherapeutic approaches in primary brain cancer and an up-to-date summary of current and ongoing checkpoint inhibitors-based clinical trials for malignant glioma. In addition, we expand on new concepts for further improving checkpoint blockade treatments, with a particular focus on the advantages of using genetically engineered mouse models for studies of immunotherapies in GBM. J. Cell. Biochem. 118: 2516-2527, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Imunoterapia/métodos , Neoplasias Experimentais , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Glioblastoma/genética , Glioblastoma/imunologia , Glioblastoma/patologia , Glioblastoma/terapia , Humanos , Camundongos , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapiaRESUMO
The possibility that chronic, multigenerational exposure to environmental estrogens selects for adaptive hormone-response phenotypes is a critical unanswered question. Embryos/larvae of killifish from an estrogenic-polluted environment (New Bedford Harbor, MA [NBH]) compared with those from a reference site overexpress estrogen receptor alpha (ERα) mRNA but are hyporesponsive to estradiol. Analysis of ERα mRNAs in the two populations revealed differences in splicing of the gene encoding ERα (esr1). Here we tested the transactivation functions of four differentially expressed ERα mRNAs and tracked their association with the hyporesponsive phenotype for three generations after transfer of NBH parents to a clean environment. Deletion variants ERαΔ6 and ERαΔ6-8 were specific to NBH killifish, had dominant negative functions in an in vitro reporter assay, and were heritable. Morpholino-mediated induction of ERαΔ6 mRNA in zebrafish embryos verified its role as a dominant negative ER on natural estrogen-responsive promoters. Alternate long (ERαL) and short (ERαS) 5'-variants were similar transcriptionally but differed in estrogen responsiveness (ERαS â« ERαL). ERαS accounted for high total ERα expression in first generation (F1) NBH embryos/larvae but this trait was abolished by transfer to clean water. By contrast, the hyporesponsive phenotype of F1 NBH embryos/larvae persisted after long-term laboratory holding but reverted to a normal or hyper-responsive phenotype after two or three generations, suggesting the acquisition of physiological or biochemical traits that compensate for ongoing expression of negative-acting ERαΔ6 and ERαΔ6-8 isoforms. We conclude that a heritable change in the pattern of alternative splicing of ERα pre-mRNA is part of a genetic adaptive response to estrogens in a polluted environment.
Assuntos
Processamento Alternativo/genética , Receptor alfa de Estrogênio/genética , Estrogênios/toxicidade , Animais , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Fundulidae , Reação em Cadeia da Polimerase , Splicing de RNA/genética , Poluentes Químicos da Água/toxicidade , Peixe-ZebraRESUMO
Increased activity of transcription factor NF-κB has been implicated in many B-cell lymphomas. We investigated effects of synthetic compound calafianin monomer (CM101) on biochemical and biological properties of NF-κB. In human 293 cells, CM101 selectively inhibited DNA binding by overexpressed NF-κB subunits REL (human c-Rel) and p65 as compared to NF-κB p50, and inhibition of REL and p65 DNA binding by CM101 required a conserved cysteine residue. CM101 also inhibited DNA binding by REL in human B-lymphoma cell lines, and the sensitivity of several B-lymphoma cell lines to CM101-induced proliferation arrest and apoptosis correlated with levels of cellular and nuclear REL. CM101 treatment induced both phosphorylation and decreased expression of anti-apoptotic protein Bcl-XL, a REL target gene product, in sensitive B-lymphoma cell lines. Ectopic expression of Bcl-XL protected SUDHL-2 B-lymphoma cells against CM101-induced apoptosis, and overexpression of a transforming mutant of REL decreased the sensitivity of BJAB B-lymphoma cells to CM101-induced apoptosis. Lipopolysaccharide-induced activation of NF-κB signaling upstream components occurred in RAW264.7 macrophages at CM101 concentrations that blocked NF-κB DNA binding. Direct inhibitors of REL may be useful for treating B-cell lymphomas in which REL is active, and may inhibit B-lymphoma cell growth at doses that do not affect some immune-related responses in normal cells.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Linfoma de Células B/tratamento farmacológico , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Tirosina/análogos & derivados , Células 3T3 , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Células L , Lipopolissacarídeos , Macrófagos/metabolismo , Camundongos , Subunidade p50 de NF-kappa B/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Fator de Transcrição RelA/antagonistas & inibidores , Tirosina/farmacologia , Proteína bcl-X/biossínteseRESUMO
BH3 mimetics such as ABT-263 induce apoptosis in a subset of cancer models. However, these drugs have shown limited clinical efficacy as single agents in small-cell lung cancer (SCLC) and other solid tumor malignancies, and rational combination strategies remain underexplored. To develop a novel therapeutic approach, we examined the efficacy of ABT-263 across >500 cancer cell lines, including 311 for which we had matched expression data for select genes. We found that high expression of the proapoptotic gene Bcl2-interacting mediator of cell death (BIM) predicts sensitivity to ABT-263. In particular, SCLC cell lines possessed greater BIM transcript levels than most other solid tumors and are among the most sensitive to ABT-263. However, a subset of relatively resistant SCLC cell lines has concomitant high expression of the antiapoptotic myeloid cell leukemia 1 (MCL-1). Whereas ABT-263 released BIM from complexes with BCL-2 and BCL-XL, high expression of MCL-1 sequestered BIM released from BCL-2 and BCL-XL, thereby abrogating apoptosis. We found that SCLCs were sensitized to ABT-263 via TORC1/2 inhibition, which led to reduced MCL-1 protein levels, thereby facilitating BIM-mediated apoptosis. AZD8055 and ABT-263 together induced marked apoptosis in vitro, as well as tumor regressions in multiple SCLC xenograft models. In a Tp53; Rb1 deletion genetically engineered mouse model of SCLC, the combination of ABT-263 and AZD8055 significantly repressed tumor growth and induced tumor regressions compared with either drug alone. Furthermore, in a SCLC patient-derived xenograft model that was resistant to ABT-263 alone, the addition of AZD8055 induced potent tumor regression. Therefore, addition of a TORC1/2 inhibitor offers a therapeutic strategy to markedly improve ABT-263 activity in SCLC.
Assuntos
Compostos de Anilina/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Sulfonamidas/uso terapêutico , Compostos de Anilina/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Engenharia Genética , Humanos , Concentração Inibidora 50 , Neoplasias Pulmonares/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Proteínas de Membrana/metabolismo , Camundongos , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Indução de Remissão , Carcinoma de Pequenas Células do Pulmão/patologia , Sulfonamidas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Human NEMO (NF-κB essential modulator) is a 419 residue scaffolding protein that, together with catalytic subunits IKKα and IKKß, forms the IκB kinase (IKK) complex, a key regulator of NF-κB pathway signaling. NEMO is an elongated homodimer comprising mostly α-helix. It has been shown that a NEMO fragment spanning residues 44-111, which contains the IKKα/ß binding site, is structurally disordered in the absence of bound IKKß. Herein we show that enforcing dimerization of NEMO1-120 or NEMO44-111 constructs through introduction of one or two interchain disulfide bonds, through oxidation of the native Cys54 residue and/or at position 107 through a Leu107Cys mutation, induces a stable α-helical coiled-coil structure that is preorganized to bind IKKß with high affinity. Chemical and thermal denaturation studies showed that, in the context of a covalent dimer, the ordered structure was stabilized relative to the denatured state by up to 3 kcal/mol. A full-length NEMO-L107C protein formed covalent dimers upon treatment of mammalian cells with H2O2. Furthermore, NEMO-L107C bound endogenous IKKß in A293T cells, reconstituted TNF-induced NF-κB signaling in NEMO-deficient cells, and interacted with TRAF6. Our results indicate that the IKKß binding domain of NEMO possesses an ordered structure in the unbound state, provided that it is constrained within a dimer as is the case in the constitutively dimeric full-length NEMO protein. The stability of the NEMO coiled coil is maintained by strong interhelix interactions in the region centered on residue 54. The disulfide-linked constructs we describe herein may be useful for crystallization of NEMO's IKKß binding domain in the absence of bound IKKß, thereby facilitating the structural characterization of small-molecule inhibitors.
Assuntos
Dissulfetos/química , Dissulfetos/metabolismo , Quinase I-kappa B/química , Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Humanos , Peróxido de Hidrogênio/farmacologia , Quinase I-kappa B/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Oxidantes/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Fator 6 Associado a Receptor de TNF/química , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismoRESUMO
Although several groups have demonstrated that concomitant use of MEK and phosphoinositide 3-kinase (PI3K) inhibitors (MEKi/PI3Ki) can induce dramatic tumor regressions in mouse models of KRAS-mutant non-small cell lung cancer (NSCLC), ongoing clinical trials investigating this strategy have been underwhelming to date. While efficacy may be hampered by a narrow therapeutic index, the contribution of biologic heterogeneity in the response of KRAS-mutant NSCLCs to MEKi/PI3Ki has been largely unexplored. In this study, we find that most human KRAS-mutant NSCLC cell lines fail to undergo marked apoptosis in response to MEKi/PI3Ki, which is key for tumor responsiveness in vivo. This heterogeneity of apoptotic response occurs despite relatively uniform induction of growth arrest. Using a targeted short hairpin RNA screen of BCL-2 family members, we identify BIM, PUMA, and BCL-XL as key regulators of the apoptotic response induced by MEKi/PI3Ki, with decreased expression of BIM and PUMA relative to BCL-XL in cell lines with intrinsic resistance. In addition, by modeling adaptive resistance to MEKi/PI3Ki both in vitro and in vivo, we find that, upon the development of resistance, tumors have a diminished apoptotic response due to downregulation of BIM and PUMA. These results suggest that the inability to induce apoptosis may limit the effectiveness of MEKi/PI3Ki for KRAS-mutant NSCLCs by contributing to intrinsic and adaptive resistance to this therapy.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas ras/genética , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Proteínas ras/metabolismoRESUMO
Colorectal cancers harboring KRAS or BRAF mutations are refractory to current targeted therapies. Using data from a high-throughput drug screen, we have developed a novel therapeutic strategy that targets the apoptotic machinery using the BCL-2 family inhibitor ABT-263 (navitoclax) in combination with a TORC1/2 inhibitor, AZD8055. This combination leads to efficient apoptosis specifically in KRAS- and BRAF-mutant but not wild-type (WT) colorectal cancer cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not WT, colorectal cancers, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS-mutant colorectal cancer xenograft and genetically engineered mouse models of colorectal cancer, but not in the corresponding KRAS-WT colorectal cancer models. These data suggest that the combination of BCL-2/BCL-XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers.
Assuntos
Compostos de Anilina/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Morfolinas/uso terapêutico , Complexos Multiproteicos/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Sulfonamidas/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Compostos de Anilina/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Mutantes , Camundongos Nus , Morfolinas/farmacologia , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras) , Sulfonamidas/farmacologia , Proteína bcl-X/antagonistas & inibidores , Proteínas ras/genéticaRESUMO
Most anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancers (NSCLCs) are highly responsive to treatment with ALK tyrosine kinase inhibitors (TKIs). However, patients with these cancers invariably relapse, typically within 1 year, because of the development of drug resistance. Herein, we report findings from a series of lung cancer patients (n = 18) with acquired resistance to the ALK TKI crizotinib. In about one-fourth of patients, we identified a diverse array of secondary mutations distributed throughout the ALK TK domain, including new resistance mutations located in the solvent-exposed region of the adenosine triphosphate-binding pocket, as well as amplification of the ALK fusion gene. Next-generation ALK inhibitors, developed to overcome crizotinib resistance, had differing potencies against specific resistance mutations. In addition to secondary ALK mutations and ALK gene amplification, we also identified aberrant activation of other kinases including marked amplification of KIT and increased autophosphorylation of epidermal growth factor receptor in drug-resistant tumors from patients. In a subset of patients, we found evidence of multiple resistance mechanisms developing simultaneously. These results highlight the unique features of TKI resistance in ALK-positive NSCLCs and provide the rationale for pursuing combinatorial therapeutics that are tailored to the precise resistance mechanisms identified in patients who relapse on crizotinib treatment.
Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Quinase do Linfoma Anaplásico , Linhagem Celular Tumoral , Crizotinibe , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Immunoblotting , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/enzimologia , Mutação , Reação em Cadeia da PolimeraseRESUMO
In this report, we investigated the effects of the natural product parthenolide on human B-lymphoma cell lines. We show that parthenolide inhibited NF-κB transcription factor c-Rel (REL). In addition, the sensitivity of several human B-lymphoma cell lines to parthenolide-induced apoptosis inversely correlated with their levels of anti-apoptosis protein Bcl-X(L). Furthermore, ectopic expression of Bcl-X(L) (but not Bcl-2) in two B-lymphoma cell lines decreased their sensitivity to parthenolide-induced apoptosis. Finally, over-expression of a transforming mutant of REL, which increased expression of endogenous Bcl-X(L), decreased the sensitivity of BJAB B-lymphoma cells to parthenolide-induced apoptosis. These results demonstrate that the NF-κB target gene products Bcl-X(L) and Bcl-2 can play different roles in protecting B-lymphoma cells from chemical-induced apoptosis.