RESUMO
For a majority of patients with non-small cell lung cancer with EGFR mutations, treatment with EGFR inhibitors (EGFRi) induces a clinical response. Despite this initial reduction in tumor size, residual disease persists that leads to disease relapse. Elucidating the preexisting biological differences between sensitive cells and surviving drug-tolerant persister cells and deciphering how drug-tolerant cells evolve in response to treatment could help identify strategies to improve the efficacy of EGFRi. In this study, we tracked the origins and clonal evolution of drug-tolerant cells at a high resolution by using an expressed barcoding system coupled with single-cell RNA sequencing. This platform enabled longitudinal profiling of gene expression and drug sensitivity in response to EGFRi across a large number of clones. Drug-tolerant cells had higher expression of key survival pathways such as YAP and EMT at baseline and could also differentially adapt their gene expression following EGFRi treatment compared with sensitive cells. In addition, drug combinations targeting common downstream components (MAPK) or orthogonal factors (chemotherapy) showed greater efficacy than EGFRi alone, which is attributable to broader targeting of the heterogeneous EGFRi-tolerance mechanisms present in tumors. Overall, this approach facilitates thorough examination of clonal evolution in response to therapy that could inform the development of improved diagnostic approaches and treatment strategies for targeting drug-tolerant cells. SIGNIFICANCE: The evolution and heterogeneity of EGFR inhibitor tolerance are identified in a large number of clones at enhanced cellular and temporal resolution using an expressed barcode technology coupled with single-cell RNA sequencing.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Recidiva Local de Neoplasia , Tolerância a MedicamentosRESUMO
It is well-known that some species of lizard have an exceptional ability known as caudal autotomy (voluntary self-amputation of the tail) as an anti-predation mechanism. After amputation occurs, they can regenerate their new tails in a few days. The new tail section is generally shorter than the original one and is composed of cartilage rather than vertebrae bone. In addition, the skin of the regenerated tail distinctly differs from its original appearance. We performed a proteomics analysis for extracts derived from regenerating lizard tail tissues after amputation and found that endoplasmin (ENPL) was the main factor among proteins up-regulated in expression during regeneration. Thus, we performed further experiments to determine whether ENPL could induce chondrogenesis of tonsil-derived mesenchymal stem cells (T-MSCs). In this study, we found that chondrogenic differentiation was associated with an increase of ENPL expression by ER stress. We also found that ENPL was involved in chondrogenic differentiation of T-MSCs by suppressing extracellular signal-regulated kinase (ERK) phosphorylation. [BMB Reports 2022; 55(5): 226-231].
Assuntos
Lagartos , Células-Tronco Mesenquimais , Animais , Diferenciação Celular , Células Cultivadas , Condrócitos/metabolismo , Condrogênese , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Lagartos/fisiologia , Glicoproteínas de Membrana , Tonsila Palatina/metabolismoRESUMO
Scleroderma is an autoimmune disease caused by the abnormal regulation of extracellular matrix synthesis and is activated by non-regulated inflammatory cells and cytokines. Echinochrome A (EchA), a natural pigment isolated from sea urchins, has been demonstrated to have antioxidant activities and beneficial effects in various disease models. The present study demonstrates for the first time that EchA treatment alleviates bleomycin-induced scleroderma by normalizing dermal thickness and suppressing collagen deposition in vivo. EchA treatment reduces the number of activated myofibroblasts expressing α-SMA, vimentin, and phosphorylated Smad3 in bleomycin-induced scleroderma. In addition, it decreased the number of macrophages, including M1 and M2 types in the affected skin, suggesting the induction of an anti-inflammatory effect. Furthermore, EchA treatment markedly attenuated serum levels of inflammatory cytokines, such as tumor necrosis factor-α and interferon-γ, in a murine scleroderma model. Taken together, these results suggest that EchA is highly useful for the treatment of scleroderma, exerting anti-fibrosis and anti-inflammatory effects.
Assuntos
Anti-Inflamatórios/farmacologia , Macrófagos/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Naftoquinonas/farmacologia , Escleroderma Sistêmico/prevenção & controle , Pele/efeitos dos fármacos , Actinas/metabolismo , Animais , Bleomicina , Colágeno/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Fibrose , Humanos , Mediadores da Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/imunologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Fosforilação , Células RAW 264.7 , Escleroderma Sistêmico/induzido quimicamente , Escleroderma Sistêmico/imunologia , Escleroderma Sistêmico/metabolismo , Pele/imunologia , Pele/metabolismo , Pele/patologia , Proteína Smad3/metabolismo , Vimentina/metabolismoRESUMO
Genomic studies are revolutionizing clinical oncology, but bridging the lab and the bedside requires the ability to efficiently interrogate rare genetic lesions in unexpected pathological settings using preclinical models. Oncogenes can exhibit intrinsic drug resistance to targeted therapy in different cells of origin, adding complexity to clinical interpretations of genomic findings. Here, we capitalize on the flexibility of engineered cell systems to rapidly profile known multi-kinase inhibitors that harbor rearranged during transfection (RET) kinase activity across multiple RET fusions. Identifying ponatinib as the most potent RET inhibitor tested, we used ponatinib to gauge therapeutic responsiveness in RET fusion-positive patient-derived xenograft (PDX) models. Using a genomics guided outlier approach, we identified 4 RET fusion PDX models with 3 different fusion partners (KIF5B, CCDC6, and NCOA4) in both non-small cell lung cancer and colorectal cancer. By comparing ponatinib activity in RET fusion-positive and RET fusion-negative PDX models alongside a standard of care chemotherapeutic agent, we show that RET fusions in colorectal tumors are therapeutically responsive to RET inhibition. Finally, we suggest that coupling engineered cell systems and genomics guided PDX model selection provides a rapid workflow to triage rare genomics findings.
RESUMO
PURPOSE: Non-small cell lung cancers (NSCLCs) harboring ALK gene rearrangements (ALK+) typically become resistant to the first-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) crizotinib through development of secondary resistance mutations in ALK or disease progression in the brain. Mutations that confer resistance to second-generation ALK TKIs ceritinib and alectinib have also been identified. Here, we report the structure and first comprehensive preclinical evaluation of the next-generation ALK TKI brigatinib. EXPERIMENTAL DESIGN: A kinase screen was performed to evaluate the selectivity profile of brigatinib. The cellular and in vivo activities of ALK TKIs were compared using engineered and cancer-derived cell lines. The brigatinib-ALK co-structure was determined. RESULTS: Brigatinib potently inhibits ALK and ROS1, with a high degree of selectivity over more than 250 kinases. Across a panel of ALK+ cell lines, brigatinib inhibited native ALK (IC50, 10 nmol/L) with 12-fold greater potency than crizotinib. Superior efficacy of brigatinib was also observed in mice with ALK+ tumors implanted subcutaneously or intracranially. Brigatinib maintained substantial activity against all 17 secondary ALK mutants tested in cellular assays and exhibited a superior inhibitory profile compared with crizotinib, ceritinib, and alectinib at clinically achievable concentrations. Brigatinib was the only TKI to maintain substantial activity against the most recalcitrant ALK resistance mutation, G1202R. The unique, potent, and pan-ALK mutant activity of brigatinib could be rationalized by structural analyses. CONCLUSIONS: Brigatinib is a highly potent and selective ALK inhibitor. These findings provide the molecular basis for the promising activity being observed in ALK+, crizotinib-resistant patients with NSCLC being treated with brigatinib in clinical trials. Clin Cancer Res; 22(22); 5527-38. ©2016 AACR.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Compostos Organofosforados/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Quinase do Linfoma Anaplásico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Crizotinibe , Células Hep G2 , Humanos , Neoplasias Pulmonares/metabolismo , Mutação/efeitos dos fármacos , Pirazóis/farmacologia , Piridinas/farmacologia , Sulfonas/farmacologia , Células U937RESUMO
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
PURPOSE: KIT is the major oncogenic driver of gastrointestinal stromal tumors (GIST). Imatinib, sunitinib, and regorafenib are approved therapies; however, efficacy is often limited by the acquisition of polyclonal secondary resistance mutations in KIT, with those located in the activation (A) loop (exons 17/18) being particularly problematic. Here, we explore the KIT-inhibitory activity of ponatinib in preclinical models and describe initial characterization of its activity in patients with GIST. EXPERIMENTAL DESIGN: The cellular and in vivo activities of ponatinib, imatinib, sunitinib, and regorafenib against mutant KIT were evaluated using an accelerated mutagenesis assay and a panel of engineered and GIST-derived cell lines. The ponatinib-KIT costructure was also determined. The clinical activity of ponatinib was examined in three patients with GIST previously treated with all three FDA-approved agents. RESULTS: In engineered and GIST-derived cell lines, ponatinib potently inhibited KIT exon 11 primary mutants and a range of secondary mutants, including those within the A-loop. Ponatinib also induced regression in engineered and GIST-derived tumor models containing these secondary mutations. In a mutagenesis screen, 40 nmol/L ponatinib was sufficient to suppress outgrowth of all secondary mutants except V654A, which was suppressed at 80 nmol/L. This inhibitory profile could be rationalized on the basis of structural analyses. Ponatinib (30 mg daily) displayed encouraging clinical activity in two of three patients with GIST. CONCLUSION: Ponatinib possesses potent activity against most major clinically relevant KIT mutants and has demonstrated preliminary evidence of activity in patients with refractory GIST. These data strongly support further evaluation of ponatinib in patients with GIST.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Tumores do Estroma Gastrointestinal/genética , Imidazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , Piridazinas/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Benzamidas/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Éxons , Feminino , Tumores do Estroma Gastrointestinal/diagnóstico , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/patologia , Humanos , Mesilato de Imatinib , Imidazóis/química , Imidazóis/uso terapêutico , Indóis/farmacologia , Concentração Inibidora 50 , Modelos Moleculares , Conformação Molecular , Mutação , Recidiva Local de Neoplasia , Piperazinas/farmacologia , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/química , Piridazinas/química , Piridazinas/uso terapêutico , Pirimidinas/farmacologia , Pirróis/farmacologia , Sunitinibe , Tomografia Computadorizada por Raios X , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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
The PI3K pathway is genetically altered in excess of 70% of breast cancers, largely through PIK3CA mutation and HER2 amplification. Preclinical studies have suggested that these subsets of breast cancers are particularly sensitive to PI3K inhibitors; however, the reasons for this heightened sensitivity are mainly unknown. We investigated the signaling effects of PI3K inhibition in PIK3CA mutant and HER2 amplified breast cancers using PI3K inhibitors currently in clinical trials. Unexpectedly, we found that in PIK3CA mutant and HER2 amplified breast cancers sensitive to PI3K inhibitors, PI3K inhibition led to a rapid suppression of Rac1/p21-activated kinase (PAK)/protein kinase C-RAF (C-RAF)/ protein kinase MEK (MEK)/ERK signaling that did not involve RAS. Furthermore, PI3K inhibition led to an ERK-dependent up-regulation of the proapoptotic protein, BIM, followed by induction of apoptosis. Expression of a constitutively active form of Rac1 in these breast cancer models blocked PI3Ki-induced down-regulation of ERK phosphorylation, apoptosis, and mitigated PI3K inhibitor sensitivity in vivo. In contrast, protein kinase AKT inhibitors failed to block MEK/ERK signaling, did not up-regulate BIM, and failed to induce apoptosis. Finally, we identified phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1) as the PI(3,4,5)P3-dependent guanine exchange factor for Rac1 responsible for regulation of the Rac1/C-RAF/MEK/ERK pathway in these cells. The expression level of P-Rex1 correlates with sensitivity to PI3K inhibitors in these breast cancer cell lines. Thus, PI3K inhibitors have enhanced activity in PIK3CA mutant and HER2 amplified breast cancers in which PI3K inhibition down-regulates both the AKT and Rac1/ERK pathways. In addition, P-Rex1 may serve as a biomarker to predict response to single-agent PI3K inhibitors within this subset of breast cancers.
Assuntos
Neoplasias da Mama/fisiopatologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Neuropeptídeos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Classe I de Fosfatidilinositol 3-Quinases , Biologia Computacional , Bases de Dados Genéticas , Feminino , Immunoblotting , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Nus , Fosfatidilinositol 3-Quinases/genética , Receptor ErbB-2/genéticaRESUMO
PURPOSE: Mutations in receptor tyrosine kinase (RTK) genes can confer resistance to receptor-targeted therapies. A T798M mutation in the HER2 oncogene has been shown to confer resistance to the tyrosine kinase inhibitor (TKI) lapatinib. We studied the mechanisms of HER2-T798M-induced resistance to identify potential strategies to overcome that resistance. EXPERIMENTAL DESIGN: HER2-T798M was stably expressed in BT474 and MCF10A cells. Mutant cells and xenografts were evaluated for effects of the mutation on proliferation, signaling, and tumor growth after treatment with combinations of inhibitors targeting the EGFR/HER2/HER3/PI3K axis. RESULTS: A low 3% allelic frequency of the T798M mutant shifted 10-fold the IC50 of lapatinib. In mutant-expressing cells, lapatinib did not block basal phosphorylation of HER2, HER3, AKT, and ERK1/2. In vitro kinase assays showed increased autocatalytic activity of HER2-T798M. HER3 association with PI3K p85 was increased in mutant-expressing cells. BT474-T798M cells were also resistant to the HER2 antibody trastuzumab. These cells were sensitive to the pan-PI3K inhibitors BKM120 and XL147 and the irreversible HER2/EGFR TKI afatinib but not the MEK1/2 inhibitor CI-1040, suggesting continued dependence of the mutant cells on ErbB receptors and downstream PI3K signaling. BT474-T798M cells showed increased expression of the EGFR ligands EGF, TGFα, amphiregulin, and HB-EGF. Addition of the EGFR neutralizing antibody cetuximab or lapatinib restored trastuzumab sensitivity of BT474-T798M cells and xenografts, suggesting that increased EGFR ligand production was causally associated with drug resistance. CONCLUSIONS: Simultaneous blockade of HER2 and EGFR should be an effective treatment strategy against HER2 gene-amplified breast cancer cells harboring T798M mutant alleles.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/metabolismo , Expressão Gênica , Mutação , Receptor ErbB-2/genética , Substituição de Aminoácidos , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Catálise , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Códon , Modelos Animais de Doenças , Receptores ErbB/antagonistas & inibidores , Feminino , Amplificação de Genes , Humanos , Ligantes , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The receptor tyrosine kinase c-MET is the high-affinity receptor for the hepatocyte growth factor (HGF). The HGF/c-MET axis is often dysregulated in tumors. c-MET activation can be caused by MET gene amplification, activating mutations, and auto- or paracrine mechanisms. Thus, c-MET inhibitors are under development as anticancer drugs. Tivantinib (ARQ 197) was reported as a small-molecule c-MET inhibitor and early clinical studies suggest antitumor activity. To assess whether the antitumor activity of tivantinib was due to inhibition of c-MET, we compared the activity of tivantinib with other c-MET inhibitors in both c-MET-addicted and nonaddicted cancer cells. As expected, other c-MET inhibitors, crizotinib and PHA-665752, suppressed the growth of c-MET-addicted cancers, but not the growth of cancers that are not addicted to c-MET. In contrast, tivantinib inhibited cell viability with similar potency in both c-MET-addicted and nonaddicted cells. These results suggest that tivantinib exhibits its antitumor activity in a manner independent of c-MET status. Tivantinib treatment induced a G(2)-M cell-cycle arrest in EBC1 cells similarly to vincristine treatment, whereas PHA-665752 or crizotinib treatment markedly induced G(0)-G(1) cell-cycle arrest. To identify the additional molecular target of tivantinib, we conducted COMPARE analysis, an in silico screening of a database of drug sensitivities across 39 cancer cell lines (JFCR39), and identified microtubule as a target of tivantinib. Tivantinib-treated cells showed typical microtubule disruption similar to vincristine and inhibited microtubule assembly in vitro. These results suggest that tivantinib inhibits microtubule polymerization in addition to inhibiting c-MET.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Pirrolidinonas/farmacologia , Quinolinas/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fase G2/efeitos dos fármacos , Humanos , Microtúbulos/efeitos dos fármacos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tubulina (Proteína)/química , Moduladores de Tubulina/farmacologiaRESUMO
KRAS is the most commonly mutated oncogene, yet no effective targeted therapies exist for KRAS mutant cancers. We developed a pooled shRNA-drug screen strategy to identify genes that, when inhibited, cooperate with MEK inhibitors to effectively treat KRAS mutant cancer cells. The anti-apoptotic BH3 family gene BCL-XL emerged as a top hit through this approach. ABT-263 (navitoclax), a chemical inhibitor that blocks the ability of BCL-XL to bind and inhibit pro-apoptotic proteins, in combination with a MEK inhibitor led to dramatic apoptosis in many KRAS mutant cell lines from different tissue types. This combination caused marked in vivo tumor regressions in KRAS mutant xenografts and in a genetically engineered KRAS-driven lung cancer mouse model, supporting combined BCL-XL/MEK inhibition as a potential therapeutic approach for KRAS mutant cancers.
Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/genética , Sulfonamidas/farmacologia , Proteína bcl-X/antagonistas & inibidores , Compostos de Anilina/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Benzimidazóis/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Neoplasias/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sulfonamidas/uso terapêuticoRESUMO
Brain metastases are a serious obstacle in the treatment of patients with human epidermal growth factor receptor-2 (HER2)-amplified breast cancer. Although extracranial disease is controlled with HER2 inhibitors in the majority of patients, brain metastases often develop. Because these brain metastases do not respond to therapy, they are frequently the reason for treatment failure. We developed a mouse model of HER2-amplified breast cancer brain metastasis using an orthotopic xenograft of BT474 cells. As seen in patients, the HER2 inhibitors trastuzumab and lapatinib controlled tumor progression in the breast but failed to contain tumor growth in the brain. We observed that the combination of a HER2 inhibitor with an anti-VEGF receptor-2 (VEGFR2) antibody significantly slows tumor growth in the brain, resulting in a striking survival benefit. This benefit appears largely due to an enhanced antiangiogenic effect: Combination therapy reduced both the total and functional microvascular density in the brain xenografts. In addition, the combination therapy led to a marked increase in necrosis of the brain lesions. Moreover, we observed even better antitumor activity after combining both trastuzumab and lapatinib with the anti-VEGFR2 antibody. This triple-drug combination prolonged the median overall survival fivefold compared with the control-treated group and twofold compared with either two-drug regimen. These findings support the clinical development of this three-drug regimen for the treatment of HER2-amplified breast cancer brain metastases.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/secundário , Neoplasias da Mama/tratamento farmacológico , Amplificação de Genes , Terapia de Alvo Molecular , Receptor ErbB-2/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/patologia , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/patologia , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Diagnóstico por Imagem , Modelos Animais de Doenças , Feminino , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/patologia , Lapatinib , Camundongos , Necrose , Neovascularização Patológica/tratamento farmacológico , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Análise de Sobrevida , Trastuzumab , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The phosphoinositide 3-kinase (PI3K)/AKT and RAF/MEK/ERK signaling pathways are activated in a wide range of human cancers. In many cases, concomitant inhibition of both pathways is necessary to block proliferation and induce cell death and tumor shrinkage. Several feedback systems have been described in which inhibition of one intracellular pathway leads to activation of a parallel signaling pathway, thereby decreasing the effectiveness of single-agent targeted therapies. In this study, we describe a feedback mechanism in which MEK inhibition leads to activation of PI3K/AKT signaling in EGFR and HER2-driven cancers. We found that MEK inhibitor-induced activation of PI3K/AKT resulted from hyperactivation of ERBB3 as a result of the loss of an inhibitory threonine phosphorylation in the conserved juxtamembrane domains of EGFR and HER2. Mutation of this amino acid led to increased ERBB receptor activation and upregulation of the ERBB3/PI3K/AKT signaling pathway, which was no longer responsive to MEK inhibition. Taken together, these results elucidate an important, dominant feedback network regulating central oncogenic pathways in human cancer.
Assuntos
Retroalimentação , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Linhagem Celular , Ativação Enzimática , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Receptores Proteína Tirosina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em TandemRESUMO
PURPOSE: Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine tumor, often metastatic at presentation, for which current chemotherapeutic regimens are largely ineffective. As its pathogenesis is still unknown, we hypothesized that deregulation of signaling pathways commonly activated in cancer may contribute to MCC tumorigenesis and may provide insights into targeted therapy approaches for this malignancy. EXPERIMENTAL DESIGN: We retrospectively profiled 60 primary MCC samples using a SNaPshot-based tumor genotyping assay to screen for common mutations in 13 cancer genes. RESULTS: We identified mutations in 9 (15%) MCC primary tumors, including mutations in TP53 (3 of 60) and activating mutations in the PIK3CA gene (6 of 60). Sanger sequencing of the primary MCC tumors detected one additional PIK3CA mutation (R19K) that had not been previously described in cancer. Merkel cell polyoma virus (MCPyV) was detected in 38 (66%) MCC cases and patients with MCPyV-positive cancers showed a trend toward better survival. With one exception, the presence of MCPyV and activating mutations in PIK3CA appeared mutually exclusive. We observed that signaling through the PI3K/pAKT pathway was active in one MCPyV-positive and in all MCPyV-negative MCC cell lines, as evidenced by AKT phosphorylation. Importantly, the presence of a PIK3CA-activating mutation was associated with sensitivity to treatment with ZST474, a specific phosphoinositide 3-kinase (PI3K) inhibitor, and to NVP-BEZ235, a dual PI3K/mTOR inhibitor, targeted agents under active clinical development. CONCLUSIONS: PI3K pathway activation may drive tumorigenesis in a subset of MCC and screening these tumors for PIK3CA mutations could help identify patients who may respond to treatment with PI3K pathway inhibitors.
Assuntos
Carcinoma de Célula de Merkel/genética , Fosfatidilinositol 3-Quinases/genética , Transdução de Sinais , Neoplasias Cutâneas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Carcinoma de Célula de Merkel/metabolismo , Carcinoma de Célula de Merkel/mortalidade , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Estadiamento de Neoplasias , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/mortalidadeRESUMO
Cancers with specific genetic mutations are susceptible to selective kinase inhibitors. However, there is a wide spectrum of benefit among cancers harboring the same sensitizing genetic mutations. Herein, we measured apoptotic rates among cell lines sharing the same driver oncogene following treatment with the corresponding kinase inhibitor. There was a wide range of kinase inhibitor-induced apoptosis despite comparable inhibition of the target and associated downstream signaling pathways. Surprisingly, pretreatment RNA levels of the BH3-only pro-apoptotic BIM strongly predicted the capacity of EGFR, HER2, and PI3K inhibitors to induce apoptosis in EGFR-mutant, HER2-amplified, and PIK3CA-mutant cancers, respectively, but BIM levels did not predict responsiveness to standard chemotherapies. Furthermore, BIM RNA levels in EGFR-mutant lung cancer specimens predicted response and duration of clinical benefit from EGFR inhibitors. These findings suggest assessment of BIM levels in treatment-naïve tumor biopsies may indicate the degree of benefit from single-agent kinase inhibitors in multiple oncogene-addiction paradigms.
Assuntos
Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular Tumoral , Estudos de Coortes , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Oncogenes , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Estudos Retrospectivos , Transdução de Sinais/efeitos dos fármacosRESUMO
Therapies inhibiting receptor tyrosine kinases (RTKs) are effective against some human cancers when they lead to simultaneous downregulation of PI3K/AKT and MEK/ERK signaling. However, mutant KRAS has the capacity to directly activate ERK and PI3K signaling, and this is thought to underlie the resistance of KRAS mutant cancers to RTK inhibitors. Here, we have elucidated the molecular regulation of both the PI3K/AKT and MEK/ERK signaling pathways in KRAS mutant colorectal cancer cells and identified combination therapies that lead to robust cancer cell apoptosis. KRAS knockdown using shRNA suppressed ERK signaling in all of the human KRAS mutant colorectal cancer cell lines examined. However, no decrease, and actually a modest increase, in AKT phosphorylation was often seen. By performing PI3K immunoprecipitations, we determined that RTKs, often IGF-IR, regulated PI3K signaling in the KRAS mutant cell lines. This conclusion was also supported by the observation that specific RTK inhibition led to marked suppression of PI3K signaling and biochemical assessment of patient specimens. Interestingly, combination of RTK and MEK inhibitors led to concomitant inhibition of PI3K and MEK signaling, marked growth suppression, and robust apoptosis of human KRAS mutant colorectal cancer cell lines in vitro and upon xenografting in mice. These findings provide a framework for utilizing RTK inhibitors in the treatment of KRAS mutant colorectal cancers.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas ras/genética , Animais , Apoptose , Sequência de Bases , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Primers do DNA/genética , Técnicas de Silenciamento de Genes , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Fosfatidilinositol 3-Quinases/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , RNA Interferente Pequeno/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Transdução de Sinais , Transplante Heterólogo , Proteínas ras/antagonistas & inibidoresRESUMO
Phosphatiditylinositide-3-kinase (PI3K) is activated in some cancers by direct mutation, but it is activated more commonly in cancer by mutation of upstream acting receptor tyrosine kinases (TK). At present, there is no systematic method to determine which TK signaling cascades activate PI3K in certain cancers, despite the likely utility of such information to help guide selection of tyrosine kinase inhibitor (TKI) drug strategies for personalized therapy. Here, we present a quantitative liquid chromatography tandem mass spectrometry approach that identifies upstream activators of PI3K both in vitro and in vivo. Using non-small cell lung carcinoma to illustrate this approach, we show a correct identification of the mechanism of PI3K activation in several models, thereby identifying the most appropriate TKI to downregulate PI3K signaling. This approach also determined the molecular mechanisms and adaptors required for PI3K activation following inhibition of the mTOR kinase TORC1. We further validated the approach in breast cancer cells with mutational activation of PIK3CA, where tandem mass spectrometry detected and quantitatively measured the abundance of a helical domain mutant (E545K) of PIK3CA connected to PI3K activation. Overall, our findings establish a mass spectrometric approach to identify functional interactions that govern PI3K regulation in cancer cells. Using this technique to define the pathways that activate PI3K signaling in a given tumor could help inform clinical decision making by helping guide personalized therapeutic strategies for different patients.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/enzimologia , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Neoplasias Pulmonares/enzimologia , Espectrometria de Massas em Tandem/métodos , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Classe I de Fosfatidilinositol 3-Quinases , Ativação Enzimática , Humanos , Neoplasias Pulmonares/genética , Camundongos , Camundongos Nus , Mutação , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Sirolimo/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
ErbB3 is a critical activator of phosphoinositide 3-kinase (PI3K) signaling in epidermal growth factor receptor (EGFR; ErbB1), ErbB2 [human epidermal growth factor receptor 2 (HER2)], and [hepatocyte growth factor receptor (MET)] addicted cancers, and reactivation of ErbB3 is a prominent method for cancers to become resistant to ErbB inhibitors. In this study, we evaluated the in vivo efficacy of a therapeutic anti-ErbB3 antibody, MM-121. We found that MM-121 effectively blocked ligand-dependent activation of ErbB3 induced by either EGFR, HER2, or MET. Assessment of several cancer cell lines revealed that MM-121 reduced basal ErbB3 phosphorylation most effectively in cancers possessing ligand-dependent activation of ErbB3. In those cancers, MM-121 treatment led to decreased ErbB3 phosphorylation and, in some instances, decreased ErbB3 expression. The efficacy of single-agent MM-121 was also examined in xenograft models. A machine learning algorithm found that MM-121 was most effective against xenografts with evidence of ligand-dependent activation of ErbB3. We subsequently investigated whether MM-121 treatment could abrogate resistance to anti-EGFR therapies by preventing reactivation of ErbB3. We observed that an EGFR mutant lung cancer cell line (HCC827), made resistant to gefitinib by exogenous heregulin, was resensitized by MM-121. In addition, we found that a de novo lung cancer mouse model induced by EGFR T790M-L858R rapidly became resistant to cetuximab. Resistance was associated with an increase in heregulin expression and ErbB3 activation. However, concomitant cetuximab treatment with MM-121 blocked reactivation of ErbB3 and resulted in a sustained and durable response. Thus, these results suggest that targeting ErbB3 with MM-121 can be an effective therapeutic strategy for cancers with ligand-dependent activation of ErbB3.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias/terapia , Receptor ErbB-3/antagonistas & inibidores , Receptor ErbB-3/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Células CHO , Linhagem Celular Tumoral , Cetuximab , Cricetinae , Cricetulus , Receptores ErbB/antagonistas & inibidores , Gefitinibe , Humanos , Ligantes , Camundongos , Neoplasias/metabolismo , Neuregulina-1/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Receptor ErbB-3/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MET amplification activates ERBB3/PI3K/AKT signaling in EGFR mutant lung cancers and causes resistance to EGFR kinase inhibitors. We demonstrate that MET activation by its ligand, HGF, also induces drug resistance, but through GAB1 signaling. Using high-throughput FISH analyses in both cell lines and in patients with lung cancer, we identify subpopulations of cells with MET amplification prior to drug exposure. Surprisingly, HGF accelerates the development of MET amplification both in vitro and in vivo. EGFR kinase inhibitor resistance, due to either MET amplification or autocrine HGF production, was cured in vivo by combined EGFR and MET inhibition. These findings highlight the potential to prospectively identify treatment naive, patients with EGFR-mutant lung cancer who will benefit from initial combination therapy.