RESUMO
DO-2 is a highly selective MNNG HOS transforming (MET) inhibitor. This deuterated drug is thought to diminish the formation of the Aldehyde Oxidase 1 inactive metabolite M3. For various reasons, quantification of DO-2 and its metabolites M3 and DO-5 is highly relevant. In this study, we present an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to quantify DO-2, M3 and DO-5. Rolipram served as the internal standard. Aliquots of 25 µL were mixed with 100 µL internal standard consisting of 10 ng/mL rolipram in acetonitrile. Separation of the analytes was achieved on an Acquity UPLC ® HSS T3 column, utilizing gradient elution with water/formic acid and acetonitrile/formic acid at a flow-rate of 0.400 mL/min. Calibration curves were linear in the range of 1.00 - 1000 ng/mL for DO-2 and DO-5, and 2.00 - 2000 ng/mL for M3 in human plasma. The within-run and between-run precisions of DO-2, DO-5 and M3, also at the level of the LLQ, were within 12.1%, while the accuracy ranged from 89.5 to 108.7%. All values for accuracy, within-run and between-run precisions met the criteria set by the Food and Drug Administration. The method was effectively employed in the analysis of samples obtained from a clinical trial.
Assuntos
Formiatos , Espectrometria de Massas em Tandem , Humanos , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Rolipram , Acetonitrilas , Reprodutibilidade dos Testes , Cromatografia Líquida de Alta Pressão/métodosRESUMO
INTRODUCTION: Targeted therapy in non-small cell lung cancer (NSCLC) patients with mesenchymal epithelial transition (MET) exon 14 skipping mutations (METex14) and MET amplifications has improved patients' outcomes. The development of more potent MET kinase inhibitors could further benefit these patients. The aim of this trial is to determine the safety and recommended phase 2 dose (RP2D) of OMO-1 (an oral dual MET kinase/OCT-2 inhibitor) and to assess preliminary clinical efficacy in METex14-positive NSCLC and other MET-positive solid tumors. MATERIALS AND METHODS: This was a first-in-patient, open-label, multicenter study of OMO-1 in patients with locally advanced or metastatic solid malignancies. A standard 3 + 3 dose escalation design was utilized starting at a dose level of 100 mg BID continuously. Preliminary efficacy was investigated in patients with METex14-positive NSCLC, and MET amplified NSCLC and other solid tumors (MET basket). RESULTS: In the dose-escalation part, 24 patients were included in 5 dose levels ranging from 100 mg twice daily (BID) to 400 mg BID. Most common adverse events (≥ 20%) were nausea, fatigue, vomiting, increased blood creatinine, and headache. The RP2D was determined at 250 mg BID. In the expansion cohorts, 15 patients were included (10 in METex14-positive NSCLC cohort and 5 in MET basket cohort) and received either 200 or 250 mg BID. Eight out of the 10 patients with METex14 positive NSCLC had stable disease as the best response. CONCLUSION: OMO-1 was tolerated at the dose of 250 mg BID and shows initial signs of MET inhibition and anti-tumor activity in METex14 mutated NSCLC patients.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Segunda Neoplasia Primária , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas Proto-Oncogênicas c-met/genética , Inibidores de Proteínas Quinases/efeitos adversos , Segunda Neoplasia Primária/genética , Éxons , MutaçãoRESUMO
Exon skipping mutations of the MET receptor tyrosine kinase (METex14), increasingly reported in cancers, occur in 3-4% of non-small-cell lung cancer (NSCLC). Only 50% of patients have a beneficial response to treatment with MET-tyrosine kinase inhibitors (TKIs), underlying the need to understand the mechanism of METex14 oncogenicity and sensitivity to TKIs. Whether METex14 is a driver mutation and whether it requires hepatocyte growth factor (HGF) for its oncogenicity in a range of in vitro functions and in vivo has not been fully elucidated from previous preclinical models. Using CRISPR/Cas9, we developed a METex14/WT isogenic model in nontransformed human lung cells and report that the METex14 single alteration was sufficient to drive MET-dependent in vitro anchorage-independent survival and motility and in vivo tumorigenesis, sensitising tumours to MET-TKIs. However, we also show that human HGF (hHGF) is required, as demonstrated in vivo using a humanised HGF knock-in strain of mice and further detected in tumour cells of METex14 NSCLC patient samples. Our results also suggest that METex14 oncogenicity is not a consequence of an escape from degradation in our cell model. Thus, we developed a valuable model for preclinical studies and present results that have potential clinical implication.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/patologia , Éxons , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias Pulmonares/patologia , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , CamundongosRESUMO
Aggressive triple-negative breast cancer (TNBC) is classically treated with chemotherapy. Besides direct tumor cell killing, some chemotherapeutics such as cisplatin provide additional disease reduction through stimulation of anti-tumor immunity. The cisplatin-induced immunomodulation in TNBC was here investigated in-depth using immunocompetent intraductal mouse models. Upon primary tumor transition to invasive carcinoma, cisplatin was injected systemically and significantly reduced tumor progression. Flow cytometric immunophenotyping was corroborated by immunohistochemical analyses and revealed both differential immune cell compositions and positivity for their programmed death (PD)-1 and PD-ligand (L)1 markers across body compartments, including the primary tumor, axillary lymph nodes and spleen. As key findings, a significant decrease in immunosuppressive and a concomitant increase in anti-tumor lymphocytic cell numbers were observed in the axillary lymph nodes and spleen, highlighting their importance in cisplatin-stimulated anti-tumor immunity. These immunomodulatory effects were already established following the first cisplatin dose, indicating that early cisplatin-mediated events may determine (immuno)therapeutic outcome. Furthermore, a single cisplatin dose sufficed to alleviate anti-PD-1 resistance in a 4T1-based model, providing add-on disease reduction without toxic side effects as seen upon multiple cisplatin dosing. Overall, these results highlight cisplatin as immunotherapeutic ally in TNBC, providing durable immunostimulation, even after a single dose.
Assuntos
Neoplasias de Mama Triplo Negativas , Animais , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Modelos Animais de Doenças , Humanos , Imunomodulação , Imunofenotipagem , Camundongos , Neoplasias de Mama Triplo Negativas/tratamento farmacológicoRESUMO
The MET oncogene encodes a tyrosine kinase (TK) receptor. Its activation protects cells from death but also stimulates DNA damage response by triggering excess replicative stress. Transcriptomic classification of cancer cell lines based on MET expression showed that response to the PARP inhibitor (PARPi) olaparib is poorer in MET overexpressing cell lines. Accordingly, a high MET expressing lung carcinoma cell line was sensitized to PARPi by MET TK inhibition. This was not linked solely to MET overexpression: other MET overexpressing cell lines were biochemically but not functionally responsive to combined inhibition. Moreover, exogenously induced MET overexpression was unable to induce resistance to PARPi. The MET overexpressing cell line, responsive to the combined PARP and MET inhibition, carried a heterozygous mutation of the ATM gene and showed an attenuated response of ATM to PARPi. Among the downstream targets of ATM activation, NuMA was phosphorylated only in response to the combined PARP and MET inhibition. Given the role played by NuMA in mitosis, data show that the latter is affected by MET and PARP inhibition in cells with haploinsufficient ATM. This is important as ATM heterozygous mutation is frequently found in human cancer and in lung carcinomas in particular.
Assuntos
Antineoplásicos , Neoplasias Pulmonares , Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Haploinsuficiência , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologiaRESUMO
c-MET is considered a driver of cancer progression, impacting tumor growth and tumor-supporting stroma. Here, we investigated the therapeutic efficacy of OMO-1, a potent and selective c-MET inhibitor, in an immunocompetent intraductal mouse model for triple-negative breast cancer (TNBC). OMO-1 reduced non-c-MET addicted 4T1 tumor progression dose dependently as monotherapeutic and provided additional disease reduction in combination with cisplatin. At the stromal level, OMO-1 significantly reduced neutrophil infiltration in 4T1 tumors, promoted immune activation, and enhanced cisplatin-mediated reduction of tumor-associated macrophages. OMO-1 treatment also reduced 4T1 tumor hypoxia and increased expression of pericyte markers, indicative for vascular maturation. Corroborating this finding, cisplatin delivery to the 4T1 primary tumor was enhanced upon OMO-1 treatment, increasing cisplatin DNA-adduct levels and tumor cell death. Although verification in additional cell lines is warranted, our findings provide initial evidence that TNBC patients may benefit from OMO-1 treatment, even in cases of non-c-MET addicted tumors.
RESUMO
BACKGROUND: Interferon-induced expression of programmed cell death ligands (PD-L1/PD-L2) may sustain tumour immune-evasion. Patients featuring MET amplification, a genetic lesion driving transformation, may benefit from anti-MET treatment. We explored if MET-targeted therapy interferes with Interferon-γ modulation of PD-L1/PD-L2 in MET-amplified tumours. METHODS: PD-L1/PD-L2 expression and signalling pathways downstream of MET or Interferon-γ were analysed in MET-amplified tumour cell lines and in patient-derived tumour organoids, in basal condition, upon Interferon-γ stimulation, and after anti-MET therapy. RESULTS: PD-L1 and PD-L2 were upregulated in MET-amplified tumour cells upon Interferon-γ treatment. This induction was impaired by JNJ-605, a selective inhibitor of MET kinase activity, and MvDN30, an antibody inducing MET proteolytic cleavage. We found that activation of JAKs/ STAT1, signal transducers downstream of the Interferon-γ receptor, was neutralised by MET inhibitors. Moreover, JAK2 and MET associated in the same signalling complex depending on MET phosphorylation. Results were confirmed in MET-amplified organoids derived from human colorectal tumours, where JNJ-605 treatment revoked Interferon-γ induced PD-L1 expression. CONCLUSIONS: These data show that in MET-amplified cancers, treatment with MET inhibitors counteracts the induction of PD-1 ligands by Interferon-γ. Thus, therapeutic use of anti-MET drugs may provide additional clinical benefit over and above the intended inhibition of the target oncogene.
Assuntos
Antígeno B7-H1/efeitos dos fármacos , Interferon gama/farmacologia , Neoplasias/genética , Proteína 2 Ligante de Morte Celular Programada 1/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Evasão Tumoral/efeitos dos fármacos , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Humanos , Janus Quinases/efeitos dos fármacos , Janus Quinases/metabolismo , Neoplasias Hepáticas/secundário , Terapia de Alvo Molecular , Neoplasias/metabolismo , Organoides , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Receptores de Interferon , Fator de Transcrição STAT1/efeitos dos fármacos , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Evasão Tumoral/genética , Receptor de Interferon gamaRESUMO
The role of paracrine Hepatocyte Growth Factor (HGF) in the resistance to angiogenesis inhibitors (AIs) is hidden in xenograft models because mouse HGF fails to fully activate human MET. To uncover it, we compared the efficacy of AIs in wild-type and human HGF knock-in SCID mice bearing orthotopic human colorectal tumors. Species-specific HGF/MET signaling dramatically impaired the response to anti-angiogenic agents and boosted metastatic dissemination. In cell-based assays mimicking the consequences of anti-angiogenic therapy, colorectal cancer cells were completely resistant to hypoxia but extremely sensitive to nutrient deprivation. Starvation-induced apoptosis could be prevented by HGF, which promoted GLUT1-mediated glucose uptake, sustained glycolysis and activated autophagy. Pharmacological inhibition of GLUT1 in the presence of glucose killed tumor cells as effectively as glucose deprivation, and this effect was antagonized by HGF. Concomitant targeting of GLUT1 and HGF potently suppressed growth and dissemination of AI-resistant human tumors in human HGF knock-in SCID mice without exacerbating tumor hypoxia. These data suggest that stroma-derived HGF protects CRC cells against glucose starvation-induced apoptosis, promoting resistance to both AIs and anti-glycolytic agents. Combined inhibition of glucose metabolism and HGF/MET signaling ('anti-METabolic therapy') may represent a more effective CRC treatment compared to utterly blocking tumor blood supply.
Assuntos
Adaptação Fisiológica/fisiologia , Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Fator de Crescimento de Hepatócito/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Xenoenxertos , Humanos , Camundongos , Camundongos SCID , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/fisiologia , Microambiente Tumoral/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Fibroblast growth factor (FGF) signaling plays critical roles in key biological processes ranging from embryogenesis to wound healing and has strong links to several hallmarks of cancer. Genetic alterations in FGF receptor (FGFR) family members are associated with increased tumor growth, metastasis, angiogenesis, and decreased survival. JNJ-42756493, erdafitinib, is an orally active small molecule with potent tyrosine kinase inhibitory activity against all four FGFR family members and selectivity versus other highly related kinases. JNJ-42756493 shows rapid uptake into the lysosomal compartment of cells in culture, which is associated with prolonged inhibition of FGFR signaling, possibly due to sustained release of the inhibitor. In xenografts from human tumor cell lines or patient-derived tumor tissue with activating FGFR alterations, JNJ-42756493 administration results in potent and dose-dependent antitumor activity accompanied by pharmacodynamic modulation of phospho-FGFR and phospho-ERK in tumors. The results of the current study provide a strong rationale for the clinical investigation of JNJ-42756493 in patients with tumors harboring FGFR pathway alterations. Mol Cancer Ther; 16(6); 1010-20. ©2017 AACR.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Quinoxalinas/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Lisossomos/metabolismo , Masculino , Camundongos , Terapia de Alvo Molecular , Fosforilação , Ligação Proteica , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Pirazóis/administração & dosagem , Pirazóis/farmacocinética , Quinoxalinas/administração & dosagem , Quinoxalinas/farmacocinética , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MET oncogene amplification is emerging as a major mechanism of acquired resistance to EGFR-directed therapy in lung and colorectal cancers. Furthermore, MET amplification predicts responsiveness to MET inhibitors currently in clinical trials. Among the anti-MET drugs available, ATP-competitive small-molecule kinase inhibitors abrogate receptor autophosphorylation and downstream activation of ERK1/2 and AKT, resulting in cell-cycle arrest. However, this antiproliferative effect allows persistence of a pool of cancer cells that are quiescent but alive. Once the inhibition is removed, rebound activation of MET-driven cell proliferative pathways and tumor growth may occur, an adverse event observed frequently in clinical settings after drug discontinuation. Here we show that inhibitor withdrawal prompts receptor phosphorylation to levels higher than those displayed at steady-state and generates a rebound effect pushing quiescent cancer cells back into the cell cycle, both in vitro and in experimental tumor models in vivo Mechanistically, we found that inhibitor treatment blocks MET endocytosis, causing a local increase in the number of receptors at the plasma membrane. Upon inhibitor washout, the receptor is readily rephosphorylated. The initial phosphorylation is not only increased but also prolonged in duration due to downmodulation of a phosphatase-mediated MET-negative feedback loop, which accompanies receptor internalization. Notably, treatment with a MET therapeutic antibody that induces proteolytic cleavage of the receptor at the cell surface substantially prevents this rebound effect, providing a rationale to combine or alternate these mechanistically different types of MET-targeted therapy. Cancer Res; 76(17); 5019-29. ©2016 AACR.
Assuntos
Anticorpos/farmacologia , Antineoplásicos/farmacologia , Recidiva Local de Neoplasia/patologia , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Imunofluorescência , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Microscopia Confocal , Recidiva Local de Neoplasia/metabolismo , Neoplasias Experimentais/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cancer stem cells (CSCs) are key players in bone metastasis. In some renal tumors CSCs overexpress the HGF receptor c-MET, speculating that c-MET targeting could lead to bone metastasis inhibition. To address this hypothesis we isolated renal CD105+/CD24-CSCs, expressing c-MET receptor from a primary renal carcinoma. Then, to study their ability to metastasize to bone, we injected renal CSCs in NOD/SCID mice implanted with a human bone and we tested the effect of a c-MET inhibitor (JNJ-38877605) on bone metastasis development. JNJ-38877605 inhibited the formation of metastases at bone implant site. We showed that JNJ-38877605 inhibited the activation of osteoclasts induced by RCC stem cells and it stimulated osteoblast activity, finally resulting in a reduction of bone turnover consistent with the inhibition of bone metastases. We measured the circulating levels of osteotropic factors induced by RCC stem cells in the sera of mice treated with c-Met inhibitor, showing that IL-11 and CCL20 were reduced in mice treated with JNJ-38877605, strongly supporting the involvement of c-MET in the regulation of this process. To address the clinical relevance of c-MET upregulation during tumor progression, we analysed c-MET in renal cancer patients detecting an increased expression in the bone metastatic lesions by IHC. Then, we dosed CCL20 serum levels resulting significantly increased in patients with bone metastases compared to non-metastatic ones. Collectively, our data highlight the importance of the c-MET pathway in the pathogenesis of bone metastases induced by RCC stem cells in mice and humans.
Assuntos
Neoplasias Ósseas/secundário , Carcinoma de Células Renais/secundário , Neoplasias Renais/patologia , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Antineoplásicos/farmacologia , Neoplasias Ósseas/metabolismo , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Humanos , Neoplasias Renais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Pirazóis/farmacologia , Piridazinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Glioblastoma (GBM) contains stem-like cells (GSCs) known to be resistant to ionizing radiation and thus responsible for therapeutic failure and rapidly lethal tumor recurrence. It is known that GSC radioresistance relies on efficient activation of the DNA damage response, but the mechanisms linking this response with the stem status are still unclear. Here, we show that the MET receptor kinase, a functional marker of GSCs, is specifically expressed in a subset of radioresistant GSCs and overexpressed in human GBM recurring after radiotherapy. We elucidate that MET promotes GSC radioresistance through a novel mechanism, relying on AKT activity and leading to (i) sustained activation of Aurora kinase A, ATM kinase, and the downstream effectors of DNA repair, and (ii) phosphorylation and cytoplasmic retention of p21, which is associated with anti-apoptotic functions. We show that MET pharmacological inhibition causes DNA damage accumulation in irradiated GSCs and their depletion in vitro and in GBMs generated by GSC xenotransplantation. Preclinical evidence is thus provided that MET inhibitors can radiosensitize tumors and convert GSC-positive selection, induced by radiotherapy, into GSC eradication.
Assuntos
Glioblastoma/radioterapia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Células-Tronco/fisiologia , Células-Tronco/efeitos da radiação , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Aurora Quinase A/metabolismo , Sobrevivência Celular , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Reparo do DNA , Xenoenxertos , Humanos , Camundongos , Proteína Oncogênica v-akt/metabolismoRESUMO
Constitutively active receptor tyrosine kinases (RTKs) are known oncogenic drivers and provide valuable therapeutic targets in many cancer types. However, clinical efficacy of RTK inhibitors is limited by intrinsic and acquired resistance. To identify genes conferring resistance to inhibition of the MET RTK, we conducted a forward genetics screen in the GTL-16 gastric cancer cell line, carrying MET amplification and exquisitely sensitive to MET inhibition. Cells were transduced with three different retroviral cDNA expression libraries and selected for growth in the presence of the MET inhibitor PHA-665752. Selected cells displayed robust and reproducible enrichment of library-derived cDNAs encoding truncated forms of RAF1 and BRAF proteins, whose silencing reversed the resistant phenotype. Transduction of naïve GTL-16 cells with truncated, but not full length, RAF1 and BRAF conferred in vitro and in vivo resistance to MET inhibitors, which could be reversed by MEK inhibition. Induction of resistance by truncated RAFs was confirmed in other MET-addicted cell lines, and further extended to EGFR-addicted cells. These data show that truncated RAF1 and BRAF proteins, recently described as products of genomic rearrangements in gastric cancer and other malignancies, have the ability to render neoplastic cells resistant to RTK-targeted therapy.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-met/metabolismo , Neoplasias Gástricas/metabolismo , Quinases raf/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , DNA Complementar/metabolismo , Receptores ErbB/metabolismo , Feminino , Biblioteca Gênica , Humanos , Indóis/química , Camundongos , Camundongos Nus , Transplante de Neoplasias , Fenótipo , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas B-raf/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Sulfonas/química , Fatores de TempoRESUMO
Cell-based drug screenings indicate that tumors displaying c-MET gene amplification are "addicted" to MET signaling and therefore are very sensitive to MET-targeted agents. However, these screenings were conducted in the absence of the MET ligand, hepatocyte growth factor (HGF), which is abundant in the tumor microenvironment. Sensitivity of six MET-addicted human tumor cells to three MET kinase inhibitors (JNJ-38877605, PHA-665752, crizotinib) and one antagonistic anti-MET antibody (DN30 Fab) was analyzed in the absence or presence of HGF, in a stroma-tumor coculture system, and by combining anti-MET drugs with an HGF neutralizing antibody (ficlatuzumab) in human HGF knock-in mice bearing c-MET-amplified tumors. In all models examined, HGF promoted resistance to MET-targeted agents, affecting both their potency and efficacy. HGF-induced resistance was due to restoration of physiologic GAB1-mediated PI3K activation that compensated for loss of aberrant HER3-dependent PI3K signaling. Ficlatuzumab restored sensitivity to MET-targeted agents in coculture systems and overcame resistance to JNJ-38877605, crizotinib, and DN30 Fab in human HGF knock-in mice. These data suggest that c-MET-amplified tumor cells-which normally exhibit ligand-independent, constitutive MET activation-become dependent on HGF for survival upon pharmacologic MET inhibition. Because HGF is frequently overexpressed in human cancer, this mechanism may represent a major cause of resistance to anti-MET therapies. The ability of ficlatuzumab to overcome HGF-mediated resistance generates proof of principle that vertical inhibition of both a tyrosine kinase receptor and its ligand can be therapeutically beneficial and opens new perspectives for the treatment of MET-dependent tumors.
Assuntos
Fator de Crescimento de Hepatócito/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Microambiente Tumoral , Animais , Anticorpos Monoclonais/farmacologia , Camundongos , Camundongos SCID , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-met/fisiologia , Receptor ErbB-3/fisiologia , Transdução de SinaisRESUMO
The tyrosine kinase encoded by the MET oncogene is activated by gene mutation or amplification in tumors, which in most instances maintain addiction, i.e., dependency, to MET activation. This makes MET an attractive candidate for targeted therapies. Here we show that, in 3/3 MET-addicted human gastric cancer cell lines, MET kinase inhibition resulted in a 3- to 4-fold increased expression of the antiapoptotic small heat-shock protein of 27 kDa (HSP27, HSPB1). HSP27 increase depended on the inhibition of the MEK/ERK pathway and on heat-shock factor 1 (HSF1) and hypoxia-inducible factor-1α (HIF-1α) regulation. Importantly, HSP27-silenced MET-addicted cells underwent 2- and 3-fold more apoptosis following MET inhibition in vitro and in vivo, respectively. Likewise, in human cancer cells susceptible to epidermal growth factor receptor (EGFR) inhibition, EGFR inhibitors induced HSP27 expression and were strengthened by HSP27 suppression. In control cell lines that were not affected by drugs targeting MET or EGFR, these drugs did not induce HSP27 increase. Therefore, in cancer therapies targeting the MET pathway, the induction of HSP27 might limit the efficacy of anti-MET agents. As HSP27 increase also impairs the effectiveness of EGFR inhibitors and is known to protect cells from chemotherapeutics, the induction of HSP27 by targeted agents might strongly affect the success of combination treatments.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Neoplasias Gástricas/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/antagonistas & inibidores , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico , Humanos , Técnicas Imunoenzimáticas , Camundongos , Chaperonas Moleculares , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/patologia , Células Tumorais Cultivadas , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
UNLABELLED: We discovered a novel somatic gene fusion, CD74-NRG1, by transcriptome sequencing of 25 lung adenocarcinomas of never smokers. By screening 102 lung adenocarcinomas negative for known oncogenic alterations, we found four additional fusion-positive tumors, all of which were of the invasive mucinous subtype. Mechanistically, CD74-NRG1 leads to extracellular expression of the EGF-like domain of NRG1 III-ß3, thereby providing the ligand for ERBB2-ERBB3 receptor complexes. Accordingly, ERBB2 and ERBB3 expression was high in the index case, and expression of phospho-ERBB3 was specifically found in tumors bearing the fusion (P < 0.0001). Ectopic expression of CD74-NRG1 in lung cancer cell lines expressing ERBB2 and ERBB3 activated ERBB3 and the PI3K-AKT pathway, and led to increased colony formation in soft agar. Thus, CD74-NRG1 gene fusions are activating genomic alterations in invasive mucinous adenocarcinomas and may offer a therapeutic opportunity for a lung tumor subtype with, so far, no effective treatment. SIGNIFICANCE: CD74NRG1 fusions may represent a therapeutic opportunity for invasive mucinous lung adenocarcinomas, a tumor with no effective treatment that frequently presents with multifocal unresectable disease.
Assuntos
Adenocarcinoma Mucinoso/genética , Adenocarcinoma/genética , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Histocompatibilidade Classe II/genética , Neoplasias Pulmonares/genética , Neuregulina-1/genética , Proteínas de Fusão Oncogênica/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Adenocarcinoma Mucinoso/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Sequência de Bases , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Células NIH 3T3 , Proteínas de Fusão Oncogênica/metabolismo , Análise de Sequência de DNA , Transdução de Sinais/genéticaRESUMO
Metastatic colorectal cancer remains largely incurable, although in a subset of patients, survival is prolonged by new targeting agents such as anti-EGF receptor (anti-EGFR) antibodies. This disease is believed to be supported by a subpopulation of stem-like cells termed colon cancer-initiating cell (CCIC), which may also confer therapeutic resistance. However, how CCICs respond to EGFR inhibition has not been fully characterized. To explore this question, we systematically generated CCICs through spheroid cultures of patient-derived xenografts of metastatic colorectal cancer. These cultures, termed "xenospheres," were capable of long-term self-propagation in vitro and phenocopied the original patient tumors in vivo, thus operationally defining CCICs. Xenosphere CCICs retained the genetic determinants for EGFR therapeutic response in vitro and in xenografts; like the original tumors, xenospheres harboring a mutated KRAS gene were resistant to EGFR therapy, whereas those harboring wild-type RAS pathway genes (RAS(wt)) were sensitive. Notably, the effects of EGFR inhibition in sensitive CCICs could be counteracted by cytokines secreted by cancer-associated fibroblasts. In particular, we found that the MET receptor ligand hepatocyte growth factor (HGF) was especially active in supporting in vitro CCIC proliferation and resistance to EGFR inhibition. Ectopic production of human HGF in CCIC xenografts rendered the xenografts susceptible to MET inhibition, which sensitized the response to EGFR therapy. By showing that RAS(wt) CCICs rely on both EGFR and MET signaling, our results offer a strong preclinical proof-of-concept for concurrent targeting of these two pathways in the clinical setting.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Neoplasias do Colo/enzimologia , Receptores ErbB/antagonistas & inibidores , Células-Tronco Neoplásicas/enzimologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Proliferação de Células , Sobrevivência Celular , Cetuximab , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Feminino , Fator de Crescimento de Hepatócito/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Transdução de Sinais , Esferoides Celulares/enzimologia , Carga Tumoral/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
EGF receptor (EGFR)-targeted monoclonal antibodies are effective in a subset of metastatic colorectal cancers. Inevitably, all patients develop resistance, which occurs through emergence of KRAS mutations in approximately 50% of the cases. We show that amplification of the MET proto-oncogene is associated with acquired resistance in tumors that do not develop KRAS mutations during anti-EGFR therapy. Amplification of the MET locus was present in circulating tumor DNA before relapse was clinically evident. Functional studies show that MET activation confers resistance to anti-EGFR therapy both in vitro and in vivo. Notably, in patient-derived colorectal cancer xenografts, MET amplification correlated with resistance to EGFR blockade, which could be overcome by MET kinase inhibitors. These results highlight the role of MET in mediating primary and secondary resistance to anti-EGFR therapies in colorectal cancer and encourage the use of MET inhibitors in patients displaying resistance as a result of MET amplification.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/farmacologia , Animais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais Humanizados/efeitos adversos , Antineoplásicos/efeitos adversos , Cetuximab , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/genética , Genes ras , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Panitumumabe , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The MET oncogene is amplified in a fraction of human gastric carcinoma cell lines, with consequent overexpression and constitutive activation of the corresponding protein product, the Met tyrosine kinase receptor. This genetically driven hyperactivation of Met is necessary for cancer cell growth and survival, so that Met pharmacological blockade results in cell-cycle arrest or apoptosis (oncogene addiction). MET gene amplification also occurs in vivo in a number of human gastric carcinomas, and clinical trials are now ongoing to assess the therapeutic efficacy of Met inhibitors in this type of malignancy. The aim of our study was to identify a preclinical algorithm of soluble surrogate biomarkers indicative of response to Met inhibition in gastric tumors, as a potential tool to integrate imaging criteria during patient follow-up. We started from a survey of candidate molecules based on antibody proteomics and gene expression profiling; after ELISA validation and analytical quantification, four biomarkers were identified that appeared to be strongly and consistently modulated by Met inhibition in a panel of Met-addicted gastric carcinoma cell lines, but not in Met-independent cell lines. Pharmacologic blockade of Met using specific small-molecule inhibitors led to reduced secretion of IL-8, GROα and the soluble form of uPAR and to increased production of IL-6 both in vitro (in culture supernatants) and in vivo (in the plasma of xenografted mice). If confirmed in patients, this information might prove useful to monitor clinical response to Met-targeted therapies in MET-amplified gastric carcinomas.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Animais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Quimiocina CXCL1/sangue , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Indóis/farmacologia , Interleucina-6/sangue , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/sangue , Interleucina-8/genética , Interleucina-8/metabolismo , Lectinas de Ligação a Manose/sangue , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Proteômica , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores de Superfície Celular/sangue , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Neoplasias Gástricas/sangue , Neoplasias Gástricas/genética , Sulfonas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Ionizing radiation (IR) is effectively used in cancer therapy. However, in subsets of patients, a few radioresistant cancer cells survive and cause disease relapse with metastatic progression. The MET oncogene encodes the hepatocyte growth factor (HGF) receptor and is known to drive "invasive growth", a regenerative and prosurvival program unduly activated in metastasis. METHODS: Human tumor cell lines (MDA-MB-231, MDA-MB-435S, U251) were subjected to therapeutic doses of IR. MET mRNA, and protein expression and signal transduction were compared in treated and untreated cells, and the involvement of the DNA-damage sensor ataxia telangiectasia mutated (ATM) and the transcription factor nuclear factor kappa B (NF-κB) in activating MET transcription were analyzed by immunoblotting, chromatin immunoprecipitation, and use of NF-κB silencing RNA (siRNA). Cell invasiveness was measured in wound healing and transwell assays, and cell survival was measured in viability and clonogenic assays. MET was inhibited by siRNA or small-molecule kinase inhibitors (PHA665752 or JNJ-38877605). Combinations of MET-targeted therapy and radiotherapy were assessed in MDA-MB-231 and U251 xenografts (n = 5-6 mice per group). All P values were from two-sided tests. RESULTS: After irradiation, MET expression in cell lines was increased up to fivefold via activation of ATM and NF-κB. MET overexpression increased ligand-independent MET phosphorylation and signal transduction, and rendered cells more sensitive to HGF. Irradiated cells became more invasive via a MET-dependent mechanism that was further enhanced in the presence of HGF. MET silencing by siRNA or inhibition of its kinase activity by treatment with PHA665752 or JNJ-38877605 counteracted radiation-induced invasiveness, promoted apoptosis, and prevented cells from resuming proliferation after irradiation in vitro. Treatment with MET inhibitors enhanced the efficacy of IR to stop the growth of or to induce the regression of xenografts (eg, at day 13, U251 xenografts, mean volume increase relative to mean tumor volume at day 0: vehicle = 438%, 5 Gy IR = 151%, 5 Gy IR + JNJ-38877605 = 76%; difference, IR vs JNJ-38877604 + IR = 75%, 95% CI = 59% to 91%, P = .01). CONCLUSION: IR induces overexpression and activity of the MET oncogene through the ATM-NF-κB signaling pathway; MET, in turn, promotes cell invasion and protects cells from apoptosis, thus supporting radioresistance. Drugs targeting MET increase tumor cell radiosensitivity and prevent radiation-induced invasiveness.