RESUMO
Although treatment advances over recent decades have significantly improved survival of patients with multiple myeloma, there is still an unmet medical need for more effective treatments. In this study, we identified G-protein-coupled receptor family C group 5 member D (GPRC5D) expression on the surface of malignant cells involved in multiple myeloma, but except for plasma cells and B cells, not at appreciable levels on normal hematopoietic cells and bone marrow progenitors, including hematopoietic stem cells. In addition, we constructed IgG-based anti-GPRC5D/CD3 bispecific T-cell-redirecting antibodies (GPRC5D TRAB), which suppressed the tumor growth of GPRC5D-positive myeloma cells through the activation of T cells in vitro and in vivo in xenograft models. Collectively, these findings suggest that GPRC5D is an antigen specific to multiple myeloma and a potential target of TRAB therapy.
Assuntos
Anticorpos Biespecíficos/imunologia , Complexo CD3/imunologia , Mieloma Múltiplo/imunologia , Receptores Acoplados a Proteínas G/imunologia , Animais , Anticorpos Biespecíficos/uso terapêutico , Especificidade de Anticorpos/imunologia , Células CHO , Linhagem Celular Tumoral , Cricetulus , Feminino , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Alectinib/CH5424802 is a known inhibitor of anaplastic lymphoma kinase (ALK) and is being evaluated in clinical trials for the treatment of ALK fusion-positive non-small cell lung cancer (NSCLC). Recently, some RET and ROS1 fusion genes have been implicated as driver oncogenes in NSCLC and have become molecular targets for antitumor agents. This study aims to explore additional target indications of alectinib by testing its ability to inhibit the activity of kinases other than ALK. We newly verified that alectinib inhibited RET kinase activity and the growth of RET fusion-positive cells by suppressing RET phosphorylation. In contrast, alectinib hardly inhibited ROS1 kinase activity unlike other ALK/ROS1 inhibitors such as crizotinib and LDK378. It also showed antitumor activity in mouse models of tumors driven by the RET fusion. In addition, alectinib showed kinase inhibitory activity against RET gatekeeper mutations (RET V804L and V804M) and blocked cell growth driven by the KIF5B-RET V804L and V804M. Our results suggest that alectinib is effective against RET fusion-positive tumors. Thus, alectinib might be a therapeutic option for patients with RET fusion-positive NSCLC.
Assuntos
Antineoplásicos/farmacologia , Carbazóis/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Rearranjo Gênico , Neoplasias Pulmonares/genética , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-ret/genética , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Carbazóis/administração & dosagem , Carbazóis/química , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Camundongos , Modelos Moleculares , Conformação Molecular , Mutação , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Piperidinas/administração & dosagem , Piperidinas/química , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-ret/química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: The clinical efficacy of the anaplastic lymphoma kinase (ALK) inhibitor crizotinib has been demonstrated in ALK fusion-positive non-small cell lung cancer (NSCLC); however, brain metastases are frequent sites of initial failure in patients due to poor penetration of the central nervous system by crizotinib. Here, we examined the efficacy of a selective ALK inhibitor alectinib/CH5424802 in preclinical models of intracranial tumors. METHODS: We established intracranial tumor implantation mouse models of EML4-ALK-positive NSCLC NCI-H2228 and examined the antitumor activity of alectinib in this model. Plasma distribution and brain distribution of alectinib were examined by quantitative whole-body autoradiography administrating a single oral dose of (14)C-labeled alectinib to rats. The drug permeability of alectinib was evaluated in Caco-2 cell. RESULTS: Alectinib resulted in regression of NCI-H2228 tumor in mouse brain and provided a survival benefit. In a pharmacokinetic study using rats, alectinib showed a high brain-to-plasma ratio, and in an in vitro drug permeability study using Caco-2 cells, alectinib was not transported by P-glycoprotein efflux transporter that is a key factor in blood-brain barrier penetration. CONCLUSIONS: We established intracranial tumor implantation models of EML4-ALK-positive NSCLC. Alectinib showed potent efficacy against intracranial EML4-ALK-positive tumor. These results demonstrated that alectinib might provide therapeutic opportunities for crizotinib-treated patients with brain metastases.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Carbazóis/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Piperidinas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Quinase do Linfoma Anaplásico , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundário , Células CACO-2 , Carbazóis/sangue , Carbazóis/farmacocinética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Humanos , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes/métodos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos Nus , Camundongos SCID , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/metabolismo , Piperidinas/sangue , Piperidinas/farmacocinética , Inibidores de Proteínas Quinases/sangue , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Ratos , Receptores Proteína Tirosina Quinases/metabolismo , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: EML4-ALK is a driver oncogene in non-small-cell lung cancer (NSCLC) and has been developed into a promising molecular target for antitumor agents. Although EML4-ALK is reported to be formed by inversion of chromosome 2, other mechanisms of this gene fusion remain unknown. This study aimed to examine the mechanism of EML4-ALK rearrangement using a novel cell line with the EML4-ALK fusion gene. METHODS: An EML4-ALK-positive cell line, termed JFCR-LC649, was established from pleomorphic carcinoma, a rare subtype of NSCLC. We investigated the chromosomal aberrations using fluorescence in situ hybridization and comparative genomic hybridization (CGH). Alectinib/CH5424802, a selective ALK inhibitor, was evaluated in the antitumor activity against JFCR-LC649 in vitro and in vivo xenograft model. RESULTS: We established an EML4-ALK-positive cell line, termed JFCR-LC649, derived from a patient with NSCLC and revealed that the JFCR-LC649 cells harbor variant 3 of the EML4-ALK fusion with twofold copy number gain. Interestingly, comparative genomic hybridization and metaphase-fluorescence in situ hybridization analysis showed that in addition to two normal chromosome 2, JFCR-LC649 cells contained two aberrant chromosome 2 that were fragmented and scattered. These observations provided the first evidence that EML4-ALK fusion in JFCR-LC649 cells was formed in chromosome 2 by a distinct mechanism of genomic rearrangement, termed chromothripsis. Furthermore, a selective ALK inhibitor alectinib/CH5424802 suppressed tumor growth of the JFCR-LC649 cells through inhibition of phospho-ALK in vitro and in vivo in a xenograft model. CONCLUSION: Our results suggested that chromothripsis may be a mechanism of oncogenic rearrangement of EML4-ALK. In addition, alectinib was effective against EML4-ALK-positive tumors with ALK copy number gain mediated by chromothripsis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Quebra Cromossômica , Neoplasias Pulmonares/genética , Micronúcleos com Defeito Cromossômico , Proteínas de Fusão Oncogênica/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Feminino , Fusão Gênica , Rearranjo Gênico , Xenoenxertos , Humanos , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Proteínas de Fusão Oncogênica/metabolismo , TransfecçãoRESUMO
The clinical efficacy of the ALK inhibitor crizotinib has been demonstrated in ALK fusion-positive NSCLC; however, resistance to crizotinib certainly occurs through ALK secondary mutations in clinical use. Here we examined the efficacy of a selective ALK inhibitor alectinib/CH5424802 in models of crizotinib resistance. Alectinib led to tumor size reduction in EML4-ALK-positive xenograft tumors that failed to regress fully during the treatment with crizotinib. In addition, alectinib inhibited the growth of some EML4-ALK mutant-driven tumors, including the G1269A model. These results demonstrated that alectinib might provide therapeutic opportunities for crizotinib-treated patients with ALK secondary mutations.
Assuntos
Antineoplásicos/farmacologia , Carbazóis/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinase do Linfoma Anaplásico , Animais , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Crizotinibe , Resistencia a Medicamentos Antineoplásicos , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos SCID , Pirazóis/farmacologia , Piridinas/farmacologia , Distribuição Aleatória , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Anaplastic lymphoma kinase (ALK) is a tyrosine kinase that is constitutively activated in certain cancers, following gene alterations such as chromosomal translocation, amplification, or point mutation. Here, we identified CH5424802, a potent, selective, and orally available ALK inhibitor with a unique chemical scaffold, showing preferential antitumor activity against cancers with gene alterations of ALK, such as nonsmall cell lung cancer (NSCLC) cells expressing EML4-ALK fusion and anaplastic large-cell lymphoma (ALCL) cells expressing NPM-ALK fusion in vitro and in vivo. CH5424802 inhibited ALK L1196M, which corresponds to the gatekeeper mutation conferring common resistance to kinase inhibitors, and blocked EML4-ALK L1196M-driven cell growth. Our results support the potential for clinical evaluation of CH5424802 for the treatment of patients with ALK-driven tumors.