RESUMEN
BACKGROUND: Effective targeted therapy for non-small-cell lung cancer (NSCLC) patients with human epidermal growth factor receptor 2 (HER2) mutations remains an unmet need. This study investigated the antitumor effect of an irreversible pan-HER receptor tyrosine kinase inhibitor, pyrotinib. PATIENTS AND METHODS: Using patient-derived organoids and xenografts established from an HER2-A775_G776YVMA-inserted advanced lung adenocarcinoma patient sample, we investigated the antitumor activity of pyrotinib. Preliminary safety and efficacy of pyrotinib in 15 HER2-mutant NSCLC patients in a phase II clinical trial are also presented. RESULTS: Pyrotinib showed significant growth inhibition of organoids relative to afatinib in vitro (P = 0.0038). In the PDX model, pyrotinib showed a superior antitumor effect than afatinib (P = 0.0471) and T-DM1 (P = 0.0138). Mice treated with pyrotinib displayed significant tumor burden reduction (mean tumor volume, -52.2%). In contrast, afatinib (25.4%) and T-DM1 (10.9%) showed no obvious reduction. Moreover, pyrotinib showed a robust ability to inhibit pHER2, pERK and pAkt. In the phase II cohort of 15 patients with HER2-mutant NSCLC, pyrotinib 400 mg resulted in a objective response rate of 53.3% and a median progression-free survival of 6.4 months. CONCLUSION: Pyrotinib showed activity against NSCLC with HER2 exon 20 mutations in both patient-derived organoids and a PDX model. In the clinical trial, pyrotinib showed promising efficacy. CLINICAL TRIAL REGISTRATION: NCT02535507.
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Acrilamidas/administración & dosificación , Aminoquinolinas/administración & dosificación , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/administración & dosificación , Receptor ErbB-2/genética , Acrilamidas/efectos adversos , Adulto , Afatinib/administración & dosificación , Anciano , Anciano de 80 o más Años , Aminoquinolinas/efectos adversos , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Mutación/genética , Organoides/efectos de los fármacos , Organoides/patología , Inhibidores de Proteínas Quinasas/efectos adversos , Receptor ErbB-2/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: NTRK1, NTRK2 and NTRK3 gene fusions (NTRK gene fusions) occur in a range of adult cancers. Larotrectinib is a potent and highly selective ATP-competitive inhibitor of TRK kinases and has demonstrated activity in patients with tumours harbouring NTRK gene fusions. PATIENTS AND METHODS: This multi-centre, phase I dose escalation study enrolled adults with metastatic solid tumours, regardless of NTRK gene fusion status. Key inclusion criteria included evaluable and/or measurable disease, Eastern Cooperative Oncology Group performance status 0-2, and adequate organ function. Larotrectinib was administered orally once or twice daily, on a continuous 28-day schedule, in increasing dose levels according to a standard 3 + 3 dose escalation scheme. The primary end point was the safety of larotrectinib, including dose-limiting toxicity. RESULTS: Seventy patients (8 with tumours with NTRK gene fusions; 62 with tumours without a documented NTRK gene fusion) were enrolled to 6 dose cohorts. There were four dose-limiting toxicities; none led to study drug discontinuation. The maximum tolerated dose was not reached. Larotrectinib-related adverse events were predominantly grade 1; none were grade 4 or 5. The most common grade 3 larotrectinib-related adverse event was anaemia [4 (6%) of 70 patients]. A dose of 100 mg twice daily was recommended for phase II studies based on tolerability and antitumour activity. In patients with evaluable TRK fusion cancer, the objective response rate by independent review was 100% (eight of the eight patients). Eight (12%) of the 67 assessable patients overall had an objective response by investigator assessment. Median duration of response was not reached. Larotrectinib had limited activity in tumours with NTRK mutations or amplifications. Pharmacokinetic analysis showed exposure was generally proportional to administered dose. CONCLUSIONS: Larotrectinib was well tolerated, demonstrated activity in all patients with tumours harbouring NTRK gene fusions, and represents a new treatment option for such patients. CLINCALTRIALS.GOV NUMBER: NCT02122913.
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Neoplasias/tratamiento farmacológico , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirazoles/uso terapéutico , Pirimidinas/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Relación Dosis-Respuesta a Droga , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Neoplasias/patología , Proteínas de Fusión Oncogénica/genética , Pronóstico , Adulto JovenRESUMEN
BACKGROUND: Response Evaluation Criteria in Solid Tumors (RECIST) permits rapid evaluation of new therapeutic strategies in cancer. However, RECIST does not capture the heterogeneity of response in highly active therapies. Depth of tumor response may provide a more granular view of response. We explored the association between, depth of response (DepOR), with overall survival (OS) and progression-free survival (PFS) for patients with NSCLC being treated with an ALK inhibitor (ALKi) or an anti-PD-1 antibody (Ab). METHODS: Experimental arms from two randomized controlled trials (RCTs) of an ALKi and two RCTs of an anti-PD-1 Ab were separately pooled. Patient responses were grouped into DepOR 'quartiles' by percentage of maximal tumor shrinkage (Q1 = 1%-25%, Q2 = 26%-50%, Q3 = 51%-75%, and Q4 = 76%-100%), Q0 had no shrinkage. We carried out a retrospective exploratory responder analysis to evaluate the association between DepOR and OS or PFS using hazard ratios (HR) generated by the Cox proportional hazards model. RESULTS: In the pooled ALK analysis there were 12, 39, 70, 144, and 40 patients in quartiles 0-4, respectively. The DepOR versus PFS/OS analyses HR were: 0.19/0.94 for Q1 0.11/0.56 for Q2, 0.05/0.28 for Q3, and 0.03/0.05 for Q4. In the PD-1 trials within quartiles 0-4 there were 168, 70, 44, 45, and 28 patients, respectively. The DepOR versus PFS/OS analyses HR were 0.3/0.52 for Q1, 0.22/0.47 for Q2, 0.09/0.07 for Q3, and 0.07/0.14 for Q4. CONCLUSIONS: Our analysis suggests a greater DepOR is associated with longer PFS and OS for patients receiving ALKi or anti-PD1 Ab. Overall, this suggests that DepOR may provide an additional outcome measure for clinical trials, and may allow better comparisons of treatment activity.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Inmunoterapia , Neoplasias Pulmonares/mortalidad , Terapia Molecular Dirigida , Adulto , Anciano , Anciano de 80 o más Años , Quinasa de Linfoma Anaplásico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/secundario , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Metástasis Linfática , Masculino , Persona de Mediana Edad , Pronóstico , Ensayos Clínicos Controlados Aleatorios como Asunto , Proteínas Tirosina Quinasas Receptoras/metabolismo , Estudios Retrospectivos , Tasa de Supervivencia , Adulto JovenRESUMEN
BACKGROUND: BIBF 1120 is an oral potent inhibitor of vascular endothelial growth factor receptor, fibroblast growth factor receptor and platelet-derived growth factor receptor, the three key receptor families involved in angiogenesis. This phase I, open-label dose-escalation study investigated BIBF 1120 combined with paclitaxel (Taxol) and carboplatin in first-line patients with advanced (IIIB/IV) non-small-cell lung cancer. PATIENTS AND METHODS: Patients received BIBF 1120 (starting dose 50 mg b.i.d.) on days 2-21 and paclitaxel (200 mg/m2) and carboplatin [area under curve (AUC)=6 mg/ml/min] on day 1 of each 21-day cycle. Primary end points were safety and BIBF 1120 maximum tolerated dose (MTD) in this combination. Pharmacokinetics (PK) profiles were evaluated. RESULTS: Twenty-six patients were treated (BIBF 1120 50-250 mg b.i.d.). BIBF 1120 MTD was 200 mg b.i.d. in combination with paclitaxel and carboplatin. Six dose-limiting toxicity events occurred during treatment cycle 1 (liver enzyme elevations, thrombocytopenia, abdominal pain, and rash). Best responses included 7 confirmed partial responses (26.9%); 10 patients had stable disease. BIBF 1120 200 mg b.i.d. had no clinically relevant influence on the PK of paclitaxel 200 mg/m2 and carboplatin AUC 6 mg/ml/min and vice versa. CONCLUSIONS: BIBF 1120 MTD was 200 mg b.i.d when given with paclitaxel and carboplatin; this combination demonstrated an acceptable safety profile. No relevant changes in PK parameters of the backbone chemotherapeutic agents or BIBF 1120 were observed.
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Carboplatino/administración & dosificación , Carboplatino/efectos adversos , Carboplatino/sangre , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/patología , Estudios de Cohortes , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Humanos , Indoles/administración & dosificación , Indoles/efectos adversos , Indoles/sangre , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Paclitaxel/administración & dosificación , Paclitaxel/efectos adversos , Paclitaxel/sangreRESUMEN
INTRODUCTION: Echinoderm microtubule-associated protein-like 4 (EML4)-Anaplastic Lymphoma Kinase (ALK) rearrangements occur in 3% to 7% of lung adenocarcinomas and are targets for treatment with tyrosine kinase inhibitors (TKIs). Here we have developed three novel EML4-ALK-positive patient-derived Non-Small-Cell-Lung-Cancer (NSCLC) cancer cell lines, CUTO8 (variant 1), CUTO9 (variant 1) and CUTO29 (variant 3) and included a fourth ALK-positive cell line YU1077 (variant 3) to study ALK-positive signaling and responses. Variants 1 and 3 are the most common EML4-ALK variants expressed in ALK-positive NSCLC, and currently cell lines representing these EML4-ALK variants are limited. MATERIALS AND METHODS: Resazurin assay was performed to evaluate cell viability. Protein levels were determined using western blotting. RNA sequencing was performed in all four cell lines to identify differentially expressed genes. Whole-genome sequencing was performed to determine the presence of EML4-ALK fusion and ALK tyrosine kinase inhibitor resistance mutations. RESULTS: In this study, we have confirmed expression of the corresponding ALK fusion protein and assessed their sensitivity to a range of ALK tyrosine kinase inhibitors. These patient derived cell lines exhibit differential sensitivity to lorlatinib, brigatinib and alectinib, with EML4-ALK variant 3 containing cell lines exhibiting increased sensitivity to lorlatinib and brigatinib as compared to alectinib. These cell lines were further characterized by whole genome sequencing and RNA-seq analysis that identified the ribonucleotide reductase regulatory subunit 2 (RRM2) as a downstream and potential therapeutic target in ALK-positive NSCLC. CONCLUSION: We provide a characterization of four novel EML4-ALK-positive NSCLC cell lines, highlighting genomic heterogeneity and differential responses to ALK TKI treatment. The RNA-Seq characterization of ALK-positive NSCLC CUTO8, CUTO9, CUTO29 and YU1077 cell lines reported here, has been compiled in an interactive ShinyApp resource for public data exploration (https://ccgg.ugent.be/shiny/nsclc_rrm2_2022/).
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Ribonucleósido Difosfato Reductasa , Quinasa de Linfoma Anaplásico/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Proteínas de Fusión Oncogénica/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Ribonucleósido Difosfato Reductasa/metabolismoRESUMEN
EGFR mutant non-small cell lung cancer patients' disease demonstrates remarkable responses to EGFR-targeted therapy, but inevitably they succumb to acquired resistance, which can be complex and difficult to treat. Analyzing acquired resistance through broad molecular testing is crucial to understanding the resistance mechanisms and developing new treatment options. We performed diverse clinical testing on a patient with successive stages of acquired resistance, first to an EGFR inhibitor with MET gene amplification and then subsequently to a combination EGFR and MET targeted therapies. A patient-derived cell line obtained at the time of disease progression was used to identify NRAS gene amplification as an additional driver of drug resistance to combination EGFR/MET therapies. Analysis of downstream signaling revealed extracellular signal-related kinase activation that could only be eliminated by trametinib treatment, while Akt activation could be modulated by various combinations of MET, EGFR, and PI3K inhibitors. The combination of an EGFR inhibitor with a MEK inhibitor was identified as a possible treatment option to overcome drug resistance related to NRAS gene amplification.
RESUMEN
ROS1 gene fusions account for approximately 1-2% of all cases of non-small cell lung cancer (NSCLC). Similarly to anaplastic lymphoma kinase (ALK)-positive NSCLC, patients with ROS1+ NSCLC tend to have minimal smoking and be of the female sex. In most cases, adenocarcinoma is the dominant histology. The ROS1 gene has homology to ALK and this structural similarity formed the basis for utilizing ALK inhibitors for ROS1+ NSCLC. On the basis of impressive progression-free survival of 19.2 months from the PROFILE 1001 trial, crizotinib obtained Food and Drug Administration (FDA) approval as first-line therapy for treatment of ROS1+ NSCLC. Since then, there has been a growing appreciation of the incidence of brain metastases in ROS1+ NSCLC and rates of central nervous system progression on crizotinib. Additionally, appreciation of novel resistance mechanisms to crizotinib has led to the development of newer tyrosine kinase inhibitors (TKIs). In this review, we highlight known and emerging TKIs for the management of ROS1+ NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Terapia Molecular Dirigida , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Humanos , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Accessory molecules, such as HLA-DM and invariant chain, modulate the ligands bound to MHC class II molecules in antigen-presenting cells. Recent investigations, including gene targeting experiments, have shed light on the functions of these molecules, their mechanisms of action, interactions with class II molecules, and the relationships with associated molecules such as tetraspanins and HLA-DO.
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Presentación de Antígeno , Antígenos de Histocompatibilidad Clase II/inmunología , Animales , Antígenos de Diferenciación de Linfocitos B/inmunología , Células Dendríticas/inmunología , Antígenos HLA-D/inmunología , Antígenos HLA-D/metabolismo , Antígeno HLA-DR3/inmunología , Antígeno HLA-DR3/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Unión ProteicaRESUMEN
We identified new gene fusions in patients with lung cancer harboring the kinase domain of the NTRK1 gene that encodes the high-affinity nerve growth factor receptor (TRKA protein). Both the MPRIP-NTRK1 and CD74-NTRK1 fusions lead to constitutive TRKA kinase activity and are oncogenic. Treatment of cells expressing NTRK1 fusions with inhibitors of TRKA kinase activity inhibited autophosphorylation of TRKA and cell growth. Tumor samples from 3 of 91 patients with lung cancer (3.3%) without known oncogenic alterations assayed by next-generation sequencing or fluorescence in situ hybridization demonstrated evidence of NTRK1 gene fusions.
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Reordenamiento Génico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Fusión de Oncogenes , Receptor trkA/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Antígenos de Diferenciación de Linfocitos B/genética , Línea Celular Tumoral , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Inhibidores de Proteínas Quinasas/farmacología , Receptor trkA/antagonistas & inhibidoresRESUMEN
Crizotinib is a potent small-molecule inhibitor of ALK tyrosine kinase receptor (anaplastic lymphoma kinase; ALK) and hepatocyte growth factor receptor (HGF receptor, proto-oncogene c-Met). A range of tumors, including subsets of non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma and inflammatory myofibroblastic tumors harbor an ALK rearrangement that leads to oncogenic activation of ALK. Crizotinib has demonstrated preclinical and clinical activity against such malignancies through inhibition of ALK, and patients harboring ALK- rearranged NSCLC have demonstrated high response rates and prolonged progression-free survival in phase I and II studies. In August 2011, crizotinib was approved for the treatment of advanced ALK-positive NSCLC.
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Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Terapia Molecular Dirigida , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirazoles/uso terapéutico , Piridinas/uso terapéutico , Quinasa de Linfoma Anaplásico , Animales , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/patología , Crizotinib , Interacciones Farmacológicas , Resistencia a Antineoplásicos , Medicina Basada en la Evidencia , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Inhibidores de Proteínas Quinasas/efectos adversos , Inhibidores de Proteínas Quinasas/farmacocinética , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/metabolismo , Pirazoles/efectos adversos , Pirazoles/farmacocinética , Piridinas/efectos adversos , Piridinas/farmacocinética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/metabolismo , Resultado del TratamientoRESUMEN
The treatment of advanced non � small cell lung cancer (NSCLC) increasingly involves the use of molecularly targeted therapy with activity against either the tumor directly, or indirectly, through activity against host-derived mechanisms of tumor support such as angiogenesis. The most well studied signaling pathway associated with angiogenesis is the vascular endothelial growth factor (VEGF) pathway, and the only antiangiogenic agent currently approved for the treatment of NSCLC is bevacizumab, an antibody targeted against VEGF. More recently, preclinical data supporting the role of fibroblast growth factor receptor (FGFR) and platelet-derived growth factor receptor (PDGFR) signaling in angiogenesis have been reported. The platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) pathways may also stimulate tumor growth directly through activation of downstream mitogenic signaling cascades. In addition, 1 or both of these pathways have been associated with resistance to agents targeting the epidermal growth factor receptor (EGFR) and VEGF. A number of agents that target FGF and/or PDGF signaling are now in development for the treatment of NSCLC. This review will summarize the potential molecular roles of PDGFR and FGFR in tumor growth and angiogenesis, as well as discuss the current clinical status of PDGFR and FGFR inhibitors in clinical development.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/efectos de los fármacos , Receptores del Factor de Crecimiento Derivado de Plaquetas/efectos de los fármacos , Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/irrigación sanguínea , Carcinoma de Pulmón de Células no Pequeñas/patología , Humanos , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/patología , Neovascularización Patológica , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Selection for suppressors of defects in the signal sequence of secretory proteins has led most commonly to identification of prlA alleles and less often to identification of prlG alleles. These genes, secY/prlA and secE/prlG, encode integral membrane components of the protein translocation system of Escherichia coli. We demonstrate that an outer membrane protein, LamB, that lacks a signal sequence can be exported with reasonable efficiency in both prlA and prlG suppressor strains. Although the signal sequence is not absolutely required for export of LamB, the level of export in the absence of prl suppressor alleles is exceedingly low. Such strains are phenotypically LamB-, and functional LamB can be detected only by using sensitive infectious-center assays. Suppression of the LamB signal sequence deletion is dependent on normal components of the export pathway, indicating that suppression is not occurring through a bypass mechanism. Our results indicate that the majority of the known prlA suppressors function by an identical mechanism and, further, that the prlG suppressors work in a similar fashion. We propose that both PrlA and PrlG suppressors lack a proofreading activity that normally rejects defective precursors from the export pathway.
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Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Genes Supresores/genética , Proteínas de la Membrana/genética , Señales de Clasificación de Proteína/fisiología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/fisiología , Análisis Mutacional de ADN , Escherichia coli/fisiología , Genes Bacterianos/genética , Proteínas de la Membrana/fisiología , Porinas , Conformación Proteica , Receptores Virales/química , Receptores Virales/genética , Receptores Virales/metabolismo , Canales de Translocación SEC , Eliminación de Secuencia/genéticaRESUMEN
HLA-DM removes CLIP and other loosely bound peptides from MHC class II molecules. The crystal structures of class II molecules and of HLA-DM have not permitted identification of their interaction sites. Here, we describe mutations in class II that impair interactions with DM. Libraries of randomly mutagenized DR3 alpha and beta chains were screened for their ability to cause cell surface accumulation of CLIP/DR3 complexes in EBV-B cells. Seven mutations were associated with impaired peptide loading in vivo, as detected by SDS stability assays. In vitro, these mutant DR3 molecules were resistant to DM-catalyzed CLIP release and showed reduced binding to DM. All mutations localize to a single lateral face of HLA-DR, which we propose interacts with DM during peptide exchange.
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Antígenos HLA-D/metabolismo , Antígenos HLA-DR/metabolismo , Secuencia de Aminoácidos , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Diferenciación de Linfocitos B/metabolismo , Línea Celular , Células Clonales , Antígenos HLA-D/genética , Antígenos HLA-D/inmunología , Antígenos HLA-DR/genética , Antígenos HLA-DR/inmunología , Antígeno HLA-DR3/genética , Antígeno HLA-DR3/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Inmunidad Innata , Inmunofenotipificación , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mapeo Peptídico , Mutación Puntual , Unión Proteica/genética , Unión Proteica/inmunología , Dodecil Sulfato de Sodio/farmacologíaRESUMEN
HLA-DM (abbreviated DM) is an MHC-encoded glycoprotein that catalyzes the selective release of peptides, including class II-associated invariant chain peptides, from MHC class II molecules. To perform its function, DM must assemble in the endoplasmic reticulum (ER), travel to endosomes, and interact productively with class II molecules. We have described previously an EBV-transformed B cell line, 7.12.6, which displays a partial Ag presentation defect and expresses a mutated DM beta-chain with Cys79 replaced by Tyr. In this study, we show that HLA-DR molecules in 7.12.6 have a defect in peptide loading and accumulate class II-associated invariant chain peptides (CLIP). Peptide loading is restored by transfection of wild-type DMB. The mutant DM molecules exit the ER slowly and are degraded rapidly, resulting in greatly reduced levels of mutant DM in post-Golgi compartments. Whereas wild-type DM forms noncovalent alphabeta dimers, such dimers form inefficiently in 7.12.6; many mutant DM beta-chains instead form a disulfide-bonded dimer with DM alpha. Homodimers of DM beta are also detected in 7.12.6 and in the alpha-chain defective mutant, 2.2.93. We conclude that during folding of wild-type DM, the native conformation is stabilized by a conserved disulfide bond involving Cys79beta and by noncovalent contacts with DM alpha. Without these interactions, DM beta can form malfolded structures containing interchain disulfide bonds; malfolding is correlated with ER retention and accelerated degradation.
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Disulfuros/metabolismo , Antígenos HLA-D/genética , Antígenos HLA-D/metabolismo , Mutagénesis Sitio-Dirigida , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Diferenciación de Linfocitos B/metabolismo , Transporte Biológico/genética , Línea Celular , Cisteína/genética , ADN Complementario/análisis , Dimerización , Antígenos HLA-D/fisiología , Antígeno HLA-DR3/genética , Antígeno HLA-DR3/metabolismo , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Péptidos/genética , Péptidos/inmunología , Péptidos/metabolismo , Mutación Puntual , Unión Proteica/genética , Análisis de Secuencia de ADN , Transfección/inmunología , Tirosina/genéticaRESUMEN
The HLA-DR hemizygous B lymphoblastoid cell line, 10.24.6, has a DRA mutation (Pro96-->Ser) that creates a novel glycosylation site at Asn94. The mutant DR molecules are primarily associated with nested fragments of invariant chain (class II-associated invariant chain peptides), and their interaction with HLA-DM is impaired. Here we further analyzed the defect in 10.24.6 cells. Expressing Ser96 mutant DRA cDNA in DRA-null cells recapitulated the 10.24.6 phenotype, indicating that the mutation causes the Ag presentation defect. A mutation to Ala96alpha, which does not introduce an extra glycan, generated a normal phenotype; the critical role of the glycan was further supported by experiments in which N-glycosylation was blocked by tunicamycin. We also evaluated whether the 10.24.6 mutation affected DR3 maturation or trafficking. Metabolic labeling and subcellular fractionation showed that assembly, endosomal transport, and invariant chain proteolysis of mutant DR3 molecules were similar to wild-type. A slight delay in export from the endoplasmic reticulum to the Golgi apparatus in 10.24.6 cells probably did not contribute significantly to the Ag presentation defect, because the abundance of DM and mutant DR in peptide-loading compartments was normal at steady state. Our results indicate that proper localization of these molecules does not depend on their interaction.