RESUMO
M3258 is an orally bioavailable, potent, selective, reversible inhibitor of the large multifunctional peptidase 7 (LMP7, ß5i, PSMB8) proteolytic subunit of the immunoproteasome, a component of the cellular protein degradation machinery, highly expressed in malignant hematopoietic cells including multiple myeloma. Here we describe the fit-for-purpose pharmacokinetic (PK)/pharmacodynamic (PD)/efficacy modeling of M3258 based on preclinical data from several species. The inhibition of LMP7 activity (PD) and tumor growth (efficacy) were tested in human multiple myeloma xenografts in mice. PK and efficacy data were correlated yielding a free M3258 concentration of 45 nM for half-maximal tumor growth inhibition (KC50). As M3258 only weakly inhibits LMP7 in mouse cells, both in vitro and in vivo bridging studies were performed in rats, monkeys, and dogs for translational modeling. These data indicated that the PD response in human xenograft models was closely reflected in dog PBMCs. A PK/PD model was established, predicting a free IC50 value of 9 nM for M3258 in dogs in vivo, in close agreement with in vitro measurements. In parallel, the human PK parameters of M3258 were predicted by various approaches including in vitro extrapolation and allometric scaling. Using PK/PD/efficacy simulations, the efficacious dose range and corresponding PD response in human were predicted. Taken together, these efforts supported the design of a phase Ia study of M3258 in multiple myeloma patients (NCT04075721). At the lowest tested dose level, the predicted exposure matched well with the observed exposure while the duration of LMP7 inhibition was underpredicted by the model. SIGNIFICANCE STATEMENT: M3258 is a novel inhibitor of the immunoproteasome subunit LMP7. The human PK and human efficacious dose range of M3258 were predicted using in vitro-in vivo extrapolation and allometric scaling methods together with a fit-for-purpose PK/PD and efficacy model based on data from several species. A comparison with data from the Phase Ia clinical study showed that the human PK was accurately predicted, while the extent and duration of PD response were more pronounced than estimated.
Assuntos
Mieloma Múltiplo , Humanos , Ratos , Camundongos , Animais , Cães , Mieloma Múltiplo/tratamento farmacológico , Modelos BiológicosRESUMO
The spleen tyrosine kinase (Syk) is a key mediator of immunoreceptor signaling in immune cells. Thus, interfering with the function of Syk by genetic deletion or pharmacological inhibition might influence a variety of allergic and autoimmune processes. Since conventional Syk knockout mice are not viable, studies addressing the effect of Syk deletion in adult animals have been limited. To further explore functions of Syk in animal models of allergy and to shed light on the role of Syk in the in vivo migration of neutrophils and monocytes, we generated inducible Syk knockout mice. These mice harbor a floxed Syk gene and a tamoxifen-inducible Cre recombinase under the control of the ubiquitously active Rosa26-promoter. Thus, treatment of mice with tamoxifen leads to the deletion of Syk in all organs. Syk-deleted mice were analyzed in mast cell-dependent models and in models focusing on neutrophil and monocyte migration. We show that Syk deletion in adult mice reduces inflammatory responses in mast cell-driven animal models of allergy and asthma but has no effect on the migration of neutrophils and monocytes. Therefore, the inducible Syk knockout mice presented here provide a valuable tool to further explore the role of Syk in disease-related animal models.
Assuntos
Movimento Celular , Quimiotaxia de Leucócito/imunologia , Hipersensibilidade/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Monócitos/imunologia , Neutrófilos/imunologia , Proteínas Tirosina Quinases/imunologia , Animais , Diferenciação Celular/imunologia , Separação Celular , Citometria de Fluxo , Inflamação/imunologia , Masculino , Mastócitos/citologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , Neutrófilos/citologia , Transdução de Sinais/imunologia , Quinase SykRESUMO
Proteasomes are broadly expressed key components of the ubiquitin-dependent protein degradation pathway containing catalytically active subunits (ß1, ß2, and ß5). LMP7 (ß5i) is a subunit of the immunoproteasome, an inducible isoform that is predominantly expressed in hematopoietic cells. Clinically effective pan-proteasome inhibitors for the treatment of multiple myeloma (MM) nonselectively target LMP7 and other subunits of the constitutive proteasome and immunoproteasome with comparable potency, which can limit the therapeutic applicability of these drugs. Here, we describe the discovery and structure-based hit optimization of novel amido boronic acids, which selectively inhibit LMP7 while sparing all other subunits. The exploitation of structural differences between the proteasome subunits culminated in the identification of the highly potent, exquisitely selective, and orally available LMP7 inhibitor 50 (M3258). Based on the strong antitumor activity observed with M3258 in MM models and a favorable preclinical data package, a phase I clinical trial was initiated in relapsed/refractory MM patients.
Assuntos
Descoberta de Drogas , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Inibidores de Proteassoma/síntese química , Inibidores de Proteassoma/química , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Relação Estrutura-AtividadeRESUMO
Amplification of fibroblast growth factor receptor 1 (FGFR1) in non-small cell lung cancer (NSCLC) has been considered as an actionable drug target. However, pan-FGFR tyrosine kinase inhibitors did not demonstrate convincing clinical efficacy in FGFR1-amplified NSCLC patients. This study aimed to characterise the molecular context of FGFR1 expression and to define biomarkers predictive of FGFR1 inhibitor response. In this study, 635 NSCLC samples were characterised for FGFR1 protein expression by immunohistochemistry and copy number gain (CNG) by in situ hybridisation (n = 298) or DNA microarray (n = 189). FGFR1 gene expression (n = 369) and immune cell profiles (n = 309) were also examined. Furthermore, gene expression, methylation and microRNA data from The Cancer Genome Atlas (TCGA) were compared. A panel of FGFR1-amplified NSCLC patient-derived xenograft (PDX) models were tested for response to the selective FGFR1 antagonist M6123. A minority of patients demonstrated FGFR1 CNG (10.5%) or increased FGFR1 mRNA (8.7%) and protein expression (4.4%). FGFR1 CNG correlated weakly with FGFR1 gene and protein expression. Tumours overexpressing FGFR1 protein were typically devoid of driver alterations (e.g. EGFR, KRAS) and showed reduced infiltration of T-lymphocytes and lower PD-L1 expression. Promoter methylation and microRNA were identified as regulators of FGFR1 expression in NSCLC and other cancers. Finally, NSCLC PDX models demonstrating FGFR1 amplification and FGFR1 protein overexpression were sensitive to M6123. The unique molecular and immune features of tumours with high FGFR1 expression provide a rationale to stratify patients in future clinical trials of FGFR1 pathway-targeting agents.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Metilação de DNA , Epigênese Genética , Neoplasias Pulmonares/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Antineoplásicos/farmacologia , Antígeno B7-H1/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Feminino , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/genética , MicroRNAs/metabolismo , Terapia de Alvo Molecular , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Large multifunctional peptidase 7 (LMP7/ß5i/PSMB8) is a proteolytic subunit of the immunoproteasome, which is predominantly expressed in normal and malignant hematolymphoid cells, including multiple myeloma, and contributes to the degradation of ubiquitinated proteins. Described herein for the first time is the preclinical profile of M3258; an orally bioavailable, potent, reversible and highly selective LMP7 inhibitor. M3258 demonstrated strong antitumor efficacy in multiple myeloma xenograft models, including a novel model of the human bone niche of multiple myeloma. M3258 treatment led to a significant and prolonged suppression of tumor LMP7 activity and ubiquitinated protein turnover and the induction of apoptosis in multiple myeloma cells both in vitro and in vivo Furthermore, M3258 showed superior antitumor efficacy in selected multiple myeloma and mantle cell lymphoma xenograft models compared with the approved nonselective proteasome inhibitors bortezomib and ixazomib. The differentiated preclinical profile of M3258 supported the initiation of a phase I study in patients with multiple myeloma (NCT04075721).
Assuntos
Ácidos Borônicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Furanos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/química , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose , Compostos de Boro/administração & dosagem , Bortezomib/administração & dosagem , Proliferação de Células , Feminino , Glicina/administração & dosagem , Glicina/análogos & derivados , Humanos , Camundongos , Camundongos Nus , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteólise , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
KRAS is the most frequently mutated driver of pancreatic, colorectal, and non-small cell lung cancers. Direct KRAS blockade has proved challenging, and inhibition of a key downstream effector pathway, the RAF-MEK-ERK cascade, has shown limited success because of activation of feedback networks that keep the pathway in check. We hypothesized that inhibiting SOS1, a KRAS activator and important feedback node, represents an effective approach to treat KRAS-driven cancers. We report the discovery of a highly potent, selective, and orally bioavailable small-molecule SOS1 inhibitor, BI-3406, that binds to the catalytic domain of SOS1, thereby preventing the interaction with KRAS. BI-3406 reduces formation of GTP-loaded RAS and limits cellular proliferation of a broad range of KRAS-driven cancers. Importantly, BI-3406 attenuates feedback reactivation induced by MEK inhibitors and thereby enhances sensitivity of KRAS-dependent cancers to MEK inhibition. Combined SOS1 and MEK inhibition represents a novel and effective therapeutic concept to address KRAS-driven tumors. SIGNIFICANCE: To date, there are no effective targeted pan-KRAS therapies. In-depth characterization of BI-3406 activity and identification of MEK inhibitors as effective combination partners provide an attractive therapeutic concept for the majority of KRAS-mutant cancers, including those fueled by the most prevalent mutant KRAS oncoproteins, G12D, G12V, G12C, and G13D.See related commentary by Zhao et al., p. 17.This article is highlighted in the In This Issue feature, p. 1.
Assuntos
Neoplasias Pulmonares , Proteínas Proto-Oncogênicas p21(ras) , Linhagem Celular Tumoral , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno , Mutação , Nucleotídeos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Spleen tyrosine kinase (Syk) binds ITAM-bearing receptors in a wide variety of cell types. One such example is the activation of mast cells, basophils and eosinophils via the stimulation of the FcepsilonRI receptor by IgE/allergen complexes. The possible role of Syk in inflammatory signaling cascades has led to the development of pharmacological agents designed to block the Syk catalytic domain as potential novel therapeutics. Whilst the enzymatic activity of Syk lends towards the design of small-molecule inhibitors, other attention has focused on the possibility of targeting Syk expression using anti-sense oligonucleotides as an alternate means of anti-inflammatory therapy. In this study, we compared the ability of multiple optimized Syk siRNA sequences and small-molecule Syk inhibitors to block FcepsilonRI-mediated signal transduction, degranulation and TNFalpha secretion in the basophilic cell line RBL-2H3. We also characterized the specificity of each siRNA sequence with regards to off-target induction of the interferon-inducible gene IFIT1. We identified a single siRNA sequence, which displayed a favorable profile of efficient Syk knockdown, blockage of FcepsilonRI-mediated signal transduction, degranulation and TNFalpha secretion and a lack of IFIT1 induction. The effect of this siRNA was comparable to that of the Syk kinase domain inhibitors BAY61-3606 and R406. The identification of an active and specific Syk siRNA could be a basis for the development of therapeutic Syk siRNAs against inflammatory diseases.
Assuntos
Antialérgicos/farmacologia , Basófilos/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Niacinamida/análogos & derivados , Oxazinas/farmacologia , Proteínas Tirosina Quinases/imunologia , Piridinas/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , Receptores de IgE/imunologia , Animais , Basófilos/efeitos dos fármacos , Basófilos/enzimologia , Linhagem Celular , Regulação Enzimológica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Niacinamida/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Ratos , Receptores de IgE/metabolismo , Transdução de Sinais , Quinase Syk , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Tepotinib is an oral MET inhibitor approved for metastatic non-small cell lung cancer (NSCLC) harboring MET exon 14 (METex14) skipping mutations. Examining treatment-naive or tepotinib-resistant cells with MET amplification or METex14 skipping mutations identifies other receptor tyrosine kinases (RTKs) that co-exist in cells prior to tepotinib exposure and become more prominent upon tepotinib resistance. In a small cohort of patients with lung cancer with MET genetic alterations treated with tepotinib, gene copy number gains of other RTKs were found at baseline and affected treatment outcome. An Src homology 2 domain-containing phosphatase 2 (SHP2) inhibitor delayed the emergence of tepotinib resistance and synergized with tepotinib in treatment-naive and tepotinib-resistant cells as well as in xenograft models. Alternative signaling pathways potentially diminish the effect of tepotinib monotherapy, and the combination of tepotinib with an SHP2 inhibitor enables the control of tumor growth in cells with MET genetic alterations.
RESUMO
Natural killer (NK) cells play a pivotal role in controlling cancer. Multiple extracellular receptors and internal signaling nodes tightly regulate NK activation. Cyclin-dependent kinases of the mediator complex (CDK8 and CDK19) were described as a signaling intermediates in NK cells. Here, we report for the first time the development and use of CDK8/19 inhibitors to suppress phosphorylation of STAT1S727 in NK cells and to augment the production of the cytolytic molecules perforin and granzyme B (GZMB). Functionally, this resulted in enhanced NK-cell-mediated lysis of primary leukemia cells. Treatment with the CDK8/19 inhibitor BI-1347 increased the response rate and survival of mice bearing melanoma and breast cancer xenografts. In addition, CDK8/19 inhibition augmented the antitumoral activity of anti-PD-1 antibody and SMAC mimetic therapy, both agents that promote T-cell-mediated antitumor immunity. Treatment with the SMAC mimetic compound BI-8382 resulted in an increased number of NK cells infiltrating EMT6 tumors. Combination of the CDK8/19 inhibitor BI-1347, which augments the amount of degranulation enzymes, with the SMAC mimetic BI-8382 resulted in increased survival of mice carrying the EMT6 breast cancer model. The observed survival benefit was dependent on an intermittent treatment schedule of BI-1347, suggesting the importance of circumventing a hyporesponsive state of NK cells. These results suggest that CDK8/19 inhibitors can be combined with modulators of the adaptive immune system to inhibit the growth of solid tumors, independent of their activity on cancer cells, but rather through promoting NK-cell function.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Quinase 8 Dependente de Ciclina/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose , Neoplasias da Mama/enzimologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Proliferação de Células , Citotoxicidade Imunológica/imunologia , Feminino , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Melanoma Experimental/enzimologia , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação , Fator de Transcrição STAT1/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The high attrition rate of oncology drug candidates can be in part explained by the disconnect between the standard preclinical models (e.g., 2D culture, xenograft tumors) commonly employed for drug discovery and the complex multicellular microenvironment of human cancers. As such, significant focus has recently shifted to the establishment of preclinical models that more closely recapitulate human tumors, such as patient-derived xenografts, 3D spheroids, humanized mice, and mixed-culture models. For these models to be suited to drug discovery, they should optimally exhibit reproducibility, high-throughput, and robust and simple assay readouts. In this article, we describe a protocol for the generation of an in vitro 3D co-culture spheroid model that recapitulates the interaction of tumor cells with stromal fibroblasts in pancreatic adenocarcinoma. We additionally describe protocols relevant to the analysis of these spheroids in high-throughput drug discovery campaigns such as the assessment of spheroid proliferation, immunofluorescence and immunohistochemistry staining of spheroids, live-cell and confocal imaging and analysis of cell surface markers.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Técnicas de Cocultura/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias Pancreáticas/tratamento farmacológico , Esferoides Celulares/efeitos dos fármacos , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas/métodos , Imunofluorescência/métodos , Humanos , Citometria por Imagem/métodos , Imuno-Histoquímica/métodos , Camundongos , Células NIH 3T3 , Neoplasias Pancreáticas/patologia , Esferoides Celulares/patologia , Células Tumorais CultivadasRESUMO
Exosomes are small membrane vesicles derived from intracellular multivescicular bodies (MVBs) that can undergo constitutive and regulated secretion from cells. Exosomes can also secrete soluble proteins through metalloprotease-dependent ectodomain shedding. In this study, we sought to determine whether ErbB1 receptors are present within exosomes isolated from the human keratinocyte cell line, HaCaT, and whether exosome-associated ErbB1 receptors can undergo further proteolytic processing. We show that full-length transmembrane ErbB1 is secreted in HaCaT exosomes. EGF treatment and calcium flux stimulated the release of phosphorylated ErbB1 in exosomes but only ligand-stimulated release was blocked by the ErbB1 kinase inhibitor, AG1478, indicating that ligand-dependent ErbB1 receptor activation can initiate ErbB1 secretion into exosomes. In addition, other immunoreactive but truncated ErbB1 isoforms were detected in exosomes suggestive of additional proteolytic processing. We demonstrate that cellular and exosomal ErbB1 receptors can undergo ectodomain shedding to generate soluble N-terminal ectodomains and membrane-associated C-terminal remnant fragments (CTFs). ErbB1 shedding was activated by calcium flux and the metalloprotease activator APMA (4-aminophenylmercuric acetate) and was blocked by a metalloprotease inhibitor (GM6001). Soluble ErbB1 ectodomains shed into conditioned medium retained the ability to bind exogenous ligand. Our results provide new insights into the proteolysis, trafficking and fate of ErbB1 receptors and suggest that the novel ErbB1 isoforms may have functions distinct from the plasma membrane receptor.
Assuntos
Membrana Celular/metabolismo , Receptores ErbB/biossíntese , Metaloproteases/metabolismo , Transporte Biológico , Linhagem Celular , Dipeptídeos/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/isolamento & purificação , Exocitose , Humanos , Membranas Intracelulares/metabolismo , Metaloproteases/antagonistas & inibidores , Acetato de Fenilmercúrio/análogos & derivados , Acetato de Fenilmercúrio/farmacologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/isolamento & purificação , Estrutura Terciária de Proteína , Quinazolinas , Transdução de Sinais , Tirfostinas/farmacologiaRESUMO
Internalization of ligand-activated type I IGF receptor (IGF1R) is followed by recycling to the plasma membrane, degradation or nuclear translocation. Nuclear IGF1R reportedly associates with clinical response to IGF1R inhibitory drugs, yet its role in the nucleus is poorly characterized. Here, we investigated the significance of nuclear IGF1R in clinical cancers and cell line models. In prostate cancers, IGF1R was predominantly membrane localized in benign glands, while malignant epithelium contained prominent internalized (nuclear/cytoplasmic) IGF1R, and nuclear IGF1R associated significantly with advanced tumor stage. Using ChIP-seq to assess global chromatin occupancy, we identified IGF1R-binding sites at or near transcription start sites of genes including JUN and FAM21, most sites coinciding with occupancy by RNA polymerase II (RNAPol2) and histone marks of active enhancers/promoters. IGF1R was inducibly recruited to chromatin, directly binding DNA and interacting with RNAPol2 to upregulate expression of JUN and FAM21, shown to mediate tumor cell survival and IGF-induced migration. IGF1 also enriched RNAPol2 on promoters containing IGF1R-binding sites. These functions were inhibited by IGF1/II-neutralizing antibody xentuzumab (BI 836845), or by blocking receptor internalization. We detected IGF1R on JUN and FAM21 promoters in fresh prostate cancers that contained abundant nuclear IGF1R, with evidence of correlation between nuclear IGF1R content and JUN expression in malignant prostatic epithelium. Taken together, these data reveal previously unrecognized molecular mechanisms through which IGFs promote tumorigenesis, with implications for therapeutic evaluation of anti-IGF drugs.Significance: These findings reveal a noncanonical nuclear role for IGF1R in tumorigenesis, with implications for therapeutic evaluation of IGF inhibitory drugs. Cancer Res; 78(13); 3497-509. ©2018 AACR.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-jun/genética , RNA Polimerase II/metabolismo , Receptores de Somatomedina/metabolismo , Idoso , Linhagem Celular Tumoral , Movimento Celular/genética , Núcleo Celular/patologia , Sobrevivência Celular/genética , Cromatina/genética , Cromatina/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Regiões Promotoras Genéticas/genética , Prostatectomia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Receptor IGF Tipo 1 , Transdução de Sinais/genética , Sítio de Iniciação de Transcrição , Regulação para CimaRESUMO
Ectodomain shedding is a proteolytic mechanism by which transmembrane molecules are converted into a soluble form. Cleavage is mediated by metalloproteases and proceeds in a constitutive or inducible fashion. Although believed to be a cell-surface event, there is increasing evidence that cleavage can take place in intracellular compartments. However, it is unknown how cleaved soluble molecules get access to the extracellular space. By analysing L1 (CD171) and CD44 in ovarian carcinoma cells, we show in the present paper that the cleavage induced by ionomycin, APMA (4-aminophenylmercuric acetate) or MCD (methyl-beta-cyclodextrin) is initiated in an endosomal compartment that is subsequently released in the form of exosomes. Calcium influx augmented the release of exosomes containing functionally active forms of ADAM10 (a disintegrin and metalloprotease 10) and ADAM17 [TACE (tumour necrosis factor a-converting enzyme)] as well as CD44 and L1 cytoplasmic cleavage fragments. Cleavage could also proceed in released exosomes, but only depletion of ADAM10 by small interfering RNA blocked cleavage under constitutive and induced conditions. In contrast, cleavage of L1 in response to PMA occurred at the cell surface and was mediated by ADAM17. We conclude that different ADAMs are involved in distinct cellular compartments and that ADAM10 is responsible for shedding in vesicles. Our findings open up the possibility that exosomes serve as a platform for ectodomain shedding and as a vehicle for the cellular export of soluble molecules.
Assuntos
Vesículas Citoplasmáticas/metabolismo , Exocitose , Receptores de Hialuronatos/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Proteína ADAM17 , Secretases da Proteína Precursora do Amiloide , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Humanos , Receptores de Hialuronatos/química , Receptores de Hialuronatos/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Molécula L1 de Adesão de Célula Nervosa/química , Molécula L1 de Adesão de Célula Nervosa/genética , Estrutura Terciária de Proteína , Interferência de RNARESUMO
PURPOSE: BI 893923 is a novel IGF1R/INSR inhibitor with promising anti-tumor efficacy. Dose-limiting hyperglycemia has been observed for other IGF1R/INSR inhibitors in clinical trials. To counterbalance anti-tumor efficacy with the risk of hyperglycemia and to determine the therapeutic window, we aimed to develop a translational pharmacokinetic/pharmacodynamics model for BI 893923. This aimed to translate pharmacokinetics and pharmacodynamics from animals to humans by an allometrically scaled semi-mechanistic model. METHODS: Model development was based on a previously published PK/PD model for BI 893923 in mice (Titze et al., Cancer Chemother Pharmacol 77:1303-1314, 13). PK and blood glucose parameters were scaled by allometric principles using body weight as a scaling factor along with an estimation of the parameter exponents. Biomarker and tumor growth parameters were extrapolated from mouse to human using the body weight ratio as scaling factor. RESULTS: The allometric PK/PD model successfully described BI 893923 pharmacokinetics and blood glucose across mouse, rat, dog, minipig, and monkey. BI 893923 human exposure as well as blood glucose and tumor growth were predicted and compared for different dosing scenarios. A comprehensive risk-benefit analysis was conducted by determining the net clinical benefit for each schedule. An oral dose of 2750 mg BI 893923 divided in three evenly distributed doses was identified as the optimal human dosing regimen, predicting a tumor growth inhibition of 90.4% without associated hyperglycemia. CONCLUSION: Our model supported human therapeutic dose estimation by rationalizing the optimal efficacious dosing regimen with minimal undesired effects. This modeling approach may be useful for PK/PD scaling of other IGF1R/INSR inhibitors.
Assuntos
Hiperglicemia/induzido quimicamente , Pirimidinas/efeitos adversos , Pirimidinas/farmacocinética , Receptor de Insulina/antagonistas & inibidores , Receptores de Somatomedina/antagonistas & inibidores , Algoritmos , Animais , Antígenos CD , Biomarcadores Tumorais/sangue , Glicemia , Peso Corporal , Simulação por Computador , Cães , Humanos , Hiperglicemia/sangue , Macaca fascicularis , Camundongos , Modelos Estatísticos , Dinâmica não Linear , Pirimidinas/farmacologia , Ratos Wistar , Receptor IGF Tipo 1 , Medição de Risco , Suínos , Porco Miniatura , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Clinical studies of pharmacologic agents targeting the insulin-like growth factor (IGF) pathway in unselected cancer patients have so far demonstrated modest efficacy outcomes, with objective responses being rare. As such, the identification of selection biomarkers for enrichment of potential responders represents a high priority for future trials of these agents. Several reports have described high IGF2 expression in a subset of colorectal cancers, with focal IGF2 amplification being responsible for some of these cases. We defined a novel cut-off value for IGF2 overexpression based on differential expression between colorectal tumors and normal tissue samples. Analysis of two independent colorectal cancer datasets revealed IGF2 to be overexpressed at a frequency of 13% to 22%. An in vitro screen of 34 colorectal cancer cell lines revealed IGF2 expression to significantly correlate with sensitivity to the IGF1R/INSR inhibitor BI 885578. Furthermore, autocrine IGF2 constitutively activated IGF1R and Akt phosphorylation, which was inhibited by BI 885578 treatment. BI 885578 significantly delayed the growth of IGF2-high colorectal cancer xenograft tumors in mice, while combination with a VEGF-A antibody increased efficacy and induced tumor regression. Besides colorectal cancer, IGF2 overexpression was detected in more than 10% of bladder carcinoma, hepatocellular carcinoma and non-small cell lung cancer patient samples. Meanwhile, IGF2-high non-colorectal cancer cells lines displayed constitutive IGF1R phosphorylation and were sensitive to BI 885578. Our findings suggest that IGF2 may represent an attractive patient selection biomarker for IGF pathway inhibitors and that combination with VEGF-targeting agents may further improve clinical outcomes. Mol Cancer Ther; 16(10); 2223-33. ©2017 AACR.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Fator de Crescimento Insulin-Like II/antagonistas & inibidores , Receptores de Somatomedina/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Fator de Crescimento Insulin-Like II/genética , Camundongos , Pirazóis/administração & dosagem , Quinazolinas/administração & dosagem , Receptor IGF Tipo 1 , Receptores de Somatomedina/genética , Fator A de Crescimento do Endotélio Vascular/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Exosomes are small microvesicles that are released from late endosomal compartments of cultured cells. Recent work has shown that exosome-like vesicles are also found in many body fluids such as blood, urine, ascites and amnionic fluid. Although the biological function of exosomes is far from being fully understood, exosomes may have general importance in cell biology and immunology. The present review aims to address some of the facets of exosome research with particular emphasis on the immunologist's perspective: (i) exosomes as a novel platform for the ectodomain shedding of membrane proteins by ADAMs and (ii) recent findings on the role of exosomes in tumor biology and immune regulation.
Assuntos
Endossomos/metabolismo , Exocitose , Imunidade/fisiologia , Animais , Endossomos/ultraestrutura , Humanos , Transporte ProteicoRESUMO
PURPOSE: BI 893923 is a novel IGF1R/INSR tyrosine kinase inhibitor demonstrating anti-tumor efficacy and good tolerability. We aimed to characterize the relationship between BI 893923 plasma concentration, tumor biomarker modulation, tumor growth and hyperglycemia in mice using in silico modeling analyses. METHODS: In vitro molecular and cellular assays were used to demonstrate the potency and selectivity of BI 893923. Diverse in vitro DMPK assays were used to characterize the compound's drug-like properties. Mice xenografted with human GEO tumors were treated with different doses of BI 893923 to demonstrate the compound's efficacy, biomarker modulation and tolerability. PK/PD analyses were performed using nonlinear mixed-effects modeling. RESULTS: BI 893923 demonstrated potent and selective molecular inhibition of the IGF1R and INSR and demonstrated attractive drug-like properties (permeability, bioavailability). BI 893923 dose-dependently reduced GEO tumor growth and demonstrated good tolerability, characterized by transient hyperglycemia and normal body weight gain. A population PK/PD model was developed, which established relationships between BI 893923 pharmacokinetics, hyperglycemia, pIGF1R reduction and tumor growth. CONCLUSION: BI 893923 demonstrates molecular properties consistent with a highly attractive inhibitor of the IGF1R/INSR. A generic PK/PD model was developed to support preclinical drug development and dose finding in mice.
Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/farmacocinética , Neoplasias do Colo/tratamento farmacológico , Modelos Biológicos , Pirimidinas/farmacologia , Pirimidinas/farmacocinética , Receptor de Insulina/antagonistas & inibidores , Receptores de Somatomedina/antagonistas & inibidores , Animais , Antígenos CD , Antineoplásicos/sangue , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Cães , Glucose/metabolismo , Humanos , Camundongos , Pirimidinas/sangue , Pirimidinas/uso terapêutico , Ratos , Receptor IGF Tipo 1 , Suínos , Porco Miniatura , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Inhibition of the IGF1R, INSRA, and INSRB receptor tyrosine kinases represents an attractive approach of pharmacologic intervention in cancer, owing to the roles of the IGF1R and INSRA in promoting cell proliferation and survival. However, the central role of the INSRB isoform in glucose homeostasis suggests that prolonged inhibition of this kinase could result in metabolic toxicity. We describe here the profile of the novel compound BI 885578, a potent and selective ATP-competitive IGF1R/INSR tyrosine kinase inhibitor distinguished by rapid intestinal absorption and a short in vivo half-life as a result of rapid metabolic clearance. BI 885578, administered daily per os, displayed an acceptable tolerability profile in mice at doses that significantly reduced the growth of xenografted human GEO and CL-14 colon carcinoma tumors. We found that treatment with BI 885578 is accompanied by increases in circulating glucose and insulin levels, which in turn leads to compensatory hyperphosphorylation of muscle INSRs and subsequent normalization of blood glucose within a few hours. In contrast, the normalization of IGF1R and INSR phosphorylation in GEO tumors occurs at a much slower rate. In accordance with this, BI 885578 led to a prolonged inhibition of cell proliferation and induction of apoptosis in GEO tumors. We propose that the remarkable therapeutic window observed for BI 885578 is achieved by virtue of the distinctive pharmacokinetic properties of the compound, capitalizing on the physiologic mechanisms of glucose homeostasis and differential levels of IGF1R and INSR expression in tumors and normal tissues.
Assuntos
Antígenos CD/biossíntese , Neoplasias do Colo/tratamento farmacológico , Homeostase/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Pirazóis/administração & dosagem , Quinazolinas/administração & dosagem , Receptor de Insulina/biossíntese , Receptores de Somatomedina/biossíntese , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Humanos , Camundongos , Receptor IGF Tipo 1 , Receptor de Insulina/antagonistas & inibidores , Receptores de Somatomedina/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Spleen tyrosine kinase (Syk) is a key regulatory factor in the IgE-mediated allergic signal transduction pathway in mast cells and basophils. Syk is phosphorylated on a number of tyrosines following the binding of IgE/allergen complexes to FcÉRI receptors leading to initiation of inflammatory signaling via downstream enzymes and scaffolding proteins. We examined the kinases responsible for the phosphorylation of key Syk tyrosines in rat RBL-2H3 basophilic cells and bone marrow-derived mast cells (BMMCs). The phosphorylation of Syk tyrosine 346 was completely blocked by the novel Src family kinase inhibitor BIRA766, suggesting this tyrosine is a pure substrate for Src family kinases. This was supported by the findings that kinase-dead (KD) Syk was efficiently phosphorylated on this tyrosine and that a specific Syk inhibitor BAY61-3606 was without effect. The phosphorylation of other Syk tyrosines 317, 342, 519 and 520 was reduced by Syk and Src family inhibitors, suggesting a role for auto- and trans-phosphorylation. Lyn was the predominant Src family kinase expressed and activated in RBL-2H3 cells, meanwhile Lyn knockdown with a specific siRNA interfered with the phosphorylation of all Syk tyrosines and the Syk substrates SLP-76 and LAT. Pharmacological inhibition of Syk completely blocked the degranulation of RBL-2H3 and BMMCs. However, Lyn knockdown sensitized RBL-2H3 cells to FcÉRI-induced degranulation. We showed that whilst interference with Lyn expression disrupts FcÉRI proximal signaling via Syk and its direct substrates including SLP-76 and LAT, distal activation of downstream proteins including Erk is enhanced. This study identifies the responsible kinases for the phosphorylation of key Syk tyrosines and the propagation of FcÉRI receptor mediated signal transduction in allergic responses.
Assuntos
Degranulação Celular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mastócitos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de IgE/metabolismo , Transdução de Sinais/imunologia , Quinases da Família src/metabolismo , Animais , Basófilos/imunologia , Basófilos/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Hipersensibilidade/imunologia , Imunoglobulina E , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Mastócitos/imunologia , Camundongos , Fosforilação , Proteínas Tirosina Quinases/imunologia , Ratos , Receptores de IgE/imunologia , Quinase Syk , Transfecção , Quinases da Família src/imunologiaRESUMO
Spleen Tyrosine Kinase (Syk) is widely expressed in the immune system and functions in the transmission of inflammatory signals via ITAM-bearing cell surface receptors. The broad expression pattern and importance of Syk in regulating innate immunity and the inflammatory response have led to significant interest from the pharmaceutical industry to developing anti-Syk therapeutics for the treatment of inflammatory disorders such as allergic rhinitis and rheumatoid arthritis. While the function and regulation of Syk has been well-described in leukocytes, where its primary role is an early transducer of signaling following immunoreceptor engagement, Syk has recently been described in non-immune cells, such as the airway epithelium, that also play an important role in mediating the inflammatory response. This manuscript will focus on the expression and function of Syk in the context of inflammatory lung diseases, and review recent data that have demonstrated novel roles for Syk in airway epithelial cells, particularly its role in mediating the human rhinovirus (HRV) induced inflammatory response and viral cell entry. In addition, data describing the efficacy of novel Syk inhibitors in the management of inflammatory diseases in animal models and early clinical trials are also reviewed.