RESUMO
Pairing immunostimulatory small molecules with the targeting capability of an antibody has emerged as a novel therapeutic modality with the potential to treat a variety of solid tumors. A series of compounds based on an imidazo-thienopyridine scaffold were synthesized and tested for their ability to agonize the innate immune sensors toll-like receptor 7 and 8 (TLR7/8). Structure-activity relationship (SAR) studies revealed that certain simple amino-substituents could enable TLR7 agonism at low nanomolar concentrations. Drug-linkers containing either payload 1 or 20h were conjugated to the HER2-targeting antibody trastuzumab at the interchain disulfide cysteine residues using a cleavable valine-citrulline dipeptide linker and stochastic thiol-maleimide chemistry. In vitro, these immune-stimulating antibody drug-conjugates (ADCs) were found to induce cytokine release in a murine splenocyte assay when co-cultured with the HER2-high NCI-N87 cancer cell line. In vivo, tumor regression was observed with a single dose in an NCI-N87 gastric carcinoma xenograft model in BALB/c nude mice.
Assuntos
Antineoplásicos , Imunoconjugados , Camundongos , Humanos , Animais , Receptor 7 Toll-Like , Imunoconjugados/química , Camundongos Nus , Trastuzumab/química , Adjuvantes Imunológicos , Linhagem Celular Tumoral , Tienopiridinas , Antineoplásicos/farmacologia , Antineoplásicos/químicaRESUMO
Neurotrophins and their receptors are frequently expressed in malignant gliomas, yet their functions are largely unknown. Previously, we have shown that p75 neurotrophin receptor is required for glioma invasion and proliferation. However, the role of Trk receptors has not been examined. In this study, we investigated the importance of TrkB and TrkC in survival of brain tumor-initiating cells (BTICs). Here, we show that human malignant glioma tissues and also tumor-initiating cells isolated from fresh human malignant gliomas express the neurotrophin receptors TrkB and TrkC, not TrkA, and they also express neurotrophins NGF, BDNF, and neurotrophin 3 (NT3). Specific activation of TrkB and TrkC receptors by ligands BDNF and NT3 enhances tumor-initiating cell viability through activation of ERK and Akt pathways. Conversely, TrkB and TrkC knockdown or pharmacologic inhibition of Trk signaling decreases neurotrophin-dependent ERK activation and BTIC growth. Further, pharmacological inhibition of both ERK and Akt pathways blocked BDNF, and NT3 stimulated BTIC survival. Importantly, attenuation of BTIC growth by EGFR inhibitors could be overcome by activation of neurotrophin signaling, and neurotrophin signaling is sufficient for long term BTIC growth as spheres in the absence of EGF and FGF. Our results highlight a novel role for neurotrophin signaling in brain tumor and suggest that Trks could be a target for combinatorial treatment of malignant glioma.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Sistema de Sinalização das MAP Quinases , Células-Tronco Neoplásicas/metabolismo , Fatores de Crescimento Neural/metabolismo , Receptor trkB/metabolismo , Receptor trkC/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Masculino , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/fisiologia , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/farmacologia , Receptor trkB/genética , Receptor trkC/genéticaRESUMO
Neural cell fate specification is well understood in the embryonic cerebral cortex, where the proneural genes Neurog2 and Ascl1 are key cell fate determinants. What is less well understood is how cellular diversity is generated in brain tumors. Gliomas and glioneuronal tumors, which are often localized in the cerebrum, are both characterized by a neoplastic glial component, but glioneuronal tumors also have an intermixed neuronal component. A core abnormality in both tumor groups is overactive RAS/ERK signaling, a pro-proliferative signal whose contributions to cell differentiation in oncogenesis are largely unexplored. We found that RAS/ERK activation levels differ in two distinct human tumors associated with constitutively active BRAF. Pilocytic astrocytomas, which contain abnormal glial cells, have higher ERK activation levels than gangliogliomas, which contain abnormal neuronal and glial cells. Using in vivo gain of function and loss of function in the mouse embryonic neocortex, we found that RAS/ERK signals control a proneural genetic switch, inhibiting Neurog2 expression while inducing Ascl1, a competing lineage determinant. Furthermore, we found that RAS/ERK levels control Ascl1's fate specification properties in murine cortical progenitors--at higher RAS/ERK levels, Ascl1(+) progenitors are biased toward proliferative glial programs, initiating astrocytomas, while at moderate RAS/ERK levels, Ascl1 promotes GABAergic neuronal and less glial differentiation, generating glioneuronal tumors. Mechanistically, Ascl1 is phosphorylated by ERK, and ERK phosphoacceptor sites are necessary for Ascl1's GABAergic neuronal and gliogenic potential. RAS/ERK signaling thus acts as a rheostat to influence neural cell fate selection in both normal cortical development and gliomagenesis, controlling Neurog2-Ascl1 expression and Ascl1 function.
Assuntos
Neoplasias Encefálicas/metabolismo , Córtex Cerebral/metabolismo , Genes ras/fisiologia , Glioma/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Neurônios/metabolismo , Animais , Neoplasias Encefálicas/patologia , Córtex Cerebral/embriologia , Córtex Cerebral/patologia , Feminino , Glioma/patologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Transgênicos , GravidezRESUMO
Malignant gliomas are highly invasive, proliferative, and resistant to treatment. Previously, we have shown that p75 neurotrophin receptor (p75NTR) is a novel mediator of invasion of human glioma cells. However, the role of p75NTR in glioma proliferation is unknown. Here we used brain tumor-initiating cells (BTICs) and show that BTICs express neurotrophin receptors (p75NTR, TrkA, TrkB, and TrkC) and their ligands (NGF, brain-derived neurotrophic factor, and neurotrophin 3) and secrete NGF. Down-regulation of p75NTR significantly decreased proliferation of BTICs. Conversely, exogenouous NGF stimulated BTIC proliferation through α- and γ-secretase-mediated p75NTR cleavage and release of its intracellular domain (ICD). In contrast, overexpression of the p75NTR ICD induced proliferation. Interestingly, inhibition of Trk signaling blocked NGF-stimulated BTIC proliferation and p75NTR cleavage, indicating a role of Trk in p75NTR signaling. Further, blocking p75NTR cleavage attenuated Akt activation in BTICs, suggesting role of Akt in p75NTR-mediated proliferation. We also found that p75NTR, α-secretases, and the four subunits of the γ-secretase enzyme were elevated in glioblastoma multiformes patients. Importantly, the ICD of p75NTR was commonly found in malignant glioma patient specimens, suggesting that the receptor is activated and cleaved in patient tumors. These results suggest that p75NTR proteolysis is required for BTIC proliferation and is a novel potential clinical target.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Neoplasias Encefálicas/metabolismo , Encéfalo/patologia , Glioma/metabolismo , Células-Tronco Neoplásicas/patologia , Fatores de Crescimento Neural/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Encéfalo/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Silenciamento de Genes , Glioma/genética , Glioma/patologia , Humanos , Mutação , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Receptor de Fator de Crescimento Neural/genéticaRESUMO
In recent years, the field of antibody drug conjugates (ADC) has seen a resurgence, largely driven by the clinical benefit observed in patients treated with ADCs incorporating camptothecin-based topoisomerase I inhibitor payloads. Herein, we present the development of a novel camptothecin ZD06519 (FD1), which has been specifically designed for its application as an ADC payload. A panel of camptothecin analogs with different substituents at the C-7 and C-10 positions of the camptothecin core was prepared and tested in vitro. Selected compounds spanning a range of potency and hydrophilicity were elaborated into drug-linkers, conjugated to trastuzumab, and evaluated in vitro and in vivo. ZD06519 was selected on the basis of its favorable properties as a free molecule and as an antibody conjugate, which include moderate free payload potency (â¼1 nmol/L), low hydrophobicity, strong bystander activity, robust plasma stability, and high-monomeric ADC content. When conjugated to different antibodies using a clinically validated MC-GGFG-based linker, ZD06519 demonstrated impressive efficacy in multiple cell line-derived xenograft models and noteworthy tolerability in healthy mice, rats, and non-human primates.
Assuntos
Camptotecina , Imunoconjugados , Ensaios Antitumorais Modelo de Xenoenxerto , Camptotecina/farmacologia , Camptotecina/química , Imunoconjugados/farmacologia , Imunoconjugados/química , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Desenho de Fármacos , Feminino , RatosRESUMO
Capicua (Cic) is a transcriptional repressor mutated in the brain cancer oligodendroglioma. Despite its cancer link, little is known of Cic's function in the brain. We show that nuclear Cic expression is strongest in astrocytes and neurons but weaker in stem cells and oligodendroglial lineage cells. Using a new conditional Cic knockout mouse, we demonstrate that forebrain-specific Cic deletion increases proliferation and self-renewal of neural stem cells. Furthermore, Cic loss biases neural stem cells toward glial lineage selection, expanding the pool of oligodendrocyte precursor cells (OPCs). These proliferation and lineage effects are dependent on de-repression of Ets transcription factors. In patient-derived oligodendroglioma cells, CIC re-expression or ETV5 blockade decreases lineage bias, proliferation, self-renewal, and tumorigenicity. Our results identify Cic as an important regulator of cell fate in neurodevelopment and oligodendroglioma, and suggest that its loss contributes to oligodendroglioma by promoting proliferation and an OPC-like identity via Ets overactivity.
Assuntos
Neoplasias Encefálicas/patologia , Células-Tronco Neurais/patologia , Oligodendroglioma/patologia , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Repressoras/metabolismo , Animais , Astrócitos/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Knockout , Neurônios/patologia , Oligodendroglia/citologia , Oligodendroglia/patologia , Cultura Primária de Células , Prosencéfalo/citologia , Prosencéfalo/patologia , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Expression of the Rac-guanine nucleotide exchange factor (RacGEF), P-Rex1 is a key determinant of progression to metastasis in a number of human cancers. In accordance with this proposed role in cancer cell invasion and metastasis, we find that ectopic expression of P-Rex1 in an immortalised human fibroblast cell line is sufficient to drive multiple migratory and invasive phenotypes. The invasive phenotype is greatly enhanced by the presence of a gradient of serum or platelet-derived growth factor, and is dependent upon the expression of functional PDGF receptor ß. Consistently, the invasiveness of WM852 melanoma cells, which endogenously express P-Rex1 and PDGFRß, is opposed by siRNA of either of these proteins. Furthermore, the current model of P-Rex1 activation is advanced through demonstration of P-Rex1 and PDGFRß as components of the same macromolecular complex. These data suggest that P-Rex1 has an influence on physiological migratory processes, such as invasion of cancer cells, both through effects upon classical Rac1-driven motility and a novel association with RTK signalling complexes.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Melanoma , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Microambiente Tumoral/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Substâncias Macromoleculares/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Invasividade Neoplásica/genética , Metástase Neoplásica , RNA Interferente Pequeno , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genéticaRESUMO
Metastases are the major cause of death from melanoma, a skin cancer that has the fastest rising incidence of any malignancy in the Western world. Molecular pathways that drive melanoblast migration in development are believed to underpin the movement and ultimately the metastasis of melanoma. Here we show that mice lacking P-Rex1, a Rac-specific Rho GTPase guanine nucleotide exchange factor, have a melanoblast migration defect during development evidenced by a white belly. Moreover, these P-Rex1(-/-) mice are resistant to metastasis when crossed to a murine model of melanoma. Mechanistically, this is associated with P-Rex1 driving invasion in a Rac-dependent manner. P-Rex1 is elevated in the majority of human melanoma cell lines and tumour tissue. We conclude that P-Rex1 has an important role in melanoblast migration and cancer progression to metastasis in mice and humans.