RESUMO
Inducing death receptor 5 (DR5) clustering holds particular promise in tumor-specific therapeutics because it could trigger an apoptotic cascade in cancerous cells. Herein, we present a tumor microenvironment H2O2-responsive self-illuminating nanoagonist, which could induce dual tumor cell death pathways through enhancing DR5 clustering. By conjugating DR5 ligand peptides onto the surfaces of self-illuminating nanoparticles with cross-linking capacity, this strategy not only provides scaffolds for ligands to bind receptors but also cross-links them through photo-cross-linking. This strategy allows for efficient activation of DR5 downstream signaling, initiating the extrinsic apoptosis pathway and immunogenic cell death of tumor cells, and contributes to improved tumor-specific immune responses, resulting in enhanced antitumor efficacy and minimized systemic adverse effects.
Assuntos
Nanopartículas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Humanos , Animais , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Nanopartículas/química , Camundongos , Apoptose/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Morte Celular/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias/metabolismo , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Peptídeos/química , Peptídeos/farmacologiaRESUMO
Clustering of type II tumor necrosis factor (TNF) receptors (TNFRs) is essential for their activation, yet currently available drugs fail to activate signaling. Some strategies aim to cluster TNFR by using multivalent streptavidin or scaffolds based on dextran or graphene. However, these strategies do not allow for control of the valency or spatial organization of the ligands, and consequently control of the TNFR activation is not optimal. DNA origami nanostructures allow nanometer-precise control of the spatial organization of molecules and complexes, with defined spacing, number and valency. Here, we demonstrate the design and characterization of a DNA origami nanostructure that can be decorated with engineered single-chain TNF-related apoptosis-inducing ligand (SC-TRAIL) complexes, which show increased cell killing compared to SC-TRAIL alone on Jurkat cells. The information in this chapter can be used as a basis to decorate DNA origami nanostructures with various proteins, complexes, or other biomolecules.
Assuntos
DNA , Nanoestruturas , Engenharia de Proteínas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Nanoestruturas/química , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/química , Humanos , Células Jurkat , DNA/químicaRESUMO
PURPOSE: Patients with unresectable/metastatic chondrosarcoma have poor prognoses; conventional chondrosarcoma is associated with a median progression-free survival (PFS) of <4 months after first-line chemotherapy. No standard targeted therapies are available. We present the preclinical characterization of INBRX-109, a third-generation death receptor 5 (DR5) agonist, and clinical findings from a phase I trial of INBRX-109 in unresectable/metastatic chondrosarcoma (NCT03715933). PATIENTS AND METHODS: INBRX-109 was first characterized preclinically as a DR5 agonist, with binding specificity and hepatotoxicity evaluated in vitro and antitumor activity evaluated both in vitro and in vivo. INBRX-109 (3 mg/kg every 3 weeks) was then evaluated in a phase I study of solid tumors, which included a cohort with any subtype of chondrosarcoma and a cohort with IDH1/IDH2-mutant conventional chondrosarcoma. The primary endpoint was safety. Efficacy was an exploratory endpoint, with measures including objective response, disease control rate, and PFS. RESULTS: In preclinical studies, INBRX-109 led to antitumor activity in vitro and in patient-derived xenograft models, with minimal hepatotoxicity. In the phase I study, INBRX-109 was well tolerated and demonstrated antitumor activity in unresectable/metastatic chondrosarcoma. INBRX-109 led to a disease control rate of 87.1% [27/31; durable clinical benefit, 40.7% (11/27)], including two partial responses, and median PFS of 7.6 months. Most treatment-related adverse events, including liver-related events, were low grade (grade ≥3 events in chondrosarcoma cohorts, 5.7%). CONCLUSIONS: INBRX-109 demonstrated encouraging antitumor activity with a favorable safety profile in patients with unresectable/metastatic chondrosarcoma. A randomized, placebo-controlled, phase II trial (ChonDRAgon, NCT04950075) will further evaluate INBRX-109 in conventional chondrosarcoma.
Assuntos
Neoplasias Ósseas , Condrossarcoma , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Humanos , Anticorpos Monoclonais/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas , Condrossarcoma/terapia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologiaRESUMO
Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) is a cytokine that initiates apoptosis upon binding to death receptor 5 (DR5) on cancer cells. Small molecule TRAIL mimetics have therefore been investigated as promising chemotherapeutic agents. Since anemia of chemotherapy is common, our goal is to investigate the hemolytic and eryptotic properties of novel DR5 agonist bioymifi (BMF) and identify the underlying molecular mechanisms. Whole blood (WB) was stimulated with 100 µM of BMF, whereas red blood cells (RBCs) were treated with 10-100 µM of BMF for 24 h at 37 °C. WB was analyzed for RBC, leukocyte, and platelet indices, while RBCs were examined for hemolysis by light absorbance of free hemoglobin, membrane scrambling by Annexin V-FITC, calcium by Fluo4/AM, cellular morphology by light scatter, and oxidative stress by 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) using flow cytometry. Caspase inhibitor Z-VAD-FMK, p38 inhibitor SB203580, casein kinase 1α inhibitor D4476, receptor-interacting protein 1 inhibitor necrostatin-2, reduced glutathione, or cyclooxygenase (COX) inhibitor aspirin were added accordingly. BMF exerted dose-responsive, calcium-independent hemolysis, reduced RBC hemoglobin, significantly increased Annexin V-, Fluo4-, and DCF-positive cells, along with a dual effect on forward and side light scatter. Notably, the cytotoxic potential of BMF was significantly mitigated upon pharmacological inhibition of p38. Furthermore, BMF exhibited selective toxicity to eosinophils and significantly diminished reticulocyte hemoglobin content. Altogether, these novel findings highlight the adverse outcomes of BMF exposure on RBC physiology and provide the first toxicological assessment of BMF as an antitumor agent.
Assuntos
Eriptose/efeitos dos fármacos , Ftalimidas/toxicidade , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Tiazolidinas/toxicidade , Cálcio/metabolismo , Eosinófilos/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
ONC201/TIC10 activates TRAIL signaling through ATF4 and the integrated stress response (ISR). ONC201 demonstrated tumor regressions and disease stability in patients with histone H3K27M-mutated midline-glioma. H3K27M-mutation prevents H3K27-methylation on the mutated allele. EZH2 inhibitors (EZH2i) reduce H3K27 methylation and have anti-tumor effects. We hypothesized ONC201 sensitivity and tumor apoptosis may increase by reducing H3K27-methylation with EZH2i or HDACi as mimics of H3K27M-mutation. EZH2i EPZ-6438 (tazemetostat) or PF-06821497 and HDACi vorinostat were combined with ONC201 to treat multiple cancer cell lines and cell viability and histone modifications were analyzed. We observed synergistic effects towards cell viability in multiple cancers by EPZ-6438 or PF-06821497 plus ONC201 or triple therapy with vorinostat, EPZ-6438, and ONC201. EPZ-6438 and vorinostat synergized with ONC201 to enhance apoptosis. Activation of the ISR and TRAIL-DR5 were observed in cells treated with ONC201 -/+ epigenetic modulators. Knockdown of ATF4 reduced DR5 induction and apoptosis following EZH2i and ONC201 treatment of U251 glioma cells. mRNA expression of dopamine-receptors did not correlate with ONC201 sensitivity in the tumor cell lines tested (N = 12), including changes after epigenetic drugs. Dopamine did not rescue apoptosis by ONC201 in different tumor cell lines (N = 10) including 2 GBM, 3 DIPG and did not prevent DR5 activation or apoptosis. DRD2 agonist sumanirole did not protect brain tumor cells (N = 6 including 4 DIPG cell lines) from ONC201 reduction in viability. Although synergy was observed with ONC201 and vorinostat, there was no significant increase in H3K27 acetylation in cell lines including DIPG as compared to vorinostat alone, and in some cases the acetylation was less than vorinostat alone at 72 H. H3K27 methylation reduction correlated with synergy from combinations of either EPZ-6438 or vorinostat with ONC201 or triple combination. Our findings provide a rationale for combination of ONC201 and epigenetic modulators including triple therapy for in vivo and clinical testing in treatment of human malignancies including brain tumors and DIPG.
Assuntos
Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Compostos de Bifenilo/farmacologia , Endopeptidase Clp/metabolismo , Histonas/metabolismo , Imidazóis/farmacologia , Morfolinas/farmacologia , Piridinas/farmacologia , Piridonas/farmacologia , Pirimidinas/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Vorinostat/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Metilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
Due to the absence of curative treatments for glioblastoma (GBM), we assessed the efficacy of single and combination treatments with a translationally relevant 2nd generation TRAIL-receptor agonist (IZI1551) and the blood-brain barrier (BBB) permeant proteasome inhibitor marizomib in a panel of patient-derived glioblastoma cell lines. These cells were cultured using protocols that maintain the characteristics of primary tumor cells. IZI1551+marizomib combination treatments synergistically induced apoptotic cell death in the majority of cases, both in 2D, as well as in 3D spheroid cultures. In contrast, single-drug treatments largely failed to induce noticeable amounts of cell death. Kinetic analyses suggested that time-shifted drug exposure might further increase responsiveness, with marizomib pre-treatments indeed strongly enhancing cell death. Cell death responses upon the addition of IZI1551 could also be observed in GBM cells that were kept in a medium collected from the basolateral side of a human hCMEC/D3 BBB model that had been exposed to marizomib. Interestingly, the subset of GBM cell lines resistant to IZI1551+marizomib treatments expressed lower surface amounts of TRAIL death receptors, substantially lower amounts of procaspase-8, and increased amounts of cFLIP, suggesting that apoptosis initiation was likely too weak to initiate downstream apoptosis execution. Indeed, experiments in which the mitochondrial apoptosis threshold was lowered by antagonizing Mcl-1 re-established sensitivity to IZI1551+marizomib in otherwise resistant cells. Overall, our study demonstrates a high efficacy of combination treatments with a latest-generation TRAIL receptor agonist and the BBB permeant proteasome inhibitor marizomib in relevant GBM cell models, as well as strategies to further enhance responsiveness and to sensitize subgroups of otherwise resistant GBM cases.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Glioma/tratamento farmacológico , Lactonas/farmacologia , Inibidores de Proteassoma/farmacologia , Pirróis/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Glioma/metabolismo , Glioma/patologia , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Pirimidinas/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transdução de Sinais , Esferoides Celulares , Tiofenos/farmacologia , Fatores de TempoRESUMO
The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) shows a promising therapeutic potential in cancer treatment as it exclusively causes apoptosis in a broad spectrum of cancer cells through triggering the extrinsic apoptosis pathway via binding to cognate death receptors, with negligible toxicity in normal cells. However, most cancers, including glioblastoma multiforme (GBM), display TRAIL resistance, hindering its application in clinical practice. Recent studies have unraveled novel mechanisms in regulating TRAIL-induced apoptosis in GBM and sought effective combinatorial modalities to sensitize GBM to TRAIL treatment, establishing pre-clinical foundations and the reasonable expectation that the TRAIL/TRAIL death receptor axis could be harnessed to treat GBM. In this review, we will revisit the status quo of the mechanisms of TRAIL resistance and emerging strategies for sensitizing GBM to TRAIL-induced apoptosis and also discuss opportunities of TRAIL-based combinatorial therapies in future clinical use for GBM treatment.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/tratamento farmacológico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Humanos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
Head and neck squamous cell carcinomas (HNSCC) induced by human papillomavirus (HPV) have increased recently in the US. However, the distinct alterations of molecules involved in the death pathways and drug effects targeting inhibitor of apoptosis proteins (IAPs) have not been extensively characterized in HPV(+) HNSCC cells. In this study, we observed the distinct genomic and expression alterations of nine genes involved in cell death in 55% HNSCC tissues, which were associated with HPV status, tumor staging, and anatomic locations. Expression of four genes was statistically correlated with copy number variation. A panel of HPV(+) HNSCC lines showed abundant TRAILR2 and IAP1 protein expression, but were not sensitive to IAP inhibitor birinapant alone, while combinatory treatment with TNFα or especially TRAIL enhanced this drug sensitivity. The death agonistic TRAILR2 antibody alone showed no cell inhibitory effects, whereas its combination with birinapant and/or TRAIL protein demonstrated additive or synergistic effects. We observed predominantly late apoptosis mode of cell death after combinatorial treatments, and pan-caspase (ZVAD) and caspase-8 (ZIETD) inhibitors attenuated treatment-induced cell death. Our genomic and expression data-driven study provides a framework for identifying relevant combinatorial therapies targeting death pathways in HPV(+) HNSCC and other squamous cancer types.
Assuntos
Proteína 3 com Repetições IAP de Baculovírus/genética , Dipeptídeos/farmacologia , Indóis/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 8/genética , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Inibidoras de Apoptose/genética , Masculino , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/tratamento farmacológico , Infecções por Papillomavirus/virologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Carcinoma de Células Escamosas de Cabeça e Pescoço/complicações , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, can induce apoptosis in cancer cells, sparing normal cells when bound to its associated death receptors (DR4/DR5). This unique mechanism makes TRAIL a potential anticancer therapeutic agent. However, clinical trials of recombinant TRAIL protein and TRAIL receptor agonist monoclonal antibodies have shown disappointing results due to its short half-life, poor pharmacokinetics and the resistance of the cancer cells. This review summarizes TRAIL-induced apoptotic and survival pathways as well as mechanisms leading to apoptotic resistance. Recent development of methods to overcome cancer cell resistance to TRAIL-induced apoptosis, such as protein modification, combination therapy and TRAIL-based gene therapy, appear promising. We also discuss the challenges and opportunities in the development of TRAIL-based therapies for the treatment of human cancers.
Assuntos
Antineoplásicos/farmacologia , Terapia Genética/métodos , Neoplasias/terapia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Ensaios Clínicos como Assunto , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Neoplasias/genética , Neoplasias/patologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Proteínas Recombinantes/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Resultado do TratamentoRESUMO
Activation of TRAILR2 has emerged as an important therapeutic concept in cancer treatment. TRAILR2 agonistic molecules have only had limited clinical success, to date, due either to lack of efficacy or hepatotoxicity. BI 905711 is a novel tetravalent bispecific antibody targeting both TRAILR2 and CDH17 and represents a novel liver-sparing TRAILR2 agonist specifically designed to overcome the disadvantages of previous strategies. Here, we show that BI 905711 effectively triggered apoptosis in a broad panel of CDH17-positive colorectal cancer tumor cells in vitro. Efficient induction of apoptosis was dependent on the presence of CDH17, as exemplified by the greater than 1,000-fold drop in potency in CDH17-negative cells. BI 905711 demonstrated single-agent tumor regressions in CDH17-positive colorectal cancer xenografts, an effect that was further enhanced upon combination with irinotecan. Antitumor efficacy correlated with induction of caspase activation, as measured in both the tumor and plasma. Effective tumor growth inhibition was further demonstrated across a series of different colorectal cancer PDX models. BI 905711 induced apoptosis in both a cis (same cell) as well as trans (adjacent cell) fashion, translating into significant antitumor activity even in xenograft models with heterogeneous CDH17 expression. In summary, we demonstrate that BI 905711 has potent and selective antitumor activity in CDH17-positive colorectal cancer models both in vitro and in vivo. The high prevalence of over 95% CDH17-positive tumors in patients with colorectal cancer, the molecule preclinical efficacy together with its potential for a favorable safety profile, support the ongoing BI 905711 phase I trial in colorectal cancer and additional CDH17-positive cancer types (NCT04137289).
Assuntos
Anticorpos Biespecíficos/farmacologia , Apoptose , Caderinas/metabolismo , Neoplasias Colorretais/patologia , Fígado/patologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Humanos , Fígado/efeitos dos fármacos , Camundongos , Metástase Neoplásica , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Indução de RemissãoRESUMO
In vivo research of the last decade revealed that the anchoring of antitumor necrosis factor (TNF) receptor superfamily (TNFRSF) receptor antibodies to cell-expressed Fcγ receptors (FcγR) can be of decisive relevance for their receptor-stimulatory activity. Indeed, FcγR anchoring may even result in the conversion of antagonistic to agonistic anti-TNFR antibody activity. The knowledge on this issue is obviously not only relevant to understand the in vivo effects of anti-TNFR antibodies but also of overwhelming importance for the rational clinical development of antibodies and antibody derivatives. Based on the fact that with exception of the decoy TNFRSF receptors (TNFRs) all TNFRs are able to trigger proinflammatory NFκB signaling, resulting in the production of chemokines and cytokines, we established an easy and broadly applicable coculture assay for the evaluation of the FcγR-dependency of the agonism of anti-TNFR antibodies. In this assay, TNFR responder cells, which produce high amounts of IL8 in response to TNFR stimulation, were pairwise incubated with empty vector- and FcγR-transfected HEK293 cells, which produce only very low amounts of IL8. This cocultures were then comparatively analyzed with respect to anti-TNFR antibody-induced IL8 production as a readout for TNFR activation to uncover proagonistic effects of FcγR binding.
Assuntos
Anticorpos Monoclonais/farmacologia , Receptores de IgG/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Citocinas/metabolismo , Humanos , NF-kappa B/metabolismo , Ligação Proteica , Transdução de SinaisRESUMO
RAS mutant (MT) metastatic colorectal cancer (mCRC) is resistant to MEK1/2 inhibition and remains a difficult-to-treat group. Therefore, there is an unmet need for novel treatment options for RASMT mCRC. RALA and RALB GTPases function downstream of RAS and have been found to be key regulators of several cell functions implicated in KRAS-driven tumorigenesis. However, their role as regulators of the apoptotic machinery remains to be elucidated. Here, we found that inhibition of RALB expression, but not RALA, resulted in Caspase-8-dependent cell death in KRASMT CRC cells, which was not further increased following MEK1/2 inhibition. Proteomic analysis and mechanistic studies revealed that RALB depletion induced a marked upregulation of the pro-apoptotic cell surface TRAIL Death Receptor 5 (DR5) (also known as TRAIL-R2), primarily through modulating DR5 protein lysosomal degradation. Moreover, DR5 knockdown or knockout attenuated siRALB-induced apoptosis, confirming the role of the extrinsic apoptotic pathway as a regulator of siRALB-induced cell death. Importantly, TRAIL treatment resulted in the association of RALB with the death-inducing signalling complex (DISC) and targeting RALB using pharmacologic inhibition or RNAi approaches triggered a potent increase in TRAIL-induced cell death in KRASMT CRC cells. Significantly, high RALB mRNA levels were found in the poor prognostic Colorectal Cancer Intrinsic Subtypes (CRIS)-B CRC subgroup. Collectively, this study provides to our knowledge the first evidence for a role for RALB in apoptotic priming and suggests that RALB inhibition may be a promising strategy to improve response to TRAIL treatment in poor prognostic RASMT CRIS-B CRC.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteínas ral de Ligação ao GTP/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzimidazóis/administração & dosagem , Neoplasias Colorretais/genética , Humanos , Mutação , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/administração & dosagem , Transfecção , Proteínas ral de Ligação ao GTP/antagonistas & inibidores , Proteínas ral de Ligação ao GTP/biossíntese , Proteínas ral de Ligação ao GTP/genéticaRESUMO
Methionine addiction is a fundamental and general hallmark of cancer. Gene expression analysis showed that methionine restriction (MR) of methionine-addicted cancer cells increases TNF-related apoptosis-induced ligand receptor-2 (TRAIL-R2) expression. Here, we determined the effects of MR on TRAIL-R2 targeted therapy in pancreatic cancer by the TRAIL-R2 agonist tigatuzumab. Human pancreatic cancer cell lines were cultured in control or methionine-free medium. The effects of MR on TRAIL-R2 expression and sensitivity to tigatuzumab were evaluated in vitro. An orthotopic pancreatic cancer mouse model was established to evaluate the efficacy of MR using oral recombinant methioninase (o-rMETase), and the efficacy of tigatuzumab and their combination. MR enabled tigatuzumab-induced apoptosis, by increasing TRAIL-R2 expression in pancreatic cancer cells in vitro. The protein expression level of the melanoma-associated antigen MAGED2, which reduces TRAIL-R2 expression, was decreased by MR. In the orthotopic pancreatic cancer mouse model, o-rMETase increased TRAIL-R2 expression level in the tumors and enabled the antitumor efficacy of tigatuzumab. MR, effected by o-rMETase, enabled the efficacy of the TRAIL-R2 agonist tigatuzumab by increasing TRAIL-R2 expression in pancreatic cancer. Our results suggest that o-rMETase has clinical potential for treating pancreatic cancer.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Liases de Carbono-Enxofre/administração & dosagem , Neoplasias Pancreáticas/tratamento farmacológico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/análise , Proteínas Adaptadoras de Transdução de Sinal/análise , Administração Oral , Animais , Antígenos de Neoplasias/análise , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Metionina/administração & dosagem , Metionina/sangue , Camundongos , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/patologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Proteínas Recombinantes/administração & dosagemRESUMO
Apoptotic cell death can be initiated through the extrinsic and intrinsic signaling pathways. While cell cycle progression promotes the responsiveness to intrinsic apoptosis induced by genotoxic stress or spindle poisons, this has not yet been studied conclusively for extrinsic apoptosis. Here, we combined fluorescence-based time-lapse monitoring of cell cycle progression and cell death execution by long-term time-lapse microscopy with sampling-based mathematical modeling to study cell cycle dependency of TRAIL-induced extrinsic apoptosis in NCI-H460/geminin cells. In particular, we investigated the interaction of cell death timing and progression of cell cycle states. We not only found that TRAIL prolongs cycle progression, but in reverse also that cell cycle progression affects the kinetics of TRAIL-induced apoptosis: Cells exposed to TRAIL in G1 died significantly faster than cells stimulated in S/G2/M. The connection between cell cycle state and apoptosis progression was captured by developing a mathematical model, for which parameter estimation revealed that apoptosis progression decelerates in the second half of the cell cycle. Similar results were also obtained when studying HCT-116 cells. Our results therefore reject the null hypothesis of independence between cell cycle progression and extrinsic apoptosis and, supported by simulations and experiments of synchronized cell populations, suggest that unwanted escape from TRAIL-induced apoptosis can be reduced by enriching the fraction of cells in G1 phase. Besides novel insight into the interrelation of cell cycle progression and extrinsic apoptosis signaling kinetics, our findings are therefore also relevant for optimizing future TRAIL-based treatment strategies.
Assuntos
Apoptose , Ciclo Celular , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Transdução de Sinais , Algoritmos , Teorema de Bayes , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Geminina/química , Células HCT116 , Humanos , Cinética , Modelos EstatísticosRESUMO
Current cancer therapeutics suffer from a lack of specificity in targeting tumor cells and cause severe side effects. Therefore, the design of highly specialized drugs comprising antibody derivatives inducing apoptosis in targeted cancer cells is considered to be a promising strategy. Drugs acting on death receptor 5 (DR5) such as DR5 agonist antibodies replacing "TNF-related apoptosis-inducing ligand" (TRAIL) offer feasible opportunities in this direction. Although such agonists provided good antitumor activity in preclinical studies, they were less effective in clinical studies, possibly due to a disturbed Fc interaction with Fc-γ receptors. Thus, multimerized antigen binding fragments without Fc have been proposed to increase their efficacy. We generated nanobodies (Nbs), recombinant variable domains of heavy chain-only antibodies of camelids, against the DR5 ectodomain. Nb24 and Nb28 had an affinity in the nM and sub-nM range, but only Nb28 competes with TRAIL for binding to DR5. Bivalent, trivalent, and tetravalent constructs were generated, as well as an innovative pentameric Nb complex, to provoke avidity effects. In our cellular assays, these trimeric, tetrameric, and pentameric Nbs have a higher apoptotic capacity than monomeric Nbs and seem to mimic the activity of the natural TRAIL ligand on various cancer cells.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Anticorpos de Domínio Único/farmacologia , Animais , Antineoplásicos Imunológicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Humanos , Camundongos , Ligação Proteica , Receptores de IgG/química , Receptores de IgG/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/química , Proteínas Recombinantes , Anticorpos de Domínio Único/química , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cardiomyocyte survival and death contributes to many cardiac diseases. A common mechanism of cardiomyocyte death is through apoptosis however, numerous death receptors (DR) have been virtually unstudied in the context of cardiovascular disease. Previous studies have identified TNF-related apoptosis inducing ligand (TRAIL) and its receptor, DR5, as being altered in a chronic catecholamine administration model of heart failure, and suggest a role of non-canonical signaling in cardiomyocytes. Furthermore, multiple clinical studies have identified TRAIL or DR5 as biomarkers in the prediction of severity and mortality following myocardial infarction and in heart failure development risk suggesting a role of DR5 signaling in the heart. While TRAIL/DR5 have been extensively studied as a potential cancer therapeutic due to their ability to selectively activate apoptosis in cancer cells, TRAIL and DR5 are highly expressed in the heart where their function is uncharacterized. However, many non-transformed cell types are resistant to TRAIL-induced apoptosis suggesting non-canonical functions in non-cancerous cell types. Our goal was to determine the role of DR5 in the heart with the hypothesis that DR5 does not induce cardiomyocyte apoptosis but initiates non-canonical signaling to promote cardiomyocyte growth and survival. Histological analysis of hearts from mice treated with a DR5 agonists showed increased hypertrophy with no differences in cardiomyocyte death, fibrosis or function. Mechanistic studies in the heart and isolated cardiomyocytes identified ERK1/2 activation with DR5 agonist treatment which contributed to hypertrophy. Furthermore, epidermal growth factor receptor (EGFR) was activated following DR5 agonist treatment through activation of MMP and HB-EGFR cleavage and specific inhibitors of MMP and EGFR prevented DR5-mediated ERK1/2 signaling and hypertrophy. Taken together, these studies identify a previously unidentified role for DR5 in the heart, which does not promote apoptosis but acts through non-canonical MMP-EGFR-ERK1/2 signaling mechanisms to contribute to cardiomyocyte hypertrophy.
Assuntos
Receptores ErbB/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Animais Recém-Nascidos , Cardiomegalia/metabolismo , Crescimento Celular , Sobrevivência Celular , Células Cultivadas , Receptores ErbB/genética , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica , Hipertrofia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Ftalimidas/farmacologia , Ratos Sprague-Dawley , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Tiazolidinas/farmacologiaRESUMO
Apoptosis is an ordered and orchestrated cellular process that occurs in physiological and pathological conditions. Resistance to apoptosis is a hallmark of virtually all malignancies. Despite being a cause of pathological conditions, apoptosis could be a promising target in cancer treatment. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), also known as Apo-2 ligand (Apo2L), is a member of TNF cytokine superfamily. It is a potent anti-cancer agent owing to its specific targeting towards cancerous cells, while sparing normal cells, to induce apoptosis. However, resistance occurs either intrinsically or after multiple treatments which may explain why cancer therapy fails. This review summarizes the apoptotic mechanisms via extrinsic and intrinsic apoptotic pathways, as well as the apoptotic resistance mechanisms. It also reviews the current clinically tested recombinant human TRAIL (rhTRAIL) and TRAIL receptor agonists (TRAs) against TRAIL-Receptors, TRAIL-R1 and TRAIL-R2, in which the outcomes of the clinical trials have not been satisfactory. Finally, this review discusses the current strategies in overcoming resistance to TRAIL-induced apoptosis in pre-clinical and clinical settings.
Assuntos
Neoplasias/tratamento farmacológico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Apoptose/efeitos dos fármacos , Humanos , Neoplasias/metabolismo , Ensaios Clínicos Controlados Aleatórios como Assunto , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Receptores Chamariz do Fator de Necrose Tumoral/agonistasRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), also known as Apo2L, has been investigated in the past decade for its promising anticancer activity due to its ability to selectively induce apoptosis in tumoral cells by binding to TRAIL receptors (TRAIL-R). Macromolecules such as agonistic monoclonal antibodies and recombinant TRAIL have not proven efficacious in clinical studies, therefore several small molecules acting as TRAIL-R agonists are emerging in the scientific literature. In this work we focus on systemizing these drug molecules described in the past years, in order to better understand and predict the requirements for a novel anti-tumoral therapy based on the TRAIL-R-induced apoptotic mechanism.
Assuntos
Neoplasias/tratamento farmacológico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Agonism of members of the tumor necrosis factor receptor superfamily (TNFRSF) with monoclonal antibodies is of high therapeutic interest due to their role in immune regulation and cell proliferation. A major hurdle for pharmacologic activation of this receptor class is the requirement for high-order clustering, a mechanism that imposes a reliance in vivo on Fc receptor-mediated crosslinking. This extrinsic dependence represents a potential limitation of virtually the entire pipeline of agonist TNFRSF antibody drugs, of which none have thus far been approved or reached late-stage clinical trials. We show that tetravalent biepitopic targeting enables robust intrinsic antibody agonism for two members of this family, OX40 and DR5, that is superior to extrinsically crosslinked native parental antibodies. Tetravalent biepitopic anti-OX40 engagement co-stimulated OX40low cells, obviated the requirement for CD28 co-signal for T cell activation, and enabled superior pharmacodynamic activity relative to native IgG in a murine vaccination model. This work establishes a proof of concept for an engineering approach that addresses a major gap for the therapeutic activation of this important receptor class.
Assuntos
Anticorpos Monoclonais/imunologia , Capeamento Imunológico , Ligante OX40/agonistas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD28/imunologia , Células CHO , Cricetulus , Humanos , Células Jurkat , Camundongos , Camundongos SCID , Camundongos Transgênicos , Ligante OX40/imunologia , Receptores Fc/imunologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Linfócitos T/citologiaRESUMO
Metabolic imaging has been widely used to measure the early responses of tumors to treatment. Here, we assess the abilities of PET measurement of [18F]FDG uptake and MRI measurement of hyperpolarized [1-13C]pyruvate metabolism to detect early changes in glycolysis following treatment-induced cell death in human colorectal (Colo205) and breast adenocarcinoma (MDA-MB-231) xenografts in mice. A TRAIL agonist that binds to human but not mouse cells induced tumor-selective cell death. Tumor glycolysis was assessed by injecting [1,6-13C2]glucose and measuring 13C-labeled metabolites in tumor extracts. Injection of hyperpolarized [1-13C]pyruvate induced rapid reduction in lactate labeling. This decrease, which correlated with an increase in histologic markers of cell death and preceded decrease in tumor volume, reflected reduced flux from glucose to lactate and decreased lactate concentration. However, [18F]FDG uptake and phosphorylation were maintained following treatment, which has been attributed previously to increased [18F]FDG uptake by infiltrating immune cells. Quantification of [18F]FDG uptake in flow-sorted tumor and immune cells from disaggregated tumors identified CD11b+/CD45+ macrophages as the most [18F]FDG-avid cell type present, yet they represented <5% of the cells present in the tumors and could not explain the failure of [18F]FDG-PET to detect treatment response. MRI measurement of hyperpolarized [1-13C]pyruvate metabolism is therefore a more sensitive marker of the early decreases in glycolytic flux that occur following cell death than PET measurements of [18F]FDG uptake. SIGNIFICANCE: These findings demonstrate superior sensitivity of MRI measurement of hyperpolarized [1-13C]pyruvate metabolism versus PET measurement of 18F-FDG uptake for detecting early changes in glycolysis following treatment-induced tumor cell death.