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1.
Nat Med ; 30(2): 443-454, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38321220

RESUMO

Compromised vascular endothelial barrier function is a salient feature of diabetic complications such as sight-threatening diabetic macular edema (DME). Current standards of care for DME manage aspects of the disease, but require frequent intravitreal administration and are poorly effective in large subsets of patients. Here we provide evidence that an elevated burden of senescent cells in the retina triggers cardinal features of DME pathology and conduct an initial test of senolytic therapy in patients with DME. In cell culture models, sustained hyperglycemia provoked cellular senescence in subsets of vascular endothelial cells displaying perturbed transendothelial junctions associated with poor barrier function and leading to micro-inflammation. Pharmacological elimination of senescent cells in a mouse model of DME reduces diabetes-induced retinal vascular leakage and preserves retinal function. We then conducted a phase 1 single ascending dose safety study of UBX1325 (foselutoclax), a senolytic small-molecule inhibitor of BCL-xL, in patients with advanced DME for whom anti-vascular endothelial growth factor therapy was no longer considered beneficial. The primary objective of assessment of safety and tolerability of UBX1325 was achieved. Collectively, our data suggest that therapeutic targeting of senescent cells in the diabetic retina with a BCL-xL inhibitor may provide a long-lasting, disease-modifying intervention for DME. This hypothesis will need to be verified in larger clinical trials. ClinicalTrials.gov identifier: NCT04537884 .


Assuntos
Diabetes Mellitus , Retinopatia Diabética , Edema Macular , Animais , Camundongos , Humanos , Edema Macular/tratamento farmacológico , Edema Macular/etiologia , Retinopatia Diabética/tratamento farmacológico , Inibidores da Angiogênese/uso terapêutico , Células Endoteliais , Senoterapia , Senescência Celular
2.
J Biol Chem ; 300(2): 105650, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38237681

RESUMO

Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient) and state 2 (active, effector binding enabled). Here, we use 31P NMR to delineate the differences in state 1 and state 2 populations present in WT and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analog GppNHp (guanosine-5'-[(ß,γ)-imido] triphosphate). Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RAS-binding domain and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small-molecule inhibitor, BBO-8956, which is effective against both GDP- and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs state 1-state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1-RAS-binding domain is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action for this novel and active-conformation inhibitor.


Assuntos
Proteínas Proto-Oncogênicas p21(ras) , Proteínas ras , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas ras/metabolismo , Guanosina Trifosfato/metabolismo , Espectroscopia de Ressonância Magnética , Transdução de Sinais , Mutação
3.
Nat Cancer ; 5(1): 66-84, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38151625

RESUMO

Chromosomal instability (CIN) is a hallmark of cancer, caused by persistent errors in chromosome segregation during mitosis. Aggressive cancers like high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) have a high frequency of CIN and TP53 mutations. Here, we show that inhibitors of the KIF18A motor protein activate the mitotic checkpoint and selectively kill chromosomally unstable cancer cells. Sensitivity to KIF18A inhibition is enriched in TP53-mutant HGSOC and TNBC cell lines with CIN features, including in a subset of CCNE1-amplified, CDK4-CDK6-inhibitor-resistant and BRCA1-altered cell line models. Our KIF18A inhibitors have minimal detrimental effects on human bone marrow cells in culture, distinct from other anti-mitotic agents. In mice, inhibition of KIF18A leads to robust anti-cancer effects with tumor regression observed in human HGSOC and TNBC models at well-tolerated doses. Collectively, our results provide a rational therapeutic strategy for selective targeting of CIN cancers via KIF18A inhibition.


Assuntos
Cinesinas , Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Cinesinas/genética , Cinesinas/metabolismo , Mitose/genética , Linhagem Celular , Pontos de Checagem da Fase M do Ciclo Celular
4.
J Immunother Cancer ; 11(5)2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37164449

RESUMO

BACKGROUND: Checkpoint inhibitors targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) have demonstrated clinical efficacy in advanced melanoma, but only a subset of patients with inflamed tumors are responsive. Talimogene laherparepvec (T-VEC), a modified herpes simplex virus type 1 (HSV-1) expressing granulocyte-macrophage colony-stimulating factor (GM-CSF), is a first-in-class oncolytic immunotherapy approved for the treatment of melanoma and has been shown to inflame the tumor microenvironment. To evaluate the potential and mechanisms of T-VEC to elicit systemic antitumor immunity and overcome resistance to checkpoint inhibitors in murine tumor models, OncoVEXmGM-CSF was developed similarly to T-VEC, except the human GM-CSF transgene was replaced with murine GM-CSF. Previous work had demonstrated that OncoVEXmGM-CSF generated systemic antitumor immunity dependent on CD8+ T cells in an immune checkpoint-sensitive tumor cell model. METHODS: A novel B16F10 syngeneic tumor model with both HSV-1-permissive subcutaneous tumors and HSV-1-refractory experimental lung metastasis was used to study the local and systemic effects of OncoVEXmGM-CSF treatment alone or in combination with checkpoint inhibitors. RESULTS: Intratumoral injection of OncoVEXmGM-CSF in combination with an anti-CTLA-4 or anti-PD-1 blocking antibody led to increased tumor growth inhibition, a reduction in the number of lung metastases, and prolonged animal survival. OncoVEXmGM-CSF induced both neoantigen-specific and tumor antigen-specific T-cell responses. Furthermore, cured mice from the combination treatment of OncoVEXmGM-CSF and anti-CTLA-4 antibody rejected tumor rechallenges. CONCLUSIONS: These data support the concept that T-VEC and checkpoint inhibition may be an effective combination to treat patients with advanced melanoma.


Assuntos
Melanoma , Terapia Viral Oncolítica , Humanos , Animais , Camundongos , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Linfócitos T CD8-Positivos/patologia , Antígenos de Neoplasias , Microambiente Tumoral
5.
Diabetes ; 70(10): 2237-2249, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34285117

RESUMO

Similar to insulin, central administration of IGF-1 can suppress hepatic glucose production (HGP), but it is unclear whether this effect is mediated via insulin receptors (InsRs) or IGF-1 receptors (IGF-1Rs) in the brain. To this end, we used pharmacologic and genetic approaches in combination with hyperinsulinemic-euglycemic clamps to decipher the role of these receptors in mediating central effects of IGF-1 and insulin on HGP. In rats, we observed that intracerebroventricular (ICV) administration of IGF-1 or insulin markedly increased the glucose infusion rate (GIR) by >50% and suppressed HGP (P < 0.001). However, these effects were completely prevented by preemptive ICV infusion with an IGF-1R and InsR/IGF-1R hybrid (HybridR) blocking antibody. Likewise, ICV infusion of the InsR antagonist, S961, which also can bind HybridRs, interfered with the ability of central insulin, but not IGF-1, to increase the GIR. Furthermore, hyperinsulinemic clamps in mice lacking IGF-1Rs in AgRP neurons revealed ∼30% reduction in the GIR in knockout animals, which was explained by an impaired ability of peripheral insulin to completely suppress HGP (P < 0.05). Signaling studies further revealed an impaired ability of peripheral insulin to trigger ribosomal S6 phosphorylation or phosphatidylinositol (3,4,5)-trisphosphate production in AgRP neurons lacking IGF-1Rs. In summary, these data suggest that attenuation of IGF-1R signaling in the mediobasal hypothalamus, and specifically in AgRP neurons, can phenocopy impaired regulation of HGP as previously demonstrated in mice lacking InsRs in these cells, suggesting a previously unappreciated role for IGF-1Rs and/or HybridRs in the regulation of central insulin/IGF-1 signaling in glucose metabolism.


Assuntos
Glucose/metabolismo , Insulina/farmacologia , Neurônios/fisiologia , Adulto , Proteína Relacionada com Agouti/genética , Proteína Relacionada com Agouti/metabolismo , Animais , Metabolismo dos Carboidratos/efeitos dos fármacos , Metabolismo dos Carboidratos/genética , Células Cultivadas , Técnica Clamp de Glucose , Humanos , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Injeções Intraventriculares , Insulina/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos F344 , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
6.
Cell Metab ; 33(4): 818-832.e7, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33548171

RESUMO

Attenuating pathological angiogenesis in diseases characterized by neovascularization such as diabetic retinopathy has transformed standards of care. Yet little is known about the molecular signatures discriminating physiological blood vessels from their diseased counterparts, leading to off-target effects of therapy. We demonstrate that in contrast to healthy blood vessels, pathological vessels engage pathways of cellular senescence. Senescent (p16INK4A-expressing) cells accumulate in retinas of patients with diabetic retinopathy and during peak destructive neovascularization in a mouse model of retinopathy. Using either genetic approaches that clear p16INK4A-expressing cells or small molecule inhibitors of the anti-apoptotic protein BCL-xL, we show that senolysis suppresses pathological angiogenesis. Single-cell analysis revealed that subsets of endothelial cells with senescence signatures and expressing Col1a1 are no longer detected in BCL-xL-inhibitor-treated retinas, yielding a retina conducive to physiological vascular repair. These findings provide mechanistic evidence supporting the development of BCL-xL inhibitors as potential treatments for neovascular retinal disease.


Assuntos
Senescência Celular , Doenças Retinianas/patologia , Proteína bcl-X/metabolismo , Animais , Apoptose/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Cadeia alfa 1 do Colágeno Tipo I/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Modelos Animais de Doenças , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Feminino , Flavonóis/química , Flavonóis/farmacologia , Flavonóis/uso terapêutico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neovascularização Patológica , Doenças Retinianas/tratamento farmacológico , Doenças Retinianas/metabolismo , Tacrolimo/análogos & derivados , Tacrolimo/farmacologia , Proteína bcl-X/antagonistas & inibidores
7.
Clin Cancer Res ; 27(5): 1526-1537, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33203642

RESUMO

PURPOSE: Small-cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with a high relapse rate, limited therapeutic options, and poor prognosis. We investigated the antitumor activity of AMG 757, a half-life extended bispecific T-cell engager molecule targeting delta-like ligand 3 (DLL3)-a target that is selectively expressed in SCLC tumors, but with minimal normal tissue expression. EXPERIMENTAL DESIGN: AMG 757 efficacy was evaluated in SCLC cell lines and in orthotopic and patient-derived xenograft (PDX) mouse SCLC models. Following AMG 757 administration, changes in tumor volume, pharmacodynamic changes in tumor-infiltrating T cells (TILs), and the spatial relationship between the appearance of TILs and tumor histology were examined. Tolerability was assessed in nonhuman primates (NHPs). RESULTS: AMG 757 showed potent and specific killing of even those SCLC cell lines with very low DLL3 expression (<1,000 molecules per cell). AMG 757 effectively engaged systemically administered human T cells, induced T-cell activation, and redirected T cells to lyse tumor cells to promote significant tumor regression and complete responses in PDX models of SCLC and in orthotopic models of established primary lung SCLC and metastatic liver lesions. AMG 757 was well tolerated with no AMG 757-related adverse findings up to the highest tested dose (4.5 mg/kg weekly) in NHP. AMG 757 exhibits an extended half-life in NHP, which is projected to enable intermittent administration in patients. CONCLUSIONS: AMG 757 has a compelling safety and efficacy profile in preclinical studies making it a viable option for targeting DLL3-expressing SCLC tumors in the clinical setting.


Assuntos
Anticorpos Biespecíficos , Anticorpos Monoclonais , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Pulmonares , Proteínas de Membrana , Carcinoma de Pequenas Células do Pulmão , Linfócitos T , Animais , Feminino , Humanos , Camundongos , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Apoptose , Proliferação de Células , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Membrana/antagonistas & inibidores , Camundongos Endogâmicos NOD , Camundongos SCID , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/imunologia , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Nat Commun ; 9(1): 2394, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29921922

RESUMO

Diminished growth factor signaling improves longevity in laboratory models, while a reduction in the somatotropic axis is favorably linked to human aging and longevity. Given the conserved role of this pathway on lifespan, therapeutic strategies, such as insulin-like growth factor-1 receptor (IGF-1R) monoclonal antibodies (mAb), represent a promising translational tool to target human aging. To this end, we performed a preclinical study in 18-mo-old male and female mice treated with vehicle or an IGF-1R mAb (L2-Cmu, Amgen Inc), and determined effects on aging outcomes. Here we show that L2-Cmu preferentially improves female healthspan and increases median lifespan by 9% (P = 0.03) in females, along with a reduction in neoplasms and inflammation (P ≤ 0.05). Thus, consistent with other models, targeting IGF-1R signaling appears to be most beneficial to females. Importantly, these effects could be achieved at advanced ages, suggesting that IGF-1R mAbs could represent a promising therapeutic candidate to delay aging.


Assuntos
Anticorpos Monoclonais/farmacologia , Longevidade/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Receptor IGF Tipo 1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/prevenção & controle , Receptor IGF Tipo 1/imunologia , Receptor IGF Tipo 1/metabolismo , Fatores Sexuais , Carga Tumoral/efeitos dos fármacos
9.
Clin Cancer Res ; 23(20): 6190-6202, 2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-28706012

RESUMO

Purpose: Talimogene laherparepvec, a new oncolytic immunotherapy, has been recently approved for the treatment of melanoma. Using a murine version of the virus, we characterized local and systemic antitumor immune responses driving efficacy in murine syngeneic models.Experimental Design: The activity of talimogene laherparepvec was characterized against melanoma cell lines using an in vitro viability assay. Efficacy of OncoVEXmGM-CSF (talimogene laherparepvec with the mouse granulocyte-macrophage colony-stimulating factor transgene) alone or in combination with checkpoint blockade was characterized in A20 and CT-26 contralateral murine tumor models. CD8+ depletion, adoptive T-cell transfers, and Enzyme-Linked ImmunoSpot assays were used to study the mechanism of action (MOA) of systemic immune responses.Results: Treatment with OncoVEXmGM-CSF cured all injected A20 tumors and half of contralateral tumors. Viral presence was limited to injected tumors and was not responsible for systemic efficacy. A significant increase in T cells (CD3+/CD8+) was observed in injected and contralateral tumors at 168 hours. Ex vivo analyses showed these cytotoxic T lymphocytes were tumor-specific. Increased neutrophils, monocytes, and chemokines were observed in injected tumors only. Importantly, depletion of CD8+ T cells abolished all systemic efficacy and significantly decreased local efficacy. In addition, immune cell transfer from OncoVEXmGM-CSF-cured mice significantly protected from tumor challenge. Finally, combination of OncoVEXmGM-CSF and checkpoint blockade resulted in increased tumor-specific CD8+ anti-AH1 T cells and systemic efficacy.Conclusions: The data support a dual MOA for OncoVEXmGM-CSF that involves direct oncolysis of injected tumors and activation of a CD8+-dependent systemic response that clears injected and contralateral tumors when combined with checkpoint inhibition. Clin Cancer Res; 23(20); 6190-202. ©2017 AACR.


Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Imunoterapia , Neoplasias/imunologia , Neoplasias/metabolismo , Terapia Viral Oncolítica , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Adenoviridae/genética , Transferência Adotiva , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Imunomodulação , Imunoterapia/métodos , Estimativa de Kaplan-Meier , Depleção Linfocítica , Melanoma/imunologia , Melanoma/metabolismo , Melanoma/patologia , Melanoma/terapia , Camundongos , Neoplasias/patologia , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Transgenes , Carga Tumoral , Replicação Viral , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Int J Cancer ; 139(6): 1340-9, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27170265

RESUMO

Gas6 and its receptors Axl, Mer and Tyro-3 (TAM) are highly expressed in human malignancy suggesting that signaling through this axis may be tumor-promoting. In pancreatic ductal adenocarcinoma (PDAC), Gas6 and the TAM receptor Axl are frequently co-expressed and their co-expression correlates with poor survival. A strategy was devised to generate fully human neutralizing antibodies against Gas6 using XenoMouse® technology. Hybridoma supernatants were selected based on their ability to inhibit Gas6 binding to the receptor Axl and block Gas6-induced Axl phosphorylation in human cells. Two purified antibodies isolated from the screened hybridomas, GMAB1 and GMAB2, displayed optimal cellular potency which was comparable to that of the soluble extracellular domain of the receptor Axl (Axl-Fc). In vivo characterization of GMAB1 was conducted using a pharmacodynamic assay that measured inhibition of Gas6-induced Akt activation in the mouse spleen. Treatment of mice with a single dose (100-1000 µg) of GMAB1 led to greater than 90% inhibition of Gas6-induced phosphorylated Akt (pAkt) for up to 72 hr. Based on the target coverage observed in the PD assay, the efficacy of GMAB1 was tested against human pancreatic adenocarcinoma xenografts. At doses of 50 µg and 150 µg, twice weekly, GMAB1 was able to inhibit 55% and 76% of tumor growth, respectively (p < 0.001 for both treatments vs. control Ig). When combined with gemcitabine, GMAB1 significantly inhibited tumor growth compared to either agent alone (p < 0.001). Together, the data suggest that Gas6 neutralization may be important as a potential strategy for the treatment of PDAC.


Assuntos
Anticorpos Neutralizantes/farmacologia , Anticorpos/farmacologia , Comunicação Autócrina/efeitos dos fármacos , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Animais , Anticorpos/imunologia , Anticorpos Neutralizantes/imunologia , Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Humanos , Camundongos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/imunologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor Tirosina Quinase Axl , Neoplasias Pancreáticas
11.
ACS Med Chem Lett ; 6(9): 987-92, 2015 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-26396685

RESUMO

In nonsmall cell lung cancer (NSCLC), the threonine(790)-methionine(790) (T790M) point mutation of EGFR kinase is one of the leading causes of acquired resistance to the first generation tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. Herein, we describe the optimization of a series of 7-oxopyrido[2,3-d]pyrimidinyl-derived irreversible inhibitors of EGFR kinase. This led to the discovery of compound 24 which potently inhibits gefitinib-resistant EGFR(L858R,T790M) with 100-fold selectivity over wild-type EGFR. Compound 24 displays strong antiproliferative activity against the H1975 nonsmall cell lung cancer cell line, the first line mutant HCC827 cell line, and promising antitumor activity in an EGFR(L858R,T790M) driven H1975 xenograft model sparing the side effects associated with the inhibition of wild-type EGFR.

12.
J Med Chem ; 58(13): 5189-207, 2015 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-25970324

RESUMO

Efforts to improve upon the physical properties and metabolic stability of Aurora kinase inhibitor 14a revealed that potency against multidrug-resistant cell lines was compromised by increased polarity. Despite its high in vitro metabolic intrinsic clearance, 23r (AMG 900) showed acceptable pharmacokinetic properties and robust pharmacodynamic activity. Projecting from in vitro data to in vivo target coverage was not practical due to disjunctions between enzyme and cell data, complex and apparently contradictory indicators of binding kinetics, and unmeasurable free fraction in plasma. In contrast, it was straightforward to relate pharmacokinetics to pharmacodynamics and efficacy by following the time above a threshold concentration. On the basis of its oral route of administration, a selectivity profile that favors Aurora-driven pharmacology and its activity against multidrug-resistant cell lines, 23r was identified as a potential best-in-class Aurora kinase inhibitor. In phase 1 dose expansion studies with G-CSF support, 23r has shown promising single agent activity.


Assuntos
Aurora Quinases/antagonistas & inibidores , Descoberta de Drogas , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Ftalazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Nus , Estrutura Molecular , Neoplasias/enzimologia , Neoplasias/patologia , Ratos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Oncotarget ; 5(19): 9007-21, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25344862

RESUMO

Inhibition of the mitogenic insulin-like growth factor receptor 1 (IGF-1R) signaling axis is a compelling treatment strategy for prostate cancer. Combining the IGF-1R inhibitor ganitumab (formerly AMG 479) with standard of care androgen-deprivation therapy greatly delays prostate cancer recurrence in xenograft models; however, a significant proportion of these tumors ultimately acquire resistance to ganitumab. Here we describe the development of a stable and reproducible ganitumab-resistant VCaP human prostate cancer cell derivative termed VCaP/GanR to investigate the mechanism of acquired resistance to IGF-1R inhibition. Unlike parental VCaP, VCaP/GanR did not undergo apoptosis following ganitumab treatment. VCaP/GanR did not express increased levels of IGF-1R, insulin receptor, or phospho-AKT compared to parental VCaP. VCaP/GanR exhibited increased levels of phospho-S6 indicative of increased mTOR activity. However, acquired resistance to ganitumab was not dependent on increased mTOR activity in VCaP/GanR. Phospho-proteomic arrays revealed alterations in several calcium-regulated signaling components in VCaP/GanR compared to VCaP. Reduction of intracellular calcium using cell-permeable calcium-specific chelators restored ganitumab sensitivity to VCaP/GanR through inhibition of cell-cycle progression. These data suggest a new mechanism of resistance to IGF-1R inhibition involving calcium-mediated proliferation effects. Such pathways should be considered in future clinical studies of IGF-1R inhibitors in prostate cancer.


Assuntos
Anticorpos Monoclonais/farmacologia , Quelantes de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Receptor IGF Tipo 1/antagonistas & inibidores , Anticorpos Monoclonais Humanizados , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Sinalização do Cálcio/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Quinase 2 de Adesão Focal/antagonistas & inibidores , Quinase 2 de Adesão Focal/metabolismo , Humanos , Masculino , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/prevenção & controle , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/biossíntese , Receptor de Insulina/biossíntese , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Clin Cancer Res ; 20(11): 2947-58, 2014 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24727326

RESUMO

PURPOSE: Insulin-like growth factor 1 receptor (IGF-IR) has been implicated in the pathogenesis of ovarian cancer. Ganitumab is an investigational, fully human monoclonal antibody against IGF-IR. Here, we explore the therapeutic potential of ganitumab for the treatment of ovarian cancer. EXPERIMENTAL DESIGN: The effects of ganitumab were tested in vitro against a panel of 23 established ovarian cancer cell lines. The ability of ganitumab to inhibit IGF-I-, IGF-II-, and insulin-mediated signaling was examined in vitro and in tumor xenografts using ovarian cancer models displaying IGF-IR/PI3K/AKT pathway activation by two distinct mechanisms, PTEN loss and IGF-II overexpression. Drug interactions between ganitumab and cisplatin, carboplatin, or paclitaxel were studied in vitro and in vivo. RESULTS: In vitro, growth inhibition varied significantly among individual ovarian cancer cell lines. IGF-II mRNA and phospho-IGF-IR protein expression were quantitatively correlated with response to ganitumab, and PTEN mutations conferred resistance to ganitumab. Ganitumab potently inhibited baseline and IGF-I-, IGF-II-, and insulin-induced IGF-IR and IGF-IR/insulin hybrid receptor signaling in vitro and in vivo. Synergistic and additive drug interactions were seen for ganitumab and carboplatin or paclitaxel in vitro. Furthermore, ganitumab significantly increased the efficacy of cisplatin in ovarian cancer xenograft models in vivo. CONCLUSIONS: These observations provide a biologic rationale to test ganitumab as a single agent or in combination with carboplatin/cisplatin and paclitaxel in patients with ovarian cancer. Moreover, assessment of tumor expression of IGF-II, phospho-IGF-IR, or PTEN status may help select patients with ovarian cancer who are most likely to benefit from ganitumab. Clin Cancer Res; 20(11); 2947-58. ©2014 AACR.


Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Ovarianas/patologia , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos Monoclonais Humanizados , Western Blotting , Carboplatina/administração & dosagem , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Sinergismo Farmacológico , Feminino , Citometria de Fluxo , Humanos , Fator de Crescimento Insulin-Like II/metabolismo , Camundongos , Camundongos Nus , Neoplasias Ovarianas/genética , PTEN Fosfo-Hidrolase/genética , Paclitaxel/administração & dosagem , Receptor IGF Tipo 1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma , Ensaios Antitumorais Modelo de Xenoenxerto
16.
J Endocrinol ; 221(1): 145-55, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24492468

RESUMO

Ganitumab is a fully human MAB to the human type 1 IGF receptor (IGF1R). Binding assays showed that ganitumab recognized murine IGF1R with sub-nanomolar affinity (KD=0.22 nM) and inhibited the interaction of murine IGF1R with IGF1 and IGF2. Ganitumab inhibited IGF1-induced activation of IGF1R in murine lungs and CT26 murine colon carcinoma cells and tumors. Addition of ganitumab to 5-fluorouracil resulted in enhanced inhibition of tumor growth in the CT26 model. Pharmacological intervention with ganitumab in naïve nude mice resulted in a number of physiological changes described previously in animals with targeted deletions of Igf1 and Igf1r, including inhibition of weight gain, reduced glucose tolerance and significant increase in serum levels of GH, IGF1 and IGFBP3. Flow cytometric analysis identified GR1/CD11b-positive cells as the highest IGF1R-expressing cells in murine peripheral blood. Administration of ganitumab led to a dose-dependent, reversible decrease in the number of peripheral neutrophils with no effect on erythrocytes or platelets. These findings indicate that acute IGF availability for its receptor plays a critical role in physiological growth, glucose metabolism and neutrophil physiology and support the presence of a pituitary IGF1R-driven negative feedback loop that tightly regulates serum IGF1 levels through Gh signaling.


Assuntos
Anticorpos Monoclonais/farmacologia , Hormônio do Crescimento/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/metabolismo , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados , Feminino , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Cinética , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Nus , Fosforilação/efeitos dos fármacos , Receptor IGF Tipo 1/química , Transdução de Sinais/efeitos dos fármacos
17.
Int J Mol Sci ; 14(7): 13782-95, 2013 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-23823800

RESUMO

Calorie restriction (CR) inhibits prostate cancer progression, partially through modulation of the IGF axis. IGF-1 receptor (IGF-1R) blockade reduces prostate cancer xenograft growth. We hypothesized that combining calorie restriction with IGF-1R blockade would have an additive effect on prostate cancer growth. Severe combined immunodeficient mice were subcutaneously injected with 22Rv1 cells and randomized to: (1) Ad libitum feeding/intraperitoneal saline (Ad-lib); (2) Ad-lib/20 mg/kg twice weekly, intraperitoneal ganitumab [anti-IGF-1R antibody (Ad-lib/Ab)]; (3) 40% calorie restriction/intraperitoneal saline (CR); (4) CR/ intraperitoneal ganitumab, (CR/Ab). CR and ganitumab treatment were initiated one week after tumor injection. Euthanasia occurred 19 days post treatment. Results showed that CR alone decreased final tumor weight, plasma insulin and IGF-1 levels, and increased apoptosis. Ganitumab therapy alone reduced tumor growth but had no effect on final tumor weight. The combination therapy (CR/Ab) further decreased final tumor weight and proliferation, increased apoptosis in comparison to the Ad-lib group, and lowered plasma insulin levels relative to the Ad-lib and Ad-lib/Ab groups. Tumor AKT activation directly correlated with plasma IGF-1 levels. In conclusion, whereas ganitumab therapy modestly affected 22Rv1 tumor growth, combining IGF-1R blockade with calorie restriction resulted in a significant decrease in final tumor weight and improved metabolic profile.


Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Antineoplásicos/farmacologia , Restrição Calórica , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias da Próstata/terapia , Receptores de Somatomedina/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos SCID , Proteínas de Neoplasias/metabolismo , Transplante de Neoplasias , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptor IGF Tipo 1 , Receptores de Somatomedina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
18.
PLoS One ; 8(2): e55135, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23383308

RESUMO

BACKGROUND: Therapeutic antibodies targeting the IGF1R have shown diverse efficacy and safety signals in oncology clinical trials. The success of these agents as future human therapeutics depends on understanding the specific mechanisms by which these antibodies target IGF1R signaling. METHODOLOGY/PRINCIPAL FINDINGS: A panel of well-characterized assays was used to investigate the mechanisms by which ganitumab, a fully human anti-IGF1R antibody undergoing clinical testing, inhibits IGF1R activity. Epitope mapping using IGF1R subdomains localized the ganitumab binding site to the L2 domain. Binding of ganitumab inhibited the high-affinity interaction of IGF-1 and IGF-2 required to activate IGF1R in cells engineered for IGF1R hypersensitivity and in human cancer cell lines, resulting in complete blockade of ligand-induced cellular proliferation. Inhibition of IGF1R activity by ganitumab did not depend on endosomal sequestration, since efficient ligand blockade was obtained without evidence of receptor internalization and degradation. Clinically relevant concentrations of ganitumab also inhibited the activation of hybrid receptors by IGF-1 and IGF-2. Ganitumab was not an agonist of homodimeric IGF1R or hybrid receptors in MCF-7 and COLO 205 cells, but low-level IGF1R activation was detected in cells engineered for IGF1R hypersensitivity. This activation seems biologically irrelevant since ganitumab completely inhibited ligand-driven proliferation. The in vivo efficacy profile of ganitumab was equivalent or better than CR and FnIII-1 domain-specific antibodies, alone or in combination with irinotecan. CR domain-specific antibodies only blocked IGF-1 binding to IGF1R but were more potent than ganitumab at inducing homodimer and hybrid receptor downregulation in vitro, however this difference was less obvious in vivo. No inhibition of hybrid receptors was observed with the FnIII-1 domain antibodies, which were relatively strong homodimer and hybrid agonists. CONCLUSIONS/SIGNIFICANCE: The safety and efficacy profile of ganitumab and other anti-IGF1R antibodies may be explained by the distinct molecular mechanisms by which they inhibit receptor signaling.


Assuntos
Anticorpos Monoclonais/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Análise de Variância , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados , Afinidade de Anticorpos , Sítios de Ligação/genética , Proliferação de Células , Mapeamento de Epitopos , Feminino , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Células MCF-7 , Camundongos , Camundongos Nus , Fosforilação , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo
19.
Mol Cancer Ther ; 12(4): 394-404, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23348048

RESUMO

Prostate cancer is the most commonly diagnosed malignancy in men. While tumors initially respond to androgen-deprivation therapy, the standard care for advanced or metastatic disease, tumors eventually recur as castration-resistant prostate cancer (CRPC). Upregulation of the insulin-like growth factor receptor type I (IGF-IR) signaling axis drives growth and progression of prostate cancer by promoting proliferation, survival, and angiogenesis. Ganitumab (formerly AMG 479) is a fully human antibody that inhibits binding of IGF-I and IGF-II to IGF-IR. We evaluated the therapeutic value of ganitumab in several preclinical settings including androgen-dependent prostate cancer, CRPC, and in combination with androgen-deprivation therapy. Ganitumab inhibited IGF-I-induced phosphorylation of the downstream effector AKT and reduced proliferation of multiple androgen-dependent and castration-resistant human prostate cancer cell lines in vitro. Ganitumab inhibited androgen-dependent VCaP xenograft growth and increased tumor-doubling time from 2.3 ± 0.4 weeks to 6.4 ± 0.4 weeks. Ganitumab blocked growth of castration-resistant VCaP xenografts for over 11.5 weeks of treatment. In contrast, ganitumab did not have appreciable effects on the castration-resistant CWR-22Rv1 xenograft model. Ganitumab was most potent against VCaP xenografts when combined with complete androgen-deprivation therapy (castration). Tumor volume was reduced by 72% after 4 weeks of treatment and growth suppression was maintained over 16 weeks of treatment. These data suggest that judicious use of ganitumab particularly in conjunction with androgen-deprivation therapy may be beneficial in the treatment of prostate cancer.


Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Androgênios/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Masculino , Camundongos , Orquiectomia , Neoplasias da Próstata/terapia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Mol Cancer Ther ; 11(7): 1539-46, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22562985

RESUMO

In preclinical models, both dietary fat reduction and insulin-like growth factor I receptor (IGF-1R) blockade individually inhibit prostate cancer xenograft growth. We hypothesized that a low-fat diet combined with IGF-1R blockade would cause additive inhibition of prostate cancer growth and offset possible untoward metabolic effects of IGF-1R blockade antibody therapy. Fifty severe combined immunodeficient mice were injected with 22Rv1 cells subcutaneously. Ten days postinjection, the animals were randomized to four groups: (i) high-fat diet + saline (HF); (ii) high-fat diet + IGF-1R blocking antibody, ganitumab (HF/Ab); (iii) low-fat diet + saline (LF); and (iv) low-fat diet + ganitumab (LF/Ab). After 19 days of treatment, the animals were euthanized, serum was collected, and tumors were weighed. Tumor Ki67, Akt and extracellular signal-regulated kinase (ERK) activation, serum insulin, IGF-I and TNF-α were measured. In vitro, ganitumab treatment inhibited growth and induced apoptosis in several prostate cancer cell lines. In vivo, tumor weights and volumes were unaffected by the different treatments. The LF/Ab therapy significantly reduced proliferation (Ki67) and ERK activation in tumors. The HF/Ab group had significantly higher serum insulin levels than the HF group. However, LF/Ab combination significantly reduced serum insulin back to normal levels as well as normalizing serum TNF-α level. Whereas the combination of low-fat diet and IGF-1R blockade did not have additive inhibitory effects on tumor weight, it led to reduced tumor cell proliferation and a reduction in serum insulin and TNF-α levels.


Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Dieta com Restrição de Gorduras , Neoplasias da Próstata/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Camundongos , Camundongos SCID , Neoplasias da Próstata/tratamento farmacológico , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
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