RESUMEN
PURPOSE: The solid tumor microenvironment (TME) drives T cell dysfunction and inhibits the effectiveness of immunotherapies such as chimeric antigen receptor-based T cell (CAR T) cells. Early data has shown that modulation of T cell metabolism can improve intratumoral T cell function in preclinical models. EXPERIMENTAL DESIGN: We evaluated GPC3 expression in human normal and tumor tissue specimens. We developed and evaluated BOXR1030, a novel CAR T therapeutic co-expressing glypican-3 (GPC3)-targeted CAR and exogenous glutamic-oxaloacetic transaminase 2 (GOT2) in terms of CAR T cell function both in vitro and in vivo. RESULTS: Cell surface expression of tumor antigen GPC3 was observed by immunohistochemical staining in tumor biopsies from hepatocellular carcinoma, liposarcoma, squamous lung cancer, and Merkel cell carcinoma patients. Compared to control GPC3 CAR alone, BOXR1030 (GPC3-targeted CAR T cell that co-expressed GOT2) demonstrated superior in vivo efficacy in aggressive solid tumor xenograft models, and showed favorable attributes in vitro including an enhanced cytokine production profile, a less-differentiated T cell phenotype with lower expression of stress and exhaustion markers, an enhanced metabolic profile and increased proliferation in TME-like conditions. CONCLUSIONS: Together, these results demonstrated that co-expression of GOT2 can substantially improve the overall antitumor activity of CAR T cells by inducing broad changes in cellular function and phenotype. These data show that BOXR1030 is an attractive approach to targeting select solid tumors. To this end, BOXR1030 will be explored in the clinic to assess safety, dose-finding, and preliminary efficacy (NCT05120271).
Asunto(s)
Neoplasias Hepáticas , Receptores Quiméricos de Antígenos , Línea Celular Tumoral , Glipicanos/genética , Glipicanos/metabolismo , Xenoinjertos , Humanos , Inmunoterapia Adoptiva/métodos , Neoplasias Hepáticas/patología , Linfocitos T , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Purpose: c-KIT overexpression is well recognized in cancers such as gastrointestinal stromal tumors (GIST), small cell lung cancer (SCLC), melanoma, non-small cell lung cancer (NSCLC), and acute myelogenous leukemia (AML). Treatment with the small-molecule inhibitors imatinib, sunitinib, and regorafenib resulted in resistance (c-KIT mutant tumors) or limited activity (c-KIT wild-type tumors). We selected an anti-c-KIT ADC approach to evaluate the anticancer activity in multiple disease models.Experimental Design: A humanized anti-c-KIT antibody LMJ729 was conjugated to the microtubule destabilizing maytansinoid, DM1, via a noncleavable linker (SMCC). The activity of the resulting ADC, LOP628, was evaluated in vitro against GIST, SCLC, and AML models and in vivo against GIST and SCLC models.Results: LOP628 exhibited potent antiproliferative activity on c-KIT-positive cell lines, whereas LMJ729 displayed little to no effect. At exposures predicted to be clinically achievable, LOP628 demonstrated single administration regressions or stasis in GIST and SCLC xenograft models in mice. LOP628 also displayed superior efficacy in an imatinib-resistant GIST model. Further, LOP628 was well tolerated in monkeys with an adequate therapeutic index several fold above efficacious exposures. Safety findings were consistent with the pharmacodynamic effect of neutropenia due to c-KIT-directed targeting. Additional toxicities were considered off-target and were consistent with DM1, such as effects in the liver and hematopoietic/lymphatic system.Conclusions: The preclinical findings suggest that the c-KIT-directed ADC may be a promising therapeutic for the treatment of mutant and wild-type c-KIT-positive cancers and supported the clinical evaluation of LOP628 in GIST, AML, and SCLC patients. Clin Cancer Res; 24(17); 4297-308. ©2018 AACR.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Inmunoconjugados/farmacología , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-kit/genética , Animales , Anticuerpos Antiidiotipos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/inmunología , Xenoinjertos , Humanos , Mesilato de Imatinib/farmacología , Inmunoconjugados/inmunología , Ratones , Mutación , Neoplasias/clasificación , Neoplasias/inmunología , Neoplasias/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-kit/inmunologíaRESUMEN
Despite an improving therapeutic landscape, significant challenges remain in treating the majority of patients with advanced ovarian or renal cancer. We identified the cell-cell adhesion molecule cadherin-6 (CDH6) as a lineage gene having significant differential expression in ovarian and kidney cancers. HKT288 is an optimized CDH6-targeting DM4-based antibody-drug conjugate (ADC) developed for the treatment of these diseases. Our study provides mechanistic evidence supporting the importance of linker choice for optimal antitumor activity and highlights CDH6 as an antigen for biotherapeutic development. To more robustly predict patient benefit of targeting CDH6, we incorporate a population-based patient-derived xenograft (PDX) clinical trial (PCT) to capture the heterogeneity of response across an unselected cohort of 30 models-a novel preclinical approach in ADC development. HKT288 induces durable tumor regressions of ovarian and renal cancer models in vivo, including 40% of models on the PCT, and features a preclinical safety profile supportive of progression toward clinical evaluation.Significance: We identify CDH6 as a target for biotherapeutics development and demonstrate how an integrated pharmacology strategy that incorporates mechanistic pharmacodynamics and toxicology studies provides a rich dataset for optimizing the therapeutic format. We highlight how a population-based PDX clinical trial and retrospective biomarker analysis can provide correlates of activity and response to guide initial patient selection for first-in-human trials of HKT288. Cancer Discov; 7(9); 1030-45. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 920.
Asunto(s)
Antineoplásicos/uso terapéutico , Cadherinas/antagonistas & inhibidores , Neoplasias Renales/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Cadherinas/genética , Cadherinas/metabolismo , Femenino , Humanos , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Macaca fascicularis , Ratones Desnudos , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Ratas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antibody-drug conjugates (ADCs) are of great interest as targeted cancer therapeutics. Preparation of ADCs for early stage screening is constrained by purification and biochemical analysis techniques that necessitate burdensome quantities of antibody. Here we describe a method, developed for the maytansinoid class of ADCs, enabling parallel conjugation of antibodies in 96-well format. The method utilizes â¼ 100 µg of antibody per well and requires <5 µg of ADC for characterization. We demonstrate the capabilities of this system using model antibodies. We also provide multiple examples applying this method to early-stage screening of maytansinoid ADCs. The method can greatly increase the throughput with which candidate ADCs can be screened in cell-based assays, and may be more generally applicable to high-throughput preparation and screening of different types of protein conjugates.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Antineoplásicos/farmacología , Inmunoconjugados/farmacología , Maitansina/farmacología , Neoplasias/tratamiento farmacológico , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Antineoplásicos/inmunología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales/métodos , Humanos , Inmunoconjugados/inmunología , Neoplasias/inmunologíaRESUMEN
Death Receptor 5 (DR5) agonists demonstrate anti-tumor activity in preclinical models but have yet to demonstrate robust clinical responses. A key limitation may be the lack of patient selection strategies to identify those most likely to respond to treatment. To overcome this limitation, we screened a DR5 agonist Nanobody across >600 cell lines representing 21 tumor lineages and assessed molecular features associated with response. High expression of DR5 and Casp8 were significantly associated with sensitivity, but their expression thresholds were difficult to translate due to low dynamic ranges. To address the translational challenge of establishing thresholds of gene expression, we developed a classifier based on ratios of genes that predicted response across lineages. The ratio classifier outperformed the DR5+Casp8 classifier, as well as standard approaches for feature selection and classification using genes, instead of ratios. This classifier was independently validated using 11 primary patient-derived pancreatic xenograft models showing perfect predictions as well as a striking linearity between prediction probability and anti-tumor response. A network analysis of the genes in the ratio classifier captured important biological relationships mediating drug response, specifically identifying key positive and negative regulators of DR5 mediated apoptosis, including DR5, CASP8, BID, cFLIP, XIAP and PEA15. Importantly, the ratio classifier shows translatability across gene expression platforms (from Affymetrix microarrays to RNA-seq) and across model systems (in vitro to in vivo). Our approach of using gene expression ratios presents a robust and novel method for constructing translatable biomarkers of compound response, which can also probe the underlying biology of treatment response.
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Linaje de la Célula/genética , Regulación Neoplásica de la Expresión Génica/genética , Expresión Génica/genética , Neoplasias Pancreáticas/genética , Biosíntesis de Proteínas/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Animales , Apoptosis/genética , Caspasa 8/genética , Línea Celular Tumoral , Humanos , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Multiple therapeutic agonists of death receptor 5 (DR5) have been developed and are under clinical evaluation. Although these agonists demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation might be that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients, particularly for dimeric antibody agonists that require secondary cross-linking via Fcγ receptors expressed on immune cells to achieve optimal clustering of DR5. To overcome this limitation, a novel multivalent Nanobody approach was taken with the goal of generating a significantly more potent DR5 agonist. In the present study, we show that trivalent DR5 targeting Nanobodies mimic the activity of natural ligand, and furthermore, increasing the valency of domains to tetramer and pentamer markedly increased potency of cell killing on tumor cells, with pentamers being more potent than tetramers in vitro. Increased potency was attributed to faster kinetics of death-inducing signaling complex assembly and caspase-8 and caspase-3 activation. In vivo, multivalent Nanobody molecules elicited superior anti-tumor activity compared to a conventional DR5 agonist antibody, including the ability to induce tumor regression in an insensitive patient-derived primary pancreatic tumor model. Furthermore, complete responses to Nanobody treatment were obtained in up to 50% of patient-derived primary pancreatic and colon tumor models, suggesting that multivalent DR5 Nanobodies may represent a significant new therapeutic modality for targeting death receptor signaling.
Asunto(s)
Caspasas/inmunología , Neoplasias/inmunología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Anticuerpos de Dominio Único/inmunología , Animales , Afinidad de Anticuerpos/inmunología , Western Blotting , Caspasas/biosíntesis , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Inducción Enzimática/efectos de los fármacos , Células HCT116 , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Ratones Endogámicos NOD , Ratones Noqueados , Ratones Desnudos , Ratones SCID , Neoplasias/tratamiento farmacológico , Multimerización de Proteína , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/farmacología , Resonancia por Plasmón de Superficie , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The bone formation inhibitor sclerostin encoded by SOST binds in vitro to low-density lipoprotein receptor-related protein (LRP) 5/6 Wnt co-receptors, thereby inhibiting Wnt/ß-catenin signaling, a central pathway of skeletal homeostasis. Lrp5/LRP5 deficiency results in osteoporosis-pseudoglioma (OPPG), whereas Sost/SOST deficiency induces lifelong bone gain in mice and humans. Here, we analyzed the bone phenotype of mice lacking Sost (Sost(-/-) ), Lrp5 (Lrp5(-/-) ), or both (Sost(-/-) ;Lrp5(-/-) ) to elucidate the mechanism of action of Sost in vivo. Sost deficiency-induced bone gain was significantly blunted in Sost(-/-) ;Lrp5(-/-) mice. Yet the Lrp5 OPPG phenotype was fully rescued in Sost(-/-) ;Lrp5(-/-) mice and most bone parameters were elevated relative to wild-type. To test whether the remaining bone increases in Sost(-/-) ;Lrp5(-/-) animals depend on Lrp6, we treated wild-type, Sost(-/-) , and Sost(-/-) ;Lrp5(-/-) mice with distinct Lrp6 function blocking antibodies. Selective blockage of Wnt1 class-mediated Lrp6 signaling reduced cancellous bone mass and density in wild-type mice. Surprisingly, it reversed the abnormal bone gain in Sost(-/-) and Sost(-/-) ;Lrp5(-/-) mice to wild-type levels irrespective of enhancement or blockage of Wnt3a class-mediated Lrp6 activity. Thus, whereas Sost deficiency-induced bone anabolism partially requires Lrp5, it fully depends on Wnt1 class-induced Lrp6 activity. These findings indicate: first, that OPPG syndrome patients suffering from LRP5 loss-of-function should benefit from principles antagonizing SOST/sclerostin action; and second, that therapeutic WNT signaling inhibitors may stop the debilitating bone overgrowth in sclerosing disorders related to SOST deficiency, such as sclerosteosis, van Buchem disease, and autosomal dominant craniodiaphyseal dysplasia, which are rare disorders without viable treatment options.
Asunto(s)
Glicoproteínas/deficiencia , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/deficiencia , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/fisiología , Vía de Señalización Wnt/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Densidad Ósea , Desarrollo Óseo/fisiología , Péptidos y Proteínas de Señalización Intercelular , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/inmunología , Ratones , Osteogénesis Imperfecta/tratamiento farmacológico , Tibia/química , Vía de Señalización Wnt/efectos de los fármacos , Microtomografía por Rayos XRESUMEN
We examined the effects of LJM716, an HER3 (ERBB3) neutralizing antibody that inhibits ligand-induced and ligand-independent HER3 dimerization, as a single agent and in combination with BYL719, an ATP competitive p110α-specific inhibitor, against HER2-overexpressing breast and gastric cancers. Treatment with LJM716 reduced HER2-HER3 and HER3-p85 dimers, P-HER3 and P-AKT, both in vitro and in vivo. Treatment with LJM716 alone markedly reduced growth of BT474 xenografts. The combination of LJM716/lapatinib/trastuzumab significantly improved survival of mice with BT474 xenografts compared with lapatinib/trastuzumab (P = 0.0012). LJM716 and BYL719 synergistically inhibited growth in a panel of HER2+ and PIK3CA mutant cell lines. The combination also inhibited P-AKT in HER2-overexpressing breast cancer cells and growth of HER2+ NCI-N87 gastric cancer xenografts more potently than LJM716 or BYL719 alone. Trastuzumab-resistant HER2+/PIK3CA mutant MDA453 xenografts regressed completely after 3 weeks of therapy with LJM716 and BYL719, whereas either single agent inhibited growth only partially. Finally, mice with BT474 xenografts treated with trastuzumab/LJM716, trastuzumab/BYL719, LJM716/BYL719, or trastuzumab/LJM716/BYL719 exhibited similar rates of tumor regression after 3 weeks of treatment. Thirty weeks after treatment discontinuation, 14% of mice were treated with trastuzumab/LJM716/BYL719, whereas >80% in all other treatment groups were sacrificed due to a recurrent large tumor burden (P = 0.0066). These data suggest that dual blockade of the HER2 signaling network with an HER3 antibody that inhibits HER2-HER3 dimers in combination with a p110α-specific inhibitor in the absence of a direct HER2 antagonist is an effective treatment approach against HER2-overexpressing cancers.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Neoplasias de la Mama/patología , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Multimerización de Proteína/efectos de los fármacos , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-3/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Proliferación Celular/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Femenino , Humanos , Immunoblotting , Inmunoprecipitación , Lapatinib , Ratones , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quinazolinas/farmacología , Receptor ErbB-2/inmunología , Receptor ErbB-2/metabolismo , Receptor ErbB-3/inmunología , Receptor ErbB-3/metabolismo , Transducción de Señal , Tasa de Supervivencia , Trastuzumab , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
HER2/HER3 dimerization resulting from overexpression of HER2 or neuregulin (NRG1) in cancer leads to HER3-mediated oncogenic activation of phosphoinositide 3-kinase (PI3K) signaling. Although ligand-blocking HER3 antibodies inhibit NRG1-driven tumor growth, they are ineffective against HER2-driven tumor growth because HER2 activates HER3 in a ligand-independent manner. In this study, we describe a novel HER3 monoclonal antibody (LJM716) that can neutralize multiple modes of HER3 activation, making it a superior candidate for clinical translation as a therapeutic candidate. LJM716 was a potent inhibitor of HER3/AKT phosphorylation and proliferation in HER2-amplified and NRG1-expressing cancer cells, and it displayed single-agent efficacy in tumor xenograft models. Combining LJM716 with agents that target HER2 or EGFR produced synergistic antitumor activity in vitro and in vivo. In particular, combining LJM716 with trastuzumab produced a more potent inhibition of signaling and cell proliferation than trastuzumab/pertuzumab combinations with similar activity in vivo. To elucidate its mechanism of action, we solved the structure of LJM716 bound to HER3, finding that LJM716 bound to an epitope, within domains 2 and 4, that traps HER3 in an inactive conformation. Taken together, our findings establish that LJM716 possesses a novel mechanism of action that, in combination with HER2- or EGFR-targeted agents, may leverage their clinical efficacy in ErbB-driven cancers.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Neoplasias de la Mama/patología , Neurregulina-1/metabolismo , Conformación Proteica/efectos de los fármacos , Receptor ErbB-3/antagonistas & inhibidores , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Proliferación Celular/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Femenino , Humanos , Immunoblotting , Inmunoprecipitación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Fosforilación/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/inmunología , Receptor ErbB-2/metabolismo , Receptor ErbB-3/química , Receptor ErbB-3/inmunología , Receptor ErbB-3/metabolismo , Transducción de Señal , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Disregulated Wnt/beta-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1- and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1- and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wnt-driven cancer.
Asunto(s)
Anticuerpos/farmacología , Proteínas Relacionadas con Receptor de LDL/inmunología , Ligandos , Proteínas Wnt/metabolismo , Animales , Anticuerpos/inmunología , Línea Celular , Transformación Celular Viral , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Immunoblotting , Proteínas Relacionadas con Receptor de LDL/genética , Proteínas Relacionadas con Receptor de LDL/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Virus del Tumor Mamario del Ratón/genética , Ratones , Ratones Desnudos , Neoplasias Experimentales/patología , Neoplasias Experimentales/prevención & control , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Proteínas Wnt/genética , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , Proteína Wnt3 , Proteína Wnt3A , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Decreased activity of osteoblasts (OBs) contributes to osteolytic lesions in multiple myeloma (MM). The production of the soluble Wnt inhibitor Dickkopf-1 (DKK1) by MM cells inhibits OB activity, and its serum level correlates with focal bone lesions in MM. Therefore, we have evaluated bone anabolic effects of a DKK1 neutralizing antibody (BHQ880) in MM. In vitro BHQ880 increased OB differentiation, neutralized the negative effect of MM cells on osteoblastogenesis, and reduced IL-6 secretion. In a severe combined immunodeficiency (SCID)-hu murine model of human MM, BHQ880 treatment led to a significant increase in OB number, serum human osteocalcin level, and trabecular bone. Although BHQ880 had no direct effect on MM cell growth, it significantly inhibited growth of MM cells in the presence of bone marrow stromal cells (BMSCs) in vitro. This effect was associated with inhibition of BMSC/MM cell adhesion and production of IL-6. In addition, BHQ880 up-regulated beta-catenin level while down-regulating nuclear factor-kappaB (NF-kappaB) activity in BMSC. Interestingly, we also observed in vivo inhibition of MM cell growth by BHQ880 treatment in the SCID-hu murine model. These results confirm DKK1 as an important therapeutic target in myeloma and provide the rationale for clinical evaluation of BHQ880 to improve bone disease and to inhibit MM growth.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Inmunoterapia , Péptidos y Proteínas de Señalización Intercelular/inmunología , Mieloma Múltiple/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Diferenciación Celular , Células Cultivadas , Progresión de la Enfermedad , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Ratones , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/inmunología , Osteogénesis/efectos de los fármacos , Osteogénesis/inmunologíaRESUMEN
Cbl proteins are ubiquitin protein ligases, which ubiquitinate activated tyrosine kinases and target them for degradation. Both c-Cbl and Cbl-b have an ubiquitin associated (UBA) domain at their C-terminal end. We observed that high molecular weight ubiquitinated proteins constitutively coimmunoprecipitated with transfected and endogenous Cbl-b, but not c-Cbl. The binding site for these ubiquitinated proteins was mapped to the UBA domain of Cbl-b (UBAb). GST-fusion proteins containing the UBAb interacted with ubiquitinated proteins and polyubiquitin chains in vitro, whereas those containing the UBA domain of c-Cbl (UBAc) did not. The UBAb had a much greater affinity for polyubiquitin chains than for monoubiquitin. Analysis of the UBAb and UBAc demonstrate that the affinity for ubiquitin is determined by multiple amino-acid differences between the two domains. Overexpression of the UBAb, but not overexpression of the UBAc, inhibited a variety of ubiquitin-mediated processes such as degradation of ubiquitinated proteins (i.e. EGFR, Mdm-2, and Siah-1). This in vivo result is consistent with the differences in ubiquitin binding observed in vitro between the UBAb and UBAc. This difference in ubiquitin-binding may reflect distinct regulatory functions of c-Cbl and Cbl-b.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Línea Celular , Células HeLa , Humanos , Riñón , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-cbl , Proteínas Recombinantes/metabolismo , Transfección , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
PURPOSE: To evaluate changes in epidermal growth factor receptor (EGFR) phosphorylation and its downstream signaling in tumor and surrogate tissue biopsies in patients with metastatic breast cancer treated with erlotinib, an EGFR tyrosine kinase inhibitor, and to assess relationships between biomarkers in tumor and normal tissues and between biomarkers and pharmacokinetics. PATIENTS AND METHODS: Eighteen patients were treated orally with 150 mg/d of erlotinib. Ki67, EGFR, phosphorylated EGFR (pEGFR), phosphorylated mitogen-activated protein kinase (pMAPK), and phosphorylated AKT (pAKT) in 15 paired tumor, skin, and buccal mucosa biopsies (at baseline and after 1 month of therapy) were examined by immunohistochemistry and analyzed quantitatively. Pharmacokinetic sampling was also obtained. RESULTS: The stratum corneum layer and Ki67 in keratinocytes of the epidermis in 15 paired skin biopsies significantly decreased after treatment (P = .0005 and P = .0003, respectively). No significant change in Ki67 was detected in 15 tumors, and no responses were observed. One was EGFR-positive and displayed heterogeneous expression of the receptor, and 14 were EGFR-negative. In the EGFR-positive tumor, pEGFR, pMAPK, and pAKT were reduced after treatment. Paradoxically, pEGFR was increased in EGFR-negative tumors post-treatment (P = .001). Although markers were reduced in surrogate and tumor tissues in the patient with EGFR-positive tumor, no apparent associations were observed in patients with EGFR-negative tumor. CONCLUSION: Erlotinib has inhibitory biologic effects on normal surrogate tissues and on an EGFR-positive tumor. The lack of reduced tumor proliferation may be attributed to the heterogeneous expression of receptor in the EGFR-positive patient and absence of target in this cohort of heavily pretreated patients.