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A majority of patients with cancer receive radiotherapy as part of their treatment regimens whether using external beam therapy or locally-delivered radioisotopes. While often effective, some tumors are inadequately controlled with radiation and radiotherapy has significant short-term and long-term toxicities for cancer survivors. Insights into molecular mechanisms involved in cellular responses to DNA breaks introduced by radiation or other cancer therapies have been gained in recent years and approaches to manipulate these responses to enhance tumor cell killing or reduce normal tissue toxicity are of great interest. Here, we report the identification and initial characterization of XRD-0394, a potent and specific dual inhibitor of two DNA damage response kinases, ATM and DNA-PKcs. This orally bioavailable molecule demonstrates significantly enhanced tumor cell kill in the setting of therapeutic ionizing irradiation in vitro and in vivo. XRD-0394 also potentiates the effectiveness of topoisomerase I inhibitors in vitro. In addition, in cells lacking BRCA1/2 XRD-0394 shows single-agent activity and synergy in combination with PARP inhibitors. A phase Ia clinical trial (NCT05002140) with XRD-0394 in combination with radiotherapy has completed. These results provide a rationale for future clinical trials with XRD-0394 in combination with radiotherapy, PARP inhibitors, and targeted delivery of topoisomerase I inhibitors.
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Proteínas de la Ataxia Telangiectasia Mutada , Proteína Quinasa Activada por ADN , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Fármacos Sensibilizantes a Radiaciones , Inhibidores de Topoisomerasa I , Humanos , Animales , Inhibidores de Topoisomerasa I/farmacología , Ratones , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Fármacos Sensibilizantes a Radiaciones/farmacología , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Proteína Quinasa Activada por ADN/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Línea Celular Tumoral , Femenino , Sinergismo FarmacológicoRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0054014.].
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[This corrects the article DOI: 10.1371/journal.pone.0054014.].
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The Translational Research Working Group (TRWG) was created as a national initiative to evaluate the current status of the National Cancer Institute's investment in translational research and envision its future. The TRWG conceptualized translational research as a set of six developmental processes or pathways focused on various clinical goals. One of those pathways describes the development of agents-both small molecules and biologics-for the treatment and prevention of cancer. The Agents Developmental Pathway was conceived not as a comprehensive description of the corresponding real-world processes, but rather as a tool designed to facilitate movement of an agent through the translational process to the point where it can begin definitive clinical testing. This article presents the Agents Developmental Pathway and discusses key challenges associated with the processes described.
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Antineoplásicos/uso terapéutico , Productos Biológicos/uso terapéutico , Vacunas contra el Cáncer/uso terapéutico , Neoplasias/terapia , Biomarcadores/análisis , Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Humanos , National Institutes of Health (U.S.) , Desarrollo de Programa , Diseño de Software , Estados UnidosRESUMEN
PURPOSE: Topotecan resistance can result from drug efflux by P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) as well as survival signals initiated by epidermal growth factor receptor family members. The present studies were done to determine the effect of combining topotecan and the dual epidermal growth factor receptor/HER2 inhibitor lapatinib in tissue culture, a murine xenograft model, and a phase I clinical trial. EXPERIMENTAL DESIGN: The effects of lapatinib on topotecan accumulation and cytotoxicity in vitro were examined in paired cell lines lacking or expressing Pgp or BCRP. Antiproliferative effects of the combination were assessed in mice bearing HER2+ BT474 breast cancer xenografts. Based on tolerability in this preclinical model, 37 patients with advanced-stage cancers received escalating doses of lapatinib and topotecan in a phase I trial. RESULTS: Lapatinib increased topotecan accumulation in BCRP- or Pgp-expressing cells in vitro, and the combination showed enhanced efficacy in HER2+ BT474 xenografts. In the phase I study, nausea, vomiting, diarrhea, and fatigue were dose limiting. The maximum tolerated doses were 1,250 mg/d lapatinib by mouth for 21 or 28 days with 3.2 mg/m2 topotecan i.v. on days 1, 8, and 15 of 28-day cycles. Pharmacokinetic analyses showed that combined drug administration resulted in decreased topotecan clearance consistent with transporter-mediated interactions. Seventeen (46%) patients had disease stabilization. CONCLUSIONS: The lapatinib/topotecan combination is well tolerated and warrants further study.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias/tratamiento farmacológico , Adulto , Anciano , Animales , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Femenino , Humanos , Técnicas In Vitro , Lapatinib , Masculino , Dosis Máxima Tolerada , Ratones , Persona de Mediana Edad , Quinazolinas/administración & dosificación , Quinazolinas/efectos adversos , Quinazolinas/farmacocinética , Topotecan/administración & dosificación , Topotecan/efectos adversos , Topotecan/farmacocinética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A common aim of pharmacogenomic studies that use genome-wide assays on panels of cancers is the unbiased discovery of genomic alterations that are associated with clinical outcome and drug response. Previous studies of lapatinib, a selective dual-kinase inhibitor of epidermal growth factor receptor (EGFR) and HER2 tyrosine kinases, have shown predictable relationships between the activity of these target genes and response. Under the hypothesis that additional genes may play a role in drug sensitivity, a predictive model for lapatinib response was constructed from genome-wide DNA copy number data from 24 cancer cell lines. An optimal predictive model which consists of aberrations at nine distinct genetic loci, includes gains of HER2, EGFR, and loss of CDKN2A. This model achieved an area under the receiver operating characteristic curve of approximately 0.85 (80% confidence interval, 0.70-0.98; P < 0.01), and correctly classified the sensitivity status of 8 of 10 head and neck cancer cell lines. This study shows that biomarkers predictive for lapatinib sensitivity, including the previously described copy number gains of EGFR and HER2, can be discovered using novel genomic assays in an unbiased manner. Furthermore, these results show the utility of DNA copy number profiles in pharmacogenomic studies.
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Antineoplásicos/uso terapéutico , Dosificación de Gen , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genoma Humano , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Quinazolinas/uso terapéutico , Proliferación Celular , Humanos , Lapatinib , Modelos Moleculares , Neoplasias/clasificación , Polimorfismo de Nucleótido Simple , Curva ROC , Células Tumorales CultivadasRESUMEN
Lapatinib (GW572016) is a selective inhibitor of both epidermal growth factor receptor (EGFR) and HER-2 tyrosine kinases. Here, we explore the therapeutic potential of lapatinib by testing its effect on tumor cell growth in a panel of 31 characterized human breast cancer cell lines, including trastuzumab-conditioned HER-2-positive cell lines. We further characterize its activity in combination with trastuzumab and analyze whether EGFR and HER-2 expression or changes induced in the activation of EGFR, HER-2, Raf, AKT, or extracellular signal-regulated kinase (ERK) are markers of drug activity. We report that concentration-dependent antiproliferative effects of lapatinib were seen in all breast cancer cell lines tested but varied significantly between individual cell lines with up to 1,000-fold difference in the IC(50)s (range, 0.010-18.6 micromol/L). Response to lapatinib was significantly correlated with HER-2 expression and its ability to inhibit HER-2, Raf, AKT, and ERK phosphorylation. Long-term in vivo lapatinib studies were conducted with human breast cancer xenografts in athymic mice. Treatment over 77 days resulted in a sustained and significant reduction in xenograft volume compared with untreated controls. For the combination of lapatinib plus trastuzumab, synergistic drug interactions were observed in four different HER-2-overexpressing cell lines. Moreover, lapatinib retained significant in vitro activity against cell lines selected for long-term outgrowth (>9 months) in trastuzumab-containing (100 microg/mL) culture medium. These observations provide a clear biological rationale to test lapatinib as a single agent or in combination with trastuzumab in HER-2-overexpressing breast cancer and in patients with clinical resistance to trastuzumab.
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Anticuerpos Monoclonales/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Quinazolinas/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales Humanizados , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Interacciones Farmacológicas , Ensayos de Selección de Medicamentos Antitumorales , Receptores ErbB/biosíntesis , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Lapatinib , Ratones , Ratones SCID , Proteína Oncogénica v-akt/metabolismo , Fosforilación , Quinazolinas/administración & dosificación , Receptor ErbB-2/biosíntesis , Trastuzumab , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasas raf/metabolismoRESUMEN
Lapatinib (GW572016) is a small-molecule dual inhibitor of epidermal growth factor receptor (ErbB1) and ErbB2 receptor kinase activities currently in phase III clinical trials. We used phosphoprotein and microarray analyses to carry out targeted pathway studies of phosphorylation and gene expression changes in human breast cancer cell lines in the presence or absence of lapatinib. Studies were done in four breast cancer cell lines, two of which were responsive and two of which were nonresponsive to lapatinib. Responsive cell lines, BT474 and SKBr3, constitutively overexpress ErbB2 and show an IC(50) of 25 or 32 nmol/L for lapatinib, respectively. In contrast, nonresponsive MDA-MB-468 and T47D cells expressed a low basal level of ErbB2 and showed IC(50) values in the micromolar range. Cells responsive to lapatinib exhibited strong differential effects on multiple genes in the AKT pathway. After 12 h of exposure to 1.0 micromol/L of lapatinib, AKT1, MAPK9, HSPCA, IRAK1, and CCND1 transcripts were down-regulated 7- to 25-fold in responsive BT474 and SKBr3 cells. In contrast, lapatinib weakly down-regulated the AKT pathway in nonresponsive breast cancer cell lines (<5-fold down-regulation of most genes in the pathway). Furthermore, the proapoptotic gene FOXO3A, which is negatively regulated by AKT, was up-regulated 7- and 25-fold in lapatinib-responsive SKBr3 and BT474 cells, respectively. Phosphorylated Akt and Akt-mediated phosphorylation of FOXO3A also decreased in responsive breast cancer cell lines exposed to lapatinib. Gene expression profiling also revealed that lapatinib stimulated the expression of estrogen and progesterone receptors and modulated the expression of genes involved in cell cycle control, glycolysis, and fatty acid metabolism. In BT474 and T47D cells, which expressed moderate basal levels of the estrogen and progesterone receptors, 1.0 micromol/L of lapatinib induced expression by 7- to 11-fold. These data provide insight into the mechanism of action of lapatinib in breast cancer cells.
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Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica , Quinazolinas/farmacología , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Receptores ErbB/metabolismo , Perfilación de la Expresión Génica , Humanos , Concentración 50 Inhibidora , Lapatinib , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteómica/métodos , Factores de TiempoRESUMEN
Polo-like kinase 1 (PLK1) plays key roles in the regulation of mitotic progression, including mitotic entry, spindle formation, chromosome segregation, and cytokinesis. PLK1 expression and activity are strongly linked to proliferating cells. Many studies have shown that PLK1 expression is elevated in a variety of tumors, and high expression often correlates with poor prognosis. Using a variety of methods, including small-molecule inhibition of PLK1 function and/or activity, apoptosis in cancer cell lines, cell cycle arrest in normal cell lines, and antitumor activity in vivo have been observed. In the present study, we have examined the in vitro biological activity of a novel and selective thiophene benzimidazole ATP-competitive inhibitor of PLK1 and PLK3 (5-(5,6-dimethoxy-1H-benzimidazol-1-yl)-3-{[2-(trifluoromethyl)-benzyl]oxy}thiophene-2-carboxamide, called compound 1). Compound 1 has low nanomolar activity against the PLK1 and PLK3 enzymes and potently inhibits the proliferation of a wide variety of tumor cell lines. In the lung adenocarcinoma cell line NCI-H460, compound 1 induces a transient G(2)-M arrest, mitotic spindle defects, and a multinucleate phenotype resulting in apoptosis, whereas normal human diploid fibroblasts arrest in G(2)-M and show little apoptosis. We also describe a cellular mechanistic assay that was developed to identify potent intracellular inhibitors of PLK1. In addition to its potential as a therapeutic agent for treating cancer, compound 1 is also a useful tool molecule for further investigation of the biological functions of PLK1 and PLK3.
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Adenocarcinoma/tratamiento farmacológico , Bencimidazoles/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Tiofenos/farmacología , Adenocarcinoma/enzimología , Adenosina Trifosfato/química , Adenosina Trifosfato/farmacología , Apoptosis/efectos de los fármacos , Bencimidazoles/química , Unión Competitiva , Western Blotting , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Neoplasias Pulmonares/enzimología , Microscopía Fluorescente , Estructura Molecular , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Tiofenos/química , Proteínas Supresoras de Tumor , Quinasa Tipo Polo 1RESUMEN
With the development of targeted therapeutics, especially for small-molecule inhibitors, it is important to understand whether the observed in vivo efficacy correlates with the modulation of desired/intended target in vivo. We have developed a small-molecule inhibitor of all three vascular endothelial growth factor (VEGF) receptors (VEGFR), platelet-derived growth factor receptor, and c-Kit tyrosine kinases, pazopanib (GW786034), which selectively inhibits VEGF-induced endothelial cell proliferation. It has good oral exposure and inhibits angiogenesis and tumor growth in mice. Because bolus administration of the compound results in large differences in C(max) and C(trough), we investigated the effect of continuous infusion of a VEGFR inhibitor on tumor growth and angiogenesis. GW771806, which has similar enzyme and cellular profiles to GW786034, was used for these studies due to higher solubility requirements for infusion studies. Comparing the pharmacokinetics by two different routes of administration (bolus p.o. dosing and continuous infusion), we showed that the antitumor and antiangiogenic activity of VEGFR inhibitors is dependent on steady-state concentration of the compound above a threshold. The steady-state concentration required for these effects is consistent with the concentration required for the inhibition of VEGF-induced VEGFR2 phosphorylation in mouse lungs. Furthermore, the steady-state concentration of pazopanib determined from preclinical activity showed a strong correlation with the pharmacodynamic effects and antitumor activity in the phase I clinical trial.
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Antineoplásicos/farmacología , Antineoplásicos/farmacocinética , Indazoles/farmacología , Indazoles/farmacocinética , Neovascularización Patológica/patología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/farmacocinética , Pirimidinas/farmacología , Pirimidinas/farmacocinética , Sulfonamidas/farmacología , Sulfonamidas/farmacocinética , Sulfonas/farmacología , Sulfonas/farmacocinética , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/sangre , Línea Celular Tumoral , Sistema Libre de Células , Córnea/patología , Relación Dosis-Respuesta a Droga , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Indazoles/administración & dosificación , Indazoles/sangre , Concentración 50 Inhibidora , Ratones , Ratones Desnudos , Fosforilación/efectos de los fármacos , Fosfotirosina/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/sangre , Pirimidinas/administración & dosificación , Pirimidinas/sangre , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Sulfonamidas/administración & dosificación , Sulfonamidas/sangre , Sulfonas/administración & dosificación , Sulfonas/sangre , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
GW572016 (Lapatinib) is a tyrosine kinase inhibitor in clinical development for cancer that is a potent dual inhibitor of epidermal growth factor receptor (EGFR, ErbB-1) and ErbB-2. We determined the crystal structure of EGFR bound to GW572016. The compound is bound to an inactive-like conformation of EGFR that is very different from the active-like structure bound by the selective EGFR inhibitor OSI-774 (Tarceva) described previously. Surprisingly, we found that GW572016 has a very slow off-rate from the purified intracellular domains of EGFR and ErbB-2 compared with OSI-774 and another EGFR selective inhibitor, ZD-1839 (Iressa). Treatment of tumor cells with these inhibitors results in down-regulation of receptor tyrosine phosphorylation. We evaluated the duration of the drug effect after washing away free compound and found that the rate of recovery of receptor phosphorylation in the tumor cells reflected the inhibitor off-rate from the purified intracellular domain. The slow off-rate of GW572016 correlates with a prolonged down-regulation of receptor tyrosine phosphorylation in tumor cells. The differences in the off-rates of these drugs and the ability of GW572016 to inhibit ErbB-2 can be explained by the enzyme-inhibitor structures.
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Inhibidores Enzimáticos/química , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/química , Quinazolinas/química , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Receptores ErbB/metabolismo , Clorhidrato de Erlotinib , Humanos , Cinética , Lapatinib , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Oncogénicas v-erbB/antagonistas & inhibidores , Conformación Proteica , Estructura Secundaria de Proteína , Quinazolinas/metabolismo , Quinazolinas/farmacología , Especificidad por SustratoRESUMEN
Mutations of the oncogene KRAS are important drivers of pancreatic cancer progression. Activation of epidermal growth factor receptor (EGFR) and human EGFR2 (HER2) is observed frequent in pancreatic adenocarcinomas. Because of co-activation of these two signaling pathways, we assessed the efficacy of inhibition of EGFR/HER2 receptors and the downstream KRAS effector, mitogen-activated protein kinase/extracellular-signal regulated kinase (ERK) kinase 1 and 2 (MEK1/2), on pancreatic cancer proliferation in vitro and in a murine orthotopic xenograft model. Treatment of established and patient-derived pancreatic cancer cell lines with the MEK1/2 inhibitor trametinib (GSK1120212) inhibited proliferation, and addition of the EGFR/HER2 inhibitor lapatinib enhanced the inhibition elicited by trametinib in three of eight cell lines. Importantly, in the orthotopic xenograft model, treatment with lapatinib and trametinib resulted in significantly enhanced inhibition of tumor growth relative to trametinib treatment alone in four of five patient-derived tumors tested and was, in all cases, significantly more effective in reducing the size of established tumors than treatment with lapatinib or trametinib alone. Acute treatment of established tumors with trametinib resulted in an increase in AKT2 phosphorylation that was blunted in mice treated with both trametinib and lapatinib. These data indicate that inhibition of the EGFR family receptor signaling may contribute to the effectiveness of MEK1/2 inhibition of tumor growth possibly through the inhibition of feedback activation of receptor tyrosine kinases in response to inhibition of the RAS-RAF-MEK-ERK pathway. These studies provide a rationale for assessing the co-inhibition of these pathways in the treatment of pancreatic cancer patients.
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Receptores ErbB/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Piridonas/administración & dosificación , Pirimidinonas/administración & dosificación , Quinazolinas/administración & dosificación , Animales , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Receptores ErbB/antagonistas & inhibidores , Humanos , Lapatinib , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/genética , Ratones , Trasplante de Neoplasias , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/administración & dosificación , Receptor ErbB-2/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
PURPOSE: The receptors for hepatocyte and vascular endothelial cell growth factors (MET and VEGFR2, respectively) are critical oncogenic mediators in gastric adenocarcinoma. The purpose is to examine the safety and efficacy of foretinib, an oral multikinase inhibitor targeting MET, RON, AXL, TIE-2, and VEGFR2 receptors, for the treatment of metastatic gastric adenocarcinoma. PATIENTS AND METHODS: Foretinib safety and tolerability, and objective response rate (ORR) were evaluated in patients using intermittent (240 mg/day, for 5 days every 2 weeks) or daily (80 mg/day) dosing schedules. Thirty evaluable patients were required to achieve alpha = 0.10 and beta = 0.2 to test the alternative hypothesis that single-agent foretinib would result in an ORR of ≥ 25%. Up to 10 additional patients could be enrolled to ensure at least eight with MET amplification. Correlative studies included tumor MET amplification, MET signaling, pharmacokinetics and plasma biomarkers of foretinib activity. RESULTS: From March 2007 until October 2009, 74 patients were enrolled; 74% male; median age, 61 years (range, 25-88); 93% had received prior therapy. Best response was stable disease (SD) in 10 (23%) patients receiving intermittent dosing and five (20%) receiving daily dosing; SD duration was 1.9-7.2 months (median 3.2 months). Of 67 patients with tumor samples, 3 had MET amplification, one of whom had SD. Treatment-related adverse events occurred in 91% of patients. Rates of hypertension (35% vs. 15%) and elevated aspartate aminotransferase (23% vs. 8%) were higher with intermittent dosing. In both patients with high baseline tumor phospho-MET (pMET), the pMET:total MET protein ratio decreased with foretinib treatment. CONCLUSION: These results indicate that few gastric carcinomas are driven solely by MET and VEGFR2, and underscore the diverse molecular oncogenesis of this disease. Despite evidence of MET inhibition by foretinib, single-agent foretinib lacked efficacy in unselected patients with metastatic gastric cancer.
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Anilidas/uso terapéutico , Antineoplásicos/uso terapéutico , Quinolinas/uso terapéutico , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/patología , Adulto , Anciano , Anciano de 80 o más Años , Anilidas/farmacología , Animales , Antineoplásicos/farmacología , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Quinolinas/farmacología , Resultado del Tratamiento , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Although BRAF and MEK inhibitors have proven clinical benefits in melanoma, most patients develop resistance. We report a de novo MEK2-Q60P mutation and BRAF gain in a melanoma from a patient who progressed on the MEK inhibitor trametinib and did not respond to the BRAF inhibitor dabrafenib. We also identified the same MEK2-Q60P mutation along with BRAF amplification in a xenograft tumor derived from a second melanoma patient resistant to the combination of dabrafenib and trametinib. Melanoma cells chronically exposed to trametinib acquired concurrent MEK2-Q60P mutation and BRAF-V600E amplification, which conferred resistance to MEK and BRAF inhibitors. The resistant cells had sustained MAPK activation and persistent phosphorylation of S6K. A triple combination of dabrafenib, trametinib, and the PI3K/mTOR inhibitor GSK2126458 led to sustained tumor growth inhibition. Hence, concurrent genetic events that sustain MAPK signaling can underlie resistance to both BRAF and MEK inhibitors, requiring novel therapeutic strategies to overcome it.
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MAP Quinasa Quinasa 2/genética , Melanoma/tratamiento farmacológico , Melanoma/genética , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Amplificación de Genes , Humanos , MAP Quinasa Quinasa 2/antagonistas & inhibidores , MAP Quinasa Quinasa 2/química , Masculino , Melanoma/enzimología , Melanoma/patología , Persona de Mediana Edad , Modelos Moleculares , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas/metabolismoRESUMEN
Recent results from clinical trials with the BRAF inhibitors GSK2118436 (dabrafenib) and PLX4032 (vemurafenib) have shown encouraging response rates; however, the duration of response has been limited. To identify determinants of acquired resistance to GSK2118436 and strategies to overcome the resistance, we isolated GSK2118436 drug-resistant clones from the A375 BRAF(V600E) and the YUSIT1 BRAF(V600K) melanoma cell lines. These clones also showed reduced sensitivity to the allosteric mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor GSK1120212 (trametinib). Genetic characterization of these clones identified an in-frame deletion in MEK1 (MEK1(K59del)) or NRAS mutation (NRAS(Q61K) and/or NRAS(A146T)) with and without MEK1(P387S) in the BRAF(V600E) background and NRAS(Q61K) in the BRAF(V600K) background. Stable knockdown of NRAS with short hairpin RNA partially restored GSK2118436 sensitivity in mutant NRAS clones, whereas expression of NRAS(Q61K) or NRAS(A146T) in the A375 parental cells decreased sensitivity to GSK2118436. Similarly, expression of MEK1(K59del), but not MEK1(P387S), decreased sensitivity of A375 cells to GSK2118436. The combination of GSK2118436 and GSK1120212 effectively inhibited cell growth, decreased ERK phosphorylation, decreased cyclin D1 protein, and increased p27(kip1) protein in the resistant clones. Moreover, the combination of GSK2118436 or GSK1120212 with the phosphoinositide 3-kinase/mTOR inhibitor GSK2126458 enhanced cell growth inhibition and decreased S6 ribosomal protein phosphorylation in these clones. Our results show that NRAS and/or MEK mutations contribute to BRAF inhibitor resistance in vitro, and the combination of GSK2118436 and GSK1120212 overcomes this resistance. In addition, these resistant clones respond to the combination of GSK2126458 with GSK2118436 or GSK1120212. Clinical trials are ongoing or planned to test these combinations.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Melanoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Apoptosis/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Imidazoles/administración & dosificación , Imidazoles/farmacología , Indoles/administración & dosificación , Indoles/farmacología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Melanoma/enzimología , Oximas/administración & dosificación , Oximas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Sulfonamidas/administración & dosificación , Sulfonamidas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , VemurafenibRESUMEN
The HER and MET receptor tyrosine kinases (RTK) are coactivated in a subset of human tumors. This study characterizes MET and HER expression and signaling in a panel of human tumor cell lines and the differential susceptibility of these cell lines to single agents or combinations of foretinib, a multikinase MET inhibitor, with HER-targeted agents, erlotinib or lapatinib. Most MET-amplified tumor lines without HER1 or HER2 amplification are sensitive to foretinib, whereas MET-amplified lines with HER1 or HER2 amplification are more sensitive to the combination of foretinib with lapatinib or erlotinib. Interestingly, MET-overexpressing tumor cell lines with HER1 or HER2 amplification also exhibited reduced sensitivity to lapatinib or erlotinib in the presence of hepatocyte growth factor (HGF), indicating MET activation can decrease the effectiveness of HER1/2 inhibitors in some cell lines. Consistent with this observation, the effect of HGF on lapatinib or erlotinib sensitivity in these cells was reversed by foretinib, other MET inhibitors, or siRNA to MET. Western blot analyses showed that combining foretinib with erlotinib or lapatinib effectively decreased the phosphorylation of MET, HER1, HER2, HER3, AKT, and ERK in these cells. Furthermore, HER2-positive advanced or metastatic breast cancer patients treated with lapatinib who had higher tumor MET expression showed shorter progression-free survival (19.29 weeks in MET-high patients vs. 28.14 weeks in MET-low patients, P < 0.0225). These data suggest that combination therapy with foretinib and HER-targeted agents should be tested as a treatment option for HER1- or HER2-positive patients with MET-amplified or -overexpressing tumors.
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Anilidas/farmacología , Receptores ErbB/metabolismo , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-met/metabolismo , Quinolinas/farmacología , Receptor ErbB-2/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Clorhidrato de Erlotinib , Femenino , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Inmunoprecipitación , Lapatinib , Neoplasias/genética , Neoplasias/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/genética , Quinazolinas/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/genética , Receptores de Factores de Crecimiento/antagonistas & inhibidores , Receptores de Factores de Crecimiento/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
HER2-directed therapies, such as trastuzumab and lapatinib, are important treatments for breast cancer. However, some tumors do not respond or develop resistance to these agents. We isolated and characterized multiple lapatinib-resistant, HER2-positive, estrogen receptor (ER)-positive breast cancer clones derived from lapatinib-sensitive BT474 cells by chronic exposure to lapatinib. We show overexpression of AXL as a novel mechanism of acquired resistance to HER2-targeted agents in these models. GSK1363089 (foretinib), a multikinase inhibitor of AXL, MET, and vascular endothelial growth factor receptor currently in phase II clinical trials, restores lapatinib and trastuzumab sensitivity in these resistant cells that exhibit increased AXL expression. Furthermore, small interfering RNA to AXL, estrogen deprivation, or fulvestrant, an ER antagonist, decreases AXL expression and restores sensitivity to lapatinib in these cells. Taken together, these data provide scientific evidence to assess the expression of AXL in HER2-positive, ER-positive patients who have progressed on either lapatinib or trastuzumab and to test the combination of HER2-targeted agents and GSK1363089 in the clinic.
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Anilidas/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/genética , Genes erbB-2 , Proteínas Oncogénicas/biosíntesis , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Quinolinas/farmacología , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Anilidas/uso terapéutico , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Células Clonales , Antagonismo de Drogas , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Lapatinib , Proteínas Oncogénicas/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas , Quinazolinas/uso terapéutico , Quinolinas/uso terapéutico , ARN Interferente Pequeño , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptores de Estrógenos/antagonistas & inhibidores , Receptores de Estrógenos/biosíntesis , Trastuzumab , Tirosina Quinasa del Receptor AxlRESUMEN
Lapatinib, a selective orally available inhibitor of epidermal growth factor receptor (EGFR) and ErbB2 receptor tyrosine kinases, is a promising agent for the treatment of breast cancer. We examined the effect of lapatinib on the development of mammary tumors in MMTV-erbB2 transgenic mice, which express wild-type ErbB2 under the control of the mouse mammary tumor virus promoter and spontaneously develop estrogen receptor (ER)-negative and ErbB2-positive mammary tumors by 14 months of age. Mice were treated from age 3 months to age 15 months with vehicle (n = 17) or lapatinib (30 or 75 mg/kg body weight; n = 16 mice per group) by oral gavage twice daily (6 d/wk). All statistical tests were two-sided. By 328 days after the start of treatment, all 17 (100%) of the vehicle-treated mice vs five (31%) of the 16 mice treated with high-dose lapatinib developed mammary tumors (P < .001). Among MMTV-erbB2 mice treated for 5 months (n = 20 mice per group), those treated with lapatinib had fewer premalignant lesions and noninvasive cancers in their mammary glands than those treated with vehicle (P = .02). Lapatinib also effectively blocked epidermal growth factor-induced signaling through the EGFR and ErbB2 receptors, suppressed cyclin D1 and epiregulin mRNA expression, and stimulated p27 mRNA expression in human mammary epithelial cells and in mammary epithelial cells from mice treated for 5 months with high-dose lapatinib. Thus, cyclin D1, epiregulin, and p27 may represent useful biomarkers of lapatinib response in patients. These data suggest that lapatinib is a promising agent for the prevention of ER-negative breast cancer.
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Antineoplásicos/farmacología , Biomarcadores de Tumor/análisis , Receptores ErbB/antagonistas & inhibidores , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/prevención & control , Lesiones Precancerosas/prevención & control , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Receptor ErbB-2/análisis , Animales , Neoplasias de la Mama/tratamiento farmacológico , Carcinoma Intraductal no Infiltrante/tratamiento farmacológico , Ciclina D1/efectos de los fármacos , Ciclina D1/metabolismo , Factor de Crecimiento Epidérmico/efectos de los fármacos , Factor de Crecimiento Epidérmico/metabolismo , Epirregulina , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lapatinib , Neoplasias Mamarias Experimentales/química , Ratones , Ratones Transgénicos , Lesiones Precancerosas/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Receptores de Estrógenos/análisis , Transducción de Señal/efectos de los fármacosRESUMEN
The goal of this study was to characterize the effects of non-small cell lung carcinoma (NSCLC)-associated mutations in epidermal growth factor receptor (EGFR/ErbB1) and HER2 (ErbB2) on interactions with the dual tyrosine kinase inhibitor lapatinib. Biochemical studies show that commonly observed variants of EGFR [G719C, G719S, L858R, L861Q, and Delta746-750 (del15)] are enzyme activating, increasing the tyrosine kinase V(max) and increasing the K(m)((app)) for ATP. The point mutations G719C and L861Q had minor effects on lapatinib K(i)s, whereas EGFR mutations L858R and del15 had a higher K(i) for lapatinib than wild-type EGFR. Structural analysis of wild-type EGFR-lapatinib complexes and modeling of the EGFR mutants were consistent with these data, suggesting that loss of structural flexibility and possible stabilization of the active-like conformation could interfere with lapatinib binding, particularly to the EGFR deletion mutants. Furthermore, EGFR deletion mutants were relatively resistant to lapatinib-mediated inhibition of receptor autophosphorylation in recombinant cells expressing the variants, whereas EGFR point mutations had a modest or no effect. Of note, EGFR T790M, a receptor variant found in patients with gefitinib-resistant NSCLC, was also resistant to lapatinib-mediated inhibition of receptor autophosphorylation. Two HER2 insertional variants found in NSCLC were less sensitive to lapatinib inhibition than two HER2 point mutants. The effects of lapatinib on the proliferation of human NSCLC tumor cell lines expressing wild-type or variant EGFR and HER2 cannot be explained solely on the basis of the biochemical activity or receptor autophosphorylation in recombinant cells. These data suggest that cell line genetic heterogeneity and/or multiple determinants modulate the role played by EGFR/HER2 in regulating cell proliferation.
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Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Genes erbB-2 , Polimorfismo de Nucleótido Simple/fisiología , Quinazolinas/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/metabolismo , Animales , Células CHO , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/genética , Proliferación Celular/efectos de los fármacos , Cricetinae , Cricetulus , Receptores ErbB/química , Gefitinib , Humanos , Lapatinib , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Modelos Moleculares , Proteínas Mutantes/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Receptor ErbB-2/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Akt kinases 1, 2, and 3 are important regulators of cell survival and have been shown to be constitutively active in a variety of human tumors. GSK690693 is a novel ATP-competitive, low-nanomolar pan-Akt kinase inhibitor. It is selective for the Akt isoforms versus the majority of kinases in other families; however, it does inhibit additional members of the AGC kinase family. It causes dose-dependent reductions in the phosphorylation state of multiple proteins downstream of Akt, including GSK3 beta, PRAS40, and Forkhead. GSK690693 inhibited proliferation and induced apoptosis in a subset of tumor cells with potency consistent with intracellular inhibition of Akt kinase activity. In immune-compromised mice implanted with human BT474 breast carcinoma xenografts, a single i.p. administration of GSK690693 inhibited GSK3 beta phosphorylation in a dose- and time-dependent manner. After a single dose of GSK690693, >3 micromol/L drug concentration in BT474 tumor xenografts correlated with a sustained decrease in GSK3 beta phosphorylation. Consistent with the role of Akt in insulin signaling, treatment with GSK690693 resulted in acute and transient increases in blood glucose level. Daily administration of GSK690693 produced significant antitumor activity in mice bearing established human SKOV-3 ovarian, LNCaP prostate, and BT474 and HCC-1954 breast carcinoma xenografts. Immunohistochemical analysis of tumor xenografts after repeat dosing with GSK690693 showed reductions in phosphorylated Akt substrates in vivo. These results support further evaluation of GSK690693 as an anticancer agent.