RESUMEN
The MAPK pathway is frequently activated in many human cancers, particularly melanomas. A single-nucleotide mutation in BRAF resulting in the substitution of glutamic acid for valine (V(600E)) causes constitutive activation of the downstream MAPK pathway. Selective BRAF and MEK inhibitor therapies have demonstrated remarkable antitumor responses in BRAF(V600) (E)-mutant melanoma patients. However, initial tumor shrinkage is transient and the vast majority of patients develop resistance. We previously reported that SCH772984, an ERK 1/2 inhibitor, effectively suppressed MAPK pathway signaling and cell proliferation in BRAF, MEK, and concurrent BRAF/MEK inhibitor-resistant tumor models. ERK inhibitors are currently being evaluated in clinical trials and, in anticipation of the likelihood of clinical resistance, we sought to prospectively model acquired resistance to SCH772984. Our data show that long-term exposure of cells to SCH772984 leads to acquired resistance, attributable to a mutation of glycine to aspartic acid (G(186D)) in the DFG motif of ERK1. Structural and biophysical studies demonstrated specific defects in SCH772984 binding to mutant ERK. Taken together, these studies describe the interaction of SCH772984 with ERK and identify a novel mechanism of ERK inhibitor resistance through mutation of a single residue within the DFG motif. Mol Cancer Ther; 15(4); 548-59. ©2016 AACR.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/genética , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/química , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Modelos Moleculares , Conformación Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , RatasRESUMEN
Antibody-drug conjugates (ADC) represent a promising therapeutic modality for managing cancer. Here, we report a novel humanized ADC that targets the tetraspanin-like protein TM4SF1. TM4SF1 is highly expressed on the plasma membranes of many human cancer cells and also on the endothelial cells lining tumor blood vessels. TM4SF1 is internalized upon interaction with antibodies. We hypothesized that an ADC against TM4SF1 would inhibit cancer growth directly by killing cancer cells and indirectly by attacking the tumor vasculature. We generated a humanized anti-human TM4SF1 monoclonal antibody, v1.10, and armed it with an auristatin cytotoxic agent LP2 (chemical name mc-3377). v1.10-LP2 selectively killed cultured human tumor cell lines and human endothelial cells that express TM4SF1. Acting as a single agent, v1.10-LP2 induced complete regression of several TM4SF1-expressing tumor xenografts in nude mice, including non-small cell lung cancer and pancreas, prostate, and colon cancers. As v1.10 did not react with mouse TM4SF1, it could not target the mouse tumor vasculature. Therefore, we generated a surrogate anti-mouse TM4SF1 antibody, 2A7A, and conjugated it to LP2. At 3 mpk, 2A7A-LP2 regressed several tumor xenografts without noticeable toxicity. Combination therapy with v1.10-LP2 and 2A7A-LP2 together was more effective than either ADC alone. These data provide proof-of-concept that TM4SF1-targeting ADCs have potential as anticancer agents with dual action against tumor cells and the tumor vasculature. Such agents could offer exceptional therapeutic value and warrant further investigation. Mol Cancer Ther; 14(8); 1868-76. ©2015 AACR.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de la Angiogénesis/toxicidad , Animales , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Antineoplásicos/toxicidad , Línea Celular Tumoral , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Expresión Génica , Humanos , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Neovascularización Patológica , Conejos , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Dinaciclib is a potent CDK1, 2, 5 and 9 inhibitor being developed for the treatment of cancer. Additional understanding of antitumor mechanisms and identification of predictive biomarkers are important for its clinical development. Here we demonstrate that while dinaciclib can effectively block cell cycle progression, in vitro and in vivo studies, coupled with mouse and human pharmacokinetics, support a model whereby induction of apoptosis is a main mechanism of dinaciclib's antitumor effect and relevant to the clinical duration of exposure. This was further underscored by kinetics of dinaciclib-induced downregulation of the antiapoptotic BCL2 family member MCL1 and correlation of sensitivity with the MCL1-to-BCL-xL mRNA ratio or MCL1 amplification in solid tumor models in vitro and in vivo. This MCL1-dependent apoptotic mechanism was additionally supported by synergy with the BCL2, BCL-xL and BCL-w inhibitor navitoclax (ABT-263). These results provide the rationale for investigating MCL1 and BCL-xL as predictive biomarkers for dinaciclib antitumor response and testing combinations with BCL2 family member inhibitors.
Asunto(s)
Apoptosis/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Neoplasias/metabolismo , Compuestos de Piridinio/farmacología , Proteína bcl-X/metabolismo , Compuestos de Anilina/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Óxidos N-Cíclicos , Modelos Animales de Enfermedad , Diterpenos/farmacología , Resistencia a Antineoplásicos/genética , Sinergismo Farmacológico , Compuestos Epoxi/farmacología , Femenino , Dosificación de Gen , Humanos , Indolizinas , Masculino , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Neoplasias/genética , Fenantrenos/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sulfonamidas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína bcl-X/genéticaRESUMEN
UNLABELLED: Next-generation sequencing was used to identify Notch mutations in a large collection of diverse solid tumors. NOTCH1 and NOTCH2 rearrangements leading to constitutive receptor activation were confined to triple-negative breast cancers (TNBC; 6 of 66 tumors). TNBC cell lines with NOTCH1 rearrangements associated with high levels of activated NOTCH1 (N1-ICD) were sensitive to the gamma-secretase inhibitor (GSI) MRK-003, both alone and in combination with paclitaxel, in vitro and in vivo, whereas cell lines with NOTCH2 rearrangements were resistant to GSI. Immunohistochemical staining of N1-ICD in TNBC xenografts correlated with responsiveness, and expression levels of the direct Notch target gene HES4 correlated with outcome in patients with TNBC. Activating NOTCH1 point mutations were also identified in other solid tumors, including adenoid cystic carcinoma (ACC). Notably, ACC primary tumor xenografts with activating NOTCH1 mutations and high N1-ICD levels were sensitive to GSI, whereas N1-ICD-low tumors without NOTCH1 mutations were resistant. SIGNIFICANCE: NOTCH1 mutations, immunohistochemical staining for activated NOTCH1, and HES4 expression are biomarkers that can be used to identify solid tumors that are likely to respond to GSI-based therapies.
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Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Antineoplásicos/farmacología , Carcinoma Adenoide Quístico/genética , Inhibidores de Proteasas/farmacología , Neoplasias de la Mama Triple Negativas/genética , Animales , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Apoptosis/genética , Biomarcadores , Carcinoma Adenoide Quístico/tratamiento farmacológico , Carcinoma Adenoide Quístico/metabolismo , Línea Celular Tumoral , Senescencia Celular/efectos de los fármacos , Óxidos S-Cíclicos/farmacología , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Exoma , Femenino , Regulación Neoplásica de la Expresión Génica , Reordenamiento Génico , Genes myc , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Modelos Moleculares , Mutación , Pronóstico , Inhibidores de Proteasas/administración & dosificación , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores Notch/antagonistas & inhibidores , Receptores Notch/química , Receptores Notch/genética , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacos , Tiadiazoles/farmacología , Resultado del Tratamiento , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
UNLABELLED: TBK1 (TANK-binding kinase 1) is a noncanonical IκB protein kinase that phosphorylates and activates downstream targets such as IRF3 and c-Rel and, mediates NF-κB activation in cancer. Previous reports demonstrated synthetic lethality of TBK1 with mutant KRAS in non-small cell lung cancer (NSCLC); thus, TBK1 could be a novel target for treatment of KRAS-mutant NSCLC. Here, the effect of TBK1 on proliferation in a panel of cancer cells by both genetic and pharmacologic approaches was evaluated. In KRAS-mutant cancer cells, reduction of TBK1 activity by knockdown or treatment with TBK1 inhibitors did not correlate with reduced proliferation in a two-dimensional viability assay. Verification of target engagement via reduced phosphorylation of S386 of IRF3 (pIRF3(S386)) was difficult to assess in NSCLC cells due to low protein expression. However, several cell lines were identified with high pIRF3(S386) levels after screening a large panel of cell lines, many of which also harbor KRAS mutations. Specifically, a large subset of KRAS-mutant pancreatic cancer cell lines was uncovered with high constitutive pIRF3(S386) levels, which correlated with high levels of phosphorylated S172 of TBK1 (pTBK1(S172)). Finally, TBK1 inhibitors dose-dependently inhibited pIRF3(S386) in these cell lines, but this did not correlate with inhibition of cell growth. Taken together, these data demonstrate that the regulation of pathways important for cell proliferation in some NSCLC, pancreatic, and colorectal cell lines is not solely dependent on TBK1 activity. IMPLICATIONS: TBK1 has therapeutic potential under certain contexts and phosphorylation of its downstream target IRF3 is a biomarker of TBK1 activity.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/terapia , Factor 3 Regulador del Interferón/antagonistas & inhibidores , Neoplasias Pulmonares/terapia , Neoplasias/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Terapia Molecular Dirigida , Neoplasias/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de SeñalRESUMEN
Inhibition of the DNA damage checkpoint kinase WEE1 potentiates genotoxic chemotherapies by abrogating cell-cycle arrest and proper DNA repair. However, WEE1 is also essential for unperturbed cell division in the absence of extrinsic insult. Here, we investigate the anticancer potential of a WEE1 inhibitor, independent of chemotherapy, and explore a possible cellular context underlying sensitivity to WEE1 inhibition. We show that MK-1775, a potent and selective ATP-competitive inhibitor of WEE1, is cytotoxic across a broad panel of tumor cell lines and induces DNA double-strand breaks. MK-1775-induced DNA damage occurs without added chemotherapy or radiation in S-phase cells and relies on active DNA replication. At tolerated doses, MK-1775 treatment leads to xenograft tumor growth inhibition or regression. To begin addressing potential response markers for MK-1775 monotherapy, we focused on PKMYT1, a kinase functionally related to WEE1. Knockdown of PKMYT1 lowers the EC(50) of MK-1775 by five-fold but has no effect on the cell-based response to other cytotoxic drugs. In addition, knockdown of PKMYT1 increases markers of DNA damage, γH2AX and pCHK1(S345), induced by MK-1775. In a post hoc analysis of 305 cell lines treated with MK-1775, we found that expression of PKMYT1 was below average in 73% of the 33 most sensitive cell lines. Our findings provide rationale for WEE1 inhibition as a potent anticancer therapy independent of a genotoxic partner and suggest that low PKMYT1 expression could serve as an enrichment biomarker for MK-1775 sensitivity.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Resistencia a Antineoplásicos/genética , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Pirazoles/administración & dosificación , Pirimidinas/administración & dosificación , Pirimidinonas , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The high frequency of activating RAS or BRAF mutations in cancer provides strong rationale for targeting the mitogen-activated protein kinase (MAPK) pathway. Selective BRAF and MAP-ERK kinase (MEK) inhibitors have shown clinical efficacy in patients with melanoma. However, the majority of responses are transient, and resistance is often associated with pathway reactivation of the extracellular signal-regulated kinase (ERK) signaling pathway. Here, we describe the identification and characterization of SCH772984, a novel and selective inhibitor of ERK1/2 that displays behaviors of both type I and type II kinase inhibitors. SCH772984 has nanomolar cellular potency in tumor cells with mutations in BRAF, NRAS, or KRAS and induces tumor regressions in xenograft models at tolerated doses. Importantly, SCH772984 effectively inhibited MAPK signaling and cell proliferation in BRAF or MEK inhibitor-resistant models as well as in tumor cells resistant to concurrent treatment with BRAF and MEK inhibitors. These data support the clinical development of ERK inhibitors for tumors refractory to MAPK inhibitors.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas Quinasa Quinasa PAM/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Mutación , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
Activation of the phosphoinositide 3-kinase pathway is commonly observed in human prostate cancer. Loss of function of phosphatase and tensin homolog (PTEN) is associated with the activation of AKT and mammalian target of rapamycin (mTOR) in many cancer cell lines as well as in other model systems. However, activation of mTOR is also dependent of kinases other than AKT. Here, we show that activation of mTOR is not dependent on AKT in a prostate-specific PTEN-deficient mouse model of prostate cancer. Pathway bifurcation of AKT and mTOR was noted in both mouse and human prostate tumors. We demonstrated for the first time that cotargeting mTOR and AKT with ridaforolimus/MK-8669 and M1K-2206, respectively, delivers additive antitumor effects in vivo when compared to single agents. Our preclinical data suggest that the combination of AKT and mTOR inhibitors might be more effective in treating prostate cancer patients than current treatment regimens or either treatment alone.
RESUMEN
We tested the use of the small-molecule Inhibitor of Apoptosis Protein (IAP) inhibitor LBW242 in combination with the standard-of-care therapies of irradiation and temozolomide for malignant gliomas. In vitro assays demonstrated that LBW242 enhanced the cytotoxic activity of radiotherapy, and clonogenic assays showed that the combination therapy led to a synergistic anti-glioma effect in multiple cell lines. Neurosphere assays revealed that the combination of radiation and LBW242 led to a pro-apoptotic effect in these glioma-initiating cell-enriched assays, with a corresponding inhibition of primary tumor cell growth. Athymic mice bearing established human malignant glioma tumor xenografts treated with LBW242 plus radiation and temozolomide demonstrated a synergistic suppression of tumor growth. Taken together, these experiments show that the pro-apoptotic and anti-glioma effects of radiotherapy and chemotherapy can be enhanced by the addition of a small-molecule IAP inhibitor. These results are readily translatable to clinical trial and offer the potential for improved treatment outcomes for patients with glioma.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Dacarbazina/análogos & derivados , Glioma/tratamiento farmacológico , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Oligopéptidos/uso terapéutico , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Western Blotting , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/radioterapia , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Terapia Combinada , Dacarbazina/uso terapéutico , Rayos gamma , Glioma/patología , Glioma/radioterapia , Humanos , Técnicas In Vitro , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ratones , Ratones Desnudos , Temozolomida , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The inhibitor of apoptosis proteins (IAPs) have recently been shown to modulate nuclear factor κB (NF-κB) signaling downstream of tumor necrosis factor (TNF) family receptors, positioning them as essential survival factors in several cancer cell lines, as indicated by the cytotoxic activity of several novel small molecule IAP antagonists. In addition to roles in cancer, increasing evidence suggests that IAPs have an important function in immunity; however, the impact of IAP antagonists on antitumor immune responses is unknown. In this study, we examine the consequences of IAP antagonism on T cell function in vitro and in the context of a tumor vaccine in vivo. We find that IAP antagonists can augment human and mouse T cell responses to physiologically relevant stimuli. The activity of IAP antagonists depends on the activation of NF-κB2 signaling, a mechanism paralleling that responsible for the cytotoxic activity in cancer cells. We further show that IAP antagonists can augment both prophylactic and therapeutic antitumor vaccines in vivo. These findings indicate an important role for the IAPs in regulating T cell-dependent responses and suggest that targeting IAPs using small molecule antagonists may be a strategy for developing novel immunomodulating therapies against cancer.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Neoplasias/inmunología , Transducción de Señal , Linfocitos T/inmunología , Animales , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer , Humanos , Inmunomodulación , Proteínas Inhibidoras de la Apoptosis/inmunología , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ratones , FN-kappa B/inmunología , FN-kappa B/metabolismo , Neoplasias/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoAsunto(s)
Neoplasias/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase I , Diseño de Fármacos , Humanos , Terapia Molecular Dirigida , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Relación Estructura-ActividadRESUMEN
PURPOSE: Tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) is a member of tumor necrosis factor family and it is important for ligand induced apoptosis in tumor cells. TRAIL has been shown to be synergistic with a variety of chemotherapies and targeted agents. In the study, a combination of TRAIL and a histone deacetylase inhibitor LBH589 was studied in mesothelioma cell lines. EXPERIMENTAL DESIGN: Five mesothelioma cell lines and two normal cell lines were tested for cell growth inhibition and apoptosis using high-throughput assays in the presence of LBH589, TRAIL and a combination of the two. Caspase induction was studied and levels of X-linked inhibitor of apoptosis (XIAP) were tested using Western blotting. A combination of a direct inhibitor of XIAP was also tested in combination with TRAIL. RESULTS: In mesothelioma cell lines, a combination of LBH589 and TRAIL markedly increased cell growth inhibition and apoptosis when compared with the effect on normal cell lines. LBH589 and TRAIL appeared to induce higher levels of caspase 3 and 7 and this appeared to be closely related to ability of LBH589 to degrade XIAP. In addition, a direct inhibitor of XIAP was also sensitized cells to TRAIL apoptosis, providing an indirect confirmation for XIAP degradation as a possible mechanism of synergy. CONCLUSIONS: In mesothelioma cell lines, LBH589 increases the sensitivity to TRAIL. In addition, at least partly, the mechanism of this induction of TRAIL sensitivity is due to LBH589 related degradation of XIAP. These results provide initial evidence for testing this combination in clinical trials.
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Apoptosis/efectos de los fármacos , Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos/farmacología , Mesotelioma/metabolismo , Mesotelioma/patología , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Acetilación , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Caspasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Histonas/metabolismo , Humanos , Immunoblotting , Indoles , Leupeptinas/farmacología , Panobinostat , Poli(ADP-Ribosa) Polimerasas/metabolismo , Células Tumorales CultivadasRESUMEN
Multiple receptor tyrosine kinases (RTKs), including PDGFR, have been validated as therapeutic targets in glioblastoma multiforme (GBM), yet inhibitors of RTKs have had limited clinical success. As various antiapoptotic mechanisms render GBM cells resistant to chemo- and radiotherapy, we hypothesized that these antiapoptotic mechanisms also confer resistance to RTK inhibition. We found that in vitro inhibition of PDGFR in human GBM cells initiated the intrinsic pathway of apoptosis, as evidenced by mitochondrial outer membrane permeabilization, but downstream caspase activation was blocked by inhibitor of apoptosis proteins (IAPs). Consistent with this, inhibition of PDGFR combined with small molecule inactivation of IAPs induced apoptosis in human GBM cells in vitro and had synergistic antitumor effects in orthotopic mouse models of GBM and in primary human GBM neurospheres. These results demonstrate that concomitant inhibition of IAPs can overcome resistance to RTK inhibitors in human malignant GBM cells, and suggest that blockade of IAPs has the potential to improve treatment outcomes in patients with GBM.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Resistencia a Antineoplásicos , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Animales , Benzamidas , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Mesilato de Imatinib , Ratones , Modelos Biológicos , Neuronas/metabolismo , Oligopéptidos/administración & dosificación , Piperazinas/administración & dosificación , Pirimidinas/administración & dosificaciónRESUMEN
Smac mimetic compounds targeting the inhibitor of apoptosis proteins (IAP) baculoviral IAP repeat-3 domain are presumed to reduce the threshold for apoptotic cell death by alleviating caspase-9 repression. We explored this tenet in an unbiased manner by searching for small interfering RNAs that are able to confer resistance to the Smac mimetic compound LBW242. Among the screening hits were multiple components of the tumor necrosis factor alpha (TNFalpha) signaling pathway as well as X-linked inhibitor of apoptosis (XIAP) itself. Here, we show that in a subset of highly sensitive tumor cell lines, activity of LBW242 is dependent on TNFalpha signaling. Mechanistic studies indicate that in this context, XIAP is a positive modulator of TNFalpha induction whereas cellular inhibitor of apoptosis protein 1 negatively regulates TNFalpha-mediated apoptosis.
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Proteínas Inhibidoras de la Apoptosis/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas Mitocondriales/fisiología , ARN Interferente Pequeño/genética , Factor de Necrosis Tumoral alfa/fisiología , Proteína Inhibidora de la Apoptosis Ligada a X/fisiología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis , Muerte Celular , Línea Celular Tumoral , Secuencia Conservada , Femenino , Humanos , Oligopéptidos/farmacología , Neoplasias Ováricas , Interferencia de ARN/fisiología , ARN Neoplásico/genética , Transducción de Señal , Proteína Inhibidora de la Apoptosis Ligada a X/genéticaRESUMEN
Members of the inhibitor of apoptosis protein (IAP) family play a role in mediating apoptosis. Studies suggest that these proteins may be a viable target in leukemia because they have been found to be variably expressed in acute leukemias and are associated with chemosensitivity, chemoresistance, disease progression, remission, and patient survival. Another promising therapeutic target, FLT3, is mutated in about one third of acute myelogenous leukemia (AML) patients; promising results have recently been achieved in clinical trials investigating the effects of the protein tyrosine kinase inhibitor PKC412 on AML patients harboring mutations in the FLT3 protein. Of growing concern, however, is the development of drug resistance resulting from the emergence of point mutations in targeted tyrosine kinases used for treatment of acute leukemia patients. One approach to overriding resistance is to combine structurally unrelated inhibitors and/or inhibitors of different signaling pathways. The proapoptotic IAP inhibitor, LBW242, was shown in proliferation studies done in vitro to enhance the killing of PKC412-sensitive and PKC412-resistant cell lines expressing mutant FLT3 when combined with either PKC412 or standard cytotoxic agents (doxorubicin and Ara-c). In addition, in an in vivo imaging assay using bioluminescence as a measure of tumor burden, a total of 12 male NCr-nude mice were treated for 10 days with p.o. administration of vehicle, LBW242 (50 mg/kg/day), PKC412 (40 mg/kg/day), or a combination of LBW242 and PKC412; the lowest tumor burden was observed in the drug combination group. Finally, the combination of LBW242 and PKC412 was sufficient to override stromal-mediated viability signaling conferring resistance to PKC412.
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Antineoplásicos/farmacología , Materiales Biomiméticos/farmacología , Proteínas Portadoras , Leucemia/tratamiento farmacológico , Proteínas Mitocondriales , Proteínas Mutantes/metabolismo , Oligopéptidos/farmacología , Tirosina Quinasa 3 Similar a fms/metabolismo , Animales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ratones , Proteínas Mutantes/genética , Oligopéptidos/química , Estaurosporina/análogos & derivados , Estaurosporina/farmacología , Células del Estroma/efectos de los fármacos , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
Second mitochondria-derived activator of caspases (Smac) promotes apoptosis via activation of caspases. Here we show that a low-molecular-weight Smac mimetic LBW242 induces apoptosis in multiple myeloma (MM) cells resistant to conventional and bortezomib therapies. Examination of purified patient MM cells demonstrated similar results, without significant cytotoxicity against normal lymphocytes and bone marrow stromal cells (BMSCs). Importantly, LBW242 abrogates paracrine MM cell growth triggered by their adherence to BMSCs and overcomes MM cell growth and drug-resistance conferred by interleukin-6 or insulinlike growth factor-1. Overexpression of Bcl-2 similarly does not affect LBW242-induced cytotoxicity. Mechanistic studies show that LBW242-induced apoptosis in MM cells is associated with activation of caspase-8, caspase-9, and caspase-3, followed by PARP cleavage. In human MM xenograft mouse models, LBW242 is well tolerated, inhibits tumor growth, and prolongs survival. Importantly, combining LBW242 with novel agents, including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the proteasome inhibitors bortezomib and NPI-0052, as well as with the conventional anti-MM agent melphalan, induces additive/synergistic anti-MM activity. Our study therefore provides the rationale for clinical protocols evaluating LBW242, alone and together with other anti-MM agents, to improve patient outcome in MM.
Asunto(s)
Antineoplásicos/farmacología , Péptidos y Proteínas de Señalización Intracelular/agonistas , Proteínas Mitocondriales/agonistas , Mieloma Múltiple/tratamiento farmacológico , Animales , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Sinergismo Farmacológico , Humanos , Melfalán/farmacología , Ratones , Imitación Molecular , Mieloma Múltiple/patología , Comunicación Paracrina/efectos de los fármacos , Células del Estroma/citología , Tasa de Supervivencia , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Inhibitor of apoptosis proteins (IAPs) such as XIAP subvert apoptosis by binding and inhibiting caspases. Because occupation of the XIAP BIR3 peptide binding pocket by Smac abolishes the XIAP-caspase 9 interaction, it is a proapoptotic event of great therapeutic interest. An assay for pocket binding was developed based on the displacement of Smac 7-mer from BIR3. Through the physical and biochemical analysis of a variety of peptides, we have determined the minimum sequence required for inhibition of the Smac-BIR3 interaction and detailed the dimensions and topology of the BIR3 peptide binding pocket. This work describes the structure-activity relationship (SAR) for peptide inhibitors of Smac-IAP binding.
RESUMEN
Tumor cells have evolved numerous mechanisms to thwart apoptosis. As our understanding of the machinery which regulates cell-death evolves, these apoptotic defects have fallen into the crosshairs of cancer drug developers. The issues raised in exploiting these alterations for therapeutic benefit are discussed.
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Apoptosis , Neoplasias/patología , Animales , Humanos , Transducción de SeñalRESUMEN
To identify genes that mediate transforming growth factor-beta (TGF-beta) signaling, a colorectal cancer cell line that was sensitive to the growth inhibitory effects of this cytokine was created. We then determined the global gene expression profiles of these cells, and those of HaCaT human keratinocytes, in the presence and absence of TGF-beta. Of the several genes identified in this screen, DEC1 was of particular note in light of the rapidity and consistency of its induction and its potential biochemical activities. We identified a consensus DNA-binding site for DEC1 and showed that DEC1 could repress the transcription of a reporter containing this binding site in its promoter. Finally, both alleles of the DEC1 locus in HaCaT cells were inactivated through targeted homologous recombination. This approach revealed that DEC1 induction was not required for the growth inhibition mediated by TGF-beta in this line. However, DEC1 may function in concert with other signaling components to mediate certain biologic effects of TGF-beta.