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Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.
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Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/genética , Animales , Dominio Catalítico/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Perros , Ensayos de Selección de Medicamentos Antitumorales/métodos , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-TransferidoraRESUMEN
BACKGROUND: Inhibition of kinases involved in the DNA damage response sensitizes cells to genotoxic agents by abrogating checkpoint-induced cell cycle arrest. CHK1 and WEE1 act in a pathway upstream of CDK1 to inhibit cell cycle progression in response to damaged DNA. Therapeutic targeting of either CHK1 or WEE1, in combination with chemotherapy, is under clinical evaluation. These studies examine the overlap and potential for synergy when CHK1 and WEE1 are inhibited in cancer cell models. METHODS: Small molecules MK-8776 and MK-1775 were used to selectively and potently inhibit CHK1 and WEE1, respectively. RESULTS: In vitro, the combination of MK-8776 and MK-1775 induces up to 50-fold more DNA damage than either MK-8776 or MK-1775 alone at a fixed concentration. This requires aberrant cyclin-dependent kinase activity but does not appear to be dependent on p53 status alone. Furthermore, DNA damage takes place primarily in S-phase cells, implying disrupted DNA replication. When dosed together, the combination of MK-8776 and MK-1775 induced more intense and more durable DNA damage as well as anti-tumor efficacy than either MK-8776 or MK-1775 dosed alone. DNA damage induced by the combination was detected in up to 40% of cells in a treated xenograft tumor model. CONCLUSIONS: These results highlight the roles of WEE1 and CHK1 in maintaining genomic integrity. Importantly, the strong synergy observed upon inhibition of both kinases suggests unique yet complimentary anti-tumor effects of WEE1 and CHK1 inhibition. This demonstration of DNA double strand breaks in the absence of a DNA damaging chemotherapeutic provides preclinical rationale for combining WEE1 and CHK1 inhibitors as a cancer treatment regimen.
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Bromodomain and extraterminal domain (BET) proteins help direct the differentiation of helper T cell subsets, but their role in activated T cell function has not been described in detail. In this study, we investigate various consequences of epigenetic perturbation in human T lymphocytes using MK-8628, a potent and highly selective inhibitor of BET proteins. MK-8628 reduces the expression of canonical transcripts directing the proliferation, activation, and effector function of T lymphocytes. Treatment with MK-8628 abolishes the expression of key cyclins required for cell cycle progression and induces G1 cell cycle arrest in TCR-activated lymphocytes. This antiproliferative phenotype partially results from T lymphocyte apoptosis, which is exacerbated by MK-8628. In naive and memory T cell subsets, MK-8628 antagonizes T cell activation and suppresses polyfunctional cytokine production. Collectively, our results describe potent immunosuppressive effects of BET inhibition on human T cell biology. These results have important implications for immune modulatory targeting of BET proteins in the settings of T cell-driven autoimmune inflammation.
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Acetanilidas/farmacología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Compuestos Heterocíclicos con 3 Anillos/farmacología , Inmunosupresores/farmacología , Proteínas/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citocinas/genética , Citocinas/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Glucólisis/efectos de los fármacos , Voluntarios Sanos , Humanos , Activación de Linfocitos/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
In cancer, autophagy is upregulated to promote cell survival and tumor growth during times of nutrient stress and can confer resistance to drug treatments. Several major signaling networks control autophagy induction, including the p53 tumor suppressor pathway. In response to DNA damage and other cellular stresses, p53 is stabilized and activated, while HDM2 binds to and ubiquitinates p53 for proteasome degradation. Thus blocking the HDM2-p53 interaction is a promising therapeutic strategy in cancer; however, the potential survival advantage conferred by autophagy induction may limit therapeutic efficacy. In this study, we leveraged an HDM2 inhibitor to identify kinases required for p53-dependent autophagy. Interestingly, we discovered that p53-dependent autophagy requires several kinases, including the myotonic dystrophy protein kinase-like alpha (MRCKα). MRCKα is a CDC42 effector reported to activate actin-myosin cytoskeletal reorganization. Overall, this study provides evidence linking MRCKα to autophagy and reveals additional insights into the role of kinases in p53-dependent autophagy.
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In response to stress, cancer cells generate nutrients and energy through a cellular recycling process called autophagy, which can promote survival and tumor progression. Accordingly, autophagy inhibition has emerged as a potential cancer treatment strategy. Inhibitors targeting ULK1, an essential and early autophagy regulator, have provided proof of concept for targeting this kinase to inhibit autophagy; however, these are limited individually in their potency, selectivity, or cellular activity. In this study, we report two small molecule ULK1 inhibitors, ULK-100 and ULK-101, and establish superior potency and selectivity over a noteworthy published inhibitor. Moreover, we show that ULK-101 suppresses autophagy induction and autophagic flux in response to different stimuli. Finally, we use ULK-101 to demonstrate that ULK1 inhibition sensitizes KRAS mutant lung cancer cells to nutrient stress. ULK-101 represents a powerful molecular tool to study the role of autophagy in cancer cells and to evaluate the therapeutic potential of autophagy inhibition.
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Constitutive NF-kappaB activity has emerged as an important cell survival component of physiological and pathological processes, including B-cell development. In B cells, constitutive NF-kappaB activity includes p50/c-Rel and p52/RelB heterodimers, both of which are critical for proper B-cell development. We previously reported that WEHI-231 B cells maintain constitutive p50/c-Rel activity via selective degradation of IkappaBalpha that is mediated by a proteasome inhibitor-resistant, now termed PIR, pathway. Here, we examined the mechanisms of PIR degradation by comparing it to the canonical pathway that involves IkappaB kinase-dependent phosphorylation and beta-TrCP-dependent ubiquitylation of the N-terminal signal response domain of IkappaBalpha. We found a distinct consensus sequence within this domain of IkappaBalpha for PIR degradation. Chimeric analyses of IkappaBalpha and IkappaBbeta further revealed that the ankyrin repeats of IkappaBalpha, but not IkappaBbeta, contained information necessary for PIR degradation, thereby explaining IkappaBalpha selectivity for the PIR pathway. Moreover, we found that PIR degradation of IkappaBalpha and constitutive p50/c-Rel activity in primary murine B cells were maintained in a manner different from B-cell-activating-factor-dependent p52/RelB regulation. Thus, our findings suggest that nonconventional PIR degradation of IkappaBalpha may play a physiological role in the development of B cells in vivo.
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Cisteína Endopeptidasas/metabolismo , Proteínas I-kappa B/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismo , Animales , Factor Activador de Células B , Quinasa I-kappa B , Proteínas I-kappa B/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Mutación , Fosforilación , Complejo de la Endopetidasa Proteasomal , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The metazoan cell cycle is driven by the timely and composite activities of cyclin-dependent kinases (CDKs). Among these, cyclin D- and cyclin E-dependent kinases phosphorylate the pRb family proteins during G(1) phase of the cell cycle and thereby advance cells beyond the restriction point. Increasing evidence suggests that cyclin D-dependent kinases might affect events other than Rb pathway-mediated entry into S phase, such as accumulation of cell mass. However, little is known about cyclin D activity toward Rb-independent pathway(s) or non-pRb substrates. In this article, we show that the tumor suppressor TSC2 is a cyclin D binding protein. Coexpression of cyclin D1-CDK4/6 in cultured cells leads to increased phosphorylation and decreased detection of both TSC2 and TSC1, and promotes the phosphorylation of the mTOR substrates, 4E-BP1 and S6K1, two key effectors of cell growth that are negatively regulated by the TSC1-TSC2 complex. At the cellular level, ectopic expression of cyclin D1 restores the cell size decrease caused by TSC1-TSC2 expression. Intriguingly, down-regulation of TSC proteins was also observed by the expression of a mutant cyclin D1 that is unable to bind to CDK4/6, or by the coexpression of cyclin D1 with either an INK4 inhibitor or with catalytically inactive CDK6, indicating that cyclin D may regulate TSC1-TSC2 independently of CDK4/6. Together, these observations suggest that mammalian D-type cyclins participate in cell growth control through negative regulation of TSC1-TSC2 function.
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Ciclina D1/metabolismo , Fase G1 , Regulación de la Expresión Génica , Proteína de Retinoblastoma/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Humanos , Riñón/metabolismo , Osteosarcoma/metabolismo , Osteosarcoma/patología , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis TuberosaRESUMEN
Deregulation of the transcription factor nuclear factor-kappaB (NF-kappaB) leading to its constitutive activation is frequently observed in human cancer. Because altered NF-kappaB activities often promote the survival of malignant cells, its inhibition is regarded as a promising anticancer strategy. Because activation of the latent cytoplasmic NF-kappaB complex can be induced by a wide variety of different stimuli, its deregulation may occur by an equally large number of distinct mechanisms. This diversity raises a conundrum in conceptualizing general approaches to attenuate NF-kappaB activity in cancer. Here, we provide evidence that inhibition of IkappaBalpha nuclear export is a viable target to generally abrogate constitutive NF-kappaB activity in different cancer cell types. We show that inhibition of IkappaBalpha nuclear export has an important course of events in cancer cells harboring constitutive NF-kappaB activity-an initial increase in the pool of stable nuclear NF-kappaB/IkappaBalpha complexes that leads to a reduction of constitutive NF-kappaB activity and subsequent induction of apoptosis. Importantly, similar effects on multiple different cancer cell types indicate that inhibition of nuclear export of IkappaBalpha leads to broad inhibition of constitutive NF-kappaB activation regardless of various deregulated, upstream events involved.
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Regulación Neoplásica de la Expresión Génica , Proteínas I-kappa B/metabolismo , FN-kappa B/metabolismo , Transporte Activo de Núcleo Celular , Animales , Apoptosis , Western Blotting , Ciclo Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Supervivencia Celular , Citoplasma/metabolismo , Fibroblastos/metabolismo , Humanos , Inmunohistoquímica , Inmunoprecipitación , Ratones , Ratones Transgénicos , Modelos Biológicos , Modelos Genéticos , Mutagénesis , Mutagénesis Sitio-Dirigida , Inhibidor NF-kappaB alfa , Neoplasias/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
Radiotherapy is commonly used to treat a variety of solid tumors but improvements in the therapeutic ratio are sorely needed. The aim of this study was to assess the Chk1 kinase inhibitor, MK-8776, for its ability to radiosensitize human tumor cells. Cells derived from NSCLC and HNSCC cancers were tested for radiosensitization by MK-8776. The ability of MK-8776 to abrogate the radiation-induced G2 block was determined using flow cytometry. Effects on repair of radiation-induced DNA double strand breaks (DSBs) were determined on the basis of rad51, γ-H2AX and 53BP1 foci. Clonogenic survival analyses indicated that MK-8776 radiosensitized p53-defective tumor cells but not lines with wild-type p53. Abrogation of the G2 block was evident in both p53-defective cells and p53 wild-type lines indicating no correlation with radiosensitization. However, only p53-defective cells entered mitosis harboring unrepaired DSBs. MK-8776 appeared to inhibit repair of radiation-induced DSBs at early times after irradiation. A comparison of MK-8776 to the wee1 inhibitor, MK-1775, suggested both similarities and differences in their activities. In conclusion, MK-8776 radiosensitizes tumor cells by mechanisms that include abrogation of the G2 block and inhibition of DSB repair. Our findings support the clinical evaluation of MK-8776 in combination with radiation.
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Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Proteína p53 Supresora de Tumor/fisiología , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Fase G2/efectos de la radiación , Histonas/análisis , Humanos , Pirimidinonas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Combination drug therapy is a widely used paradigm for managing numerous human malignancies. In cancer treatment, additive and/or synergistic drug combinations can convert weakly efficacious monotherapies into regimens that produce robust antitumor activity. This can be explained in part through pathway interdependencies that are critical for cancer cell proliferation and survival. However, identification of the various interdependencies is difficult due to the complex molecular circuitry that underlies tumor development and progression. Here, we present a high-throughput platform that allows for an unbiased identification of synergistic and efficacious drug combinations. In a screen of 22,737 experiments of 583 doublet combinations in 39 diverse cancer cell lines using a 4 by 4 dosing regimen, both well-known and novel synergistic and efficacious combinations were identified. Here, we present an example of one such novel combination, a Wee1 inhibitor (AZD1775) and an mTOR inhibitor (ridaforolimus), and demonstrate that the combination potently and synergistically inhibits cancer cell growth in vitro and in vivo This approach has identified novel combinations that would be difficult to reliably predict based purely on our current understanding of cancer cell biology. Mol Cancer Ther; 15(6); 1155-62. ©2016 AACR.
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Ensayos de Selección de Medicamentos Antitumorales/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Neoplasias Experimentales/tratamiento farmacológico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Ratones , Pirazoles/administración & dosificación , Pirazoles/farmacología , Pirimidinas/administración & dosificación , Pirimidinas/farmacología , Pirimidinonas , Distribución Aleatoria , Sirolimus/administración & dosificación , Sirolimus/análogos & derivados , Sirolimus/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Purpose AZD1775 is a WEE1 kinase inhibitor targeting G2 checkpoint control, preferentially sensitizing TP53-deficient tumor cells to DNA damage. This phase I study evaluated safety, tolerability, pharmacokinetics, and pharmacodynamics of oral AZD1775 as monotherapy or in combination with chemotherapy in patients with refractory solid tumors. Patients and Methods In part 1, patients received a single dose of AZD1775 followed by 14 days of observation. In part 2, patients received AZD1775 as a single dose (part 2A) or as five twice per day doses or two once per day doses (part 2B) in combination with one of the following chemotherapy agents: gemcitabine (1,000 mg/m2), cisplatin (75 mg/m2), or carboplatin (area under the curve, 5 mg/mLâ min). Skin biopsies were collected for pharmacodynamic assessments. TP53 status was determined retrospectively in archival tumor tissue. Results Two hundred two patients were enrolled onto the study, including nine patients in part 1, 43 in part 2A (including eight rollover patients from part 1), and 158 in part 2B. AZD1775 monotherapy given as single dose was well tolerated, and the maximum-tolerated dose was not reached. In the combination regimens, the most common adverse events consisted of fatigue, nausea and vomiting, diarrhea, and hematologic toxicity. The maximum-tolerated doses and biologically effective doses were established for each combination. Target engagement, as a predefined 50% pCDK1 reduction in surrogate tissue, was observed in combination with cisplatin and carboplatin. Of 176 patients evaluable for efficacy, 94 (53%) had stable disease as best response, and 17 (10%) achieved a partial response. The response rate in TP53-mutated patients (n = 19) was 21% compared with 12% in TP53 wild-type patients (n = 33). Conclusion AZD1775 was safe and tolerable as a single agent and in combination with chemotherapy at doses associated with target engagement.
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias/tratamiento farmacológico , Neoplasias/mortalidad , Pirazoles/administración & dosificación , Pirimidinas/administración & dosificación , Adulto , Anciano , Análisis de Varianza , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Carboplatino/administración & dosificación , Carboplatino/efectos adversos , Cisplatino/administración & dosificación , Cisplatino/efectos adversos , Desoxicitidina/administración & dosificación , Desoxicitidina/efectos adversos , Desoxicitidina/análogos & derivados , Supervivencia sin Enfermedad , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/epidemiología , Femenino , Humanos , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Neoplasias/patología , Pronóstico , Pirazoles/efectos adversos , Pirimidinas/efectos adversos , Pirimidinonas , Medición de Riesgo , Análisis de Supervivencia , Resultado del Tratamiento , GemcitabinaRESUMEN
Inducible activation of the transcription factor NF-kappaB (nuclear factor kappaB) is classically mediated by proteasomal degradation of its associated inhibitors, IkappaBalpha (inhibitory kappaBalpha) and IkappaBbeta. However, certain B-lymphocytes maintain constitutively nuclear NF-kappaB activity (a p50-c-Rel heterodimer) which is resistant to inhibition by proteasome inhibitors. This activity in the WEHI-231 B-cell line is associated with continual and preferential degradation of IkappaBalpha, which is also unaffected by proteasome inhibitors. Pharmacological studies indicated that there was a correlation between inhibition of IkappaBalpha degradation and constitutive p50-c-Rel activity. Domain analysis of IkappaBalpha by deletion mutagenesis demonstrated that an N-terminal 36-amino-acid sequence of IkappaBalpha represented an instability determinant for constitutive degradation. Moreover, domain grafting studies indicated that this sequence was sufficient to cause IkappaBbeta, but not chloramphenicol acetyltransferase, to be rapidly degraded in WEHI-231 B-cells. However, this sequence was insufficient to target IkappaBbeta to the non-proteasome degradation pathway, suggesting that there was an additional cis-element(s) in IkappaBalpha that was required for complete targeting. Nevertheless, the NF-kappaB pool associated with IkappaBbeta now became constitutively active by virtue of IkappaBbeta instability in these cells. These findings further support the notion that IkappaB instability governs the maintenance of constitutive p50-c-Rel activity in certain B-cells via a unique degradation pathway.
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Proteínas I-kappa B/fisiología , FN-kappa B/metabolismo , Proteínas de Neoplasias/fisiología , Animales , Calpaína/antagonistas & inhibidores , Calpaína/fisiología , Línea Celular Tumoral/metabolismo , Cisteína Endopeptidasas , Dimerización , Proteínas I-kappa B/química , Lipopolisacáridos/farmacología , Linfoma de Células B/patología , Sustancias Macromoleculares , Ratones , Complejos Multienzimáticos/antagonistas & inhibidores , Inhibidor NF-kappaB alfa , Subunidad p50 de NF-kappa B , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Proteínas Proto-Oncogénicas c-rel/metabolismo , Eliminación de Secuencia , Proteína bcl-XRESUMEN
PURPOSE: We determined the safety, pharmacokinetics, pharmacodynamics, and recommended phase II dose of MK-8776 (SCH 900776), a potent, selective checkpoint kinase 1 (Chk1) inhibitor, as monotherapy and in combination with gemcitabine in a first-in-human phase I clinical trial in patients with advanced solid tumor malignancies. PATIENTS AND METHODS: Forty-three patients were treated by intravenous infusion with MK-8776 at seven dose levels ranging from 10 to 150 mg/m(2) as monotherapy and then in combination with gemcitabine 800 mg/m(2) (part A, n = 26) or gemcitabine 1,000 mg/m(2) (part B, n = 17). Forty percent of patients had three or more prior treatment regimens, and one third of patients had previously received gemcitabine. RESULTS: As monotherapy, MK-8776 was well tolerated, with QTc prolongation (19%), nausea (16%), fatigue (14%), and constipation (14%) as the most frequent adverse effects. Combination therapy demonstrated a higher frequency of adverse effects, predominantly fatigue (63%), nausea (44%), decreased appetite (37%), thrombocytopenia (32%), and neutropenia (24%), as well as dose-related, transient QTc prolongation (17%). The median number of doses of MK-8776 administered was five doses, with relative dose-intensity of 0.96. Bioactivity was assessed by γ-H2AX ex vivo assay. Of 30 patients evaluable for response, two showed partial response, and 13 exhibited stable disease. CONCLUSION: MK-8776 was well tolerated as monotherapy and in combination with gemcitabine. Early evidence of clinical efficacy was observed. The recommended phase II dose is MK-8776 200 mg plus gemcitabine 1,000 mg/m(2) on days 1 and 8 of a 21-day cycle.
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Desoxicitidina/análogos & derivados , Neoplasias/tratamiento farmacológico , Proteínas Quinasas/metabolismo , Pirazoles/administración & dosificación , Pirazoles/uso terapéutico , Pirimidinas/administración & dosificación , Pirimidinas/uso terapéutico , Adenocarcinoma/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Estudios de Cohortes , Citarabina/administración & dosificación , Desoxicitidina/administración & dosificación , Femenino , Histonas/metabolismo , Humanos , Infusiones Intravenosas , Células K562 , Masculino , Melanoma/tratamiento farmacológico , Persona de Mediana Edad , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/uso terapéutico , Sarcoma/tratamiento farmacológico , Factores de Tiempo , GemcitabinaRESUMEN
PURPOSE: Wee1 regulates key DNA damage checkpoints, and in this study, the efficacy of the Wee1 inhibitor MK-1775 was evaluated in glioblastoma multiforme (GBM) xenograft models alone and in combination with radiation and/or temozolomide. EXPERIMENTAL DESIGN: In vitro MK-1775 efficacy alone and in combination with temozolomide, and the impact on DNA damage, was analyzed by Western blotting and γH2AX foci formation. In vivo efficacy was evaluated in orthotopic and heterotopic xenografts. Drug distribution was assessed by conventional mass spectrometry (MS) and matrix-assisted laser desorption/ionization (MALDI)-MS imaging. RESULTS: GBM22 (IC50 = 68 nmol/L) was significantly more sensitive to MK-1775 compared with five other GBM xenograft lines, including GBM6 (IC50 >300 nmol/L), and this was associated with a significant difference in pan-nuclear γH2AX staining between treated GBM22 (81% cells positive) and GBM6 (20% cells positive) cells. However, there was no sensitizing effect of MK-1775 when combined with temozolomide in vitro. In an orthotopic GBM22 model, MK-1775 was ineffective when combined with temozolomide, whereas in a flank model of GBM22, MK-1775 exhibited both single-agent and combinatorial activity with temozolomide. Consistent with limited drug delivery into orthotopic tumors, the normal brain to whole blood ratio following a single MK-1775 dose was 5%, and MALDI-MS imaging demonstrated heterogeneous and markedly lower MK-1775 distribution in orthotopic as compared with heterotopic GBM22 tumors. CONCLUSIONS: Limited distribution to brain tumors may limit the efficacy of MK-1775 in GBM.
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Barrera Hematoencefálica/metabolismo , Dacarbazina/análogos & derivados , Glioblastoma/metabolismo , Glioblastoma/patología , Pirazoles/farmacología , Pirimidinas/farmacología , Animales , Proteínas de Ciclo Celular/antagonistas & inhibidores , Daño del ADN/efectos de los fármacos , Dacarbazina/farmacocinética , Dacarbazina/farmacología , Modelos Animales de Enfermedad , Glioblastoma/tratamiento farmacológico , Glioblastoma/mortalidad , Humanos , Ratones , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/farmacocinética , Pirimidinas/farmacocinética , Pirimidinonas , Temozolomida , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Neuroblastoma is uniquely sensitive to single-agent inhibition of the DNA damage checkpoint kinase Chk1, leading us to examine downstream effectors of this pathway and identify mitotic regulator Wee1 as an additional therapeutic target in this disease. Wee1 was overexpressed in both neuroblastoma cell lines and high-risk patient tumors. Genetic or pharmacologic abrogation of Wee1 signaling results in marked cytotoxicity in 10 of 11 neuroblastoma cell lines with a median IC(50) of 300 nmol/L for the Wee1-selective small-molecule inhibitor MK-1775. Murine tumor lines derived from mice that were either heterozygous or homozygous for MycN were particularly sensitive to single-agent inhibition of Wee1 (IC(50)s of 160 and 62 nmol/L, respectively). Simultaneous pharmacologic inhibition of Chk1 and Wee1 acted in a synergistic fashion to further impede neuroblastoma cell growth in vitro, in a manner greater than the individual inhibitors either alone or combined with chemotherapy. Combination Chk1 and Wee1 inhibition also revealed in vivo efficacy in neuroblastoma xenografts. Taken together, our results show that neuroblastoma cells depend on Wee1 activity for growth and that inhibition of this kinase may serve as a therapeutic for patients with neuroblastoma.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proteínas de Ciclo Celular/antagonistas & inhibidores , Neuroblastoma/tratamiento farmacológico , Proteínas Nucleares/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Quinasas/efectos de los fármacos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/administración & dosificación , Pirimidinas/administración & dosificación , Animales , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Femenino , Humanos , Ratones , Ratones SCID , Pirimidinonas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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
Inhibition of the protein kinase WEE1 synergizes with chemotherapy in preclinical models and WEE1 inhibitors are being explored as potential cancer therapies. Here, we investigate the mechanism that underlies this synergy. We show that WEE1 inhibition forces S-phase-arrested cells directly into mitosis without completing DNA synthesis, resulting in highly abnormal mitoses characterized by dispersed chromosomes and disorganized bipolar spindles, ultimately resulting in mitotic exit with gross micronuclei formation and apoptosis. This mechanism of cell death is shared by CHK1 inhibitors, and combined WEE1 and CHK1 inhibition forces mitotic entry from S-phase in the absence of chemotherapy. We show that p53/p21 inactivation combined with high expression of mitotic cyclins and EZH2 predispose to mitotic entry during S-phase with cells reliant on WEE1 to prevent premature cyclin-dependent kinase (CDK)1 activation. These features are characteristic of aggressive breast, and other, cancers for which WEE1 inhibitor combinations represent a promising targeted therapy.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/uso terapéutico , Pirimidinas/uso terapéutico , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Ciclinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Proteína Potenciadora del Homólogo Zeste 2 , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones SCID , Mitosis/efectos de los fármacos , Complejo Represivo Polycomb 2 , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Pirazoles/farmacología , Pirimidinas/farmacología , Pirimidinonas , Quinolinas/farmacología , Quinolinas/uso terapéutico , Fase S/efectos de los fármacos , Tiazoles/farmacología , Tiazoles/uso terapéutico , Tiofenos/farmacología , Tiofenos/uso terapéutico , Factores de Transcripción/metabolismo , Carga Tumoral/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Urea/análogos & derivados , Urea/farmacología , Urea/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
The purpose of this study was to determine the capacity of MK-1775, a potent Wee-1 inhibitor, to abrogate the radiation-induced G(2) checkpoint arrest and modulate radiosensitivity in glioblastoma cell models and normal human astrocytes. The radiation-induced checkpoint response of established glioblastoma cell lines, glioblastoma neural stem (GNS) cells, and astrocytes were determined in vitro by flow cytometry and in vivo by mitosis-specific staining using immunohistochemistry. Mechanisms underlying MK-1775 radiosensitization were determined by mitotic catastrophe and γH2AX expression. Radiosensitivity was determined in vitro by the clonogenic assay and in vivo by tumor growth delay. MK-1775 abrogated the radiation-induced G(2) checkpoint and enhanced radiosensitivity in established glioblastoma cell lines in vitro and in vivo, without modulating radiation response in normal human astrocytes. MK-1775 appeared to attenuate the early-phase of the G(2) checkpoint arrest in GNS cell lines, although the arrest was not sustained and did not lead to increased radiosensitivity. These results show that MK-1775 can selectively enhance radiosensitivity in established glioblastoma cell lines. Further work is required to determine the role Wee-1 plays in checkpoint activation of GNS cells.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de la radiación , Glioblastoma/tratamiento farmacológico , Pirazoles/uso terapéutico , Pirimidinas/uso terapéutico , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular Tumoral , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Ratones , Ratones Desnudos , Terapia Molecular Dirigida , Proteínas Nucleares/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/farmacocinética , Pirazoles/farmacología , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Pirimidinonas , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/farmacocinética , Fármacos Sensibilizantes a Radiaciones/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/efectos de la radiación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Investigate the efficacy and pharmacodynamic effects of MK-1775, a potent Wee1 inhibitor, in both monotherapy and in combination with gemcitabine (GEM) using a panel of p53-deficient and p53 wild-type human pancreatic cancer xenografts. EXPERIMENTAL DESIGN: Nine individual patient-derived pancreatic cancer xenografts (6 with p53-deficient and 3 with p53 wild-type status) from the PancXenoBank collection at Johns Hopkins were treated with MK-1775, GEM, or GEM followed 24 hour later by MK-1775, for 4 weeks. Tumor growth rate/regressions were calculated on day 28. Target modulation was assessed by Western blotting and immunohistochemistry. RESULTS: MK-1775 treatment led to the inhibition of Wee1 kinase and reduced inhibitory phosphorylation of its substrate Cdc2. MK-1775, when dosed with GEM, abrogated the checkpoint arrest to promote mitotic entry and facilitated tumor cell death as compared to control and GEM-treated tumors. MK-1775 monotherapy did not induce tumor regressions. However, the combination of GEM with MK-1775 produced robust antitumor activity and remarkably enhanced tumor regression response (4.01-fold) compared to GEM treatment in p53-deficient tumors. Tumor regrowth curves plotted after the drug treatment period suggest that the effect of the combination therapy is longer-lasting than that of GEM. None of the agents produced tumor regressions in p53 wild-type xenografts. CONCLUSIONS: These results indicate that MK-1775 selectively synergizes with GEM to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamiento farmacológico , Pirazoles/administración & dosificación , Pirimidinas/administración & dosificación , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular Tumoral , Desoxicitidina/administración & dosificación , Progresión de la Enfermedad , Sinergismo Farmacológico , Femenino , Genes p53 , Humanos , Ratones , Ratones Desnudos , Mutación/fisiología , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Proteínas Nucleares/antagonistas & inhibidores , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Pirimidinonas , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Tuberous sclerosis (TSC) is an autosomal dominant disease characterized by hamartoma formation in various organs and is caused by mutations targeting either the TSC1 or TSC2 genes. TSC1 and TSC2 proteins form a functionally interdependent dimeric complex. Phosphorylation of either TSC subunit by different kinases regulates the function of TSC and represents a major mechanism to integrate various signals into a centralized cell growth pathway. The majority of disease-associated mutations targeting either TSC1 or TSC2 results in a substantial decrease in protein level, suggesting that protein turnover also plays a critical role in TSC regulation. Here we report that TSC2 protein binds to FBW5, a DDB1-binding WD40 (DWD) protein, and is recruited by FBW5 to the DDB1-CUL4-ROC1 E3 ubiquitin ligase. Overexpression of FBW5 or CUL4A promotes TSC2 protein degradation, and this is abrogated by the coexpression of TSC1. Conversely, depletion of FBW5, DDB1, or CUL4A/B stabilizes TSC2. Ddb1 or Cul4 mutations in Drosophila result in Gigas/TSC2 protein accumulation and cause growth defects that can be partially rescued by Gigas/Tsc2 reduction. These results indicate that FBW5-DDB1-CUL4-ROC1 is an E3 ubiquitin ligase regulating TSC2 protein stability and TSC complex turnover.