RESUMEN
p21-activated kinase 1 (PAK1) has an important role in transducing signals in several oncogenic pathways. The concept of inhibiting this kinase has garnered significant interest over the past decade, particularly for targeting cancers associated with PAK1 amplification. Animal studies with the selective group I PAK (pan-PAK1, 2, 3) inhibitor G-5555 from the pyrido[2,3-d]pyrimidin-7-one class uncovered acute toxicity with a narrow therapeutic window. To attempt mitigating the toxicity, we introduced significant structural changes, culminating in the discovery of the potent pyridone side chain analogue G-9791. Mouse tolerability studies with this compound, other members of this series, and compounds from two structurally distinct classes revealed persistent toxicity and a correlation of minimum toxic concentrations and PAK1/2 mediated cellular potencies. Broad screening of selected PAK inhibitors revealed PAK1, 2, and 3 as the only overlapping targets. Our data suggest acute cardiovascular toxicity resulting from the inhibition of PAK2, which may be enhanced by PAK1 inhibition, and cautions against continued pursuit of pan-group I PAK inhibitors in drug discovery.
Asunto(s)
Enfermedades Cardiovasculares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Quinasas p21 Activadas/antagonistas & inhibidores , Enfermedad Aguda , Animales , Relación Dosis-Respuesta a Droga , Femenino , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Piridinas/síntesis química , Piridinas/química , Piridonas , Pirimidinas/síntesis química , Pirimidinas/química , Relación Estructura-Actividad , Quinasas p21 Activadas/metabolismoRESUMEN
INTRODUCTION: Breast cancer, the most common cause of cancer-related deaths worldwide among women, is a molecularly and clinically heterogeneous disease. Extensive genetic and epigenetic profiling of breast tumors has recently revealed novel putative driver genes, including p21-activated kinase (PAK)1. PAK1 is a serine/threonine kinase downstream of small GTP-binding proteins, Rac1 and Cdc42, and is an integral component of growth factor signaling networks and cellular functions fundamental to tumorigenesis. METHODS: PAK1 dysregulation (copy number gain, mRNA and protein expression) was evaluated in two cohorts of breast cancer tissues (n=980 and 1,108). A novel small molecule inhibitor, FRAX1036, and RNA interference were used to examine PAK1 loss of function and combination with docetaxel in vitro. Mechanism of action for the therapeutic combination, both cellular and molecular, was assessed via time-lapse microscopy and immunoblotting. RESULTS: We demonstrate that focal genomic amplification and overexpression of PAK1 are associated with poor clinical outcome in the luminal subtype of breast cancer (P=1.29×10(-4) and P=0.015, respectively). Given the role for PAK1 in regulating cytoskeletal organization, we hypothesized that combination of PAK1 inhibition with taxane treatment could be combined to further interfere with microtubule dynamics and cell survival. Consistent with this, administration of docetaxel with either a novel small molecule inhibitor of group I PAKs, FRAX1036, or PAK1 small interfering RNA oligonucleotides dramatically altered signaling to cytoskeletal-associated proteins, such as stathmin, and induced microtubule disorganization and cellular apoptosis. Live-cell imaging revealed that the duration of mitotic arrest mediated by docetaxel was significantly reduced in the presence of FRAX1036, and this was associated with increased kinetics of apoptosis. CONCLUSIONS: Taken together, these findings further support PAK1 as a potential target in breast cancer and suggest combination with taxanes as a viable strategy to increase anti-tumor efficacy.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Microtúbulos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Moduladores de Tubulina/farmacología , Quinasas p21 Activadas/antagonistas & inhibidores , Apoptosis/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN , Docetaxel , Sinergismo Farmacológico , Femenino , Amplificación de Genes , Expresión Génica , Humanos , Pronóstico , Transducción de Señal/efectos de los fármacos , Taxoides/farmacología , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismoRESUMEN
Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder characterized by the development of multiple tumors in the central nervous system, most notably schwannomas and meningiomas. Mutational inactivation of NF2 is found in 40-60% of sporadic meningiomas, but the molecular mechanisms underlying malignant changes of meningioma cells remain unclear. Because group I p21-activated kinases (Paks) bind to and are inhibited by the NF2-encoded protein Merlin, we assessed the signaling and anti-tumor effects of three group-I specific Pak inhibitors - Frax597, 716 and 1036 - in NF2-/- meningiomas in vitro and in an orthotopic mouse model. We found that these Pak inhibitors suppressed the proliferation and motility of both benign (Ben-Men1) and malignant (KT21-MG1) meningiomas cells. In addition, we found a strong reduction in phosphorylation of Mek and S6, and decreased cyclin D1 expression in both cell lines after treatment with Pak inhibitors. Using intracranial xenografts of luciferase-expressing KT21-MG1 cells, we found that treated mice showed significant tumor suppression for all three Pak inhibitors. Similar effects were observed in Ben-Men1 cells. Tumors dissected from treated animals exhibited an increase in apoptosis without notable change in proliferation. Collectively, these results suggest that Pak inhibitors might be useful agents in treating NF2-deficient meningiomas.
Asunto(s)
Meningioma/metabolismo , Neurofibromina 2/deficiencia , Quinasas p21 Activadas/metabolismo , Animales , Antineoplásicos/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Doxiciclina/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Immunoblotting , Meningioma/genética , Meningioma/terapia , Ratones SCID , Neurofibromina 2/genética , Piridonas/farmacología , Pirimidinas/farmacología , Interferencia de ARN , Tratamiento con ARN de Interferencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Quinasas p21 Activadas/antagonistas & inhibidores , Quinasas p21 Activadas/genéticaRESUMEN
Pancreatic adenocarcinoma (PDAC) is a major unmet medical need and a deeper understanding of molecular drivers is needed to advance therapeutic options for patients. We report here that p21-activated kinase 1 (PAK1) is a central node in PDAC cells downstream of multiple growth factor signalling pathways, including hepatocyte growth factor (HGF) and MET receptor tyrosine kinase. PAK1 inhibition blocks signalling to cytoskeletal effectors and tumour cell motility driven by HGF/MET. MET antagonists, such as onartuzumab and crizotinib, are currently in clinical development. Given that even highly effective therapies have resistance mechanisms, we show that combination with PAK1 inhibition overcomes potential resistance mechanisms mediated either by activation of parallel growth factor pathways or by direct amplification of PAK1. Inhibition of PAK1 attenuated in vivo tumour growth and metastasis in a model of pancreatic adenocarcinoma. In human tissues, PAK1 is highly expressed in a proportion of PDACs (33% IHC score 2 or 3; n = 304) and its expression is significantly associated with MET positivity (p < 0.0001) and linked to a widespread metastatic pattern in patients (p = 0.067). Taken together, our results provide evidence for a functional role of MET/PAK1 signalling in pancreatic adenocarcinoma and support further characterization of therapeutic inhibitors in this indication.
Asunto(s)
Adenocarcinoma/metabolismo , Movimiento Celular , Resistencia a Antineoplásicos/fisiología , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Quinasas p21 Activadas/metabolismo , Adenocarcinoma/patología , Animales , Anticuerpos Monoclonales/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Azetidinas/farmacología , Movimiento Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Inmunohistoquímica , Ratones , Neoplasias Pancreáticas/patología , Piperidinas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Although K-Ras, Cdc42, and PAK4 signaling are commonly deregulated in cancer, only a few studies have sought to comprehensively examine the spectrum of phosphorylation-mediated signaling downstream of each of these key signaling nodes. In this study, we completed a label-free quantitative analysis of oncogenic K-Ras, activated Cdc42, and PAK4-mediated phosphorylation signaling, and report relative quantitation of 2152 phosphorylated peptides on 1062 proteins. We define the overlap in phosphopeptides regulated by K-Ras, Cdc42, and PAK4, and find that perturbation of these signaling components affects phosphoproteins associated with microtubule depolymerization, cytoskeletal organization, and the cell cycle. These findings provide a resource for future studies to characterize novel targets of oncogenic K-Ras signaling and validate biomarkers of PAK4 inhibition.
Asunto(s)
Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Quinasas p21 Activadas/metabolismo , Animales , Ratones , Células 3T3 NIH , Fosfopéptidos/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Proteómica , Transducción de Señal , Quinasas p21 Activadas/genéticaRESUMEN
BACKGROUND: Although remarkable clinical response rates in melanoma have been observed using vemurafenib or dabrafenib in patients with tumors carrying oncogenic mutations in BRAF, a substantial unmet medical need remains for the subset of patients with wild-type BRAF tumors. METHODS: To investigate the role of p21-activated kinases (PAKs) in melanoma, we determined PAK1 genomic copy number and protein expression for a panel of human melanoma tissues. PAK1 was inhibited in vitro and in vivo using RNA interference or PF-3758309 inhibitor treatment in a panel of melanoma cell lines with known BRAF and RAS (rat sarcoma) genotype to better understand its role in melanoma cell proliferation and migration. Tumorigenesis was assessed in vivo in female NCR nude mice and analyzed with cubic spline regression and area under the curve analyses. All statistical tests were two-sided. RESULTS: Strong cytoplasmic PAK1 protein expression was prevalent in melanomas (27%) and negatively associated with activating mutation of the BRAF oncogene (P < .001). Focal copy number gain of PAK1 at 11q13 was also observed in 9% of melanomas (n = 87; copy number ≥ 2.5) and was mutually exclusive with BRAF mutation (P < .005). Selective PAK1 inhibition attenuated signaling through mitogen-activated protein kinase (MAPK) as well as cytoskeleton-regulating pathways to modulate the proliferation and migration of BRAF wild-type melanoma cells. Treatment of BRAF wild-type melanomas with PF-3758309 PAK inhibitor decreased tumor growth for SK-MEL23 and 537MEL xenografts (91% and 63% inhibition, respectively; P < .001) and MAPK pathway activation in vivo. CONCLUSIONS: Taken together, our results provide evidence for a functional role of PAK1 in BRAF wild-type melanoma and therapeutic use of PAK inhibitors in this indication.
Asunto(s)
Melanoma/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Quinasas p21 Activadas/efectos de los fármacos , Quinasas p21 Activadas/metabolismo , Animales , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Proliferación Celular , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Inmunoprecipitación , Ipilimumab , Melanoma/tratamiento farmacológico , Ratones , Ratones Desnudos , Proteínas Proto-Oncogénicas B-raf , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/metabolismo , Trasplante HeterólogoRESUMEN
The p21-activated kinases (PAKs) are central players in growth factor signaling networks and morphogenetic processes that control proliferation, cell polarity, invasion and actin cytoskeleton organization. This raises the possibility that interfering with PAK activity may produce significant anti-tumor activity. In this perspective, we summarize recent data concerning the contribution of the PAK family member, PAK1, in growth factor signaling and tumorigenesis. We further discuss mechanisms by which inhibition of PAK1 can arrest tumor growth and promote cell apoptosis, and the types of cancers in which PAK1 inhibition may hold promise.
Asunto(s)
Antineoplásicos/uso terapéutico , Descubrimiento de Drogas , Neoplasias/tratamiento farmacológico , Quinasas p21 Activadas/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/fisiología , Proliferación Celular/efectos de los fármacos , Descubrimiento de Drogas/métodos , Humanos , Modelos Biológicos , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinasas p21 Activadas/antagonistas & inhibidores , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismoRESUMEN
p21-activated kinases (PAKs) are serine/threonine protein kinases that serve as important mediators of Rac and Cdc42 GTPase function as well as pathways required for Ras-driven tumorigenesis. PAK1 has been implicated in signaling by growth factor receptors and morphogenetic processes that control cell polarity, invasion, and actin cytoskeleton organization. To better understand the role of PAK1 in tumorigenesis, PAK1 genomic copy number and expression were determined for a large panel of breast, lung, and head and neck tumors. PAK1 genomic amplification at 11q13 was prevalent in luminal breast cancer, and PAK1 protein expression was associated with lymph node metastasis. Breast cancer cells with PAK1 genomic amplification rapidly underwent apoptosis after inhibition of this kinase. Strong nuclear and cytoplasmic PAK1 expression was also prevalent in squamous nonsmall cell lung carcinomas (NSCLCs), and selective PAK1 inhibition was associated with delayed cell-cycle progression in vitro and in vivo. NSCLC cells were profiled using a library of pathway-targeted small-molecule inhibitors, and several synergistic combination therapies, including combination with antagonists of inhibitor of apoptosis proteins, were revealed for PAK1. Dual inhibition of PAK1 and X chromosome-linked inhibitor of apoptosis efficiently increased effector caspase activation and apoptosis of NSCLC cells. Together, our results provide evidence for dysregulation of PAK1 in breast and squamous NSCLCs and a role for PAK1 in cellular survival and proliferation in these indications.
Asunto(s)
Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacocinética , Quinasas p21 Activadas/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Activación Enzimática/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ganglios Linfáticos/enzimología , Ganglios Linfáticos/patología , Metástasis Linfática , Masculino , Ratones , Ratones Desnudos , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimología , Neoplasias/patología , Quinasas p21 Activadas/metabolismoRESUMEN
Apoptosis is important in controlling hematopoietic stem cell (HSC) numbers. However, the specific BCL-2 family member(s) that regulate HSC homeostasis are not precisely defined. We tested myeloid leukemia-1 (MCL-1) as an attractive candidate that is highly expressed in HSCs and regulated by growth factor signals. Inducible deletion of Mcl-1 in mice resulted in ablation of bone marrow. This resulted in the loss of early bone marrow progenitor populations, including HSCs. Moreover, growth factors including stem cell factor increased transcription of the Mcl-1 gene and required MCL-1 to augment survival of purified bone marrow progenitors. Deletion of Mcl-1 in other tissues, including liver, did not impair survival. Thus, MCL-1 is a critical and specific regulator essential for ensuring the homeostasis of early hematopoietic progenitors.
Asunto(s)
Células Madre Hematopoyéticas/fisiología , Proteínas de Neoplasias/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Animales , Apoptosis , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Recuento de Células , Linaje de la Célula , Forma de la Célula , Supervivencia Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Eliminación de Gen , Expresión Génica , Células Madre Hematopoyéticas/citología , Homeostasis , Interleucina-6/farmacología , Hígado/citología , Hígado/fisiología , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/genética , Poli I-C/farmacología , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Factor de Células Madre/farmacología , Transducción GenéticaRESUMEN
Regulated apoptosis is essential for both the development and the subsequent maintenance of the immune system. Interleukins, including IL-2, IL-4, IL-7 and IL-15, heavily influence lymphocyte survival during the vulnerable stages of VDJ rearrangement and later in ensuring cellular homeostasis, but the genes specifically responsible for the development and maintenance of lymphocytes have not been identified. The antiapoptotic protein MCL-1 is an attractive candidate, as it is highly regulated, appears to enhance short-term survival and functions at an apical step in genotoxic deaths. However, Mcl-1 deficiency results in peri-implantation lethality. Here we show that mice conditional for Mcl-1 display a profound reduction in B and T lymphocytes when MCL-1 is removed. Deletion of Mcl-1 during early lymphocyte differentiation increased apoptosis and arrested the development at pro-B-cell and double-negative T-cell stages. Induced deletion of Mcl-1 in peripheral B- and T-cell populations resulted in their rapid loss. Moreover, IL-7 both induced and required MCL-1 to mediate lymphocyte survival. Thus, MCL-1, which selectively inhibits the proapoptotic protein BIM, is essential both early in lymphoid development and later on in the maintenance of mature lymphocytes.
Asunto(s)
Apoptosis , Linfocitos B/citología , Linfocitos B/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Linfocitos T/citología , Linfocitos T/metabolismo , Alelos , Animales , Antígenos CD19/genética , Apoptosis/efectos de los fármacos , Sitios de Ligazón Microbiológica/genética , Linfocitos B/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/farmacología , Eliminación de Gen , Integrasas/genética , Integrasas/metabolismo , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/genética , Especificidad de Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Bazo/citología , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Linfocitos T/efectos de los fármacos , Timo/citología , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The proper expansion and contraction of hematopoietic cells requires tight regulation of cell death. BID, a "BH3-only" molecule, amplifies death receptor signals connecting the extrinsic to intrinsic pathways by triggering the mitochondrial pathway of apoptosis. Bid-deficient mice, as they age, spontaneously develop a myeloproliferative disorder, which progresses from myeloid hyperplasia to a fatal, clonal malignancy closely resembling chronic myelomonocytic leukemia (CMML). Thus, an apoptotic defect can result in myeloid leukemogenesis. Premalignant Bid-/- myeloid precursor cells are resistant to death receptor-induced apoptosis. Furthermore, a competitive reconstitution assay demonstrates that Bid-deficient long-term repopulating cells give rise to expanded myelomonocytic cells in vivo. Surprisingly, a single BH3-only molecule operating in the extrinsic death receptor pathway proved essential in vivo for physiologic cell death required to maintain myeloid homeostasis. Moreover, progression to CMML indicates that an upstream BH3-only molecule, BID, is required to suppress tumorigenesis.