RESUMEN
BACKGROUND: Drugs targeting the spindle assembly checkpoint (SAC), such as inhibitors of Aurora kinase B (AURKB) and dual specific protein kinase TTK, are in different stages of clinical development. However, cell response to SAC abrogation is poorly understood and there are no markers for patient selection. METHODS: A panel of 53 tumor cell lines of different origins was used. The effects of drugs were analyzed by MTT and flow cytometry. Copy number status was determined by FISH and Q-PCR; mRNA expression by nCounter and RT-Q-PCR and protein expression by Western blotting. CRISPR-Cas9 technology was used for gene knock-out (KO) and a doxycycline-inducible pTRIPZ vector for ectopic expression. Finally, in vivo experiments were performed by implanting cultured cells or fragments of tumors into immunodeficient mice. RESULTS: Tumor cells and patient-derived xenografts (PDXs) sensitive to AURKB and TTK inhibitors consistently showed high expression levels of BH3-interacting domain death agonist (BID), while cell lines and PDXs with low BID were uniformly resistant. Gene silencing rendered BID-overexpressing cells insensitive to SAC abrogation while ectopic BID expression in BID-low cells significantly increased sensitivity. SAC abrogation induced activation of CASP-2, leading to cleavage of CASP-3 and extensive cell death only in presence of high levels of BID. Finally, a prevalence study revealed high BID mRNA in 6% of human solid tumors. CONCLUSIONS: The fate of tumor cells after SAC abrogation is driven by an AURKB/ CASP-2 signaling mechanism, regulated by BID levels. Our results pave the way to clinically explore SAC-targeting drugs in tumors with high BID expression.
Asunto(s)
Neoplasias , Proteínas Serina-Treonina Quinasas , Humanos , Animales , Ratones , Proteínas Serina-Treonina Quinasas/genética , Aurora Quinasa B/genética , Aurora Quinasa B/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Línea Celular Tumoral , ARN Mensajero , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas de Ciclo Celular/genéticaRESUMEN
Activated B-cell-like (ABC) and germinal center B-cell-like diffuse large B-cell lymphoma (DLBCL) represent the 2 major molecular DLBCL subtypes. They are characterized by differences in clinical course and by divergent addiction to oncogenic pathways. To determine activity of novel compounds in these 2 subtypes, we conducted an unbiased pharmacologic in vitro screen. The phosphatidylinositol-3-kinase (PI3K) α/δ (PI3Kα/δ) inhibitor AZD8835 showed marked potency in ABC DLBCL models, whereas the protein kinase B (AKT) inhibitor AZD5363 induced apoptosis in PTEN-deficient DLBCLs irrespective of their molecular subtype. These in vitro results were confirmed in various cell line xenograft and patient-derived xenograft mouse models in vivo. Treatment with AZD8835 induced inhibition of nuclear factor κB signaling, prompting us to combine AZD8835 with the Bruton's tyrosine kinase inhibitor ibrutinib. This combination was synergistic and effective both in vitro and in vivo. In contrast, the AKT inhibitor AZD5363 was effective in PTEN-deficient DLBCLs through downregulation of the oncogenic transcription factor MYC. Collectively, our data suggest that patients should be stratified according to their oncogenic dependencies when treated with PI3K and AKT inhibitors.
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Antineoplásicos/farmacología , Regulación Neoplásica de la Expresión Génica , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Oxadiazoles/farmacología , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Adenina/análogos & derivados , Agammaglobulinemia Tirosina Quinasa , Animales , Apoptosis/efectos de los fármacos , Combinación de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Humanos , Linfoma de Células B Grandes Difuso/clasificación , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Ratones , FN-kappa B/antagonistas & inhibidores , FN-kappa B/genética , FN-kappa B/metabolismo , Especificidad de Órganos , Fosfohidrolasa PTEN/deficiencia , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Starting from potent inhibitors of PI3Kα having poor general kinase selectivity (e.g., 1 and 2), optimisation of this series led to the identification of 25, a potent inhibitor of PI3Kα (wild type, E545K and H1047R mutations) and PI3Kδ, selective versus PI3Kß and PI3Kγ, with excellent general kinase selectivity. Compound 25 displayed low metabolic turnover and suitable physical properties for oral administration. In vivo, compound 25 showed pharmacodynamic modulation of AKT phosphorylation and near complete inhibition of tumour growth (93% tumour growth inhibition) in a murine H1047R PI3Kα mutated SKOV-3 xenograft tumour model after chronic oral administration at 25mg/kg b.i.d. Compound 25, also known as AZD8835, is currently in phase I clinical trials.
Asunto(s)
Antineoplásicos/farmacología , Oxadiazoles/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Perros , Humanos , Ratones , Ratones Desnudos , Ratones SCID , Simulación del Acoplamiento Molecular , Oxadiazoles/síntesis química , Piperidinas/síntesis química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Ratas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cancerous stroma coevolves alongside tumour progression, thereby promoting the malignant conversion of epithelial carcinoma cells. To date, an abundance of data have supported crucial roles of the tumour microenvironment (TME) in providing cancer cells with proliferative, migratory, survival and invasive propensities favouring the processes of tumourigenesis. The cancerous reactive stroma is frequently populated by a large number of myofibroblasts (MFs), which are activated, non-transformed fibroblasts expressing α-smooth muscle actin (α-SMA). MFs together with non-MF cells present in the tumour-associated stroma are collectively referred to as carcinoma-associated fibroblasts (CAFs), one of the major stromal cell types recognised in various human carcinomas. Recruitment of fibroblasts and/or their progenitors to a tumour mass and their subsequent transdifferentiation into MFs, as well as ongoing maintenance of their activated state, are believed to be essential processes facilitating tumour progression. However, the complex networks of signalling pathways mediating the phenotypic conversion into CAFs, as well as those underlying their tumour-promoting interactions with other tumour-constituting cells, have yet to be fully explored. Histopathological confirmation of the presence of large numbers of CAF MFs within TME and their altered gene expression profiles are known to be associated with disease progression and to serve as independent negative prognostic factors for a wide range of tumour types. In this review, we examine the current evidence shedding light on the emerging roles of tumour-promoting CAFs, cells that are pivotal for epithelial cancer development and progression, and discuss the therapeutic potential of targeting these cells.
Asunto(s)
Carcinoma/patología , Fibroblastos/patología , Células Madre Mesenquimatosas/patología , Microambiente Tumoral , Animales , Carcinoma/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Fibroblastos/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patologíaRESUMEN
PURPOSE: Therapeutic resistance to frontline therapy develops rapidly in small cell lung cancer (SCLC). Treatment options are also limited by the lack of targetable driver mutations. Therefore, there is an unmet need for developing better therapeutic strategies and biomarkers of response. Aurora kinase B (AURKB) inhibition exploits an inherent genomic vulnerability in SCLC and is a promising therapeutic approach. Here, we identify biomarkers of response and develop rational combinations with AURKB inhibition to improve treatment efficacy. EXPERIMENTAL DESIGN: Selective AURKB inhibitor AZD2811 was profiled in a large panel of SCLC cell lines (n = 57) and patient-derived xenograft (PDX) models. Proteomic and transcriptomic profiles were analyzed to identify candidate biomarkers of response and resistance. Effects on polyploidy, DNA damage, and apoptosis were measured by flow cytometry and Western blotting. Rational drug combinations were validated in SCLC cell lines and PDX models. RESULTS: AZD2811 showed potent growth inhibitory activity in a subset of SCLC, often characterized by, but not limited to, high cMYC expression. Importantly, high BCL2 expression predicted resistance to AURKB inhibitor response in SCLC, independent of cMYC status. AZD2811-induced DNA damage and apoptosis were suppressed by high BCL2 levels, while combining AZD2811 with a BCL2 inhibitor significantly sensitized resistant models. In vivo, sustained tumor growth reduction and regression was achieved even with intermittent dosing of AZD2811 and venetoclax, an FDA-approved BCL2 inhibitor. CONCLUSIONS: BCL2 inhibition overcomes intrinsic resistance and enhances sensitivity to AURKB inhibition in SCLC preclinical models.
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Antineoplásicos , Aurora Quinasa B , Neoplasias Pulmonares , Proteínas Proto-Oncogénicas c-bcl-2 , Carcinoma Pulmonar de Células Pequeñas , Humanos , Antineoplásicos/uso terapéutico , Apoptosis , Aurora Quinasa B/antagonistas & inhibidores , Línea Celular Tumoral , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Proteómica , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
FGFs have traditionally been associated with cell proliferation, morphogenesis, and development; yet, a subfamily of FGFs (FGF19, -21, and -23) functions as hormones to regulate glucose, lipid, phosphate, and vitamin D metabolism with impact on energy balance and aging. In mammals, Klotho and beta-Klotho are type 1 transmembrane proteins that function as obligatory co-factors for endocrine FGFs to bind to their cognate FGF receptors (FGFRs). Mutations in Klotho/beta-Klotho or fgf19, -21, or -23 are associated with a number of human diseases, including autosomal dominant hypophosphatemic rickets, premature aging disorders, and diabetes. The Caenorhabditis elegans genome contains two paralogues of Klotho/beta-Klotho, klo-1, and klo-2. klo-1 is expressed in the C. elegans excretory canal, which is structurally and functionally paralogous to the vertebrate kidney. KLO-1 associates with EGL-15/FGFR, suggesting a role for KLO-1 in the fluid homeostasis phenotype described previously for egl-15/fgfr mutants. Altered levels of EGL-15/FGFR signaling lead to defects in excretory canal development and function in C. elegans. These results suggest an evolutionarily conserved function for the FGFR-Klotho complex in the development of excretory organs such as the mammalian kidney and the worm excretory canal. These results also suggest an evolutionarily conserved function for the FGFR-Klotho axis in metabolic regulation.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Glucuronidasa/metabolismo , Homeostasis/fisiología , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Evolución Molecular , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/metabolismo , Glucuronidasa/genética , Humanos , Proteínas Klotho , Mutación , Receptores de Factores de Crecimiento de Fibroblastos/genéticaRESUMEN
Drug-induced cytopenias are a prevalent and significant issue that worsens clinical outcomes and hinders the effective treatment of cancer. While reductions in blood cell numbers are classically associated with traditional cytotoxic chemotherapies, they also occur with newer targeted small molecules and the factors that determine the hematotoxicity profiles of oncologic drugs are not fully understood. Here, we explore why some Aurora kinase inhibitors cause preferential neutropenia. By studying drug responses of healthy human hematopoietic cells in vitro and analyzing existing gene expression datasets, we provide evidence that the enhanced vulnerability of neutrophil-lineage cells to Aurora kinase inhibition is caused by early developmental changes in ATP-binding cassette (ABC) transporter expression. These data show that hematopoietic cell-intrinsic expression of ABC transporters may be an important factor that determines how some Aurora kinase inhibitors affect the bone marrow.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Neutrófilos , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato , Aurora Quinasas/metabolismo , Hematopoyesis/genética , Humanos , Proteínas de Neoplasias/metabolismo , Neutrófilos/metabolismo , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
PURPOSE: PARP inhibitors (PARPi) induce synthetic lethality in homologous recombination repair (HRR)-deficient tumors and are used to treat breast, ovarian, pancreatic, and prostate cancers. Multiple PARPi resistance mechanisms exist, most resulting in restoration of HRR and protection of stalled replication forks. ATR inhibition was highlighted as a unique approach to reverse both aspects of resistance. Recently, however, a PARPi/WEE1 inhibitor (WEE1i) combination demonstrated enhanced antitumor activity associated with the induction of replication stress, suggesting another approach to tackling PARPi resistance. EXPERIMENTAL DESIGN: We analyzed breast and ovarian patient-derived xenoimplant models resistant to PARPi to quantify WEE1i and ATR inhibitor (ATRi) responses as single agents and in combination with PARPi. Biomarker analysis was conducted at the genetic and protein level. Metabolite analysis by mass spectrometry and nucleoside rescue experiments ex vivo were also conducted in patient-derived models. RESULTS: Although WEE1i response was linked to markers of replication stress, including STK11/RB1 and phospho-RPA, ATRi response associated with ATM mutation. When combined with olaparib, WEE1i could be differentiated from the ATRi/olaparib combination, providing distinct therapeutic strategies to overcome PARPi resistance by targeting the replication stress response. Mechanistically, WEE1i sensitivity was associated with shortage of the dNTP pool and a concomitant increase in replication stress. CONCLUSIONS: Targeting the replication stress response is a valid therapeutic option to overcome PARPi resistance including tumors without an underlying HRR deficiency. These preclinical insights are now being tested in several clinical trials where the PARPi is administered with either the WEE1i or the ATRi.
Asunto(s)
Antineoplásicos , Neoplasias Ováricas , Antineoplásicos/uso terapéutico , Proteínas de la Ataxia Telangiectasia Mutada , Proteína BRCA1/genética , Biomarcadores , Carcinoma Epitelial de Ovario/tratamiento farmacológico , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Femenino , Humanos , Nucleósidos/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Ftalazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismoRESUMEN
The regulation of cell function by fibroblast growth factors (FGFs) classically occurs through a dual receptor system of a tyrosine kinase receptor (FGFR) and a heparan sulfate proteoglycan co-receptor. Mutations in some consensus N-glycosylation sites in human FGFR result in skeletal disorders and craniosynostosis syndromes, and biophysical studies in vitro suggest that N-glycosylation of FGFR alters ligand and heparan sulfate binding properties. The evolutionarily conserved FGFR signaling system of Caenorhabditis elegans has been used to assess the role of N-glycosylation in the regulation of FGFR signaling in vivo. The C. elegans FGF receptor, EGL-15, is N-glycosylated in vivo, and genetic substitution of specific consensus N-glycosylation sites leads to defects in the maintenance of fluid homeostasis and differentiation of sex muscles, both of which are phenotypes previously associated with hyperactive EGL-15 signaling. These phenotypes are suppressed by hypoactive mutations in EGL-15 downstream signaling components or activating mutations in the phosphatidylinositol 3-kinase pathway, respectively. The results show that N-glycans negatively regulate FGFR activity in vivo supporting the notion that mutation of N-glycosylation sites in human FGFR may lead to inappropriate activation of the receptor.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Sitios de Unión/genética , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Diferenciación Celular , Femenino , Glicosilación , Humanos , Masculino , Microscopía Fluorescente , Datos de Secuencia Molecular , Músculo Liso/citología , Músculo Liso/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Mioblastos/citología , Mioblastos/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Homología de Secuencia de AminoácidoRESUMEN
Tumours defective in the DNA homologous recombination repair pathway can be effectively treated with poly (ADP-ribose) polymerase (PARP) inhibitors; these have proven effective in clinical trials in patients with BRCA gene function-defective cancers. However, resistance observed in both pre-clinical and clinical studies is likely to impact on this treatment strategy. Over-expression of phosphoglycoprotein (P-gp) has been previously suggested as a mechanism of resistance to the PARP inhibitor olaparib in mouse models of Brca1/2-mutant breast cancer. Here, we report that in a Brca2 model treated with olaparib, P-gp upregulation is observed but is not sufficient to confer resistance. Furthermore, resistant/relapsed tumours do not show substantial changes in PK/PD of olaparib, do not downregulate PARP1 or re-establish double stranded DNA break repair by homologous recombination, all previously suggested as mechanisms of resistance. However, resistance is strongly associated with epithelial-mesenchymal transition (EMT) and treatment-naïve tumours given a single dose of olaparib upregulate EMT markers within one hour. Therefore, in this model, olaparib resistance is likely a product of an as-yet unidentified mechanism associated with rapid transition to the mesenchymal phenotype.
RESUMEN
Loss of the tumor suppressor PTEN confers a tumor cell dependency on the PI3Kß isoform. Achieving maximal inhibition of tumor growth through PI3K pathway inhibition requires sustained inhibition of PI3K signaling; however, efficacy is often limited by suboptimal inhibition or reactivation of the pathway. To select combinations that deliver comprehensive suppression of PI3K signaling in PTEN-null tumors, the PI3Kß inhibitor AZD8186 was combined with inhibitors of kinases implicated in pathway reactivation in an extended cell proliferation assay. Inhibiting PI3Kß and mTOR gave the most effective antiproliferative effects across a panel of PTEN-null tumor cell lines. The combination of AZD8186 and the mTOR inhibitor vistusertib was also effective in vivo controlling growth of PTEN-null tumor models of TNBC, prostate, and renal cancers. In vitro, the combination resulted in increased suppression of pNDRG1, p4EBP1, as well as HMGCS1 with reduced pNDRG1 and p4EBP1 more closely associated with effective suppression of proliferation. In vivo biomarker analysis revealed that the monotherapy and combination treatment consistently reduced similar biomarkers, while combination increased nuclear translocation of the transcription factor FOXO3 and reduction in glucose uptake. These data suggest that combining the PI3Kß inhibitor AZD8186 and vistusertib has potential to be an effective combination treatment for PTEN-null tumors. Mol Cancer Ther; 17(11); 2309-19. ©2018 AACR.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias/patología , Fosfohidrolasa PTEN/deficiencia , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Compuestos de Anilina/farmacología , Animales , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Cromonas/farmacología , Femenino , Fluorodesoxiglucosa F18/farmacocinética , Proteína Forkhead Box O3/metabolismo , Glucosa/metabolismo , Humanos , Ratones Desnudos , Neoplasias/enzimología , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The cyclin dependent kinase (CDK)-retinoblastoma (RB)-E2F pathway plays a critical role in the control of cell cycle in estrogen receptor-positive (ER+) breast cancer. Small-molecule inhibitors of CDK4/6 have shown promise in this tumor type in combination with hormonal therapies, reflecting the particular dependence of this subtype of cancer on cyclin D1 and E2F transcription factors. mTOR inhibitors have also shown potential in clinical trials in this disease setting. Recent data have suggested cooperation between the PI3K/mTOR pathway and CDK4/6 inhibition in preventing early adaptation and eliciting growth arrest, but the mechanisms of the interplay between these pathways have not been fully elucidated. Here we show that profound and durable inhibition of ER+ breast cancer growth is likely to require multiple hits on E2F-mediated transcription. We demonstrate that inhibition of mTORC1/2 does not affect ER function directly, but does cause a decrease in cyclin D1 protein, RB phosphorylation, and E2F-mediated transcription. Combination of an mTORC1/2 inhibitor with a CDK4/6 inhibitor results in more profound effects on E2F-dependent transcription, which translates into more durable growth arrest and a delay in the onset of resistance. Combined inhibition of mTORC1/2, CDK4/6, and ER delivers even more profound and durable regressions in breast cancer cell lines and xenografts. Furthermore, we show that CDK4/6 inhibitor-resistant cell lines reactivate the CDK-RB-E2F pathway, but remain sensitive to mTORC1/2 inhibition, suggesting that mTORC1/2 inhibitors may represent an option for patients that have relapsed on CDK4/6 therapy. Mol Cancer Ther; 17(5); 908-20. ©2018 AACR.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Factores de Transcripción E2F/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Benzamidas , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Factores de Transcripción E2F/metabolismo , Femenino , Humanos , Células MCF-7 , Ratones SCID , Morfolinas/administración & dosificación , Piperazinas/administración & dosificación , Piridinas/administración & dosificación , Pirimidinas , Receptores de Estrógenos/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
BACKGROUND: The phosphatidyl inositol 3 kinase (PI3K), AKT and mammalian target of rapamycin (mTOR) signal transduction pathway is frequently de-regulated and activated in human cancer and is an important therapeutic target. AZD8835 is a PI3K inhibitor, with selectivity against PI3K α and δ isoforms, which is currently in Phase 1 clinical trials. 18F-Fluoro-deoxy-glucose positron emission tomography (18F-FDG PET) is a non-invasive pharmacodynamic imaging biomarker that has become an integral part of drug development. It has been used widely with PI3K inhibitors both clinically and pre-clinically because of the role of the PI3K pathway in glucose metabolism. In this study we investigated the potential of 18F-FDG PET as a non-invasive pharmacodynamic biomarker for AZD8835. We sought to understand if 18F-FDG PET could determine the minimally effective dose of AZD8835 and correlate with other pharmacodynamic biomarkers for validation of its use in clinical development. 18F-FDG PET scans were performed in nude mice in the BT474C breast xenograft model. Mice were fasted prior to imaging and static 18F-FDG PET was performed. Treatment groups received AZD8835 by oral gavage at a dose volume of 10ml/kg. Treatment groups received either 3, 6, 12.5, 25 or 50mg/kg AZD8835. Tumour growth was monitored throughout the study, and at the end of the imaging procedure, tumours were taken and a full pharmacodynamic analysis was performed. RESULTS: Results showed that AZD8835 reduced 18F-FDG uptake at a dose of 12.5, 25 and 50mg/kg with no significant reduction at doses of 3 and 6mg/kg. These results were consistent with other pharmacodynamics biomarkers measured and show 18F-FDG PET as a sensitive biomarker with the ability to determine the minimal effective dose of AZD8835. CONCLUSIONS: Our pre-clinical studies support the use of 18F-FDG PET imaging as a sensitive and non- invasive pharmacodynamic biomarker (understanding the role of PI3K signalling in glucose uptake) for AZD8835 with a decrease in 18F-FDG uptake observed at only two hours post treatment. The decrease in 18F-FDG uptake was dose dependent and data showed excellent PK/PD correlation. This data supports and parallels observations obtained with this class of compounds in patients.
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Fluorodesoxiglucosa F18/metabolismo , Oxadiazoles/farmacología , Oxadiazoles/farmacocinética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Piperidinas/farmacología , Piperidinas/farmacocinética , Tomografía de Emisión de Positrones/métodos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/farmacocinética , Animales , Biomarcadores de Tumor/metabolismo , Glucemia/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , Técnicas de Silenciamiento del Gen , Homeostasis/efectos de los fármacos , Humanos , Ratones Desnudos , Oxadiazoles/administración & dosificación , Fosfatidilinositol 3-Quinasas/metabolismo , Piperidinas/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Purpose: PTEN-null tumors become dependent on the PI3Kß isoform and can be targeted by molecules such as the selective PI3Kß inhibitor AZD8186. However, beyond the modulation of the canonical PI3K pathway, the consequences of inhibiting PI3Kß are poorly defined.Experimental Design: To determine the broader impact of AZD8186 in PTEN-null tumors, we performed a genome-wide RNA-seq analysis of PTEN-null triple-negative breast tumor xenografts treated with AZD8186. Mechanistic consequences of AZD8186 treatment were examined across a number of PTEN-null cell lines and tumor models.Results: AZD8186 treatment resulted in modification of transcript and protein biomarkers associated with cell metabolism. We observed downregulation of cholesterol biosynthesis genes and upregulation of markers associated with metabolic stress. Downregulation of cholesterol biosynthesis proteins, such as HMGCS1, occurred in PTEN-null cell lines and tumor xenografts sensitive to AZD8186. Therapeutic inhibition of PI3Kß also upregulated PDHK4 and increased PDH phosphorylation, indicative of reduced carbon flux into the TCA cycle. Consistent with this, metabolomic analysis revealed a number of changes in key carbon pathways, nucleotide, and amino acid biosynthesis.Conclusions: This study identifies novel mechanistic biomarkers of PI3Kß inhibition in PTEN-null tumors supporting the concept that targeting PI3Kß may exploit a metabolic dependency that contributes to therapeutic benefit in inducing cell stress. Considering these additional pathways will guide biomarker and combination strategies for this class of agents. Clin Cancer Res; 23(24); 7584-95. ©2017 AACR.
Asunto(s)
Compuestos de Anilina/administración & dosificación , Cromonas/administración & dosificación , Fosfatidilinositol 3-Quinasas Clase II/genética , Fosfohidrolasa PTEN/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Compuestos de Anilina/efectos adversos , Animales , Línea Celular Tumoral , Cromonas/efectos adversos , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Hidroximetilglutaril-CoA Sintasa/genética , Redes y Vías Metabólicas/genética , Ratones , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The phosphatidylinositol 3 kinase (PI3K) signalling pathway is frequently altered in human cancer and a promising therapeutic target. AZD8186 (AstraZeneca) is a PI3Kß/δ inhibitor, currently in phase 1 clinical trials. (18)F-fluorodeoxyglucose positron emission tomography ((18)F-FDG PET) is often used as a biomarker for inhibitors targeting the PI3K axis because of the association of this pathway with glucose metabolism. In this study, we assessed if (18)F-FDG PET could be used as a pharmacodynamic marker to monitor PI3Kß inhibition by AZD8186, and hence have potential as a clinical biomarker of PI3Kß pathway activation, and for patient selection. (18)F-FDG PET scans were performed in nude mice bearing 786-0 renal, U87-MG glioma, and BT474C breast xenograft models. Mice were fasted prior to imaging and static (18)F-FDG PET imaging was performed. Tumour growth was monitored throughout each study, and at the end of the imaging procedure, tumours were taken and a full pharmacodynamic analysis performed. RESULTS: Results showed that in PTEN null tumour xenograft models, 786-0 and U87-MG, the PI3Kß inhibitor AZD8186 reduces (18)F-FDG uptake at a dose of 50 mg/kg, the same dose which causes tumour inhibition, while it has no impact in a PI3Kα mutant tumour xenograft BT474C. Consistent with the change in (18)F-FDG uptake, AZD8186 also modulated AKT and associated glucose pathway biomarkers in the PTEN null tumour xenografts but not in PTEN wild-type tumours. CONCLUSIONS: Our pre-clinical studies support the use of (18)F-FDG PET imaging as a sensitive and non-invasive pharmacodynamic biomarker for use in clinical studies with AZD8186.
RESUMEN
The PIK3CA gene, encoding the p110α catalytic unit of PI3Kα, is one of the most frequently mutated oncogenes in human cancer. Hence, PI3Kα is a target subject to intensive efforts in identifying inhibitors and evaluating their therapeutic potential. Here, we report studies with a novel PI3K inhibitor, AZD8835, currently in phase I clinical evaluation. AZD8835 is a potent inhibitor of PI3Kα and PI3Kδ with selectivity versus PI3Kß, PI3Kγ, and other kinases that preferentially inhibited growth in cells with mutant PIK3CA status, such as in estrogen receptor-positive (ER(+)) breast cancer cell lines BT474, MCF7, and T47D (sub-µmol/L GI50s). Consistent with this, AZD8835 demonstrated antitumor efficacy in corresponding breast cancer xenograft models when dosed continuously. In addition, an alternative approach of intermittent high-dose scheduling (IHDS) was explored given our observations that higher exposures achieved greater pathway inhibition and induced apoptosis. Indeed, using IHDS, monotherapy AZD8835 was able to induce tumor xenograft regression. Furthermore, AZD8835 IHDS in combination with other targeted therapeutic agents further enhanced antitumor activity (up to 92% regression). Combination partners were prioritized on the basis of our mechanistic insights demonstrating signaling pathway cross-talk, with a focus on targeting interdependent ER and/or CDK4/6 pathways or alternatively a node (mTOR) in the PI3K-pathway, approaches with demonstrated clinical benefit in ER(+) breast cancer patients. In summary, AZD8835 IHDS delivers strong antitumor efficacy in a range of combination settings and provides a promising alternative to continuous dosing to optimize the therapeutic index in patients. Such schedules merit clinical evaluation. Mol Cancer Ther; 15(5); 877-89. ©2016 AACR.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Oxadiazoles/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Piperidinas/farmacología , Animales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Análisis por Conglomerados , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Perfilación de la Expresión Génica , Humanos , Isoenzimas , Ratones , Oxadiazoles/química , Piperidinas/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Efforts to apply nanotechnology in cancer have focused almost exclusively on the delivery of cytotoxic drugs to improve therapeutic index. There has been little consideration of molecularly targeted agents, in particular kinase inhibitors, which can also present considerable therapeutic index limitations. We describe the development of Accurin polymeric nanoparticles that encapsulate the clinical candidate AZD2811, an Aurora B kinase inhibitor, using an ion pairing approach. Accurins increase biodistribution to tumor sites and provide extended release of encapsulated drug payloads. AZD2811 nanoparticles containing pharmaceutically acceptable organic acids as ion pairing agents displayed continuous drug release for more than 1 week in vitro and a corresponding extended pharmacodynamic reduction of tumor phosphorylated histone H3 levels in vivo for up to 96 hours after a single administration. A specific AZD2811 nanoparticle formulation profile showed accumulation and retention in tumors with minimal impact on bone marrow pathology, and resulted in lower toxicity and increased efficacy in multiple tumor models at half the dose intensity of AZD1152, a water-soluble prodrug of AZD2811. These studies demonstrate that AZD2811 can be formulated in nanoparticles using ion pairing agents to give improved efficacy and tolerability in preclinical models with less frequent dosing. Accurins specifically, and nanotechnology in general, can increase the therapeutic index of molecularly targeted agents, including kinase inhibitors targeting cell cycle and oncogenic signal transduction pathways, which have to date proved toxic in humans.
Asunto(s)
Aurora Quinasas/antagonistas & inhibidores , Nanopartículas/química , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Aurora Quinasas/metabolismo , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Línea Celular Tumoral , Liberación de Fármacos , Femenino , Humanos , Masculino , Espectrometría de Masas , Ratones , Ratones SCID , Organofosfatos/química , Organofosfatos/farmacocinética , Organofosfatos/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Quinazolinas/química , Quinazolinas/farmacocinética , Quinazolinas/farmacología , Ratas Desnudas , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PI3K/AKT/mTOR signaling plays an important role in breast cancer. Its interaction with estrogen receptor (ER) signaling becomes more complex and interdependent with acquired endocrine resistance. Targeting mTOR combined with endocrine therapy has shown clinical utility; however, a negative feedback loop exists downstream of PI3K/AKT/mTOR. Direct blockade of AKT together with endocrine therapy may improve breast cancer treatment. AZD5363, a novel pan-AKT kinase catalytic inhibitor, was examined in a panel of ER(+) breast cancer cell lines (MCF7, HCC1428, T47D, ZR75.1) adapted to long-term estrogen deprivation (LTED) or tamoxifen (TamR). AZD5363 caused a dose-dependent decrease in proliferation in all cell lines tested (GI50 < 500 nmol/L) except HCC1428 and HCC1428-LTED. T47D-LTED and ZR75-LTED were the most sensitive of the lines (GI50 â¼ 100 nmol/L). AZD5363 resensitized TamR cells to tamoxifen and acted synergistically with fulvestrant. AZD5363 decreased p-AKT/mTOR targets leading to a reduction in ERα-mediated transcription in a context-specific manner and concomitant decrease in recruitment of ER and CREB-binding protein (CBP) to estrogen response elements located on the TFF1, PGR, and GREB1 promoters. Furthermore, AZD5363 reduced expression of cell-cycle-regulatory proteins. Global gene expression highlighted ERBB2-ERBB3, ERK5, and IGFI signaling pathways driven by MYC as potential feedback-loops. Combined treatment with AZD5363 and fulvestrant showed synergy in an ER(+) patient-derived xenograft and delayed tumor progression after cessation of therapy. These data support the combination of AZD5363 with fulvestrant as a potential therapy for breast cancer that is sensitive or resistant to E-deprivation or tamoxifen and that activated AKT is a determinant of response, supporting the need for clinical evaluation.
Asunto(s)
Antineoplásicos Hormonales/farmacología , Resistencia a Antineoplásicos , Estradiol/análogos & derivados , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Pirimidinas/farmacología , Pirroles/farmacología , Receptores de Estrógenos/metabolismo , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Análisis por Conglomerados , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Estradiol/farmacología , Femenino , Fulvestrant , Perfilación de la Expresión Génica , Humanos , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Activación Transcripcional , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
mTOR is an atypical serine threonine kinase involved in regulating major cellular functions, such as nutrients sensing, growth, and proliferation. mTOR is part of the multiprotein complexes mTORC1 and mTORC2, which have been shown to play critical yet functionally distinct roles in the regulation of cellular processes. Current clinical mTOR inhibitors only inhibit the mTORC1 complex and are derivatives of the macrolide rapamycin (rapalogs). Encouraging effects have been observed with rapalogs in estrogen receptor-positive (ER(+)) breast cancer patients in combination with endocrine therapy, such as aromatase inhibitors. AZD2014 is a small-molecule ATP competitive inhibitor of mTOR that inhibits both mTORC1 and mTORC2 complexes and has a greater inhibitory function against mTORC1 than the clinically approved rapalogs. Here, we demonstrate that AZD2014 has broad antiproliferative effects across multiple cell lines, including ER(+) breast models with acquired resistance to hormonal therapy and cell lines with acquired resistance to rapalogs. In vivo, AZD2014 induces dose-dependent tumor growth inhibition in several xenograft and primary explant models. The antitumor activity of AZD2014 is associated with modulation of both mTORC1 and mTORC2 substrates, consistent with its mechanism of action. In combination with fulvestrant, AZD2014 induces tumor regressions when dosed continuously or using intermittent dosing schedules. The ability to dose AZD2014 intermittently, together with its ability to block signaling from both mTORC1 and mTORC2 complexes, makes this compound an ideal candidate for combining with endocrine therapies in the clinic. AZD2014 is currently in phase II clinical trials.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Morfolinas/farmacología , Complejos Multiproteicos/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Benzamidas , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Esquema de Medicación , Estradiol/administración & dosificación , Estradiol/análogos & derivados , Estradiol/farmacología , Femenino , Fulvestrant , Células HEK293 , Humanos , Immunoblotting , Células MCF-7 , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Morfolinas/administración & dosificación , Morfolinas/química , Complejos Multiproteicos/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas , Receptores de Estrógenos/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Human carcinomas frequently exhibit significant stromal reactions such as the so-called "desmoplastic stroma" or "reactive stroma", which is characterised by the existence of large numbers of stromal cells and extracellular matrix proteins. Carcinoma-associated fibroblasts (CAFs), which are rich in activated fibroblast populations exemplified by myofibroblasts, are among the predominant cell types present within the tumour-associated stroma. Increased numbers of stromal myofibroblasts are often associated with high-grade malignancies with poor prognoses in humans. CAF myofibroblasts possess abilities to promote primary tumour development, growth and progression by stimulating the processes of neoangiogenesis as well as tumour cell proliferation, survival, migration and invasion. Moreover, it has been demonstrated that CAFs serve as a niche supporting the metastatic colonisation of disseminated carcinoma cells in distant organs. Their contribution to primary and secondary malignancies makes these fibroblasts a potential therapeutic target and they also appear to be relevant to the development of drug resistance and tumour recurrence. This review summarises our current knowledge of tumour-promoting CAFs and discusses the therapeutic feasibility of targeting these cells as well as disrupting heterotypic interactions with other cell types in tumours that may improve the efficacy of current anti-tumour therapies.