RESUMEN
PURPOSE: The combination of targeting the CDK4/6 and estrogen receptor (ER) signaling pathways with palbociclib and fulvestrant is a proven therapeutic strategy for the treatment of ER-positive breast cancer. However, the poor physicochemical properties of fulvestrant require monthly intramuscular injections to patients, which limit the pharmacokinetic and pharmacodynamic activity of the compound. Therefore, an orally available compound that more rapidly reaches steady state may lead to a better clinical response in patients. Here, we report the identification of G1T48, a novel orally bioavailable, non-steroidal small molecule antagonist of ER. METHODS: The pharmacological effects and the antineoplastic mechanism of action of G1T48 on tumors was evaluated using human breast cancer cells (in vitro) and xenograft efficacy models (in vivo). RESULTS: G1T48 is a potent and efficacious inhibitor of estrogen-mediated transcription and proliferation in ER-positive breast cancer cells, similar to the pure antiestrogen fulvestrant. In addition, G1T48 can effectively suppress ER activity in multiple models of endocrine therapy resistance including those harboring ER mutations and growth factor activation. In vivo, G1T48 has robust antitumor activity in a model of estrogen-dependent breast cancer (MCF7) and significantly inhibited the growth of tamoxifen-resistant (TamR), long-term estrogen-deprived (LTED) and patient-derived xenograft tumors with an increased response being observed with the combination of G1T48 and the CDK4/6 inhibitor lerociclib. CONCLUSIONS: These data show that G1T48 has the potential to be an efficacious oral antineoplastic agent in ER-positive breast cancer.
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Anticuerpos Monoclonales Humanizados/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Anticuerpos Anti-VIH/farmacología , Neoplasias Hormono-Dependientes/tratamiento farmacológico , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Antagonistas de Estrógenos/farmacología , Femenino , Humanos , Ratones , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias Hormono-Dependientes/patología , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Estrógenos/metabolismo , Tamoxifeno/farmacología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Immune checkpoint blockade, exemplified by antibodies targeting the PD-1 receptor, can induce durable tumor regressions in some patients. To enhance the efficacy of existing immunotherapies, we screened for small molecules capable of increasing the activity of T cells suppressed by PD-1. Here, we show that short-term exposure to small-molecule inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) significantly enhances T-cell activation, contributing to antitumor effects in vivo, due in part to the derepression of NFAT family proteins and their target genes, critical regulators of T-cell function. Although CDK4/6 inhibitors decrease T-cell proliferation, they increase tumor infiltration and activation of effector T cells. Moreover, CDK4/6 inhibition augments the response to PD-1 blockade in a novel ex vivo organotypic tumor spheroid culture system and in multiple in vivo murine syngeneic models, thereby providing a rationale for combining CDK4/6 inhibitors and immunotherapies.Significance: Our results define previously unrecognized immunomodulatory functions of CDK4/6 and suggest that combining CDK4/6 inhibitors with immune checkpoint blockade may increase treatment efficacy in patients. Furthermore, our study highlights the critical importance of identifying complementary strategies to improve the efficacy of immunotherapy for patients with cancer. Cancer Discov; 8(2); 216-33. ©2017 AACR.See related commentary by Balko and Sosman, p. 143See related article by Jenkins et al., p. 196This article is highlighted in the In This Issue feature, p. 127.
Asunto(s)
Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Neoplasias/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Animales , Antineoplásicos/farmacología , Antineoplásicos Inmunológicos/farmacología , Línea Celular Tumoral , Humanos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Ratones , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Linfocitos T/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Conventional cytotoxic chemotherapy is highly effective in certain cancers but causes dose-limiting damage to normal proliferating cells, especially hematopoietic stem and progenitor cells (HSPCs). Serial exposure to cytotoxics causes a long-term hematopoietic compromise ("exhaustion"), which limits the use of chemotherapy and success of cancer therapy. We show that the coadministration of G1T28 (trilaciclib), which is a small-molecule inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6), contemporaneously with cytotoxic chemotherapy protects murine hematopoietic stem cells (HSCs) from chemotherapy-induced exhaustion in a serial 5-fluorouracil treatment model. Consistent with a cell-intrinsic effect, we show directly preserved HSC function resulting in a more rapid recovery of peripheral blood counts, enhanced serial transplantation capacity, and reduced myeloid skewing. When administered to healthy human volunteers, G1T28 demonstrated excellent in vivo pharmacology and transiently inhibited bone marrow (BM) HSPC proliferation. These findings suggest that the combination of CDK4/6 inhibitors with cytotoxic chemotherapy should provide a means to attenuate therapy-induced BM exhaustion in patients with cancer.
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Células Madre Hematopoyéticas/efectos de los fármacos , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/metabolismo , Femenino , Fluorouracilo/farmacología , Voluntarios Sanos , Células Madre Hematopoyéticas/citología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Inhibition of the p16INK4a/cyclin D/CDK4/6/RB pathway is an effective therapeutic strategy for the treatment of estrogen receptor positive (ER+) breast cancer. Although efficacious, current treatment regimens require a dosing holiday due to severe neutropenia potentially leading to an increased risk of infections, as well as tumor regrowth and emergence of drug resistance. Therefore, a next generation CDK4/6 inhibitor that can inhibit proliferation of CDK4/6-dependent tumors while minimizing neutropenia could reduce both the need for treatment holidays and the risk of inducing drug resistance.Here, we describe the preclinical characterization and development of G1T38; a novel, potent, selective, and orally bioavailable CDK4/6 inhibitor. In vitro, G1T38 decreased RB1 (RB) phosphorylation, caused a precise G1 arrest, and inhibited cell proliferation in a variety of CDK4/6-dependent tumorigenic cell lines including breast, melanoma, leukemia, and lymphoma cells. In vivo, G1T38 treatment led to equivalent or improved tumor efficacy compared to the first-in-class CDK4/6 inhibitor, palbociclib, in an ER+ breast cancer xenograft model. Furthermore, G1T38 accumulated in mouse xenograft tumors but not plasma, resulting in less inhibition of mouse myeloid progenitors than after palbociclib treatment. In larger mammals, this difference in pharmacokinetics allowed for 28 day continuous dosing of G1T38 in beagle dogs without producing severe neutropenia. These data demonstrate G1T38 has unique pharmacokinetic and pharmacodynamic properties, which result in high efficacy against CDK4/6 dependent tumors while minimizing the undesirable on-target bone marrow activity, thus potentially allowing G1T38 to be used as a continuous, daily oral antineoplastic agent.
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Antineoplásicos/farmacología , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Antineoplásicos/farmacocinética , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Ratones , Estructura Molecular , Fosfatidilinositol 3-Quinasas/metabolismo , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Piridinas/farmacología , Receptores de Estrógenos/metabolismo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chemotherapy-induced myelosuppression continues to represent the major dose-limiting toxicity of cytotoxic chemotherapy, which can be manifested as neutropenia, lymphopenia, anemia, and thrombocytopenia. As such, myelosuppression is the source of many of the adverse side effects of cancer treatment including infection, sepsis, bleeding, and fatigue, thus resulting in the need for hospitalizations, hematopoietic growth factor support, and transfusions (red blood cells and/or platelets). Moreover, clinical concerns raised by myelosuppression commonly lead to chemotherapy dose reductions, therefore limiting therapeutic dose intensity, and reducing the antitumor effectiveness of the treatment. Currently, the only course of treatment for myelosuppression is growth factor support which is suboptimal. These treatments are lineage specific, do not protect the bone marrow from the chemotherapy-inducing cytotoxic effects, and the safety and toxicity of each agent is extremely specific. Here, we describe the preclinical development of G1T28, a novel potent and selective CDK4/6 inhibitor that transiently and reversibly regulates the proliferation of murine and canine bone marrow hematopoietic stem and progenitor cells and provides multilineage protection from the hematologic toxicity of chemotherapy. Furthermore, G1T28 does not decrease the efficacy of cytotoxic chemotherapy on RB1-deficient tumors. G1T28 is currently in clinical development for the reduction of chemotherapy-induced myelosuppression in first- and second-line treatment of small-cell lung cancer. Mol Cancer Ther; 15(5); 783-93. ©2016 AACR.
Asunto(s)
Antineoplásicos/efectos adversos , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Mielopoyesis/efectos de los fármacos , Sustancias Protectoras/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Animales , Apoptosis/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Daño del ADN , Modelos Animales de Enfermedad , Perros , Evaluación Preclínica de Medicamentos , Femenino , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Ratones , Neoplasias/complicaciones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Sustancias Protectoras/química , Inhibidores de Proteínas Quinasas/química , Proteína de Retinoblastoma/deficienciaRESUMEN
BACKGROUND: Cyclin-dependent kinases (CDKs) regulate cell proliferation and coordinate the cell cycle checkpoint response to DNA damage. Although inhibitors with varying selectivity to specific CDK family members have been developed, selective CDK4/6 inhibitors have emerged as the most attractive antineoplastic agents because of the importance of CDK4/6 activity in regulating cell proliferation and the toxic effects associated with inhibition of other CDKs (eg, CDK1 and CDK2). METHODS: FVB/N wild-type mice (n = 13) were used to evaluate carboplatin-induced myelosuppression in bone marrow by complete blood cell counts after treatment with the CDK4/6 inhibitor PD0332991. Genetically engineered murine models of retinoblastoma (Rb)-competent (MMTV-c-neu) and Rb-incompetent (C3-TAg) breast cancer (n = 16 MMTV-c-neu mice in the carboplatin plus vehicle control group, n = 17 MMTV-c-neu mice in the carboplatin plus PD0332991 group, n = 17 C3-TAg mice in the carboplatin plus vehicle control group, and n = 14 C3-TAg mice in the carboplatin plus PD0332991 group) were used to investigate the antitumor activity of PD0332991 alone or in combination with chemotherapy. All statistical tests were two-sided. RESULTS: Coadministration of PD0332991 with carboplatin compared with carboplatin alone in FVB/N wild-type mice increased hematocrit (51.2% vs 33.5%, difference = 17.7%, 95% confidence interval [CI] = -26.7% to -8.6%, P < .001), platelet counts (1321 vs 758.5 thousand cells per µL, difference = 562.5 thousand cells per µL, 95% CI = -902.8 to -222.6, P = .002), myeloid cells (granulocytes and monocytes; 3.1 vs 1.6 thousand cells per µL, difference = 1.5 thousand cells per µL, 95% CI = -2.23 to -0.67, P < .001), and lymphocytes (7.9 vs 5.4 thousand cells per µL, difference = 2.5 thousand cells per µL, 95% CI = -4.75 to -0.18, P = .02). Daily administration of PD0332991 exhibited antitumor activity in MMTV-c-neu mice as a single agent. However, the combination of carboplatin plus PD0332991 decreased antitumor activity compared with carboplatin alone in Rb-competent mice (mean percent change in tumor volume at day 21 = -52.6% vs 3.7% for carboplatin and carboplatin plus PD0332991, respectively, difference = 56.3%, 95% CI = -109.0% to -3.6%, P = .04). In contrast, Rb-deficient tumors in C3-Tag mice were resistant to PD0332991, and coadministration of PD0332991 plus carboplatin had no effect on in vivo tumor growth (mean percent change in tumor volume at day 21 = 118.8% and 109.1% for carboplatin and carboplatin plus PD0332991, respectively, difference = 9.7%, 95% CI = -183.5% to 202.9%, P = .92). Finally, in tumor-bearing mice, coadministration of PD0332991 with carboplatin provided statistically significant protection of platelets (P = .04). CONCLUSION: We believe that the present data support a possible role for CDK4/6 inhibitors in a majority of patients with advanced cancer: to either inhibit tumor growth in CDK4/6-dependent tumors or ameliorate the dose-limiting toxicities of chemotherapy in CDK4/6-indepdendent tumors. Our data also suggest CDK4/6 inhibitors should not be combined with DNA-damaging therapies, such as carboplatin, to treat tumors that require CDK4/6 activity for proliferation.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Apoptosis/efectos de los fármacos , Plaquetas/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carboplatino/farmacología , Caspasas/metabolismo , Proliferación Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , ADN de Neoplasias/efectos de los fármacos , Doxorrubicina/farmacología , Activación Enzimática , Femenino , Citometría de Flujo , Ratones , Piperazinas/efectos adversos , Sustancias Protectoras/farmacología , Piridinas/efectos adversos , Receptor ErbB-2/metabolismo , Retinoblastoma/tratamiento farmacológicoRESUMEN
The orphan nuclear receptor liver receptor homolog-1 (LRH-1) has been reported to play a role in bile acid biosynthesis and reverse cholesterol transport. In this study, we examined the role of LRH-1 in the regulation of the apolipoprotein AI (APOAI) gene. Using RNA interference and adenovirus-mediated overexpression, we show that LRH-1 directly regulates APOAI gene transcription. Transient transfection experiments and EMSAs revealed that LRH-1 directly regulates APOAI transcription by binding to an LRH-1 response element located in the proximal APOAI promoter region. Chromatin immunoprecipitation experiments revealed that LRH-1 binds to the human APO AI promoter in vivo. Finally, we show that the transcriptional repressor SHP (small heterodimer partner) suppressed APOAI gene expression by inhibiting LRH-1 transcriptional activity. Taken together, our results demonstrate that LRH-1 is a novel regulator of APOAI transcription and underscore the role of this receptor in cholesterol homeostasis.
Asunto(s)
Apolipoproteína A-I/genética , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Receptores Citoplasmáticos y Nucleares/fisiología , Transcripción Genética , Animales , Secuencia de Bases , Línea Celular , Línea Celular Tumoral , Cartilla de ADN , Hepatocitos/fisiología , Humanos , Ratones , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Factores de TranscripciónRESUMEN
Peroxisome proliferator-activated receptor-gamma (PPARgamma) serves as a target for the thiazolidinedione class of antidiabetic drugs and is an important regulator of adipose tissue differentiation. By contrast, the principal target genes for PPARgamma in the pancreatic islet and the impact of their induction on insulin secretion are largely undefined. Here, we show that mRNAs encoding both isoforms of rodent PPARgamma, gamma1 and gamma2, are expressed in primary rat islets and are upregulated by overexpresssion of the lipogenic transcription factor sterol response element-binding protein 1c. Unexpectedly, however, oligonucleotide microarray analysis demonstrates that graded activation of PPARgamma achieved with 1) the thiazolidinedione GW-347845, 2) transduction with adenoviral PPARgamma1, or 3) a combination of both treatments progressively enhances the expression of genes involved in fatty acid oxidation and transport. Moreover, maximal activation of PPARgamma1 reduces islet triglyceride levels and enhances the oxidation of exogenous palmitate while decreasing glucose oxidation, cellular ATP content, and glucose-, but not depolarization-stimulated, insulin secretion. We conclude that, in the context of the pancreatic islet, the principal response to PPARgamma expression and activation is the activation of genes involved in the disposal, rather than the synthesis, of fatty acids. Although fatty acid oxidation may have beneficial effects on beta-cell function in the longer term by countering beta-cell "lipotoxicity," the acute response to this metabolic shift is a marked inhibition of insulin secretion.
Asunto(s)
Perfilación de la Expresión Génica , Expresión Génica/fisiología , Islotes Pancreáticos/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/fisiología , Animales , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas de Unión al ADN/metabolismo , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Técnicas In Vitro , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Masculino , Cloruro de Potasio/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
The promyelocytic leukaemia (PML) gene is translocated in most acute promyelocytic leukaemias and encodes a tumour suppressor protein. PML is involved in multiple apoptotic pathways and is thought to be pivotal in gamma irradiation-induced apoptosis. The DNA damage checkpoint kinase hCds1/Chk2 is necessary for p53-dependent apoptosis after gamma irradiation. In addition, gamma irradiation-induced apoptosis also occurs through p53-independent mechanisms, although the molecular mechanism remains largely unknown. Here, we report that hCds1/Chk2 mediates gamma irradiation-induced apoptosis in a p53-independent manner through an ataxia telangiectasia-mutated (ATM)-hCds1/Chk2-PML pathway. Our results provide the first evidence of a functional relationship between PML and a checkpoint kinase in gamma irradiation-induced apoptosis.
Asunto(s)
Apoptosis , Proteínas de Neoplasias/fisiología , Proteínas Nucleares , Proteínas Serina-Treonina Quinasas/fisiología , Factores de Transcripción/fisiología , Adenoviridae/genética , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Quinasa de Punto de Control 2 , Daño del ADN , Proteínas de Unión al ADN , Relación Dosis-Respuesta en la Radiación , Electroporación , Rayos gamma , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Microscopía Confocal , Proteínas de Neoplasias/metabolismo , Fosforilación , Plásmidos/metabolismo , Pruebas de Precipitina , Proteína de la Leucemia Promielocítica , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Serina/química , Factores de Tiempo , Factores de Transcripción/metabolismo , Transfección , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor , Células U937RESUMEN
Using alternative reading frames, the human ARF-INK4a locus encodes two unrelated proteins that both function in tumor suppression. p16(INK4a) maintains the retinoblastoma protein in its growth-suppressive state through inhibition of cyclin D-dependent kinase activity, whereas ARF binds with MDM2 and stabilizes p53. The majority of the activity of ARF to date is ascribed to its ability to activate p53, resulting in a G(1) cell cycle arrest or apoptosis. We show here that ARF colocalizes with DNA replication protein A (RPA32) and that overexpression of ARF reduces the rate of DNA synthesis resulting in accumulation of an S-phase cell population. Impediment of DNA synthesis by ARF can occur and becomes more evident in the absence of p53. Hence, the biological consequence of ARF induction varies dependent on cellular p53 status, inducing predominantly a G(1) arrest or apoptosis in p53-positive cells or causing S-phase retardation when p53 function is comprised.