RESUMEN
Clinical successes demonstrated by chimeric antigen receptor T-cell immunotherapy have facilitated further development of T-cell immunotherapy against wide variety of diseases. One approach is the development of "off-the-shelf" T-cell sources. Technologies to generate T-cells from pluripotent stem cells (PSCs) may offer platforms to produce "off-the-shelf" and synthetic allogeneic T-cells. However, low differentiation efficiency and poor scalability of current methods may compromise their utilities. Here we show improved differentiation efficiency of T-cells from induced PSCs (iPSCs) derived from an antigen-specific cytotoxic T-cell clone, or from T-cell receptor (TCR)-transduced iPSCs, as starting materials. We additionally describe feeder-free differentiation culture systems that span from iPSC maintenance to T-cell proliferation phases, enabling large-scale regenerated T-cell production. Moreover, simultaneous addition of SDF1α and a p38 inhibitor during T-cell differentiation enhances T-cell commitment. The regenerated T-cells show TCR-dependent functions in vitro and are capable of in vivo anti-tumor activity. This system provides a platform to generate a large number of regenerated T-cells for clinical application and investigate human T-cell differentiation and biology.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Inmunoterapia Adoptiva/métodos , Células Madre Pluripotentes Inducidas/fisiología , Neoplasias/terapia , Linfocitos T Citotóxicos/trasplante , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Quimiocina CXCL12/metabolismo , Medios de Cultivo/metabolismo , Medios de Cultivo/farmacología , Femenino , Humanos , Imidazoles/farmacología , Ratones , Neoplasias/inmunología , Piridinas/farmacología , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T Citotóxicos/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Protein arginine methyltransferase (PRMT) 4 (also known as coactivator-associated arginine methyltransferase 1; CARM1) is involved in a variety of biological processes and is considered as a candidate oncogene owing to its overexpression in several types of cancer. Selective PRMT4 inhibitors are useful tools for clarifying the molecular events regulated by PRMT4 and for validating PRMT4 as a therapeutic target. Here, we report the discovery of TP-064, a potent, selective, and cell-active chemical probe of human PRMT4 and its co-crystal structure with PRMT4. TP-064 inhibited the methyltransferase activity of PRMT4 with high potency (half-maximal inhibitory concentration, IC50 < 10 nM) and selectivity over other PRMT family proteins, and reduced arginine dimethylation of the PRMT4 substrates BRG1-associated factor 155 (BAF155; IC50= 340 ± 30 nM) and Mediator complex subunit 12 (MED12; IC50 = 43 ± 10 nM). TP-064 treatment inhibited the proliferation of a subset of multiple myeloma cell lines, with affected cells arrested in G1 phase of the cell cycle. TP-064 and its negative control (TP-064N) will be valuable tools to further investigate the biology of PRMT4 and the therapeutic potential of PRMT4 inhibition.
RESUMEN
BACKGROUND: Recent evidence suggests that androgen receptor (AR) splice variants, including AR-V7, play a pivotal role in resistance to androgen blockade in prostate cancer treatment. The development of new therapeutic agents that can suppress the transcriptional activities of AR splice variants has been anticipated as the next generation treatment of castration-resistant prostate cancer. METHODS: High-throughput screening of AR-V7 signaling inhibitors was performed using an AR-V7 reporter system. The effects of a glycogen synthase kinase-3 (GSK3) inhibitor, LY-2090314, on endogenous AR-V7 signaling were evaluated in an AR-V7-positive cell line, JDCaP-hr, by quantitative reverse transcription polymerase chain reaction. The relationship between AR-V7 signaling and ß-catenin signaling was assessed using RNA interference. The effect of LY-2090314 on cell growth in various prostate cancer cell lines was also evaluated. RESULTS: We identified GSK3 inhibitors as transcriptional suppressors of AR-V7 using a high-throughput screen with an AR-V7 reporter system. LY-2090314 suppressed the reporter activity and endogenous AR-V7 activity in JDCaP-hr cells. Because silencing of ß-catenin partly rescued the suppression, it was evident that the suppression was mediated, at least partially, via the activation of ß-catenin signaling. AR-V7 signaling and ß-catenin signaling reciprocally regulate each other in JDCaP-hr cells, and therefore, GSK3 inhibition can repress AR-V7 transcriptional activity by accumulating intracellular ß-catenin. Notably, LY-2090314 selectively inhibited the growth of AR-V7-positive prostate cancer cells in vitro. CONCLUSIONS: Our findings demonstrate the potential of GSK3 inhibitors in treating advanced prostate cancer driven by AR splice variants. In vivo evaluation of AR splice variant-positive prostate cancer models will help illustrate the overall significance of GSK3 inhibitors in treating prostate cancer.
Asunto(s)
Glucógeno Sintasa Quinasa 3 , Neoplasias de la Próstata Resistentes a la Castración , Receptores Androgénicos/metabolismo , beta Catenina/metabolismo , Empalme Alternativo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Masculino , Próstata , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Neoplasias de la Próstata Resistentes a la Castración/terapia , Transducción de Señal/efectos de los fármacosRESUMEN
Dysregulation of lysine (K)-specific demethylase 1A (LSD1), also known as KDM1A, has been implicated in the development of various cancers, including leukemia. Here, we describe the antileukemic activity and mechanism of action of T-3775440, a novel irreversible LSD1 inhibitor. Cell growth analysis of leukemia cell lines revealed that acute erythroid leukemia (AEL) and acute megakaryoblastic leukemia cells (AMKL) were highly sensitive to this compound. T-3775440 treatment enforced transdifferentiation of erythroid/megakaryocytic lineages into granulomonocytic-like lineage cells. Mechanistically, T-3775440 disrupted the interaction between LSD1 and growth factor-independent 1B (GFI1B), a transcription factor critical for the differentiation processes of erythroid and megakaryocytic lineage cells. Knockdown of LSD1 and GFI1B recapitulated T-3775440-induced transdifferentiation and cell growth suppression, highlighting the significance of LSD1-GFI1B axis inhibition with regard to the anti-AML effects of T-3775440. Moreover, T-3775440 exhibited significant antitumor efficacy in AEL and AMKL xenograft models. Our findings provide a rationale for evaluating LSD1 inhibitors as potential treatments and indicate a novel mechanism of action against AML, particularly AEL and AMKL. Mol Cancer Ther; 16(2); 273-84. ©2016 AACR.
Asunto(s)
Antineoplásicos/farmacología , Transdiferenciación Celular/efectos de los fármacos , Histona Demetilasas/antagonistas & inhibidores , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Complejos Multiproteicos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Animales , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Análisis por Conglomerados , Biología Computacional/métodos , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Femenino , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Hematopoyesis/genética , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Ratones , Terapia Molecular Dirigida , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mounting evidence suggests that constitutively active androgen receptor (AR) splice variants, typified by AR-V7, are associated with poor prognosis and resistance to androgen deprivation therapy in prostate cancer patients. However, mechanisms governing the generation of AR splice variants are not fully understood. In this study, we aimed to investigate the dynamics of AR splice variant generation using the JDCaP prostate cancer model that expresses AR splice variants under androgen depletion. Microarray analysis of JDCaP xenografts before and after expression of AR splice variants suggested that dysregulation of RNA processing pathways is likely involved in AR splice variant generation. To explore factors contributing to generation of AR-V7 mRNA, we conducted a focused RNA interference screen in AR-V7-positive JDCaP-hr cells using an shRNA library targeting spliceosome-related genes. This screen identified DDX39B as a regulator of AR-V7 mRNA expression. Simultaneous knockdown of DDX39B and its paralog DDX39A drastically and selectively downregulated AR-V7 mRNA expression in multiple AR-V7-positive prostate cancer cell lines. DDX39B was upregulated in relapsed JDCaP xenografts expressing AR splice variants, suggesting its role in expression of AR splice variants. Taken together, our findings offer insight into the mechanisms of AR splice variant generation and identify DDX39 as a potential drug target for the treatment of AR splice variant-positive prostate cancer.
Asunto(s)
Empalme Alternativo , ARN Helicasas DEAD-box/química , Regulación Neoplásica de la Expresión Génica , Receptores Androgénicos/genética , Animales , Línea Celular Tumoral , Silenciador del Gen , Variación Genética , Humanos , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias de la Próstata/metabolismo , ARN/análisis , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , TranscriptomaRESUMEN
BACKGROUND: Castration resistance creates a significant problem in the treatment of prostate cancer. Constitutively active splice variants of androgen receptor (AR) have emerged as drivers for resistance to androgen deprivation therapy, including the next-generation androgen-AR axis inhibitors abiraterone and enzalutamide. In this study, we describe the characteristics of a novel castration-resistant prostate cancer (CRPC) model, designated JDCaP-hr (hormone refractory). METHODS: JDCaP-hr was established from an androgen-dependent JDCaP xenograft model after surgical castration. The expression of AR and its splice variants in JDCaP-hr was evaluated by immunoblotting and quantitative reverse transcription-polymerase chain reaction. The effects of AR antagonists and testosterone on JDCaP-hr were evaluated in vivo and in vitro. The roles of full-length AR (AR-FL) and AR-V7 in JDCaP-hr cell growth were evaluated using RNA interference. RESULTS: JDCaP-hr acquired a C-terminally truncated AR protein during progression from the parental JDCaP. The expression of AR-FL and AR-V7 mRNA was upregulated by 10-fold in JDCaP-hr compared with that in JDCaP, indicating that the JDCaP and JDCaP-hr models simulate castration resistance with some clinical features, such as overexpression of AR and its splice variants. The AR antagonist bicalutamide did not affect JDCaP-hr xenograft growth, and importantly, testosterone induced tumor regression. In vitro analysis demonstrated that androgen-independent prostate-specific antigen secretion and cell proliferation of JDCaP-hr were predominantly mediated by AR-V7. JDCaP-hr cell growth displayed a bell-shaped dependence on testosterone, and it was suppressed by physiological concentrations of testosterone. Testosterone induced rapid downregulation of both AR-FL and AR-V7 expression at physiological concentrations and suppressed expression of the AR target gene KLK3. CONCLUSIONS: Our findings support the clinical value of testosterone therapy, including bipolar androgen therapy, in the treatment of AR-overexpressed CRPC driven by AR splice variants that are not clinically actionable at present. Prostate 76:1536-1545, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Empalme del ARN/genética , Receptores Androgénicos/genética , Testosterona/farmacología , Antagonistas de Receptores Androgénicos/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Variación Genética , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Orquiectomía , Antígeno Prostático Específico/metabolismo , ARN Mensajero/análisis , Receptores Androgénicos/fisiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Nuclei are anatomical units of the central nervous system (CNS). Their formation sets the structural basis for the functional organization of the brain, a process known as nucleogenesis. In the present study, we investigated the role of the transmembrane immunoglobulin superfamily molecule Neph2 in the nucleogenesis of the pontine nucleus (PN). Neph2 expression is turned on in migrating PN neurons only after they enter the presumptive nuclear region. Neph2 knockdown disrupted the nuclear organization of PN presumably by changing the migratory behavior of PN neurons inside the nuclear region. Moreover, overexpression of the cytoplasmic region of Neph2, which can sequester intracellular signaling of endogenous Neph2, resulted in similar phenotypes. Overall, these results suggest Neph2 is involved in the nucleogenesis of the PN through the control of neuronal migration inside the nucleus.