RESUMEN
mTOR activation leads to enhanced survival signaling in acute myeloid leukemia (AML) cells. The active-site mTOR inhibitors (asTORi) represent a promising new approach to targeting mTOR in AKT/mTOR signaling. MLN0128 is an orally-administered, second-generation asTORi, currently in clinical development. We examined the anti-leukemic effects and the mechanisms of action of MLN0128 in AML cell lines and primary samples, with a particular focus on its effect in AML stem/progenitor cells. MLN0128 inhibited cell proliferation and induced apoptosis in AML by attenuating the activity of mTOR complex 1 and 2. Using time-of-flight mass cytometry, we demonstrated that MLN0128 selectively targeted and functionally inhibited AML stem/progenitor cells with high AKT/mTOR signaling activity. Using the reverse-phase protein array technique, we measured expression and phosphorylation changes in response to MLN0128 in 151 proteins from 24 primary AML samples and identified several pro-survival pathways that antagonize MLN0128-induced cellular stress. A combined blockade of AKT/mTOR signaling and these pro-survival pathways facilitated AML cell killing. Our findings provide a rationale for the clinical use of MLN0128 to target AML and AML stem/progenitor cells, and support the use of combinatorial multi-targeted approaches in AML therapy.
Asunto(s)
Apoptosis/efectos de los fármacos , Benzoxazoles/farmacología , Leucemia Mieloide/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Pirimidinas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Enfermedad Aguda , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Leucemia Mieloide/metabolismo , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Células U937 , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
The central role of phosphoinositide 3-kinase (PI3K) activation in tumour cell biology has prompted a sizeable effort to target PI3K and/or downstream kinases such as AKT and mammalian target of rapamycin (mTOR) in cancer. However, emerging clinical data show limited single-agent activity of inhibitors targeting PI3K, AKT or mTOR at tolerated doses. One exception is the response to PI3Kδ inhibitors in chronic lymphocytic leukaemia, where a combination of cell-intrinsic and -extrinsic activities drive efficacy. Here, we review key challenges and opportunities for the clinical development of inhibitors targeting the PI3K-AKT-mTOR pathway. Through a greater focus on patient selection, increased understanding of immune modulation and strategic application of rational combinations, it should be possible to realize the potential of this promising class of targeted anticancer agents.
Asunto(s)
Antineoplásicos/uso terapéutico , Descubrimiento de Drogas , Inhibidores Enzimáticos/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Ensayos Clínicos como Asunto , Descubrimiento de Drogas/métodos , Descubrimiento de Drogas/tendencias , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Humanos , Neoplasias/patología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Receptor Cross-Talk/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
Phosphoinositide-3 kinase (PI3K)-δ and PI3K-γ are preferentially expressed in immune cells, and inhibitors targeting these isoforms are hypothesized to have anti-inflammatory activity by affecting the adaptive and innate immune response. We report on a potent oral PI3K-δ and PI3K-γ inhibitor (IPI-145) and characterize this compound in biochemical, cellular, and in vivo assays. These studies demonstrate that IPI-145 exerts profound effects on adaptive and innate immunity by inhibiting B and T cell proliferation, blocking neutrophil migration, and inhibiting basophil activation. We explored the therapeutic value of combined PI3K-δ and PI3K-γ blockade, and IPI-145 showed potent activity in collagen-induced arthritis, ovalbumin-induced asthma, and systemic lupus erythematosus rodent models. These findings support the hypothesis that inhibition of immune function can be achieved through PI3K-δ and PI3K-γ blockade, potentially leading to significant therapeutic effects in multiple inflammatory, autoimmune, and hematologic diseases.
Asunto(s)
Artritis/tratamiento farmacológico , Asma/tratamiento farmacológico , Modelos Animales de Enfermedad , Isoquinolinas/farmacología , Lupus Eritematoso Sistémico/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Purinas/farmacología , Animales , Artritis/inducido químicamente , Artritis/inmunología , Asma/inducido químicamente , Asma/inmunología , Colágeno Tipo II , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Isoquinolinas/química , Lupus Eritematoso Sistémico/inmunología , Estructura Molecular , Ovalbúmina , Fosfatidilinositol 3-Quinasas/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Purinas/química , Ratas , Ratas Endogámicas Lew , Ratas Wistar , Relación Estructura-ActividadRESUMEN
Class IA phosphoinositide 3-kinase (PI3K) is essential for clonal expansion, differentiation, and effector function of B and T lymphocytes. The p110δ catalytic isoform of PI3K is highly expressed in lymphocytes and plays a prominent role in B and T cell responses. Another class IA PI3K catalytic isoform, p110α, is a promising drug target in cancer but little is known about its function in lymphocytes. Here we used highly selective inhibitors to probe the function of p110α in lymphocyte responses in vitro and in vivo. p110α inhibition partially reduced B cell receptor (BCR)-dependent AKT activation and proliferation, and diminished survival supported by the cytokines BAFF and IL-4. Selective p110δ inhibition suppressed B cell responses much more strongly, yet maximal suppression was achieved by targeting multiple PI3K isoforms. In mouse and human T cells, inhibition of single class IA isoforms had little effect on proliferation, whereas pan-class I inhibition did suppress T cell expansion. In mice, selective p110α inhibition using the investigational agent MLN1117 (previously known as INK1117) did not disrupt the marginal zone B cell compartment and did not block T cell-dependent germinal center formation. In contrast, the selective p110δ inhibitor IC87114 strongly suppressed germinal center formation and reduced marginal zone B cell numbers, similar to a pan-class I inhibitor. These findings show that although acute p110α inhibition partially diminishes AKT activation, selective p110α inhibitors are likely to be less immunosuppressive in vivo compared with p110δ or pan-class I inhibitors.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica , Linfocitos/citología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Animales , Calcio/metabolismo , Línea Celular Tumoral , Proliferación Celular , Diseño de Fármacos , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Inmunosupresores/farmacología , Linfocitos/enzimología , Ratones , Ratones Endogámicos BALB C , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Isoformas de Proteínas , Transducción de Señal , Bazo/citología , Linfocitos T/citología , Linfocitos T/enzimologíaRESUMEN
Aberrant activation of the mammalian target of rapamycin (mTOR) signaling plays an important role in breast cancer progression and represents a potential therapeutic target for breast cancer. In this study, we report the impact of the investigational drug MLN0128, a potent and selective small molecule active-site TORC1/2 kinase inhibitor, on tumor growth and metastasis using human breast cancer xenograft models. We assessed in vitro antiproliferative activity of MLN0128 in a panel of breast cancer cell lines. We next evaluated the impact of MLN0128 on tumor growth, angiogenesis and metastasis using mammary fat pad xenograft models of a non-VEGF (ML20) and a VEGF-driven (MV165) MCF-7 sublines harboring PIK3CA mutations. MLN0128 potently inhibited cell proliferation in various breast cancer cell lines harboring PIK3CA (IC(50): 1.5-53 nM), PTEN (IC(50): 1-149 nM), KRAS, and/or BRAF mutations (IC(50): 13-162 nM), and in human endothelial cells (IC(50): 33-40 nM) in vitro. In vivo, MLN0128 decreased primary tumor growth significantly in both non-VEGF (ML20; p = 0.05) and VEGF-driven MCF-7 (MV165; p = 0.014) xenograft models. MLN0128 decreased the phosphorylation of Akt, S6, 4E-BP1, and NDRG1 in both models. In contrast, rapamycin increased Akt activity and failed to reduce the phosphorylation of 4E-BP1, PRAS40, and NDRG1. VEGF-induced lung metastasis in MV165 is inhibited by MLN0128 and rapamycin. In conclusion, MLN0128 inhibits TORC1/2-dependent signaling in preclinical models of breast cancer. MLN0128 appears to be superior in blocking mTORC1/2 signaling in contrast to rapamycin. Our findings support the clinical research of MLN0128 in patients with breast cancer and metastasis.
Asunto(s)
Antineoplásicos/farmacología , Benzoxazoles/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Complejos Multiproteicos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas/antagonistas & inhibidores , Pirimidinas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Antineoplásicos/sangre , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase I , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Desnudos , Complejos Multiproteicos/metabolismo , Mutación , Neovascularización Patológica/tratamiento farmacológico , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/sangre , Proteínas/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The interactions between the bone marrow (BM) microenvironment and acute myeloid leukemia (AML) is known to promote survival of AML cells. In this study, we used reverse phase-protein array (RPPA) technology to measure changes in multiple proteins induced by stroma in leukemic cells. We then investigated the potential of an mTOR kinase inhibitor, PP242, to disrupt leukemia/stroma interactions, and examined the effects of PP242 in vivo using a mouse model. Using RPPA, we confirmed that multiple survival signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), were up-regulated in primary AML cells cocultured with stroma. PP242 effectively induced apoptosis in primary samples cultured with or without stroma. Mechanistically, PP242 attenuated the activities of mTORC1 and mTORC2, sequentially inhibited phosphorylated AKT, S6K, and 4EBP1, and concurrently suppressed chemokine receptor CXCR4 expression in primary leukemic cells and in stromal cells cultured alone or cocultured with leukemic cells. In the in vivo leukemia mouse model, PP242 inhibited mTOR signaling in leukemic cells and demonstrated a greater antileukemia effect than rapamycin. Our findings indicate that disrupting mTOR/AKT signaling with a selective mTOR kinase inhibitor can effectively target leukemic cells within the BM microenvironment.
Asunto(s)
Apoptosis/efectos de los fármacos , Médula Ósea/metabolismo , Indoles/uso terapéutico , Leucemia Experimental/prevención & control , Leucemia Mieloide Aguda/prevención & control , Células Madre Mesenquimatosas/patología , Complejos Multiproteicos/antagonistas & inhibidores , Purinas/uso terapéutico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Antibióticos Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica , Western Blotting , Médula Ósea/patología , Proliferación Celular , Técnicas de Cocultivo , Citometría de Flujo , Humanos , Leucemia Experimental/mortalidad , Leucemia Experimental/patología , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones SCID , Complejos Multiproteicos/metabolismo , Fosforilación/efectos de los fármacos , Análisis por Matrices de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Sirolimus/uso terapéutico , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
PURPOSE: The PI3K/Akt/mTOR pathway is an attractive target in HER2-positive breast cancer that is refractory to anti-HER2 therapy. The hypothesis is that the suppression of this pathway results in sensitization to anti-HER2 agents. However, this combinatorial strategy has not been comprehensively tested in models of trastuzumab and lapatinib resistance. EXPERIMENTAL DESIGN: We analyzed in vitro cell viability and induction of apoptosis in five different cell lines resistant to trastuzumab and lapatinib. Inhibition of HER2/HER3 phosphorylation, PI3K/Akt/mTOR, and extracellular signal-regulated kinase (ERK) signaling pathways was evaluated by Western blotting. Tumor growth inhibition after treatment with lapatinib, INK-128, or the combination of both agents was evaluated in three different animal models: two cell-based xenograft models refractory to both trastuzumab and lapatinib and a xenograft derived from a patient who relapsed on trastuzumab-based therapy. RESULTS: The addition of lapatinib to INK-128 prevented both HER2 and HER3 phosphorylation induced by INK-128, resulting in inhibition of both PI3K/Akt/mTOR and ERK pathways. This dual blockade produced synergistic induction of cell death in five different HER2-positive cell lines resistant to trastuzumab and lapatinib. In vivo, both cell line-based and patient-derived xenografts showed exquisite sensitivity to the antitumor activity of the combination of lapatinib and INK-128, which resulted in durable tumor shrinkage and exhibited no signs of toxicity in these models. CONCLUSIONS: The simultaneous blockade of both PI3K/Akt/mTOR and ERK pathways obtained by combining lapatinib with INK-128 acts synergistically in inducing cell death and tumor regression in breast cancer models refractory to anti-HER2 therapy.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos , Proteínas/antagonistas & inhibidores , Receptor ErbB-2/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Benzoxazoles/farmacología , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Sinergismo Farmacológico , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Lapatinib , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Desnudos , Complejos Multiproteicos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirimidinas/farmacología , Quinazolinas/farmacología , Receptor ErbB-2/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Factores de Transcripción/metabolismo , Trastuzumab , Células Tumorales CultivadasRESUMEN
Class I phosphoinositide 3 kinase (PI3K) δ is a promising therapeutic target for rheumatoid arthritis (RA) because of its contribution to leukocyte biology. However, its contribution in fibroblasts has not been studied as a mechanism that contributes to efficacy. We investigated the expression and function of PI3Kδ in synovium and cultured fibroblast-like synoviocytes (FLS). Immunohistochemistry demonstrated that PI3Kδ is highly expressed in RA synovium, especially in the synovial lining. Using quantitative PCR and Western blot analysis, we found that PI3Kδ mRNA and protein expression is higher in RA than in osteoarthritis (OA) synovium. PI3Kδ was also expressed in cultured FLS, along with PI3Kα and PI3Kß, whereas PI3Kγ was not detectable. PI3Kδ mRNA expression was selectively induced by inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) but not by growth factors platelet-derived growth factor (PDGF) and transforming growth factor ß (TGFß). The use of inhibitors that block individual PI3K isoforms, including the novel selective PI3Kδ inhibitor INK007, showed that PI3Kδ is required for PDGF- and TNF-induced Akt activation. PI3Kδ inhibition also diminished PDGF-mediated synoviocyte growth and sensitized cells to H(2)O(2)-induced apoptosis. These data are the first documentation of increased PI3Kδ expression in both RA synovium and cultured synoviocytes. Furthermore, these are the first data demonstrating that PI3Kδ is a major regulator of PDGF-mediated fibroblast growth and survival via Akt. Thus, targeting PI3Kδ in RA could modulate synoviocyte function via anti-inflammatory and disease-altering mechanisms.
Asunto(s)
Artritis Reumatoide/enzimología , Fosfatidilinositol 3-Quinasas/fisiología , Membrana Sinovial/enzimología , Apoptosis/fisiología , Artritis Reumatoide/patología , División Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Fosfatidilinositol 3-Quinasa Clase I , Citocinas/farmacología , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Mediadores de Inflamación/farmacología , Osteoartritis/enzimología , Fosfatidilinositol 3-Quinasas/biosíntesis , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , Membrana Sinovial/patología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
The mammalian target of rapamycin (mTOR) kinase is a master regulator of protein synthesis that couples nutrient sensing to cell growth and cancer. However, the downstream translationally regulated nodes of gene expression that may direct cancer development are poorly characterized. Using ribosome profiling, we uncover specialized translation of the prostate cancer genome by oncogenic mTOR signalling, revealing a remarkably specific repertoire of genes involved in cell proliferation, metabolism and invasion. We extend these findings by functionally characterizing a class of translationally controlled pro-invasion messenger RNAs that we show direct prostate cancer invasion and metastasis downstream of oncogenic mTOR signalling. Furthermore, we develop a clinically relevant ATP site inhibitor of mTOR, INK128, which reprograms this gene expression signature with therapeutic benefit for prostate cancer metastasis, for which there is presently no cure. Together, these findings extend our understanding of how the 'cancerous' translation machinery steers specific cancer cell behaviours, including metastasis, and may be therapeutically targeted.
Asunto(s)
Metástasis de la Neoplasia , Neoplasias de la Próstata/patología , Biosíntesis de Proteínas , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Benzoxazoles/farmacología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Genoma/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Invasividad Neoplásica/genética , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/genética , Fosfoproteínas/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Pirimidinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
Obligate intracellular pathogens such as Leishmania specifically target host phagocytes for survival and replication. Phosphoinositide 3-kinase γ (PI3Kγ), a member of the class I PI3Ks that is highly expressed by leukocytes, controls cell migration by initiating actin polymerization and cytoskeletal reorganization, which are processes also critical for phagocytosis. In this study, we demonstrate that class IB PI3K, PI3Kγ, plays a critical role in pathogenesis of chronic cutaneous leishmaniasis caused by L. mexicana. Using the isoform-selective PI3Kγ inhibitor, AS-605240 and PI3Kγ gene-deficient mice, we show that selective blockade or deficiency of PI3Kγ significantly enhances resistance against L. mexicana that is associated with a significant suppression of parasite entry into phagocytes and reduction in recruitment of host phagocytes as well as regulatory T cells to the site of infection. Furthermore, we demonstrate that AS-605240 is as effective as the standard antileishmanial drug sodium stibogluconate in treatment of cutaneous leishmaniasis caused by L. mexicana. These findings reveal a unique role for PI3Kγ in Leishmania invasion and establishment of chronic infection, and demonstrate that therapeutic targeting of host pathways involved in establishment of infection may be a viable strategy for treating infections caused by obligate intracellular pathogens such as Leishmania.
Asunto(s)
Resistencia a la Enfermedad/efectos de los fármacos , Leishmania mexicana , Leishmaniasis Cutánea/parasitología , Fosfatidilinositol 3-Quinasas/metabolismo , Quinoxalinas/farmacología , Tiazolidinedionas/farmacología , Animales , Gluconato de Sodio Antimonio/uso terapéutico , Citometría de Flujo , Interacciones Huésped-Parásitos/efectos de los fármacos , Humanos , Leishmaniasis Cutánea/fisiopatología , Macrófagos , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Neutrófilos , Fagocitos/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Quinoxalinas/uso terapéutico , Tiazolidinedionas/uso terapéuticoRESUMEN
Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment to regulate multiple cellular processes. Rapamycin and its analogs have not shown significant activity in multiple myeloma (MM), likely because of the lack of inhibition of TORC2. In the present study, we investigated the baseline activity of the PI3K/Akt/mTOR pathway TORC1/2 in MM cell lines with different genetic abnormalities. TORC1/2 knock-down led to significant inhibition of the proliferation of MM cells, even in the presence of BM stromal cells. We also tested INK128, a dual TORC1/2 inhibitor, as a new therapeutic agent against these MM cell lines. We showed that dual TORC1/2 inhibition is much more active than TORC1 inhibition alone (rapamycin), even in the presence of cytokines or stromal cells. In vitro and in vivo studies showed that p-4EBP1 and p-Akt inhibition could be predictive markers of TORC2 inhibition in MM cell lines. Dual TORC1/2 inhibition showed better inhibition of adhesion to BM microenvironmental cells and inhibition of homing in vivo. These studies form the basis for further clinical testing of TORC1/2 inhibitors in MM.
Asunto(s)
Mieloma Múltiple/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones SCID , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Complejos Multiproteicos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas/antagonistas & inhibidores , Proteínas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Proteína Asociada al mTOR Insensible a la Rapamicina , Proteína Reguladora Asociada a mTOR , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
UNLABELLED: Several phosphoinositide 3-kinase (PI3K) inhibitors are in the clinic and many more are in preclinical development. CAL-101, a selective inhibitor of the PI3Kδ isoform, has shown remarkable success in certain hematologic malignancies. Although PI3Kδ signaling plays a central role in lymphocyte biology, the degree of single-agent therapeutic activity of CAL-101 during early-phase development has been somewhat unexpected. CAL-101 works in part by blocking signals from the microenvironment that normally sustain leukemia and lymphoma cells in a protective niche. As PI3Ks enter the arena of molecular-targeted therapies, CAL-101 provides proof of principle that isoform-selective compounds can be effective in selected cancer types and patient populations. SIGNIFICANCE: A key question is whether compounds targeting a single PI3K catalytic isoform can provide meaningful single agent efficacy in cancer cells that express multiple isoforms. Clinical studies of the drug CAL-101 have provided a significant advance by showing that selective targeting of PI3Kδ achieves efficacy in chronic lymphocytic leukemia, in part through targeting the tumor microenvironment.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Purinas/farmacología , Quinazolinonas/farmacología , Animales , Antineoplásicos/uso terapéutico , Ensayos Clínicos Fase I como Asunto , Evaluación Preclínica de Medicamentos , Humanos , Neoplasias/enzimología , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Purinas/uso terapéutico , Quinazolinonas/uso terapéuticoRESUMEN
The multiple roles of PI3Kδ (p110δ) and PI3Kγ (p110γ) in various immune cells have encouraged the development of small-molecule inhibitors of both PI3K isoforms, alone or in combination, for the treatment of immune-mediated inflamatory diseases. Recent findings suggest a previously unrecognized interdependent cooperativity between p110δ and p110γ, if not all class I PI3K isoforms, expressed and activated in leukocytes and endothelium. For example, the activity of p110δ and p110γ in combination appears to be necessary for mediating efficient (velocity and direction) trafficking of immune competent cells to sites of inflammation. This chapter will focus on the emerging evidence of the dynamic interplay of p110δ and p110γ supporting the hypothesis that dual-blockade of both, p110δ and p110γ, presents a unique therapeutic opportunity in that pharmacological inhibition of the two PI3K isoforms simultaneously may yield superior clinical results in the treatment of a variety of complex immune-mediated inflammatory diseases.
Asunto(s)
Inflamación/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas/fisiología , Artritis/tratamiento farmacológico , Movimiento Celular , Humanos , Células Asesinas Naturales/fisiología , Linfocitos/fisiología , Mastocitos/fisiología , Neutrófilos/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Neumonía/tratamiento farmacológicoRESUMEN
From humble beginnings over 25 years ago as a lipid kinase activity associated with certain oncoproteins, PI3K (phosphoinositide 3-kinase) has been catapulted to the forefront of drug development in cancer, immunity and thrombosis, with the first clinical trials of PI3K pathway inhibitors now in progress. Here, we give a brief overview of some key discoveries in the PI3K area and their impact, and include thoughts on the current state of the field, and where it could go from here.PI3K has become a very intense area of research, with over 2,000 publications on PI3K in PubMed for 2009 alone. The expectations for a therapeutic impact of intervention with PI3K activity are high, and progress in the clinical arena is being monitored by many. However, targeted therapies almost invariably encounter roadblocks, often exposing unresolved questions in the basic understanding of the target. PI3K will most likely be no exception. Below, we describe some of these early "surprises" and how these inform and shape basic science investigations.
Asunto(s)
Fosfatidilinositol 3-Quinasas/fisiología , Animales , Humanos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/uso terapéutico , Transducción de SeñalRESUMEN
Oligodendrocytes generate and maintain myelin, which is essential for axonal function and protection of the mammalian central nervous system. To advance our molecular understanding of differentiation by these cells, we screened libraries of pharmacologically active compounds and identified inducers of differentiation of Oli-neu, a stable cell line of mouse oligodendrocyte precursors (OPCs). We identified four broad classes of inducers, namely, forskolin/cAMP (protein kinase A activators), steroids (glucocorticoids and retinoic acid), ErbB2 inhibitors, and nucleoside analogs, and confirmed the activity of these compounds on rat primary oligodendrocyte precursors and mixed cortical cultures. We also analyzed transcriptional responses in the chemically induced mouse and rat OPC differentiation processes and compared these with earlier studies. We confirm the view that ErbB2 is a natural signaling component that is required for OPC proliferation, whereas ErbB2 inhibition or genetic knockdown results in OPC differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Proliferación Celular/efectos de los fármacos , Corteza Cerebral/metabolismo , Oligodendroglía/metabolismo , Transducción de Señal/fisiología , Células Madre/metabolismo , Animales , Animales Recién Nacidos , Biomarcadores/análisis , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Corteza Cerebral/citología , Colforsina/metabolismo , Colforsina/farmacología , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Ratones , Oligodendroglía/citología , Interferencia de ARN/fisiología , Ratas , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/deficiencia , Receptor ErbB-2/genética , Transducción de Señal/efectos de los fármacos , Células Madre/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/farmacologíaRESUMEN
Targeting the mammalian target of rapamycin (mTOR) protein is a promising strategy for cancer therapy. The mTOR kinase functions in two complexes, TORC1 (target of rapamycin complex-1) and TORC2 (target of rapamycin complex-2); however, neither of these complexes is fully inhibited by the allosteric inhibitor rapamycin or its analogs. We compared rapamycin with PP242, an inhibitor of the active site of mTOR in both TORC1 and TORC2 (hereafter referred to as TORC1/2), in models of acute leukemia harboring the Philadelphia chromosome (Ph) translocation. We demonstrate that PP242, but not rapamycin, causes death of mouse and human leukemia cells. In vivo, PP242 delays leukemia onset and augments the effects of the current front-line tyrosine kinase inhibitors more effectively than does rapamycin. Unexpectedly, PP242 has much weaker effects than rapamycin on the proliferation and function of normal lymphocytes. PI-103, a less selective TORC1/2 inhibitor that also targets phosphoinositide 3-kinase (PI3K), is more immunosuppressive than PP242. These findings establish that Ph(+) transformed cells are more sensitive than normal lymphocytes to selective TORC1/2 inhibitors and support the development of such inhibitors for leukemia therapy.
Asunto(s)
Antineoplásicos/farmacología , Sistemas de Liberación de Medicamentos , Leucemia/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Animales , Humanos , Ratones , Sirolimus/farmacologíaRESUMEN
Deregulation of the phosphoinositide-3-OH kinase (PI(3)K) pathway has been implicated in numerous pathologies including cancer, diabetes, thrombosis, rheumatoid arthritis and asthma. Recently, small-molecule and ATP-competitive PI(3)K inhibitors with a wide range of selectivities have entered clinical development. In order to understand the mechanisms underlying the isoform selectivity of these inhibitors, we developed a new expression strategy that enabled us to determine to our knowledge the first crystal structure of the catalytic subunit of the class IA PI(3)K p110 delta. Structures of this enzyme in complex with a broad panel of isoform- and pan-selective class I PI(3)K inhibitors reveal that selectivity toward p110 delta can be achieved by exploiting its conformational flexibility and the sequence diversity of active site residues that do not contact ATP. We have used these observations to rationalize and synthesize highly selective inhibitors for p110 delta with greatly improved potencies.
Asunto(s)
Dominio Catalítico , Fosfatidilinositol 3-Quinasas/química , Inhibidores de Proteínas Quinasas/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Simulación por Computador , Cristalografía por Rayos X , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Fosfatidilinositol 3-Quinasas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Spodoptera , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
This study aimed at identifying transcriptional changes associated to neuronal differentiation induced by six distinct stimuli using whole-genome microarray hybridization analysis. Bioinformatics analyses revealed the clustering of these six stimuli into two categories, suggesting separate gene/pathway dependence. Treatment with specific inhibitors demonstrated the requirement of both Janus kinase and microtubule-associated protein kinase activation to trigger differentiation with nerve growth factor (NGF) and dibutyryl cAMP. Conversely, activation of protein kinase A, phosphatidylinositol-3-kinase alpha, and mammalian target of rapamycin, although required for dibutyryl cAMP-induced differentiation, exerted a negative feedback on NGF-induced differentiation. We identified Polo-like kinase 2 (Plk2) and poliovirus receptor (PVR) as indispensable for NGF-driven neuronal differentiation and alphaB-crystallin (Cryab) as an inhibitor of this process. Silencing of Plk2 or PVR blocked NGF-triggered differentiation and Cryab down-regulation, while silencing of Cryab enhanced NGF-induced differentiation. Our results position both Plk2 and PVR upstream of the negative regulator Cryab in the pathway(s) leading to neuronal differentiation triggered by NGF.