RESUMEN
Glucocorticoid (GC) resistance is a poor prognostic factor in T-cell acute lymphoblastic leukaemia (T-ALL). Interleukin-7 (IL-7) mediates GC resistance via GC-induced upregulation of IL-7 receptor (IL-7R) expression, leading to increased pro-survival signalling. IL-7R reaches the cell surface via the secretory pathway, so we hypothesized that inhibiting the translocation of IL-7R into the secretory pathway would overcome GC resistance. Sec61 is an endoplasmic reticulum (ER) channel that is required for insertion of polypeptides into the ER. Here, we demonstrate that KZR-445, a novel inhibitor of Sec61, potently attenuates the dexamethasone (DEX)-induced increase in cell surface IL-7R and overcomes IL-7-induced DEX resistance.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Canales de Translocación SEC , Citocinas/metabolismo , Dexametasona/farmacología , Glucocorticoides/farmacología , Humanos , Interleucina-7 , Errores Innatos del Metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptores de Glucocorticoides/deficiencia , Canales de Translocación SEC/metabolismo , Linfocitos T/metabolismoRESUMEN
The immunological synapse (IS) is a cell-cell junction formed between CD4(+) T cells and dendritic cells (DCs). Here we show in vitro and in vivo that IS formation inhibits apoptosis of DCs. Consistent with these results, IS formation induced antiapoptotic signaling events, including activation of the kinase Akt1 and localization of the prosurvival transcription factor NF-kappaB and the proapoptotic transcription factor FOXO1 to the nucleus and cytoplasm, respectively. Inhibition of phosphatidylinositol 3-OH kinase and Akt1 partially prevented the antiapoptotic effects of IS formation. Direct stimulation of the IS component CD40 on DCs leads to the activation of Akt1, suggesting the involvement of this receptor in the antiapoptotic effects observed upon IS formation.
Asunto(s)
Apoptosis/inmunología , Células Dendríticas/inmunología , Factores de Transcripción Forkhead/metabolismo , Sinapsis Inmunológicas/inmunología , FN-kappa B/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Antígenos CD40/inmunología , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Humanos , Immunoblotting , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Transporte de Proteínas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Early T-cell precursor (ETP) acute lymphoblastic leukemia (ALL) is a recently described subtype of T-ALL characterized by a unique immunophenotype and genomic profile, as well as a high rate of induction failure. Frequent mutations in cytokine receptor and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways led us to hypothesize that ETP-ALL is dependent on JAK/STAT signaling. Here we demonstrate aberrant activation of the JAK/STAT pathway in ETP-ALL blasts relative to non-ETP T-ALL. Moreover, ETP-ALL showed hyperactivation of STAT5 in response to interleukin-7, an effect that was abrogated by the JAK1/2 inhibitor ruxolitinib. In vivo, ruxolitinib displayed activity in 6 of 6 patient-derived murine xenograft models of ETP-ALL, with profound single-agent efficacy in 5 models. Ruxolitinib treatment decreased peripheral blast counts relative to pretreatment levels and compared with control (P < .01) in 5 of 6 ETP-ALL xenografts, with marked reduction in mean splenic blast counts (P < .01) in 6 of 6 samples. Surprisingly, both JAK/STAT pathway activation and ruxolitinib efficacy were independent of the presence of JAK/STAT pathway mutations, raising the possibility that the therapeutic potential of ruxolitinib in ETP-ALL extends beyond those cases with JAK mutations. These findings establish the preclinical in vivo efficacy of ruxolitinib in ETP-ALL, a biologically distinct subtype for which novel therapies are needed.
Asunto(s)
Quinasas Janus/antagonistas & inhibidores , Células Precursoras de Linfocitos T/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Factores de Transcripción STAT/antagonistas & inhibidores , Adolescente , Animales , Niño , Preescolar , Femenino , Humanos , Interleucina-7/metabolismo , Quinasas Janus/genética , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mutación , Nitrilos , Células Precursoras de Linfocitos T/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Pirazoles/farmacología , Pirimidinas , Factores de Transcripción STAT/genética , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto JovenRESUMEN
The outcome for pediatric acute lymphoblastic leukemia (ALL) patients who relapse is dismal. A hallmark of relapsed disease is acquired resistance to multiple chemotherapeutic agents, particularly glucocorticoids. In this study, we performed a genome-scale short hairpin RNA screen to identify mediators of prednisolone sensitivity in ALL cell lines. The incorporation of these data with an integrated analysis of relapse-specific genetic and epigenetic changes allowed us to identify the mitogen-activated protein kinase (MAPK) pathway as a mediator of prednisolone resistance in pediatric ALL. We show that knockdown of the specific MAPK pathway members MEK2 and MEK4 increased sensitivity to prednisolone through distinct mechanisms. MEK4 knockdown increased sensitivity specifically to prednisolone by increasing the levels of the glucocorticoid receptor. MEK2 knockdown increased sensitivity to all chemotherapy agents tested by increasing the levels of p53. Furthermore, we demonstrate that inhibition of MEK1/2 with trametinib increased sensitivity of ALL cells and primary samples to chemotherapy in vitro and in vivo. To confirm a role for MAPK signaling in patients with relapsed ALL, we measured the activation of the MEK1/2 target ERK in matched diagnosis-relapse primary samples and observed increased phosphorylated ERK levels at relapse. Furthermore, relapse samples have an enhanced response to MEK inhibition compared to matched diagnosis samples in xenograft models. Together, our data indicate that inhibition of the MAPK pathway increases chemosensitivity to glucocorticoids and possibly other agents and that the MAPK pathway is an attractive target for prevention and/or treatment of relapsed disease.
Asunto(s)
Resistencia a Antineoplásicos , Sistema de Señalización de MAP Quinasas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Prednisolona , Piridonas/farmacología , Pirimidinonas/farmacología , Adolescente , Animales , Línea Celular Tumoral , Niño , Preescolar , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Técnicas de Silenciamiento del Gen , Estudio de Asociación del Genoma Completo , Humanos , MAP Quinasa Quinasa 2/antagonistas & inhibidores , MAP Quinasa Quinasa 2/genética , MAP Quinasa Quinasa 2/metabolismo , MAP Quinasa Quinasa 4/antagonistas & inhibidores , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Masculino , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The chemokine receptor CCR7 directs mature dendritic cells (mDCs) to the lymph nodes where these cells control the initiation of the immune response. CCR7 regulates chemotaxis, endocytosis, survival, migratory speed, and cytoarchitecture in mDCs. The molecular mechanisms used by CCR7 to regulate these functions in mDCs are not completely understood. The mammalian sterile 20-like 1 kinase (Mst1) plays a proapoptotic role under stress conditions; however, recently, it has been shown that Mst1 can also control homeostatic cell functions under normal conditions. In this study, we show that stimulation of CCR7 in mDCs induces Gαi-dependent activation of Mst1, suggesting the involvement of this kinase in the control of CCR7-dependent functions. Analysis of the mDCs in which Mst1 expression levels were reduced with small interfering RNA shows that this kinase mediates CCR7-dependent effects on cytoarchitecture, endocytosis and migratory speed but not on chemotaxis or survival. In line with these results, biochemical analysis indicates that Mst1 does not control key signaling regulators of CCR7-dependent chemotaxis or survival. In contrast, Mst1 regulates downstream of CCR7 and, of note, independently of Gα13, the RhoA pathway. Reduction of Mst1 inhibits CCR7-dependent phosphorylation of downstream targets of RhoA, including cofilin, myosin L chain, and myosin L chain phosphatase. Consistent with the role of the latter molecules as modulators of the actin cytoskeleton, mDCs with reduced Mst1 also displayed a dramatic reduction in actin barbed-end formation that could not be recovered by stimulating CCR7. The results indicate that the kinase Mst1 controls selective CCR7-dependent functions in human mDCs.
Asunto(s)
Células Dendríticas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores CCR7/inmunología , Transducción de Señal/inmunología , Citoesqueleto de Actina/metabolismo , Apoptosis/genética , Apoptosis/inmunología , Supervivencia Celular/genética , Células Cultivadas , Quimiotaxis/genética , Cofilina 1/metabolismo , Endocitosis/genética , Activación Enzimática , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ganglios Linfáticos/inmunología , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , ARN Interferente Pequeño , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Chemokine receptor CCR7 directs mature dendritic cells (mDCs) to secondary lymph nodes where these cells regulate the activation of T cells. CCR7 also promotes survival in mDCs, which is believed to take place largely through Akt-dependent signaling mechanisms. We have analyzed the involvement of the AMP-dependent kinase (AMPK) in the control of CCR7-dependent survival. A pro-apoptotic role for AMPK is suggested by the finding that pharmacological activators induce apoptosis, whereas knocking down of AMPK with siRNA extends mDC survival. Pharmacological activation of AMPK also induces apoptosis of mDCs in the lymph nodes. Stimulation of CCR7 leads to inhibition of AMPK, through phosphorylation of Ser-485, which was mediated by G(i)/Gßγ, but not by Akt or S6K, two kinases that control the phosphorylation of AMPK on Ser-485 in other settings. Using selective pharmacological inhibitors, we show that CCR7-induced phosphorylation of AMPK on Ser-485 is mediated by MEK and ERK. Coimmunoprecipitation analysis and proximity ligation assays indicate that AMPK associates with ERK, but not with MEK. These results suggest that in addition to Akt-dependent signaling mechanisms, CCR7 can also promote survival of mDCs through a novel MEK1/2-ERK1/2-AMPK signaling axis. The data also suggest that AMPK may be a potential target to modulate mDC lifespan and the immune response.
Asunto(s)
Proteínas Quinasas Activadas por AMP/genética , Inmunidad Innata/genética , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 2/genética , Receptores CCR7/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Apoptosis/genética , Supervivencia Celular , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Humanos , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Fosforilación , Receptores CCR7/genética , Transducción de Señal/genética , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Chemokines control several cell functions in addition to chemotaxis. Although much information is available on the involvement of specific signaling molecules in the control of single functions controlled by chemokines, especially chemotaxis, the mechanisms used by these ligands to regulate several cell functions simultaneously are completely unknown. Mature dendritic cells (maDCs) migrate through the afferent lymphatic vessels to the lymph nodes, where they regulate the initiation of the immune response. As maDCs are exposed to chemokine CXCL12 (receptors CXCR4 and CXCR7) during their migration, its functions are amenable to be regulated by this ligand. We have used maDCs as a model system to analyze the mechanisms whereby CXCL12 simultaneously controls chemotaxis and survival in maDCs. We show that CXCL12 uses CXCR4, but not CXCR7, and the components of a signaling core that includes G(i)/Gßγ, PI3K-α/-δ/-γ, Akt, ERK1/2 and mammalian target of rapamycin complex 1 (mTORC1), which organize hierarchically to control both functions. Downstream of Akt, Forkhead box class O (FOXO) regulates CXCL12-dependent survival, but not chemotaxis, suggesting that downstream of the aforementioned signaling core, additional signaling molecules may control more selectively CXCL12-dependent chemotaxis or survival. Finally, the data obtained also show that CXCR4 uses a signaling signature that is different from that used by CCR7 to control similar functions.
Asunto(s)
Quimiocina CXCL12/metabolismo , Quimiotaxis/fisiología , Células Dendríticas/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores CXCR4/metabolismo , Animales , Supervivencia Celular/fisiología , Células Cultivadas , Células Dendríticas/citología , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Complejos Multiproteicos , Receptores CCR7/metabolismo , Serina-Treonina Quinasas TORRESUMEN
Migration of mature dendritic cells (mDCs) to secondary lymphoid organs is required for the development of immunity. Recently, we reported that polysialic acid (PSA) and the transmembrane glycoprotein neuropilin-2 (NRP2) control mDC chemotaxis to CCL21 and that this process is dependent on the C-terminal basic region of the chemokine. Herein, we provide further insight into the molecular components controlling PSA regulated chemotaxis in mDCs. In the present study, we demonstrate that human mDCs express the NRP2 isoforms NRP2a and NRP2b, that both of them are susceptible to polysialylation and that polysialylation is required to specifically enhance chemotaxis toward CCL21 in mDCs. The results presented suggest that PSA attached to NRP2 isoforms acts as a binding module for the CCL21 chemokine, thereby facilitating its presentation to the chemokine receptor CCR7. To investigate the relevance of polysialylation on mDC migration, a xenograft mouse model was used and the migration of human DCs to mouse lymph nodes analyzed. Here, we demonstrate that the depletion of PSA from mDCs results in a drastic reduction in the migration of the cells to draining popliteal lymph nodes. With this finding, we provide first evidence that PSA is a crucial factor for in vivo migration of mDCs to lymph nodes.
Asunto(s)
Quimiocina CCL21/fisiología , Quimiotaxis , Células Dendríticas/fisiología , Neuropilina-2/metabolismo , Ácidos Siálicos/metabolismo , Animales , Células Cultivadas , Quimiocina CCL19/farmacología , Quimiocina CCL21/farmacología , Células Dendríticas/efectos de los fármacos , Glicosilación , Humanos , Ratones , Neuropilina-2/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Chemokine receptor CCR7 regulates chemotaxis and survival in mature dendritic cells (DCs). We studied the role of glycogen synthase kinase-3beta (GSK3beta) in the regulation of CCR7-dependent survival. We show that GSK3beta behaves as a proapoptotic regulator in cultured monocyte-derived human DCs and murine splenic DCs in vitro, and in lymph node DCs in vivo. In keeping with its prosurvival role, stimulation of CCR7 induced phosphorylation/inhibition of GSK3beta, which was mediated by the prosurvival regulator Akt1, but it was independent of ERK1/2, a key regulator of chemotaxis. Stimulation of CCR7 also induced translocation of two transcription-factor targets of Akt, prosurvival NF-kappaB and proapoptotic FOXO1, to the nucleus and cytosol, respectively, resulting in DCs with a phenotype more resistant to apoptotic stimuli. We analyzed if GSK3beta was able to modulate the mobilizations of these transcription factors. Using pharmacological inhibitors, small interfering RNA, and a construct encoding constitutively active GSK3beta, we show that active GSK3beta fosters and hampers the translocations to the nucleus of FOXO and NF-kappaB, respectively. Inhibition of GSK3beta resulted in the degradation of the NF-kappaB inhibitor IkappaB, indicating a mechanism whereby GSK3 can control the translocation of NF-kappaB to the nucleus. GSK3beta and FOXO interacted in vivo, suggesting that this transcription factor could be a substrate of GSK3. The results provide a novel mechanism whereby active GSK3beta contributes to regulate apoptosis in DCs. They also suggest that upon stimulation of CCR7, Akt-mediated phosphorylation/inhibition of GSK3beta may be required to allow complete translocations of FOXO and NF-kappaB that confer DCs an extended survival.
Asunto(s)
Células Dendríticas/inmunología , Glucógeno Sintasa Quinasa 3/inmunología , Receptores CCR7/inmunología , Adyuvantes Inmunológicos/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Quimiocina CCL19/farmacología , Quimiocina CCL21/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/efectos de los fármacos , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Quinasa I-kappa B/inmunología , Quinasa I-kappa B/metabolismo , Cloruro de Litio/farmacología , Ratones , Ratones Endogámicos C57BL , FN-kappa B/inmunología , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/inmunología , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores CCR7/agonistas , Receptores CCR7/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Tiazoles/farmacología , Urea/análogos & derivados , Urea/farmacologíaRESUMEN
Dendritic cell (DC) migration to secondary lymphoid organs is a critical step to properly exert its role in immunity and predominantly depends on the interaction of the chemokine receptor CCR7 with its ligands CCL21 and CCL19. Polysialic acid (PSA) has been recently reported to control CCL21-directed migration of mature DCs. Here, we first demonstrate that PSA present on human mature monocyte-derived dendritic cells did not enhance chemotactic responses to CCL19. We have also explored the molecular mechanisms underlying the selective enhancing effect of PSA on CCL21-driven chemotaxis of DCs. In this regard, we found out that prevention of DC polysialylation decreased CCL21 activation of JNK and Akt signaling pathways, both associated with CCR7-mediated chemotaxis. We also report that the enhanced PSA-mediated effect on DC migration towards CCL21 relied on the highly basic C-terminal region of this chemokine and depended on the PSA acceptor molecule neuropilin-2 (NRP2) and on the polysialyltransferase ST8SiaIV. Altogether, our data indicate that the CCR7/CCL21/NRP2/ST8SiaIV functional axis constitutes an important guidance clue for DC targeting to lymphoid organs.
Asunto(s)
Movimiento Celular/fisiología , Quimiocina CCL21/química , Quimiocina CCL21/metabolismo , Células Dendríticas/fisiología , Neuropilina-2/metabolismo , Neuropilina-2/fisiología , Secuencia de Aminoácidos , Aminoácidos Básicos/química , Aminoácidos Básicos/metabolismo , Animales , Células COS , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Células Cultivadas , Quimiocina CCL21/farmacología , Quimiocina CCL21/fisiología , Chlorocebus aethiops , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Neuropilina-2/antagonistas & inhibidores , Neuropilina-2/genética , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas/fisiología , Procesamiento Proteico-Postraduccional/fisiología , ARN Interferente Pequeño/farmacología , Homología de Secuencia de Aminoácido , Ácidos Siálicos/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Glucocorticoids (GCs) are a central component of therapy for patients with T cell acute lymphoblastic leukemia (T-ALL), and although resistance to GCs is a strong negative prognostic indicator in T-ALL, the mechanisms of GC resistance remain poorly understood. Using diagnostic samples from patients enrolled in the frontline Children's Oncology Group (COG) T-ALL clinical trial AALL1231, we demonstrated that one-third of primary T-ALLs were resistant to GCs when cells were cultured in the presence of IL-7, a cytokine that is critical for normal T cell function and that plays a well-established role in leukemogenesis. We demonstrated that in these T-ALLs and in distinct populations of normal developing thymocytes, GCs paradoxically induced their own resistance by promoting upregulation of IL-7 receptor (IL-7R) expression. In the presence of IL-7, this augmented downstream signal transduction, resulting in increased STAT5 transcriptional output and upregulation of the prosurvival protein BCL-2. Taken together, we showed that IL-7 mediates an intrinsic and physiologic mechanism of GC resistance in normal thymocyte development that is retained during leukemogenesis in a subset of T-ALLs and is reversible with targeted inhibition of the IL-7R/JAK/STAT5/BCL-2 axis.
Asunto(s)
Resistencia a Antineoplásicos , Glucocorticoides/farmacología , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Transducción de Señal , Timocitos , Animales , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/inmunología , Humanos , Interleucina-7/inmunología , Subunidad alfa del Receptor de Interleucina-7/inmunología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Factor de Transcripción STAT5/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Timocitos/inmunología , Timocitos/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) is a genetically heterogeneous subtype of B-cell ALL characterized by chromosomal rearrangements and mutations that result in aberrant cytokine receptor and kinase signaling. In particular, chromosomal rearrangements resulting in the overexpression of cytokine receptor-like factor 2 (CRLF2) occur in 50% of Ph-like ALL cases. CRLF2 overexpression is associated with particularly poor clinical outcomes, though the molecular basis for this is currently unknown. Glucocorticoids (GCs) are integral to the treatment of ALL and GC resistance at diagnosis is an important negative prognostic factor. Given the importance of GCs in ALL therapy and the poor outcomes for patients with CRLF2 overexpression, we hypothesized that the aberrant signal transduction associated with CRLF2 overexpression might mediate intrinsic GC insensitivity. To test this hypothesis, we exposed Ph-like ALL cells from patient-derived xenografts to GCs and found that CRLF2 rearranged (CRLF2R) leukemias uniformly demonstrated reduced GC sensitivity in vitro. Furthermore, targeted inhibition of signal transduction with the MEK inhibitor trametinib and the Akt inhibitor MK2206, but not the JAK inhibitor ruxolitinib, was sufficient to augment GC sensitivity. These data suggest that suboptimal GC responses may in part underlie the poor clinical outcomes for patients with CRLF2 overexpression and provide rationale for combination therapy involving GCs and signal transduction inhibitors as a means of enhancing GC efficacy.
Asunto(s)
Resistencia a Antineoplásicos/genética , Reordenamiento Génico , Glucocorticoides/farmacología , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Receptores de Citocinas/genética , Línea Celular Tumoral , Regulación Leucémica de la Expresión Génica , Glucocorticoides/uso terapéutico , Humanos , Cromosoma Filadelfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Inhibidores de Proteínas Quinasas/uso terapéutico , Transducción de Señal/efectos de los fármacosRESUMEN
Poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors are a class of anticancer drugs that block the catalytic activity of PARP proteins. Optimization of our lead compound 1 (( Z)-2-benzylidene-3-oxo-2,3-dihydrobenzofuran-7-carboxamide; PARP-1 IC50 = 434 nM) led to a tetrazolyl analogue (51, IC50 = 35 nM) with improved inhibition. Isosteric replacement of the tetrazole ring with a carboxyl group (60, IC50 = 68 nM) gave a promising new lead, which was subsequently optimized to obtain analogues with potent PARP-1 IC50 values (4-197 nM). PARP enzyme profiling revealed that the majority of compounds are selective toward PARP-2 with IC50 values comparable to clinical inhibitors. X-ray crystal structures of the key inhibitors bound to PARP-1 illustrated the mode of interaction with analogue appendages extending toward the PARP-1 adenosine-binding pocket. Compound 81, an isoform-selective PARP-1/-2 (IC50 = 30 nM/2 nM) inhibitor, demonstrated selective cytotoxic effect toward breast cancer gene 1 ( BRCA1)-deficient cells compared to isogenic BRCA1-proficient cells.
Asunto(s)
Adenosina/metabolismo , Benzofuranos/síntesis química , Benzofuranos/farmacología , Diseño de Fármacos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/síntesis química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Secuencias de Aminoácidos , Benzofuranos/química , Benzofuranos/metabolismo , Biocatálisis , Línea Celular Tumoral , Técnicas de Química Sintética , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Poli(ADP-Ribosa) Polimerasas/química , Relación Estructura-ActividadRESUMEN
Kinase inhibitors have dramatically increased patient survival in a multitude of cancers, including hematological malignancies. However, kinase inhibitors have not yet been integrated into current clinical trials for patients with T-cell-lineage acute lymphoblastic leukemia (T-ALL). In this study, we used a high-throughput flow cytometry (HTFC) approach to test a collection of small-molecule inhibitors, including 26 FDA-approved tyrosine kinase inhibitors in a panel of T-ALL cell lines and patient-derived xenografts. Because hypoxia is known to cause resistance to chemotherapy, we developed a synthetic niche that mimics the low oxygen levels found in leukemic bone marrow to evaluate the effects of hypoxia on the tested inhibitors. Drug sensitivity screening was performed using the Agilent BioCel automated liquid handling system integrated with the HyperCyt HT flow cytometry platform, and the uptake of propidium iodide was used as an indication of cell viability. The HTFC dose-response testing identified several compounds that were efficacious in both normal and hypoxic conditions. This study shows that some clinically approved kinase inhibitors target T-ALL in the hypoxic niche of the bone marrow.
Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , Reposicionamiento de Medicamentos , Citometría de Flujo , Ensayos Analíticos de Alto Rendimiento , Bibliotecas de Moléculas Pequeñas , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas/métodos , Reposicionamiento de Medicamentos/métodos , Citometría de Flujo/métodos , Humanos , Ratones , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
The liver X receptors (LXRs) are ligand-activated nuclear receptors with established roles in the maintenance of lipid homeostasis in multiple tissues. LXRs exert additional biological functions as negative regulators of inflammation, particularly in macrophages. However, the transcriptional responses controlled by LXRs in other myeloid cells, such as dendritic cells (DCs), are still poorly understood. Here we used gain- and loss-of-function models to characterize the impact of LXR deficiency on DC activation programs. Our results identified an LXR-dependent pathway that is important for DC chemotaxis. LXR-deficient mature DCs are defective in stimulus-induced migration in vitro and in vivo Mechanistically, we show that LXRs facilitate DC chemotactic signaling by regulating the expression of CD38, an ectoenzyme important for leukocyte trafficking. Pharmacological or genetic inactivation of CD38 activity abolished the LXR-dependent induction of DC chemotaxis. Using the low-density lipoprotein receptor-deficient (LDLR-/-) LDLR-/- mouse model of atherosclerosis, we also demonstrated that hematopoietic CD38 expression is important for the accumulation of lipid-laden myeloid cells in lesions, suggesting that CD38 is a key factor in leukocyte migration during atherogenesis. Collectively, our results demonstrate that LXRs are required for the efficient emigration of DCs in response to chemotactic signals during inflammation.
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Quimiotaxis/fisiología , Células Dendríticas/fisiología , Receptores X del Hígado/fisiología , ADP-Ribosil Ciclasa 1/metabolismo , Animales , Células Cultivadas , Células Dendríticas/citología , Inflamación , Metabolismo de los Lípidos , Receptores X del Hígado/genética , Macrófagos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Nucleares Huérfanos , Receptores Citoplasmáticos y Nucleares , Transducción de SeñalRESUMEN
Although there are multiple methods for analyzing apoptosis in cultured cells, methodologies for analyzing apoptosis in vivo are sparse. In this protocol, we describe how to detect apoptosis of leukocytes in mouse lymph nodes (LNs) via the detection of apoptotic caspases. We have previously used this protocol to study factors that modulate dendritic cell (DC) survival in LNs; however, it can also be used to analyze other leukocytes that migrate to the LNs. DCs labeled with a fluorescent cell tracker are subcutaneously injected in the posterior footpads of mice. Once the labeled DCs reach the popliteal LN (PLN), the animals are intravenously injected with FLIVO, a permeant fluorescent reagent that selectively marks active caspases and consequently apoptotic cells. Explanted PLNs are then examined under a two-photon microscope to look for the presence of apoptotic cells among the DCs injected. The protocol requires 6-6.5 h for preparation and analysis plus an additional 34-40 h to allow apoptosis of the injected DCs in the PLN.
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Apoptosis/inmunología , Leucocitos/citología , Ganglios Linfáticos/citología , Animales , Bencimidazoles , Inhibidores de Caspasas/metabolismo , Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/metabolismo , Leucocitos/inmunología , Ganglios Linfáticos/inmunología , Ratones , Modelos BiológicosRESUMEN
The adaptive immune response requires the formation of a specialized interface called the immunological synapse (IS), which is formed between a mature dendritic cell (DC) and a CD4(+) T cell in the lymph node. The IS involves organized motifs formed by cell-surface and cytoplasmic molecules at both the DC side (IS-DC) and the T cell side (IS-T) of the IS. Most studies of the functions of the IS have focused on the IS-T; however, to understand the function(s) of the entire IS, it is also necessary to gain insight into the role(s) of the IS-DC. Unlike T cells, which upon their activation leave the lymph node and return to the circulation, DCs largely become apoptotic and die in the node region. This latter observation and the known stability of the IS, which may last for hours, is consistent with the hypothesis that one of the functions of the IS-DC could be the temporal inhibition of the apoptosis of DCs, which would enable the activation of clonal T cells in the lymph nodes. Here, we discuss experimental data supporting the latter hypothesis, as well as the concept that the IS-DC is a signaling region that contributes to the functions of the IS.
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Células Dendríticas/inmunología , Sinapsis Inmunológicas/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Humanos , Transducción de Señal/inmunologíaRESUMEN
Immunological synapses (IS) are emerging as highly organized 3D structures -formed by surface and cytoplasmic signalling and cytoskeletal molecules - that assemble at the zone of contact between a T cell and an antigen presenting cell (APC). The IS control functions that allow APC and T cells modulate the immune response.
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Sinapsis Inmunológicas/fisiología , Animales , Células Presentadoras de Antígenos/inmunología , Comunicación Celular/inmunología , Proteínas del Citoesqueleto/inmunología , Células Dendríticas/inmunología , Humanos , Transducción de Señal , Linfocitos T/inmunologíaRESUMEN
The activation and clonal expansion of naive T cells by antigen-loaded dendritic cells (DCs) in the lymph nodes is a key event during immune response. This activation involves the formation of a specialized cell-cell contact region, formed between a mature DC and a CD4 T cell, which is called immunological synapse (IS). The IS includes a DC and a T cell side that we call IS(DC) and IS(T cell), respectively. Most studies on the IS have focused on the IS(T cell) and the information gathered on the IS(DC) is sparse. However, lines of emerging evidence indicate that the IS(DC), likewise the IS(T cell), is a signaling and functional region that makes important contribution to T cell activation and immune response.