RESUMEN
Background: Because CHARGE syndrome is characterized by high clinical variability, molecular confirmation of the clinical diagnosis is of pivotal importance. Most patients have a pathogenic variant in the CHD7 gene; however, variants are distributed throughout the gene and most cases are due to de novo mutations. Often, assessing the pathogenetic effect of a variant can be challenging, requiring the design of a unique assay for each specific case. Method: Here we describe a new CHD7 intronic variant, c.5607+17A>G, identified in two unrelated patients. In order to characterize the molecular effect of the variant, minigenes were constructed using exon trapping vectors. Results: The experimental approach pinpoints the pathogenetic effect of the variant on CHD7 gene splicing, subsequently confirmed using cDNA synthetized from RNA extracted from patient lymphocytes. Our results were further corroborated by the introduction of other substitutions at the same nucleotide position, showing that c.5607+17A>G specifically alters splicing possibly due to the generation of a recognition motif for the recruitment of a splicing effector. Conclusion: Here we identify a novel pathogenetic variant affecting splicing, and we provide a detailed molecular characterization and possible functional explanation.
RESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological disorder that results from the clonal transformation of T-cell precursors. Phosphatidylinositol 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) and canonical Wnt/ß-catenin signaling pathways play a crucial role in T-cell development and in self-renewal of healthy and leukemic stem cells. Notably, ß-catenin is a transcriptional regulator of several genes involved in cancer cell proliferation and survival. In this way, aberrations of components belonging to the aforementioned networks contribute to T-ALL pathogenesis. For this reason, inhibition of both pathways could represent an innovative strategy in this hematological malignancy. Here, we show that combined targeting of Wnt/ß-catenin pathway through ICG-001, a CBP/ß-catenin transcription inhibitor, and of the PI3K/Akt/mTOR axis through ZSTK-474, a PI3K inhibitor, downregulated proliferation, survival, and clonogenic activity of T-ALL cells. ICG-001 and ZSTK-474 displayed cytotoxic effects, and, when combined together, induced a significant increase in apoptotic cells. This induction of apoptosis was associated with the downregulation of Wnt/ß-catenin and PI3K/Akt/mTOR pathways. All these findings were confirmed under hypoxic conditions that mimic the bone marrow niche where leukemic stem cells are believed to reside. Taken together, our findings highlight potentially promising treatment consisting of cotargeting Wnt/ß-catenin and PI3K/Akt/mTOR pathways in T-ALL settings.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Linfocitos T/efectos de los fármacos , beta Catenina/genética , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/genética , Pirimidinonas/farmacología , Linfocitos T/metabolismo , Serina-Treonina Quinasas TOR/genética , Triazinas/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genética , beta Catenina/antagonistas & inhibidoresRESUMEN
Familial aggregation is a significant risk factor for the development of thyroid cancer and familial non-medullary thyroid cancer (FNMTC) accounts for 5-7% of all NMTC. Whole exome sequencing analysis in the family affected by FNMTC with oncocytic features where our group previously identified a predisposing locus on chromosome 19p13.2, revealed a novel heterozygous mutation (c.400G > A, NM_012335; p.Gly134Ser) in exon 5 of MYO1F, mapping to the linkage locus. In the thyroid FRTL-5 cell model stably expressing the mutant MYO1F p.Gly134Ser protein, we observed an altered mitochondrial network, with increased mitochondrial mass and a significant increase in both intracellular and extracellular reactive oxygen species, compared to cells expressing the wild-type (wt) protein or carrying the empty vector. The mutation conferred a significant advantage in colony formation, invasion and anchorage-independent growth. These data were corroborated by in vivo studies in zebrafish, since we demonstrated that the mutant MYO1F p.Gly134Ser, when overexpressed, can induce proliferation in whole vertebrate embryos, compared to the wt one. MYO1F screening in additional 192 FNMTC families identified another variant in exon 7, which leads to exon skipping, and is predicted to alter the ATP-binding domain in MYO1F. Our study identified for the first time a role for MYO1F in NMTC.
Asunto(s)
Proliferación Celular , Embrión no Mamífero/patología , Mitocondrias/patología , Mutación , Miosina Tipo I/genética , Cáncer Papilar Tiroideo/patología , Neoplasias de la Tiroides/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apoptosis , Células Cultivadas , Niño , Cromosomas Humanos Par 19 , Embrión no Mamífero/metabolismo , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias/genética , Mitocondrias/metabolismo , Miosina Tipo I/química , Miosina Tipo I/metabolismo , Consumo de Oxígeno , Linaje , Conformación Proteica , Cáncer Papilar Tiroideo/genética , Cáncer Papilar Tiroideo/metabolismo , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Adulto Joven , Pez CebraRESUMEN
Despite remarkable progress in polychemotherapy protocols, pediatric B-cell acute lymphoblastic leukemia (B-ALL) remains fatal in around 20% of cases. Hence, novel targeted therapies are needed for patients with poor prognosis. Glucocorticoids (GCs) are drugs commonly administrated for B-ALL treatment. Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin signaling pathway is frequently observed in B-ALL and contributes to GC-resistance. Here, we analyzed for the first time to our knowledge, the therapeutic potential of pan and isoform-selective PI3K p110 inhibitors, alone or combined with dexamethasone (DEX), in B-ALL leukemia cell lines and patient samples. We found that a pan PI3K p110 inhibitor displayed the most powerful cytotoxic effects in B-ALL cells, by inducing cell cycle arrest and apoptosis. Both a pan PI3K p110 inhibitor and a dual γ/δ PI3K p110 inhibitor sensitized B-ALL cells to DEX by restoring nuclear translocation of the GC receptor and counteracted stroma-induced DEX-resistance. Finally, gene expression analysis documented that, on one hand the combination consisting of a pan PI3K p110 inhibitor and DEX strengthened the DEX-induced up- or down-regulation of several genes involved in apoptosis, while on the other, it rescued the effects of genes that might be involved in GC-resistance. Overall, our findings strongly suggest that PI3K p110 inhibition could be a promising strategy for treating B-ALL patients by improving GC therapeutic effects and/or overcoming GC-resistance.
Asunto(s)
Antineoplásicos/farmacología , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Dexametasona/farmacología , Glucocorticoides/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Linfocitos B/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Niño , Preescolar , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Isoquinolinas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Purinas/farmacología , Quinazolinonas/farmacología , Quinoxalinas/farmacología , Tiazolidinedionas/farmacología , Triazinas/farmacologíaRESUMEN
BACKGROUND: Although in recent years, the introduction of novel chemotherapy protocols has improved the outcome of T cell acute lymphoblastic leukemia (T-ALL) patients, refractory and/or relapsing disease remains a foremost concern. In this context, a major contribution was provided by the introduction of the nucleoside analog nelarabine, approved for salvage treatment of T-ALL patients with refractory/relapsed disease. However, nelarabine could induce a life-threatening, dose-dependent neurotoxicity. To improve nelarabine efficacy, we have analyzed its molecular targets, testing selective inhibitors of such targets in combination with nelarabine. METHODS: The effectiveness of nelarabine as single agent or in combination with PI3K, Bcl2, and MEK inhibitors was evaluated on human T-ALL cell lines and primary T-ALL refractory/relapsed lymphoblasts. The efficacy of signal modulators in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed by flow cytometry, western blotting, and quantitative real-time PCR in T-ALL settings. RESULTS: Treatment with nelarabine as a single agent identified two groups of T-ALL cell lines, one sensitive and one resistant to the drug. Whereas sensitive T-ALL cells showed a significant increase of apoptosis and a strong down-modulation of PI3K signaling, resistant T-ALL cells showed a hyperactivation of AKT and MEK/ERK1/2 signaling pathways, not caused by differences in the expression of nelarabine transporters or metabolic activators. We then studied the combination of nelarabine with the PI3K inhibitors (both pan and dual γ/δ inhibitors), with the Bcl2 specific inhibitor ABT199, and with the MEK inhibitor trametinib on both T-ALL cell lines and patient samples at relapse, which displayed constitutive activation of PI3K signaling and resistance to nelarabine alone. The combination with the pan PI3K inhibitor ZSTK-474 was the most effective in inhibiting the growth of T-ALL cells and was synergistic in decreasing cell survival and inducing apoptosis in nelarabine-resistant T-ALL cells. The drug combination caused AKT dephosphorylation and a downregulation of Bcl2, while nelarabine alone induced an increase in p-AKT and Bcl2 signaling in the resistant T-ALL cells and relapsed patient samples. CONCLUSIONS: These findings indicate that nelarabine in combination with PI3K inhibitors may be a promising therapeutic strategy for the treatment of T-ALL relapsed patients.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidad , Apoptosis/efectos de los fármacos , Arabinonucleósidos/uso terapéutico , Arabinonucleósidos/toxicidad , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/complicaciones , Proteínas Proto-Oncogénicas c-akt , Piridonas/uso terapéutico , Pirimidinonas/uso terapéutico , Sulfonamidas/uso terapéutico , Triazinas/uso terapéutico , Células Tumorales CultivadasRESUMEN
The bone marrow (BM) microenvironment regulates the properties of healthy hematopoietic stem cells (HSCs) localized in specific niches. Two distinct microenvironmental niches have been identified in the BM, the "osteoblastic (endosteal)" and "vascular" niches. Nevertheless, these niches provide sanctuaries where subsets of leukemic cells escape chemotherapy-induced death and acquire a drug-resistant phenotype. Moreover, it is emerging that leukemia cells are able to remodel the BM niches into malignant niches which better support neoplastic cell survival and proliferation. This review focuses on the cellular and molecular biology of microenvironment/leukemia interactions in acute lymphoblastic leukemia (ALL) of both B- and T-cell lineage. We shall also highlight the emerging role of exosomes/microvesicles as efficient messengers for cell-to-cell communication in leukemia settings. Studies on the interactions between the BM microenvironment and ALL cells have led to the discovery of potential therapeutic targets which include cytokines/chemokines and their receptors, adhesion molecules, signal transduction pathways, and hypoxia-related proteins. The complex interplays between leukemic cells and BM microenvironment components provide a rationale for innovative, molecularly targeted therapies, designed to improve ALL patient outcome. A better understanding of the contribution of the BM microenvironment to the process of leukemogenesis and leukemia persistence after initial remission, may provide new targets that will allow destruction of leukemia cells without adversely affecting healthy HSCs. This article is part of a Special Issue entitled: Tumor Microenvironment Regulation of Cancer Cell Survival, Metastasis,Inflammation, and Immune Surveillance edited by Peter Ruvolo and Gregg L. Semenza.
Asunto(s)
Médula Ósea/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Nicho de Células Madre , Microambiente Tumoral , Antineoplásicos/uso terapéutico , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Moléculas de Adhesión Celular/metabolismo , Quimiocinas/metabolismo , Humanos , Modelos Biológicos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Sphingosine kinase (there are two isoforms, SK1 and SK2) catalyses the formation of sphingosine 1-phosphate (S1P), a bioactive lipid that can be released from cells to activate a family of G protein-coupled receptors, termed S1P1-5. In addition, S1P can bind to intracellular target proteins, such as HDAC1/2, to induce cell responses. There is increasing evidence of a role for S1P receptors (e.g. S1P4) and SK1 in cancer, where high expression of these proteins in ER negative breast cancer patient tumours is linked with poor prognosis. Indeed, evidence will be presented here to demonstrate that S1P4 is functionally linked with SK1 and the oncogene HER2 (ErbB2) to regulate mitogen-activated protein kinase pathways and growth of breast cancer cells. Although much emphasis is placed on SK1 in terms of involvement in oncogenesis, evidence will also be presented for a role of SK2 in both T-cell and B-cell acute lymphoblastic leukemia. In patient T-ALL lymphoblasts and T-ALL cell lines, we have demonstrated that SK2 inhibitors promote T-ALL cell death via autophagy and induce suppression of c-myc and PI3K/AKT pathways. We will also present evidence demonstrating that certain SK inhibitors promote oxidative stress and protein turnover via proteasomal degradative pathways linked with induction of p53-and p21-induced growth arrest. In addition, the SK1 inhibitor, PF-543 exacerbates disease progression in an experimental autoimmune encephalomyelitis mouse model indicating that SK1 functions in an anti-inflammatory manner. Indeed, sphingosine, which accumulates upon inhibition of SK1 activity, and sphingosine-like compounds promote activation of the inflammasome, which is linked with multiple sclerosis, to stimulate formation of the pro-inflammatory mediator, IL-1ß. Such compounds could be exploited to produce antagonists that diminish exaggerated inflammation in disease. The therapeutic potential of modifying the SK-S1P receptor pathway in cancer and inflammation will therefore, be reviewed.
Asunto(s)
Inflamación/enzimología , Neoplasias/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/metabolismo , Animales , Humanos , Inflamación/genética , Inflamación/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Receptores de Lisoesfingolípidos/genéticaRESUMEN
The proteasome inhibitor bortezomib is a new targeted treatment option for refractory or relapsed acute lymphoblastic leukemia (ALL) patients. However, a limited efficacy of bortezomib alone has been reported. A terminal pro-apoptotic endoplasmic reticulum (ER) stress/unfolded protein response (UPR) is one of the several mechanisms of bortezomib-induced apoptosis. Recently, it has been documented that UPR disruption could be considered a selective anti-leukemia therapy. CX-4945, a potent casein kinase (CK) 2 inhibitor, has been found to induce apoptotic cell death in T-ALL preclinical models, via perturbation of ER/UPR pathway. In this study, we analyzed in T- and B-ALL preclinical settings, the molecular mechanisms of synergistic apoptotic effects observed after bortezomib/CX-4945 combined treatment. We demonstrated that, adding CX-4945 after bortezomib treatment, prevented leukemic cells from engaging a functional UPR in order to buffer the bortezomib-mediated proteotoxic stress in ER lumen. We documented that the combined treatment decreased pro-survival ER chaperon BIP/Grp78 expression, via reduction of chaperoning activity of Hsp90. Bortezomib/CX-4945 treatment inhibited NF-κB signaling in T-ALL cell lines and primary cells from T-ALL patients, but, intriguingly, in B-ALL cells the drug combination activated NF-κB p65 pro-apoptotic functions. In fact in B-cells, the combined treatment induced p65-HDAC1 association with consequent repression of the anti-apoptotic target genes, Bcl-xL and XIAP. Exposure to NEMO (IKKγ)-binding domain inhibitor peptide reduced the cytotoxic effects of bortezomib/CX-4945 treatment. Overall, our findings demonstrated that CK2 inhibition could be useful in combination with bortezomib as a novel therapeutic strategy in both T- and B-ALL.
Asunto(s)
Apoptosis/efectos de los fármacos , Bortezomib/farmacología , Proteínas de Choque Térmico/metabolismo , Naftiridinas/farmacología , Factor de Transcripción ReIA/metabolismo , Antineoplásicos/farmacología , Western Blotting , Quinasa de la Caseína II/antagonistas & inhibidores , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Células Jurkat , Microscopía Fluorescente , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Fenazinas , Leucemia-Linfoma Linfoblástico de Células Precursoras B/sangre , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangre , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/sangre , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
Class I phosphatidylinositol 3-kinases (PI3Ks) are frequently activated in T-cell acute lymphoblastic leukemia (T-ALL), mainly due to the loss of PTEN function. Therefore, targeting PI3Ks is a promising innovative approach for T-ALL treatment, however at present no definitive evidence indicated which is the better therapeutic strategy between pan or selective isoform inhibition, as all the four catalytic subunits might participate in leukemogenesis. Here, we demonstrated that in both PTEN deleted and PTEN non deleted T-ALL cell lines, PI3K pan-inhibition exerted the highest cytotoxic effects when compared to both selective isoform inhibition or dual p110γ/δ inhibition. Intriguingly, the dual p110γ/δ inhibitor IPI-145 was effective in Loucy cells, which are representative of early T-precursor (ETP)-ALL, a T-ALL subtype associated with a poor outcome. PTEN gene deletion did not confer a peculiar reliance of T-ALL cells on PI3K activity for their proliferation/survival, as PTEN was inactivated in PTEN non deleted cells, due to posttranslational mechanisms. PI3K pan-inhibition suppressed Akt activation and induced caspase-independent apoptosis. We further demonstrated that in some T-ALL cell lines, autophagy could exert a protective role against PI3K inhibition. Our findings strongly support clinical application of class I PI3K pan-inhibitors in T-ALL treatment, with the possible exception of ETP-ALL cases.
Asunto(s)
Inhibidores de las Quinasa Fosfoinosítidos-3 , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Isoenzimas , Células Jurkat , Fosfatidilinositol 3-Quinasas/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Análisis de SupervivenciaRESUMEN
BACKGROUND: Thyroid neoplasias with oncocytic features represent a specific phenotype in non-medullary thyroid cancer, reflecting the unique biological phenomenon of mitochondrial hyperplasia in the cytoplasm. Oncocytic thyroid cells are characterized by a prominent eosinophilia (or oxyphilia) caused by mitochondrial abundance. Although disruptive mutations in the mitochondrial DNA (mtDNA) are the most significant hallmark of such tumors, oncocytomas may be envisioned as heterogeneous neoplasms, characterized by multiple nuclear and mitochondrial gene lesions. We investigated the nuclear mutational profile of oncocytic tumors to pinpoint the mutations that may trigger the early oncogenic hit. METHODS: Total DNA was extracted from paraffin-embedded tissues from 45 biopsies of oncocytic tumors. High-resolution melting was used for mutation screening of mitochondrial complex I subunits genes. Specific nuclear rearrangements were investigated by RT-PCR (RET/PTC) or on isolated nuclei by interphase FISH (PAX8/PPARγ). Recurrent point mutations were analyzed by direct sequencing. RESULTS: In our oncocytic tumor samples, we identified rare TP53 mutations. The series of analyzed cases did not include poorly- or undifferentiated thyroid carcinomas, and none of the TP53 mutated cases had significant mitotic activity or high-grade features. Thus, the presence of disruptive TP53 mutations was completely unexpected. In addition, novel mutations in nuclear-encoded complex I genes were identified. CONCLUSIONS: These findings suggest that nuclear genetic lesions altering the bioenergetics competence of thyroid cells may give rise to an aberrant mitochondria-centered compensatory mechanism and ultimately to the oncocytic phenotype.
Asunto(s)
Complejo I de Transporte de Electrón/genética , Genes Supresores de Tumor , Mutación , Oncogenes , Neoplasias de la Tiroides/genética , Proteína p53 Supresora de Tumor/genética , Análisis Mutacional de ADN , Complejo I de Transporte de Electrón/metabolismo , Genes Microbianos , Genotipo , Humanos , Recombinación Genética , Neoplasias de la Tiroides/diagnóstico , Neoplasias de la Tiroides/patología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive molecule expressed in some subsets of normal and neoplastic cells. Mature human dendritic cells (DCs) have been shown to express IDO1, but little is known about its expression and function during DC differentiation from bone marrow hematopoietic stem/progenitor cells (HSPCs). Here, we show that during in vitro differentiation along the myeloid DC lineage, CD34(+) HSPCs acquire IDO1 expression, which acts in a tolerogenic manner by inducing a population of fully functional CD4(+)CD25(+) FOXP3(+) T-regulatory cells. Phenotypically, CD1a(+)CD14(-) HPSC-derived DCs expressed IDO1, langerin, CD11b, and CD1c. Cell-sorting experiments demonstrated that IDO1 expression is found in a subset of CD1a(+)CD14(-)langerin(+) cells, expressing CD103, which is capable of inducing T-regulatory cells in an IDO1-dependent manner. In conclusion, DC differentiation from CD34(+) HSPCs results in the expression of a functionally active IDO1 protein in CD1a(+)langerin(+), CD103-expressing DCs. These data point toward IDO1 expression as part of a tolerogenic signature during DC development.
Asunto(s)
Antígenos CD34/inmunología , Antígenos CD/inmunología , Células Dendríticas/inmunología , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Cadenas alfa de Integrinas/inmunología , Lectinas Tipo C/inmunología , Lectinas de Unión a Manosa/inmunología , Linfocitos T Reguladores/inmunología , Western Blotting , Células Cultivadas , Humanos , Microscopía FluorescenteRESUMEN
Macroautophagy, usually referred to as autophagy, is a degradative pathway wherein cytoplasmatic components such as aggregated/misfolded proteins and organelles are engulfed within double-membrane vesicles (autophagosomes) and then delivered to lysosomes for degradation. Autophagy plays an important role in the regulation of numerous physiological functions, including hematopoiesis, through elimination of aggregated/misfolded proteins, and damaged/superfluous organelles. The catabolic products of autophagy (amino acids, fatty acids, nucleotides) are released into the cytosol from autophagolysosomes and recycled into bio-energetic pathways. Therefore, autophagy allows cells to survive starvation and other unfavorable conditions, including hypoxia, heat shock, and microbial pathogens. Nevertheless, depending upon the cell context and functional status, autophagy can also serve as a death mechanism. The cohort of proteins that constitute the autophagy machinery function in a complex, multistep biochemical pathway which has been partially identified over the past decade. Dysregulation of autophagy may contribute to the development of several disorders, including acute leukemias. In this kind of hematologic malignancies, autophagy can either act as a chemo-resistance mechanism or have tumor suppressive functions, depending on the context. Therefore, strategies exploiting autophagy, either for activating or inhibiting it, could find a broad application for innovative treatment of acute leukemias and could significantly contribute to improved clinical outcomes. These aspects are discussed here after a brief introduction to the autophagic molecular machinery and its roles in hematopoiesis.
Asunto(s)
Autofagia , Leucemia/patología , Enfermedad Aguda , Autofagia/fisiología , Hematopoyesis , Humanos , Leucemia/terapia , Leucemia Mieloide Aguda/patología , Leucemia Promielocítica Aguda/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologíaRESUMEN
Sphingosine 1-phosphate (S1P) is a bioactive lipid that is formed by the phosphorylation of sphingosine and catalysed by sphingosine kinase 1 (SK1) or sphingosine kinase 2 (SK2). Sphingosine kinases play a fundamental role in many signaling pathways associated with cancer, suggesting that proteins belonging to this signaling network represent potential therapeutic targets. Over the last years, many improvements have been made in the treatment of T-cell acute lymphoblastic leukemia (T-ALL); however, novel and less toxic therapies are still needed, especially for relapsing and chemo-resistant patients. Here, we analyzed the therapeutic potential of SKi and ROMe, a sphingosine kinase 1 and 2 inhibitor and SK2-selective inhibitor, respectively. While SKi induced apoptosis, ROMe initiated an autophagic cell death in our in vitro cell models. SKi treatment induced an increase in SK1 protein levels in Molt-4 cells, whereas it activated the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) pathway in Jurkat and CEM-R cells as protective mechanisms in a sub-population of T-ALL cells. Interestingly, we observed a synergistic effect of SKi with the classical chemotherapeutic drug vincristine. In addition, we reported that SKi affected signaling cascades implicated in survival, proliferation and stress response of cells. These findings indicate that SK1 or SK2 represent potential targets for treating T-ALL.
Asunto(s)
Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Glicoles de Propileno/farmacología , Esfingosina/análogos & derivados , Tiazoles/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , Antineoplásicos/farmacología , Western Blotting , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Clorhidrato de Fingolimod , Humanos , Microscopía Electrónica de Transmisión , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Esfingosina/farmacologíaRESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous neoplastic disorder of immature hematopoietic precursors committed to the T-cell lineage. T-ALL comprises about 15% of pediatric and 25% of adult ALL cases. Even if the prognosis of T-ALL has improved especially in the childhood due to the use of new intensified treatment protocols, the outcome of relapsed patients who are resistant to conventional chemotherapeutic drugs or who relapse is still poor. For this reason, there is a need for novel and less toxic targeted therapies against signaling pathways aberrantly activated in T-ALL, such as the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR). Small molecules designed to target key components of this signaling axis have proven their efficacy both in vitro and in vivo in pre-clinical settings of T-ALL. In particular, different classes of mTOR inhibitors have been disclosed by pharmaceutical companies, and they are currently being tested in clinical trials for treating T-ALL patients. One of the most promising approaches for the treatment of T-ALL seems to be the combination of mTOR inhibitors with traditional chemotherapeutic agents. This could lead to a lower drug dosage that may circumvent the systemic side effects of chemotherapeutics. In this review, we focus on the different classes of mTOR inhibitors that will possibly have an impact on the therapeutic arsenal we have at our disposal against T-ALL.
Asunto(s)
Antineoplásicos/farmacología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica , Humanos , Terapia Molecular Dirigida , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologíaRESUMEN
Polo-like kinases (PLKs) and Aurora kinases (AKs) act as key cell cycle regulators in healthy human cells. In cancer, these protein kinases are often overexpressed and dysregulated, thus contributing to uncontrolled cell proliferation and growth. T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous malignancy arising in the thymus from T-cell progenitors. Primary chemoresistant and relapsed T-ALL patients have yet a poor outcome, therefore novel therapies, targeting signaling pathways important for leukemic cell proliferation, are required. Here, we demonstrate the potential therapeutic effects of BI6727, MK-5108, and GSK1070916, three selective inhibitors of PLK1, AK-A, and AK-B/C, respectively, in a panel of T-ALL cell lines and primary cells from T-ALL patients. The drugs were both cytostatic and cytotoxic to T-ALL cells by inducing G2/M-phase arrest and apoptosis. The drugs retained part of their pro-apoptotic activity in the presence of MS-5 bone marrow stromal cells. Moreover, we document for the first time that BI6727 perturbed both the PI3K/Akt/mTORC2 and the MEK/ERK/mTORC1 signaling pathways, and that a combination of BI6727 with specific inhibitors of the aforementioned pathways (MK-2206, CCI-779) displayed significantly synergistic cytotoxic effects. Taken together, our findings indicate that PLK1 and AK inhibitors display the potential for being employed in innovative therapeutic strategies for improving T-ALL patient outcome.
Asunto(s)
Antineoplásicos/farmacología , Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa B/antagonistas & inhibidores , Proteínas de Ciclo Celular/antagonistas & inhibidores , Diseño de Fármacos , Terapia Molecular Dirigida , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Aurora Quinasa A/metabolismo , Aurora Quinasa B/metabolismo , Compuestos Aza/farmacología , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Ácidos Ciclohexanocarboxílicos/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Indoles/farmacología , Células Jurkat , Ratones , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/efectos de los fármacos , Tiazoles/farmacología , Células Tumorales Cultivadas , Quinasa Tipo Polo 1RESUMEN
BACKGROUND: An early age at Breast Cancer (BC) onset may be a hallmark of inherited predisposition, but BRCA1/2 mutations are only found in a minority of younger BC patients. Among the others, a fraction may carry mutations in rarer BC genes, such as TP53, STK11, CDH1 and PTEN. As the identification of women harboring such mutations allows for targeted risk-management, the knowledge of associated manifestations and an accurate clinical and family history evaluation are warranted. CASE PRESENTATION: We describe the case of a woman who developed an infiltrating ductal carcinoma of the right breast at the age of 32, a contralateral BC at age 36 and another BC of the right breast at 40. When she was 39 years-old, during a dermatological examination, mucocutaneous features suggestive of Cowden Syndrome, a disorder associated to germ-line PTEN mutations, were noticed. PTEN genetic testing revealed the novel c.71A > T (p.Asp24Val) mutation, whose deleterious effect, suggested by conservation data and in silico tools, was definitely demonstrated by the incapacity of mutant PTEN to inhibit Akt phosphorylation when used to complement PTEN-null cells. In BC tissue, despite the absence of LOH or somatic mutations of PTEN, Akt phosphorylation was markedly increased in comparison to normal tissue, thus implying additional somatic events into the deregulation of the PI3K/Akt/mTOR pathway and, presumably, into carcinogenesis. Hence, known oncogenic mutations in PIK3CA (exons 10 and 21) and AKT1 (exon 2) were screened in tumor DNA with negative results, which suggests that the responsible somatic event(s) is a different, uncommon one. CONCLUSION: This case stresses the importance of clinical/genetic assessment of early-onset BC patients in order to identify mutation carriers, who are at high risk of new events, so requiring tailored management. Moreover, it revealed a novel PTEN mutation with pathogenic effect, pointing out, however, the need for further efforts to elucidate the molecular steps of PTEN-associated carcinogenesis.
Asunto(s)
Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/genética , Síndrome de Hamartoma Múltiple/genética , Mutación , Fosfohidrolasa PTEN/genética , Adulto , Edad de Inicio , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Carcinoma Ductal de Mama/enzimología , Carcinoma Ductal de Mama/patología , Carcinoma Ductal de Mama/terapia , Análisis Mutacional de ADN , Femenino , Predisposición Genética a la Enfermedad , Síndrome de Hamartoma Múltiple/enzimología , Síndrome de Hamartoma Múltiple/patología , Síndrome de Hamartoma Múltiple/terapia , Humanos , Fenotipo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
Dendritic cells (DCs) are professional APCs that have a role in the initiation of adaptive immune responses and tolerance. Among the tolerogenic mechanisms, the expression of the enzyme IDO1 represents an effective tool to generate T regulatory cells. In humans, different DC subsets express IDO1, but less is known about the IDO1-related enzyme IDO2. In this study, we found a different pattern of expression and regulation between IDO1 and IDO2 in human circulating DCs. At the protein level, IDO1 is expressed only in circulating myeloid DCs (mDCs) and is modulated by PGE2, whereas IDO2 is expressed in both mDCs and plasmacytoid DCs and is not modulated by PGE2. In healthy subjects, IDO1 expression requires the presence of PGE2 and needs continuous transcription and translation, whereas IDO2 expression is constitutive, independent from suppressor of cytokine signaling 3 activity. Conversely, in patients suffering from inflammatory arthritis, circulating DCs express both IDO1 and IDO2. At the functional level, both mDCs and plasmacytoid DCs generate T regulatory cells through an IDO1/IDO2-dependent mechanism. We conclude that, in humans, whereas IDO1 provides an additional mechanism of tolerance induced by proinflammatory mediators, IDO2 is stably expressed in steady-state conditions and may contribute to the homeostatic tolerogenic capacity of DCs.
Asunto(s)
Células Dendríticas/inmunología , Indolamina-Pirrol 2,3,-Dioxigenasa/fisiología , Linfocitos T Reguladores/inmunología , Artritis Psoriásica/inmunología , Artritis Psoriásica/patología , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Células Cultivadas , Células Dendríticas/clasificación , Células Dendríticas/enzimología , Dinoprostona/farmacología , Dinoprostona/fisiología , Inducción Enzimática/efectos de los fármacos , Homeostasis , Humanos , Tolerancia Inmunológica , Indolamina-Pirrol 2,3,-Dioxigenasa/biosíntesis , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Monocitos/citología , Monocitos/efectos de los fármacos , Especificidad de Órganos , Biosíntesis de Proteínas/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Espondilitis Anquilosante/inmunología , Espondilitis Anquilosante/patología , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/antagonistas & inhibidores , Transcripción Genética/efectos de los fármacos , Triptófano/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
INTRODUCTION: Despite continuous advances in our knowledge of the biology of acute myelogenous leukemia (AML), the prognosis of AML patients treated with standard chemotherapy is still poor, especially in the elderly. Therefore, there is a need for novel targeted and less toxic therapies, particularly for patients who develop resistance to traditional chemotherapeutic drugs. Constitutively active phosphatidylinositol 3-kinase (PI3K) signaling characterizes many types of tumors, including AML, where it negatively influences response to therapeutic treatments. AREAS COVERED: The literature data showed that small inhibitor molecules targeting PI3K signaling induced cell cycle arrest, apoptosis and decreased drug-resistance in AML cells. PI3K inhibitors were also capable of targeting leukemic initiating cells (LICs), the most relevant target for leukemia eradication, whereas they tended to spare healthy hematopoietic stem cells. EXPERT OPINION: Data emerging from pre-clinical settings suggest that the PI3K pathway is critically involved in regulating proliferation, survival and drug-resistance of AML cells. Therefore, we propose that novel drugs targeting this signaling pathway may offer a novel and less toxic treatment option for AML patients, most likely in combination with a lower dosage of traditional chemotherapeutic agents or other innovative therapeutic agents.
Asunto(s)
Leucemia Mieloide Aguda/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Animales , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Transducción de SeñalRESUMEN
Along with their immunogenic role, dendritic cells (DCs) are also critical in maintaining tolerance to self-antigens by inducing regulatory T cells (Tregs) via the expression of the immunomodulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1). In turn, Tregs modulate the maturation and/or function of DCs. In immune thrombocytopenia (ITP), the interaction between DCs and Tregs has never been investigated although decreased number/function of Tregs as well as altered DCs have been described. Here, we ask whether, in ITP: (1) IDO1 expression/activity is decreased in mature DCs; (2) IDO1-mediated Treg generation is impaired; and (3) DC maturation is abnormally modulated by Tregs. We found that in ITP, DCs show reduced capability of upregulating the expression/activity of IDO1. This finding results in the reduced ability of mature DCs of converting T cells into Tregs. In turn, Tregs are characterized by decreased interleukin-10 production and show lower ability of inhibiting DC maturation. In conclusion, these data point out the role of IDO1 in the impaired regulatory T cell development of ITP patients and suggest that the cross-talk between Tregs and DCs is hampered and plays a pathogenetic role.
Asunto(s)
Células Dendríticas/enzimología , Indolamina-Pirrol 2,3,-Dioxigenasa/biosíntesis , Púrpura Trombocitopénica Idiopática/enzimología , Linfocitos T Reguladores/patología , Adulto , Linfocitos T CD4-Positivos/patología , Diferenciación Celular , Técnicas de Cocultivo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Interleucina-10/biosíntesis , Interleucina-10/genética , Subunidad alfa del Receptor de Interleucina-2/análisis , Activación de Linfocitos , Prueba de Cultivo Mixto de Linfocitos , Púrpura Trombocitopénica Idiopática/genética , Púrpura Trombocitopénica Idiopática/inmunología , ARN Mensajero/biosíntesis , Subgrupos de Linfocitos T/patología , Regulación hacia ArribaRESUMEN
BACKGROUND: The immunoregulatory enzyme indoleamine 2,3-dioxygenase, which catalyzes the conversion of tryptophan into kynurenine, is expressed in a significant subset of patients with acute myeloid leukemia, resulting in the inhibition of T-cell proliferation and the induction of regulatory T cells. Acute myeloid leukemia cells can be differentiated into dendritic cells, which have increased immunogenicity and have been proposed as vaccines against leukemia. DESIGN AND METHODS: Leukemic dendritic cells were generated from acute myeloid leukemia cells and used as stimulators in functional assays, including the induction of regulatory T cells. Indoleamine 2,3-dioxygenase expression in leukemic dendritic cells was evaluated at molecular, protein and enzymatic levels. RESULTS: We demonstrate that, after differentiation into dendritic cells, both indoleamine 2,3-dioxygenase-negative and indoleamine 2,3-dioxygenase-positive acute myeloid leukemia samples show induction and up-regulation of indoleamine 2,3-dioxygenase gene and protein, respectively. Indoleamine 2,3-dioxygenase-positive acute myeloid leukemia dendritic cells catabolize tryptophan into kynurenine metabolite and inhibit T-cell proliferation through an indoleamine 2,3-dioxygenase-dependent mechanism. Moreover, indoleamine 2,3-dioxygenase-positive leukemic dendritic cells increase the number of allogeneic and autologous CD4(+)CD25(+) Foxp3(+) T cells and this effect is completely abrogated by the indoleamine 2,3-dioxygenase-inhibitor, 1-methyl tryptophan. Purified CD4(+)CD25(+) T cells obtained from co-culture with indoleamine 2,3-dioxygenase-positive leukemic dendritic cells act as regulatory T cells as they inhibit naive T-cell proliferation and impair the complete maturation of normal dendritic cells. Importantly, leukemic dendritic cell-induced regulatory T cells are capable of in vitro suppression of a leukemia-specific T cell-mediated immune response, directed against the leukemia-associated antigen, Wilms' tumor protein. CONCLUSIONS: These data identify indoleamine 2,3-dioxygenase-mediated catabolism as a tolerogenic mechanism exerted by leukemic dendritic cells and have clinical implications for the use of these cells for active immunotherapy of leukemia.