RESUMEN
Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of diseases primarily involving the skin that could have an aggressive course with circulating blood cells, especially in Sézary syndrome and transformed mycosis fungoides. So far, few CTCL cell lines have been adapted for in vivo experiments and their tumorigenicity has not been adequately assessed, hampering the use of a reproducible model for CTCL biological evaluation. In fact, both patient-derived xenografts and cell line xenografts at subcutaneous sites failed to provide a robust tool, because engraftment was dependent on mice strain and cell line subtype. Herein, we describe an original method of intrahepatic injection into adult NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ mice liver of both aggressive (My-La, HUT78, HH, MAC2A, and MAC2B) and indolent (FE-PD and MAC1) CTCL cell lines. Six of the seven CTCL cell lines were grafted with a high rate of success (80%). Moreover, this model provided a quick (15 days) and robust assay for in vivo evaluation of CTCL cell lines tumorigenicity and therapeutic response in preclinical studies. Such a reproducible model can be therefore used for further functional studies and in vivo drug testing.
Asunto(s)
Línea Celular Tumoral/trasplante , Hígado , Linfoma Cutáneo de Células T/patología , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos NODRESUMEN
White adipose tissue (WAT) is the focus of new interest because of the presence of an abundant and complex immune cell population that is involved in key pathologies such as metabolic syndrome. Based on in vivo reconstitution assays, it is thought that these immune cells are derived from the bone marrow (BM). However, previous studies have shown that WAT exhibits specific hematopoietic activity exerted by an unknown subpopulation of cells. In the present study, we prospectively isolated a peculiar hematopoietic stem/progenitor cell population from murine WAT. The cells are phenotypically similar to BM hematopoietic stem cells and are able to differentiate into both myeloid and lymphoid lineages in vitro. In competitive repopulation assays in vivo, they reconstituted the innate immune compartment in WAT preferentially and more efficiently than BM cells, but did not reconstitute hematopoietic organs. They were also able to give rise to multilineage engraftment in both secondary recipients and in utero transplantation. Therefore, we propose that WAT hematopoietic cells constitute a population of immature cells that are able to renew innate immune cell populations. Considering the amount of WAT in adults, our results suggest that WAT hematopoietic activity controls WAT inflammatory processes and also supports innate immune responses in other organs.
Asunto(s)
Tejido Adiposo Blanco/citología , Tejido Adiposo Blanco/inmunología , Células Madre Hematopoyéticas/citología , Linfocitos/citología , Células Mieloides/citología , Tejido Adiposo Blanco/trasplante , Animales , Antígenos Ly/análisis , Diferenciación Celular , Femenino , Células Madre Hematopoyéticas/inmunología , Inmunidad Innata , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Linfocitos/inmunología , Masculino , Proteínas de la Membrana/análisis , Ratones , Ratones Endogámicos C57BL , Células Mieloides/inmunología , Proteínas Proto-Oncogénicas c-kit/análisisRESUMEN
As a major cancer hallmark, there is a sustained interest in understanding the telomerase contribution to carcinogenesis in order to therapeutically target this enzyme. This is particularly relevant in primary cutaneous T-cell lymphomas (CTCL), a malignancy showing telomerase dysregulation with few investigative data available. In CTCL, we examined the mechanisms involved in telomerase transcriptional activation and activity regulation. We analyzed 94 CTCL patients from a Franco-Portuguese cohort, as well as 8 cell lines, in comparison to 101 healthy controls. Our results showed that not only polymorphisms (SNPs) located at the promoter of human telomerase reverse transcriptase (hTERT) gene (rs2735940 and rs2853672) but also an SNP located within the coding region (rs2853676) could influence CTCL occurrence. Furthermore, our results sustained that the post-transcriptional regulation of hTERT contributes to CTCL lymphomagenesis. Indeed, CTCL cells present a different pattern of hTERT spliced transcripts distribution from the controls, mostly marked by an increase in the hTERT ß+ variants proportion. This increase seems to be associated with CTCL development and progression. Through hTERT splicing transcriptome modulation with shRNAs, we observed that the decrease in the α-ß+ transcript induced a decrease in the cell proliferation and tumorigenic capacities of T-MF cells in vitro. Taken together, our data highlight the major role of post-transcriptional mechanisms regulating telomerase non canonical functions in CTCL and suggest a new potential role for the α-ß+ hTERT transcript variant.
Asunto(s)
Linfoma Cutáneo de Células T , Telomerasa , Humanos , Línea Celular , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Telomerasa/genéticaRESUMEN
Cutaneous T-cell lymphoma (CTCL) such as Sézary syndrome or mycosis fungoides corresponds to an abnormal infiltration of T lymphocytes in the skin. CTCL cells have a heterogeneous phenotype and express cell adhesion molecules such as cutaneous lymphocyte antigen (CLA) supporting skin homing. The use of a mAb (HECA-452) against CLA significantly decreased transendothelial migration and survival of CTCL cells from patient samples and My-La cell line. The decrease of CLA expression by inhibition of its maturation enzyme, ST3 ß-galactoside α-2,3-sialyltransferase 4, also impaired CTCL cell migration, proliferation, and survival. We confirmed in vivo that treatment with anti-CLA mAb decreased the tumorigenicity as well as dissemination of CTCL cells in different tissues compared with the control group. Our findings provide evidence of the involvement of CLA in CTCL cell migration and survival, supporting that CLA inhibition could represent an actionable therapy in patients with CTCL.
Asunto(s)
Linfoma Cutáneo de Células T , Micosis Fungoide , Síndrome de Sézary , Neoplasias Cutáneas , Humanos , Neoplasias Cutáneas/patología , Linfoma Cutáneo de Células T/patología , Micosis Fungoide/patología , Síndrome de Sézary/tratamiento farmacológico , Síndrome de Sézary/patologíaRESUMEN
Cutaneous T-cell lymphomas (CTCLs) are telomerase-positive tumors expressing hTERT, although neither gene rearrangement/amplification nor promoter hotspot mutations could explain the hTERT re-expression. As the hTERT promoter is rich in CpG, we investigated the contribution of epigenetic mechanisms in its re-expression. We analyzed hTERT promoter methylation status in CTCL cells compared with healthy cells. Gene-specific methylation analyses revealed a common methylation pattern exclusively in tumor cells. This methylation pattern encompassed a hypermethylated distal region from -650 to -150 bp and a hypomethylated proximal region from -150 to +150 bp. Interestingly, the hypermethylated region matches with the recently named TERT hypermethylated oncogenic region (THOR). THOR has been associated with telomerase reactivation in many cancers, but it has so far not been reported in cutaneous lymphomas. Additionally, we assessed the effect of THOR on two histone deacetylase inhibitors (HDACi), romidepsin and vorinostat, both approved for CTCL treatment and a DNA methyltransferase inhibitor (DNMTi) 5-azacytidine, unapproved for CTCL. Contrary to our expectations, the findings reported herein revealed that THOR methylation is relatively stable under these epigenetic drugs' pressure, whereas these drugs reduced the hTERT gene expression.
Asunto(s)
Linfoma Cutáneo de Células T , Telomerasa , Metilación de ADN/genética , Epigénesis Genética , Humanos , Linfoma Cutáneo de Células T/genética , Regiones Promotoras Genéticas/genética , Telomerasa/genética , Telomerasa/metabolismoRESUMEN
White adipose tissue (WAT) is a heterogeneous tissue, found in various locations throughout the body, containing mature adipocytes and the stroma-vascular fraction (SVF). The SVF includes a large proportion of immune hematopoietic cells, among which, mast cells that contribute to diet-induced obesity. In this study, we asked whether mast cells present in mice adipose tissue could derive from hematopoietic stem/progenitor cells (HSPC) identified in the tissue. We therefore performed both in vitro and in vivo experiments dedicated to monitoring the progeny of WAT-derived HSPC. The entire study was conducted in parallel with bone marrow-derived cells, considered the gold standard for hematopoietic-lineage studies. Here, we demonstrate that adipose-derived HSPC contain a precursor-cell population committed to the mast cell lineage, and able to efficiently home to peripheral organs such as intestine and skin, where it acquires properties of functional tissue mast cells. Additionally, WAT contains a significant mast cell progenitor population, suggesting that the entire mast cell lineage process take place in WAT. Considering the quantitative importance of WAT in the adult organism and the increasing roles recently assigned to mast cells in physiopathology, WAT may represent an important source of mast cells in physiological and pathological situations.
Asunto(s)
Tejido Adiposo/citología , Células Madre Hematopoyéticas/citología , Mastocitos/citología , Células Madre/citología , Tejido Adiposo Blanco/citología , Animales , Diferenciación Celular/fisiología , Linaje de la Célula , Células Cultivadas , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Sézary Syndrome (SS) is a rare aggressive epidermotropic cutaneous T-cell lymphoma (CTCL) defined by erythroderma, pruritis, and a circulating atypical CD4 + T-cell clonal population. The diversity of Sézary cell (SC) phenotype and genotype may reflect either plasticity or heterogeneity, which was difficult to evaluate dynamically until the achievement of long-term SC expansion. Therefore, we developed six defined culture conditions allowing for the expansion of SC defined by their phenotype and monoclonality in four of seven SS cases. Engraftment of SC through the intrafemoral route into immunodeficient NOD.Cg-Prkdc(scid)Il2rg(tm1Wjll)/SzJ (NSG) mice was achieved in 2 of 14 SS cases. Secondary xenograft by percutaneous injection mimicked most of the features of SS with dermal infiltration, epidermotropism, and blood spreading. These models also allowed assessing the intra-individual heterogeneity of patient SC. Subclones sharing the same TCR gene rearrangement evolved independently according to culture conditions and/or after xenografting. This clonal selection was associated with some immunophenotypic plasticity and limited genomic evolution both in vitro and in vivo. The long-term amplification of SC allowed us to develop eight new SC lines derived from four different patients. These lines represent the cell of origin diversity of SC and provide new tools to evaluate their functional hallmarks and response to therapy.
Asunto(s)
Células Clonales/patología , Genes Codificadores de los Receptores de Linfocitos T , Síndrome de Sézary/patología , Neoplasias Cutáneas/patología , Adulto , Animales , Apoptosis , Técnicas de Cultivo de Célula , Proliferación Celular , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Pronóstico , Síndrome de Sézary/genética , Neoplasias Cutáneas/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphomas (CTCL) in which the human Telomerase Reverse Transcriptase (hTERT) gene is re-expressed. Current available treatments do not provide long-term response. We previously reported that Histone deacetylase inhibitors (HDACi, romidespin and vorinostat) and a DNA methyltransferase inhibitor (DNMTi, 5-azacytidine) can reduce hTERT expression without altering the methylation level of hTERT promoter. Romidepsin and vorinostat are approved for CTCL treatment, while 5-azacytidine is approved for the treatment of several hematological disorders, but not for CTCL. Here, using the soft agar assay, we analyzed the functional effect of the aforementioned epidrugs on the clonogenic capacities of Sézary cells. Our data revealed that, besides hTERT downregulation, epidrugs' pressure reduced the proliferative and the tumor formation capacities in Sézary cells in vitro.
RESUMEN
Resistance to chemotherapy, a major therapeutic challenge in the treatment of T-cell acute lymphoblastic leukemia (T-ALL), can be driven by interactions between leukemic cells and the microenvironment that promote survival of leukemic cells. The bone marrow, an important leukemia niche, has low oxygen partial pressures that highly participate in the regulation of normal hematopoiesis. Here we show that hypoxia inhibits T-ALL cell growth by slowing down cell cycle progression, decreasing mitochondria activity, and increasing glycolysis, making them less sensitive to antileukemic drugs and preserving their ability to initiate leukemia after treatment. Activation of the mammalian target of rapamycin (mTOR) was diminished in hypoxic leukemic cells, and treatment of T-ALL with the mTOR inhibitor rapamycin in normoxia mimicked the hypoxia effects, namely decreased cell growth and increased quiescence and drug resistance. Knocking down (KD) hypoxia-induced factor 1α (HIF-1α), a key regulator of the cellular response to hypoxia, antagonized the effects observed in hypoxic T-ALL and restored chemosensitivity. HIF-1α KD also restored mTOR activation in low O2 concentrations, and inhibiting mTOR in HIF1α KD T-ALL protected leukemic cells from chemotherapy. Thus, hypoxic niches play a protective role of T-ALL during treatments. Inhibition of HIF-1α and activation of the mTORC1 pathway may help suppress the drug resistance of T-ALL in hypoxic niches.
Asunto(s)
Preparaciones Farmacéuticas , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Resistencia a Antineoplásicos , Humanos , Hipoxia , Diana Mecanicista del Complejo 1 de la Rapamicina , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Microambiente TumoralRESUMEN
PURPOSE: Ultra-high-dose-rate FLASH radiation therapy has been shown to minimize side effects of irradiation in various organs while keeping antitumor efficacy. This property, called the FLASH effect, has caused enthusiasm in the radiation oncology community because it opens opportunities for safe dose escalation and improved radiation therapy outcome. Here, we investigated the impact of ultra-high-dose-rate FLASH versus conventional-dose-rate (CONV) total body irradiation (TBI) on humanized models of T-cell acute lymphoblastic leukemia (T-ALL) and normal human hematopoiesis. METHODS AND MATERIALS: We optimized the geometry of irradiation to ensure reproducible and homogeneous procedures using eRT6/Oriatron. Three T-ALL patient-derived xenografts and hematopoietic stem/progenitor cells (HSPCs) and CD34+ cells isolated from umbilical cord blood were transplanted into immunocompromised mice, together or separately. After reconstitution, mice received 4 Gy FLASH and CONV-TBI, and tumor growth and normal hematopoiesis were studied. A retrospective study of clinical and gene-profiling data previously obtained on the 3 T-ALL patient-derived xenografts was performed. RESULTS: FLASH-TBI was more efficient than CONV-TBI in controlling the propagation of 2 cases of T-ALL, whereas the third case of T-ALL was more responsive to CONV-TBI. The 2 FLASH-sensitive cases of T-ALL had similar genetic abnormalities, and a putative susceptibility imprint to FLASH-RT was found. In addition, FLASH-TBI was able to preserve some HSPC/CD34+ cell potential. Interestingly, when HSPC and T-ALL were present in the same animals, FLASH-TBI could control tumor development in most (3 of 4) of the secondary grafted animals, whereas among the mice receiving CONV-TBI, treated cells died with high leukemia infiltration. CONCLUSIONS: Compared with CONV-TBI, FLASH-TBI reduced functional damage to human blood stem cells and had a therapeutic effect on human T-ALL with a common genetic and genomic profile. The validity of the defined susceptibility imprint needs to be investigated further; however, to our knowledge, the present findings are the first to show benefits of FLASH-TBI on human hematopoiesis and leukemia treatment.
Asunto(s)
Hematopoyesis/efectos de la radiación , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/efectos de la radiación , Leucemia-Linfoma Linfoblástico de Células T Precursoras/radioterapia , Irradiación Corporal Total/métodos , Animales , Perfil Genético , Humanos , Huésped Inmunocomprometido , Ratones , Órganos en Riesgo/efectos de la radiación , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Traumatismos por Radiación/prevención & control , Tolerancia a Radiación , Dosificación Radioterapéutica , Reproducibilidad de los Resultados , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Treatment of cancer using radiation can be significantly compromised by the development of severe acute and late damage to normal tissue. Treatments that either reduce the risk and severity of damage or that facilitate the healing of radiation injuries are being developed, including autologous adipose tissue grafts to repair tissue defects or involutional disorders that result from tumor resection. Adipose tissue is specialized in energy storage and contains different cell types, including preadipocytes, which could be used for autologous transplantation. It has long been considered a poorly proliferative connective tissue; however, the acute effects of ionizing radiation on adipose tissue have not been investigated. Therefore, the aim of this study was to characterize the alterations induced in adipose tissue by total body irradiation. A severe decrease in proliferating cells, as well as a significant increase in apoptotic cells, was observed in vivo in inguinal fat pads following irradiation. Additionally, irradiation altered the hematopoietic population. Decreases in the proliferation and differentiation capacities of non-hematopoietic progenitors were also observed following irradiation. Together, these data demonstrate that subcutaneous adipose tissue is very sensitive to irradiation, leading to a profound alteration of its developmental potential. This damage could also alter the reconstructive properties of adipose tissue and, therefore, calls into question its use in autologous fat transfer following radiotherapy.
Asunto(s)
Adipocitos/efectos de la radiación , Tejido Adiposo/efectos de la radiación , Diferenciación Celular/efectos de la radiación , Proliferación Celular/efectos de la radiación , Traumatismos Experimentales por Radiación/patología , Adipocitos/citología , Adipocitos/metabolismo , Tejido Adiposo/citología , Animales , Citometría de Flujo , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Ratones , Estrés Oxidativo/efectos de la radiación , Fenotipo , Reacción en Cadena de la Polimerasa , Traumatismos Experimentales por Radiación/metabolismo , Células Madre/metabolismo , Células Madre/patología , Células Madre/efectos de la radiaciónRESUMEN
The SATB1 protein has been the focus of two recent studies of cutaneous T-cell lymphomas. Fredholm et al. observed a stage-related decrease of SATB1 expression in epidermotropic cutaneous T-cell lymphomas. SATB1 was negatively regulated by STAT5 through microRNA-155, which in turn triggered enhanced expression of T helper type 2 cytokines such as IL-5 and IL-9. In parallel, Sun et al. found that SATB1 expression was up-regulated by promoter demethylation in a subset of cutaneous anaplastic lymphoma and was associated with T helper type 17 polarization in patients with better therapeutic responses.
Asunto(s)
Linfoma Cutáneo de Células T , Proteínas de Unión a la Región de Fijación a la Matriz , MicroARNs , Neoplasias Cutáneas , Biomarcadores , Humanos , Linfocitos TRESUMEN
Ca2+ release-activated Ca2+ channels, composed of Orai1 and STIM1 (stromal interaction molecule 1) proteins, are the main Ca2+ entry mechanism in lymphocytes. Their role in cell migration and metastasis is demonstrated in solid cancers but it remains elusive in malignant hemopathies. Diffuse large B cell lymphoma (DLBCL) is characterized by the dissemination of neoplastic B cells throughout the organism which is under the control of chemokines such as Stromal Derived Factor 1 (SDF-1) and its receptor CXCR4. CXCR4 activation triggers a complex intracellular signaling including an increase in intracellular Ca2+ concentration whose role is still unclear. Using pharmacological and genetic approaches, we revealed that STIM1 and Orai1 were responsible for Ca2+ influx induced by SDF-1. Furthermore, we provide in vitro and in vivo evidence that they are necessary for basal or SDF-1-induced DLBCL cell migration which is independent of Ca2+ entry. We identify that they act as effectors coupling RhoA and ROCK dependent signaling pathway to MLC2 phosphorylation and actin polymerization. Finally, we revealed an alteration of Orai1 and STIM1 expression in extra-nodal DLBCL. Thus, we discovered a novel Ca2+-independent but Orai1 and STIM1-dependent signaling pathway involved in basal and CXCR4 dependent cell migration, which could be relevant for DLBCL physiopathology.
RESUMEN
Recent massive parallel sequencing data have evidenced the genetic diversity and complexity of Sézary syndrome mutational landscape with TP53 alterations being the most prevalent genetic abnormality. We analyzed a cohort of 35 patients with SS and a control group of 8 patients with chronic inflammatory dermatoses. TP53 status was analyzed at different clinical stages especially in 9 patients with a past-history of mycosis fungoides (MF), coined secondary SS. TP53 mutations were only detected in 10 patients with either primary or secondary SS (29%) corresponding to point mutations, small insertions and deletions which were unique in each case. Interestingly, TP53 mutations were both detected in sequential unselected blood mononuclear cells and in skin specimens. Cytogenetic analysis of blood specimens of 32 patients with SS showed a TP53 deletion in 27 cases (84%). Altogether 29 out of 35 cases exhibited TP53 mutation and/or deletion (83%). No difference in prognosis was observed according to TP53 status while patients with secondary SS had a worse prognosis than patients with primary SS. Interestingly, patients with TP53 alterations displayed a younger age and the presence of TP53 alteration at initial diagnosis stage supports a pivotal oncogenic role for TP53 mutation in SS as well as in erythrodermic MF making TP53 assessment an ancillary method for the diagnosis of patients with erythroderma as patients with inflammatory dermatoses did not display TP53 alteration.
Asunto(s)
Dermatitis Exfoliativa/complicaciones , Genes p53 , Síndrome de Sézary/genética , Neoplasias Cutáneas/genética , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Variaciones en el Número de Copia de ADN , Femenino , Citometría de Flujo , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Síndrome de Sézary/complicaciones , Neoplasias Cutáneas/complicacionesRESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) expands in various bone marrow (BM) sites of the body. We investigated whether different BM sites could differently modulate T-ALL propagation using in vivo animal models. We observed that mouse and human T-ALL develop slowly in the BM of tail vertebrae compared with the BM from thorax vertebrae. T-ALL recovered from tail BM displays lower cell-surface marker expression and decreased metabolism and cell-cycle progression, demonstrating a dormancy phenotype. Functionally, tail-derived T-ALL exhibit a deficient short-term ex vivo growth and a delayed in vivo propagation. These features are noncell-autonomous because T-ALL from tail and thorax shares identical genomic abnormalities and functional disparities disappear in vivo and in prolonged in vitro assays. Importantly tail-derived T-ALL displays higher intrinsic resistance to cell-cycle-related drugs (ie, vincristine sulfate and cytarabine). Of note, T-ALL recovered from gonadal adipose tissues or from cocultures with adipocytes shares metabolic, cell-cycle, and phenotypic or chemoresistance features, with tail-derived T-ALL suggesting adipocytes may participate in the tail BM imprints on T-ALL. Altogether these results demonstrate that BM sites differentially orchestrate T-ALL propagation stamping specific features to leukemic cells such as quiescence and decreased response to cell-cycle-dependent chemotherapy.
RESUMEN
T cell acute lymphoblastic leukemia (T-ALL) develops through accumulation of multiple genomic alterations within T-cell progenitors resulting in clonal heterogeneity among leukemic cells. Human T-ALL xeno-transplantation in immunodeficient mice is a gold standard approach to study leukemia biology and we recently uncovered that the leukemia development is more or less rapid depending on T-ALL sample. The resulting human leukemia may arise through genetic selection and we previously showed that human T-ALL development in immune-deficient mice is significantly enhanced upon CD7+/CD34+ leukemic cell transplantations. Here we investigated the genetic characteristics of CD7+/CD34+ and CD7+/CD34- cells from newly diagnosed human T-ALL and correlated it to the speed of leukemia development. We observed that CD7+/CD34+ or CD7+/CD34- T-ALL cells that promote leukemia within a short-time period are genetically similar, as well as xenograft-derived leukemia resulting from both cell fractions. In the case of delayed T-ALL growth CD7+/CD34+ or CD7+/CD34- cells were either genetically diverse, the resulting xenograft leukemia arising from different but branched subclones present in the original sample, or similar, indicating decreased fitness to mouse micro-environment. Altogether, our work provides new information relating the speed of leukemia development in xenografts to the genetic diversity of T-ALL cell compartments.
Asunto(s)
Variación Genética , Trasplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Trasplante Heterólogo , Animales , Antígenos CD34/metabolismo , Línea Celular Tumoral , Niño , Progresión de la Enfermedad , Heterogeneidad Genética , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Ratones Transgénicos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Factores de Tiempo , Adulto JovenRESUMEN
Monoallelic 6p25.3 rearrangements associated with DUSP22 (Dual Specificity Phosphatase 22) gene silencing have been reported in CD30+ peripheral T-cell lymphomas (PTCL), mostly with anaplastic morphology and of cutaneous origin. However, the mechanism of second allele silencing and the putative tumor suppressor function of DUSP22 have not been investigated so far. Here, we show that the presence, in most individuals, of an inactive paralog hampers genetic and epigenetic evaluation of the DUSP22 gene. Identification of DUSP22-specific single-nucleotide polymorphisms haplotypes and fluorescence in situ hybridization and epigenetic characterization of the paralog status led us to develop a comprehensive strategy enabling reliable identification of DUSP22 alterations. We showed that one cutaneous anaplastic large T-cell lymphomas (cALCL) case with monoallelic 6p25.3 rearrangement and DUSP22 silencing harbored exon 1 somatic mutations associated with second allele inactivation. Another cALCL case carried an intron 1 somatic splice site mutation with predicted deleterious exon skipping effect. Other tested PTCL cases with 6p25.3 rearrangement exhibited neither mutation nor deletion nor methylation accounting for silencing of the non-rearranged DUSP22 allele, thus inactivated by a so far unknown mechanism. We also characterized the expression status of four DUSP22 splice variants and found that they are all silenced in cALCL cases with 6p25.3 breakpoints. We finally showed that restoring expression of the physiologically predominant isoform in DUSP22-deficient malignant T cells inhibits cellular expansion by stimulating apoptosis and impairs soft agar clonogenicity and tumorigenicity. This study therefore shows that DUSP22 behaves as a tumor suppressor gene in PTCL.
Asunto(s)
Fosfatasas de Especificidad Dual/genética , Linfoma de Células T/genética , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/genética , Polimorfismo de Nucleótido Simple , Proteínas Supresoras de Tumor/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , Cromosomas Humanos Par 6/genética , Metilación de ADN , Fosfatasas de Especificidad Dual/metabolismo , Femenino , Reordenamiento Génico , Humanos , Hibridación Fluorescente in Situ , Linfoma Anaplásico de Células Grandes/enzimología , Linfoma Anaplásico de Células Grandes/genética , Linfoma de Células T/metabolismo , Linfoma Cutáneo de Células T/enzimología , Linfoma Cutáneo de Células T/genética , Linfoma de Células T Periférico/embriología , Linfoma de Células T Periférico/genética , Masculino , Persona de Mediana Edad , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Mutación , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Development of novel therapies is critical for T-cell acute leukaemia (T-ALL). Here, we investigated the effect of inhibiting the MAPK/MEK/ERK pathway on T-ALL cell growth. Unexpectedly, MEK inhibitors (MEKi) enhanced growth of 70% of human T-ALL cell samples cultured on stromal cells independently of NOTCH activation and maintained their ability to propagate in vivo. Similar results were obtained when T-ALL cells were cultured with ERK1/2-knockdown stromal cells or with conditioned medium from MEKi-treated stromal cells. Microarray analysis identified interleukin 18 (IL-18) as transcriptionally up-regulated in MEKi-treated MS5 cells. Recombinant IL-18 promoted T-ALL growth in vitro, whereas the loss of function of IL-18 receptor in T-ALL blast cells decreased blast proliferation in vitro and in NSG mice. The NFKB pathway that is downstream to IL-18R was activated by IL-18 in blast cells. IL-18 circulating levels were increased in T-ALL-xenografted mice and also in T-ALL patients in comparison with controls. This study uncovers a novel role of the pro-inflammatory cytokine IL-18 and outlines the microenvironment involvement in human T-ALL development.
Asunto(s)
Interleucina-18/inmunología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/inmunología , Células del Estroma/inmunología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Regulación Leucémica de la Expresión Génica , Silenciador del Gen , Humanos , Interleucina-18/sangre , Interleucina-18/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos NOD , Leucemia-Linfoma Linfoblástico de Células T Precursoras/sangre , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Células del Estroma/citología , Células del Estroma/metabolismo , Células del Estroma/patología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/patologíaRESUMEN
T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer that is frequently associated with activating mutations in NOTCH1 and dysregulation of MYC. Here, we performed 2 complementary screens to identify FDA-approved drugs and drug-like small molecules with activity against T-ALL. We developed a zebrafish system to screen small molecules for toxic activity toward MYC-overexpressing thymocytes and used a human T-ALL cell line to screen for small molecules that synergize with Notch inhibitors. We identified the antipsychotic drug perphenazine in both screens due to its ability to induce apoptosis in fish, mouse, and human T-ALL cells. Using ligand-affinity chromatography coupled with mass spectrometry, we identified protein phosphatase 2A (PP2A) as a perphenazine target. T-ALL cell lines treated with perphenazine exhibited rapid dephosphorylation of multiple PP2A substrates and subsequent apoptosis. Moreover, shRNA knockdown of specific PP2A subunits attenuated perphenazine activity, indicating that PP2A mediates the drug's antileukemic activity. Finally, human T-ALLs treated with perphenazine exhibited suppressed cell growth and dephosphorylation of PP2A targets in vitro and in vivo. Our findings provide a mechanistic explanation for the recurring identification of phenothiazines as a class of drugs with anticancer effects. Furthermore, these data suggest that pharmacologic PP2A activation in T-ALL and other cancers driven by hyperphosphorylated PP2A substrates has therapeutic potential.