RESUMEN
BACKGROUND: The prognosis for high-risk childhood acute leukaemias remains dismal and established treatment protocols often cause long-term side effects in survivors. This study aims to identify more effective and safer therapeutics for these patients. METHODS: A high-throughput phenotypic screen of a library of 3707 approved drugs and pharmacologically active compounds was performed to identify compounds with selective cytotoxicity against leukaemia cells followed by further preclinical evaluation in patient-derived xenograft models. RESULTS: Auranofin, an FDA-approved agent for the treatment of rheumatoid arthritis, was identified as exerting selective anti-cancer activity against leukaemia cells, including patient-derived xenograft cells from children with high-risk ALL, versus solid tumour and non-cancerous cells. It induced apoptosis in leukaemia cells by increasing reactive oxygen species (ROS) and potentiated the activity of the chemotherapeutic cytarabine against highly aggressive models of infant MLL-rearranged ALL by enhancing DNA damage accumulation. The enhanced sensitivity of leukaemia cells towards auranofin was associated with lower basal levels of the antioxidant glutathione and higher baseline ROS levels compared to solid tumour cells. CONCLUSIONS: Our study highlights auranofin as a well-tolerated drug candidate for high-risk paediatric leukaemias that warrants further preclinical investigation for application in high-risk paediatric and adult acute leukaemias.
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Auranofina/administración & dosificación , Citarabina/administración & dosificación , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Especies Reactivas de Oxígeno/metabolismo , Animales , Auranofina/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Niño , Citarabina/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Bibliotecas de Moléculas Pequeñas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: NUT carcinoma (NC), previously known as NUT midline carcinoma, is a rare and very aggressive cancer that occurs in both children and adults. NC is largely chemoresistant, with an overall survival of less than 7 months. Because the carcinoma is not restricted to a particular organ, diagnosis is often a challenge. In the absence of a clearly determined incidence for NC, we sought to study the diagnosis of patients in a well-defined population. METHODS: We systematically reviewed records of all patients that presented to the Oncology Department of the Princess Margaret Hospital for Children from 1989 to 2014. This institution in the geographically isolated state of Western Australia has a catchment population of around 2 million. We then identified all high grade undifferentiated sarcomas or carcinomas in the 0-16 year age group. RESULTS: Over 26 years, we found 14 patients of 16 years or younger with undifferentiated malignant tumors. Of these, five tumors were positive by immunohistochemistry for the NUT/NUTM1 (Nuclear Protein in Testis) protein and/or the translocation t(15;19). Three patients presented with thoracic tumors, one with a para-spinal tumor, and one had an upper airway nasopharyngeal carcinoma. In all five cases, there was an initial response to therapy and then progression. This 26-year survey was conducted in a geographically isolated state with a well-defined population, and we determined an estimated incidence of NC of around 0.41 per million child years (0-16 yrs. of age) at risk. From three patients it was feasible to derive cell lines for further genetic analyses and drug screening. CONCLUSIONS: For the first time, the incidence of NC could be determined in a well-defined geographic area. The calculated rate of NC incidence is consistent with a history of under-recognition for this malignancy. These findings indicate that improved diagnostic detection of NC would enable better management and counselling of patients. Our findings emphasize the heterogeneity of NC, and they highlight the need to develop personalised therapy options, and to consider a diagnosis of NC in undifferentiated malignant tumors.
Asunto(s)
Neoplasias de Células Escamosas/epidemiología , Sarcoma/epidemiología , Adolescente , Niño , Preescolar , Femenino , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Lactante , Recién Nacido , Masculino , Australia OccidentalRESUMEN
Around 10% of acute leukemias harbor a rearrangement of the MLL/KMT2A gene, and the presence of this translocation results in a highly aggressive, therapy-resistant leukemia subtype with survival rates below 50%. There is a high unmet need to identify safer and more potent therapies for MLL-rearranged (MLL-r) leukemia that can be combined with established chemotherapeutics to decrease treatment-related toxicities. The curaxin, CBL0137, has demonstrated nongenotoxic anticancer and chemopotentiating effects in a number of preclinical cancer models and is currently in adult Phase I clinical trials for solid tumors and hematological malignancies. The aim of our study was to investigate whether CBL0137 has potential as a therapeutic and chemopotentiating compound in MLL-r leukemia through a comprehensive analysis of its efficacy in preclinical models of the disease. CBL0137 decreased the viability of a panel of MLL-r leukemia cell lines (n = 12) and xenograft cells derived from patients with MLL-r acute lymphoblastic leukemia (ALL, n = 3) in vitro with submicromolar IC50s. The small molecule drug was well-tolerated in vivo and significantly reduced leukemia burden in a subcutaneous MV4;11 MLL-r acute myeloid leukemia model and in patient-derived xenograft models of MLL-r ALL (n = 5). The in vivo efficacy of standard of care drugs used in remission induction for pediatric ALL was also potentiated by CBL0137. CBL0137 exerted its anticancer effect by trapping Facilitator of Chromatin Transcription (FACT) into chromatin, activating the p53 pathway and inducing an Interferon response. Our findings support further preclinical evaluation of CBL0137 as a new approach for the treatment of MLL-r leukemia.
Asunto(s)
Antineoplásicos/farmacología , Carbazoles/farmacología , Reordenamiento Génico , N-Metiltransferasa de Histona-Lisina/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Animales , Antineoplásicos/uso terapéutico , Apoptosis/genética , Carbazoles/uso terapéutico , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/genética , Humanos , Estimación de Kaplan-Meier , Leucemia Bifenotípica Aguda/diagnóstico , Leucemia Bifenotípica Aguda/tratamiento farmacológico , Leucemia Bifenotípica Aguda/genética , Leucemia Bifenotípica Aguda/mortalidad , Ratones , Transducción de Señal/efectos de los fármacos , Factores de Elongación Transcripcional/genética , Transcriptoma , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Connective tissue growth factor (CTGF/CCN2) has long been associated with human cancers. The role it plays in these neoplasms is diverse and tumour specific. Recurring patterns in clinical outcome, histological desmoplasia and mechanisms of action have been found. When CTGF is overexpressed compared to low-expressing normal tissue or is underexpressed compared to high-expressing normal tissue, the functional outcome favours tumour survival and disease progression. CTGF acts by altering proliferation, drug resistance, angiogenesis, adhesion and migration contributing to metastasis. The pattern of CTGF expression and tumour response helps to clarify the role of this matricellular protein across a multitude of human cancers.
Asunto(s)
Factor de Crecimiento del Tejido Conjuntivo/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Neoplasias/mortalidad , Adhesión Celular/genética , Movimiento Celular/genética , Proliferación Celular , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Resistencia a Antineoplásicos/genética , Humanos , Metástasis de la Neoplasia , Neoplasias/patología , Neovascularización Patológica/genética , Especificidad de Órganos/genética , Evaluación del Resultado de la Atención al Paciente , PronósticoRESUMEN
The class 1A aldehyde dehydrogenase (ALDH1A) subfamily of genes encode enzymes that function at the apex of the retinoic acid (RA) signalling pathway. We detected aberrant expression of ALDH1A genes, particularly ALDH1A2, in a majority (72%) of primary paediatric T cell acute lymphoblastic leukaemia (T-ALL) specimens. ALDH1A expression was almost exclusive to T-lineage, but not B-lineage, ALL. To determine whether ALDH1A expression may have relevance to T-ALL cell growth and survival, the effect of inhibiting ALDH1A function was measured on a panel of human ALL cell lines. This revealed that T-ALL proliferation had a higher sensitivity to modulation of ALDH1A activity and RA signalling as compared to ALL cell lines of B-lineage. Consistent with these findings, the genes most highly correlated with ALDH1A2 expression were involved in cell proliferation and apoptosis. Evidence that such genes may be targets of regulation via RA signalling initiated by ALDH1A activity was provided by the TNFRSF10B gene, encoding the apoptotic death receptor TNFRSF10B (also termed TRAIL-R2), which negatively correlated with ALDH1A2 and showed elevated transcription following treatment of T-ALL cell lines with the ALDH1A inhibitor citral (3,7-dimethyl-2,6-octadienal). These data indicate that ALDH1A expression is a common event in T-ALL and supports a role for these enzymes in the pathobiology of this disease.
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Aldehído Deshidrogenasa/biosíntesis , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Aldehído Deshidrogenasa/genética , Familia de Aldehído Deshidrogenasa 1 , Proliferación Celular/fisiología , Perfilación de la Expresión Génica , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Retinal-Deshidrogenasa , Transducción de SeñalRESUMEN
Drug-resistant forms of acute lymphoblastic leukaemia (ALL) are a leading cause of death from disease in children. Up to 25% of patients with T-cell ALL (T-ALL) develop resistance to chemotherapeutic agents, particularly to glucocorticoids (GCs), a class of drug to which resistance is one of the strongest indicators of poor clinical outcome. Despite their clinical importance, the molecular mechanisms that underpin GC resistance and leukaemia relapse are not well understood. Recently, we demonstrated that GC-resistance is associated with a proliferative metabolism involving the up-regulation of glycolysis, oxidative phosphorylation and cholesterol biosynthesis. Here we confirm that resistance is directly associated with a glycolytic phenotype and show that GC-resistant T-ALL cells are able to shift between glucose bioenergetic pathways. We evaluated the potential for targeting these pathways in vitro using a glycolysis inhibitor, 2-deoxyglucose (2DG), and the oxidative phosphorylation inhibitor oligomycin in combination with methylprednisolone (MPRED). We found that oligomycin synergized with MPRED to sensitize cells otherwise resistant to GCs. Similarly we observed synergy between MPRED and simvastatin, an inhibitor of cholesterol metabolism. Collectively, our findings suggest that dual targeting of bioenergetic pathways in combination with GCs may offer a promising therapeutic strategy to overcome drug resistance in ALL.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Sinergismo Farmacológico , Galactosa/metabolismo , Glucocorticoides/farmacología , Glucocorticoides/uso terapéutico , Glucólisis/efectos de los fármacos , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Metilprednisolona/farmacología , Metilprednisolona/uso terapéutico , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oligomicinas/farmacología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Transducción de Señal/efectos de los fármacosRESUMEN
Germ-line RB-1 mutations predispose to pineoblastoma (PinB), but other predisposing genetic factors are not well established. We recently identified a germ-line DICER1 mutation in a child with a PinB. This was accompanied by loss of heterozygosity (LOH) of the wild-type allele within the tumour. We set out to establish the prevalence of DICER1 mutations in an opportunistically ascertained series of PinBs. Twenty-one PinB cases were studied: Eighteen cases had not undergone previous testing for DICER1 mutations; three patients were known carriers of germ-line DICER1 mutations. The eighteen PinBs were sequenced by Sanger and/or Fluidigm-based next-generation sequencing to identify DICER1 mutations in blood gDNA and/or tumour gDNA. Testing for somatic DICER1 mutations was also conducted on one case with a known germ-line DICER1 mutation. From the eighteen PinBs, we identified four deleterious DICER1 mutations, three of which were germ line in origin, and one for which a germ line versus somatic origin could not be determined; in all four, the second allele was also inactivated leading to complete loss of DICER1 protein. No somatic DICER1 RNase IIIb mutations were identified. One PinB arising in a germ-line DICER1 mutation carrier was found to have LOH. This study suggests that germ-line DICER1 mutations make a clinically significant contribution to PinB, establishing DICER1 as an important susceptibility gene for PinB and demonstrates PinB to be a manifestation of a germ-line DICER1 mutation. The means by which the second allele is inactivated may differ from other DICER1-related tumours.
Asunto(s)
Neoplasias Encefálicas/genética , ARN Helicasas DEAD-box/genética , Mutación de Línea Germinal/genética , Glándula Pineal/patología , Pinealoma/genética , Ribonucleasa III/genética , Adolescente , Niño , Preescolar , Análisis Mutacional de ADN , Salud de la Familia , Femenino , Humanos , Lactante , Masculino , Adulto JovenAsunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Sinergismo Farmacológico , Reordenamiento Génico , Histona Desacetilasas/química , N-Metiltransferasa de Histona-Lisina/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Animales , Apoptosis , Proliferación Celular , Citarabina/administración & dosificación , Depsipéptidos/administración & dosificación , Femenino , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Lactante , Ratones , Ratones Endogámicos NOD , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hematopoiesis occurs in a complex bone marrow microenvironment in which bone marrow stromal cells provide critical support to the process through direct cell contact and indirectly through the secretion of cytokines and growth factors. We report that connective tissue growth factor (Ctgf, also known as Ccn2) is highly expressed in murine bone marrow stromal cells. In contrast, connective tissue growth factor is barely detectable in unfractionated adult bone marrow cells. While connective tissue growth factor has been implicated in hematopoietic malignancies, and is known to play critical roles in skeletogenesis and regulation of bone marrow stromal cells, its role in hematopoiesis has not been described. Here we demonstrate that the absence of connective tissue growth factor in mice results in impaired hematopoiesis. Using a chimeric fetal liver transplantation model, we show that absence of connective tissue growth factor has an impact on B-cell development, in particular from pro-B to more mature stages, which is linked to a requirement for connective tissue growth factor in bone marrow stromal cells. Using in vitro culture systems, we demonstrate that connective tissue growth factor potentiates B-cell proliferation and promotes pro-B to pre-B differentiation in the presence of interleukin-7. This study provides a better understanding of the functions of connective tissue growth factor within the bone marrow, showing the dual regulatory role of the growth factor in skeletogenesis and in stage-specific B lymphopoiesis.
Asunto(s)
Subgrupos de Linfocitos B/efectos de los fármacos , Subgrupos de Linfocitos B/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/genética , Expresión Génica , Interleucina-7/farmacología , Linfopoyesis , Células Madre Mesenquimatosas/metabolismo , Animales , Animales Recién Nacidos , Subgrupos de Linfocitos B/citología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Linaje de la Célula/genética , Proliferación Celular/efectos de los fármacos , Factor de Crecimiento del Tejido Conjuntivo/deficiencia , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Hepatocitos/metabolismo , Hepatocitos/trasplante , Activación de Linfocitos/efectos de los fármacos , Linfopoyesis/genética , Ratones , Ratones Noqueados , Fenotipo , Fosforilación , Factor de Transcripción STAT5/metabolismoRESUMEN
The connective tissue growth factor gene (CTGF) is aberrantly expressed in 75% of precursor B-cell acute lymphoblastic leukaemias (pre-B ALL) and is associated with poor outcome. We identified consistent hypomethylation of the CTGF locus in primary pre-B ALL specimens regardless of CTGF expression. By contrast, primary T-cell ALL specimens, which do not express CTGF, exhibited distinctive patterns of hypermethylation. Furthermore, we confirmed that global changes in DNA methylation and histone acetylation can both functionally modulate CTGF expression in pre-B ALL cell lines. These data suggest that hypomethylation of the CTGF locus is an essential prerequisite for aberrant CTGF expression in pre-B ALL.
Asunto(s)
Factor de Crecimiento del Tejido Conjuntivo/genética , Metilación de ADN , Regulación Leucémica de la Expresión Génica , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Azacitidina/análogos & derivados , Azacitidina/farmacología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Niño , Factor de Crecimiento del Tejido Conjuntivo/biosíntesis , Islas de CpG , Decitabina , Humanos , Ácidos Hidroxámicos/farmacología , Células Jurkat/efectos de los fármacos , Células Jurkat/metabolismo , Proteínas de Neoplasias/biosíntesis , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
Infants with KMT2A-rearranged B-cell acute lymphoblastic leukemia (ALL) have a dismal prognosis. Survival outcomes have remained static in recent decades despite treatment intensification and novel therapies are urgently required. KMT2A-rearranged infant ALL cells are characterized by an abundance of promoter hypermethylation and exhibit high BCL-2 expression, highlighting potential for therapeutic targeting. Here, we show that hypomethylating agents exhibit in vitro additivity when combined with most conventional chemotherapeutic agents. However, in a subset of samples an antagonistic effect was seen between several agents. This was most evident when hypomethylating agents were combined with methotrexate, with upregulation of ATP-binding cassette transporters identified as a potential mechanism. Single agent treatment with azacitidine and decitabine significantly prolonged in vivo survival in KMT2A-rearranged infant ALL xenografts. Treatment of KMT2A-rearranged infant ALL cell lines with azacitidine and decitabine led to differential genome-wide DNA methylation, changes in gene expression and thermal proteome profiling revealed the target protein-binding landscape of these agents. The selective BCL-2 inhibitor, venetoclax, exhibited in vitro additivity in combination with hypomethylating or conventional chemotherapeutic agents. The addition of venetoclax to azacitidine resulted in a significant in vivo survival advantage indicating the therapeutic potential of this combination to improve outcome for infants with KMT2A-rearranged ALL.
Asunto(s)
Leucemia Mieloide Aguda , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Lactante , Azacitidina/farmacología , Azacitidina/uso terapéutico , Decitabina/farmacología , Decitabina/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogénicas c-bcl-2 , Leucemia Mieloide Aguda/genéticaRESUMEN
BACKGROUND: Infants with KMT2A-rearranged B-cell precursor acute lymphoblastic leukemia (ALL) have poor outcomes. There is an urgent need to identify novel agents to improve survival. Proteasome inhibition has emerged as a promising therapeutic strategy for several hematological malignancies. The aim of this study was to determine the preclinical efficacy of the selective proteasome inhibitor carfilzomib, for infants with KMT2A-rearranged ALL. METHODS: Eight infant ALL cell lines were extensively characterized for immunophenotypic and cytogenetic features. In vitro cytotoxicity to carfilzomib was assessed using a modified Alamar Blue assay with cells in logarithmic growth. The Bliss Independence model was applied to determine synergy between carfilzomib and the nine conventional chemotherapeutic agents used to treat infants with ALL. Established xenograft models were used to identify the maximal tolerated dose of carfilzomib and determine in vivo efficacy. RESULTS: Carfilzomib demonstrated low IC50 concentrations within the nanomolar range (6.0-15.8 nm) across the panel of cell lines. Combination drug testing indicated in vitro synergy between carfilzomib and several conventional chemotherapeutic agents including vincristine, daunorubicin, dexamethasone, L-asparaginase, and 4-hydroperoxycyclophosphamide. In vivo assessment did not lead to a survival advantage for either carfilzomib monotherapy, when used to treat both low or high disease burden, or for carfilzomib in combination with multi-agent induction chemotherapy comprising of vincristine, dexamethasone, and L-asparaginase. CONCLUSIONS: Our study highlights that in vitro efficacy does not necessarily translate to benefit in vivo and emphasizes the importance of in vivo validation prior to suggesting an agent for clinical use. Whilst proteasome inhibitors have an important role to play in several hematological malignancies, our findings guard against prioritization of carfilzomib for treatment of KMT2A-rearranged infant ALL in the clinical setting.
RESUMEN
BACKGROUND: Pre-clinical models that effectively recapitulate human disease are critical for expanding our knowledge of cancer biology and drug resistance mechanisms. For haematological malignancies, the non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse is one of the most successful models to study paediatric acute lymphoblastic leukaemia (ALL). However, for this model to be effective for studying engraftment and therapy responses at the whole genome level, careful molecular characterisation is essential. RESULTS: Here, we sought to validate species-specific gene expression profiling in the high engraftment continuous ALL NOD/SCID xenograft. Using the human Affymetrix whole transcript platform we analysed transcriptional profiles from engrafted tissues without prior cell separation of mouse cells and found it to return highly reproducible profiles in xenografts from individual mice. The model was further tested with experimental mixtures of human and mouse cells, demonstrating that the presence of mouse cells does not significantly skew expression profiles when xenografts contain 90% or more human cells. In addition, we present a novel in silico and experimental masking approach to identify probes and transcript clusters susceptible to cross-species hybridisation. CONCLUSIONS: We demonstrate species-specific transcriptional profiles can be obtained from xenografts when high levels of engraftment are achieved or with the application of transcript cluster masks. Importantly, this masking approach can be applied and adapted to other xenograft models where human tissue infiltration is lower. This model provides a powerful platform for identifying genes and pathways associated with ALL disease progression and response to therapy in vivo.
Asunto(s)
Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Animales , Médula Ósea/metabolismo , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Especificidad de la Especie , Bazo/metabolismoRESUMEN
BACKGROUND: Aberrant promoter DNA methylation has been reported in childhood acute lymphoblastic leukaemia (ALL) and has the potential to contribute to its onset and outcome. However, few reports demonstrate consistent, prevalent and dense promoter methylation, associated with tumour-specific gene silencing. By screening candidate genes, we have detected frequent and dense methylation of the TESTIN (TES) promoter. RESULTS: Bisulfite sequencing showed that 100% of the ALL samples (n = 20) were methylated at the TES promoter, whereas the matched remission (n = 5), normal bone marrow (n = 6) and normal PBL (n = 5) samples were unmethylated. Expression of TES in hyperdiploid, TEL-AML+, BCR-ABL+, and E2A-PBX+ subtypes of B lineage ALL was markedly reduced compared to that in normal bone marrow progenitor cells and in B cells. In addition TES methylation and silencing was demonstrated in nine out of ten independent B ALL propagated as xenografts in NOD/SCID mice. CONCLUSION: In total, 93% of B ALL samples (93 of 100) demonstrated methylation with silencing or reduced expression of the TES gene. Thus, TES is the most frequently methylated and silenced gene yet reported in ALL. TES, a LIM domain-containing tumour suppressor gene and component of the focal adhesion complex, is involved in adhesion, motility, cell-to-cell interactions and cell signalling. Our data implicate TES methylation in ALL and provide additional evidence for the involvement of LIM domain proteins in leukaemogenesis.
Asunto(s)
Alelos , Metilación de ADN , Silenciador del Gen , Proteínas de Homeodominio/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Regiones Promotoras Genéticas , Proteínas Supresoras de Tumor/genética , Animales , Línea Celular Tumoral , Proteínas del Citoesqueleto , Humanos , Proteínas con Dominio LIM , Pérdida de Heterocigocidad , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteínas de Unión al ARN , Trasplante HeterólogoRESUMEN
BACKGROUND: Continuous complete clinical remission in T-cell acute lymphoblastic leukemia (T-ALL) is now approaching 80% due to the implementation of aggressive chemotherapy protocols but patients that relapse continue to have a poor prognosis. Such patients could benefit from augmented therapy if their clinical outcome could be more accurately predicted at the time of diagnosis. Gene expression profiling offers the potential to identify additional prognostic markers but has had limited success in generating robust signatures that predict outcome across multiple patient cohorts. This study aimed to identify robust gene classifiers that could be used for the accurate prediction of relapse in independent cohorts and across different experimental platforms. RESULTS: Using HG-U133Plus2 microarrays we modeled a five-gene classifier (5-GC) that accurately predicted clinical outcome in a cohort of 50 T-ALL patients. The 5-GC was further tested against three independent cohorts of T-ALL patients, using either qRT-PCR or microarray gene expression, and could predict patients with significantly adverse clinical outcome in each. The 5-GC featured the interleukin-7 receptor (IL-7R), low-expression of which was independently predictive of relapse in T-ALL patients. In T-ALL cell lines, low IL-7R expression was correlated with diminished growth response to IL-7 and enhanced glucocorticoid resistance. Analysis of biological pathways identified the NF-kappaB and Wnt pathways, and the cell adhesion receptor family (particularly integrins) as being predictive of relapse. Outcome modeling using genes from these pathways identified patients with significantly worse relapse-free survival in each T-ALL cohort. CONCLUSIONS: We have used two different approaches to identify, for the first time, robust gene signatures that can successfully discriminate relapse and CCR patients at the time of diagnosis across multiple patient cohorts and platforms. Such genes and pathways represent markers for improved patient risk stratification and potential targets for novel T-ALL therapies.
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Biomarcadores de Tumor/genética , Perfilación de la Expresión Génica/métodos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Adolescente , Algoritmos , Biomarcadores de Tumor/análisis , Niño , Preescolar , Árboles de Decisión , Femenino , Expresión Génica , Humanos , Estimación de Kaplan-Meier , Masculino , FN-kappa B/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/mortalidad , Pronóstico , Receptores de Interleucina-7/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Resultado del Tratamiento , Proteínas Wnt/genéticaRESUMEN
BACKGROUND: Rearrangement of the mixed-lineage leukemia gene (MLL) is found in 80% of infant acute lymphoblastic leukemia (ALL) and is associated with poor prognosis and resistance to glucocorticoids (GCs). We have recently observed that GC resistance in T-ALL cell lines is associated with a proliferative metabolism and reduced expression of MLL. In this study we have further explored the relationship between MLL status and GC sensitivity. RESULTS: Negative correlation of MLL expression with GC resistance in 15 T-ALL cell lines was confirmed by quantitative RT-PCR. The absence of MLL-rearrangements suggested that this relationship represented expression of wild-type MLL. Analysis of MLL expression patterns revealed a negative relationship with cellular metabolism, proliferation and anti-apoptotic transcriptional networks. In silico analysis of published data demonstrated that reduced levels of MLL mRNA are associated with relapse and prednisolone resistance in T-ALL patients and adverse clinical outcome in children with MLL-rearranged ALL. RNAi knockdown of MLL expression in T-ALL cell lines significantly increased resistance to dexamethasone and gamma irradiation indicating an important role for wild-type MLL in the control of cellular apoptosis. CONCLUSIONS: The data suggests that reduced expression of wild-type MLL can contribute to GC resistance in ALL patients both with and without MLL-translocations.
Asunto(s)
Daño del ADN/genética , Resistencia a Antineoplásicos/genética , Glucocorticoides/farmacología , Linfocitos/efectos de los fármacos , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Línea Celular Tumoral , Humanos , Proteína de la Leucemia Mieloide-Linfoide/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The bone marrow microenvironment (BMM) plays a key role in leukemia progression, but its molecular complexity in pre-B cell acute lymphoblastic leukemia (B-ALL), the most common cancer in children, remains poorly understood. To gain further insight, we used single-cell RNA sequencing to characterize the kinetics of the murine BMM during B-ALL progression. Normal pro- and pre-B cells were found to be the most affected at the earliest stages of disease and this was associated with changes in expression of genes regulated by the AP1-transcription factor complex and regulatory factors NELFE, MYC and BCL11A. Granulocyte-macrophage progenitors show reduced expression of the tumor suppressor long non-coding RNA Neat1 and disruptions in the rate of transcription. Intercellular communication networks revealed monocyte-dendritic precursors to be consistently active during B-ALL progression, with enriched processes including cytokine-mediated signaling pathway, neutrophil-mediated immunity and regulation of cell migration and proliferation. In addition, we confirmed that the hematopoietic stem and progenitor cell compartment was perturbed during leukemogenesis. These findings extend our understanding of the complexity of changes and molecular interactions among the normal cells of the BMM during B-ALL progression.
Asunto(s)
Linfocitos B/patología , Células de la Médula Ósea/metabolismo , Médula Ósea/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Microambiente Tumoral , Animales , Linfocitos B/metabolismo , Médula Ósea/metabolismo , Progresión de la Enfermedad , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Represoras/metabolismoRESUMEN
OBJECTIVES: Natural killer (NK) cells are an attractive source of cells for an 'off the shelf' cellular therapy because of their innate capacity to target malignant cells, and ability to be transferred between donors and patients. However, since not all NK cells are equally effective at targeting cancer, selecting the right donor for cellular therapy is critical for the success of the treatment. Recently, cellular therapies utilising NK cells from cytomegalovirus (CMV)-seropositive donors have been explored. However, whether these NK cells are the best source to treat paediatric acute lymphoblastic leukaemia (ALL) remains unclear. METHODS: Using a panel of patient-derived paediatric B- and T-ALL, we assessed the ability of NK cells from 49 healthy donors to mount an effective functional response against these two major subtypes of ALL. RESULTS: From this cohort, we have identified a pool of donors with superior activity against multiple ALL cells. While these donors were more likely to be CMV+, we identified multiple CMVneg donors within this group. Furthermore, NK cells from these donors recognised B- and T-ALL through different activating receptors. Dividing functional NK cells into 29 unique subsets, we observed that within each individual the same NK cell subsets dominated across all ALL cells. Intriguingly, this occurred despite the ALL cells in our panel expressing different combinations of NK cell ligands. Finally, we can demonstrate that cellular therapy products derived from these superior donors significantly delayed leukaemia progression in preclinical models of ALL. CONCLUSIONS: We have identified a pool of superior donors that are effective against a range of ALL cells, representing a potential pool of donors that can be used as an adoptive NK cell therapy to treat paediatric ALL.
RESUMEN
The prognosis for children diagnosed with high-risk acute lymphoblastic leukemia (ALL) remains suboptimal, and more potent and less toxic treatments are urgently needed. We investigated the efficacy of a novel nicotinamide phosphoribosyltransferase inhibitor, OT-82, against a panel of patient-derived xenografts (PDXs) established from high-risk and poor outcome pediatric ALL cases. OT-82 was well-tolerated and demonstrated impressive single agent in vivo efficacy, achieving significant leukemia growth delay in 95% (20/21) and disease regression in 86% (18/21) of PDXs. In addition, OT-82 enhanced the efficacy of the established drugs cytarabine and dasatinib and, as a single agent, showed similar efficacy as an induction-type regimen combining three drugs used to treat pediatric ALL. OT-82 exerted its antileukemic action by depleting NAD+ and ATP, inhibiting the NAD+-requiring DNA damage repair enzyme PARP-1, increasing mitochondrial ROS levels and inducing DNA damage, culminating in apoptosis induction. OT-82 sensitivity was associated with the occurrence of mutations in major DNA damage response genes, while OT-82 resistance was characterized by high expression levels of CD38. In conclusion, our study provides evidence that OT-82, as a single agent, and in combination with established drugs, is a promising new therapeutic strategy for a broad spectrum of high-risk pediatric ALL for which improved therapies are urgently needed.
Asunto(s)
Antineoplásicos/farmacología , Citocinas/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Medulloblastoma (MB) is the most common type of brain tumor affecting children. These tumors are a significant cause of childhood mortality and morbidity, and more effective and less invasive treatment options are urgently required. To achieve these aims, it will be critical to develop a more comprehensive understanding of the molecular pathogenesis of MB. At present, there are relatively few well-characterized MB cell lines available to the research community for the study of MB molecular and cellular biology. Here we present the case reports of two children diagnosed with classic and desmoplastic MB, and describe the characteristics of two new MB cell lines derived from these individuals. A number of genes encoding components of the sonic hedgehog (SHH) and WNT pathways were up-regulated in the desmoplastic relative to the classic MB cell line consistent with aberrant activation of these pathways in desmoplastic MB. These cell lines represent an additional resource for the analysis of diverse aspects of MB biology.