RESUMO
The use of genome-wide copy-number analysis and massive parallel sequencing has revolutionized the understanding of the clonal architecture of pediatric acute lymphoblastic leukemia (ALL) by demonstrating that this disease is composed of highly variable clonal ancestries following the rules of Darwinian selection. The current study aimed to analyze the molecular composition of childhood ALL biopsies and patient-derived xenografts with particular emphasis on mechanisms associated with acquired chemoresistance. Genomic DNA from seven primary pediatric ALL patient samples, 29 serially passaged xenografts, and six in vivo selected chemoresistant xenografts were analyzed with 250K single-nucleotide polymorphism arrays. Copy-number analysis of non-drug-selected xenografts confirmed a highly variable molecular pattern of variegated subclones. Whereas primary patient samples from initial diagnosis displayed a mean of 5.7 copy-number alterations per sample, serially passaged xenografts contained a mean of 8.2 and chemoresistant xenografts a mean of 10.5 copy-number alterations per sample, respectively. Resistance to cytarabine was explained by a new homozygous deletion of the DCK gene, whereas methotrexate resistance was associated with monoallelic deletion of FPGS and mutation of the remaining allele. This study demonstrates that selecting for chemoresistance in xenografted human ALL cells can reveal novel mechanisms associated with drug resistance.
Assuntos
Antineoplásicos/farmacologia , Células Clonais/patologia , Células-Tronco Neoplásicas/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Animais , Antineoplásicos/uso terapêutico , Biópsia , Citarabina/farmacologia , Citarabina/uso terapêutico , DNA de Neoplasias/genética , Desoxicitidina Quinase/genética , Dexametasona/uso terapêutico , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Dosagem de Genes , Xenoenxertos , Humanos , Masculino , Metotrexato/farmacologia , Metotrexato/uso terapêutico , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Peptídeo Sintases/genética , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Quimera por Radiação , Análise de Sequência de DNA , Vincristina/uso terapêuticoRESUMO
Most patients with acute lymphoblastic leukemia (ALL) respond well to standard chemotherapy-based treatments. However a significant proportion of patients, particularly adult patients, relapse with the majority dying of leukemia. FTY720 is an immunosuppressive drug that was recently approved for the treatment of multiple sclerosis and is currently under pre-clinical investigation as a therapy for a number of hematological malignancies. Using human ALL xenografts in NOD/SCIDγc(-/-) mice, we show for the first time that three Ph(+) human ALL xenografts responded to FTY720 with an 80 ± 12% (p = 0.048) reduction in overall disease when treatment was commenced early. In contrast, treatment of mice with FTY720 did not result in reduced leukemia compared to controls using four separate human Ph(-) ALL xenografts. Although FTY720 reactivated PP2A in vitro, this reactivation was not required for death of Ph(-) ALL cells. The plasma levels of FTY720 achieved in the mice were in the high nanomolar range. However, the response seen in the Ph(+) ALL xenografts when treatment was initiated early implies that in vivo efficacy may be obtained with substantially lower drug concentrations than those required in vitro. Our data suggest that while FTY720 may have potential as a treatment for Ph(+) ALL it will not be a useful agent for the treatment of Ph(-) B-ALL.
Assuntos
Antineoplásicos/uso terapêutico , Cromossomo Filadélfia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Propilenoglicóis/uso terapêutico , Esfingosina/análogos & derivados , Animais , Antineoplásicos/administração & dosagem , Progressão da Doença , Esquema de Medicação , Cloridrato de Fingolimode , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Estadiamento de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Propilenoglicóis/administração & dosagem , Proteína Fosfatase 2/metabolismo , Esfingosina/administração & dosagem , Esfingosina/uso terapêutico , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Modelos Animais de Doenças , Perfilação da Expressão Gênica , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Animais , Medula Óssea/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Especificidade da Espécie , Baço/metabolismoRESUMO
Due to the development of neurological toxicity and resistance to methotrexate (MTX), other antifolates have been evaluated for its potential replacement in the treatment of childhood acute lymphoblastic leukaemia (ALL). Aminopterin (AMT) has been suggested to provide clinical advantages over MTX and other antifolates. AMT activity, compared with MTX, was evaluated in ALL and lymphoma preclinical models. The minimum survival fraction at the range of concentrations tested was lower with AMT than with MTX in 3 out of 15 cell lines. Both AMT and MTX significantly extended the event-free survival of mice bearing 3 out of 4 xenografts with equivalent activity.
Assuntos
Aminopterina/uso terapêutico , Antagonistas do Ácido Fólico/uso terapêutico , Linfoma/tratamento farmacológico , Metotrexato/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Aminopterina/efeitos adversos , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Antagonistas do Ácido Fólico/efeitos adversos , Humanos , Concentração Inibidora 50 , Metotrexato/efeitos adversos , Camundongos , Camundongos SCID , Recidiva , Análise de Regressão , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Modelos Animais de Doenças , Proteína de Leucina Linfoide-Mieloide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação Genética , Animais , Feminino , Histona-Lisina N-Metiltransferase , Humanos , Lactente , Cariotipagem , Camundongos , Camundongos SCID , Transplante de Neoplasias , Transplante HeterólogoRESUMO
The microtubule-depolymerizing drug, vincristine, is effective in the treatment of acute lymphoblastic leukemia (ALL). Although vincristine resistance mechanisms have been extensively characterized in cell lines, their clinical relevance is poorly understood. The aim of the current study was to define clinically relevant mechanisms of vincristine resistance in a panel of childhood ALL xenografts established in immune-deficient (nonobese diabetic/severe combined immunodeficient) mice. We also studied two independent xenograft sublines that were selected by in vivo vincristine exposure. In vitro vincristine sensitivity determined by a stromal coculture, murine bone marrow stromal cell line (MS-5), assay, but not methyl-thiazolyl-tetrazolium metabolic activity assay, significantly correlated (P = 0.05) with the length of the patients' first remission. Investigations into mechanisms of resistance revealed no association with steady-state vincristine accumulation or increased activity and/or expression of ATP-binding cassette transporters, although increased intracellular levels of polymerized tubulin significantly correlated with resistance (r = 0.85; P = 0.0019). Two xenograft sublines selected by in vivo vincristine exposure exhibited a 2-fold increase in polymerized tubulin levels compared with the parental subline (P < 0.05), reflecting their in vivo vincristine resistance. In this study, a vincristine-resistant xenograft with high levels of polymerized tubulin was relatively sensitive to the microtubule-polymerizing drug paclitaxel. These results indicate that the balance between polymerized and nonpolymerized tubulin may be an important determinant of response to Vinca alkaloid-based chemotherapy regimens in childhood ALL.
Assuntos
Resistencia a Medicamentos Antineoplásicos/fisiologia , Microtúbulos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Vincristina/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Camundongos , Microtúbulos/efeitos dos fármacos , Polímeros/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Vincristina/farmacologiaRESUMO
Cell line models of glucocorticoid resistance in childhood acute lymphoblastic leukemia (ALL) almost invariably exhibit altered glucocorticoid receptor (GR) function. However, these findings are incongruous with those using specimens derived directly from leukemia patients, in which GR alterations are rarely found. Consequently, mechanisms of glucocorticoid resistance in the clinical setting remain largely unresolved. We present a novel paradigm of glucocorticoid resistance in childhood ALL, in which patient biopsies have been directly established as continuous xenografts in immune-deficient mice, without prior in vitro culture. We show that the GRs from six highly dexamethasone-resistant xenografts (in vitro IC(50) >10 micromol/L) exhibit no defects in ligand-induced nuclear translocation and binding to a consensus glucocorticoid response element (GRE). This finding contrasts with five commonly used leukemia cell lines, all of which exhibited defective GRE binding. Moreover, whereas the GRs of dexamethasone-resistant xenografts were transcriptionally active, as assessed by the ability to induce the glucocorticoid-induced leucine zipper (GILZ) gene, resistance was associated with failure to induce the bim gene, which encodes a proapoptotic BH3-only protein. Furthermore, the receptor tyrosine kinase inhibitor, SU11657, completely reversed dexamethasone resistance in a xenograft expressing functional GR, indicating that pharmacologic reversal of glucocorticoid resistance in childhood ALL is achievable.
Assuntos
Dexametasona/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Receptores de Glucocorticoides/metabolismo , Animais , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Criança , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Compostos Orgânicos/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores de Glucocorticoides/biossíntese , Receptores de Glucocorticoides/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transcrição Gênica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Acute lymphoblastic leukemia (ALL) is predominantly a disease of the bone marrow that disseminates to multiple organ sites throughout the body and, without aggressive treatment, eventually results in multiorgan failure and death. Experimental models that mimic the dissemination of ALL have been difficult to establish, principally due to the poor engraftment efficiency of normal and malignant human hematopoietic cells in various strains of immune-deficient mice. The recent availability of mouse strains that are even more immunocompromised than established strains such as the nude (nu/nu) or severe combined immunodeficient (SCID) mouse has presented opportunities to establish improved experimental models of human leukemia. In this chapter we outline the methodology to (1) establish continuous xenografts from primary childhood ALL biopsies in nonobese diabetic/SCID (NOD/SCID) mice and (2) utilize these xenograft models of systemic disease to test established and experimental drugs while monitoring leukemia progression in "real time" by serial monitoring of murine peripheral blood. These experimental models will be useful for the preclinical evaluation of novel therapies.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Animais , Biópsia , Progressão da Doença , Humanos , Técnicas In Vitro , Camundongos , Camundongos Nus , Camundongos SCID , Transplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologiaRESUMO
Continuous xenografts from 10 children with acute lymphoblastic leukemia (ALL) were established in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Relative to primary engrafted cells, negligible changes in growth rates and immunophenotype were observed at second and third passage. Analysis of clonal antigen receptor gene rearrangements in 2 xenografts from patients at diagnosis showed that the pattern of clonal variation observed following tertiary transplantation in mice exactly reflected that in bone marrow samples at the time of clinical relapse. Patients experienced diverse treatment outcomes, including 5 who died of disease (median, 13 months; range, 11-76 months, from date of diagnosis), and 5 who remain alive (median, 103 months; range, 56-131 months, following diagnosis). When stratified according to patient outcome, the in vivo sensitivity of xenografts to vincristine and dexamethasone, but not methotrexate, differed significantly (P =.028, P =.029, and P =.56, respectively). The in vitro sensitivity of xenografts to dexamethasone, but not vincristine, correlated significantly with in vivo responses and patient outcome. This study shows, for the first time, that the biologic and genetic characteristics, and patterns of chemosensitivity, of childhood ALL xenografts accurately reflect the clinical disease. As such, they provide powerful experimental models to prioritize new therapeutic strategies for future clinical trials.