RESUMO
A large body of literature highlights the importance of energy metabolism in the response of haematological malignancies to therapy. In this review, we are particularly interested in acute myeloid leukaemia, where mitochondrial metabolism plays a key role in response and resistance to treatment. We describe the new concept of mitohormesis in the response to therapy-induced stress and in the initiation of relapse in this disease.
De nombreux travaux de la littérature illustrent l'importance de l'étude du métabolisme énergétique pour la compréhension de la réponse aux thérapies des hémopathies malignes. Dans cette revue, nous nous sommes particulièrement intéressés aux leucémies aiguës myéloïdes pour lesquelles le métabolisme mitochondrial joue un rôle clé dans la réponse et la résistance aux traitements. Nous avons décrit le nouveau concept de mitohormesis dans la réponse aux stress induits par les thérapies et dans l'initiation des rechutes dans cette pathologie.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Mitocôndrias , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Metabolismo Energético/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Neoplasias/tratamento farmacológico , Antineoplásicos/farmacologia , AnimaisRESUMO
Mass cytometry by time-of-flight (CyTOF) is an emerging technology allowing for in-depth characterization of cellular heterogeneity in cancer and other diseases. Unfortunately, high-dimensional analyses of CyTOF data remain quite demanding. Here, we deploy a bioinformatics framework that tackles two fundamental problems in CyTOF analyses namely (1) automated annotation of cell populations guided by a reference dataset and (2) systematic utilization of single-cell data for effective patient stratification. By applying this framework on several publicly available datasets, we demonstrate that the Scaffold approach achieves good trade-off between sensitivity and specificity for automated cell type annotation. Additionally, a case study focusing on a cohort of 43 leukemia patients reported salient interactions between signaling proteins that are sufficient to predict short-term survival at time of diagnosis using the XGBoost algorithm. Our work introduces an automated and versatile analysis framework for CyTOF data with many applications in future precision medicine projects.
RESUMO
Dependency on mitochondrial oxidative phosphorylation (OxPhos) is a potential weakness for leukemic stem cells (LSC) that can be exploited for therapeutic purposes. Fatty acid oxidation (FAO) is a crucial OxPhos-fueling catabolic pathway for some acute myeloid leukemia (AML) cells, particularly chemotherapy-resistant AML cells. Here, we identified cold sensitivity at 4°C (cold killing challenge; CKC4), commonly used for sample storage, as a novel vulnerability that selectively kills AML LSCs with active FAO-supported OxPhos while sparing normal hematopoietic stem cells. Cell death of OxPhos-positive leukemic cells was induced by membrane permeabilization at 4°C; by sharp contrast, leukemic cells relying on glycolysis were resistant. Forcing glycolytic cells to activate OxPhos metabolism sensitized them to CKC4. Lipidomic and proteomic analyses showed that OxPhos shapes the composition of the plasma membrane and introduces variation of 22 lipid subfamilies between cold-sensitive and cold-resistant cells. Together, these findings indicate that steady-state energy metabolism at body temperature predetermines the sensitivity of AML LSCs to cold temperature, suggesting that cold sensitivity could be a potential OxPhos biomarker. These results could have important implications for designing experiments for AML research to avoid cell storage at 4°C. SIGNIFICANCE: Mitochondrial metabolism fueled by FAO alters the membrane composition and introduces membrane fragility upon cold exposure in OxPhos-driven AML and in LSCs. See related commentary by Jones, p. 2441.
Assuntos
Leucemia Mieloide Aguda , Fosforilação Oxidativa , Humanos , Temperatura Baixa , Proteômica , Leucemia Mieloide Aguda/tratamento farmacológico , Células-Tronco Hematopoéticas/metabolismo , Ácidos Graxos/metabolismo , Células-Tronco Neoplásicas/metabolismoRESUMO
The treatment of acute leukemia is challenging because of the genetic heterogeneity between and within patients. Leukemic stem cells (LSCs) are relatively drug-resistant and frequently relapse. Their plasticity and capacity to adapt to extracellular stress, in which mitochondrial metabolism and autophagy play important roles, further complicates treatment. Genetic models of phosphatidylinositol-5-phosphate 4-kinase type 2 protein (PIP4K2s) inhibition have demonstrated the relevance of these enzymes in mitochondrial homeostasis and autophagic flux. Here, we uncovered the cellular and molecular effects of THZ-P1-2, a pan-inhibitor of PIP4K2s, in acute leukemia cells. THZ-P1-2 reduced cell viability and induced DNA damage, apoptosis, loss of mitochondrial membrane potential, and the accumulation of acidic vesicular organelles. Protein expression analysis revealed that THZ-P1-2 impaired autophagic flux. In addition, THZ-P1-2 induced cell differentiation and showed synergistic effects with venetoclax. In primary leukemia cells, LC-MS/MS-based proteome analysis revealed that sensitivity to THZ-P1-2 is associated with mitochondrial metabolism, cell cycle, cell-of-origin (hematopoietic stem cell and myeloid progenitor), and the TP53 pathway. The minimal effects of THZ-P1-2 observed in healthy CD34+ cells suggest a favorable therapeutic window. Our study provides insights into the pharmacological inhibition of PIP4K2s targeting mitochondrial homeostasis and autophagy, shedding light on a new class of drugs for acute leukemia.
Assuntos
Leucemia Mieloide Aguda , Espectrometria de Massas em Tandem , Humanos , Cromatografia Líquida , Autofagia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Apoptose , HomeostaseAssuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Animais , Citocinas , Humanos , CamundongosRESUMO
Mitochondria are crucial organelles that not only regulate the energy metabolism, but also the survival and fate of eukaryotic cells. Mitochondria were recently discovered to be able to translocate from one cell to the other. This phenomenon was observed in vitro and in vivo, both in physiological and pathophysiological conditions including tissue injury and cancer. Mitochondria trafficking was found to exert prominent biological functions. In particular, several studies pointed out that this process governs some of the therapeutic effects of mesenchymal stem cells (MSCs). In this review, we give an overview of the current knowledge on MSC-dependent intercellular mitochondria trafficking and further discuss the recent findings on the intercellular mitochondria transfer between differentiated and mesenchymal stem cells, their biological significance and the mechanisms underlying this process.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Animais , Diferenciação Celular , DNA Mitocondrial/metabolismo , Humanos , Inflamação/prevenção & controle , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/terapiaRESUMO
Patients with acute myeloid leukemia and a high white blood cell count are at increased risk of early death and relapse. Because mediators of inflammation contribute to leukostasis and chemoresistance, dexamethasone added to chemotherapy could improve outcomes. This retrospective study evaluated the impact of adding or not adding dexamethasone to chemotherapy in a cohort of 160 patients with at least 50×109 white blood cells. In silico studies, primary samples, leukemic cell lines, and xenograft mouse models were used to explore the antileukemic activity of dexamethasone. There was no difference with respect to induction death rate, response, and infections between the 60 patients in the dexamethasone group and the 100 patients in the no dexamethasone group. Multivariate analysis showed that dexamethasone was significantly associated with improved relapse incidence (adjusted sub-HR: 0.30; 95% CI: 0.14-0.62; P=0.001), disease-free survival (adjusted HR: 0.50; 95% CI: 0.29-0.84; P=0.010), event-free survival (adjusted HR: 0.35; 95% CI: 0.21-0.58; P<0.001), and overall survival (adjusted HR: 0.41; 95% CI: 0.22-0.79; P=0.007). In a co-culture system, dexamethasone reduced the frequency of leukemic long-term culture initiating cells by 38% and enhanced the cytotoxicity of doxorubicin and cytarabine. In a patient-derived xenograft model treated with cytarabine, chemoresistant cells were enriched in genes of the inflammatory response modulated by dexamethasone. Dexamethasone also demonstrated antileukemic activity in NPM1-mutated samples. Dexamethasone may improve the outcome of acute myeloid leukemia patients receiving intensive chemotherapy. This effect could be due to the modulation of inflammatory chemoresistance pathways and to a specific activity in acute myeloid leukemia with NPM1 mutation.
Assuntos
Antineoplásicos Hormonais/uso terapêutico , Dexametasona/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Leucocitose/tratamento farmacológico , Leucocitose/patologia , Adolescente , Adulto , Idoso , Antineoplásicos Hormonais/administração & dosagem , Antineoplásicos Hormonais/efeitos adversos , Dexametasona/administração & dosagem , Dexametasona/efeitos adversos , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Leucocitose/genética , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Nucleares/genética , Nucleofosmina , Prognóstico , Recidiva , Indução de Remissão , Resultado do Tratamento , Adulto JovemRESUMO
Xenograft assay allows functional analysis of leukemia-initiating cells of acute myeloid leukemia primary samples. However, 40% of samples derived from patients with better outcomes fail to engraft in immunodeficient mouse recipients when conventional protocols are followed. At diagnosis, the engraftment of intermediate-risk group samples cannot be anticipated. In this study, we decided to further explore the reasons for xenograft success and failure. No differences in extracellular phenotype, apoptosis, or cell cycle profile could distinguish samples that engraft (engrafter [E]) from samples that do not engraft (nonengrafter [NE]) in NSG mice. In addition, ex vivo long-term culture assay revealed, after 5 weeks, a lower content of leukemic-LTC-initiating cells in the NE samples associated with a lower expansion rate capacity. One-week co-cultures with mesenchymal or osteoblastic or endothelial cells did not influence the proliferation rate, suggesting that E and NE samples are genuinely rapidly or slowly expanding independent of external cue. Engraftment success for some NE samples was consistently observed in recipient mice analyzed 6 months later than the conventional 3-month period. Eventually we implemented a flow cytometry-based assay, which allowed us to predict, in 1 week, the fast or delayed engraftment potential of a noncharacterized acute myeloid leukemia sample. This approach will be especially useful in selecting intermediate-risk-group patient samples and restricting the experimental duration to a 3-month period and, eventually, in reducing the number of animals and the cost and effort of unnecessary xenograft failures.
Assuntos
Leucemia Mieloide Aguda , Transplante de Neoplasias , Células-Tronco Neoplásicas , Animais , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/transplanteRESUMO
The bone marrow microenvironment (BMME) is a complex ecosystem that instructs and protects hematopoietic stem cells (HSCs) and their malignant counterparts, the leukemia-initiating cells (LICs). Within the physical and functional crosstalk that takes place between HSCs, LICs, and the BMME, the transfer of organelles and of mitochondria in particular is an important new intercellular communication mode in addition to adhesion molecules, tunneling nanotubes (TNTs), and the paracrine secretion of cytokines, (onco)metabolites, and extracellular vesicles (EVs). In this review we discuss the functional roles of mitochondrial transfer between BMME and leukemic cells, and give insights into this new mechanism of drug resistance whose understanding will open the way to innovative anticancer adjuvant treatments.
Assuntos
Células da Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Leucemia/genética , Mitocôndrias/metabolismo , Células da Medula Óssea/patologia , Citocinas/genética , Citocinas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia/metabolismo , Leucemia/patologia , Mitocôndrias/patologia , Comunicação Parácrina/genética , Microambiente Tumoral/genéticaRESUMO
Chemotherapy-resistant human acute myeloid leukemia (AML) cells are thought to be enriched in quiescent immature leukemic stem cells (LSC). To validate this hypothesis in vivo, we developed a clinically relevant chemotherapeutic approach treating patient-derived xenografts (PDX) with cytarabine (AraC). AraC residual AML cells are enriched in neither immature, quiescent cells nor LSCs. Strikingly, AraC-resistant preexisting and persisting cells displayed high levels of reactive oxygen species, showed increased mitochondrial mass, and retained active polarized mitochondria, consistent with a high oxidative phosphorylation (OXPHOS) status. AraC residual cells exhibited increased fatty-acid oxidation, upregulated CD36 expression, and a high OXPHOS gene signature predictive for treatment response in PDX and patients with AML. High OXPHOS but not low OXPHOS human AML cell lines were chemoresistant in vivo. Targeting mitochondrial protein synthesis, electron transfer, or fatty-acid oxidation induced an energetic shift toward low OXPHOS and markedly enhanced antileukemic effects of AraC. Together, this study demonstrates that essential mitochondrial functions contribute to AraC resistance in AML and are a robust hallmark of AraC sensitivity and a promising therapeutic avenue to treat AML residual disease.Significance: AraC-resistant AML cells exhibit metabolic features and gene signatures consistent with a high OXPHOS status. In these cells, targeting mitochondrial metabolism through the CD36-FAO-OXPHOS axis induces an energetic shift toward low OXPHOS and strongly enhanced antileukemic effects of AraC, offering a promising avenue to design new therapeutic strategies and fight AraC resistance in AML. Cancer Discov; 7(7); 716-35. ©2017 AACR.See related commentary by Schimmer, p. 670This article is highlighted in the In This Issue feature, p. 653.
Assuntos
Citarabina/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Animais , Antígenos CD36/genética , Linhagem Celular Tumoral , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Citarabina/efeitos adversos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Iron is an essential nutrient, acting as a catalyst for metabolic reactions that are fundamental to cell survival and proliferation. Iron complexed to transferrin is delivered to the metabolism after endocytosis via the CD71 surface receptor. We found that transformed cells from a murine PTEN-deficient T-cell lymphoma model and from T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/T-LL) cell lines overexpress CD71. As a consequence, the cells developed an addiction toward iron whose chelation by deferoxamine (DFO) dramatically affected their survival to induce apoptosis. Interestingly, DFO displayed synergistic activity with three ALL-specific drugs: dexamethasone, doxorubicin, and L-asparaginase. DFO appeared to act through a reactive oxygen species-dependent DNA damage response and potentiated the action of an inhibitor of the PARP pathway of DNA repair. Our results demonstrate that targeting iron metabolism could be an interesting adjuvant therapy for acute lymphoblastic leukemia.
Assuntos
Quelantes de Ferro/farmacologia , Ferro/metabolismo , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , PTEN Fosfo-Hidrolase/deficiência , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Apoptose/efeitos dos fármacos , Asparaginase/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quimioterapia Adjuvante , Dano ao DNA , Desferroxamina/farmacologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Expressão Gênica , Humanos , Quelantes de Ferro/uso terapêutico , Linfoma de Células T/tratamento farmacológico , Linfoma de Células T/mortalidade , Camundongos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/mortalidade , Espécies Reativas de Oxigênio/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismoRESUMO
Multiple myeloma (MM) evolves from a premalignant condition known as monoclonal gammopathy of undetermined significance (MGUS). However, the factors underlying the malignant transformation of plasmocytes in MM are not fully characterized. We report here that Eµ-directed expression of the antiapoptotic Bcl-B protein in mice drives an MM phenotype that reproduces accurately the human disease. Indeed, with age, Eµ-bcl-b transgenic mice develop the characteristic features of human MM, including bone malignant plasma cell infiltration, a monoclonal immunoglobulin peak, immunoglobulin deposit in renal tubules, and highly characteristic bone lytic lesions. In addition, the tumors are serially transplantable in irradiated wild-type mice, underlying the tumoral origin of the disease. Eµ-bcl-b plasmocytes show increased expression of a panel of genes known to be dysregulated in human MM pathogenesis. Treatment of Eµ-bcl-b mice with drugs currently used to treat patients such as melphalan and VELCADE efficiently kills malignant plasmocytes in vivo. Finally, we find that Bcl-B is overexpressed in plasmocytes from MM patients but neither in MGUS patients nor in healthy individuals, suggesting that Bcl-B may drive MM. These findings suggest that Bcl-B could be an important factor in MM disease and pinpoint Eµ-bcl-b mice as a pertinent model to validate new therapies in MM.
Assuntos
Mieloma Múltiplo/etiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Hipergamaglobulinemia/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mieloma Múltiplo/terapia , Proteínas Proto-Oncogênicas c-bcl-2/análise , Sindecana-1/análise , Proteína bcl-X/fisiologiaRESUMO
Here we demonstrate that in a niche-like coculture system, cells from both primary and cultured acute myeloid leukemia (AML) sources take up functional mitochondria from murine or human bone marrow stromal cells. Using different molecular and imaging approaches, we show that AML cells can increase their mitochondrial mass up to 14%. After coculture, recipient AML cells showed a 1.5-fold increase in mitochondrial adenosine triphosphate production and were less prone to mitochondrial depolarization after chemotherapy, displaying a higher survival. This unidirectional transfer enhanced by some chemotherapeutic agents required cell-cell contacts and proceeded through an endocytic pathway. Transfer was greater in AML blasts compared with normal cord blood CD34(+) cells. Finally, we demonstrate that mitochondrial transfer was observed in vivo in an NSG immunodeficient mouse xenograft model and also occurred in human leukemia initiating cells and progenitors. As mitochondrial transfer provides a clear survival advantage following chemotherapy and a higher leukemic long-term culture initiating cell potential, targeting mitochondrial transfer could represent a future therapeutic target for AML treatment.
Assuntos
Células da Medula Óssea/metabolismo , Leucemia Mieloide Aguda/metabolismo , Mitocôndrias/metabolismo , Animais , Células da Medula Óssea/patologia , Técnicas de Cocultura , Células HL-60 , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Nus , Mitocôndrias/patologia , Transplante de Neoplasias , Células Estromais/metabolismo , Células Estromais/patologia , Células U937RESUMO
Acute myeloid leukemia (AML) is sustained by a subpopulation of rare leukemia-initiating cells (LIC) detected in the xenograft assay by their capacity to self-renew and to generate non-LICs in vivo The xenotransplantation model captures functional properties of LICs that have clinical prognostic value. However, the long duration of this in vivo assay has hampered its use as a prognostic tool. Here, we show, using an ex vivo coculture system, that intermediate and poor risk AML patient samples at diagnosis have a 5 to 7 times higher frequency of leukemic long-term culture-initiating cells (L-LTC-IC) compared with the good risk group. We defined a fluorescence dilution factor (FDF) parameter that monitors sample proliferation over 1 week and established a strong correlation of this parameter with the L-LTC-IC frequency. A higher FDF was found for poor prognostic AMLs or for samples capable of engrafting NSG mice compared with good risk AMLs or nonengrafters. Importantly, FDF could classify normal karyotype intermediate risk patients into two groups with a significant difference in their overall survival, thus making this nongenetic and non-in vivo approach a new clinically relevant tool for better diagnosis of AML patients. Cancer Res; 76(8); 2082-6. ©2016 AACR.
Assuntos
Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/patologia , Proliferação de Células/fisiologia , Feminino , Humanos , Masculino , Prognóstico , Células Tumorais CultivadasRESUMO
Classic deamination mRNA changes, including cytidine to uridine (C-to-U) and adenosine to inosine (A-to-I), are important exceptions to the central dogma and lead to significant alterations in gene transcripts and products. Although there are a few reports of non-classic mRNA alterations, as yet there is no molecular explanation for these alternative changes. Wilms Tumor 1 (WT1) mutations and variants are implicated in several diseases, including Wilms tumor and acute myeloid leukemia (AML). We observed two alternative G-to-A changes, namely c.1303G>A and c.1586G>A in cDNA clones and found them to be recurrent in a series of 21 umbilical cord blood mononuclear cell (CBMC) samples studied. Two less conserved U-to-C changes were also observed. These alternative changes were found to be significantly higher in non-progenitor as compared to progenitor CBMCs, while they were found to be absent in a series of AML samples studied, indicating they are targeted, cell type-specific mRNA editing modifications. Since APOBEC/ADAR family members are implicated in RNA/DNA editing, we screened them by RNA-interference (RNAi) for WT1-mRNA changes and observed near complete reversal of WT1 c.1303G>A alteration upon APOBEC3A (A3A) knockdown. The role of A3A in mediating this change was confirmed by A3A overexpression in Fujioka cells, which led to a significant increase in WT1 c.1303G>A mRNA editing. Non-progenitor CBMCs showed correspondingly higher levels of A3A-mRNA and protein as compared to the progenitor ones. To our knowledge, this is the first report of mRNA modifying activity for an APOBEC3 protein and implicates A3A in a novel G-to-A form of editing. These findings open the way to further investigations into the mechanisms of other potential mRNA changes, which will help to redefine the RNA editing paradigm in both health and disease.
Assuntos
Citidina Desaminase/genética , Proteínas/genética , Edição de RNA , RNA Mensageiro/metabolismo , Proteínas WT1/genética , Adenosina/metabolismo , Sequência de Bases , Citidina Desaminase/antagonistas & inibidores , Citidina Desaminase/metabolismo , Guanina/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Interferência de RNA , RNA Mensageiro/química , RNA Interferente Pequeno/metabolismo , Análise de Sequência de DNA , Cordão Umbilical/citologia , Proteínas WT1/metabolismo , Tumor de Wilms/genética , Tumor de Wilms/patologiaRESUMO
Acute myeloid leukemia-initiating cells (LICs) are responsible for the emergence of leukemia and relapse after chemotherapy. Despite their identification more than 15 years ago, our understanding of the mechanisms responsible for their self-renewal activity and their chemoresistance remains poor. The slow progress in this area is partly due to the difficulty of studying these cells ex vivo. Indeed, current studies are reliant on xenotransplantation assays in immunodeficient mice. In this paper, we report that by modeling key elements of the bone marrow niche using different stromal feeder layers and hypoxic culture conditions, we can maintain LICs over at least 3 weeks and support their self-renewal properties demonstrated through primary and secondary successful xenograft. We provide a proof of principle that this niche-like culture system can be used to study LIC chemoresistance following in vitro cytarabine treatment similarly to the xenograft chemotherapy model. We found that although LICs are believed to be more chemoresistant than non-LICs, functionally defined LICs are not enriched after cytarabine treatment, and heterogeneity in their resistance to treatment can be seen between patients and even within the same patient. We present a culture system that can be used as an in vitro surrogate for xenotransplantation and that has the potential to dramatically increase the throughput of the investigation of LICs. This would further provide the means by which to identify and target the functionality of the different signaling pathways involved in the maintenance and resistance of LICs to improve acute myeloid leukemia treatments.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Citarabina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Células-Tronco Neoplásicas/metabolismo , Nicho de Células-Tronco , Animais , Feminino , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/transplante , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Acute myeloid leukemia (AML) induces bone marrow (BM) failure in patients, predisposing them to life-threatening infections and bleeding. The mechanism by which AML mediates this complication is unknown but one widely accepted explanation is that AML depletes the BM of hematopoietic stem cells (HSCs) through displacement. We sought to investigate how AML affects hematopoiesis by quantifying residual normal hematopoietic subpopulations in the BM of immunodeficient mice transplanted with human AML cells with a range of genetic lesions. The numbers of normal mouse HSCs were preserved whereas normal progenitors and other downstream hematopoietic cells were reduced following transplantation of primary AMLs, findings consistent with a differentiation block at the HSC-progenitor transition, rather than displacement. Once removed from the leukemic environment, residual normal hematopoietic cells differentiated normally and outcompeted steady-state hematopoietic cells, indicating that this effect is reversible. We confirmed the clinical significance of this by ex vivo analysis of normal hematopoietic subpopulations from BM of 16 patients with AML. This analysis demonstrated that the numbers of normal CD34(+)CD38(-) stem-progenitor cells were similar in the BM of AML patients and controls, whereas normal CD34(+)CD38(+) progenitors were reduced. Residual normal CD34(+) cells from patients with AML were enriched in long-term culture, initiating cells and repopulating cells compared with controls. In conclusion the data do not support the idea that BM failure in AML is due to HSC depletion. Rather, AML inhibits production of downstream hematopoietic cells by impeding differentiation at the HSC-progenitor transition.
Assuntos
Células da Medula Óssea/patologia , Diferenciação Celular/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Leucemia Mieloide Aguda/fisiopatologia , ADP-Ribosil Ciclase 1/metabolismo , Animais , Antígenos CD34/metabolismo , Proliferação de Células , Citometria de Fluxo , Imunofluorescência , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Microscopia Confocal , Estatísticas não ParamétricasRESUMO
The membrane-bound carbonic anhydrase isoforms CAIX and CAXII, underpin a pH-regulating system that enables hypoxic tumor cell survival. Here, we observed for the first time an upregulation of CAXII in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LL) cells. First we showed that CAXII is overexpressed in thymocytes from tPTEN-/- mice suffering of T lymphoma and that its pharmacological inhibition decreased cell proliferation and induced apoptosis. The same results were observed with the SupT1 human T cell lymphoma line. In addition we observed an upregulation of CAXII in human T-ALL samples supporting the case that CAXII may represent a new therapeutic target for T-ALL/LL.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/fisiologia , Proliferação de Células/efeitos dos fármacos , Linfoma de Células T/tratamento farmacológico , Animais , Anidrases Carbônicas/efeitos dos fármacos , Anidrases Carbônicas/genética , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , Linfoma de Células T/enzimologia , Camundongos , PTEN Fosfo-Hidrolase/fisiologiaRESUMO
The research reported in this communication demonstrates the emerging direct cell handling technology now widely referred to as aerodynamically assisted bio-jetting. This is a non-electric field driven approach which directly competes with bio-electrosprays. The technology in these investigations has been explored for the direct handling of live murine primary hematopoietic stem cells. The viability studies demonstrate the complete inertness of this technology for handling such cells for a wide range of applications in both basic biology and clinical medicine. Interestingly these studies pave the way for this technology to undergo development as a flow cell for utility as a sheathless cell most useful in flow cytometry.
Assuntos
Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Células-Tronco Hematopoéticas/citologia , Microfluídica/instrumentação , Microfluídica/métodos , Animais , Técnicas de Cultura de Células , Células Cultivadas , Humanos , Camundongos , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodosRESUMO
Dysregulation of the Wnt/ß-catenin pathway has been observed in various malignancies, including acute myeloid leukemia (AML), where the overexpression of ß-catenin is an independent adverse prognostic factor. ß-catenin was found upregulated in the vast majority of AML samples and more frequently localized in the nucleus of leukemic stem cells compared with normal bone marrow CD34(+) cells. The knockdown of ß-catenin, using a short hairpin RNA (shRNA) lentiviral approach, accelerates all-trans retinoic acid-induced differentiation and impairs the proliferation of HL60 leukemic cell line. Using in vivo quantitative tracking of these cells, we observed a reduced engraftment potential after xenotransplantation when ß-catenin was silenced. However, when studying primary AML cells, despite effective downregulation of ß-catenin we did not observe any impairment of their in vitro long-term maintenance on MS-5 stroma nor of their engraftment potential in vivo. Altogether, these results show that despite a frequent ß-catenin upregulation in AML, leukemia-initiating cells might not be 'addicted' to this pathway and thus targeted therapy against ß-catenin might not be successful in all patients.