RESUMEN
Hematopoietic stem cells (HSCs) sustain blood cell homeostasis throughout life and can regenerate all blood lineages after transplantation. Despite this clear functional definition, highly enriched isolation of human HSCs can currently only be achieved through combinatorial assessment of multiple surface antigens. Although several transgenic HSC reporter mouse strains have been described, no analogous approach to prospectively isolate human HSCs has been reported. To identify genes with the most selective expression in human HSCs, we profiled population and single-cell transcriptomes of unexpanded and ex vivo cultured cord blood-derived hematopoietic stem and progenitor cells as well as peripheral blood, adult bone marrow, and fetal liver. On the basis of these analyses, we propose the master transcription factor HLF (hepatic leukemia factor) as one of the most specific HSC marker genes. To directly track its expression in human hematopoietic cells, we developed a genomic HLF reporter strategy, capable of selectively labeling the most immature blood cells on the basis of a single engineered parameter. Most importantly, HLF-expressing cells comprise all stem cell activity in culture and in vivo during serial transplantation. Taken together, these results experimentally establish HLF as a defining gene of the human HSC state and outline a new approach to continuously mark these cells with high fidelity.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Transcriptoma , Hematopoyesis , Células Madre Hematopoyéticas/citología , Humanos , Análisis de la Célula IndividualRESUMEN
Chromatin modulators are emerging as attractive drug targets, given their widespread implication in human cancers and susceptibility to pharmacological inhibition. Here we establish the histone methyltransferase G9a/EHMT2 as a selective regulator of fast proliferating myeloid progenitors with no discernible function in hematopoietic stem cells (HSCs). In mouse models of acute myeloid leukemia (AML), loss of G9a significantly delays disease progression and reduces leukemia stem cell (LSC) frequency. We connect this function of G9a to its methyltransferase activity and its interaction with the leukemogenic transcription factor HoxA9 and provide evidence that primary human AML cells are sensitive to G9A inhibition. Our results highlight a clinical potential of G9A inhibition as a means to counteract the proliferation and self-renewal of AML cells by attenuating HoxA9-dependent transcription.
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Regulación Neoplásica de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas de Homeodominio/metabolismo , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Células HEK293 , Células Madre Hematopoyéticas/enzimología , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Leucemia Mieloide Aguda/patología , Ratones Endogámicos C57BL , Quinazolinas/farmacologíaRESUMEN
Neomorphic missense mutations affecting crucial lysine residues in histone H3 genes significantly contribute to a variety of solid cancers. Despite the high prevalence of H3K27M mutations in pediatric glioblastoma and their well-established impact on global histone H3 lysine 27 di- and trimethylation (H3K27me2/3), the relevance of these mutations has not been studied in acute myeloid leukemia (AML). Here, we report the first identification of H3K27M and H3K27I mutations in patients with AML. We find that these lesions are major determinants of reduced H3K27me2/3 in these patients and that they are associated with common aberrations in the RUNX1 gene. We demonstrate that H3K27I/M mutations are strong disease accelerators in a RUNX1-RUNX1T1 AML mouse model, suggesting that H3K27me2/3 has an important and selective leukemia-suppressive activity in this genetic context.
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Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Histonas/genética , Leucemia Mieloide Aguda/genética , Mutación Missense , Transformación Genética , Adolescente , Anciano de 80 o más Años , Animales , Metilación de ADN , Femenino , Humanos , Lisina/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Proteínas de Fusión Oncogénica/genética , Proteína 1 Compañera de Translocación de RUNX1/genética , Análisis de Secuencia de ADNRESUMEN
A small subset of human cord blood CD34+ cells express endothelial protein C receptor (EPCR/CD201/PROCR) when exposed to the hematopoietic stem cell (HSC) self-renewal agonist UM171. In this article, we show that EPCR-positive UM171-treated cells, as opposed to EPCR-negative cells, exhibit robust multilineage repopulation and serial reconstitution ability in immunocompromised mice. In contrast to other stem cell markers, such as CD38, EPCR expression is maintained when cells are introduced in culture, irrespective of UM171 treatment. Although engineered overexpression of EPCR fails to reproduce the effects of UM171 on HSC activity, its expression is required for the repopulating activity of human HSCs. Altogether, our results indicate that EPCR is a reliable and cell culture-compatible marker of UM171-expanded human cord blood HSCs.
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Antígenos CD34/análisis , Antígenos CD/análisis , Sangre Fetal/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Indoles/farmacología , Pirimidinas/farmacología , Receptores de Superficie Celular/análisis , Animales , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de los fármacos , Receptor de Proteína C Endotelial , Femenino , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Humanos , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
Methylation of specific lysine residues in the C terminus of p53 is thought to govern p53-dependent transcription following genotoxic and oncogenic stress. In particular, Set7/9 (KMT7)-mediated monomethylation of human p53 at lysine 372 (p53K372me1) was suggested to be essential for p53 activation in human cell lines. This finding was confirmed in a Set7/9 knockout mouse model (Kurash et al., 2008). In an independent knockout mouse strain deficient in Set7/9, we have investigated its involvement in p53 regulation and find that cells from these mice are normal in their ability to induce p53-dependent transcription following genotoxic and oncogenic insults. Most importantly, we detect no impairment in canonical p53 functions in these mice, indicating that Set7/9-mediated methylation of p53 does not seem to represent a major regulatory event and does not appreciably control p53 activity in vivo.
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Proteína Metiltransferasas/genética , Transcripción Genética , Proteína p53 Supresora de Tumor/fisiología , Animales , Apoptosis/genética , Ciclo Celular , Senescencia Celular/genética , Regulación de la Expresión Génica , N-Metiltransferasa de Histona-Lisina , Ratones , Ratones Endogámicos C57BL , Proteína Metiltransferasas/metabolismo , Proteína Metiltransferasas/fisiología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The ubiquitin-associated protein 2-like (UBAP2L) gene remains poorly studied in human and mouse development. UBAP2L interacts with the Polycomb group protein B lymphoma Mo-MLV insertion region 1 homolog (BMI1) and determines the activity of mouse hematopoietic stem cells in vivo Here we show that loss of Ubap2l leads to disorganized respiratory epithelium of mutant neonates, which die of respiratory failure. We also show that UBAP2L overexpression leads to epithelial-mesenchymal transition-like phenotype in a non-small cell lung carcinoma (NSCLC) cell line. UBAP2L is amplified in 15% of human primary lung adenocarcinoma specimens. Such patients express higher levels of UBAP2L and show a reduction in survival when compared with those who do not have this gene amplification. Supporting a possible role for UBAP2L in lung tumor progression, NSCLC cells engineered to express low levels of this gene produce much smaller tumors in vivo than wild-type control cells. Together, these results suggest that UBAP2L contributes to epithelial lung cell identity in mice and that it plays an important role in human lung adenocarcinoma.-Aucagne, R., Girard, S., Mayotte, N., Lehnertz, B., Lopes-Paciencia, S., Gendron, P., Boucher, G., Chagraoui, J., Sauvageau, G. UBAP2L is amplified in a large subset of human lung adenocarcinoma and is critical for epithelial lung cell identity and tumor metastasis.
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Adenocarcinoma/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteínas Portadoras/biosíntesis , Células Epiteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/biosíntesis , Mucosa Respiratoria/metabolismo , Células A549 , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas Portadoras/genética , Células Epiteliales/patología , Transición Epitelial-Mesenquimal/genética , Femenino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Ratones , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Mucosa Respiratoria/patologíaRESUMEN
Methylation on lysine 9 of histone H3 (H3K9me) and DNA methylation play important roles in the transcriptional silencing of specific genes and repetitive elements. Both marks are detected on class I and II endogenous retroviruses (ERVs) in murine embryonic stem cells (mESCs). Recently, we reported that the H3K9-specific lysine methyltransferase (KMTase) Eset/Setdb1/KMT1E is required for H3K9me3 and the maintenance of silencing of ERVs in mESCs. In contrast, G9a/Ehmt2/KMT1C is dispensable, despite the fact that this KMTase is required for H3K9 dimethylation (H3K9me2) and efficient DNA methylation of these retroelements. Transcription of the exogenous retrovirus (XRV) Moloney murine leukemia virus is rapidly extinguished after integration in mESCs, concomitant with de novo DNA methylation. However, the role that H3K9 KMTases play in this process has not been addressed. Here, we demonstrate that G9a, but not Suv39h1 or Suv39h2, is required for silencing of newly integrated Moloney murine leukemia virus-based vectors in mESCs. The silencing defect in G9a(-/-) cells is accompanied by a reduction of H3K9me2 at the proviral LTR, indicating that XRVs are direct targets of G9a. Furthermore, de novo DNA methylation of newly integrated proviruses is impaired in the G9a(-/-) line, phenocopying proviral DNA methylation and silencing defects observed in Dnmt3a-deficient mESCs. Once established, however, maintenance of silencing of XRVs, like ERVs, is dependent exclusively on the KMTase Eset. Taken together, these observations reveal that in mESCs, the H3K9 KMTase G9a is required for the establishment, but not for the maintenance, of silencing of newly integrated proviruses.
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Metilación de ADN/genética , Células Madre Embrionarias/virología , N-Metiltransferasa de Histona-Lisina/metabolismo , Virus de la Leucemia Murina de Moloney/genética , Animales , Western Blotting , Inmunoprecipitación de Cromatina , ADN (Citosina-5-)-Metiltransferasas/genética , Retrovirus Endógenos/genética , Citometría de Flujo , Silenciador del Gen , Vectores Genéticos/genética , N-Metiltransferasa de Histona-Lisina/genética , Ratones , Ratones Noqueados , Provirus/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Precision gene editing in primary hematopoietic stem and progenitor cells (HSPCs) would facilitate both curative treatments for monogenic disorders as well as disease modelling. Precise efficiencies even with the CRISPR/Cas system, however, remain limited. Through an optimization of guide RNA delivery, donor design, and additives, we have now obtained mean precise editing efficiencies >90% on primary cord blood HSCPs with minimal toxicity and without observed off-target editing. The main protocol modifications needed to achieve such high efficiencies were the addition of the DNA-PK inhibitor AZD7648, and the inclusion of spacer-breaking silent mutations in the donor in addition to mutations disrupting the PAM sequence. Critically, editing was even across the progenitor hierarchy, did not substantially distort the hierarchy or affect lineage outputs in colony-forming cell assays or the frequency of high self-renewal potential long-term culture initiating cells. As modelling of many diseases requires heterozygosity, we also demonstrated that the overall editing and zygosity can be tuned by adding in defined mixtures of mutant and wild-type donors. With these optimizations, editing at near-perfect efficiency can now be accomplished directly in human HSPCs. This will open new avenues in both therapeutic strategies and disease modelling.
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Edición Génica , Células Madre Hematopoyéticas , Humanos , Edición Génica/métodos , Sistemas CRISPR-Cas , ARN Guía de Sistemas CRISPR-Cas/genética , Sangre Fetal/citología , Células CultivadasRESUMEN
Leukemia stem cells (LSCs) share numerous features with healthy hematopoietic stem cells (HSCs). G-protein coupled receptor family C group 5 member C (GPRC5C) is a regulator of HSC dormancy. However, GPRC5C functionality in acute myeloid leukemia (AML) is yet to be determined. Within patient AML cohorts, high GPRC5C levels correlated with poorer survival. Ectopic Gprc5c expression increased AML aggression through the activation of NF-κB, which resulted in an altered metabolic state with increased levels of intracellular branched-chain amino acids (BCAAs). This onco-metabolic profile was reversed upon loss of Gprc5c, which also abrogated the leukemia-initiating potential. Targeting the BCAA transporter SLC7A5 with JPH203 inhibited oxidative phosphorylation and elicited strong antileukemia effects, specifically in mouse and patient AML samples while sparing healthy bone marrow cells. This antileukemia effect was strengthened in the presence of venetoclax and azacitidine. Our results indicate that the GPRC5C-NF-κB-SLC7A5-BCAAs axis is a therapeutic target that can compromise leukemia stem cell function in AML.
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Aminoácidos de Cadena Ramificada , Leucemia Mieloide Aguda , Receptores Acoplados a Proteínas G , Animales , Humanos , Ratones , Aminoácidos de Cadena Ramificada/uso terapéutico , Transportador de Aminoácidos Neutros Grandes 1/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , FN-kappa B/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
High-mobility group AT-hook 2 (HMGA2) is a nonhistone chromatin-binding protein that is normally expressed in stem cells of various tissues and aberrantly detected in several tumor types. We recently observed that one-fourth of human acute myeloid leukemia (AML) specimens express HMGA2, which associates with a very poor prognosis. We present results indicating that HMGA2+ AMLs share a distinct transcriptional signature representing an immature phenotype. Using single-cell analyses, we showed that HMGA2 is expressed in CD34+ subsets of stem cells and early progenitors, whether normal or derived from AML specimens. Of interest, we found that one of the strongest gene expression signatures associated with HMGA2 in AML is the upregulation of G2/M checkpoint genes. Whole-genome CRISPR/Cas9 screening in HMGA2 overexpressing cells further revealed a synthetic lethal interaction with several G2/M checkpoint genes. Accordingly, small molecules that target G2/M proteins were preferentially active in vitro and in vivo on HMGA2+ AML specimens. Together, our findings suggest that HMGA2 is a key functional determinant in AML and is associated with stem cell features, G2/M status, and related drug sensitivity.
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Leucemia Mieloide Aguda , Antígenos CD34 , Puntos de Control del Ciclo Celular , Humanos , Leucemia Mieloide Aguda/patología , Regulación hacia ArribaRESUMEN
Bone marrow haematopoietic stem cells (HSCs) are vital for lifelong maintenance of healthy haematopoiesis. In inbred mice housed in gnotobiotic facilities, the top of the haematopoietic hierarchy is occupied by dormant HSCs, which reversibly exit quiescence during stress. Whether HSC dormancy exists in humans remains debatable. Here, using single-cell RNA sequencing, we show a continuous landscape of highly purified human bone marrow HSCs displaying varying degrees of dormancy. We identify the orphan receptor GPRC5C, which enriches for dormant human HSCs. GPRC5C is also essential for HSC function, as demonstrated by genetic loss- and gain-of-function analyses. Through structural modelling and biochemical assays, we show that hyaluronic acid, a bone marrow extracellular matrix component, preserves dormancy through GPRC5C. We identify the hyaluronic acid-GPRC5C signalling axis controlling the state of dormancy in mouse and human HSCs.
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Células Madre Hematopoyéticas , Ácido Hialurónico , Animales , Médula Ósea , Hematopoyesis , Humanos , Ratones , Transducción de SeñalRESUMEN
Cholesterol homeostasis has been proposed as one mechanism contributing to chemoresistance in AML and hence, inclusion of statins in therapeutic regimens as part of clinical trials in AML has shown encouraging results. Chemical screening of primary human AML specimens by our group led to the identification of lipophilic statins as potent inhibitors of AMLs from a wide range of cytogenetic groups. Genetic screening to identify modulators of the statin response uncovered the role of protein geranylgeranylation and of RAB proteins, coordinating various aspect of vesicular trafficking, in mediating the effects of statins on AML cell viability. We further show that statins can inhibit vesicle-mediated transport in primary human specimens, and that statins sensitive samples show expression signatures reminiscent of enhanced vesicular trafficking. Overall, this study sheds light into the mechanism of action of statins in AML and identifies a novel vulnerability for cytogenetically diverse AML.
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Inhibidores de Hidroximetilglutaril-CoA Reductasas , Leucemia Mieloide Aguda , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genéticaRESUMEN
Acute myeloid leukemias (AML) with mutations in the NPM1 gene (NPM1c+) represent a large AML subgroup with varying response to conventional treatment, highlighting the need to develop targeted therapeutic strategies for this disease. We screened a library of clinical drugs on a cohort of primary human AML specimens and identified the BCL2 inhibitor ABT-199 as a selective agent against NPM1c+ AML. Mutational analysis of ABT-199-sensitive and -resistant specimens identified mutations in NPM1, RAD21, and IDH1/IDH2 as predictors of ABT-199 sensitivity. Comparative transcriptome analysis further uncovered BCL2A1 as a potential mediator of ABT-199 resistance in AML. In line with our observation that RAD21 mutation confers sensitivity to ABT-199, we provide functional evidence that reducing RAD21 levels can sensitize AML cells to BCL2 inhibition. Moreover, we demonstrate that ABT-199 is able to produce selective anti-AML activity in vivo toward AML with mutations associated with compound sensitivity in PDX models. Overall, this study delineates the contribution of several genetic events to the response to ABT-199 and provides a rationale for the development of targeted therapies for NPM1c+ AML.
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Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Resistencia a Antineoplásicos/genética , Leucemia Mieloide Aguda/genética , Antígenos de Histocompatibilidad Menor/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Sulfonamidas/farmacología , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Mutación , Proteínas Nucleares/genética , Nucleofosmina , Células Tumorales CultivadasRESUMEN
BACKGROUND: Benefits of cord blood transplantation include low rates of relapse and chronic graft-versus-host disease (GVHD). However, the use of cord blood is rapidly declining because of the high incidence of infections, severe acute GVHD, and transplant-related mortality. UM171, a haematopoietic stem cell self-renewal agonist, has been shown to expand cord blood stem cells and enhance multilineage blood cell reconstitution in mice. We aimed to investigate the safety and feasibility of single UM171-expanded cord blood transplantation in patients with haematological malignancies who do not have a suitable HLA-matched donor. METHODS: This single-arm, open-label, phase 1-2 safety and feasibility study was done at two hospitals in Canada. The study had two parts. In part 1, patients received two cord blood units (one expanded with UM171 and one unmanipulated cord blood) until UM171-expanded cord blood demonstrated engraftment. Once engraftment was documented we initiated part 2, reported here, in which patients received a single UM171-expanded cord blood unit with a dose de-escalation design to determine the minimal cord blood unit cell dose that achieved prompt engraftment. Eligible patients were aged 3-64 years, weighed 12 kg or more, had a haematological malignancy with an indication for allogeneic hematopoietic stem cell transplant and did not have a suitable HLA-matched donor, and a had a Karnofsky performance status score of 70% or more. Five clinical sites were planned to participate in the study; however, only two study sites opened, both of which only treated adult patients, thus no paediatric patients (aged <18 years) were recruited. Patients aged younger than 50 years without comorbidities received a myeloablative conditioning regimen (cyclophosphamide 120 mg/kg, fludarabine 75 mg/m2, and 12 Gy total body irradiation) and patients aged older than 50 years and those with comorbidities received a less myeloablative conditioning regimen (cyclophosphamide 50 mg/kg, thiotepa 10 mg/kg, fludarabine 150 mg/m2, and 4 Gy total body irradiation). Patients were infused with the 7-day UM171-expanded CD34-positive cells and the lymphocyte-containing CD34-negative fraction. The primary endpoints were feasibility of UM171 expansion, safety of the transplant, kinetics of hematopoietic reconstitution (time to neutrophil and platelet engraftment) of UM171-expanded cord blood, and minimal pre-expansion cord blood unit cell dose that achieved prompt engraftment. We analysed feasibility in all enrolled patients and all other primary outcomes were analysed per protocol, in all patients who received single UM171-expanded cord blood transplantation. This trial has been completed and was registered with ClinicalTrials.gov, NCT02668315. FINDINGS: Between Feb 17, 2016, and Nov 11, 2018, we enrolled 27 patients, four of whom received two cord blood units for safety purposes in part 1 of the study. 23 patients were subsequently enrolled in part 2 to receive a single UM171-expanded cord blood transplant and 22 patients received a single UM171-expanded cord blood transplantation. At data cutoff (Dec 31, 2018), median follow-up was 18 months (IQR 12-22). The minimal cord blood unit cell dose at thaw that achieved prompt engraftment as a single cord transplant after UM171 expansion was 0·52â×â105 CD34-positive cells. We successfully expanded 26 (96%) of 27 cord blood units with UM171. Among the 22 patients who received single UM171-expanded cord blood transplantation, median time to engraftment of 100 neutrophils per µL was 9·5 days (IQR 8-12), median time to engraftment of 500 neutrophils per µL was 18 days (12·5-20·0), and no graft failure occurred. Median time to platelet recovery was 42 days (IQR 35-47). The most common non-haematological adverse events were grade 3 febrile neutropenia (16 [73%] of 22 patients) and bacteraemia (nine [41%]). No unexpected adverse events were observed. One (5%) of 22 patients died due to treatment-related diffuse alveolar haemorrhage. INTERPRETATION: Our preliminary findings suggest that UM171 cord blood stem cell expansion is feasible, safe, and allows for the use of small single cords without compromising engraftment. UM171-expanded cord blood might have the potential to overcome the disadvantages of other cord blood transplants while maintaining the benefits of low risk of chronic GVHD and relapse, and warrants further investigation in randomised trials. FUNDING: Canadian Institutes of Health Research, Canadian Cancer Society and Stem Cell Network.
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Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/efectos de los fármacos , Indoles/farmacología , Pirimidinas/farmacología , Adolescente , Adulto , Autorrenovación de las Células/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Células Cultivadas/trasplante , Trasplante de Células Madre de Sangre del Cordón Umbilical/efectos adversos , Supervivencia sin Enfermedad , Estudios de Factibilidad , Neutropenia Febril/etiología , Femenino , Supervivencia de Injerto , Enfermedad Injerto contra Huésped/etiología , Neoplasias Hematológicas/terapia , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Células Madre Hematopoyéticas/citología , Humanos , Recién Nacido , Masculino , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , Resultado del Tratamiento , Adulto JovenRESUMEN
Transplantation of expanded hematopoietic stem cells (HSCs) and gene therapy based on HSC engineering have emerged as promising approaches for the treatment of hematological diseases. Nevertheless, the immunophenotype of cultured HSCs remains poorly defined. Here, we identify Integrin-α3 (ITGA3) as a marker of cultured human HSCs. Exploiting the pyrimidoindole derivative UM171 to expand cord blood (CB) cells, we show that ITGA3 expression is sufficient to separate the primitive EPCR+CD90+CD133+CD34+CD45RA- HSC population into two functionally distinct fractions presenting mostly short-term (ITGA3-) and both short-term and long-term (ITGA3+) repopulating potential. ITGA3+ cells exhibit robust multilineage differentiation potential, serial reconstitution ability in immunocompromised mice, and an HSC-specific transcriptomic signature. Moreover, ITGA3 expression is functionally required for the long-term engraftment of CB cells. Altogether, our results indicate that ITGA3 is a reliable marker of cultured human long-term repopulating HSCs (LT-HSCs) and represents an important tool to improve the accuracy of prospective HSC identification in culture.
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Biomarcadores/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Integrina alfa3/metabolismo , Animales , Antígenos CD34/metabolismo , Proliferación Celular , Autorrenovación de las Células , Regulación hacia Abajo , Sangre Fetal/citología , Perfilación de la Expresión Génica , Humanos , Ratones , Fenotipo , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
Elucidation of the molecular cues required to balance adult stem cell self-renewal and differentiation is critical for advancing cellular therapies. Herein, we report that the hematopoietic stem cell (HSC) self-renewal agonist UM171 triggers a balanced pro- and anti-inflammatory/detoxification network that relies on NFKB activation and protein C receptor-dependent ROS detoxification, respectively. We demonstrate that within this network, EPCR serves as a critical protective component as its deletion hypersensitizes primitive hematopoietic cells to pro-inflammatory signals and ROS accumulation resulting in compromised stem cell function. Conversely, abrogation of the pro-inflammatory activity of UM171 through treatment with dexamethasone, cAMP elevating agents or NFkB inhibitors abolishes EPCR upregulation and HSC expansion. Together, these results show that UM171 stimulates ex vivo HSC expansion by establishing a critical balance between key pro- and anti-inflammatory mediators of self-renewal.
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Autorrenovación de las Células/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Homeostasis/efectos de los fármacos , Indoles/farmacología , Pirimidinas/farmacología , Biomarcadores , Diferenciación Celular , Proliferación Celular , Perfilación de la Expresión Génica , Humanos , Fase I de la Desintoxicación Metabólica , Especies Reactivas de Oxígeno , Transducción de Señal/efectos de los fármacos , TranscriptomaRESUMEN
Patients diagnosed with acute myeloid leukemia with complex karyotype (CK AML) have an adverse prognosis using current therapies, especially when accompanied by TP53 alterations. We hereby report the RNA-sequencing analysis of the 68 CK AML samples included in the Leucegene 415 patient cohort. We confirm the frequent occurrence of TP53 alterations in this subgroup and further characterize the allele expression profile and transcript alterations of this gene. We also document that the RAS pathway (N/KRAS, NF1, PTPN11, BRAF) is frequently altered in this disease. Targeted chemical interrogation of genetically characterized primary CK AML samples identifies polo-like kinase 1 (PLK1) inhibitors as the most selective agents for this disease subgroup. TP53 status did not alter sensitivity to PLK1 inhibitors. Interestingly, CK AML specimens display a G2/M transcriptomic signature that includes higher expression levels of PLK1 and correlates with PLK1 inhibition sensitivity. Together, our results highlight vulnerability in CK AML. In line with these in vitro data, volasertib shows a strong anti-AML activity in xenotransplantation mouse models of human adverse AML. Considering that PLK1 inhibitors are currently being investigated clinically in AML and myelodysplastic syndromes, our results provide a new rationale for PLK1-directed therapy in patients with adverse cytogenetic AML.
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Proteínas de Ciclo Celular/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Pteridinas/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Animales , Línea Celular Tumoral , Femenino , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Cariotipo , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Masculino , Ratones , Persona de Mediana Edad , Proteína p53 Supresora de Tumor/genética , Adulto Joven , Quinasa Tipo Polo 1RESUMEN
To identify therapeutic targets in acute myeloid leukemia (AML), we chemically interrogated 200 sequenced primary specimens. Mubritinib, a known ERBB2 inhibitor, elicited strong anti-leukemic effects in vitro and in vivo. In the context of AML, mubritinib functions through ubiquinone-dependent inhibition of electron transport chain (ETC) complex I activity. Resistance to mubritinib characterized normal CD34+ hematopoietic cells and chemotherapy-sensitive AMLs, which displayed transcriptomic hallmarks of hypoxia. Conversely, sensitivity correlated with mitochondrial function-related gene expression levels and characterized a large subset of chemotherapy-resistant AMLs with oxidative phosphorylation (OXPHOS) hyperactivity. Altogether, our work thus identifies an ETC complex I inhibitor and reveals the genetic landscape of OXPHOS dependency in AML.
Asunto(s)
Antineoplásicos/farmacología , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Leucemia Mieloide Aguda/metabolismo , Oxazoles/farmacología , Fosforilación Oxidativa/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Triazoles/farmacología , Animales , Biomarcadores , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Hematopoyesis/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Ratones , Modelos Biológicos , Receptor ErbB-2/antagonistas & inhibidoresRESUMEN
BACKGROUND: Histone H3 lysine 9 (H3-K9) methylation and DNA methylation are characteristic hallmarks of mammalian heterochromatin. H3-K9 methylation was recently shown to be a prerequisite for DNA methylation in Neurospora crassa and Arabidopsis thaliana. Currently, it is unknown whether a similar dependence exists in mammalian organisms. RESULTS: Here, we demonstrate a physical and functional link between the Suv39h-HP1 histone methylation system and DNA methyltransferase 3b (Dnmt3b) in mammals. Whereas in wild-type cells Dnmt3b interacts with HP1 alpha and is concentrated at heterochromatic foci, it fails to localize to these regions in Suv39h double null (dn) mouse embryonic stem (ES) cells. Consistently, the Suv39h dn ES cells display an altered DNA methylation profile at pericentric satellite repeats, but not at other repeat sequences. In contrast, H3-K9 trimethylation at pericentric heterochromatin is not impaired in Dnmt1 single- or Dnmt3a/Dnmt3b double-deficient ES cells. We also show that pericentric heterochromatin is not transcriptionally inert and can give rise to transcripts spanning the major satellite repeats. CONCLUSIONS: These data demonstrate an evolutionarily conserved pathway between histone H3-K9 methylation and DNA methylation in mammals. While the Suv39h HMTases are required to direct H3-K9 trimethylation and Dnmt3b-dependent DNA methylation at pericentric repeats, DNA methylation at centromeric repeats occurs independent of Suv39h function. Thus, our data also indicate a more complex interrelatedness between histone and DNA methylation systems in mammals. Both methylation systems are likely to be important in reinforcing the stability of heterochromatic subdomains and thereby in protecting genome integrity.