RESUMEN
RUNX1 is mutated in â¼10% of adult acute myeloid leukemia (AML). Although most RUNX1 mutations in this disease are believed to be acquired, they can also be germline. Indeed, germline RUNX1 mutations result in the well-described autosomal-dominant familial platelet disorder with predisposition to hematologic malignancies (RUNX1-FPD, FPD/AML, FPDMM); â¼44% of affected individuals progress to AML or myelodysplastic syndromes. Using the Leucegene RUNX1 AML patient group, we sought to investigate the proportion of germline vs acquired RUNX1 mutations in this cohort. Our results showed that 30% of RUNX1 mutations in our AML cohort are germline. Molecular profiling revealed higher frequencies of NRAS mutations and other mutations known to activate various signaling pathways in these patients with RUNX1 germline-mutated AML. Moreover, 2 patients (mother and son) had co-occurrence of RUNX1 and CEBPA germline mutations, with variable AML disease onset at 59 and 27 years, respectively. Together, these data suggest a higher than anticipated frequency of germline RUNX1 mutations in the Leucegene cohort and further highlight the importance of testing for RUNX1 mutations in instances in which allogeneic stem cell transplantation using a related donor is envisioned.
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Biomarcadores de Tumor/genética , Proteínas Potenciadoras de Unión a CCAAT/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Factor de Transcripción GATA2/genética , Mutación de Línea Germinal , Leucemia Mieloide Aguda/genética , Mutación , Adulto , Anciano , Femenino , Estudios de Seguimiento , Humanos , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
BACKGROUND: Next-generation sequencing (NGS) allows unbiased, in-depth interrogation of cancer genomes. Many somatic variant callers have been developed yet accurate ascertainment of somatic variants remains a considerable challenge as evidenced by the varying mutation call rates and low concordance among callers. Statistical model-based algorithms that are currently available perform well under ideal scenarios, such as high sequencing depth, homogeneous tumor samples, high somatic variant allele frequency (VAF), but show limited performance with sub-optimal data such as low-pass whole-exome/genome sequencing data. While the goal of any cancer sequencing project is to identify a relevant, and limited, set of somatic variants for further sequence/functional validation, the inherently complex nature of cancer genomes combined with technical issues directly related to sequencing and alignment can affect either the specificity and/or sensitivity of most callers. RESULTS: For these reasons, we developed SNooPer, a versatile machine learning approach that uses Random Forest classification models to accurately call somatic variants in low-depth sequencing data. SNooPer uses a subset of variant positions from the sequencing output for which the class, true variation or sequencing error, is known to train the data-specific model. Here, using a real dataset of 40 childhood acute lymphoblastic leukemia patients, we show how the SNooPer algorithm is not affected by low coverage or low VAFs, and can be used to reduce overall sequencing costs while maintaining high specificity and sensitivity to somatic variant calling. When compared to three benchmarked somatic callers, SNooPer demonstrated the best overall performance. CONCLUSIONS: While the goal of any cancer sequencing project is to identify a relevant, and limited, set of somatic variants for further sequence/functional validation, the inherently complex nature of cancer genomes combined with technical issues directly related to sequencing and alignment can affect either the specificity and/or sensitivity of most callers. The flexibility of SNooPer's random forest protects against technical bias and systematic errors, and is appealing in that it does not rely on user-defined parameters. The code and user guide can be downloaded at https://sourceforge.net/projects/snooper/ .
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Biología Computacional/métodos , Variación Genética , Aprendizaje Automático , Programas Informáticos , Algoritmos , Secuenciación de Nucleótidos de Alto Rendimiento , Mutación , Polimorfismo de Nucleótido Simple , Reproducibilidad de los Resultados , Navegador Web , Flujo de TrabajoRESUMEN
One of the most rapidly evolving genes in humans, PRDM9, is a key determinant of the distribution of meiotic recombination events. Mutations in this meiotic-specific gene have previously been associated with male infertility in humans and recent studies suggest that PRDM9 may be involved in pathological genomic rearrangements. In studying genomes from families with children affected by B-cell precursor acute lymphoblastic leukemia (B-ALL), we characterized meiotic recombination patterns within a family with two siblings having hyperdiploid childhood B-ALL and observed unusual localization of maternal recombination events. The mother of the family carries a rare PRDM9 allele, potentially explaining the unusual patterns found. From exomes sequenced in 44 additional parents of children affected with B-ALL, we discovered a substantial and significant excess of rare allelic forms of PRDM9. The rare PRDM9 alleles are transmitted to the affected children in half the cases; nonetheless there remains a significant excess of rare alleles among patients relative to controls. We successfully replicated this latter observation in an independent cohort of 50 children with B-ALL, where we found an excess of rare PRDM9 alleles in aneuploid and infant B-ALL patients. PRDM9 variability in humans is thought to influence genomic instability, and these data support a potential role for PRDM9 variation in risk of acquiring aneuploidies or genomic rearrangements associated with childhood leukemogenesis.
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Alelos , N-Metiltransferasa de Histona-Lisina/genética , Leucemia Bifenotípica Aguda/genética , Leucemia Bifenotípica Aguda/patología , Adolescente , Niño , Preescolar , Estudios de Cohortes , Intercambio Genético , Exoma , Femenino , Frecuencia de los Genes , Reordenamiento Génico , Inestabilidad Genómica , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Lactante , Masculino , Meiosis , Análisis por Micromatrices , Mutación , Linaje , Polimorfismo de Nucleótido Simple , Recombinación Genética , Análisis de Secuencia de ADN , Translocación GenéticaRESUMEN
The most common rearrangement in childhood precursor B-cell acute lymphoblastic leukemia is the t(12;21)(p13;q22) translocation resulting in the ETV6-AML1 fusion gene. A frequent concomitant event is the loss of the residual ETV6 allele suggesting a critical role for the ETV6 transcriptional repressor in the etiology of this cancer. However, the precise mechanism through which loss of functional ETV6 contributes to disease pathogenesis is still unclear. To investigate the impact of ETV6 loss on the transcriptional network and to identify new transcriptional targets of ETV6, we used whole transcriptome analysis of both pre-B leukemic cell lines and patients combined with chromatin immunoprecipitation. Using this integrative approach, we identified 4 novel direct ETV6 target genes: CLIC5, BIRC7, ANGPTL2 and WBP1L To further evaluate the role of chloride intracellular channel protein CLIC5 in leukemogenesis, we generated cell lines overexpressing CLIC5 and demonstrated an increased resistance to hydrogen peroxide-induced apoptosis. We further described the implications of CLIC5's ion channel activity in lysosomal-mediated cell death, possibly by modulating the function of the transferrin receptor with which it colocalizes intracellularly. For the first time, we showed that loss of ETV6 leads to significant overexpression of CLIC5, which in turn leads to decreased lysosome-mediated apoptosis. Our data suggest that heightened CLIC5 activity could promote a permissive environment for oxidative stress-induced DNA damage accumulation, and thereby contribute to leukemogenesis.
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Canales de Cloruro/genética , Regulación Leucémica de la Expresión Génica , Proteínas de Microfilamentos/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogénicas c-ets/metabolismo , Proteínas Represoras/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/genética , Biomarcadores de Tumor , Línea Celular Tumoral , Niño , Preescolar , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Peróxido de Hidrógeno/farmacología , Lisosomas/metabolismo , Masculino , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Translocación Genética , Proteína ETS de Variante de Translocación 6RESUMEN
Whole-exome or gene targeted resequencing in hundreds to thousands of individuals has shown that the majority of genetic variants are at low frequency in human populations. Rare variants are enriched for functional mutations and are expected to explain an important fraction of the genetic etiology of human disease, therefore having a potential medical interest. In this work, we analyze the whole-exome sequences of French-Canadian individuals, a founder population with a unique demographic history that includes an original population bottleneck less than 20 generations ago, followed by a demographic explosion, and the whole exomes of French individuals sampled from France. We show that in less than 20 generations of genetic isolation from the French population, the genetic pool of French-Canadians shows reduced levels of diversity, higher homozygosity, and an excess of rare variants with low variant sharing with Europeans. Furthermore, the French-Canadian population contains a larger proportion of putatively damaging functional variants, which could partially explain the increased incidence of genetic disease in the province. Our results highlight the impact of population demography on genetic fitness and the contribution of rare variants to the human genetic variation landscape, emphasizing the need for deep cataloguing of genetic variants by resequencing worldwide human populations in order to truly assess disease risk.
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Susceptibilidad a Enfermedades , Exoma/genética , Mutación , Análisis de Secuencia de ADN/métodos , Canadá , Demografía , Francia , Frecuencia de los Genes , Genética de Población , Humanos , Polimorfismo de Nucleótido Simple , Población Blanca/genéticaRESUMEN
The contributions of the five (mv4)Int- and two (mv4)Xis arm-binding sites to the spatial intasome organization of bacteriophage mv4 were found not to be equivalent. The 8-bp overlap region was mapped to the left extremity of the core region and is directly flanked by the P2 Int arm-binding site. These results and the absence of characteristic Int core-binding sites suggest that the P2 site is the determinant for integrase positioning and recognition of the core region.
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Bacteriófagos/enzimología , Bacteriófagos/genética , ADN Viral/metabolismo , Integrasas/metabolismo , Lactobacillus delbrueckii/virología , Recombinación Genética , Proteínas Virales/metabolismo , Sitios de Ligazón Microbiológica , Bacteriófagos/química , Bacteriófagos/fisiología , Secuencia de Bases , Sitios de Unión , ADN Viral/química , ADN Viral/genética , Integrasas/genética , Datos de Secuencia Molecular , Proteínas Virales/genética , Integración ViralRESUMEN
BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. While the multi-step model of pediatric leukemogenesis suggests interplay between constitutional and somatic genomes, the role of inherited genetic variability remains largely undescribed. Nonsyndromic familial ALL, although extremely rare, provides the ideal setting to study inherited contributions to ALL. Toward this goal, we sequenced the exomes of a childhood ALL family consisting of mother, father and two non-twinned siblings diagnosed with concordant pre-B hyperdiploid ALL and previously shown to have inherited a rare form of PRDM9, a histone H3 methyltransferase involved in crossing-over at recombination hotspots and Holliday junctions. We postulated that inheritance of additional rare disadvantaging variants in predisposing cancer genes could affect genomic stability and lead to increased risk of hyperdiploid ALL within this family. METHODS: Whole exomes were captured using Agilent's SureSelect kit and sequenced on the Life Technologies SOLiD System. We applied a data reduction strategy to identify candidate variants shared by both affected siblings. Under a recessive disease model, we focused on rare non-synonymous or frame-shift variants in leukemia predisposing pathways. RESULTS: Though the family was nonsyndromic, we identified a combination of rare variants in Fanconi anemia (FA) genes FANCP/SLX4 (compound heterozygote - rs137976282/rs79842542) and FANCA (rs61753269) and a rare homozygous variant in the Holliday junction resolvase GEN1 (rs16981869). These variants, predicted to affect protein function, were previously identified in familial breast cancer cases. Based on our in-house database of 369 childhood ALL exomes, the sibs were the only patients to carry this particularly rare combination and only a single hyperdiploid patient was heterozygote at both FANCP/SLX4 positions, while no FANCA variant allele carriers were identified. FANCA is the most commonly mutated gene in FA and is essential for resolving DNA interstrand cross-links during replication. FANCP/SLX4 and GEN1 are involved in the cleavage of Holliday junctions and their mutated forms, in combination with the rare allele of PRDM9, could alter Holliday junction resolution leading to nondisjunction of chromosomes and segregation defects. CONCLUSION: Taken together, these results suggest that concomitant inheritance of rare variants in FANCA, FANCP/SLX4 and GEN1 on the specific genetic background of this familial case, could lead to increased genomic instability, hematopoietic dysfunction, and higher risk of childhood leukemia.
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Exoma , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Predisposición Genética a la Enfermedad , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Alelos , Preescolar , Secuenciación de Nucleótidos de Alto Rendimiento , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Masculino , Linaje , Polimorfismo de Nucleótido Simple , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , HermanosRESUMEN
BACKGROUND: The identification of oncogenic driver mutations has largely relied on the assumption that genes that exhibit more mutations than expected by chance are more likely to play an active role in tumorigenesis. Major cancer sequencing initiatives have therefore focused on recurrent mutations that are more likely to be drivers. However, in specific genetic contexts, low frequency mutations may also be capable of participating in oncogenic processes. Reliable strategies for identifying these rare or even patient-specific (private) mutations are needed in order to elucidate more personalized approaches to cancer diagnosis and treatment. METHODS: Here we performed whole-exome sequencing on three cases of childhood pre-B acute lymphoblastic leukemia (cALL), representing three cytogenetically-defined subgroups (high hyperdiploid, t(12;21) translocation, and cytogenetically normal). We applied a data reduction strategy to identify both common and rare/private somatic events with high functional potential. Top-ranked candidate mutations were subsequently validated at high sequencing depth on an independent platform and in vitro expression assays were performed to evaluate the impact of identified mutations on cell growth and survival. RESULTS: We identified 6 putatively damaging non-synonymous somatic mutations among the three cALL patients. Three of these mutations were well-characterized common cALL mutations involved in constitutive activation of the mitogen-activated protein kinase pathway (FLT3 p.D835Y, NRAS p.G13D, BRAF p.G466A). The remaining three patient-specific mutations (ACD p.G223V, DOT1L p.V114F, HCFC1 p.Y103H) were novel mutations previously undescribed in public cancer databases. Cytotoxicity assays demonstrated a protective effect of the ACD p.G223V mutation against apoptosis in leukemia cells. ACD plays a key role in protecting telomeres and recruiting telomerase. Using a telomere restriction fragment assay, we also showed that this novel mutation in ACD leads to increased telomere length in leukemia cells. CONCLUSION: This study identified ACD as a novel gene involved in cALL and points to a functional role for ACD in enhancing leukemia cell survival. These results highlight the importance of rare/private somatic mutations in understanding cALL etiology, even within well-characterized molecular subgroups.
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Apoptosis/genética , Análisis Mutacional de ADN/métodos , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Homeostasis del Telómero/genética , Proteínas de Unión a Telómeros/genética , Niño , Preescolar , Exoma/genética , Femenino , Humanos , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Complejo ShelterinaRESUMEN
Monosomy 5 and deletions of the chromosome 5q (-5/del(5q)) are recurrent events in de novo adult acute myeloid leukemia (AML), reaching up to 40% of cases in secondary AML. These chromosome anomalies are associated with TP53 mutations and with very poor prognosis. Using the large Leucegene genomic and transcriptomic dataset composed of 48 -5/del(5q) patient specimens and 367 control AML, we identified DELE1 - located in the common deleted region - as the most consistently downregulated gene in these leukemias. DELE1 encodes a mitochondrial protein recently characterized as the relay of mitochondrial stress to the cytosol through a newly defined OMA1-DELE1-HRI pathway which ultimately leads to the activation of ATF4, the master transcription factor of the integrated stress response. Here, we showed that the partial loss of DELE1 expression observed in -5/del(5q) patients was sufficient to significantly reduce the sensitivity to mitochondrial stress in AML cells. Overall, our results suggest that DELE1 haploinsufficiency could represent a new driver mechanism in -5/del(5q) AML.
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Haploinsuficiencia , Leucemia Mieloide Aguda , Proteínas Mitocondriales , Monosomía , Adulto , Humanos , Apoptosis/genética , Deleción Cromosómica , Cromosomas Humanos Par 5/genética , Leucemia Mieloide Aguda/genética , Proteínas Mitocondriales/genéticaRESUMEN
In a phenotypical screen of 56 acute myeloid leukemia (AML) patient samples and using a library of 10,000 compounds, we identified a hit with increased sensitivity toward SF3B1-mutated and adverse risk AMLs. Through structure-activity relationship studies, this hit was optimized into a potent, specific, and nongenotoxic molecule called UM4118. We demonstrated that UM4118 acts as a copper ionophore that initiates a mitochondrial-based noncanonical form of cell death known as cuproptosis. CRISPR-Cas9 loss-of-function screen further revealed that iron-sulfur cluster (ISC) deficiency enhances copper-mediated cell death. Specifically, we found that loss of the mitochondrial ISC transporter ABCB7 is synthetic lethal to UM4118. ABCB7 is misspliced and down-regulated in SF3B1-mutated leukemia, creating a vulnerability to copper ionophores. Accordingly, ABCB7 overexpression partially rescued SF3B1-mutated cells to copper overload. Together, our work provides mechanistic insights that link ISC deficiency to cuproptosis, as exemplified by the high sensitivity of SF3B1-mutated AMLs. We thus propose SF3B1 mutations as a biomarker for future copper ionophore-based therapies.
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Cobre , Leucemia Mieloide Aguda , Humanos , Cobre/metabolismo , Factores de Empalme de ARN/genética , Mutación , Leucemia Mieloide Aguda/genética , Ionóforos/farmacología , Fosfoproteínas/metabolismoRESUMEN
Immunotherapy remains underexploited in acute myeloid leukemia (AML) compared to other hematological malignancies. Currently, gemtuzumab ozogamicin is the only therapeutic antibody approved for this disease. Here, to identify potential targets for immunotherapeutic intervention, we analyze the surface proteome of 100 genetically diverse primary human AML specimens for the identification of cell surface proteins and conduct single-cell transcriptome analyses on a subset of these specimens to assess antigen expression at the sub-population level. Through this comprehensive effort, we successfully identify numerous antigens and markers preferentially expressed by primitive AML cells. Many identified antigens are targeted by therapeutic antibodies currently under clinical evaluation for various cancer types, highlighting the potential therapeutic value of the approach. Importantly, this initiative uncovers AML heterogeneity at the surfaceome level, identifies several antigens and potential primitive cell markers characterizing AML subgroups, and positions immunotherapy as a promising approach to target AML subgroup specificities.
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Inmunoterapia , Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/patología , Inmunoterapia/métodos , Proteínas de la Membrana/metabolismoRESUMEN
ABSTRACT: The leukemic stem cell (LSC) score LSC-17 based on a stemness-related gene expression signature is an indicator of poor disease outcome in acute myeloid leukemia (AML). However, it is not known whether "niche anchoring" of LSC affects disease evolution. To address this issue, we conditionally inactivated the adhesion molecule JAM-C (Junctional Adhesion Molecule-C) expressed by hematopoietic stem cells (HSCs) and LSCs in an inducible mixed-lineage leukemia (iMLL)-AF9-driven AML mouse model. Deletion of Jam3 (encoding JAM-C) before induction of the leukemia-initiating iMLL-AF9 fusion resulted in a shift from long-term to short-term HSC expansion, without affecting disease initiation and progression. In vitro experiments showed that JAM-C controlled leukemic cell nesting irrespective of the bone marrow stromal cells used. RNA sequencing performed on leukemic HSCs isolated from diseased mice revealed that genes upregulated in Jam3-deficient animals belonged to activation protein-1 (AP-1) and tumor necrosis factor α (TNF-α)/NF-κB pathways. Human orthologs of dysregulated genes allowed to identify a score that was distinct from, and complementary to, the LSC-17 score. Substratification of patients with AML using LSC-17 and AP-1/TNF-α genes signature defined 4 groups with median survival ranging from <1 year to a median of "not reached" after 8 years. Finally, coculture experiments showed that AP-1 activation in leukemic cells was dependent on the nature of stromal cells. Altogether, our results identify the AP-1/TNF-α gene signature as a proxy of LSC anchoring in bone marrow niches, which improves the prognostic value of the LSC-17 score. This trial was registered at www.ClinicalTrials.gov as #NCT02320656.
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Moléculas de Adhesión Celular , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda , Células Madre Neoplásicas , Animales , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular/genética , Eliminación de Gen , Modelos Animales de Enfermedad , Células Madre Hematopoyéticas/metabolismo , InmunoglobulinasRESUMEN
The joint sequencing of related genomes has become an important means to discover rare variants. Normal-tumor genome pairs are routinely sequenced together to find somatic mutations and their associations with different cancers. Parental and sibling genomes reveal de novo germline mutations and inheritance patterns related to Mendelian diseases.Acute lymphoblastic leukemia (ALL) is the most common paediatric cancer and the leading cause of cancer-related death among children. With the aim of uncovering the full spectrum of germline and somatic genetic alterations in childhood ALL genomes, we conducted whole-exome re-sequencing on a unique cohort of over 120 exomes of childhood ALL quartets, each comprising a patient's tumor and matched-normal material, and DNA from both parents. We developed a general probabilistic model for such quartet sequencing reads mapped to the reference human genome. The model is used to infer joint genotypes at homologous loci across a normal-tumor genome pair and two parental genomes.We describe the algorithms and data structures for genotype inference, model parameter training. We implemented the methods in an open-source software package (QUADGT) that uses the standard file formats of the 1000 Genomes Project. Our method's utility is illustrated on quartets from the ALL cohort.
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Análisis Mutacional de ADN/métodos , Técnicas de Genotipaje , Mutación de Línea Germinal , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Algoritmos , Niño , Exoma , Genoma Humano , Genotipo , Humanos , Programas InformáticosRESUMEN
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
While oncogenes promote tumorigenesis, they also induce deleterious cellular stresses, such as apoptosis, that cancer cells must combat by coopting adaptive responses. Whether tumor suppressor gene haploinsufficiency leads to such phenomena and their mechanistic basis is unclear. Here, we demonstrate that elevated levels of the anti-apoptotic factor, CASP8 and FADD-like apoptosis regulator (CFLAR), promotes apoptosis evasion in acute myeloid leukemia (AML) cells haploinsufficient for the cut-like homeobox 1 (CUX1) transcription factor, whose loss is associated with dismal clinical prognosis. Genome-wide CRISPR/Cas9 screening identifies CFLAR as a selective, acquired vulnerability in CUX1-deficient AML, which can be mimicked therapeutically using inhibitor of apoptosis (IAP) antagonists in murine and human AML cells. Mechanistically, CUX1 deficiency directly alleviates CUX1 repression of the CFLAR promoter to drive CFLAR expression and leukemia survival. These data establish how haploinsufficiency of a tumor suppressor is sufficient to induce advantageous anti-apoptosis cell survival pathways and concurrently nominate CFLAR as potential therapeutic target in these poor-prognosis leukemias.
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Apoptosis/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Haploinsuficiencia , Proteínas de Homeodominio/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/genética , Inmunoprecipitación de Cromatina , Dipéptidos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ontología de Genes , Genes Supresores de Tumor , Células Madre Hematopoyéticas/metabolismo , Proteínas de Homeodominio/genética , Humanos , Indoles/farmacología , Estimación de Kaplan-Meier , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Leucemia Mielomonocítica Crónica/genética , Leucemia Mielomonocítica Crónica/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Análisis por Matrices de Proteínas , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
Human hematopoietic stem cells (HSCs) exhibit attrition of their self-renewal capacity when cultured ex vivo, a process that is partially reversed upon treatment with epigenetic modifiers, most notably inhibitors of histone deacetylases (HDACs) or lysine-specific demethylase LSD1. A recent study showed that the human HSC self-renewal agonist UM171 modulates the CoREST complex, leading to LSD1 degradation, whose inhibition mimics the activity of UM171. The mechanism underlying the UM171-mediated loss of CoREST function remains undetermined. We now report that UM171 potentiates the activity of a CULLIN3-E3 ubiquitin ligase (CRL3) complex whose target specificity is dictated by the poorly characterized Kelch/BTB domain protein KBTBD4. CRL3KBTBD4 targets components of the LSD1/RCOR1 corepressor complex for proteasomal degradation, hence re-establishing H3K4me2 and H3K27ac epigenetic marks, which are rapidly decreased upon ex vivo culture of human HSCs.
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Proteínas Co-Represoras , Epigénesis Genética , Células Madre Hematopoyéticas , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/metabolismo , Células Madre Hematopoyéticas/metabolismo , Histona Desacetilasas/metabolismo , HumanosRESUMEN
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
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
Acute megakaryoblastic leukemia (AMKL) represents â¼10% of pediatric acute myeloid leukemia cases and typically affects young children (<3 years of age). It remains plagued with extremely poor treatment outcomes (<40% cure rates), mostly due to primary chemotherapy refractory disease and/or early relapse. Recurrent and mutually exclusive chimeric fusion oncogenes have been detected in 60% to 70% of cases and include nucleoporin 98 (NUP98) gene rearrangements, most commonly NUP98-KDM5A. Human models of NUP98-KDM5A-driven AMKL capable of faithfully recapitulating the disease have been lacking, and patient samples are rare, further limiting biomarkers and drug discovery. To overcome these impediments, we overexpressed NUP98-KDM5A in human cord blood hematopoietic stem and progenitor cells using a lentiviral-based approach to create physiopathologically relevant disease models. The NUP98-KDM5A fusion oncogene was a potent inducer of maturation arrest, sustaining long-term proliferative and progenitor capacities of engineered cells in optimized culture conditions. Adoptive transfer of NUP98-KDM5A-transformed cells into immunodeficient mice led to multiple subtypes of leukemia, including AMKL, that phenocopy human disease phenotypically and molecularly. The integrative molecular characterization of synthetic and patient NUP98-KDM5A AMKL samples revealed SELP, MPIG6B, and NEO1 as distinctive and novel disease biomarkers. Transcriptomic and proteomic analyses pointed to upregulation of the JAK-STAT signaling pathway in the model AMKL. Both synthetic models and patient-derived xenografts of NUP98-rearranged AMKL showed in vitro therapeutic vulnerability to ruxolitinib, a clinically approved JAK2 inhibitor. Overall, synthetic human AMKL models contribute to defining functional dependencies of rare genotypes of high-fatality pediatric leukemia, which lack effective and rationally designed treatments.
Asunto(s)
Biomarcadores , Modelos Animales de Enfermedad , Leucemia Megacarioblástica Aguda/etiología , Leucemia Megacarioblástica Aguda/patología , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Fusión Oncogénica/genética , Proteína 2 de Unión a Retinoblastoma/genética , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Biología Computacional/métodos , Susceptibilidad a Enfermedades , Expresión Génica , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunofenotipificación , Leucemia Megacarioblástica Aguda/terapia , Ratones , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteína 2 de Unión a Retinoblastoma/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.