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[This corrects the article DOI: 10.18632/oncotarget.27206.].
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The DNA methylation regulators DNMT3A and TET2 are recurrently mutated in hematological disorders. Despite possessing antagonistic biochemical activities, loss-of-function murine models show overlapping phenotypes in terms of increased hematopoietic stem cell (HSC) fitness. Here, we directly compared the effects of these mutations on hematopoietic progenitor function and disease initiation. In contrast to Dnmt3a-null HSCs, which possess limitless self-renewal in vivo, Tet2-null HSCs unexpectedly exhaust at the same rate as control HSCs in serial transplantation assays despite an initial increase in self-renewal. Moreover, loss of Tet2 more acutely sensitizes hematopoietic cells to the addition of a common co-operating mutation (Flt3ITD) than loss of Dnmt3a, which is associated with a more rapid expansion of committed progenitor cells. The effect of Tet2 mutation manifests more profound myeloid lineage skewing in committed hematopoietic progenitor cells rather than long-term HSCs. Molecular characterization revealed divergent transcriptomes and chromatin accessibility underlying these functional differences.
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ADN (Citosina-5-)-Metiltransferasas/genética , Proteínas de Unión al ADN/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Mutación/genética , Proteínas Proto-Oncogénicas/genética , Adaptación Fisiológica , Animales , Diferenciación Celular , Autorrenovación de las Células , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Proteínas de Unión al ADN/metabolismo , Dioxigenasas , Ratones Endogámicos C57BL , Células Mieloides/citología , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
Kinases such as MEK are attractive targets for novel therapy in cancer, including acute myeloid leukaemia (AML). Acquired and inherent resistance to kinase inhibitors, however, is becoming an increasingly important challenge for the clinical success of such therapeutics, and often arises from mutations in the drug-binding domain of the target kinase. To identify possible causes of resistance to MEK inhibition, we generated a model of resistance by long-term treatment of AML cells with AZD6244 (selumetinib). Remarkably, resistance to MEK inhibition was due to acquired PTEN haploinsufficiency, rather than mutation of MEK. Resistance via this mechanism was confirmed using CRISPR/Cas9 technology targeting exon 5 of PTEN. While PTEN loss has been previously implicated in resistance to a number of other therapeutic agents, this is the first time that it has been shown directly and in AML.
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Epidemiological sequencing studies have revealed that somatic mutations characteristic of myeloid neoplasms can be detected in the blood of asymptomatic individuals decades prior to presentation of any clinical symptoms. This premalignant condition is known as clonal hematopoiesis of indeterminate potential (CHIP). Despite the fact these mutant clones become readily detectable in the blood of elderly individuals (â¼10% of people over the age of 65), the overall rate of disease progression remains relatively low. Thus, in addition to genetic mutations, there are likely environmental factors that contribute to clonal evolution in people with CHIP. One environmental stress that increases with age is inflammation. Although chronic inflammation is detrimental to the long-term function of normal hematopoietic stem cells, several recent studies in animal models have indicated hematopoietic stem cells with CHIP mutations may be resistant to these deleterious effects. However, direct evidence indicating a correlation between increased inflammation and accelerated CHIP in humans is currently lacking. In this study, we sequenced the peripheral blood cells of a cohort of patients with ulcerative colitis, an autoimmune disease characterized by increased levels of pro-inflammatory cytokines. This analysis revealed that the inflammatory environment of ulcerative colitis promoted CHIP with a distinct mutational spectrum, notably positive selection of clones with DNMT3A and PPM1D mutations. We also show a specific association between elevated levels of serum interferon gamma and DNMT3A mutations. These data add to our understanding of how cell extrinsic factors select for clones with specific mutations to promote clonal hematopoiesis.
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Colitis Ulcerosa/patología , Hematopoyesis/genética , Interferón gamma/sangre , Mutación , Factor de Necrosis Tumoral alfa/análisis , Anciano , Anciano de 80 o más Años , Células Clonales/citología , Colitis Ulcerosa/sangre , Colitis Ulcerosa/genética , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Metiltransferasa 3A , Femenino , Humanos , Masculino , Persona de Mediana Edad , Lesiones Precancerosas/sangre , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Proteína Fosfatasa 2C/genéticaRESUMEN
Mixed lineage leukemia (MLL) gene rearrangements characterize approximately 70% of infant and 10% of adult and therapy-related leukemia. Conventional clinical diagnostics, including cytogenetics and fluorescence in situ hybridization (FISH) fail to detect MLL translocation partner genes (TPG) in many patients. Long-distance inverse (LDI)-PCR, the "gold standard" technique that is used to characterize MLL breakpoints, is laborious and requires a large input of genomic DNA (gDNA). To overcome the limitations of current techniques, a targeted next-generation sequencing (NGS) approach that requires low RNA input was tested. Anchored multiplex PCR-based enrichment (AMP-E) was used to rapidly identify a broad range of MLL fusions in patient specimens. Libraries generated using Archer FusionPlex Heme and Myeloid panels were sequenced using the Illumina platform. Diagnostic specimens (n = 39) from pediatric leukemia patients were tested with AMP-E and validated by LDI-PCR. In concordance with LDI-PCR, the AMP-E method successfully identified TPGs without prior knowledge. AMP-E identified 10 different MLL fusions in the 39 samples. Only two specimens were discordant; AMP-E successfully identified a MLL-MLLT1 fusion where LDI-PCR had failed to determine the breakpoint, whereas a MLL-MLLT3 fusion was not detected by AMP-E due to low expression of the fusion transcript. Sensitivity assays demonstrated that AMP-E can detect MLL-AFF1 in MV4-11 cell dilutions of 10-7 and transcripts down to 0.005 copies/ng.Implications: This study demonstrates a NGS methodology with improved sensitivity compared with current diagnostic methods for MLL-rearranged leukemia. Furthermore, this assay rapidly and reliably identifies MLL partner genes and patient-specific fusion sequences that could be used for monitoring minimal residual disease. Mol Cancer Res; 16(2); 279-85. ©2017 AACR.