RESUMO
Cohesin shapes the chromatin architecture, including enhancer-promoter interactions. Its components, especially STAG2, but not its paralog STAG1, are frequently mutated in myeloid malignancies. To elucidate the underlying mechanisms of leukemogenesis, we comprehensively characterized genetic, transcriptional, and chromatin conformational changes in acute myeloid leukemia (AML) patient samples. Specific loci displayed altered cohesin occupancy, gene expression, and local chromatin activation, which were not compensated by the remaining STAG1-cohesin. These changes could be linked to disrupted spatial chromatin looping in cohesin-mutated AMLs. Complementary depletion of STAG2 or STAG1 in primary human hematopoietic progenitors (HSPCs) revealed effects resembling STAG2-mutant AML-specific changes following STAG2 knockdown, not invoked by the depletion of STAG1. STAG2-deficient HSPCs displayed impaired differentiation capacity and maintained HSPC-like gene expression. This work establishes STAG2 as a key regulator of chromatin contacts, gene expression, and differentiation in the hematopoietic system and identifies candidate target genes that may be implicated in human leukemogenesis.
RESUMO
The sensitivity of conventional techniques for reliable quantification of minimal/measurable residual disease (MRD) in chronic lymphocytic leukemia (CLL) is limited to MRD 10-4. Measuring MRD <10-4 could help to further distinguish between patients with CLL with durable remission and those at risk of early relapse. We herein present an academically developed immunoglobulin heavy-chain variable (IGHV) leader-based next-generation sequencing (NGS) assay for the quantification of MRD in CLL. We demonstrate, based on measurements in contrived MRD samples, that the linear range of detection and quantification of our assay reaches beyond MRD 10-5. If provided with sufficient DNA input, MRD can be detected down to MRD 10-6. There was high interassay concordance between measurements of the IGHV leader-based NGS assay and allele-specific oligonucleotide quantitative polymerase chain reaction (PCR) (r = 0.92 [95% confidence interval {CI}, 0.86-0.96]) and droplet digital PCR (r = 0.93 [95% CI, 0.88-0.96]) on contrived MRD samples. In a cohort of 67 patients from the CLL11 trial, using MRD 10-5 as a cutoff, undetectable MRD was associated with superior progression-free survival (PFS) and time to next treatment. More important, deeper MRD measurement allowed for additional stratification of patients with MRD <10-4 but ≥10-5. PFS of patients in this MRD range was significantly shorter, compared with patients with MRD <10-5 (hazard ratio [HR], 4.0 [95% CI, 1.6-10.3]; P = .004), but significantly longer, compared with patients with MRD ≥10-4 (HR, 0.44 [95% CI, 0.23-0.87]; P = .018). These results support the clinical utility of the IGHV leader-based NGS assay.
Assuntos
Leucemia Linfocítica Crônica de Células B , Humanos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/terapia , Prognóstico , Cadeias Pesadas de Imunoglobulinas/genética , Reação em Cadeia da Polimerase , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasia Residual/diagnóstico , Neoplasia Residual/genéticaRESUMO
Substantial heterogeneity within mutant TP53 acute myeloid leukemia (AML) and myelodysplastic syndrome with excess of blast (MDS-EB) precludes the exact assessment of prognostic impact for individual patients. We performed in-depth clinical and molecular analysis of mutant TP53 AML and MDS-EB to dissect the molecular characteristics in detail and determine its impact on survival. We performed next-generation sequencing on 2200 AML/MDS-EB specimens and assessed the TP53 mutant allelic status (mono- or bi-allelic), the number of TP53 mutations, mutant TP53 clone size, concurrent mutations, cytogenetics, and mutant TP53 molecular minimal residual disease and studied the associations of these characteristics with overall survival. TP53 mutations were detected in 230 (10.5%) patients with AML/MDS-EB with a median variant allele frequency of 47%. Bi-allelic mutant TP53 status was observed in 174 (76%) patients. Multiple TP53 mutations were found in 49 (21%) patients. Concurrent mutations were detected in 113 (49%) patients. No significant difference in any of the aforementioned molecular characteristics of mutant TP53 was detected between AML and MDS-EB. Patients with mutant TP53 have a poor outcome (2-year overall survival, 12.8%); however, no survival difference between AML and MDS-EB was observed. Importantly, none of the molecular characteristics were significantly associated with survival in mutant TP53 AML/MDS-EB. In most patients, TP53 mutations remained detectable in complete remission by deep sequencing (73%). Detection of residual mutant TP53 was not associated with survival. Mutant TP53 AML and MDS-EB do not differ with respect to molecular characteristics and survival. Therefore, mutant TP53 AML/MDS-EB should be considered a distinct molecular disease entity.
Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Citogenética , Humanos , Leucemia Mieloide Aguda/diagnóstico , Mutação , Síndromes Mielodisplásicas/diagnóstico , Proteína Supressora de Tumor p53/genéticaRESUMO
In acute myeloid leukemia (AML) with inv(3)(q21;q26) or t(3;3)(q21;q26), a translocated GATA2 enhancer drives oncogenic expression of EVI1. We generated an EVI1-GFP AML model and applied an unbiased CRISPR/Cas9 enhancer scan to uncover sequence motifs essential for EVI1 transcription. Using this approach, we pinpointed a single regulatory element in the translocated GATA2 enhancer that is critically required for aberrant EVI1 expression. This element contained a DNA-binding motif for the transcription factor MYB, which specifically occupied this site at the translocated allele and was dispensable for GATA2 expression. MYB knockout as well as peptidomimetic blockade of CBP/p300-dependent MYB functions resulted in downregulation of EVI1 but not of GATA2. Targeting MYB or mutating its DNA-binding motif within the GATA2 enhancer resulted in myeloid differentiation and cell death, suggesting that interference with MYB-driven EVI1 transcription provides a potential entry point for therapy of inv(3)/t(3;3) AMLs. SIGNIFICANCE: We show a novel paradigm in which chromosomal aberrations reveal critical regulatory elements that are nonfunctional at their endogenous locus. This knowledge provides a rationale to develop new compounds to selectively interfere with oncogenic enhancer activity.This article is highlighted in the In This Issue feature, p. 2659.
Assuntos
Elementos Facilitadores Genéticos , Genes myb , Leucemia Mieloide Aguda , Translocação Genética , Fator de Transcrição GATA2 , Humanos , Leucemia Mieloide Aguda/genética , Proteína do Locus do Complexo MDS1 e EVI1 , OncogenesAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Mieloide Aguda/patologia , Mutação , Recidiva Local de Neoplasia/patologia , Proteína Fosfatase 2C/genética , Seguimentos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Prognóstico , Indução de RemissãoAssuntos
Medula Óssea/patologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leucemia Mieloide Aguda/genética , Mutação , Calreticulina/genética , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Frequência do Gene , Humanos , Janus Quinase 2/genética , Leucemia Mieloide Aguda/patologiaRESUMO
B-cell precursors (BCP) arise from hematopoietic stem cells in bone marrow (BM). Identification and characterization of the different BCP subsets has contributed to the understanding of normal B-cell development. BCP first rearrange their immunoglobulin (Ig) heavy chain (IGH) genes to form the pre-B-cell receptor (pre-BCR) complex together with surrogate light chains. Appropriate signaling via this pre-BCR complex is followed by rearrangement of the Ig light chain genes, resulting in the formation, and selection of functional BCR molecules. Consecutive production, expression, and functional selection of the pre-BCR and BCR complexes guide the BCP differentiation process that coincides with corresponding immunophenotypic changes. We studied BCP differentiation in human BM samples from healthy controls and patients with a known genetic defect in V(D)J recombination or pre-BCR signaling to unravel normal immunophenotypic changes and to determine the effect of differentiation blocks caused by the specific genetic defects. Accordingly, we designed a 10-color antibody panel to study human BCP development in BM by flow cytometry, which allows identification of classical preB-I, preB-II, and mature B-cells as defined via BCR-related markers with further characterization by additional markers. We observed heterogeneous phenotypes associated with more than one B-cell maturation pathway, particularly for the preB-I and preB-II stages in which V(D)J recombination takes place, with asynchronous marker expression patterns. Next Generation Sequencing of complete IGH gene rearrangements in sorted BCP subsets unraveled their rearrangement status, indicating that BCP differentiation does not follow a single linear pathway. In conclusion, B-cell development in human BM is not a linear process, but a rather complex network of parallel pathways dictated by V(D)J-recombination-driven checkpoints and pre-BCR/BCR mediated-signaling occurring during B-cell production and selection. It can also be described as asynchronous, because precursor B-cells do not differentiate as full population between the different stages, but rather transit as a continuum, which seems influenced (in part) by V-D-J recombination-driven checkpoints.
Assuntos
Diferenciação Celular/genética , Diferenciação Celular/imunologia , Síndromes de Imunodeficiência/imunologia , Células Precursoras de Linfócitos B/imunologia , Criança , Feminino , Citometria de Fluxo/métodos , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Síndromes de Imunodeficiência/genética , Masculino , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Recombinação V(D)J/genética , Recombinação V(D)J/imunologiaRESUMO
The tendency of 5-methylcytosine (5mC) to undergo spontaneous deamination has had a major role in shaping the human genome, and this methylation damage remains the primary source of somatic mutations that accumulate with age. How 5mC deamination contributes to cancer risk in different tissues remains unclear. Genomic profiling of 3 early-onset acute myeloid leukemias (AMLs) identified germ line loss of MBD4 as an initiator of 5mC-dependent hypermutation. MBD4-deficient AMLs display a 33-fold higher mutation burden than AML generally, with >95% being C>T in the context of a CG dinucleotide. This distinctive signature was also observed in sporadic cancers that acquired biallelic mutations in MBD4 and in Mbd4 knockout mice. Sequential sampling of germ line cases demonstrated repeated expansion of blood cell progenitors with pathogenic mutations in DNMT3A, a key driver gene for both clonal hematopoiesis and AML. Our findings reveal genetic and epigenetic factors that shape the mutagenic influence of 5mC. Within blood cells, this links methylation damage to the driver landscape of clonal hematopoiesis and reveals a conserved path to leukemia. Germ line MBD4 deficiency enhances cancer susceptibility and predisposes to AML.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Endodesoxirribonucleases/genética , Regulação Leucêmica da Expressão Gênica , Hematopoese , Leucemia Mieloide Aguda/genética , Adulto , DNA Metiltransferase 3A , Feminino , Deleção de Genes , Células Germinativas/metabolismo , Células Germinativas/patologia , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Mutação , Acúmulo de MutaçõesRESUMO
Immunological aging remodels the immune system at several levels. This has been documented in particular for the T-cell receptor (TCR)αß+ T-cell compartment, showing reduced naive T-cell outputs and an accumulation of terminally differentiated clonally expanding effector T-cells, leading to increased proneness to autoimmunity and cancer development at older age. Even though TCRαß+ and TCRγδ+ T-cells follow similar paths of development involving V(D)J-recombination of TCR genes in the thymus, TCRγδ+ T-cells tend to be more subjected to peripheral rather than central selection. However, the impact of aging in shaping of the peripheral TRG/TRD repertoire remains largely elusive. Next-generation sequencing analysis methods were optimized based on a spike-in method using plasmid vector DNA-samples for accurate TRG/TRD receptor diversity quantification, resulting in optimally defined primer concentrations, annealing temperatures and cycle numbers. Next, TRG/TRD repertoire diversity was evaluated during TCRγδ+ T-cell ontogeny, showing a broad, diverse repertoire in thymic and cord blood samples with Gaussian CDR3-length distributions, in contrast to the more skewed repertoire in mature circulating TCRγδ+ T-cells in adult peripheral blood. During aging the naive repertoire maintained its diversity with Gaussian CDR3-length distributions, while in the central and effector memory populations a clear shift from young (Vγ9/Vδ2 dominance) to elderly (Vγ2/Vδ1 dominance) was observed. Together with less clear Gaussian CDR3-length distributions, this would be highly suggestive of differentially heavily selected repertoires. Despite the apparent age-related shift from Vγ9/Vδ2 to Vγ2/Vδ1, no clear aging effect was observed on the Vδ2 invariant T nucleotide and canonical Vγ9-Jγ1.2 selection determinants. A more detailed look into the healthy TRG/TRD repertoire revealed known cytomegalovirus-specific TRG/TRD clonotypes in a few donors, albeit without a significant aging-effect, while Mycobacterium tuberculosis-specific clonotypes were absent. Notably, in effector subsets of elderly individuals, we could identify reported TRG and TRD receptor chains from TCRγδ+ T-cell large granular lymphocyte leukemia proliferations, which typically present in the elderly population. Collectively, our results point to relatively subtle age-related changes in the human TRG/TRD repertoire, with a clear shift in Vγ/Vδ usage in memory cells upon aging.
Assuntos
Envelhecimento/imunologia , Mycobacterium tuberculosis/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Subpopulações de Linfócitos T/fisiologia , Linfócitos T/fisiologia , Tuberculose/imunologia , Adulto , Idoso , Biodiversidade , Células Cultivadas , Tolerância Central , Seleção Clonal Mediada por Antígeno , Regiões Determinantes de Complementaridade/genética , Feminino , Citometria de Fluxo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Memória Imunológica , Masculino , Pessoa de Meia-Idade , Tolerância Periférica , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Recombinação GenéticaRESUMO
BACKGROUND: Patients with acute myeloid leukemia (AML) often reach complete remission, but relapse rates remain high. Next-generation sequencing enables the detection of molecular minimal residual disease in virtually every patient, but its clinical value for the prediction of relapse has yet to be established. METHODS: We conducted a study involving patients 18 to 65 years of age who had newly diagnosed AML. Targeted next-generation sequencing was carried out at diagnosis and after induction therapy (during complete remission). End points were 4-year rates of relapse, relapse-free survival, and overall survival. RESULTS: At least one mutation was detected in 430 out of 482 patients (89.2%). Mutations persisted in 51.4% of those patients during complete remission and were present at various allele frequencies (range, 0.02 to 47%). The detection of persistent DTA mutations (i.e., mutations in DNMT3A, TET2, and ASXL1), which are often present in persons with age-related clonal hematopoiesis, was not correlated with an increased relapse rate. After the exclusion of persistent DTA mutations, the detection of molecular minimal residual disease was associated with a significantly higher relapse rate than no detection (55.4% vs. 31.9%; hazard ratio, 2.14; P<0.001), as well as with lower rates of relapse-free survival (36.6% vs. 58.1%; hazard ratio for relapse or death, 1.92; P<0.001) and overall survival (41.9% vs. 66.1%; hazard ratio for death, 2.06; P<0.001). Multivariate analysis confirmed that the persistence of non-DTA mutations during complete remission conferred significant independent prognostic value with respect to the rates of relapse (hazard ratio, 1.89; P<0.001), relapse-free survival (hazard ratio for relapse or death, 1.64; P=0.001), and overall survival (hazard ratio for death, 1.64; P=0.003). A comparison of sequencing with flow cytometry for the detection of residual disease showed that sequencing had significant additive prognostic value. CONCLUSIONS: Among patients with AML, the detection of molecular minimal residual disease during complete remission had significant independent prognostic value with respect to relapse and survival rates, but the detection of persistent mutations that are associated with clonal hematopoiesis did not have such prognostic value within a 4-year time frame. (Funded by the Queen Wilhelmina Fund Foundation of the Dutch Cancer Society and others.).