Mutation of DNMT3A, encoding a de novo methyltransferase essential for cytosine methylation, is a common early event in clonal hematopoiesis (CH) and adult acute myeloid leukemia (AML). Spontaneous deamination of methylated cytosines damages DNA, which is repaired by the base excision repair (BER) enzymes MBD4 and TDG. Congenital MBD4-deficiency has been linked to early-onset CH and AML, and is marked by exceedingly high levels of DNA damage and mutation of DNMT3A. Strikingly, wildtype (WT) DNMT3A binds TDG, thereby potentiating its repair activity. Since TDG is the only remaining BER enzyme in MBD4-deficient AML patients capable of repairing methylation damage, we investigated whether mutant DNMT3A negatively affects the repair function of TDG. We found that, whereas WT DNMT3A stimulates TDG function, mutant DNMT3A impairs TDG-mediated repair of DNA damage in vitro. In light of this finding and to extrapolate our observations to the broader AML patient population, we investigate here the genetic profiles and survival outcomes of AML patients with single (SM) versus double mutant (DM) DNMT3A. DM DNMT3A AML patients show a characteristic driver mutation landscape and reduced overall survival when compared to SM DNMT3A AML patients. Importantly, whole-genome sequencing showed a trend for increased DNA damage in primary DM DNMT3A AML samples, especially when DNMT3A mutations are located at the DNMT3A-TDG interaction interface.

Introduction: Mutation of DNMT3A, encoding a de novo methyltransferase essential for cytosine methylation, is a common early event in clonal hematopoiesis (CH) and adult acute myeloid leukemia (AML). Spontaneous deamination of methylated cytosines incurs DNA damage (methylation damage), which is repaired by the base excision repair enzymes MBD4 and TDG. Wildtype DNMT3A binds TDG, thereby potentiating its repair activity. In previous work (Massaar et al., EHA 2024), we had found that whereas wildtype DNMT3A stimulates TDG activity, mutant DNMT3A impairs TDG-mediated repair of methylation damage in vitro. Besides its role in the repair of methylation damage, TDG is also involved in another branch of the DNA demethylation pathway. In this branch, TET enzymes catalyze a stepwise hydroxylation process in which 5mC is converted to 5-hydroxymethylcytosin (5hmC). This is then further hydroxylated to form 5-formylcytosine (5fC), followed by 5-carboxylcytosine (5caC). Based on our previous findings, we aimed to further dissect the mechanisms by which mutant DNMT3A inhibits TDG activity. In addition, based on our initial results, we investigated whether the hydroxylation-demethylation pathway is similarly disrupted by mutation of DNMT3A. Finally, we hypothesized that in both branches of the demethylation pathway, the inhibiting effect of mutant DNMT3A on TDG was the result of decreased binding activity of TDG to its canonical substrates. Methods: AlphaFold 2.0 was used to predict changes in the interacting residues between TDG and mutant DNMT3A. Glycosylase activity assays with 5caC as a substrate were performed to confirm the role of TDG in demethylation through hydroxylation. Band shift assays were performed to assess the binding affinity of TDG to oligoduplexes in the presence of wildtype or mutant DNMT3A, using either the G:T mismatch or 5caC as substrate, to represent the two branches of the demethylation pathway in which TDG acts. Results: Alphafold 2.0 predicted that mutation of DNMT3A results in novel interactions with TDG. Interestingly, most forms of mutant DNMT3A are predicted to interact with residues located within the uracil-DNA glycosylase domain of TDG, whereas wildtype DNMT3A is predicted not to interact with this specific domain. Given the significance of this domain for TDG repair capacity, such changes may further impact TDG repair activity. Glycosylase activity assays including 5caC as a substrate confirmed that TDG acts in the hydroxylation-demethylation pathway. Using a band shift assay we observed that increasing concentrations of recombinant human wildtype DNMT3A stimulates TDG binding to oligoduplexes containing a G:T-mismatch. Of note, we confirmed a decreased binding affinity of TDG to the same oligoduplexes when co-titrated with mutant DNMT3A (R635W, R688C, R882C and A884V) compared to wildtype DNMT3A. This could potentially explain how mutant DNMT3A inhibits TDG repair activity. Since TDG is an integral part of the hydroxylation-demethylation pathway, we next investigated whether wildtype and mutant DNMT3A differentially impact TDG binding activity to oligoduplexes containing a 5caC as substrate. Strikingly, similar to our previous experiments, we observed that mutant DNMT3A negatively impacts TDG binding activity towards this pivotal epigenetic derivative. Finally, similar to previous work, we observe that the DNMT3A R635W mutation, not involved as residue in the TDG-DNMT3A interaction interface, only moderately impacts TDG binding activity, independent of its substrate. This indicates that the effect on TDG binding may depend on whether the mutated DNMT3A residue is involved in the TDG-DNMT3A interaction. Conclusion & Discussion: We here provide insights in the potential mechanisms underlying TDG inhibition by mutant DNMT3A. Our results show that mutated DNMT3A causes alterations in the predicted interaction between DNMT3A and TDG, which may interfere with the glycosylase function of TDG. We also confirmed decreased binding affinity of TDG to its canonical substrates in the presence of mutant DNMT3A, representing a potential mechanism behind the observed inhibitory effect of mutant DNMT3A on TDG activity. Finally, we showed that mutant DNMT3A not only impairs methylation damage repair, but likely also active demethylation through hydroxylation, uncovering another potential mechanistic link between mutant DNMT3A and leukemogenesis.

Context Mutations in DNMT3A, a de novo methyltransferase essential for the methylation of cytosines in a CpG dinucleotide context, are common early events in adult acute myeloid leukemia (AML). Methylation of cytosines can lead to DNA damage through spontaneous deanimation, which is normally repaired by the base excision repair enzymes MBD4 and TDG. Germline deficiency of MBD4 has been linked to increased risk of clonal hematopoiesis (CH) and AML with exceedingly high levels of methylation damage and mutations in DNMT3A. In MBD4-deficient AML, TDG does not compensate for MBD4 loss. Previous investigation suggests that DNMT3A and TDG interact, thereby positively influencing the repair activity of TDG. Nevertheless, it is unknown whether mutant DNMT3A could negatively impact the function of TDG. Objective We investigated the impact of common DNMT3A mutations on TDG function to learn whether methylation damage repair is impaired. Targeted next-generation sequencing was performed to compare the driver landscape of single- (SM) versus double-mutated (DM) DNMT3A AML. Finally, we investigated whether overall methylation damage is increased in DM compared to SM. Methods MBD4 and TDG repair activity was assessed in the presence of wildtype (WT) or mutant DNMT3A using glycosylase assays. Differences in survival and co-mutations were assessed in our AML cohort comprising 1,872 DNMT3A WT, 585 SM, and 88 DM patients. Whole-genome sequencing (WGS) was performed on diagnosis bone marrow aspirates from 6 DNMT3A WT, 7 SM, and 9 DM AML patients. Results WT DNMT3A potentiates the capacity of TDG to repair methylation damage. Mutant DNMT3A cotitrated with WT DNMT3A demonstrated weaker potentiation of TDG and resulted in inhibition at higher concentration. DNMT3A SM had a strong enrichment for DNMT3A R882 hotspot and NPM1 mutations, whereas there was a 10-fold increase in the IDH2 R172 hotspot mutation in DM. DNMT3A DM had a significantly worse overall survival (OS) compared to SM (HR=1.33, P=0.045). WGS uncovered a genome-wide increase in methylation damage when comparing DNMT3A DM to SM, which becomes even more prominent when DNMT3A mutations are positioned at the DNMT3A-TDG interaction interface. Conclusion Double mutant DNMT3A is linked to increased methylation damage, diverging driver landscape and unfavorable overall survival.

In acute myeloid leukemia (AML), acquired genetic aberrations carry prognostic implications and guide therapeutic decisions. Clinical algorithms have been improved by the incorporation of novel aberrations. Here, we report the presence and functional characterization of mutations in the transcription factor NFE2 in patients with AML and in a patient with myelosarcoma. We previously described NFE2 mutations in patients with myeloproliferative neoplasms and demonstrated that expression of mutant NFE2 in mice causes a myeloproliferative phenotype. Now, we show that, during follow-up, 34% of these mice transform to leukemia presenting with or without concomitant myelosarcomas, or develop isolated myelosarcomas. These myelosarcomas and leukemias acquired AML-specific alterations, including the murine equivalent of trisomy 8, loss of the AML commonly deleted region on chromosome 5q, and mutations in the tumor suppressor Trp53 Our data show that mutations in NFE2 predispose to the acquisition of secondary changes promoting the development of myelosarcoma and/or AML.

Key Points The DNA glycosylase MBD4 acts as a safeguard against damage from 5mC deamination. Germ line MBD4 deficiency stimulates clonal hematopoiesis and guides the development of leukemia via recurrent mutations in DNMT3A.

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.).

Acute myeloid leukemia (AML) is a heterogeneous disease with a great variety of somatic driver mutations. Each AML carries specific (combinations of) acquired mutations, which enables minimal residual disease (MRD) detection in virtually every patient with next-generation sequencing (NGS). In recent years there has been an increasing interest to conduct longitudinal molecular MRD detection to better estimate prognosis, monitor treatment efficacy, as well as prediction of an impending relapse. The development of molecular monitoring on archived samples has been slow because only limited numbers of samples collected during the course of the disease were biobanked. We have systematically investigated whether archived May-Giemsa Grünwald (MGG)-stained bone marrow slides could be an alternative source of material to conduct NGS-based molecular MRD detection in AML. Since MGG-stained bone marrow evaluations are performed at high frequency to evaluate the response on therapy and repopulation of the bone marrow, we focused on these bone marrow slides taken at regular intervals during treatment.

Abstract Spontaneous methylcytosine (5mC) deamination is a common source of cytosine to thymine (C>T) mutations. These mutations accumulate over time, serving as a “molecular clock” that tracks cellular age. MBD4 is a thymine glycosylase that recognises sites of 5mC deamination and initiates base excision repair. Three patients (of whom 2 are siblings) with germline MBD4 loss of function (LOF) mutations developed acute myeloid leukemia (AML) at a young age (<35 years old). Whole exome (WES) and whole genome sequencing (WGS) showed an elevated C>T mutation rate ~33 times the average for AML. The mutations occurred nearly exclusively at CG dinucleotides. Reduced representation bisulfite sequencing showed the mutations occurred at previously methylated cytosines. The 3 AMLs had somatic C>T mutations in the same driver genes (DNMT3A and either IDH1 or IDH2), suggesting the methylation damage charts a recurrent path towards malignancy in the hematopoietic system. MBD4 is inactivated in other cancers, although this appears to be a rare event. Nine of 10,638 cancers in the TCGA database had MBD4 LOF mutations. In a uveal melanoma and a glioblastoma multiforme, there was also loss of heterozygosity of the wild-type MBD4. These 2 cancers with MBD4 deficiency exhibit the same mutational signature, with a high C>T mutation rate at CG dinucleotides. To verify the link between MBD4 and the 5mC damage signature, Mbd4-/- and Mbd4+/+ mouse bone marrow were cultured in semi-solid agar and WGS was performed on individual myeloid progenitor colonies. Mbd4-/- myeloid progenitor colonies displayed the same increase in C>T mutations at CG dinucleotides. This highlights the importance of MBD4 across species. Mbd4 deficient mice offer a model system to investigate mutation acquisition and cancer pathogenesis. Work on the interaction of MBD4 with other leukaemia initiating genes is underway. Citation Format: Edward Chew, Mathijs A. Sanders, Christoffer Flensburg, Annelieke Zeilemaker, Sarah E. Miller, Adil S. al Hinai, Ashish Bajel, Bram Luiken, Melissa Rijken, Tamara Mclennan, Remco M. Hoogenboezem, François G. Kavelaars, Marnie E. Blewitt, Eric M. Bindels, Warren S. Alexander, Bob Löwenberg, Andrew W. Roberts, Ian J. Majewski, Peter J. Valk. MBD4 guards against DNA damage from methylcytosine deamination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1366.

Cytosine methylation is essential for normal mammalian development, yet also provides a major mutagenic stimulus. Methylcytosine (5mC) is prone to spontaneous deamination, which introduces cytosine to thymine transition mutations (C>T) upon replication 1 . Cells endure hundreds of 5mC deamination events each day and an intricate repair network is engaged to restrict this damage. Central to this network are the DNA glycosylases MBD4 2 and TDG 3 , 4 , which recognise T:G mispairing and initiate base excision repair (BER). Here we describe a novel cancer predisposition syndrome resulting from germline biallelic inactivation of MBD4 that leads to the development of acute myeloid leukaemia (AML). These leukaemias have an extremely high burden of C>T mutations, specifically in the context of methylated CG dinucleotides (CG>TG). This dependence on 5mC as a source of mutations may explain the remarkable observation that MBD4-deficient AMLs share a common set of driver mutations, including biallelic mutations in DNMT3A and hotspot mutations in IDH1/IDH2 . By assessing serial samples taken over the course of treatment, we highlight a critical interaction with somatic mutations in DNMT3A that accelerates leukaemogenesis and accounts for the conserved path to AML. MBD4-deficiency was also detected, rarely, in sporadic cancers, which display the same mutational signature. Collectively these cancers provide a model of 5mC-dependent hypermutation and reveal factors that shape its mutagenic influence.

Abstract Introduction: Although the majority of Acute Myeloid Leukemia (AML) patients achieve a complete morphological remission (CR) after induction therapy, relapse rates remain high. Molecular Minimal Residual Disease (MRD) detection by PCR-based technologies has been shown to improve relapse prediction but has been restricted to specific genetically-defined subsets of AML only. Next-Generation Sequencing (NGS) has the advantage that it allows for the assessment of a broad range of disease-related gene mutations in a single assay. Residual leukemia-specific mutations in bone marrow in morphological CR after induction therapy are supposed to represent the source of relapse. However, persistent mutations may also represent clonal hematopoiesis, analogous to age-related clonal hematopoiesis of indeterminate potential (CHIP) present in healthy individuals. It is currently unknown which and to what extent the persisting somatic mutations after induction therapy contribute to AML relapse. Here, we present a comprehensive study, detailing the value of molecular MRD detection by NGS, in a large prospective cohort of newly diagnosed AML. Methods: 482 AML patients (<65 years) were treated with 2 cycles of standard induction chemotherapy followed by consolidation in HOVON-SAKK clinical trials (www.hovon.nl). NGS was performed to detect mutations in a panel of 54 genes frequently mutated in myeloid malignancies (Illumina) at diagnosis and in bone marrow in morphological CR after completion of induction therapy. Thompson-Tau outlier testing was performed to reliably detect persisting mutations above background error rates. The primary and secondary endpoints of the study were relapse and overall survival, respectively. To establish and subsequently test our definition of NGS MRD, the cohort was split into a representative training (n=283) and validation cohort (n=147). The Cumulative Incidence of Relapse (CIR) was estimated with competing-risks regression analyses according to the method of Fine & Gray. The Cox proportional hazard model was used to calculate overall survival estimates. P-values <0.05 were considered significant. Results: In 430 out of 482 (89.2%) AML patients somatic driver mutations were present at diagnosis. In 51.4% of subjects persisting mutations were detected in bone marrow in morphological CR at highly variable variant allele frequencies (VAF 0.0002-0.47), predominantly persisting in DNMT3A (78.7%), TET2 (54.2%) and ASXL1 (51.6%). These persistent mutations in DNMT3A, TET2 and ASXL1 (DTA) in the training cohort did not associate with the incidence of relapse at any VAF cut-off, indicating a stage of clonal hematopoiesis rather than a condition of impending relapse. In contrast, in the subset of AML patients with persisting DTA mutations, a significant correlation with relapse was observed when any other persistent non-DTA mutation was considered (training cohort: 5-years CIR 76.4% vs. 39.4%; p=0.002). In the training cohort NGS MRD, as defined by persistent non-DTA mutations, was found to be highly associated with the risk of relapse (SHR:1.85 [95%CI 1.27-2.70]; p=0.001), which was confirmed in the validation set (SHR:2.81 [95%CI 1.64-4.79]; p<0.001) (Fig. 1). In fact, NGS MRD was significantly associated with CIR when the training and validation series were combined (5-years CIR 58.3% versus 33.9% (p<0.001)) (Fig. 2). In addition, NGS MRD predicted for reduced survival in both cohorts (training: HR:1.64 [95%CI 1.12-2.42]; p=0.012 and validation: HR:3.08 [95%CI 1.87-5.08]; p<0.001). Finally, multivariable analysis including the data of all 430 AML patients, with adjustment for age, WBC, ELN2017 risk and number of induction cycles needed to achieve CR, revealed that NGS MRD expresses profound independent prognostic significance for relapse (SHR:1.89 [95%CI:1.34-2.65]; p<0.001) and overall survival (HR:1.64 [95%CI:1.18-2.27]; p=0.003). In sensitivity analysis with time-dependent correction for allogeneic stem cell transplantation NGS MRD remained highly prognostic for relapse and survival. Conclusions: In an unprecedentedly large prospective study including training and validation cohorts, targeted NGS MRD detection is established as a powerful and independent predictor for relapse and survival. NGS MRD is applicable in virtually all newly diagnosed adults with AML while persistent CHIP-related mutations lack prognostic value. Disclosures Ossenkoppele: J&J: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; Karyopharm: Consultancy, Research Funding; Roche: Honoraria; Novartis: Research Funding.

Clonal hematopoiesis of indeterminate potential (CHIP) is a clonal disorder characterized by preleukemic mutations and increases in prevalence during aging. Infrequently CHIP progresses to hematological cancer implying that preleukemic mutations subtly affect leukemogenesis but a mechanistic explanation is lacking. Exceedingly, preleukemic mutations are acquired in genes encoding for DNA methylation modifiers, predominantly in DNMT3A and members of the active DNA demethylation pathway. DNMT3A encodes a de novo methyltransferase establishing 5-methylcytosine (5mC) and mutations in this gene are linked to impaired DNA methylation and DNA damage sensing.

BCR-ABL1 and BCR-ABL1-like acute lymphoblastic leukemia (ALL) are two major pre-B cell acute leukemia subtypes characterized by genetic alterations affecting lymphoid-specific transcription factors. Studies examining the chain of genetic events necessary to develop leukemia established that the BCR-ABL1 fusion gene and kinase-activating BCR-ABL1-like lesions are initiating events, however, insufficient for leukemia development. Secondary genetic events targeting B cell development genes are therefore an essential requirement for overt ALL. A recent study (Papaemmanuil et al, Nat. Genet., 2014) revealed that illegitimate RAG-mediated recombination is the predominant mutational mechanism establishing these secondary genetic events in ETV6-RUNX1 ALL. Of note, ETV6-RUNX1ALL is mainly restricted to pediatric cases and it remains unanswered whether this mutational process also plays a prominent role in adult ALL pathogenesis.

Myeloid malignancies bearing chromosomal inv(3)/t(3; 3) abnormalities are among the most therapy-resistant leukemias. Deregulated expression of EVI1 is the molecular hallmark of this disease; however, the genome-wide spectrum of cooperating mutations in this disease subset has not been systematically elucidated. Here, we show that 98% of inv(3)/t(3; 3) myeloid malignancies harbor mutations in genes activating RAS/receptor tyrosine kinase (RTK) signaling pathways. In addition, hemizygous mutations in GATA2, as well as heterozygous alterations in RUNX1, SF3B1, and genes encoding epigenetic modifiers, frequently co-occur with the inv(3)/t(3; 3) aberration. Notably, neither mutational patterns nor gene expression profiles differ across inv(3)/t(3; 3) acute myeloid leukemia, chronicmyeloid leukemia, andmyelodysplastic syndromecases, suggestingrecognitionof inv(3)/t(3; 3) myeloid malignancies as a single disease entity irrespective of blast count. The high incidence of activating RAS/RTK signalingmutations may provide a target for a rational treatment strategy in this high-risk patient group.

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are heterogeneous malignancies characterized by a variety of acquired genetic abnormalities and variable response to treatment.[1][1],[2][2] In the last decade, a number of novel molecular genetic abnormalities have been revealed in MDS

Introduction: Acute myeloid leukemia (AML) with inv(3)(q21q26) or t(3;3)(q21;q26) [inv(3)/t(3;3)] is associated with aberrant expression of the stem cell regulator EVI1 and dismal prognosis. Recently, we and others (Gröschel et al, Cell, 2014; Yamazaki et al, Cancer Cell, 2014) have shown, as a consequence of the inv(3)/t(3;3) rearrangements, that the proto-oncogene EVI1 is activated upon the structural repositioning of a distal GATA2 enhancer from 3q21 to EVI1, coinciding with loss of GATA2 expression from the rearranged allele. Notably, GATA2 deficiency has been shown to impair hematopoietic stem cell frequency and function (Lim et al, J. Clin. Invest., 2012) and Evi1 activation in inv(3) murine models is followed by leukemia onset after a long latency of 6 months (Yamazaki et al). We therefore hypothesize that additional cooperating genetic lesions, other than EVI1 activation and GATA2 deregulation, are required for full leukemic transformation. We sought to extend the molecular characterization of inv(3)/t(3;3) myeloid malignancies through next-generation sequencing.

Acute myeloid leukemia is a neoplasm characterized by recurrent molecular aberrations traditionally demonstrated by cytogenetic analyses. We used high density genome-wide genotyping and gene expression profiling to reveal acquired cryptic abnormalities in acute myeloid leukemia. By genome-wide genotyping of 137 cases of primary acute myeloid leukemia, we disclosed a recurrent focal amplification on chromosome 14q32, which included the genes BCL11B, CCNK, C14orf177 and SETD3, in two cases. In the affected cases, the BCL11B gene showed consistently high mRNA expression, whereas the expression of the other genes was unperturbed. Fluorescence in situ hybridization on 40 cases of acute myeloid leukemia with high BCL11B mRNA expression [2.5-fold above median; 40 out of 530 cases (7.5%)] revealed 14q32 abnormalities in two additional cases. In the four BCL11B-rearranged cases the 14q32 locus was fused to different partner chromosomes. In fact, in two cases, we demonstrated that the focal 14q32 amplifications were integrated into transcriptionally active loci. The translocations involving BCL11B result in increased expression of full-length BCL11B protein. The BCL11B-rearranged acute myeloid leukemias expressed both myeloid and T-cell markers. These biphenotypic acute leukemias all carried FLT3 internal tandem duplications, a characteristic marker of acute myeloid leukemia. BCL11B mRNA expression in acute myeloid leukemia appeared to be strongly associated with expression of other T-cell-specific genes. Myeloid 32D(GCSF-R) cells ectopically expressing Bcl11b showed decreased proliferation rate and less maturation. In conclusion, by an integrated approach involving high-throughput genome-wide genotyping and gene expression profiling we identified BCL11B as a candidate oncogene in acute myeloid leukemia.

The prevalence, the prognostic effect, and interaction with other molecular markers of DNMT3A mutations was studied in 415 patients with acute myeloid leukemia (AML) younger than 60 years. We show mutations in DNMT3A in 96 of 415 patients with newly diagnosed AML (23.1%). Univariate Cox regression analysis showed that patients with DNMT3A(mutant) AML show significantly worse overall survival (OS; P = .022; hazard ratio [HR], 1.38; 95% confidence interval [CI], 1.04-1.81), and relapse-free survival (RFS; P = .005; HR, 1.52; 95% CI, 1.13-2.05) than DNMT3A(wild-type) AMLs. In a multivariable analysis, DNMT3A mutations express independent unfavorable prognostic value for OS (P = .003; HR, 1.82; 95% CI, 1.2-2.7) and RFS (P < .001; HR, 2.2; 95% CI, 1.4-3.3). In a composite genotypic subset of cytogenetic intermediate-risk AML without FLT3-ITD and NPM1 mutations, this association is particularly evident (OS: P = .013; HR, 2.09; 95% CI, 1.16-3.77; RFS: P = .001; HR, 2.65; 95% CI, 1.48-4.89). The effect of DNMT3A mutations in human AML remains elusive, because DNMT3A(mutant) AMLs did not express a methylation or gene expression signature that discriminates them from patients with DNMT3A(wild-type) AML. We conclude that DNMT3A mutation status is an important factor to consider for risk stratification of patients with AML.

Somatic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) were recently demonstrated in acute myeloid leukemia (AML), but their prevalence and prognostic impact remain to be explored in large extensively characterized AML series, and also in various other hematologic malignancies. Here, we demonstrate in 893 newly diagnosed cases of AML mutations in the IDH1 (6%) and IDH2 (11%) genes. Moreover, we identified IDH mutations in 2 JAK2 V617F myeloproliferative neoplasias (n = 96), a single case of acute lymphoblastic leukemia (n = 96), and none in chronic myeloid leukemias (n = 81). In AML, IDH1 and IDH2 mutations are more common among AML with normal karyotype and NPM1(mutant) genotypes. IDH1 mutation status is an unfavorable prognostic factor as regards survival in a composite genotypic subset lacking FLT3(ITD) and NPM1(mutant). Thus, IDH1 and IDH2 mutations are common genetic aberrations in AML, and IDH1 mutations may carry prognostic value in distinct subtypes of AML.