DNMT3A R882 Mutations Research Articles

Quizartinib with donor lymphocyte infusion for post-transplant relapse of FLT3-ITD-positive acute myeloid leukemia.

FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD)-positive acute myeloid leukemia (AML) has a poor prognosis, particularly with DNMT3A and NPM1 mutations. Quizartinib, a FLT3 inhibitor showing clinical benefit in FLT3-ITD-positive AML, has unclear safety and efficacy when combined with donor lymphocyte infusion (DLI). We report a case of FLT3-ITD-positive AML with DNMT3A and NPM1 mutations that relapsed after allogeneic hematopoietic stem cell transplantation (allo-HCT) and was treated with quizartinib and DLI. A 49-year-old man was diagnosed with AML. Target-sequencing analysis of the bone marrow revealed FLT3-ITD, DNMT3A R882, and NPM1 mutations. Although the patient achieved complete remission (CR) through induction therapy and received allo-HCT, he relapsed on day 71. Quizartinib was initiated on day 79, and the patient achieved CR with incomplete recovery on day 106. He did not desire a second allo-HCT and continued quizartinib in combination with DLI, which was started on day 156 and administered eight times every 2 to 3months. The patient achieved hematological CR on day 163 and remained in molecular CR 3years after allo-HCT without adverse effects. Quizartinib combined with DLI may be a feasible treatment for early relapse of FLT3-ITD-positive AML after allo-HCT, even with concurrent DNMT3A and NPM1 mutations.

Structure-guided functional suppression of AML-associated DNMT3A hotspot mutations

DNA methyltransferases DNMT3A- and DNMT3B-mediated DNA methylation critically regulate epigenomic and transcriptomic patterning during development. The hotspot DNMT3A mutations at the site of Arg822 (R882) promote polymerization, leading to aberrant DNA methylation that may contribute to the pathogenesis of acute myeloid leukemia (AML). However, the molecular basis underlying the mutation-induced functional misregulation of DNMT3A remains unclear. Here, we report the crystal structures of the DNMT3A methyltransferase domain, revealing a molecular basis for its oligomerization behavior distinct to DNMT3B, and the enhanced intermolecular contacts caused by the R882H or R882C mutation. Our biochemical, cellular, and genomic DNA methylation analyses demonstrate that introducing the DNMT3B-converting mutations inhibits the R882H-/R882C-triggered DNMT3A polymerization and enhances substrate access, thereby eliminating the dominant-negative effect of the DNMT3A R882 mutations in cells. Together, this study provides mechanistic insights into DNMT3A R882 mutations-triggered aberrant oligomerization and DNA hypomethylation in AML, with important implications in cancer therapy.

Identification of Cell Type-Specific Effects of DNMT3A Mutations on Relapse in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease caused by distinctive mutations in individual patients; therefore, each patient may display different cell-type compositions. Although most patients with AML achieve complete remission (CR) through intensive chemotherapy, the likelihood of relapse remains high. Several studies have attempted to characterize the genetic and cellular heterogeneity of AML; however, our understanding of the cellular heterogeneity of AML remains limited. In this study, we performed single-cell RNA sequencing (scRNAseq) of bone marrow-derived mononuclear cells obtained from same patients at different AML stages (diagnosis, CR, and relapse). We found that hematopoietic stem cells (HSCs) at diagnosis were abnormal compared to normal HSCs. By improving the detection of the DNMT3A R882 mutation with targeted scRNAseq, we identified that DNMT3A-mutant cells that mainly remained were granulocyte-monocyte progenitors (GMPs) or lymphoid-primed multipotential progenitors (LMPPs) from CR to relapse and that DNMT3A-mutant cells have gene signatures related to AML and leukemic cells. Copy number variation analysis at the single-cell level indicated that the cell type that possesses DNMT3A mutations is an important factor in AML relapse and that GMP and LMPP cells can affect relapse in patients with AML. This study advances our understanding of the role of DNMT3A in AML relapse and our approach can be applied to predict treatment outcomes.

Clinical features and prognostic significance of DNMT3A, FLT3, and NPM1 mutations in de novo acute myeloid leukemia patients.

Different co-mutation patterns are associated with varied clinical manifestations and prognosis. The purpose of this research was to explore the clinical characteristics and prognosis of individuals with AML who had DNMT3A, FLT3, and NPM1 mutations. A total of 259 newly diagnosed AML patients were investigated in this study, including 148 AMLFLT3mutDNMT3Awt , 48 AMLFLT3wtDNMT3Amut , and 63 AMLFLT3mutDNMT3Amut patients. Mutations were detected by targeted next-generation sequencing and Sanger sequencing. In addition, we utilized the publicly available data to analyze the expression profiles of AML. Correlation analysis showed NPM1 mutations were positively associated with FLT3-ITD and DNMT3A, but negatively with CEBPA and RUNX1 mutations. In the presence of both DNMT3A and FLT3 mutations, patients were associated with typical clinical manifestations such as heavy disease burden and old age. Patients with both FLT3 and DNMT3A mutations had lower complete remission rates and poorer clinical outcomes than those with FLT3 or DNMT3A mutation alone. Univariate analysis showed that age, response to treatment, DNMT3A R882 mutation, NPM1 mutation, and consolidation treatment options were associated with OS. According to multivariate analysis, only consolidation treatment options could be considered as an independent prognostic factor. In addition, the percentage of AMLFLT3mutDNMT3AmutNPM1mut patients in our study was about 5.9%. Interestingly, the expression profile of this subgroup was significantly related to HOX family and histone H1 family, and enriched pathways associated with transcriptional misregulation. We comprehensively evaluated the clinical and genetic characteristics, and expression profiles of AML patients with common mutations, and found that AML patients with triple mutations might be a distinct AML subtype, which should be redefined.

DNMT3A mutation promotes leukemia development through NAM-NAD metabolic reprogramming

BackgroundDNA methyltransferase 3A (DNMT3A) is frequently mutated in acute myeloid leukemia (AML) with Arg882His (R882H) as the hotspot mutation. It has been reported that DNMT3A mutation plays a key role in leukemogenesis through hypomethylation of some target genes associated with cell growth and differentiation. In this study, we investigated the function of DNMT3A R882H in the malignant progression of AML by regulating metabolic reprogramming.MethodsUltra-High Performance Liquid Chromatography–High Resolution Tandem Mass Spectrometry (UHPLC-HRMS/MS) was used to detect metabolites in the serum of mice harboring Dnmt3a R878H mutation and the wild-type Dnmt3a. Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) and RNA sequencing (RNA-seq) were used to analyze the levels of DNA methylation and mRNA expression of genes in mouse Gr1+ bone marrow cells respectively. The TCGA and GO databases were used to analyze the differential genes between human samples carrying the DNMT3A R882 mutation and the wild-type DNMT3A. Co-immunoprecipitation and immunoblotting were used to illustrate the binding levels of Cyclins-CDKs and CDK inhibitors including CDKN1A and CDKN1B. Flow cytometry was used to analyze the cell differentiation, division, apoptosis and cell cycle. The effect of NAMPT inhibition on leukemia was evaluated by using in vivo fluorescence imaging in NOG mouse model bearing OCI-AML3 cells.ResultsDNMT3A mutation caused high expression of nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in the nicotinamide adenine dinucleotide (NAD) salvage synthetic pathway, through DNA hypomethylation, and finally led to abnormal nicotinamide (NAM) metabolism and NAD synthesis. The NAM-NAD metabolic abnormalities caused accelerated cell cycle progression. Inhibition of NAMPT can reduce the binding degree between Cyclins-CDKs, and increase the binding interaction of the CDK inhibitors with Cyclins-CDKs complexes. Moreover, cells with high expression of NAMPT were more sensitive to the NAMPT inhibitor FK866 with a lower IC50. The inhibition of NAMPT can remarkably extend the survival time of tumor-bearing mice and reduce the infiltration of tumor cells.ConclusionsTaken together, our data showed that DNMT3A mutation caused NAMPT overexpression to induce the reprogramming of NAM-NAD metabolism and contribute to abnormal proliferation, which provided a potential direction for targeted therapy at the metabolic level in AML with DNMT3A mutation.

Clinical Value of Translocator Protein Gene in Evaluating the Efficacy of FLT3-ITD/DNMT3A R882 Double-Mutated Acute Myeloid Leukemia

To observe the clinical significance of translocator proteins (TSPO) gene in the treatment of FLT3-ITD/DNMT3A R882 double-mutated acute myeloid leukemia (AML). Seventy-six patients with AML hospitalized in the Department of Hematology of the Affiliated People's Hospital of Ningbo University from June 2018 to June 2020 were selected, including 34 patients with FLT3-ITD mutation, 27 patients with DNMT3A R882 mutation, 15 patients with FLT3-ITD/DNMT3A R882 double mutation, as well as 19 patients with immune thrombocytopenia (ITP) hospitalized during the same period as control group. RNA was routinely extracted from 3 ml bone marrow retained during bone puncture, and TSPO gene expression was detected by transcriptome sequencing (using 2-deltadeltaCt calculation). The expression of TSPO gene in FLT3-ITD group and DNMT3A R882 group at first diagnosis was 2.02±1.04 and 1.85±0.76, respectively, which were both higher than 1.00±0.06 in control group, but the differences were not statistically significant (P=0.671, P=0.821). The expression of TSPO gene in the FLT3-ITD/DNMT3A R882 group was 3.98±1.07, wich was significantly higher than that in the FLT3-ITD group and DNMT3A R882 group, the differences were statistically significant (P=0.032, P=0.021). The expression of TSPO gene in patients who achieved complete response after chemotherapy in the FLT3-ITD/DNMT3A R882 group was 1.19±0.87, which was significantly lower than that at first diagnosis, and the difference was statistically significant (P=0.011). TSPO gene may be used as an indicator of efficacy in FLT3-ITD /DNMT3A R882 double-mutated AML.

Mutational analysis of DNMT3A improves the prognostic stratification of patients with acute myeloid leukemia.

Nucleophosmin1 (NPM1) mutations are the most frequently detected gene mutations in acute myeloid leukemia (AML) and are considered a favorable prognostic factor. We retrospectively analyzed the prognosis of 605 Japanese patients with de novo AML, including 174 patients with NPM1-mutated AML. Although patients with NPM1-mutated AML showed a high remission rate, this was not a favorable prognostic factor for overall survival (OS); this is contrary to generally accepted guidelines. Comprehensive gene mutation analysis showed that mutations in codon R882 of DNA methyltransferase 3A (DNMT3AR882 mutations) were a strong predicative factor indicating poor prognosis in all AML (p < 0.0001) and NPM1-mutated AML cases (p=0.0020). Furthermore, multivariate analysis of all AML cases showed that DNMT3AR882 mutations and the co-occurrence of internal tandem duplication in FMS-like tyrosine kinase 3 (FLT3-ITD), NPM1 mutations, and DNMT3AR882 mutations (triple mutations) were independent factors predicting a poor prognosis related to OS, with NPM1 mutations being an independent factor for a favorable prognosis (hazard ratios: DNMT3AR882 mutations, 1.946; triple mutations, 1.992, NPM1 mutations, 0.548). Considering the effects of DNMT3AR882 mutations and triple mutations on prognosis and according to the classification of NPM1-mutated AML into three risk groups based on DNMT3AR882 /FLT3-ITD genotypes, we achieved the improved stratification of prognosis (p < 0.0001). We showed that DNMT3AR882 mutations are an independent factor for poor prognosis; moreover, when confounding factors that include DNMT3AR882 mutations were excluded, NPM1 mutations were a favorable prognostic factor. This revealed that ethnological prognostic discrepancies in NPM1 mutations might be corrected through prognostic stratification based on the DNMT3A status.

Mutational Analysis of DNMT3A Improves the Prognostic Stratification Using NPM1/FLT3-ITD Genotypes in Patients with Acute Myeloid Leukemia

Background: Nucleophosmin 1 (NPM1) mutations are considered a favorable factor of acute myeloid leukemia (AML). However, their clinical significance in non-European regions remains unclear. DNA methyltransferase 3A (DNMT3A) mutations have been reported as a factor for poor prognosis in many cohort analyses. However, they were excluded among major prognostic factors even in the European Leukemia Net 2017 (ELN2017) classification. In this study, we analyzed the effects of NPM1 and concurrent mutations, particularly DNMT3A mutations, on prognosis. Methods: All patients in this analysis were enrolled and selected by the gene sequencing of Japanese AML (GS-JAML) conducted by Nippon Medical School. We targeted only patients aged 70 years or younger who were diagnosed after 2010 and who received standard induction therapy consisting of 7 days of standard-dose cytarabine (100-200 mg/m2 continuous infusion) and 3 days of an anthracycline antibiotic infusion (idarubicin 12 mg/m2 or daunorubicin 60-90 mg/m2). Mutation screening were performed with target-captured sequencing for the AML gene panel. We also conducted agarose gel electrophoresis for the FMS-like tyrosine kinase 3 (FLT3-ITD) mutations and used the Sanger method to screen for CEBPA and NPM1 mutations. This study was reviewed and approved by the Human Subjects Institutional Review Board (project approval number 29-07-783) of the Nippon Medical School (Tokyo, Japan). Informed consent was obtained in accordance with the Declaration of Helsinki from all participants. Results: We retrospectively analyzed the prognosis in 605 Japanese patients with de novo AML, including 174 patients with NPM1-mutated, 125 patients with DNMT3A-mutated and 127 patients with FLT3-ITD mutated AML. NPM1 mutation was not a prognostic factor either overall or in subgroups based on NPM1/tandem duplication in FLT3-ITD genotypes. Comprehensive gene mutation analysis showed that mutations in codon R882 of DNMT3A(DNMT3AR882) were a strong factor of poor prognosis for AML overall and NPM1-mutated AML. Furthermore, multivariate analysis of all AML showed that DNMT3AR882 mutations and the cooccurrence of FLT3-ITD, NPM1 mutations, and DNMT3AR882 mutations (triple mutation) were independent factors for poor prognosis related to overall survival, with NPM1 mutations being an independent factor for favorable prognosis (hazard ratio: DNMT3AR882 mutations, 1.946; triple mutation, 1.992, NPM1 mutations, 0.548). Furthermore, we evaluated the prognostic impacts of DNMT3AR882 and triple mutation and demonstrated that NPM1-mutated AML patients could be more clearly stratified into three risk groups based on DNMT3AR882/FLT3-ITD genotypes than the ELN 2017 classification. Conclusion: Our study showed that the addition of DNMT3AR882 mutations to existing prognostic models allowed for further stratification of NPM1-mutated AML. This new prognostic model might provide important clinical guidance. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Integrating Mosaic Chromosomal Alterations and Single-Nucleotide Variants to Predict Evolution of Clonal Hematopoiesis

Background: Clonal hematopoiesis (CH) is an age-related expansion of mutated hematopoietic stem cells. Multiple studies have previously described distinct types of CH, CHIP (mutations in genes commonly associated with myeloid malignancies) as well as mosaic chromosomal alterations (mCA), that have been associated with increased risk of hematologic malignancies and cardiovascular morbidity. We hypothesized that the presence of both mCA and CHIP mutations may have an additive impact vis-à-vis risk of hematologic malignancy. Methods: Data on 91,335 patients from the BioVU repository, a Vanderbilt University Medical Center biobank with linked de-identified EHRs, spanning 2006 to 2021 were included in the analysis. Peripheral blood-derived DNA of 91,335 subjects from BioVU were genotyped on the Illumina Multi-Ethnic Global Array (MEGAEX) platform, which contains more than 2 million SNPs. CHIP mutation calls (DNMT3A R882/JAK2 V617F) were made by deconvoluting the MEGAEX array intensity data. Predetermined array intensity scores greater than a defined threshold for DNMT3A R882 and JAK2 V617F mutations were highly predictive of the corresponding mutation, so these mutations were included in the analysis. Individuals with a diagnosis of hematologic malignancy (HM) prior to blood draw were excluded, while those that went on to develop HM post-blood draw were included in the analyses. The relationship between independent variables and disease progression and death was assessed using Cox proportional hazards model. All statistical analyses were performed using R. Results: In this large cohort (n=91,335), CH is strongly age-related with an increased frequency in the elderly, as has been previously shown (eg., Bick et al, Nature 2021; Jaiswal et al NEJM 2014). In total, we identified mCA in 765 (0.8%) patients. DNMT3A R882 and JAK2 V617F SNV/indels were identified in 1167 (1.3%) and 347 (0.4%) patients, respectively. Most frequent mCA detected in this cohort include 9p CN-LOH (n=105, 6.9%), del (5q) (n=99, 6.5%), del (20q) (n=98, 6.4%), del (7q) (n=94, 6.2%), and del (13q) (n=67, 4.4%). Combined mCA and DNMT3A R882/JAK2 V617F mutations were reported in 147 (0.2%). Co-occurring JAK2 and 9p CN-LOH (n= 100, OR=5658, q<0.001) are consistent with the well-known mechanisms of biallelic alterations involved in the pathogenesis of HM. There were significant correlations between abnormal blood counts and CH lesions, for example, high platelet count had a higher frequency of JAK2 V617F and 9p CN-LOH (OR=2.57, q=3.41e-141). Individuals with mCA are associated with lower WBC and platelet count, and any blood count abnormality (Figure 1A). Finally, we looked at how presence of both mCA and JAK2/DNMT3A impact the risk of HM. In total, 1615 cases in the entire BioVU cohort were reported to develop HM: 947 myeloid, AML (n=210), CML (n=73), MDS (n=315), MPN (n=349) and 668 lymphoid, ALL (n=72), B-NHL (n=252), T-NHL (n=22), MM (n=322). The presence of mCA was associated with a higher incidence of HM (hazard ratio (HR) = 6.85, 95% confidence interval (CI) = 5.9-7.95, P < 0.001), and DNMT3A/JAK2 was associated with higher incidence of HM ( HR=5.7, 95% CI= 4.75-6.85, P<0.001), and presence of both (mCA and DNMT3A/JAK2) were significantly associated with higher incidence of HM (HR=26.67, 95% CI= 19.48-36.5, P<0.001) than observed in no CH patients (Figure 1B). Conclusion: In this large cohort of patients, the presence of CH (both mCA and DNMT3A/JAK2) in apparently healthy individuals positively correlated with blood count abnormalities and increased incidence of hematologic malignancy. The results from this large cohort of patients emphasize the significance of detecting both mCA and SNV/indels in clonal hematopoiesis. These results should be validated in large datasets to determine if detecting both mCAs and CH can augment clinical care and outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DNMT3A R882H mutation drives daunorubicin resistance in acute myeloid leukemia via regulating NRF2/NQO1 pathway

BackgroundDNA methyltransferase 3A (DNMT3A) often mutate on arginine 882 (DNMT3AR882) in acute myeloid leukemia (AML). AML patients with DNMT3A R882 mutation are usually resistant to daunorubicin treatment; however, the associated mechanism is still unclear. Therefore, it is urgent to investigate daunorubicin resistance in AML patients with DNMT3A R882 mutant.MethodAML cell lines with DNMT3A-wild type (DNMT3A-WT), and DNMT3A-Arg882His (DNMT3A-R882H) mutation were constructed to investigate the role of DNMT3A R882H mutation on cell proliferation, apoptosis and cells’ sensitivity to Danunorubin. Bioinformatics was used to analyze the role of nuclear factor-E2-related factor (NRF2) in AML patients with DNMT3A R882 mutation. The regulatory mechanism of DNMT3A R882H mutation on NRF2 was studied by Bisulfite Sequencing and CO-IP. NRF2 inhibitor Brusatol (Bru) was used to explore the role of NRF2 in AML cells carried DNMT3A R882H mutation.ResultsAML cells with a DNMT3A R882H mutation showed high proliferative and anti-apoptotic activities. In addition, mutant cells were less sensitive to daunorubicin and had a higher NRF2 expression compared with those in WT cells. Furthermore, the NRF2/NQO1 pathway was activated in mutant cells in response to daunorubicin treatment. DNMT3A R882H mutation regulated the expression of NRF2 via influencing protein stability rather than decreasing methylation of NRF2 promoter. Also, NRF2/NQO1 pathway inhibition improved mutant cells’ sensitivity to daunorubicin significantly.ConclusionOur findings identified NRF2 as an important player in the regulation of cell apoptosis through which helps mediate chemoresistance to daunorubicin in AML cells with DNMT3A R882H mutation. Targeting NRF2 might be a novel therapeutic approach to treat AML patients with a DNMT3A R882H mutation.9jD1n7ooVY8uHe1s9Su8KLVideo abstract

Clinical Outcomes and Prognostic Factors of Allogeneic Hematopoietic Stem Cell Transplantation in the Treatment of Refractory/Relapsed Acute Myeloid Leukemia

To investigate the clinical outcomes and prognostic factors of refractory/relapsed acute myeloid leukemia (AML) patients who received allogeneic hematopoietic stem cell transplantation (allo-HSCT). The clinical data of 80 refractory/relapsed AML patients who received allo-HSCT from December 2013 to June 2020 were retrospectively analyzed, including the overall survival (OS) rate, disease-free survival (DFS) rate, relapse rate, incidence of transplant-related mortality (TRM), and the related risk factors were explored. Hematopoietic reconstitution was obtained in all 80 patients after transplantation, the 3-year OS and DFS rates were (48.8±6.3)% and (40.8±6.7)%, respectively. The 3-year cumulative incidence of relapse and TRM were 33.8% (95%CI: 0.254-0.449) and 15.0%(95%CI: 0.114-0.198), respectively. Univariate analysis showed that non-remission (NR) status before transplantation, DNMT3A R882 mutations and grade II-IV acute graft-versus-host disease (aGVHD) had negative effects on OS and DFS. Multivariate analysis indicated that the DNMT3A R882 mutations and grade II-IV aGVHD were independent risk factors for OS (HR=0.253, 95%CI: 0.092-0.695, P=0.008; HR=5.681, 95%CI: 2.101-15.361, P=0.001) and DFS (HR=0.200, 95%CI: 0.071-0.569, P=0.003; HR=7.117, 95%CI: 2.556-19.818, P<0.001). The 3-year cumulative incidence of relapse was 71.4%(95%CI: 0.610-0.836) in genetic high-risk group, which was higher than 23.3%(95%CI: 0.147-0.370) in intermediate-risk group and 23.5%(95%CI: 0.127-0.437) in favorable-risk group (P=0.006). Allo-HSCT is an effective and safe choice for refractory/relapsed AML patients. DNMT3A R882 mutations and grade II-IV aGVHD are negative prognostic factors of allo-HSCT for refractory/relapsed AML patients.

Single-cell multi-omics of human clonal hematopoiesis reveals that DNMT3A R882 mutations perturb early progenitor states through selective hypomethylation.

Somatic mutations in cancer genes have been detected in clonal expansions across healthy human tissue, including in clonal hematopoiesis. However, because mutated and wild-type cells are admixed, we have limited ability to link genotypes with phenotypes. To overcome this limitation, we leveraged multi-modality single-cell sequencing, capturing genotype, transcriptomes and methylomes in progenitors from individuals with DNMT3A R882 mutated clonal hematopoiesis. DNMT3A mutations result in myeloid over lymphoid bias, and an expansion of immature myeloid progenitors primed toward megakaryocytic-erythroid fate, with dysregulated expression of lineage and leukemia stem cell markers. Mutated DNMT3A leads to preferential hypomethylation of polycomb repressive complex 2 targets and a specific CpG flanking motif. Notably, the hypomethylation motif is enriched in binding motifs of key hematopoietic transcription factors, serving as a potential mechanistic link between DNMT3A mutations and aberrant transcriptional phenotypes. Thus, single-cell multi-omics paves the road to defining the downstream consequences of mutations that drive clonal mosaicism.

Prognostic impact of early minimal residual disease combined with complete molecular evaluation in acute myeloid leukemia with mutated NPM1: a single center study

We evaluated prognostic factors in 83 intensively treated adult patients with NPM1 mutated AML. Targeted next-generation sequencing revealed high frequency of co-mutations in epigenetic modifiers or proliferation pathways. NPM1 minimal residual disease (MRD), assessed in bone marrow by specific polymerase chain reaction after one chemotherapy course, was >0.01% in 50 patients considered poor responders (PR). On multivariate analysis, including all variables with a p value <.1 on univariate analysis, age >60, performance status (PS) ≥1, presence of FLT3 mutations, DNMT3A-R882, and PR status, were independently associated with lower leukemia-free survival. Age >60, a previous hematological disease and PR status were independent negative predictive factors for overall survival. In our study, early NPM1 MRD was confirmed as an important prognostic factor. All FLT3 and DNMT3A-R882 mutations have also an independent prognostic value. We support the rational for in-depth investigations for a better approach in patients who achieving a first complete remission.

DNMT3A R882 Mutations Confer Unique Clinicopathologic Features in MDS Including a High Risk of AML Transformation

DNMT3A mutations play a prominent role in clonal hematopoiesis and myeloid neoplasms with arginine (R)882 as a hotspot, however the clinical implications of R882 vs. non-R882 mutations in myeloid neoplasms like myelodysplastic syndrome (MDS) is unclear. By data mining with publicly accessible cancer genomics databases and a clinical genomic database from a tertiary medical institution, DNMT3A R882 mutations were found to be enriched in AML (53% of all DNMT3A mutations) but decreased in frequency in clonal hematopoiesis of indeterminate potential (CHIP) (10.6%) or other myeloid neoplasms including MDS (27%) (p<.001). Next with the largest cohort of patients with DNMT3A R882 mutant MDS known to date from multiple institutions, DNMT3A R882 mutant MDS cases were shown to have more severe leukopenia, enriched SRSF2 and IDH2 mutations, increased cases with excess blasts (47% vs 22.5%, p=.004), markedly increased risk of AML transformation (25.8%, vs. 1.7%, p=.0001) and a worse progression-free survival (PFS) (median 20.3, vs. >50 months, p=.009) than non-R882 mutant MDS cases. DNMT3A R882 mutation is an independent risk factor for worse PFS, and importantly the differences in the risk of AML transformation between R882 vs. non-R882 mutant patients cannot be explained by different treatment approaches. Interestingly the higher risk of AML transformation and the worse PFS in DNMT3A R882 mutant MDS cases are mitigated by coexisting SF3B1 or SRSF2 mutations. The unique clinicopathologic features of DNMT3A R882 mutant MDS shed light on the prognostic and therapeutic implications of DNMT3A R882 mutations.

3214 – DNMT3A R882 MUTATION IN HUMAN HAEMATOPOIETIC STEM CELLS ALTERS DIFFERENTIATION TOWARDS NEUTROPHILS AND MONOCYTES

Age related clonal haematopoiesis (ARCH) results from clonal expansion of hematopoietic stem cells (HSCs), driven by recurrent leukemic mutations, and carries an increased risk of haematological malignancies and cardiometabolic disease. DNMT3A R882 is the most common ARCH mutation, and we hypothesise that it affects the differentiation dynamics of HSCs, potentially contributing to disease risk. To comprehensively characterise changes in HSC differentiation driven by DNMT3A R882 at single cell resolution, 2785 single phenotypic HSC/MPPs (CD33-/CD34+/CD45dim/CD38-/CD45RA-) from 6 individuals were cultured in media supporting differentiation into all major blood lineages. DNMT3A R882 HSC derived colonies were compared with WT colonies within each individual. Outcomes were analysed using flow phenotyping combined with novel multiparameter in silico analysis pipelines, targeted genotyping and for one sample RNA-seq and whole genome sequencing followed by phylogenetic analysis based on somatic mutations. The number of HSCs committing to the erythroid, myeloid or lymphoid (NK) lineages were similar between DNMT3A R882 and WT HSCs. DNMT3A R882 HSCs however, produced more mature granulocytes and fewer mature monocytes than WT HSCs (as measured by proportions and MFI of CD14+, CD15+ & CD66b+ cells in mature colonies). Transcriptional profiles from RNAseq of monocytic colonies confirmed the presence of less mature monocytes in DNMT3A R882 colonies compared to WT in one ARCH sample. Overall, our data indicate that DNMT3A R882 mutation in HSCs alters monocyte and neutrophil differentiation dynamics. We speculate these defects respectively contribute to the early changes that may lead to phenotypic AML following secondary somatic mutations; and increased risk of cardiovascular disease observed in DNMT3A R882 carriers, for which clear mechanisms have not been identified to date. Age related clonal haematopoiesis (ARCH) results from clonal expansion of hematopoietic stem cells (HSCs), driven by recurrent leukemic mutations, and carries an increased risk of haematological malignancies and cardiometabolic disease. DNMT3A R882 is the most common ARCH mutation, and we hypothesise that it affects the differentiation dynamics of HSCs, potentially contributing to disease risk. To comprehensively characterise changes in HSC differentiation driven by DNMT3A R882 at single cell resolution, 2785 single phenotypic HSC/MPPs (CD33-/CD34+/CD45dim/CD38-/CD45RA-) from 6 individuals were cultured in media supporting differentiation into all major blood lineages. DNMT3A R882 HSC derived colonies were compared with WT colonies within each individual. Outcomes were analysed using flow phenotyping combined with novel multiparameter in silico analysis pipelines, targeted genotyping and for one sample RNA-seq and whole genome sequencing followed by phylogenetic analysis based on somatic mutations. The number of HSCs committing to the erythroid, myeloid or lymphoid (NK) lineages were similar between DNMT3A R882 and WT HSCs. DNMT3A R882 HSCs however, produced more mature granulocytes and fewer mature monocytes than WT HSCs (as measured by proportions and MFI of CD14+, CD15+ & CD66b+ cells in mature colonies). Transcriptional profiles from RNAseq of monocytic colonies confirmed the presence of less mature monocytes in DNMT3A R882 colonies compared to WT in one ARCH sample. Overall, our data indicate that DNMT3A R882 mutation in HSCs alters monocyte and neutrophil differentiation dynamics. We speculate these defects respectively contribute to the early changes that may lead to phenotypic AML following secondary somatic mutations; and increased risk of cardiovascular disease observed in DNMT3A R882 carriers, for which clear mechanisms have not been identified to date.

Focal disruption of DNA methylation dynamics at enhancers in IDH-mutant AML cells

Recurrent mutations in IDH1 or IDH2 in acute myeloid leukemia (AML) are associated with increased DNA methylation, but the genome-wide patterns of this hypermethylation phenotype have not been comprehensively studied in AML samples. We analyzed whole-genome bisulfite sequencing data from 15 primary AML samples with IDH1 or IDH2 mutations, which identified ~4000 focal regions that were uniquely hypermethylated in IDHmut samples vs. normal CD34+ cells and other AMLs. These regions had modest hypermethylation in AMLs with biallelic TET2 mutations, and levels of 5-hydroxymethylation that were diminished in IDH and TET-mutant samples, indicating that this hypermethylation results from inhibition of TET-mediated demethylation. Focal hypermethylation in IDHmut AMLs occurred at regions with low methylation in CD34+ cells, implying that DNA methylation and demethylation are active at these loci. AML samples containing IDH and DNMT3AR882 mutations were significantly less hypermethylated, suggesting that IDHmut-associated hypermethylation is mediated by DNMT3A. IDHmut-specific hypermethylation was highly enriched for enhancers that form direct interactions with genes involved in normal hematopoiesis and AML, including MYC and ETV6. These results suggest that focal hypermethylation in IDH-mutant AML occurs by altering the balance between DNA methylation and demethylation, and that disruption of these pathways at enhancers may contribute to AML pathogenesis.

DNMT3A R882 Mutation in Human Haematopoietic Stem Cells Alters Differentiation Towards Neutrophils and Monocytes

Age related clonal haematopoiesis (ARCH) is defined as the clonal expansion of hematopoietic stem cells (HSCs), driven by recurrent mutations. Many of these are also commonly seen in haematological malignancies, hence termed pre-leukemic mutations (pLM). ARCH carries an increased risk of haematological malignancies and cardiometabolic disease. Further understanding of the role of pLMs in HSC function is necessary to predict and possibly prevent leukemia. DNMT3A R882 is the most common pLM observed in ARCH and is associated with poor outcomes in acute myeloid leukemia (AML). It is acquired early in life and is positively selected in HSCs. HSCs of Dnmt3a knock-out or knock-in mouse models have increased self-renewal capacity and can differentiate towards all lineages. DNMT3A R882 mutation is observed in all mature blood lineages in ARCH individuals, indicating their multipotential differentiation capacity, but the dynamics of differentiation are not known. We hypothesise that DNMT3A R882 mutation affects the differentiation dynamics of HSCs, potentially contributing to disease risk.We characterised the functional changes in HSC differentiation driven by DNMT3A R882 at single cell resolution. Single phenotypic HSCs (CD33-/CD34+/CD45dim/CD38-/CD45RA-) from 9 individuals (Table 1) were cultured in media supporting differentiation into all major blood lineages. To assess changes in differentiation capacity between DNMT3A R882 and wild-type (WT) HSCs within each individual, each single-cell colony was genotyped by targeted DNA sequencing and scored by high-throughput flow cytometry. Flow cytometry data was analysed using a novel unbiased analytical pipeline, which we have named ‘FlowPAC’, allowing generation of a two-dimensional representation of the global output of HSC differentiation by identifying clusters based on fluorochrome intensity. Four samples were underpowered for either DNMT3A R882 or WT colonies and were excluded from this analysis.We did not observe any difference in erythroid versus myeloid or lymphoid (NK) differentiation capacity between DNMT3A R882 and WT HSC. DNMT3A R882 and WT HSCs also produced similar proportions of monocytic and neutrophil colonies. However, FlowPAC analysis identified differences in their level of maturity. CD15 (neutrophil marker) expression was significantly increased in myeloid clusters of DNMT3A R882 colonies compared to WT (p=0.0043), whereas CD14 (monocyte marker) expression was decreased compared to WT (p=0.0645). We further analysed mature neutrophil differentiation using CD66b expression. Consistent with promotion of neutrophil differentiation, CD66b median fluorescence intensity was significantly increased in the CD15+ population of DNMT3A R882 colonies compared to WT. To further investigate monocyte differentiation, we performed RNAseq on colonies containing only monocytes from 1 ARCH sample. Analysis of transcriptional profiles confirmed less mature monocytes in DNMT3A R882 colonies compared to WT. Overall, our data indicate that DNMT3A R882 mutation in HSCs alters neutrophil and monocyte production.Additionally, we observed that in contrast to samples with high LSC content leading to leukemic engraftment in mice, in samples where DNMT3A R882 HSCs were able to reconstitute normal blood production, the expression of the cell surface marker CD49f, a marker of the most potent HSCs, was significantly increased in DNMT3A R882 HSCs compared to WT at the time of sorting. This may suggest unequal distribution of this pLM within the preserved normal HSC pool prior to leukemic transformation.In conclusion, DNMT3A R882 alters the dynamics of human HSC myeloid differentiation. The propensity of DNMT3A R882 derived monocytes to retain an immature phenotype relative to WT cells could represent an early change later facilitating the complete differentiation block seen in AML, driven by the acquisition of additional mutations such as NPM1. DNMT3A R882 also promoted neutrophil maturation. Altered neutrophil differentiation has been observed in patients with germline DNMT3A mutation. As altered neutrophil function plays a key role in cardiovascular disease, future studies are required to investigate whether the effect of DNMT3A R882 on neutrophil differentiation may contribute to the increased cardiovascular disease risk associated with this mutation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hotspot DNMT3A mutations in clonal hematopoiesis and acute myeloid leukemia sensitize cells to azacytidine via viral mimicry response.

Somatic mutations in DNA methyltransferase 3A (DNMT3A) are among the most frequent alterations in clonal hematopoiesis (CH) and acute myeloid leukemia (AML), with a hotspot in exon 23 at arginine 882 (DNMT3AR882). Here, we demonstrate that DNMT3AR882H-dependent CH and AML cells are specifically susceptible to the hypomethylating agent azacytidine (AZA). Addition of AZA to chemotherapy prolonged AML survival solely in individuals with DNMT3AR882 mutations, suggesting its potential as a predictive marker for AZA response. AML and CH mouse models confirmed AZA susceptibility specifically in DNMT3AR882H-expressing cells. Hematopoietic stem cells (HSCs) and progenitor cells expressing DNMT3AR882H exhibited cell autonomous viral mimicry response as a result of focal DNA hypomethylation at retrotransposon sequences. Administration of AZA boosted hypomethylation of retrotransposons specifically in DNMT3AR882H-expressing cells and maintained elevated levels of canonical interferon-stimulated genes (ISGs), thus leading to suppressed protein translation and increased apoptosis.

Using an in-Vivo Degron-Based Approach to Interrogate Dependencies of Serially Acquired Mutations - Including DNMT3a-R882 and NPM1c - in Acute Myeloid Leukemia

While a number of targeted therapies have recently become available in the treatment of acute myeloid leukemia (AML), it remains to be determined whether early acquired mutations are essential for ongoing leukemia maintenance in the face of sequential acquisition of additional mutations. In some cases, our ability to answer this question is hampered by the lack of targeted therapies to a number of these mutations. Of all AML mutations, those involving DNMT3a and NPM1c are some of the most frequent - often acquired before the final transforming event. Here we report the development of an in-vivo degron-based approach to interrogate the requirement for DNMT3a-R882 and NPM1c after leukemia development. Using an IKZF-based degron system offers us the opportunity to address the ongoing requirement of particular oncogenes for AML maintenance - by way of a conditional system that can be rapidly modulated. Thalidomide and its related analogues act by recruiting various substrates to the CRL4CRBN E3-ubiquitin ligase complex mediating subsequent ubiquitination and proteasomal degradation. In a majority of cases - the basis of this degradation has been determined to be the result of recognition and binding to a degron motif within C2H2 zinc fingers. Using more detailed analysis with single amino-acid resolution, we have engineered 'degrons' sensitive or resistant to degradation by thalidomide and its related analogues. In-vivo degradation using pomalidomide revealed a dependence of GMP cells on the presence of NPM1c but not DNMT3a R882. Using our cereblon (CRBN) knock-in mice engineered to mediate sensitivity to these compounds, we generated triple-mutant acute-myeloid leukemia - sequentially introducing DNMT3a-R882, NPM1c and N-RAS G12D mutations whilst altering the site of the degron tag in each case. Degradation-resistant degron constructs were used as a control in each case. Findings were subsequently correlated and validated with in-vitro treatments of triple mutant cells. RNA-seq was also performed after both in-vitro and in-vivo treatments to further delineate acute transcriptional changes in the absence of NPM1c - highlighting a number of changes including those within the Hox family of genes Together, these findings suggest triple mutant leukemia remains dependent on the leukemia-inducing mutation of NPM1c. In contrast, whilst epigenetic alterations induced by DNMT3a mutations are essential for leukemia development - these do not appear to be essential for ongoing leukemia maintenance. Further work is currently underway to determine how modulating NPM1c impacts long-term survival from leukemia and whether the epigenetic changes associated with DNMT3a R882 mutations are indeed reversible in the context of leukemia. This work highlights an approach to interrogate a protein of interest in-vivo using a conditional system that is reversible, rapidly acting and that uses a series of FDA-approved compounds with known kinetics. We envisage this will be generalisable as a tool to be used in other similar contexts. Disclosures No relevant conflicts of interest to declare.

Single-Cell Multi-Omics in Human Clonal Hematopoiesis Reveals That DNMT3A R882 Mutations Perturb Early Progenitor States through Selective Hypomethylation

Leukemia driver mutations have been identified in clonal hematopoiesis (CH; Jaiswal et al, NEJM, 2014). This provides a window of opportunity to interrogate the downstream impact of driver mutations in the earliest stages of neoplasia, before the accumulation of additional drivers that lead to frank malignancy. However, CH mutated cells are morphologically and phenotypically similar to normal cells. Thus, previous characterization in primary human samples have largely focused on genetic identification of these clonal outgrowths. To overcome this limitation and define the downstream effects of CH mutations, we leveraged multi-omic single-cell sequencing to profile the mutation status and whole transcriptome for the same cells at high-throughput (GoT; Nam et al, Nature, 2019). To identify CH samples, we screened 136 stem cell grafts collected for autologous transplant from multiple myeloma patients in remission (Mouhieddine et al, Nat Comm, 2020). We identified four individuals with DNMT3AR882 mutations with VAF &amp;gt; 0.05, allowing the profiling of 27,324 CD34+ cells (Fig. 1a-b). DNMT3A R882 mutations in these human CH samples did not result in a significant differentiation block in the hematopoietic stem cells, as assessed by pseudotime analysis (Fig. 1c). However, specific differentiation skews were apparent in multi-lineage progenitors, corresponding to the highest expression of DNMT3A in mutated immature myeloid progenitors (IMP, i.e. common myeloid progenitors/granulocyte-monocyte progenitors, Fig. 1d). We observed the expansion of mutated IMPs (Fig. 1e) that showed priming toward the megakaryocytic-erythroid (ME) lineage (Fig. 1f). DNMT3AR882 lympho-myeloid primed progenitors showed a myeloid bias and disfavored lymphoid development (Fig. 1g). In these CD34+ progenitors, we identified dysregulated expressions of megakaryocytic lineage markers, such as CD9 and PLEK, consistent with the ME-bias (Fig. 1h). We also observed downregulation of lymphoid differentiation gene ZBTB1, corresponding to the myeloid over lymphoid skewing (Fig. 1h). Finally, we identified dysregulated expression of genes involved in TNF-alpha signaling (e.g. TNFRSF4, TNFSF13B), suggesting a pro-inflammatory state. To identify activated pathways, we performed a gene set enrichment analysis that revealed activation of MYC and IL-6 signaling (FDR &amp;lt;0.05, Hallmark). A focused analysis of regulatory networks identified enhanced activity of key transcription factors (TFs) involved in ME differentiation such as GATA1 and FLI1, as well as those involved in inflammation such as NFKB/REL (FDR &amp;lt;0.05). We also identified increased expression of polycomb repression complex 2 (PRC2) target genes (FDR &amp;lt;0.05). To determine how aberrant DNA methylation may serve as a link between DNMT3A mutations and the observed transcriptional dysregulation, we performed single-cell multi-omics that simultaneously profiles the cells' methylome and transcriptome, linked with mutation status (Gaiti et al, Nature, 2019; Fig. 1i). By comparing the methylation rates in mutated vs. wildtype CD34+ cells within the same sample, we found that DNMT3AR882 result in selective hypomethylation that preferentially impacts CpG islands (Fig. 1j) and PRC2 targets (Fig. 1k), which may underlie the dysregulated expression of PRC2 targets in DNMT3AR882 progenitors. Notably, DNMT3AR882-induced hypomethylation was enriched in a specific sequence context in which the CpG is followed by a T nucleotide (Fig. 1l). This DNMT3AR882 motif was enriched in the DNA binding motifs of central hematopoietic TFs, including MYC/MAX (Fig. 1m), suggesting that DNMT3AR882-induced hypomethylation may serve as a mechanism to preferentially increase the activity of these TFs. Consistent with this hypothesis, joint single-cell methylome and transcriptome data revealed that the expression of MYC targets increased with hypomethylation of its binding motifs (Fig. 1n), providing a mechanism of enhanced MYC activity due to DNMT3AR882 mutations. Altogether, we report the direct examination of the consequences of DNMT3AR882 mutations in primary CD34+ cells in CH. We discovered that DNMT3A is most highly expressed in mutated multi-lineage progenitors, promoting their expansion and biasing their downstream differentiation. This is accompanied by a disruption to differentiation through PRC2 and MYC target reactivation via selective hypomethylation. Disclosures Abdel-Wahab: Envisagenics Inc.: Current equity holder in private company; H3 Biomedicine Inc.: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy. Ghobrial:Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Karyopharm Therapeutics: Consultancy, Honoraria; Cellectar: Honoraria; Adaptive Biotechnologies: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Novartis: Consultancy; Noxxon Pharma: Consultancy; Genentech: Consultancy; GlaxoSmithKline: Consultancy; GNS Healthcare: Consultancy; AbbVie: Consultancy. Landau:Bristol Myers Squibb: Research Funding; Illumina: Research Funding.