Loss of Dnmt3a increases self-renewal and resistance to pegIFN-α in JAK2-V617F-positive myeloproliferative neoplasms.

ABSTRACT: Pegylated interferon alfa (pegIFN-α) can induce molecular remissions in patients with JAK2-V617F-positive myeloproliferative neoplasms (MPNs) by targeting long-term hematopoietic stem cells (LT-HSCs). Additional somatic mutations in genes regulating LT-HSC self-renewal, such as DNMT3A, have been reported to have poorer responses to pegIFN-α. We investigated whether DNMT3A loss leads to alterations in JAK2-V617F LT-HSC functions conferring resistance to pegIFN-α treatment in a mouse model of MPN and in hematopoietic progenitors from patients with MPN. Long-term treatment with pegIFN-α normalized blood parameters and reduced splenomegaly and JAK2-V617F chimerism in single-mutant JAK2-V617F (VF) mice. However, pegIFN-α in VF;Dnmt3aΔ/Δ (VF;DmΔ/Δ) mice worsened splenomegaly and failed to reduce JAK2-V617F chimerism. Furthermore, LT-HSCs from VF;DmΔ/Δ mice compared with VF were less prone to accumulate DNA damage and exit dormancy upon pegIFN-α treatment. RNA sequencing showed that IFN-α induced stronger upregulation of inflammatory pathways in LT-HSCs from VF;DmΔ/Δ than from VF mice, indicating that the resistance of VF;DmΔ/Δ LT-HSC was not due to failure in IFN-α signaling. Transplantations of bone marrow from pegIFN-α-treated VF;DmΔ/Δ mice gave rise to more aggressive disease in secondary and tertiary recipients. Liquid cultures of hematopoietic progenitors from patients with MPN with JAK2-V617F and DNMT3A mutation showed increased percentages of JAK2-V617F-positive colonies upon IFN-α exposure, whereas in patients with JAK2-V617F alone, the percentages of JAK2-V617F-positive colonies decreased or remained unchanged. PegIFN-α combined with 5-azacytidine only partially overcame resistance in VF;DmΔ/Δ mice. However, this combination strongly decreased the JAK2-mutant allele burden in mice carrying VF mutation only, showing potential to inflict substantial damage preferentially to the JAK2-mutant clone.

Mitochondrial genome undergoes de novo DNA methylation that protects mtDNA against oxidative damage during the peri-implantation window.

Mitochondrial remodeling during the peri-implantation stage is the hallmark event essential for normal embryogenesis. Among the changes, enhanced oxidative phosphorylation is critical for supporting high energy demands of postimplantation embryos, but increases mitochondrial oxidative stress, which in turn threatens mitochondrial DNA (mtDNA) stability. However, how mitochondria protect their own histone-lacking mtDNA, during this stage remains unclear. Concurrently, the mitochondrial genome gain DNA methylation by this stage. Its spatiotemporal coincidence with enhanced mitochondrial stress led us to ask if mtDNA methylation has a role in maintaining mitochondrial genome stability. Herein, we report that mitochondrial genome undergoes de novo mtDNA methylation that can protect mtDNA against enhanced oxidative damage during the peri-implantation window. Mitochondrial genome gains extensive mtDNA methylation during transition from blastocysts to postimplantation embryos, thus establishing relatively hypermethylated mtDNA from hypomethylated state in blastocysts. Mechanistic study revealed that DNA methyltransferase 3A (DNMT3A) and DNMT3B enter mitochondria during this process and bind to mtDNA, via their unique mitochondrial targeting sequences. Importantly, loss- and gain-of-function analyses indicated that DNMT3A and DNMT3B are responsible for catalyzing de novo mtDNA methylation, in a synergistic manner. Finally, we proved, in vivo and in vitro, that increased mtDNA methylation functions to protect mitochondrial genome against mtDNA damage induced by increased mitochondrial oxidative stress. Together, we reveal mtDNA methylation dynamics and its underlying mechanism during the critical developmental window. We also provide the functional link between mitochondrial epigenetic remodeling and metabolic changes, which reveals a role for nuclear-mitochondrial crosstalk in establishing mitoepigenetics and maintaining mitochondrial homeostasis.

Downregulation of C1R promotes hepatocellular carcinoma development by activating HIF-1α-regulated glycolysis.

C1R has been identified to have a distinct function in cutaneous squamous cell carcinoma that goes beyond its role in the complement system. However, it is currently unknown whether C1R is involved in the progression of hepatocellular carcinoma (HCC). HCC tissues were used to examine C1R expression in relation to clinical and pathological factors. Malignant characteristics of HCC cells were assessed through in vitro and in vivo experiments. The mechanism underlying the role of C1R in HCC was explored through RNA-seq, methylation-specific PCR, immuno-precipitation, and dual-luciferase reporter assays. This study found that the expression of C1R decreased as the malignancy of HCC increased and was associated with poor prognosis. C1R promoter was highly methylated through DNMT1 and DNMT3a, resulting in a decrease in C1R expression. Downregulation of C1R expression resulted in heightened malignant characteristics of HCC cells through the activation of HIF-1α-mediated glycolysis. Additionally, decreased C1R expression was found to promote xenograft tumor formation. We found that C-reactive protein (CRP) binds to C1R, and the free CRP activates the NF-κB signaling pathway, which in turn boosts the expression of HIF-1α. This increase in HIF-1α leads to higher glycolysis levels, ultimately promoting aggressive behavior in HCC. Methylation of the C1R promoter region results in the downregulation of C1R expression in HCC. C1R inhibits aggressive behavior in HCC in vitro and in vivo by inhibiting HIF-1α-regulated glycolysis. These findings indicate that C1R acts as a tumor suppressor gene during HCC progression, opening up new possibilities for innovative therapeutic approaches.

Clonal Hematopoiesis and Bone Marrow Infiltration in Patients With Follicular Helper T-Cell Lymphoma of Angioimmunoblastic Type.

Follicular helper T-cell (TFH) lymphoma harbors recurrent mutations of RHOAG17V, IDH2R172, TET2, and DNMT3A. TET2 and DNMT3A mutations are the most frequently affected genes in clonal hematopoiesis (CH). The aim of our study was to investigate the frequency of CH in bone marrow biopsies (BMB) of TFH/angioimmunoblastic T-cell lymphoma (TFH-AITL) patients and its association with myeloid neoplasms. A total of 29 BMB from 22 patients with a diagnosis of TFH-AITL were analyzed by next-generation sequencing (NGS) with a custom panel. Morphologically, 5 BMB revealed that TFH-AITL infiltrates of >5% of bone marrow (BM) cellularity confirmed in 4 cases by NGS-based T-cell clonality. IDH2R172 was demonstrated only in 1 (3%) of 29, and RHOAG17V in 2 (7%) of 29 samples. TET2 and DNMT3A were identified in 24 (83%) of 29 and 17 (59%) of 29 BMB, respectively. In the parallel lymph node the frequencies of mutations were 27% (IDH2R172), 64% (RHOAG17V), 86% (TET2), and 50% (DNMT3A). TET2 and/or DNMT3A mutations identical in lymph node and BMB were present in 18 (82%) of 22 patients, regardless of BM infiltration. In 3 cases the CH mutations were detected 13, 41, and 145 months before TFH-AITL diagnosis. Cases with TET2/DNMT3A mutations and BM variant allele frequencies >40% (7/18, 39%) showed lower blood counts. However, only low platelet count was statistically significant (P = .024). Myeloid neoplasms and/or myelodysplastic syndrome-related mutations were identified in 4 cases (4/22; 18%); all with high TET2 variant allele frequencies (>40%; P = .0114). In conclusion, CH is present in 82% of TFH-AITL and can be demonstrated up to 145 months before TFH-AITL diagnosis. NGS T-cell clonality analysis is an excellent tool to confirm TFH-AITL BM infiltration. Concurrent myeloid neoplasms were identified in 18% of the cases and were associated with TET2 mutations with high allelic burden (>40%). We demonstrated that myeloid neoplasms might occur simultaneously or precede the diagnosis of TFH lymphoma.

Suppression of DNMT2/3 by proinflammatory cytokines inhibits CtBP1/2-dependent genes to promote the occurrence of atrophic nonunion.

Failure of bone healing after fracture often results in nonunion, but the underlying mechanism of nonunion pathogenesis is poorly understood. Herein, we provide evidence to clarify that the inflammatory microenvironment of atrophic nonunion (AN) mice suppresses the expression levels of DNA methyltransferases 2 (DNMT2) and 3A (DNMT3a), preventing the methylation of CpG islands on the promoters of C-terminal binding protein 1/2 (CtBP1/2) and resulting in their overexpression. Increased CtBP1/2 acts as transcriptional corepressors that, along with histone acetyltransferase p300 and Runt-related transcription factor 2 (Runx2), suppress the expression levels of six genes involved in bone healing: BGLAP (bone gamma-carboxyglutamate protein), ALPL (alkaline phosphatase), SPP1 (secreted phosphoprotein 1), COL1A1 (collagen 1a1), IBSP (integrin binding sialoprotein), and MMP13 (matrix metallopeptidase 13). We also observe a similar phenomenon in osteoblast cells treated with proinflammatory cytokines or treated with a DNMT inhibitor (5-azacytidine). Forced expression of DNMT2/3a or blockage of CtBP1/2 with their inhibitors can reverse the expression levels of BGLAP/ALPL/SPP1/COL1A1/IBSP/MMP13 in the presence of proinflammatory cytokines. Administration of CtBP1/2 inhibitors in fractured mice can prevent the incidence of AN. Thus, we demonstrate that the downregulation of bone healing genes dependent on proinflammatory cytokines/DNMT2/3a/CtBP1/2-p300-Runx2 axis signaling plays a critical role in the pathogenesis of AN. Disruption of this signaling may represent a new therapeutic strategy to prevent AN incidence after bone fracture.

Epigenetic mechanisms and oral mucositis in children with acute lymphoblastic leukaemia.

This study aimed to investigate the relationship between epigenetic mechanisms and oral mucositis (OM) in paediatric patients with acute lymphoblastic leukaemia. Oral cells were collected from 76 participants, including 15 healthy individuals, 10 patients with acute lymphoblastic leukaemia but without a history of OM and 51 acute lymphoblastic leukaemia patients with a history of OM (35 with active OM and 16 who had recovered from OM). Global DNA methylation in the miR-9-1 and miR-9-3 genes was performed. Seven polymorphisms rs1801131, rs1801133 (MTHFR), rs2228611 (DNMT1), rs7590760, rs1550117 (DNMT3A), rs6087990, rs2424913 (DNMT3B) were genotyped and an analysis of association with global DNA methylation was performed. The global methylation levels were lower in cancer patients recovered from OM than in the other groups. A higher frequency of unmethylated profile for miR-9-1 and partially methylated profile for miR-9-3 was observed in cancer patients regardless of OM history compared to healthy patients. The GG genotype of the rs2228611 (DNMT1) polymorphism was associated with higher levels of global methylation in cancer patients irrespective of OM. It was concluded that global methylation is associated with mucosal recovery. The effect of DNMT1 genotype on the global DNA methylation profile, as well as the methylation profile of miR-9-1 and miR-9-3 in cancer patients is independent of OM.

Dnmt3a deficiency in the skin causes focal, canonical DNA hypomethylation and a cellular proliferation phenotype.

DNA hypomethylation is a feature of epidermal cells from aged and sun-exposed skin, but the mechanisms responsible for this methylation loss are not known. Dnmt3a is the dominant de novo DNA methyltransferase in the skin; while epidermal Dnmt3a deficiency creates a premalignant state in which keratinocytes are more easily transformed by topical mutagens, the conditions responsible for this increased susceptibility to transformation are not well understood. Using whole genome bisulfite sequencing, we identified a focal, canonical DNA hypomethylation phenotype in the epidermal cells of Dnmt3a-deficient mice. Single-cell transcriptomic analysis revealed an increased proportion of cells with a proliferative gene expression signature, while other populations in the skin were relatively unchanged. Although total DNMT3A deficiency has not been described in human disease states, rare patients with an overgrowth syndrome associated with behavioral abnormalities and an increased risk of cancer often have heterozygous, germline mutations in DNMT3A that reduce its function (Tatton-Brown Rahman syndrome [TBRS]). We evaluated the DNA methylation phenotype of the skin from a TBRS patient with a germline DNMT3AR882H mutation, which encodes a dominant-negative protein that reduces its methyltransferase function by ∼80%. We detected a focal, canonical hypomethylation phenotype that revealed considerable overlap with hypomethylated regions found in Dnmt3a-deficient mouse skin. Together, these data suggest that DNMT3A loss creates a premalignant epigenetic state associated with a hyperproliferative phenotype in the skin and further suggest that DNMT3A acts as a tumor suppressor in the skin.

Relationship between clone metrics and clinical outcome in clonal cytopenia.

Clonal cytopenia of undetermined significance (CCUS) is associated with an increased risk of developing a myeloid neoplasm with myelodysplasia (MN). To identify the features of the mutant clone(s) that is associated with clinical phenotype and progression, we studied the following cohorts of individuals: 311 patients with idiopathic cytopenia of undetermined significance (ICUS), 532 community-dwelling individuals without hematologic phenotype (n = 355) or with unexplained anemia (n = 177), and 592 patients with overt MN. Ninety-two of 311 (30%) patients with ICUS carried a somatic genetic lesion that signaled CCUS. Clonal hematopoiesis (CH) was detected in 19.7% and 27.7% of nonanemic and anemic community-dwelling individuals, respectively. Different mutation patterns and variant allele frequencies (VAFs) (clone metrics parameters) were observed in the conditions studied. Recurrent mutation patterns exhibited different VAFs associated with marrow dysplasia (0.17-0.48), indicating variable clinical expressivity of mutant clones. Unsupervised clustering analysis based on mutation profiles identified 2 major clusters, characterized by isolated DNMT3A mutations (CH-like cluster) or combinatorial mutation patterns (MN-like cluster), and showing different overall survival (HR, 1.8). In patients with CCUS, the 2 clusters had different risk of progression to MN (HR, 2.7). Within the MN-like cluster, distinct subsets with different risk of progression to MN were identified based on clone metrics. These findings unveil marked variability in the clinical expressivity of myeloid driver genes and underline the limitations of morphologic dysplasia for clinical staging of mutant hematopoietic clones. Clone metrics appears to be critical for informing clinical decision-making in patients with clonal cytopenia.

CHIP and hips: clonal hematopoiesis is common in patients undergoing hip arthroplasty and is associated with autoimmune disease.

Clonal hematopoiesis (CH) is an age-related condition predisposing to blood cancer and cardiovascular disease (CVD). Murine models demonstrate CH-mediated altered immune function and proinflammation. Low-grade inflammation has been implicated in the pathogenesis of osteoarthritis (OA), the main indication for total hip arthroplasty (THA). THA-derived hip bones serve as a major source of healthy hematopoietic cells in experimental hematology. We prospectively investigated frequency and clinical associations of CH in 200 patients without known hematologic disease who were undergoing THA. Prevalence of CH was 50%, including 77 patients with CH of indeterminate potential (CHIP, defined as somatic variant allele frequencies [VAFs] ≥2%), and 23 patients harboring CH with lower mutation burden (VAF, 1% to 2%). Most commonly mutated genes were DNMT3A (29.5%), TET2 (15.0%), and ASXL1 (3.5%). CHIP is significantly associated with lower hemoglobin, higher mean corpuscular volume, previous or present malignant disease, and CVD. Strikingly, we observed a previously unreported association of CHIP with autoimmune diseases (AIDs; multivariable adjusted odds ratio, 6.6; 95% confidence interval, 1.7-30; P = .0081). These findings underscore the association between CH and inflammatory diseases. Our results have considerable relevance for managing patients with OA and AIDs or mild anemia and question the use of hip bone-derived cells as healthy experimental controls.

Prognostic significance of concurrent gene mutations in intensively treated patients with IDH-mutated AML: an ALFA study.

In patients with isocitrate dehydrogenase (IDH)-mutated acute myeloid leukemia (AML) treated by intensive chemotherapy (IC), prognostic significance of co-occurring genetic alterations and allogeneic hematopoietic stem cell transplantation (HSCT) are of particular interest with the advent of IDH1/2 mutant inhibitors. We retrospectively analyzed 319 patients with newly diagnosed AML (127 with IDH1, 135 with IDH2R140, and 57 with IDH2R172 mutations) treated with IC in 3 Acute Leukemia French Association prospective trials. In each IDH subgroup, we analyzed the prognostic impact of clinical and genetic covariates, and the role of HSCT. In patients with IDH1 mutations, the presence of NPM1 mutations was the only variable predicting improved overall survival (OS) in multivariate analysis (P < .0001). In IDH2R140-mutated AML, normal karyotype (P = .008) and NPM1 mutations (P = .01) predicted better OS. NPM1 mutations were associated with better disease-free survival (DFS; P = .0009), whereas the presence of DNMT3A mutations was associated with shorter DFS (P = .0006). In IDH2R172-mutated AML, platelet count was the only variable retained in the multivariate model for OS (P = .002). Among nonfavorable European LeukemiaNet 2010-eligible patients, 71 (36%) underwent HSCT in first complete remission (CR1) and had longer OS (P = .03) and DFS (P = .02) than nontransplanted patients. Future clinical trials testing frontline IDH inhibitors combined with IC may consider stratification on NPM1 mutational status, the primary prognostic factor in IDH1- or IDH2R140-mutated AML. HSCT improve OS of nonfavorable IDH1/2-mutated AML and should be fully integrated into the treatment strategy.

DNMT3A low-expression is correlated to poor prognosis in childhood B-ALL and confers resistance to daunorubicin on leukemic cells.

BACKGROUND: Little is known about DNMT3A expression and its prognostic significance in childhood B cell acute lymphoblastic leukemia (B-ALL). METHODS: We determined DNMT3A mRNA expression in 102 children with B-ALL. Correlations with relapse-free survival (RFS) and common clinical characteristics were analyzed. DNMT3A was stably knocked out by CRISPR/Cas9 gene editing technology in Reh and 697 B-ALL cell lines. Cell proliferation activity after treated with daunorubicin (DNR) was determined by CCK8 assay in DNMT3A KO Reh and 697 cell lines. RESULTS: DNMT3A expression in B-ALL patients who were in continuous complete remission (CCR) was higher than in those who got relapse (P = 0.0111). Receiver operating characteristic curve showed prognostic significance of DNMT3A expression (P = 0.003). Low expression of DNMT3A (≤ 0.197) was significantly correlated with poor RFS (P < 0.001) in children with B-ALL. Knock-out of DNMT3A in Reh and 697 cell lines significantly increased IC50 of DNR (P = 0.0201 and 0.0022 respectively), indicating elevated resistance to DNR. CONCLUSION: Low expression of DNMT3A associates with poor prognosis in children with B-ALL. Knock-out of DNMT3A confers resistance to DNR on leukemic cells.

FAK Activation Promotes SMC Dedifferentiation via Increased DNA Methylation in Contractile Genes.

RATIONALE: Vascular smooth muscle cells (SMCs) exhibit remarkable plasticity and can undergo dedifferentiation upon pathological stimuli associated with disease and interventions. OBJECTIVE: Although epigenetic changes are critical in SMC phenotype switching, a fundamental regulator that governs the epigenetic machineries regulating the fate of SMC phenotype has not been elucidated. METHODS AND RESULTS: Using SMCs, mouse models, and human atherosclerosis specimens, we found that FAK (focal adhesion kinase) activation elicits SMC dedifferentiation by stabilizing DNMT3A (DNA methyltransferase 3A). FAK in SMCs is activated in the cytoplasm upon serum stimulation in vitro or vessel injury and active FAK prevents DNMT3A from nuclear FAK-mediated degradation. However, pharmacological or genetic FAK catalytic inhibition forced FAK nuclear localization, which reduced DNMT3A protein via enhanced ubiquitination and proteasomal degradation. Reduced DNMT3A protein led to DNA hypomethylation in contractile gene promoters, which increased SMC contractile protein expression. RNA-sequencing identified SMC contractile genes as a foremost upregulated group by FAK inhibition from injured femoral artery samples compared with vehicle group. DNMT3A knockdown in injured arteries reduced DNA methylation and enhanced contractile gene expression supports the notion that nuclear FAK-mediated DNMT3A degradation via E3 ligase TRAF6 (TNF [tumor necrosis factor] receptor-associated factor 6) drives differentiation of SMCs. Furthermore, we observed that SMCs of human atherosclerotic lesions exhibited decreased nuclear FAK, which was associated with increased DNMT3A levels and decreased contractile gene expression. CONCLUSIONS: This study reveals that nuclear FAK induced by FAK catalytic inhibition specifically suppresses DNMT3A expression in injured vessels resulting in maintaining SMC differentiation by promoting the contractile gene expression. Thus, FAK inhibitors may provide a new treatment option to block SMC phenotypic switching during vascular remodeling and atherosclerosis.

Clonal Hematopoiesis Is Associated With Higher Risk of Stroke.

BACKGROUND AND PURPOSE: Clonal hematopoiesis of indeterminate potential (CHIP) is a novel age-related risk factor for cardiovascular disease-related morbidity and mortality. The association of CHIP with risk of incident ischemic stroke was reported previously in an exploratory analysis including a small number of incident stroke cases without replication and lack of stroke subphenotyping. The purpose of this study was to discover whether CHIP is a risk factor for ischemic or hemorrhagic stroke. METHODS: We utilized plasma genome sequence data of blood DNA to identify CHIP in 78 752 individuals from 8 prospective cohorts and biobanks. We then assessed the association of CHIP and commonly mutated individual CHIP driver genes (DNMT3A, TET2, and ASXL1) with any stroke, ischemic stroke, and hemorrhagic stroke. RESULTS: CHIP was associated with an increased risk of total stroke (hazard ratio, 1.14 [95% CI, 1.03-1.27]; P=0.01) after adjustment for age, sex, and race. We observed associations with CHIP with risk of hemorrhagic stroke (hazard ratio, 1.24 [95% CI, 1.01-1.51]; P=0.04) and with small vessel ischemic stroke subtypes. In gene-specific association results, TET2 showed the strongest association with total stroke and ischemic stroke, whereas DMNT3A and TET2 were each associated with increased risk of hemorrhagic stroke. CONCLUSIONS: CHIP is associated with an increased risk of stroke, particularly with hemorrhagic and small vessel ischemic stroke. Future studies clarifying the relationship between CHIP and subtypes of stroke are needed.

Helicobacter pylori infection and DNMT3a polymorphism are associated with the presence of premature coronary artery disease and subclinical atherosclerosis. Data from the GEA Mexican Study.

BACKGROUND: The association between H. pylori infection and coronary artery disease (CAD) is well-known. Alterations in DNA methylation in CAD have been reported, which can be induced by H. pylori through the DNA demethylases (DNMTs). The objective was to analyze the association and interaction of H. pylori infection and DMNT3a gene polymorphisms with premature CAD (pCAD) and subclinical atherosclerosis (SA). METHODS: The study included 561 patients with pCAD, 318 subjects with SA, and 599 healthy controls. Antibodies against H. pylori and DNMT3a rs13420827, rs752208, and rs1550117 polymorphisms were determined. RESULTS: The pCAD group presented the highest seroprevalence of H. pylori infection (87.7%) compared to the SA (74.5%, p = 1 × 10-6) and the control group (63.1%, p = 7 × 10-23). A significant association was observed between H. pylori infection and pCAD (OR = 2.729, p = 1.0 × 10-6). The rs13420827 polymorphism was associated with a high risk of H. pylori infection in the whole population (padditive = 0.009, pdominant = 0.018, and pcodominant2 = 0.013) and in individuals with SA (padditive = 0.003, pdominant = 0.020, precessive = 0.013, and pcodominant2 = 0.005). The coexistence of H. pylori infection and the rs13420827GG genotype increases the risk of pCAD (pinteraction = 1.1 × 10-5). CONCLUSIONS: According to the model adjusted for more confounding variables, H. pylori infection was associated with almost three times the risk of developing pCAD. The rs13420827G allele was associated with an increased risk of H. pylori infection in the whole population and in individuals with SA. Individuals in whom H. pylori infection and the rs13420827GG genotype coexist are at increased risk of pCAD.

Parallel evolution of two distinct lymphoid proliferations in clonal haematopoiesis.

AIMS: Angioimmunoblastic T-cell lymphoma (AITL) is genetically characterized by TET2 and DNMT3A mutations occurring in haematopoietic progenitor cells, and late events (e.g. the RHOA-G17V mutation) associated with malignant transformation. As TET2/DNMT3A-mutated progenitor cells can differentiate into multilineage progenies and give rise to both AITL and myeloid neoplasms, they may also have the potential to lead to other metachronous/synchronous neoplasms. We report two cases showing parallel evolution of two distinct potentially neoplastic lymphoid proliferations from a common mutated haematopoietic progenitor cell population. METHODS AND RESULTS: Both cases presented with generalized lymphadenopathy. In case 1 (a 67-year-old female), an initial lymph node (LN) biopsy was dismissed as reactive, but a repeat biopsy showed a nodal marginal zone lymphoma (NMZL)-like proliferation with an increase in the number of T-follicular helper (TFH) cells. Immunohistochemistry, and clonality and mutational analyses by targeted sequencing of both whole tissue sections and microdissected NMZL-like lesions, demonstrated a clonal B-cell proliferation that harboured the BRAF-G469R mutation and shared TET2 and DNMT3A mutations with an underlying RHOA-G17V-mutant TFH proliferation. Review of the original LN biopsy showed histological and immunophenotypic features of AITL. In case 2 (a 66-year-old male), cytotoxic T-cell lymphoma with an increase in the number of Epstein-Barr virus-positive large B cells was diagnosed on initial biopsy. On review together with the relapsed biopsy, we identified an additional occult neoplastic TFH proliferation/smouldering AITL. Both T-cell proliferations shared TET2 and DNMT3A mutations while RHOA-G17V was confined to the smouldering AITL. CONCLUSIONS: In addition to demonstrating diagnostic challenges, these cases expand the potential of clonal haematopoiesis in the development of different lineage neoplastic proliferations.

Intracellular matrix metalloproteinase-9 mediates epigenetic modifications and autophagy to regulate differentiation in human cardiac stem cells.

Epigenetic reprogramming and autophagy have critical roles in differentiation of stem cells. However, very little is known about how epigenetic modifications are mediated and how they contribute to autophagy and differentiation in human cardiac stem cells (hCSCs). Previously, we have reported that intracellular matrix metalloproteinase-9 (MMP9), a collagenase, mediates cell death in hCSCs. Here, we investigated whether intracellular MMP9 mediates epigenetic modifications and autophagy in hCSCs. We created MMP9KO hCSCs and treated them with 5-azacytidine, an inhibitor of DNA methylation, and bafilomycin A1, an inhibitor of autophagosome degradation, and evaluated epigenetic modifications, autophagic flux, and differentiation. Our results showed compromised epigenetic modifications, reduced autophagy, and impaired differentiation in MMP9KO hCSCs. Remarkably, paracrine MMP9 supplementation restored epigenetic modifications but further reduced autophagy in MMP9KO hCSCs. We conclude that intracellular MMP9 is a critical mediator of epigenetic modifications and autophagy in hCSCs. Furthermore, the endocrine and paracrine effects of MMP9 vary for regulating autophagy in hCSCs. These novel roles of MMP9 are valuable for stem cell therapy.

Reduction of DNMT3a and RORA in the nucleus accumbens plays a causal role in post-traumatic stress disorder-like behavior: reversal by combinatorial epigenetic therapy.

Post-traumatic stress disorder (PTSD) is an incapacitating trauma-related disorder, with no reliable therapy. Although PTSD has been associated with epigenetic alterations in peripheral white blood cells, it is unknown where such changes occur in the brain, and whether they play a causal role in PTSD. Using an animal PTSD model, we show distinct DNA methylation profiles of PTSD susceptibility in the nucleus accumbens (NAc). Data analysis revealed overall hypomethylation of different genomic CG sites in susceptible animals. This was correlated with the reduction in expression levels of the DNA methyltransferase, DNMT3a. Since epigenetic changes in diseases involve different gene pathways, rather than single candidate genes, we next searched for pathways that may be involved in PTSD. Analysis of differentially methylated sites identified enrichment in the RAR activation and LXR/RXR activation pathways that regulate Retinoic Acid Receptor (RAR) Related Orphan Receptor A (RORA) activation. Intra-NAc injection of a lentiviral vector expressing either RORA or DNMT3a reversed PTSD-like behaviors while knockdown of RORA and DNMT3a increased PTSD-like behaviors. To translate our results into a potential pharmacological therapeutic strategy, we tested the effect of systemic treatment with the global methyl donor S-adenosyl methionine (SAM), for supplementing DNA methylation, or retinoic acid, for activating RORA downstream pathways. We found that combined treatment with the methyl donor SAM and retinoic acid reversed PTSD-like behaviors. Thus, our data point to a novel approach to the treatment of PTSD, which is potentially translatable to humans.

Identification of three novel DNMT3A mutations with compromising methylation capacity in human acute myeloid leukaemia.

BACKGROUND: Acute myeloid leukaemia (AML) is a complex and heterogeneous hematopoietic stem and progenitor cell malignancy characterised by the accumulation of immature blast cells in the bone marrow, blood, and other organs linked to environmental, genetic, and epigenetic factors. Somatic mutations in the gene DNA (cytosine-5)-methyltransferase 3A (DNMT3A; NM_022552.4) are common in AML patients. METHODS: In this study, we used Sanger sequencing to detect the mutations in the DNMT3A gene in 61 Iraqi AML patients, Hence, the protein function and stability within alterations were identified and analyzed using a variety of computational tools with the goal of determining how these changes affect total protein stability, and then the capacity of methylation was analyzed by methylation specific PCR MSP status at CpG islands. RESULTS: Three novel mutations in exon 23 of DNMT3A were identified in 14 patients (22.9%; V877I, N879delA, and L888Q). The V877I and L888Q substitutions are caused by heterozygous C2629G > A and C2663T > A mutations, respectively, while frameshift mutation C2635delA caused protein truncation with stop codon N879T*. Methylation was detected in the DNMT3A promoter region in 9 patients carrying DNMT3A mutations (64.28%) by MSP, and we found significant correlations between DNMT3A mutations and promoter methylation (p = 8.52 × 105). In addition, we found a significant overrepresentation of DNMT3A methylation status in patients ≥ 50 years old (p = 0.025). CONCLUSION: Our findings highlight the importance of studying the effects of DNMT3A methylation alteration in Iraqi populations beyond R882 substitutions in the leukemogenic pathway so that patient treatment can be tailored to prevent therapeutic resistance and relapse.

Clinical and genomic characterization of patients diagnosed with the provisional entity acute myeloid leukemia with BCR-ABL1, a Swedish population-based study.

Acute myeloid leukemia (AML) with t(9;22)(q34;q11), also known as AML with BCR-ABL1, is a rare, provisional entity in the WHO 2016 classification and is considered a high-risk disease according to the European LeukemiaNet 2017 risk stratification. We here present a retrospective, population-based study of this disease entity from the Swedish Acute Leukemia Registry. By strict clinical inclusion criteria we aimed to identify genetic markers further distinguishing AML with t(9;22) as a separate entity. Twenty-five patients were identified and next-generation sequencing using a 54-gene panel was performed in 21 cases. Interestingly, no mutations were found in NPM1, FLT3, or DNMT3A, three frequently mutated genes in AML. Instead, RUNX1 was the most commonly mutated gene, with aberrations present in 38% of the cases compared to around 10% in de novo AML. Additional mutations were identified in genes involved in RNA splicing (SRSF2, SF3B1) and chromatin regulation (ASXL1, STAG2, BCOR, BCORL1). Less frequently, mutations were found in IDH2, NRAS, TET2, and TP53. The mutational landscape exhibited a similar pattern as recently described in patients with chronic myeloid leukemia (CML) in myeloid blast crisis (BC). Despite the concomitant presence of BCR-ABL1 and RUNX1 mutations in our cohort, both features of high-risk AML, the RUNX1-mutated cases showed a superior overall survival compared to RUNX1 wildtype cases. Our results suggest that the molecular characteristics of AML with t(9;22)/BCR-ABL1 and CML in myeloid BC are similar and do not support a distinction of the two disease entities based on their underlying molecular alterations.

Clonal hematopoiesis of indeterminate potential (CHIP): Linking somatic mutations, hematopoiesis, chronic inflammation and cardiovascular disease.

Clonal hematopoiesis of indeterminate potential (CHIP) is the presence of a clonally expanded hematopoietic stem cell caused by a leukemogenic mutation in individuals without evidence of hematologic malignancy, dysplasia, or cytopenia. CHIP is associated with a 0.5-1.0% risk per year of leukemia. Remarkably, it confers a two-fold increase in cardiovascular risk independent of traditional risk factors. Roughly 80% of patients with CHIP have mutations in epigenetic regulators DNMT3A, TET2, ASXL1, DNA damage repair genes PPM1D, TP53, the regulatory tyrosine kinase JAK2, or mRNA spliceosome components SF3B1, and SRSF2. CHIP is associated with a pro-inflammatory state that has been linked to coronary artery disease, myocardial infarction, and venous thromboembolic disease, as well as prognosis among those with aortic stenosis and heart failure. Heritable and acquired risk factors are associated with increased CHIP prevalence, including germline variation, age, unhealthy lifestyle behaviors (i.e. smoking, obesity), inflammatory conditions, premature menopause, HIV and exposure to cancer therapies. This review aims to summarize emerging research on CHIP, the mechanisms underlying its important role in propagating inflammation and accelerating cardiovascular disease, and new studies detailing the role of associated risk factors and co-morbidities that increase CHIP prevalence.