DNMT3A Mutation Leads to Hematopoietic Dysregulation.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2382-2382
Author(s):  
Jie Xu ◽  
Wei-na Zhang ◽  
Tao Zhen ◽  
Yang Li ◽  
Jing-yi Shi ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Epigenetic Modification ◽  
Hematopoietic Cells ◽  
In Vitro Assays ◽  
Clinical Samples ◽  
Hematopoietic Stem

Abstract Abstract 2382 Epigenetic modification process is required for the development of hematopoietic cells. DNA methyltransferase DNMT3A, responsible for de novo DNA methylation, was newly reported to have a high frequency of mutations in hematopoietic malignancies. Conditional knock-out of DNMT3A promoted self-renewal activity of murine hematopoietic stem cells (HSCs). However, the role of mutated DNMT3A in hematopoiesis and its regulative mechanism of epigenetic network mostly remain unknown. Here we showed that the Arg882His (R882H) hotspot locus on DNMT3A impaired the normal function of this enzyme and resulted in an abnormal increase of primitive hematopoietic cells. In both controlled in vivo and in vitro assays, we found that the cells transfected by R882H mutant promoted cell proliferation, while decreased the differentiation of myeloid lineage compared to those with wild type. Analysis of bone marrow (BM) cells from mice transduced by R882H reveals an expansion of Lin−Sca-1+C-kit+ populations and a reduction of mature myeloid cells. Meanwhile, a cluster of upregulated genes and downregulated lineage-specific differentiation genes associated with hematopoiesis were discovered in mice BM cells with R882H mutation. We further evaluated the association of mutated DNMT3A and HOXB4 which was previously detected to be highly expressed in clinical samples carrying R882 mutation. Compared with wildtype DNMT3A, R882H mutation disrupted the repression of HOXB4 by largely recruiting tri-methylated histone 3 lysine 4 (H3K4). Taken together, our results showed that R882H mutation disturbed HSC activity through H3K4 tri-methylation, and transcriptional activation of HSC-related genes. Disclosures: No relevant conflicts of interest to declare.

Recombinant mammalian DNA methyltransferase activity on model transcriptional gene silencing short RNA–DNA heteroduplex substrates

2010 ◽  
Vol 432 (2) ◽  
pp. 323-332 ◽  
Author(s):  
Jason P. Ross ◽  
Isao Suetake ◽  
Shoji Tajima ◽  
Peter L. Molloy
Keyword(s):  
Gene Silencing ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Double Helix ◽  
In Vitro Assays ◽  
Enzyme Analysis ◽  
Transcriptional Gene Silencing ◽  
Restriction Enzyme Analysis

The biochemical mechanism of short RNA-induced TGS (transcriptional gene silencing) in mammals is unknown. Two competing models exist; one suggesting that the short RNA interacts with a nascent transcribed RNA strand (RNA–RNA model) and the other implying that short RNA forms a heteroduplex with DNA from the unwound double helix, an R-loop structure (RNA–DNA model). Likewise, the requirement for DNA methylation to enact TGS is still controversial. In vitro assays using purified recombinant murine Dnmt (DNA methyltransferase) 1-dN (where dN indicates an N-terminal truncation), 3a and 3b enzymes and annealed oligonucleotides were designed to question whether Dnmts methylate DNA in a RNA–DNA heteroduplex context and whether a RNA–DNA heteroduplex R-loop is a good substrate for Dnmts. Specifically, model synthetic oligonucleotides were used to examine methylation of single-stranded oligonucleotides, annealed oligonucleotide duplexes, RNA–DNA heteroduplexes, DNA bubbles and R-loops. Dnmt methylation activity on the model substrates was quantified with initial velocity assays, novel ARORA (annealed RNA and DNA oligonucleotide-based methylation-sensitive restriction enzyme analysis), tBS (tagged-bisulfite sequencing) and the quantitative PCR-based method MethylQuant. We found that RNA–DNA heteroduplexes and R-loops are poor substrates for methylation by both the maintenance (Dnmt1) and de novo (Dnmt3a and Dnmt3b) Dnmts. These results suggest the proposed RNA/DNA model of TGS in mammals is unlikely. Analysis of tagged-bisulfite genomic sequencing led to the unexpected observation that Dnmt1-dN can methylate cytosines in a non-CpG context in DNA bubbles. This may have relevance in DNA replication and silencing of transcriptionally active loci in vivo.

scholarly journals Variants ofDNMT3Acause transcript-specific DNA methylation patterns and affect hematopoiesis

2018 ◽  
Vol 1 (6) ◽  
pp. e201800153 ◽  
Author(s):  
Tanja Božić ◽  
Joana Frobel ◽  
Annamarija Raic ◽  
Fabio Ticconi ◽  
Chao-Chung Kuo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem ◽  
Acute Myeloid

De novo DNA methyltransferase 3A (DNMT3A) plays pivotal roles in hematopoietic differentiation. In this study, we followed the hypothesis that alternative splicing ofDNMT3Ahas characteristic epigenetic and functional sequels. SpecificDNMT3Atranscripts were either down-regulated or overexpressed in human hematopoietic stem and progenitor cells, and this resulted in complementary and transcript-specific DNA methylation and gene expression changes. Functional analysis indicated that, particularly, transcript 2 (coding for DNMT3A2) activates proliferation and induces loss of a primitive immunophenotype, whereas transcript 4 interferes with colony formation of the erythroid lineage. Notably, in acute myeloid leukemia expression of transcript 2 correlates with its in vitro DNA methylation and gene expression signatures and is associated with overall survival, indicating thatDNMT3Avariants also affect malignancies. Our results demonstrate that specificDNMT3Avariants have a distinct epigenetic and functional impact. Particularly, DNMT3A2 triggers hematopoietic differentiation and the corresponding signatures are reflected in acute myeloid leukemia.

scholarly journals Two-dimensional electrophoresis protein profiles of HL-60 and CCRF-CEM cell lines treated with epigenetic modification drugs

2019 ◽  
pp. 10-23
Author(s):  
Aziee Sudin ◽  
Haiyuni Mohd Yassim ◽  
Shafini Mohamed Yusoff ◽  
Shaharum Shamsuddin ◽  
Ridhwan Abdul Wahab ◽  
...  
Keyword(s):  
Cell Lines ◽  
Dna Methyltransferase ◽  
Epigenetic Modification ◽  
Trichostatin A ◽  
Lymphoblastic Leukemia ◽  
Genetic Alterations ◽  
Epigenetic Modifications ◽  
Hematopoietic Stem ◽  
2D Gel

Leukemia is classified as a malignant disease of hematopoietic stem cells (HSCs) that fails in cell differentiation but preserve their self-renewal. It is caused by genetic alterations and epigenetic modifications resulting in the activation or inactivation of particular genes for transcription. Epigenetic causes changes in gene expression without any alteration in the DNA sequence. The most common epigenetic modifications are DNA methylation and histone acetylation. 5-Azacitidine (5-Aza) is a DNA methytransferase inhibitor (DNMTi) that inhibits DNA methyltransferase enzymes resulting in hypomethylation. Trichostatin A (TSA) is a histone deacetylase inhibitor which inhibits deacetylation of both histone and non-histone proteins resulting in chromatin relaxation. This present study focused on the alteration of proteome profile on 2D gel electrophoresis (2-DE) induced by 5-Aza and TSA in HL-60 and CCRF-CEM cell lines as in vitro model to represent acute promyelocytic leukemia (APL) and T-lymphoblastic leukemia (T-ALL), respectively. Total proteins of untreated and 5-Aza/TSA-treated HL-60 and CCRF-CEM cell lines were extracted using urea/thiourea buffer and stained with Coomassie Blue. Comparative analysis of untreated and 5-Aza/TSA-treated HL-60 and CCRF-CEM was performed by PDQuest software. Qualitative analysis identified 190-659 protein spots detected in untreated, 5-Aza and TSA-treated HL-60 and CCRF-CEM. Quantitative comparison analysis was analyzed by over 2-fold change in 5-Aza/TSA-treated cells compared to untreated. One and eight upregulated proteins were detected in 5-Aza and TSA-treated HL-60, respectively. While five and one upregulated proteins were detected in 5-Aza and TSA-treated CCRF-CEM, respectively. These preliminary results suggested that 5-Aza and TSA induced proteome profiles alterations due to their inhibition effects in HL-60 and CCRF-CEM cell lines.

Chromosomal Instability in Cultured Mouse Hematopoietic Cells Associated with Oxidative Stress

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4819-4819
Author(s):  
Alice M. Liu ◽  
William W. Qu ◽  
Xia Liu
Keyword(s):  
Oxidative Stress ◽  
Bone Marrow ◽  
Genomic Instability ◽  
Chromosomal Instability ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Genetic Alterations ◽  
In Vitro Test ◽  
Hematopoietic Stem

Abstract Abstract 4819 Hematopoietic stem cells (HSCs) that give rise to all blood cell types are important vehicles for cell-based and gene therapies. After isolation from the bone marrow, HSCs are often cultured in laboratory settings for purposes of ex vivo expansion, gene transduction, and bone marrow transplantation for the treatment of various disorders of the blood and immune systems. While undergoing proliferation and differentiation in vitro, test tube and dish culturing can potentially induce genomic instability in HSCs due to prolonged culturing periods or the exposure to increased levels of oxygen. Here we demonstrate that in vitro culturing outside their bone marrow niches, HSCs may change even under very short durations of time. Lineage− Scal-1+ c-Kit+ (LSK) cells that are enriched with HSCs revealed significant levels of genomic instability in culture, as evidenced by the emergence of aneuploidy cells. To further determine the effects of in vitro culturing conditions, whole bone marrow cells were cultured in a hypoxic environment of 2–3% oxygen, mimicking conditions inside the body's bone marrow. In this case, cells proved to undergo less genetic alterations. Proper dosages of the antioxidant N-Acetyl-Cysteine (NAC) similarly decreased occurrences of chromosomal changes. Furthermore, in vitro normoxic culture-induced chromosomal instability was enhanced in aged hematopoietic cells compared to that in young hematopoietic cells due to noted increased oxidative stress in aged cells. These results reveal that in vitro cell culturing does indeed cause genomic instability in hematopoietic cells. Reduced oxygen levels and additions of antioxidants can be employed as a possible agent to lower oxidative stress and decrease chances of transformation. Additionally, since hematopoietic cells are commonly developed in laboratory settings before transplantation for patient treatment, our findings raise a concern for using cultured hematopoietic cells for therapeutic purposes. Note: Alice Liu and William Qu contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

Modeling MLL-PTD Related Myelodysplastic Syndromes in Mouse.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2811-2811
Author(s):  
Xiaomei Yan ◽  
Yue Zhang ◽  
Goro Sashida ◽  
Aili Chen ◽  
Xinghui Zhao ◽  
...  
Keyword(s):  
De Novo ◽  
Conflicts Of Interest ◽  
Gene Dosage ◽  
Fetal Liver ◽  
Loss Of Function ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
De Novo Aml

Abstract Abstract 2811 MLL partial tandem duplication (MLL-PTD) is found in 5–8% of human MDS, secondary acute myeloid leukemia (s-AML) and de novo AML. The molecular and clinical features of MLL-PTD+ AML are different from MLL-fusion+ AML, although they share similar worse outcomes. Mouse knock-in model of Mll-PTD has been generated to understand its underlining mechanism (Dorrance et al. JCI. 2006). Using this model, we've recently reported hematopoietic stem/progenitor cell (HSPC) phenotypes of MllPTD/WT mice. Their HSPCs showed increased apoptosis and reduced cell number, but they have a proliferative advantage over wild-type HSPCs. Furthermore, the MllPTD/WT–derived phenotypic ST-HSCs/MPPs and even GMPs have self-renewal capabilities. However, MllPTD/WT HSPCs never develop MDS or s-AML in primary or transplanted recipient mice, suggesting that additional genetic and/or epigenetic defects are necessary for transformation (Zhang et al. Blood. 2012). Recently, high frequent co-existences of both MLL-PTD and RUNX1 mutations have been reported in several MDS, s-AML and de novo AML clinical cohorts, which strongly suggest a potential cooperation for transformation between these two mutations. Our previous study has shown that MLL interacts with and stabilizes RUNX1 (Huang et al. Blood. 2011). Thus, we hypothesize that reducing RUNX1 dosage may facilitate the MLL-PTD mediated transformation toward MDS and/or s-AML. We first generated the mice containing one allele of Mll-PTD in a Runx1+/− background and assessed HSPCs of MllPTD/wt/Runx1+/− double heterozygous (DH) mice. The DH newborns are runty; they frequently die in early postnatal stage and barely survive to adulthood, compared to the normal life span of wild type (WT) or single heterozygous (Mllwt/wt/Runx1+/− and MllPTD/wt/Runx1+/+) mice. We studied DH embryos fetal liver hematopoiesis and found reduced LSK and LSK/SLAM+ cells, partly because of increased apoptosis. Enhanced proliferation was found in DH fetal liver cells (FLCs) in vitro CFU replating assays over WT and MllPTD/wt/Runx1+/+ controls. DH FLCs also showed dominant expansion in both serial competitive and serial non-competitive BMT assays compared to WT controls. The DH derived phenotypic ST-HSCs/MPPs and GMPs also have enhanced self-renewal capabilities, rescuing hematopoiesis by giving rise to long-term repopulating cells in recipient mice better than cells derived from MllPTD/wt/Runx1+/+ mice. However, DH HSPCs didn't develop MDS or s-AML in primary or in serial BMT recipient mice. We further generated MllPTD/wt/Runx1Δ/Δ mice using Mx1-Cre mediated deletion. These mice showed thrombocytopenia 1 month after pI-pC injection, and developed pancytopenia 2–4 months later. All these MllPTD/wt/Runx1Δ/Δ mice died of MDS induced complications within 7–8 months, and tri-lineages dysplasias (TLD) were found in bone marrow aspirate. However, there are no spontaneous s-AML found in MllPTD/wt/Runx1Δ/Δ mice, which suggests that RUNX1 mutants found in MLL-PTD+ patients may not be simply loss-of-function mutations and present gain-of-function activities which cooperate with MLL-PTD in human diseases onsets. In conclusion, our study demonstrates that: 1) RUNX1 gene dosage reverse-correlates with HSPCs self-renewal activity; 2) Runx1 complete deletion causes MDS in Mll-PTD background. Future studies are needed to fully understand the collaboration between MLL-PTD and RUNX1 mutations for MDS development and leukemic transformation, which should facilitate improved therapies and patient outcomes. Disclosures: No relevant conflicts of interest to declare.

Developing a Model of Human Pluripotent to Hematopoietic Stem Cell Development in Mistrg Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4755-4755
Author(s):  
John Astle ◽  
Yangfei Xiang ◽  
Anthony Rongvaux ◽  
Carla Weibel ◽  
Henchey Elizabeth ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Biology ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice

Abstract De novo generation of HSCs has been described as a "holy grail" of stem cell biology, however the factors required for converting human pluripotent stem cells (PSCs) to true hematopoietic stem cells (HSCs) capable of robust long-term engraftment have yet to be fully characterized. Two groups have shown that injection of PSCs into immunodeficient mice leads to teratomas containing niches producing hematopoietic progenitors capable of long-term engraftment. Once these hematopoietic progenitors and their microenvironments are better characterized, this system could be used as a model to help direct in vitro differentiation of PSCs to HSCs. Toward this end, we have injected human PSCs into immunodeficient mice expressing human rather than mouse M-CSF, IL-3, GM-CSF, and thrombopoietin, as well as both human and mouse versions of the "don't eat me signal" Sirpa (collectively termed MISTRG mice). These cytokines are known to support different aspects of hematopoiesis, and thrombopoietin in particular has been shown to support HSC maintenance, suggesting these mice may provide a better environment for human PSC-derived HSCs than the more traditional mice used for human HSC engraftment. The majority of teratomas developed so far in MISTRG contain human hematopoietic cells, and the CD34+ population isolated from over half of the teratomas contained hematopoietic colony forming cells by colony forming assay. These findings further corroborate this approach as a viable method for studying human PSC to HSC differentiation. Disclosures No relevant conflicts of interest to declare.

scholarly journals 41BB-based and CD28-based CD123-redirected T-cells ablate human normal hematopoiesis in vivo

2020 ◽  
Vol 8 (1) ◽  
pp. e000845 ◽  
Author(s):  
Matteo Libero Baroni ◽  
Diego Sanchez Martinez ◽  
Francisco Gutierrez Aguera ◽  
Heleia Roca Ho ◽  
Maria Castella ◽  
...  
Keyword(s):  
De Novo ◽  
In Vitro Assays ◽  
Target Antigen ◽  
Hematopoietic Stem ◽  
Hematopoietic Malignancy ◽  
Hematopoietic Reconstitution ◽  
Bona Fide ◽  
Normal Human

BackgroundAcute myeloid leukemia (AML) is a hematopoietic malignancy which is biologically, phenotypically and genetically very heterogeneous. Outcome of patients with AML remains dismal, highlighting the need for improved, less toxic therapies. Chimeric antigen receptor T-cell (CART) immunotherapies for patients with refractory or relapse (R/R) AML are challenging because of the absence of a universal pan-AML target antigen and the shared expression of target antigens with normal hematopoietic stem/progenitor cells (HSPCs), which may lead to life-threating on-target/off-tumor cytotoxicity. CD33-redirected and CD123-redirected CARTs for AML are in advanced preclinical and clinical development, and they exhibit robust antileukemic activity. However, preclinical and clinical controversy exists on whether such CARTs are myeloablative.MethodsWe set out to comparatively characterize in vitro and in vivo the efficacy and safety of 41BB-based and CD28-based CARCD123. We analyzed 97 diagnostic and relapse AML primary samples to investigate whether CD123 is a suitable immunotherapeutic target, and we used several xenograft models and in vitro assays to assess the myeloablative potential of our second-generation CD123 CARTs.ResultsHere, we show that CD123 represents a bona fide target for AML and show that both 41BB-based and CD28-based CD123 CARTs are very efficient in eliminating both AML cell lines and primary cells in vitro and in vivo. However, both 41BB-based and CD28-based CD123 CARTs ablate normal human hematopoiesis and prevent the establishment of de novo hematopoietic reconstitution by targeting both immature and myeloid HSPCs.ConclusionsThis study calls for caution when clinically implementing CD123 CARTs, encouraging its preferential use as a bridge to allo-HSCT in patients with R/R AML.

scholarly journals Aorta-associated CD34+ hematopoietic cells in the early human embryo

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 67-72 ◽  
Author(s):  
M Tavian ◽  
L Coulombel ◽  
D Luton ◽  
HS Clemente ◽  
F Dieterlen-Lievre ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Embryo ◽  
Hematopoietic Cells ◽  
Yolk Sac ◽  
Blood System ◽  
In Vitro Assays ◽  
Avian Embryo ◽  
Hematopoietic Stem

Abstract Hematopoiesis is established from circulating blood stem cells that seed the embryonic rudiments of blood-forming tissues, a basic notion in developmental hematology. However, the assumption that these stem cells originate from the extraembryonic mesoderm, where primitive hematopoiesis is initiated by intrinsic precursors, has been reconsidered after analysis of blood cell development in avian embryo chimeras: yolk-sac-derived stem cells do not contribute significantly to the definitive blood system, whose first forerunners develop independently along the ventral aspect of the embryonic aorta. Recently, the homologous intraembryonic tissues of the mouse have been submitted to sensitive in vivo and in vitro assays, which showed that they also harbor multipotential hematopoietic stem cells. We have now identified a dense population of hematogenous cells, marked by the surface expression of the CD34 glycoprotein, associated with the ventral endothelium of the aorta in the 5-week human embryo. Therefore, we extend to the human species the growing evidence that intraembryonic hematopoietic cells developing independently of the yolk sac might be the real stem of the whole blood system.

scholarly journals NPM1 Mutation Contributes to Hematological Dysfunction By Disrupting H3K79 Methylation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2702-2702
Author(s):  
Jie Xu ◽  
Wu Zhang ◽  
Xiaojing Yan ◽  
Chen Zhao ◽  
Jiang Zhu ◽  
...  
Keyword(s):  
Target Genes ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Gene Profiling ◽  
In Vitro Assays ◽  
Hoxa Cluster ◽  
Hematopoietic Stem ◽  
Npm1 Mutation ◽  
H3k79 Methylation

Abstract NPM1 is one of the most frequent acquired mutated genes in acute myeloid leukemia (AML). Previous studies have shown that NPM1 mutation (NPMc+) established the distinctive gene expression signatures, which were associated with mixed lineage leukemia (MLL)-target genes, like MEIS1 and HOXA cluster. In AML carrying MLL fusion-oncoproteins, DOT1L-mediated histone 3 lysine 79 (H3K79) methylation is implicated in the regulation of MLL-target genes. Compared with MLL abnormalities, NPM1 variants preserve the similar transcriptional characteristics. However, whether NPM1 mutation could affect the histone modification of H3K79 methylation is unknown. In this study, we showed that NPM1 mutation dysregulated the homeostasis of hematopoietic stem and progenitor cells and resulted in ageing-related myeloproliferation in NPMc+ transgenic mouse model. Interestingly, through scanning the chromatin modification related gene profiling, di- and tri- methylated H3K79 were significantly elevated in bone marrow (BM) Lin-Sca-1+c-Kit+ cells (LSKs) of NPMc+ mice comparing to wild type (WT). Meanwhile, in the leukemia cell lines and AML primary BM samples, we confirmed that NPM1 mutated cells expressed the higher level of H3K79 methylation. In vitro assays also indicated that the decrease or increase of methylated H3K79 could be regulated respectively by knockdown or overexpression of NPM1 mutant but not WT. Importantly, with DOT1L inhibitor treatment, reduced di- and tri- methylated H3K79 was observed in OCI-AML3 (NPMc+) strains but not OCI-AML2 (NPM1 WT) cells. In contrast with OCI-AML2, DOT1L inhibitor significantly promoted the cell apoptosis and restrained the cell cycle of OCI-AML3. Moreover, by the means of murine BM colony formation assay, DOT1L inhibitor obviously weakened myeloid cell proliferation in NPMc+ mice, while colony number in WT group did not change. Also, leukemia development was repressed in OCI-AML3-xenografted NOD/SCID mice with the treatment of DOT1L inhibitor. Taken together, NPM1 mutation contributes to hematological dysfunction by disrupting H3K79 methylation, which could be largely attenuated by DOT1L inhibitor. Disclosures No relevant conflicts of interest to declare.

scholarly journals Activation of JAK2/STAT5 Pathway Reduces Expression Level of DNMT3a in MPN Cell Line

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5394-5394
Author(s):  
Jie Zhou ◽  
Aibin Liang ◽  
Shaoguang Li ◽  
Wenjun Zhang ◽  
Jianfei FU
Keyword(s):  
Protein Expression ◽  
Cell Line ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Expression Level ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Increased Risk

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by overproduction of mature blood cells and increased risk of transformation to acute myeloid leukemia (AML), and JAK2V167F is the most frequent MPN driving mutation detected in >95% of PV and 50-60% ET and PMF. DNMT3A is a de novo DNA methyltransferase that catalyzes the addition of methyl groups into active chromatin in CpG-rich regions leading to gene inactivation. Dnmt3a-/- HSC have enhanced self-renewal and a block in differentiation in vivo. Previous study showed that JAK2V617F and Dnmt3a loss cooperate to induce myelofibrosis through activated enhancer-driven inflammation, while whether JAK2V617F regulates DNMT3a still remains unclear. AZ960 is a potent and selective ATP competitive inhibitor of the JAK2 kinase, and previous studies reported that AZ960 possessed the activity selectively against JAK2. LY2784544 has been identified as a selective inhibitor of JAK2V617F and has undergone clinical trials for the treatment of several myeloproliferative disorders. Methods: Empty vector (control) and mutant JAK2V617F were transduced into BaF3 cells using a lentivirus system. JAK2V617F-expressing BaF3 cells grow IL-3 independent and were selected by fluorescence-activated cell sorting (FACS) for GFP expression. The protein expression levels of p-STAT5 and DNMT3a were detected by western blotting. JAK2V617F-expressing and control BaF3 cells were incubated with gradient concentration of LY2784544 or AZ960 to inhibit JAK2/STAT5 pathway. Results: The expression levels of p-STAT5 were obviously up-regulated in the JAK2V617F-expressing BaF3 cells, and DNMT3a was down-regulated. After 1-hour incubation in the serial diluted LY2784544, p-STAT5 were reduced in JAK2V617F-expressing BaF3 cells, with expression of DNMT3a elevated. To further confirm the correlation between JAK2/STAT5 pathway and expression of DNMT3a, another JAK2 inhibitor AZ960 was tested similar to LY2784544. With p-STAT5 expression suppressed, protein level of DNMT3a showed significantly promotion. Conclusion: We observed that JAK2V167F mutation suppresses protein expression levels of DNMT3a in MPN cell lines. JAK2 inhibition by AZ960 and LY2784544 significantly improved expression levels of DNMT3a. The activation of JAK2/STAT5 pathway reduces expression level of DNMT3a in MPN cell line, and the specific mechanism still needs to be explored. Figure Disclosures No relevant conflicts of interest to declare.

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