Deletion Of Tet2 Research Articles

DNA demethylase Tet2 promotes the terminal maturation of natural killer cells.

The cytotoxicity feature to eliminate malignant cells makes natural killer (NK) cells a candidate for tumor immunotherapy. However, this scenario is currently hampered by inadequate understanding of the regulatory mechanisms of NK cell development. Ten-Eleven-Translocation 2 (Tet2) is a demethylase whose mutation was recently shown to cause phenotypic defects in NK cells. However, the role of Tet2 in the development and maturation of NK cells is not entirely clear. Here we studied the modulatory role of Tet2 in NK cell development and maturation by generating hematopoietic Tet2 knockout mice and mice with Tet2 conditional deletion in NKp46+ NK cells. The results showed that both hematopoietic and NK cell conditional deletion of Tet2 had no effect on the early steps of NK cell development, but impaired the terminal maturation of NK cells defined by CD11b, CD43, and KLRG1 expression. In the liver, Tet2 deletion not only prevented the terminal maturation of NK cells, but also increased the proportion of type 1 innate lymphoid cells (ILC1s) and reduced the proportion of conventional NK cells (cNK). Moreover, hematopoietic deletion of Tet2 lowered the protein levels of perforin in NK cells. Furthermore, hematopoietic deletion of Tet2 downregulated the protein levels of Eomesodermin (Eomes), but not T-bet, in NK cells. In conclusion, our results demonstrate that Tet2 plays an important role in the terminal maturation of NK cells, and the Eomes transcription factor may be involved.

Clonal hematopoiesis related TET2 loss-of-function impedes IL1β-mediated epigenetic reprogramming in hematopoietic stem and progenitor cells

Clonal hematopoiesis (CH) is defined as a single hematopoietic stem/progenitor cell (HSPC) gaining selective advantage over a broader range of HSPCs. When linked to somatic mutations in myeloid malignancy-associated genes, such as TET2-mediated clonal hematopoiesis of indeterminate potential or CHIP, it represents increased risk for hematological malignancies and cardiovascular disease. IL1β is elevated in patients with CHIP, however, its effect is not well understood. Here we show that IL1β promotes expansion of pro-inflammatory monocytes/macrophages, coinciding with a failure in the demethylation of lymphoid and erythroid lineage associated enhancers and transcription factor binding sites, in a mouse model of CHIP with hematopoietic-cell-specific deletion of Tet2. DNA-methylation is significantly lost in wild type HSPCs upon IL1β administration, which is resisted by Tet2-deficient HSPCs, and thus IL1β enhances the self-renewing ability of Tet2-deficient HSPCs by upregulating genes associated with self-renewal and by resisting demethylation of transcription factor binding sites related to terminal differentiation. Using aged mouse models and human progenitors, we demonstrate that targeting IL1 signaling could represent an early intervention strategy in preleukemic disorders. In summary, our results show that Tet2 is an important mediator of an IL1β-promoted epigenetic program to maintain the fine balance between self-renewal and lineage differentiation during hematopoiesis.

Mutant Natural Killer Cell Dysfunction Enables the Immune Escape of Premalignant MDS Cell Clones

Myelodysplastic syndromes (MDS) cannot be cured by standard pharmacological therapies owing to their inability to eradicate aberrant hematopoietic stem cells (HSCs). Given that developing early intervention strategies for MDS patients requires an improved understanding of the mechanisms underlying aberrant HSC expansion, we sought to elucidate how MDS HSCs evade immune surveillance and expand in patients with clonal cytopenias of undetermined significance (CCUS), the pre-malignant stage of MDS. To characterize transcriptomic changes induced by CCUS in the aged immune microenvironment, we used single-cell RNA sequencing (scRNA-seq) of bone marrow (BM) mononuclear cells (MNCs) isolated from 2 young healthy donors (yHDs), 2 elderly healthy donors (eHDs), and 3 CCUS patients harboring mutations in MDS driver genes such as DNMT3A or TET2. CCUS immune cells were enriched in CD8 + T effector and NK cells, which were in activated states, characterized by the significant (P adj<0.05) upregulation of genes involved in protein translation, mitochondrial metabolism, and response to interferon signaling. A computational interactome analysis of ligand-receptor pair expression predicted gained intercellular communications among myeloid cells and CD8 + T and NK cells in CCUS patients compared with yHDs and eHDs, consistent with a tumor recognition process. However, CCUS CD8 + T and NK cells had downregulated NF-kB-mediated inflammatory signaling genes, which suggested a loss of proinflammatory capacity. Because an efficient antitumor CD8 + T-cell response requires functional NK cells, we further dissected their functional state by performing scRNA-seq of CD3 + and/or CD56 + BM cells isolated from 2 yHDs, 2 eHDs, and 3 CCUS patients. A trajectory reconstruction analysis showed that CCUS NK cells were in more advanced differentiation states than HD NK cells were, which was confirmed by the significant downregulation of the transcription factor FOXO1 (P adj=1.17 x 10 -81). Differential gene expression analyses revealed that CCUS NK cells had significantly (P adj<0.05) upregulated MYC and mTOR target genes and genes involved in oxidative metabolism, protein synthesis, and immune signaling (consistent with an NK cell proliferative burst) compared with eHD NK cells, which suggests that CCUS NK cells are hyperactivated. However, cytokine secretion assays with K562 cells revealed that NK cells from 5 CCUS patients secreted significantly (P=0.002) less interferon gamma than those from 6 eHDs did. CCUS NK cells and eHD NK cells produced similar levels of granzyme and perforin, and CCUS NK cells could degranulate (based on CD107a expression); however, CCUS NK cells had significantly lower CD16 expression (P=0.008), which suggests an impaired capacity to form stable immune synapses and degranulate in a polarized manner. Indeed, compared with those from 3 yHDs and 4 eHDs, NK cells from 3 CCUS patients had a significantly (P<0.0001) impaired cytolytic capacity against leukemic cells that could not be rescued by prolonged culture with feeder cells. Moreover, NK cells from 4 CCUS patients failed to kill both heterotypic and isotypic CCUS CD34 + cells ex vivo (Fig. 1). To investigate the molecular mechanisms of NK cell dysfunction, we performed joint targeted single-cell DNA sequencing and immunophenotypic analyses of MNCs from 5 CCUS patients with DNMT3A or TET2 mutations, which revealed that NK cells were enriched in aberrant clones and had mutational burdens similar to those of myelomonocytes (Fig. 2). Deletion of DNMT3A or TET2 by CRISPR/Cas9 in cord blood-derived NK cells and subsequent killing assays showed a significant (P<0.0001) progressive loss of cytolytic capacity with tumor re-challenging in knock-out NK cells, which demonstrated that loss of function mutations in those genes are a direct cause of NK cell dysfunction in the context of sustained immune activation. This study demonstrates that MDS driver mutations in CCUS NK cells induce immune dysfunction, thus enabling the expansion of malignant cells and thereby contributing to MDS onset. Our results suggest that immunotherapy with adoptive NK cell transfer could restore the anti-tumor immune response in patients with CCUS and may be an effective approach to arrest disease progression and prevent MDS onset.

Reprogramming of ovarian aging epigenome by resveratrol.

Resveratrol is an antiaging, antioxidant, and anti-inflammatory natural polyphenolic compound. Growing evidence indicates that resveratrol has potential therapeutic effects for improving aging ovarian function. However, the mechanisms underlying prolonged reproductive longevity remain elusive. We found that resveratrol ameliorates ovarian aging transcriptome, some of which are associated with specific changes in methylome. In addition to known aging transcriptome of oocytes and granulosa cells such as decline in oxidoreductase activity, metabolism and mitochondria function, and elevated DNA damage and apoptosis, actin cytoskeleton are notably downregulated with age, and these defects are mostly rescued by resveratrol. Moreover, the aging-associated hypermethylation of actin cytoskeleton is decreased by resveratrol. In contrast, deletion of Tet2, involved in DNA demethylation, abrogates resveratrol-reprogrammed ovarian aging transcriptome. Consistently, Tet2 deficiency results in additional altered pathways as shown by increased mTOR and Wnt signaling, as well as reduced DNA repair and actin cytoskeleton with mouse age. Moreover, genes associated with oxidoreductase activity and oxidation-reduction process were hypermethylated in Tet2-deficient oocytes from middle-age mice treated with resveratrol, indicating that loss of Tet2 abolishes the antioxidant effect of resveratrol. Taking together, our finding provides a comprehensive landscape of transcriptome and epigenetic changes associated with ovarian aging that can be reprogrammed by resveratrol administration, and suggests that aberrantly increased DNA methylation by Tet2 deficiency promotes additional aging epigenome that cannot be effectively restored to younger state by resveratrol.

Tet2- and Tet3- Mediated Cytosine Hydroxymethylation in Six2 Progenitor Cells in Mice Is Critical for Nephron Progenitor Differentiation and Nephron Endowment.

Epigenetic changes have been proposed to mediate nephron endowment during development, a critical determinant of future renal disease development. Hydroxymethyl cytosine, an epigenetic modification important for gene regulation, is abundant in the human kidney, but its physiologic role and the role of DNA demethylase enzymes encoded by the Tet1 , Tet2 , or Tet3 , which mediate cytosine hydroxymethylation, are unclear. By genetically deleting Tet1 , Tet2 , or Tet3 in nephron progenitors in mice, the authors showed that combined Tet2 and Tet3 loss in nephron progenitors cause defective kidney development, leading to kidney failure and perinatal death. Tet2 and Tet3 deletion also caused an alteration in demethylation and expression of genes critical for nephron formation. These findings establish that Tet2- and Tet3 -mediated cytosine hydroxymethylation in nephron progenitors plays a critical role in nephron endowment. Nephron endowment is a key determinant of hypertension and renal disease in later life. Epigenetic changes have been proposed to mediate fetal programming and nephron number. DNA cytosine methylation, which plays a critical role in gene regulation, is affected by proteins encoded by the ten-eleven translocation (TET) DNA demethylase gene family ( Tet1 , Tet2 , and Tet3 ), but the roles of TET proteins in kidney development and nephron endowment have not been characterized . To study whether epigenetic changes-specifically, active DNA hydroxymethylation mediated by Tet1 , Tet2 , and Tet3- are necessary for nephron progenitor differentiation and nephron endowment, we generated mice with deletion of Tet1 , Tet2 , or Tet3 in Six2-positive nephron progenitors cells (NPCs). We then performed unbiased omics profiling, including whole-genome bisulfite sequencing on isolated Six2-positive NPCs and single-cell RNA sequencing on kidneys from newborn mice. We did not observe changes in kidney development or function in mice with NPC-specific deletion of Tet1 , Tet2 , Tet3 or Tet1 / Tet2 , or Tet1 / Tet3 . On the other hand, mice with combined Tet2 and Tet3 loss in Six2-positive NPCs failed to form nephrons, leading to kidney failure and perinatal death. Tet2 and Tet3 loss in Six2 -positive NPCs resulted in defective mesenchymal to epithelial transition and renal vesicle differentiation. Whole-genome bisulfite sequencing, single-cell RNA sequencing, and gene and protein expression analysis identified a defect in expression in multiple genes, including the WNT- β -catenin signaling pathway, due to a failure in demethylation of these loci in the absence of Tet2 and Tet3 . These findings suggest that Tet2- and Tet3 -mediated active cytosine hydroxymethylation in NPCs play a key role in kidney development and nephron endowment.

Demonstration That Loss of TET2 Impairs DNA Mismatch Repair Resulting in Clonally Restricted Genomic Instability

Epigenetic dysregulation is a common feature of myeloid neoplasia (MN). TET2, along with TET1 and TET3 are the only known enzymes, which progressively oxidize 5-methyl cytosine and thus facilitate active demethylation of CpG islands, a process critical for efficient gene expression and maintenance of genomic integrity. Loss of function TET2 mutations (TET2MT) are one of the most common somatic lesions in MN. To date, the precise mechanisms of TET2MT in leukemogenesis remain unclear. However, two potential mechanisms have been postulated; i) TET2MT may lead to expansion of stem cells by differentiation block and/or increased self-renewal ii) clonally restricted hyper mutations resulting in acquisition of secondary driver mutations. This functional duality may explain proleukemogenic consequences of TET2MT. Both mechanisms are compatible with their relatively moderate driver phenotype. Increased CàT transitions via deamination of 5mC in TET2MT has been postulated; however this report focuses on a novel mechanism of TET2MT- mediated impairment of mismatch repair (MMR). WES was used for analysis of the mutational status of 435 MN cases to test whether TET2MT (n= 82/435; 19%) predispose to acquisition of additional mutations. Following exclusion of dysfunction in known DNA repair genes, we compared the median SNVs in TET2MT vs. TET2WT. TET2MT cases had nearly a 2-fold increased median SNVs (p<0.0001) vs. TET2WT including a 2.1-fold increase in truncations (p=0.03) with a linear correlation (r=0.7) of SNVs vs. clonality, consistent with a gene-dose effect. ShRNA knockdown of TET2 in HeLa and CRISPR knockout in HEK293 cells resulted in a 24- and 30-fold increase in spontaneous mutations in the HPRT assay. The heightened spontaneous mutability in TET2KO/KD cells were reversed by ectopic expression of TET2. In Tet2-/- mice, ~1.5-fold increased mutagenicity at TET2-dependent active demethylation sites were detected suggesting its direct or indirect involvement in one or more DNA repair pathways. Consistent with our hypothesis, endogenous co-immunoprecipitation of TET2 followed by tandem-affinity purification and characterization of protein complexes with LCMS/MS in 4 different systems including human leukemic cell lines (K562 and CMK), murine embryonic stem cell and erythroid leukemia cells MEL identified all the major components of DNA MMR complex as a part of TET2 interactome. In every cell line, MSH6, a main component of MMR complex was an abundant TET2 interacting protein. This result was further confirmed by reverse co-immunoprecipitation using anti-MSH6 antibodies in different cell lines. Using truncating deletion constructs of TET2, MSH6 binding site was mapped on TET2 N-terminal amino acid segment 460-942. Functional MMR fluorescence reporter assay, wherein a point mutant of GFP, if repaired, is converted to BFP, demonstrated that loss of TET2 indeed compromised the rate of MMR in CRISPR generated deletion of TET2 in K562 and HEK293 cells. Ectopic expression of N-terminal (aa 460-942) MSH6 binding domain rescued this defect. The genomic instability due to MMR dysfunction in TET2MT cells may allow therapeutic targeting of DNA repair proteins in a subset of TET2MT MN. Therefore, we tested the therapeutic vulnerability of TET2MT cells against small molecule DNA repair inhibitors (e.g., clinically approved PARP antagonists) in engineered isogenic and natural TET2MT cell lines. The TET2KO/KD cells were 2-10X more sensitive to olaparib and rukaparib compared to parental TET2 proficient cells, both under steady state and under conditions of oxidative stress as observed in gH2AX assays and various apoptotic readouts. In summary, for the first time here we report that TET2 directly binds to MSH6 and recruits MMR complex to the site of mismatch around CpG islands and facilitate the repair. Loss of TET2impairs this process, explaining the accumulation of secondary hits in mutant clones, which by this virtue, progressively increases their leukemogenic fitness. Given that MSH6 is known to interact 5hmC, TET2 may guide MMR to TET2-dependent DNA loci directly linking TET2MTto genomic instability a biologic feature that can be therapeutically exploited. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Vitamin C epigenetically controls osteogenesis and bone mineralization

Vitamin C deficiency disrupts the integrity of connective tissues including bone. For decades this function has been primarily attributed to Vitamin C as a cofactor for collagen maturation. Here, we demonstrate that Vitamin C epigenetically orchestrates osteogenic differentiation and function by modulating chromatin accessibility and priming transcriptional activity. Vitamin C regulates histone demethylation (H3K9me3 and H3K27me3) and promotes TET-mediated 5hmC DNA hydroxymethylation at promoters, enhancers and super-enhancers near bone-specific genes. This epigenetic circuit licenses osteoblastogenesis by permitting the expression of all major pro-osteogenic genes. Osteogenic cell differentiation is strictly and continuously dependent on Vitamin C, whereas Vitamin C is dispensable for adipogenesis. Importantly, deletion of 5hmC-writers, Tet1 and Tet2, in Vitamin C-sufficient murine bone causes severe skeletal defects which mimic bone phenotypes of Vitamin C-insufficient Gulo knockout mice, a model of Vitamin C deficiency and scurvy. Thus, Vitamin C’s epigenetic functions are central to osteoblastogenesis and bone formation and may be leveraged to prevent common bone-degenerating conditions.

LB964 TET2 participates in the dysregulation of skin barrier in psoriasis

Psoriasis is a chronic inflammatory skin disease. Dysregulation of the epidermal barrier in psoriasis has been demonstrated and epigenetic modifications have been found to be associated with psoriasis. TET2 has dioxygenase activity and oxidizes 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and is a key regulator of DNA demethylation. There is a lack of studies on the effect of TET2 on the epidermal barrier in psoriasis. We observed reduced expression of TET2 and 5hmc in the lesions of psoriasis patients. In imiquimod psoriatic dermatitis mice, we obtained similar results by immunohistochemistry. We constructed epidermal TET2 conditional knockout mice (TET2CKO). In the imiquimod psoriasis dermatitis model, TET2CKO mice presented more severe erythema, scaling and skin thickness. The TEWL assay was performed on imiquimod-treated mice and was elevated in the TET2CKO group. This indicates that epidermal TET2 deficiency exacerbates psoriatic dermatitis in mice. We next examined the expression of K14, K10, filaggrin, involucrin, which represent proliferation and differentiation, TET2CKO mice showed K14, involucrin increased and K10, filaggrin decreased. TET2 absence may accelerate the proliferation and inhibit differentiation of keratinocytes. We subsequently observed that intercellular linker molecules Claudin-1 and E-cadherin were both downregulated in TET2CKO mice. In conclusion, deletion of epidermal TET2 is involved in dysregulation of the skin barrier. By RNAseq, we found that FLG mRNA is reduced after TET2 knockdown. Deficiency of filaggrin encoded by the FLG gene, leads to impaired epidermal barrier. Filaggrin expression reduced but mutations in the FLG gene have been reported to be rarely observed in psoriasis, therefore we suppose that FLG gene expression might be regulated by TET2, which is an alternative mechanism for barrier dysregulation in non-mutant FLG patients.

TET deficiency perturbs mature B cell homeostasis and promotes oncogenesis associated with accumulation of G-quadruplex and R-loop structures.

Enzymes of the TET family are methylcytosine dioxygenases that undergo frequent mutational or functional inactivation in human cancers. Recurrent loss-of-function mutations in TET proteins are frequent in human Diffuse Large B-Cell Lymphoma (DLBCL). Here we investigate the role of TET proteins in B-cell homeostasis and development of B cell lymphomas with features of DLBCL. We show that deletion of Tet2 and Tet3 genes in mature B cells in mice perturbs B-cell homeostasis and results in spontaneous development of germinal center-derived B cell lymphomas with increased G-quadruplexes and R-loops. At a genome-wide level, G-quadruplexes and R-loops were associated with increased DNA double strand breaks at immunoglobulin switch regions. Deletion of the DNA methyltransferase DNMT1 in TET-deficient B cells prevented expansion of germinal center B cells, diminished the accumulation of G-quadruplexes and R-loops, and delayed B lymphoma development, consistent with the opposing functions of DNMT and TET enzymes in DNA methylation and demethylation. CRISPR-mediated depletion of nucleases and helicases that regulate G-quadruplexes and R-loops decreased the viability of TET-deficient B cells. Our studies suggest a molecular mechanism by which TET loss-of-function might predispose to development of B cell malignancies.

TET Deficiency Is Associated with Accumulation of G-Quadruplex and R-Loop Structures during Oncogenesis

The three members of TET family of Fe(II) and alpha-ketoglutarate-dependent dioxygenases mediate DNA demethylation by sequentially oxidizing 5-methylcytosine (5mC) to 5-hydroxymethyl- (5hmC), 5-formyl- (5fC) and 5-carboxyl-cytosine (5caC). TET enzymes are required for normal development, and loss of TET function due to mutations, metabolic perturbations and hypoxia, among other mechanisms, occurs frequently in many hematological malignancies and solid tumors. Recent studies have identified mutations in TET proteins (TET2, most commonly) and metabolic enzymes which regulate TET catalytic activity in a large cohort of patients with Diffuse Large B-cell Lymphoma (DLBCL). However, the clinical significance of these mutations in DLBCL and the molecular mechanisms through which TET proteins suppress development of malignancies in general, are not fully-understood.To investigate the role of TET loss-of-function in the pathogenesis of DLBCL, we generated mice with B-cell-specific deletion of TET2 and TET3, the major TET homologs expressed in mature B cells. TET deficiency in B cells perturbed mature B cell homeostasis resulting in spontaneous development of Germinal Center-derived B cell lymphomas. Moreover, B cells with TET deficiency demonstrated increased genomic instability, a feature previously associated with TET loss-of-function in other hematopoietic lineages. Transcriptional profiling of TET-deficient expanded B cells revealed altered expression of genes and proteins involved in modulating the levels of secondary DNA structures, G-quadruplexes and DNA:RNA hybrids (R-loops) which have been linked to genomic instability and transcriptional perturbations in many different cancers. Using previously described methods and newer approaches, we observed a substantial increase in the levels of G-quadruplex and R-loop structures in TET-deficient B cells compared with control B cells. The increase in G-quadruplex and R-loop structures was evident in naïve, activated and GC B cells following acute TET deletion as well as in TET-deficient myeloid cells and T cells. Genome-wide mapping studies and high-throughput genome-wide translocation sequencing (HTGTS) showed a strong correlation of increased G-quadruplex and R-loop structures with increased DNA DSBs in switch regions of immunoglobulin heavy chain locus in TET-deficient B cells. Deletion of the DNA methyltransferase DNMT1 in TET-deficient B cells prevented the expansion of germinal center B cells, diminished the accumulation of G-quadruplexes and R-loops, and caused a notable delay in lymphoma development, consistent with the opposing functions of DNMT and TET enzymes in DNA methylation and demethylation. CRISPR-mediated depletion of nucleases and helicases that regulate G-quadruplexes and R-loops decreased the viability of TET-deficient B cells. Our studies suggest a molecular mechanism by which TET loss-of-function might predispose to development of B cell-derived and other malignancies, and highlight novel therapeutic avenues that could be further explored. DisclosuresRao: Cambridge Epigenetix: Membership on an entity's Board of Directors or advisory committees.

Abstract MP245: Tet Proteins Regulate Second Heart Field Multipotent Progenitors Differentiating To Myocytes

DNA methylation and demethylation play an important role in shaping the epigenetic landscape and chromatin accessibility to control gene expression during development in mammals. Ten-eleven Translocation (Tet1, Tet2 and Tet3) is a family of dioxygenases that catalyze DNA methylation oxidation with ultimate DNA demethylation. Our previous study showed that cardiac-specific deletion of Tet2 and Tet3 could disrupt YY1-mediated long range chromatin interactions during heart development and lead to ventricular non-compaction cardiomyopathy. However, it is still unclear whether and how Tet protein mediated epigenetic modifications contribute to cardiac lineage specification during embryonic development. In this study, we generated cardiac specific Tet1-3 triple deficient (Tet-TKO) mouse lines using various cardiac specific Cres to evaluate the function of Tet protein in regulating cardiac lineage specification. We observed developmental defects at outflow tract (OFT) in Tet-TKO embryos, suggesting that Tet deficiency affects the second heart field (SHF) development. Single cell RNA-seq analysis further revealed the accumulation of multipotent SHF progenitors and subsequent halt of myocyte differentiation upon Tet depletion. At the molecular level, we found that Tet ablation perturbs the transcriptional network of Islet1, a transcription factor that is crucial for cardiac development in embryos. Overall, our study demonstrates a critical role of Tet-mediated epigenetic regulation for embryonic cardiac development.

Acute Deletion of Tet2 or Tet3 Transiently Dysregulate Murine Erythropoiesis

The majority of palindromic CpG dinucleotides in mammalian DNA are symmetrically methylated on cytosine (5mC). Erythroid differentiation is associated with replication-dependent genome-wide loss of ~30-50% of all 5mC (1). One potential mechanism of demethylation involves Tet dioxygneases, which oxidize 5 methylcytosine (5mC) to 5-hydroxy-methylcytosine (5hmC), thereby interfering with maintenance methylation by DNMT1 (2). Tet2 and Tet3 are the two major Tet proteins expressed in hematopoietic cells. Tet2 is one of the most commonly mutated genes in hematological malignancies. Germline deletion of Tet2 results in increased hematopoietic stem cell self-renewal, erythroid and myeloid hyperplasia and myeloid and lymphoid malignancies (3, 4). Germline deletion of Tet3 is embryonic lethal (5). Here we investigated the potential role of Tet2 and Tet3 in the global loss of DNA methylation during erythroid differentiation, using mouse genetic models.We bred Tet2fl/fl and Tet3fl/fl mice onto the Rosa26-CreERT2 background, enabling acute deletion in vivo of either Tet2, Tet3 or both genes in adult and fetal mice. Within 4 to 10 days of deletion, we noted a rapid decline in total bone marrow cellularity in all genotypes; bone-marrow erythroblasts decreased to 15% of control value in Tet2-deleted (Tet2del) mice (p<0.0001), and to 43% (p=0.05) and 20% (p<0.0001) of controls in the Tet3 del and Tet2/Tet3 double deleted mice (DKO), respectively. These findings were associated with decreased reticulocyte counts in peripheral blood, and a decline in hematocrit, in Tet2del (36% vs. 46% in controls, p<0.004) and the DKO (31%, p=0.03). Bone-marrow CFU-e progenitors, but not BFU-e progenitors, also decreased in number. These losses partially improved over the next 2 to 4 weeks in mice deleted for either Tet2 or Tet3, but not in the DKO mice, in which anemia persisted. Megakaryocyte CFU-Mk progenitors increase in the DKO but are unaffected in the single deletions. Lymphoid lineage cells (CD4+, CD8+ and CD19+) showed similar transient losses, but the numbers of granylocytic and monocytic progenitors in the bone marrow was not affected. Similarly, fetal liver cell number decreased in all genotypes within 4 days of deletion, compared with controls, in all genotypes, and most severely in the DKO.We used three approaches to determine the potential effect of Tet proteins on erythroid DNA demethylation: Mass spectrometry, to measure global levels of 5mC and 5hmC in fetal liver erythroblasts; 5hMe-DIP-seq (5hyrdoxymethyl-DNA immune precipitation- sequencing), to determine the genomic distribution of hydroxymethylation in erythroblasts; and pyrosequencing for DNA methylation/hydroxymethylation at specific genomic loci including repetitive LINE_1 elements. We found that 5hmC is highly enriched in previously identified erythroid enhancers. Tet2 accelerates, but is not essential, for DNA demethylation of specific erythroid enhancers. Neither Tet2 nor Tet3 are required for global DNA demethylation.Taken together, we conclude that Tet2 and Tet3 are required during erythropoiesis, where they play partially redundant roles, possibly contributing to the changing chromatin environment during activation of erythroid terminal differentiation. Their acute deletion leads to transient loss of erythroblast viability and to anemia, but this is followed by a process of adaptation and chronically compensated erythropoiesis. These results raise the possibility that the adaptation required for the rapid partial recovery in the erythroid and lymphoid lineages following acute Tet2 loss may contribute, over the long term, to the development of myeloid and lymphoid malignancies.1. J. R. Shearstone et al., Global DNA demethylation during mouse erythropoiesis in vivo. Science334, 799-802 (2011).2. M. Ko et al., Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2. Nature468, 839-843 (2010).3. M. Ko et al., TET proteins and 5-methylcytosine oxidation in hematological cancers. Immunol Rev263, 6-21 (2015).4. J. An et al., Acute loss of TET function results in aggressive myeloid cancer in mice. Nature communications6, 10071 (2015).5. T. P. Gu et al., The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature477, 606-610 (2011). DisclosuresNo relevant conflicts of interest to declare.

Spliceosomal Dysfunction Is a Critical Mediator of IDH2 Mutant Leukemogenesis

Mutations affecting RNA splicing factors are commonly found in leukemias and mutations in SF3B1, SRSF2, and U2AF1 impart specific changes to global splicing. We analyzed RNA-seq data from acute myeloid leukemia (AML) patients from The Cancer Genome Atlas (TCGA) to evaluate for spliceosomal alterations characteristic of mutant SRSF2, SF3B1, and U2AF1 . Although only 1 patient with an SRSF2 mutation was reported in the TCGA AML publication, we identified 19 additional patients to have the characteristic splicing changes of mutant SRSF2 . Mutational analysis of the RNA-seq data identified the SRSF2 proline 95 hotspot mutation in each of these 19 patients (19/178, 11%), making SRSF2 amongst the most commonly mutated genes in the AML TCGA dataset. Interestingly, 47% of SRSF2 mutant patients also had a co-existing IDH2 mutation (and conversely, 50% of IDH2 mutant patients had a co-existing SRSF2 mutation). These data are similar to recent observation that SRSF2 mutations were the most common accompanying mutation in IDH2 mutant myeloid leukemia patients treated on the phase I clinical trial of AG-221.We next sought to understand the functional contribution of SRSF2 mutations to co-existing IDH2 mutations. Overexpression of IDH2 R140Q and IDH2 R172K in Vav -cre Srsf2 P95H or Vav- cre Srsf2 wildtype(WT) backgrounds revealed a clear collaborative effect of mutant IDH2 and mutant Srsf2 (Figure A-B). As early as 4 weeks post-transplant, the peripheral blood of Srsf2 mutant mice transplanted with IDH2 R140Q or IDH2 R172K forms had a substantially greater percentage of GFP+ cells than seen in an Srsf2 WT background. Moreover, these mice exhibited significant myeloid skewing, macrocytic anemia, and thrombocytopenia of greater magnitude than seen with mutant IDH2 expressed in the SRSF2 WT background. IDH2 / Srsf2 double-mutant mice developed a lethal, mixed myelodysplastic/myeloproliferative neoplasm that was serially transplantable (Figure B). Effects from overexpression of mutant IDH2 were recapitulated in mice expressing Idh2 R140Q and Srsf2 P95H from endogenous loci(Mx1 -cre Idh2R140Q/+Srsf2P95H/+ double knock-in mice) (Figure C), providing the first model where spliceosomal gene mutations promote leukemogenesis in vivo .Given prior data identifying inhibition of TET2 as a mediator of IDH2 -mutant leukemogenesis, we evaluated if loss of TET2 might promote transformation of SRSF2 mutant cells. However, deletion of TET2 in an SRSF2 mutant background (Mx1 -cre Tet2fl/flSrsf2P95H/+) was insufficient to promote transformation (Figure D). We next analyzed RNA-seq data from the TCGA, Leucegene, and our own unpublished AML cohort using a novel analysis method to detect unannotated as well as complex splicing events that might explain the basis for the collaboration of mutant IDH2 and SRSF2 . This revealed that IDH2/SRSF2 double-mutant AML had increased aberrant splicing events compared to SRSF2 single-mutant AML and also that IDH2 -mutations are associated with a small but reproducible change in RNA splicing (Figure E). The vast majority of splicing changes affected by SRSF2 and/or IDH2 mutations were a change in cassette exon splicing with SRSF2 mutations promoting inclusion of C-rich containing mRNA sequences, regardless of the presence of concomitant IDH2 mutations (Figure F). One of the most robust mis-splicing events in IDH2/SRSF2 mutant AML was an intron retention event in INTS3 resulting in its reduced mRNA and protein expression (Figure G-H). INTS3 encodes a component of the integrator complex that participates in small nuclear RNA (snRNA) processing. Consistent with this, IDH2 / SRSF2 double-mutant cells exhibited alterations in processing of U2 and U4 snRNAs similar to that seen with direct INTS3 downregulation. Biologically, attenuation of INTS3 expression caused a blockade of myeloid differentiation in HL-60 cells upon ATRA treatment, an effect which was further enhanced in an IDH2 mutant background.These data uncover an important role for spliceosomal gene mutations in the pathogenesis of IDH2 mutant leukemogenesis and identify perturbations in the integrator complex as a novel mediator driving the transformation of IDH2 and SRSF2 mutant cells. The frequent coexistence of IDH2 and SRSF2 mutations underscores the enormous therapeutic potential for modulation of splicing in the ~50% of IDH2 -mutant leukemia patients who also harbor a spliceosomal gene mutation. [Display omitted] DisclosuresDe Botton:Servier: Honoraria; Agios: Honoraria, Research Funding; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Stein:Pfizer: Consultancy, Other: Travel expenses; Novartis: Consultancy, Research Funding; Agios Pharmaceuticals, Inc.: Consultancy, Research Funding; Celgene Corporation: Consultancy, Other: Travel expenses, Research Funding; Seattle Genetics: Research Funding; GSK: Other: Advisory Board, Research Funding; Constellation Pharma: Research Funding. Peng:H3 Biomedicine Inc.: Employment. Buonamici:H3 Biomedicine Inc.: Employment. Palacino:H3 Biomedicine Inc.: Employment. Smith:H3 Biomedicine Inc.: Employment. Thompson:Merck: Membership on an entity's Board of Directors or advisory committees; Charles River Laboratories: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Founder. Levine:Roche: Research Funding; Roche: Research Funding; Qiagen: Equity Ownership; Qiagen: Equity Ownership; Celgene: Research Funding; Celgene: Research Funding.

TET2 Regulates CD4+ T Follicular Helper Differentiation

DNA methylation is a key epigenetic mechanism that controls T cell differentiation. The ten-eleven translocation (TET) family of methylcytosine dioxygenases converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and other oxidized methylcytosines. TET2 mutations have been identified in angioimmunoblastic T cell lymphomas (AITL), which are derived from CD4+ T follicular helper (TFH) cells. CD4+ TFH cells are a T cell subset that provides essential help for the germinal center reaction and class switching. Here we demonstrate that TET2 regulates murine CD4+ TFH differentiation in vivo . At steady-state, mice with a T cell specific deletion of TET2 (TET2cKO) have intact thymic and peripheral T cell populations. Following acute viral infection with LCMV-Armstrong, TET2 loss promotes CD4+ TFH cell expansion. Examination of the Peyer's patches (a site of TFH differentiation in response to gut microbes) of wild-type and TET2cKO mice demonstrate an increase in the absolute number of the CD4+ TFH (CXCR5+PD1+) population in TET2cKO mice. Furthermore, there is an increase in the frequency and absolute number of germinal center B cells (characterized by PNA+GL7+ B cells) and an increase in IgA+ B cells in the Peyer's patches of TET2cKO mice compared to wild-type mice. Ongoing studies are examining the mechanism by which TET2 controls CD4+ TFH expansion. Our data indicate that TET2 is an important regulator of CD4+ TFH cells. Understanding the role of TET2 in normal CD4+ TFH differentiation and its contribution to T cell lymphomagenesis will lead to important insights into the epigenetic and molecular mechanisms underlying AITL pathogenesis DisclosuresNo relevant conflicts of interest to declare.

CXCL12 Knock-out Enhances Leukemia Stem Cell Response to Combination Chemotherapy Plus Tyrosine Kinase Inhibition in Flt3-ITD Acute Myeloid Leukemia

One of the most common mutations in adult AML is a constitutively activating internal tandem duplication in the juxtamembrane domain of the Flt3 receptor (Flt3-ITD), which portends poor prognosis due to high recurrence rates and defines a distinct subtype of disease with unique features and biology. Although several FLT3 TKIs have been developed for clinical use, responses to these drugs, especially as single agents, are limited and are not sustained. The objective of our study was to determine the contribution of bone marrow stromal populations to LSC resistance to Flt3-targeted TKI in Flt3-ITD AML. We utilized a newly generated Flt3-ITD TET2flox/flox Mx1-cre mouse model of AML, as well as primary Flt3-ITD TET2 mutant AML patient samples, to identify phenotypic populations with leukemia initiating capacity (LIC) in Flt3-ITD AML. In the animal model, administration of pIpC leads to deletion of TET2 and development of AML characterized by leukocytosis, accumulation of blasts, splenomegaly, anemia, thrombocytopenia, and lethality. Limiting dilution transplantation of FACS-sorted ST-HSC, MPP and GMP populations revealed that LIC were absent from GMPs and almost exclusively limited to the phenotypic ST-HSC population (calculated stem cell frequencies: &amp;lt;1:180,000, 1:63,635, and 1:2,730 for GMP, MPP, and ST-HSC, respectively). We similarly found that in samples from human Flt3-ITD TET2 mutant AML patients LIC capacity was restricted to primitive HSPC populations (Lin-CD34+CD38-), and was not seen in committed GMP (Lin-CD34+CD38+CD123+CD45RA+). We characterized bone marrow stromal cells in Flt3-ITD AML mice by flow cytometry on collagenase digested bone fragments. We also transplanted murine AML cells into CXCL12-GFP mice to assess alterations in CXCL12-expressing stromal populations in AML bone marrow. We found expansion of several stromal populations in AML vs. WT mice, including a 3.5-fold increase in mesenchymal stem cells (CD45-Ter119-CD31-VECadherin-Sca1+CD51+) and a 1.5-fold increase in osteoprogenitors (CD45-Ter119-CD31-VECadherin-Sca1-CD51+). CXCL12 expression, however, was greater than 2-fold higher in osteoprogenitors and 2-fold lower in mesenchymal stem cells in AML vs. WT mice. We also showed that Flt3-ITD AML HSPCs have nearly 2-fold higher CXCR4 expression than WT HSPCs. These data taken together supported further exploration of the role of a CXCL12-expressing niche in supporting Flt3-ITD AML LSC. To assess the effect of CXCL12 deletion from the marrow microenvironment on AML TKI response, we transplanted murine AML cells into CXCL12flox/flox UBC-cre mice and control Cre-ve mice. We found that AML developing in Cre-ve control mice was resistant to single agent Flt3 TKI (AC220, Quizartinib) treatment, but that CXCL12 deletion modestly improved response to TKI. We next tested a combination of standard-of-care "7+3" chemotherapy (cytarabine + doxorubicin) and AC220, and found that this approach resulted in more effective and selective, but only partial, reduction of leukemia cells in this model. We found that control AML mice showed an initial response to combination chemo + TKI, but developed disease recurrence by 3 weeks of treatment. In contrast, CXCL12-deleted AML mice maintained peripheral blood response for up to 3 weeks, and showed enhanced suppression of LIC-containing populations compared to control mice. We are now performing secondary transplants using BM cells harvested from these treated mice to assess long-term effects on leukemia stem cell capacity. We are also testing the effect of the combination of chemotherapy and TKI following osteoblast-specific deletion of CXCL12, using CXCL12flox/flox BGLAP-cre mice, to assess whether osteoblastic cells are the source of CXCL12 responsible for this effect. In conclusion, our results suggest that LSC in Flt3-ITD AML are found within a primitive phenotypic ST-HSC population as opposed to GMP populations as seen in some other types of AML. Furthermore, CXCL12-expressing bone marrow microenvironmental cells contribute to drug resistance in AML LSC and global knockout of CXCL12 enhances drug response in these populations. Our studies support a potential role for a CXCL12-expressing osteoprogenitor niche in supporting Flt3-ITD AML LSC growth and drug resistance, targeting of which could improve responses and outcomes in Flt3-ITD AML. Disclosures No relevant conflicts of interest to declare.

Abstract PO-05: TET2 deficiency alters the epigenome of germinal center B cells, contributing to lymphoma formation

Abstract Diffuse large B-cell lymphomas (DLBCLs) are aggressive tumors derived from germinal center (GC) B cells. Despite progress in the treatment of DLBCL, approximately 40% of patients relapse or are refractory to the treatment, which usually leads to fatality. DLBCLs are characterized by aberrant DNA methylation and this feature correlates with poor clinical outcome. We and others have shown that deregulated epigenetic mechanisms contribute to lymphoma formation. Particularly, we have demonstrated that TET2, an enzyme that converts methylcytosine (mC) into hydroxymethylcytosine (hmC) and is mutated in ~10% of DLBCLs, is a B-cell tumor suppressor. GC-specific deletion of TET2 (Cg1Cre/Tet2-/-) resulted in accelerated lymphomagenesis in DLBCL mouse models driven by BCL6 overexpression, with 100% Cg1Cre/Tet2-/-;ImBcl6 mice developing lymphoma at 7 months compared to only 50% in ImBcl6 control mice. In addition, TET2 deletion in hematopoietic stem cells (VavCre/Tet2-/-) induced GC B-cell hyperplasia (B220+GL7+CD95+ cells; 10% VavCre/Tet2-/- vs 5% VavCre/Tet2+/+), promoting malignant transformation. Further analysis of the GC reaction revealed that TET2-deficient GC B cells displayed anomalous patterns of DNA methylation. GC B cells from VavCre/Tet2-/- mice presented 1) focal loss of hmC—using hMeDIPseq—with 25,000 differentially hydroxymethylated regions (DHMR) lost compared to VavCre/Tet2+/+ GC B cells and 2) increased mC—using RRBS—with almost 11,000 differentially methylated cytosines (DMCs), 84% hypermethylated, compared to VavCre/Tet2+/+ GC B cells. TET2-mediated reduction of hmC and hypermethylation affected enhancers and promoters, respectively, of genes mediating GC exit and terminal differentiation of GC B cells, especially those regulated at enhancers by the opposing functions of CREBBP and HDAC3. We are currently investigating the potential cooperative role between TET2-mediated hmC and CREBBP-mediated H3K27Ac, supported by reduced H3K27Ac at enhancers activated by CREBBP in VavCre/Tet2-/- GC B cells and mutual exclusion between TET2-mutant and CREBBP-mutant primary DLBCL. RNA sequencing analysis revealed that the genes epigenetically regulated by TET2 were aberrantly repressed in VavCre/Tet2+/+ GC B cells, explaining the observed GC hyperplasia in TET2-deficient GC B cells since these genes control the differentiation of GC B cells into plasma cells. Importantly, TET2-mutant DLBCL primary samples display a similar repressive transcriptional signature associated with GC B-cell terminal differentiation. Our data show how TET2-induced epigenetic changes contribute to lymphoma development and highlight the multilayered nature of the epigenome, which can be therapeutically exploited. We are evaluating the therapeutic potential in TET2-mutant DLBCL of a combinatorial therapy consisting of DNA methylation inhibitors (DNMTi), to revert hypermethylation at promoters, plus specific HDAC3 inhibitors, to compensate for the loss of hmC at enhancers. Citation Format: Pilar M. Dominguez, Wojciech Rosikiewicz, Xiaowen Chen, Hussein Ghamlouch, Said Aoufouchi, Olivier A. Bernard, Ari M. Melnick, Sheng Li, Ricky W. Johnstone. TET2 deficiency alters the epigenome of germinal center B cells, contributing to lymphoma formation [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-05.

TET2 promotes anti-tumor immunity by governing G-MDSCs and CD8+ T-cell numbers.

The host immune response is a fundamental mechanism for attenuating cancer progression. Here we report a role for the DNA demethylase and tumor suppressor TET2 in host anti-tumor immunity. Deletion of Tet2 in mice elevates IL-6 levels upon tumor challenge. Elevated IL-6 stimulates immunosuppressive granulocytic myeloid-derived suppressor cells (G-MDSCs), which in turn reduce CD8+ T cells upon tumor challenge. Consequently, systematic knockout of Tet2 in mice leads to accelerated syngeneic tumor growth, which is constrained by anti-PD-1 blockade. Removal of G-MDSCs by the anti-mouse Ly6g antibodies restores CD8+ T-cell numbers in Tet2-/- mice and reboots their anti-tumor activity. Importantly, anti-IL-6 antibody treatment blocks the expansion of G-MDSCs and inhibits syngeneic tumor growth. Collectively, these findings reveal a TET2-mediated IL-6/G-MDSCs/CD8+ T-cell immune response cascade that safeguards host adaptive anti-tumor immunity, offering a cell non-autonomous mechanism of TET2 for tumor suppression.

Hmga2 Functions As an Oncogene upon the Deletion of Tet2 and Promotes the Pathogenesis of Myelodysplastic Syndrome

High Mobility Group AT-hook 2 (HMGA2) is a chromatin modifier and its overexpression has been found in a subset of patients with myelodysplastic syndrome (MDS). The high level of HMGA2 expression appears to predict poor prognosis in various tumors; however, it remains unclear how HMGA2 dysregulates expression of target genes to facilitate the transformation. To elucidate the mechanisms by which the overexpression of Hmga2 promotes the development of MDS, we generated an Hmga2-expressing Tet2-deficient (Hmga2-Tet2Δ/Δ) mouse model showing the progressive phenotype of MDS. We found that Hmga2-Tet2Δ/Δ mice had neutropenia and anemia, but variable platelet counts, accompanied by elevated frequencies of mutant cells in myeloid cells. Hmga2-Tet2Δ/Δ mice showed a similar median survival to Tet2Δ/Δ mice (274 days vs 290 days), but shorter survival than Hmga2-Tet2wt/wt mice (274 days vs undetermined). Moribund Hmga2-Tet2Δ/Δ mice showed progressive leukopenia and anemia, accompanied by the emergence of dysplastic neutrophils, myeloblasts and anisocytosis in the PB and BM and dysplastic megakaryocytes in the BM. Hmga2-Tet2Δ/Δ mice had mildly increased spleen weights, and expanded myeloid cells and HSPCs in the spleen without the deposition of fibrosis. During a 12-month observation, we found that Hmga2-Tet2Δ/Δ mice developed lethal MDS/MPN overlap disease (47%), MDS (33%), MPN (13%), and AML (7%), while 6 out of 11 Tet2Δ/Δ mice developed MPN (55%). Hmga2-Tet2wt/wt mice subsequently showed similar blood counts in PB and died without the expansion of leukemic or dysplastic blood cells. Therefore, Hmga2 overexpression did not transform wild-type HSCs but promoted the development of MDS in the absence of Tet2 in vivo. In order to elucidate the molecular mechanisms underlying the transformation of Hmga2-Tet2Δ/Δ cells, we initially performed gene expression profiling by a RNA sequencing analysis in LSK HSPCs isolated from WT, Hmga2-Tet2wt/wt, Tet2Δ/Δ, and Hmga2-Tet2Δ/Δ mice at a pre-disease stage and those isolated from two Hmga2-Tet2Δ/Δ MDS/MPN and AML mice. Hmga2-Tet2Δ/Δ leukemic cells were placed closer to one out of two Hmga2-Tet2Δ/Δ cells at the pre-disease stage, but clearly apart from the other genotype cells, indicating that Hmga2 overexpression and Tet2 loss result in the accumulation of alterations in the transcriptional program during the development of MDS.In order to clarify the mechanisms by which the overexpression of Hmga2 alters the transcriptional program in Tet2-deficient cells, we performed the ChIP-sequencing of FLAG-tagged Hmga2 in bone marrow progenitor cells isolated from WT, Hmga2-Tet2wt/wt, and Hmga2-Tet2Δ/Δ mice. The numbers of Hmga2-binding peaks were markedly lower in Tet2-deficient cells than in Hmga2-Tet2wt/wt cells (2227 peaks versus 11500 peaks). Furthermore, annotated genes adjacent to Hmga2-binding sites partially overlapped in both genotype cells, whereas 2965 out of 3843 genes identified in Tet2 wild-type cells lost the binding peaks of Hmga2 upon the deletion of Tet2. Based on the DNA-binding capacity of Hmga2, the loss of Tet2 remodeled the binding sites of Hmga2 via change in DNA methylation in Hmga2-binding flanking regions, which were not observed in the presence of Tet2, leading to significant enrichments in genes involved in cell-to-cell adhesion and cell morphogenesis in Hmga2-Tet2Δ/Δ cells. Furthermore, we found that the overexpression of Hmga2 and loss of Tet2 resulted in the activation of oncogenic pathways (e.g. TGF-b, TNF-a), but suppressed the expression of genes in the unfolded protein response. Notably, the inhibition of bile acid metabolism to reactivate the unfolded protein response markedly attenuated the proliferation of Hmga2-Tet2Δ/Δ cells. These combinatory effects on the transcriptional program and cellular functions were not redundant to those in either single mutant cell, supporting Hmga2 being a proto-oncogene because its overexpression alone was not sufficient to develop MDS in vivo. Thus, Hmga2 overexpression exerts synergistic, but also gain-of-function effects with the loss of Tet2 to target these key biological pathways and promotes the transformation of Tet2-deficient stem cells. This study also provides a new rationale for targeting the unfolded protein response in MDS cells expressing HMGA2. Disclosures No relevant conflicts of interest to declare.

Identifying the Leukemia Stem Cell Niche Mediating Drug Resistance in Flt3-ITD Acute Myeloid Leukemia

The Flt3-ITD is one of the most common mutations in AML, and defines a distinct subtype of disease with unique features and biology, and with poor prognosis related to high rates of recurrence. Although several FLT3 TKIs have been developed for clinical use, responses are limited and are not sustained. The objective of our study was to determine the contribution of bone marrow stromal populations to LSC drug resistance to Flt3-targeted TKI in Flt3-ITD AML. We utilized a newly generated Flt3-ITD TET2flox/flox Mx1-cre mouse model of AML to identify phenotypic populations with leukemia initiating capacity (LIC) in Flt3-ITD AML. Administration of pIpC leads to deletion of TET2 and development of AML-like disease characterized by leukocytosis, accumulation of blasts, anemia and thrombocytopenia. Transplantation of selected STHSC, MPP and GMP populations revealed that LIC were almost exclusively found within the phenotypic ST-HSC population (calculated stem cell frequencies: &amp;lt;1:180,000, 1:63,635, and 1:2,730 for GMP, MPP, and ST-HSC, respectively). We similarly found that in samples from human Flt3-ITD AML patients LIC capacity was similarly restricted to more primitive HSPC populations (Lin-CD34+CD38-) and was not seen in committed GMP (Lin-CD34+CD38+CD123+CD45RA+). We performed flow cytometry on collagenase-digested bone fragments from AML mice to characterize bone marrow stromal cells in Flt3-ITD AML mice. We also transplanted murine AML cells into CXCL12-GFP mice to assess alterations in CXCL12-expressing stromal populations in AML bone marrow. We found that several stromal populations are expanded in AML vs. WT mice, including a 3.5-fold increase in mesenchymal stem cells (CD45-Ter119-CD31-VECadherin-Sca1+CD51+), a 3.9-fold increase in endothelial cells (CD45-Ter119-CD31+), and a 1.5-fold increase in osteoprogenitors (CD45-Ter119-CD31-VECadherin-Sca1-CD51+). The expression of CXCL12, a key factor that mediates niche localization of HSC and LSC, was greater than 2-fold higher in osteoprogenitors, but not significantly different in endothelial cells, and 2-fold lower in mesenchymal stem cells in AML vs. WT mice. We also showed that Flt3-ITD AML HSPCs have nearly 2-fold higher CXCR4 expression than WT HSPCs. These data taken together supported further exploration of the role of a CXCL12-expressing osteoprogenitor niche in supporting Flt3-ITD AML LSC. We transplanted murine AML cells into CXCL12flox/flox UBC-cre or CXCL12flox/flox Osx-cre mice to assess the effect of global or osteoprogenitor-specific CXCL12-KO, respectively, on AML progression and TKI response. We found that this model of AML was resistant to single agent Flt3 TKI (AC220) treatment. Global CXCL12-KO using CXCL12flox/flox UBC-cre mice modestly improved response to TKI. We show that a combination regimen including standard-of-care "7+3" chemotherapy (cytarabine + doxorubicin) and a Flt3 TKI (AC220) results in more effective and selective targeting of leukemia cells in this model. We are currently treating osteoprogenitor-specific CXCL12-KO AML mice with the combination chemotherapy + TKI regimen to investigate the contribution of osteoprogenitors to disease progression and drug resistance in Flt3-ITD AML LSC. In conclusion, our results suggest that LSC in Flt3-ITD AML are present within a primitive phenotypic ST-HSC population more so than in MPP and GMP populations as often seen in some other types of AML. Our studies support a potential role for a CXCL12-expressing osteoprogenitor niche in supporting Flt3-ITD AML LSC growth and drug resistance, targeting of which could improve responses and outcomes in Flt3-ITD AML. Disclosures No relevant conflicts of interest to declare.

Tet inactivation disrupts YY1 binding and long-range chromatin interactions during embryonic heart development

Tet-mediated DNA demethylation plays an important role in shaping the epigenetic landscape and chromatin accessibility to control gene expression. While several studies demonstrated pivotal roles of Tet in regulating embryonic development, little is known about their functions in heart development. Here we analyze DNA methylation and hydroxymethylation dynamics during early cardiac development in both human and mice. We find that cardiac-specific deletion of Tet2 and Tet3 in mice (Tet2/3-DKO) leads to ventricular non-compaction cardiomyopathy (NCC) with embryonic lethality. Single-cell RNA-seq analyses reveal a reduction in cardiomyocyte numbers and transcriptional reprogramming in cardiac tissues upon Tet2/3 depletion. Impaired DNA demethylation and reduced chromatin accessibility in Tet2/3-DKO mice further compromised Ying-yang1 (YY1) binding to its genomic targets, and perturbed high-order chromatin organization at key genes involved in heart development. Our studies provide evidence of the physiological role of Tet in regulating DNA methylation dynamics and chromatin organization during early heart development.