Tet2 Loss Research Articles

Loss of TET2 Uncouples Proliferative and Effector Functions in CAR T Cells

Chimeric antigen receptor (CAR) T cells have opened a new paradigm for the treatment of leukemia and lymphoma. Their production, however, is laborious, requiring tens of millions of CAR T cells per infusion. This constraint could be significantly alleviated if safe and more efficacious T cells could be generated. In a patient with chronic lymphocytic leukemia, treated with anti-CD19 CAR T cells, a recent report described the emergence of a single T cell clone that at its expansion peak accounted for 94% of circulating CAR T cells, coinciding with the development of cytokine release syndrome and tumor regression (Fraietta et. al. Nature 2018). Insertional mutagenesis in this T cell had disrupted an allele of TET2, an epigenetic regulator mediating the oxidation of 5-methylcytosine. The other allele appeared to bear an inherited hypomorphic variant, resulting in the near complete loss of TET2 function in this clone. To understand the mechanisms accounting for this chance clinical finding, we investigated the effect of TET2 loss in human T cells engineered to express different chimeric receptors. Using CRISPR/Cas9, we edited TET2 in T cells engineered to express a CD19-specific second-generation CAR encompassing the costimulatory domain of either CD28 or 4-1BB (Rv-1928z and Rv-19BBz). TET2 disruption enhanced the in vivo anti-tumor activity of Rv-19BBz but not Rv-1928z CAR T cells tested under stress test conditions using limiting CAR T cell doses (as previously described in a human B cell acute lymphoblastic leukemia (B-ALL) NALM6 model, Zhao et. al. Cancer Cell 2015). Since Rv-1928z induces potent effector differentiation but limited persistence compared to Rv-19BBz, we hypothesized that loss of TET2 could amplify the expansion and persistence of 4-1BB-costimulated T cells but not override the differentiation program imparted by Rv-1928z. To test this hypothesis, we utilized two orthogonal approaches known to limit exhaustion and increase persistence of CD28-costimulated CAR T cells, Rv-1928z co-expressed with 4-1BB ligand (Rv-1928z-41BBL) and 1928z driven by the TRAC promoter (TRAC-1928z). Disruption of TET2 enhanced the anti-tumor efficacy of both these CAR T cells and promoted acquisition of a central memory phenotype. However, over time (50-200 days), TET2-edited TRAC-1928z and Rv-1928z-41BBL attained a hyper-proliferative phenotype ultimately requiring euthanasia due to splenomegaly and extensive CAR T cell accumulation in various organs. Post-mortem analysis found no evidence of NALM6 in these mice. This was in contrast to stress test studies with Rv-1928z and Rv-19BBz where most mice succumbed to NALM6 progression. These observations established an essential role for CAR signaling in determining the phenotypic outcome of TET2 loss in T cells. To examine the long-term effects of TET2 disruption in the context of all 4 receptors, we treated human B-ALL bearing mice with curative doses of all 4 CAR T cells and followed them for up to 200 days. We found that all 4 CAR expressing TET2-edited T cells could eventually attain a hyper-proliferative phenotype, but with varying frequency depending on the CAR design (Rv-1928z-41BBL and TRAC-1928z > Rv-19BBz > Rv-1928z). To assess their effector function, NALM6-bearing mice were infused with adoptively transferred hyper-proliferative TET2-edited CAR T cells. Strikingly, these T cells were unable to elicit any tumor control, despite their maintaining a central memory phenotype as assessed by flow cytometry. This loss of effector function was observed for all 4 CAR T cell types, suggesting a discrepancy between function and flow cytometric phenotype. Transcriptional, methylation and genome accessibility studies revealed a unique T cell state wherein the proliferative program is uncoupled from effector response. We identified a unique transcriptional and epigenetic signature that is manifested in a loss of effector function while maintaining robust proliferation. This state stands in contrast to the classically described T cell exhaustion state where loss of effector function is preceded by loss of proliferative ability. TET2 disruption thus promotes a CAR T cell proliferative program that depends on the CAR design but does not in itself enhance anti-tumor activity. Disclosures Sadelain: Atara: Patents & Royalties, Research Funding; Mnemo: Patents & Royalties; Minerva: Other: Biotechnologies , Patents & Royalties; Fate Therapeutics: Patents & Royalties, Research Funding; Takeda: Patents & Royalties, Research Funding.

TET2 Inhibitory Effects of Eltrombopag Contribute Its Hematopoietic Activity

Severe aplastic anemia (sAA) is bone marrow (BM) failure syndrome characterized by immune mediated BM hypoplasia and pancytopenia usually treated with immunosuppressive therapy (IST) or allogeneic BM transplant. Despite the successes of IST in treating acquired, idiopathic sAA, most of the available treatment options only convert severe into non-severe forms of disease. Recently, eltrombopag (Epag), a small molecule thrombopoietin receptor (TPOR) agonist has been introduced into the routine treatment of de-novo and refractory AA with robust clinical response. Epag treatment showed remarkable tri-lineage responses in AA, suggesting an effect on hematopoietic stem and progenitor cells (HSPCs) amplification. The use of hematopoietic growth factors in AA that increases HSPCs has been associated with justified fears of facilitating AA evolution to MDS. Similarly, Epag has parallel effects on the amplification of HSPCs and there has been recent signals that long term treatment of Epag may increase the MDS risk. Using an in-house developed highly stable cell free high throughput screening of bioactive small molecule chemical library, we identified Epag as one of the most potent inhibitor of TET2. TET2 is the most abundant DNA dioxygenase in HSPCs. The loss-of-function mutations of TET2 frequently occurs in myeloid neoplasia. Here using in vitro and in vivo model system we report that effect of Epag, in part, is mediated by its ability to inhibit TET-dioxygenase activity. We demonstrate that Epag directly binds to TET2 in the presence of Fe2+ and inhibits its dioxygenase function in cell free system. In cell culture model we observed that nearly 40% of Epag partitioned into to nucleus suggesting its potential TET2 inhibitory role in cells. To test if TPOR signaling activation by Epag has any impact on TET activity we engineered murine cell lines BaF3 and 32D by introducing human TPOR. Irrespective of the status of TPOR-JAK-STAT pathway activation, as reflected in STAT5 phosphorylation, Epag demonstrated a robust TET-inhibition activity. However, in similar experiments peptide agonist of TPOR or another small molecule TPOR agonist Avotrombopag did not have any TET inhibitory effects. Consistent with these observations, Epag but not TPO or Avotrombopag inhibited TET activity in murine BM. Thus, Epag mediated TET2 inhibition in these cells are specific and independent of TPOR signaling. Consistent with previous reports, we observed that Epag treatment can significantly expand murine HSPCs even in the absence of TPOR signaling activation. The hematopoietic activity of Epag in murine system is dependent on its ability to inhibit TET2 as confirmed by the lack of Epag effects on Tet2-/- HSPCs. We further confirmed that TET-inhibition by Epag is central for its ability to expand HSPCs in murine model under in vivo settings using BM transplant experiments. We transplanted a mixture of cells derived from Tet2+/+ Pep Boy (CD45.1, 95%) and Tet2-/- C57BL/6 (CD45.2, 5%) into lethally irradiated recipient PepBoy mice. Epag treatment of mice reconstituted with the mixture of BM cells increases the fraction of Tet2+/+ BM cells only. Interestingly, Epag mimic loss of Tet2 in vivo as observed in the expansion of myeloid compartments of Tet2+/+ fractions reflected in CD11b-CD11c+, CD11b+CD11c-, CD11b+CD11c-Ly6C+Ly6G- (monocytes) and CD11b+CD11c-Ly6ClowLy6G+ (neutrophils). We did not observe any significant change in B220+, CD4+ or CD8+ lymphoid populations. On the contrary, we observed a growth restrictive effect of Epag treatment on Tet2-/- myeloid cells. Consistent with our hypothesis that Epag inhibits TET-dioxygenase activity, we observed hypermethylation in the mononuclear cells derived AA patients (n= 16) after Epag treatment. This was further confirmed in ex vivo Epag treatment of BM cells derived from healthy donor. Epag treatment significant increase clonogenic expansion of normal BM cells but not TET2 mutant myeloid cells. In summary, here we demonstrate that Epag is a potent inhibitor of TET-dioxygenase and its biological consequences are independent of TPOR-JAK-STAT activation. Epag treatment mimics TET2 loss of function in AA cells, however it restricts clonal expansion of TET2 mutant myeloid cells. Disclosures Patel: Alexion: Other: educational speaker. Nazha:Jazz: Research Funding; Incyte: Speakers Bureau; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau. Saunthararajah:EpiDestiny: Consultancy, Current equity holder in private company, Patents & Royalties: University of Illinois at Chicago. Sekeres:Pfizer: Consultancy; BMS: Consultancy; Takeda/Millenium: Consultancy. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

Mechanisms of Therapeutic Response to Tipifarnib in a Mouse Model of Angioimmunoblastic T-Cell Lymphoma

Angioimmunoblastic T-cell lymphomas (AITLs) are aggressive non-Hodgkin lymphomas arising from malignant transformation of follicular helper T-cells (Tfh) and are associated with limited response to intensified chemotherapy and poor prognosis. The genomic landscape of AITL is characterized by loss of function mutations in epigenetic regulators, including TET2, and the highly prevalent RHOA G17V mutation identified in almost 70% of AITL cases (Palomero et al., 2014). Using a combination of loss of TET2 and expression of RHOA G17V in CD4+ T-cells, our group developed a new mouse model of AITL, which accurately recapitulates the features of the human disease (Cortes et al., 2018). Our model constitutes a relevant platform for studying AITL pathogenesis and developing experimental therapeutic approaches for the treatment of this disease. Tipifarnib is a farnesyltransferase inhibitor that has been linked to reduced cell proliferation and increased apoptosis in a variety of solid tumors and leukemias (Alsina et al., 2004; Kirschbaum et al., 2011). Interestingly, a phase II study of tipifarnib in patients with relapsed/refractory lymphoma indicated that, while tipifarnib had a modest anti-lymphoma activity within the whole cohort, it elicited an overall response rate of 31% within the Peripheral T-cell Lymphoma cases, particularly AITL (Witzig et al, 2011). However, the molecular bases for the therapeutic activity of tipifarnib in AITL are still unknown. To understand the role and the mechanisms of action of tipifarnib in AITL, we have used our TET2-/-RHOA G17V mouse AITL lymphoma model. Using this model, we first identified that treatment in vitro with increasing doses of tipifarnib showed limited effect on the viability of AITL lymphoma cells; however, when mouse lymphoma cells were co-cultured in the presence of splenocytes as supporting cells, tipifarnib induced a strong decrease in cell viability and proliferation, suggesting that tipifarnib might be exerting its therapeutic effects not only on the tumor cells, but also indirectly by regulating the tumor microenvironment. Indeed, tipifarnib treatment in a lymphoma model in vivo led to significantly decreased tumor load and substantial reduction of lymphoma cell infiltration in solid organs, demonstrating a consistent and strong anti-tumor effect of tipifarnib in AITL in vivo. Mechanistic analysis of signaling pathways regulated by tipifarnib indicated that tipifarnib induced downregulation of surface expression of the CXCR4 receptor in tumor cells and increased the circulating levels of its ligand, CXCL12, resulting in an effective increase of the CXCL12/CXCR4 ratio. In addition, our data revealed a significant tipifarnib-induced decrease in the levels of IFN-G, TNF-A, IL-10 and IL-17 cytokines. We also performed gene expression profiling in sorted AITL lymphoma cells and sorted stroma splenocytes from mice that had been treated with tipifarnib or vehicle control. Interestingly, most of the genes specifically affected by tipifarnib were expressed in stromal cells, where we detected an upregulation of Fas-L and, more notably, of multiple genes associated with NK cell function and differentiation (CD226, CD244a, Klre1, Xcl1 and Eomes). CYBERSORT analysis of hematopoietic cell populations indicated that treatment with tipifarnib in our mouse lymphoma model is associated with enrichment in NK signatures and a potential decrease of monocytes and neutrophils within the tumor microenvironment. In summary, we have demonstrated that tipifarnib has a strong anti-lymphoma effect on a mouse model of AITL and that this effect might be mediated by the recruitment and/or activation of different hematopoietic cell populations in the tumor microenvironment that can alter the survival and proliferation of lymphoma cells. Disclosures Palomero: Kura Onclology: Research Funding.

Inhibition of Critical DNA Dioxygenase Activity in IDH1/2 Mutant Myeloid Neoplasms

Neomorphic mutations in IDH1/2 producing R-2-Hydroxyglutrate (R-2HG), are common in myeloid malignancies and in various solid cancers. A diffuse hypermethylated status is the biological consequence of the R-2HG-mediated inhibition of several α-ketoglutarate (αKG)-dependent enzymes including DNA dioxygenases TET1, TET2 and TET3.1,2 Specifically, the inhibition of TET2, either induced by the interaction with R-2HG or by direct genomic silencing (as in case of TET2 loss of function mutations) is responsible for the block of the DNA cytosine demethylation pathway, inducing changes in expression patterns, (e.g. decreasing expression of tumor suppressor genes) and impairing execution of differentiation programs. Analysis of genomic data from a Cleveland Clinic (CCF) cohort of AML/MDS patients combined in a meta-analytic fashion with BeatAML3 and Tumor Cancer Genome Atlas (TCGA) cohorts (1119 profiled patients) showed that IDH1/2 mutations are mutually exclusive (only 3% [N=4/106] of AML IDH1/2 mutated cases had TET2 mutations, expected to be at a frequency of 18% [N=110/585] in IDH1/2 wild type cases, p=.000125). In this scenario we suggest that the loss of TET2 activity due to mutations prevents the expansion of IDH1/2 mutant myeloid neoplasms (MNs) because of phenotypic redundancies inducing synthetic lethality. With this premise we stipulated that a critical level of DNA dioxygenase activity exists and thus cells with low TET2 activity will not tolerate further inhibition by R-2HG. Here we propose to apply pharmacologic inhibition of TET2 to produce an additive effect on DNA dioxygenases to investigate whether this will result in a synthetic lethality of IDH1/2 mutant cells. Specifically we hypothesize that TET-dioxygenase inhibition may be implemented as a possible therapeutic strategy in neomorphic IDH1/2 mutant MNs. To explore this hypothesis we conducted a series of in vitro experiments in different isogenic cell lines expressing either mutant or wild type IDH1 or IDH2, that were simultaneously mutant, wild type (WT) or knock down (KD) for TET2 (TF1-IDH2R140Q, K562-IDH1R132C both WT for TET2 gene, and K18-IDH1R132CTET2KD and SIGM5-IDH1R132C TET2MT, both with a doxycycline inducible promoter for mutant IDH1). First we found that the doxycycline induction of ectopic IDH1R132C expression led to R-2HG increase (~10,000-fold over the baseline) and induced cell death in TET2-deficient cells (experiments conducted in SIGM5-IDH1R132C cells showing 70% of decrease in cell growth after five days of IDH induction with doxycycline), confirming the cytotoxic effect of cellular R-2HG. We then tested in IDH1/2MT cells sensitivity towards TETi76, a specific TET inhibitor designed on R-2HG scaffold (with more than 200 fold potency compared to R-2HG in cell-free assays of 5-hydroxy-methyl cytosine [5hMC] production).4 This compound showed particular selectivity towards inhibition of DNA dioxygenases when a set of 23 other dioxygenase inhibitors were screened. Most importantly, consistent with our hypothesis, TETi76 preferentially inhibited the proliferation of IDH1/2MT cells either following doxycycline-induction both in TET2WTand TET2 deficient models (K562 TET2WT, K18 TET2kD, SIGM-5 TET2MT cell lines), or in models not carrying the inducible promoter (TF1 TETWT) (Growth inhibition: 20-25% in IDHWT vs 70-80% in IDHMT cell lines after 72h of co-culture with TETi76 treatment for concentrations ranging between 1 and 5 µM. P-value range: 0.04-0.001 in pairwise comparisons with untreated controls). Overall, our findings are consistent with the idea that neomorphic IDH1/2MT phenocopies loss of function TET2MT, through R-2HG, down-modulating pathways fundamental for cell homeostasis, division and differentiation. If a residual TET-activity is needed for the function of IDH1/2MT cells, the complete block of the residual activity appears to inevitably disrupt this phenotype impairing cell growth and proliferation. This is also in agreement with the paucity of TET3 and TET1 mutation in the context of TET2MT carriers. In summary, results shown here represent an important proof of concept that the increased inhibition of DNA dioxygenase activity, instead of being more leukemogenic, can be synthetically lethal. Our observations may have implications with regard to the therapy of IDH1/2 mutated neoplasms including AML and MDS Disclosures Saunthararajah: EpiDestiny: Consultancy, Current equity holder in private company, Patents & Royalties: University of Illinois at Chicago. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

ZRSR2 Mutation Induced Minor Intron Retention Drives MDS and Diverse Cancer Predisposition Via Aberrant Splicing of LZTR1

Mutations in RNA splicing factors are amongst the most common genetic alterations in myeloid malignancies. Mutations in the splicing factors SF3B1, SRSF2, and U2AF1 occur as heterozygous, missense mutations and have been shown to confer a change-of-function. In contrast, the X chromosome encoded ZRSR2 is enriched in nonsense/frameshift mutations in males, consistent with loss of function. To date however, we do not understand the basis for enrichment of ZRSR2 mutations in leukemia. Moreover, ZRSR2 is the only one of these factors that primarily functions in the minor spliceosome. While most introns are spliced by the major spliceosome, a small subset (<1%) of introns are recognized by a separate complex, the minor spliceosome. Although minor (or "U12") introns are present in only ~800 genes in humans, their sequences and positions are highly evolutionarily conserved - more so than their U2 counterparts. The high conservation of minor introns suggests key regulatory roles yet few functional roles for the minor spliceosome in regulating biological phenotypes are known. The rarity and conservation of minor introns offered a unique opportunity to investigate splicing factor mutations and identify potential tissue-specific roles of the minor spliceosome. Modeling loss-of-function mutations in ZRSR2 via a mouse model for induced deletion of Zrsr2 revealed strikingly enhanced self-renewal of Zrsr2-deficient male and female hematopoietic cells (Fig. A-C). This was in stark contrast to the effects of hotspot mutations in Sf3b1and Srsf2 and similar to those of Tet2 loss on increasing self-renewal and numbers of HSCs. Zrsr2 loss was also associated increased myeloid cells in the blood and long-term hematopoietic stem cells (HSCs) in the marrow (Fig. C). To understand the mechanistic basis by which ZRSR2 loss causes aberrant HSC self-renewal, we quantified transcriptome-wide splicing patterns in MDS patients. ZRSR2-mutant samples had widespread, dysfunctional recognition of minor introns- 48% of minor introns exhibiting significantly increased retention (Fig. D). We next systematically mimicked the effects of nonsense-mediated decay caused by minor intron retention in ZRSR2-mutants. Every gene containing a ZRSR2-regulated minor intron was targeted by 4 sgRNAs via a positive-enrichment CRISPR screen using pools of lentiviral sgRNAs in cytokine-dependent human and mouse hematopoietic cell lines. This identified several minor intron-containing genes whose downregulation conferred cytokine independence. Strikingly, just one gene was enriched in all lines (Fig. E): LZTR1, a cullin-3 adaptor for ubiquitin-mediated suppression of RAS-related GTPases which is subject to loss-of-function mutations in several cancers and the RASopathy Noonan Syndrome. Minor intron retention in LZTR1 correlated with reduced LZTR1 protein in MDS patients (Fig. F-G). Inducing mutations in either the protein-coding region of LZTR1 or its minor intron resulted in cytokine independence (Fig. H), reduced LZTR1, and dramatic accumulation of RIT1, a RAS GTPase substrate of LZTR1. In a Noonan Syndrome family wherein one child died of AML, the mother and all children carried an intronic mutation within LZTR1's minor intron (Fig. I-J). Fibroblasts from each family member revealed clear LZTR1 minor intron retention with impaired LZTR1 protein expression and RIT1 accumulation in subjects bearing the LZTR1 minor intron mutation (Fig. J). We next interrogated LZTR1 minor intron splicing across all cancers in the TCGA. While LZTR1's minor intron was efficiently excised in normal samples, a notable subset of tumors in almost all cancer types exhibited significantly increased retention within LZTR1's minor intron. These data indicate LZTR1 is frequently dysregulated via perturbed minor intron splicing - much more so than by protein-coding mutations alone. Here we uncover a heretofore unrecognized role of minor intron excision in regulating HSC self-renewal, a molecular link between ZRSR2 mutations and aberrant LZTR1 splicing and expression, and frequent LZTR1 minor intron retention in diverse cancers and cancer predisposition syndromes. Given frequent post-transcriptional disruption of LZTR1 in the absence of protein-coding mutations, our data additionally motivate study of other cancer-associated minor intron-containing genes which may be dysregulated via similar, and as-yet-undetected, aberrant splicing. Figure Disclosures Abdel-Wahab: Merck: Consultancy; Envisagenics Inc.: Current equity holder in private company; H3 Biomedicine Inc.: Consultancy, Research Funding; Janssen: Consultancy.

Loss of Tet2 affects platelet function but not coagulation in mice.

Ten-eleven translocation 2 (TET2) functions as a methylcytosine dioxygenase that catalyzes the iterative oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. TET2 has been shown to be crucial for the maintenance and differentiation of hematopoietic stem cells, and its deletion and/or mutations results in the expansion of HSPCs, and leads to hematological malignancies. TET2 mutations were found in a variety of hematological disorders such as CMML (60%), MDS (30%), MPN (13%) and AML (20%). Interestingly, it was shown that CMML patients with TET2 mutation exhibited fewer platelets than CMML patients without TET2 mutation. However, the role and function of TET2 in platelet hemostasis and thrombogenesis is not well defined. Here in this study, using a genetically engineered Tet2 deletion mouse model, we found that the absence of Tet2 caused a decrease in the proportion of MEP cells and hyperploid megakaryocytes. Additionally, Tet2-deficient mice displayed impaired platelet activation and aggregation under stimulation of ADP and low concentrations of thrombin, although the modestly compromised platelet function and MEP differentiation in Tet2-deficient mice could be compensated without affecting blood coagulation function. Our study indicate that Tet2 deficiency leads to mild impairment of platelet function and thrombopoiesis in mice.

Degradation of 5hmC-marked stalled replication forks by APE1 causes genomic instability.

Synthetic lethality between poly(ADP-ribose) polymerase (PARP) inhibition and BRCA deficiency is exploited to treat breast and ovarian tumors. However, resistance to PARP inhibitors (PARPis) is common. To identify potential resistance mechanisms, we performed a genome-wide RNAi screen in BRCA2-deficient mouse embryonic stem cells and validation in KB2P1.21 mouse mammary tumor cells. We found that resistance to multiple PARPi emerged with reduced expression of TET2 (ten-eleven translocation), which promotes DNA demethylation by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethycytosine (5hmC) and other products. TET2 knockdown in BRCA2-deficient cells protected stalled replication forks (RFs). Increasing 5hmC abundance induced the degradation of stalled RFs in KB2P1.21 and human cancer cells by recruiting the base excision repair-associated apurinic/apyrimidinic endonuclease APE1, independent of the BRCA2 status. TET2 loss did not affect the recruitment of the repair protein RAD51 to sites of double-strand breaks (DSBs) or the abundance of proteins associated with RF integrity. The loss of TET2, of its product 5hmC, and of APE1 recruitment to stalled RFs promoted resistance to the chemotherapeutic cisplatin. Our findings reveal a previously unknown role for the epigenetic mark 5hmC in maintaining the integrity of stalled RFs and a potential resistance mechanism to PARPi and cisplatin.

Epigenomic analysis of Parkinson's disease neurons identifies Tet2 loss as neuroprotective.

Parkinson's disease (PD) pathogenesis may involve the epigenetic control of enhancers that modify neuronal functions. Here, we comprehensively examine DNA methylation at enhancers, genome-wide, in neurons of patients with PD and of control individuals. We find a widespread increase in cytosine modifications at enhancers in PD neurons, which is partly explained by elevated hydroxymethylation levels. In particular, patients with PD exhibit an epigenetic and transcriptional upregulation of TET2, a master-regulator of cytosine modification status. TET2 depletion in a neuronal cell model results in cytosine modification changes that are reciprocal to those observed in PD neurons. Moreover, Tet2 inactivation in mice fully prevents nigral dopaminergic neuronal loss induced by previous inflammation. Tet2 loss also attenuates transcriptional immune responses to an inflammatory trigger. Thus, widespread epigenetic dysregulation of enhancers in PD neurons may, in part, be mediated by increased TET2 expression. Decreased Tet2 activity is neuroprotective, in vivo, and may be a new therapeutic target for PD.

Abstract MP153: TET2 Regulates Type I Interferon Signaling in the Hematopoietic Response to Myocardial Infarction

Myocardial infarction (MI) elicits a rapid and vigorous reaction from the bone marrow hematopoietic compartment, inducing a massive efflux of myeloid first responders into the bloodstream. These cells traffic to the infarct, where they mediate cardiac remodeling and repair through inflammatory signaling and recruitment of additional immune cells to the injured myocardium. A hyperinflammatory myeloid compartment, as is produced by mutations in epigenetic regulator TET2 associated with clonal hematopoiesis, can thus drive adverse cardiac remodeling after MI and accelerate progression to heart failure. Whether loss of TET2 alters the transcriptional landscape of MI-induced myelopoiesis remains to be investigated in an unbiased fashion. Here, we performed single-cell RNA sequencing of >16,000 bone marrow myeloid cells isolated from wild-type and Tet2 -/- mice after MI to characterize the emergency hematopoietic response in the presence and absence of TET2. Our data capture distinct transitional states of myeloid lineage commitment and maturation, originating from myeloid progenitors and progressing along divergent granulocytic and monocytic differentiation trajectories. Additionally, we delineate a subpopulation of interferon (IFN)-activated myeloid progenitors, monocytes, and neutrophils characterized by the concerted upregulation of various Type I IFN-stimulated genes, and find the fraction of IFN-activated cells, as well as the degree of activation, to be markedly higher in Tet2 -/- mice. We have previously described activation of this pathway after MI in mice, and demonstrated cardioprotective effects of its genetic or pharmacological inhibition. Our findings reveal heightened activation of the antiviral Type I interferon response among bone marrow myeloid cells of Tet2 -/- mice during MI-induced emergency hematopoiesis. This highlights IFN signaling as a potential candidate driver of cardiovascular pathologies (including atherosclerosis, myocardial infarction, and heart failure) associated with TET2-mediated clonal hematopoiesis. Further studies are necessary to investigate whether Tet2 -/- mice exhibit enhanced response to blockade of Type I IFN signaling after MI, and to determine whether myeloid cells of TET2 -mutant humans are similarly activated.

HDAC4 inhibition disrupts TET2 function in high-risk MDS and AML.

Aberrant DNA methylation often silences transcription of tumor-suppressor genes and is considered a hallmark of myeloid neoplasms. Similarly, histone deacetylation represses transcription of genes responsible for cell differentiation/death. A previous clinical study suggested potential pharmacodynamic antagonism between histone deacetylase inhibitors (HDACi) and DNA hypomethylating agents (HMA). Herein, to determine such antagonism, we used MDS/AML lines and NHD13 transgenic mice, and demonstrated that treatment with the pan-HDACi suberoylanilide hydroxamic acid (SAHA) significantly decreased TET2 expression and global 5-hydroxymethylcytosine (5hmC) levels. Mechanistically, our RNAi screen revealed that HDAC4 was responsible for maintaining TET2 levels. Accordingly, HDAC4 knockout reduced expression levels of MTSS1, a known TET2 target, an event associated with decreased 5hmC enrichment on the MTSS1 enhancer. Retrospective analysis of GEO datasets demonstrated that lower HDAC4 levels predict worse prognosis for AML patients. In an MDS-L xenografted immunodeficient mouse model, vitamin C co-treatment prevented TET2 loss of activity seen following SAHA treatment. Accordingly, vitamin C co-treatment further reduced MDS-L cell engraftment relative to SAHA alone. In summary, our findings suggest that co-administration of a TET2 agonist with pan-HDACi treatment could effectively counter potential diminution in TET2 activity resulting from pan-HDACi treatment alone, providing a rationale for evaluating such combinations against high-risk MDS/AML.

Loss of TET2 boosts cell fitness

The loss of TET2 favors a pathogenic switch to a stem cell–like state in cancer.

Divergent Effects of Dnmt3a and Tet2 Mutations on Hematopoietic Progenitor Cell Fitness.

SummaryThe DNA methylation regulators DNMT3A and TET2 are recurrently mutated in hematological disorders. Despite possessing antagonistic biochemical activities, loss-of-function murine models show overlapping phenotypes in terms of increased hematopoietic stem cell (HSC) fitness. Here, we directly compared the effects of these mutations on hematopoietic progenitor function and disease initiation. In contrast to Dnmt3a-null HSCs, which possess limitless self-renewal in vivo, Tet2-null HSCs unexpectedly exhaust at the same rate as control HSCs in serial transplantation assays despite an initial increase in self-renewal. Moreover, loss of Tet2 more acutely sensitizes hematopoietic cells to the addition of a common co-operating mutation (Flt3ITD) than loss of Dnmt3a, which is associated with a more rapid expansion of committed progenitor cells. The effect of Tet2 mutation manifests more profound myeloid lineage skewing in committed hematopoietic progenitor cells rather than long-term HSCs. Molecular characterization revealed divergent transcriptomes and chromatin accessibility underlying these functional differences.

DNA methylation disruption reshapes the hematopoietic differentiation landscape.

Mutations in genes involved in DNA methylation (DNAme; e.g., TET2, DNMT3A), are frequently observed in hematological malignancies1–3 and clonal hematopoiesis4,5. Applying single-cell sequencing to murine hematopoietic stem and progenitor cells, we observed that these mutations disrupt hematopoietic differentiation, causing opposite shifts in the frequencies of erythroid vs. myelo-monocytic progenitors upon Tet2 or Dnmt3a loss. Notably, these shifts trace back to transcriptional priming skews in uncommitted hematopoietic stem cells (HSCs). To reconcile genome-wide DNAme changes with specific erythroid vs. myelo-monocytic skews, we provide evidence in support of differential sensitivity of transcription factors due to biases in CpG enrichment in their binding motif. Single-cell transcriptomes with targeted genotyping showed similar skews in transcriptional priming of DNMT3A-mutated human clonal hematopoiesis bone marrow progenitors. These data show that DNAme shapes the hematopoietic differentiation topography, and support a model in which genome-wide methylation changes are transduced to differentiation skews through biases in transcription factor binding-motif CpG enrichment.

ABCA1 Exerts Tumor-Suppressor Function in Myeloproliferative Neoplasms

SUMMARYDefective cholesterol efflux pathways in mice promote the expansion of hematopoietic stem and progenitor cells and a bias toward the myeloid lineage, as observed in chronic myelomonocytic leukemia (CMML). Here, we identify 5 somatic missense mutations in ABCA1 in 26 patients with CMML. These mutations confer a proliferative advantage to monocytic leukemia cell lines in vitro. In vivo inactivation of ABCA1 or expression of ABCA1 mutants in hematopoietic cells in the setting of Tet2 loss demonstrates a myelosuppressive function of ABCA1. Mechanistically, ABCA1 mutations impair the tumor-suppressor functions of WT ABCA1 in myeloproliferative neoplasms by increasing the IL-3Rβ signaling via MAPK and JAK2 and subsequent metabolic reprogramming. Overexpression of a human apolipoprotein A-1 transgene dampens myeloproliferation. These findings identify somatic mutations in ABCA1 that subvert its anti-proliferative and cholesterol efflux functions and permit the progression of myeloid neoplasms. Therapeutic increases in HDL bypass these defects and restore normal hematopoiesis.

Reduction of Tet2 exacerbates early stage Alzheimer's pathology and cognitive impairments in 2×Tg-AD mice.

Abnormal modification of 5-hydroxymethylcytosine (5hmC) is closely related to the occurrence of Alzheimer's disease (AD). However, the role of 5hmC and its writers, ten-eleven translocation (Tet) proteins, in regulating the pathogenesis of AD remains largely unknown. We detected a significant decrease in 5hmC and Tet2 levels in the hippocampus of aged APPswe/PSEN1 double-transgenic (2×Tg-AD) mice that coincides with abundant amyloid-β (Aβ) plaque accumulation. On this basis, we examined the reduction of Tet2 expression in the hippocampus at early disease stages, which caused a decline of 5hmC levels and led young 2×Tg-AD mice to present with advanced stages of AD-related pathological hallmarks, including Aβ accumulation, GFAP-positive astrogliosis and Iba1-positive microglia overgrowth as well as the overproduction of pro-inflammatory factors. Additionally, the loss of Tet2 in the 2×Tg-AD mice at 5months of age accelerated hippocampal-dependent learning and memory impairments compared to age-matched control 2×Tg-AD mice. In contrast, restoring Tet2 expression in adult neural stem cells isolated from aged 2×Tg-AD mice hippocampi increased 5hmC levels and increased their regenerative capacity, suggesting that Tet2 might be an exciting target for rejuvenating the brain during aging and AD. Further, hippocampal RNA sequencing data revealed that the expression of altered genes identified in both Tet2 knockdown and control 2×Tg-AD mice was significantly associated with inflammation response. Finally, we demonstrated that Tet2-mediated 5hmC epigenetic modifications regulate AD pathology by interacting with HDAC1. These results suggest a combined approach for the regulation and treatment of AD-related memory impairment and cognitive symptoms by increasing Tet2 via HDAC1 suppression.

EP300 Suppresses Leukemia Development in Myelodysplastic Syndrome through Myb Repression

Background Epigenetic dysregulation is a hallmark feature of myelodysplastic syndrome (MDS) as epigenetic regulator genes, such as ASXL1, TET2, and SRSF2, are highly mutated in MDS patients. Tet2 (ten-eleven translocation-2, a methylcytosine deoxygenase) mutations are found in ~20% of MDS patients; they are also seen in patients with acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPN). Loss of Tet2 in hematopoietic cells leads hematopoietic defects including enhanced hematopoietic stem cell (HSC) self-renewal, myeloid expansion and an increased propensity to develop MDS or AML. The lysine acetyltransferase (KAT) p300 (encoded by the EP300 gene) plays pivotal roles in essential cellular functions including proliferation, differentiation and signal transduction. However, the function of p300 in hematopoietic malignancies is clearly cell-context dependent, as we have shown that p300 functions as an oncogene in AML1-ETO-driven AML, and a tumor suppressor gene in NUP98-HOXD13-driven MDS. The EP300 and the related CREBBP genes are infrequently mutated in MDS patients. Methods To investigate the role of p300 in MDS driven by lack of Tet2, we generated a mouse model carrying a hematopoietic-specific EP300 conditional knockout on a Tet2-null background and characterized the effect of p300 loss on HSC function and the pathogenesis of MDS and AML. We also explored the therapeutic potential of manipulating p300 KAT activity to affect the course of MDS driven by Tet2 loss. Results Compared to the Tet2-/- mice, the EP300Δ/ΔTet2-/- (DKO) mice had shortened survival and an accelerated onset of AML, with increased blasts in the bone marrow and peripheral blood, anemia, splenomegaly, and universal presence of granulocytic sarcomas, demonstrating that depletion of p300 enhances the leukemogenicity of Tet2-/- HSC. The DKO mice showed enhanced hematopoietic stem/progenitor cell (HSPC, defined as Lin-Sca1+Kit+ cell) proliferation and an increased HSPC pool only 2 weeks after p300 deletion, far in advance of the development of any disease manifestations. In contrast, deletion of p300 in wild type (wt) mice affected neither HSPC proliferation, nor pool size. To further define the mechanisms by which these two genetic events cooperate to drive the development of MDS/AML, we assessed the DNA methylation status and gene expression signatures of HSPCs isolated from wt, Tet2-/- and DKO mice. As expected, loss of Tet2 leads to a genome-wide decrease in 5-hmC, whereas loss of p300 does not change 5-hmC distribution across the genome. Interestingly, DKO HSPCs showed local gains of 5-hmC at a variety of genes (compared to Tet2-null HSPCs), including at the c-Myb gene, which is a known oncogenic driver of AML. Gene Set Enrichment Analysis (GSEA) revealed that p300 loss in Tet2-/- HSPCs interferes with leukocyte differentiation, enhances proliferation and inhibits apoptosis. ChIP-X Enrichment Analysis (ChEA) further also indicated that among the differentially expressed genes, between DKO HSPCs and Tet2-null HSPCs, are potential Myb target genes. Given this data and the RNA-Seq and Q-PCR results that showed elevated levels of Myb mRNA in the DKO HSPCs compared to the Tet2-/- HSPCs, we examined the biological effects of knocking down (KD) Myb. Myb KD reduced the colony-forming capacity of the DKO HSPCs, but not the Tet2 -/- HSPCs, identifying its potential role in mediating the effect of p300 on MDS prone HSPCs. These results indicate that loss of p300 in Tet2-null HSPCs enhances their leukemogenicity through elevated Myb expression and activity. To determine whether increasing p300 KAT activity could have an opposite effect on MDS cells, we utilized I-CBP112, which binds p300 and increases its KAT activity and ability to stimulate H3K18 acetylation. Exposure to I-CBP112 did in fact eliminate the indefinite self-renewal capacity of Tet2-/- HSPCs, as measured by serial replating assays, further highlighting the importance of p300 KAT activity in regulating the behavior of Tet2-/- HSPCs. Additional in vivo studies of the effects of I-CBP112 on the course of MDS/AML driven by the absence of Tet2 are ongoing. Conclusions This work clearly indicates that the EP300 gene can play a tumor suppressor role in MDS/AML driven by loss of Tet2- by repressing Myb expression and activity. It also suggests that the enhancement of p300's KAT activity by use of small molecules, could be an effective therapeutic strategy for MDS/AML. Disclosures Cole: Abbvie: Consultancy; Acylin Therapeutics: Equity Ownership.

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.

Talazoparib Treatment Preferentially Depletes Cohesin-Mutant Clones in New In Vivo Models of Cohesin-Mutant Myeloid Diseases

Cohesin is a multisubunit protein complex that is involved in sister chromatid cohesion, post-replicative DNA repair and transcriptional regulation, and is frequently mutated in cancer. STAG2 is the most recurrently mutated cohesin gene in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (AML) but no STAG2-mutant disease models currently exist to study the natural evolution of cohesin-mutant myeloid disease, or to test therapeutic vulnerabilities. Our previous workimplicates PARP1 inhibition as a therapeutic vulnerability in STAG2-mutant AML cell lines, which display impaired replication fork progression and accumulation of double-strand DNA breaks.We developed a syngeneic mouse model of cohesin-mutant myeloid disease mimicking the phenotype seen in human MDS, in which STAG2 mutations arise as clonal secondary lesions in the background of clonal hematopoiesis. We used this model to test the effect of sequential acquisition of Tet2 and Stag2 mutations on mouse hematopoiesis, and in vivo therapeutic vulnerabilities including sensitivity to PARP1 inhibition. In parallel, we developed patient-derived xenograft (PDX) models of cohesin-mutant AML to extend our findings. Hematopoietic stem and progenitor cells (Lineage-, Sca1+, c-Kit+ cells) harvested from Mx1-Cre;Cas9 heterozygous C57BL.6 mice were transduced with sgRNA targeting Tet2 or non-targeting sgRNA and transplanted into lethally irradiated SJL recipient mice. Engraftment and clonal expansion of Tet2-mutant cells was confirmed with fluorescent protein reporter expression and next-generation sequencing (NGS), and was not associated with any overt phenotype, similar to human clonal hematopoiesis of indeterminate potential (CHIP). c-Kit enriched bone marrow cells from mice with Tet2-mutant CHIP were subsequently transduced with sgRNA targeting Stag2 or non-targeting sgRNAs and re-transplanted into recipient mice. Mice were serially followed for up to 9 months and showed increased clonal expansion of Tet2/Stag2-mutant cells over Tet2-only mutant cells. In contrast to Tet2-only mutant mice, Tet2/Stag2-mutant mice developed leukocytosis, absolute monocytosis, anemia, and thrombocytopenia. Bone marrow evaluation of Tet2/Stag2 mice demonstrated increased hemophagocytosis and fewer megakaryocytes in comparison to Tet2-only mice and NGS confirmed predicted loss of function frameshift mutations in Tet2 and Stag2. We therefore generated a new model of cohesin-mutant myeloid disease recapitulating sequential acquisition of Stag2mutations in the context of an initiating epigenetic lesion as seen in patients with cohesin-mutant MDS. Tet2/Stag2 andTet2-mutant clones and the associated hematologic phenotypes were serially transplantable and allowed for genotype-specific in vivo drug testing of the PARP1 inhibitor talazoparib. Forty recipient mice transplanted with Tet2 or Tet2/Stag2 mutant bone marrow cells were stratified into treatment groups with talazoparib or vehicle. Expression of congenic markers and fluorescent reporters linked to Tet2 and Stag2 sgRNA expression were used to monitor mice during 4 weeks of treatment. We observed normalization of leukocytosis, monocytosis and thrombocytopenia in Tet2/Stag2 mutant mice treated with talazoparib. In addition, Tet2/Stag2-mutant mice but not Tet2-only mice treated with talazoparib demonstrated a significant loss of mutant clones as determined by flow cytometry and NGS. To further extend these findings in primary human cells, we developed serially transplantable PDX models of cohesin-mutant AML and performed in vivo drug testing with talazoparib. We noted increased survival of cohesin-mutant PDX models treated with talazoparib as compared to vehicle. We demonstrate the development of a syngeneic mouse model of Stag2-mutated myeloid disease through sequential introduction of Tet2 or Stag2 mutations in primary mouse hematopoietic stem and progenitor cells. Mice with loss of Tet2 and Stag2 recapitulate hematologic parameters mimicking myelodysplastic/myeloproliferative neoplasms in humans. In vivo treatment with talazoparib demonstrates selective depletion of cohesin mutations in syngeneic and PDX models. The effect of talazoparib monotherapy in cohesin-mutated AML or MDS with excess blasts is under investigation in a pilot proof-of-concept study (ClinicalTrials.gov Identifier NCT03974217). Disclosures Garcia: Abbvie: Research Funding; Genentech: Research Funding.

TET2 Loss Accelerates Leukemogenesis By Disrupting Mismatch Repair Proteins

TET2 mutations (TET2MT) occur around 40% of myeloid neoplasms (MN) and often constitute founder hits, as concluded from patient's clonal architecture and subclinical TET2MT clones in healthy individuals at risk for MN. The mechanisms of TET2MT in leukemogenesis involve at least two potential mechanisms: i) TET2 mutations could lead to stem cell expansion and altered differentiation and/or ii) they may convey a mutator phenotype with progression due to a higher rate of subsequent genetic hits. Both mechanisms are compatible with the weak driver function of TET2MT. Here, we studied whether TET2MT predispose to additional oncogenic mutations through faulty DNA repair,i.e., whether they produce a clonally acquired mutator phenotype. TET2MT lead to the inhibition of passive demethylation. Alternatively, they impair demethylation via excision repair of 5fC/5caC. Moreover, global 5mC accumulation may increase background C>T mutation rates via 5mC deamination linking TET2 to base excision repair machinery. We investigated this hypothesis because of the finding of increased mutability found in Tet2KO and Tet2kdin the mouse as determined by the numbers of coexisting subclonal hits in Tet2MTdisease (Pan F et.al., Nature Comm, 2017). Using WES analyses (n=435), we identified 95 cases with TET2MT stringently selected for high VAF and most damaging effects as confirmed by 2D-UPLC-MS/MS assay showing low levels of the TET2-dependent DNA oxidation products. To test whether these TET2MT indeed predisposed to additional mutations, we enumerated somatic SNV vs. TET2WT cases. Sequencing of MSH2, MSH3, MSH6, MLH1, CHEK2, and BRCA2 excluded the potential effects of alteration in these genes on our results. Similarly, the MMR gene expression were not significantly different among 20 TET2MT MDS, 71 TET2WT MDS pts, and 17 controls, indicating MMR downregulation was not the culprit. Consistent with our theory, TET2MT pts had a 1.5-fold increase in median WES SNV (p<.0001), particularly at 5hmC sites. Pts with a TET2MT VAF>50% had a 2.1-fold increase in median WES SNV (p=.03), consistent with a gene-dose effect. HeLa TET2kd cells also had a 24-fold increase in spontaneous mutations, reversed with TET2 cDNA knock-in. In mice, 1.4-fold increased mutagenicity at Tet2-dependent active demethylation sites was found, in a manner also suggestive of a defective MMR. TET2MT cases (n=5) were shown to be microsatellite-stable at 5 TET2-independent poly-dA microsatellite loci, suggesting that hypermutagenicity was not driven by global MMR dysfunction and may occur only at CpG-containing microsatellites. MOLM-13 TET2kd increased PARP inhibitor sensitivity by almost 3-fold, suggesting that TET2kd renders cells vulnerable to DNA damage. In murine cells, by overexpressing Flag/V5-tagged TET2 in MEL cells and subjecting them to protein affinity purification, we identified MSH6 as a novel TET2 binding partner. Using HPRT mutability assay, we measured mutational frequency in the HPRT1 gene in control, TET2kd, MSH6kd,and TET2kd/MSH6kd HeLa cells. Moreover, HPRT1 mutational frequency in TET2kd HeLa cells increased 24-fold compared to parental controls while MSH6kd HeLa cells had a further 10-fold increase in HPRT1, likely reflecting the presence of TET2-independent mechanisms for MSH6-mediated MMR. Intriguingly, the dual TET2kd/MSH6kd HeLa cells had equivalent MSI and mutational frequencies to the MSH6kd HeLa cells, supporting MSH6-dependence for the mutator phenotype induced by TET2 loss. TET2:MSH6 interactions were validated in co-immunofluorescence experiments in MEL and MOLM-13 cells, and TET2kd altered MSH6 nuclear co-localization. TET2 immunoprecipitation from human CD34+ cells demonstrated abundant MSH2 binding to TET2, although we were unable to co-precipitate MSH6 in the same experiment. In sum, we uncovered novel connections among TET2, MMR proteins, epigenetic modifications, and genomic instability. Given that MSH2/6 is known to preferentially bind 5hmC, TET2 may target MSH2/6 to TET2-dependent DNA loci. Genomic instability due to TET2 dysfunction may allow therapeutic targeting of DNA repair proteins in the subset of pts with TET2MT-driven MN. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

Innate Immune Signaling Suppresses Acute Leukemia By Modifying MYC Oncogenic Activity

Individuals with clonal hematopoiesis of indeterminant potential (CHIP) are healthy, however they are at an increased risk of developing hematopoietic malignancies. The most frequent mutations in CHIP target DNMT3A and TET2, also are observed in acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), and myelodysplastic syndromes (MDS). These findings indicate that additional alterations are needed for the transition from a pre-leukemic stage to frank leukemia, although the identity of such molecular events remains uncharacterized. To identify cellular states that cooperate with Tet2 loss, we used in vivo RNAi screening and identified the ubiquitin ligase TRAF6 required for malignant transformation of pre-leukemic TET2-deficient hematopoietic stem/progenitor cell (HSPC). Importantly, TRAF6 expression is significantly reduced in 25-50% of AML and MPN patients as compared to healthy controls. Furthermore, TET2 mutations are more strongly correlated with lower expression of TRAF6 as compared to patients with higher TRAF6 expression in certain subsets of AML. To evaluate the consequences of TRAF6 deletion on TET2-deficienct pre-leukemic cells, we generated mice in which TRAF6 and TET2 are conditionally deleted in hematopoietic cells (VavCre;Traf6fl/fl;Tet2fl/fl[DKO]). Traf6KO mice developed a lethal phenotype with signs of MPN, including lymphopenia, neutrophilia, and increased hemoglobin levels; however, this disease was not transplantable. In striking contrast, deletion of TRAF6 in the context of TET2-deficient HSPC resulted in a rapid, penetrant, aggressive, and transplantable MPN/AML. To firmly establish that TRAF6 exhibits tumor suppressor functions, we determined whether physiological levels of TRAF6 overexpression could prevent malignant transformation. Overexpression of TRAF6 in FLT3-ITD mice inhibited malignant myeloid cell expansion in FLT3-ITD mice, and rescued the survival of the animals. To uncover the molecular basis of TRAF6's tumor suppressor function, we performed gene expression profiling and proteomic characterization of TRAF6 ubiquitination substrates in leukemic cells. RNA-sequencing of HSPC revealed that deletion of TRAF6 resulted in a significant overexpression of MYC regulated genes in pre-leukemic HSPC. In support of these findings, the proteomic screen along with extensive in vitro validation experiments identified MYC as a substrate of TRAF6. Unlike the majority of reported ubiquitin-dependent post-translational modifications of MYC, we found that ubiquitination of MYC on Lysine (K) 148 by TRAF6 does not affect its protein stability but rather antagonizes acetylation of MYC on the same lysine and thus suppresses MYC oncogenic activity. We extended these observations to investigate whether inflammatory signaling via Toll-like receptors (TLRs) can antagonize MYC function and suppress leukemic cells. Stimulation of TLRs on leukemic cells resulted in TRAF6-dependent ubiquitination of MYC at K148, which coincided with repositioning of MYC off of its target gene promoters and enhancers, and ultimately in the suppression of leukemic cell viability. Our results demonstrate that TRAF6 functions as a tumor suppressor via its ubiquitination activity that antagonizes K148 acetylation leading to a decrease of MYC transcriptional activity without affecting its protein abundance. Our findings identify TRAF6 as a novel, context-dependent tumor suppressor in myeloid neoplasms, and suggest that innate immune signaling via TLR/TRAF6 could explain why some of the clonal hematopoiesis patients develop AML and others do not. Disclosures Lowe: Blueprint Medicines: Consultancy, Equity Ownership; PMV Pharmaceuticals: Consultancy, Equity Ownership; Petra Pharmaceuticals: Consultancy, Equity Ownership; Constellation Pharma: Consultancy, Equity Ownership; Mirimus: Consultancy, Equity Ownership; ORIC pharmaceuticals: Consultancy, Equity Ownership; Faeth Therapeutics: Consultancy, Equity Ownership. Starczynowski:Kurome Therapeutics: Consultancy.