Therapeutic Targeting of TET-Dioxygenase Deficiency in Myeloid Malignancies

Background: TET-dioxygenases (TET1, TET2, and TET3) are key epigenetic regulators, which require molecular oxygen, αKG, and Fe 2+ to progressively oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine, leading to the demethylation of mCpGs at promoter and enhancer, a key step for mounting an efficient transcription profile and thereby differentiation. Loss of function TET2 mutation (TET2 MT) is frequently observed in myeloid neoplasms (MN). Despite extensive studies of the biochemical mechanisms underlying distorted myeloid differentiation and neoplastic evolution of TET2 MT HSPCs, targeted therapies are lagging. Here, we report the therapeutic utility of preferential targeting of TET2 mutant and TET-dioxygenase deficient cells either by restoring the lost activity to induce differentiation and death or by inhibiting the residual activity to induce synthetic lethality. Methods: Genetic as well as pharmacologic in vitro and in vivo models of AML were used to validate specific targeting of TET2 mutant cells. Knockout and over-expression in the isogenic background of different cells were used to understand the pro-survival and proliferative mechanism of TET2 loss in myeloid cells. The efficacy of small molecule pharmacophore targeting TET-dioxygenase was evaluated in vitro in cell free and cell culture as well as in vivo in a mouse competitive transplant model to demonstrate the proof of therapeutic concept. Result: Ascorbate, used as an activator of αKG/Fe 2+ dependent dioxygenase including TETs, functions via maintaining the redox state of iron (II) in the dioxygenase catalytic site with a half-maximal effective concentration of 13.8 ± 1.3 µM for enhancing TET2activity as measured by an ELISA for 5hmC. However, the reported intracellular cellular concentrations of ascorbate in human remains in the millimolar range, well above the saturating concentration required for TET-dioxygenases activation. Interestingly, ascorbate failed to activate TET in primary mononuclear cells isolated from myeloid neoplasia patients with TET2 mutations. Consistent with the in-vitro observation, we found that ascorbate treatment in physiologically relevant doses has no significant effect in syngeneic murine models (p=0.1). TET-inactivation is a complex phenomenon that is controlled by context-dependent post-translational modifications including lysine acetylation by acetyltransferases and class I and II deacetylases. Therefore, the anti-leukemic effect of the supraphysiological doses of ascorbate in humans and mice leukemia models may not be dependent on TET activation by ascorbate. On the other hand, a comprehensive analysis of the configurations of TET2 MT in myeloid neoplasia (n= 2617) demonstrated a remarkable exclusivity with 2-hydroxyglutarate (2-HG) producing neomorphic IDH1/2 MT. Ectopic inducible expression of IDH1/2 MT induced synthetic lethality of malignant TET2 MT cells. Therefore, themutual exclusivity of TET2 MT and neomorphic IDH1/2 MT is due to synthetic lethality caused by 2-HG production. In addition, we demonstrate that sequential deletion of TET1 and TET3 leads to growth arrest in cellular models, which has never been observed in any myeloid malignancies. Altogether, these observations suggest that malignant TET2 mutant myeloid cells may be vulnerable to inhibition of residual TET-activity coming from TET1 and TET3. Therefore, we synthesized TET-specific inhibitor on 2HG scaffold using iterative computer-aided design, characterized in cell-free as well as cell culture model systems, and selected TETi76 with no cytotoxic effect in normal bone marrow-derived CD34 + cells. TETi76 selectively inhibits TET-dioxygenase activity with IC50 of 1.5, 9.4, and 8.8 μmol/L for TET1/2/3, and it restricts clonal outgrowth of TET2 MT both in vitro and in vivo in malignant and nonmalignant clonal hematopoiesis of indeterminate potential (CHIP). Conclusion: Taken together, here we demonstrated that anti-cancer activity of ascorbate may not come from its role as a TET activator and that a minimum level of TET-dioxygenase activity is required for cell survival, rendering TET2 -mutant malignant cells selectively vulnerable to inhibitors of TET-function. Disclosures Maciejewski: Regeneron: Consultancy; Novartis: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Alexion: Consultancy.

Phage-encoded ten-eleven translocation dioxygenase (TET) is active in C5-cytosine hypermodification in DNA

TET/JBP (ten-eleven translocation/base J binding protein) enzymes are iron(II)- and 2-oxo-glutarate–dependent dioxygenases that are found in all kingdoms of life and oxidize 5-methylpyrimidines on the polynucleotide level. Despite their prevalence, few examples have been biochemically characterized. Among those studied are the metazoan TET enzymes that oxidize 5-methylcytosine in DNA to hydroxy, formyl, and carboxy forms and the euglenozoa JBP dioxygenases that oxidize thymine in the first step of base J biosynthesis. Both enzymes have roles in epigenetic regulation. It has been hypothesized that all TET/JBPs have their ancestral origins in bacteriophages, but only eukaryotic orthologs have been described. Here we demonstrate the 5mC-dioxygenase activity of several phage TETs encoded within viral metagenomes. The clustering of these TETs in a phylogenetic tree correlates with the sequence specificity of their genomically cooccurring cytosine C5-methyltransferases, which install the methyl groups upon which TETs operate. The phage TETs favor Gp5mC dinucleotides over the 5mCpG sites targeted by the eukaryotic TETs and are found within gene clusters specifying complex cytosine modifications that may be important for DNA packaging and evasion of host restriction.

Development and Application of a High Throughput Assay System for the Detection of Rieske Dioxygenase Activity

Herein we report the development of a new periodate-based reactive assay system for the fluorescent detection of the cis-diol metabolites produced by Rieske dioxygenases. This sensitive and diastereoselective assay system...

Statistical Optimisation of Phenol Degradation and Pathway Identification through Whole Genome Sequencing of the Cold-Adapted Antarctic Bacterium, Rhodococcus sp. Strain AQ5-07

Study of the potential of Antarctic microorganisms for use in bioremediation is of increasing interest due to their adaptations to harsh environmental conditions and their metabolic potential in removing a wide variety of organic pollutants at low temperature. In this study, the psychrotolerant bacterium Rhodococcus sp. strain AQ5-07, originally isolated from soil from King George Island (South Shetland Islands, maritime Antarctic), was found to be capable of utilizing phenol as sole carbon and energy source. The bacterium achieved 92.91% degradation of 0.5 g/L phenol under conditions predicted by response surface methodology (RSM) within 84 h at 14.8 °C, pH 7.05, and 0.41 g/L ammonium sulphate. The assembled draft genome sequence (6.75 Mbp) of strain AQ5-07 was obtained through whole genome sequencing (WGS) using the Illumina Hiseq platform. The genome analysis identified a complete gene cluster containing catA, catB, catC, catR, pheR, pheA2, and pheA1. The genome harbours the complete enzyme systems required for phenol and catechol degradation while suggesting phenol degradation occurs via the β-ketoadipate pathway. Enzymatic assay using cell-free crude extract revealed catechol 1,2-dioxygenase activity while no catechol 2,3-dioxygenase activity was detected, supporting this suggestion. The genomic sequence data provide information on gene candidates responsible for phenol and catechol degradation by indigenous Antarctic bacteria and contribute to knowledge of microbial aromatic metabolism and genetic biodiversity in Antarctica.

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.

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.

Context dependent effects of ascorbic acid treatment in TET2 mutant myeloid neoplasia

Loss-of-function TET2 mutations (TET2MT) are common in myeloid neoplasia. TET2, a DNA dioxygenase, requires 2-oxoglutarate and Fe(II) to oxidize 5-methylcytosine. TET2MT thus result in hypermethylation and transcriptional repression. Ascorbic acid (AA) increases dioxygenase activity by facilitating Fe(III)/Fe(II) redox reaction and may alleviate some biological consequences of TET2MT by restoring dioxygenase activity. Here, we report the utility of AA in the prevention of TET2MT myeloid neoplasia (MN), clarify the mechanistic underpinning of the TET2-AA interactions, and demonstrate that the ability of AA to restore TET2 activity in cells depends on N- and C-terminal lysine acetylation and nature of TET2MT. Consequently, pharmacologic modulation of acetyltransferases and histone deacetylases may regulate TET dioxygenase-dependent AA effects. Thus, our study highlights the contribution of factors that may enhance or attenuate AA effects on TET2 and provides a rationale for novel therapeutic approaches including combinations of AA with class I/II HDAC inhibitor or sirtuin activators in TET2MT leukemia.