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.