Lessons from Nature: A Novel Class of TET Inhibitors for TET2 Mutant Associated Diseases

Discovery of many somatic lesions in leukemia enables development of targeted therapies. TET2 is one of the most frequently mutated genes in MDS/related disorders and is also present in a significant proportion of CHIP (clonal hematopoiesis of indeterminate potential) carriers. TET2 mutations (TET2^mt) are mostly loss of function and occur in biallelic, heterozygous and hemi/homozygous configurations. TET2 encodes for Fe²⁺-dependent DNA dioxygenase that utilizes 2-ketoglutarate (αKG) for oxidation of 5-methylcytosine (mC) that results in demethylation either actively, by base excision repair of further oxidation products (fC or caC), or passively, via replication, due to DNA methyltransferase's inability to recognize 5-hydroxymethylcytosine (hmC). TET2^mt are good targets for drug discovery because they often initiate the clonal evolution and are present in a large fraction of patients. However, except for ascorbic acid (AA) applied to augment TET2 activity and hypomethylating agents to which TET2^mtmay be more susceptible, no specific therapies have been conceptualized for TET2^mt disease.

Synthetic lethality can be applied to genetic loci affected by loss of function mutations never occurring in homo/hemi/homozygous configuration. However, many tumor suppressor genes (TSG) are similar to TET2, where biallelic inactivation promotes oncogenicity and thus synthetic lethality would not be directly applicable. However, TET2 has TET1/TET3 homologs and a transient inhibition of TET enzymes could still yield a synthetic lethality (particularly in TET2^mt). The idea for the proposed therapeutic strategy of TET inhibition was conceptualized based on in vivo observations of mutual exclusivity of TET2^mt and IDH1/2 mutations which produces 2HG as a bone-fide natural TET inhibitor. Indeed, in our study of 485 TET2^mt cases only 9 carried IDH1/2^mt (mostly tiny subclones). Conversely, among 157 IDH1/2^mtcases, there were only 11TET2^mt (p=5.5x10^-9) of which 4 were non-deleterious missense alterations or had a small clonal burden. To further support our hypothesis, we knocked in IDH1^mt controlled by the doxycycline-inducible promoter into TET2^mt cells. Induction of IDH1^R132C or IDH1^R132H (or IDH2^mt), expression resulted in rapid cytotoxicity in TET2^mt, and no growth perturbation was observed for TET2^wt cells. Similar results have been observed in mice, where knockdown of TET3 in TET2 background shortened the life span. These observations led us to the idea of developing an aKG antagonist TET specific inhibitors (TETi). Using a structure-guided targeted discovery approach we designed, synthesized and characterized TETi, which demonstrated dose dependent inhibition of dioxygenase activity in a cell-free system. Using an iterative approach of design synthesis and activity, we selected the most potent 'hit', designated as TETi76, for further evaluation. Esterified TETi76 decreased 5hmC production and selectively induced cell death in TET2^mt leukemia cell lines, SIGM5 (TET2^-/-) and OCI-AML5 (TET2^+/-), while a minimal effect was observed in K562 and CMK which are TET2^+/+. The LD50 of TETi was 250-fold lower than 2HG in TET2^mt cells. In TET2^-/-engineered K562, TETi76 also showed cytotoxicity leaving a therapeutic window as compared with wild type K562. Normal bone marrows were resistant to TETi76 in clonogenic assay. In vitro mixing experiments in which Tet2^mt/Tet2^wt were subjected to methocult cultures at fixed ratios to mimic evolving Tet2^mt clones, CD45.2 (either Tet2^+/-or Tet2^-/-) outcompeted CD45.1 marrow in a control setting, while treatment with TETi76 led to a gradual elimination of mutant marrow. This result was further recapitulated in vivo. In a competitive repopulation transplantation model using graft consist of a mixture of CD45 isotypes mismatched Tet2^+/- or Tet2^-/-and Tet2^+/+marrow cells, TETi76 treatment selectively eliminated Tet2 deficient marrow.

Our data have several important implications. It is likely that compensatory function from other TETs or remaining allele is needed for survival and elimination of dioxygenase appears to be lethal. This explains the exclusivity of TET2/IDH1/2 mutations. Dioxygenase inhibitors may have therapeutic applicability for selective elimination of TET2^mt cells in MDS or potentially as a preventive measure in CHIP. TETi may represent a novel class of antileukemic agents.