PROJECT SUMMARY/ABSTRACT Ten Eleven Translocation (TET1, TET2 and TET3) are α-ketoglutarate (αKG) and Fe2+ dependent DNA- dioxygenases that is a key regulator of epigenetic landscape. These enzymes progressively oxidize 5- methylcytosine to 5-hydroxymethylcytosine and further to 5-formylcytosine and 5-carboxylcytosine in DNA culminating into DNA demethylation essential for efficient transcription over large time scales. Loss-of-function TET2 mutations (TET2MT), is one of the most frequent pathogenic lesion in MDS and related hematologic malignancies in humans. TET2MT are found in all disease stages and levels of aggressiveness. In addition, recent studies have demonstrated that somatic TET2MT are very frequently found in “healthy” elderly. The presence of TET2MT in CHIP implies that it is an early event in the creation of hematologic disorders. It also supports murine study conclusions that TET2MT cause expansions of hematopoietic stem and progenitor cells (HSPC). Early events in the evolution of defective clones are rational targets for preventative strategies since clones in such stages are likely to be dependent on these events. Their presence in all cells can, however, also be exploited in late stages of the disease. For example, the combined loss of TET1 and TE2 in hematopoietic cells in murine models extends life substantially relative to TET2 loss alone. In addition, the proof of principle is also derived from observation in patients with IDH1/2MT whereby, these neomorphic mutations lead to the production of a weak TET inhibitor, 2-hydroxyglutyrate (2-HG), known inhibitor of dioxygenases. Our observations that IDH1/2MT are mutually exclusive with TET2MT, strongly supports our hypotheses that the TET inhibitor, 2-HG, prevents evolution of TET2MT clones. This observation was further substantiated in a cellular model of myeloid malignant cells and supports that synthetic lethality can be achieved through elimination of remaining TET-activity essential for efficient transcription for proliferation in TET-dioxygenase deficient TET2MT clones. Our overarching hypothesis is that rationally designed and synthesized small molecules TETi76 can be utilized to impede compensatory TET dioxygenase activity originating from TET3 and TET1, to cause either synthetic lethality or lineage redirection in cases with TET2MT inactivation. Our goal is to develop a novel therapeutic approach for TET2MT MDS by evaluating the potential use of TETi76 as targeted treatments in preclinical models. More specifically, we aim to: i) Study the mechanistic consequences of TET inhibition in normal and malignant hematopoiesis in vitro. ii) Establish the effects of TETi compounds in vitro using human and murine cellular models and iii) Characterize TETi preventative and therapeutic efficacy as well as tolerability in pre-clinical murine models. Our proposal, if successful, will lead to a novel class of therapeutic agents for TET2MT associated hematopoietic disorders, and perhaps also other dioxygenase mutations. This work could also have implications for cancers mutated in histone demethylase KDM6A, which is similar to TET2 and frequently mutated in bladder cancer.

PROJECT NARRATIVE TET-dioxygenase are the gatekeeper of efficient transcription that regulate cell lineage fate, proliferation and survival. TET2 is one of the major TET-dixygenase that is most frequently mutated in many healthy elderly, wherein its presence put them at significantly higher risk of developing myelodysplastic syndromes and associated disorders. Naturally existing 2HG producing neomorhic mutations in IDH1/2 causes TET-inhibition which in turn leads to the preferential elimination of TET deficient TET2MT hematopoietic clones in human body as observed upon analysis of large cohort of patient data. Hence, establishing TET enzymes as a therapeutic targets. This proposal will enable us to develop small molecule lead compound for early as well as late therapeutic intervention and allow us to gain insights into strategies by which this might be achieved.