Recently, mutations in the TET family of oxygenases have been described in a subset of acute myeloid leukemias (AML). TET enzymes catalyze the addition of a hydroxyl group to methylated cytosine (5mC) within DNA to generate 5-hydroxymethylcytosine (5hmC). Additionally, these enzymes can further oxidize 5hmC to produce 5-formylcytosine (5fc) and 5-carboxylcytosine (5caC), which are the key intermediates in a newly defined active demethylation pathway in mammals. We hypothesize that the disruption of demethylation is a key contributor to the leukemic phenotype in TET-mutant hematopoiesis. To test our hypothesis, we plan to use novel techniques to identify the locations of 5hmC and 5fC on a genome-wide scale. Additionally, we will identify genome-wide occupancy maps for TET1 and TET2, as well as thymine DNA glycosylase, the enzyme responsible for removing 5fC and 5caC from DNA to allow for replacement with an unmethylated cytosine. Along with global gene expression analysis, in the presence and absence of TET1/2, these datasets will point us to the genes that are most significantly and directly affected by loss of specific TET enzymes in hematopoiesis. Finally, we will test the role of these identified genes in hematopoietic cells and validate our findings in primary human AML samples.
Recently, mutations in TET enzymes that reversibly modify the chemical properties of DNA have been identified in patients with certain types of cancer, including acute leukemias. This research proposal aims to understand how these TET mutations affect blood cell development and lead to leukemia by characterizing the specific and direct effects of the DNA modifications generated by the normal and mutant TET proteins in human blood cells. Together, the experiments proposed will define fundamental mechanisms of normal and malignant blood cell development, identify novel contributors to leukemia, and provide the rationale for developing treatments for leukemia that target the TET family or pathway.