TET-family of dioxygenases mediate DNA demethylation by sequentially oxidizing 5-methylcytosine (5mC) to 5- hydroxymethyl- (5hmC), 5-formyl- (5fC) and 5-carboxyl-cytosine (5caC). TET enzymes are required for normal development and loss-of TET function due to mutations, metabolic reprogramming, hypoxia and other additional mechanisms occurs frequently in many hematological malignancies and solid tumors. Recent studies have identified mutations in TET proteins and metabolic enzymes which regulate TET catalytic activity in a large cohort of patients with Diffuse Large B-cell Lymphoma (DLBCL). However, the clinical significance of these mutations in DLBCL and the molecular mechanisms through which TET proteins suppress development of malignancies in general, is not well-understood. Studies from us and others have highlighted that TET activity is required for differentiation of mature B cells; the cell-of-origin for DLBCL. To investigate the role of TET loss-of-function in the pathogenesis of DLBCL, I recently generated mice with B-cell-specific deletion of TET2 and TET3 proteins. Deletion of TET2 and TET3 in mouse B cells caused development of aggressive lymphomas and rapid mortality. Preliminary analysis of TET-deficient B cells revealed increased genomic instability and a striking accumulation of unusual secondary DNA structures called G-quadruplexes (G-quads). The accumulation of G-quads occurred at early stages of B-cell expansion and was also a feature associated with TET-deficiency in other cellular lineages. G-quad structures have been linked to genomic instability and transcriptional alterations in cancers but their physiological functions in normal cells and pathological roles in malignant cells remain poorly understood. For the studies proposed here, I hypothesize that TET proteins function as tumor suppressors by limiting the accumulation of G-quad structures. Studies in Aim1 will examine the association of G-quad structures with TET activity, genomic instability and transcriptional alterations during TET loss-of-function induced lymphomagenesis.
Aim2 will interrogate molecular mechanisms by which TET proteins limit the accumulation of G-quad structures.
Aim3 will focus on delineating mechanisms by which G-quads regulate chromatin structure and function in normal and malignant B cells. Studies in Aim2 and Aim3 will be performed in R00 phase and will likely prime the emergence of many new lines of investigations. Together, these studies will use B cells as a model system to understand the mechanistic basis for the broad tumor suppressive functions of TET proteins and will reveal novel facets about the biological functions of G-quad structures. The proposal outlines a specific training plan for the K99 and R00 phases, to acquire the skillset necessary to perform the proposed studies and successfully transition to an independent position. During this process, I anticipate to develop an independent line of research through my continued training in an excellent research environment, facilitated by an outstanding mentoring team.
TET enzymes remove DNA methylation and their activity is often lost or diminished in many different cancers, including B cell lymphomas. Why loss-of TET function causes development of cancers remains unclear. This proposal will investigate mechanisms by TET proteins prevent the accumulation of unusual structures in the DNA, called G-quadruplexes, which cause genomic instability and transcriptional perturbations; that are hallmarks of cancer cells. These studies will be pivotal in understanding how TET proteins suppress development of cancers and will pave way for new therapeutic regimens for treatment of B-cell and other malignancies.
