TET Deficiency Is Associated with Accumulation of G-Quadruplex and R-Loop Structures during Oncogenesis

The three members of TET family of Fe(II) and alpha-ketoglutarate-dependent 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 perturbations and hypoxia, among other mechanisms, occurs frequently in many hematological malignancies and solid tumors. Recent studies have identified mutations in TET proteins (TET2, most commonly) 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, are not fully-understood.

To investigate the role of TET loss-of-function in the pathogenesis of DLBCL, we generated mice with B-cell-specific deletion of TET2 and TET3, the major TET homologs expressed in mature B cells. TET deficiency in B cells perturbed mature B cell homeostasis resulting in spontaneous development of Germinal Center-derived B cell lymphomas. Moreover, B cells with TET deficiency demonstrated increased genomic instability, a feature previously associated with TET loss-of-function in other hematopoietic lineages. Transcriptional profiling of TET-deficient expanded B cells revealed altered expression of genes and proteins involved in modulating the levels of secondary DNA structures, G-quadruplexes and DNA:RNA hybrids (R-loops) which have been linked to genomic instability and transcriptional perturbations in many different cancers. Using previously described methods and newer approaches, we observed a substantial increase in the levels of G-quadruplex and R-loop structures in TET-deficient B cells compared with control B cells. The increase in G-quadruplex and R-loop structures was evident in naïve, activated and GC B cells following acute TET deletion as well as in TET-deficient myeloid cells and T cells. Genome-wide mapping studies and high-throughput genome-wide translocation sequencing (HTGTS) showed a strong correlation of increased G-quadruplex and R-loop structures with increased DNA DSBs in switch regions of immunoglobulin heavy chain locus in TET-deficient B cells. Deletion of the DNA methyltransferase DNMT1 in TET-deficient B cells prevented the expansion of germinal center B cells, diminished the accumulation of G-quadruplexes and R-loops, and caused a notable delay in lymphoma development, consistent with the opposing functions of DNMT and TET enzymes in DNA methylation and demethylation. CRISPR-mediated depletion of nucleases and helicases that regulate G-quadruplexes and R-loops decreased the viability of TET-deficient B cells. Our studies suggest a molecular mechanism by which TET loss-of-function might predispose to development of B cell-derived and other malignancies, and highlight novel therapeutic avenues that could be further explored.