ABOVE: Parallel G-quadruplexes WIKIMEDIA COMMONS, Thomas Splettstoesser

A loss of TET enzymes, which demethylate DNA, may cause cancer. In TET deficient mice, DNA forms strange structures called G-quadruplexes (G4s) and R-loops that may drive cancer development, a December 22 study in Nature Immunology suggests. 

The paper “is a great contribution to the field of G4/R-loop biology,” Giovanni Capranico, a molecular biologist at the University of Bologna who was not involved in the study, writes in an email to The Scientist. “The major advance is the strong and convincing evidence that TET gene deletions cause a B cell malignancy, at least in mice, [which is] associated with . . . G4s and R-loops,” he says. Non-canonical nucleic acid structures were noticed to be increased in cancer cells already, but this paper has established a better-defined connection.” 

Anjana Rao, a cellular and molecular biologist at the La Jolla Institute for Immunology, and her team first described the role of TET proteins in DNA demethylation in mammals in 2009. Since then, Rao’s and other groups have found that TET proteins are involved in regulating gene expression, embryonic development, and cancer. Specifically, studies have correlated TET deficiencies with white blood cell cancers. So Rao and colleagues wanted to see what happens when TET is absent from mature B cells. 

To investigate, the researchers used a targeted DNA modification tool to create mice in which the genes for two of the three mammalian TET enzymes, Tet2 and Tet3, are knocked out in just mature (CD-19 positive) B cells. These dual TET knockout mice developed B cell lymphoma within weeks, more quickly than mice that had either Tet2 or Tet 3 removed from their B cells. “It turned out to look like this human disease called DLBCL [diffuse large B cell lymphoma]. DLBCL starts in the germinal centers, which are the place where T cells and B cells get together to make antibodies,” Rao describes. The stimulation of B cells that occurs there prompts proliferation, but when the dual TET knockout B cells are stimulated, they proliferate more rapidly than normal B cells do, thus giving rise to the DLBCL-like cancer, Rao adds.

When the team focuses on the molecular level, they detected uncommon DNA structures called G-quadruplexes and R-loops. R-loops appear in DNA when RNA slips in between the two DNA strands, while G-quadruplexes are “knots” in the DNA strands themselves. The DNA of mice lacking TET2 and TET3 formed these structures more frequently than mice with intact TET enzymes. And G-quadruplexes and R-loops hinder replication forks, leading to replication stress—one of the hallmarks of cancer. 

These G-quadruplex and R-loops are going to be driving this genome instability.

—Luisa Cimmino, University of Miami

TET enzymes demethylate DNA, but their counterpart DNMT1 methylates DNA—and in TET-deficient B cells, where this balance was upset, DNMT1 was upregulated. When the researchers deleted the Dnmt1 gene from the mature B cells of TET knockout mice, the DLBCL-like lymphoma developed later and the mice survived for longer. At the same time, G-quadruplexes and R-loops became scarcer. According to Rao, however, the B cells that did grow out eventually were ones where the DNMT deletion failed while the two TET deletions succeeded. 

This is “one of the first papers to definitely show how TET deficiency can cause genomic instability. These G-quadruplex and R-loops are going to be driving this genome instability,” Luisa Cimmino, a biochemist at the University of Miami who was not involved with the study, tells The Scientist. “This is some of the first evidence to show that in a cancer model.” 

Robert Hänsel-Hertsch, a biochemist at the Center for Molecular Medicine Cologne in Germany who was not involved in this study, says he similarly finds the connection between TET and the formation of destabilizing DNA structures and the counterbalance by removing the opposing enzyme DNMT1 convincing and novel. He adds the connection is especially interesting because the enzymes’ activities (demethylating and methylating cytosines) “[do] not have anything to do with G-quadruplexes and R-loops directly.” He notes that the mechanism behind how TET enzymes cause the structures to form remains unclear. 

Cimmino suggests that the study “has huge potential for any TET-deficient cancer,” but would have been more therapeutically relevant if an inhibitor of DNMT1 had been used instead of a DNMT1 deletion. While Rao says she plans to focus on lingering basic research questions surrounding TET deficiencies, she notes that first author Vipul Shukla, who recently started his own lab at Northwestern University, plans to investigate that idea further. “If you apply DNMT1 inhibitors and G-quadruplex-stabilizers together, can we cure the cancer? Maybe we can,” she says.