B-cell lymphoma is a type of non-Hodgkin lymphoma that originates in B cells. It is the most common type of lymphoma and about 85% of all lymphomas in the United States is B cell. Depending on the type and stage of the lymphoma, treatment options include chemotherapy, radiation therapy, targeted drug therapy, immunotherapy, and more. Now, scientists at the La Jolla Institute for Immunology (LJI) have uncovered how the loss of TET enzymes can lead to B-cell lymphoma.

Their findings are published in Nature Immunology, in a paper titled “TET deficiency perturbs mature B cell homeostasis and promotes oncogenesis associated with accumulation of G-quadruplex and R-loop structures,” and led by LJI professor Anjana Rao, PhD, in the LJI Center for Cancer Immunotherapy. Experiments were spearheaded by LJI instructor Vipul Shukla, PhD, (soon to be an assistant professor at Northwestern University) and University of California, San Diego, graduate student Daniela Samaniego-Castruita.

“Enzymes of the TET family are methylcytosine dioxygenases that undergo frequent mutational or functional inactivation in human cancers,” the researchers wrote. “Recurrent loss-of-function mutations in TET proteins are frequent in human diffuse large B-cell lymphoma (DLBCL). Here, we investigate the role of TET proteins in B-cell homeostasis and development of B-cell lymphomas with features of DLBCL.”

Previously, scientists discovered mutations that cause TET enzymes to lose their function in many patients with blood cancers and solid cancers. Researchers have also found that double-stranded breaks in the DNA code, are a common feature in cancer cells.

In this study, researchers explored a potential way in which TET deficiency is connected to genomic instability.

“This study provides insights about an important question in the field,” said Shukla.

The researchers used a mouse model of lymphoma and observed that deletion of TET2 and TET3 enzymes in mature B cells had huge consequences for B-cell homeostasis. “The TET-deficient mice developed lymphoma, and we observed an increase in marks associated with genomic instability, such as double-strand breaks,” said Samaniego-Castruita.

To observe what was happening on the molecular level, the researchers performed genomic analysis for clues as to what was happening at the molecular level.

Diagrammatic representation of a G-quadruplex (G4) with an associated R-loop structure, illustrating the reagents used for detection of G-quadruplexes and R-loops. [Shukla et al]
“We show that deletion of Tet2 and Tet3 genes in mature B cells in mice perturbs B-cell homeostasis and results in spontaneous development of germinal center (GC)-derived B cell lymphomas with increased G-quadruplexes and R-loops,” the researchers wrote.

DNA normally has two strands running parallel to each other. R-loops appear when a third rail, made of RNA, slips in and forces a gap between the two DNA strands. G-quadruplexes act like knots on the DNA rails. Both R-loops and G-quadruplexes make it hard for the original two rails of DNA to “unzip.”

“These structures represent sites in the DNA that are much more fragile than other regions,” said Shukla. “With this study, we found that TET enzymes are perhaps related to the regulation of these structures, which could, in turn, explain one mechanism for acquisition of genomic instability in the absence of TET enzymes.”

Shukla and Samaniego-Castruita observed that DNMT1 was upregulated in TET-deficient B cells. DNMT1 is a key enzyme responsible for maintaining marks on DNA called DNA methylation. DNA methylation is an important regulatory mark in the genome, and is normally removed through the activity of TET enzymes.

The researchers then deleted the Dnmt1 gene in TET-deficient B cells in mice to test if levels of G quadruplexes and R-loops could be altered upon removal of DNMT1 protein.

“At a genome-wide level, G-quadruplexes and R-loops were associated with increased DNA double-strand breaks (DSBs) at immunoglobulin switch regions,” the researchers wrote. “Deletion of the DNA methyltransferase DNMT1 in TET-deficient B cells prevented expansion of GC B cells, diminished the accumulation of G-quadruplexes and R-loops, and delayed B lymphoma development, consistent with the opposing functions of DNMT and TET enzymes in DNA methylation and demethylation.”

The researchers hope to devise strategies through which G-quadruplexes and R-loops could be targeted to help cancer patients. Their findings may pave a way for designing drug treatment strategies to target malignant cells in many cancers.

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