Studying Mutations in Non-Hodgkin Lymphoma Yields Clues for Potential New Therapies

DNA mutations found in a type of non-Hodgkin lymphoma that has a poor prognosis has led researchers at the National Cancer Institute (NCI), part of the National Institutes of Health, and their colleagues to a better understanding of how the cancer develops and how it might be treated.

Diffuse large B-cell lymphoma (DLBCL), a cancer that originates in white blood cells called B lymphocytes, is the most common form of non-Hodgkin lymphoma, accounting for 30 to 40 percent of all such cases. DLBCL can affect people of any age, although it is most often observed in adults 60 years or older. Roughly one half of DLBCLs are curable by current therapies and response, or non-response, to therapy has been traced to molecular differences in the tumors. The least curable subtype of DLBCL is termed activated B cell-like (ABC) DLBCL, and the more responsive subtype is termed germinal center B cell-like (GCB) DLBCL.

Previous studies by researchers at NCI have demonstrated that malignant cells of the ABC DLBCL subtype rely on the nuclear factor-kappa B (NF-kB) signaling pathway to prevent cell death. A signaling pathway is a series of specific molecular interactions in a cell in which a signal is passed from one molecule to the next. Signaling pathways can regulate important cellular functions, such as cellular proliferation or survival.

The NF-kB signaling pathway is activated when normal B lymphocytes encounter a foreign antigen, a process that involves a protein called CARD11. Previously, NCI researchers reported that CARD11 stimulates the NF-kB signaling pathway in ABC DLBCL, but not in GCB DLBCL, in the absence of activation by antigens, although the mechanism underlying this abnormal cellular signaling were not understood.

To determine how CARD11 contributes to cancer, researchers sequenced the "CARD11" gene in human DLBCL tumors and detected mutations in about 10 percent of ABC DLBCL biopsy specimens. According to lead investigator, Louis Staudt, M.D., Ph.D., deputy chief of the Metabolism Branch and head of the Lymphoid Malignancies Section in NCI's Center of Cancer Research, "Our results demonstrate that "CARD11" is a bona fide cancer-causing gene in DLBCL, thus providing a genetic rationale for the development of drugs that could block the CARD11 pathway."

Gene sequencing is a process in which the order of the four building blocks, or bases, of DNA -- adenine, guanine, cytosine, and thymine -- is determined within a gene in an automated fashion. For each gene, this sequence of bases is the code that specifies the production of a unique protein. A mutation is a change in the DNA sequence or code of a gene that may lead to the production an abnormal form of the encoded protein.

At the start of their study, the researchers postulated that the malignant nature of ABC DLBCLs might be due to cancer-causing mutations in genes that encode the protein components of the NF-kB signaling pathway. The researchers initially sequenced all coding gene regions of the "CARD11" gene in 16 ABC DLBCL biopsy specimens and four ABC DLBCL laboratory cell lines and discovered mutations in three biopsy specimens and one cell line.

The scientists found that all of the mutations caused changes in amino acids in a small segment of the CARD11 protein known as the coiled-coil domain, which is required for proper CARD11 function. Amino acids are the building blocks of proteins, and the sequence of amino acids in a protein is determined by the sequence of consecutive three-base codes in the DNA of that protein's gene.

The researchers next sequenced the coiled-coil domain in 136 additional DLBCL biopsy specimens and, overall, detected mutations altering amino acids in or immediately adjacent to the coiled-coil region in 9.6 percent (7/73) of ABC DLBCL specimens but in only 3.8 percent (3/79) of GCB DLBCL specimens.

A coiled-coil domain is a structural design feature of some proteins in which stretches of protein are coiled together like strands of a rope. Many coiled-coil proteins are involved in important biological functions, such as the regulation of gene expression and cellular signaling.

The researchers discovered that all of the coiled-coil mutations caused the CARD11 protein to spontaneously activate the NF-kB signaling pathway, thereby promoting survival of the malignant cell.

By demonstrating that a subset of ABC DLBCLs acquire "CARD11" mutations that activate the NF-kB pathway, Staudt's team believes that they have uncovered a key to the evolution of these lymphomas and to the normal function of CARD11. The authors hypothesized that the coiled-coil domain normally keeps CARD11 in a latent state in unstimulated B lymphocytes, but, when these cells encounter foreign antigen, the coiled-coil region switches from its latent state to one that causes the CARD11 protein to aggregate and stimulate the NF-kB pathway. This process is mimicked pathologically by the "activating" mutations in DLBCL tumors.

The scientists also speculated how CARD11 could be attacked therapeutically. By genetically interfering with the function of the CARD11 coiled-coil domain, the researchers were able to kill ABC DLBCL cells in the laboratory. This result provides a rationale for the development of drugs aimed at the coiled-coil domain of CARD11, which could have activity in ABC DLBCL, the subtype of DLBCL that is most resistant to current therapies.

"This study highlights the exciting potential of The Cancer Genome Atlas project, which is trying to identify all genes that are altered in cancer cells" said Staudt. "Cancer genome sequencing will not only illuminate the development of cancer but could also uncover mutated genes that will provide insights into the normal function of proteins."