Using a new and unbiased CRISPR-Cas9-based screen, scientists at Vanderbilt University Medical Center and their collaborators have zeroed in on a metabolic checkpoint enzyme called MTHFD2 as a potential target for anti-inflammatory immunotherapy in multiple inflammatory diseases. MTHFD2 is consistently upregulated in individuals with a variety of inflammatory diseases, including cancer.

In an article in the journal Immunity this week, titled “MTHFD2 is a metabolic checkpoint controlling effector and regulatory T cell fate and function,” the authors report pharmaceutically inhibiting or genetically deleting the enzyme reduced disease severity in multiple animal models of inflammatory diseases, including asthma, multiple sclerosis, inflammatory bowel disease, and a model of general allergic response.

One-carbon metabolism is needed for nucleotide synthesis required for T cell division, specialization and function. This study identifies a metabolic checkpoint enzyme as a therapeutic target for inflammatory diseases. [Sugiura et al, Immunity, 2021].
Mobilizing an effective adaptive immune response needs the rapid growth and division of a group of immune cells called CD4 positive T cells into specialized effector and regulatory subsets. This requires overhauling the cellular metabolic machinery to meet the needs for biosynthesis, bioenergy and cellular communications. Balancing effector and regulatory T cell subsets is critical in preventing inflammation and autoimmune diseases.

“We examined the role of one-carbon metabolism in CD4 positive T effector and T regulatory cell subsets,” the authors note. One-carbon metabolism is a series of reactions that generate chemical building blocks for the biosynthesis of DNA and other molecules.

Jeffrey Rathmell, PhD, Cornelius Vanderbilt Professor of Immunobiology and the senior author of the paper said, “One-carbon metabolism has been a target for drug development for years, but it really hasn’t been explored in an unbiased way.” The immunosuppressant drug methotrexate, for example, inhibits an enzyme in the one-carbon metabolism pathway, but it may not be the “right target or the right drug” for optimal therapeutic activity, he said.

Jeff Rathmell, PhD, and MD-PhD student Ayaka Sugiura at Vanderbilt University [Susan Urmy]
To systematically study the one-carbon pathway in T cell subsets, Ayaka Sugiura, an MD PhD student in Rathmell’s group, developed an unbiased CRISPR-Cas9-based screening approach, designing CRISPR guideRNAs to selectively inactivate each gene in the one-carbon metabolism pathway. She then introduced this library of inactivators into isolated T cells, controlling the experimental conditions so that each cell harbored only one or no inactivated gene.

“This screening strategy and whole approach to look for important disease genes, which might be therapeutic targets, in an unbiased way is really valuable and has been very impactful for our group,” Rathmell said.

Using the approach on effector and regulatory T cells isolated from animal model of asthma and a variety of other inflammatory diseases, Sugiura identified genes important to T cell function during the respective disease processes. In this multi-model analysis, MTHFD2 stood out. It was highly expressed during embryonic development and in diseased states, but it was expressed negligibly or not at all, in adult tissues.

Sugiura said, “MTHFD2 is important for nucleotide synthesis not only for DNA, but also for proper signaling required for T cell function” MTHFD2 deficiency reduced overall proliferation of CD4 T cells and suppressed immune responses.

However, the researchers discovered, MTHFD2 inhibition had distinct effects on different T cell subsets, suggesting immune cell subsets rely on one-carbon metabolism and MTHFD2 function in different ways. Whereas inhibiting MTHFD2 promoted the activity of regulatory CD4 T cells, which suppresses immune response, it converted inflammatory CD4 T cells (Th17) into an anti-inflammatory cell type.

“This was pretty surprising,” Rathmell said. “Ayaka was able to show that inhibiting MTHFD2 doesn’t just stop an immune response, it actually switches it from inflammatory to anti-inflammatory.”

Sugiura added, “It was promising that while the [MTHFD2] inhibitor suppressed inflammation in multiple disease models of hyperactive T cell activity, it did not affect desirable T cell responses, such as the response to vaccination.”

MTHFD2 had previously been a target for anti-cancer drug development because of its overexpression in many cancers, but preclinical studies did not support further anti-cancer development of MTHFD2 inhibitors. Although MTHFD2 inhibitors were not successful as general anti-cancer agents they might be useful for cancers driven by inflammation, such as colorectal cancer. An MTHFD2 inhibitor would potentially slow down cancer cell proliferation and block “the specific inflammatory T cells that can promote that type of cancer,” Rathmell said.

The Rathmell group is currently working with collaborators to develop MTHFD2 inhibitors with improved clinical characteristics, exploring multiple sets of genes in various disease models using the new CRISPR-based approach and building a core resource for other Vanderbilt investigators.

The study was supported by grants from the Lupus Research Alliance and the National Institutes of Health.