A Master Regulator of Gene Expression

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Immunotherapies such as chimeric antigen receptor (CAR) T cell therapy are promising approaches in the fight against cancer. How well the therapies work depends on T cell function, which is determined by the network of genes that these immune cells express. Recently, researchers at Duke University developed a CRISPR-based screening platform to identify key epigenetic regulators of human T cell function, and discovered the central role of the transcription factor Basic leucine zipper transcription factor ATF-like 3 (BATF3) in reprogramming the expression of several genes and improving the efficacy of CAR T cells in eliminating cancer cells.1 Their findings, published in Nature Genetics, may aid in the development of more effective T cell-based immunotherapies. 

The illustration shows an artistic rendering of CRISPR-enhanced T cells attacking a tumor. 

Researchers used a CRISPR-based platform to identify master regulators of T cell function. 

Ella Maru Studio

“It’s a very elegant study. It’s really interesting to see how this field of CRISPR screen is developed here using primary human T cells, which are not the easiest to work with,” said Fredrik Wermeling, an immunologist at the Karolinska Institute who was not involved in the research.

For years, biomedical engineer and study author Charles Gersbach from Duke University and his team have developed technologies to screen and manipulate the expression of genes in cells. In previous studies, they used these epigenome editing tools to reprogram fibroblasts to become neuronal cells and to control cell differentiation in human neuronal and pluripotent stem cell populations.2-4

Interested in exploring a more therapeutic application of these tools, the researchers turned their attention to T cell-based immunotherapies, specifically CAR T cells. According to Gersbach, the use of such epigenetic enhancement approaches on T cells may help expand T cell-based therapies beyond the cancer types, such as blood cancers, in which they have been effective.

For their study, the team developed a CRISPR-based screening approach in primary human CD8+ T cells. The researchers fused a catalytically inactive Cas9 nuclease with either a transcriptional repressor for CRISPR interference (CRISPRi) or an activator for CRISPR activation (CRISPRa). Using guide RNAs targeting specific genes, the researchers directed the inactive Cas9 to either silence or activate expression.  

To select the target genes, the scientists analyzed public datasets describing changes in chromatin accessibility as T cells differentiate, and homed in on the transcription factors that bound to these genomic regions. They identified 120 transcription factors enriched in chromatin regions that change accessibility during T cell differentiation. They then directed CRISPRi and CRISPRa to the genes encoding each of these transcription factors to determine which, if any, of these proteins would act as a master regulator. 

By coupling loss-of-function and gain-of-function perturbations, the team identified several potential modulators of T cell function. One of the most promising was BATF3, which promotes CD8+ T cell survival.5 

In subsequent in vitro experiments, the team found that overexpressing BATF3 regulated a broad network of genes. While some of the upregulated genes related to T cell survival, some of the downregulated genes associated with T cell exhaustion, a natural mechanism that evolved to keep T cells under control but that can hinder their ability to mount an antitumor response in a cancer setting. 

They also found that overexpressing BATF3 enhanced the ability of CAR T cells to eliminate cancer cells in vitro. Treating solid tumors has been a major challenge for CAR T cell therapy, so the team next tested whether this strategy worked against tumors. In a humanized breast cancer mouse model, the researchers found that CAR T cells engineered to overexpress BATF3 reduced tumor size more than standard CAR T cells did. 

“We knew that BATF3 was important to T cells. What we didn’t know is that by overexpressing it, we could profoundly change their phenotypes and make them incredibly more potent against these tumor models,” Gersbach said.

To assess whether gene expression changes induced by BATF3 associated with positive clinical outcomes, the team compared the transcriptional signatures of T cells with or without BATF3 overexpression with the transcriptional profiles of patients who received CAR T cell therapy in a recent clinical trial.6 They found that BATF3 overexpression silenced more than 30 percent of the genes associated with nonresponse and activated 20 percent of the genes associated with response to CAR T cell therapy. “If these gene expression patterns could be very well established, that would be very useful both for CAR T cells, but also cancer vaccines or other types of T cell-based therapies,” Wermeling pointed out. 

According to Wermeling, an important next step for this research is to translate these findings into clinical trials, a goal that Gersbach and his team are already pursuing. “We are very excited that we could actually impact cancer patients’ outcomes with these types of enhancements and interventions,” he said.

Charles Gersbach is a cofounder and scientific advisor at Tune Therapeutics.

References

  1. McCutcheon SR, et al. Transcriptional and epigenetic regulators of human CD8+ T cell function identified through orthogonal CRISPR screens. Nat Genet. 2023;55(12):2211-2223.
  2. Black JB, et al. Targeted epigenetic remodeling of endogenous loci by CRISPR/Cas9-based transcriptional activators directly converts fibroblasts to neuronal cells. Cell Stem Cell. 2016;19(3):406-414. 
  3. Black JB, et al. Master regulators and cofactors of human neuronal cell fate specification identified by CRISPR gene activation screens. Cell Rep. 2020;33(9):108460. 
  4. Kwon JB, et al. Myogenic progenitor cell lineage specification by CRISPR/Cas9-based transcriptional activators. Stem Cell Rep. 2020;14(5):755-769.
  5. Ataide MA, et al. BATF3 programs CD8+ T cell memory. Nat Immunol. 2020;21(11):1397-1407.
  6. Haradhvala NJ, et al. Distinct cellular dynamics associated with response to CAR-T therapy for refractory B cell lymphoma. Nat Med. 2022;28(9):1848-1859. 
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