ABOVE: High FOXO1 activity can make CAR T cells more potent. ©iStock, Christoph Burgstedt
Chimeric antigen receptor (CAR) T cells have a tough job. Engineered with a receptor that recognizes molecules decorating the surface of cancer cells, CAR T cells find and kill malignancies. However, if they don’t have the right disposition, this task can wear them out.
Researchers have observed that certain types of CAR T cells are more effective at eradicating cancer. One especially advantageous quality is a memory-like state.1 This molecular program gives T cells the ability to remember threats that they’ve seen before and mount an immune response for a long period of time—key assets for a CAR T cell training to fight a long battle against cancer.
“People have appreciated this important paradigm for a long time, but there’s been one piece of missing information,” said Evan Weber, an immunologist at the University of Pennsylvania. “What are the major transcription factors that are responsible for driving this beneficial phenotype?”
In a new study published in Nature, Weber and his colleagues showed that the transcription factor forkhead box protein O1 (FOXO1) may be the key to activating memory programs in a CAR T cell.2 By turning the gene on and off, the researchers changed not only the cells’ gene expression patterns, but also their behavior, suggesting that increasing FOXO1 expression is one way to boost the effectiveness of CAR T cell therapies.
Weber first became interested in FOXO1 during his postdoctoral research on how to prevent CAR T cell burnout after long-term exposure to cancer cells. One solution that worked was pharmacologically suppressing T cell activity to give the CAR T cells a break.3 During this resting period, the chromatin that protects regions of the DNA that FOXO1 binds became more accessible, suggesting that the gene may be active.
In their recent work, Weber and his team overexpressed the gene in human CAR T cells and found that the cells had more molecular markers of memory functions and prolonged survival of a mouse model of leukemia compared to CAR T cells with normal FOXO1 levels. When they reduced FOXO1 levels using CRISPR-Cas9 or blocked its activity with a small molecule inhibitor, it had the opposite effect: markers of memory function waned, and mice succumbed to the cancer more quickly. Weber was surprised to see how drastic the difference was, especially in the functionality of the T cells.
“To me, that was a bit of a lightbulb moment because it suggested that we were really fundamentally reprogramming the cells,” he said.
His team wasn’t sure exactly how FOXO1 elicited these changes. Luckily, they weren’t the only ones studying this transcription factor. Thousands of miles away, researchers at the University of Melbourne also suspected that FOXO1 was the key to boosting CAR T cell attacks. They focused their investigation on the molecular pathways driven by FOXO1, and when the two teams, an ocean apart, learned about their mutual interest in FOXO1, they began to share their progress and findings with each other. In the same issue of Nature, the Australian researchers revealed that FOXO1 controls metabolism and T cell trafficking to key locations in the body.4 Importantly, the transcription factor also made CAR T cells more like stem cells, which improved their longevity and effectiveness. “Our stories are very complementary,” Weber said.
To build on what the Australian team learned about the molecular pathways, Weber’s team sifted through RNA sequencing data from patients who had received CAR T cell therapies to explore how FOXO1-related genes might affect patient outcomes. When the researchers zeroed in on 41 genes controlled by FOXO1, they found that people with higher expression of these genes, which indicates higher FOXO1 activity, tended to survive much longer than people with fewer signs of FOXO1 activity.
“The Holy Grail of cell therapy is to come up with an actionable strategy to improve the intrinsic qualities of T cells,” said Christopher Klebanoff, an immunologist at Memorial Sloan Kettering Cancer Center who was not involved in this study. “[This paper] shows an actionable strategy: overexpression of the wild-type form of FOXO1.”
Klebanoff was also interested to see that T cell factor 1 (TCF1), a transcription factor that previous studies had pointed to as a driver of helpful memory phenotypes in checkpoint blockade immunotherapy, didn’t seem to influence the CAR T cells’ effectiveness.5 This might point to the context-specific importance of different memory-driving factors, according to Weber.
“We're not saying that TCF1 isn’t important in cancer immunotherapy,” he said. “It might not be relevant for engineering T cells outside of the body and then putting them back.”
Weber’s team continues to investigate what makes these FOXO1-overexpressing CAR T cells so potent, and they are optimistic that this will improve the outcomes of CAR T cell therapy for patients. Weber imagines that they could engineer CAR T cells to express a cancer-recognizing receptor and high levels of FOXO1. Klebanoff wonders whether overexpressing FOXO1 in other T cell therapies might also give them a boost.
In his own research, Klebanoff discovered that shutting down a molecular pathway driven by protein kinase B can induce a memory state in CAR T cells, and the pathway happens to interact with FOXO1.6 This transcription factor may be “just the tip of the iceberg,” Weber said. “As we start to develop a blueprint for the perfect therapeutic T cell, FOXO1 can give us a clearer idea of what that blueprint should look like.”
References
1. Fraietta JA, et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat Med. 2018;24(5):563-571.
2. Doan AE, et al. FOXO1 is a master regulator of memory programming in CAR T cells. Nature. 2024;629(8010):211-218.
3. Weber EW, et al. Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling. Science. 2021;372(6537):eaba1786.
4. Chan JD, et al. FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy. Nature. 2024;629(8010):201-210.
5. Sade-Feldman M, et al. Defining T cell states associated with response to checkpoint immunotherapy in melanoma. Cell. 2018;175(4):998-1013.e20.
6. Klebanoff CA, et al. Inhibition of AKT signaling uncouples T cell differentiation from expansion for receptor-engineered adoptive immunotherapy. JCI Insight. 2017;2(23):e95103.