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247 FOXO1 is a master regulator of CAR T memory programming
  1. Alexander E Doan1,
  2. Katherine P Mueller2,
  3. Andy Chen1,
  4. Geoffrey Rouin2,
  5. Bence Daniel1,
  6. John Lattin3,
  7. Yingshi Chen4,
  8. Brett Mozarsky2,
  9. Martina Markovska2,
  10. Jose Arias-Umana2,
  11. Robert Hapke2,
  12. Inyoung Jung2,
  13. Peng Xu1,
  14. Dorota Klysz1,
  15. Malek Bashti1,
  16. Patrick Quinn1,
  17. Katalin Sandor1,
  18. Wenxi Zhang1,
  19. Junior Hall4,
  20. Caleb A Lareau1,
  21. Stephan Grupp4,
  22. Joseph A Fraietta2,
  23. Elena Sotillo1,
  24. Ansuman T Satpathy1,
  25. Crystal L Mackall1 and
  26. Evan W Weber4
  1. 1Stanford University, Stanford, CA, USA
  2. 2University of Pennsylvania, Philadelphia, PA, USA
  3. 3Washington University in St. Louis, St. Louis, MO, USA
  4. 4Children’s Hospital of Philadelphia, Philadelphia, PA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background CAR T cell therapy is a promising therapeutic modality for cancer treatment, but poor CAR T cell persistence limits efficacy in patients.1 CAR T cells with a memory-like phenotype are associated with durable persistence in patients and response to therapy,2 thereby implicating memory as an important therapeutic axis. Thus, strategies to stably promote CAR T memory differentiation are urgently needed. Here, we demonstrate that FOXO1 is required for the development of memory CAR T cells and that FOXO1 overexpression maintains memory gene expression programs and enhances CAR T cell antitumor activity in liquid and solid tumor models.

Methods T cells were co-transduced to express a CAR and a transcription factor, and in relevant experiments were CRISPR-edited using Cas9. CAR T cell function was assessed in vitro using tumor co-culture assays wherein cytokine secretion, killing, and metabolic fitness (via Seahorse) were measured. In vivo studies were performed using murine xenograft tumor models in NSG mice. CAR T phenotyping experiments were performed using flow cytometry, RNA-seq, and ATAC-seq. Public datasets were reanalyzed to evaluate FOXO1 activity in patients treated with CAR T cell therapy.

Results FOXO1 knockout in human CAR T cells prevented the development of a memory-like phenotype, and instead, promoted an exhausted phenotype and gene expression profile and attenuated antitumor activity in vitro and in vivo. FOXO1 overexpression in CAR T cells dramatically enhanced functionality in vitro and in vivo, especially in models of chronic antigen stimulation, and promoted a memory-like phenotype via flow cytometry, RNA-seq, and ATAC-seq. A FOXO1 regulon consisting of 41 putative FOXO1 target genes, which were unbiasedly identified in our knockout and overexpression studies, highly correlated with long-term persistence and positive outcomes in patients treated with CD19 CAR T cells.

Conclusions Our results demonstrate that FOXO1 is a master regulator of CAR T cell memory programming and that overexpression of FOXO1 triggers both transcriptional and epigenetic changes in CAR T cells that enhance memory differentiation, persistence, and potency. Our findings further demonstrate the potential for transcription factor engineering as an approach to generate highly effective CAR T cell products for antitumor therapy.


  1. Weber EW, Maus MV, Mackall CL. The Emerging Landscape of Immune Cell Therapies. Cell. 2020;181:46–62.

  2. 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;24:563–571.

Ethics Approval All animal studies were undertaken under Stanford University APLAC #31287 and Children’s Hospital of Philadelphia ACUP-approved protocols.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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