Dasatinib enhances anti-leukemia efficacy of chimeric antigen receptor T cells by inhibiting cell differentiation and exhaustion
Journal of Hematology & Oncology volume 14, Article number: 113 (2021)
Relapses of CD19-expressing leukemia in patients who achieved initial remission after CART cell treatment have been reported to correlate with poor CART cells persistence. Sustained tonic signaling or strong activation drives CART cell differentiation and exhaustion, which limit the therapeutic efficacy and persistence of CART cells. Here, we identified dasatinib as the optimal candidate to prevent or reverse both CD28/CART and 4-1BB/CART cell differentiation and exhaustion during ex vivo expansion, which profoundly enhanced the therapeutic efficacy and in vivo persistence. Moreover, strong activation-induced CART cells differentiation, exhaustion and apoptosis driven by CD3/CD28 stimulation or antigen exposure were dramatically prevented or reversed by dasatinib treatment. Mechanistically, dasatinib markedly reduced the phosphorylation of Src and Lck, and downregulated the expression of genes involved in CAR signaling pathways, which resulted in the optimization of cell differentiation, exhaustion and apoptosis-related gene expression. Our study proposes a promising pharmacological approach for optimizing CART cells manufacture, and provides an experimental basis for reinvigorating CART cells in clinical application.
To the editor,
Chimeric antigen receptor T cells (CART) emerges as a promising therapeutic approach for adoptive immunotherapy of cancer in recent years. The most impressive responses have been achieved in patients with refractory or relapsed B acute lymphoblastic leukemia (B-ALL) by CART cells targeting CD19 [1,2,3], which provides a potential curative option for patients who are refractory to standard treatments. However, approximately 30–50% of patients experienced leukemia relapse, the majority within one year after CART cells therapy . Relapses of CD19-expressing leukemia in patients who achieved initial remission after CART cell treatment have been reported to correlate with poor CART cell persistence. Sustained tonic signaling or strong activation in manufacture or clinical therapy drives CART cells terminal differentiation , exhaustion  and even apoptosis, which limits their anti-tumor efficacy and in vivo persistence. Calibrating the activation potential of CAR signaling by modifying the configuration of CD3ζ immunoreceptor tyrosine-based activation motifs (ITAMs) reduced terminal differentiation and exhaustion of CART cells and thus increased their persistence in vivo . Therefore, modulation of T cell activation signaling may be an important and feasible approach to enhance the efficacy and in vivo persistence of CART cells.
The traditional concepts that tyrosine kinase inhibitors (TKIs) inhibit T cell activation [8, 9] inspired us to explore their effects on CART cells differentiation and exhaustion. We systematically evaluated the effects of clinical commonly used TKIs including imatinib, dasatinib and nilotinib on CART cells, and surprisingly identified dasatinib as the potential candidate to strongly reverse differentiation and exhaustion of CD28/CART cells during ex vivo expansion (Fig. 1a, Additional file 1: Fig. S1a-e) with the optimal effective concentration of 30 nM in consideration of relative less impact on cell proliferation (Fig. 1b–d, Additional file 1: Fig. S1f). As expected, Nalm6 bearing mice with treatment of dasatinib pre-treated CD28/CART cells demonstrated significant lower tumor burden and prolonged survival compared to untreated counterparts (Fig. 1e–h). Despite the less tendency of exhaustion, 4-1BB/CART cells exhibited downstream differentiation with prolonged cell culture (Fig. 1i), which also could be effectively prevented in the presence of dasatinib 30 nM without significant impact on cell expansion (Fig. 1j–n), and showed an improved efficacy in xenograft mice models (Fig. 1o–r).
T-cell activation is a necessary step in the manufacture of CART cells, and is also the prerequisite of robust cytotoxicity. However, excessive activation drives CART cells differentiation, exhaustion and even apoptosis. Dasatinib significantly protected CART cells from CD3/CD28 signaling induced apoptosis, differentiation and high expression of inhibitory receptors (eg.PD1 and LAG3) (Fig. 2a–g). Moreover, in differentiated CART cells with high expression of inhibitory receptors induced by tumor antigen exposure (Fig. 2h), dasatinib strongly reversed their stages back to TN and TCM (Fig. 2i), markedly abolished the increased expression of PD1 and LAG3 (Fig. 2j), and promoted cell proliferation (Fig. 2k).
Mechanically, we showed that dasatinib, not imatinib significantly reduced the phosphorylation of Src and Lck (Fig. 2l), downregulated T cell activation associated signaling pathways (T cell receptor, Jak-STAT, MAPK and PI3K-Akt) (Fig. 2m, Additional file 1: Fig. S4b-d), inhibitory receptors (PD1, LAG3) and exhaustion-related regulators (NR4A1, BATF3, ATF4 and FOS), whereas increased expression of naive/memory-associated genes (TCF7, CCR7) (Fig. 2n, Additional file 1: Fig. S3). Besides, p53 signaling pathway which upregulated in Nalm6 stimulated CART cells significantly downregulated in dasatinib treated group (Additional file 1: Fig. S4a). Thus, we reckoned that dasatinib modulates CART cells differentiation, exhaustion and apoptosis by inhibiting cell activation pathway. Interestingly, a recent study demonstrated that transient cessation of CAR signaling by dasatinib could reverse dysfunction and induce epigenetic reprogramming in exhausted CART cells . Future studies are warranted to determine the relationship between epigenetic modifiers, T cell activation, differentiation and exhaustion.
Pharmacologic inhibition of T-cell activation signaling with dasatinib during ex vivo expansion successfully reduced CART cell differentiation and exhaustion, thus enhanced their therapeutic efficacy and in vivo persistence. On the other hand, dasatinib could effectively prevent or reverse the strong activation-induced CART cells differentiation and exhaustion by CD3/CD28 stimulation or antigen exposure, which proposed a potential clinical application of drug for functional reinvigoration of CART cells. Of note, simultaneous application of CART cells with dasatinib limited the efficacy both in vitro and in vivo (Additional file 1: Fig. S2). These findings indicate that the mode and duration of drug administration may be critical for its positive effects on CART cells, which is consistent with the notion proposed by Mestermann et al. . Collectively, dasatinib is a promising pharmacological approach which can be incorporated into CART cells production, and also be potentially applied for functional reinvigoration of CART cells in clinical.
Availability of data and materials
RNA-seq data that support the findings of this study have been deposited in the GEO under accession number GSE151774. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.
Chimeric antigen receptor
Single-chain variable fragment
B acute lymphoblastic leukemia
Chronic myeloid leukemia
Tyrosine kinase inhibitor
Activation-induced cell death
Immunoreceptor tyrosine-based activation motifs
T cell receptor
Peripheral blood mononuclear cells
Naive T cell
Central memory T cell
Memory effector T cell
Effector T cell
Differential expressed genes
False discovery rate
Principal component analysis
Visualization and integrated discovery
Gene set enrichment analysis
Mitogen-activated protein kinase
Janus kinase and signal transducer and activator of transcription
Phosphatidylinositol 3 kinase
Programmed death 1
T cell immunoglobulin domain and mucin domain 3
Lymphocyte activating 3
Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med. 2013;368(16):1509–18.
Gu R, Liu F, Zou D, Xu Y, Lu Y, Liu B, et al. Efficacy and safety of CD19 CAR T constructed with a new anti-CD19 chimeric antigen receptor in relapsed or refractory acute lymphoblastic leukemia. J Hematol Oncol. 2020;13(1):122.
Hu Y, Wu Z, Luo Y, Shi J, Yu J, Pu C, et al. Potent anti-leukemia activities of chimeric antigen receptor–modified T cells against CD19 in Chinese patients with relapsed/refractory acute lymphocytic leukemia. Clin Cancer Res. 2017;23(13):3297–306.
Shah NN, Fry TJ. Mechanisms of resistance to CAR T cell therapy. Nat Rev Clin Oncol. 2019.
Zheng W, O’Hear CE, Alli R, Basham JH, Abdelsamed HA, Palmer LE, et al. PI3K orchestration of the in vivo persistence of chimeric antigen receptor-modified T cells. Leukemia. 2018;32(5):1157–67.
Long AH, Haso WM, Shern JF, Wanhainen KM, Murgai M, Ingaramo M, et al. 4–1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat Med. 2015;21(6):581–90.
Feucht J, Sun J, Eyquem J, Ho YJ, Zhao Z, Leibold J, et al. Calibration of CAR activation potential directs alternative T cell fates and therapeutic potency. Nat Med. 2019;25(1):82–8.
Schade AE, Schieven GL, Townsend R, Jankowska AM, Susulic V, Zhang R, et al. Dasatinib, a small-molecule protein tyrosine kinase inhibitor, inhibits T-cell activation and proliferation. Blood. 2008;111(3):1366–77.
Lee KC, Ouwehand I, Giannini AL, Thomas NS, Dibb NJ, Bijlmakers MJ. Lck is a key target of imatinib and dasatinib in T-cell activation. Leukemia. 2010;24(4):896–900.
Weber EW, Parker KR, Sotillo E, Lynn RC, Anbunathan H, Lattin J, et al. Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling. Science. 2021;372(6537).
Mestermann K, Giavridis T, Weber J, Rydzek J, Frenz S, Nerreter T, et al. The tyrosine kinase inhibitor dasatinib acts as a pharmacologic on/off switch for CAR T cells. Science Transl Med. 2019;11(499).
Wu KN, Wang YJ, He Y, Hu YX, Fu HR, Sheng LX, et al. Dasatinib promotes the potential of proliferation and antitumor responses of human gammadeltaT cells in a long-term induction ex vivo environment. Leukemia. 2014;28(1):206–10.
We thank the Center for Drug Safety Evaluation at Zhejiang University for animal technical support.
This work was supported by grants from the National Natural Science Foundation of China (81730008, 81520108002, 81770201, 81970137, 81870080, 91949115), the National Key R&D Program of China, Stem Cell and Translation Research (2018YFA0109300), Key Project of Science and Technology Department of Zhejiang Province (2018C03016-2, 2019C03016), and Zhejiang Province Science Foundation for Distinguished Young Scholars (LR19H080001).
Ethics approval and consent to participate
The animal study was performed in compliance with relevant regulatory standards. All animal studies are approved by ZJU IRB committee.
Consent for publication
Written informed consent for publication was obtained from all participants.
The authors declare that they have no competing interest.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Zhang, H., Hu, Y., Shao, M. et al. Dasatinib enhances anti-leukemia efficacy of chimeric antigen receptor T cells by inhibiting cell differentiation and exhaustion. J Hematol Oncol 14, 113 (2021). https://doi.org/10.1186/s13045-021-01117-y