Demethylating therapy increases cytotoxicity of CD44v6 CAR-T cells against acute myeloid leukemia

Ling Tang; Yingjie Kong; Haobing Wang; Ping Zou; Ting Sun; Ying Liu; Juan Zhang; Na Jin; Hanwen Mao; Xiaojian Zhu; Jue Wang; Fankai Meng; Yong You

doi:10.3389/fimmu.2023.1145441

Demethylating therapy increases cytotoxicity of CD44v6 CAR-T cells against acute myeloid leukemia

Front Immunol. 2023 Apr 27:14:1145441. doi: 10.3389/fimmu.2023.1145441. eCollection 2023.

Authors

Ling Tang¹, Yingjie Kong¹, Haobing Wang¹, Ping Zou¹, Ting Sun², Ying Liu³, Juan Zhang³, Na Jin⁴, Hanwen Mao⁵, Xiaojian Zhu², Jue Wang², Fankai Meng², Yong You¹

Affiliations

¹ Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
² Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
³ Department of R&D, Wuhan Biological Sample Bank Co., Ltd., Wuhan, China.
⁴ Department of Oncology, The First People's Hospital of Jiangxia District, Wuhan City and Union Jiangnan Hospital, Huazhong University of Science and Technology, Wuhan, China.
⁵ Oncology Department, Wuhan Dongxihu District People's Hospital, Wuhan, China.

Abstract

Background: CD44v6 chimeric antigen receptor T (CD44v6 CAR-T) cells demonstrate strong anti-tumor ability and safety in acute myeloid leukemia (AML). However, the expression of CD44v6 on T cells leads to transient fratricide and exhaustion of CD44v6 CAR-T cells, which affect the application of CD44v6 CAR-T. The exhaustion and function of T cells and CD44v6 expression of AML cells are associated with DNA methylation. Hypomethylating agents (HAMs) decitabine (Dec) and azacitidine (Aza) have been widely used to treat AML. Therefore, there may be synergy between CD44v6 CAR-T cells and HAMs in the treatment of AML.

Methods: CD44v6 CAR-T cells pretreated with Dec or Aza were co-cultured with CD44v6+ AML cells. Dec or aza pretreated AML cells were co-cultured with CD44v6 CAR-T cells. The cytotoxicity, exhaustion, differentiation and transduction efficiency of CAR-T cells, and CD44v6 expression and apoptosis in AML cells were detected by flow cytometry. The subcutaneous tumor models were used to evaluate the anti-tumor effect of CD44v6 CAR-T cells combined with Dec in vivo. The effects of Dec or Aza on gene expression profile of CD44v6 CAR-T cells were analyzed by RNA-seq.

Results: Our results revealed that Dec and Aza improved the function of CD44v6 CAR-T cells through increasing the absolute output of CAR+ cells and persistence, promoting activation and memory phenotype of CD44v6 CAR-T cells, and Dec had a more pronounced effect. Dec and Aza promoted the apoptosis of AML cells, particularly with DNA methyltransferase 3A (DNMT3A) mutation. Dec and Aza also enhanced the CD44v6 CAR-T response to AML by upregulating CD44v6 expression of AML cells regardless of FMS-like tyrosine kinase 3 (FLT3) or DNMT3A mutations. The combination of Dec or Aza pretreated CD44v6 CAR-T with pretreated AML cells demonstrated the most potent anti-tumor ability against AML.

Conclusion: Dec or Aza in combination with CD44v6 CAR-T cells is a promising combination therapy for AML patients.

Keywords: CD44v6 chimeric antigen receptor-T cells; acute myeloid leukemia; azacitidine; decitabine; demethylating therapy.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Azacitidine / pharmacology
Azacitidine / therapeutic use
Cell Line, Tumor
DNA Modification Methylases
Humans
Leukemia, Myeloid, Acute* / genetics
Receptors, Chimeric Antigen* / genetics
T-Lymphocytes

Substances

Receptors, Chimeric Antigen
Azacitidine
DNA Modification Methylases

Grants and funding

This work was supported by National Key R&D Program of China (2021YFF0703704), National Natural Science Foundation of China (82270183, 81570110), the Excellent Young Science Foundation Project of Tongji Hospital (No.2020YQ0012) and the Wuhan Joint Fund Project 2019 (WJ2019H383), Natural Sciences Foundation of Hubei Province of China (2021CFB561).