去甲基化治疗增强了针对急性髓系白血病的 CD44v6 CAR-T 细胞的细胞毒性。
Demethylating therapy increases cytotoxicity of CD44v6 CAR-T cells against acute myeloid leukemia.
机构信息
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
出版信息
Front Immunol. 2023 Apr 27;14:1145441. doi: 10.3389/fimmu.2023.1145441. eCollection 2023.
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 . 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.
背景
嵌合抗原受体 T（CAR-T）细胞表达 CD44v6 在治疗急性髓系白血病（AML）中显示出强大的抗肿瘤能力和安全性。然而，T 细胞上 CD44v6 的表达导致了 CD44v6 CAR-T 细胞的短暂自相残杀和耗竭，这影响了 CD44v6 CAR-T 的应用。T 细胞的耗竭和功能以及 AML 细胞的 CD44v6 表达与 DNA 甲基化有关。低甲基化剂（HAMs）地西他滨（Dec）和阿扎胞苷（Aza）已广泛用于治疗 AML。因此，CD44v6 CAR-T 细胞与 HAMs 在治疗 AML 中可能存在协同作用。
方法
用 Dec 或 Aza 预处理 CD44v6 CAR-T 细胞，然后与 CD44v6+AML 细胞共培养。用 Dec 或 aza 预处理 AML 细胞与 CD44v6 CAR-T 细胞共培养。通过流式细胞术检测 CAR-T 细胞的细胞毒性、耗竭、分化和转导效率，以及 AML 细胞中 CD44v6 的表达和凋亡。利用皮下肿瘤模型评价 CD44v6 CAR-T 细胞联合 Dec 的抗肿瘤作用。通过 RNA-seq 分析 Dec 或 Aza 对 CD44v6 CAR-T 细胞基因表达谱的影响。
结果
我们的结果表明，Dec 和 Aza 通过增加 CAR+细胞的绝对输出和持久性来改善 CD44v6 CAR-T 细胞的功能，促进 CD44v6 CAR-T 细胞的激活和记忆表型，Dec 的效果更为显著。Dec 和 Aza 促进 AML 细胞凋亡，特别是在 DNA 甲基转移酶 3A（DNMT3A）突变的情况下。Dec 和 Aza 还通过上调 AML 细胞的 CD44v6 表达来增强 CD44v6 CAR-T 对 AML 的反应，无论 FMS 样酪氨酸激酶 3（FLT3）或 DNMT3A 突变如何。用 Dec 或 Aza 预处理 CD44v6 CAR-T 与预处理 AML 细胞联合应用显示出对 AML 最有效的抗肿瘤能力。
结论
Dec 或 Aza 联合 CD44v6 CAR-T 细胞是治疗 AML 患者的一种很有前途的联合治疗方法。
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