CD44v6 CAR-T Cells Target DNMT3A-Mutant AML: Synergistic Enhancement by Decitabine

Abstract

Objective

Acute myeloid leukemia (AML) is a highly heterogeneous disease, and molecular events such as DNMT3A gene mutations are associated with poor prognosis in AML patients. Consequently, there is an urgent need for a novel therapeutic approach for AML.

Methods

DNMT3A mRNA and protein expression were confirmed in DNMT3A-mutant AML cells via RT-qPCR and Western blotting. Cell proliferation and apoptosis were assessed via CCK-8 and Annexin V/PI staining, respectively. Flow cytometry was used to analyze surface antigens and CD44v6 CAR-T-cell transfection efficiency. CD44v6-directed CAR plasmids were constructed, and lentiviruses were packaged. Methylation-specific PCR was used to evaluate differences in promoter methylation, whereas ELISA was used to measure cytokine secretion.

Results

In this study, we found that the DNMT3A-mutant group presented significantly increased expression of CD44v6 on the cell surface. Methylation of the CD44 promoter region was lower in the mutant group than in the control group. CD44v6 CAR-T cells exhibited specific cytotoxicity against DNMT3A-mutant AML cells. Furthermore, pretreatment with low concentrations of decitabine significantly enhanced the killing effect of CD44v6 CAR-T cells on DNMT3A-mutant AML cells (P < 0.05). Additionally, decitabine treatment upregulated the expression of CD44v6 on the surface of DNMT3A-mutant AML cells (P < 0.05).

Conclusion

CD44v6 is a promising CAR-T-cell therapy target in AML patients with DNMT3A mutations. Notably, treatment with decitabine resulted in increased CD44v6 expression on the cell surface of DNMT3A-mutant AML cells. This increase in CD44v6 expression facilitates improved recognition and targeting by CD44v6 CAR-T cells.

Keywords

Acute myeloid leukemia / DNMT3A mutation / Chimeric antigen receptor / CD44v6 / Decitabine / Epigenetic regulation / Combination therapy / DNA methylation

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Funding

the National Natural Science Foundation of China(82270177)

Chen Xiaoping Foundation for the development of Science and Technology of Hubei Province(CXPJJH122001-2221)

RIGHTS & PERMISSIONS

The Author(s), under exclusive licence to the Huazhong University of Science and Technology