Abstract 3286: Targeting ALKBH5 to enhance the anti-tumor immunity of natural killer cells

Abstract Natural killer (NK) cell-based immunotherapy is emerging to be a promising strategy to treat cancers due to its high safety profile. Compared with traditional chimeric antigen receptor (CAR) T therapy, NK cell-based therapy has much lower side effects such as cytokine release syndrome (CRS) or neurotoxicity. However, the limited persistence and lower levels of infiltration to the tumor microenvironment has impeded the outcomes for NK cell-based clinical trials. How to genetically engineer NK cells to a better priming activation state has yet to be explored. Here, we identify ALKBH5, a well-established RNA N6-methyladenosine (m6A) demethylase, as a key regulator that restricts NK cells cytotoxicity. Xenograft model studies using B16F10 and MC38 tumor cells demonstrated that tumor progression was dramatically inhibited upon Alkbh5 knockout (Alkbh5-/-) in the recipient mice. Single-cell RNA sequencing (scRNA-seq) revealed the enriched abundance of NK cells among tumor-infiltrating lymphocytes in Alkbh5-/- mice. Also, NK cells extracted from Alkbh5-/- spleen are hyperactivated, exhibiting enhanced cytotoxic function and proliferation rate, facilitating robust tumor suppression. Using MeRIP-seq, RIP-seq and lipid metabolomics, we demonstrated that Alkbh5 deficiency in NK cells reprogramed lipid metabolism pathway, enhancing the conjugation rate between NK cells and tumor cells. This increased synapse formation leaded to elevated release of perforin and granzyme, further improving NK cell killing capacity. Based on these findings, we genetically reprogrammed NK cells by conjugating anti-NK1.1 antibodies to lipid nanoparticles (LNPs) embedding Alkbh5-targeting oligonucleotides. Using the in vivo LNPs target delivery system, we want to explore whether this NK-based immunotherapy could inhibit tumor growth/progression in mice. Altogether, our findings uncover a previously unrecognized role for ALKBH5 in regulating NK cell cytotoxicity and proliferation, providing a new therapeutic strategy by targeting ALKBH5 in NK cell-based immunotherapies for solid tumors. Citation Format: Yuan Che, Tingting Tang, Zunsong Hu, Chao Shen, Zhenhua Chen, Wei Li, Kitty Wang, Lillian Sau, Ying Qing, Dong Wu, Keith Leung, Zhaoxu Xu, Xiaolan Deng, Jianjun Chen. Targeting ALKBH5 to enhance the anti-tumor immunity of natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3286.