Background: Natural killer (NK) cells are key effectors in cancer immunotherapy, and their activa-tion can be enhanced by cationic compounds. While 25KDa branched polyethylenimine (25KbPEI) has been shown to prime NK cells, the underlying molecular mechanisms remain poorly understood. Here, we investigated how 25KbPEI-mediated chemical priming enhances NK cell function and elu-cidated its clinical potential in combination with daratumumab (DARA).
Methods: We examined 25KbPEI-induced NK cell priming via the CD38-TRPM2-Ca2+-AMPK sig-naling axis by assessing CD38 internalization, Ca2+ influx, perforin accumulation, and AMPK activa-tion using flow cytometry, confocal microscopy, and biochemical assays. To verify the signaling pathways, NK cells isolated from TRPM2 knockout (KO) and CD38 KO mice were analyzed. NK cell cytotoxicity was evaluated in vitro against MM.1S multiple myeloma cells in the presence of DARA and in vivo using a multiple myeloma xenograft model in NSG mice.
Results: 25KbPEI induced CD38 internalization, promoting ADPR production and TRPM2-mediated Ca2+ influx, leading to enhanced perforin accumulation and AMPK activation. AMPK facilitated per-forin accumulation and lytic granule convergence, further amplifying NK cell cytotoxicity. In vivo, Chem_NK synergized with DARA, significantly suppressing tumor growth and extending survival compared to control NK cells combined with DARA.
Conclusions: This study defines the CD38-TRPM2-Ca2+-AMPK axis as a key regulatory pathway in 25KbPEI-induced NK cell priming, driving perforin accumulation and lytic granule convergence. By enhancing NK cell-mediated cytotoxicity while preventing CD38-mediated fratricide, these findings offer a clinically viable alternative to genetic engineering for optimizing DARA combination therapy and expanding NK cell-based cancer treatments.