DRP1 depletion protects NK cells against hypoxia-induced dysfunction

Verhezen, T.; Van den Eynde, A.; Verstraelen, P.; Gehrcken, L.; Palmiotto, G.; Lau, H. W.; De Vos, W.; van der Heijden, S.; Brants, L.; Melis, J.; Van Audenaerde, J.; Van Laere, S.; Lardon, F.; Deben, C.; Wouters, A.; Smits, E.; De Waele, J.

doi:10.1101/2025.06.23.661011

bioRxiv · 10.1101/2025.06.23.661011

DRP1 depletion protects NK cells against hypoxia-induced dysfunction

Verhezen, T.·Van den Eynde, A.·Verstraelen, P.·Gehrcken, L.·Palmiotto, G.·Lau, H. W.·De Vos, W.·van der Heijden, S.·Brants, L.·Melis, J.·Van Audenaerde, J.·Van Laere, S.·Lardon, F.·Deben, C.·Wouters, A.·Smits, E.·De Waele, J.

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Abstract

Hypoxia within the tumor microenvironment poses a major barrier to the efficacy of NK cell-based immunotherapies for solid tumors. In this study, we investigated the influence of hypoxia on NK cell function and mitochondria. We found that hypoxia reduced NK cell cytotoxicity, mitochondrial content, and membrane potential, while increasing mtROS and inducing broad transcriptional changes in metabolic and stress response pathways. CAR engineering with CD70 and IL-15, while designed to enhance persistence and metabolic fitness, did not prevent hypoxia-induced impairment. Given the mitochondrial disruption, we then explored whether DRP1 ablation could mitigate hypoxia-induced dysfunction. Pharmacological inhibition of DRP1 restored mitochondrial content and cytotoxic function. To confirm the role of DRP1, we generated CRISPR-Cas9-mediated DRP1 KO NK cells, which preserved mitochondrial load and membrane potential under hypoxia. When armed with CD70-CAR-IL-15, DRP1KO cells retained cytotoxic activity under hypoxic conditions. These findings show that DRP1 inactivation can support NK cell function in hypoxic environments, and that metabolic engineering may enhance CAR NK cell efficacy in solid tumors. Graphical abstractNK cells become dysfunctional in hypoxic conditions, while DRP1KO NK cells retain their function. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/661011v2_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@2c3a96org.highwire.dtl.DTLVardef@1ba7d20org.highwire.dtl.DTLVardef@6ceeecorg.highwire.dtl.DTLVardef@1b9e0ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Verhezen, T., Van den Eynde, A., Verstraelen, P., Gehrcken, L., Palmiotto, G., Lau, H. W., De Vos, W., van der Heijden, S., Brants, L., Melis, J., Van Audenaerde, J., Van Laere, S., Lardon, F., Deben, C., Wouters, A., Smits, E., De Waele, J.. 2025-06-27. DRP1 depletion protects NK cells against hypoxia-induced dysfunction. https://doi.org/10.1101/2025.06.23.661011

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