Unraveling the functional responses of natural killer cells through functional genomics and genetic engineering

Natural killer (NK) cells have emerged as potent cytotoxic lymphocytes in cellular immunotherapies in the recent decades. 50 years of extensive research into their multidimensional nature have led to their substantial characterization, however, the genetic circuits governing their cytotoxic and cytokine programs, and the constraints limiting their therapeutic engineering and in-tumor function, remain incompletely defined. This thesis combines genome-wide functional genomics, small molecule-assisted gene delivery, and translational NK cell engineering to dissect and therapeutically exploit NK-cell biology in cancer. In Study I, a genome-wide loss-of-function CRISPR-Cas9 screen in NK-92 cells simultaneously interrogated degranulation and interferon-gamma (IFNy) production. The results mapped 914 regulators of NK effector functions, recovering known genes in cytotoxic granule trafficking and primary immunodeficiencies, and revealing a predominance of negative regulators limiting degranulation versus extensive positive regulators sustaining IFNy production. Pathway-level analyses highlighted vesicle trafficking, mitochondrial metabolism, translational control, and signaling modules downstream of activating receptors, and an updated NK cell cytotoxicity gene set was curated to better align CRISPR-based functional data with existing transcriptomic resources. To overcome innate antiviral barriers that limit NK-cell engineering, RIG-I-pathway inhibitor, 5Z-7-oxozeaenol, was identified as a transient, low-toxicity enhancer of lentiviral gene delivery in Study II. Short-term exposure to 5Z-7-oxozeaenol increased transduction efficiencies up to eight-fold in NK cell lines and up to four-fold in primary NK and T cells across multiple multiplicities of infection, without impairing viability, degranulation, or IFNy responses, and with activity extending to B and T cell lines in an envelope-dependent manner. Study III explored the translational potential of engineered NK cells in ovarian cancer models using KHYG-1 cells modified with a DR5-selective TRAIL variant (TRAILV-KHYG-1). TRAILv-KHYG-1 cells exhibited enhanced apoptosis induction and reduced viability in TRAIL-sensitive OVCAR-3 cells compared to parental KHYG-1, associated with increased granzyme and IFNy secretion and elevated soluble TRAIL, whereas both NK cell products showed minimal cytotoxicity against TRAIL-resistant SKOV-3 cells despite higher DR5 surface expression, indicating that DR5 abundance alone is insufficient to overcome intrinsic TRAIL resistance. Finally, in Study IV, the thesis outlines a regulatory role of NKs, whereby tumor-associated NK cells promote myeloid-derived suppressor cell (MDSC)- mediated immune tolerance through IL-6/STAT3 signaling. Tumor-experienced human NK cells acquired a CD69+perforin phenotype and reprogrammed monocytes and neutrophils toward suppressive, MDSC-like states characterized by defective antigen presentation, up-regulation of PD-L1 and ER-stress markers, and enhanced suppression of CD8+ T cells. In patient tumors and multiple murine models, NK cell-derived IL-6 correlated with MDSC accumulation in an MHC class I-dependent manner. Genetic or siRNA-mediated IL-6 ablation in NK cells, or pharmacologic blockade of the IL-6/STAT3 axis, reduced MDSC-associated suppression, limited metastatic lesions, and improved T-cell activity in xenograft and zebrafish models. To conclude, these studies provide a systemwide map of NK effector functions, introduce a strategy to enhance NK genetic modification, demonstrate the opportunities and limits of death receptor-targeted NK cell therapy in solid tumors, and uncover a role of NK cells in actively shaping tumor-associated immune suppression that can be therapeutically overcome. List of scientific papers I. Burduli N, Klarenbeek K, Zhou ZH, Norman A, Dhanjal S, Gilljam M, Lundqvist A, Schmierer B, Ljunggren HG, Alici E, Wagner AK. Genome-wide loss-of-function CRISPR-Cas9 screen reveals redundancies in NK cell functional landscape. [Manuscript] II. Burduli N, Wagner AK, Dharmaraj DS, Gilljam M, Hållstrand C, Chrobok M, Meinke S, Lundqvist A, Ljunggren HG, Alici E. 5Z-7-Oxozeaenol enhances lentiviral gene delivery across NK, T and B cell populations. [Manuscript] III. Sheedy AM, Burduli N, Prakash A, Gurney M, Hanley S, Prendeville H, Sarkar S, O'Dwyer J, O'Dwyer M, Dolan EB. NK cell line modified to express a potent, DR5 specific variant of TRAIL, show enhanced cytotoxicity in ovarian cancer models Heliyon. 2024 Jul 19;10(15):e34976. PMID: 39170449. https://doi.org/10.1016/j.heliyon.2024.e34976 IV. Neo SY, Tong L, Chong J, Liu Y, Jing X, Oliveira MMS, Chen Y, Chen Z, Lee K, Burduli N, Chen X, Gao J, Ma R, Lim JP, Huo J, Xu S, Alici E, Wickström SL, Haglund F, Hartman J, Wagner AK, Cao Y, Kiessling R, Lam KP, Westerberg LS, Lundqvist A. Tumor-associated NK cells drive MDSC-mediated tumor immune tolerance through the IL-6/STAT3 axis. Sci Transl Med. 2024 May 15;16(747):eadi2952. Epub 2024 May 15. PMID: 38748775. https://doi.org/10.1126/scitranslmed.adi2952