Comprehensive Drug Profiling and CRISPR Screening Reveal Essential Pathways for NK Cell Cytotoxicity

NK cell therapies offer great promise in treating hematological cancers due to their "off-the-shelf" applicability and reduced toxicity. However, challenges in specificity, potency, and longevity persist compared to T cell therapies. Genetic engineering has shown promise in enhancing NK cell function, and recent research indicates that certain oncology drugs can further potentiate NK cell cytotoxicity against leukemic cells. Despite the synergistic potential of individual drugs with NK cells, systematic, large-scale high-throughput testing of NK cell-drug combinations is still largely unexplored. Our study integrates high-throughput drug sensitivity and resistance testing (DSRT), single-cell transcriptomics, cytokine analyses, and CRISPR-Cas9 genome wide screening with a combination of oncology drugs and expanded NK cells (exNK) to provide a detailed understanding of the underlying biological mechanisms.

We conducted a comprehensive analysis of over 500 drug compounds to explore synergistic approaches to enhance NK cell effectiveness against hematological cancers. Overall, this method was applied to 10 blood cancer cell lines, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM). Drugs showing synergistic effects with NK cells through high-throughput DSRT were further tested using multiplexed single-cell RNA sequencing (scRNA seq) and cytokine analysis. Key hits were validated by assessing the effectiveness of drug-NK combinations using NK cells derived from three different donors. Essential pathways for NK cell-mediated killing were identified using genome-wide CRISPR-Cas9 screening. Additionally, we applied DSRT to drug combinations with CD19-targeting CAR NK cells in ALL and validated our findings using primary leukemia samples in vitro.

Among the compounds tested, pevonedistat (NAE inhibitor), daporinad (NAMPT inhibitor), and PKC pathway-targeting compounds sotrastaurin (inhibitor) and bryostatin 1 (activator) exhibited the most notable synergistic effects with NK cells. In THP-1 cells (AML), the combination with NK cells significantly increased target cell killing, while these compounds had little to no effect alone. In addition, pevonedistat, daporinad, and bryostatin 1 enhanced NK cell activity, whereas sotrastaurin showed strong NK cell-inhibiting effects. Similar trends were observed in other AML cell lines, with some effects in CML, ALL, DLBCL, and MM cell lines. Various NAMPT inhibitors (KPT-9274, GMX1778 and LSN3154567) not included in the original screens showed similar effects to daporinad in AML and ALL cell lines. CAR NK cells demonstrated similar efficacy against CD19+ cell lines as non-CAR exNK cells, with more potent enhancing effect of NK cell cytotoxicity in the case of bryostatin 1 and pevonedistat. Our validation results using primary AML patient samples showed strong synergistic effects with the combination of bryostatin 1 and exNK cells, with effects observed with pevonedistat and daporinad as well.

ScRNA-seq results after combination treatments revealed drug-specific clusters of treated NK cells. For pevonedistat, genes associated with NEDD8 and downstream of NRF2, such as TXN, TALDO1, TXNRD1, NQO1, and SRXN1, were upregulated, suggesting increased resistance to oxidative and metabolic stress. TNF was notably upregulated, which was also reflected in secreted cytokines. The daporinad-specific cluster showed upregulation of IFNG, CXCR4, and glucose metabolism-related genes. Bryostatin 1 upregulated IL2RA, CTSW, and ACTB, while sotrastaurin downregulated ACTB and upregulated LGALS3.

Genome-wide CRISPR screens provided further insights into the mechanisms of drug-NK cell synergy. Our results from treating THP-1-Cas9 cells transduced with the Brunello library using pevonedistat (50 nM) and exNK cells at 1:2 effector-target ratio revealed, among other pathways and mechanisms, the importance of ubiquitination in enhancing NK cell cytotoxicity.

In conclusion, our study identifies PKC, NAMPT, and NEDD8 having an essential role in controlling NK cell-mediated killing and suggests potential compounds to enhance NK cell effectiveness in treating hematological malignancies. These findings offer insights into developing combination immunotherapy strategies to improve treatment outcomes.