Deletion of CISH in Human Pluripotent Stem Cell-Derived Natural Killer Cells Enhances Anti-Tumor Activity Via Metabolic Reprogramming

Cellular metabolism is now recognized to play a key role in regulating function and differentiation of immune cells. Immune cells undergo dynamic metabolic shifts to support their activity. Recent studies have shown the importance of metabolism to regulate NK cell function under different conditions. IL-15 can regulate NK cell metabolism in a dose- and time- dependent manner. However, whether improving metabolic fitness of NK cells can lead to improved anti-tumor activity is largely unknown. Here, we deleted CIS (encoded by the CISH gene in human induced pluripotent stem cells (iPSC)-derived NK cells (CISH^-/- iPSC-NK) using CRISPR/Cas9 technology. CIS is known to function as a critical negative regulator of IL-15 signaling and a potent inhibitory checkpoint in NK cell-mediated anti-tumor activity. Surprisingly, more than 3000 differentially expressed genes were identified in CISH^-/- iPSC-NK cells through RNA sequencing analysis. Gene ontology (GO) enrichment analysis showed that the top 2 up-regulated signaling pathways were associated with leukocyte differentiation and activation. Notably, more than 50 genes that involved in positive regulation of lymphocyte activation including genes associated with NK cell effector functions were significantly up-regulated in CISH^-/- iPSC-NK cells compared with unmodified iPSC-NK cells. As expected, CISH^-/- iPSC-NK cells exhibited increased sensitivity to IL-15 stimulation. Specifically, IL-15 stimulated JAK1 tyrosine phosphorylation, STAT3 and STAT5 phosphorylation were all dramatically increased in CISH^-/- iPSC-NK cells, compared with unmodified iPSC-NK cells. In addition, when incubated with low concentrations of IL-15 (5 ng/ml), a concentration that does not support the growth and functions of unmodified iPSC-NK cells, CISH^-/- iPSC-NK cells maintained significantly better expansion with >10 fold expansion for CISH^-/- iPSC-NK cells in 3 weeks, whereas most of WT iPSC-NK cells died under these conditions (p<0.001). These CISH^-/- iPSC-NK cells exhibited improved cytotoxic against acute myeloid leukemia (AML) cell lines K562 and Molm-13. More importantly, in an AML (MOLM-13) xenograft model, CISH^-/- iPSC-NK cells displayed significantly increased in vivo persistence thus lead to significantly better control of tumor progression. To further understand the underling mechanisms contributing to the increased functionality in CISH^-/- iPSC-NK cells, we then analyzed the metabolic profiles of these cells using Agilent Seahorse assays. After treatment with low concentration IL-15 for 7 days, CISH^-/- iPSC-NK cells displayed both increased glucose metabolism and increased mitochondrial respiration profiles when compared with unmodified iPSC-NK cells. Increased glucose metabolism include a significant increase in basal glycolysis (p=0.0003, 84.5 ± 1.0 vs 61.1 ± 1.8 mpH/min, n=6), and glycolytic capacity (p=0.0049, 15.2±0.4 vs 11.7±0.5 mpH/min, n=6). And increased mitochondrial respiration profile is characterized by an increase in the spare respiratory capacity (p=0.0009, 16.3±1.9 vs 6.9±0.5 pmol/min, n=6), maximal respiration (p<0.0001, 31.6±1.9 vs 15.7±0.8 pmol/min, n=6) and ATP-linked respiration (p<0.0001, 11.5 ± 0.4 vs 7.0±0.3 pmol/min, n=6). Interestingly, limiting the increase of glucose metabolism and mitochondrial respiration via utilization of an mTOR inhibitor (rapamycin) attenuated the increased functionality in CISH^-/- iPSC-NK cells. The findings presented here show that deletion of CISH in iPSC-NK cells enhance anti-tumor activity, at least in part via metabolic reprogramming. These findings suggest that improving NK cell functions for cancer immunotherapy through metabolic reprogramming provides a promising therapeutic strategy.