Hypoxia-Driven HIF-1α Activation Reprograms Pre-Activated NK Cells towards Highly Potent Effector Phenotypes via ERK/STAT3 Pathways

SIMPLE SUMMARY: In patients with advanced cancer, hypoxic stress shapes NK cells toward tumor-resistant and immunosuppressive phenotypes. Therefore, a strategy to restore NK cell function within hypoxia would be crucial for successful tumor immunotherapy. By manipulating pO(2) exposure to naïve vs. pre-activated NK cells, we found that HIF-1α-dependent metabolic reprogramming of NK cells is the key to overcoming hypoxia-mediated NK cell impairment. Exposure of pre-activated NK cells to hypoxia with 1.5% pO(2) initiated metabolic shift from oxidative phosphorylation to glycolysis and reduction of p21/p53-dependent apoptotic pathways, with concomitant upregulation of cell cycle-promoting genes and downregulation of cell cycle-arrest genes via HIF-1a/ERK/STAT3 activation. Furthermore, upregulation of NKp44 activating receptor in hypoxia-exposed pre-activated NK cells elevated cytotoxicity of K562, CEM, and A375 tumor cells, in both in-vitro and in-vivo tumor-clearance assays. Therefore, HIF-1α-mediated metabolic reprogramming of NK cells could reverse their impaired phenotype, generating functionally robust NK cells for adoptive therapy and clinical evaluation. ABSTRACT: NK cells are the predominant innate lymphocyte subsets specialized to kill malignant tumor cells. In patients with advanced cancer, hypoxic stress shapes NK cells toward tumor-resistant and immunosuppressive phenotypes, hence a strategy to restore NK function is critical for successful tumor immunotherapy. Here, we present evidence that pre-activation and subsequent HIF-1α-dependent metabolic shift of NK cells from oxidative phosphorylation into glycolysis are keys to overcome hypoxia-mediated impairment in NK cell survival, proliferation, and tumor cytotoxicity. Specifically, exposing NK cells to 7–9 days of normoxic culture followed by a pO(2) of 1.5% hypoxia led to a highly potent effector phenotype via HIF-1α stabilization and upregulation of its target genes, BNIP3, PDK1, VEGF, PKM2, and LDHA. RNA sequencing and network analyses revealed that concomitant reduction of p21/p53 apoptotic pathways along with upregulation of cell cycle-promoting genes, CCNE1, CDC6, CDC20, and downregulation of cell cycle-arrest genes, CDKN1A, GADD45A, and MDM2 were accountable for superior expansion of NK cells via ERK/STAT3 activation. Furthermore, HIF-1α-dependent upregulation of the NKp44 receptor in hypoxia-exposed NK cells resulted in increased killing against K562, CEM, and A375 tumor targets both in-vitro and in-vivo tumor clearance assays. Therefore, hypoxic exposure on pre-activated proliferating NK cells triggered HIF-1α-dependent pathways to initiate coordinated regulation of cell cycle, apoptosis, and cytotoxicity at the global gene transcription level. Our results uncover a previously unidentified role of HIF-1α-mediated metabolic reprogramming that can reverse impaired NK effector phenotypes to generate requisite numbers of functionally robust NK cells for adoptive cellular therapy for clinical evaluation.