Associate Professor USC, California, United States
Introduction: Glioblastoma (GBM) is the most common and deadly primary adult brain tumor. It is notoriously difficult to treat, with minimal improvements in patient survival and no new therapeutic options available over the last two decades. Chimeric Antigen Receptor Natural Killer (CAR-NK) cell therapy has recently gained attraction for GBM treatment. While clinical trials have established the safety and anti-tumor efficacy of CAR-NK therapies in GBM patients, durable remission remains to be achieved, highlighting the need for optimizing CAR-NK for GBM therapy. Nutrient competition and hypoxia within the solid tumor microenvironment (TME) drive mitochondrial dysfunction, metabolic insufficiency, and severe oxidative damage in NK cells, which lead to poor antitumoral efficacy. The transcription factor, Nuclear factor erythroid 2-related factor 2 (NRF2), is a master regulator of the cellular antioxidant response. We hypothesize that improving NRF2 function can boost CAR-NK cell functions in hypoxic TME. Here, we utilized a 3D hypoxic tumor model previously established by our group to investigate the impact of NRF2 activation on CAR-NK cell infiltration and killing in GBM microenvironment. Furthermore, we are investigating NRF2 overexpression as a strategy for boosting CAR-NK cell function against hypoxic GBM tumors.
Materials and
Methods: U251 GBM cells expressing a hypoxia-driven biosensor, HRE-UnaG, were cultured in a Gelatin-Methacrylate (GelMA) hydrogel in microfluidic devices to create normoxia or hypoxia tumor models. UnaG expression levels were determined via imaging at the 24-hour time point to confirm the generation of hypoxia within the hypoxic tumor devices. Omaveloxolone (RTA-408) mediated NRF2 activation has been shown to reduce cellular oxidative stress and confer metabolic flexibility in NK cells. Hypoxic devices were treated with EGFR CAR-NK cells in combination with or without RTA-408. NK cell cytotoxicity (Propidium Iodide (PI)) and infiltration (CD45) were assessed 24 hours after treating the tumor model. We further created a lentiviral construct to drive the over-expression (OE) of NRF2 in CAR-NK cells for NK-specific NRF2 activation.
Results, Conclusions, and Discussions: UnaG expression in the engineered U251 cells depends on HIF1a activity, which is only stabilized under hypoxic oxygen levels (Figure 1A). In line with this, normoxia cultured U251 cells showed no expression of UnaG, whereas those in the hypoxic devices showed upregulation of UnaG signal within the tumor model in 24 hours (Figure 1A). In the 3D hypoxic killing assay, RTA-408 or CAR-NK cell treatment alone did not show significant cancer cell death. In contrast, we observed increased cancer cell death in the CAR-NK cell and RTA-408 co-treatment group, indicating increased CAR-NK cytotoxicity upon NRF2 activation (Figure 1B). Interestingly, we did not observe a significant difference in NK cell infiltration between treatment groups, warranting further investigation into the mechanism of action of NRF2 activation (Figure 1C). Finally, we created a NRF2 construct and showed successful expression in Jurkat T-cells (Figure 1D).
Overall, this data serves as proof-of-concept for enhancing NRF2 activation to boost CAR-NK cell antitumoral function against GBM tumors. We are validating NRF2 construct in primary CAR-NK cells and investigating its impact on CAR-NK cytotoxicity as well as mitochondrial and metabolic fitness.
