Abstract We0043: DUSP1-dependent regulation of the mTOR/TFEB axis as a common dysregulated signaling pathway in obesity as well as LDL-induced NK cell dysfunction

Background: Obesity and hyperlipidemia disproportionally affect African American (AA) women due to lifelong exposure to adverse social determinants of health (aSDoH), leading to greater cardiovascular disease (CVD) risk. In a previous study we identified Dual Specificity Protein Phosphatase 1 (DUSP1) as the only commonly upregulated gene in NK cells isolated from AA women with obesity and LDL-treated NK cells and that inhibition of DUSP1 with a small molecule inhibitor rescued LDL-induced NK cell dysfunction. In this study, we aimed to determine the pathways underlying the NK cell dysfunction observed in LDL-treated NK cells and their dependency on Dusp1. Methods: NK cells (CD3-/CD56+) were isolated from healthy donor buffy coats or AA women with/without obesity (W/O vs WO/O). NK cells were treated with vehicle ctr or LDL (50mg/dl) overnight ± a Dusp1 inhibitor (DUSP1-i) at 5mM. Autophagic flux and TFEB dephosphorylation were assessed by western blotting. RT-qPCR was used for mRNA expression analysis, while flow cytometry was used to determine lysosomal biogenesis. TFEB localization was visualized using confocal microscopy. Results: Treatment of NK cells with LDL induced inhibition of late-stage autophagy as determined by a reduced autophagic flux (p=0.01) and a significant 18±4.6% reduction of intracellular CD107a expression. Interestingly, the addition of a Dusp1-i did not rescue the LDL-induced autophagic flux but increased LAMP1 mRNA expression and lysosome count. A key pathway of lysosomal biogenesis is the mTOR/TFEB axis. We determined that mTOR mRNA is decreased by 27% after LDL incubation. Additionally, LDL prohibited de-phosphorylation and subsequent nuclear translocation of transcription factor EB (TFEB). The LDL-induced impact on mTOR and TFEB was abrogated in the presence of the Dusp1-i. Interestingly, a 45% lower mTOR gene expression and disturbed TFEB phosphorylation pattern was also detected in NK cells of women W/O when compared to NK cells of women WO/O. Conclusions: Taken together, our data suggest that LDL and obesity inhibit lysosomal biogenesis by inhibiting the mTOR-dependent TFEB nuclear translocation in a DUSP1-dependent manner. Future studies should determine the potential of therapeutic targeting of the mTOR/TFEB/DUSP1 axis to improve NK cell function in individuals at high risk for obesity in the setting of aSDoH.