(651) Hyperglycemia Contributes to Oxidative Stress and Impairs Potency of Highly Effective Modulator Therapies.

Background: Cystic fibrosis-related diabetes (CFRD) is the most prevalent co-morbidity among people with CF. An estimated 30-50% of CF adults are diagnosed with CFRD, which develops during adolescence and leads to increased frequency of acute pulmonary exacerbations together with a six-fold higher risk of mortality. Improved understanding of mechanisms by which systemic hyperglycemia contributes to accelerated pulmonary decline represents a significant unmet medical need. Major drivers implicated in CFRD etiology include inflammation, oxidative stress, and CF transmembrane conductance regulator (CFTR) variant severity. Redox poise is dysregulated from the earliest stages of CF disease as evidenced by diminished levels of the antioxidant glutathione (GSH). This leads to decreased buffering of pro-inflammatory reactive oxygen species (ROS) and enhanced protease activity in the airways. Although highly effective modulator therapies (HEMTs) were recently developed to specifically restore functional defects in CFTR, treatment outcomes with regard to CFRD pathogenesis have not been well delineated. A major objective of our work is to determine impact of HEMTs on mutant CFTR biogenesis, glucose metabolism, and redox poise in cells altered by CFRD sequelae.

Methods: We hypothesize chronic hyperglycemia depletes GSH levels by increasing ROS (electron leak) and decreasing NADPH (polyol pathway), resulting in CFTR turnover by accumulation of advanced glycation end products and redox stress. We used CF bronchial epithelia (CFBE) stably transduced with wild-type (WT) or F508del-CFTR, F508del homozygous primary human bronchial epithelia (HBE), and 16HBE14o^- edited with CRISPR/Cas9 to express WT- or F508del-CFTR. Polarized cells were grown in normoglycemia (1g/L) or hyperglycemia (2.5g/L), and with or without chronic treatment of HEMTs. These included: ivacaftor; lumacaftor/ivacaftor; or elexacaftor/tezacaftor/ivacaftor. CFTR short-circuit currents (Ussing chamber) and GSH redox state (liquid chromatography-mass spectrometry, LC-MS) were quantified.

Results: In CFBE, hyperglycemia diminished WT- and F508del-CFTR transepithelial ion transport by approximately 31% or 32%, respectively. Elevated glucose also reduced HEMT-mediated correction of F508del channel activity by ~30%. In 16HBE encoding WT- or F508del-CFTR, hyperglycemia decreased intracellular GSH by 21% or 9.8%, respectively. In primary HBE with CFTR^{F508del/F508del} genotype, apical fluid GSH was increased ~2-fold after 8-hour HEMT application. Ongoing studies in human bronchial and pancreatic epithelia will ascertain effects of hyperglycemia and HEMTs on GSH redox state (LC-MS), glucose metabolism (¹³C₆-glucose tracing), and CFTR functional expression (western/Ussing).

Conclusions: Our results suggest that in the airway environment: (1) elevated glucose impairs CFTR ion transport, HEMT rescue of mutant CFTR activity, and cellular redox state; and (2) HEMTs augment CFTR-dependent redox buffering. Enhanced understanding of mechanisms by which HEMTs affect GSH regulation, CFTR biogenesis, and oxidative stress in the setting of hyperglycemia would improve clinical resolution regarding implications of long-term HEMT use. Further research is needed to elucidate the impact of hyperglycemia and HEMTs on redox state to strengthen precision medicine-based approaches to CFRD complications.

Acknowledgements: This work was supported by the National Institutes of Health (HL151965 to KEO; HL150658 to JDC) and CF Foundation (OLIVER22A0-KB to KEO; MCCART21G0 to NAM).

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