LIPID-BASED APPROACHES TO OPTIMIZE THE EFFECTIVENESS OF INNOVATIVE DRUG MODULATORS IN CYSTIC FIBROSIS

Cystic Fibrosis (CF), a life-threatening hereditary disease prevalent among Caucasians, results from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. This gene encodes a chloride-conducting transmembrane channel crucial for ion transport across epithelial cells. CFTR mutations lead to impaired expression and function of the channel, causing disrupted chloride ion transport in secretory epithelia. Severe manifestations affect many organs, primarily the respiratory system, with impaired mucociliary clearance and airway surface liquid dehydration, leading to recurrent infections and high morbidity. The most common mutation, the deletion of phenylalanine at position 508 (F508del), induces misfolding and retention of CFTR in the endoplasmic reticulum, causing premature degradation. In airway epithelial cells, CFTR associates with proteins in a macromolecular complex, and its channel activity and surface expression are regulated by local signaling and recycling through endosomal compartments. The CFTR interactome also includes lipids, such as cholesterol and sphingolipids, that play a significant role in stabilizing CFTR within the cell membrane, particularly in specialized regions known as lipid rafts. In fact, the ganglioside GM1 is an important bioactive sphingolipid that has been reported to play a crucial role in the control of several plasma membrane (PM) proteins and its correlation with CFTR prompts speculations about its possible role in the CFTR interactome. Despite therapeutic improvements that extended CF patients' life expectancy, a cure remains elusive. Promising strategies involve CFTR-modulators, such as correctors, enhancing CFTR biosynthesis and trafficking, and potentiators, ameliorating mutated channel function. Treatment of patients having F508del mutation requires a combination of correctors and potentiators. Indeed, recent advancements in therapy encompass the triple combination Kaftrio (comprising two correctors, VX-661, VX-445, and a potentiator, VX-770) as a pharmacological intervention for CF patients who have at least one F508del mutation in the CFTR gene. In my PhD project, I explored the impact of Kaftrio on the lipid composition of bronchial epithelial cells expressing WT and F508del-CFTR. I found that it induced significant changes reducing LacCer levels and increasing the content of GM1 and GD1a, respectively. The modifications were attributed to the effect of Kaftrio on the activity of some enzymes related to sphingolipid metabolism, specially the GM3 synthase and sialidase. Additionally, the administration of GM1, previously noted for improving the rescue of F508del-CFTR with Orkambi (consisting of the corrector VX-809 and potentiator VX-770), has also shown effectiveness with Kaftrio, mitigating the side effect of VX-770 on the stability of F508del-CFTR rescued at the cell surface by the correctors. This additive effect of GM1 to Kaftrio was maintained also in the presence of Pseudomonas Aeruginosa infection. On the other hand, I demonstrated that Kaftrio induces a further decrease of already reduced levels of cholesterol in F508del-CFTR mutated cells. However, exogenously administering cholesterol, in particular with low-density-lipoprotein, I observed a cumulative effect of this lipid to the triple drug combination on CFTR correction at the PM. Understanding the CFTR interactome, including its interactions with lipids, is important for unraveling the molecular mechanisms of cystic fibrosis and may provide insights into possible therapeutic strategies given the ongoing challenge of developing new correctors and potentiators. These findings highlight the potential of exogenous GM1 and cholesterol administration in restoring plasma membrane properties conducive to CFTR correction, suggesting novel therapeutic approaches to enhance the effectiveness of CFTR modulators in the treatment of the disease.