WS04.01 A PI3Kγ Mimetic Peptide Promotes F508del-CFTR Plasma Membrane Stabilization Through Protein Kinase D1

Objectives: Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). For most CF patients, FDA approved ELX-TEZ-IVA (ETI), a combination therapy restoring F508del-CFTR folding and gating. Although ETI has shown high effectiveness in clinics, it increases CFTR function, including CFTR stability, by only 50% compared to fully functional channels, underlying the need to find new and/or add-on treatments. Our results indicate that a PI3Kγ mimetic peptide (PI3Kγ MP; Patent n° WO2016103176) could represent an alternative strategy to restore F508del-CFTR stability thanks to its ability to increase the PM amount of channel (unpublished). However, the molecular mechanism behind this PI3Kγ MP-mediated effect remains unclear. Methods: To unravel how PI3Kγ MP controls F508del-CFTR stability, we performed biochemical and microscopy-based assays in human bronchial epithelial cells upon peptide treatment. Results: A phospho-proteomics approach revealed protein kinase D1 (PKD1), a well-established protein secretion orchestrator, as being activated by PI3Kγ MP and thus a putative mediator of its effects on PM F508del-CFTR stabilization. Western blot analysis confirmed PI3Kγ MP-mediated PKD1 phosphorylation, while cell surface biotinylation upon treatment with a mutagenized peptide unveiled the key role of PI3Kγ MP arginine residues for PKD1 activation and CFTR stabilization at the PM. Accordingly, PKD1 inhibitors abolished the ability of PI3Kγ MP to increase F508del-CFTR PM density. Finally, we demonstrated that, by increasing CFTR stabilization, PI3Kγ MP maximizes the effects of ETI on Cl– conductance in both F508del homozygous and heterozygous primary bronchial epithelial cells. Conclusion: We identified PI3Kγ MP as a new effective approach to stabilize PM F508del-CFTR and to increase ETI therapeutic effect. Supported by the Italian CF Research Foundation and by CFE and ECFS (postdoctoral fellowship to A.M.)