(688) Lipid based therapeutic strategies in addition to CFTR modulators for Cystic Fibrosis treatment

Background: Combined therapy with modulators, based on both correctors and potentiators, is considered mandatory to efficiently rescue F508del-CFTR. Co-presence of different small organic molecules interacting with F508del-CFTR may however result in decreasing the efficiency of mutant protein rescue, as shown in the case of the negative effect produced by the potentiator ivacaftor when associated with the corrector lumacaftor [1]. Moreover, it has been demonstrated that the stability of mutated CFTR at the plasma membrane (PM) upon rescue with modulators is strongly impaired by P.aeruginosa (P.a) infection [2]. Thus, the discovery of novel stabilizers of rescued F508del-CFTR is gaining further relevance. Recently, we have shown that sphingolipids, GM1 and cholesterol, which are all downregulated in CF bronchial epithelial cells, can stabilize and improve the function of rescued F508del-CFTR protein [3]. Since CFTR stability and function are the result of a fine coordination of a molecular complex composed by lipids and proteins, in this study we sought to investigate the effect of elexacaftor/tezacaftor/ivacaftor on lipid microenvironment surrounding the CFTR. In addition, we analyzed the effect of GM1 on CFTR protein expression upon treatment with CFTR modulators, in the presence of P.a in CF cell models.

Methods: To study the effect of elexacaftor/tezacaftor/ivacaftor on lipid expression and metabolism we used tritiated sphingosine to label membrane lipids of CFBE41o- F508del CFTR epithelial respiratory cells. Lipids have been extracted and analyzed with HPTLC and visualized by digital autoradiography. CF bronchial epithelial cells treated with elexacaftor/tezacaftor/ivacaftor alone or in combination with P.a, GM1 or its derivative LIGA20 were used to investigate the expression and stability of CFTR protein by WB upon biotinylation of proteins associated with the external leaflet of PM or by WB on total cell lysates.

Results: We observed that both GM1 and LIGA20 promoted the stabilization of F508del-CFTR protein rescued by elexacaftor/tezacaftor/ivacaftor treatment. Interestingly, P.a reduced the expression of rescued CFTR upon treatment with both elexacaftor and tezacaftor, or the combination of elexacaftor/tezacaftor/ivacaftor. Of note, GM1 partially counteracted the P.a-dependent impairment of CFTR rescue upon CFTR modulator therapy.

Conclusions: Taken together, these results suggest that GM1 and its pharmacological derivative LIGA20 play a critical role in stabilization of CFTR at PM, also in the presence of infection. The association of these sphingolipids with current therapeutic treatments could therefore increase the efficacy of CFTR modulators.

Acknowledgements: This work was supported by the Italian Cystic Fibrosis Foundation [grant number 2/2020]

References: [1] Matthes E, Goepp J, Carlile GW, Luo Y, Dejgaard K, Billet A, et al. Low free drug concentration prevents inhibition of F508del CFTR functional expression by the potentiator VX-770 (ivacaftor). British Journal of Pharmacology. 2016;173(3):459-70.

[2] Stanton BA, Coutermarsh B, Barnaby R, Hogan D. Pseudomonas aeruginosa Reduces VX-809 Stimulated F508del-CFTR Chloride Secretion by Airway Epithelial Cells. PLOS ONE. 2015;10(5):e0127742.

[3] Mancini G, Loberto N, Olioso D, Dechecchi MC, Cabrini G, Mauri L, et al. GM1 as Adjuvant of Innovative Therapies for Cystic Fibrosis Disease. International Journal of Molecular Sciences. 2020;21(12):4486.