Treatment of Polarized Cystic Fibrosis Airway Cells With HGF Prevents VX-661-Rescued F508del-CFTR Destabilization Caused by Prolonged Co-exposure to VX-770.

Ana M Matos,Paulo Matos,Peter Jordan

doi:10.3389/fmolb.2021.812101

Abstract

Cystic fibrosis (CF), the most common inherited disease in Caucasians, is caused by mutations in CFTR, the most frequent of which is F508del. F508del causes ER retention and degradation of the mutant CFTR protein, but also defective channel gating and decreased half-life at the plasma membrane. Despite the recent successes with small-molecule CFTR modulator drugs, the folding-corrector/gating-potentiator drug combinations approved for CF individuals carrying F508del-CFTR have sometimes produced severe side effects. Previously, we showed that a prolonged, 15-days treatment of polarized bronchial epithelial monolayers with the VX-809+VX-770 combination resulted in epithelial dedifferentiation effects that we found were caused specifically by VX-809. Moreover, prolonged VX-770 exposure also led to the destabilization of VX-809-rescued F508del-CFTR. Notably, co-treatment with the physiological factor HGF prevented VX-809-mediated epithelial differentiation and reverted the destabilizing effect of VX-770 on VX-809-rescued CFTR. Here, we show that prolonged treatment with VX-661, a second-generation corrector developed based on VX-809 structure, does not perturb epithelial integrity of polarized bronchial epithelial monolayers. Yet, its efficacy is still affected by co-exposure to VX-770, the potentiator present in all VX-661-containing combination therapies approved in the United States and Europe for treatment of F508del-CFTR carriers. Importantly, we found that co-treatment with HGF still ameliorated the impact of VX-770 in F508del-CFTR functional rescue by VX-661, without increasing cell proliferation (Ki-67) or altering the overall expression of epithelial markers (ZO-1, E-cadherin, CK8, CK18). Our findings highlight the importance of evaluating the cellular effects of prolonged exposure to CFTR modulators and suggest that the benefits of adding HGF to current combination therapies should be further investigated.

Highlights

Cystic fibrosis (CF) is one of the most common lifeshortening inherited diseases in Caucasian populations (De Boeck, 2020)
In contrast to the commonly used 48 h in vitro treatments, prolonged exposure of F508del-CFTRexpressing CFBE cells to 3 μM VX-809 resulted in decreased transepithelial resistance and concomitant downregulation of epithelial differentiation markers, namely the tight junction protein Zonula occludens-1 (ZO-1) (Martin and Jiang, 2009) and the pro-differentiative marker cytokeratin 18 (CK18) (Zhang et al, 2014), whereas the lung cancer pro-dedifferentiation marker cytokeratin 8 (CK8) (Fukunaga et al, 2002) became upregulated (Matos et al, 2018)
We found that in contrast to VX-809 treatment, which progressively decreased transepithelial electrical resistance (TEER) reaching a significant reduction over control conditions at 12 days of treatment, TEER values for VX-661-treated cells showed no significant difference from control cells during the entire 15 days of treatment and were significantly higher than VX809-treated cells at day 15 (Figure 1A)

Summary

Introduction

Cystic fibrosis (CF) is one of the most common lifeshortening inherited diseases in Caucasian populations (De Boeck, 2020). CF is a monogenic disease caused by mutations in CF transmembrane conductance regulator (CFTR) gene (Saint-Criq and Gray, 2017). It encodes the CFTR protein, an anion channel expressed at the apical plasma membrane (PM) of epithelial cells, responsible for the transport of chloride and bicarbonate across different epithelia (SaintCriq and Gray, 2017; De Boeck, 2020). The most frequent mutation, F508del, is present in at least one allele of 80–85% of CF individuals worldwide and causes the protein to misfold and be prematurely degraded by the ER quality control mechanism (ERQC) (Farinha and Matos, 2016). Despite significant clinical progress in the last decades, with symptomatic therapies enabling the delay of disease progression, CF individuals inevitably develop severe chronic complications, in the lungs, which greatly impact their quality of life and life expectancy (Saint-Criq and Gray, 2017; McBennett et al, 2021)

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