Combination of Correctors Rescues CFTR Transmembrane-Domain Mutants by Mitigating their Interactions with Proteostasis

Abstract

Background/Aims: Premature degradation of mutated cystic fibrosis transmembrane conductance regulator (CFTR) protein causes cystic fibrosis (CF), the commonest Mendelian disease in Caucasians. Despite recent advances in precision medicines for CF patients, many CFTR mutants have not been characterized and the effects of these new therapeutic approaches are still unclear for those mutants. Methods: Cells transfected or stably expressing four CFTR transmembrane-domain mutants (G85E, E92K, L1077P, and M1101K) were used to: 1) characterize the mutants according to their protein expression, thermal sensitivity, and degradation pathways; 2) evaluate the effects of correctors in rescuing them; and 3) explore the effects of correctors on CFTR interactions with proteostasis components. Results: All four mutants exhibited lower protein expression than did wild type-CFTR, and they were degraded by proteasomes and aggresomes. At low temperature, only cells expressing the mutants L1077P and M1101K exhibited increased CFTR maturation. Co-administration of C4 and C18 showed the greatest effect, restoring functional expression and partial stability of CFTR bearing E92K, L1077P, or M1101K at the cell surface. However, this treatment was inefficient in rectifying the defect of CFTR bearing G85E. Correctors rescued CFTR mutants by reducing their interactions with proteostasis components associated with protein retention in the endoplasmic reticulum and ubiquitination. Conclusion: Co-administration of C4 and C18 rescued CFTR transmembrane-domain mutants by remodeling the CFTR interactome.

HDAC Inhibitors Rescue Multiple Disease-Causing CFTR Variants

“…The substitution of glycine for glutamic acid at this position drastically impacts CFTR structure both locally, given the interaction of G85 with M82, and globally, given the interaction of G85 with H199. These data are in agreement with the observations that G85E is a severe class II variant disrupting the ER targeting, integration and topology of the TMD1 [108,109], which cannot be overcome with low temperature nor with well-established correctors such as C4, C18 and Vx809 [110]. While the impact of Vx809 alone was hard to detect, we observed a synergistic correction of the variants in response to HDACi + Vx809, an effect that is most pronounced with LBH-589.…”

“…To characterize the 10 variants described above in response to HDACi in the presence or absence of Lumacaftor, we transduced the CFBE41o-null cells with adenoviral particles carrying the different CFTR variant cDNAs. While the transient expression level of the F508del variant in CFBE41o-null cells was much lower than seen in CFBE41o-stable cell lines, the stability and trafficking of the variants observed is consistent with what has been previously reported for these variants [105,106] (Figure 6B & 7).…”

“…While most efforts using HDACi have focused on killing cancer cells, a number of approaches now demonstrate their value in providing benefit to human disease (see Introduction). For example, LBH-589 has been reported to rescue sickle cell disease (SCD) [110] and its clinical benefits for patients with SCD are under investigation. While the toxicity of these compounds has been well documented, they have been shown to be sufficiently well tolerated by patients even at doses toxic to cancer cells, which far exceed the doses at which we see functional correction of CF-causing variants, suggesting that a therapeutic index may exist for currently FDAapproved HDACi that could provide significant benefit to a broad spectrum CF patients based on future VSP analyses [2].…”

Regulation of CFTR Biogenesis by the Proteostatic Network and Pharmacological Modulators

“…Although class II CFTR variants are retained in the ER and targeted for ERAD (see above), simply shifting cells to a lower temperature corrects the misfolding defect for many variants, including F508del, and enables the release of maturely glycosylated, partially functional channels to the cell surface [90,164]. Though of limited therapeutic benefit, this observation was significant because it implied that if mutant CFTR could be rescued by hypothermia, it might also be rescued by treatment with a compound or set of compounds that successfully recapitulates the effect of low temperature.…”

“…For example, F508del, E92K, and G85E CFTR are all class II variants that exhibit severe misfolding defects (Figure 3), yet only F508del and E92K are approved for treatment. In contrast, G85E is completely intractable to correction, whether by low temperature or by treatment with small molecules [164]. The nature of this phenomenon remains mysterious.…”