Strategies for the etiological therapy of cystic fibrosis

Abstract

Etiological therapies aim at repairing the underlying cause of cystic fibrosis (CF), which is the functional defect of the cystic fibrosis transmembrane conductance regulator (CFTR) protein owing to mutations in the CFTR gene. Among these, the F508del CFTR mutation accounts for more than two thirds of CF cases worldwide. Two somehow antinomic schools of thought conceive CFTR repair in a different manner. According to one vision, drugs should directly target the mutated CFTR protein to increase its plasma membrane expression (correctors) or improve its ion transport function (potentiators). An alternative strategy consists in modulating the cellular environment and proteostasis networks in which the mutated CFTR protein is synthesized, traffics to its final destination, the plasma membrane, and is turned over. We will analyze distinctive advantages and drawbacks of these strategies in terms of their scientific and clinical dimensions, and we will propose a global strategy for CF research and development based on a reconciliatory approach. Moreover, we will discuss the utility of preclinical biomarkers that may guide the personalized, patient-specific implementation of CF therapies.

Figure 1

Classes of CFTR gene mutations. (a) Epithelial cells bearing wild-type CFTR. CFTR traffics from the endoplasmic reticulum (ER) to the Golgi apparatus (GA) and finally to the plasma membrane. (b) Impact of different classes of CFTR mutations (numbered 1–6) on synthesis, trafficking and function of CFTR. Class I mutations include nonsense, frameshift and splicing variants that prevent CFTR biosynthesis by creating in-frame stop signals (premature termination codons, PTCs) that generate a mechanism of mRNA surveillance, the nonsense-mediated decay. This causes premature arrest of translation and accelerated mRNA decay, thus reducing or abolishing the production of CFTR protein. The most common class I mutations are G542X in Mediterranean countries, R1162X and W1282X in Ashkenazy Jews. Class II mutations cause retention of misfolded proteins at the ER by local quality control mechanism. This is followed by premature ubiquitination and degradation of the misfolded protein, hence preventing its trafficking to the PM.^, F508del affects the region of NBD1 crucial for the interdomain interaction with ICL4 of MSD2, thereby causing changes in the loop configuration between residues 509–511 with consequent topography changes of NBD1 surface.^, Small amounts of F508del-CFTR can reach the PM, where the CFTR mutant protein manifests poor stability and defective gating.^, Class III mutations are compatible with the synthesis and trafficking of CFTR mutants to the PM, but impair conduction and permeation properties of the channel. G551D, the third most common CFTR mutation accounting for 4–5% of CFTR mutations worldwide, is located in NBD1 at the ATP-binding site 2, thus affecting ATP binding and hydrolysis, and mostly the conformational changes subsequent to ATP binding that favour channel gating. In spite of some differences, both G551D and G1349D, a mirror image of G551D at ATP-binding site 1, decrease the duration of channel openings and hence prolong closure periods. Class IV mutations, located mostly in the MSDs, including R117H in MSD1 [M2] and R334W or R347P in MSD1 [M6], perturb anion flow through reducing the single-channel conductance of CFTR. In these mutants, the cAMP-stimulated Cl⁻ current is greatly reduced as compared to wild-type CFTR (up to 70% for R334W) by perturbing ion-ion interaction whit in the CFTR pore. Class V mutations reduce protein synthesis. They include mutations that promote alternative splicing with generation of aberrant mRNA transcripts and reduced amounts of normal mRNA transcripts, as well as mutations in the promoter that reduce gene transcription. Class VI mutations destabilize CFTR at the PM and compromise its half-life, either by favoring CFTR disposal by endocytosis or by impairing its recycling to the PM. An additional class of mutations causing large deletions, named class VII, has been proposed to categorize untreatable mutations

Figure 2

Manipulating the cellular quality control (QC) systems to circumvent F508del-CFTR defect. (a) ERQC checkpoints. Major ERQC checkpoints test whether CFTR is eligible for ER exiting.^, ERQC1: chaperone trap. Nascent F508del-CFTR polypeptide chains interact the Hsp70/Hsp40 chaperone/co-chaperones that regulate, together with Hsc/Hdj-2 system, the early stages of the folding process. Hsp90 and its co-chaperones Aha1, which strongly interacts with F508del-CFTR, and FKP8 manage later stages of CFTR folding.^, Recruitment of the the Ub-ligases and ubiquitin adaptor proteins, as the E3 Ub ligase CHIP, E2 enzyme UbcH5, Derlin-1, E2 ligase Ubc6e or Gp78, leads to F508del ubiquitination and proteasome degradation. The Hsp70 co-chaperone Bag-1 can assist CFTR folding. ERQC2: calnexin cycle. Small amounts of F508del that escape ERQC1, can undergo glycan-mediated ER associated degradation (GERAD) in the chaperone/lectin cycle of calnexin which strongly binds F508del-CFTR. ERQC3 and ERQC4: ER exit. Either exposure of arginine-framed tripeptides (AFTs) ER-retention motifs or disruption of positive cargo signals (di-acidic code, DAD motif) in NBD1 compromise ER exit, Sec24-mediated packaging of CFTR into coat protein (COP) II-coated vesicles and CFTR transport to the Golgi. Moreover, alternative routes of CFTR transport (unconventional secretory pathways), either COPII-independent or bypassing Golgi through syntaxin 13 (ref. 148) or through GRASPs may deliver CFTR to the plasma membrane. Potential ERQC-targeted interventions to prevent CFTR degradation are indicated by dashed red lines and box: (i) depleting Aha1 (ref. 69); decreasing HOP by GNSOR inhibitors;^, overexpressing Bag-1 (ref. 150); decreasing Hsp70/CFTR interaction by Corr-4a; (ii) antagonizing CFTR ubiquitination by USP19 or soluble UCH-L1 (ref. 151). (b) Peripheral QC system. Additional QC mechanisms check F508del-CFTR at the PM (PQC). Chaperone/co-chaperones (including Hsc70/Hsp70/Hsp90, HOP, Hdjs, Aha1, CHIP and Bag-1), Ub-ligases and several adaptor proteins, including the assembly polypeptide-2 (AP-2), lead to the incorporation of poly-/multiple-mono ubiquitinated F508del-CFTR into clathrin-positive vesicles.^{, , ,} Endocytosis. Clathrin-mediated endocytosis is facilitated by Myosins Vb/VI and Rab small GTPases, which orchestrate the fusion of early endocytic vesicles (mainly through Rab5), PM recycling (Rab4 or Rab11), sorting to late endosomes (Rab7), transport back to TGN (Rab9 and/or Rab11). Instead of being recycled to the PM, the unstable ubiquitinated F508del-CFTR is rapidly sorted to the late (Rab7 positive) endosomes and lysosomes.^, CFTR anchoring: Up to 50% of CFTR pool is anchored to F-actin filaments through several PDZ proteins including NHERF-1, ezrin/radixin/moesin (ERM) and annexin 5A.^{, ,} RhoA, Rac1 and Cdc42 GTPases modulate actin cytoskeleton reoganization and NHERF-1 binding thus regulating F508del-CFTR thetering and PM stability. Potential PQC-targeted interventions to stabilize F508del-CFTR and prevent PM disposal are indicated by dashed red lines and box: (i) CHIP or Aha1 ablation;^, (ii) USP 10 overexpression; (iii) NHERF-1 overexpression, which stimulates RhoA, ROCK, Rac1 signaling, ezrin phosphorylation, tight-junction organization; manipulation of cytocheratyn-8/F508del-CFTR association; modulation of Rac1 signaling through HGF, which enhances the rescuing efficacy of CFTR correctors corr-4a and VX-809 (ref. 161). Hsp, heath shock protein; Hsc, heat shock cognate protein; Aha1, Hsp90 co-chaperones activator of Hsp90 ATPase homolog 1; HOP, Hsp70/Hsp90 organizing protein; GSNOR, S-nitrosoglutathione reductase; Bag-1, Bcl-2- associated athanogenes; Ub, ubiquitin; USP, ubiquitin protease; SAHA, suberoylanilide hydroxamic acid; CHIP, Carboxy-Terminal of Hsp70 Interacting Protein; GRASP, Golgi Re-assembling and Stacking Protein; AFT, four arginine-framed RXR tripeptides; NHERF-1, Na+/H+ exchanger regulator factor isoform-1; ROCK, RhoA-activated kinase; HGF, Hepatocyte Growth Factor; ESCRT, endosomal sorting complex required for transport; MVB, multivesicular bodies

Figure 3

Defective autophagy impacts on most F508del-CFTR quality control checkpoints. (a) Defective CFTR induces ROS-mediated TG2 activation. ROS-induced PIASy-mediated TG2 SUMOylation sustains Ca²⁺-dependent TG2 activation leading to crosslinking and aggregation of substrate proteins, including PPARγ and IKBα^, . Most TG2 interactor proteins are molecular chaperones which impact on CFTR processing. (b) TG2 activation disables autophagy. Activated TG2 crosslinks BECN1 and dislodges the phosphatidyl-inositol-3-kinase complex 3 (PI3KC3) away from the ER, thus inhibiting autophagosome formation and disabling autophagy. Defective autophagy induces accumulation of the autophagic substrate SQSTM1/p62, that targets ubiquitinated proteins, including F508del-CFTR, leading to proteasome overload and sequestration of aggregated proteins within HDAC6+/SQSTM1+ aggresomes. (c) Accumulation of SQSTM1/p62 at the PM favors F508del-CFTR disposal. SQSTM1/p62, a critical regulator of internalization, trafficking and sorting of ubiquitinated surface proteins, accumulates at the epithelial surface, binds PM-located ubiquitinated F508del-CFTR and colocalizes with mutant CFTR within enlarged early endosomal antigen (EEA) 1⁺ vesicles.^, (d) Sequestration of PI3KC3 impairs endosomal trafficking. The sequestration of PI3KC3 reduces the abundance of phosphatidyl-inositol-3-Phosphate (PtdIns3P) at the EEA1⁺ endosomes, thus impairing endosomal fusion/maturation, and hence CFTR recycling. The PI3KC3 sequestration reduces the availability of the BECN1-interactor UVRAG that interacts with the HOP complex, thus recruiting and activating Rab7 and favoring Rab5 to Rab7 transition. Moreover, SQSTM1/p62 targeting reduces Rab5 levels at the EEA1⁺ vesicles.^, Defective autophagy compromises CFTR recycling through Rab11+ vesicles and diverts CFTR recycling to lysosomal degradation. Moreover, it impairs Rab5-Rab7 transition, thus delaying CFTR trafficking to the late endosomes.^{, ,} Potential targeted interventions to circumvent F508del-CFTR defect are indicated by dashed red lines and box. (i) TG2 depletion or inhibition by cysteamine or (ii) BECN1 overexpression can re-establish autophagy flux, increase PI3P availability and prevent SQSTM1/p62 accumulation, thus ultimately favouring F508del-CFTR rescue and PM stability. (iii) direct SQSTM1/p62 depletion or the enforced expression of SQSTM1/p62 mutants lacking the UBA domain, increases F508del-CFTR PM stability and autophagy flux.^{, ,} BECN1, Beclin 1; SQSTM/p62, sequestrosome 1; ROS,reactive oxygen species; TG2, transglutaminase 2; UVRAG, UV-irradiation-resistant-associated-gene