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Individuals with Cystic Fibrosis (CF) suffer from severe respiratory disease due to a genetic defect in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene, which impairs airway epithelial ion and fluid secretion. New CFTR modulators that restore mutant CFTR function have been recently approved for a large group of people with CF (pwCF), but ∼19% of pwCF cannot benefit from CFTR modulators [1]. Restoration of epithelial fluid secretion through non-CFTR pathways might be an effective treatment for all pwCF. Here we developed a medium-throughput 384-wells screening assay using nasal CF airway epithelial organoids, with the aim to repurpose FDA-approved drugs as modulators of non-CFTR dependent epithelial fluid secretion. From a ∼1400 FDA-approved drug library, we identified and validated 12 FDA-approved drugs that induced CFTR-independent fluid secretion. Among the hits were several cAMP-mediating drugs, including β2-adrenergic agonists. The hits displayed no effects on chloride conductance measured in Ussing chamber, and fluid secretion was not affected by TMEM16A as demonstrated by knockout (KO) experiments in primary nasal epithelial cells. Altogether, our results demonstrate the use of primary nasal airway cells for mediumscale drug screening, target validation with a highly efficient protocol for generating CRISPR-Cas9 KO cells and identification of compounds which induce fluid secretion in a CFTR- and TMEM16A-indepent manner.

The solute carrier 26 family member A9 (SLC26A9) is an epithelial anion transporter that is assumed to contribute to airway chloride secretion and surface hydration. Whether SLC26A9 or CFTR is responsible for airway Cl

Despite the major advances and successes in finding and establishing new treatments that tackle the basic defect in Cystic Fibrosis (CF), there is still an unmet need to bring these potentially curative therapies to all individuals with CF. Here, we review aspects of what is still missing to treat all individuals with CF by such approaches. On the one hand, we discuss novel holistic (high-throughput) approaches to elucidate mechanistic defects caused by distinct classes of mutations to identify novel drug targets. On the other hand, we examine therapeutic approaches to correct the gene in its own environment, i.e., in the genome.

Background The Brazilian population has a tri-hybrid composition with a high degree of ethnic admixture. We hypothesized that Brazilian individuals with CF from different Brazilian regions have a specific distribution of CFTR variants. Methods Individuals with CF with data available in the Patient Registry and without an established genotype were submitted to CFTR sequencing by Next Generation Sequencing (NGS) methodology, and results were anonymously incorporated to the Registry Database. Genotyping results were expressed as ‘positive’, ‘inconclusive’ or ‘negative’. Logistic regression models were performed to investigate the association between demographic/clinical variables and genotyping results. Mediation analysis was conducted to estimate direct and indirect effects of Brazilian region on a binary positive genotyping response. Results In October 2017, data from 4,654 individuals with CF were available, and 3,104(66.7%) of them had a genotyping result. A total of 236 variants (114 new variants) were identified, with F508del identified in 46% of the alleles tested. Genotyping revealed 2,002(64.5%) individuals positive, 757(24.4%) inconclusive and 345(11.1%) negative. Distribution of genotype categories was markedly different across Brazilian Regions, with greater proportions of negative individuals in the North (45%) and Northeast (26%) regions. Newborn screening (CF-NBS) and age at diagnosis were identified as mediators of the effect of Brazilian region on a positive genotyping result. Conclusions This large initiative of CFTR genotyping showed significant regional discrepancies in Brazil, probably related to socio-economic conditions, lack of adequate CF-NBS and poor access to reliable sweat testing. These results may be useful to indicate Regions where CF care demands more attention.

MotivationThe forskolin-induced swelling (FIS) assay has become the preferential assay to predict the efficacy of approved and investigational CFTR-modulating drugs for individuals with cystic fibrosis (CF). Currently, no standardized quantification method of FIS data exists thereby hampering inter-laboratory reproducibility.ResultsWe developed a complete open-source workflow for standardized high-content analysis of CFTR function measurements in intestinal organoids using raw microscopy images as input. The workflow includes tools for (i) file and metadata handling; (ii) image quantification and (iii) statistical analysis. Our workflow reproduced results generated by published proprietary analysis protocols and enables standardized CFTR function measurements in CF organoids.Availability and implementationAll workflow components are open-source and freely available: the htmrenamer R package for file handling https://github.com/hmbotelho/htmrenamer; CellProfiler and ImageJ analysis scripts/pipelines https://github.com/hmbotelho/FIS_image_analysis; the Organoid Analyst application for statistical analysis https://github.com/hmbotelho/organoid_analyst; detailed usage instructions and a demonstration dataset https://github.com/hmbotelho/FIS_analysis. Distributed under GPL v3.0.Supplementary informationSupplementary data are available at Bioinformatics online.

In cystic fibrosis (CF), the correction of splicing defects represents an interesting therapeutic approach to restore normal CFTR function. In this study, we focused on 10 common mutations/variants 711+3A>G/C, 711+5G>A, TG13T3, TG13T5, TG12T5, 1863C>T, 1898+3A>G, 2789+5G>A, and 3120G>A that induce skipping of the corresponding CFTR exons 5, 10, 13, 16, and 18. To rescue the splicing defects we tested, in a minigene assay, a panel of modified U1 small nuclear RNAs (snRNAs), named Exon Specific U1s (ExSpeU1s), that was engineered to bind to intronic sequences downstream of each defective exon. Using this approach, we show that all 10 splicing mutations analyzed are efficiently corrected by specific ExSpeU1s. Using complementary DNA-splicing competent minigenes, we also show that the ExspeU1-mediated splicing correction at the RNA level recovered the full-length CFTR protein for 1863C>T, 1898+3A>G, 2789+5G>A variants. In addition, detailed mutagenesis experiments performed on exon 13 led us to identify a novel intronic regulatory element involved in the ExSpeU1-mediated splicing rescue. These results provide a common strategy based on modified U1 snRNAs to correct exon skipping in a group of disease-causing CFTR mutations.

CFTR modulators associated with substantial clinical benefit are expected to rapidly improve the baseline condition of people with cystic fibrosis (PWCF) as well as decrease the rate of lung function decline, the occurrence of pulmonary exacerbations and likely even other disease complications. These changes in clinical status of PWCF introduced by clinically effective modulator therapy will have major repercussions on modalities of future CF drug development. As part of its 'Strategic Plan to speed up Access to new Drugs', the European Cystic Fibrosis Society (ECFS) convened a meeting in Brussels on November 27th 2019 with relevant stakeholders (CF researchers and clinicians, patient organization and pharmaceutical company representatives, regulators, health technology assessors; see Acknowledgments for list of attendees) to discuss the future of clinical trials in cystic fibrosis (CF) in the context of HEMT entering the clinical arena. The following is the conclusion of the presentations and discussions. It is hoped that these concepts will be considered in future regulatory guidelines and may provide rationale and support for alternative trial designs.

Cystic Fibrosis (CF), the most common lethal autosomic recessive disorder among Caucasians, is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein, a cAMP-regulated chloride channel expressed at the apical surface of epithelial cells. Cyclic AMP regulates both CFTR channel gating through a protein kinase A (PKA)-dependent process and plasma membane (PM) stability through activation of the exchange protein directly activated by cAMP1 (EPAC1). This cAMP effector, when activated promotes the NHERF1:CFTR interaction leading to an increase in CFTR at the PM by decreasing its endocytosis. Here, we used protein interaction profiling and bioinformatic analysis to identify proteins that interact with CFTR under EPAC1 activation as possible regulators of this CFTR PM anchoring. We identified an enrichment in cytoskeleton related proteins among which we characterized CAPZA2 and INF2 as regulators of CFTR trafficking to the PM. We found that CAPZA2 promotes wt-CFTR trafficking under EPAC1 activation at the PM whereas reduction of INF2 levels leads to a similar trafficking promotion effect. These results suggest that CAPZA2 is a positive regulator and INF2 a negative one for the increase of CFTR at the PM after an increase of cAMP and concomitant EPAC1 activation. Identifying the specific interactions involving CFTR and elicited by EPAC1 activation provides novel insights into late CFTR trafficking, insertion and/or stabilization at the PM and highlighs new potential therapeutic targets to tackle CF disease.

Early eukaryotic cells emerged from the compartmentalization of metabolic processes into specific organelles through the development of an endomembrane system (ES), a precursor of the endoplasmic reticulum (ER), which was essential for their survival. Recently, substantial evidence emerged on how organelles communicate among themselves and with the plasma membrane (PM) through contact sites (CSs). From these studies, the ER-the largest single structure in eukaryotic cells-emerges as a central player communicating with all organelles to coordinate cell functions and respond to external stimuli to maintain cellular homeostasis. Herein we review the functional insights into the ER-CSs with other organelles in a physiological perspective. We hypothesize that, in addition to the primitive role by the ES in the appearance of proto-eukaryotes, its successor-the ER-emerges as the key coordinator of inter-organelle/PM communication. The ER thus appears to be the 'maestro' driving eukaryotic cell evolution by incorporating new functions/organelles, while remaining the real coordinator overarching cellular functions and orchestrating them with the external milieu.

The European Cystic Fibrosis Society (ECFS) Basic Science Working Group (BSWG) organized a session on the topic “Activating Alternative Chloride Channels to Treat CF: Friends or Foes?”, within the 16th ECFS Basic Science Conference gathering ∼200 researchers from CF Basic Science. The session was organized into two round tables, each chaired by Margarida Amaral (BioISI, University of Lisboa, Portugal) and Jeffrey Beekman (University Medical Centre Utrecht, Netherlands) as Chair and Vice-Chair of the BSWG, respectively. The purpose of this session was to bring attention of participants of the ECFS Basic Science Conference to alternative chloride channels as important therapeutic targets for CF which may treat all individuals with CF, independently of their CFTR genotype. The session had various speakers who had presented abstracts to the conference on this topic and discussants with different views. Here we try to convey the presentations as well as ideas that emerged during the discussion.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride and bicarbonate channel in secretory epithelia with a critical role in maintaining fluid homeostasis. Mutations in CFTR are associated with Cystic Fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasians. While remarkable treatment advances have been made recently in the form of modulator drugs directly rescuing CFTR dysfunction, there is still considerable scope for improvement of therapeutic effectiveness. Here, we report the application of a high-throughput screening variant of the Mammalian Membrane Two-Hybrid (MaMTH-HTS) to map the protein–protein interactions of wild-type (wt) and mutant CFTR (F508del), in an effort to better understand CF cellular effects and identify new drug targets for patient-specific treatments. Combined with functional validation in multiple disease models, we have uncovered candidate proteins with potential roles in CFTR function/CF pathophysiology, including Fibrinogen Like 2 (FGL2), which we demonstrate in patient-derived intestinal organoids has a significant effect on CFTR functional expression.

The most prevalent cystic fibrosis (CF)-causing mutation - F508del - impairs the folding of CFTR protein, resulting in its defective trafficking and premature degradation. Small molecules termed correctors may rescue F508del-CFTR and therefore constitute promising pharmacotherapies acting on the fundamental cause of the disease. Here, we screened a collection of triazole compounds to identify novel F508del-CFTR correctors. The functional primary screen identified four hit compounds (LSO-18, LSO-24, LSO-28, and LSO-39), which were further validated and demonstrated to rescue F508del-CFTR processing, plasma membrane trafficking, and function. To interrogate their mechanism of action (MoA), we examined their additivity to the clinically approved drugs VX-661 and VX-445, low temperature, and genetic revertants of F508del-CFTR. Rescue of F508del-CFTR processing and function by LSO-18, LSO-24, and LSO-28, but not by LSO-39, was additive to VX-661, whereas LSO-28 and LSO-39, but not LSO-18 nor LSO-24, were additive to VX-445. All compounds under investigation demonstrated additive rescue of F508del-CFTR processing and function to low temperature as well as to rescue by genetic revertants G550E and 4RK. Nevertheless, none of these compounds was able to rescue processing nor function of DD/AA-CFTR, and LSO-39 (similarly to VX-661) exhibited no additivity to genetic revertant R1070W. From these findings, we suggest that LSO-39 (like VX-661) has a putative binding site at the NBD1:ICL4 interface, LSO-18 and LSO-24 seem to share the MoA with VX-445, and LSO-28 appears to act by a different MoA. Altogether, these findings represent an encouraging starting point to further exploit this chemical series for the development of novel CFTR correctors.

SLC26A9, a constitutively active Cl− transporter, has gained interest over the past years as a relevant disease modifier in several respiratory disorders including Cystic Fibrosis (CF), asthma, and non-CF bronchiectasis. SLC26A9 contributes to epithelial Cl− secretion, thus preventing mucus obstruction under inflammatory conditions. Additionally, SLC26A9 was identified as a CF gene modifier, and its polymorphisms were shown to correlate with the response to drugs modulating CFTR, the defective protein in CF. Here, we aimed to investigate the relationship between SLC26A9 and CFTR, and its role in CF pathogenesis. Our data show that SLC26A9 expression contributes to enhanced CFTR expression and function. While knocking-down SLC26A9 in human bronchial cells leads to lower wt- and F508del-CFTR expression, function, and response to CFTR correctors, the opposite occurs upon its overexpression, highlighting SLC26A9 relevance for CF. Accordingly, F508del-CFTR rescue by the most efficient correctors available is further enhanced by increasing SLC26A9 expression. Interestingly, SLC26A9 overexpression does not increase the PM expression of non-F508del CFTR traffic mutants, namely those unresponsive to corrector drugs. Altogether, our data indicate that SLC26A9 stabilizes CFTR at the ER level and that the efficacy of CFTR modulator drugs may be further enhanced by increasing its expression.

Airway inflammation, mucus hyperproduction and epithelial remodelling are hallmarks of many chronic airway diseases, including asthma, COPD and cystic fibrosis. While several cytokines are dysregulated in these diseases, most studies focus on the response of airways to interleukin (IL)-4 and IL-13, which have been shown to induce mucus hyperproduction and shift the airway epithelium towards a hypersecretory phenotype. We hypothesised that other cytokines might induce the expression of chloride (Cl−) channels/transporters, and regulate epithelial differentiation and mucus production. To this end, fully differentiated human airway basal cells (BCi-NS1.1) were treated with cytokines identified as dysregulated in those diseases, namely IL-8, IL-1β, IL-4, IL-17A, IL-10 and IL-22, and tumour necrosis factor-α. Our results show that the cystic fibrosis transmembrane conductance regulator (CFTR) is the main Cl− channel modulated by inflammation, in contrast to transmembrane protein 16A (TMEM16A), whose levels only changed with IL-4. Furthermore, we identified novel roles for IL-10 and IL-22 by influencing epithelial differentiation towards ciliated cells and away from pulmonary ionocytes. In contrast, IL-1β and IL-4 reduced the number of ciliated cells while increasing club cells. Interestingly, while IL-1β, IL-4 and IL-10 upregulated CFTR expression, IL-4 was the only cytokine that increased both its function and the number of CFTR-expressing club cells, suggesting that this cell type may be the main contributor for CFTR function. Additionally, all cytokines assessed increased mucus production through a differential upregulation of MUC5AC and MUC5B transcript levels. This study reveals a novel insight into differentiation resulting from the cross-talk of inflammatory mediators and airway epithelial cells, which is particularly relevant for chronic airway diseases., Disease-specific inflammatory cytokines induce airway mucus hyperproduction and changes in chloride channels/transporters, and modulate epithelial differentiation https://bit.ly/2WaeV1z

Mutations in the CFTR anion channel cause cystic fibrosis (CF) and have also been related to higher cancer incidence. Previously we proposed that this is linked to an emerging role of functional CFTR in protecting against epithelial–mesenchymal transition (EMT). However, the pathways bridging dysfunctional CFTR to EMT remain elusive. Here, we applied systems biology to address this question. Our data show that YAP1 is aberrantly active in the presence of mutant CFTR, interacting with F508del, but not with wt-CFTR, and that YAP1 knockdown rescues F508del-CFTR processing and function. Subsequent analysis of YAP1 interactors and roles in cells expressing either wt- or F508del-CFTR reveal that YAP1 is an important mediator of the fibrotic/EMT processes in CF. Alongside, five main pathways emerge here as key in linking mutant CFTR to EMT, namely, (1) the Hippo pathway; (2) the Wnt pathway; (3) the TGFβ pathway; (4) the p53 pathway; and (5) MYC signaling. Several potential hub proteins which mediate the crosstalk among these pathways were also identified, appearing as potential therapeutic targets for both CF and cancer.

Major advances have recently been made in the development and application of CFTR (cystic fibrosis transmembrane conductance regulator) mutation class-specific modulator therapies, but to date, there are no approved modulators for Class I mutations, i.e., those introducing a premature termination codon (PTC) into the CFTR mRNA. Such mutations induce nonsense-mediated decay (NMD), a cellular quality control mechanism that reduces the quantity of PTC bearing mRNAs, presumably to avoid translation of potentially deleterious truncated CFTR proteins. The NMD-mediated reduction of PTC-CFTR mRNA molecules reduces the efficacy of one of the most promising approaches to treatment of such mutations, namely, PTC readthrough therapy, using molecules that induce the incorporation of near-cognate amino acids at the PTC codon, thereby enabling translation of a full-length protein. In this study, we measure the effect of three different PTC mutations on the abundance, integrity, and stability of respective CFTR mRNAs, using CFTR specific RT-qPCR-based assays. Altogether, our data suggest that optimized rescue of PTC mutations has to take into account (1) the different steady-state levels of the CFTR mRNA associated with each specific PTC mutation, (2) differences in abundance between the 3′ and 5′ regions of CFTR mRNA, even following PTC readthrough or NMD inhibition, and (3) variable effects on CFTR mRNA stability for each specific PTC mutation.

Cystic fibrosis (CF) is a life-threatening autosomal recessive disease caused by more than 2100 mutations in the CF transmembrane conductance regulator (CFTR) gene, generating variability in disease severity among individuals with CF sharing the same CFTR genotype. Systems biology can assist in the collection and visualization of CF data to extract additional biological significance and find novel therapeutic targets. Here, we present the CyFi-MAP—a disease map repository of CFTR molecular mechanisms and pathways involved in CF. Specifically, we represented the wild-type (wt-CFTR) and the F508del associated processes (F508del-CFTR) in separate submaps, with pathways related to protein biosynthesis, endoplasmic reticulum retention, export, activation/inactivation of channel function, and recycling/degradation after endocytosis. CyFi-MAP is an open-access resource with specific, curated and continuously updated information on CFTR-related pathways available online at https://cysticfibrosismap.github.io/. This tool was developed as a reference CF pathway data repository to be continuously updated and used worldwide in CF research.

Cystic fibrosis (CF) is a life-shortening monogenic disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, an anion channel that transports chloride and bicarbonate across epithelia. Despite clinical progress in delaying disease progression with symptomatic therapies, these individuals still develop various chronic complications in lungs and other organs, which significantly restricts their life expectancy and quality of life. The development of high-throughput assays to screen drug-like compound libraries have enabled the discovery of highly effective CFTR modulator therapies. These novel therapies target the primary defect underlying CF and are now approved for clinical use for individuals with specific CF genotypes. However, the clinically approved modulators only partially reverse CFTR dysfunction and there is still a considerable number of individuals with CF carrying rare CFTR mutations who remain without any effective CFTR modulator therapy. Accordingly, additional efforts have been pursued to identify novel and more potent CFTR modulators that may benefit a larger CF population. The use of ex vivo individual-derived specimens has also become a powerful tool to evaluate novel drugs and predict their effectiveness in a personalized medicine approach. In addition to CFTR modulators, pro-drugs aiming at modulating alternative ion channels/transporters are under development to compensate for the lack of CFTR function. These therapies may restore normal mucociliary clearance through a mutation-agnostic approach (ie, independent of CFTR mutation) and include inhibitors of the epithelial sodium channel (ENaC), modulators of the calcium-activated channel transmembrane 16A (TMEM16, or anoctamin 1) or of the solute carrier family 26A member 9 (SLC26A9), and anionophores. The present review focuses on recent progress and challenges for the development of ion channel/transporter-modulating drugs for the treatment of CF.

As highly effective CFTR modulator therapies (HEMT) emerge, there is an unmet need to find effective drugs for people with CF (PwCF) with ultra-rare mutations who are too few for classical clinical trials and for whom there are no drug discovery programs. Therefore, biomarkers reliably predicting the benefit from CFTR modulator therapies are essential to find effective drugs for PwCF through personalized approaches termed theranostics. Here, we assess CFTR basal function and the individual responses to CFTR modulators in primary human nasal epithelial (pHNE) cells from PwCF carrying rare mutations and compare these measurements with those in native rectal biopsies and intestinal organoids, respectively, in the same individual. The basal function in pHNEs shows good correlation with CFTR basal function in rectal biopsies. In parallel, CFTR rescue in pHNEs by CFTR modulators correlates to that in intestinal organoids. Altogether, results show that pHNEs are a bona fide theranostic model to assess CFTR rescue by CFTR modulator drugs, in particular for PwCF and rare mutations.

Cystic fibrosis (CF) is a monogenetic disease resulting from mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene encoding an anion channel. Recent evidence indicates that CFTR plays a role in other cellular processes, namely in development, cellular differentiation and wound healing. Accordingly, CFTR has been proposed to function as a tumour suppressor in a wide range of cancers. Along these lines, CF was recently suggested to be associated with epithelial–mesenchymal transition (EMT), a latent developmental process, which can be re-activated in fibrosis and cancer. However, it is unknown whether EMT is indeed active in CF and if EMT is triggered by dysfunctional CFTR itself or a consequence of secondary complications of CF. In this study, we investigated the occurrence of EMT in airways native tissue, primary cells and cell lines expressing mutant CFTR through the expression of epithelial and mesenchymal markers as well as EMT-associated transcription factors. Transepithelial electrical resistance, proliferation and regeneration rates, and cell resistance to TGF-β1induced EMT were also measured. CF tissues/cells expressing mutant CFTR displayed several signs of active EMT, namely: destructured epithelial proteins, defective cell junctions, increased levels of mesenchymal markers and EMT-associated transcription factors, hyper-proliferation and impaired wound healing. Importantly, we found evidence that the mutant CFTR triggered EMT was mediated by EMT-associated transcription factor TWIST1. Further, our data show that CF cells are over-sensitive to EMT but the CF EMT phenotype can be reversed by CFTR modulator drugs. Altogether, these results identify for the first time that EMT is intrinsically triggered by the absence of functional CFTR through a TWIST1 dependent mechanism and indicate that CFTR plays a direct role in EMT protection. This mechanistic link is a plausible explanation for the high incidence of fibrosis and cancer in CF, as well as for the role of CFTR as tumour suppressor protein.

In Cystic Fibrosis (CF), correction of splicing defects represents an interesting therapeutic approach to restore normal CFTR function. In this study, we focused on ten common mutations/variants, 711+3A>G/C, 711+5G>A, 1863C>T, 1898+3A>G, 2789+5G>A, TG13T3, TG13T5, TG12T5 and 3120G>A that induce skipping of the corresponding CFTR exons 5, 9, 13, 16 and 18. To rescue the splicing defects we tested, in a minigene assay, a panel of modified U1 snRNAs, named Exon Specific U1s (ExSpeU1) that were engineered to bind to intronic sequences downstream of each defective exon. Using this approach, we show that all ten splicing mutations analysed are efficiently corrected by specific ExSpeU1s. Using cDNA-splicing competent minigenes, we also show that the ExspeU1-mediated splicing correction at the RNA level recovered the full-length CFTR protein for 1863C>T, 1898+3A>G, 2789+5G>A variants. In addition, detailed mutagenesis experiments performed on exon 13 led us to identify a novel intronic regulatory element involved in the ExSpeU1-mediated splicing rescue. These results provide a common strategy based on modified U1 snRNAs to correct exon skipping in a group of disease-causing CFTR mutations.

Cystic Fibrosis (CF) is caused by >2000 mutations in the CF transmembrane conductance regulator (CFTR) gene, but one mutation—F508del—occurs in ~80% of patients worldwide. Besides its main function as an anion channel, the CFTR protein has been implicated in epithelial differentiation, tissue regeneration, and, when dysfunctional, cancer. However, the mechanisms that regulate such relationships are not fully elucidated. Krüppel-like factors (KLFs) are a family of transcription factors (TFs) playing central roles in development, stem cell differentiation, and proliferation. Herein, we hypothesized that these TFs might have an impact on CFTR expression and function, being its missing link to differentiation. Our results indicate that KLF4 (but not KLF2 nor KLF5) is upregulated in CF vs. non-CF cells and that it negatively regulates wt-CFTR expression and function. Of note, F508del–CFTR expressing cells are insensitive to KLF4 modulation. Next, we investigated which KLF4-related pathways have an effect on CFTR. Our data also show that KLF4 modulates wt-CFTR (but not F508del–CFTR) via both the serine/threonine kinase AKT1 (AKT) and glycogen synthase kinase 3 beta (GSK3β) signaling. While AKT acts positively, GSK3β is a negative regulator of CFTR. This crosstalk between wt-CFTR and KLF4 via AKT/ GSK3β signaling, which is disrupted in CF, constitutes a novel mechanism linking CFTR to the epithelial differentiation.

Cystic Fibrosis (CF) is caused by mutations in the CF Transmembrane conductance Regulator (CFTR), the only ATP-binding cassette (ABC) transporter functioning as a channel. Unique to CFTR is a regulatory domain which includes a highly conformationally dynamic region&mdash, the regulatory extension (RE). The first nucleotide-binding domain of CFTR contains another dynamic region&mdash, regulatory insertion (RI). Removal of RI rescues the trafficking defect of CFTR with F508del, the most common CF-causing mutation. Here we aimed to assess the impact of RE removal (with/without RI or genetic revertants) on F508del-CFTR trafficking and how CFTR modulator drugs VX-809/lumacaftor and VX-770/ivacaftor rescue these variants. We generated cell lines expressing &Delta, RE and &Delta, RI CFTR (with/without genetic revertants) and assessed CFTR expression, stability, plasma membrane levels, and channel activity. Our data demonstrated that &Delta, RI significantly enhanced rescue of F508del-CFTR by VX-809. While the presence of the RI seems to be precluding full rescue of F508del-CFTR processing by VX-809, this region appears essential to rescue its function by VX-770, suggesting some contradictory role in rescue of F508del-CFTR by these two modulators. This negative impact of RI removal on VX-770-stimulated currents on F508del-CFTR can be compensated by deletion of the RE which also leads to the stabilization of this mutant. Despite both regions being conformationally dynamic, RI precludes F508del-CFTR processing while RE affects mostly its stability and channel opening.

Despite progress in developing pharmacotherapies to rescue F508del-CFTR, the most prevalent Cystic Fibrosis (CF)-causing mutation, individuals homozygous for this mutation still face several disease-related symptoms. Thus, more potent compound combinations are still needed. Here, we investigated the mechanism of action (MoA) of RDR01752, a novel F508del-CFTR trafficking corrector. F508del-CFTR correction by RDR01752 was assessed by biochemical, immunofluorescence microscopy and functional assays in cell lines and in intestinal organoids. To determine the MoA of RDR01752, we assessed its additive effects to those of genetic revertants of F508del-CFTR, the FDA-approved corrector drugs VX-809 and VX-661, and low temperature. Our data demonstrated that RDR01752 rescues F508del-CFTR processing and plasma membrane (PM) expression to similar levels of VX-809 in cell lines, although RDR01752 produced lower functional rescue. However, in functional assays using intestinal organoids (F508del/F508del), RDR01752, VX-809 and VX-661 had similar efficacy. RDR01752 demonstrated additivity to revertants 4RK and G550E, but not to R1070W, as previously shown for VX-809. RDR01752 was also additive to low temperature. Co-treatment of RDR01752 and VX-809 did not increase F508del-CFTR PM expression and function compared to each corrector alone. The lack of additivity of RDR01752 with the genetic revertant R1070W suggests that this compound has the same effect as the insertion of tryptophan at 1070, i.e., filling the pocket at the NBD1:ICL4 interface in F508del-CFTR, similarly to VX-809. Combination of RDR01752 with correctors mimicking the rescue by revertants G550E or 4RK could thus maximize rescue of F508del-CFTR.

One of the key features associated with the substantial increase in life expectancy for individuals with CF is an elevated predisposition to cancer, firmly established by recent studies involving large cohorts. With the recent advances in cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies and the increased long-term survival rate of individuals with cystic fibrosis (CF), this is a novel challenge emerging at the forefront of this disease. However, the mechanisms linking dysfunctional CFTR to carcinogenesis have yet to be unravelled. Clues to this challenging open question emerge from key findings in an increasing number of studies showing that CFTR plays a role in fundamental cellular processes such as foetal development, epithelial differentiation/polarization, and regeneration, as well as in epithelial–mesenchymal transition (EMT). Here, we provide state-of-the-art descriptions on the moonlight roles of CFTR in these processes, highlighting how they can contribute to novel therapeutic strategies. However, such roles are still largely unknown, so we need rapid progress in the elucidation of the underlying mechanisms to find the answers and thus tailor the most appropriate therapeutic approaches.

Cystic fibrosis (CF) cells display a more cancer-like phenotype vs. non-CF cells. KLF4 overexpression has been described in CF and this transcriptional factor acts as a negative regulator of wt-CFTR. KLF4 is described as exerting its effects in a cell-context-dependent fashion, but it is generally considered a major regulator of proliferation, differentiation, and wound healing, all the processes that are also altered in CF. Therefore, it is relevant to characterize the differential role of KLF4 in these processes in CF vs. non-CF cells. To this end, we used wt- and F508del-CFTR CFBE cells and their respective KLF4 knockout (KO) counterparts to evaluate processes like cell proliferation, polarization, and wound healing, as well as to compare the expression of several epithelial differentiation markers. Our data indicate no major impact of KLF4 KO in proliferation and a differential impact of KLF4 KO in transepithelial electrical resistance (TEER) acquisition and wound healing in wt- vs. F508del-CFTR cells. In parallel, we also observed a differential impact on the levels of some differentiation markers and epithelial-mesencymal transition (EMT)-associated transcription factors. In conclusion, KLF4 impacts TEER acquisition, wound healing, and the expression of differentiation markers in a way that is partially dependent on the CFTR-status of the cell.

BACKGROUND: For most of the >2,000 CFTR gene variants reported, neither the associated disease liability nor the underlying basic defect are known, and yet these are essential for disease prognosis and CFTR-based therapeutics. Here we aimed to characterize two ultra-rare mutations - 1717–2A>G (c.1585–2A>G) and S955P (p.Ser955Pro) - as case studies for personalized medicine. METHODS: Patient-derived rectal biopsies and intestinal organoids from two individuals with each of these mutations and F508del (p.Phe508del) in the other allele were used to assess CFTR function, response to modulators and RNA splicing pattern. In parallel, we used cellular models to further characterize S955P independently of F508del and to assess its response to CFTR modulators. RESULTS: Results in both rectal biopsies and intestinal organoids from both patients evidence residual CFTR function. Further characterization shows that 1717–2A>G leads to alternative splicing generating

Background and purpose Rescue of F508del-cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the most common CF mutation, requires small molecules that overcome protein processing, stability and channel gating defects. Here, we investigate F508del-CFTR rescue by CFFT-004, a small molecule designed to independently correct protein processing and channel gating defects. Experimental approach Using CFTR-expressing recombinant cells and CF patient-derived bronchial epithelial cells, we studied CFTR expression by Western blotting and channel gating and stability with the patch-clamp and Ussing chamber techniques. Key results Chronic treatment with CFFT-004 improved modestly F508del-CFTR processing, but not its plasma membrane stability. By contrast, CFFT-004 rescued F508del-CFTR channel gating better than C18, an analogue of the clinically used CFTR corrector lumacaftor. Subsequent acute addition of CFFT-004, but not C18, potentiated F508del-CFTR channel gating. However, CFFT-004 was without effect on A561E-CFTR, a CF mutation with a comparable mechanism of CFTR dysfunction as F508del-CFTR. To investigate the mechanism of action of CFFT-004, we used F508del-CFTR revertant mutations. Potentiation by CFFT-004 was unaffected by revertant mutations, but correction was abolished by the revertant mutation G550E. These data suggest that correction, but not potentiation, by CFFT-004 might involve nucleotide-binding domain 1 of CFTR. Conclusions and implications CFFT-004 is a dual-acting small molecule with independent corrector and potentiator activities that partially rescues F508del-CFTR in recombinant cells and native airway epithelia. The limited efficacy and potency of CFFT-004 suggests that combinations of small molecules targeting different defects in F508del-CFTR might be a more effective therapeutic strategy than a single agent.

Most of the ~2100 CFTR variants so far reported are very rare and still uncharacterized regarding their cystic fibrosis (CF) disease liability. Since some may respond to currently approved modulators, characterizing their defect and response to these drugs is essential. Here we aimed characterizing the defect associated with four rare missense (likely Class II) CFTR variants and assess their rescue by corrector drugs. We produced CFBE cell lines stably expressing CFTR with W57G, R560S, H1079P and Q1100P, assessed their effect upon CFTR expression and maturation and their rescue by VX-661/VX-445 correctors. Results were validated by forskolin-induced swelling assay (FIS) using intestinal organoids from individuals bearing these variants. Finally, knock-down (KD) of genes previously shown to rescue F508del-CFTR was assessed on these mutants. Results show that all the variants preclude the production of mature CFTR, confirming them as Class II mutations. None of the variants responded to VX-661 but the combination rescued H1079P- and Q1100P-CFTR. The KD of factors that correct F508del-CFTR retention only marginally rescued R560S- and H1079P-CFTR. Overall, data evidence that Class II mutations induce distinct molecular defects that are neither rescued by the same corrector compounds nor recognized by the same cellular machinery, thus requiring personalized drug discovery initiatives.