Theratyping of the Rare CFTR Variants E193K and R334W in Rectal Organoid-Derived Epithelial Monolayers.

BACKGROUND: The effect of presently available CFTR modulator combinations, such as elexacaftor (ELX), tezacaftor (TEZ), and ivacaftor (IVA), on rare CFTR alleles is often unknown. Several assays have been developed, such as forskolin-induced swelling (FIS), to evaluate the rescue of such uncommon CFTR alleles both by established and novel modulators in patient-derived primary cell cultures (organoids). Presently, we assessed the CFTR-mediated electrical current across rectal organoid-derived epithelial monolayers. This technique, which allows separate measurement of CFTR-dependent chloride or bicarbonate transport, was used to assess the effect of ELX/TEZ/IVA on two rare CFTR variants. METHODS: Intestinal organoid cultures were established from rectal biopsies of CF patients carrying the rare missense mutations E193K or R334W paired with F508del. The effect of the CFTR modulator combination ELX/TEZ/IVA on CFTR-mediated Cl- and HCO3- secretion was assessed in organoid-derived intestinal epithelial monolayers. Non-CF organoids were used for comparison. Clinical biomarkers (sweat chloride, FEV1) were monitored in patients receiving modulator therapy. RESULTS: ELX/TEZ/IVA markedly enhanced CFTR-mediated bicarbonate and chloride transport across intestinal epithelium of both patients. Consistent with the rescue of CFTR function in cultured intestinal cells, ELX/TEZ/IVA therapy improved biomarkers of CFTR function in the R334W/F508del patient. CONCLUSIONS: Current measurements in organoid-derived intestinal monolayers can readily be used to monitor CFTR-dependent epithelial Cl- and HCO3- transport. This technique can be explored to assess the functional consequences of rare CFTR mutations and the efficacy of CFTR modulators. We propose that this functional CFTR assay may guide personalized medicine in patients with CF-like clinical manifestations as well as in those carrying rare CFTR mutations.

Rescue of chloride and bicarbonate transport by elexacaftor-ivacaftor-tezacaftor in organoid-derived CF intestinal and cholangiocyte monolayers.

BACKGROUND: In cystic fibrosis (CF), loss of CF transmembrane conductance regulator (CFTR)-dependent bicarbonate secretion precipitates the accumulation of viscous mucus in the lumen of respiratory and gastrointestinal epithelial tissues. We investigated whether the combination of elexacaftor (ELX), ivacaftor (IVA) and tezacaftor (TEZ), apart from its well-documented effect on chloride transport, also restores Phe508del-CFTR-mediated bicarbonate transport. METHODS: Epithelial monolayers were cultured from intestinal and biliary (cholangiocyte) organoids of homozygous Phe508del-CFTR patients and controls. Transcriptome sequencing was performed, and bicarbonate and chloride transport were assessed in the presence or absence of ELX/IVA/TEZ, using the intestinal current measurement technique. RESULTS: ELX/IVA/TEZ markedly enhanced bicarbonate and chloride transport across intestinal epithelium. In biliary epithelium, it failed to enhance CFTR-mediated bicarbonate transport but effectively rescued CFTR-mediated chloride transport, known to be requisite for bicarbonate secretion through the chloride-bicarbonate exchanger AE2 (SLC4A2), which was highly expressed by cholangiocytes. Biliary but not intestinal epithelial cells expressed an alternative anion channel, anoctamin-1/TMEM16A (ANO1), and secreted bicarbonate and chloride upon purinergic receptor stimulation. CONCLUSIONS: ELX/IVA/TEZ has the potential to restore both chloride and bicarbonate secretion across CF intestinal and biliary epithelia and may counter luminal hyper-acidification in these tissues.

A Potential Treatment of Congenital Sodium Diarrhea in Patients With Activating GUCY2C Mutations.

INTRODUCTION: Gain-of-function mutations in guanylyl cyclase C (GCC) result in persistent diarrhea with perinatal onset. We investigated a specific GCC inhibitor, SSP2518, for its potential to treat this disorder. METHODS: We investigated the effect of SSP2518 on GCC-mediated intracellular cyclic guanosine monophosphate (cGMP) levels and on GCC-mediated chloride secretion in intestinal organoids from 3 patients with distinct activating GCC mutations and from controls, with and without stimulation of GCC with heat-stable enterotoxin. RESULTS: Patient-derived organoids had significantly higher basal cGMP levels than control organoids, which were lowered by SSP2518 to levels found in control organoids. In addition, SSP2518 significantly reduced cGMP levels and chloride secretion in patient-derived and control organoids (P < 0.05 for all comparisons) after heat-stable enterotoxin stimulation. DISCUSSION: We reported in this study that the GCC inhibitor SSP2518 normalizes cGMP levels in intestinal organoids derived from patients with GCC gain-of-function mutations and markedly reduces cystic fibrosis transmembrane conductance regulator-dependent chloride secretion, the driver of persistent diarrhea.

Impaired Intestinal Farnesoid X Receptor Signaling in Cystic Fibrosis Mice.

BACKGROUND & AIMS: The bile acid (BA)-activated farnesoid X receptor (FXR) controls hepatic BA synthesis and cell proliferation via the intestinal hormone fibroblast growth factor 19. Because cystic fibrosis (CF) is associated with intestinal dysbiosis, anomalous BA handling, and biliary cirrhosis, we investigated FXR signaling in CF. METHODS: Intestinal and hepatic expression of FXR target genes and inflammation markers was assessed in Cftr null mice and controls. Localization of the apical sodium-dependent BA transporter was assessed, and BAs in gastrointestinal tissues were analyzed. The CF microbiota was characterized and FXR signaling was investigated in intestinal tissue and organoids. RESULTS: Ileal murine fibroblast growth factor 19 ortholog (Fgf15) expression was strongly reduced in CF mice, compared with controls. Luminal BA levels and localization of apical sodium-dependent BA transporter was not affected, and BAs induced Fgf15 up to normal levels in CF ileum, ex vivo, and CF organoids. CF mice showed a dysbiosis that was associated with a marked up-regulation of genes involved in host-microbe interactions, including those involved in mucin glycosylation, antimicrobial defense, and Toll-like receptor signaling. Antibiotic treatment reversed the up-regulation of inflammatory markers and restored intestinal FXR signaling in CF mice. Conversely, FXR-dependent gene induction in ileal tissue and organoids was repressed by bacterial lipopolysaccharide and proinflammatory cytokines, respectively. Loss of intestinal FXR activity was associated with a markedly blunted hepatic trophic response to oral BA supplementation, and with impaired repression of Cyp7a1, the gene encoding the rate-limiting enzyme in BA synthesis. CONCLUSIONS: In CF mice, the gut microbiota represses intestinal FXR activity, and, consequently, FXR-dependent hepatic cell proliferation and feedback control of BA synthesis.

Transcriptome analysis of the distal small intestine of Cftr null mice.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CFTR anion channel. Loss of CFTR function in pancreatic, biliary and intestinal epithelia, severely affects gastrointestinal function. Transcriptome analysis indicated the activation of an innate and adaptive immune response in the distal small intestine of Cftr null mice. Inflammation was associated with differential regulation of numerous genes involved in the transport and metabolism of nutrients and, particularly, lipids, that are targeted by ligand-dependent nuclear receptors and/or HNF4α. Among the most strongly down-regulated genes are the FXR targets Fgf15 and Nr0b2, the PPARα target Pdk4, and the PXR target Ces2a, whereas expression of the CF modifier gene Slc6a14 was strongly increased. Most changes in gene expression were reversed by bacterial containment. Our data suggest that the gut microbiota has a pervasive effect on gene expression in CF mice, affecting enterocyte maturation, lipid metabolism, and nutrient absorption in CF.

Selective inhibition of intestinal guanosine 3',5'-cyclic monophosphate signaling by small-molecule protein kinase inhibitors.

The guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase II (cGKII) serine/threonine kinase relays signaling through guanylyl cyclase C (GCC) to control intestinal fluid homeostasis. Here, we report the discovery of small-molecule inhibitors of cGKII. These inhibitors were imidazole-aminopyrimidines, which blocked recombinant human cGKII at submicromolar concentrations but exhibited comparatively little activity toward the phylogenetically related protein kinases cGKI and cAMP-dependent protein kinase (PKA). Whereas aminopyrimidyl motifs are common in protein kinase inhibitors, molecular modeling of these imidazole-aminopyrimidines in the ATP-binding pocket of cGKII indicated an unconventional binding mode that directs their amine substituent into a narrow pocket delineated by hydrophobic residues of the hinge and the αC-helix. Crucially, this set of residues included the Leu-530 gatekeeper, which is not conserved in cGKI and PKA. In intestinal organoids, these compounds blocked cGKII-dependent phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). In mouse small intestinal tissue, cGKII inhibition significantly attenuated the anion secretory response provoked by the GCC-activating bacterial heat-stable toxin (STa), a frequent cause of infectious secretory diarrhea. In contrast, both PKA-dependent VASP phosphorylation and intestinal anion secretion were unaffected by treatment with these compounds, whereas experiments with T84 cells indicated that they weakly inhibit the activity of cAMP-hydrolyzing phosphodiesterases. As these protein kinase inhibitors are the first to display selective inhibition of cGKII, they may expedite research on cGMP signaling and may aid future development of therapeutics for managing diarrheal disease and other pathogenic syndromes that involve cGKII.