RFFL-mediated protein quality control limits functional rescue of TRID-CFTR modulator combination therapy for cystic fibrosis nonsense mutations
Hazuki Tateishi, Yukako Doi, Yuka Kamada, Tsukasa Okiyoneda
TL;DR
This study shows that RFFL, a protein involved in quality control, limits the effectiveness of combination therapies for cystic fibrosis caused by nonsense mutations.
Contribution
The study identifies RFFL as a key regulator in degrading TRID-induced CFTR proteins and suggests targeting RFFL to improve therapy efficacy.
Findings
RFFL targets TRID-induced full-length CFTR for ubiquitination and degradation.
RFFL knockdown stabilizes mature CFTR at the plasma membrane and enhances functional rescue.
Combining TRID and CFTR modulators with RFFL inhibition improves therapeutic outcomes.
Abstract
Cystic fibrosis (CF) is a monogenic disorder caused by mutations in the CFTR gene, which encodes a cAMP-regulated anion channel at the apical plasma membrane (PM) of epithelial cells. CFTR modulators have recently been approved as effective therapies for folding-defective mutations, including the most common variant, F508del. However, no clinically effective treatments are available for nonsense mutations such as G542X, the second most frequent CF-causing mutation. Translational readthrough-inducing drugs (TRIDs), such as G418, can suppress premature termination codons (PTCs) and partially restore full-length CFTR expression, but their therapeutic efficacy remains limited. Notably, combining TRIDs with CFTR modulators enhances functional rescue, suggesting that the restored full-length CFTR may be targeted by protein quality control (QC) pathways. Here, we investigated the QC mechanisms…
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Figure 7- —JSPS KAKENHI
- —http://dx.doi.org/10.13039/100007449Takeda Science Foundation
- —http://dx.doi.org/10.13039/100012044Kwansei Gakuin University
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Taxonomy
TopicsCystic Fibrosis Research Advances · Endoplasmic Reticulum Stress and Disease · Bacterial Genetics and Biotechnology
Introduction
Cystic fibrosis (CF) is a monogenic disorder most prevalent in Caucasians and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated anion channel localized to the apical plasma membrane (PM) of epithelial cells [1, 2]. To date, approximately 1,000 disease-causing CFTR mutations have been identified and are categorized into several classes based on their effects on CFTR protein synthesis, processing, and function [3, 4]. Class I mutations introduce premature termination codons (PTCs) due to nonsense mutations, frameshifts, or splicing defects, resulting in little to no production of full-length CFTR protein [5]. Class II mutations, exemplified by F508del, the most common CF-causing mutation, disrupt protein folding within the endoplasmic reticulum (ER), leading to recognition by the ER quality control (ERQC) system and degradation via the ER-associated degradation (ERAD) pathway, thereby severely reducing CFTR expression at the cell surface [2, 6, 7]. Class III and IV mutations do not impair protein maturation or trafficking to the PM, but disrupt channel function: Class III mutations impair gating, whereas Class IV mutations reduce ion conductance. Class V mutations typically affect transcription or splicing, leading to reduced amounts of functional CFTR protein. Class VI mutations allow normal trafficking and function but impair the protein's stability at the PM, leading to accelerated turnover and degradation [8].
In recent years, the development of CFTR modulators has enabled mutation class-specific therapies for CF [9, 10]. The first approved modulator, ivacaftor (IVA, VX-770), is a CFTR potentiator that improves channel gating and is clinically effective in patients with class III mutations, such as G551D, which accounts for ~ 5% of CF cases [11]. For the most prevalent mutation, F508del, which causes a class II folding and trafficking defect, a combination therapy has been developed [12–14]. This regimen comprising the correctors elexacaftor (ELX, VX-445) and tezacaftor (TEZ, VX-661) along with the potentiator IVA is marketed as Trikafta® (or Kaftrio®) and referred to here as ELX/TEZ/IVA (ETI) [15]. However, approximately 10% of CF patients carry nonsense mutations, including the second most common variant, G542X-CFTR. These mutations introduce PTCs into CFTR mRNA, leading to truncated, non-functional proteins that are not amenable to correction by currently available CFTR modulators [10, 16, 17].
Promoting translational readthrough at PTCs represents a promising therapeutic strategy for restoring the production of full-length, functional CFTR protein in CF patients with class I mutations. Various small molecules, collectively known as translational readthrough-inducing drugs (TRIDs), have been developed to suppress nonsense codon recognition and enhance PTC readthrough efficiency [18]. Aminoglycoside antibiotics such as gentamicin and G418 can induce PTC readthrough in CFTR; however, their clinical application is limited due to significant toxicity [19, 20]. Gentamicin has been associated with acute renal toxicity, while G418 exhibits broad cytotoxicity by interfering with protein synthesis in both prokaryotic and eukaryotic cells. Ataluren (PTC124), developed by PTC Therapeutics, received conditional approval in 2014 for the treatment of nonsense mutation Duchenne muscular dystrophy. Although Ataluren has demonstrated some in vitro efficacy in restoring G542X-CFTR function, its clinical benefit in CF patients has been minimal or inconsistent [21, 22]. Recent advances have introduced alternative TRIDs targeting the translation termination machinery. SRI-41315, an inhibitor of eukaryotic release factor 1 (eRF1), has been shown to promote PTC readthrough in CFTR while sparing normal termination codons [21]. Similarly, CC-90009, a cereblon (CRBN) E3 ligase modulator, enhances readthrough by promoting degradation of eRF3a, a class 2 translation termination factor [23], and has demonstrated efficacy in restoring G542X- and W1282X-CFTR function [22]. Notably, CC-90009 also synergizes with G418 to further improve readthrough efficiency. ELX-02 (NB-124), a next-generation aminoglycoside derivative with enhanced specificity for eukaryotic ribosomes, has been developed to reduce toxicity and improve readthrough capacity compared to conventional aminoglycosides. However, in clinical trials, ELX-02 alone or in combination with the CFTR potentiator ivacaftor failed to show statistically significant improvement in sweat chloride concentration or forced expiratory volume in 1 s (FEV₁) [24]. These findings underscore the need for further optimization of TRID-based therapies to achieve clinically meaningful restoration of CFTR function in patients with class I nonsense mutations.
Recent studies have demonstrated that neither TRIDs nor CFTR modulators alone provide substantial rescue of G542X-CFTR function; however, their combination can result in additive or even synergistic effects. For example, co-treatment with G418 and CFTR corrector and/or potentiator significantly enhanced the production of mature, full-length G542X-CFTR protein and restored channel activity [25, 26]. Similarly, the use of CFTR correctors such as VX-809 or VX-445 (ELX) in combination with TRIDs including G418 or ELX-02 has been shown to improve the functional expression of class I CFTR mutants such as G542X-, R1162X-, and W1282X-CFTR [17, 27]. Furthermore, it has been reported that the G542X nonsense mutation (UGA) can undergo translational readthrough upon G418 treatment, resulting in the incorporation of amino acids such as cysteine (C), tryptophan (W), or arginine (R) at position 542 [27–29]. These amino acid substitutions effectively generate missense CFTR variants, G542C, G542W, and G542R, that may be structurally unstable or misfolded. Based on these findings, we hypothesize that while TRIDs enable the production of full-length CFTR protein in class I mutants, the resulting proteins may be structurally abnormal and subject to recognition and degradation by the cellular PQC system. This could account for the limited efficacy of TRID monotherapy and the enhanced rescue observed when combined with CFTR modulators, which are effective in correcting the folding defects seen in class II mutations such as F508del-CFTR. Importantly, although TRID-CFTR modulator combination therapy has provided some benefit, its overall efficacy remains limited [17, 24]. Targeting PQC factors such ubiquitin (Ub) E3 ligase could further enhance these therapeutic effects, as previously demonstrated for F508del-CFTR [30–33].
In this study, we aimed to elucidate the PQC mechanisms acting on full-length G542X-CFTR generated by TRIDs, with a particular focus on the Ub E3 ligases that restrict the functional expression of misfolded CFTR [30, 33–36]. Our findings demonstrate that G418-induced full-length G542X-CFTR is predominantly targeted for degradation by the E3 ligase RFFL, which plays a key role in peripheral QC mechanisms that eliminate misfolded CFTR proteins from the PM [33]. Additionally, the ER-associated E3 ligases RNF5 and RNF185 contribute partially to this degradation process. RFFL KD stabilized the mature form of full-length G542X-CFTR by reducing its ubiquitination, leading to enhanced PM expression and improved channel function, particularly under combined treatment with G418 and CFTR modulators. Importantly, this effect was not restricted to G542X-CFTR but was also observed in other class I nonsense mutants, including W1282X-, R553X-, and R1162X-CFTR. These results indicate that TRID-induced full-length CFTR proteins derived from class I mutations remain conformationally defective and are subjected to clearance by RFFL-mediated peripheral QC, even in the presence of CFTR modulators. Therefore, targeting RFFL represents a promising therapeutic strategy to augment the efficacy of TRID and modulator combination therapies for CF patients harboring various class I CFTR mutations.
Materials and methods
Plasmids and reagents
G542X (c.1624G > T), G542C, G542R, G542W, W1282X (c.3846G > A), R553X (c.1657C > T), and R1162X (c.3484C > T) CFTR variants were generated by PCR-based mutagenesis. Each CFTR construct was engineered with a HiBiT tag in the extracellular region as previously described [30, 31, 37] and cloned into the pLX303 vector (Addgene #25897) via LR recombination using Gateway™ LR Clonase™ II Enzyme Mix (ThermoFisher). HBH-tagged CFTR-3HA variants were cloned into the pLIX402 vector (Addgene #41394) by the same procedure. G418 was purchased from FUJIFILM Wako Pure Chemicals, SRI-41315 from Sigma-Aldrich (#SML3312), and CC-90009 from Cayman Chemical (#CAY-33692). Elexacaftor (ELX, VX-445; Cat# S8851) and tezacaftor (TEZ, VX-661; Cat# S7059) were obtained from Selleck Chemicals (Houston, TX), and ivacaftor (IVA, VX-770; Cat# CS-0497) was purchased from Chemscene LLC (Monmouth Junction, NJ).
Cell culture
The human bronchial epithelial cell line BEAS-2B stably expressing G542X-, G542C-, G542R-, and G542W-CFTR-HiBiT(Ex) was established by lentiviral transduction as previously described [30, 33, 37]. BEAS-2B Tet-on cells stably expressing His_6_-Biotin-His_6_ (HBH) [38] -tagged G542X-, G542C-, G542R-, and G542W-CFTR-3HA were also generated by lentiviral transduction [33]. BEAS-2B cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; FUJIFILM Wako Pure Chemicals) supplemented with 10% fetal bovine serum (FBS), penicillin–streptomycin, and 10 µg/mL blasticidin S (KAKEN Pharmaceutical). CFBE Tet-on cells stably expressing HBH-G542X-CFTR-3HA were generated by lentiviral transduction and cultured in Minimum Essential Medium (MEM; FUJIFILM Wako Pure Chemicals) supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES, and 10 µg/mL blasticidin S. For propagation, CFBE cells were grown on plastic dishes coated with an extracellular matrix (ECM mix) consisting of 10 mg/mL human fibronectin (FUJIFILM Wako Pure Chemicals, Cat# 641–54943), 30 mg/mL collagen (FUJIFILM Wako Pure Chemicals, Cat# 387–21361), and 100 mg/mL bovine serum albumin (Sigma-Aldrich). Expression of HBH-CFTR-3HA was typically induced by treatment with 1 µg/mL doxycycline (Dox) for 4 days. All cell lines were maintained at 37 °C in a hu’Idified Incubator with 5% CO₂.
Transfection
siRNA and dsiRNA transfections in BEAS-2B and CFBE cells were performed using Lipofectamine RNAiMax transfection reagent (ThermoFisher), as described previously [33]. Unless otherwise specified, siRNA-transfected cells were used for experiments 4 days post-transfection. siRNA and dsiRNA used in this study were listed in Table S1.
Measurement of PM expression of CFTR-HiBiT
Cell-surface expression of CFTR-HiBiT in BEAS-2B cells seeded in 96-well plates was measured using the Nano-Glo HiBiT Extracellular System (Promega, Madison, WI), as previously described [31]. To enhance CFTR-HiBiT expression in stable cells, cultures were pre-treated with 2 mM sodium butyrate (NaB) at 37 °C for 2 days prior to analysis. Readthrough drug G418 was typically applied for 4 days, whereas CC-90009 or SRI-41315 was administered for 2 days, with or without ETI (1 μM ELX/3 μM TEZ/1 μM IVA) for 2 days at 37 °C. Luminescence was measured using either the Luminoskan or Varioskan Flash microplate reader (ThermoFisher Scientific, Waltham, MA).
Western blotting
Western blotting was performed as previously described [33]. Cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris–HCl, pH 8.0, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 1 mM phenylmethanesulfonyl fluoride (PMSF), 5 µg/mL leupeptin, and 5 µg/mL pepstatin. Total protein was visualized on membranes by Ponceau S staining (Sigma-Aldrich). HBH-CFTR-3HA variants were detected using anti-HA (16B12, BioLegend, Cat# 901515) antibody or NeutrAvidin protein–HRP (ThermoFisher). RFFL and RNF185 were detected using rabbit anti-RFFL (Sigma-Aldrich, Cat# HPA019492) or anti-RNF185 [36], followed by Peroxidase AffiniPure Donkey Anti-Rabbit IgG (H + L) secondary antibody (Jackson ImmunoResearch, Cat# 711–035–152). Antigen–antibody complexes were visualized with SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher) or ImmunoStar Zeta (FUJIFILM Wako Pure Chemicals) and imaged using the Fusion chemiluminescence imaging system (Vilber Bio Imaging, France). Densitometric quantification was performed using Evolution-Capt software (Vilber Bio Imaging).
G542X-CFTR ubiquitination measurement
Full-length HBH-G542X-CFTR was produced in BEAS-2B Tet-on cells transfected with 25 nM dsiNC or dsiRFFL and incubated with Dox (1 µg/mL) and G418 (700 µM), with or without ETI (1 μM ELX/3 μM TEZ/1 μM IVA), for 2 days at 37 °C. To accumulate ubiquitinated CFTR, cells were treated with MG-132 (10 µM) for 3 h at 37 °C prior to lysis. Four days post-siRNA transfection, cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris–HCl, pH 8.0, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with PMSF (1 mM), leupeptin (5 µg/mL), pepstatin (5 µg/mL), MG-132 (10 µM), and N-ethylmaleimide (5 mM). HBH-G542X-CFTR was purified under denaturing conditions using NeutrAvidin agarose (ThermoFisher) and analyzed by Western blotting with anti-ubiquitin antibody (P4D1, Santa Cruz Biotechnology, Cat# sc-8017) and NeutrAvidin protein–HRP (ThermoFisher). The level of CFTR ubiquitination was quantified by densitometry and normalized to the total CFTR level in the precipitate.
Halide-sensitive YFP quenching assay
CFBE Tet-on cells stably expressing G542X-CFTR-3HA were seeded into black 96-well plates at 2.5 × 10^4^ cells per well and transfected with 25 nM dsiRNA as indicated in the figure legend. Full-length G542X-CFTR expression was induced with Dox (1 µg/mL) and G418 (500 µM) for 4 days, followed by treatment with ETI (1 µM ELX, 3 µM TEZ, 1 µM IVA) for 2 days at 37 °C. Adenovirus encoding the halide-sensitive YFP variant (F46L/H148Q/I152L, Ad-YFP) was introduced at a multiplicity of infection (MOI) of 0.5, as described previously [39]. Four days after ASO transfection and 2 days after Ad-YFP infection, cells were washed three times with PBS-chloride buffer (140 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 1.1 mM MgCl₂, 0.7 mM CaCl₂, and 5 mM glucose). Each well was then incubated with 50 µL of PBS-chloride, followed by the addition of 50 µL of activator solution (20 µM forskolin, 0.5 mM IBMX, 0.5 mM cpt-cAMP, and 0.1 mM genistein) and incubated for 57 s. Fluorescence was recorded continuously at 200 ms intervals for 3 s (baseline) and for 32 s after rapid addition of 100 µL PBS-iodide (NaCl replaced with NaI). Measurements were obtained using a VICTOR Nivo multimode microplate reader (PerkinElmer) equipped with a dual syringe pump (excitation/emission: 500/535 nm). The iodide influx rate was calculated by fitting the YFP fluorescence decay curve using GraphPad Prism 8 (GraphPad Software, San Diego, CA).
Statistical analysis
Data from at least three independent experiments were used for quantification and are presented as mean ± standard deviation (SD). Statistical significance was assessed using either a two-tailed unpaired Student’s t-test, a one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test, or a two-way ANOVA with Holm–Sidak multiple comparisons, as indicated in the figure legends. All analyses were performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA). A P value < 0.05 was considered statistically significant.
Results
CFTR modulators improve the limited efficacy of TRIDs
To evaluate the effects of TRIDs and CFTR modulators on the PM expression of G542X-CFTR, we generated a human airway epithelial cell line BEAS-2B stably expressing G542X-CFTR fused with a HiBiT tag at the extracellular domain. The HiBiT system enables sensitive quantification of CFTR surface expression through luminescence detection using LgBiT and a luciferase substrate [31]. We tested three TRIDs: G418, CC-90009, and SRI-41315. G418 is an aminoglycoside antibiotic known to promote translational readthrough and induce production of full-length CFTR protein [40]. CC-90009 is a cereblon (CRBN) E3 ubiquitin ligase modulator that inhibits eukaryotic release factor 3a (eRF3a), a class II translation termination factor, thereby enhancing PTC readthrough [23]. SRI-41315 promotes degradation of eRF1, a class I termination factor, and increases PTC readthrough by reducing its abundance [21]. The HiBiT assay revealed that luminescence signals were comparable between parental BEAS-2B cells and G542X-CFTR-HiBiT stable cells under vehicle-treated conditions, indicating negligible PM expression of G542X-CFTR in the absence of treatment (Fig. 1A). Signal from parental BEAS-2B cells was defined as the background (BG) level. Neither individual TRIDs nor the CFTR modulator triple combination therapy, Elaxacaftor (ELX/VX-445), Tezacaftor (TEZ/VX-661), and Ivacaftor (IVA/VX-770), referred to as ETI, significantly increased G542X-CFTR-HiBiT PM levels when used alone (Fig. 1B–D). Interestingly, only the combination of G418 with ETI resulted in a notable increase in surface expression of G542X-CFTR-HiBiT, suggesting a synergistic effect (Fig. 1B–D). Based on these findings, we focused on G418 for subsequent experiments.Fig. 1. Combination effects of TRIDs and CFTR modulators on the PM expression of G542X-CFTR. (A) Basal PM levels of G542X-CFTR-HiBiT(Ex) in BEAS-2B cells were measured by HiBiT assay (n = 12). (B–D) PM levels of G542X-CFTR-HiBiT(Ex) in BEAS-2B cells treated with G418 (B, n = 3) for 4 days, CC-90009 (C, n = 3), or SRI-41315 (D, n = 3) for 2 days at the concentrations indicated, with or without ETI (1 µM ELX, 3 µM TEZ, 1 µM IVA, for 2 days) at 37 °C. As indicated, 0.1 µM CC-90009 or 5 µM SRI-41315 were administered for 4 days. The background (BG) level, determined from parental BEAS-2B cells lacking CFTR-HiBiT expression, is shown as a reference in panels (A–D). (E) Western blot analysis of HBH-G542X-CFTR-3HA expression in BEAS-2B Tet-on cells treated with 1 µg/ml Dox and G418 for 4 days, and ETI for 2 days at 37 °C. CFTR was detected using anti-HA (αHA) or NeutrAvidin-HRP (NA-HRP). Ponceau S staining was used as a loading control. Band B and Band C represent the immature and mature forms of full-length G542X-CFTR, respectively. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test (B-D). ***p < 0.001; ****p < 0.0001
Western blot analysis using BEAS-2B cells stably expressing HBH-G542X-CFTR-3HA further supported the HiBiT assay results. An N-terminal HBH tag enabled detection using NeutrAvidin-HRP [38]. G418 alone modestly increased full-length G542X-CFTR expression, while combination treatment with ETI further enhanced expression, particularly of the mature, complex-glycosylated form (band C) (Fig. 1E). These results suggest that G418-induced full-length G542X-CFTR protein likely retains conformational defects that can be corrected by CFTR modulators such as ETI. In this study, we employed a higher concentration of G418 (700 µM) than that used in previous reports [25–27] in order to reliably detect full-length G542X-CFTR protein by Western blot analysis.
Ub E3 ligases limit the PM levels of full-length G542X-CFTR
Because G418-induced full-length G542X-CFTR responds to ETI treatment in a manner similar to F508del-CFTR, we hypothesized that it might also be degraded by PQC pathways, including Ub E3 ligases known to target misfolded F508del-CFTR. To test this, we examined Ub ligases previously implicated in F508del-CFTR PQC [30, 33–36]. HiBiT assay results showed that knockdown (KD) of RFFL or UBE3C significantly increased the PM levels of G542X-CFTR following G418 treatment (Fig. 2A). Moreover, when cells were treated with both G418 and ETI, KD of RFFL or RNF5/185 markedly elevated G542X-CFTR PM expression (Fig. 2B).Fig. 2. Identification of Ub ligases limiting the PM expression of G542X-CFTR. (A, B) PM levels of G542X-CFTR-HiBiT(Ex) in BEAS-2B cells transfected with the indicated siRNAs: 25 nM dsiNC, 25 nM dsiRFFL, 25 nM dsiRNF34, 25 nM dsiAMFR, 25 nM UBE3C, 100 nM siNT, 50 nM siRNF5 and 50 nM siRNF185 (combined as siRNF5/185), 50 nM siSTUB1, and 50 nM siHERC3. Cells were treated with 250 µM G418 for 4 days (A), or G418 for 4 days with or without ETI treatment (1 µM ELX, 3 µM TEZ, 1 µM IVA, for 2 days) at 37 °C (B). PM levels were measured 4 days post-siRNA transfection. (C) Combined effects of RFFL KD and RNF5/185 KD on PM levels of G542X-CFTR-HiBiT(Ex) in BEAS-2B cells. Cells were transfected with siRNAs (25 nM dsiNC, 25 nM dsiRFFL, 50 nM siRNF5/185; total 100 nM), then treated with 250 µM G418 for 4 days and ETI for the last 2 days at 37 °C as above. (D) Combined effects of RFFL KD and RNF5/185 KD on PM levels of F508del-CFTR-HiBiT(Ex) in BEAS-2B cells. Cells were transfected with siRNAs (25 nM dsiNC, 25 nM dsiRFFL, 50 nM siRNF5/185; total 100 nM), then treated with ETI for 2 days at 37 °C. The BG level is shown as a reference in panels (A–D). Data represent mean ± SD (n = 6 for A–D). Statistical analyses: One-way ANOVA with Dunnett’s multiple comparisons (A, B), unpaired Student’s t-test (A, B), or two-way ANOVA with Holm–Sidak multiple comparisons (C, D). The interaction between RFFL KD and RNF5/185 KD is shown as P interaction (P_int_). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant
To evaluate whether combined KD of these ligases would yield additive or synergistic effects, we performed triple KD of RFFL, RNF5, and RNF185. Unexpectedly, this did not further enhance PM expression beyond that observed with RFFL KD alone (Fig. 2C). This phenotype resembled that of F508del-CFTR, where KD of either RFFL or RNF5/185 increased PM levels, particularly in the presence of ETI. However, simultaneous KD of RFFL, RNF5/185 did not produce any additive effect (Fig. 2D). These findings suggest that G418-induced full-length G542X-CFTR is eliminated via PQC pathways mediated by either RFFL or RNF5/185, with RFFL playing a dominant role.
We next investigated the mechanism by which RFFL KD enhances G418-induced G542X-CFTR expression. HiBiT assays showed that, in the presence of 250 µM or 500 µM G418, combining ETI with RFFL KD additively increased G542X-CFTR PM levels, indicating that ETI and RFFL KD act through independent mechanisms (Fig. 3A and B). Consistent with these findings, Western blot analysis showed that treatment with 700 µM G418 alone (control, left lane) resulted in low-level expression of the mature form of G542X-CFTR. In contrast, ETI treatment and RFFL KD each independently increased the expression of G418-induced full-length G542X-CFTR, and their combination produced an additive effect. Notably, this enhancement was most pronounced for the mature, complex-glycosylated form (band C), which localizes to post-Golgi compartments, including the plasma membrane (Fig. 3C).Fig. 3RFFL KD and ETI treatment additively improve PM expression and function of G542X-CFTR. (A, B) PM levels of G542X-CFTR-HiBiT(Ex) in BEAS-2B cells transfected with 25 nM dsiNC or 25 nM dsiRFFL and treated with 250 µM G418 (A, n = 12) or 500 µM G418 (B, n = 12) for 4 days. ETI (1 µM ELX, 3 µM TEZ, 1 µM IVA) was added during the last 2 days at 37 °C. The BG level is shown as a reference in panels. (C) Western blot analysis of HBH-G542X-CFTR-3HA in BEAS-2B Tet-on cells treated with 25 nM dsiRFFL, 1 µg/ml Dox, 700 µM G418 for 4 days, and ETI for the last 2 days. CFTR and RFFL were detected using Neutravidin-HRP (NA-HRP) and anti-RFFL antibody, respectively. Ponceau S staining was used as a loading control. Bands B and C correspond to the immature and mature forms of full-length G542X-CFTR, respectively. The immature (B band) and mature forms (C band) of full-length G542X-CFTR were quantified by densitometry (bottom, n = 4). Each biological replicate (n) is color-coded. (D, E) Functional analysis of HBH-G542X-CFTR-3HA channel activity in CFBE Tet-on cells using the halide-sensitive YFP quenching assay. (D) Representative traces of YFP fluorescence over time. (E) Quantification of the initial YFP quenching rate (n = 6), representing CFTR channel activity. Cells were transfected with 25 nM dsiRFFL and treated with 1 µg/ml Dox and 500 µM G418 for 4 days, and ETI for the last 2 days. Halide-sensitive YFP was introduced via adenoviral infection (MOI 0.5). The assay was performed 4 days after siRNA transfection and 2 days after adenovirus infection. Data are presented as mean ± SD. Statistical analyses were performed using two-way ANOVA (A, B), two-way repeated-measures ANOVA (C), or two-way ANOVA with Holm–Sidak multiple comparisons (E). No significant interaction between RFFL KD and ETI was detected (P_int_ > 0.05). *p < 0.05, ****p < 0.0001, ns, not significant
To determine whether this increase in mature protein translated into functional rescue, we performed halide-sensitive YFP quenching assays in CFBE Tet-on cells stably expressing G542X-CFTR-3HA. In this assay, CFTR channel activity is quantified by the rate of YFP fluorescence quenching upon iodide influx [33, 41, 42]. Co-administration of 250 µM G418 and ETI did not result in a significant improvement in G542X-CFTR function compared with the G418 monotherapy control (DMSO), indicating that, under these conditions, meaningful functional rescue cannot be achieved by G418–ETI combination therapy alone (Fig. 3D and E). Similarly, combined treatment with 250 µM G418 and RFFL dsiRNA did not significantly enhance G542X-CFTR function. In contrast, simultaneous RFFL KD in the presence of both G418 and ETI led to a significant improvement in G542X-CFTR function relative to G418 monotherapy (Fig. 3D and E). Together, these results indicate that ETI and RFFL KD act through complementary mechanisms to improve the expression and function of G418-induced full-length G542X-CFTR, highlighting the therapeutic potential of combined strategies that target both protein folding and degradation pathways.
RFFL KD reduced ubiquitination and peripheral degradation of full-length G542X-CFTR.
We next investigated the mechanism by which RFFL KD enhances the functional PM expression of G418-induced full-length G542X-CFTR. Since RFFL is implicated in peripheral QC of misfolded CFTR [33], its KD likely reduces ubiquitination and subsequent degradation of full-length G542X-CFTR protein. Western blotting following cycloheximide (CHX) chase experiments revealed that the mature form of G418-induced G542X-CFTR was degraded over time (Fig. 4A). As expected, RFFL KD markedly delayed this degradation, stabilizing the mature form (Fig. 4A). In contrast, under combined G418 and ETI treatment, full-length G542X-CFTR remained stable throughout the 24-h CHX chase, and we were unable to detect any additional stabilizing effect of RFFL KD (Fig. 4B).Fig. 4RFFL KD reduces ubiquitination and peripheral degradation of G542X-CFTR. (A, B) Metabolic stability of HBH-G542X-CFTR-3HA in BEAS-2B Tet-on cells transfected with 25 nM dsiNC or dsiRFFL and treated with DMSO (A, n = 3) or ETI (1 µM ELX, 3 µM TEZ, 1 µM IVA) (B, n = 4) was assessed by cycloheximide (CHX) chase at 37 °C, followed by Western blotting using NA-HRP. The remaining mature form (band C) of full-length G542X-CFTR was quantified and expressed as a percentage of the amount at time 0. G418 (700 µM) was treated for 4 days. (C–E) PM levels of HBH-G542X-CFTR-3HA in BEAS-2B Tet-on cells transfected with 25 nM dsiRFFL and treated with ETI were measured by PM ELISA using an anti-HA antibody before (C, n = 6), and after 6 h (D, n = 6) or 24 h (E, n = 6) of chase at 37 °C. Cells were pre-treated with Dox (1 µg/ml) and G418 (500 µM) for 4 days. (F, G) Ubiquitination of HBH-G542X-CFTR-3HA in BEAS-2B Tet-on cells was assessed by Neutravidin pull-down under denaturing conditions and Western blotting. Cells were treated with 25 nM dsiRFFL, Dox (1 µg/ml), G418 (500 µM) for 4 days, and ETI for the last 2 days. CFTR ubiquitination levels were quantified by densitometry and normalized to the total amount of CFTR in the precipitates (G, n = 3). Data represent mean ± SD. Each biological replicate (n) is color-coded (A, B, G). Statistical analyses: paired t-test (A, B), two-way ANOVA with Holm–Sidak multiple comparisons (C–E), and two-way RM ANOVA (G). No significant interaction between RFFL KD and ETI was detected (P_int_ > 0.05). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant
To further evaluate the impact of RFFL KD on the PM stability of G418-induced G542X-CFTR, we performed PM ELISA [34]. Cell-surface G542X-CFTR-3HA was selectively labeled with anti-HA antibody at 4 °C to block endocytosis, followed by a chase at 37 °C for 6 or 24 h to allow CFTR internalization.
At the initial time point (T = 0 h), the combination of ETI and RFFL KD increased PM G542X-CFTR-3HA levels by approximately six-fold (Fig. 4C). Notably, after 6 and 24 h of chase, conditions that promote CFTR endocytosis and peripheral degradation, the combined treatment led to ~ 20-fold increases in PM G542X-CFTR-3HA, whereas ETI or RFFL KD alone had no significant effect (Fig. 4D-E). These findings indicate that G418-induced full-length G542X-CFTR is eliminated from the PM during the 6–24 h chase period, likely through peripheral QC mechanisms. However, the combination of ETI and RFFL KD stabilized this protein at the cell surface, probably by reducing peripheral CFTR degradation, thereby enhancing its PM expression.
To investigate how RFFL KD stabilizes G418-induced full-length G542X-CFTR, we examined the ubiquitination of the protein in BEAS-2B cells stably expressing HBH-G542X-CFTR-3HA. Cells were treated with Dox and G418 to induce full-length CFTR expression, followed by siRNA-mediated RFFL KD and/or CFTR modulator treatment (ETI). HBH-G542X-CFTR-3HA proteins were isolated under denaturing conditions, and ubiquitination was analyzed by Western blotting. A smear of ubiquitinated bands was detected in Dox- and G418-treated cells, but not in untreated controls, indicating that these bands correspond to ubiquitinated forms of full-length HBH-G542X-CFTR-3HA (Fig. 4F). Ubiquitination levels were quantified by densitometry and normalized to the total precipitated CFTR. Both RFFL KD and ETI treatment modestly reduced ubiquitination, and the combination of RFFL KD with ETI further decreased ubiquitination in an additive manner (Fig. 4G). These results suggest that RFFL KD and ETI independently stabilize G418-induced full-length G542X-CFTR by reducing its ubiquitination.
RFFL limits the PM expression of G542C-, G542R-, and G542W-CFTR
The nonsense CFTR mutation G542X (UGA) is known to undergo translational readthrough by G418, leading to insertion of cysteine I, tryptophan (W), or arginine I at position 542 [27–29]. These amino acid substitutions likely represent the molecular species generated by G418 treatment and may subsequently be recognized as missense mutants by the cellular PQC machinery. To test whether these G542 missense variants are targeted by RFFL-mediated PQC, we established BEAS-2B cell lines stably expressing G542C-, G542R-, or G542W-CFTR-HiBiT.
HiBiT assays showed that, consistent with a previous study [27, 28], G542C-CFTR was intrinsically expressed at the PM, whereas G542W-CFTR showed only weak PM expression, comparable to BG levels in parental BEAS-2B cells. G542R-CFTR exhibited moderate PM localization (Fig. 5A). Upon either RFFL KD or ETI treatment, PM expression of all three mutants increased, and the combination of both treatments resulted in the most pronounced enhancement (Fig. 5B). Western blotting confirmed efficient RFFL KD in each cell line (Fig. 5C) and showed that mature, fully glycosylated forms of the CFTR variants were increased by either RFFL KD or ETI treatment, with maximal effects observed when both were applied together (Fig. 5D). We also investigated the role of RNF5 and RNF185 by performing DKD. This modestly increased PM expression (Fig. 5E) and maturation of all three CFTR variants (Fig. 5F and G), although the effects were less prominent than those seen with RFFL KD alone. These findings suggest that full-length G542X-CFTR generated via G418-induced readthrough is primarily targeted by RFFL through peripheral QC pathways, thereby limiting its PM expression. RNF5 and RNF185 may additionally contribute to ER-associated PQC of these variants, further restricting their PM expression.Fig. 5. Combination of RFFL KD and ETI treatment robustly improves the PM expression of G542C-, G542R-, and G542W-CFTR. (A) Basal PM levels of G542C-, G542R-, and G542W-CFTR-HiBiT(Ex) in BEAS-2B cells were measured by HiBiT assay (n = 12). (B) PM levels of G542C-, G542R-, and G542W-CFTR-HiBiT(Ex) in BEAS-2B cells transfected with 25 nM dsiNC or dsiRFFL and treated with ETI (1 µM ELX, 3 µM TEZ, 1 µM IVA) for 2 days at 37 °C (n = 6). BG level is shown as a reference in (A-B). (C, D) Western blot analysis of HBH-G542C-, G542R-, and G542W-CFTR-3HA in BEAS-2B Tet-on cells transfected with 25 nM dsiRFFL and treated with 1 µg/mL Dox and ETI for 2 days at 37 °C. RFFL was detected with anti-RFFL antibody (C), and CFTR with Neutravidin-HRP (D). Ponceau S staining was used as a loading control. Bands B and C indicate immature and mature CFTR, respectively. (E) Effects of siRNF5/185 in combination with ETI on PM levels of G542C-, G542R-, and G542W-CFTR-HiBiT(Ex) in BEAS-2B cells were measured as in (B) (n = 6). (F, G) Effects of siRNF5/185 in combination with ETI on HBH-G542C-, G542R-, and G542W-CFTR-3HA expression in BEAS-2B Tet-on cells were measured as in (D). RNF5/185 KD was confirmed (F). Data represent mean ± SD. Statistical significance was determined by two-way ANOVA. The interaction between KD of RFFL or RNF5/185 and ETI is shown as P_int_. **p < 0.01, ***p < 0.001, ****p < 0.0001
Effects of RFFL KD and ETI on other class I CFTR mutants with TRIDs
To assess whether the susceptibility to PQC is a general feature of TRID-induced full-length CFTR class I mutants, we established BEAS-2B cell lines stably expressing representative variants other than G542X. W1282X, R553X, and R1162X are common PTC mutations in CF patients, all caused by the UGA stop codon. W1282X-CFTR undergoes partial maturation and can reach the PM to some extent, whereas R1162X-CFTR shows little to no detectable PM expression [43]. The R553X mutation results from a C > T substitution in exon 11, introducing a PTC that produces a truncated, non-functional CFTR protein unresponsive to CFTR modulators [44, 45]. HiBiT assays confirmed that W1282X-CFTR was detectable at the PM under basal conditions, whereas R553X- and R1162X-CFTR were not (Fig. 6A). Among the TRIDs tested, only G418 modestly increased PM expression of all three mutants, while CC-90009 and SRI-41315 had no significant effect (Fig. 6B-D). ETI treatment alone enhanced PM expression of W1282X-CFTR but not R553X- or R1162X-CFTR. Notably, combined treatment with G418 and ETI significantly increased the PM levels of all three variants (Fig. 6B-D), supporting the idea that, like G542X-CFTR, G418-induced full-length class I CFTR mutants retain structural defects and are targeted by PQC.Fig. 6. Combination effects of TRIDs and ETI treatment on PM expression of various CFTR class I mutants. (A) Basal PM levels of W1282X-, R553X-, and R1162X-CFTR-HiBiT(Ex) in BEAS-2B cells were measured by HiBiT assay (n = 12). (B–D) PM levels of W1282X- (B, n = 3), R553X- (C, n = 6), or R1162X-CFTR-HiBiT(Ex) (D, n = 6) in BEAS-2B cells treated with G418 for 4 days, or with CC-90009 or SRI-41315 for 2 days at the indicated concentrations, with or without ETI (1 µM ELX, 3 µM TEZ, 1 µM IVA) at 37 °C. As indicated, 0.1 µM CC-90009 or 5 µM SRI-41315 were administered for 4 days. The BG level is shown as a reference in panels (A–D). Data represent mean ± SD. Statistical significance was assessed using one-way ANOVA with Dunnett’s multiple comparisons test (B-D). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
To evaluate whether class I CFTR mutants are subject to PQC mediated by RFFL and RNF5/185, we examined the effects of their KD on PM expression. As expected, RFFL KD markedly increased the PM levels of W1282X-, R553X-, and R1162X-CFTR, particularly in the presence of G418 (Fig. 7A-C). This enhancement was further amplified by ETI treatment. KD of RNF5/185 also elevated the PM expression of these mutants under the same conditions, although the effect was less pronounced than that of RFFL KD (Fig. 7D-F). Collectively, these findings highlight a shared mechanism whereby diverse full-length CFTR class I mutants generated by G418 readthrough are targeted for degradation by RFFL- and RNF5/185-mediated PQC, thereby limiting the therapeutic benefit of TRID-CFTR modulator combinations (Fig. 7G).Fig. 7. Combination of RFFL KD and ETI treatment enhances TRID-mediated PM expression of multiple CFTR class I mutants. (A–C) PM levels of W1282X- (A, n = 6), R553X- (B, n = 3), and R1162X-CFTR-HiBiT(Ex) (C, n = 3) in BEAS-2B cells treated with dsiRFFL (25 nM), G418 (250 µM) for 4 days, and ETI (1 µM ELX, 3 µM TEZ, 1 µM IVA) for the last 2 days at 37 °C. (D–F) PM levels of W1282X- (D, n = 6), R553X- (E, n = 3), and R1162X-CFTR-HiBiT(Ex) (F, n = 3) in BEAS-2B cells treated with siRNF5 and siRNF185 (50 nM each), along with G418 and ETI as above. BG level is shown as a reference in panels (A-F). Data represent mean ± SD. Statistical significance was assessed by two-way ANOVA (A-F). The interaction between KD of RFFL or RNF5/185 and ETI is shown as P_int_. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant. (G) Schematic model of the predicted PQC mechanisms targeting TRID-induced full-length CFTR class I mutant proteins
Discussion
In this study, we demonstrated that Ub E3 ligases involved in the PQC of misfolded CFTR also contribute to the elimination of full-length CFTR class I mutants generated by G418 readthrough, including G542X-CFTR, the second most common CF-causing mutation. TRIDs such as G418 suppress PTCs to produce full-length CFTR, while modulators such as ETI enhance folding and promote PM expression as functional channels. However, our results strongly suggest that these TRID-induced full-length CFTR proteins retain structural defects and are therefore targeted by PQC pathways in both the ER and post-ER compartments, similar to class II mutants such as F508del-CFTR.
Previous studies have shown that TRIDs can insert alternative amino acids such as cysteine I, tryptophan (W), or arginine I at position 542 of G542X-CFTR [27–29], effectively converting the nonsense mutation into missense variants (G542C, G542W, and G542R). These missense proteins are likely ubiquitinated by RNF5/185 in the ER and degraded via the ERAD pathway, similar to F508del-CFTR [36, 46, 47]. Although ETI treatment can partially bypass this ERQC checkpoint by stabilizing folding, TRID-induced CFTR proteins remain highly vulnerable to peripheral QC mediated by RFFL. This mirrors the fate of F508del-CFTR, which retains residual conformational defects even after ETI treatment, undergoes enhanced ubiquitination compared with the wild-type protein, and is rapidly cleared from the PM by RFFL-dependent degradation [30, 33]. Supporting this model, KD of RNF5/185 or RFFL increased the expression and PM levels of G542C-, G542W-, and G542R-CFTR, with effects further amplified by ETI. Notably, RFFL KD produced a stronger increase in PM expression than RNF5/185 KD, suggesting that TRID-induced missense CFTR proteins are primarily eliminated through RFFL-mediated peripheral QC. Thus, RFFL inhibition preferentially enhances the PM expression of functional G542X-CFTR when combined with G418 and ETI. Previous studies using cell-line models reported that combination therapy with G418 and CFTR modulators such as elexacaftor/tezacaftor (ELX/TEZ) restored G542X-CFTR function to only ~ 10% of wild-type levels [17, 24]. RFFL KD has the potential to substantially augment this limited rescue, offering a strategy to improve the therapeutic benefit of TRID-modulator combination therapy.
We showed that RFFL, and to a lesser extent RNF5/185, contribute to the elimination of G418-induced full-length W1282X-, R553X-, and R1162X-CFTR, in addition to G542X-CFTR. Previous studies have reported that G418 promotes the insertion of cysteine I, tryptophan (W), or leucine (L) at the PTC of W1282X-CFTR [29], and arginine I, cysteine I, or tryptophan (W) at the PTC of R1162X-CFTR [27]. Although the precise amino acids inserted at the PTC of R553X-CFTR remain undefined, our findings suggest a similar mechanism, since ETI treatment alone or in combination with RFFL or RNF5/185 KD improved the PM levels of R553X-CFTR after G418 treatment, comparable to the effects observed with G542X-, R1162X-, and W1282X-CFTR. Together, these results support a general principle: TRID-induced full-length CFTR class I mutants are subject to PQC mediated by specific E3 ligases, analogous to the degradation of misfolded missense variants such as F508del-CFTR.
In this study, we did not observe clear effects of CC-90009 or SRI-41315 on G542X-CFTR, in contrast to previous reports [21, 22]. CC-90009 is an eRF3a degrader that inhibits nonsense-mediated mRNA decay (NMD) by modulating the SURF (SMG1–UPF1–eRF1–eRF3) complex [23]. Its effect has primarily been reported in endogenous CFTR class I mutant-expressing cells, such as patient-derived nasal epithelial cells [22]. By contrast, the class I CFTR mutants we used were expressed from heterologous cDNA in BEAS-2B cells, which lack introns and are therefore not subject to exon junction complex (EJC)-dependent NMD [48]. SRI-41315 reduces the abundance of the termination factor eRF1 (21), and although it has been proposed to promote readthrough, our data suggest its effect is mainly mediated by NMD inhibition. This would explain why, like CC-90009, SRI-41315 had no effect in our cDNA-based model system. The reported synergistic effect of SRI-41315 or CC-90009 with G418 [21, 22] also supports the idea that their primary action is NMD suppression, distinct from G418’s direct readthrough activity. Although our cell models are less susceptible to NMD, which limits the ability to capture such effects, they offer the advantage of allowing a more direct evaluation of translational readthrough efficiency and protein expression. CC-90009 and SRI-41315 unexpectedly attenuated the effect of ETI on W1282X-CFTR. The mechanism underlying this antagonistic effect remains unclear. Although no significant reduction in cell numbers was observed under our experimental conditions, the possibility of subtle cytotoxic effects cannot be excluded. In addition, degradation of eRF3a or eRF1, the respective targets of CC-90009 and SRI-41315, may alter CFTR biosynthesis, translational efficiency, or protein stability, thereby counteracting the beneficial effects of ETI.
G418 and other aminoglycoside antibiotics, such as gentamicin, have not progressed as viable clinical readthrough therapies because of off-target toxicities, including nephrotoxicity and ototoxicity in humans [49]. ELX-02 (NB-124) is a next-generation aminoglycoside derivative designed to selectively bind eukaryotic ribosomes [50], thereby reducing toxicity and improving readthrough efficiency compared with conventional aminoglycosides [51]. While the present study focused on G418, future work will be required to determine whether RFFL inhibition also enhances the efficacy of ELX-02. Importantly, if RFFL inhibition can potentiate the activity of readthrough agents, it may allow therapeutic benefit to be achieved at lower TRID concentrations. The modest efficacy of ELX-02 observed in Phase 2 trials was attributed in part to insufficient drug concentrations in the respiratory tract, a limitation that RFFL inhibition could help overcome. This study used an airway epithelial cell line with exogenous CFTR expression. Although efficacy was validated across multiple model systems, future evaluation in primary nasal epithelial cells from CF patients carrying endogenous CFTR class I mutations will be critical. Moreover, the strategy of combining TRIDs and ETI with PQC inhibition may extend beyond CF, providing a therapeutic approach for other genetic diseases caused by PTC mutations in membrane proteins [52–56]. Because many membrane proteins are subject to PQC pathways similar to CFTR, this approach has broad translational potential.
Conclusions
TRID-induced full-length CFTR class I mutants are eliminated by PQC pathways mediated primarily by the E3 Ub ligase RFFL. Inhibiting these ligases prevents degradation, thereby stabilizing the rescued proteins at the PM and enhancing the efficacy of TRID–ETI combination therapy.
Supplementary Information
Below is the link to the electronic supplementary material.Supplementary file1 (PDF 14 KB)