RNA Interference Screen to Identify Kinases That Suppress Rescue of ΔF508-CFTR*

Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene encoding the Cystic fibrosis transmembrane conductance regulator (CFTR). ΔF508-CFTR, the most common disease-causing CF mutant, exhibits folding and trafficking defects and is retained in the endoplasmic reticulum, where it is targeted for proteasomal degradation. To identify signaling pathways involved in ΔF508-CFTR rescue, we screened a library of endoribonuclease-prepared short interfering RNAs (esiRNAs) that target ∼750 different kinases and associated signaling proteins. We identified 20 novel suppressors of ΔF508-CFTR maturation, including the FGFR1. These were subsequently validated by measuring channel activity by the YFP halide-sensitive assay following shRNA-mediated knockdown, immunoblotting for the mature (band C) ΔF508-CFTR and measuring the amount of surface ΔF508-CFTR by ELISA. The role of FGFR signaling on ΔF508-CFTR trafficking was further elucidated by knocking down FGFRs and their downstream signaling proteins: Erk1/2, Akt, PLCγ-1, and FRS2. Interestingly, inhibition of FGFR1 with SU5402 administered to intestinal organoids (mini-guts) generated from the ileum of ΔF508-CFTR homozygous mice resulted in a robust ΔF508-CFTR rescue. Moreover, combination of SU5402 and VX-809 treatments in cells led to an additive enhancement of ΔF508-CFTR rescue, suggesting these compounds operate by different mechanisms. Chaperone array analysis on human bronchial epithelial cells harvested from ΔF508/ΔF508-CFTR transplant patients treated with SU5402 identified altered expression of several chaperones, an effect validated by their overexpression or knockdown experiments. We propose that FGFR signaling regulates specific chaperones that control ΔF508-CFTR maturation, and suggest that FGFRs may serve as important targets for therapeutic intervention for the treatment of CF.

Cystic fibrosis (CF) 1 is a pleiotropic disease caused by an abnormal ion transport in the secretory epithelia lining the tubular organs of the body such as lungs, intestines, pancreas, liver, and male reproductive tract. In the airways of CF patients, reduced Cl Ϫ and bicarbonate secretion caused by lack of functional Cystic fibrosis transmembrane conductance regulator (CFTR) on the apical surface, and hyper-absorption of Na ϩ because of elevated activity of ENaC (1), lead to a dehydration of the airway surface liquid (ASL). This reduces the viscosity of the mucus layer and the deposited layer of thickened mucus creates an environment that promotes bacterial colonization, which eventually leads to chronic infection of the lungs and death (2,3).
CFTR is a transmembrane protein that functions as a cAMP-regulated, ATP-dependent Cl Ϫ channel that also allows passage of bicarbonate through its pore (4,5). It also possesses ATPase activity important for Cl Ϫ conductance (6,7). The CFTR structure is predicted to consist of five domains: two membrane spanning domains (MSD1, MSD2), each composed of six putative transmembrane helices, two nucleotide binding domains (NBD1, NBD2), and a unique regulatory (R) region (8).
More than 1900 CFTR mutations have been identified to date (www.genet.sickkids.on.ca/cftr). The most common mutation is a deletion of phenylalanine at position 508 (⌬F508 or ⌬F508-CFTR) in NBD1 (9). The ⌬F508 mutation causes severe defects in the processing and function of CFTR. The protein exhibits impaired trafficking from the endoplasmic reticulum (ER) to the plasma membrane (PM), impaired intramolecular interactions between NBD1 and the transmembrane domain, and cell surface instability (10 -15). Nevertheless, the ⌬F508 defect can be corrected, because treating cells expressing ⌬F508-CFTR with low temperature or chemical chaperones (e.g. glycerol) can restore some surface expression of the mutant (11,16). blasticidin, and 50 g/ml zeocin, at 37°C, 5% CO 2 in humidified atmosphere.
The Cellomics halide-exchange assay was performed as described previously (29). Briefly, 5 ϫ 10 4 ⌬F508-CFTR cells (i.e. 293MSR-GT cells stably coexpressing eYFP(H148Q/I152L) and ⌬F508-CFTR) per well were seeded in 96-well plates. The next day the cells were transfected with esiRNA duplexes from the library (final concentration 40 nM), luciferase (nonsilencing control), EG5 (transfection control), or AHA1 esiRNA (assay control), using Lipofectamine 2000. Medium was changed 6 h after transfection, and the cells were placed at 37°C, 5% CO 2 for 72 h. The 96-well transfection protocol was optimized using EG5 (KIF11) esiRNA as a transfection control. The transfection was considered successful if more than 80% of ⌬F508-CFTR cells exhibited round-shape phenotype 72 h post-transfection (see "Results"). After (Thermo Fisher) and a modified Target Activation algorithm, objects (individual cells or sometimes clusters of cells) were defined by eYFP(H148Q/I152L) fluorescence intensity, and the fluorescence quenching over 24-s time course at 30°C, 5% CO 2 , was recorded. Valid wells contained between 70 and 300 objects per field (single field per well). Genes that displayed a difference in the YFP fluorescence intensity (between FIG-stimulated sample and nonsilencing control) lower than 0.09 were rejected after the first two runs of the screen. This cut-off value equaled three times the standard deviation from the mean value of the control (AHA1). The rest of the esiRNA duplexes (56 genes) were subjected to the third run of the screen. Twenty top hits of the screen were subjected to further validation of ⌬F508-CFTR rescue by functional assay (Cellomics ArrayScan VTI platform) immunoblotting and ELISA following shRNA-mediated knockdown.
For drug combination testing, 8 ϫ 10 4 ⌬F508-CFTR cells (i.e. 293MSR-GT cells stably coexpressing eYFP(H148Q/I152L) and ⌬F508-CFTR) per well were seeded in 96-wells plates. The next day, the cells were treated with either SU5402ϩVX809 or AZD4547ϩ VX809 with concentration ranging from 1 M to 10 M. The cells were then incubated at 37°C, 5% CO 2 for 48 h, and analyzed by the Cellomics halide-exchange assay, as above.
Validation of the esiRNA Screen-Cellomics Analysis Following shRNA Knockdown-Prior to the Cellomics halide-exchange assay ⌬F508-CFTR cells (stably expressing eYFP(H148Q/I152L)) were transfected with shRNA constructs targeting the identified genes or luciferase (nonsilencing control), using Lipofectamine 2000, according to the manufacturer's instructions. Medium was changed 6 h after transfection and ⌬F508-CFTR cells were placed at 37°C, 5% CO 2 . Forty-eight hours after transfection the cells were incubated with media containing puromycin (5 g/ml, 3 days). Cellomics halide-exchange assay was performed as described above, using Cellomics ArrayScan VTI platform (data from three fields per well). A total number of 133 shRNA clones was screened (multiple shRNA clones per gene) (supplemental Table S2). Knockdown efficiency was validated by two-step RT-qPCR as described previously (29). Briefly, total RNA was isolated using the RNeasy 96 kit (Qiagen, Venlo, Netherlands), and cDNA was prepared using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Real-time PCR reactions were performed using Platinum® SYBR® Green qPCR-SuperMix-UDG (Invitrogen) and CFX96 Real-Time System (BioRad). Primers were obtained from Integrated DNA Technologies. For standard curves, real-time PCR was performed on a fivefold dilution series of pooled cDNA.
ELISA-⌬F508-CFTR 3HA cells were transfected with shRNA constructs (pGIPZ) for the identified genes or nonsilencing control, using Lipofectamine 2000, according to the manufacturer's instructions. Forty-eight hours after transfection the cells were incubated with media containing puromycin (5 g/ml, 3 days). The cells were then biotinylated with 0.5 mg/ml biotin in PBS (15 min), washed with ice-cold PBS and lysed. To capture CFTR or ⌬F508-CFTR, 50 g of total lysate protein (per well) were incubated with anti HA antibody (1:400) in 96-well plate, for 2 h at 4°C. The plates were then washed with PBST (PBS ϩ 0.05% Tween) and SA-HRP (1:1000) was added in ELISA buffer (PBST ϩ 0.5% BSA) into each well (20 min). After washing, the plates were developed with TMB substrate. The reaction was stopped with 1N H 2 SO 4 and the plates were read at 450 nm.
Salivary Secretion Assay (SSA)-The salivary secretion assay, described previously (31), was modified as follows. Male ⌬F508 mice (CFTR tm1Eur on a 129/FVB background) and their wild-type littermates (kindly provided by Dr. C. Bear) of 9 -12 weeks were intra-peritoneally injected with DMSO or SU5402 (dissolved in DMSO at the concentration of 6 mg/ml) at 25 mg/kg body weight, every day for 1 week. The mice were weighed daily and the dosages adjusted accordingly. The mice were then anesthetized by inhaling isoflurane until the end of the procedure. Cholinergic antagonist, atropine (1 mM, 50 l) was subcutaneously injected into the right cheek to block potential cholinergic stimulation of the salivary gland. A small strip of filter paper was placed against the injected cheek, for 4 min. Isoprenaline (10 mM, 37.5 l) was subsequently injected in the same spot to stimulate an adrenergic secretion of saliva (time 0). Filter strips (preweighed in an Eppendorf tube) were replaced every 5 min, over a period of 30 min. All six filter strips were weighed at the end of the collection and the results were normalized relative to mg/g body weight. All animal work was done in accordance with SickKids Institutional guidelines and approval of the Animal Care Committee.
Chaperone Array Screen-Human Bronchial Epithelial (HBE) cells from ⌬F508/⌬F508-CFTR transplant patients (P2 cells) were obtained from the University of Iowa Cell Culture Facility and grown on collagen-coated permeable millicell inserts. The cells were treated with DMSO (control), 1 M or 10 M SU5402 for 48 h prior to RNA extraction. Total RNA was extracted using the PureLink RNA Mini Kit (Invitrogen) and cDNA was synthesized from 1 g of mRNA using the High capacity cDNA reverse transcription kit (Applied Bioscience) according to the manufacturer's instructions. Array analysis was performed using the RT 2 Profiler™ PCR Array Human Heat Shock Proteins & Chaperones kit (Qiagen). mRNA expression levels were determined relative to actin, GAPDH and B2M using the ⌬C t method. Changes in chaperone expression level relative to DMSO control were determined using the ⌬⌬C t method. The chaperone array experiment was performed three times and average values are shown in a heat map.
Validation of the Chaperone Array Hits-7 ϫ 10 5 ⌬F508-CFTR 3HA cells per well were seeded in a 6-well plate format. The next day the cells were transfected with the clones for the analyzed chaperone genes (shRNA or overexpression) or luciferase control, using PolyJet™ DNA In Vitro Transfection Reagent according to the manufacturer's instructions. Forty-eight hours post-transfection, the cells that were transfected with shRNA were further incubated with media containing puromycin (5 g/ml, 3 days). The cells that were transfected with the chaperone overexpression clones were biotinylated, and ELISA was performed as described above.
HSF1 Experiments-The pcDNA3.1(eYFP H148Q/I152L) plasmid containing the wild-type HSF1 was used to construct a constitutively active mutant of HSF1 (34) using site directed mutagenesis consisting of one-step PCR with two overlapping internal primers at the mutagenic site. The internal primers used were 5ЈGAA-CGACAGTGGCTCAGCACATGGGCGCCCATCTTCCGTGGAC3Ј and 5ЈGTCCACGGAAGATGGGCGCCCATGTGCTGAGCCACTGTCGTT-C3Ј. DNA sequencing was performed to verify the constructs. 7 ϫ 10 5 ⌬F508-CFTR 3HA cells per well were seeded in 6-well plates. The next day the cells were transfected with the constructs for wild-type HSF1, mutant HSF1, or luciferase control using PolyJet™ DNA In Vitro Transfection Reagent, according to the manufacturer's instructions. Forty-eight hours after transfection, the cells were biotinylated, and ELISA was performed as described above.

Identification of Kinases and Associated Signaling Proteins
That Suppress Rescue of ⌬F508-CFTR-Delineation of pathways and proteins that prevent rescue of ⌬F508-CFTR is important for the identification of drugs that target these pathways. Our group previously developed a high-content functional screen to identify ⌬F508-CFTR correctors (and poten-tiators) in multiple individual cells simultaneously, using Cellomics KineticScan KSR and ArrayScan VTI platforms (28,29). The kinome esiRNA screen presented in this study complements the small-molecule kinase inhibitor screen described previously (29).
We screened a library of esiRNA duplexes targeting 759 different kinases and associated proteins (supplemental Table  S1). Because esiRNA transfection efficiency is critical to performing this screen, and the esiRNA molecules do not carry a selection marker, the 96-well transfection protocol was optimized using EG5 (KIF11) esiRNA as a transfection control added to parallel wells on the same plate. Knockdown of EG5, a kinesin-related motor protein, leads to mitotic arrest and rounded-up phenotype of the cells. The transfection was considered successful if more than 80% of ⌬F508-CFTR cells exhibited round-shape phenotype 72 h post-transfection.
In the Cellomics halide-exchange assay the fluorescence quenching of YFP, corresponding to Cl Ϫ /I Ϫ exchange via ⌬F508-CFTR, was quantified in cells stably expressing ⌬F508-CFTR and transfected with esiRNA duplexes from the library. Fig. 1 depicts several representative "hit" suppressors that when knocked-down exhibited various degrees of correction of the ⌬F508-CFTR defect. The complete list of the hits, defined as those exhibiting a difference in average fluorescence intensity ⌬FI avg (between Forskolin-IBMX-Genistein  Table I. The cut-off value of 9% (0.09) was chosen as it equals three times the standard deviation from the mean value of the control (AHA1). Interestingly, the "hit" list reveals novel suppressors involved in Ras/ Raf/MEK/Erk and PI3K/Akt signaling pathways, along with the kinases previously identified by our small-molecule kinome inhibitor screen (e.g. FGFR1, Raf) (29), which further validates our approach.
As both the transfection and knockdown efficiency can influence the level of rescue observed in our esiRNA screen, we further validated the hits (and possible off-target effects) by carrying out an independent high-content screen, using shRNA.
Validation of the Hits-To validate the effect of knockdown of the suppressor genes on ⌬F508-CFTR chloride channel activity with esiRNA, we tested the top "hits" with shRNA. For this we screened 133 shRNA clones targeting 20 suppressors identified in our kinome esiRNA screen (supplemental Table  S2). ⌬F508-CFTR cells transfected with shRNA constructs for the suppressor genes or luciferase (nonsilencing control) were analyzed in the Cellomics halide-exchange assay. In parallel, qPCR was performed to determine the knockdown efficiency of these constructs. Although the shRNA clones yielded varied degrees of knockdown, most of them resulted in more than 50% knockdown of target genes (supplemental Table  S2), and in general, the degree of ⌬F508-CFTR rescue correlated with knockdown efficiency. In the case of MET and BRAF genes, cell death was observed upon knockdown higher than 60 -70% and, therefore, shRNA clones that resulted in the best rescue exhibited knockdown of 30% (B-Raf)-60% (MET).
The results of the shRNA screen corroborate the esiRNA hits ( Fig. 2 and Table II). Out of 20 analyzed genes, 14, when knocked-down by shRNA, produced reproducible rescue of ⌬F508-CFTR function (⌬FI avg of 12-30%). These included genes encoding the receptor Tyr kinases FGFR1 and MET, as well as B-Raf and the MAPK kinase kinase MAP3K13. Exam-ples of other hits are the receptor interacting protein kinase RIPK4, the sedoheptulokinase SHPK, the cyclin-dependent kinase CDK10, the suppressor of cytokine signaling SOCS1, and the PKA regulatory subunit PRKAR2B. The knockdown of six genes, PANK1, NEK10, CLK3, DTYMK, ERN1, and CAMK2B, led to a lower degree of rescue (⌬FI avg Ͻ 10%) (Table II).
To further validate the hits, we analyzed maturation of ⌬F508-CFTR in response to knockdown of the identified suppressors, using immunoblotting for the mature (Band C) protein. supplemental Fig. S1B shows that knockdown of most of the analyzed suppressor genes led to at least 10% increase of band C/B ratio relative to nonsilencing control, except PRKAR2B and DTYMK, where only 7 and 9% increase of band C/B ratio was observed, respectively.
Akt, PLC-␥1, and FRS2) led to an increase in ⌬F508-CFTR channel activity in the halide-exchange assay (⌬FI avg Յ 17%), as well as an appearance of band C in the immunoblot (the increase of band C/B ratio of 25-75%), which further confirms our hypothesis that FGFR signaling normally inhibits rescue of ⌬F508-CFTR.
Interestingly, inhibition of FGFR1 with the small molecule compound SU5402 administered to ⌬F508-CFTR homozy-gous mice resulted in partial ⌬F508-CFTR rescue, as shown by an increase in saliva secretion (supplemental Fig. S3), a surrogate "sweat test" assay in mice (35). As salivary secretion is often sex dependent, only male mice were chosen for these experiments (35). Our results indicate that treatment of the ⌬F508-CFTR mice with SU5402 restores the saliva secretion level to ϳ10% of that observed for the wild-type CFTR mice, which suggests that indolinone derivatives (such as SU5402) could have therapeutic benefits to CF.
To investigate the effect of FGFR1 inhibition in a highly relevant tissue, we tested the effect of SU5402 on rescue of ⌬F508-CFTR function in intestinal organoids (mini-guts) derived from the ileum of ⌬F508-CFTR mice (36). As seen in Fig.  5, SU5402 (10 M) treatment of these organoids resulted in rescue level equivalent to temperature rescue, and ϳ85% of WT-CFTR (at 50 min) (Fig. 5C, 5D). This rescue was equivalent or stronger than that observed with VX-809 (3 M) (Fig. 5D).
Given the role of FGFR1 suppression in rescue of ⌬F508-CFTR, we reasoned that FGFR1 (and its downstream signaling) might inhibit chaperones that promote maturation of ⌬F508-CFTR, or promote suppressors of this maturation. To identify these downstream chaperones, we treated ⌬F508/ ⌬F508-CFTR HBE cells from lung explants of two CF patients with 1 or 10 M SU5402 for 48 h. Our parallel testing of cells from these two patients for SU5402 sensitivity in an Ussing chamber revealed that they exhibit differential sensitivity to this compound at 1 M; We thus termed these HBEs as responder and poor responder (Fig. 6A). mRNA isolated from these SU5402-treated (or not) cells incubated with chaperone arrays revealed a strikingly different response pattern in the responder versus poor responder (Fig. 6B), underscoring the variability in drug response of patients with the same CF mutation (⌬F508/⌬F508). Analysis of the stronger responder identified several chaperones and regulatory proteins with enhanced expression after treatment (e.g. HSPA 4, 1A, 5, 14, 9; DNAJA2; HSF 1, 4; CRYAB; CCT 3, 5, 7, 6B; HSPE1; SERPINH1), and a few with reduced expression (Bag4; DNAJ C1, C14, C21; TOR1A) (supplemental Table S3). The effect of these chaperones on cell surface expression of ⌬F508-CFTR following their overexpression or knockdown with shRNA was then validated using an ELISA assay (Fig. 7A, 7B). Curiously, several of the chaperones exhibiting enhanced expression following SU5402 treatment are known effectors of the transcription factor HSF1 (see below), and accordingly, constitutively active HSF1 led to elevated cell surface expression of ⌬F508-CFTR relative to nonactivated HSF1 (supplemental Fig. S4).
Additive Effect of FGFR Inhibitors and VX-809 on Rescue of ⌬F508-CFTR-Clinical tests of VX-809 administered alone to ⌬F508-CFTR patients have not yielded a significant improvement in lung function of these patients (27), although its combination with VX-770 yielded a small improvement  3. Effect of shRNA-mediated knockdown of the hit genes on surface expression of ⌬F508-CFTR. 293MSR-GT cells stably expressing ⌬F508-CFTR (bearing a 3HA tag at the ectodomain) were transfected with shRNA for the analyzed genes or nonsilencing control (as indicated), grown at 37°C for 48 h, selected on puromycin, and quantitation of surface expression of ⌬F508-CFTR was carried out by ELISA assay. SU5402 represents cells treated with SU5402 and serves as a positive control. Data are mean Ϯ S.E. from three independent experiments.
Increase in band C/B ratio relative to non-silencing control anti vinculin L u c i f e r a s e F G F R 1 P L C -γ 1 KD stands for knockdown efficiency (%). I, Immunoblot analysis for maturation of ⌬F508-CFTR in 293MSR-GT cells (stably expressing ⌬F508-CFTR) transfected with shRNA for FGFR1, 2, and 3, Erk1 and 2, Akt, PLC-␥1, or luciferase (nonsilencing control). 27°C represents temperature rescue of ⌬F508-CFTR at 27°C, and WT CFTR represents wild-type CFTR. The 27°C and WT CFTR lanes were loaded with a half of the amount of protein in comparison to the other analyzed samples. (J) Quantitation of rescue (increase in the band C/B ratio) of ⌬F508-CFTR, following shRNA-mediated knockdown of the indicated signaling proteins.
FIG. 5. Rescue of ⌬F508-CFTR in intestinal organoids from ⌬F508/⌬F508 mice. A-D, Rescue of ⌬F508-CFTR with SU5402 analyzed in intestinal organoids from ⌬F508/⌬F508 mice by organoids swelling: A and B, Intestinal organoids derived from crypts isolated from the terminal ileum of ⌬F508 mice (and WT littermate controls) were treated (at 37°C) with 5 M forskolin for 50 min to activate CFTR, leading to swelling via chloride efflux and lumenal fluid accumulation by WT-CFTR but not ⌬F508-CFTR. Scale bars ϭ 50 m. C, ⌬F508 organoids do not exhibit increased surface area following forskolin treatment, but swelling can be rescued at low temp: As in B, except one batch of ⌬F508 organoids were preincubated at 27°C for 24 h prior to assay, revealing temp. rescue. D, Treatment with SU5402 or VX-809 partially rescued swelling in ⌬F508-CFTR organoids. ⌬F508 organoids were pretreated with VX-809 (3 M; green) or SU-5402 (10 M; red), administered to the organoid culture media. Treatment with either compound (at 37°C) partially rescued CFTR-mediated swelling in the ⌬F508 organoids, a rescue augmented by combining both SU5402 and VX-809. Error bars for panels B-D represent mean Ϯ S.D. n ϭ 20 -30 organoids per treatment. Forsk: Forskolin. DISCUSSION Our previous identification, using small-molecule kinase inhibitors, of several signaling cascades (e.g. Ras/Raf/MEK/Erk, Wnt/GSK-3␤, PI3K/Akt/mTOR, TAK1/p38) (29) that regulate ⌬F508-CFTR trafficking and maturation prompted us to perform a more systematic screen for all human kinases and select associated signaling proteins that may regulate ⌬F508-CFTR rescue. We therefore performed an esiRNA human kinome screen using our Cellomics high-content assay to search for suppressors that block maturation of ⌬F508-CFTR and hence, when knocked-down, lead to rescue of this mutant. The top hits (20 genes) were validated with another RNAi technology, shRNA, to ensure that the rescue observed in the original esiRNA screen was not caused by off-target effects. In parallel, shRNA constructs were used to test the effect of knockdown of these genes on ⌬F508-CFTR maturation and surface expression by immunoblotting and ELISA, respectively. The knockdown of several of the analyzed genes (e.g. FGFR1, SHPK, MAP3K13, DUSP22, CDK10, IPMK, and PANK4) led to a substantial rescue of ⌬F508-CFTR activity in the halide-exchange assay, and a corresponding robust increase in cell surface amount of ⌬F508-CFTR.
The results of both RNAi screens (esiRNA and shRNA) lend further support to the results obtained in our small-molecule kinase inhibitor screen (29). Several of the hits are kinases that are either direct targets, or are involved in the signaling cas-cades by the kinase inhibitors discovered in our earlier compound screen. For example, FGFR1 was the target of several ⌬F508-CFTR correctors that were previously identified in our kinase inhibitor screen (e.g. SU5402, SU6668, PD173074) (29), and in accord, its knockdown here also led to a substantial rescue of this CFTR mutant.
In this study, we show that knockdown of FGF receptors (FGFR 1, 2, and 3), or their intermediate signaling proteins such as PLC-␥1, Erk1/2, Akt, and FRS2␣, can stimulate rescue of ⌬F508-CFTR trafficking. Furthermore, our results suggest that FGFR1 inhibitors (e.g. SU5402) may prove useful in treatment of CF: The treatment of the ⌬F508-CFTR mice with SU5402 led to the increase of the isoprenaline-stimulated salivary secretion (ϳ10% of the wild-type CFTR mice level), which corresponds to a partial correction of the ⌬F508-CFTR defect, and a strong rescue of ⌬F508-CFTR with SU5402 was observed in intestinal organoids harvested from these mice. Given the importance of even a partial (10 -25%) rescue of FIG. 7. Validation of chaperone hits. ELISA analysis of cell surface expression of ⌬F508-CFTR (relative to luciferase control) expressed in 293-GT cells following, A, overexpression of the top chaperones that exhibited elevated expression following SU5402 treatment in the heat map (Fig. 6), or B, shRNA-mediated knockdown of chaperones that exhibited reduced expression in the heat map following SU5402 treatment. The extent of knockdown (two clones per gene) is shown in Supplemental Table  S3. Data in A and B are mean Ϯ S.E., n ϭ three independent experiments. ⌬F508-CFTR for improvement in CF patients' health (39,40), our findings could have significant clinical implications. Moreover, modifications of drug dose, scheduling, mode of administration, and the development of SU5402 more potent analogs, may further improve the efficacy of this (or related) compound for the treatment of CF. In addition, the additive effect of SU5402 and VX-809 on ⌬F508-CFTR rescue suggests that these compounds act on different cellular targets and hence drug combination may be useful for future CF treatment.
The results of our RNAi screen also support a negative role of the NFB signaling in the maturation of ⌬F508-CFTR. We identified RIPK4, a member of the Receptor Interacting Protein Kinase family, which is known to activate NFB (75). The elevated NFB-mediated IL8 signaling is one of the main contributors to the chronic hyper-inflammation in the CF lung (76 -78). However, how the NFB pathway inhibits maturation of ⌬F508-CFTR needs to be elucidated. We also identified SOCS1 (Suppressor of Cytokine Signaling 1) as an inhibitor of ⌬F508-CFTR rescue. SOCS proteins block STAT phosphorylation by inhibiting Janus kinases (JAKs) activity or competing with STATs for binding sites on cytokine receptors (79 -81). In support, we previously demonstrated that overexpression of STAT1 promotes the rescue of ⌬F508-CFTR (28).
Another interesting gene identified in our esiRNA screen is PRKAR2B, which encodes the RII␤ regulatory subunit of cAMP-dependent kinase PKA. Stimulation by PKA is known to promote CFTR channel activity, enhance CFTR trafficking, and to decrease CFTR endocytosis from the plasma membrane (82,83). It was shown that the cAMP-binding domain B of RII␤ subunit inhibits the PKA holoenzyme activation (84). Therefore, the knockdown of this inhibitory subunit could lead to an increase of PKA activity, which in turn promotes ⌬F508-CFTR rescue.
Our screen also identified ERN1 (Serine/threonine-protein kinase/endoribonuclease IRE1 or Inositol-requiring protein 1) and IPMK (Inositol Polyphosphate Multikinase) as suppressors of ⌬F508-CFTR rescue. ERN1/IRE1 signaling is known for its role in the unfolded protein response in the ER lumen and was reported to reduce the level of misfolded CFTR (85). IPMK is a PI3-kinase that acts as a molecular switch for Akt, inhibiting or stimulating PI3K/Akt signaling (86). PI3K/Akt cascade, along with Ras/Raf/MEK/ERK, is known to affect expression and function of numerous chaperones (87). However, the exact role of IPMK in CFTR maturation and trafficking requires further investigation.
In summary, this study has identified several novel suppressors of ⌬F508-CFTR rescue. These suppressors modulate or belong to important cellular signaling pathways such as Ras/ Raf/MEK/ERK, PI3K/Akt, p38, and NFB. The effect of receptor Tyr kinases (especially FGFRs) on ⌬F508-CFTR maturation and chaperone expression in ⌬F508/⌬F508-CFTR HBE was further elucidated. Thus, identifying proteins and their respective pathways that control rescue of ⌬F508-CFTR is a powerful approach to identify drugs that target these same proteins/pathways, and thus can provide new potential treatment avenues for CF.