Research ArticleCell biologyPulmonology Open Access | 10.1172/jci.insight.198846

Alessandra Murabito,1 Marco Mergiotti,1 Valeria Capurro,2 Alessia Loffreda,3 Mingchuan Li,1 Paola Peretto,1 Kai Ren,1 Andrea Raimondi,3 Carlo Tacchetti,3,4 Dario Diviani,5 Nicoletta Pedemonte,2 Emilio Hirsch,1,6 and Alessandra Ghigo1,6

1Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center “Guido Tarone”, University of Torino, Torino, Italy.

2UOC Genetica Medica, IRCCS Istituto Giannina Gaslini, Genova, Italy.

3Experimental Imaging Center, IRCCS Ospedale San Raffaele, Milan, Italy.

4Università Vita-Salute San Raffaele, Milan, Italy.

5Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne; Lausanne, Switzerland.

6Kither Biotech, Torino, Italy.

Address correspondence to: Alessandra Ghigo, Via Nizza 52, 10126, Torino, Italy. Phone: 39.011.670.6335; Email: alessandra.ghigo@unito.it. AM’s present address is: Candiolo Cancer Institute, FPO - IRCCS, Candiolo, Torino, Italy. AR’s present address is: Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera Italiana, Bellinzona, Switzerland. ML’s present address is: Guangzhou Institute of Cancer Research, the Affiliated Cancer Hospital & State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Guangzhou, China.

Authorship note: AM and MM contributed equally to this work.

Find articles by Murabito, A. in: PubMed | Google Scholar

1Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center “Guido Tarone”, University of Torino, Torino, Italy.

2UOC Genetica Medica, IRCCS Istituto Giannina Gaslini, Genova, Italy.

3Experimental Imaging Center, IRCCS Ospedale San Raffaele, Milan, Italy.

4Università Vita-Salute San Raffaele, Milan, Italy.

5Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne; Lausanne, Switzerland.

6Kither Biotech, Torino, Italy.

Address correspondence to: Alessandra Ghigo, Via Nizza 52, 10126, Torino, Italy. Phone: 39.011.670.6335; Email: alessandra.ghigo@unito.it. AM’s present address is: Candiolo Cancer Institute, FPO - IRCCS, Candiolo, Torino, Italy. AR’s present address is: Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera Italiana, Bellinzona, Switzerland. ML’s present address is: Guangzhou Institute of Cancer Research, the Affiliated Cancer Hospital & State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Guangzhou, China.

Authorship note: AM and MM contributed equally to this work.

Find articles by Mergiotti, M. in: PubMed | Google Scholar

1Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center “Guido Tarone”, University of Torino, Torino, Italy.

2UOC Genetica Medica, IRCCS Istituto Giannina Gaslini, Genova, Italy.

3Experimental Imaging Center, IRCCS Ospedale San Raffaele, Milan, Italy.

4Università Vita-Salute San Raffaele, Milan, Italy.

5Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne; Lausanne, Switzerland.

6Kither Biotech, Torino, Italy.

Address correspondence to: Alessandra Ghigo, Via Nizza 52, 10126, Torino, Italy. Phone: 39.011.670.6335; Email: alessandra.ghigo@unito.it. AM’s present address is: Candiolo Cancer Institute, FPO - IRCCS, Candiolo, Torino, Italy. AR’s present address is: Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera Italiana, Bellinzona, Switzerland. ML’s present address is: Guangzhou Institute of Cancer Research, the Affiliated Cancer Hospital & State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Guangzhou, China.

Authorship note: AM and MM contributed equally to this work.

Find articles by Capurro, V. in: PubMed | Google Scholar

1Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center “Guido Tarone”, University of Torino, Torino, Italy.

2UOC Genetica Medica, IRCCS Istituto Giannina Gaslini, Genova, Italy.

3Experimental Imaging Center, IRCCS Ospedale San Raffaele, Milan, Italy.

4Università Vita-Salute San Raffaele, Milan, Italy.

5Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne; Lausanne, Switzerland.

6Kither Biotech, Torino, Italy.

Address correspondence to: Alessandra Ghigo, Via Nizza 52, 10126, Torino, Italy. Phone: 39.011.670.6335; Email: alessandra.ghigo@unito.it. AM’s present address is: Candiolo Cancer Institute, FPO - IRCCS, Candiolo, Torino, Italy. AR’s present address is: Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera Italiana, Bellinzona, Switzerland. ML’s present address is: Guangzhou Institute of Cancer Research, the Affiliated Cancer Hospital & State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Guangzhou, China.

Authorship note: AM and MM contributed equally to this work.

Find articles by Loffreda, A. in: PubMed | Google Scholar

1Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center “Guido Tarone”, University of Torino, Torino, Italy.

2UOC Genetica Medica, IRCCS Istituto Giannina Gaslini, Genova, Italy.

3Experimental Imaging Center, IRCCS Ospedale San Raffaele, Milan, Italy.

4Università Vita-Salute San Raffaele, Milan, Italy.

5Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne; Lausanne, Switzerland.

6Kither Biotech, Torino, Italy.

Address correspondence to: Alessandra Ghigo, Via Nizza 52, 10126, Torino, Italy. Phone: 39.011.670.6335; Email: alessandra.ghigo@unito.it. AM’s present address is: Candiolo Cancer Institute, FPO - IRCCS, Candiolo, Torino, Italy. AR’s present address is: Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera Italiana, Bellinzona, Switzerland. ML’s present address is: Guangzhou Institute of Cancer Research, the Affiliated Cancer Hospital & State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Guangzhou, China.

Authorship note: AM and MM contributed equally to this work.

Find articles by Li, M. in: PubMed | Google Scholar

1Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center “Guido Tarone”, University of Torino, Torino, Italy.

2UOC Genetica Medica, IRCCS Istituto Giannina Gaslini, Genova, Italy.

3Experimental Imaging Center, IRCCS Ospedale San Raffaele, Milan, Italy.

4Università Vita-Salute San Raffaele, Milan, Italy.

5Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne; Lausanne, Switzerland.

6Kither Biotech, Torino, Italy.

Address correspondence to: Alessandra Ghigo, Via Nizza 52, 10126, Torino, Italy. Phone: 39.011.670.6335; Email: alessandra.ghigo@unito.it. AM’s present address is: Candiolo Cancer Institute, FPO - IRCCS, Candiolo, Torino, Italy. AR’s present address is: Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera Italiana, Bellinzona, Switzerland. ML’s present address is: Guangzhou Institute of Cancer Research, the Affiliated Cancer Hospital & State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Guangzhou, China.

Authorship note: AM and MM contributed equally to this work.

Find articles by Peretto, P. in: PubMed | Google Scholar

1Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center “Guido Tarone”, University of Torino, Torino, Italy.

2UOC Genetica Medica, IRCCS Istituto Giannina Gaslini, Genova, Italy.

3Experimental Imaging Center, IRCCS Ospedale San Raffaele, Milan, Italy.

4Università Vita-Salute San Raffaele, Milan, Italy.

5Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne; Lausanne, Switzerland.

6Kither Biotech, Torino, Italy.

Address correspondence to: Alessandra Ghigo, Via Nizza 52, 10126, Torino, Italy. Phone: 39.011.670.6335; Email: alessandra.ghigo@unito.it. AM’s present address is: Candiolo Cancer Institute, FPO - IRCCS, Candiolo, Torino, Italy. AR’s present address is: Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera Italiana, Bellinzona, Switzerland. ML’s present address is: Guangzhou Institute of Cancer Research, the Affiliated Cancer Hospital & State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Guangzhou, China.

Authorship note: AM and MM contributed equally to this work.

Find articles by Ren, K. in: PubMed | Google Scholar

1Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center “Guido Tarone”, University of Torino, Torino, Italy.

2UOC Genetica Medica, IRCCS Istituto Giannina Gaslini, Genova, Italy.

3Experimental Imaging Center, IRCCS Ospedale San Raffaele, Milan, Italy.

4Università Vita-Salute San Raffaele, Milan, Italy.

5Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne; Lausanne, Switzerland.

6Kither Biotech, Torino, Italy.

Address correspondence to: Alessandra Ghigo, Via Nizza 52, 10126, Torino, Italy. Phone: 39.011.670.6335; Email: alessandra.ghigo@unito.it. AM’s present address is: Candiolo Cancer Institute, FPO - IRCCS, Candiolo, Torino, Italy. AR’s present address is: Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera Italiana, Bellinzona, Switzerland. ML’s present address is: Guangzhou Institute of Cancer Research, the Affiliated Cancer Hospital & State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Guangzhou, China.

Authorship note: AM and MM contributed equally to this work.

Find articles by Raimondi, A. in: PubMed | Google Scholar

1Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center “Guido Tarone”, University of Torino, Torino, Italy.

2UOC Genetica Medica, IRCCS Istituto Giannina Gaslini, Genova, Italy.

3Experimental Imaging Center, IRCCS Ospedale San Raffaele, Milan, Italy.

4Università Vita-Salute San Raffaele, Milan, Italy.

5Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne; Lausanne, Switzerland.

6Kither Biotech, Torino, Italy.

Address correspondence to: Alessandra Ghigo, Via Nizza 52, 10126, Torino, Italy. Phone: 39.011.670.6335; Email: alessandra.ghigo@unito.it. AM’s present address is: Candiolo Cancer Institute, FPO - IRCCS, Candiolo, Torino, Italy. AR’s present address is: Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera Italiana, Bellinzona, Switzerland. ML’s present address is: Guangzhou Institute of Cancer Research, the Affiliated Cancer Hospital & State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Guangzhou, China.

Authorship note: AM and MM contributed equally to this work.

Find articles by Tacchetti, C. in: PubMed | Google Scholar

1Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center “Guido Tarone”, University of Torino, Torino, Italy.

2UOC Genetica Medica, IRCCS Istituto Giannina Gaslini, Genova, Italy.

3Experimental Imaging Center, IRCCS Ospedale San Raffaele, Milan, Italy.

4Università Vita-Salute San Raffaele, Milan, Italy.

5Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne; Lausanne, Switzerland.

6Kither Biotech, Torino, Italy.

Address correspondence to: Alessandra Ghigo, Via Nizza 52, 10126, Torino, Italy. Phone: 39.011.670.6335; Email: alessandra.ghigo@unito.it. AM’s present address is: Candiolo Cancer Institute, FPO - IRCCS, Candiolo, Torino, Italy. AR’s present address is: Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera Italiana, Bellinzona, Switzerland. ML’s present address is: Guangzhou Institute of Cancer Research, the Affiliated Cancer Hospital & State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Guangzhou, China.

Authorship note: AM and MM contributed equally to this work.

Find articles by Diviani, D. in: PubMed | Google Scholar

1Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center “Guido Tarone”, University of Torino, Torino, Italy.

2UOC Genetica Medica, IRCCS Istituto Giannina Gaslini, Genova, Italy.

3Experimental Imaging Center, IRCCS Ospedale San Raffaele, Milan, Italy.

4Università Vita-Salute San Raffaele, Milan, Italy.

5Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne; Lausanne, Switzerland.

6Kither Biotech, Torino, Italy.

Address correspondence to: Alessandra Ghigo, Via Nizza 52, 10126, Torino, Italy. Phone: 39.011.670.6335; Email: alessandra.ghigo@unito.it. AM’s present address is: Candiolo Cancer Institute, FPO - IRCCS, Candiolo, Torino, Italy. AR’s present address is: Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera Italiana, Bellinzona, Switzerland. ML’s present address is: Guangzhou Institute of Cancer Research, the Affiliated Cancer Hospital & State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Guangzhou, China.

Authorship note: AM and MM contributed equally to this work.

Find articles by Pedemonte, N. in: PubMed | Google Scholar |

1Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center “Guido Tarone”, University of Torino, Torino, Italy.

2UOC Genetica Medica, IRCCS Istituto Giannina Gaslini, Genova, Italy.

3Experimental Imaging Center, IRCCS Ospedale San Raffaele, Milan, Italy.

4Università Vita-Salute San Raffaele, Milan, Italy.

5Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne; Lausanne, Switzerland.

6Kither Biotech, Torino, Italy.

Address correspondence to: Alessandra Ghigo, Via Nizza 52, 10126, Torino, Italy. Phone: 39.011.670.6335; Email: alessandra.ghigo@unito.it. AM’s present address is: Candiolo Cancer Institute, FPO - IRCCS, Candiolo, Torino, Italy. AR’s present address is: Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera Italiana, Bellinzona, Switzerland. ML’s present address is: Guangzhou Institute of Cancer Research, the Affiliated Cancer Hospital & State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Guangzhou, China.

Authorship note: AM and MM contributed equally to this work.

Find articles by Hirsch, E. in: PubMed | Google Scholar

1Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center “Guido Tarone”, University of Torino, Torino, Italy.

2UOC Genetica Medica, IRCCS Istituto Giannina Gaslini, Genova, Italy.

3Experimental Imaging Center, IRCCS Ospedale San Raffaele, Milan, Italy.

4Università Vita-Salute San Raffaele, Milan, Italy.

5Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne; Lausanne, Switzerland.

6Kither Biotech, Torino, Italy.

Address correspondence to: Alessandra Ghigo, Via Nizza 52, 10126, Torino, Italy. Phone: 39.011.670.6335; Email: alessandra.ghigo@unito.it. AM’s present address is: Candiolo Cancer Institute, FPO - IRCCS, Candiolo, Torino, Italy. AR’s present address is: Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera Italiana, Bellinzona, Switzerland. ML’s present address is: Guangzhou Institute of Cancer Research, the Affiliated Cancer Hospital & State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Guangzhou, China.

Authorship note: AM and MM contributed equally to this work.

Find articles by Ghigo, A. in: PubMed | Google Scholar |

Authorship note: AM and MM contributed equally to this work.

Published March 23, 2026 - More info

Published in Volume 11, Issue 6 on March 23, 2026
JCI Insight. 2026;11(6):e198846. https://doi.org/10.1172/jci.insight.198846.
© 2026 Murabito et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published March 23, 2026 - Version history
Received: August 6, 2025; Accepted: January 22, 2026 View PDF Abstract

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-activated chloride channel, cause cystic fibrosis (CF), the most common life-threatening inherited disorder among White individuals. Current CFTR correctors and potentiators, such as elexacaftor-tezacaftor-ivacaftor (ETI), only partially restore the function of the most prevalent mutant, F508del-CFTR, resulting in residual disease in people with CF. Here, we demonstrate that a mimetic peptide targeting the A-kinase–anchoring protein (AKAP) function of PI3Kγ (PI3Kγ MP), and driving localized cAMP elevation, enhances F508del-CFTR membrane localization, maximizing ETI efficacy in restoring chloride secretion. Mechanistically, PI3Kγ MP activates an AKAP-Lbc–anchored pool of PKD1, a known regulator of membrane trafficking. Consistently, PKD1 inhibition prevents PI3Kγ MP from enhancing the membrane expression of ETI-corrected F508del-CFTR. Overall, our findings reveal a regulatory pathway controlling CFTR membrane abundance via the AKAP function of PI3Kγ, which can be targeted to overcome the limitations of current CFTR modulator therapies.

Graphical Abstract Introduction

Cystic fibrosis (CF) is the most common life-threatening inherited disorder among the White population. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-activated anion channel expressed on the surface of epithelial cells, regulating mucus hydration and clearance (1). CFTR dysfunction results in impaired chloride (Cl−) and water secretion across the airway epithelium, producing a viscous mucus that traps pathogens, causing chronic infections and inflammation (2). These complications are responsible for a progressive decline in airway function, ultimately leading to respiratory failure (3).

In the United States, about 85% of people with CF carry at least 1 copy of the F508del allele, resulting in a mutant CFTR channel, F508del-CFTR, characterized by defective folding, impaired gating, and reduced stability at the plasma membrane (1). The standard of care for patients carrying at least 1 F508del allele is a combination of 2 “correctors,” elexacaftor (VX-445) and tezacaftor (VX-661), and the “potentiator” ivacaftor (VX-770), which correct the folding and improve the gating of the mutant channel, respectively (1). Despite its high clinical efficacy, resulting in an average lung function improvement of 9.8% in F508del heterozygous patients after 6 months of treatment (4), the triple-combination therapy of elexacaftor-tezacaftor-ivacaftor (ETI) restores F508del-CFTR activity to only 50% of that of the WT channel (5). As a result, patients with CF on ETI therapy continue to experience residual mucus dysfunction, airway infection, and inflammation (6–8), underscoring the need for strategies to enhance treatment efficacy.

Boosting cAMP signaling enhances the efficacy of CFTR modulators by activating PKA, which phosphorylates CFTR, a step essential for optimal potentiation by VX-770 (9, 10). On the other hand, cAMP can enhance the effectiveness of correctors like lumacaftor (VX-809) in rescuing F508del-CFTR, by tethering the channel to the actin cytoskeleton and reducing its endocytosis (11, 12). Hence, agents that elevate cAMP near the F508del-CFTR may help to prolong the residence time of the channel at the plasma membrane, ultimately enhancing the efficacy of CFTR modulator therapies (13).

Our previous research demonstrated that a physiological and compartment-restricted increase in cAMP near the CFTR can be achieved by targeting the A-kinase–anchoring protein (AKAP) function of PI3Kγ. We showed that a PI3Kγ mimetic peptide (PI3Kγ MP) disrupts the interaction between PI3Kγ and PKA. This disruption inhibits a specific phosphodiesterase 4 (PDE4) pool responsible for hydrolyzing cAMP in the vicinity of CFTR, thereby enhancing PKA-mediated phosphorylation and gating of the channel. Accordingly, PI3Kγ MP promotes the opening of WT CFTR and augments the efficacy of the VX-770 potentiator in rescuing the function of F508del-CFTR (10). However, the role of the PI3Kγ/PKA signalosome in regulating CFTR membrane expression has remained unexplored.

Here, we demonstrate that PI3Kγ MP increases the plasma membrane density of F508del-CFTR, thereby enhancing the efficacy of the CFTR modulator combination ETI. This effect is mediated by PKD1, which acts downstream of cAMP elevation and PKA activation induced by PI3Kγ MP. Overall, these findings reveal a regulatory mechanism governing CFTR membrane expression that can be harnessed to enhance the efficacy of current CFTR modulator therapies.

Results

PI3Kγ MP increases the plasma membrane density of F508del-CFTR in combination with ETI therapy. First, we assessed the extent to which PI3Kγ MP influences CFTR membrane localization and augments the effect of ETI on the surface expression of F508del-CFTR. Immunogold labeling in HEK293T cells expressing GFP-tagged F508del-CFTR revealed that ETI treatment in combination with PI3Kγ MP resulted in a 2-fold increase in F508del-CFTR at the plasma membrane compared with ETI administered with the inactive control peptide (CP) (Figure 1, A and B). These findings were corroborated in F508del-CFBE41o- cells using surface protein biotinylation followed by streptavidin pulldown, which showed a 3-fold increase in mature (C band) F508del-CFTR at the cell surface in cells treated with PI3Kγ MP plus ETI compared with ETI alone (Figure 1, C and D). Notably, global cAMP elevation induced by the cell-permeable, non-hydrolysable analog CPTcAMP failed to phenocopy the effect of the peptide (Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.198846DS1), suggesting that the ability of PI3Kγ MP to increase the plasma membrane abundance of F508del-CFTR depends on its capacity to elevate cAMP locally (10). Enhanced trafficking of CFTR to the plasma membrane may increase its functional lifetime by improving its membrane stability and reducing degradation (14, 15). Consistent with this, cycloheximide (CHX) chase experiments showed that, 6 hours after inhibition of de novo protein synthesis, the level of mature F508del-CFTR in CFBE41o- cells treated with both ETI and PI3Kγ MP was 47% higher than in cells treated with ETI alone (Figure 1, E and F). Altogether, these data indicate that PI3Kγ MP enhances the plasma membrane density of F508del-CFTR under ETI therapy.

Figure 1

PI3Kγ MP increases the plasma membrane density of F508del-CFTR in combination with ETI. (A) Representative immunogold electron microscopy images showing the distribution of overexpressed F508del-CFTR-GFP in HEK293T cells. Cells were treated for 24 hours with DMSO (Veh), ETI (3 μM VX-445, 10 μM VX-661, and 1 μM VX-770) plus control peptide (ETI + 25 μM CP), or ETI plus PI3Kγ MP (ETI + 25 μM PI3Kγ MP). HEK293T cells expressing WT CFTR-GFP served as positive controls. Arrowheads: CFTR channels located at the plasma membrane. Insets: higher magnification of selected regions. Scale bar: 1 μm. (B) Quantification of plasma membrane gold density as shown in A; 99–118 cells from 19–20 images were quantified. ****P < 0.0001 by Kruskal-Wallis test with Dunn’s multiple-comparison test. (C) Representative Western blot of CFTR and GAPDH in plasma membrane fractions and total lysates (TLs) of F508del-CFBE41o- cells treated for 24 hours with ETI alone or ETI + PI3Kγ MP (25 μM). GAPDH was absent in plasma membrane fractions and used as a loading control in TLs. (D) Quantification of plasma membrane CFTR band C intensity as shown in C, expressed as fold-change relative to ETI alone. n = 4–6. *P < 0.05, ****P < 0.0001 by 1-way ANOVA with Dunnett’s multiple-comparison test. (E) Representative Western blot of CFTR and GAPDH levels in F508del-CFBE41o- cells treated for 24 hours with ETI alone or ETI + PI3Kγ MP (25 μM) and cycloheximide (CHX, 1 μg/mL) exposure. Cells were lysed at the indicated times after CHX treatment. (F) Quantification of CFTR band C as shown in E, expressed as fold-change relative to t = 0. n = 3. ##P < 0.01, ###P < 0.001 versus t = 0 by 1-way ANOVA with Bonferroni’s post hoc test; *P < 0.05 for ETI versus ETI + PI3Kγ MP by Welch’s t test. Data shown as mean ± SEM. n: number of independent experiments. Data points represent independent biological replicates.

PI3Kγ MP does not promote F508del-CFTR maturation. Next, we investigated the mechanism by which PI3Kγ MP enhances the plasma membrane density of F508del-CFTR. To assess whether the peptide promotes maturation of the mutant channel, we performed immunogold staining in GFP-F508del-CFTR–expressing HEK293T cells in the absence of correctors, thereby excluding any confounding effects from pharmacological chaperones. This analysis revealed a 2-fold increase in CFTR membrane density in cells treated with PI3Kγ MP compared with those treated with CP, reaching approximately 20% of the levels observed in cells expressing WT CFTR (Figure 2, A and B). Cell-surface biotinylation experiments in F508del-CFBE41o- cells confirmed a significant increase in CFTR levels at the cell membrane at 15 and 30 minutes after treatment with PI3Kγ MP compared with CP (Figure 2, C and D). Notably, PI3Kγ MP increased the membrane abundance of the immature core-glycosylated (B band) but not the mature complex-glycosylated (C band) CFTR (Figure 2, C and D). These observations suggest that the peptide does not replicate the mechanism of CFTR correctors in rescuing the folding and defective trafficking of F508del-CFTR from the endoplasmic reticulum (ER) to the Golgi and plasma membrane. As further confirmation, we treated biotin-labeled cell lysates with endoglycosidase H (Endo H), an enzyme that removes ER core-glycosylation but not complex sugars added in the Golgi. After Endo H treatment, a shift in the molecular weight of F508del-CFTR was observed in CFBE41o- cells treated with the peptide (Figure 2E), indicating that the CFTR at the membrane was sensitive to Endo H and therefore in its immature, ER core-glycosylated form. Further supporting the inability of PI3Kγ MP to promote F508del-CFTR maturation, neither the cAMP analog CPTcAMP nor the CFTR potentiator VX-770 elicited CFTR-mediated currents (ISC) in F508del/F508del human bronchial epithelial (HBE) cells chronically treated with the peptide (Supplemental Figure 2, A and B), indicating a lack of functional CFTR channels at the cell surface. In contrast, cells treated with the CFTR corrector VX-809 showed robust CPTcAMP/VX-770–stimulated currents (Supplemental Figure 2, A and B). These results confirm that PI3Kγ MP does not enhance F508del-CFTR maturation.

Figure 2

PI3Kγ MP does not affect F508del-CFTR maturation. (A) Representative immunogold electron microscopy images showing the distribution of overexpressed F508del-CFTR-GFP in HEK293T cells after 15 minutes of treatment with DMSO (Veh), control peptide (CP; 25 μM), or PI3Kγ MP (25 μM). HEK293T cells expressing WT-CFTR-GFP served as positive controls. Arrowheads indicate CFTR channels located at the plasma membrane. Insets: Higher magnification of selected regions. Scale bar: 1 μm. (B) Quantification of plasma membrane gold density as shown in A. For Veh, CP, and PI3Kγ MP, 68 cells were quantified; 32 cells were quantified for WT. ***P < 0.001, ****P < 0.0001 by Kruskal-Wallis test with Dunn’s multiple-comparison test. (C) Representative Western blot of CFTR and tubulin in plasma membrane fractions and total lysates (TLs) of F508del-CFBE41o- cells treated with CP or PI3Kγ MP for 5, 15, or 30 minutes. WT-CFTR-CFBE41o- cells served as controls for mature complex-glycosylated CFTR (band C). Tubulin was absent in plasma membrane fractions and used as a loading control in TLs. (D) Quantification of immature core-glycosylated CFTR (band B) in plasma membrane fractions as in C. n = 5. ##P < 0.01, ####P < 0.0001 versus t = 0, and **P < 0.01 versus CP by 1-way ANOVA with Bonferroni’s post hoc test. (E) Representative Western blot of CFTR and vinculin in plasma membrane fractions and TLs from F508del-CFBE41o- cells treated with PI3Kγ MP (25 μM; 15 minutes). Plasma membrane proteins were biotinylated, isolated with streptavidin beads, and incubated with Endo H for 1 hour. Vinculin was absent in plasma membrane fractions and used as a loading control in TLs. Throughout, data are mean ± SEM. n: number of independent experiments. Data points represent independent biological replicates.

PI3Kγ MP activates PKD1. To gain further mechanistic insight into the PI3Kγ MP action, a targeted phosphoproteomics approach was performed in F508del-CFBE41o- cells (Figure 3A), with the aim of identifying kinases and regulatory proteins modulated by the peptide. PI3Kγ MP induced phosphorylation changes in a subset of proteins implicated in membrane remodeling and calcium (Ca2+) homeostasis (Figure 3, B and C) as well as protein trafficking (Figure 3D), like small GTPases from the Rho/Rac/Cdc42 families (16). Notably, PKD1, a well-established regulator of Golgi-to–plasma membrane trafficking, endocytosis, and recycling of membrane proteins (17), exhibited the most significant relative increase in phosphorylation in cells treated with PI3Kγ MP compared with those exposed to CP (Figure 3E). Phosphoproteomics results were confirmed by Western blot assays showing that PI3Kγ MP but not CP triggered PKD1 phosphorylation on activating serines 744 and 748 in HEK293T cells overexpressing the kinase (Figure 4, A and B).

Figure 3

Phosphoproteomic analysis reveals that PI3Kγ MP modulates proteins involved in membrane remodeling and trafficking. (A) Experimental workflow used for phosphoproteomic analysis of F508del-CFBE41o- cells treated with control peptide (CP) or PI3Kγ MP (25 μM, 30 minutes). A phospho-specific microarray, containing 875 phosphosite-specific and 451 pan-specific antibodies, was used. Thirty-six proteins showing phosphorylation changes exceeding ±60% compared with the control (expressed as percentage CFC, i.e., percentage fold-change compared with control) were selected for downstream analysis. (B and C) Panther Gene Ontology (GO) slim-term enrichment analysis of proteins with altered phosphorylation after PI3Kγ MP treatment. Significantly enriched GO terms (FDR < 0.05) were categorized under (B) biological processes and (C) cellular components. (D) Reactome pathway enrichment analysis of differentially phosphorylated proteins after PI3Kγ MP treatment. Lines represent the top 20 pathways; x axis shows the –log10(FDR). Color intensity reflects fold enrichment, and circle size indicates the number of proteins; color intensity (yellow to red) indicates increasing fold enrichment. The full phospho-array protein list served as background reference. (E) Proteins with CFC greater than 60% (green) or less than −60% (red). A CFC of 100% corresponds to a 2-fold increase in signal intensity after PI3Kγ MP treatment relative to CP.

Figure 4

PI3Kγ MP activates AKAP-Lbc–anchored PKD1. (A and B) Representative Western blot (A) and relative quantification (B) of phospho-PKD1 (pPKD1 S744-748) in HEK293T cells overexpressing PKD1 and treated with CP or PI3Kγ MP (25 μM). Poly-L-arginine (5 μM, 10 minutes) served as positive control. n = 3. (C) Proposed mechanism of PKD1 activation: PI3Kγ MP elevates cAMP levels, leading to PKA activation, phosphorylation of AKAP-Lbc, and full activation and release of PKD1 from signalosome. (D and E) Representative Western blot (D) and relative quantification (E) of pPKD1 S744-748 in HEK293T cells overexpressing FLAG-PKD1 and treated with PI3Kγ MP (25 μM, 30 minutes) ± PKC inhibitor GO6983 (0.5 μM, 30 min preincubation). n = 4. (F and G) Representative Western blot (F) and relative quantification (G) of phosphorylated AKAP-Lbc in HEK293T cells overexpressing GFP-AKAP-Lbc and treated with vehicle, PI3Kγ MP (25 μM, 30 minutes), or Fsk (1.5 μM, 10 minutes; positive control). AKAP-Lbc was immunoprecipitated and IP pellets probed with PKA substrate antibody. TL, total lysate. n = 3. (H and I) Representative Western blot (H) and relative quantification (I) of PKD1 bound to AKAP-Lbc in HEK293T cells overexpressing GFP-AKAP-Lbc and FLAG-PKD1, treated with vehicle, PI3Kγ MP, or forskolin as in F. n = 3. (J and K) Representative Western blot (J) and relative quantification (K) of pPKD1 S744-748 in HEK293T cells overexpressing FLAG-PKD1 alone or with AKAP-Lbc PH domain, treated with vehicle or PI3Kγ MP (25 μM, 30 minutes). n = 4. In B, ##P < 0.01 for PI3Kγ MP at t = 30 versus t = 0 minutes; *P < 0.05 PI3Kγ MP versus CP at t = 30 minutes by 2-tailed Student’s t test. In G and I, *P < 0.05 PI3Kγ MP versus vehicle by Student’s t test. In E and K, *P < 0.05, **P < 0.01 by 1-way ANOVA with Dunnett’s multiple-comparison test. Data shown as mean ± SEM; n = independent experiments; data points = independent biological replicates.

Next, we aimed to investigate how the increase in cAMP triggered by PI3Kγ MP could lead to PKD1 activation. Previous work demonstrated that PKD1 activation requires the coordinated action of PKC and PKA, which is facilitated by AKAP-Lbc. Within the AKAP-Lbc complex, PKC phosphorylates PKD1 at serines 744 and 748, and PKA-mediated phosphorylation of the AKAP promotes the release of the activated kinase (18) (Figure 4C). Pharmacological PKC inhibition with GO6983 significantly reduced the PKD1 phosphorylation on serines 744 and 748 elicited by PI3Kγ MP (Figure 4, D and E), indicating that the ability of the peptide to trigger PKD1 depends on PKC. Furthermore, PI3Kγ MP increased PKA-mediated phosphorylation of AKAP-Lbc by 4-fold (Figure 4, F and G), which corresponded with an approximately 50% reduction in binding with PKD1 in HEK293T cells coexpressing GFP-AKAP-Lbc and PKD1 (Figure 4, H and I). To further demonstrate that PI3Kγ MP activates an AKAP-Lbc–anchored pool of PKD1, HEK293T cells were transfected with a plasmid encoding for a soluble version of the pleckstrin homology (PH) domain of AKAP-Lbc, which antagonizes PKD1 activation by displacing PKC from the complex (18). In these cells, PI3Kγ MP failed to significantly raise PKD1 phosphorylation over basal values (Figure 4, J and K), demonstrating that the peptide controls a pool of PKD1 tethered to AKAP-Lbc. Overall, these data support a model in which PI3Kγ MP–induced cAMP elevation activates a localized pool of PKA, which in turn mediates full PKD1 activation and its subsequent release from the AKAP-Lbc signalosome.

PKD1 activation by PI3Kγ MP enhances F508del-CFTR membrane localization, maximizing the efficacy of ETI therapy. Next, we evaluated whether the pool of PKD1 activated by the peptide and released from the AKAP-Lbc signalosome localizes to compartments that contain CFTR, potentially influencing its plasma membrane localization. To this end, we tested the association between PKD1 and CFTR by proximity ligation assay (PLA), a test detecting proteins in close vicinity (<40 nm). Our results showed that in CFBE-F508del-CFBE41o- cells under ETI therapy, the number of PLA puncta significantly increased upon treatment with PI3Kγ MP (Figure 5, A and B). To assess the extent to which PI3Kγ MP-activated PKD1 contributes to increased plasma membrane expression of F508del-CFTR, we inhibited PKD1 using the inhibitor CRT0066101. PKD1 inhibition completely abolished the increase in F508del-CFTR plasma membrane density elicited by PI3Kγ MP in CFBE-F508del-CFBE41o- cells treated with ETI (Figure 5, C and D), demonstrating that the ability of PI3Kγ MP to maximize the effect of ETI on F508del-CFTR plasma membrane localization requires PKD1.

Figure 5

PKD1 activation by PI3Kγ MP enhances F508del-CFTR membrane localization. (A) Representative images showing red proximity ligation assay (PLA) signals indicating the interaction between F508del-CFTR and PKD1 in F508del-CFBE41o- cells. Cells were treated with ETI (3 μM VX-445, 10 μM VX-661, and 1 μM VX-770), alone or with PI3Kγ MP (25 μM) for 24 hours. Nuclei were stained with DAPI (blue). Scale bar: 15 μm. (B) Quantification of PLA dots per cell as in A. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA with Bonferroni’s post hoc correction. n = 3. (C) Representative Western blot of CFTR and GAPDH in plasma membrane fractions and total lysates (TLs) of F508del-CFBE41o- cells treated with ETI, PI3Kγ MP (25 μM), or the PKD inhibitor CRT0066101 (10 μM) for 24 hours. GAPDH was absent in plasma membrane fractions and used as a loading control in TLs. (D) Quantification of plasma membrane CFTR band C intensity as shown in C, expressed as fold-change relative to ETI + PI3Kγ MP. **P < 0.01, ****P < 0.0001 by 1-way ANOVA with Bonferroni’s post hoc test. n = 3. Data are shown as mean ± SEM. n: number of independent experiments. Data points represent independent biological replicates.

Finally, we aimed to assess the functional impact of this regulatory mechanism. To this end, we measured CFTR-dependent Cl– secretion in F508del/F508del primary human bronchial epithelial (HBE) cells treated with ETI alone or combined with PI3Kγ MP. The membrane-permeable cAMP analog CPTcAMP was used to induce maximal CFTR activation, which is directly proportional to the channel plasma membrane density. In F508del/F508del HBE cells from 3 independent donors, the maximal CFTR activity elicited by CPTcAMP, and the relative current drop induced by the specific CFTR inhibitor CFTRinh-172 at the end of the experiment, were 30% higher in cells co-treated with PI3Kγ MP than in those exposed to ETI alone (Figure 6, A and B, and Supplemental Figure 3, A and B). Similar results were obtained in HBE cells derived from 2 patients with a more severe genotype, carrying the F508del mutation and the nonsense G542X mutation (Figure 6, C and D, and Supplemental Figure 3C). Overall, these results demonstrate that PI3Kγ MP maximizes the efficacy of the triple-combination ETI by increasing the plasma membrane density of F508del channels through the activation of PKD1.

Figure 6

PI3Kγ MP enhances ETI efficacy in human bronchial epithelial cells from individuals with CF. (A) Representative short-circuit current (ISC) traces in primary human bronchial epithelial (HBE) cells from a F508del/F508del CF donor (patient BE93) grown at the air-liquid interface (ALI). Cells were corrected for 24 hours with 10 μM VX-661 and 3 μM VX-445, alone or with PI3Kγ MP (10 μM), and then acutely exposed to amiloride (100 μM), VX-770 (1 μM), CPTcAMP (100 μM), and CFTRinh-172 (10 μM). (B) Average current inhibition by CFTRinh-172 (10 μM) in primary HBE cells from 3 F508del/F508del donors. For BE93, n = 12 (ETI) and n = 9 (ETI + PI3Kγ MP) technical replicates. For BE91 and BE86, n = 5 and n = 3 technical replicates per treatment, respectively. *P < 0.05, **P < 0.01 by 2-tailed Student’s t test. (C) Representative ISC traces in primary HBE cells from a F508del/G542X CF donor (patient BE146) grown at the ALI. Cells were corrected for 24 hours with VX-661 (10 μM) and VX-445 (3 μM), alone or with PI3Kγ MP (10 μM), followed by acute application of amiloride (100 μM), VX-770 (1 μM), CPTcAMP (100 μM), and CFTRinh-172 (10 μM) at the indicated times. (D) Average current inhibition by CFTRinh-172 (10 μM) in HBE cells from 2 F508del/G542X donors (BE146 and BE157); n = 4 technical replicates each. *P < 0.05 by Mann-Whitney U test. Data are shown as mean ± SEM.

Discussion

Our findings revealed a regulatory mechanism governing CFTR membrane localization under the control of the AKAP function of PI3Kγ. Furthermore, our results support the pharmacological targeting of this pathway using PI3Kγ MP (10) as a promising strategy to overcome the limitations of current CFTR modulator therapies.

The triple-combination therapy ETI, the standard of care for patients with CF, reinstates only partially the activity of F508del-CFTR, up to a maximum of 50% of WT CFTR activity (5). This limitation arises from the inability of correctors to fully stabilize the mutant channel and restore its trafficking to the plasma membrane (19). Our findings not only support this notion but also propose a potential solution. We demonstrate that the physiological and compartment-restricted increase in cAMP achieved with PI3Kγ MP (10) enhances the ability of ETI to augment the surface expression of F508del-CFTR. Importantly, this effect is not due to the peptide acting as a CFTR corrector itself since, in the absence of pharmacological chaperones that rescue folding and ER-to-Golgi trafficking defects, the peptide fails to increase the levels of mature, fully glycosylated CFTR at the plasma membrane. Our data support a model in which CFTR correctors are necessary to enable the mutant channel to pass the ER quality control, thereby allowing PI3Kγ MP to exert its effect. This underscores the therapeutic importance of combining PI3Kγ MP with CFTR correctors. In the absence of correction, the misfolded channel accumulates in the ER and can trigger an alternativ