Response to elexacaftor/tezacaftor/ivacaftor in intestinal organoids derived from people with cystic fibrosis

Eva Furstova a,b, Tereza Dousova a, Jakub Beranek b, Malgorzata Libikc, Libor Fila d,
Martin Modrake, Ondrej Cinek a, Milan Macek Jr c, Pavel Drevinek b,∗
a Department of Paediatrics, 2nd Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic
b Department of Medical Microbiology, 2nd Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic
c Department of Biology and Medical Genetics, 2nd Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic
d Department of Pulmonology, 2nd Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic
e Department of Bioinformatics, 2nd Faculty of Medicine, Charles University, Prague, Czech Republic

a r t i c l e i n f o
Article history:
Received 29 March 2021
Revised 11 June 2021
Accepted 14 July 2021 Available online xxx
∗ Corresponding author at: Department of Medical Microbiology, 2nd Faculty of Medicine, Motol University Hospital, V Uvalu 84, 15000 Prague 5, Czech Republic.
E-mail address: [email protected] (P. Drevinek).

Keywords:
Cystic fibrosis Intestinal organoids Elexacaftor Tezacaftor
Ivacaftor
CFTR sequence

Abstract:

Superior efficacy of elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) over tezacaftor/ivacaftor (TEZ/IVA) in people with cystic fibrosis (CF) and Phe508del/Phe508del genotype was shown in clinical trials. We uti- lized intestinal organoid approach to compare in vitro responses to these 2 CFTR modulator drug com- binations and to check potential inter-individual variability in therapeutic response to the triple com- bination. Organoids from 17 subjects with Phe508del/Phe508del were screened with forskolin induced swelling assay. Significantly larger swelling, when exposed to ELX/TEZ/IVA as compared to TEZ/IVA, was observed in 16 of them. However, 1 sample showed no additional effect of ELX. The finding of unique CFTR variants in this sample indicates that genetic traits other than CF-causing CFTR mutation are worth exploring as they may have an impact on the definitive modulator drug response.

Abbreviations: ELX, elexacaftor; TEZ, tezacaftor; LUM, lumacaftor; IVA, ivacaftor; CF, cystic fibrosis; FIS, forskolin induced swelling; VUS, variant of unknown signifi- cance; ppFEV1, percent predicted force expiratory volume in 1 second; CFTR, cystic fibrosis conductance regulator; ∆F/∆F, Phe508del/Phe508del.

1. Introduction

New pharmacotherapies targeting the underlying cause of cystic fibrosis (CF) have a significant impact on clinical outcome as well as on the quality of life and life expectancy of people with CF [1]. These drugs termed CFTR modulators can restore the CFTR ion-channel function, albeit their efficacy is determined by the presence of certain CFTR mutations. Three genotype-specific CFTR modulator drugs combining 2 to 3 small molecules were approved for treatment of people carrying Phe508del/Phe508del (∆F/∆F): lumacaftor/ivacaftor (LUM/IVA, Orkambi®, Vertex Pharmaceuticals, 2015), tezacaftor/ivacaftor (TEZ/IVA, Symkevi®, Vertex Pharma- ceuticals, 2018) and elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA, Kaftrio®, Vertex Pharmaceuticals, 2020). Their overall efficacy has been proven in multiple clinical trials [2–4], but inter-individual differences in response to the treatment have been also shown.
Similarly, inter-individual variability was observed during in vitro experiments on intestinal organoids with TEZ/IVA and LUM/IVA [5]. Patient-derived intestinal organoids, a tool to predict the treatment response to different CFTR modulators using forskolin induced swelling (FIS) assay [6–8], indicated that there was a moderate to high correlation between in vitro CFTR function and clinical biomarkers such as ppFEV1 (percent predicted force expiratory volume in 1 second) and sweat chloride concentration [7,9]. Interestingly, de Winter et al. suggested that even small differences in CFTR function may affect disease progression [5].
We aimed to test the biological response to the latest approved CFTR modulator combination ELX/TEZ/IVA in intestinal organoids as it has not been established yet. Based on clinical trial data [4], we hypothesized that ELX/TEZ/IVA was superior in the extent of this in vitro response to other approved CFTR modulators eligible for ∆F/∆F individuals.

2. Methods

We collected rectal biopsies from 17 CF subjects with ∆F/∆F genotype upon obtaining their informed consent, approved by the Motol University Hospital Ethics committee (for subject charac- teristics see Supplemental Table 1). Biopsy samples were cultured according to the previously described protocols [10–11]. Grown intestinal organoids were mechanically split and plated in 96-well plate, treated approx. for 20 hours with a) 3 μM TEZ (Selleckchem, USA), b) 3 μM TEZ and 2 μM ELX (Selleckchem, USA) or c) without any modulator added. The concentration of ELX was established based on previous experiments on human bronchial epithelial cells [12]. Organoids were stained with 3μM calcein green (Invitrogen, USA) for 30 min prior to the FIS assay that was performed im- mediately after adding the solutions with 3μM IVA (Selleckchem, USA) and/or forskolin of various concentrations (Selleckchem, USA) [13]. Each experimental condition was run in triplicates. Swelling of organoids was visualized with a confocal microscope (Leica TCS SP8, Leica Microsystems, Germany). Acquired images were anal- ysed using CellProfiler software [14]. We calculated the AUC (ab- solute area under the curve) from mean percentage of organoids swelling (baseline = 100%, t = 60 min). We employed a linear model with the full interaction of the forskolin level and drug as

Fig. 1. FIS assays in intestinal organoids from subjects carrying Phe508del/Phe508del CFTR genotype. Average AUC during 1 hour experiment and standard deviation using 3 different conditions: forskolin only (solid line), TEZ/IVA (dashed line), ELX/TEZ/IVA (dotted line). n = replicates of the experiment per subject. The error bars represent +/- 1 standard deviation.
E. Furstova, T. Dousova, J. Beranek et al. Journal of Cystic Fibrosis xxx (xxxx) xxx
JB; Data analysis: EF; Statistics: MM; Writing: EF; Genetic analysis: ML, MMJr; Review and editing: all.

3. Results and discussion

Significantly larger swelling was observed in organoids exposed to ELX/TEZ/IVA compared to TEZ/IVA in all forskolin concentra- tions (all p < 0.001) (Fig. 1). However, we noted differences in the response between individual organoids, which suggest that the extent of organoids swelling may reflect the inter-individual variability seen in clinical trials [4]. The most striking and some- what unexpected finding in our study was essentially no addi- tional response to ELX on top of TEZ/IVA (Fig. 1, subject 58-CF) as both TEZ/IVA and ELX/TEZ/IVA combinations resulted in similar organoid swelling. The summarized differences between subjects’ responses to ELX/TEZ/IVA at a single forskolin concentration are shown in Fig. 2.
The CFTR gene analysis (Supplemental Table 2) showed that subject 58-CF harboured 4 CFTR variants, not present in any of 14 remaining subjects whose DNA analysis results were available. Moreover, the variant c.2658-37T>C has not yet been described elsewhere; we classified it as a variant of unknown significance (VUS) by using several independent bioinformatics tools and in ac- cordance with the guidelines of American College of Medical Ge- netics (Supplemental Table 2). The presence of the variants in the only organoid sample that failed to prove the beneficial effect of ELX may mean that they affect the target site for ELX, leading to the unresponsiveness to this molecule. Although we cannot rule out the impact of other genetic factors such as disease modify- ing genes [16] on the final effect of ELX, our results underlines the need to explore the relationship between the genetic background and individual drug responses. Such knowledge is crucial for fur- ther development of the precision medicine concept in CF [17,18]. It is of note that until now, none of the studied subjects has been on the CFTR modulator therapy; thus no clinical data can be pro- vided to compare in vitro and in vivo treatment response.
In conclusion, this is to the best of our knowledge the first re- port comparing the TEZ/IVA and ELX/TEZ/IVA effects on intestinal organoids. We observed in vitro superiority of ELX/TEZ/IVA over TEZ/IVA in all but 1 subjects carrying Phe508del/Phe508del. The unique CFTR variants may play a role in the lack of in vitro re- sponse to the ELX.

Funding
This work was supported by the Charles University, project GA UK no 1034819; Ministry of Health of the Czech Republic, grant no NU20-07-00049.

Supplementary materials
predictors and used the Tukey method to correct for multiple com- parisons. The analysis was performed using the R language [15], see Supplementary statistical analysis material for the full code.

Declaration of Competing Interest
All authors declare no conflict of interest.

Author statement

Methodology: EF, TD, PD, OC; Recruiting subjects: TD, LF; Collection of biopsies: EF; Organoids cultures and FIS assays: EF,
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jcf.2021.07.006.

References

[1] Balfour-Lynn IM, King JA. CFTR modulator therapies – effect on life expectancy in people with cystic fibrosis. Paediatric respiratory reviews. WB Saunders Ltd; 2020.
[2] Wainwright CE, Elborn JS, Ramsey BW, Marigowda G, Huang X, Cipolli M, et al. Lumacaftor–ivacaftor in patients with cystic fibrosis homozygous for Phe508del CFTR. N Engl J Med Jul 2015;373(3):220–31.
[3] Taylor-Cousar JL, Munck A, McKone EF, van der Ent CK, Moeller A, Simard C, et al. Tezacaftor–Ivacaftor in patients with cystic fibrosis homozygous for Phe508del. N Engl J Med Nov 2017;377(21):2013–23.
[4] Heijerman HGM, McKone EF, Downey DG, van Braeckel E, Rowe SM, Tullis E, et al. Efficacy and safety of the elexacaftor plus tezacaftor plus ivacaftor com- bination regimen in people with cystic fibrosis homozygous for the F508del mutation: a double-blind, randomised, phase 3 trial. Lancet North Am Ed Nov 2019;394(10212):1940–8.
[5] de Winter – de Groot KM, Berkers G, Marck – van der Wilt REP, van der Meer R, Vonk A, Dekkers JF, et al. Forskolin-induced swelling of in- testinal organoids correlates with disease severity in adults with cystic fi- brosis and homozygous F508del mutations. J Cyst Fibros Jul 2020;19(4): 614–619.
[6] de Poel E, Lefferts JW, Beekman JM. Intestinal organoids for Cystic Fibrosis re- search. Vol. 19, Journal of Cystic Fibrosis. Elsevier B.V.; 2020. p. S60–4.
[7] Ramalho AS, Fürstová E, Vonk AM, Ferrante M, Verfailli C, Dupont L, et al. Cor- rection of CFTR function in intestinal organoids to guide treatment of cystic fibrosis. Eur Respir J Jan 2021;57(1).
[8] Noordhoek J, Gulmans V, van der Ent K, Beekman JM. Intestinal organoids and personalized medicine in cystic fibrosis: A successful patient-oriented re- search collaboration. Vol. 22, Current Opinion in Pulmonary Medicine. Lippin- cott Williams and Wilkins; 2016. p. 610–16.
[9] Berkers G, van Mourik P, Vonk AM, Kruisselbrink E, Dekkers JF, de Winter-de Groot KM, et al. Rectal organoids enable personalized treatment of cystic fi- brosis. Cell Rep Feb 2019;26(7):1701–8 e3.
[10] Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature May 2009;459(7244):262–5.
[11] Dekkers JF, Berkers G, Kruisselbrink E, Vonk A, de Jonge HR, Janssens HM, et al. Characterizing responses to CFTR-modulating drugs using rectal organoids derived from subjects with cystic fibrosis [Internet]. Available from: www.CFTR2.org
[12] Keating D, Marigowda G, Burr L, Daines C, Mall MA, McKone EF, et al. VX-445–tezacaftor–ivacaftor in patients with cystic fibrosis and one or two Phe508del alleles. N Engl J Med Oct 2018;379(17):1612–20.
[13] Dekkers JF, Wiegerinck CL, de Jonge HR, Bronsveld I, Janssens HM, de Win- ter-De Groot KM, et al. A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat Med 2013;19(7):939–45 Jul.
[14] Jones TR, Kang IH, Wheeler DB, Lindquist RA, Papallo A, Sabatini DM, et al. CellProfiler Analyst: Data exploration and analysis software for complex image-based screens. BMC Bioinformatics Nov 2008;9.
[15] Core Team R. R: A language and environment for statistical comput- ing, Vienna, Austria: R Foundation for Statistical Computing; 2020. URL https://www.R-project.org/.
[16] Strug LJ, Gonska T, He G, Keenan K, Ip W, Böelle PY, et al. Cystic fibrosis gene modifier SLC26A9 modulates airway response to CFTR-directed therapeutics. Hum Mol Genet 2016;25(20):4590–600.
[17] Chen KG, Zhong P, Zheng W, Beekman JM. Pharmacological analysis of CFTR variants of cystic fibrosis using stem cell-derived organoids. Vol. 24, Drug Dis- covery Today. Elsevier Ltd; 2019. p. 2126–38.
[18] Marson FAL, Bertuzzo CS, Ribeiro JD. Personalized or precision medicine?. The example of cystic fibrosis. Vol. 8, Frontiers in Pharmacology. Frontiers Media SA; 2017.