ABCMdb

PMID: 26137539

Awatade NT, Uliyakina I, Farinha CM, Clarke LA, Mendes K, Sole A, Pastor J, Ramos MM, Amaral MD
Measurements of Functional Responses in Human Primary Lung Cells as a Basis for Personalized Therapy for Cystic Fibrosis.
EBioMedicine. 2014 Dec 17;2(2):147-53. doi: 10.1016/j.ebiom.2014.12.005. eCollection 2015 Feb., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Tyr1092* ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu Methods: The effect of lumacaftor was investigated in primary HBE cells from non-CF and CF patients with F508del/F508del, A561E/A561E, N1303K/G542X, F508del/G542X and F508del/Y1092X genotypes by measurements of Forskolin plus Genistein-inducible equivalent short-circuit current (Ieq-SC-Fsk + Gen) in perfused open-circuit Ussing chambers. Login to comment 4 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu Efficacy of corrector C18 was also assessed on A561E/A561E and F508del/F508del cells. Login to comment 5 ABCC7 p.Asn1303Lys ABCC7 p.Tyr1092* ABCC7 p.Ala561Glu Results: Our data indicate that A561E (when present in both alleles) responds positively to lumacaftor treatment at equivalent efficacy of F508del in primary HBE cells. Similarly, lumacaftor has a positive impact on Y1092X, but not on N1303K. Login to comment 8 ABCC7 p.Ala561Glu Compound C18 failed to rescue A561E-CFTR but not in F508del-CFTR, thus plausibly it has a different mechanism of action distinct from lumacaftor. Login to comment 9 ABCC7 p.Ala561Glu Conclusions: CF patients with A561E (and likely also those with Y1029X) can potentially benefit from lumacaftor. Login to comment 29 ABCC7 p.Gly551Asp Potentiator ivacaftor, the first CFTR-targeting drug, was recently approved by FDA/EMA, albeit for a rare mutation - G551D (Ramsey et al., 2011) and for other Class III CFTR mutations (Van Goor et al., 2014; De Boeck et al., 2013), which, altogether only target ~5% of CF patients worldwide. Login to comment 34 ABCC7 p.Asn1303Lys ABCC7 p.Ala561Glu Mutations tested here include: A561E, quite frequent in Southern-European and South-American countries like in Portugal (Mendes et al., 2003), Spain (Moya-Quiles et al., 2009) and Brazil (Servidoni et al., 2013) and N1303K, linked to ancient Mediterranean populations (Bobadilla et al., 2002). Login to comment 35 ABCC7 p.Gly542* ABCC7 p.Tyr1092* In addition we tested VX-809 in HBE cells bearing 2 nonsense mutations: G542X and Y1092X, both in heterozygosity with F508del. Login to comment 36 ABCC7 p.Asn1303Lys ABCC7 p.Ala561Glu Our data in primary HBE cells show that lumacaftor rescues A561E at equivalent efficacy of F508del, but N1303K is not significantly rescued. Login to comment 38 ABCC7 p.Ala561Glu Compound C18 (lumacaftor analogue, also reported to rescue F508del) failed to rescue A561E-CFTR, thus plausibly rescuing CFTR by a different mechanism of action than lumacaftor. Login to comment 39 ABCC7 p.Ala561Glu We conclude that CF patients with the A561E mutation can potentially benefit from lumacaftor and personalized medicine is the way forward to tackle CF. Login to comment 42 ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Tyr1092* ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu Culture Conditions of Primary Human Bronchial Epithelial Cells Human lung tissues from CF donors with the F508del/F508del (2 donors), A561E/A561E, N1303K/G542X, F508del/G542X and F508del/ Y1092X genotypes, were obtained from the Cardio-Thoracic Surgery Department (University Hospital la Fe, Valencia, Spain) after receiving patient's written consent and approval by the hospital Ethics Committee. Login to comment 58 ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Tyr1092* ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu Response to lumacaftor for Class II Mutants Assessed by CFTR-Mediated Chloride Secretion The effects of 24 h-treatment with lumacaftor were assessed here by determining CFTR-mediated Cl-secretion in HBE cells from CF donors with the following genotypes (Fig. 1): wt/wt control (a, b); F508del/F508del-Donor 1 (c, d); F508del/F508del-Donor 2 (e, f); A561E/A561E (Fig. 1g, h) and also on the additional genotypes (Fig. 2): N1303K/G542X (a, b), F508del/G542X (c, d); F508del/ Y1092X (e, f). Login to comment 59 ABCC7 p.Asn1303Lys ABCC7 p.Gly542* Since G542X is a "null" variant (i.e., generating no protein) results on the latter are representative of the N1303K variant, albeit in a single dose. Login to comment 61 ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Tyr1092* ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu These results show that Fsk + Gen responses of F508del/F508del (2 donors), A561E/A561E F508del/G542X and F508del/Y1092X cells after VX-809/lumacaftor treatment were significantly different from those under DMSO, while that of N1303K/G542X cells was not significantly different (Fig. 3b). Login to comment 63 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu Effect of lumacaftor (VX-809) on cAMP-induced Isc-eq in primary cultures of HBE cells from CF patients with class II mutations. Original Ussing chamber (open-circuit) recordings showing transepithelial voltage measurements (Vte) obtained for CF primary airway HBE monolayers with different CFTR genotypes: wt/wt control (a, b); F508del/F508del-donor 1 (c, d); F508del/F508del-donor 2 (e, f); and A561E/A561E (g, h). Login to comment 69 ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Tyr1092* ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu These data again clearly show a positive effect of VX-809 on HBE cells with genotypes F508del/ F508del (both donors), A561E/A561E, F508del/G542X and F508del/ Y1092X but not on N1303K/G542X cells. Login to comment 71 ABCC7 p.Gly542* ABCC7 p.Tyr1092* It is also interesting to note the difference in responses by the F508del/G542X and F508del/Y1092X cells. Login to comment 72 ABCC7 p.Gly542* ABCC7 p.Tyr1092* Since F508del/ Y1092X cells (but not F508del/G542X cells) already exhibit levels of Ieq-sc-Fsk or Ieq-sc-Fsk + Gen before VX-809, we assessed Fig. 2. Login to comment 73 ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Tyr1092* Effect of lumacaftor (VX-809) on cAMP-induced Isc-eq in primary cultures of HBE cells from CF patients with different CFTR mutations. Original Ussing chamber (open-circuit) recordings showing transepithelial voltage measurements (Vte) obtained for CF primary airway HBE monolayers with different genotypes: N1303K/G542X (a, b); F508del/G542X (c, d); and F508del/Y1092X (e, f). Login to comment 77 ABCC7 p.Gly542* ABCC7 p.Tyr1092* ABCC7 p.Tyr1092* Data, show that the levels of Y1092X-transcripts are higher than those from G542X (Table S3), indicating that Y1092X transcripts are less prone to degradation through nonsense-mediated decay. Login to comment 83 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu Original tracings and summary of the effect of C18 in A561E/A561E and F508del/F508del primary HBE cells. Login to comment 84 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu (a, b) represent original Ussing chamber (open-circuit) recordings obtained for the analysis of CF primary airway HBE monolayers with A561E/A561E and F508del/F508del treated with 5 bc;M C18 for 24 h. Login to comment 88 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu Response of A561E/A561E HBE Cells to lumacaftor and Compound C18 A561E/A561E HBE cells were also treated with C18 compound, described as a lumacaftor analogue (Eckford et al., 2014). Login to comment 89 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu As demonstrated by the original tracing in Fig. 4a, the responses elicited by either Fsk or Fsk + Gen in A561E/A561E cells pre-incubated with C18 are lower than those in F508del/F508del cells (Fig. 4b) and this difference is statistically different (Fig. 4c). Login to comment 90 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu Moreover, the response of A561E/ A561E cells after C18 treatment is also significantly lower than that in lumacaftor-treated cells, while those of F508del/F508del cells after C18 and VX-809 are similar (Fig. 1c, Table S4). Login to comment 91 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu Indeed, the Fold rescue of Ieq-sc-Fsk + Gen in A561E/A561E cells after C18 treatment was 1.93&#d7;, while this value was 6.51&#d7; F508del/F508del cells. Similarly, the percentages of rescue by C18 vs non-CF cells (wt/wt) were ~0.8% and ~5.0% for A561E/A561E F508del/F508del cells, respectively. Login to comment 92 ABCC7 p.Ala561Glu These data also indicate that the response of A561/A561E HBE cells to C18 is lower than to lumacaftor, when these cells are stimulated by Gen, but interestingly, not when stimulated only by Fsk. Login to comment 93 ABCC7 p.Ala561Glu To confirm these data, Western blot was performed in BHK cells stably expressing F508del or A561E mutant protein. Login to comment 94 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu Data show that VX-809 rescues both F508del and A561E-CFTR, while C18 failed to rescue A561E-CFTR but not in F508del-CFTR protein (Fig. S2). Login to comment 95 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu These data are thus consistent with those obtained for A561E/A561E cells treated with C18. Login to comment 103 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu Our data show that the effect of lumacaftor on A561E/A561E HBE cells was equivalent to that of this investigational drug in F508del/ F508del cells. Login to comment 104 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu Indeed, after the incubation of A561E/A561E cells with 3 bc;M lumacaftor for 24 h, responses obtained in the Ussing chamber were 7-fold higher than when cells were incubated with DMSO-vehicle, representing ~6% of rescue vs non-CF cells. Login to comment 106 ABCC7 p.Ala561Glu These data seem to indicate that the previously characterized trafficking defect of the A561E-CFTR protein (Mendes et al., 2003) can be, as least partially, corrected by lumacaftor. Login to comment 107 ABCC7 p.Ala561Glu Interestingly, a previous study showed that A561E-CFTR can be rescued to the cell surface by the same genetic revertants as F508del-CFTR (Roxo-Rosa et al., 2006). Login to comment 108 ABCC7 p.Ala561Glu In another more recent study, the A561E-CFTR channel was also described to have similar mechanisms of dysfunction and response to potentiators as F508del-CFTR (Wang et al., 2014). Login to comment 110 ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Tyr1092* Data presented here also show a positive effect of VX-809 on HBE cells with genotypes F508del/G542X (~4% vs non-CF) and F508del/Y1092X (~7% vs non-CF) but not on N1303K/G542X cells. Login to comment 111 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu Our data also lead to the conclusion that the A561E responses to lumacaftor and its analogue C18 do not totally overlap, as observed from the significantly lower Fsk + Gen response of A561E/A561E cells pre-incubated with C18 vs those under lumacaftor. Login to comment 113 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu Noticeably, however, the Fsk-response of C18-treated A561E/A561E cells is significantly higher than in the DMSO-treated cells (Fig. 4c). Login to comment 115 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu Nonetheless, C18 also failed to rescue A561E-CFTR as assessed by Western blot, while VX-809 induces a detectable levels of mature A561E-CFTR (Fig. S2). Login to comment 117 ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu In contrast to the effect on A561E/A561E HBE cells, the magnitude of the response of lumacaftor-treated N1303K/G542X cells was just slightly higher by ~2-fold (both under Fsk and Gen) and not statistically different from that in DMSO-treated cells. Login to comment 118 ABCC7 p.Asn1303Lys Moreover, the percentage of rescue vs non-CF cells was barely 0.5%, thus showing a lack of an effect by VX-809 on N1303K. Login to comment 120 ABCC7 p.Asn1303Lys ABCC7 p.Ala561Glu Firstly, N1303K located in the second nucleotide binding domain (NBD2) of CFTR protein, may cause a different structural defect from that of F508del or A561E, both located in NBD1. Login to comment 122 ABCC7 p.Asn1303Lys Plausibly, NBD2-located N1303K creates a distinct defect which unlikely would be corrected by the lumacaftor. Login to comment 123 ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu Secondly, it is possible that the response of a single copy of N1303K (the other CFTR allele is G542X, a "null" variant) may be insufficient to observe an effect similar in magnitude to that of A561E/A561E or F508del/F508del cells. Login to comment 124 ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Tyr1092* Contradicting the latter hypothesis are the positive responses of the F508del/G542X and F508del/Y1092X cells, showing that VX-809 can elicit a detectable effect on a single dose of F508del, in contrast to N1303K. Login to comment 125 ABCC7 p.Gly542* ABCC7 p.Tyr1092* Interestingly, the response of F508del/Y1092X cells is almost double to that of F508del/G542X. Login to comment 126 ABCC7 p.Gly542* ABCC7 p.Tyr1092* While difference could be due to intrinsic responses of the F508del alleles from each of these donors, it is also plausible that the Y1092X mutation, given its localization towards the C-terminus of the protein, does not totally abolish the production of functional CFTR protein, in contrast to G542X. Login to comment 127 ABCC7 p.Tyr1092* Indeed, these HBE cells already exhibit levels of Ieq-sc-Fsk or Ieq-sc-Fsk + Gen before VX-809, which are higher than those of F508del/F508del cells, suggesting that Y1092X-CFTR protein may elicit such response. Login to comment 128 ABCC7 p.Gly542* ABCC7 p.Tyr1092* ABCC7 p.Tyr1092* Moreover, the levels of Y1092X-transcripts (Table S3) are higher than those from G542X, again indicating that Y1092X transcripts are less prone to degradation through nonsense-mediated decay. Login to comment 129 ABCC7 p.Tyr1092* It is thus likely that Y1092X originates CFTR protein with residual function with some positive response to VX-809. Login to comment 130 ABCC7 p.Ala561Glu In conclusion, our data suggest that CF patients bearing the A561E mutation, which is associated with a severe clinical phenotype and quite common in some countries (Mendes et al., 2003), can potentially benefit from lumacaftor treatment. Login to comment 131 ABCC7 p.Tyr1092* Similarly, lumacaftor seems to have a positive impact on Y1092X. Login to comment