Correction of nonsense BMPR2 and SMAD9 mutations by ataluren in pulmonary arterial hypertension

Abstract

Heritable pulmonary arterial hypertension (HPAH) is a serious lung vascular disease caused by heterozygous mutations in the bone morphogenetic protein (BMP) pathway genes, BMPR2 and SMAD9. One noncanonical function of BMP signaling regulates biogenesis of a subset of microRNAs. We have previously shown that this function is abrogated in patients with HPAH, making it a highly sensitive readout of BMP pathway integrity. Ataluren (PTC124) is an investigational drug that permits ribosomal readthrough of premature stop codons, resulting in a full-length protein. It exhibits oral bioavailability and limited toxicity in human trials. Here, we tested ataluren in lung- or blood-derived cells from patients with HPAH with nonsense mutations in BMPR2 (n = 6) or SMAD9 (n = 1). Ataluren significantly increased BMP-mediated microRNA processing in six of the seven cases. Moreover, rescue was achieved even for mutations exhibiting significant nonsense-mediated mRNA decay. Response to ataluren was dose dependent, and complete correction was achieved at therapeutic doses currently used in clinical trials for cystic fibrosis. BMP receptor (BMPR)-II protein levels were normalized and ligand-dependent phosphorylation of downstream target Smads was increased. Furthermore, the usually hyperproliferative phenotype of pulmonary artery endothelial and smooth muscle cells was reversed by ataluren. These results indicate that ataluren can effectively suppress a high proportion of BMPR2 and SMAD9 nonsense mutations and correct BMP signaling in vitro. Approximately 29% of all HPAH mutations are nonsense point mutations. In light of this, we propose ataluren as a potential new personalized therapy for this significant subgroup of patients with PAH.

Figure 1.

Ataluren significantly increases microRNA (miR) processing across a range of nonsense pulmonary arterial hypertension (PAH) mutations. Cells were treated with 3 μM ataluren for 24 hours, then the appropriate bone morphogenetic protein (BMP) ligand for 4 hours. RNA was analyzed for miR-27a, normalized to RNU48. Results are shown as fold change of miR-27a with BMP treatment compared with vehicle. (A) Pulmonary artery endothelial cells (PAECs; R321X and R294X) or late-outgrowth endothelial progenitor cells (L-EPCs; W9X) treated with BMP9. (B) Lymphoblastoid cell line (LCL) treated with BMP4. Means (±SEM) of nine replicates are shown. *P < 0.05; **P < 0.001.

Figure 2.

Response of PAH PAECs to ataluren is dose dependent. PAECs with the BMPR2 R321X mutation were treated for 24 hours with a range of ataluren concentrations as indicated, followed by the addition of BMP9 for 4 hours. (A) miR-27a normalized to RNU48. Induction of miR-27a is dose dependent to 20 μM, then plateaus. (B) ID1 mRNA normalized to GAPDH, as a readout of canonical BMP signaling. R321X shows no deficit in ID1 induction and no change with ataluren treatment. Means (±SEM) of nine replicates are shown.

Figure 3.

Ataluren corrects miR processing in endothelial cells and pulmonary artery smooth muscle cells (PASMCs). Cells were treated with 15 μM ataluren for 24 hours, then BMP9 (PAEC, L-EPC) or BMP4 (PASMC) for 4 hours. RNA was analyzed for miR-27a, normalized to RNU48. Results are shown as fold change of miR-27a with BMP treatment compared with vehicle. Means (±SEM) of nine replicates are shown.

Figure 4.

Ataluren restores bone morphogenetic protein receptor (BMPR)-II protein levels and increases Smad phosphorylation. (A) Human endothelial cells were treated with 20 μM ataluren for 24 hours. Total BMPR-II protein levels were assessed using immunoblotting. Blots were reprobed for α-tubulin and normalized to account for loading differences. Quantification was performed across two replicate blots, and is indicated by the values and plots below the blot. (B) W9X L-EPCs were treated with 20 μM ataluren for 24 hours and then induced with BMP9 for 4 hours. Immunoblotting was performed with antibodies against phospho-Smad1/5 or total-Smad1/5/8. Blots were quantitated and the phospho-Smad signal was normalized to total-Smad levels. Bars indicate the fold change of phospho-Smad in BMP9-treated cells compared with vehicle.

Figure 5.

Effects of ataluren readthrough on the proliferation of PAECs. Live cell counts show that ataluren treatment leads to growth suppression of R321X (A) and restores BMP-mediated growth repression in response to BMP9 (B) (post hoc P values versus control at 72-h: R321X + ataluren, P < 0.001; R294X + ataluren + BMP9 versus control + BMP9, P = 0.42). In cell proliferation assays using XTT: (C) untreated PAECs from heritable PAH (HPAH) proliferate significantly faster than controls (*post hoc pairwise P values at 72 h; all P < 0.001); (D) BMP9 treatment slows proliferation in all cells, but HPAH cases still proliferate faster than untreated control cells (*P < 0.001); (E) Ataluren treatment significantly represses the growth of R321X and R294X cells, but has no effect on Δex1–8 cells (*P < 0.001); (F) BMP9 induction after ataluren treatment shows restoration of BMP growth repression in R321X and R294X, but not Δex1–8 cells. Means (±SD) of triplicate wells are shown.