An mTOR Signaling Modulator Suppressed Heterotopic Ossification of Fibrodysplasia Ossificans Progressiva

Abstract

Fibrodysplasia ossificans progressiva (FOP) is a rare and intractable disorder characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor gain-of-function mutations in ACVR1 (FOP-ACVR1), a type I receptor for bone morphogenetic proteins. Despite numerous studies, no drugs have been approved for FOP. Here, we developed a high-throughput screening (HTS) system focused on the constitutive activation of FOP-ACVR1 by utilizing a chondrogenic ATDC5 cell line that stably expresses FOP-ACVR1. After HTS of 5,000 small-molecule compounds, we identified two hit compounds that are effective at suppressing the enhanced chondrogenesis of FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) and suppressed the heterotopic ossification (HO) of multiple model mice, including FOP-ACVR1 transgenic mice and HO model mice utilizing FOP-iPSCs. Furthermore, we revealed that one of the hit compounds is an mTOR signaling modulator that indirectly inhibits mTOR signaling. Our results demonstrate that these hit compounds could contribute to future drug repositioning and the mechanistic analysis of mTOR signaling.

Keywords: ACVR1; activin A; bone morphogenetic protein (BMP); endochondral ossification; fibrodysplasia ossificans progressiva (FOP); heterotopic ossification; high-throughput screening (HTS); induced pluripotent stem cell (iPSC); mammalian target of rapamycin (mTOR); transforming growth factor β (TGF-β).

Graphical abstract

Figure 1

Construction and Validation of the Compound Screening System (A) Vector map of the Dox-inducible ACVR1 expression vector. (B) The expression of ACVR1 and mCherry in ATDC5/FOP-ACVR1 24 hr after 2 ng/mL Dox treatment. Scale bar, 100 μm. (C) ALP activity of ATDC5/WT-ACVR1 or FOP-ACVR1 72 hr after Dox treatment. (D) Concentration response curves of BMP-4 and activin A in ATDC5/WT-ACVR1 or FOP-ACVR1 72 hr after 3 ng/mL Dox and ligand treatment. (E) DMH-1 (ACVR1 kinase inhibitor) inhibited the ALP activity but not the viability (AlamarBlue) of ATDC5/FOP-ACVR1. ALP and AlamarBlue assays were performed 72 hr after Dox and DMH-1 treatment. Results are the mean ± SE, n = 1 (C) or biological triplicate in three independent experiments (D and E).

Figure 2

Schematic and Detailed Results of High-Throughput Screening (A) Schematic of the first screening. (B–E) Scatterplot distribution of ALP activity (B), viability (C), Z′ factor (D), and S/B ratio (E) from the first screening against 4,892 compounds. (F) Classification of 79 hit compounds through the second screening. (G) Results of the HTS campaign and follow-up screens. Biological duplicates (B–F).

Figure 3

Detailed Dose-Response Assay Results of 14 Hit Compounds (A) Dose-response curves of 14 hit compounds. ALP assay and AlamarBlue assay were performed using the same protocol as the HTS. (B) IC₅₀ values and viability (%) at 10 μM in the dose-response assay, highest stage, and putative mechanism of 14 hit compounds are shown. Seven compounds (red) satisfied the criteria (IC₅₀ of ALP assay <500 nM and viability at 10 μM >50%). Results are the mean ± SE, biological triplicates.

Figure 4

AZD0530, PD 161570, and TAK 165 Suppressed the Chondrogenic Induction of FOP-iMSCs (A) The inhibitory effect of seven hit compounds on the chondrogenic induction of FOP-iMSCs. The cells were harvested 7 days after chondrogenic induction, which was performed with or without activin A and inhibitors (1 μM). (B) AZD0530, PD 161570, and TAK 165 suppressed the chondrogenic induction of FOP-iMSCs in a dose-dependent manner. (C) Alcian blue staining of DMH-1, AZD0530, PD 161570, and TAK 165. Results are the mean ± SE, biological triplicates used FOP-iPSCs (vFOP4-1) (A and B). n.s., no significant difference; ^∗∗∗p < 0.001 by Dunnett's multiple comparisons t test compared with the DMSO treatment control with activin A. Scale bar, 200 μm.

Figure 5

AZD0530 and TAK 165 Suppressed HO in FOP-ACVR1 Conditional Transgenic Mice (A) Schematic of the in vivo efficacy study utilized FOP-ACVR1 conditional transgenic mice. Intraperitoneal (i.p.) administration of 5 mg/kg AZD0530 and TAK 165 (once daily, five times a week) suppressed the HO in FOP-ACVR1 (R206H) conditional transgenic mice. HO was induced by muscle injury triggered by cardiotoxin (CTX) injection and oral administration of Dox. Three weeks after CTX injection and drug administration, mice were analyzed. (B) X-rays (upper panels) and μCT (lower panels) observations. Scale bars, 10 mm. (C) Average heterotopic bone volume. (D) Histological analysis of the CTX-injected region. H&E staining, safranin O staining (acidic proteoglycan), von Kossa staining (calcium), and anti-COL1 (bone) staining are shown. Scale bars, 100 μm (H&E, safranin O, and von Kossa) and 500 μm (COL1 and hVimentin). (E) Body weight change (%) of mice administered compounds. Results are the mean ± standard error (SE), n = 6 (vehicle), n = 7 (AZD0530), or n = 5 (TAK 165). ^∗∗p < 0.01 by Dunnett's multiple comparisons t test compared with vehicle treatment group (C). No significant differences between the AZD- or TAK-administered group compared with the vehicle group in two-way repeated-measures ANOVA followed by Dunnett's multiple comparisons t test (E).

Figure 6

AZD0530 and TAK 165 Suppressed HO Derived from FOP-iMSCs In Vivo (A) Schematic of the in vivo efficacy study utilized a human FOP-iPSC-based in vivo model. Intraperitoneal (i.p.) administration of 5 mg/kg AZD0530 or TAK 165 (once daily, five times a week) suppressed the HO derived from FOP-iMSCs triggered by activin A. Eight weeks after transplantation and drug administration, mice were analyzed. (B) X-ray (upper panels) and μCT (lower panels) observations. Scale bars, 10 mm. (C) Average heterotopic bone volume. (D) Histological analysis of the cell-transplanted region. H&E, safranin O, von Kossa, anti-COL1, and anti-human vimentin staining are shown. Scale bars, 100 μm (H&E, safranin O, and von Kossa) and 500 μm (COL1 and hVimentin). (E) Body weight change (%) of mice administered compounds. Results are the mean ± SE, n = 8 (vehicle), n = 10 (AZD0530), or n = 12 (TAK165). ^∗p < 0.05 by Dunnett's multiple comparisons t test compared with vehicle treatment group (C). No significant differences between the AZD- or TAK-administered group compared with the vehicle group in two-way repeated-measures ANOVA followed by Dunnett's multiple comparisons t test (E).

Figure 7

Mechanism of Action of AZD0530, PD 161570, and TAK 165 (A and B) AZD0530 and PD 161570, but not TAK 165, inhibited both BMP signaling (A) and TGF-β signaling (B). FOP-iMSCs transiently transfected with BRE-Luc (A) or CAGA-Luc (B) with CMV-Renilla were stimulated with activin A and compounds for 16 hr (A) or 3 hr (B). (C) ERBB2 knockdown did not reduce GAG content in the chondrogenic assay of FOP-iMSCs. One day after siRNA transfection, chondrogenic induction with activin A was initiated, and after 7 days the cells were harvested. (D–F) TAK 165 indirectly inhibited mTOR signaling. (D) TAK 165 did not inhibit mTOR signaling directly, as assessed by western blotting of the phosphorylation of S6 (p-S6), a surrogate marker of mTORC1 activity. FOP-iMSCs cultured with 10% FBS were treated with 100 nM rapamycin (Rapa) or TAK 165 (TAK) for 2 hr, and the cells were harvested. (E and F) TAK 165 indirectly inhibited p-S6 during chondrogenic induction with activin A. After 24 hr or 7 days of chondrogenic induction of FOP-iMSCs with activin A and test compounds, the cells were harvested. 1 μM TAK, 1 μM CP (CP-724714, another selective ERBB2 inhibitor), or 10 nM Rapa were applied in the experiments. Results are the mean ± SE of biological quadruplicates (A and B) or triplicates (C–F) using FOP-iPSCs (vFOP4-1). n.s., no significant difference; ^∗∗p < 0.01, ^∗∗∗p < 0.001 by Dunnett's multiple comparisons t test compared with the siRNA-transfected negative control and activin A (C) or with the DMSO treatment control and activin A (A, B, D–F).