(A) Heterotopic ossification was induced at P7 injecting adenovirus-Cre (Ad.Cre) and cardiotoxin in the mice hindlimb. The pattern of 5 administrations of DMSO or BYL719 (25 mg/kg) is indicated by grey dots started at P8 (start day 1), P10 (delayed start day 3), and P14 (delayed start day 7). One group only received the administration of DMSO or BYL719 (25 mg/kg) at P8, P9, and P10 (first 3 days post-HO-induction), as indicated by grey dots. The final time-point, P30, was conserved between experimental groups. (B) Quantification of heterotopic ossifications bone volume (mm3) of each experimental group. Colored symbols indicate the presence of heterotopic ossifications. Black symbols indicate the absence of heterotopic ossifications. Data shown are of each individual mouse with group median. *P<0.05, **P<0.01, Kruskal–Wallis test with Dunn’s multiple comparison test. (C) Representative 3D microtomography images of the injected hindlimbs of 3 different mice for each experimental group. White arrows show heterotopic ossification.

(A) Heterotopic ossification was induced at P7 injecting adenovirus-Cre (Ad.Cre) and cardiotoxin in the mice hindlimb. Starting at P8, mice were treated with DMSO or BYL719 (25 mg/kg) as indicated by grey dots for 5 administrations. (B) Quantification of heterotopic ossifications bone volume (mm3) of each experimental group. Colored symbols indicate the presence of heterotopic ossifications. Black symbols indicate the absence of heterotopic ossifications. Data shown are of each individual mouse with group median. *P<0.05, **P<0.01, Kruskal–Wallis test with Dunn’s multiple comparison test. (C) Quantification of the ratio of bone volume per tissue volume (BV/TV) within heterotopic ossifications of each experimental group, including only mice with detected HO. Data shown are of each individual mouse with the group median. **P<0.01, ***P<0.001, Kruskal-Wallis test with Dunn’s multiple comparison test. (D) Representative 3D frontal microtomography images of the injected hindlimbs of mice for each experimental group and a detailed close-up image for each selected mouse. White arrows show heterotopic ossification in the close-up images.

mRNA expression of Acvr1 (A) or p110α (B) genes in BM-MSCs from p110αfl/fl mice, infected with virus expressing wild-type Acvr1 (WT) or Acvr1R206H (RH) and/or Cre co-infection. (A) Comparison to the endogenous expression level of Acvr1 gene in mock-transfected BM-MSCs is shown as a dotted horizontal line. Data are shown as mean ± SD (n = 12 per group). Unpaired t-test between transfected groups. (B) Data are shown as mean ± SD (n = 3 per group). ***P<0.001, two-way ANOVA with Tukey’s multiple comparisons test. (C) mRNA expression of canonical (Id1 and Sp7), non-canonical (Ptgs2) target genes, and activin A (Inhba) in BM-MSCs p110αfl/fl transfected with Acvr1 (wild type or R206H) with or without Cre recombinase. Cells were NT (not treated), or treated with BYL719 (2µM) and/or activin A (2nM). Expression data was normalized to those of control cells which were transfected only with Acvr1 WT without any treatment, shown as a dotted horizontal line. Asterisks (*) refer to the differences between Acvr1 RH cells compared to control cells or Acvr1 RH cells without Cre recombinase treated with BYL719 or activin A. Hash signs (#) refer to the differences between Acvr1 RH cells without Cre recombinase treated with activin A compared to the same condition with Cre recombinase, or compared with Acvr1 RH cells treated with BYL719 and/or activin A. Data are shown as mean ± SD (n = 6 per group). * or # P<0.05, ** or ## P<0.01, *** or ### P<0.001, two-way ANOVA with Tukey’s multiple comparisons test.

Transforming growth factor-β receptor (TGFβR) kinase assays. (A) A schematic depiction of nano-bioluminescence resonance energy transfer (nanoBRET) target engagement assays. TGFβR-Nanoluciferase fusion proteins are expressed in live COS-1 cells and an ATP-like tracer analogue causes proximity-based BRET from the Nanoluciferase donor to the fluorescent tracer acceptor. Competition of the tracer with a possible ATP-pocket inhibitor would result in decreased nanoBRET. (B) The nanoBRET emission spectra consist of the Nanoluciferase donor (460nm) and the fluorescent acceptor (610nm). The nanoBRET ratio is shown in milliBRET units (mBU) by dividing the acceptor emission by the donor emission times 1000. (C) NanoBRET target engagement analyses of ALK1, ALK2 (ACVR1), ALK2R206H, ALK3, ALK4, ALK5, TGFBR2, ACVR2A, ACVR2B and BMPR2 testing 1 or 10 µM BYL719 with n=4. As controls, LDN193189 (0,5 µM), SB431542 (10 µM), and ML347 (10 µM) were used. Data are shown as mean ± SD. One-way ANOVA with Dunnett’s multiple comparisons test. (D) Casein phosphorylation by ACVR1R206H kinase. Phosphorylation was performed in the presence of ACVR1R206H kinase and increasing concentrations of the PI3Kα inhibitor BYL719. Quantification of kinase activity. Data shown as mean ± SD (n=4 independent experiments). One-way ANOVA with Tukey’s multiple comparisons test. * P<0.05, ** P<0.01, *** P<0.001.

Analyses of chondrogenic progenitor specification (using differentiation medium; DM) of hMSC-ACVR1/ALK2WT or hMSC-ACVR1/ALK2R206H treated with BYL719 (10 µM) with and without Activin A (50 ng/ml), compared to growth medium (GM). (A) Representative images of the ALP staining after one week of chondrogenic progenitor specification. The images were obtained at 5x magnification and the scale bar represents 1000 µm. (B) Quantification of the ALP staining by densitometry (n=3 per group). (C) Representative images of Alcian Blue staining of micromass cultures after 3 weeks of chondrogenic progenitor specification. The images were taken at 2.5x magnification and the scale bar represents 500 µm. (D) Quantification of the solubilized Alcian Blue staining via the measurement of absorbance (595nm) (n=3 per group). (E) RT-qPCR results of COL1A1 and ALPL after 3 days of chondrogenic progenitor specification (n=3 per condition). Data are shown as mean ± SD. In all figure panels, significance is detailed between DM and DM with BYL719, and between DM with Act A and DM with Act A and BYL719. * P<0.05, ** P<0.01, *** P<0.001, two-way ANOVA with Tukeýs multiple comparisons test.

Bulk RNA sequencing of hMSC-ACVR1/ALK2R206H upon overnight starvation, 30 minutes pre-treatment with 1 µM BYL719 and 1 hour Activin A (50 ng/ml) stimulation including continued BYL719 treatment. (A) Schematic depiction of the experimental setup. (B) Gene Set Enrichment Analysis (GSEA) with the groups described in (B), showing the significant enrichment plots of the gene ontology sets ossification (GO: 0001503, padj=3.83e-5) and osteoblast differentiation (GO:0001649, padj=3.10e-5). (C) A heatmap of the top 40 most relevant genes within the ossification GO geneset derived from the leading-edge subset of the enrichment plot (n=4 per group). The statistics are described in the methods section.

(A, B, C) Proliferation assays of THP1 (A), RAW264.7 (B), and HMC-1 (C) cells. Cells were cultured for 6 days and in control conditions, with BMP6 (2nM) and/or BYL719 (2 or 10µM). Areas under the proliferation curves were compared by one-way ANOVA with Dunnett’s multiple comparisons test against the control. An asterisk (*) refers to significant differences between control and single treatments (BMP6 or BYL719). A hash sign (#) refers to significant differences between control and combined treatments. Data shown as mean ± SD (n = 4 per group). ** or # # P<0.01, *** or # # # P<0.001. (D) Migration assay of THP1 cells with control conditions, or with FBS or BMP6 as chemotactic agents with or without BYL719 treatment. Data are shown as mean ± SD (n = 4 per group). ***P<0.001, two-way ANOVA with Tukey’s multiple comparisons test. (E, F, G) Gene expression assays of THP1 (E), RAW264.7 (F) and HMC-1 (G) cells. Cells were treated for 48 hours with BYL719 (2µM) and/or BMP6 (2nM). Data are shown as mean ± SD (n = 4 per group). *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA with Dunnett’s multiple comparisons test, significance shown between control group and other groups.

(A) Heterotopic ossification was induced at P7 injecting adenovirus-Cre (Ad.Cre) and cardiotoxin in the mice hindlimb. Starting at P8, mice were treated with DMSO or BYL719 (25 mg/kg) as indicated by grey dots. The final time-point changed between experimental groups, stopping at P9 (2 days post-HO-induction), P11 (4 days post-HO-induction), P16 (9 days post-HO-induction), P23 (16 days post-HO-induction), and P30 (23 days post-HO-induction). (B) The quantification of F4/80-positive monocytes/macrophages per field view from immunohistochemistry staining, with 20x amplification as shown in the representative images depicted in Figure 8C. Five images were randomly acquired per mice, from 2 mice per group, for a total of 10 quantified images per group. F4/80 positive cells were detected as cells with dark brown staining. Data are shown as mean ± SD. *P<0.05, **P<0.01, ***P<0.001, two-way ANOVA with Sidak’s multiple comparisons test, comparing both groups on each day (n=10). (C) Representative images from the quantified immunohistochemistry staining for F4/80 to detect monocytes/macrophages. Representative images are shown for each final time-point at day 2, 4, 9, 16, and 23 post-HO-induction. All images were obtained at 20x, with a representative scale bar at 100 µm. (D) Quantification of CEM-positive mast cells per field view from CEM staining, with 20x amplification as shown in the representative images depicted in Figure 8E. Three images were obtained per mice, from 4 mice per group, for a total of 12 quantified images per group. Mast cells were detected as cells with bright blue staining. Data are shown as mean ± SD. ***P<0.001, two-way ANOVA with Sidak’s multiple comparisons test, comparing both groups in each day (n=12). (E) Representative images from the quantified C.E.M. staining to detect mast cells, highlighted with black arrows. Representative images are shown for each final time-point at day 2, 4, 9, 16, and 23 post-HO-induction. All the images were obtained at 20x magnification, with a representative scale bar at 100 µm.