MEK-SHP2 inhibition prevents tibial pseudarthrosis caused by NF1 loss in Schwann cells and skeletal stem/progenitor cells

Abstract

Congenital pseudarthrosis of the tibia (CPT) is a severe pathology marked by spontaneous bone fractures that fail to heal, leading to fibrous nonunion. Half of patients with CPT are affected by the multisystemic genetic disorder neurofibromatosis type 1 (NF1) caused by mutations in the NF1 tumor suppressor gene, a negative regulator of RAS-mitogen-activated protein kinase (MAPK) signaling pathway. Here, we analyzed patients with CPT and Prss56-Nf1 knockout mice to elucidate the pathogenic mechanisms of CPT-related fibrous nonunion and explored a pharmacological approach to treat CPT. We identified NF1-deficient Schwann cells and skeletal stem/progenitor cells (SSPCs) in pathological periosteum as affected cell types driving fibrosis. Whereas NF1-deficient SSPCs adopted a fibrotic fate, NF1-deficient Schwann cells produced critical paracrine factors including transforming growth factor-β and induced fibrotic differentiation of wild-type SSPCs. To counteract the elevated RAS-MAPK signaling in both NF1-deficient Schwann cells and SSPCs, we used MAPK kinase (MEK) and Src homology 2 containing protein tyrosine phosphatase 2 (SHP2) inhibitors. Combined MEK-SHP2 inhibition in vivo prevented fibrous nonunion in the Prss56-Nf1 knockout mouse model, providing a promising therapeutic strategy for the treatment of fibrous nonunion in CPT.

Figure 1:. Schwann cells and SSPCs within periosteum harbor NF1 biallelic inactivation in CPT

A. X-ray of the tibia and fibula pseudarthrosis (white arrows) of patient with CPT P15. B. Experimental design. DNA was extracted from tissues or periosteal SSPCs (pSSPCs) collected at the pseudarthrosis (PA) site and the iliac crest (IC), and from blood of patients with CPT undergoing surgery, and NF1 targeted sequencing was performed. C. NF1 genotyping of tissues from 17 patients with CPT shows the absence of NF1 biallelic inactivation in blood and IC and the presence of NF1 biallelic inactivation in the periosteum at PA site in 13/17 patients. NF1 biallelic inactivation was also detected in 6/17 patients in fibrous tissue and bone, in 4/17 in bone marrow, in 3/17 in muscle, and in 2/17 in PA site skin. D. Number of patients with NF1-related CPT and isolated CPT carrying NF1 biallelic inactivation. E. NF1 genotyping of periosteum and SSPCs from PA site shows the presence of NF1 biallelic inactivation in 9/13 patients. F. Left: experimental design. Cell populations were digested and sorted from PA periosteum of patient P15 and the frequency of the 2 NF1 point mutations (c.574C>T and c.5839C>T) was determined using droplet digital PCR (ddPCR). Right: Percentage of the 2 mutations in the different cell populations shows that Schwann cells and SSPCs carry both NF1 hits, but not endothelial and immune cells. (n= 3 replicates). G. Phospho-ERK (pERK) immunofluorescence on periosteum sections showing the number of pERK+ cells in the periosteum from PA site compared to the periosteum from IC (white arrowheads). Quantification of the percentage of pERK+ cells in the periosteum from PA and IC (n=5–6 patients per group). H. Co-immunofluorescence of pERK and CD90, SOX10, CD31, CD68 and αSMA on PA periosteum sections. I. Quantification of pERK+ cells in PA periosteum compared to IC periosteum (n= 5–6 patients per group). ** p < 0.01. BM: Bone marrow. Endo: endothelial cells. Scale bars: 50μm.

Figure 2:. Tibial pseudarthrosis in mice lacking Nf1 gene in boundary cap-derived pSSPCs and Schwann cells

A. Longitudinal sections of uninjured tibia periosteum (po) from 3-month-old Prss56-Nf1^+/+ mice stained with Hematoxylin-Eosin and immunofluorescence on adjacent sections showing tdTom+ periosteal skeletal stem/progenitor cells (pSSPCs) expressing PDGFRα and tdTom+ Schwann cells (SCs) expressing SOX10 along TH+ nerves (orange box: transverse imaging). B. Left: Experimental design of tibial fracture in Prss56^Cre; R26^tdTom; Nf1^+/+ (Prss56-Nf1^+/+) control, Prss56^Cre; R26^tdTom; Nf1^fl/fl (Prss56-Nf1^fl/fl) and Prss56^Cre; R26^tdTom; Nf1^fl/− (Prss56-Nf1^fl/−) mutant mice. Right: Histomorphometric quantification of the volume of callus fibrosis at days 7, 14, 21 and 28 post-fracture in Prss56-Nf1^+/+, Prss56-Nf1^fl/fl and Prss56-Nf1^fl/− mice (n=5–6 mice per group). C. Top: Representative microCT images of calluses from Prss56-Nf1^+/+, Prss56-Nf1^fl/fl and Prss56-Nf1^fl/− mice at 28 days post-fracture, showing absence of bone bridging in Prss56-Nf1^fl/fl and Prss56-Nf1^fl/− mutant mice (white arrows). Bottom, high magnification of callus periphery showing bone bridging (black arrows) in Prss56-Nf1^+/+ control mice, and fibrosis and unresorbed cartilage (red, Safranin’O (SO)) in Prss56-Nf1^fl/fl and Prss56-Nf1^fl/− mutant mice. D. Percentage of calluses from Prss56-Nf1^+/+, Prss56-Nf1^fl/fl and Prss56-Nf1^fl/− mice showing bone union (white), semi-union (grey), or nonunion (black) on microCT scan at day 28 post-fracture (n=6 mice per group). Bone union was significantly different between mutant and control mice (***, p=0.0067) but not between the mutant groups (p=0.52). E. Lineage tracing of Prss56-expressing Boundary Cap (BC)-derived tdTom+ cells (white arrowheads) in callus cartilage (labelled by SOX9) and fibrosis (labelled by POSTN) of Prss56-Nf1^+/+ and Prss56-Nf1^fl/fl mice 14 days after tibial fracture. F. Quantification of tdTom+ signal in cartilage and fibrosis of Prss56-Nf1^+/+ and Prss56-Nf1^fl/fl mice 14- and 28-days post-fracture (n=5 mice per group). G. RNAscope and immunofluorescence on callus sections of Prss56-Nf1^fl/fl mice day 14 post-fracture show the presence of Postn-expressing tdTom⁺ fibroblasts and SOX10⁺tdTom⁺ SCs in fibrotic tissue. TdTom⁺ SC are SOX2⁺ and MBP⁻ and are localized along TH⁺ nerves. po: periosteum, b: bone, fib: fibrosis, cart: cartilage, bm: bone marrow, MBP, Myelin Basic Protein; TH, Tyrosine Hydroxylase. p-value: * p < 0.05, ** p < 0.01. Scale bars: Panel A: 25μm. Panel C-microCT: 1mm. Panel E/C-histology: 100μm. Panel G: 10μm.

Figure 3:. Fibrotic fate of NF1-deficient periosteal SSPCs in patients with CPT and Prss56-Nf1 KO mice

A. Experimental design. Nuclei were extracted from PA or IC periosteum, sorted, and processed for single-nuclei RNAseq. The datasets were integrated for analyses. B. UMAP projection of color-coded clustering (top) and sampling (bottom) of the integration of the datasets of IC periosteum from P15 (IC-P15, green), IC periosteum from P13 (IC-P13, blue), PA periosteum from P13 (PA-P13, red) and PA periosteum from P5 (PA-P5, yellow). C. Feature plots of the SSPC/fibroblast lineage score and ADAM12/NCAM1 (Neural Cell Adhesion Molecule 1), PDGFRA, ACAN, and RUNX2 gene expression. D. Percentage of cells from PA and IC samples in SSPC/fibroblast populations and in ADAM12+, PDGFRA+, and osteochondral clusters. E. Violin plots of the fibrotic, osteogenic, chondrogenic, MAPK activation, and cellular response to TGFβ lineage score in IC and PA. F. Experimental design. PA or IC pSSPCs from patients P3 and P4 were transplanted at the fracture site of immunodeficient mice. G. Representative callus sections stained with Picrosirius (PS). High magnification of cartilage stained with Safranin’O and fibrosis stained with PS and immunofluorescence of the human KU80 protein at day 14 post-fracture showing that IC pSSPC-derived cells are located mostly in cartilage while PA pSSPC-derived cells are located in fibrosis (white arrowheads). H. Left: Percentage of callus grafted with PA or IC pSSPCs showing union, semi-union, and nonunion at day 28 post-fracture. Right: Volume of fibrosis in day 14 and 28 post-fracture callus of immunodeficient mice grafted with human pSSPCs from IC and PA (n=6–8 mice per group). I. Left: Experimental design. Periosteum or cultured tdTom⁺ periosteal skeletal stem/progenitor cells (pSSPCs) were isolated from Prss56-Nf1^+/+ or Prss56-Nf1^fl/fl mice and transplanted at the fracture site of wild-type hosts. Middle: Representative images of the contribution of grafted tdTom+ cells (white arrowheads) showing cells from Prss56-Nf1^+/+ mice detected in cartilage (labelled by SOX9) and cells from Prss56-Nf1^fl/fl mice detected in fibrosis (labelled by POSTN). Right: Percentage of grafted tdTom+ cells in cartilage and fibrosis (n=5 mice per group). SMC: smooth muscle cells, cal: callus, fib: fibrosis, cart: cartilage. p-value: * p < 0.05, ** p < 0.01, **** p < 0.0001. Scale bars: Panel G: Low magnification: 1mm. High magnification: 100μm. Panel I: 100μm.

Figure 4:. Overactivation of MAPK pathway causes fibrotic differentiation of Nf1-deficient pSSPCs

A. Experimental design of single nuclei RNAseq (snRNAseq) experiment. Nuclei were isolated from uninjured periosteum, or periosteum and hematoma of wild-type mice at days 3, 5, and 7 post- tibial fracture, sorted, and processed for snRNAseq. B. UMAP projection of clustering and monocle pseudotime trajectory of the subset of SSPCs, injury-induced fibrogenic cells, osteoblasts, and chondrocytes from integrated uninjured, day 3, day 5, and day 7 post-fracture samples. The four populations are delimited by black dashed lines. C. Scatter plot of MAPK score along pseudotime. D. Scatter plots of MAPK score along chondrogenic lineage score, Sox9 expression, fibrogenic, and osteogenic lineage scores. E. Immunofluorescence of SOX9 and phospho-ERK (pERK) in day 7 post-fracture callus section of wild type (WT) mice. Quantification and correlation of SOX9 and pERK signal per cell (red line) (n = 397 cells from 8 callus sections of 4 mice). Scale bars: low magnification, 150μm; high magnification, 25μm. F. Left: Quantification of SOX9 and pERK fluorescent signal per tdTom+ cells in day 7 post-fracture callus of Prss56-Nf1^+/+ and Prss56-Nf1^fl/fl mice. Right: Correlation analysis of pERK and SOX9 signals in tdTom+ cells in Prss56-Nf1^+/+ (top) and Prss56-Nf1^fl/fl (bottom) mice (n = 209 to 238 cells from 9 sections of 3 mice per group). cart: cartilage, fib: fibrosis. ****: p < 0.0001.

Figure 5:. Pro-fibrotic effect of Nf1-deficient Schwann cells in fibrous nonunion

A. Left: Experimental design. tdTom+ Schwann cells (SCs) were isolated from Prss56-Nf1^+/+ or Prss56-Nf1^fl/fl mice and transplanted at the fracture site of wild-type hosts. Middle: MicroCT images of 28 days post-fracture calluses of wild-type mice grafted with SCs from Prss56-Nf1^+/+ or Prss56-Nf1^fl/fl mice, showing absence of bridging in mice grafted with Nf1-deficient SCs (white arrow). Right: Percentage of day 28 post-fracture calluses showing union, semi-union, or nonunion. Volume of callus fibrosis at 14 and 28 days post-fracture (n=4–5 mice per group). B. Experimental design. Nuclei were isolated from periosteum and hematoma at day 7 post-fracture of control or Prss56-Nf1^fl/fl mice and processed for snRNAseq. Datasets were integrated. C. UMAP projection of SSPC, injury-induced fibrogenic, chondrogenic, and osteogenic cell subsets from the integrated day 7 post-fracture control and Prss56-Nf1^fl/fl datasets. D. Violon plot of chondrogenic and fibrogenic lineage scores per dataset. Percentage of cells per cluster. E. RNAscope experiment on day 7 post-fracture callus from Prss56-Nf1^fl/fl mice shows the expression of Tgfb1, Pdgfa, and Osm by Sox10-, tdTom-expressing Schwann cells in callus fibrosis. F. Relative expression of Tgfb1 in day 7 post-fracture callus of Prss56-Nf1^+/+ or Prss56-Nf1^fl/fl mice (n=5 mice per group). G. Percentage of phospho-SMAD2 positive (pSMAD2+) cells in the day 7 post-fracture callus of Prss56-Nf1^+/+ or Prss56-Nf1^fl/fl mice (n=4 mice per group). Representative pSMAD2 immunofluorescence of Prss56-Nf1^+/+ or Prss56-Nf1^fl/fl callus. H. Top: Experimental design. Wild-type mice grafted with tdTom+ Schwann cells from Prss56-Nf1^fl/fl mice were treated with blocking TGFβ blocking antibody or IgG1 control isotype at days 5, 8, and 11 post-fracture. Middle: Picrosirius staining of fracture calluses at 14 days post-fracture. Bottom: Volume of callus fibrosis (n=4–5 mice per group). I. Top: Experimental design. Prss56-Nf1^fl/fl mice were treated with blocking TGFβ blocking antibody or IgG1control isotype at days 5, 8, and 11 post-fracture. Middle: MicroCT images of callus of Prss56-Nf1^fl/fl mice treated with IgG1 isotype control or TGFβ blocking antibody at 28 days post-fracture. High magnification of the callus periphery stained with Picrosirius. Bottom left: percentage of day 28 post-fracture calluses showing union (white), semi-union (grey) or nonunion (black) on microCT scan. Bottom right: Volume of callus fibrosis of Prss56-Nf1^fl/fl mice treated with blocking TGFβ blocking antibody or IgG1 isotype control at 28 days post-fracture. (n=4–5 mice per group). p-value: * p < 0.05, ** p < 0.01. Scale bars: Panel A/H 1mm. Panel E: 10μm. Panel G 50μm, panel I: Low magnification: 1mm, High magnification: 250 μm.

Figure 6:. Combined MEK and SHP2 inhibition prevents fibrous nonunion in Prss56-Nf1 KO mice

A. Experimental design. Periosteal SSPCs from PA site of patients with CPT were treated with MEK inhibitor (selumetinib), SHP2 inhibitor (SHP099), MEK and SHP2 inhibitors (selumetinib and SHP099), or vehicle (DMSO) for in vitro analyses. B. MAPK pathway activation in pSSPCs from PA site treated with selumetinib, SHP099, selumetinib and SHP099, or DMSO measured by the pERK/ERK ratio on Western blot. Statistical significance was determined compared to DMSO control (n=3 patients). C. Reduced in vitro proliferation of pSSPCs from PA site treated with combined selumetinib and SHP099 (n=3 patients). D. Increased in vitro chondrogenic differentiation measured by SOX9 expression of pSSPCs from PA site treated with combined selumetinib and SHP099 (n=3 patients in duplicates). E. Experimental design. Prss56-Nf1^fl/− mice were treated by oral gavage with selumetinib, SHP099, selumetinib and SHP099, or vehicle from days 5 to 14 post-fracture. F. Representative microCT images of callus from Prss56-Nf1^fl/− mice at 28 days post-fracture, with bone bridging indicated by white arrows. G. Percentage of calluses from treated and control Prss56-Nf1^fl/− mice showing bone union (white), semi-union (grey), or nonunion (black) on microCT scan at day 28 post-fracture (n=4–6 mice per group). H. Volume of callus, cartilage, bone, and fibrosis at days 14 and 28 post-fracture in treated and control Prss56-Nf1^fl/− mice (n=4–6 mice per group). I. Surface of tdTom signal in cartilage and fibrosis of day 14 and 28 callus from treated and control Prss56-Nf1^fl/− mice. Scale bars: 1mm. p-value: * p < 0.05, ** p < 0.01, *** p < 0.001.