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The neurofibromatosis type 1 (NF1) RASopathy is associated with persistent fibrotic nonunions (pseudarthrosis) in human and mouse skeletal tissue. Here, we performed spatial transcriptomics to define the molecular signatures occurring during normal endochondral healing following fracture in mice. Within the control fracture callus, we observed spatially restricted activation of morphogenetic pathways, such as TGF-β, WNT, and BMP. To investigate the molecular mechanisms contributing to Nf1-deficient delayed fracture healing, we performed spatial transcriptomic analysis on a Postn-cre;Nf1fl/- (Nf1Postn) fracture callus. Transcriptional analyses, subsequently confirmed through phospho-SMAD1/5/8 immunohistochemistry, demonstrated a lack of BMP pathway induction in Nf1Postn mice. To gain further insight into the human condition, we performed spatial transcriptomic analysis of fracture pseudarthrosis tissue from a patient with NF1. Analyses detected increased MAPK signaling at the fibrocartilaginous-osseus junction. Similar to that in the Nf1Postn fracture, BMP pathway activation was absent within the pseudarthrosis tissue. Our results demonstrate the feasibility of delineating the molecular and tissue-specific heterogeneity inherent in complex regenerative processes, such as fracture healing, and reconstructing phase transitions representing endochondral bone formation in vivo. Furthermore, our results provide in situ molecular evidence of impaired BMP signaling underlying NF1 pseudarthrosis, potentially informing the clinical relevance of off-label BMP2 as a therapeutic intervention.

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.

Neurofibromatosis type 1 (NF1) is a tumor predisposition syndrome caused by heterozygous NF1 gene mutations. Patients with NF1 present with pleiotropic somatic secondary manifestations, including development of bone pseudarthrosis after fracture. Somatic NF1 gene mutations were reproducibly identified in patient-derived pseudarthrosis specimens, suggesting a local mosaic cell population including somatic pathologic cells. The somatic cellular pathogenesis of NF1 pseudarthroses remains unclear, though defects in osteogenesis have been posited. Here, we applied time-series single-cell RNA-sequencing (scRNA-seq) to patient-matched control and pseudarthrosis-derived primary bone stromal cells (BSCs). We show that osteogenic specification to an osteoblast progenitor cell population was evident for control bone-derived cells and haploinsufficient pseudarthrosis-derived cells. Similar results were observed for somatic patient fracture-derived NF1 -/- cells; however, expression of genetic pathways associated with skeletal mineralization were significantly reduced in NF1 -/- cells compared with fracture-derived NF1 +/- cells. In mice, we show that Nf1 expressed in bone marrow osteoprogenitors is required for the maintenance of the adult skeleton. Results from our study implicate impaired Clec11a-Itga11-Wnt signaling in the pathogenesis of NF1-associated skeletal disease. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)., (© 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).)