C-type natriuretic peptide analog treatment of craniosynostosis in a Crouzon syndrome mouse model

RESEARCH ARTICLE C-type natriuretic peptide analog treatment of craniosynostosis in a Crouzon syndrome mouse model Greg Holmes1*, Lening Zhang2, Joshua Rivera1, Ryan Murphy2, Claudia Assouline1, Lorraine Sullivan2, Todd Oppeneer2, Ethylin Wang Jabs1 1 Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America, 2 BioMarin Pharmaceutical, Novato, California, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Holmes G, Zhang L, Rivera J, Murphy R, Assouline C, Sullivan L, et al. (2018) C-type natriuretic peptide analog treatment of craniosynostosis in a Crouzon syndrome mouse model. PLoS ONE 13(7): e0201492. https://doi. org/10.1371/journal.pone.0201492 Editor: Damian Christopher Genetos, University of California Davis, UNITED STATES Received: April 26, 2018 Accepted: July 16, 2018 Published: July 26, 2018 Copyright: © 2018 Holmes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. * Abstract Activating mutations of fibroblast growth factor receptors (FGFRs) are a major cause of skeletal dysplasias, and thus they are potential targets for pharmaceutical intervention. BMN 111, a C-type natriuretic peptide analog, inhibits FGFR signaling at the level of the RAF1 kinase through natriuretic peptide receptor 2 (NPR2) and has been shown to lengthen the long bones and improve skull morphology in the Fgfr3Y367C/+ thanatophoric dysplasia mouse model. Here we report the effects of BMN 111 in treating craniosynostosis and aberrant skull morphology in the Fgfr2cC342Y/+ Crouzon syndrome mouse model. We first demonstrated that NPR2 is expressed in the murine coronal suture and spheno-occipital synchondrosis in the newborn period. We then gave Fgfr2cC342Y/+ and Fgfr2c+/+ (WT) mice once-daily injections of either vehicle or reported therapeutic levels of BMN 111 between post-natal days 3 and 31. Changes in skeletal morphology, including suture patency, skull dimensions, and long bone length, were assessed by micro-computed tomography. Although BMN 111 treatment significantly increased long bone growth in both WT and mutant mice, skull dimensions and suture patency generally were not significantly affected. A small but significant increase in the relative length of the anterior cranial base was observed. Our results indicate that the differential effects of BMN 111 in treating various skeletal dysplasias may depend on the process of bone formation targeted (endochondral or intramembranous), the specific FGFR mutated, and/or the specific signaling pathway changes due to a given mutation. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Funding was provided by BioMarin Pharmaceutical, Inc. BioMarin Pharmaceutical, Inc., provided support in the form of partial salaries (GH, EWJ), reagents, equipment, and facilities, and had a role in the study design, data collection and analysis, decision to publish, and preparation of the manuscript. The specific roles of the authors employed by BioMarin Pharmaceutical included Introduction Skeletal dysplasias comprise a diverse group of disorders. The skeleton forms via both intramembranous ossification, by which osteoblasts differentiate directly from mesenchyme to form bone, and endochondral ossification, by which a cartilage template of individual bones is first established and then replaced by osteoblasts to form the final bone. The flat bones of the skull form via intramembranous ossification, while the base of the skull, vertebrae, and long PLOS ONE | https://doi.org/10.1371/journal.pone.0201492 July 26, 2018 1 / 14 BMN 111 treatment of Crouzon syndrome mice imaging methodology, data curation, visualization, and analysis, which were performed by LZ and RM, who along with LS and TO reviewed and edited the manuscript. Their specific roles are articulated in the ‘Author Contributions’ section. Additional funding was supplied by National Institute of Health (NIH) National Institute of Dental and Craniofacial Research (https://www.nidcr.nih.gov/) R01 DE022988 (to EWJ) and NIH National Institute of Child Health and Human Development (https:// www.nichd.nih.gov/) P01 HD078233 (to EWJ). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH, which had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: LZ, RM, LS, and TO are employees of BioMarin Pharmaceutical, Inc., that sponsored this study. GH and EWJ received funding from BioMarin, but do not have any commercial interest in BioMarin Pharmaceutical, Inc. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. The internal publication review committee of BioMarin Pharmaceutical, Inc., approved the manuscript for publication. bones of the limbs form via endochondral ossification. Common skeletal dysplasias include craniosynostosis, in which the sutures separating the skull bones fuse prematurely [1], and chondrodysplasias resulting in dwarfism [2]. Activating mutations of fibroblast growth factor receptors (FGFRs) are a major cause of skeletal dysplasias, including syndromic craniosynostosis and chondrodysplasias [3]. For many of these conditions complex surgical intervention is the only therapeutic strategy, and thus pharmacological attenuation of FGFR activity is an attractive potential alternative for treatment of these skeletal dysplasias. C-type natriuretic peptide (CNP) binds the guanyl cyclase natriuretic peptide receptor 2 (NPR2) and activation of NPR2 results in the inhibition of the FGFR signaling pathway at the level of the RAF1 kinase [4, 5]. Total or chondrocyte-specific genetic deletion of Nppc (the gene encoding CNP) [6–8] or Npr2 [8, 9] in mice severely impairs endochondral ossification, resulting in dwarfism characterized by shortening of the vertebrae, long bones, and skull. Conversely, overexpression of CNP in chondrocytes using a collagen 2 promoter sequence [10] or systemically from the liver using a human serum amyloid P promoter [11] results in skeletal overgrowth. Chondrocyte-specific or systemic overexpression of CNP from these transgenic alleles was also able to ameliorate the dwarfism phenotype of the transgenic Fgfr3ACH/+ achondroplasia mouse model [10, 12], in which Fgfr3G380R expression was also targeted to cartilage using a collagen 2 promoter sequence [13]. Loss-of-function mutations in the human NPR2 gene result in short stature and the dwarfism syndrome acromesomelic dysplasia, Maroteaux type [14, 15]. CNP or modified CNP analogs are considered a potential therapeutic strategy for the treatment of h (...truncated)