Second-Generation SYK Inhibitor Entospletinib Ameliorates Fully Established Inflammation and Bone Destruction in the Cherubism Mouse Model

Abstract

Cherubism is a craniofacial disorder characterized by maxillary and mandibular bone destruction. Gain-of-function mutations in the SH3-domain binding protein 2 (SH3BP2) are responsible for the excessive bone resorption caused by fibrous inflammatory lesions. A homozygous knock-in (KI) mouse model for cherubism (Sh3bp2KI/KI ) develops autoinflammation resulting in systemic bone destruction. Although administration of the TNF-α blocker etanercept to neonatal Sh3bp2KI/KI mice prevented the disease onset, this therapy was not effective for adult Sh3bp2KI/KI mice or human cherubism patients who already had lesions. Because genetic ablation of spleen tyrosine kinase (SYK) in myeloid cells rescues Sh3bp2KI/KI mice from inflammation, we examined whether SYK inhibitor administration can improve fully developed cherubism symptoms in adult Sh3bp2KI/KI mice. Entospletinib (GS-9973) was intraperitoneally injected into 10-week-old Sh3bp2KI/KI mice every day for 6 weeks. Treatment with GS-9973 improved facial swelling and histomorphometric analysis of lung and liver tissue showed that GS-9973 administration significantly reduced inflammatory infiltrates associated with decreased levels of serum TNF-α. Micro-computed tomography (μCT) analysis showed that GS-9973 treatment reduced bone erosion in mandibles, calvariae, and ankle and elbow joints of Sh3bp2KI/KI mice compared to Sh3bp2KI/KI mice treated with dimethyl sulfoxide (DMSO). Taken together, the results demonstrate that administration of the SYK inhibitor ameliorates an already established cherubism phenotype in mice, suggesting that pharmacological inhibition of SYK may be a treatment option for cherubism patients with active disease progression. © 2018 American Society for Bone and Mineral Research.

Keywords: AUTOINFLAMMATION; BONE DESTRUCTION; CHERUBISM; ENTOSPLETINIB/GS-9973; SH3BP2; SYK.

Conflict of interest statement

Disclosures

All authors declare that they have no conflicts of interest.

© 2018 American Society for Bone and Mineral Research.

Figures

Fig. 1.

GS-9973 administration to actively inflamed 10-week-old Sh3bp2KI/KI mice improves facial swelling, body weight loss, and systemic autoinflammation. (A) Experimental sequence of GS-9973 administration. 10-week-old Sh3bp2+/+ and Sh3bp2KI/KI mice were treated with 100 mg/kg of GS-9973 or DMSO every day for 6 weeks. Mice were euthanized at 16 weeks of age for analysis. (B) Facial appearance of GS-9973- treated or DMSO-treated mice (top: before treatment at 10 weeks of age; bottom: after treatment at 16 weeks of age). Blue arrows indicate closed eyelids due to facial skin inflammation. GS-9973 treatment improved eyelid closure in Sh3bp2KI/KI mice at 16 weeks old (red arrows). Numbers represent the percentage of Sh3bp2KI/KI mice with improved facial swelling. (C) Body weight changes in GS-9973-administered or DMSO-administered Sh3bp2+/+ and Sh3bp2KI/KI mice. Body weight in GS-9973-administered Sh3bp2KI/KI mice (red lines) was increased, whereas weight of DMSO-administered Sh3bp2KI/KI mice (blue lines) continued to decrease. Numbers in parentheses represent the number of the mice weighed. (D) H&E-stained lung tissue sections. Arrows indicate inflammatory nodules. Scale bar = 1 mm. (E) Quantitative analysis of total inflamed area in the lung. (F) Liver sections from GS-9973-treated or DMSO-treated Sh3bp2+/+ and Sh3bp2KI/KI mice (H&E). Arrows indicate inflammatory infiltrates. Scale bar = 500 μm. (G) Quantitative measurement of total area of inflammatory infiltrates in the liver. (H) Quantitative-PCR analysis of TNF-α mRNA expression in the liver. Average expression level in Sh3bp2+/+ mice treated with DMSO was set as 1. (I) Serum TNF-α levels of GS-9973-treated or DMSO-treated Sh3bp2+/+ and Sh3bp2KI/KI mice at 16 weeks old. Data are presented as mean ± SD. *p < 0.05. ANOVA with Tukey-Kramer post hoc test. ND = not detectable; NS = not significant.

Fig. 2

Decreased craniofacial bone erosion and ankle joint destruction in adult Sh3bp2KI/KI mice treated with GS-9973. (A) Reconstructed 3D μCT images of the mandibular bone (top) and reconstructed 2D μCT images showing the coronal plane at the center of the mandibular first molar (bottom) from 16-week-old Sh3bp2+/+ and Sh3bp2KI/KI mice treated with GS-9973 or DMSO for 6 weeks. Black arrows indicate CEJ and ABC. Red line indicates the distance between CEJ and ABC. White arrows indicate erosion pits. (B) Quantitative measurement of the CEJ-ABC distance at the distal lingual surface of the mandibular first molar. (C) BV/TV. Alveolar bone between roots of the mandibular first molar was segmented and subjected for analysis. (D) Reduction rate of alveolar BV/TV in Sh3bp2KI/KI mice treated with GS-9973 or DMSO relative to Sh3bp2+/+ mice treated with DMSO. (E) Reconstructed μCT images of calvarial bone from GS-9973-treated or DMSO-treated 16-week-old Sh3bp2+/+ and Sh3bp2KI/KI mice. Note that overall numbers and areas of erosion pits in GS-9973-treated Sh3bp2KI/KI mutant mice are decreased compared to DMSO-treated 16-weeks-old Sh3bp2KI/KI mice. (F) Quantitative measurement of calvarial bone erosion. Proportion (%) of bone erosion area including suture to total calvarial bone area (6 mm × 6 mm) was calculated. (G) BS/BV in the 6-mm × 6-mm area. (H) BV/TV in the 6-mm × 6-mm area. (I) 3D μCT images of the ankle joint (top) and calcaneus (bottom) from 16-week-old Sh3bp2+/+ and Sh3bp2KI/KI mice treated with GS-9973 or DMSO. (J, K) BV and BV/TV of calcaneus. (L) Reduction rate of calcaneus BV/TV in Sh3bp2KI/KI mice treated with GS-9973 or DMSO relative to Sh3bp2+/+ mice treated with DMSO. Data are presented as mean ± SD. *p < 0.05. ANOVA with Tukey-Kramer post hoc test. #p < 0.05 with two-tailed t test. NS = not significant; M1 = first molar; M2 = second molar; CEJ = cementoenamel junction; ABC = alveolar bone crest; BV/TV = bone volume/tissue volume; BS/BV = bone surface/bone volume.