Inhibition of FGFR signaling with infigratinib improves linear bone growth in the female Aga2/+ mouse model of osteogenesis imperfecta

Abstract

Osteogenesis imperfecta, a genetically heterogeneous disorder, is characterized by brittle bones with recurrent fractures as well as short stature. A recent study investigating cellular signaling in growth plate chondrocytes identified an increased expression of FGF receptors and elevated FGF signaling. We inhibited FGF receptor activation with the small molecule infigratinib in growing Aga2/+ mice and then characterized linear growth, cartilage growth plate histology, and bone morphometrics. Infigratinib treatment resulted in increased female femur length, increased growth plate lengths in both sexes, specifically in the proliferative zone, and reduced phospho-ERK1/2 as well as SOX9 expression in lower hypertrophic chondrocytes. Infigratinib had no impact on cortical bone but reduced male trabecular bone parameters in Aga2/+ mice. Three point bending biomechanical tests showed that Aga2/+-treated males had increased bone strength for some parameters, but reduced bone strength in treated WT males, correlating with the negative impact on trabecular bone parameters. WT and Aga2-treated male mice also showed increased serum testosterone levels. Together, these data establish FGFR as a therapeutic target for short stature in osteogenesis imperfecta and identify potential negative effects of pan-FGFR inhibition, particularly in the male skeleton.

Graphical Abstract

Graphical Abstract.

Introduction

Osteogenesis imperfecta (OI) is a genetically heterogeneous condition characterized by brittle bones and recurrent fractures. The majority of OI cases are caused by heterozygosity for variants in COL1A1 or COL1A2 encoding the structural proteins of type I collagen.¹ Additionally, 20 other genes have been implicated in producing overlapping phenotypes.^1,2 Classical OI is also phenotypically heterogeneous, with mild (type I, OMIM#166200), moderate (type IV, OMIM#166220), severe (type III, OMIM#259420), and perinatal lethal forms (type II, OMIM#166210). In addition to brittle bones with progressive skeletal deformities, the OI phenotype includes blue/gray sclera, joint hypermobility, dentinogenesis imperfecta, respiratory compromise, hearing loss, and short stature.² Short stature is present in all forms of OI,^3,4 and has negative psychosocial impacts reported by individuals with OI.^5,6 Multiple mouse models of OI recapitulate short stature with reduced body size as well as long bone length, and are described to have abnormalities in the endochondral or cartilage growth plate.^7–9

Linear growth is achieved through chondrocyte proliferation and differentiation within the endochondral growth plate. Resting zone chondrocytes transition to flattened proliferative chondrocytes, which divide and form orthogonal columns, eventually differentiating into prehypertrophic and hypertrophic chondrocytes. Hypertrophic chondrocytes then undergo apoptosis or transdifferentiate into osteoblasts to form bone.¹⁰ Throughout the endochondral growth plate, the proliferation and differentiation of chondrocytes is regulated, in part, by factors secreted by the perichondrium.¹¹ Disruptions to this orderly chondrocyte maturation lead to short stature, primarily through alterations of proliferation or differentiation. The Aga2/+ mouse model of moderate to severe OI (Col1a1^+/− c.-16 T > A, exon 50) demonstrates a smaller body size with both a shorter distal femur proliferative zone and overall growth plate compared to the WT littermates.^7,12 Similarly, the Col1a2^G610C/+ model has reduced hypertrophic cell maturation, resulting in a longer distal femur hypertrophic zone,⁸ and Col1a2^oim/+ tibial growth plate chondrocytes have decreased proliferative capacity.⁹

As growth plate chondrocyte dynamics, including proliferation and differentiation, are disrupted in multiple OI mouse models, Zieba et al.⁷ performed single-cell RNA sequencing analysis using growth plate tissue from Aga2/+ mice and identified dysregulated FGF receptor 1 and 2 (FGFR1/2) signaling in chondrocytes, particularly in the proliferative zone. These findings indicated that chondrocyte proliferation was not altered in these mice but that proliferative zone chondrocytes went through accelerated differentiation.⁷ FGFRs are tyrosine kinase receptors that bind to extracellular ligands and activate numerous downstream signaling pathways, including MAPK.¹³ MAPK signaling plays an important role in chondrocyte behavior, in part through the activity of MAPK 3 and 1 (ERK1/2), which positively regulate the expression of SRY-box transcription factor 9 (SOX9), a critical transcription factor controlling chondrocyte differentiation.¹⁴

Pathogenic variants that elevate FGFR1, 2, and 3 signaling are implicated in multiple skeletal dysplasias: Activating FGFR1 variants cause osteoglophonic dysplasia (OMIM#166250), characterized by rhizomelic dwarfism;¹⁵ variants in FGFR2 produce Apert syndrome (OMIM#101200), characterized by the deceleration of linear growth in childhood, among other skeletal abnormalities; and activating variants in FGFR3 cause achondroplasia (OMIM#100800), the most common heritable form of dwarfism that is associated with significant short stature. Reducing FGFR signaling directly with tyrosine kinase inhibitors or indirectly with a C-natriuretic peptide (CNP) analog has been a successful treatment in achondroplasia. The CNP analog vosoritide increases linear height and is an FDA-approved treatment for achondroplasia.¹⁶ Infigratinib (BGJ398), a small molecule pan-FGFR inhibitor that functions as an ATP-competitive inhibitor of FGFR1-3,¹⁷ improved axial and appendicular growth in a mouse model of achondroplasia.^18–20 A recent phase 2 clinical study found that infigratinib treatment increased the annualized height velocity of children with achondroplasia.²¹ Together, reducing elevated FGFR signaling appears to be a promising strategy to treat short stature in some skeletal dysplasias.

In this study, we tested whether inhibiting elevated FGFR signaling in growing Aga2/+ mice with infigratinib restored growth plate morphology and improved linear growth. Infigratinib treatment lengthened the Aga2/+ proliferative zones and overall growth plates, while also reducing both phospho-ERK1/2 and SOX9 expression in lower hypertrophic chondrocytes. In addition, we identified sex differences in response to infigratinib treatment. Infigratinib-treated female Aga2/+ femora were significantly longer compared to the VEH control, but this effect was not seen in males. As infigratinib is a pan-FGFR inhibitor and FGFRs play a role in bone homeostasis, we evaluated bone parameters by microCT after treatment and found effect in cortical bone but reduced trabecular bone parameters specifically in males. Biomechanical data via 3-point bending showed significantly reduced bone strength in WT males but increased strength in Aga2/+ males following treatment for certain parameters. Because of sex differences in response to treatment, we also measured serum testosterone in WT and Aga2/+ males and found elevated levels in both genotypes following treatment. These data demonstrate that pharmacologically reducing elevated FGF signaling can increase linear growth in females and bone strength, measured by 3 point-bending, in the Aga2/+ male mice; however, systemic treatment may negatively impact bone health, particularly in growing males.

Materials and methods

Animals

Aga2/+ mice (Col1a1^+/− c.-16 T > A, exon 50) were received as a gift from the C. Jacobsen laboratory and maintained on a C57BL/6 J background.¹² Mice were housed in the University of California Los Angeles (UCLA) vivarium under a 12 h light/dark cycle and provided water and standard chow ad libitum (LabDiet, 5053). These animal studies were performed under a protocol approved by the UCLA Research Safety and Animal Welfare Committee (ARC Committee).

Infigratinib (Selleck, S2183) was dissolved in DMSO (MedChemExpress, HY-Y0320) to create a 20 mM stock solution, which was then diluted with 0.9% NaCl, 3.5 mM HCl, 20 mM cyclodextrin (MedChemExpress, HY-107201) for injections. Twenty-one-day-old WT and Aga2/+ mice were randomized into groups that received daily subcutaneous infigratinib (2 mg/kg)¹⁹ or VEH control injections for 28 d. This was the earliest time point that we could handle the fragile Aga2/+ mice. Body weights were collected every second day. Seven and 2 d prior to sacrifice, mice received i.p. calcein (10 mg/kg). On d 28, 2 h after receiving the final dose, mice were euthanized, radiographed, measured, and dissected for tissue collection. Using dorsalventral radiographs, fractures were counted as the number of long bones with fractures or calluses by 2 independent blinded observers. The long bones analyzed for fractures were the humeri, radii with ulnae (considered together), femora, and tibiae. Each bone was considered fractured regardless of the number of fractures per bone; thus, the maximum possible number of counted fractures per mouse was 8.

Histology, immunohistochemistry, and RNAscope

Formalin fixed legs were decalcified using Immunocal decalcification solution (StatLab, 141432), paraffin embedded, and 5 μm thick sagittal sections were then collected. Sections were stained with picrosirius red and hematoxylin using standard methods. 10X images of distal femur growth plates were used to measure growth plate zone lengths in ImageJ by 2 independent blinded observers. Growth plates were measured parallel to the chondrocyte columns at approximately the center of the growth plate at a region with no tissue folding. The resting zone was defined as immediately adjacent to the epiphyses with irregularly scattered chondrocytes, the proliferative zone contained flattened chondrocytes organized into columns, and the hypertrophic zone was characterized by enlarged and vacuolated columns of chondrocytes ending at the primary spongiosa or the newly formed network of trabecular bone. Results were compared between observers, and results from a single observer were used for statistical testing and are presented.

For immunohistochemistry (IHC), sections were rehydrated and heated in citrate antigen retrieval buffer (Vector Laboratories, H3300) at 95 °C for 20 min, quenched in methanol with 3% H₂O₂ for 10 min, and then stained using either anti-phospho-ERK1/2 (Cell Signaling Technology; 9101; 1:250) or anti-SOX9 (Cell Signaling Technology; 82630; 1:500) overnight at 4 °C. Slides were then incubated with biotinylated anti-rabbit IgG (Vector Laboratories, BP-9100-50) followed by streptavidin peroxidase (Vector Laboratories, SA-5704) for 30 min each before incubation with 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories, SK-4105). Phospho-ERK1/2 immunostaining was imaged at 10X with and analyzed by 2 independent blind observers in ImageJ by counting positive hypertrophic chondrocytes normalized by the hypertrophic zone area. SOX9 immunostained growth plates were imaged at 20X in bright field microscopy.

RNAscope analysis was performed on growth plate tissue samples fixed in 4% paraformaldehyde overnight followed by paraffin embedding. Sections were probed using the RNAscope Multiplex Fluorescent V2 assay (ACDBio) following the manufacturer’s protocol. RNAscope-processed slides were imaged at 20× original magnification using the Echo Revolution microscope, and images were processed using Adobe Photoshop. Probes used were Sox9 (401051), and Col10a1 (426181). Sample sizes included 4 WT treated, 4 WT untreated, 5 Aga2/+ treated, and 6 Aga2/+ untreated.

Micro-CT

Formalin fixed femora were imaged with a Quantum GX2 μCT system (Perkin Elmer). Bones were scanned at 90 kV and 88 μA using an Al 0.5 mm + Cu 0.06 mm filter. A hydroxyapatite phantom containing 0, 50, 200, 800, and 1200 mg/cm³ standards (QRM, MHA-379) was imaged under the same settings to calibrate the BMD. Reconstructions were generated with a voxel size of 50 μm in each spatial direction for femur length analysis and 8 μm in each spatial direction for trabecular and cortical bone analysis. Two independent blinded observers analyzed the μCT images using CTAn (Brucker). Observers measured femur length using transaxial femur slices, femora with callus or fractures were excluded from the analysis. Trabecular bone was analyzed at the distal femur starting 0.5 mm proximal to the growth plate, using a 1 mm long ROI containing the trabecular compartment. Cortical bone was analyzed at the mid-femur with an ROI starting at the most distal edge of the third trochanter and advancing distally for 1 mm. Femora with callus or fractures in the ROI were excluded from the analysis. The BMD, tissue mineral density, trabecular microarchitecture, and cortical bone geometry were analyzed using standard methods. Thresholds corresponding to 508 and 745 mg/cm³ were used for trabecular and cortical analyses, respectively.

3-point bending

Dissected femurs were loaded to failure using an Instron 5943 using a 100 N load cell and a displacement rate of 0.05 mm/s. The femurs were supported on 2 points with a span of 1 cm, and a third point was lowered to load the bone, placing the anterior surface in compression and the posterior surface in tension. Compressive force and displacement were measured at 50 Hz. Stiffness, yield, post-yield displacement, maximum load, work to failure were manually determined from force vs displacement curves using custom MATLAB code.²²

Serum biochemistry

Serum was isolated from blood collected at sacrifice (BD, 365967). Male serum testosterone and female serum estradiol were measured with a competitive binding ELISA (R&D Systems, KGE010 and KGE014), in triplicate, according to the manufacturer’s instructions.

Statistical analysis

Prism 10 (GraphPad) was used for statistical analysis. Comparisons between WT VEH vs WT infigratinib, as well as Aga2/+ VEH vs Aga2/+ infigratinib, were conducted by first testing for normality using Shaprio-Wilk tests, followed by t-tests if normally distributed or Mann-Whitney U tests otherwise. Phopsho-ERK1/2 and serum testosterone were log transformed for significance testing. p values are shown for 0.0001 < p < .05 and p < .05 was considered statistically significant. Due to sexual dimorphism in the skeleton, males and females were analyzed and presented separately. Data are presented as group means ± SEM. In a secondary analysis, 2-way ANOVA with post hoc Šidák corrected pairwise comparisons was employed with results detailed in Table S3.

Results

Infigratinib increases female long bone length

Starting at 21 d of age, and over 28 d of treatment, female and male Aga2/+ mice had consistently lower BWs compared to the WT littermates (Figure 1A, Table S1). Infigratinib treatment had no significant effect on BW compared to the VEH control. On d 28, female and male Aga2/+ mice had shorter body lengths compared to the WT littermates but there were no significant body length differences between infigratinib- and VEH-treated mice (Figure 1B). Femora measured by μCT from both female and male Aga2/+ mice were significantly shorter compared to those of WT littermates (Figure 1C and D). There was a statistically significant difference in female Aga2/+ femur lengths with the infigratinib-treated average length of 12.61 mm (95%CI, 12.22-13.00), compared to the VEH-treated average length of 11.98 mm (95%CI, 11.67-12.29), an increase of 5.2%. However, the effect of infigratinib on male Aga2/+ femur lengths was not significant with the infigratinib-treated average length of 12.88 mm (95%CI, 12.17-13.59) and a VEH-treated average length of 12.45 mm (95%CI, 11.93-12.98). These results suggest that after 28 d of treatment infigratinib leads to a statistically significant increase in long bone linear growth in Aga2/+ females, but not males.

Figure 1.

Infigratinib improves appendicular skeletal growth in female Aga2/+ mice. (A) Female and male BWs over the course of the study. Data represent means ± SEM, sample size, and detailed statistical test results are provided in Tables S1 and S2. (B) Nose to tail-tip body length at d 28 of treatment. For females, WT VEH n = 11, WT infigratinib n = 10, Aga2/+ VEH n = 15, Aga2/+ infigratinib n = 13. For males, n = WT VEH n = 9, WT infigratinib n = 8, Aga2/+ VEH and infigratinib n = 13. (C) Femur length at d 28 of treatment. For females, WT VEH n = 11, WT infigratinib n = 10, Aga2/+ VEH n = 12, Aga2/+ infigratinib n = 9. For males, WT VEH and infigratinib n = 10, Aga2/+ VEH n = 12, Aga2/+ infigratinib n = 9. (D) Representative slices of ex vivo femur μCT reconstruction. Bar plots represent means ± SEM and dots represent independent replicates. p values above bars are from 2-sided T-tests, p < .05 are shown.

Infigratinib treatment increased overall growth plate and proliferative zone length

Long bone linear growth is achieved through chondrocyte proliferation and differentiation in the endochondral growth plate. Picrosirius-red-stained distal femur growth plates were used to analyze growth plate lengths and identify the effect of infigratinib on chondrocyte proliferation and/or differentiation (Figure 2A). Infigratinib treatment significantly increased the overall growth plate length in both females and males, as compared to the VEH control (Figure 2B).

Figure 2.

Infigratinib lengthens the endochondral growth plate. (A) Representative images of female picrosirius red stained distal femur endochondral growth plate (representative males presented in Figure S1). Estimated resting, proliferative, and hypertrophic zones indicated by blue bars. (B) Total growth plate length. (C) Resting zone length. (D) Proliferative zone length. (E) Hypertrophic zone length. For females, WT VEH and infigratinib n = 9, Aga2/+ VEH n = 10, Aga2/+ infigratinib n = 8. For males, n = 10/group. Bar plots represent means ± SEM and dots represent independent replicates. p values above bars are from 2-sided T-tests, p < .05 are shown.

Two-way ANOVA identified that female Aga2/+ resting zones were significantly longer than WT, but no significant differences were detected when comparing VEH and infigratinib treatment (Figure 2C). Infigratinib treatment led to a significant increase in both female and male Aga2/+ proliferative zone lengths compared to the VEH (Figure 2D). Infigratinib-treated female Aga2/+ mice had significantly longer proliferative zones compared to the VEH but no significant differences between treatment groups were detected in WT. In males, the proliferative zone was significantly longer in infigratinib-treated Aga2/+ mice compared to the VEH but infigratinib had no significant effect on WT mice when compared to the VEH. Female Aga2/+ hypertrophic zones were significantly longer than those of the WT and infigratinib significantly increased the hypertrophic zone lengths in females, but not males (Figure 2E). There was no significant difference identified in male hypertrophic zone lengths. One limitation to this study is that the proliferative zone was not evaluated by BrdU/EDU incorporation. Together, these data indicate that infigratinib treatment increased Aga2/+ long bone linear growth primarily through increased proliferative zone length in females.

Infigratinib restores growth plate chondrocyte signaling

When bound to a ligand, the tyrosine kinase domains of FGFRs can activate several signaling pathways, including MAPK, an important signaling cascade in growth plate chondrocytes. In chondrocytes, there are 3 main MAPK pathways: extracellular regulated kinase (ERK), c-Jun N-terminal (JNK), and p38 MAPK (p38 MAPK).²³ As ERK signaling has been reported to regulate growth plate chondrocyte differentiation and proliferation, the activated form phospho-ERK1/2 expression was interrogated.^24,25 Immunohistochemistry analysis showed that compared to the VEH, infigratinib treatment led to a significant decrease in the number of phospho-ERK1/2 expressing hypertrophic chondrocytes in female Aga2/+ mice (Figure 3A and B). ERK1/2 signaling regulates, in part, the expression of SOX9, a master chondrocyte transcription factor. SOX9 has been reported to be expressed in proliferative, prehypertrophic, and upper hypertrophic chondrocytes²⁶; and single-cell RNA sequencing of the Aga2/+ growth plate tissues identified increased Sox9 expression, particularly in hypertrophic chondrocytes.⁷ Immunohistochemistry analysis showed that VEH-treated Aga2/+ mice had inappropriate SOX9 expression in lower hypertrophic chondrocytes relative to the WT, which was reduced with infigratinib treatment (Figure 3C). To determine how infigratinib treatment affected Sox9 expression at the RNA level, we performed RNAscope analysis using a Sox9 probe as well as a Col10a1 probe to mark late hypertrophic chondrocytes (Figure S3). Unlike at the protein level, we saw late hypertrophic chondrocyte Sox9 RNA expression in both WT and Aga2/+ growth plates, though the signal was reduced when compared to early hypertrophic chondrocytes (Figure S3A). However, we found that infigratinib treatment clearly reduced the Sox9 RNA expression signal in late hypertrophic chondrocytes of both WT and Aga2/+ growth plates. (Figure S3B) Together, these data suggest that blocking FGFR signaling in Aga2/+ mice reduced MAPK signaling and ERK1/2 activation, while also reducing lower hypertrophic zone SOX9 expression.

Figure 3.

Infigratinib restores ERK1/2 signaling and proper SOX9 localization in growth plate chondrocytes. (A) Representative 10X images of distal femur growth plates stained with anti-phospho-ERK1/2 by IHC. Arrows indicate phospho-ERK1/2 positive hypertrophic chondrocytes. (B) Quantification of the phospho-ERK1/2 expressing cells/mm2 of hypertrophic zone. WT VEH and infigratinib n = 11, Aga2/+ VEH n = 9, Aga2/+ infigratinib n = 11. Bar plot represents means ± SEM and dots represent independent replicates. Data were log transformed for analysis, p values above bars are from 2-sided T-tests, p < .05 are shown. (C) Representative 20X images of distal femur growth plates stained with anti-SOX9 by IHC. Brackets indicate lower hypertrophic zone with increased SOX9 in VEH-treated Aga2/+ mice that is reduced in infigratinib-treated Aga2/+ mice. All replicates (3-5/group) included in Figures S2-S3.

Infigratinib treatment leads to a sex specific bone response

Brittle bone and recurrent fractures are key features of the OI phenotype, and the endochondral growth plate is an important source of osteoblasts. Aga2/+ mice experience spontaneous fractures and infigratinib treatment had no effect on the number of fractured long bones present at sacrifice (Figure 4A). The skeletal phenotype was further characterized using μCT analysis of femur cortical and trabecular bone.

Figure 4.

Infigratinib has a minor effect on cortical bone. (A) Long bone fractures at d 28 of treatment. No fractures were detected in WT mice (n = 10-11/group). For females, Aga2/+ VEH n = 15 and Aga2/+ infigratinib n = 13. For males, Aga2/+ VEH n = 14 and Aga2/+ infigratinib n = 13. Mann-Whitney U tests identified p > .1 for both comparisons between Aga2/+ VEH and infigratinib treatment. (B) Representative 2D cross section reconstruction of mid-femur cortical bone μCT. (C) Cortical tissue mineral density (TMD). (D) Cortical thickeness (CtTh). (E) Cortical area per tissue area (CtAr/TtAr). (F) Polar moment of inertia (pMOI). For females, WT VEH n = 11, WT infigratinib n = 10, Aga2/+ VEH and infigratinib n = 11. For males, WT VEH and infigratinib n = 10, Aga2/+ VEH n = 11, Aga2/+ infigratinib n = 10. Bar plots represent means ± SEM and dots represent independent replicates.

At the mid-femur, a 2-way ANOVA confirmed both male and female Aga2/+ mice had significantly decreased tissue mineral density (TMD), cortical thickness (CtTh), cortical area/tissue area (CtAr/TtAr), and polar moment of inertia (pMOI) when compared to the WT (Figure 4B-F). There were no significant differences between infigratinib and VEH treatment for TMD, CtTh, CtAr/TtAr, and pMOI (Figure 4C-F). Infigratinib treatment had no significant effect on male and female cortical bone.

At the distal femur, 2-way ANOVA identified that both female and male Aga2/+ mice had significantly decreased BMD, bone volume fraction (BV/TV), trabecular thickness (TbTh), and trabecular number (TbN) as well as increased trabecular separation (TbSp) when compared to the WT (Figure 5A-F). Infigratinib treatment had a sex dependent effect on trabecular bone. Compared to the VEH, infigratinib treatment significantly reduced BMD in both WT and Aga2/+ male mice and reduced BV/TV and TbTh in WT male mice (Figure 5B-D). Infigratinib treatment also increased TbSp in male Aga2/+ mice (Figure 5F; Šidák corrected pairwise comparisons). Overall, infigratinib treatment had no effect on cortical bone but reduced both trabecular bone quantity and microarchitecture parameters, particularly in males, but not in Aga2/+ females.

Figure 5.

Infigratinib treatment negatively impacted male trabecular bone. (A) Representative reconstructions of distal femur trabecular bone μCT analysis. (B) BMD. (C) Bone volume fraction (BV/TV). (D) Trabecular thickens (TbTh). (E) Trabecular number (TbN). (F) Trabecular separation (TbSp). For females, WT VEH n = 11, WT infigratinib n = 10, Aga2/+ VEH n = 13, Aga2/+ infigratinib n = 10. For males, WT VEH and infigratinib n = 10, Aga2/+ VEH n = 12, Aga2/+ infigratinib n = 9. Bar plots represent means ± SEM and dots represent independent replicates. p values above bars are from 2-sided T-tests, p < .05 are shown.

To determine the effect of infigratinib treatment on bone strength, biomechanical analysis via 3-point bending was performed on WT and Aga2/+ femurs. (Figure 6) No significant differences were observed in female femurs, suggesting that mechanical properties remained unchanged after treatment in female groups. In males, WT mice showed significant decreases in stiffness, yield point, and ultimate force after treatment. However, male Aga2/+ mice treated with infigratinib exhibited a statistically significant increase in ultimate force and work to failure, indicating a potential treatment-related improvement in bone mechanical strength and less brittleness. Together, though there were negative effects on bone CT parameters, particularly in males. Treatment with infigratinib improved bone strength (ability to withstand force and in a measure of brittleness) in Aga2/+ males while significantly worsening multiple parameters of bone strength in WT males.

Figure 6.

Infigratinib treatment improved Aga2/+ bone strength but negatively impacted WT bone. (A) Stiffness. (B) Yield point. (C) Post-yield displacement. (D) Ultimate force. (E) Work to failure. For 3-point bending, sample sizes were as follows, females: WT VEH n = 9, WT Infigratinib n = 10, Aga2/+ VEH n = 7, Aga2/+ Infigratinib n = 10, males: WT VEH n = 10, WT Infigratinib n = 9, Aga2/+ VEH n = 11, Aga2/+ Infigratinib n = 9. Bar plots represent means ± SEM, and individual dots represent independent replicates. p values above bars are from 2-sided T-tests, p < .05 are shown.

Infigratinib treatment increases serum testosterone, but not estrogen

As this study identified sex differences in the impact of infigratinib on bone, we investigated whether infigratinib impacted serum sex steroid levels. While infigratinib is a selective inhibitor of FGFR1-3, its bioactive intermediates also act as reversible inhibitors and mechanism-based inactivators of cytochrome P450 3A4 (CYP3A4).²⁷ CYP3A4 is an enzyme involved in the oxidation of a large number of pharmaceuticals, and it is estimated to be involved in the metabolism of 40%-50% of all clinically used drugs, as well as of sex steroids and growth hormone.^28–30 Additionally, FGF signaling is critical for testis development^31–33 and is reported to impact testosterone release from postnatal Leydig cells.³⁴

To assess whether infigratinib treatment influenced serum sex steroid levels, testosterone levels in male mice and estradiol levels in females were analyzed using a competitive ELISA. Female estradiol measurements were not statistically different between genotypes and treatment groups (Figure 7A). Two-way ANOVA analyses identified that infigratinib-treated males (both WT and Aga2/+) had a statistically significant increase in testosterone (Figure 7B). These results suggest that infigratinib treatment increases circulating testosterone levels in adolescent male mice, which were about to reach sexual maturity.

Figure 7.

Infigratinib treatment increased serum testosterone, but not estradiol. (A) Female serum estradiol. WT VEH n = 9, WT infigratinib n = 10, Aga2/+ VEH and infigratinib n = 10. (B) Male serum testosterone. Data were log-transformed for analysis. WT VEH n = 9, WT infigratinib n = 10, Aga2/+ VEH and infigratinib n = 13. Bar plots represent means ± SEM, dots represent independent replicates, p values above bars are from 2-sided T-tests, p < .05 are shown.

Discussion

Short stature is part of the OI phenotype, and recent work has highlighted elevated FGFR1/2 signaling as a potential therapeutic target.⁷ In this study, we treated growing Aga2/+ mice with the pan-FGFR inhibitor infigratinib and found that the treatment led to increased long bone linear growth in females, as well as increased growth plate lengths in both sexes, particularly in the proliferative zone. These results support the single-cell RNA sequencing data reported by Zieba et al.⁷, which suggested that Aga2/+ growth plates were shorter in part to increased FGFR1/2 signaling activation. Infigratinib treatment reduced lower hypertrophic zone phosphorylated ERK1/2 as well as SOX9 expression, restoring the localization of this critical chondrocyte transcription factor to be comparable to the WT. Inappropriate expression of Sox9 in late hypertrophic chondrocytes is predicted to suppress the transdifferentiation of these cells into osteoblasts; and in this study, reducing this mislocalized Sox9 expression corresponded to a correction of the expression of this key transcription factor, suggesting an improvement in growth dynamics.³⁵ Together, this work furthers the idea that a dominant Col1a1 variant can impact intracellular signaling in tissues not thought to express high levels of type I collagen, such as cartilage.

This study identified greater linear growth effects of infigratinib in Aga2/+ compared to the WT mice. For example, infigratinib treatment led to significantly increased female femur lengths, female overall growth plate lengths, as well as proliferative zone lengths in both sexes when compared to the VEH in Aga2/+, but not WT mice. Infigratinib treatment also reduced late hypertrophic chondrocyte phospho-ERK1/2 and SOX9 expression in only Aga2/+ mice at the protein level, but it did reduce Sox9 expression in both genotypes at the RNA level. This could be indicative of a change in SOX9 post-translational protein stabilization, which is known to be increased by transforming growth factor β (TGFβ) signaling.³⁶ TGFβ signaling is increased in Aga2/+ growth plate tissues, which may explain the increase in late hypertrophic protein localization compared to the WT.⁷ It is possible that in the Aga2/+ growth plate, TGFβ signaling is more susceptible to changes in FGF activation, which is known to regulate TGFβ signaling in growth plate tissues.³⁷ Additionally, studies on achondroplasia mouse models with gain-of-function variants in Fgfr3 and significant activation of the FGF-FGFR3 signaling pathway found that infigratinib treatment increased femur lengths approximately 10%-20% over VEH treatment when treating 1 d-old mice for 15 d, which is greater than the increase seen in Aga2/+ females.^19,20 This suggests that FGFR inhibition has a greater effect on linear growth based on the degree of elevated FGFR signaling at baseline. Additionally, analyses of single-cell RNA sequencing in Aga2/+ chondrocytes showed dysregulation of multiple signaling pathways including MAPK, PTH, Wnt, TGFβ, and hedgehog.⁷ MAPK and ERK in chondrocytes are activated by many other stimuli including TGFβ, IGFs, retinoic acid, and extracellular matrix components, thus the effectiveness of infigratinib on linear growth may differ among disease states or genotypes.²³

FGF signaling is important for bone homeostasis.^13,38,39 In this study, WT and Aga2/+ males treated with infigratinib had decreased trabecular bone quantity and microarchitecture compared to the VEH. Further, though Aga2/+ male bone strength for some parameters improved with infigratinib treatment, male WT bone strength was overall reduced. The impact of FGF signaling on bone has been investigated using genetic mouse models as well as pharmacologic inhibition. The global Fgfr3 knockout mouse had reduced cortical bone thickness and trabecular bone mineralization.⁴⁰ Similarly, the Fgfr2 mesenchymal condensation conditional knockout led to decreased femur and lumbar spine BMD.⁴¹ These genetic mouse models are consistent with a study of WT female rats where infigratinib reduced craniofacial bone volume and alveolar bone density.⁴² Future studies investigating the impact of pharmacological FGFR inhibition on bone, particularly during rapid growth before attaining peak bone mass are warranted given the potential of FGFR inhibitors to treat skeletal dysplasias. This is particularly relevant if treatment is to be considered to improve linear growth in disorders with underlying bone mineralization defects.

In addition to bone homeostasis, FGF signaling plays an important role in bone regeneration. Fgf9⁴³ and Fgf18⁴⁴ heterozygous knockout mice have impaired fracture repair while FGF2 treatment in WT mice improves fracture repair.^45–48 In this study, there were no differences in the number of fractured long bones between infigratinib and VEH-treated Aga2/+ mice. However, these fractures occur spontaneously at an unknown timepoint, at different skeletal sites, under uncontrolled loading conditions thus we were unable to assess whether infigratinib negatively impacted fracture healing. Future studies of FGFR inhibition’s potential impact on fracture healing are warranted, especially as non-union is reported in adults and children with OI.⁴⁹

We identified multiple sex differences in response to infigratinib treatment, highlighting the importance of considering sex as a biological variable, particularly when analyzing the skeleton. Infigratinib treatment led to significantly longer female, but not male Aga2/+ femora. This could reflect a sex-specific response to FGFR inhibition, sex differences in response to the infigratinib dose, or increased femur length variance in male Aga2/+ mice as males exhibited a more severe OI phenotype and tended to have more fractures relative to their female littermates. Importantly, both female and male Aga2/+ growth plates were longer with infigratinib treatment, suggesting that infigratinib increased growth rates in both sexes. In contrast, both WT and Aga2/+ males treated with infigratinib had reduced trabecular bone quantity and microarchitectural properties, while female trabecular bone was not affected by infigratinib treatment. Additionally, while Aga2/+ males showed some parameters of improved bones ability to withstand force and a decrease in brittleness following treatment as determined by 3-point bending, which assesses cortical bone strength. WT male femurs became weaker. The mechanism for the discrepancy between WT and Aga2/+ males for the 3 point bending results are not appreciated, but could represent a threshold effect since Aga2/+ had elevated FGF signaling at baseline and WT mice did not. Further, sex differences in the skeletal response to infigratinib have been previously reported: In a study of rat dentoalveolar and craniofacial bones, both males and females were negatively impacted, but the females were more sensitive to infigratinib compared to males.⁴² This discrepancy could be due to the difference in infigratinib dose, species, or skeletal site used for analysis.

As this study identified sex differences in response to infigratinib treatment, we analyzed serum sex steroid levels. FGFRs are reported to be involved in postnatal testicular development and are expressed by the testosterone producing Leydig cells, negatively regulating testosterone release in response to luteinizing hormone stimulation.^31–34 Inhibiting FGFRs with infigratinib could reduce this negative regulation and lead to elevated serum testosterone. Alternatively, CYP3A4 and its role in drug and hormone metabolism were considered as infigratinib acts as a mechanism-based inactivator of CYP3A4, an important enzyme in sex steroid metabolism that is associated with sexual dimorphism, with higher levels of activity in females compared to males; thus, males could be more sensitive to inhibitory effects.^27,29,30 CYP3A4 is involved in the metabolism of testosterone through hydroxylation at multiple sites, which are important to its inactivation. Thus, infigratinib treatment can reduce CYP3A4 activity and lead to reduced serum testosterone metabolism, leading to elevated total testosterone which is what was seen in both WT and Aga2/+ mice. However, the dynamics of CYP3A4 inactivation are complex with substrate specificity. Infigratinib is a potent noncompetitive reversible inhibitor of CYP3A4 and it can inactivate it in a time-dependent, concentration, and NADPH-dependent manners, generating concerns about mechanisms of inactivation, particularly around multiple drug-drug interactions.²⁷ In this study, infigratinib treatment at doses proposed in humans led to increased levels of testosterone in both WT and Aga2/+ animals. Although the implication of this finding is unknown, it suggests that the effects of long term CYP3A4 activation merit follow-up. Testosterone is thought to be osteoanabolic and both sex steroids are important for maintaining BMD.⁵⁰ Unexpectedly, infigratinib-treated male mice had higher testosterone levels and reduced trabecular BMD and microarchitecture.

This study was limited by technical challenges in handling the Aga2/+ mice which have brittle bone and experience spontaneous fractures. To reduce the risk of injuring mice while administering subcutaneous injections, we began treating them at d 21. At this timepoint, mice had already gained approximately 50% of their final BW before treatment began. Additionally, although mice received treatment for 28 d, approximately 90% of their growth had occurred by d 20 (Female WT 92%, Aga2/+ 87%; Male WT 90%, Aga2/+ 85%). Treating younger mice could have provided more time for infigratinib to act and may have resulted in detectable BW/length differences. Additionally, previous work has shown that FGF signaling is increased in Aga2/+ growth plate tissues at P5; however, it is unclear if FGF signaling remains elevated at later timepoints.⁷ In this study, growth plate lengths and levels of phospho-ERK1/2 were comparable between VEH-treated WT and Aga2/+ mice. However, there was an Aga2/+ specific response to infigratinib treatment in female proliferative zone length and phospho-ERK1/2 levels, which suggests that FGFR inhibition may have a greater impact on Aga2/+ chondrocytes.

Next, infigratinib is orally bioavailable and given clinically as a daily tablet.²¹ Previous studies treating mice with infigratinib have used subcutaneous injections;²⁰ however, the parenteral route could have important differences compared to the oral route. The infigratinib dose was selected based on previous work treating a murine model of achondroplasia,¹⁹ and it is intermediate between a recent study of low-dose 0.2 mg/kg to 0.5 mg/kg subcutaneous injections.²⁰

Finally, Infigratinib was selected due to its established safety profile and its use in clinical trials of other skeletal dysplasias.²¹ Infigratinib predominantly inhibits FGFR1, FGFR2, and FGFR3, followed by FGFR4 and VEGF receptor 2 (VEGFR2).¹⁸ Cells in the Aga2/+ growth plate were reported to have increased FGFR1 and FGFR2 signaling in cartilage and it is possible that inhibiting non-dysregulated receptors had a negative impact on the bone. FGFR3¹³ is reported to have important regulatory effects in the skeleton and inhibiting this receptor that is not known to be dysregulated in OI could have contributed to the observed negative bone effects.

Despite limitations, this work demonstrates that inhibiting elevated FGFR signaling in the Aga2/+ growth plate increased endochondral growth plate lengths, corrected the inappropriate expression of SOX9, and increased long bone linear growth. However, treatment negatively impacted trabecular bone parameters, particularly in males, and when evaluating treatment options for patients who have disorders associated with reduced bone mass, these factors should be considered.

Author contributions

Alexander Kot (Conceptualization, Data curation, Formal analysis, Investigation, Project administration, Visualization, Writing—original draft, Writing—review & editing), Caroline Wight (Investigation, Writing—review & editing), Roya Bagheri (Investigation, Writing—review & editing), Davis Wachtell (Investigation, Writing—review & editing), Alma Rios (Investigation), Benjamin Bober (Investigation, Writing—review & editing), Cora Chun (Investigation, Writing—review & editing), Pavel Krejci (Conceptualization, Methodology, Writing—review & editing), Jennifer Zieba (Conceptualization, Investigation, Writing—review & editing), and Deborah Krakow (Conceptualization, Funding acquisition, Supervision, Writing—review & editing)

Funding

This work was supported by National Institutes of Health F30HD113414 (A.K.), T32GM008042 (A.K.), and T32GM152342 (A.K.). P.K. is supported by Praemium Academiae of the Czech Academy of Sciences. D.K. is supported by the Joshua S. and Beth C. Friedman Chair in Women’s Genetic Research.

Conflicts of interest

The authors have no conflicts of interest to disclose.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics approval

These animal studies were performed under a protocol approved by the UCLA Research Safety and Animal Welfare Committee (ARC Committee).