Eleni Douni

Background: Receptor activator of nuclear factor-κB ligand (RANKL) plays a central role in regulating osteoclast formation and bone resorption, while its inhibition by the monoclonal antibody denosumab serves as an effective antiresorptive treatment for postmenopausal osteoporosis. However, denosumab discontinuation triggers a severe rebound effect involving rapid bone mineral density (BMD) loss, accompanied by an overshooting of bone turnover markers (BTMs), and increased risk of multiple fractures. Preclinical studies investigating this rebound phenomenon after denosumab discontinuation have been limited, mainly because denosumab does not cross-react with murine RANKL. This study explores the rebound phenomenon in a transgenic mouse model of osteoporosis expressing human RANKL (TgRANKL) and evaluates the impact of sequential zoledronate therapy. Methods: TgRANKL mice were divided into four experimental groups: vehicle control, continuous denosumab treatment, denosumab withdrawal, and sequential denosumab followed by zoledronate, including an additional follow-up phase after zoledronate discontinuation. Skeletal alterations were characterized using microCT, histomorphometric assessments, serum bone turnover markers (BTMs), and bone gene expression analyses. Results: Denosumab therapy rescued the osteoporotic phenotype of TgRANKL mice, whereas its discontinuation resulted in a rebound bone loss accompanied by elevated bone turnover markers. Denosumab also inhibited bone marrow adipose tissue formation in TgRANKL mice, while its discontinuation led to moderate reformation of marrow adiposity. Sequential administration of zoledronate effectively prevented the rebound bone loss response. However, discontinued therapy after denosumab-zoledronate sequence, showed that the protective effects of zoledronate were not persistent. Conclusions: Our findings establish TgRANKL mice as a unique osteoporotic model for investigating the mechanisms driving denosumab rebound and testing sequential antiresorptive strategies.

Receptor activator of nuclear factor-κB ligand (RANKL) is essential for osteoclast formation and bone resorption, in osteolytic conditions such as osteoporosis and bone metastases. However, its role in metastasis progression remains incompletely understood. Herein, we examined whether the overexpression of human RANKL in transgenic mice (TgRANKL) affects their susceptibility to breast cancer bone metastasis compared to their wild-type (WT) littermates. Bone metastasis was induced by injecting EO771 mouse mammary adenocarcinoma cells into the caudal artery of syngeneic WT and TgRANKL mice. RANKL overexpression led to an earlier onset and increased burden of bone metastasis in EO771-bearing TgRANKL mice compared to WT mice. It also exacerbated the bone destruction caused by metastasis-associated osteolysis. The prophylactic inhibition of RANKL activity with denosumab, a monoclonal antibody targeting human RANKL, prevented osteolysis and significantly reduced the incidence and progression of bone metastases in TgRANKL mice. However, the therapeutic denosumab treatment had no effect on metastasis incidence or tumor burden, although it alleviated osteolysis. The treatment with zoledronic acid, an anti-resorptive agent inhibiting osteoclast activity, yielded results similar to those of denosumab. These findings emphasize the significance of initiating early treatment with anti-resorptive agents such as denosumab or zoledronic acid to reduce the risk of bone metastasis in patients at high risk.

Bone marrow adipose tissue (BMAT) accrues in osteoporosis, whereas its contribution to the progression of bone resorption remains insufficiently understood. To understand the mechanisms that promote BMAT expansion in osteoporosis, in the present study, we performed extensive analysis of the spatiotemporal pattern of BMAT expansion during the progression of bone resorption in TgRANKL transgenic mouse models of osteoporosis expressing human RANKL (receptor activator of nuclear factor‐κB ligand). Our results showed that TgRANKL mice of both sexes developed dramatically increased BMAT expansion compared to wild‐type (WT) littermates, that was analogous to the levels of RANKL expression and the severity of the bone loss phenotype. BMAT was formed at close proximity to areas undergoing active bone remodelling and bone resorption, whereas bone resorption preceded BMAT development. Expression analysis in bone fractions demonstrated that BMAT constitutes a major source for RANKL production. Ex vivo analysis of isolated bone marrow stromal cells from TgRANKL mice showed an increased adipogenic differentiation capacity compared to WT, while osteoclast supernatants further exaggerated adipogenesis, supporting a critical role of the osteoclast‐derived secretome in the differentiation of bone marrow adipocytes. Furthermore, the effectiveness of an antiosteoporosis treatment in BMAT development was investigated upon treatment of TgRANKL models with the bisphosphonate alendronate. Notably, alendronate effectively improved bone mass and attenuated BMAT expansion, indicating a possible involvement of osteoclasts and bone resorption in BMAT development. On the contrary, inhibition of BMAT with PPARγ antagonists (GW9662 or BADGE) effectively ameliorated BMAT expansion but failed to reverse the osteoporotic phenotype of TgRANKL mice. Overall, our data demonstrate that TgRANKL mice constitute unique genetic mouse models for investigating the pathogenic mechanisms that regulate the development and expansion of BMAT in osteolytic diseases.

Receptor activator of nuclear factor-κB ligand (RANKL) is critically involved in mammary gland pathophysiology, while its pharmaceutical inhibition is being currently investigated in breast cancer. Herein, we investigated whether the overexpression of human RANKL in transgenic mice affects hormone-induced mammary carcinogenesis, and evaluated the efficacy of anti-RANKL treatments, such as OPG-Fc targeting both human and mouse RANKL or Denosumab against human RANKL. We established novel MPA/DMBA-driven mammary carcinogenesis models in TgRANKL mice that express both human and mouse RANKL, as well as in humanized humTgRANKL mice expressing only human RANKL, and compared them to MPA/DMBA-treated wild-type (WT) mice. Our results show that TgRANKL and WT mice have similar levels of susceptibility to mammary carcinogenesis, while OPG-Fc treatment restored mammary ductal density, and prevented ductal branching and the formation of neoplastic foci in both genotypes. humTgRANKL mice also developed MPA/DMBA-induced tumors with similar incidence and burden to those of WT and TgRANKL mice. The prophylactic treatment of humTgRANKL mice with Denosumab significantly prevented the rate of appearance of mammary tumors from 86.7% to 15.4% and the early stages of carcinogenesis, whereas therapeutic treatment did not lead to any significant attenuation of tumor incidence or tumor burden compared to control mice, suggesting the importance of RANKL primarily in the initial stages of tumorigenesis. Overall, we provide unique genetic tools for investigating the involvement of RANKL in breast carcinogenesis, and allow the preclinical evaluation of novel therapeutics that target hormone-related breast cancers.

Receptor activator of nuclear factor-κB ligand (RANKL) has been actively pursued as a therapeutic target for osteoporosis, given that RANKL is the master mediator of bone resorption as it promotes osteoclast differentiation, activity and survival. We employed a structure-based virtual screening approach comprising two stages of experimental evaluation and identified 11 commercially available compounds that displayed dose-dependent inhibition of osteoclastogenesis. Their inhibitory effects were quantified through TRAP activity at the low micromolar range (IC₅₀ < 5 μΜ), but more importantly, 3 compounds displayed very low toxicity (LC₅₀ > 100 μΜ). We also assessed the potential of an N-(1-aryl-1H-indol-5-yl)aryl-sulfonamide scaffold that was based on the structure of a hit compound, through synthesis of 30 derivatives. Their evaluation revealed 4 additional hits that inhibited osteoclastogenesis at low micromolar concentrations; however, cellular toxicity concerns preclude their further development. Taken together with the structure–activity relationships provided by the hit compounds, our study revealed potent inhibitors of RANKL-induced osteoclastogenesis of high therapeutic index, which bear diverse scaffolds that can be employed in hit-to-lead optimization for the development of therapeutics against osteolytic diseases.

Multidrug resistance is a significant barrier that makes anticancer therapies less effective. Glutathione transferases (GSTs) are involved in multidrug resistance mechanisms and play a significant part in the metabolism of alkylating anticancer drugs. The purpose of this study was to screen and select a lead compound with high inhibitory potency against the isoenzyme GSTP1-1 from Mus musculus (MmGSTP1-1). The lead compound was selected following the screening of a library of currently approved and registered pesticides that belong to different chemical classes. The results showed that the fungicide iprodione [3-(3,5-dichlorophenyl)-2,4-dioxo-N-propan-2-ylimidazolidine-1-carboxamide] exhibited the highest inhibition potency (ΙC₅₀ = 11.3 ± 0.5 μΜ) towards MmGSTP1-1. Kinetics analysis revealed that iprodione functions as a mixed-type inhibitor towards glutathione (GSH) and non-competitive inhibitor towards 1-chloro-2,4-dinitrobenzene (CDNB). X-ray crystallography was used to determine the crystal structure of MmGSTP1-1 at 1.28 Å resolution as a complex with S-(p-nitrobenzyl)glutathione (Nb-GSH). The crystal structure was used to map the ligand-binding site of MmGSTP1-1 and to provide structural data of the interaction of the enzyme with iprodione using molecular docking. The results of this study shed light on the inhibition mechanism of MmGSTP1-1 and provide a new compound as a potential lead structure for future drug/inhibitor development.

PurposeEven though current techniques provide two-dimensional (2D) imaging of the mouse mammary gland, they fail to achieve high-resolution three-dimensional (3D) reconstruction and quantification. The objective of this study is to establish and evaluate quantitative visualization of the mouse mammary epithelium through microcomputed tomography (microCT) using phosphotungstic acid (PTA) as a contrast agent.ApproachEx vivo microCT scan images of the mouse mammary glands were obtained following staining by PTA, whereas for quantification we adapted volumetric parameters that are used for assessing trabecular bone morphometry and can be structurally applicable in the mammary ductal system. The proposed method was validated in distinct developmental stages and upon short-term treatment with synthetic progesterone, using the carmine alum staining for comparison.ResultsWe demonstrate a simple PTA staining procedure that allows high contrast 3D imaging of mammary glands and quantitation of mammary duct structures using microCT. We validated the proposed method in distinct developmental stages, such as at puberty, adult mice, pregnancy as well as upon progesterone treatment. Compared with carmine alum staining, the microCT analysis provided higher resolution 2D and 3D images of the mammary gland morphology, with lower background that enabled the detection of subtle changes.ConclusionsThis work is the first study that employs PTA-enhanced microCT for 3D imaging and volumetric analysis of mouse mammary glands. Our results establish PTA-enhanced microCT as a useful tool for comparative studies of the mouse mammary gland morphology that can apply in mutant mice and for the preclinical evaluation of pharmaceuticals in breast cancer models.

The outer mitochondrial membrane protein SLC25A46 has been recently identified as a novel genetic cause of a wide spectrum of neurological diseases. The aim of the present work was to elucidate the physiological role of SLC25A46 through the identification of its interactome with immunoprecipitation and proteomic analysis in whole cell extracts from the cerebellum, cerebrum, heart, and thymus of transgenic mice expressing ubiquitously SLC25A46-FLAG. Our analysis identified 371 novel putative interactors of SLC25A46 and confirmed 17 known ones. A total of 79 co-immunoprecipitated proteins were common in two or more tissues, mainly participating in mitochondrial activities such as oxidative phosphorylation (OXPHOS) and ATP production, active transport of ions or molecules, and the metabolism. Tissue-specific co-immunoprecipitated proteins were enriched for synapse annotated proteins in the cerebellum and cerebrum for metabolic processes in the heart and for nuclear processes and proteasome in the thymus. Our proteomic approach confirmed known mitochondrial interactors of SLC25A46 including MICOS complex subunits and also OPA1 and VDACs, while we identified novel interactors including the ADP/ATP translocases SLC25A4 and SLC25A5, subunits of the OXPHOS complexes and F₁F_o-ATP synthase, and components of the mitochondria–ER contact sites. Our results show that SLC25A46 interacts with a large number of proteins and protein complexes involved in the mitochondria architecture, energy production, and flux and also in inter-organellar contacts.

Glucocorticoids are used widely on a long-term basis in autoimmune and inflammatory diseases. Their adverse effects include the development of hyperglycemia and osteoporosis, whose molecular mechanisms have been only partially studied in preclinical models. Both these glucocorticoid-induced pathologies have been shown to be mediated at least in part by oxidative stress. The transcription factor nuclear erythroid factor 2-like 2 (NRF2) is a central regulator of antioxidant and cytoprotective responses. Thus, we hypothesized that NRF2 may play a role in glucocorticoid-induced metabolic disease and osteoporosis. To this end, WT and Nrf2 knockout (Nrf2KO) mice of both genders were treated with 2 mg/kg dexamethasone or vehicle 3 times per week for 13 weeks. Dexamethasone treatment led to less weight gain during the treatment period without affecting food consumption, as well as to lower glucose levels and high insulin levels compared to vehicle-treated mice. Dexamethasone also reduced cortical bone volume and density. All these effects of dexamethasone were similar between male and female mice, as well as between WT and Nrf2KO mice. Hepatic NRF2 signaling and gluconeogenic gene expression were not affected by dexamethasone. A 2-day dexamethasone treatment was also sufficient to increase insulin levels without affecting body weight and glucose levels. Hence, dexamethasone induces hyperinsulinemia, which potentially leads to decreased glucose levels, as well as osteoporosis, both independently of NRF2.