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Novel mechanisms of site-specific regulation of bone strength for the prevention of osteoporotic fractures: a translational project

Periodic Reporting for period 1 - GOTBONE (Novel mechanisms of site-specific regulation of bone strength for the prevention of osteoporotic fractures: a translational project)

Reporting period: 2017-12-01 to 2019-11-30

With increasing age of the population, european countries are facing a substantial increase in osteoporotic fractures (occuring in 53% of women and 20% of men after 50 years1), that are associated with mortality, especially at the hip2. Nearly 4 million osteoporotic bone fractures cost the European health system more than 30 billion Euro per year. This figure could double by 20501. The long term aim of this research project is to improve the prevention, diagnosis and treatment of osteoporosis and related fractures. Fracture risk is influenced by both bone strength and risk of falls. Bone strength is determined by its density, size and quality. Currently available osteoporosis drugs are effective in reducing vertebral fracture risk while they are less effective in reducing non-vertebral and hip fracture risk. Most osteoporosis-related fractures actually occur at non-vertebral bone sites. In addition, there is increasing concern regarding side effects associated with currently available drugs (mainly atypical femoral fractures and osteonecrosis of the jaw).
The purpose of this project was to identify new possible targets to improve bone strength by performing mechanistic preclinical studies on the most promising target genes that were identified in humans.
Among the targets that were identified by the genome-wide association studies, we were particularly interested by WNT16, a major regulator of cortical bone mass (Moverare-Sktric, Nature Med, 2014). WNTs are secreted ligands that bind to its receptor Frizzled, thereby activating a signaling pathway that promotes bone formation by osteoblasts. However, in order to be secreted out of the cell, and to activate the signaling pathway, the palmitoleoylation (addition of lipid called palmitoleic acid) of WNTs ligands is required. Palmitoleoylation is performed by Porcupine in the endoplasmic retinaculum, whereas the removal of the lipid is performed by NOTUM, a secreted lipase. As we observed in preliminary studies that NOTUM inhibition also leads to an increase in cortical bone mass and strength in mice, we hypothesized that the NOTUM’s effect on cortical bone was dependent on WNT16 or vice versa.

Effect of Porcupine inhibition on bone mass. (Funck-Brentano T et al, J Endocrinol, 2018)

During this first part of the scientific project, we focused on the role of palmitolyeolation of WNTs by Porcupine on bone density and bone strength. Using a murine pharmacological approach, we inhibited either Porcupine (that adds palmitoleate to WNTs) or NOTUM (that removes palmitoleate from WNTs). We found that Porcupine inhibitors, currently in Phase I trials for cancer therapy, dramatically reduced bone density and bone strength. Both cortical and trabecular bone was affected due to a decrease in bone formation and an increase in bone resorption. On the other hand, NOTUM inhibition increased cortical bone mass and strength, mainly by enhancing endocortical bone formation. Altogether, these results demonstrate that palmitolyeolation of WNTs is a key factor for the site-specific regulation of bone mass and strength. This mechanism could be further tested as pharmacologic targets for the prevention of osteoporosis, or as biomarkers to identify patients at risk of fractures.

Methods

Twelve-week-old female mice were treated for three weeks by oral gavage with two different Porcupine inhibitors (PORCN_I : LGK974 at 3 [LGK_Lo] or 6 mg/kg [LGK_Hi] or C59 at 10 mg/kg, Selleckchem), a NOTUM inhibitor (30 mg/kg, Lexicon) or vehicle (n= 10 per group). Bone density was evaluated by DXA (PIXIMUS). Cortical bone was evaluated at the femur by µCT and trabecular bone was evaluated at the spine. Static and dynamic histomorphometry was also performed at the femur and spine. Strength was evaluated at the tibia by the 3-point bending test.

Results

Total body BMD was decreased by PORCN_I while it was increased by NOTUM inhibition. Cortical thickness at the femur was also decreased by PORCN_I and increased by NOTUM inhibition. Vertebral trabecular bone volume fraction was substantially decreased by PORCN_I but unchanged by NOTUM inhibition.

In conclusion, palmitoleoylation of WNTs by Porcupine is a major determinant of both trabecular and cortical bone mass. In addition, our findings suggest that Porcupine inhibitors, under development for cancer treatment, may have deleterious skeletal side-effects.

This study was published in Journal of Endocrinology in May 2018

Studies in progress:

Based on the results of this first part of the project, we are currently studying the effect of co-treatment by Porcupine and NOTUM inhibitors. The aim of this study is to determine whether NOTUM inhibitors may affect cortical bone in a WNT-independent manner. If in this setting NOTUM inhibition exerts no effect on cortical bone mass and density, we will conclude that the effect of NOTUM is solely driven by the Wnt signaling pathway. On the contrary, if NOTUM inhibition still increases cortical bone mass despite the absence of functional WNTs, we will conclude that NOTUM inhibition is partly independent of the Wnt signaling pathway.

Ageing and senescence: In a second set of experiments, we aimed to determine if the age-dependent bone loss is mediated by NOTUM. We treated young and old female mice by our NOTUM inhibitor according to the same methods presented above. The goal of this experiment is to assess the skeletal effects of NOTUM inhibition in aging mice and to determine if NOTUM can affect senescent markers.
This EU project was terminated prematurely after 10 months due to personal evolution in my academic carrier with a position as Associate professor. However, the experiments are still ongoing. We have already demonstrated in a preclinical study that Porcupine inhibitors are deleterious for the bone, and based on these results, one can speculate that these small molecules in trial for cancer therapy may increase patients risk of fracture. On the other hand, our preliminary results suggest that NOTUM inhibitors may represent a key therapeutic target for the prevention of peripheric fractures such as the femoral neck or the humerus. With ageing of the population in the EU, reducing the incidence of major osteoporotic fractures would significantly improve the morbidity and mortality linked to these events, in addition to reducing societal costs.