Periodic Reporting for period 1 - TAMYOCAL (Tamoxifen mediated protection on X-linked centronuclear myopathy: a mechanistic and pre-clinical study) Reporting period: 2018-05-01 to 2020-04-30 Summary of the context and overall objectives of the project X-linked myotubular myopathy (XLMTM) is a severe congenital myopathy caused by the absence of lipid phosphatase myotubularin. XLMTM affects 1/50.000 male births, there is no treatment and many cases lead to premature death. Thus, treatments are needed.Tamoxifen (TAM) is a selective estrogen receptor modulator that mimics estrogen signalling in skeletal muscle. Pilot study and previous results from the hosting group, have shown that TAM improve muscle symptoms and survival rate in Mtm1-/y mice, a validate model of XLMTM. Results strongly support the idea that TAM protects skeletal muscle via enhancement of estrogenic signalling positively modulating multiple pathways linked to muscle function. However, the exact mechanism(s) is not yet understood. This project aims at elucidating the mechanism(s) of action of TAM and evaluate further its efficacy in Mtm1-/y mice. Two specific aims are proposed. First, to determine how TAM acts on pathways and key players involved in XLCNM. Estrogen signalling, phosphoinositide balance, autophagy and mTORC1 signalling were targeted in Mtm1+/y (WT from now on) and Mtm1-/y (KO from now on) mice treated and non-treated by TAM. Second, to investigate the pre-clinical efficacy of TAM combined with other compounds in XLMTM mice. TAM extends the lifespan of XLMTM mice according to the pilot study but it prevents only partly leg muscle atrophy, restricting complete functional rescue. Thus, TAM is combined with other approved drugs that target muscle atrophy (L-Citrullin and IGF).These parallel approaches will provide knowledge about not-yet explained events related to XLMTM and TAM effects, from molecular to in-vivo level, and instrumental information for other myopathies and rare diseases where lipid phosphatases are missing and phosphoinositide are unbalanced. This knowledge might provide the rationale for new therapeutic options for these dramatic conditions, being highly relevant to patients and families.The overarching objective of the project was to understand the mechanisms of action of tamoxifen and to evaluate further its efficacy in XLMTM. Two specific aims were determined to accomplish this main objective. First aim was the determination of the mechanisms underlying tamoxifen protection on XLMTM muscles. Second aim was the pre-clinical study of the combination of tamoxifen with other compounds in order to achieve a complete rescue of the XLMTM phenotype, targeting the observed muscle atrophy. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far "To accomplished the first objective, an extension of the previous pilot study was started before the beginning of the project. The performed work included included colony management, in vivo test, mice dissection and analysis of samples. The main part of this first aim was published on the paper ""Tamoxifen prolongs survival and alleviates symptoms in mice with fatal X-linked myotubular myopathy"" on Nature Communications journal, with Gold Open Access. Summarizing the main results, as it was observed on the pilot study, lifespan of treated KO mice was expanded compared to the not treated ones, clinical stage ameliorated and grid test score (in vivo test to measure muscle performance) of KO treated mice was close to WT values. Tamoxifen treatment also improved the force generated by the triceps surae, a large muscle group of the lower leg that makes up most of the calf volume and contains gastrocnemius, plantaris and soleus muscles. Tamoxifen enhanced the absolute and specific phasic and tetanic force of KO treated mice. Histology analyses were performed and it showed how tamoxifen treatment reduced by 50% the number of myofibers showing nuclei in abnormal position (a major histological feature of XLMTM). Muscle samples from WT, KO and KO treated mice allowed us to analyze the consequences of tamoxifen treatment on muscular transcript and protein levels of selected targets for their known contribution to XLMTM pathogenesis. In general, tamoxifen helps to normalize molecular hallmarks of the disease. The second aim of the project was the pre-clinical study of tamoxifen treatment with other compounds. After the good results of tamoxifen extending lifespan, combination therapy seemed to be the next step to prevent leg muscle atrophy and achieve complete functional rescue. The selected compounds were L-citrullin and IGF. L-citrullin is used by body-builders to increase muscle mass. To determine the optimal dose for mice, a pilot study was carried out and 3 mg/kg/day was choses as the minimum dose having beneficial effects on muscle mass. This compound can be added to the food and taken orally without manipulation of the mice. A modified version of IGF-1, growth factor similar to insulin, was the second compound. It stimulates proliferation and differentiation of satellite cells during muscle regeneration. Increasing IGF-1 levels on animal models affected by loose and degradation of muscle fibers has shown beneficial effects. For the moment, only a pilot study with few animals per group has been carried out. Summarizing, animals were followed-up during the whole treatment (from 21 to 45/90 day-old depending on the group). Mice weight and clinical state were determined twice a week and grid test, once a week. When arriving at the stablished time point, final measurements of respiratory function (plethysmography), body composition (echo-MRI), bone density and kyphosis (micro-CT) were performed. In general, no differences were observed between obtained results from mice treated with tamoxifen and those treated with a combined treatment. Tamoxifen combined with L-citrullin seemed to increase muscle mass in comparison with the other treatments at 45-days-old. This effect was only noticeable after muscle dissection and sample weighting. The results for bone density and the kyphose are still being analyzed." Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) XLMTM patients present with profound and generalized muscle weakness from birth and most of them die in the first 2 years of life. Some develop a milder form and may survive into adulthood and female carriers display a more heterogeneous disease. Until the moment, no specific treatment exists for XLMTM. Repurposing well-known drugs like tamoxifen might offer time- and cost-effective therapeutic options in comparison with other innovative therapies. Tamoxifen has been used for almost 40 years to treat breast cancers in both women and men and is proved safe in pediatric patients. After the described results, tamoxifen is the first EMA and FDA-approved drug to show such a promising therapeutic potential for patients with XLMTM. The combination therapy is one of the main options for this kind of serious diseases. The pilot study allowed us to explore the combination of tamoxifen with two of the possible compounds capable to recover the muscle atrophy detected on the mice treated with tamoxifen. This study will be completed in the near future and a complete analysis of obtained data will be performed. This will increase the information about these compounds and its real capacity to ameliorate muscle atrophy. Often, rare diseases like XLMTM share symptoms, pathogenic mechanisms of affected pathways. Thus, all the obtained results could be useful to understand or to increase the knowledge of other myopathies. Paper describing the effects of Tamoxifen on XLMTM
