401 Dermal fibroblasts with loss of TSC2 express increased galectin-3, a potential biomarker for tuberous sclerosis complex

400 Amlexanox: Efficacy and mechanism for potential therapy for RDEB V Atanasova, Q Jiang, J Uitto and A South Thomas Jefferson University, Philadelphia, PA Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin blistering disorder caused by loss-of-function mutations in the COL7A1 gene, which encodes type VII collagen (ColVII). ColVII is the main, if not exclusive, component of anchoring fibrils that adhere the epidermis to the dermal layer of the skin. Mutations in COL7A1 lead to defective anchoring fibrils and subsequent perturbed adhesion between the two skin layers resulting in severe skin fragility and constant wounding without ability to heal. A subgroup of RDEB patients harbors nonsense mutations in COL7A1 which create premature termination codons (PTCs) of translation in the mRNA molecule. This leads to mRNA instability and lack of functional protein synthesized. A group of drugs have been reported for their ability to induce “readthrough” and allow for insertion of an amino acid at the site of PTC, thus producing fulllength protein. The mechanism by which these drugs act is currently not known. Here we focus on a PTC read-through drug, amlexanox, previously approved by the FDA for mouth ulcers. Our data show that amlexanox is able to induce full-length synthesis of ColVII in HpV immortalized RDEB patient derived keratinocytes and fibroblasts, as shown by western blotting. After 48 hours of treatment keratinocytes showed up to 14 fold increase in full-length ColVII protein synthesis as compared to non-treated controls. The same treatment in HpV fibroblasts led up to 3 fold increase in ColVII as compared to non-treated controls, or 25% of normal human fibroblasts. While the mechanism by which amlexanox is able to recover protein synthesis from the PTC containing COL7A1 gene is currently unclear, activation of UPF1 by its phosphorylation has been shown to be required for assembly of mRNA degradation machinery. Our data show that amlexanox treatment increases UPF1 phosphorylation, which may be part of the PTC read-through mechanism. In conclusion, we present data to support amlexanox as a read-through agent for the treatment of RDEB in cases with PTC in COL7A1. 401 Dermal fibroblasts with loss of TSC2 express increased galectin-3, a potential biomarker for tuberous sclerosis complex P Klover, R Thangapazham, J Kato, J Wang, S Li, C Dalgard, M Wilkerson, E Mccart, J Moss and T Darling 1 Dermatology, Uniformed Services University of the Health Sciences, Bethesda, MD, 2 Cardiovascular and Pulmonary Branch, NIH/NHLBI, Bethesda, MD and 3 Anatomy, Physiology, and Genetics, Uniformed Services University of the Health Sciences, Bethesda, MD Tuberous sclerosis complex (TSC) is an autosomal genetic disorder in which tumors form in multiple organs due to biallelic inactivating mutations in TSC1 or TSC2 and increased mTORC1 signaling. In order to discover biomarkers for TSC, we studied our mouse model of TSC with conditional deletion of Tsc2 in dermal mesenchyme (Tsc2cKO), testing the potential clinical relevance of findings using human TSC skin tumors. RNA-sequencing analysis of cultured Tsc2-null and wild-type mouse dermal fibroblasts identified galectin-3 as overexpressed by the mutant cells. Levels of both intracellular and secreted galectin-3 protein were approximately 3-fold greater in Tsc2-null fibroblasts than wild-type fibroblasts. Tsc2-null cells incubated in the presence of 20 nM sirolimus, an mTORC1-inhibitor, showed decreased galectin-3 expression but not to the level of wild-type fibroblasts, suggesting that overexpression of galectin-3 is only partially mTORC1-dependent. Serum levels of galectin-3 were 68% greater (p1⁄40.0015) in Tsc2 cKO than wild-type mice and sirolimus treatment for 4 weeks decreased galectin-3 serum levels 25% (p1⁄40.036) in the Tsc2cKO. Fibroblast-like cells grown from human TSC skin tumors, including angiofibromas, fibrous cephalic plaque, and periungual fibromas, showed greater intracellular and secreted galectin-3 levels than patient normal fibroblasts. Immunohistochemical analysis showed abundant galectin-3 positive dermal cells in TSC skin tumors but nearly absent dermal staining in paired samples of patient normal-appearing skin. Our results demonstrate that loss of TSC2 in dermal fibroblasts is associated with increased galectin-3 and suggest that this molecule should be investigated as a biomarker for TSC and possibly other diseases associated with increased mTORC1 activation.