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Chronic pain is a major health issue, and the search for new analgesics has become increasingly important because of the addictive properties and unwanted side effects of opioids. To explore potentially new drug targets, we investigated mutations in the NTRK1 gene found in individuals with congenital insensitivity to pain with anhidrosis (CIPA). NTRK1 encodes tropomyosin receptor kinase A (TrkA), the receptor for nerve growth factor (NGF) and that contributes to nociception. Molecular modeling and biochemical analysis identified mutations that decreased the interaction between TrkA and one of its substrates and signaling effectors, phospholipase Cγ (PLCγ). We developed a cell-permeable phosphopeptide derived from TrkA (TAT-pQYP) that bound the Src homology domain 2 (SH2) of PLCγ. In HEK-293T cells, TAT-pQYP inhibited the binding of heterologously expressed TrkA to PLCγ and decreased NGF-induced, TrkA-mediated PLCγ activation and signaling. In mice, intraplantar administration of TAT-pQYP decreased mechanical sensitivity in an inflammatory pain model, suggesting that targeting this interaction may be analgesic. The findings demonstrate a strategy to identify new targets for pain relief by analyzing the signaling pathways that are perturbed in CIPA.

Epigenetic dysregulation is an important emerging hallmark of cutaneous melanoma development. The global loss of DNA methylation in gene-poor regions and transposable DNA elements of cancer cells contributes to increased genomic instability. Long interspersed element-1 (LINE-1) sequences are the most abundant repetitive sequence of the genome and can be evaluated as a surrogate marker of the global level of DNA methylation. In this work, LINE-1 methylation levels were evaluated in cutaneous melanomas and normal melanocyte primary cell cultures to investigate their possible association with both distinct clinicopathological characteristics and tumor mutational profile. A set of driver mutations frequently identified in cutaneous melanoma was assessed by sequencing (actionable mutations in BRAF, NRAS, and KIT genes, and mutations affecting the TER T promoter) or multiplex ligation-dependent probe amplification (MLPA) (CDKN2A deletions). Pyrosequencing was performed to investigate the methylation level of LINE-1 and CDKN2A promoter sequences. The qualitative analysis showed a trend toward an association between LINE-1 hypomethylation and CDKN2A inactivation (p=0.05). In a quantitative approach, primary tumors, mainly the thicker ones (>4 mm), exhibited a trend toward LINE-1 hypomethylation when compared with control melanocytes. To date, this is the first study reporting in cutaneous melanomas a possible link between the dysregulation of LINE-1 methylation and the presence of driver mutations., (Copyright © 2016 American Federation for Medical Research.)