P24 Folliculin silencing in human hair follicles ex vivo upregulates intrafollicular mechanistic target of rapamycin complex 1 activity: a new model for studying Birt–Hogg–Dubé syndrome

Abstract Introduction and aims Birt–Hogg–Dubé (BHD) syndrome is a rare multisystem disorder caused by germline pathogenic variants in the FLCN gene. BHD skin tumours predominantly affect facial skin, confer a significant psychosocial burden, and there is a lack of nonsurgical therapies. Here we report a novel ex vivo human model to study the impact of FLCN loss in human hair follicles (HFs), the putative origin of BHD skin tumours. Methods Organ-cultured human anagen HFs and scalp skin were treated with small interfering RNA targeting FLCN (n = 2 donors) for 6 days ex vivo, followed by quantitative real-time polymerase chain reaction (qRT-PCR), and quantitative immunohistomorphometry (qIHM). Results Successful FLCN knockdown was confirmed at both the mRNA (34% reduction compared with nontargeting control levels measured by qRT-PCR) and protein level (12% reduction in FLCN expression measured by qIHM). In FLCN-silenced HFs (n = 14, two donors), our preliminary Results demonstrated an increase in p-S6 expression, a direct downstream kinase of mechanistic target of rapamycin complex (mTORC)1 signalling. This was spatially restricted to the hair bulb and proximal outer root sheath keratinocytes, and was significantly greater than quantified in nontargeting control HFs (P < 0.05). Conclusions FLCN silencing in ex vivo cultured, healthy human HFs and scalp skin provides a novel, instructive and accessible preclinical research model. We aim to delineate whether FLCN interacts with the key endogenous inhibitor of mTORC1 activity, TSC2, in future work, to gain insight into the role of FLCN in the pathobiology of BHD and the maintenance of healthy human HFs.