Autophagy induction as a new therapeutic target for pachyonychia congenita

Summary: Abstract Body: Introduction: Pachyonychia Congenita (PC) is an ultrarare dermatologic disorder affecting an estimated 10,000 people worldwide. PC is caused by an autosomal dominant mutation in a series of keratin genes (KRT6A/B/C, KRT16, & KRT17). PC manifests with blisters, ichthyosis, deformed, thickened, or discolored nails and constant plantar pain. Plantar pain has the greatest impact on quality of life. PC is an unmet need with no approved therapies. PC and epidermolysis bullosa simplex (EBS) are caused by mutated keratins that form structurally weakened keratin intermediate filaments (KIFs). Stress collapses the KIFs into aggregates weakening the suprabasal keratinocytes causing premature cell death which contributes to ichthyosis. Clinical trials for oral rapamycin were not well-tolerated. Topical rapamycin trials did not reach significance. Our research suggests a potential benefit of autophagy induction by mTOR inhibitors to treat EBS. The purpose of this research was to evaluate mTOR inhibitors including BM-3103 (4-Hydroxy 4'-methyoxytolan) and rapamycin in vitro in PC keratinocytes. Specific aims: 1) autophagy induction, 2) KIF stability, 3) uptake of keratin by the autophagosome. Methods: In vitro analysis was performed using immortalized cell lines (K6a N171K, K16, N125D) derived from PC patients. Autophagy signaling was evaluated using western blotting and immunocytochemistry. A heat stress assay was used to assess KIF aggregation. Colocalization using immunofluorescence of keratin (K6a/K16) uptake by the autophagosome (LC3-II) was done. Results: At 25uM BM-3103 was significantly better than rapamycin at inducing autophagy(p=0.03). BM3103 increased LC3-II levels 5-12-fold vs 0.5-1.6-fold for rapamycin (0.4μM). BM-3103 treated cells resisted KIF collapse after heat stress. Keratin was colocalized with LC3-II staining of autophagosomes. Conclusions: Induction of autophagy in PC cells increasing KIF network stability through the degradation of mutant and damaged keratins. mTOR inhibitors like BM-3103 need to be clinically tested to assess impact on blistering and ichthyosis. Karen McGuire¹, Theresa Rowe¹, Laurie Broadwater¹, Aleesha McCormick¹ 1. BioMendics, LLC, Rootstown, OH, United States. Translational Studies: Cell and Molecular Biology