Preclinical validation of PKC412 as a therapy candidate for epidermolysis bullosa simplex across multiple keratin pathogenic variants.

Abstract

Epidermolysis bullosa simplex (EBS) is a hereditary skin fragility disorder caused by missense pathogenic variants in KRT5 or KRT14. These variants trigger the collapse of the cytoskeleton into cytoplasmic protein aggregates, rendering the epidermis highly susceptible to mechanical stress, leading to intraepidermal blistering and the loss of intercellular cohesion. No molecular therapies for EBS currently exist. To characterize keratin 5 (K5) or keratin 14 (K14) mutant keratinocytes from patients with EBS in response to PKC412 treatment in monolayer culture and epidermal equivalents, to clarify the potential of PKC412 as a drug repurposing therapy approach in EBS. We conducted a comprehensive characterization of K5 and K14 mutant keratinocytes in response to PKC412, examining its effects on proliferation, wound closure and apoptosis. Additionally, we evaluated the improvement of intercellular cohesion through stretch assays, epithelial sheet assays and assessment of desmosomal organization. Finally, we investigated the efficacy of PKC412 application both in skin explants and EBS-derived epidermal equivalent cultures. We found that PKC412 is effective in various keratinocytes carrying pathogenic variants associated with localized, generalized or intermediate forms of EBS. PKC412 enhanced intercellular adhesion both in immortalized healthy and EBS keratinocytes, as well as healthy primary keratinocytes, and under stretch conditions. Immunoblot analyses revealed a concentration-dependent reduction in desmoplakin phosphorylation, which remained stable over the course of 3 days at the sites investigated. Additionally, application of PKC412 in epidermal equivalent cultures restored desmoplakin distribution in the epidermal basal layer. PKC412 markedly enhanced intercellular cohesion and stress resilience in patient-derived EBS keratinocytes, both in monolayer and three-dimensional culture systems. These findings highlight PKC412 as a promising therapeutic candidate for the treatment of EBS.