Delineating pathogenic effects of ATP2C1 loss-of-function in human keratinocytes and organotypic epidermis to identify therapeutic strategies for Hailey-Hailey disease

Abstract: The epidermis provides a critical barrier between the body and its environment; it is composed of multiple layers of keratinocytes that form robust connections by assembling desmosomes between neighboring cells. Breakdown of desmosomes induces separation between keratinocytes, called acantholysis, a hallmark of

several skin blistering disorders, most notably Hailey-Hailey disease (HHD). This inherited disease manifests as recurrent skin erosions causing super-infections, chronic pain, and reduced quality of life for patients. The advent of biologic and molecular therapies has brought relief to many patients with inflammatory skin

diseases, but comparable advances for HHD and other genetic cutaneous pathologies remain elusive. Despite linkage to the ATP2C1 gene more than two decades ago, there are no FDA-approved therapies for HHD and ablating Atp2c1 in mice did not replicate HHD, hampering translational work. ATP2C1 encodes a Golgi calcium

ATPase SPCA1, but our limited understanding of how SPCA1 loss compromises skin integrity limits rational drug development for HHD. Using CRISPR/Cas9, we ablated ATP2C1 in human keratinocytes to build cellular and tissue models of HHD with the goal of delineating its pathogenesis and identifying putative drug targets.

Our preliminary data show that SPCA1-deficient cells model HHD pathology with impaired cohesion in epithelial sheets and organotypic epidermis; this is consistent with our finding impaired expression and trafficking of adhesive proteins as well as abnormal cell morphology suggesting cytoskeletal dysregulation. To

identify pathogenic drivers downstream of SPCA1 loss, we performed RNA sequencing. Our data revealed up- regulation of (1) Rho GTPase signaling and actin modifiers, which modulate cell-cell adhesion, and (2) stress- mitigating pathways, including autophagy, the unfolded protein response (UPR), and antioxidant metabolism.

Our aims will determine how these cellular dysfunctions compromise keratinocyte cohesion and determine if targeting them can restore epidermal integrity in our HHD model. Aim 1 will test the hypothesis that SPCA1 deficiency compromises cell-cell adhesion due to faulty cadherin trafficking and impaired regulation of RhoA

and actin. Aim 2 will test the hypothesis that up-regulation of autophagy, induction of the UPR, and dampening of reactive oxygen species (ROS) could restore tissue integrity in HHD. Combining our established systems for live cell and tissue imaging with novel biosensors, we will delineate the pathogenic signals downstream of

SPCA1 loss. To validate the functional role of these pathways in adhesion, we will evaluate candidate drugs using a mechanical dissociation assay, then test if lead compounds restore integrity in our HHD tissue model. In sum, the proposed work leverages new human cellular and tissue models to identify pathways that drive

epidermal breakdown in HHD. Results from the planned studies will provide a platform for future work to validate druggable targets in patient-derived cells and de-identified HHD biopsies with the ultimate goal of delivering lead drugs for clinical trials aiming to improve skin integrity in patients living with this rare disorder.