Merkel cell carcinoma (MCC), a rare skin cancer, is mostly driven by integration of the Merkel cell polyomavirus which encodes T-antigen (T-Ag) proteins. Previous research has shown that T & B cells target T-Ag. Indeed, patients with virus-driven MCC produce T-Ag-specific antibodies that are useful to track disease progression. These antibodies do not play a direct role in MCC immunity as T-Ag proteins are intracellular. Our group has recently found that in tumors, T-Ag-specific B cells with germinal center or antibody-secreting phenotypes strongly predict improved MCC outcomes. These intratumoral B cell phenotypes reflect a robust cancer-specific T cell response. In contrast, T-Ag-specific B cells in the blood of MCC patients are predicted to predominantly have a memory or naive phenotype, and it is unknown if they contribute to anti-tumor immunity. We used fluorescently labeled T-Ag-proteins and flow cytometry to assess B cell responses in blood at the time of MCC diagnosis. In total, we analyzed samples from 23 patients whose MCC recurred within 3 years of diagnosis and 24 samples from stage- and age-matched MCC patients whose disease did not recur. We found no difference in the frequency of all circulating B cells (regardless of T-Ag-specificity) between patients who did and did not develop MCC recurrence. In contrast, higher frequencies of total memory B cells (CD27+IgD-IgM-) were associated with an increased risk of disease recurrence (HR 3.67 [1.58- 8.55], p=0.003). Intriguingly, T-Ag-specific memory B cells were also more abundant in the blood of patients who ultimately developed MCC recurrence (HR 2.82 [1.22- 6.53], p=0.012). Together, our results demonstrate that higher frequencies of circulating memory B cells associate with worse MCC outcomes. These findings suggest that the functional state of total and T-Ag-specific circulating B cells reflect their immune response within MCC tumors.

Hailey-Hailey disease (HHD) is a rare autosomal dominant skin disorder characterized by recurrent epidermal blistering and infections, significantly impacting quality of life. The disease results from ATP2C1 mutations causing deficiency of SPCA1. This Golgi-embedded calcium pump is critical for folding and trafficking proteins, including cadherins that mediate desmosomal cell-to-cell adhesion. Although its genetic cause was identified 25 years ago, HHD remains an orphan disease without any FDA-approved treatments. Drug development for HHD has been hindered by the lack of pre-clinical models as knockout mice failed to replicate the disease. To overcome this barrier, we engineered human cellular and tissue models of HHD to elucidate its pathogenesis and identify therapeutic strategies. Utilizing CRISPR/Cas9, we ablated ATP2C1 in TERT-immortalized human epidermal keratinocytes and generated cell lines with haploinsufficiency or total loss of SPCA1. Mechanical dissociation assays showed SPCA1-depleted keratinocyte sheets had weakened intercellular adhesion. Explaining this phenotype, immunoblots of SPCA1-deficient keratinocyte lysates revealed lower expression of desmosomal cadherins, while immunofluorescent staining showed reduced concentration of adhesive proteins at cell-cell junctions. These effects induced tissue splitting within 3D organotypic epidermis, replicating the key feature of HHD pathology. To define the mechanisms impairing cadherin localization in our model, we performed RNA sequencing, which showed dysregulation of the actin cytoskeleton in SPCA1-deficient cells. We used live confocal microscopy to confirm that SPCA1 depletion altered actin organization in keratinocytes and we hypothesize that this disrupts cadherin trafficking and cell-cell junction stability. Using image analysis software to quantify cytoskeletal morphology, we found that SPCA1-deficient cells had reduced linear actin filaments, which could impact intracellular protein trafficking and keratinocyte migration during wound healing. In sum, our novel pre-clinical model replicated HHD pathology and revealed that actin dysregulation may undermine intercellular adhesion and could represent a new therapeutic target for this orphan blistering disorder.

Cutaneous squamous cell carcinoma (cSCC) is the second most common cancer in the United States. Distant metastasis serves as the primary cause of mortality. It is therefore important to identify molecular signatures as potential therapeutic targets that contribute to metastatic phenotypes of cSCC. To profile gene expression in cSCCs, we performed RNA-seq analyses of normal skin (n = 6), primary cSCCs (n = 12), and metastatic cSCCs (n = 3). To identify differentially expressed genes, we created a hierarchical clustering heatmap of top 5,000 most variable genes across these 21 samples. This analysis identified multiple clusters of coordinately expressed genes, and we selected three clusters that showed robust upregulation or downregulation in metastatic cSCCs, compared to normal skin and primary cSCCs. Gene set enrichment analysis for each of these three clusters revealed what pathways were associated with upregulation or downregulation of the genes in each cluster. Cluster 1 includes 786 genes that were upregulated in metastatic cSCCs, and Cluster 1 was associated with mitotic cell cycle, indicating hyperproliferation of cancer cells. Cluster 2 includes 932 genes that were downregulated in metastatic cSCCs, and Cluster 2 was associated with lipid biosynthetic pathway. The downregulation of this pathway may reflect the loss of differentiated skin cells during cancer progression. Cluster 3 includes 440 genes that were also downregulated in metastatic cSCCs, and Cluster 3 was associated with extracellular matrix organization pathway. The extracellular matrix is a structural scaffold for tissues, and its dysregulation is related to tumor growth and metastasis. The sets of identified genes and pathways provide novel insights into potential biomarkers and therapeutic targets for patients with cSCC.