Biomarkers of UV irradiated notch-deficient keratinocytes driving neoplasia.

Summary: Abstract Body: Actinic keratosis (AK) and squamous cell carcinoma in situ (SCCIS) are precancerous lesions that can progress to cutaneous squamous cell carcinoma (cSCC). To better understand cSCC development, we used omics platforms to a murine skin cancer model and human SCCIS samples to identify biomarkers of disease. We used K14-CreERT2-DNMAML;GFP mice to generate random Notch-deficient keratinocytes which were subjected to UV irradiation or sham followed by in vivo fluorescence microscopy to track clone development. UV irradiated Notch-deficient keratinocytes demonstrated both positive and negative selection. Positive selection of large clones, >2000um2, were significantly increased in irradiated Notch-deficient cells compared to non-irradiated Notch-deficient cells, p <0.01 . Notch-deficient keratinocytes showed negative selection manifested by fewer clones in areas of UV irradiated skin compared to unirradiated skin, p <0.0001. UV Irradiated skin containing GFP-labeled clones was subjected to spatial transcriptomics followed by qRT-PCR and RNAscope to identify differentially expressed genes. Twenty-six differentially expressed genes in the Notch-deficient clones including calmodulin isoform CALML3 (p<4.6x10-6). To validate this data, RNAscope was performed on human SCCIS and adjacent epidermis. CALML3 was markedly overexpressed in SCCIS (Score 4>) in the lesion compared to adjacent epidermis (Score 1), N=8. To better assess the expression of all six human calmodulin genes in human SCCIS and adjacent epidermis we analyzed expression of all 6 genes using the Xenium platform. Xenium analysis revelated that CALM1 (p<0.0001), CALM3 (p<0.001) and CALML3 (p<0.01) were overexpressed in SCCIS compared to adjacent epidermis. Expression of CALM2, CALML4 and CALML6 did not statistically differ between SCCIS and epidermis. These data confirm that calmodulin genes are biomarkers for human SCCIS and further experiments will determine the mechanism associated with upregulation of these genes. Yoko Suzuki-Horiuchi¹, Matthew L. Hedberg¹, Qi Zheng¹, Eun K. Ko¹, Stephen Prouty¹, Pantelimon Rompolas¹, Elizabeth Grice¹, Brian Capell¹, Vivian Lee¹, John T. Seykora¹ 1. Dermatology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, United States. UV Biology/Injury and Non-melanoma Cancers