299 Biomarkers of early UV-induced skin cancer

Actinic keratosis (AK) and squamous cell carcinomain situ (SCCIS) are the most common precancerous lesions and may progress to cutaneous squamous cell carcinoma (cSCC), the second most common cancer in the US. Biomarker identification associated with keratinocyte clones undergoing UV-induced selection may provide new targets to treat AK/SCCIS. Prior studies using comparative whole exome-seq and RNA-seq studies of SCCIS and epidermis identified UV-signature loss-of-function mutations in NOTCH1-3 in keratinocyte clones that may represent AK/SCCIS precursors and upregulation of calcium signaling genes in SCCIS. To identify genes that cooperate with loss of NOTCH function during UV-induced clonal selection, we performed spatial transcriptomics on Notch-deficient keratinocyte clones in UV-irradiated K14-CreER;ROSA26-loxp-STOP-loxp-tTA;TRE-H2BGFP;TRE-DNMAML transgenic mice which permit visualization of UV-induced epidermal clones in Notch-deficient keratinocytes. Mice harboring scattered Notch-deficient keratinocytes in skin were UV-irradiated and followed by in vivo macrofluorescence microscopy to track clone development. Representative samples of irradiated skin containing were subjected to spatial transcriptomic analysis followed by qRT-PCR and RNAscope. Spatial transcriptomics identified 26 differentially expressed genes in the Notch-deficient clones (FDR<1x10-5), including calmodulin isoform CALM3 (p<4.6x10-6). RNAscope in situ hybridization using semi-quantitative scoring (range 0-4, 4 is strong signal) showed CALM3 overexpression (Score 4>) in AK/SCCIS compared with epidermis (Score 1). CALM1 and CALM2 were also overexpressed (Score 3) in SCCIS. High resolution spatial transcriptomics of human SCCIS and epidermis confirmed increased CALM3 (FDR<0.003) and CALM2 (FDR<1x10-5) expression in SCCIS lesions (N=34). These data indicate that calcium signaling pathways regulated by calmodulin are upregulated in AK/SCCIS.