092 Merkel cell polyomavirus small T antigen co-expressed with Atoh1 drives development of intraepidermal MCC

Abstract

092 Merkel cell polyomavirus small T antigen co-expressed with Atoh1 drives development of intraepidermal MCC ME Verhaegen, D Mangelberger, PW Harms, D Wilbert, J Meireles, T Saunders and AA Dlugosz 1 Dermatology, University of Michigan, Ann Arbor, MI, 2 Pathology, University of Michigan, Ann Arbor, MI and 3 Internal Medicine, University of Michigan, Ann Arbor, MI Merkel cell carcinoma (MCC) is a rare and deadly neuroendocrine skin tumor that frequently expresses viral transforming antigens, sTAg and tLTAg, encoded by Merkel cell polyomavirus (MCPyV). While the cell of origin is unknown, MCCs express markers detected in normal Merkel cells, including Keratin (K) 20, K8, and synaptophysin. Most MCCs and MCC cell lines also express ATOH1, a bHLH protein that is required for Merkel cell specification, and when expressed ectopically, drives de novo development of Merkel cells from epidermis. To test the potential of MCPyV viral oncoproteins to drive MCC development, we co-expressed MCPyV TAgs and Atoh1 in epidermis of transgenic mice. Combined expression of sTAg and Atoh1 in skin of multiple independent founders induced striking alterations that mimicked the appearance of human intraepidermal MCC, including numerous foci of small blue cells with nuclear hyperchromasia, mitotic activity and epidermal clefting. These tumor-like collections of cells expressed typical Merkel cell and MCC markers, including synaptophysin, K8 and K20. In contrast to normal Merkel cells, however, K20 was present in a paranuclear dot-like pattern which is pathognomonic of MCC. Moreover, many tumor cells expressed both K8 and the proliferation maker Ki67, which is characteristic of MCC tumor cells but not Merkel cells, which are post-mitotic. Addition of tLTAg did not appreciably alter the MCC-like phenotype produced by co-expression of sTAg and Atoh1. Thus, expression of MCPyV sTAg in Atoh1expressing epidermal cells of pre-term mouse embryos leads to widespread development of MCC-like tumors, strongly supporting the concept that sTAg is the primary oncogenic driver of MCC when expressed in the appropriate progenitor population. 093 Cell fate determination in the initiation of high-risk skin carcinoma WK Ryan, J Fernandez and EC Torchia Dermatology, University of Colorado Anschutz Medical Campus, Aurora, CO There are few effective treatment options for high-risk Squamous Cell Carcinomas (SCCs). Our goal in this study was to define the early signaling pathways in “initiated” Keratinocyte (KC) progenitors that contribute to the progression of high-risk tumors in a well-characterized mouse SCC model. Here, the activation of a Gain-Of-Function (GOF) p53 mutant resulted in tumors with centrosome amplification, dysregulation of the mitotic Kinase Aurora-A (Aur-A), genomic DNA instability, and enhanced metastasis, implicating abnormal mitotic regulation in the progression to high-risk SCCs. We initially observed a marked S-phase increase in both GOF and Loss-Of-Function (LOF) p53-KCs after a short course of the tumor promoter, TPA. However, histological presentation remained similar. The genetic deletion of Aur-A (Aur-A) in GOF or LOF p53 skin led to dramatic morphological changes associated with diffused hyperplasia, cellular pleomorphism, cell karyomegaly, prominent nucleoli, and multinucleation. These effects were more pronounced in Aur-A; GOF p53 skin, but surprisingly, there was minimal induction of DNA damage or cell death. By RNA seq, subtle differences between GOF and LOF p53 skin were observed. However, co-deletion of Aur-A markedly altered gene expression profiles. Analysis by the IPA software predicted the activation of the PI3K/mTOR pathway in AurA;GOF p53 skin. By immunoblotting, phospho/ total S6 protein ratios were increased in GOF vs LOF p53 skin, but markedly elevated after Aur-A deletion, whereas Bcl-xl protein levels remained unchanged. We confirmed p-S6 levels by IF and noted its predominantly suprabasal expression in untreated controls that extended to the basal layer in Aur-A skin. Lastly, we sorted KCs from TPA treated skin and perform single cell RNA sequencing. Pathway analysis revealed the enrichment of mTOR pathway signatures in GOF p53 KCs, suggesting that the involvement of the pathway in GOF p53 skin was cell-autonomous to KCs. Our data indicate that the PI3K/mTOR pathway may have role in the initiation of high-risk SCCs.