SUMMARY Mutations that activate proto-oncogenes or inactivate tumor suppressor genes are the root causes of tumor initiation, but recent genomic analyses also detect these mutations in many cells of healthy tissues. These data and classic skin carcinogenesis studies suggest that mutations in proto-oncogenes or tumor-suppressor genes are tolerated and maintained in epidermal progenitor cells (EPCs) until elusive mechanisms transform these precancerous cells into stem-cell-like tumor propagating cells (TPCs) that can support tumor formation and growth. We transcriptionally profiled these TPCs in squamous cell carcinoma (SCC) models and defined a gene expression signature that distinguishes TPCs from EPCs. Within this signature, we identified the transcription factors PITX1 and SOX2 in >60% of mouse and human SCCs, even though they are epigenetically repressed and not detectable in normal skin epithelial cells. We showed PITX1 and SOX2 are required for SCC growth in mouse and patient-derived SCC models highly enriched on gene regulatory enhancers accessible in TPCs but not EPCs, and responsible for the expression of SCC signature genes. Although PITX1 and SOX2 are pivotal for squamous carcinogenesis, it is still unclear what events trigger their de novo expression in TPCs, and whether their ability to bind condensed (inactive) chromatin and open it to activate SCC-specific gene expression reprograms EPCs into TPCs. Here, we propose to test the hypothesis that oncogenic RAS and inflammation together or independently activate RUNX1, which initiates expression of PITX1 and then SOX2, allowing them to transcriptionally reprogram EPCs into TPCs that promote squamous carcinogenesis. To test this hypothesis, we propose to: 1) determine whether and how inflammation-induced RUNX1 activity promotes de novo PITX1 expression in normal and pre-cancerous skin epithelial cells; and 2) determine whether and how ectopically expressed RUNX1, PITX1, and/or SOX2 reprogram EPCs into TPCs to promote SCC initiation. To accomplish these aims, we already established genetic gain- and loss-of-function approaches in autochthonous mouse and human SCC models along with genome-wide analyses (ATAC-seq, ChIP-seq, 4C-seq, RNA-seq) and fluorescent transcriptional reporter assays. We are uniquely positioned to reveal the molecular events that explain: 1) how PITX1 and SOX2 become expressed de novo in mouse and human SCCs, 2) whether SOX2 and PITX1 establish or use SCC-defining gene regulatory enhancers to control the expression of SCC signature genes, and 3) how these changes in gene expression transform precancerous cells into TPCs that promote squamous carcinogenesis. Our proposed research will provide molecular insights that promise to guide the development of approaches to prevent or treat SCCs in patients and/or define mechanisms that may promote the initiation of other cancers including but not limited to lung, esophageal, and pancreatic SCCs, which are among the most common de...