PROJECT SUMMARY/ABSTRACT: Basal cell carcinoma (BCC) represents the most common type of skin cancer with more than four million cases in the US each year. BCCs have the ability to circumvent therapeutics due to their intrinsic tumor plasticity, allowing for incredible tumor evolution. The identification of non-canonical pathways that drive tumor resistance has dramatically impacted the understanding of BCC resistance mechanisms, however, the complex tumor heterogeneity generates a significant knowledge gap, which continues to hamper clinical outcomes. The observation that BCCs can undergo tumor evolution events (referred to as pathway switching) to squamous cell carcinoma (SCC) represents a resistance mechanism involving complete alterations in gene expression programs leading to cancer-type switching. The long-term goal of this proposal is to elucidate the molecular and transcriptional mechanisms underlying pathway switching with particular emphasis on understanding the epigenetic regulation at single cell resolution. Preliminary data indicate that SCC-signatures develop early in tumor progression, which is in part due to differential transcriptional regulation of AP-1 family members, but what specific transcription factor is not clear. Therefore, the central hypothesis underlying this proposal is that dynamic epigenetic regulation promotes the acquisition of SCC-like features in BCCs, potentially early on in tumor development. Aim 1 will uncover the key AP-1 family transcription factor(s) that drive BCC to SCC pathway switching utilizing a novel in vitro system which can faithfully model this process. Aim 2 takes advantage of scRNA-Seq datasets from naïve tumors and proposes additional scRNA-Seq along with scATAC-Seq experiments to define a multi-omic temporal progression of BCC to SCC pathway switching. With a pipeline to acquire fresh human BCC tumors established and the ability to leverage expertise in single-cell genomic analysis, the proposed research is highly feasible in the allotted time. The overall rationale for the proposal is to adequately address a clinically unmet need to understand a key resistant mechanism in BCC. This highly innovative proposal uses novel tools, coupling an in vitro system to model pathway switching with single cell gene expression and chromatin analysis of patient samples. This work has great promise to fill a significant knowledge gap concerning mechanisms of BCC tumor evolution with goals to clear the path for more targeted and rationale drug therapies.