The lineage master transcription factor p63 cooperates with FOXK1, a cancer-specific cofactor, to mediate oncogenic 3D chromatin dynamics in squamous cell carcinoma.

Summary: Abstract Body: Transcriptional dysregulation by lineage-specific master transcription factors (TFs) is critical in cancer, often altering three-dimensional (3D) chromatin architecture. However, the mechanisms through which master TFs drive these changes remain unclear. Here, we investigate the role of the epidermal master TF, p63, in remodeling 3D chromatin in squamous cell carcinomas (SCCs) compared to normal keratinocytes (KCs). Using a multi-omic approach—integrating chromatin looping, accessibility, transcriptomics, p63 binding, and proteomics—we show that p63 increases connectivity between prognostic genes and cis-regulatory elements (CREs). Gene Ontology term enrichment confirmed a critical role for p63 in regulation of cell proliferation in progenitor KC and SCC, with >67% cancer-specific p63 binding sites enriched for target genes related to the cell cycle pathway. p63-dependent genomic accessibility was also increased in SCC lines and up to 40% was not detected in primary KCs, suggesting p63 may uniquely regulate chromatin architecture in cancer. Integrative analysis of p63 cancer-specific binding, accessibility and looping led to the identification of a unique type of 3D chromatin regulation, termed ‘Secondary’ mode, where p63 indirectly regulates SCC-specific tumor-promoting genes through an additional CRE lacking direct p63 epigenetic signatures. Proximity proteomics was employed to identify the FOXK1 TF as an SCC-specific p63 cofactor. The p63-FOXK1 interaction was validated in SCC cells, in which FOXK1 loss phenocopied p63 loss, as well in human SCCs. FOXK1 binding was found in 32% of p63-dependent chromatin loops, indicating FOXK1 collaborates with p63 to shape these oncogenic chromatin interactions and drive cell proliferation. These findings uncover the p63-FOXK1 axis as a potential therapeutic target in SCCs. Vanessa Lopez-Pajares¹, Namyoung Jung¹, Laura Donohue¹, Margaret Guo¹, Paul Khavari^{1, 2} 1. Stanford University, Stanford, CA, United States. 2. VA Palo Alto Health Care System, Palo Alto, CA, United States. Genetic Disease, Gene Regulation, Gene Therapy & Epigenetics