Connecting liquid-liquid phase separation with super-enhancers and key oncogene transcription in HNSCC - Project Summary/Abstract Cells can perform their functions by spatiotemporally enriching biomacromolecues to form membrane-less condensates through liquid-liquid phase separation (LLPS). The formation of phase-separated condensates allows the cell to concentrate regulatory proteins with interaction partners to accelerate biological processes not only for cellular homeostasis but also for disease progression. Targeting condensates that promote dysregulated cellular processes has been suggested as a novel approach to developing new drugs. Studies have linked LLPS to super-enhancers (SEs), a transcriptional mechanism driven by an extremely high density of master regulators and Mediator complex that maintains the high expression of cell-type specific genes to determine cell identity, disease states, and most notably, oncogenesis. It has been implicated that the formation of phase-separated condensates of master regulators with Mediators is critical for activating the transcription of cell identity genes driven by SEs. However, how the master regulators undergo LLPS at SEs to maintain key oncogene expression is still largely under investigation, especially in HNSCC. In addition, no therapeutics are available for targeting SEs in cancer treatment, indicating the need for alternative strategies to be developed. We recently discovered that FOSL1 serves as a master regulator to promote the malignant progression of HNSCC through establishing SEs with Mediator complex (MED1) at key oncogenes. However, how FOSL1 drives key oncogene expression without a transcriptional activation domain is still not fully understood. Furthermore, the crystal structure of FOSL1 still remains unsolved and the structure-based drug design for FOSL1 inhibitors has been severely stifled. Currently, there are no FOSL1 inhibitors for cancer treatment. Using the AlphaFold program, we identified two intrinsically disordered regions (IDRs) in FOSL1. The presence of IDRs in a protein is frequently diagnostic of its ability to phase separate. We found that purified FOSL1 recombinant proteins can phase separate at in vitro conditions. Moreover, phase-separated condensates containing FOSL1 can be visualized in live HNSCC cells. Based on these results, we hypothesize that FOSL1 associates with MED1 to undergo phase separation to establish super-enhancers at key oncogenes and maintain their high expression and consequent malignant phenotype of HNSCC. We proposed the following two specific aims to validate our hypothesis. In Aim 1, we will determine whether FOSL1 cooperates with MED1 to assemble phase-separated condensates at in vitro conditions and also in live HNSCC cells. Furthermore, we will also examine these condensate formations at key oncogenes driven by SEs in HNSCC cells and patient-derived xenografts. In Aim 2, we will explore whether the deletion of IDRs in FOSL1 will abolish condensate formation to disrupt SEs to suppress the expression of key oncogenes and the consequent malignant phenotype of HNSCC. The successful accomplishment of this study will provide solid evidence to link LLPS with SEs and key oncogene expression in HNSCC, support a notion that targeting FOSL1 phase separation suppresses HNSCC tumorigenesis, and lay the foundation for future studies.
