FOSL1-super-enhancers define cisplatin-enriched cancer stem cells in HNSCC

Project Summary/Abstract The chemotherapeutic drug cisplatin is a first-line drug for the treatment of many solid tumors, especially head and neck squamous cell carcinoma (HNSCC). However, resistance to cisplatin has become a major obstacle to effective cancer therapy. Cisplatin is well-known to enrich the cancer stem cell (CSC) population, which can contribute to chemoresistance. Accordingly, targeting CSCs may represent an effective strategy to overcome cisplatin resistance. Previously, we demonstrated that FOSL1 promotes tumorigenesis and metastasis of HNSCC through establishing Super-Enhancers (SEs) at key cancer stemness and pro-metastatic genes, and furthermore, disruption of FOSL1-SEs suppresses HNSCC growth and metastasis. Additionally, we have discovered that increased FOSL1 activity facilitates cisplatin resistance of CSCs in a spontaneous mouse model of HNSCC. Taken together, we believe that the FOSL1-SE-dependent transcription program may represent a novel target for overcoming cisplatin resistance in HNSCC. To validate this, the key scientific questions of whether FOSL1-SEs are present in cisplatin-enriched CSCs and how to determine their functional properties must be addressed, as these FOSL1-SEs were originally characterized using whole HNSCC cell populations only. Currently, no effective FOSL1 inhibitors are available, which hinders our further mechanistic and functional studies. Proteolysis Targeting Chimeras (PROTAC) is a novel approach to selectively and potently degrade a protein of interest (POI) both in vitro and in vivo. In this approach, a hetero-functional molecule is designed to contain an inhibitor that binds to a POI, another small-molecule ligand which binds to an E3 ubiquitin ligase complex, and a linker to tether these two ligands together. In our preliminary studies, we have designed and synthesized a group of novel PROTAC-based FOSL1 degraders, from a parental FOSL1 inhibitor. The top compound can potently degrade FOSL1 to eliminate CSCs in vivo, displaying advantages over the parent inhibitor. Therefore, we hypothesize that FOSL1-SEs determine the functional properties of cisplatin-enriched CSCs in HNSCC, and serve as a promising target for overcoming cisplatin resistance in HNSCC, which can be validated by utilizing the effective PROTAC-based FOSL1 degraders. We propose the following specific aims to test our hypotheses. Aim 1: Investigate the mechanism of action of FOSL1-SEs in determining the functional properties of cisplatin-enriched CSCs in HNSCC and apply PROTACs to validate such a mechanism of action. Aim 2: Initiate SAR studies and a functional screening to identify the top PROTACs to target FOSL1-SEs effectively in HNSCC from a library of 100 candidates. The goal is to identify optimal compounds for mechanistic (Aim 1) and functional validations (Aim 3). Aim 3: Validate whether targeting FOSL1-SEs with PROTAC molecules (top 2 compounds from Aim 2) can efficiently eliminate CSCs and overcome cisplatin resistance using mouse models of HNSCC. The successful completion of this study will provide a novel and validated drug target and contribute to the development of new strategies for improving HNSCC chemotherapy outcome.