Establishing PROTACs as a Novel Approach to Targeting Cancer Stem Cells in Colorectal Cancer and Head and Neck Squamous Cell Carcinoma

Cancer stem cells (CSCs) are crucial components of tumorigenesis, metastasis and chemotherapy. The activity of CSCs occurs through certain oncogenes that maintain stemness of the cells, promote proliferation and stimulate epithelial-mesenchymal transition (EMT). The expression of these genes is regulated by pathways like Wnt signaling, Notch signaling, Hedgehog pathway and AP-1 signaling. Typically, these signaling networks are involved in normal stem cell maintenance and tissue homeostasis and therefore strictly regulated. But abnormal activation of these signaling leads to cancer development. Drug development against these pathways remains a challenge due to intrinsic involvement of these pathways in normal functions of the body and due to “undruggable” nature of many of the targets within these pathways. The theme of this work is the use of proteolysis target chimera (PROTAC) technology to selectively inhibit these signaling and eliminate CSCs in colorectal cancer (CRC) and head and neck squamous cell carcinoma (HNSCC). The first project investigates the viability of PROTACs in targeting KDM3 proteins in CRC. Previous studies have provided strong evidence that KDM3 regulates Wnt signaling in CRC to maintain CSC activity. PROTAC analogs of IOX1, a broad-spectrum KDM inhibitor were designed, and this work demonstrates that some of these degraders selectively degraded KDM3 proteins, inhibited Wnt signaling and eliminated CSC activity to suppress CRC tumorigenesis both in vitro and in vivo. In the second project, an AP-1 inhibitor T-5224 was modified to produce PROTAC analogs that can degrade FOSL1. FOSL1 is uniquely overexpressed member of AP-1 transcription factors in HNSCC and previous studies have demonstrated that it is a master regulator of certain oncogenes involved in CSC activity. This project successfully established one of these PROTACs as a potent degrader of FOSL1 in vitro and in vivo and showed its pronounced efficacy in eliminating HNSCC-CSCs. In addition, certain tools (e.g. purified proteins) were developed in this project that are important to establish a rigorous screening methodology for FOSL1-targeting PROTACs. These tools were used in a target engagement assay and is expected to aid in setting up the necessary framework for monitoring degradation in live cells. Overall, the research presented in this dissertation is an important addition to the growing field of cancer stem cell research and PROTAC development investigations.