Abstract 6587: Discovery of molecular glue of FBXW7:MYC interaction using small molecule microarray

Abstract

Abstract MYC deregulation is a key driver and feature of many human cancers. Yet, despite its importance in cancer progression, MYC is considered “undruggable” by small molecules due to its disordered conformation. Therefore, interfering with its interactions with protein partners to modulate MYC activity appears viable. FBXW7 is an E3 ligase, which serves as a constituent of the SCF (Skp1-Cul1-F box) ubiquitin ligase complex. It mediates the degradation of both c-Myc and Cyclin E through ubiquitin-dependent proteolysis. FBXW7 mutations are found in various human tumor types, establishing its role as a general human tumor suppressor. Specifically, FBXW7 down-regulation increases MYC level, and its expression correlates to lethal outcomes of breast epithelial cells, poor prognosis in glioblastoma, and T cell acute lymphoblastic leukemia remission. Therefore, our project aims to discover potent small molecule glues that would enhance the interaction between FBXW7 and MYC, leading to MYC degradation through proteolysis and eventually suppression of various cancers. Taking advantage of our high throughput screening technology, Small Molecule Microarrays (SMMs), we successfully identified 29 compounds that potently bind to both c-Myc and FBXW7 after screening our 65,000 ‘drug-like’ small molecule library. We then performed secondary assays using a mCherry-GFP MYC reporter cell line to identify our lead compound, KI-FBX-001, which decreases MYC’s expression:transcription ratio by over 50% compared to control. Additionally, this down-regulation can be rescued by protease inhibitor MG132, indicating that the effect is achieved via proteolysis. In vitro dose-response and time-course treatment revealed an optimal drugging concentration at 15uM in K562 cells, with inhibition reaching its peak at the 4-hour time point at over 60%. Using the Qiagen MYC reporter assay, the in vitro IC50 of KI-FBX-001 is found at 7.42uM. Beyond the protein level, the qPCR result also reveals a decrease in MYC mRNA level at over 70%. Surface Plasmon Resonance (SPR) is used to confirm direct binding between FBXW7 and KI-FBX-001, which showed promising binding with kd=0.17µM. Proximity Ligation Assay further confirms KI-FBX-001’s effect in enhancing FBXW7:MYC interaction in the cell. In summary, our findings proved that KI-FBX-001 is a promising molecular glue in the FBXW7:MYC interaction, and can effectively modulate the MYC level for future therapeutic purposes. Citation Format: Shenghao Guo, Morgan Stilgenbauer, Bocheng Wu, Yatin Mankan, Angela N. Koehler. Discovery of molecular glue of FBXW7:MYC interaction using small molecule microarray [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6587.