Abstract 6054: UPPGRADER: a bioinformatics-based novel E3 ligase discovery platform
Authors: Kyungsik Ha, Woojeung Song, Woochang Choi, Gi Bbeum Lee, Soohee Ryu, Jun Kyu Lee, Hwajin Lee
Journal: Cancer Research (2024)

Abstract In Target Protein Degradation (TPD) field, conventional E3 ligases utilized in the clinic are limited to CRBN and VHL, which opens the need for exploration of novel E3 ligases for the advancement of TPD. To address this, we have developed a proprietary platform, UPPGRADER, for systematic discovery of novel E3 ligases based on -omics data including single cell RNA sequencing (scRNA-seq) and whole genome sequencing (WGS), in conjunction with proteomics data and bioinformatics tools.UPPGRADER enables systematic analyses on alterations occurred in all genomic areas including E3 ligase genes and potential target genes. For example, analyzing Pan-Cancer Analysis of Whole Genomes (PCAWG) data identified amino acid changes into Cysteine or Lysine, which can be targeted by covalent binders with cancer specificity. Through such analysis, we discovered several novel E3 ligases, including but not limited to FBXW7, with the categorization of the findings based on specific cancer types.At the single cell expression level, UPPGRADER provides the capability to assess gene expression levels of E3 ligases and target proteins by proprietary measurements based on gene detection rate. This system also allows assessment of E3 ligase-Target gene co-expression per single cell. In colorectal cancer, UPPGRADER has shown that the co-expression of E3 ligase FBXW7 and well-known colorectal cancer target (KRAS) increases from 1% (4 out of 334) in normal epithelial cells to 17% (2,967 out of 17,458) in cancerous epithelial cells, highlighting a significant change in their simultaneous expression patterns during cancer progression.UPPGRADER also identified E3 ligase X which showed high co-expression level with KRAS gene alongside universal expression properties and high cancer cell dependencies. For E3 ligase X, we developed novel small molecule binders (nM binding affinity) with bioavailability &amp;gt; 20% in animal pharmacokinetics. Bifunctional degraders utilizing these binders displayed BRD4 degradation in more than 20 cell lines with double to triple digit nM DC50 and DC90 values. Other targets (AURKA, CRBN and others) were potently degraded with anticipated mechanism of action (assessed by E3 ligase KO cell line and proteasomal inhibitors).To conclude, UPPGRADER is a comprehensive bioinformatics tool for discovering novel E3 ligases with biological validations. Through recent explosion of scRNA-seq and WGS datasets in different diseases and mechanism-based identification of additional molecular targets, UPPGRADER is poised to advance the field of TPD. Citation Format: Kyungsik Ha, Woojeung Song, Woochang Choi, Gi Bbeum Lee, Soohee Ryu, Jun Kyu Lee, Hwajin Lee. UPPGRADER: a bioinformatics-based novel E3 ligase discovery platform [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 6054.