Title: Ubiquitination and degradation by small-molecule degraders of SUMO1 extends survival of mice with patient-derived tumors

Authors: Anita C. Bellail, Hong Ri Jin, Ho-Yin Lo, Sung Han Jung, Chafiq Hamdouchi, Daeho Kim, Ryan K. Higgins, Maximilian Blanck, Carlos le Sage, Benedict C.S. Cross, Jing Li, Amber L. Mosley, Aruna B. Wijeratne, Wen Jiang, Manali Ghosh, Yin Quan Zhao, Paula M. Hauck, Anantha Shekhar, Chunhai Hao

Abstract: Discovery of small-molecule degraders that activate ubiquitin (UB) ligase-mediated ubiquitination and degradation of targeted proteins in cancer cells has been an elusive therapeutic strategy. Here, we report a cancer cell-based drug screen of the NCI drug-like compounds library that enabled identification of small-molecule degraders of the small ubiquitin-related modifier 1 (SUMO1). Structure-activity relationship studies of analogs of the hit compound CPD1 led to identification of a lead compound HB007 with improved properties and anticancer potency in vitro and in vivo . A genome-scale CRISPR-Cas9 knockout screen identified the substrate receptor F-box protein 42 (FBXO42) of cullin 1 (CUL1) E3 ligase that is required for the HB007 activity. Using HB007 pulldown proteomics assays we were further able to pinpoint HB007’s binding protein as the cytoplasmic activation/proliferation-associated protein 1 (CAPRIN1). Biolayer interferometry and compound competitive immunoblot assays confirmed the selectivity of HB007 binding to the CAPRIN1. Upon binding of CAPRIN1, HB007 induced the interaction of CAPRIN1 with FBXO42; FBXO42 then recruited SUMO1 to the CAPRIN1-CUL1-FBXO42 UB ligase complex where SUMO1 was ubiquitinated in a subset of human cancer cells. The compounds CPD1 and HB007 selectively degraded SUMO1 protein in patient-derived xenografts implanted in mice. Systemic administration of the compounds inhibited the progression of patient-derived brain, breast, colon and lung cancers in mice and increased survival of the animals. This cancer cell-based approach enabled discovery of a new small-molecule degrader of SUMO1 and may be useful for identifying other small-molecule degraders of oncoproteins.

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• Data_File_S1_CRISPR_based_resistance_screen.xlsx - List of all genes in the genome-wide HB007 screen in the HCT116 human colon cancer cell line. Significant gene hits in this positive selection screen were selected based on an FDR < 0.05.