Abstract 7023: A phenotypic screening method identifies a potent anti-cancer PROTAC targeting MAP3K1

Abstract The identification of Thalidomide, Lenalidomide, and Pomalidomide as Cereblon (CRBN) binders spurred the development of proteolysis-targeting chimeras (PROTACs). PROTACs with CRBN-binding moieties are heterobifunctional molecules that form a ternary complex with CRBN, which recruits the CUL4-DDB1 ubiquitin ligase protein complex, and a protein of interest (POI) and targets it for degradation. Despite numerous ongoing efforts, the structure-based design of PROTACs remains elusive. The current approach relies on the synthesis of a PROTAC library and screening the library in the chosen assay format to identify viable hits. Here, we propose a viability-based phenotypic screening system using HCT116 wild type (WT) POI knock-out (POI-/-) and CRBN knock-out (CRBN-/-) cells to identify PROTACs that display activity in both a POI and CRBN-dependent manner. For this study, we selected MAP3K1, an oncogenic signaling activator, as our POI and conjugated an ATP-competitive MAP3K1 inhibitor with thalidomide using various linkers to generate the library. The library was subjected to a 3-day viability screen using the above WT, MAP3K1-/-, and CRBN-/- cell lines. The screen identified 50-008 as a hit that selectively inhibited the viability of the WT cells but not MAP3K1-/- or CRBN-/- cells. 50-008 was chemically validated in a colorectal cancer cell line and organoid model by making single changes that affect either CRBN binding, MAP3K1 binding, or the linker. Follow-up studies showed that 50-008 inhibits cancer cell proliferation, induces apoptosis, and an S-phase cell cycle arrest in a CRBN and MAP3K1-dependent manner. Furthermore, A Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) screen of 50-008 across ∼900 cancer cell lines identified 267 sensitive cancer cell lines (IC50 < 0.1μM), including luminal B breast cancer cell line T47D and 131 resistant cancer cell lines (IC50 > 10 μM). Mechanistic studies in T47D cells demonstrated that 50-008 degrades MAP3K1 and modulates the activation of key downstream effectors, such as ERK and IKKβ. Citation Format: Lelisse T. Umeta, Jayapal R. Mallareddy, Sarbjit Singh, Smitha Kizhake, Lidia Boghean, Amritha Kizhake, Suchita Vishwakarma, Katelyn L. O’Neill, Lisa Humphrey-Brattain, Adrian Black, Xu Luo, Andrew S. Boghossian, Matthew G. Rees, Melissa M. Ronan, Jennifer A. Roth, Amarnath Natarajan. A phenotypic screening method identifies a potent anti-cancer PROTAC targeting MAP3K1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7023.