Abstract 4694: In charge: Targeting neddylation of UBE2M for anticancer therapy

Abstract

Abstract Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules orchestrates a vast number of processes in the cell. Consequently, post-translational modification pathways are considered major targets for therapeutic intervention of many diseases, including cancers. Neddylation is a post-translational mechanism like ubiquitination, but instead of ubiquitin, NEDD8 is conjugated to protein targets via an E1-E2-E3 multi-enzymatic cascade. Neddylation is upregulated in multiple cancer types and the development of therapeutics targeting neddylation pathways are gaining increasing popularity. UBE2M, one of the two E2 neddylation conjugation enzymes, takes part in two types of reactions: transthiolation-transfer of NEDD8 from a thioester to a thiol group and aminolysis-transfer of NEDD8 from a thioester to an amino group. These activities require both a 26-residue N-terminal docking peptide and a conserved E2 catalytic core domain, which is the basis for the transfer of NEDD8. Here, we describe findings in the quest to understand the UBE2M interaction with NEDD8 and effects on cancer cells by modifying UBE2M at the N-terminal docking peptide or at the catalytic site. Cancer cells were transfected with ΔN or catalytic site UBE2M mutant constructs and analyzed by immunoprecipitation for charging of NEDD8 onto UBE2M. The catalytic site mutants are deficient in charging (C111A) or discharging (C111S) of UBE2M by NEDD8, which leads to a decrease in neddylation of downstream substrates, such as CULLIN family members. Elimination of the N-terminal docking peptide by introduction of catalytically competent ΔN UBE2M mutant constructs resulted in reduced reaction kinetics. Structural modeling suggests that the loss of the N-terminal docking region likely weakens protein-protein interactions between UBE2M and the UBA3-APPBP1 E1 complex. Interestingly, ΔN UBE2M mutants were observed to be slightly charged with NEDD8, but appeared to have a defect in NEDD8 discharge, resulting in reduced neddylation of substrates. Moreover, further phenotypic evaluation of UBE2M mutant transfected cancer cells revealed a divergence in phenotypic effect: while both mutant types led to a deficiency in NEDD8 charge or discharge from UBE2M, only the manipulation of the active site of UBE2M led to a DNA re-replication phenotype with subsequent cell death. These observations reveal the complexity of the neddylation cascade and raise the question if slowing down discharge of NEDD8 from UBE2M is a more viable therapeutic option than slowing down the charging of UBE2M. Citation Format: Alex G. Batrouni, Jacob P. Matson, Andressa L. Mota, Archna Ravi, Marcus O'Hara, Micholas D. Smith, Jeremy C. Smith, Vivek K. Vishnudas, Anjan Thakurta, Stephane Gesta, Maria D. Nastke. In charge: Targeting neddylation of UBE2M for anticancer therapy [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 4694.