144PD - Optimizing Gemcitabine Efficacy Through Degradation of RRM1

Abstract

ABSTRACT Objectives RRM1 (ribonucleotide reductase M1) is the molecular target and key efficacy determinant of gemcitabine. Gemcitabine binds directly to active sites resulting in irreversible inactivation. Mechanisms that control RRM1 abundance are largely unknown, but may provide an opportunity for optimization of gemcitabine efficacy. Methods and results We have identified that the E3 ubiquitin-protein ligases RNF2 (RING finger protein 2, Ring1B) and Bmi1 (B cell-specific moloney murine leukemia virus insertion site 1) interact with RRM1 using yeast two-hybrid screening. We confirmed that RNF2and Bmi1 interact with RRM1 in vivo by immunoprecipitation. Confocal immunofluorescence microscopy revealed that RNF2 and Bmi1 completely co-localized with RRM1 in nucleus. RRM1 undergoes proteasome-mediated polyubiquitination and degradation. The proteasome inhibitor MG132 blocked the turnover of RRM1. We found that RNF2 and Bmi1 can induce polyubiquitination of RRM1 in vitro and in vivo. Lysine (K) 548 and 224 are major polyubiquitination sites of RRM1. Substitution of the lysine residues 548 and 224, either alone or together, significantly reduced RRM1 polyubiquitination. Depletion of RNF2 and Bmi1 by siRNA increased RRM1 protein level and promoted chemoresistance to gemcitabine in vitro. Conclusions These results establish that RNF2 and Bmi1 are E3 ubiquitin ligases of RRM1 that regulate RRM1 ubiquitination, degradation, and cellular response to gemcitabine. This suggests that RNF2 and Bmi1 might be attractive therapeutic targets to overcome gemcitabine resistance in malignancies. Disclosure All authors have declared no conflicts of interest.