Development and Characterization of Third Generation MDM2 PROTACs for the Treatment of Acute Myeloid Leukemia Utilizing an Achiral Ligand for E3 Ligase Cereblon

Abstract

<b>Abstract ID 52646</b> <b>Poster Board 520</b> Tumor suppressor protein p53 plays a pivotal role in the regulation of cell processes and the prevention of cancer development. The function of p53 is highly regulated by murine double minute 2 protein (MDM2); thus, the inhibition or elimination of MDM2 is a validated and robust therapeutic strategy to restore wild-type p53 function. In this work, we aim to regulate the proteostasis of MDM2 by developing PROteolysis TArgeting Chimera (PROTAC) degraders. PROTACs are heterobifunctional molecules that co-opt the ubiquitin-proteasome system to induce the degradation of target proteins. PROTACs link one ligand that recruits the protein of interest (POI) to another ligand which recruits an E3 ligase to facilitate the polyubiquitination and subsequent proteasomal degradation of the POI. Previous efforts to develop MDM2 PROTACs in our lab have yielded molecules that are capable of degrading MDM2; however, they also degrade G1 to S Phase Transition 1 (GSPT1) (degrader WB156) and/or p53 itself (degrader WB214). Due to their off-target effects, these degraders are not suitable for further therapeutic advancement. Our ongoing work focuses on the development of the third generation of MDM2 degraders featuring a novel and achiral ligand for the E3 ligase component cereblon (CRBN). The discovery of an achiral CRBN ligand from our lab provides a more stable CRBN ligand than the currently widely used molecules such as lenalidomide and has been validated for BRD4 degradation in a PROTAC setting. A combination of NanoLuc luciferase assay and western blotting were used to screen compounds and quantify MDM2 degradation. Degrader-induced antiproliferation was quantified using Alamar Blue cell viability assay and binding affinity was measured using a fluorescence polarization assay. Preliminary results show that four compounds (QF 2-010, 2-016, 2-049, and 2-097) are capable of degrading MDM2 between 10 and 1 μM in MOLT-4 and RS4;11 leukemia cells and have less GSPT1 degradation activity than first generation degrader WB156. Surprisingly, however, the degradation induced by third generation MDM2 PROTACs does not correspond to an upregulation in p53 levels. The PROTACs also display a limited anti-proliferation effect and low binding affinity for MDM2. Taken together, the data suggests that these degraders may work in an unexpected mechanism and will require further evaluation to understand the lack of impact on p53 protein levels. Future work will also involve the development of more achiral CRBN ligand-based MDM2 degraders in order to find a more potent PROTAC, as well as expanding our MDM2 degraders to other cancers where MDM2 is a key therapeutic target. Despite the unusual mechanism of action of the degraders, this work shows that achiral CRBN ligand-based PROTACs are capable of degrading different POIs and may be widely applicable for PROTAC development. I.T. thanks NIH T32 GM141013 for predoctoral fellowship support. W.T. thanks the financial support from the University of Wisconsin– Madison Office of the Vice Chancellor for Research and Graduate Education with funding from the Wisconsin Alumni Research Foundation (WARF) through a UW2020 award.