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The ubiquitin-proteasome system (UPS) plays an important role in cellular processes for protein quality control and homeostasis and indeed, dysregulation within the pathway has implications in numerous diseases, especially cancer. Here, we introduce BRG399, a novel modulator of UBE2K - a unique E2 conjugating enzyme of the UPS, that we previously identified as a potential therapeutic target using BERG’s Interrogative Biology® platform. The anticancer potential of UBE2K modulation was validated by gene manipulation using siRNA mediated knockdown of UBE2K in several cancer cell models. Knockdown of UBE2K resulted in a 50% decrease in cell number in MIA PaCa2 cells and a 30% decrease in cell number in SKHEP1 and HepG2 cells at 96h post transfection. This effect was the result of a robust G2/M cell cycle arrest associated with increased CyclinB1 expression. In addition, a modest increase in apoptosis/necrosis (6-8%) was observed in cells with UBE2K knockdown. Using Fragment-Based Ligand Discovery and structure-based drug design, BRG399 was developed as a new chemical entity (NCE) which binds to UBE2K, resulting in modulation of several established canonical functions and an anti-cancer phenotype. In a panel of 102 cancer cell lines, BRG399 exhibited an anti-cancer activity in all cell lines tested as evident by extrapolation of IC50 and %Max Effect values from the dose response curves. In all but one cell line, double to triple digit nanomolar potency was observed ranging from 17nM to 318 nM and viability of 76 cell lines was reduced by more than 70%. The anti-cancer potency of BRG399 was also established in a multi-tumor type panel of patient-derived organoids (PDO) of which 68% were highly or partially responsive to BRG399 as indicated by 37-73% decrease in PDO viability. Mechanistic studies demonstrated that BRG399’s anti-cancer effect was a result of a robust G2/M cell cycle arrest and apoptosis mediated cell death. Molecular events associated with G2/M arrest included stabilization of CyclinB1 and sustained activation of cyclin-dependent kinase CDK1, while those associated with apoptosis involved decreased expression of anti-apoptotic proteins Bcl-XL and Mcl-1 and increased expression of cleaved PARP (cPARP) and Capase 3. Together, these results support a novel role for UBE2K in the regulation of the cell cycle in cancer cells and demonstrate that its modulation by BRG399 influences phenotypic end-points consistent with an anti-cancer effect. Citation Format: Maria-Dorothea Nastke, Marisa Permatteo, Pragalath Sundararajan, Shefali Sharma, Jacob Matson, Kaleigh Gray, Arcan Guven, Shiva Kazerounian, Anne Diers, Rangaprasad Sarangarajan, Niven R. Narain, Vivek K. Vishnudas, Stephane Gesta. BRG399: A novel potent small molecule modulator of UBE2K for the treatment of various cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5321. |
