Abstract 1822: Selective inhibition of ZCCHC11/ZCCHC6 TUTases with genetic and pharmacological tools supports a role in glioblastoma cell growth

Abstract

Abstract The purpose of the study was to determine if genetic and pharmacological inhibition of terminal uridyltransferases (TUTases) ZCCHC11 and ZCCHC6 could modulate Glioblastoma (GBM) cell growth in vitro. ZCCHC11 (also known as TUT4 and TENT3A) and ZCCHC6 (also known as TUT7 and TENT3B) catalyze uridylation of diverse RNA species. Recent evidence indicates that genetic perturbation of ZCCHC11/6 expression can disrupt cell proliferation of both immortalized and patient-derived primary GBM cell lines (Kim et al., Mol Cell 2020). We confirmed that genetic inhibition of ZCCHC11/6 with siRNA or CRISPR can decrease viability of U-87 MG and A-172 GBM cell lines, and we identified DK-MG as another GBM cell line with sensitivity to ZCCHC11/6 knockdown. Herein, we report the first novel, potent, and selective inhibitor of ZCCHC11/6, TS-1. Biochemically, TS-1 can block ZCCHC11/6-mediated uridylation of an RNA substrate in vitro (IC50 = 0.65 nM for recombinant ZCCHC11 protein; IC50 = 9.6 nM for recombinant ZCCHC6 protein). ZCCHC11/6 inhibitor TS-1 could decrease cell viability in U-87 MG, A-172 and DK-MG cells (cell viability IC50s ranging between 0.7-1.5 μM) and induce apoptosis and cell cycle arrest. A cellular assay detecting uridylation of miR-191 as a marker for ZCCHC11/6 activity was developed and validated. TS-1 strongly decreased uridylation of miR-191 in U-87 MG, A-172 and DK-MG cells (IC50 ranging between 10-40 nM). In contrast, TS-2, a weakly active enantiomer of TS-1 (enzyme IC50 = 0.15 μM and 1.8 μM against ZCCHC11 and ZCCHC6, respectively), had less impact on cell viability, apoptosis, cell cycle and uridylation in cells. In conclusion, we have verified ZCCHC11/6-dependency in a set of GBM cell lines and we have also developed a first-in class potent and selective small molecule that reduces in vitro Glioblastoma cell proliferation through selective inhibition of TUTases activity. Citation Format: Robinson Triboulet, Khikmet Sadykov, Jessica L. Johnson, Andrew R. Snyder, Sarah K. Knutson, Pavan Kumar, Christopher B. Mayo, Dillon Hawley, Andrew Madanjian, Ross L. Stein, David M. Wilson, Darren M. Harvey, Shomir Ghosh, Robert M. Campbell. Selective inhibition of ZCCHC11/ZCCHC6 TUTases with genetic and pharmacological tools supports a role in glioblastoma cell growth [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 1822.