Abstract B196: Evaluation of the ubiquitin activating enzyme (UBA1) as a therapeutic target via inducible RNA interference in human tumor xenografts.

Abstract

Abstract The successful transition of the Nedd8-activating enzyme inhibitor, MLN4924, into clinical trials has greatly increased optimism for the application of other E1-activating enzyme inhibitor therapies in cancer. UBA1 is an essential E1 enzyme that controls the first step in the activation of ubiquitin. Ubiquitin is a 9 KDa protein that is involved in multiple cellular processes through conjugation to a large range of target proteins. Several of these processes are important in cancer including ubiquitin-dependent protein turnover, cell cycle progression, apoptosis and the DNA damage response. Thus, inhibiting UBA1 with small molecule inhibitors may prove to be a viable anticancer strategy. To validate UBA1 as an oncology target we utilized a lentiviral vector-mediated inducible knockdown of UBA1 by a microRNA-based shRNA (shRNAmir) in cells and xenografts derived from human cancer cells. Our data show that shRNAmir-mediated knockdown of UBA1 is tightly regulated by the inducer doxycycline in a concentration- and time-dependent manner in HCT-116 cells. Furthermore, UBA1 expression is restored upon withdrawal of the inducing agent, which coincides with decreased shRNAmir expression as marked by turboRFP. The shRNAmir-mediated depletion of UBA1 in cells resulted in reduced abundance of ubiquitin conjugates and decreased proliferation of HCT-116 cells, ultimately resulting in apoptosis. Next, we established inducible shRNAmir HCT-116 xenograft models in nude mice to examine the in vivo efficacy of inhibiting UBA1. Our results showed knockdown of UBA1 protein levels, decreased abundance of ubiquitin conjugates and apoptosis in vivo following doxycycline administration to mice. The effects of UBA1 knockdown and inhibition of ubiquitin conjugation resulted in tumor xenograft regressions in all mice. Withdrawal of doxycycline treatment resulted in regrowth of tumors with concomitant recovery of UBA1 levels and detectable ubiquitin conjugates in tumor xenografts. This study demonstrates that UBA1 knockdown effectively inhibits tumor growth and mediates tumor regression in a human xenograft model, implicating UBA1 as a novel therapeutic target for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B196.