Abstract PR09: High-throughput chemical screening identifies a novel class of proteasome inhibitors with significant activity against Ewing sarcoma

Abstract

Abstract To identify novel small molecules with potential therapeutic activity against Ewing sarcoma (ES) and to define critical biological pathways in ES, we performed a high-throughput chemical screen, followed by functional, biochemical and genetic studies of 2 compounds of interest. We screened a library of 309,989 compounds for agents with selective activity against a panel of 5 ES cell lines. We identified 23 agents with significant activity (IC50 < 1uM) against all ES cell lines. Eight compounds showed relative selectivity against ES cell lines when compared to 11 cell lines from different tumor types. Two of the 8 compounds (compounds P and W) were analogs. The expression profiles of ES cell lines treated with P and W showed highly significant overlap with the profiles generated for cell lines treated with MG-262 (Connectivity Map analysis) and its structural analog bortezomib. Cell lines treated with compounds P and W resulted in accumulation of poly-ubiquitinated proteins, similar to bortezomib and MG-262 treated cells. However, unlike bortezomib and MG-262, compounds P and W did not target the 20S proteasome in vitro. To further characterize these compounds, we synthesized additional analogs for in vitro and in vivo studies. One analog (compound E) was 10-fold more potent than compounds P and W in growth inhibition assays, and induced apoptosis and cell cycle arrest in multiple ES cell lines. Treatment of mice with ES xenograft tumors resulted in tumor growth regression, with significantly more efficacy than mice treated with bortezomib. A compound structurally very similar to compound E (b-AP15) has recently been demonstrated to inhibit the deubiquitinating enzymes USP14 and UCHL5 (D'Arcy P et al, Nat Med 2011). These enzymes associate with the 19S proteasome, and promote 20S proteasomal function. In a previous genome wide loss-of-function shRNA screen against ES cell line CHP100, we identified both USP14 and UCHL5 as hits, and have now validated these findings in multiple additional ES cell lines. To further investigate compound E as an alternative proteasome inhibitor, we studied its effect in a colon cancer cell line (HCT-116) with an established resistance mutation (PSMB5 M104V) to bortezomib. While the cell line demonstrated > 10-fold resistance to bortezomib as compared to the wild-type cell line, there was no significant difference in susceptibility to compounds E and P. We have identified a class of proteasome inhibitors with significant activity against in vitro and in vivo models of ES. Previous in vitro studies of 20S proteasome inhibitors, such as bortezomib, have demonstrated efficacy in ES and other solid tumor types. However in vivo studies and clinical trials of bortezomib in patients with solid tumors have yielded only modest effects. Alternative proteasome inhibitors may therefore be promising candidates for future therapy against ES as well as other solid tumor types. This abstract is also presented as Poster A36. Citation Format: Neerav Shukla, Romel Somwar, Srikanth Ambati, Roger S. Smith, Melinda Merchant, Rachel Kobos, Ouathek Ouerfelli, Hakim Djaballah, Marc Ladanyi. High-throughput chemical screening identifies a novel class of proteasome inhibitors with significant activity against Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR09.