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A. Tumors were undetectable at 4 weeks by IVIS imaging when b-AP15 was administered from day 1 after injecting A673 cells expressing GFP-luciferase. NSG mice bearing A673 xenografts were treated with DMSO, bortezomib or b-AP15 after they reached 80-100mm3 and weekly tumor size measurements were taken. B. b-AP15 slowed the growth of the established A673 xenografts and C. prolonged survival of A673 tumor bearing mice compared to bortezomib. D. VLX-1570 and b-AP15 treatment did not cause significant weight loss in xenograft models. (two way Anova with Tukey's multiple comparison test).

Ewing sarcoma is a primitive round cell sarcoma with a peak incidence in adolescence that is driven by a chimeric oncogene created from the fusion of the EWSR1 gene with a member of the ETS family of genes. Patients with metastatic and recurrent disease have dismal outcomes and need better therapeutic options. We screened a library of 309,989 chemical compounds for growth inhibition of Ewing sarcoma cells to provide the basis for the development of novel therapies and to discover vulnerable pathways that might broaden our understanding of the pathobiology of this aggressive sarcoma. This screening campaign identified a class of benzyl-4-piperidone compounds that selectively inhibit the growth of Ewing sarcoma cell lines by inducing apoptosis. These agents disrupt 19S proteasome function through inhibition of the deubiquitinating enzymes USP14 and UCHL5. Functional genomic data from a genome-wide shRNA screen in Ewing sarcoma cells also identified the proteasome as a node of vulnerability in Ewing sarcoma cells, providing orthologous confirmation of the chemical screen findings. Furthermore, shRNA-mediated silencing of USP14 or UCHL5 in Ewing sarcoma cells produced significant growth inhibition. Finally, treatment of a xenograft mouse model of Ewing sarcoma with VLX1570, a benzyl-4-piperidone compound derivative currently in clinical trials for relapsed multiple myeloma, significantly inhibited in vivo tumor growth. Overall, our results offer a preclinical proof of concept for the use of 19S proteasome inhibitors as a novel therapeutic strategy for Ewing sarcoma. Cancer Res; 76(15); 4525–34. ©2016 AACR.

Cell lines were treated with the indicated compounds for 96 h and then growth determined. Results represent the mean {plus minus} SD of 2-4 experiments in which each condition was assayed in triplicate.

Results and analysis of a genome-wide functional genomic screen using the Sigma-Aldrich shRNA Human Genome Library consisting of 63,093 shRNAs targeting 11,748 human genes.

A. Pair�wise expression profile comparisons of EWS cell lines treated with the known proteasome inhibitors bortezomib and MG�262 demonstrated highly significant overlap with profiles treated with compounds EWS-P and EWS-W. B. Overlap included 13 core genes established through published studies as being upregulated by 20S inhibitors such as bortezomib and MG-262. C. EWS cell lines TC-71 and CHP100 show demonstrate an increase in polyubiquitinated proteins following treatment with proteasome inhibitors MG-262, bortezomib, EWS-P, and EWS-W.

General Procedures for synthesis of 2,6-Bis-arylidenepiperidone scaffold

Ingenuity Pathway Analysis of positive hits identifies enrichment of genes involved in the ubiquitin-proteasome pathway.

72 hour IC50 growth inhibition levels (μM) of 23 compounds (labeled EWS-A through EWS-X) against five EWS cell lines and 11 non-EWS cell lines

Panel upper left: CHP100 cells exposed 3 hours to different concentrations of b-AP15. Extracts were labeled with HA-Ub-VS and subjected to western blot. Filters probed with HA antibody. Note the lack of inhibition of any of the DUBs labeled by HA-Ub-VS. Panel upper right: CHP100 cells exposed 3 hours to 1 uM b-AP15 Extracts were labeled with Ub-VS and subjected to western blot. Filters probed with USP14, UCHL5 or UCHL1 antibodies. b-AP15 treated extracts demonstrate inhibition of USP14, weak inhibition of UCHL5, and no inhibition of UCHL1. Middle Panels: Purified 26S proteasomes (5 nM) were treated with indicate concentrations for b-AP15 and DUB activity was determined by cleavage of Ub-AMC. Note the reduction in 26S associated proteasome activity following treatment with b-AP15. Lower Panels: Recombinant UCHL1 or UCHL3 was treated with b-AP15 and DUB activity was monitored using Ub-AMC cleavage as above. No inhibition of either UCHL1 or UCHL3 was observed.

Ewing sarcoma is a primitive round cell sarcoma with a peak incidence in adolescence that is driven by a chimeric oncogene created from the fusion of the EWSR1 gene with a member of the ETS family of genes. Patients with metastatic and recurrent disease have dismal outcomes and need better therapeutic options. We screened a library of 309,989 chemical compounds for growth inhibition of Ewing sarcoma cells to provide the basis for the development of novel therapies and to discover vulnerable pathways that might broaden our understanding of the pathobiology of this aggressive sarcoma. This screening campaign identified a class of benzyl-4-piperidone compounds that selectively inhibit the growth of Ewing sarcoma cell lines by inducing apoptosis. These agents disrupt 19S proteasome function through inhibition of the deubiquitinating enzymes USP14 and UCHL5. Functional genomic data from a genome-wide shRNA screen in Ewing sarcoma cells also identified the proteasome as a node of vulnerability in Ewing sarcoma cells, providing orthologous confirmation of the chemical screen findings. Furthermore, shRNA-mediated silencing of USP14 or UCHL5 in Ewing sarcoma cells produced significant growth inhibition. Finally, treatment of a xenograft mouse model of Ewing sarcoma with VLX1570, a benzyl-4-piperidone compound derivative currently in clinical trials for relapsed multiple myeloma, significantly inhibited in vivo tumor growth. Overall, our results offer a preclinical proof of concept for the use of 19S proteasome inhibitors as a novel therapeutic strategy for Ewing sarcoma. Cancer Res; 76(15); 4525–34. ©2016 AACR.