Abstract 343: ERG oncogene-specific inhibitors for prostate cancer
Authors: Mohamed Ahmed , Sukumar Gauthaman , Xavier Charles , Katta Shilpa , Ravindranath Lakshmi , Jamal Muhammad , Sreenath Taduru , Petrovics Gyorgy , Dobi Albert , Srivastava Meera , Dalgard Clifton , Srivastava Shiv 
Published: July 2016

Abstract
Introduction: Treatment for advanced prostate cancer (CaP) remains challenging due to accumulation of numerous genomic alterations or treatment-related mutations during disease continuum. Therefore, targeting early CaP driver genes may introduce new approaches in complementing current therapeutic paradigms. ERG oncogenic activation by recurrent gene fusions is the most validated driver gene alteration in CaP and represents a high priority therapeutic target. However, the transcription factor nature of ERG poses challenges. Current ERG-targeted strategies include direct or indirect inhibition of ERG/ETS transcription factor activity. Due to high homology of ERG to a large number of ETS-related proteins the specificity of these strategies remains to be established. In order to screen for ERG-specific inhibitors, we developed a new approach for screening of small molecules by assessing inhibition of ERG oncoprotein expression using an In-cell-Western based assay employing a specific anti-ERG monoclonal antibody (9FY). Here we report the characterization of a highly selective small molecule ERG inhibitor (ERGi-USU).
Methods: A small molecule library from the National Cancer Institute was screened for ERG protein inhibition in TMPRSS2-ERG harboring CaP cells (VCaP) using an In-Cell-Western assay employing ERG-MAb (9FY). Among the promising candidates NSC139021 (ERGi-USU) was selected for further characterization in cell culture-based (VCaP, COLO320, MOLT4, KG1, LNCaP, LAPC4, MDAPCa2b, BPH1, RWPE and HUVEC) and VCaP nude mouse xenograft assays. Effect of ERGi-USU was determined using cell growth, cell cycle and apoptosis assays and whole transcriptome sequencing.
Result: Among the cell lines evaluated, ERGi-USU exhibited exclusive inhibitory effects on ERG protein as well as cell growth including TMPRSS2-ERG harboring CaP (VCaP) and ERG positive colon cancer and leukemia cells (COLO320, MOLT4, KG1) (IC50, 50-400 nM). There was no inhibitory effect on cell growth of ERG negative CaP cells or normal prostate-derived epithelial cells. Importantly, ERGi-USU did not inhibit the growth of primary endothelial cells with endogenous ERG. Molecular and cellular mechanisms of ERGi-USU included inhibition of G2 phase of cell cycle, induction of apoptosis. RNA-Seq analyses revealed activation of the ER stress pathway. Importantly, significant inhibitory effect of ERGi-USU was shown on VCaP xenograft growth in nude mice without any significant toxicity.
Conclusion: ERG inhibition through ERGi-USU showed high specificity for ERG expressing tumor cells in experimental models and has potential in further developing ERG-targeted therapeutic strategies for CaP and other ERG positive cancers.
Citation Format: Ahmed Mohamed, Gauthaman Sukumar, Charles Xavier, Shilpa Katta, Lakshmi Ravindranath, Muhammad Jamal, Taduru Sreenath, Gyorgy Petrovics, Albert Dobi, Meera Srivastava, Clifton Dalgard, Shiv Srivastava. ERG oncogene-specific inhibitors for prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 343.