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Genomic alterations involving translocations of the ETS-related gene ERG occur in approximately half of prostate cancer cases. These alterations result in aberrant, androgen-regulated production of ERG protein variants that directly contribute to disease development and progression. This study describes the discovery and characterization of a new class of small molecule ERG antagonists identified through rational in silico methods. These antagonists are designed to sterically block DNA binding by the ETS domain of ERG and thereby disrupt transcriptional activity. We confirmed the direct binding of a lead compound, VPC-18005, with the ERG-ETS domain using biophysical approaches. We then demonstrated VPC-18005 reduced migration and invasion rates of ERG expressing prostate cancer cells, and reduced metastasis in a zebrafish xenograft model. These results demonstrate proof-of-principal that small molecule targeting of the ERG ETS domain can suppress transcriptional activity and reverse transformed characteristics of prostate cancers aberrantly expressing ERG. Clinical advancement of the developed small molecule inhibitors may provide new therapeutic agents for use as alternatives to, or in combination with, current therapies for men with ERG-expressing metastatic castration-resistant prostate cancer. Citation Format: Miriam S. Butler, Mani Roshan-Moniri, Michael Hsing, Desmond Lau, Ari Kim, Paul Yen, Marta Mroczek, Mannan Nouri, Scott Lien, Peter Axerio-Cilies, Kush Dalal, Clement Yau, Fariba Ghaidi, Yubin Guo, Takeshi Yamazaki, Sam Lawn, Martin Gleave, Cheryl Y. Gregory-Evans, Lawrence P. McIntosh, Paul S. Rennie, Artem Cherkasov, Michael E. Cox. Discovery and characterization of small molecules targeting the DNA-binding ETS domain of ERG in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 130. doi:10.1158/1538-7445.AM2017-130

Prostate cancer (PCa) is one of the leading causes of cancer-related death in men worldwide. The common treatment option for recurring and advanced PCa focuses on inhibiting the androgen receptor (AR). Unfortunately, despite an initial response to this treatment, drug resistance occurs, and the cancer relapses to an incurable, castration-resistant form; thus, there is a pressing need for new therapeutics. Previous research has shown that in up to 50% of all prostate cancer cases, the cause of the disease may be attributed to a common genomic rearrangement, the fusion between TMPRSS2 and ERG (ETS-related gene). ERG is a transcription factor mainly involved in hematopoiesis regulation during embryonic development, and it is not normally expressed in prostate cells in adults. However, its fusion with the TMPRSS2 promoter puts ERG under the regulation of AR, and as a consequence, ERG is one of the most commonly overexpressed genes in PCa. ERG overexpression in prostate epithelium has been shown to induce transformation and promote epithelial-mesenchymal transition (EMT) that gives cancer cells enhanced migratory and invasive characteristics. Currently, there is no approved therapeutic targeting ERG or any other member of the ETS family. While targeting transcription factors has been challenging, our integrated research team is specialized in targeting protein-DNA interaction sites. Using our established computer-aided drug discovery pipeline, we have identified several small molecules that can bind to and inhibit ERG. A total of 133 candidate compounds, pre-selected from the in silico screening of millions of chemical structures, were tested using a luciferase-based transcriptional reporter assay across two cell lines: the TMPRSS2-ERG fusion positive VCaP cell line, and the human prostate epithelial cell line (PNT1B) engineered to constitutively express ERG. In addition to transcriptional inhibition, the most potent compounds inhibited migration of PNT1B-ERG cells as demonstrated by a Real-Time Cell Analyzer. Significantly, both compounds shifted the binding spectra in protein NMR assays, indicating their direct interactions with residues located in the DNA binding domain of the ERG protein. We anticipate that results from this project will lead to the development of new drugs that can be used alternatively or synergistically with current anti-AR therapy to benefit patients with the most deadly forms of prostate cancer. (Supported by a grant from Prostate Cancer Canada) Citation Format: Mani Roshan-Moniri, Michael Hsing, Miriam S. Butler, Desmond Lau, Peter Axerio-Cilies, Paul Yen, Ari Kim, Scott Lien, Marta Mroczek, Dennis Ma, Huifang Li, Yubin Guo, Fuqiang Ban, Fariba Ghaidi, Eric LeBlanc, Lawrence McIntosh, Michael Cox, Artem Cherkasov, Paul S. Rennie. Therapeutic targeting of ETS factor ERG for the treatment of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1652. doi:10.1158/1538-7445.AM2015-1652

Despite the development of several successful targeted therapies, drug resistance and metastasis remain a significant challenge in the treatment of both breast (BCa) and prostate (PCa) cancers. For these reasons, there is an immediate need to identify a novel class of therapeutics targeting alternative factors, such as those that promote the metastatic capacity of tumour cells. ETS translocation variant 4 (ETV4) is a member of the ETS transcription factor family and is a significant mediator of tumorigenesis through its activation of several downstream pathways that are associated with migration and invasion. ETV4 is overexpressed in breast tumours and is associated with distant metastasis and poor prognosis particularly in triple negative BCa, which still lacks an approved targeted therapy. Similarly in PCa, overexpression of ETV4 is associated with the deregulation of the PI3K and Ras signalling pathways that are commonly implicated in metastatic disease. Like other ETS factors such as ERG and ETV1, fusion of the ETV4 gene can be found in a subtype of PCa cases and is associated with the disease progression. Thus, ETV4 is an important therapeutic candidate with potential applications in both advanced BCa and PCa. Drug development against ETV4 is made even more significant due to the lack of any approved therapy that directly targets it or any other members of the ETS family. Using a combination of in silico screening and in vitro assays, we have identified several small molecules with strong binding affinities and selectivity toward the DNA binding domain of ETV4. The prostate cell line, PC3, and the triple negative breast cancer cell line, MDA-MB-231, were used for in vitro studies as they endogenously express moderate to high levels of ETV4. Over 100 candidate compounds, which were selected from virtual screening against millions of small-molecular structures, were tested across the two cell lines using a luciferase-based transcriptional reporter assay. From this assay approximately 30 compounds were identified with significant inhibition on the transcriptional activity of ETV4 without a cytotoxic effect. The most potent of these compounds was shown by protein nuclear magnetic resonance (NMR) to directly interact with specific residues within the DNA binding domain. These compounds were also able to inhibit the migratory capacity of cancer cells. This data provides evidence for the direct targeting of ETV4 by small molecules, and future work will aim to further characterize their mechanism of action and effects on downstream targets in an effort to create a novel therapeutic strategy to treat metastatic breast and prostate cancers. (Supported by grants from the Canadian Cancer Society Research Initiative and Prostate Cancer Canada) Citation Format: Miriam S. Butler, Michael Hsing, Mani Roshan Moniri, Desmond Lau, Paul Yen, Ari Kim, Scott Lien, Marta Mroczek, Fariba Ghaidi, Eric LeBlanc, Lawrence McIntosh, Michael Cox, Artem Cherkasov, Paul S. Rennie. Targeting ETS factor ETV4 as a novel therapeutic for the management of breast and prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1648. doi:10.1158/1538-7445.AM2015-1648