Abstract 3651: The development of anti-estrogens targeting the activation function 2 site of estrogen receptor alpha for treatment of Tamoxifen resistant breast cancer
Authors: Singh Kriti , Munuganti Ravi Shashi Nayana , Leblanc Eric , Cherkasov Artem , Rennie Paul S. 
Published: August 2015

Abstract
Estrogen receptor alpha positive disease constitutes approximately 75% of all breast cancer (BCa) cases and its treatment with anti-estrogens such as Tamoxifen has been the main therapeutic avenue for more than 30 years. However, one third of women treated with Tamoxifen for 5 years develop recurrent disease. Conventional anti-estrogens share similar chemical scaffold and target the estrogen binding site (EBS) on the ER alpha. The effectiveness of these drugs is hampered due to mutations that occur around the EBS. Moreover, cross-talk between ERα and activated growth factor receptors and their downstream kinases such as HER2 mediated Ras/Raf/MEK/MAPK or MAPK pathway have shown to play a major role in activating ERα and its downstream target genes which leads to accelerated tumor growth. Thus, drugs that target ERα EBS can become ineffective with time. Therefore, there is an urgent need to develop entirely novel alternative therapeutics for instance, directly targeting co-activator binding pocket on ER alpha, the Activation Function-2 (AF2) site, and not affecting the EBS of the protein.
Using in silico screen approach we identified several promising small-molecule inhibitors as AF2 binders. One of our best lead compounds, VPC-16417 exhibited an IC50 of 0.31 micro-molar in ER alpha luciferase transcriptional assay. VPC-16417 did not displace estrogen but block ER alpha-coactivator interaction as measured by TR-FRET assay, thereby confirming that inhibition of coactivator recruitment is not by the allosteric mechanism of conventional antagonists. VPC-16417 exhibited strong anti-proliferative effect against MCF7 with no effect on ER alpha negative HeLa and MDA-MB-453 cells, confirming its ER alpha specific activity. Inhibition of ER alpha activity by blocking co-activator recruitment resulted in down-regulation of ER alpha regulated genes, Ps2, Cathepsin-D and CDC2 both at mRNA and protein levels. Since the ultimate goal of developing new AF2 inhibitors is to target Tamoxifen resistant (TamR) BCa, VPC-16417 was tested for its anti-proliferative effect on E2 responsive TamR3, a model cell line which closely resembles the clinical scenario in BCa patients who have relapsed on Tamoxifen. As anticipated, VPC-16417 successfully reduced the growth of TamR3 cells. More importantly, the combination treatment of VPC-16417 and a MAPK inhibitor synergistically inhibited growth of TamR3 cells very effectively. Altogether, these studies helped to identify a novel class of ER alpha AF2 inhibitors which have the potential to effectively inhibit ER alpha activity by a unique mechanism and circumvent the issue of Tamoxifen resistance in BCa patients. This could potentially decrease the time to cancer remission in BCa patients eventually having a substantial impact on patient survival.
Citation Format: Kriti Singh, Ravi Shashi Nayana Munuganti, Eric Leblanc, Artem Cherkasov, Paul S. Rennie. The development of anti-estrogens targeting the activation function 2 site of estrogen receptor alpha for treatment of Tamoxifen resistant breast 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 3651. doi:10.1158/1538-7445.AM2015-3651