Abstract A170: Discovery of a potent and selective ATAD2 inhibitor with antiproliferative activity in breast cancer models
Authors: Kulkarni Meghana M. , Winter Jon , Whalley David , Dale ian , Denz Christopher , Guan Nin , Bhavsar Deepa , Zhang Andrew , Rasmusson Timothy , Fretland Adrian , Guichard Sylvie M. 
Published: August 2018

Abstract
ATAD2 is a bromodomain (BD)-containing protein binding to chromatin at sites enriched in acetylated histones to modulate chromatin-related processes including transcription, DNA replication, and repair. ATAD2 is frequently amplified and/or overexpressed in breast cancer and is associated with aggressive disease, tumor metastasis, and poor outcome. Given the importance of the BD in targeting ATAD2 to relevant sites on the chromatin, we and others have hypothesized that the BD is essential for its mechanism of action and its targeting would have antiproliferative effects in cancer cells. The ATAD2 BD is one of the least tractable and to date reported inhibitors have been weak, nonselective, or exhibit poor cell permeability. Here we describe a potent, selective, and cell-permeable inhibitor of ATAD2 AZ4374 that modulates ATAD2 interaction with chromatin in cells and induces growth inhibition in breast cancer cell lines. AZ4374 has a potency (IC50) of 6.3 nM in the biochemical assay with &gt;100-fold selectivity against other bromodomain-containing proteins. Chemoproteomic analysis of AZ4374 confirmed its selectivity. ATAD2 and BRD4 inhibition in cells was determined using displacement assays (NanoBRETTM) against the bromodomains. The IC50 for AZ4374 against ATAD2 was 80 nM and no inhibition of BRD4 was observed. The recently published GSK8814 was equipotent to AZ4374 in biochemical assays (IC50 ~6 nM) but less potent in the cell-based assay (IC50 ~600 nM). AZ4374 (10 µM) modulated the mRNA levels of CDK1, CDK2, and CCNE1 in EVSA-T cells (ATAD2 amplified). CRISPR/Cas9-based knockout of ATAD2 resulted in significant decrease in colony formation in breast cancer cell lines. Exposure of these cell lines to AZ4374 also reduced colony formation in a dose-dependent manner with IC50s ranging from 100 nM in SK-BR-3 to ~1µM in EVSA-T, so consistent with the potency observed in the cell-based assay. In addition, ATAD2 protacs caused a similar reduction of colony formation as AZ4374. Considering the role of chromatin modifiers in DNA damage response (DDR), we evaluated the impact of AZ4374 on the repair pathways HR and NHEJ using a CRISPR-reporter assay (traffic light reporter). A reduction in homology-directed repair was observed with 1 uM AZ4374, consistent with its biologic effect. Studies are ongoing to probe further the impact of ATAD2 inhibition on DNA repair. This is the first demonstration of highly potent and selective ATAD2 inhibition showing consistent cellular activity and biologic effect in cancer cell lines.
Citation Format: Meghana M. Kulkarni, Jon Winter, David Whalley, ian Dale, Christopher Denz, Nin Guan, Deepa Bhavsar, Andrew Zhang, Timothy Rasmusson, Adrian Fretland, Sylvie M. Guichard. Discovery of a potent and selective ATAD2 inhibitor with antiproliferative activity in breast cancer models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A170.