Abstract A121: Preclinical evaluation of OTX015 a novel BET-BRD inhibitor across a panel of solid tumor cell lines of different lineages.
Authors: Riveiro Maria Eugenia , Astorgues-Xerri Lucile , Odore Elodie , Bekradda Mohamed , Cvitkovic Esteban , Noel Kay , Raymond Eric 
Published: August 2014

Background: The human BET family bromodomains which consists of BRD2, BRD3, BRD4 and BRDT proteins has become a druggable target for the development of specific gene transcription inhibitors. Here, we report anti-proliferative activity of OTX015, an orally bioavailable small-molecule BRD-inhibitor which displays high potency and specificity to BRDs 2, 3, and 4, across a large panel of human solid tumor cell lines.

Material and Methods: Established 20 human cancer cell lines derived from head and neck, lung, liver, colorectal, renal and pancreatic cancers, were treated with increasing doses of OTX015 (OncoEthix SA, Switzerland). MTT assays were performed after 72 hours exposure. GI50 (CI95%) values were estimated using GraphPad Prism 3.0. Cell lines displaying GI50 values500nM were considered insensitive. Genomic DNA from the cell lines was PCR-amplified, sequenced, and assessed for potential sequence alterations (ABI BigDye Terminator Sequencing kit) in the genes KRAS (exon 2 and 3); BRAF (exon 11 and 15); EGFR (exon 20) and PI3KCA (exon 20). Protein levels were analyzed by Western Blot using commercial antibodies. For cell cycle analysis, cells were stained with propidium iodide and analyzed for DNA content using a FACScan flow cytometer. RNA was extracted using the Qiagen RNAEasy kit and reverse-transcribed using the Superscript First-Strand Synthesis System for RT-PCR kit following manufacturer's instructions. RT- PCR was performed using Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System.

Results: Twenty cell lines derived from a wide range of solid tumor types, belonging to head and neck squamous cell carcinoma (HNSCC), lung adenocarcinoma (LA), hepatocarcinoma (HCC), colorectal carcinoma (CRC), renal (RC), pancreatic (PC) and triple-negative breast cancer (BC) cells were exposed to various concentrations of OTX015 (from 3µM to 9 nM) for 72 h. Eight cell lines displayed GI50 values lower than 500 nM, ranging from 45.9 (15.5-135.1)nM in HT-29 cells (CRC) to 432.0 (38.0-618.7)nM in A-549 (LA) cells. Baseline BRD4/2, c-MYC, BCL-2, CyclinD1 m-RNA leves and protein were characterized in our panel of cell lines and no difference in BRD4/2, c-MYC or BCL-2 basal levels was observed between OTX015 sensitive or resistant cell lines. In addition, no correlation was found between OTX015 anti-proliferative activity and the most prevalent mutations seen in cancer cells (KRAS, BRAF, EGFR and PI3K). In sensitive cell lines, OTX015 caused a cell cycle arrest in G1 in a dose-dependent manner without an increase in cell death (sub-G0 increased). No c-MYC protein level down-regulation was observed after treatment (500nM; 72h) in most of the OTX015 sensitive cell lines, suggesting that alternative pathways can be affected by BRD-inhibition. Noteworthy, after 72 h exposure, significant changes in the cell morphology such as expanded cytoplasm, cytoskeleton reorganization and pyknotic nuclei observed in OTX015 treated cells compared to vehicle treated group, further studies are being conducted to characterize these findings.

Conclusion: Together, these findings suggest that OTX015 is active across a wide range of tumor types but this activity could not be restricted only cancers with genomic alterations resulting in MYC overexpression.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A121.

Citation Format: Maria Eugenia Riveiro, Lucile Astorgues-Xerri, Elodie Odore, Mohamed Bekradda, Esteban Cvitkovic, Kay Noel, Eric Raymond. Preclinical evaluation of OTX015 a novel BET-BRD inhibitor across a panel of solid tumor cell lines of different lineages. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A121.