Abstract 5530: OTX015, a novel pan BET-BRD inhibitor is active in non-small-cell lung cancer (NSCLC) cell lines bearing the fusion protein EML4-ALK
Authors: Vázquez Ramiro , Astorgues-Xerri Lucile , Bekradda Mohamed , Cvitkovic Esteban , Herait Patrice , Boi Michela , Inghirami Giorgio , D'Incalci Maurizio , Riveiro María E. , Raymond Eric 
Published: May 2015

Abstract
Background: Various inhibitors targeting the activity of BET proteins have been recently developed and have shown potent anti-proliferative effects in several tumors, including NSCLC. The fusion of the echinoderm microtubule-associated protein-like 4 (EML4) and the anaplastic lymphoma kinase (ALK) genes results in the chimeric oncogene EML4-ALK, identified as a distinct entity of NSCLC patients that enables effective ALK-targeted therapy. However, most of these patients invariably acquire resistance within few months. Herein, we report preclinical findings obtained with a novel oral pan-BET-BRD inhibitor, OTX015, in a panel of NSCLC cell lines, some of which bear the fusion protein EML4-ALK.
Materials and Methods: Five established NSCLC cell lines (i.e. H2228, H3122, A549, HOP62 and HOP92) were exposed to increasing concentrations of OTX015 (OncoEthix SA, Switzerland). In each case, the effect on cell viability was determined by the MTT assay after 72 h of exposure. GI50 values, representing 50% growth inhibition concentration, were calculated using GraphPad Prism 5.0 software. Protein levels were determined by Western blot using commercial antibodies. RNA was extracted with 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: OTX015 displayed anti-proliferative activity in EML4-ALK-positive H2228 and H3122 cells after a 72-h treatment, with GI50 values of 629 and 627nM, respectively. The corresponding GI50 values for the benchmark compound, JQ-1, were 2161 and 1265 nM. Interestingly, OTX015 was also active in the EML4-ALK-negative A549 cell line, with a GI50 of 432 nM. BRD4/3/2, c-MYC, BCL-2, p21 and CyclinD1 were detected at protein and mRNA levels in all cell lines. Both OTX015-sensitive and -resistant cells exhibited similar basal expression levels for the aforementioned proteins. EML4-ALK variants 1 and 3 were identified in H3122 and H2228 cells, respectively. Assessment of the signaling pathways involved in the anti-proliferative activity of OTX015 in these cells showed a transient up-regulation of STAT3, followed by a down-regulation after 24 h and up to 72 h of exposure. These results indicate that the key downstream effector of OTX015 is STAT3, frequently found to be overexpressed in crizotinib-resistant cell lines. Interestingly, c-MYC protein and mRNA levels were not altered by OTX015. EML4-ALK-positive H3122 cells showed down-regulation of n-MYC mRNA levels after OTX015 treatment.
Conclusion: Our results indicate that NSCLC cell lines with genomic ALK alterations are sensitive to BET-BRD inhibition by OTX015, suggesting its potential clinical use as anticancer agent in EML4-ALK positive NSCLC patients.
Citation Format: Ramiro Vázquez, Lucile Astorgues-Xerri, Mohamed Bekradda, Esteban Cvitkovic, Patrice Herait, Michela Boi, Giorgio Inghirami, Maurizio D'Incalci, María E. Riveiro, Eric Raymond. OTX015, a novel pan BET-BRD inhibitor is active in non-small-cell lung cancer (NSCLC) cell lines bearing the fusion protein EML4-ALK. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5530. doi:10.1158/1538-7445.AM2014-5530