Abstract A219: OTX015, a bromodomain and extraterminal inhibitor, represents a novel agent for ALK positive anaplastic large cell lymphoma.
Authors: Boi Michela , Todaro Maria , Vurchio Valentina , Cvitkovic Esteban , Riveiro Eugenia , Bertoni Francesco , Inghirami Giorgio 
Published: August 2014

Abstract
Introduction: Inhibitors of the bromodomain and extraterminal (BET) family (BRD2, BRD3, BRD4, and BRDT) are a promising new class of anticancer agents. Here, we assessed the activity and the mechanism of action of a novel molecule OTX015, a selective orally bioavailable BRD2/3/4 inhibitor, in a panel of anaplastic large cell lymphoma (ALCL) cell lines.
Material and Methods: Human cell lines derived from ALKpos ALCL (SUPM2/TS, SU-DHL-1, L82, JB-6, Karpas 299) were treated with increasing doses of OTX015 (OncoEthix SA, Swtizerland). Cell proliferation was evaluated by ATPlite and MTT methods over time. For cell cycle analysis cells were treated and stained with citrate buffer and PI and analyzed for DNA content using a FACScan flow cytometer. RNA was extracted by TRIzol and reverse-transcribed using the Superscript First-Strand Synthesis System kit according to the manufacturer's instructions. RT-qPCR was performed using SYBR Green Master Mix on a Bio-RAD Real-Time PCR System. For WB analysis, cell lysates were fractionated by 8% polyacrilamide gels and membranes incubated with specific antibodies.
Results: ALKpos ALCL cell lines (5) treated with different doses of OTX015 (ranging from 100nM to 1µM) underwent to a proliferation arrest as compare to 0.1% DMSO-treated cells. This was already detectable after 24h of treatment, and more pronounced at 48h and 72h. Cell cycle analysis showed a rapid G1 cell cycle arrest of all OTX015 treated ALCLpos cells as early as 24hrs. In 2 cell lines (SUPM2/TS, and JB-6) an increment in cell death rate was observed. Dose-curve studies and kinetics experiments demonstrated that a single exposure of 250nM of OTX015, without any subsequent refills, could sustain the OTX015 mediated changes (cell cycle arrest and c-MYC down-regulation) over time (up to 72-96 hrs). A time and dose dependent decreased of c-MYC m-RNA and protein levels was observed after OTX015 exposure in all ALCL cell lines, even after an exposure of 24h. Loss of c-MYC expression was associated with a concomitant down-regulation of known c-MYC regulated genes (CAD, NUC). The treatment with OTX015 (250nM) and suboptimal concentration of selective ALK inhibitor (5nM of CEP28122) led to a rapid and more significant down-regulation of c-MYC expression than those seen after each individual drug treatment. Once OTX015 was compared with OTX015 analog and a bona fine BET-inhibitor, JQ1, similar activity were observed.
Conclusion: OTX015 is a new potent BRD-inhibitor with cytostatic anti-proliferative activity in several ALCL cell lines. The rapid down-regulation of c-MYC expression, which precedes the cell cycle G1 arrest, may provide a model of action in this experimental context. However, the identification of other biomarkers may be relevant in determining the appropriate drug dose and schedule and to predict clinical responses. The synergistic effects of OTX015 with anti-ALK inhibitors provide the rational for more efficacious and possibly less toxic protocols. This could represent a desirable strategy in patients partially responsive or refractory to ALK therapy. Overall, these findings demonstrated that BET bromodomains inhibitors represent promising therapeutic agents for the treatment of ALKpos ALCL patients.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A219.
Citation Format: Michela Boi, Maria Todaro, Valentina Vurchio, Esteban Cvitkovic, Eugenia Riveiro, Francesco Bertoni, Giorgio Inghirami. OTX015, a bromodomain and extraterminal inhibitor, represents a novel agent for ALK positive anaplastic large cell lymphoma. [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 A219.