Abstract 5531: OTX015, a novel BET inhibitor, is a promising anticancer agent for multiple myeloma
Authors: Todaro Maria , Boi Michela , Vurchio Valentina , Ercole Elisabetta , Machiorlatti Rodolfo , Messana Katia , Landra Indira , Urigu Susanna , Aliberti Sabrina , Riveiro Eugenia , Bertoni Francesco , Inghirami Giorgio 
Published: May 2015

Abstract
Introduction. The activity of BET inhibitors in several models human hematologic disorders, including multiple myeloma (MM) has recently been demonstrated. Exposure of cancer cells to BET inhibitors results in a significant down-regulation of c-Myc expression, and suppression of transcriptional programs associated with proliferation and survival. OTX015 is a new selective orally bioavailable inhibitor of the BET family proteins in clinical development. We evaluated its activity in a panel of MM models with known upregulation of the c-MYC pathway.
Materials and Methods. Human cell lines derived from MM (RPMI 8226, KMM1, JJN3, OPM2, KMS27, U266, KMS18) were treated with increasing doses of OTX015 (OncoEthix SA, Switzerland). Cell proliferation was evaluated by ATPlite and MTT assays over time, and cell cycle analysis by FACScan flow cytometer. RNA was extracted using TRIzol and reverse-transcribed with the Superscript First-Strand Synthesis System kit. RT-qPCR was performed using SYBR Green Master Mix on a Bio-RAD Real-Time PCR System. For Western blot analysis, lysates were fractionated on 8% polyacrilamide gels and membranes incubated with commercial antibodies. OPM2 cells were inoculated (1*10^6 cells both flanks) in NSG mice and 72h after treatments started (vehicle; OTX015 50 mg/kg bid, po, 5 days on-2 days off).Tumor growth was measured over time.
Results. OTX105 treatment resulted in G1 cell cycle arrest in a dose-dependent manner in all MM cell lines. Increased percentages of G1 cells (∼20%) were found after 24h exposure, reaching a maximum after 72h. OTX015 treatment for 24h followed by drug washout induced an initial cell cycle arrest, which was then overcome at 72-96h. RPMI 8226 and KMS27 cell lines also displayed increased apoptosis after prolonged exposure (&gt; 48h) at 500 nM of OTX015. In 7 out of 9 MM cell lines, OTX015 treatment resulted in rapid downregulation of c-MYC mRNA and protein, while two cell lines (U266 and KMM1) displayed dose and time dependent c-MYC upregulation. Equivalent activity was observed with OTX015 (250 nM) and the OTX015 analog and bona fide BET-inhibitor, JQ1. In OPM2-bearing tumor mice, OTX015 treatment showed impaired tumor growth respect to controls after 4 weeks of treatment.
Conclusions. OTX015 is a novel potent BRD-inhibitor with cytostatic anti-proliferative or pro-apoptotic activity in MM demonstrated by in vitro and in vivo experiments. The different changes of c-MYC expression in MM cell lines after OTX015 exposure suggest that the biological effects could be due to multiple and/or alternative mechanisms, which are not strictly linked to down-regulation of this transcription factor. These findings support the ongoing clinical development of OTX015 in MM patients.
Citation Format: Maria Todaro, Michela Boi, Valentina Vurchio, Elisabetta Ercole, Rodolfo Machiorlatti, Katia Messana, Indira Landra, Susanna Urigu, Sabrina Aliberti, Eugenia Riveiro, Francesco Bertoni, Giorgio Inghirami. OTX015, a novel BET inhibitor, is a promising anticancer agent for multiple myeloma. [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 5531. doi:10.1158/1538-7445.AM2014-5531