The Brd-Inhibitor OTX015 Is Active in Pre-Clinical Models of Mature B-Cell Lymphoid Tumors

• Published in: Blood 2012-11, Vol.120 (21), p.1657-1657
• Main Authors: Bonetti, Paola, Boi, Michela, Ponzoni, Maurilio, Tibiletti, Maria Grazia, Stahis, Anastasios, Inghirami, Giorgio, Noel, Kay, Zucca, Emanuele, Bertoni, Francesco
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V120.21.1657.1657

Abstract 1657 Bromodomain-containing proteins play an important role in gene expression regulation, via chromatin structure remodelling. Antitumor activity has been reported in acute and chronic hematological malignancies using inhibitors of BRD2/3/4, members of the Bromodomain and Extraterminal (BET) family. Here, we report anti-proliferative activity of OTX015, a novel selective orally bioavailable BRD2/3/4 inhibitor, in a large panel of cell lines derived from mature B-cell lymphoid tumors. Established human cell lines derived from 13 diffuse large B-cell lymphoma (DLBCL), 4 mantle cell lymphoma (MCL), three splenic marginal zone lymphoma (SMZL) and from three multiple myeloma (MM) were treated with increasing doses of OTX015 (OncoEthix SA) and MTT assays were performed after 72 hours exposure. For cell cycle analysis, cells were treated and stained with Click-iT Edu Flow Cytometry Assay Kits (Invitrogen) and 7-AAD and analyzed for DNA content using a FACScan flow cytometer. Results were analyzed with FlowJo 7.6.3 software. RNA extracted using the Qiagen RNAEasy kit and reverse-transcribed using the Superscript First-Strand Synthesis System for RT-PCR kit according to the manufacturer's instructions. RT-PCR was performed using Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. For senescence detection, cells were stained using a b-Galactosidase Staining Kit (Calbiochem). OTX015 demonstrated anti-proliferative activity in DLBCL cell lines (median IC50 0.192μM; range 0.069–12.68μM). Similar results were obtained on SMZL (median IC50 0.165μM, range 0.105–0.24μM), and on MM cell lines (median IC50 0.449μM; range 0.06–0.7μM). Conversely, MCL cell lines appeared less sensitive to OTX015 (median IC50 2.01μM; range 1.22- >15μM). Among DLBCL cell lines, there was no significant difference based upon the cell of origin of the cell lines. OTX105 caused a cell cycle arrest in G1 in a dose-dependent manner in 5/5 DLBCL and 3/3 MM cell lines, without an increase in cell death. An increase in the percentage of senescent cells after treatment with the BRD-inhibitor was observed in 1/1 sensitive DLBCL cell line. In order to understand the mechanism of action of OTX015, we assessed MYC mRNA levels before and after 24h treatment with increasing doses. We observed a dose-dependent suppression of MYC mRNA by OTX015 in 4/5 DLBCL and in 2/2 MM cell lines. In DLBCL, down-regulation of MYC mRNA was observed within 1h after treatment with OTX015, suggesting a dire