Introduction: The BET bromodomain proteins, including BRD2, BRD3, and BRD4, have emerged as major epigenetic regulators of proliferation and differentiation and also have been associated with predisposition to dyslipidemia or improper regulation of adipogenesis, elevated inflammatory profile, and increased susceptibility to autoimmune disease. OTX015, a novel thienotriazolodiazepine compound, was identified in a cell-based screen for inhibitors of cell adhesion. Subsequently, it was evaluated for inhibition of the binding between acetylated histone and BRD2, BRD3, and BRD4 and antiproliferative effects in both in vitro and in vivo tumor models.
Material and Methods: To assess binding of OTX015 to BRD2, BRD3, and BRD4, BRD-expressing CHO cell lysate (from CHO cells transfected with expression plasmids for Flag-tagged BRD2, BRD3, or BRD4 or vector alone), europium-conjugated anti-Flag antibody, XL-665-conjugated streptavidin, and biotinylated OTX015 were incubated at room temperature for 0.2 to 2h. Fluorescence was measured by TR-FRET using an EnVision 2103 Multilabel Reader and EC50 for binding was calculated by nonlinear regression using PRISM version 5.02. Using a similar system, the effect of OTX015 on binding of BRD2, BRD3, and BRD4 to acetylated histone H4 (AcH4) was evaluated by incubating biotin-conjugated -AcH4, BRD-expressing CHO cell lysate, europium-conjugated anti-Flag antibody, and XL-665-conjugated streptavidin. Fluorescence was measured by TR-FRET using an EnVision 2103 Multilabel Reader and percent binding was calculated by defining the value of the sample without biotin conjugate dAcH4 as 0% and the sample without OTX015 as 100%. The IC50 value was calculated by nonlinear regression using PRISM version 5.02. Effects of OTX015 on cancer cell proliferation were evaluated by incubating human tumor cells for 72 h with increasing concentrations of OTX015 and assessing proliferation using a tetrazolium salt (WST-8)-based colorimetric assay. To assess antiproliferative effects in vivo, BLAB/c-nu/nu mice bearing established Ty82 BRD-NUT midline carcinoma xenografts were given OTX015 (0, 10, 30 or 100 mg/kg qd or 10 mg/kg bid) by oral gavage over 14 days. Animals were sacrificed on day 15 and tumors were extracted and weighed.
Results: OTX015 was a potent inhibitor of BRD2, BRD3, and BRD4, with EC50 values from 10 to 19 nM. Binding of OTX015 to BRD2, BRD3, and BRD4 was inhibited by addition of OTX015 in a concentration-dependent manner, suggesting competitive inhibition. OTX015 inhibited the binding of BRD2, BRD3, and BRD4 to AcH4, with IC50 values from 92 to 112 nM. OTX015 inhibited the growth of a variety of human cancer cell lines; for most hematologic malignancies tested, GI50 values ranged from 60 to 200 nM. Oral administration of OTX015 significantly inhibited the growth of Ty82 BRD-NUT midline carcinoma tumors in nude mice, showing 79% TGI at 100 mg/kg qd and 61% TGI at 10 mg/kg bid.
Conclusions: OTX015 is a potent inhibitor of BRD2, BRD3, and BRD4 and inhibits the binding of BRD2, BRD3, and BRD4 to AcH4. OTX015 showed significant anti-tumor activity both in vitro and in vivo tumor models. These findings encouraged the clinical development of OTX015, which is currently in Phase 1 studies in patients with advanced hematologic malignancies (ClinicalTrials.gov Identifier NCT01713582).
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C244.
Citation Format: J. Kay Noel, Kazunori Iwata, Shinsuke Ooike, Kunio Sugahara, Hideo Nakamura, Masanori Daibata. Development of the BET bromodomain inhibitor OTX015. [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 C244.
- Copyright © November 2013, American Association for Cancer Research