Abstract 5529: In vitro evaluation of OTX015, a novel pan-BET-bromodomain (BET-BRD) inhibitor, as single agent and in combination with standard chemotherapy drugs in human leukemic cell lines
Authors: Astorgues-Xerri Lucile , Vázquez Ramiro , Bekradda Mohamed , Cvitkovic Esteban , Herait Patrice , Raymond Eric , Riveiro María E. 
Published: May 2015

Abstract
Background: The human BET family bromodomains, including BRD2-4 and BRDT proteins, has become a druggable target for the development of specific gene transcription inhibitors. Here, we report the preclinical anti-leukemic activity of OTX015 (OncoEthix SA, Switzerland), a novel pan-BET-BRD inhibitor, as single agent and in combination with different drugs.
Material and Methods: Eight established human cell lines from acute and chronic myeloid leukemia (AML, i.e. HL-60 and U-937; CML, i.e. K-562 and NALM-1) and acute lymphoblastic leukemia (ALL, i.e. Jurkat, CCRF-CEM, MOLT-3 and -4) were treated by increasing doses of OTX015. The growth inhibition 50% (GI50) values of OTX015 were evaluated by MTT the assay at 72 h. Protein levels were analyzed by Western blot using commercial antibodies. RNA was extracted using the Qiagen RNAEasy and RT-PCR was performed using Fast SYBR Green on a StepOnePlus Real-Time PCR System. Combination effects were evaluated in HL60, U937, Jurkat and K562 cell lines; OTX015 was administered with daunorubicin, azacytidine, dexamethasone, cytarabine or methotrexate. The 48-h combination index (CI) was determined by median effect plot analysis using CalcuSyn software expressed as the median and range of CI values among cell lines (Chou & Talalay analysis). CI&lt;1: synergism, CI=1: additivity and CI&gt;1: antagonism.
Results: Firstly, the anti-proliferative effect of OTX015 was assessed. In HL60, U937 and Jurkat cells, GI50s values were between 230 and 384 nM, while ≥ 1,000 nM for the remaining cell lines. Both OTX015-sensitive and -resistant cells showed similar basal expression levels of BRD2-4, c-MYC, BCL-2, p21 and Cyclin D1 proteins. In sensitive cell lines, OTX015 caused cell cycle arrest in G1 in a time-dependent manner without induction of apoptosis. Likewise, c-MYC mRNA and protein levels were down-regulated after 6-h and up to 72-h treatments with 500 nM OTX015. In Jurkat cells, OTX015 induced up-regulation of BRD2 and 4 mRNA without modifying protein levels. On the other hand, although the OTX015-resistant cell line K562 showed decreased levels of c-MYC mRNA after 4-h and 24-h exposure, protein levels were constant even after 72 h of treatment. Synergy was observed with daunorubicin in OTX015-sensitive and -resistant cell lines (CI=0.8;0.4-0.9). The combination of OTX015 with azacytine or cytarabine was synergistic (CI=0.6;0.6-0.7 and CI=0.8;0.4-0.9, respectively) despite primary resistance of Jurkat and K562 lines to either of the individual drugs. Methotrexate, dexamethasone and panobinostat exerted synergistic effects with OTX015 in 3 out of 4 cell lines (CI=0.5;0.2-0.9; CI=0.3;0.1-0.7 and CI=0.6;0.5-0.7).
Conclusion: Our findings indicate that OTX015 enhances in vitro anti-proliferative effects of standard antileukemic agents supporting combination therapy as an important aspect of the clinical development plan.
Citation Format: Lucile Astorgues-Xerri, Ramiro Vázquez, Mohamed Bekradda, Esteban Cvitkovic, Patrice Herait, Eric Raymond, María E. Riveiro. In vitro evaluation of OTX015, a novel pan-BET-bromodomain (BET-BRD) inhibitor, as single agent and in combination with standard chemotherapy drugs in human leukemic cell lines. [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 5529. doi:10.1158/1538-7445.AM2014-5529