Abstract 5528: The BET Bromodomain inhibitor OTX015 targets the NFKB, TLR and JAK/STAT pathways and shows pre-clinical activity as single agent and in combination in mature B-cell tumors
Authors: Gaudio Eugenio , Bernasconi Elena , Kwee Ivo , Boi Michela , Bonetti Paola , Tarantelli Chiara , Rinaldi Andrea , Testoni Monica , Ponzoni Maurilio , Stathis Anastasios , Stüssi Georg , Riveiro Eugenia , Herait Patrice , Zucca Emanuele , Bertoni Francesco 
Published: May 2015

Abstract
Background:
Lymphomas are still incurable in many patients and novel active compounds are actively being sought. We previously reported single agent activity of the BET bromodomain OTX015 in lymphoma cell lines and in vivo (AACR 2012; ICML 2013). Here, we report a study of the mechanism of action of OTX015 and its activity in combination with other anti-cancer compounds.
Methods:
Cell lines: 22 diffuse large B-cell lymphoma (DLBCL), 4 mantle cell lymphomas, 3 multiple myelomas, 3 splenic marginal zone lymphoma and 1 prolymphocytic leukemia. Anti-proliferative of OTX015 (OncoEthix SA, Switzerland) was assessed by MTT and its cytotoxic activity by Annexin V staining and gene expression profiling (GEP) with Illumina HumanHT-12 Expression BeadChips. Data mining was done with LIMMA, GSEA, Metacore. Synergy was assessed in cells (2-5 cell lines) exposed for 72 h to increasing doses of OTX015 alone or in combination with increasing doses of other agents. MTT assays were performed and Chou-Talalay combination index (CI) calculated.
Results:
OTX015 (500 nM, 72h) showed cytostatic activity in 29/33 (88%) cell lines and apoptosis in 3/22 (14%). Mutations in genes coding for MYD88 and components of BCR (P=0.027), and ABC signaling phenotypes (P=0.008) were significantly associated with apoptosis induction. We performed GEP on 2 cell lines (SU-DHL-6, SU-DHL-2), treated with DMSO or OTX015 (500 nM) for 1, 2, 4, 8 or 12 hours. Most upregulated genes were histones. MYC target genes were highly significantly enriched among all OTX015 regulated transcripts and MYC was the most frequently downregulated gene. OTX015 also downregulated MYD88, IRAK1, TLR6, IL6, STAT3, and TNFRSF17, members of the NFKB, TLR and JAK/STAT pathways. NFKB target genes (IRF4, TNFAIP3 and BIRC3) were also downregulated (PCR). Immunoblotting and immunohistochemistry showed a reduction of transcriptionally active pSTAT3 in 2 ABC cell lines, and a reduction in nuclear localization of p50 (NFKB1), indicating an inhibitory effect of OTX015 on the canonical NFKB pathway. Finally, IL10 and IL4 production was reduced after 24 hours OTX015 treatment.
Synergy was observed with everolimus (median CI=0.1; range 0.1-0.2), ibrutinib in ABC-DLBCL (CI=0.04; 0.02-0.1), idelalisib (CAL101) (CI=0.5; 0.04-2.4), vorinostat (CI=0.5; 0.3-0.6), rituximab (CI=0.5; 0.4-0.5), decitabine (CI=0.6; 0.6-0.7), lenalidomide (CI=0.7; 0.6-0.7), and all-trans retinoic acid (CI=0.4; .1-1.6). Additive effects were observed for combinations with romidepsin (CI=1.08; 1-1.22), bendamustine (CI=0.92; 0.83-1.1), and doxorubicin (CI=0.83; 0.71-0.96).
Conclusions:
OTX015 is a promising candidate for targeted combination therapies. A phase I study (NCT01713582) in patients with hematological neoplasias is underway and together with our preclinical data, may support further clinical investigations.
Citation Format: Eugenio Gaudio, Elena Bernasconi, Ivo Kwee, Michela Boi, Paola Bonetti, Chiara Tarantelli, Andrea Rinaldi, Monica Testoni, Maurilio Ponzoni, Anastasios Stathis, Georg Stüssi, Eugenia Riveiro, Patrice Herait, Emanuele Zucca, Francesco Bertoni. The BET Bromodomain inhibitor OTX015 targets the NFKB, TLR and JAK/STAT pathways and shows pre-clinical activity as single agent and in combination in mature B-cell tumors. [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 5528. doi:10.1158/1538-7445.AM2014-5528