Abstract 4731: Targeting super-enhancer induced gene expression with the novel BRD4 inhibitor OTX015 in preclinical models of MYCN-amplified neuroblastoma
Authors: Henssen Anton , Althoff Kristina , Koche Richard , Odersky Andrea , Beckers Anneleen , Speleman Frank , Schäfers Simon , De Preter Katleen , Florin Alexandra , Heukamp Lukas , Spruessel Annika , Astrahanseff Kathy , Sadowski Natalie , Schramm Alexander , Eggert Angelika , Astorgues-Xerri Lucile , Riveiro Eugenia , Cvitkovic Esteban , Schulte Johannes H. 
Published: August 2015

Bromodomain-containing protein 4 (BRD4) functions as an epigenetic reader and binds to promoter super-enhancer regions driving oncogenes such as MYC. Neuroblastomas (NB) harboring MYCN amplifications are highly lethal tumors often resistant to standard chemotherapy. OTX015 is a novel BRD2/3/4 inhibitor currently in clinical Phase Ib studies in hematologic malignancies and solid tumors. We have previously reported that OTX015 displayed in vitro and in vivo antitumor effects, together with MYCN transcription attenuation in NB models (Henssen et al; AACR 2014). Here, we investigated OTX015 targeting of super-enhancer regulated genes in MYCN-amplified NB in vitro and in vivo models. Protein-DNA interactions were analyzed using ChipSeq in IMR 5 cells. We identified super-enhancers associated with a variety of genes of known importance in NB, including MYCN, as well as some previously undescribed genes. OTX015 inhibited cell proliferation in Chp-212, Chp-134, Gimen, IMR-32, NB69, SK-N-AS, SK-N-BE, and SK-N-BE2 NB cell lines after 72 h exposure. OTX015 reduced tumor burden in IMR 5 xenograft mice and in a genetically engineered model of MYCN-amplified NB LSL MYCN;Dbh-iCre, when administered by oral gavage at a dose of 25 mg/kg daily for 3 weeks. Antitumoral effects of OTX015 were coupled with decreased binding of BRD4 to chromatin and subsequent global transcriptional changes. Moreover, OTX015 exposure led to significant transcriptional downregulation of genes associated with super-enhancers, supporting the notion that BRD4 preferentially acts at these chromatin sites. Interestingly, BRD inhibition not only attenuated MYCN transcription but most significantly affected MYCN-regulated transcriptional programs. Ectopic expression of MYCN was not able to abrogate the antitumoral effects of BRD4 inhibition, indicating direct involvement of MYCN in super-enhancer regulated gene expression and possibly explaining the increased susceptibility of MYCN-amplified NB to OTX015 inhibition. We describe here for the first time that BRD inhibition by OTX015 selectively and preferentially targets global super-enhancer induced transcription in MYCN-driven NB. These new insights will serve as a rationale for a clinical trial in pediatric MYCN-amplified NB patients with OTX015.

Citation Format: Anton Henssen, Kristina Althoff, Richard Koche, Andrea Odersky, Anneleen Beckers, Frank Speleman, Simon Schäfers, Katleen De Preter, Alexandra Florin, Lukas Heukamp, Annika Spruessel, Kathy Astrahanseff, Natalie Sadowski, Alexander Schramm, Angelika Eggert, Lucile Astorgues-Xerri, Eugenia Riveiro, Esteban Cvitkovic, Johannes H. Schulte. Targeting super-enhancer induced gene expression with the novel BRD4 inhibitor OTX015 in preclinical models of MYCN-amplified neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4731. doi:10.1158/1538-7445.AM2015-4731