Abstract B75: Defining the antitumor activity and sensitivity profiles of BET inhibitors in neuroblastoma
Authors: Schnepp Robert , Hart Lori , Raman Pichai , Danielson Laura , Gagliardi Maria , Kinsey Ryan , Wyce Anastasia , Barbash Olena , Tummino Peter , Chesler Louis , Maris John 
Published: May 2015

Background: MYCN amplification is the most frequent somatically acquired genomic alteration in neuroblastoma and is a potent oncogenic driver. Targeting MYCN therapeutically has been complicated by the challenges inherent in targeting transcription factors. Recently, several groups have shown that inhibiting bromodomain and extra-terminal (BET) proteins (BRD2, BRD3, BRD4, and BRDT) and their ability to bind to acetylated lysine residues within histone tails resulted in silencing of MYC family protein expression and demonstrated therapeutic efficacy in preclinical cancer models. We hypothesized that potent inhibition of bromodomain-chromatin interactions would show anti-tumor activity in preclinical models of neuroblastoma and that reliable biomarkers of activity could be discovered.

Methods: GlaxoSmithKline compounds GSK726 and GSK762 potently inhibit BET proteins (BETi) and were used for in vitro cytotoxicity and in vivo therapeutic studies, respectively. We exposed a panel of 15 cell lines to a 4-log dose range of GSK726 to determine IC50s in a luminescence-based cell viability assay. We also measured cell cycle changes by flow cytometry and apoptosis by immunoblotting for cleaved PARP. We tested the in vivo efficacy of the GSK762 in subcutaneous xenograft models (3 sensitive and 2 resistant cell lines) and in genetically engineered neuroblastoma mouse models (overexpressing MYCN and MYCN/ALK F1174L in the neural crest). For biomarker discovery, gene expression data from the neuroblastoma cell lines generated on HuGene1.0ST expression microarrays (Affymetrix) were utilized, and data were analyzed using the Limma package in R/Bioconductor.

Results: Neuroblastoma cell lines (N=15) were differentially sensitive to GSK726, with a median IC50 of 128nM and a range of 17nM to &gt;10uM. In sensitive cell lines, GSK726 treatment resulted in MYCN depletion, G1 arrest within 24 hours, and apoptosis. In subcutaneous xenograft models and genetically engineered neuroblastoma mouse models, GSK762 treatment resulted in tumor growth delay. Importantly, MYCN amplification status did not fully predict sensitivity to GSK726 or GSK762. Thus, to determine additional biomarkers of sensitivity, we examined baseline gene expression data at the extremes of IC50s by comparing sensitive (N=6; IC50&lt;128 nM) and resistant (N=4; IC50&gt;940 nM) neuroblastoma cell lines. Univariate analysis, with an FDR &lt; 0.25, revealed 6 genes (PTER, PCDHB14, RFTN1, JAK2, MYCBP, and DACH1) differentially expressed between sensitive and resistant cell lines in the MYCN amplified setting. While all 6 genes predicted BETi sensitivity in the MYCN-amplified subset of cell lines, only DACH1 expression predicted sensitivity to BET inhibition irrespective of MYCN amplification status (7.92x10-7). Moreover, DACH1 levels were heterogeneously expressed in a panel of 88 primary neuroblastoma tumors (Versteeg-88) and high expression was correlated with poor patient outcome (1.74x10-5).

Conclusions: BET inhibitors demonstrated significant anti-tumor activity in a subset of neuroblastoma preclinical models. Although MYCN protein levels decreased in neuroblastoma cell lines that were sensitive to BETi, MYCN amplification status alone did not fully explain BETi sensitivity. DACH1 expression serves as a candidate biomarker for sensitivity to BET inhibition. These studies will help to optimize the clinical utility of BETi in neuroblastoma and perhaps other MYC-driven malignancies.

Citation Format: Robert Schnepp, Lori Hart, Pichai Raman, Laura Danielson, Maria Gagliardi, Ryan Kinsey, Anastasia Wyce, Olena Barbash, Peter Tummino, Louis Chesler, John Maris. Defining the antitumor activity and sensitivity profiles of BET inhibitors in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B75.