Abstract B82: MEK 1/2 inhibition and biomarkers of response in preclinical models of neuroblastoma
Authors: Hart Lori S. , Batra Vandana , Raman Pichai , Gagliardi Maria , Rader JulieAnn , Chen Lucy , Fritsch Christine , Caponigro Giordano , Peters Malte , Boehm Markus , Maris John M. 
Published: May 2015

Abstract
Background: Despite an aggressive treatment regime, 40% of patients with high-risk neuroblastoma die of their disease. The mitogen-activated protein kinase (MAPK) signaling cascade is hyperactive in many cancers, including a subset of high-risk neuroblastomas. We, therefore, sought to determine the efficacy of MEK inhibition in neuroblastoma and develop a responder hypothesis of anti-tumor activity based on molecular genetic features.
Methods: MEK162 (Novartis Pharmaceuticals), an orally available MEK1/2 inhibitor, is currently in adult clinical trials for advanced malignancies harboring RAS/RAF mutations. In determining MEK162 IC50 values, we employed the RT-CES cell impedance assay as it measures both cytostatic and cytotoxic responses in a temporal manner. Additional in vitro analysis of MEK162 activity included immunoblotting, cell cycle analysis with flow cytometric methods, and phosphorylation arrays across a panel of 23 neuroblastoma cell lines. Four of these cell lines (2 sensitive and 2 resistant) were also tested in vivo in subcutaneous xenograft models with tumor volume endpoints. The cell line IC50 ranking was cross-referenced with HuGene1.0ST expression microarrays (Affymetrix), phosphorylation arrays (Full Moon Biosystems), and exome sequencing in order to identify the genetic and proteomic underpinnings predictive of response to MEK inhibition. Microarray analysis was performed using the Limma package (Bioconductor/R), unsupervised clustering, and gene set enrichment (GSEA).
Results: Neuroblastoma cell lines (N=23) showed a wide range of sensitivity to MEK162 across a 4-log dose range (median IC50 = 771 nM, range 5 nM-10 μM). Sensitive cell lines demonstrated G1 arrest within 24 hours of exposure to MEK162. In vivo subcutaneous xenograft experiments recapitulated the in vitro response of MEK162 sensitivity in each of the cell lines tested. In order to identify determinants of sensitivity, we profiled the genomic signature of 23 cell lines in the exponential growth phase prior to confluence. Of the ten most sensitive cell lines, six possessed mutations predicted to be damaging and indicative of RAS/MAPK pathway hyperactivation (NRAS-Q61K, NF1 copy number loss, KRAS-G12D). No resistant lines possessed genetic evidence of RAS hyperactivation. However, baseline phosphorylated-ERK status was not robustly predictive of drug activity. For the purpose of biomarker discovery, we considered a total of 4 cells lines as sensitive (IC50&lt; 200 nM) and 4 resistant (IC50&gt; 3,000 nM). Unsupervised clustering and GSEA of the most differentially expressed genes derived from comparing sensitive (N=4) and resistant (N=4) cell lines confirmed increased baseline MAPK pathway activity in the sensitive cell lines. In addition, resistance to MEK162 was found to be associated with MYCN expression (R=.70/p=0.00043), suggesting that MYCN may serve as a biomarker of MEK162 resistance. Independently, we have demonstrated a correlation between MYCN-amplification and sensitivity to CDK4/6 inhibition (p=0.0227), suggesting the combination of MEK and CDK4/6 inhibition may synergize in the inhibition of neuroblastoma growth.
Conclusions: MEK inhibition is effective in a definable subset of human-derived neuroblastoma preclinical models. Canonical activation of the RAS/MAPK pathway via RAS mutation and/or NF1 inactivation partially account for sensitivity to MEK inhibition, however, biomarkers predictive of sensitivity to MEK inhibition remain to be identified. We demonstrate that both MYCN expression and CDK4/6 activity are associated with MEK resistance, providing a basis for defining MEK162 combination strategies in the treatment of neuroblastoma.
Citation Format: Lori S. Hart, Vandana Batra, Pichai Raman, Maria Gagliardi, JulieAnn Rader, Lucy Chen, Christine Fritsch, Giordano Caponigro, Malte Peters, Markus Boehm, John M. Maris. MEK 1/2 inhibition and biomarkers of response in preclinical models of 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 B82.