Abstract C111: Pediatric Preclinical Testing Program (PPTP): Evaluation of the MEK1/2 inhibitor AZD6244 against juvenile pilocytic astrocytoma (JPA) xenografts
Authors: Smith Malcolm A. , Morton Christopher L. , Phelps Doris , Neale Geoffrey A. , Houghton Peter J. 
Published: June 2012

Background: AZD6244 (ARRY-142886) is a potent small molecule inhibitor of MEK1/2 that is in phase 2 clinical development. Against the PPTP in vivo tumor panels, AZD6244 induced significant differences in EFS distribution in 10 of 37 (27%) solid tumor models and 0 of 6 acute lymphoblastic leukemia (ALL) models, but there were no objective responses. Recently, molecular characterization of JPAs has shown tandem duplication producing a novel fusion gene (KIAA1549-BRAF) that lacks the BRAF regulatory domain and leads to constitutive activation of BRAF. Activating point mutations in BRAF are less frequent in JPA, accounting for only 5 percent of cases. AZD6244 was evaluated against two JPA xenografts, BT-35 and BT-40, that are used for secondary testing by the PPTP.

Methods: AZD6244 was dissolved in 0.5% hydroxypropyl methyl cellulose, 0.1% Polysorbate 80 and administered p.o., using a twice daily schedule (excepting weekends, for which a once daily schedule was used) for 6 weeks at a dose of 100 or 75 mg/kg. Tumors were followed for an additional 6 weeks. Three measures of antitumor activity were used: 1) response criteria modeled after the clinical setting; 2) treated to control (T/C) tumor volume at day 21; and 3) a time to event (4-fold increase in tumor volume) measure based on the median EFS of treated and control lines (intermediate activity required EFS T/C &gt; 2, and high activity additionally required a net reduction in median tumor volume at the end of the experiment). Phosphorylation of ERK1/2, as a surrogate for in vivo inhibition of MEK1/2 was determined by immunoblotting. Genomic DNA from BT-35 and BT-40 was screened for BRAF mutations with primers designed to amplify exons 1–18. Purified BRAF BAC DNA was labeled with digoxigenin-11-dUTP (Roche Molecular Biochemicals, Indianapolis, IN) by nick translation and used for FISH analysis.

Results: Both JPA models have additional copies of chromosome 7, but lack evidence of focal duplication of BRAF. Sequencing showed that BRAF is wild type in BT-35 but that BT-40 harbors an activating mutation [V600E]. Pharmacodynamic studies indicated that AZD6244 administered at 100 mg/kg twice-daily completely inhibited ERK1/2 phosphorylation. AZD6244 was evaluated against two JPA xenografts; BT-40 xenografts completely regressed on treatment with AZD6244, but re-grew subsequent to stopping therapy. In contrast, BT-35 xenografts progressed on AZD6244 treatment.

Conclusions: The PPTP previously demonstrated that AZD6244 had limited in vivo activity against the pediatric preclinical models evaluated, despite inhibition of MEK1/2 activity. However, AZD6244 was highly active at the same dose and schedule against BT-40 JPA xenografts that harbor constitutively activated BRAF. The BT-40 xenograft model may be valuable for developing rational combinations of molecularly-targeted agents against JPAs with BRAF activation.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C111.