Activating mutation of BRAF is a common finding in pediatric gliomas. As many as 14% of high grade and up to 66% of certain subtypes of low grade pediatric glioma have the BRAFV600E mutation. Small molecule inhibitors that selectively target BRAFV600E are FDA approved for melanoma and have shown significant efficacy in treating BRAFV600E glioma in pre-clinical trials. Despite showing initial anti-tumor activity, acquired drug resistance significantly limits the benefit from being treated with BRAFV600E inhibitors. Here, we have identified molecular responses to BRAFV600E inhibitor treatment in human glioma models that have substantial clinical implications. Specifically, we show that BRAFV600E inhibitor resistant cells upregulate pro-survival mediators such as Wnt, and additionally increase receptor tyrosine kinase activity, including EGFR and Axl, promoting resistance to BRAFV600E inhibition. Our results suggest strategies to circumvent acquired resistance to BRAFV600E inhibitor therapy, and thereby improve outcomes for patients with BRAFV600E gliomas.

BRAF^V600E is a common finding in glioma (about 10-60% depending on histopathologic subclassification). BRAF^V600E monotherapy shows modest preclinical efficacy against BRAF^V600E gliomas and also induces adverse secondary skin malignancies. Here, we examine the molecular mechanism of intrinsic resistance to BRAF^V600E inhibition in glioma. Furthermore, we investigate BRAF^V600E/MEK combination therapy that overcomes intrinsic resistance to BRAF^V600E inhibitor and also prevents BRAF^V600E inhibitor induced secondary malignancies. Immunoblotting and Human Phospho-Receptor Tyrosine Kinase Array assays were used to interrogate MAPK pathway activation. The cellular effect of BRAF^V600E and MEK inhibition was determined by WST-1 viability assay and cell cycle analysis. Flanked and orthotopic GBM mouse models were used to investigate the in vivo efficacy of BRAF^V600E/MEK combination therapy and the effect on secondary malignancies. BRAF^V600E inhibition leads to recovery of ERK phosphorylation. Combined BRAF^V600E and MEK inhibition prevents reactivation of the MAPK signaling, which correlates with decreased cell viability and augmented cell cycle arrest. Similarly, mice bearing BRAF^V600E glioma showed reduced tumor growth when treated with a combination of BRAF^V600E and MEK inhibitor compared to BRAF^V600E inhibition alone. Additional benefit of BRAF^V600E/MEK inhibition was reflected by reduced cutaneous squamous-cell carcinoma (cSCC) growth (a surrogate for RAS-driven secondary maligancies). In glioma, recovery of MAPK signaling upon BRAF inhibition accounts for intrinsic resistance to BRAF^V600E inhibitor. Combined BRAF^V600E and MEK inhibition prevents rebound of MAPK activation, resulting in enhanced antitumor efficacy and also reduces the risk of secondary malignancy development.

This report describes results from our analysis of the activity and biodistribution of a novel pan-ERBB inhibitor, NT113, when used in treating mice with intracranial glioblastoma (GBM) xenografts. Approaches used in this investigation include: bioluminescence imaging (BLI) for monitoring intracranial tumor growth and response to therapy; determination of survival benefit from treatment; analysis of tumor IHC reactivity for indication of treatment effect on proliferation and apoptotic response; Western blot analysis for determination of effects of treatment on ERBB and ERBB signaling mediator activation; and high-performance liquid chromatography for determination of NT113 concentration in tissue extracts from animals receiving oral administration of inhibitor. Our results show that NT113 is active against GBM xenografts in which wild-type EGFR or EGFRvIII is highly expressed. In experiments including lapatinib and/or erlotinib, NT113 treatment was associated with the most substantial improvement in survival, as well as the most substantial tumor growth inhibition, as indicated by BLI and IHC results. Western blot analysis results indicated that NT113 has inhibitory activity, both in vivo and in vitro, on ERBB family member phosphorylation, as well as on the phosphorylation of downstream signaling mediator Akt. Results from the analysis of animal tissues revealed significantly higher NT113 normal brain-to-plasma and intracranial tumor-to-plasma ratios for NT113, relative to erlotinib, indicating superior NT113 partitioning to intracranial tissue compartments. These data provide a strong rationale for the clinical investigation of NT113, a novel ERBB inhibitor, in treating patients with GBM.