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Background: BRAF V600E mutation is present in a subset of pediatric brain tumors. Vemurafenib is an oral, selective ATP-competitive inhibitor of BRAF V600E kinase. The goal of this multi-center study conducted through the Pacific Pediatric Neuro-Oncology Consortium (PNOC) was to determine the recommended phase 2 dose (RP2D) and dose limiting toxicities (DLTs) in children < 18 years with recurrent or progressive BRAF V600E mutant brain tumors. Results: Nineteen eligible patients were enrolled. Eleven patients had received three or more prior therapies. Data reported are from the start of treatment for the first patient (April 30 2014) through August 31 2019. The RP2D was defined as 550 mg/m 2 twice daily after DLT criteria adjustment for rash. Related grade ≥ 3 adverse events included secondary keratoacanthoma ( n = 1); rash ( n =16); and fever ( n = 5). Subjects received a median of 23 cycles (range 3-63). Four patients remain on treatment. Centrally reviewed best radiographic responses included 1 complete response, 5 partial responses, and 13 stable disease. The steady-state area under the curve (AUC 0-∞ median) was 604 mg*h/L (range 329-1052). Methods: Vemurafenib was given starting at 550 mg/m 2 , twice daily which corresponds to the adult RP2D. Adverse events were graded using the NIH Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. Central imaging review was performed. Pharmacokinetic sampling was performed. Conclusions: Vemurafenib has promising anti-tumor activity in recurrent BRAF V600E-positive brain tumors with manageable toxicity. A phase 2 study is ongoing (NCT01748149)., Competing Interests: CONFLICTS OF INTEREST There are no conflicts of interest for any authors., (Copyright: © 2020 Nicolaides et al.)

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.

Mutational activation of BRAF(BRAF(V600E)) occurs in pediatric glioma and drives aberrant MAPK signaling independently of upstream cues. Targeted monotherapy against BRAF(V600E) displays efficacy in pre-clinical models of glioma, however xenograft tumors adapt rapidly and escape from the growth-inhibitory effects of BRAF-targeted therapy. Here, we show that intrinsic resistance to a BRAF(V600E) specific inhibitor stems, in part, from feedback activation of EGFR and downstream signaling pathways. BRAF(V600E) inhibition suppresses MAPK signaling, which in turn downregulates the EGFR phosphatase PTPN9, resulting in sustained EGFR phosphorylation and enhanced EGFR activity. We demonstrated that overexpression of PTPN9 reduces EGFR phosphorylation and cooperates with BRAF(V600E) inhibitor PLX4720 to reduce MAPK and Akt signaling, resulting in decreased glioma cell viability. Moreover, pharmacologic inhibition of EGFR combined with inhibition of BRAF(V600E) to reduce growth of glioma cell lines and orthotopic glioma xenograft by decreasing tumor cell proliferation while increasing apoptosis, with resultant significant extension of animal subject survival. Our data support clinical evaluation of BRAF(V600E) and EGFR targeted therapy in treating BRAF(V600E) glioma.