plx-4720 and Glioma

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.

Topics: Antineoplastic Agents; Apoptosis; Axl Receptor Tyrosine Kinase; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Resistance, Neoplasm; ErbB Receptors; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glioma; Humans; Indoles; Mutation; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; ras Proteins; Receptor Protein-Tyrosine Kinases; Signal Transduction; Sulfonamides

Radiation (RT) is critical to the treatment of high-grade gliomas (HGGs) but cures remain elusive. The BRAF mutation V600E is critical to the pathogenesis of 10-20% of pediatric gliomas, and a small proportion of adult HGGs. Here we aim to determine whether PLX4720, a specific BRAF V600E inhibitor, enhances the activity of RT in human HGGs in vitro and in vivo. Patient-derived HGG lines harboring wild-type BRAF or BRAF V600E were assessed in vitro to determine IC50 values, cell cycle arrest, apoptosis and senescence and elucidate mechanisms of combinatorial activity. A BRAF V600E HGG intracranial xenograft mouse model was used to evaluate in vivo combinatorial efficacy of PLX4720+RT. Tumors were harvested for immunohistochemistry to quantify cell cycle arrest and apoptosis. RT+PLX4720 exhibited greater anti-tumor effects than either monotherapy in BRAF V600E but not in BRAF WT lines. In vitro studies showed increased Annexin V and decreased S phase cells in BRAF V600E gliomas treated with PLX4720+RT, but no significant changes in β-galactosidase levels. In vivo, concurrent and sequential PLX4720+RT each significantly prolonged survival compared to monotherapies, in the BRAF V600E HGG model. Immunohistochemistry of in vivo tumors demonstrated that PLX4720+RT decreased Ki-67 and phospho-MAPK, and increased γH2AX and p21 compared to control mice. BRAF V600E inhibition enhances radiation-induced cytotoxicity in BRAF V600E-mutated HGGs, in vitro and in vivo, effects likely mediated by apoptosis and cell cycle, but not senescence. These studies provide the pre-clinical rationale for clinical trials of concurrent radiotherapy and BRAF V600E inhibitors.

Topics: Animals; Apoptosis; Brain Neoplasms; Cell Cycle; Cell Proliferation; Chemoradiotherapy; Gamma Rays; Glioma; Humans; Immunoenzyme Techniques; Indoles; Mice; Mice, Nude; Mutation; Neoplasm Grading; Proto-Oncogene Proteins B-raf; Sulfonamides; Survival Rate; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Topics: Adolescent; Brain Neoplasms; Child; Child, Preschool; Drug Therapy, Combination; Drugs, Investigational; Female; Follow-Up Studies; Glioma; Humans; Indoles; Male; Molecular Targeted Therapy; Pediatrics; Proto-Oncogene Proteins B-raf; Sulfonamides; United States; United States Food and Drug Administration

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.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; ErbB Receptors; Female; Glioma; Humans; Indoles; MAP Kinase Signaling System; Mice; Mice, Nude; Molecular Targeted Therapy; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Random Allocation; Signal Transduction; Sulfonamides; Transfection; Xenograft Model Antitumor Assays