plx-4720 and Brain-Neoplasms

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

Topics: Animals; Antineoplastic Agents; Astrocytoma; Brain Neoplasms; Disease Models, Animal; Humans; Indoles; Mice; Pediatrics; Piperazines; Proto-Oncogene Proteins B-raf; Pyridines; Sulfonamides

Malignant astrocytomas (MA) are aggressive central nervous system tumors with poor prognosis. Activating mutation of BRAF (BRAF(V600E)) has been reported in a subset of these tumors, especially in children. We have investigated the incidence of BRAF(V600E) in additional pediatric patient cohorts and examined the effects of BRAF blockade in preclinical models of BRAF(V600E) and wild-type BRAF MA.. BRAF(V600E) mutation status was examined in two pediatric MA patient cohorts. For functional studies, BRAF(V600E) MA cell lines were used to investigate the effects of BRAF shRNA knockdown in vitro, and to investigate BRAF pharmacologic inhibition in vitro and in vivo.. BRAF(V600E) mutations were identified in 11 and 10% of MAs from two distinct series of tumors (six of 58 cases total). BRAF was expressed in all MA cell lines examined, among which BRAF(V600E) was identified in four instances. Using the BRAF(V600E)-specific inhibitor PLX4720, pharmacologic blockade of BRAF revealed preferential antiproliferative activity against BRAF(V600E) mutant cells in vitro, in contrast to the use of shRNA-mediated knockdown of BRAF, which inhibited cell growth of glioma cell lines regardless of BRAF mutation status. Using orthotopic MA xenografts, we show that PLX4720 treatment decreases tumor growth and increases overall survival in mice-bearing BRAF(V600E) mutant xenografts, while being ineffective, and possibly tumor promoting, against xenografts with wild-type BRAF.. Our results indicate a 10% incidence of activating BRAF(V600E) among pediatric MAs. With regard to implications for therapy, our results support evaluation of BRAF(V600E)-specific inhibitors for treating BRAF(V600E) MA patients.

Topics: Adolescent; Amino Acid Substitution; Animals; Astrocytoma; Base Sequence; Blotting, Western; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Child; Child, Preschool; DNA Mutational Analysis; Female; Humans; Indoles; Infant; Kaplan-Meier Estimate; Mice; Mice, Nude; Mutation; Proto-Oncogene Proteins B-raf; RNA Interference; Sulfonamides; Tumor Burden; Xenograft Model Antitumor Assays; Young Adult