Abstract
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.
Similar content being viewed by others
References
Stupp R et al (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352(10):987–996
Bamford S et al (2004) The COSMIC (catalogue of somatic mutations in cancer) database and website. Br J Cancer 91(2):355–358
Basto D et al (2005) Mutation analysis of B-RAF gene in human gliomas. Acta Neuropathol 109(2):207–210
Knobbe CB, Reifenberger J, Reifenberger G (2004) Mutation analysis of the Ras pathway genes NRAS, HRAS, KRAS and BRAF in glioblastomas. Acta Neuropathol 108(6):467–470
Myung JK et al (2012) Analysis of the BRAF(V600E) mutation in central nervous system tumors. Transl Oncol 5(6):430–436
Schindler G et al (2011) Analysis of BRAF V600E mutation in 1,320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma, ganglioglioma and extra-cerebellar pilocytic astrocytoma. Acta Neuropathol 121(3):397–405
Schiffman JD et al (2010) Oncogenic BRAF mutation with CDKN2A inactivation is characteristic of a subset of pediatric malignant astrocytomas. Cancer Res 70(2):512–519
Lyustikman Y et al (2008) Constitutive activation of Raf-1 induces glioma formation in mice. Neoplasia 10(5):501–510
Horbinski C et al (2012) Interplay among BRAF, p16, p53, and MIB1 in pediatric low-grade gliomas. Neurooncology 14(6):777–789
Kasid U, Dritschilo A (2003) RAF antisense oligonucleotide as a tumor radiosensitizer. Oncogene 22(37):5876–5884
Weidhaas JB et al (2006) A conserved RAS/mitogen-activated protein kinase pathway regulates DNA damage-induced cell death postirradiation in Radelegans. Cancer Res 66(21):10434–10438
Chapman PB et al (2011) Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 364(26):2507–2516
Hauschild A et al (2012) Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet 380(9839):358–365
Long GV et al (2012) Dabrafenib in patients with Val600Glu or Val600Lys BRAF-mutant melanoma metastatic to the brain (BREAK-MB): a multicentre, open-label, phase 2 trial. Lancet Oncol 13(11):1087–1095
Dasgupta T et al. (2013) Genotype-dependent cooperation of ionizing radiation with BRAF inhibition in BRAF V600E-mutated carcinomas. Investig New Drugs 31(5):1136–1141
Sambade MJ et al (2011) Melanoma cells show a heterogeneous range of sensitivity to ionizing radiation and are radiosensitized by inhibition of B-RAF with PLX-4032. Radiother Oncol 98(3):394–399
Michaud K et al (2010) Pharmacologic inhibition of cyclin-dependent kinases 4 and 6 arrests the growth of glioblastoma multiforme intracranial xenografts. Cancer Res 70(8):3228–3238
Franken NA et al (2006) Clonogenic assay of cells in vitro. Nat Protoc 1(5):2315–2319
Debacq-Chainiaux F et al (2009) Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo. Nat Protoc 4(12):1798–1806
Sarkaria JN et al (2007) Identification of molecular characteristics correlated with glioblastoma sensitivity to EGFR kinase inhibition through use of an intracranial xenograft test panel. Mol Cancer Ther 6(3):1167–1174
Hashizume R et al (2010) Morphologic and molecular characterization of ATRT xenografts adapted for orthotopic therapeutic testing. Neurooncology 12(4):366–376
Wang J et al (2009) A reproducible brain tumour model established from human glioblastoma biopsies. BMC Cancer 9:465
Poulikakos PI et al (2010) RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature 464(7287):427–430
Merten R et al (2014) Increased skin and mucosal toxicity in the combination of vemurafenib with radiation therapy. Strahlentherapie Onkologie 190(12):1169–1172
Nicolaides TP et al (2011) Targeted therapy for BRAFV600E malignant astrocytoma. Clin Cancer Res 17(24):7595–7604
Hidalgo M (2003) Erlotinib: preclinical investigations. Oncology 17(11 Suppl 12):11–16
Seiwert TY, Salama JK, Vokes EE (2007) The concurrent chemoradiation paradigm–general principles. Nat Clin Pract Oncol 4(2):86–100
Hecht M et al (2015) Radiosensitization by BRAF inhibitor therapy-mechanism and frequency of toxicity in melanoma patients. Ann Oncol 26(6):1238–1244
Boussemart L et al (2013) Vemurafenib and radiosensitization. JAMA Dermatol 149(7):855–857
Peuvrel L et al (2013) Severe radiotherapy-induced extracutaneous toxicity under vemurafenib. Eur J Dermatol 23(6):879–881
Narayana A et al (2013) Vemurafenib and radiation therapy in melanoma brain metastases. J Neurooncol 113(3):411–416
Forschner A et al (2014) Radiation recall dermatitis and radiation pneumonitis during treatment with vemurafenib. Melanoma Res 24(5):512–516
Stein AP et al (2015) Xenograft assessment of predictive biomarkers for standard head and neck cancer therapies. Cancer Med 4(5):699–712
Ryu CH et al (2012) Valproic acid downregulates the expression of MGMT and sensitizes temozolomide-resistant glioma cells. J Biomed Biotechnol 2012:987495
Acknowledgments
The authors acknowledge a Young Investigator Award to TD from the American Society of Clinical Oncology, NIH R01NS091620 (DHK, WAW), NS080619 (CDJ), Grand Philanthropic Fund (DHK), and University of California Cancer Research Coordinating Committee (DHK).
Author information
Authors and Affiliations
Corresponding author
Additional information
Tina Dasgupta and Aleksandra K. Olow have contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
11060_2015_1939_MOESM1_ESM.tiff
Supplemental Figure 1. Radiation cooperates with PLX4720 to reduce S phase and induce apoptosis in BRAF V600E glioma cells. Flow cytometry analyses of (A-B) cell cycle using BrdU and 7-AAD staining; and (C-D) apoptosis using Annexin V staining. Cells analyzed include (A, C) BRAF WT GBM36 and (B, D) BRAF V600E DBTRG-05MG cells. These additional cell lines complement the data shown in Figures 2 and 3 and utilize identical methods to those delineated in Figures 2 and 3 (TIFF 3736 kb)
11060_2015_1939_MOESM2_ESM.tiff
Supplemental Figure 2. Flow cytometric in vitro measurement of β-galactosidase levels in (A) GBM36 (BRAF WT) and (B) AM-38 (BRAF V600E) cells reveals higher background levels of senescence in BRAF V600E cells compared to BRAF WT cells but no pronounced effects of PLX4720, radiation or their combination on levels of senescent cells (TIFF 6527 kb)
11060_2015_1939_MOESM3_ESM.tiff
Supplemental Figure 3. Immunohistochemical analyses of survival study mice reveals combination (concurrent) therapy of PLX4720 and radiation significantly decreases tumor proliferation (Ki-67 staining) and increases cleaved caspase 3 (CC3) compared to control mice. Comparisons of combination therapy to each monotherapy were not statistically significant (TIFF 2927 kb)
Rights and permissions
About this article
Cite this article
Dasgupta, T., Olow, A.K., Yang, X. et al. Survival advantage combining a BRAF inhibitor and radiation in BRAF V600E-mutant glioma. J Neurooncol 126, 385–393 (2016). https://doi.org/10.1007/s11060-015-1939-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11060-015-1939-2