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Survival advantage combining a BRAF inhibitor and radiation in BRAF V600E-mutant glioma

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An Erratum to this article was published on 19 January 2016

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.

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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).

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Correspondence to Tina Dasgupta.

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Tina Dasgupta and Aleksandra K. Olow have contributed equally to this work.

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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)

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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

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  • DOI: https://doi.org/10.1007/s11060-015-1939-2

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