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. 2012 May 29;109(22):8710-5.
doi: 10.1073/pnas.1117255109. Epub 2012 May 14.

Cooperative interactions of BRAFV600E kinase and CDKN2A locus deficiency in pediatric malignant astrocytoma as a basis for rational therapy

Emmanuelle Huillard  1 , Rintaro Hashizume, Joanna J Phillips, Amélie Griveau, Rebecca A Ihrie, Yasuyuki Aoki, Theodore Nicolaides, Arie Perry, Todd Waldman, Martin McMahon, William A Weiss, Claudia Petritsch, C David James, David H Rowitch
Affiliations

Cooperative interactions of BRAFV600E kinase and CDKN2A locus deficiency in pediatric malignant astrocytoma as a basis for rational therapy

Emmanuelle Huillard et al. Proc Natl Acad Sci U S A. .

Abstract

Although malignant astrocytomas are a leading cause of cancer-related death in children, rational therapeutic strategies are lacking. We previously identified activating mutations of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) (BRAF(T1799A) encoding BRAF(V600E)) in association with homozygous cyclin-dependent kinase inhibitor 2A (CDKN2A, encoding p14ARF and p16Ink4a) deletions in pediatric infiltrative astrocytomas. Here we report that BRAF(V600E) expression in neural progenitors (NPs) is insufficient for tumorigenesis and increases NP cellular differentiation as well as apoptosis. In contrast, astrocytomas are readily generated from NPs with additional Ink4a-Arf deletion. The BRAF(V600E) inhibitor PLX4720 significantly increased survival of mice after intracranial transplant of genetically relevant murine or human astrocytoma cells. Moreover, combination therapy using PLX4720 plus the Cyclin-dependent kinase (CDK) 4/6-specific inhibitor PD0332991 further extended survival relative to either monotherapy. Our findings indicate a rational therapeutic strategy for treating a subset of pediatric astrocytomas with BRAF(V600E) mutation and CDKN2A deficiency.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Concurrent BRAFV600E activation and Ink4a-arf deletion induces malignant astrocytoma from mouse neural progenitors. (A) Mouse model for pediatric malignant astrocytoma. GE, ganglionic eminence; ctx, cortex. (B) Kaplan–Meier survival curves of SCID mice transduced with hGFAP-cre BrafCA/+ Ink4a-Arf−/− (n = 6) or Ad:cre BrafCA/+ Ink4a-Arf−/− (n = 5) neural progenitor cells (P = 0.64). (C) Injection of an adenovirus encoding ubiquitous cre expression into BrafCA/+ or BrafCA/+ Ink4a-arfLoxP/LoxP adult mice. (D) Kaplan–Meier survival curves of BrafCA/+ (n = 6) and BrafCA/+ Ink4a-arfLoxP/LoxP (n = 9) mice injected with Ad:cre into the lateral ventricle (P = 0.0004).
Fig. 2.
Fig. 2.
hGFAP-cre; BrafCA/+Ink4a-Arf−/− cells are more proliferative and less differentiated than hGFAP-cre; BrafCA/+ cells. (A) Immunocytochemical analysis of proliferation (BrdU) and apoptosis (cleaved caspase 3) markers in BrafCA/+ and BrafCA/+ Ink4a-Arf−/− neural progenitors cultures. (Scale bars, 100 µm.) (B) Quantification of the numbers of BrdU+ and Cleaved-caspase 3+ cells. (C) Corresponding analysis of the differentiation markers βIII-tubulin (neuronal), GFAP (astrocyte), O4 (oligodendrocyte), and the proliferation marker Ki67 in neural progenitor cultures under differentiation culture conditions. (Scale bars, 100 µm.) (D) Quantification of the numbers of cells expressing the differentiation markers (mean ± SEM). ***P < 0.0005, **P < 0.005.
Fig. 3.
Fig. 3.
CDK4/6 activity is required in the context of dual BRAFV600E; Ink4a-Arf−/− genetic alterations for cell cycle progression in murine astrocytoma cells. BrafCA/+; Ink4a-Arf+/+ and BrafCA/+; Ink4a-Arf−/− cells were incubated in the presence of vehicle (sodium lactate) or 10 μM PD0332991 for 48 h. (A) Flow cytometry analysis of treated BrafCA/+; Ink4a-Arf+/+ (+/+) and BrafCA/+; Ink4a-Arf−/− (−/−) cells, pulsed with BrdU for 1 h and costained with FITC-conjugated BrdU and 7-amino-actinomycin D (7-AAD). The intensity of cell incorporated BrdU (logarithmic mode) vs. total DNA content with 7-AAD (linear signal amplification mode) is shown. (B) Cell cycle distribution of control and treated BrafCA/+; Ink4a-Arf+/+ (+/+) and BrafCA/+; Ink4a-Arf−/− (−/−) cells. (C) Immunoblotting analysis of pRb and Rb in treated BrafCA/+; Ink4a-Arf+/+ and BrafCA/+; Ink4a-Arf−/− cells.
Fig. 4.
Fig. 4.
Mono- and combination therapy of mice transduced with BrafCA/+ Ink4a-Arf−/− murine cells using BRAFV600E and CDK4/6 inhibitors. (A) Histological characterization of luciferase-modified BrafCA/+ Ink4a-Arf−/− orthotopic allografts. (Scale bars, 20 µm.) (B–E) BrafCA/+ Ink4a-Arf−/− orthotopic allografts were treated with PD0332991, PLX4720, or PLX4720 + PD0332991 for 14 consecutive days (pink area). (B) Bioluminescence imaging (BLI); P = 0.0110 for control vs. PLX + PD, P = 0.0341 for PLX vs. PLX + PD, P = 0.0222 for PD vs. PLX + PD. There are no statistically significant differences in tumor growth rate between control vs. PLX (P = 0.1866) or PD (P = 0.1971). (C) Kaplan–Meier survival curves; P = 0.0057 for control vs. PLX, P = 0.0069 for control vs. PD0332991, P = 0.0005 for control vs. PLX4720 + PD0332991. Although there is a trend for the combination being superior, there are no statistically significant differences in survival between PD or PLX vs. PD/PLX treatment groups: P = 0.0637 for combination vs. PLX only, and P = 0.0698 for combination vs. PD only. (D) Immunostaining of the proliferation marker Ki67 in control vs. treated (PLX, PD, and PLX/PD) tumors. (Scale bars, 20 µm.) (E) Quantification of the percentage of Ki67+ cells in untreated as well as treated tumor sections (mean ± SEM); ***P < 0.001, **P = 0.006.
Fig. 5.
Fig. 5.
Importance of BRAFV600E mutation and Ink4a-Arf deletion in determining orthotopic xenograft response to BRAF and CDK4/6 treatment. Tumors generated from the DBTRG05-MG astrocytoma line were treated with PD0332991, PLX4720, or PLX4720 + PD0332991 therapies for 14 consecutive days (pink area). (A) Histological features of DBTRG xenografts. (Scale bars, 20 µm.) (B) Bioluminescence imaging (BLI); P = 0.0013 for control vs. PLX, P = 0.0120 for control vs. PD, P < 0.0001 for control vs. PLX + PD combination therapy on day 77. P = 0.0018 for PLX vs. PLX + PD, P = 0.0018 for PD vs. PLX + PD. (C) Kaplan–Meier survival curves; P = 0.0028 for control vs. PLX, P = 0.0005 for control vs. PD0332991; P < 0.001 for PLX or PD vs. PLX4720 + PD0332991 combination therapy. (D) Quantification of the number of Ki67+ cells in treated tumors (mean ± SEM); ***P < 0.0001; NS, not significant. (E and F) BLI and survival curves of mice transplanted with the GS2 astrocytoma line, which has wild-type BRAF and intact Ink4a-Arf.
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