plx-4720 and Astrocytoma

Astrocytomas are the most common type of brain tumors in children. Activated BRAF protein kinase mutations are characteristic of pediatric astrocytomas with KIAA1549-BRAF fusion genes typifying low-grade astrocytomas and (V600E)BRAF alterations characterizing distinct or higher-grade tumors. Recently, BRAF-targeted therapies, such as vemurafenib, have shown great promise in treating V600E-dependent melanomas. Like (V600E)BRAF, BRAF fusion kinases activate MAPK signaling and are sufficient for malignant transformation; however, here we characterized the distinct mechanisms of action of KIAA1549-BRAF and its differential responsiveness to PLX4720, a first-generation BRAF inhibitor and research analog of vemurafenib. We found that in cells expressing KIAA1549-BRAF, the fusion kinase functions as a homodimer that is resistant to PLX4720 and accordingly is associated with CRAF-independent paradoxical activation of MAPK signaling. Mutagenesis studies demonstrated that KIAA1549-BRAF fusion-mediated signaling is diminished with disruption of the BRAF kinase dimer interface. In addition, the KIAA1549-BRAF fusion displays increased binding affinity to kinase suppressor of RAS (KSR), an RAF relative recently demonstrated to facilitate MEK phosphorylation by BRAF. Despite its resistance to PLX4720, the KIAA1549-BRAF fusion is responsive to a second-generation selective BRAF inhibitor that, unlike vemurafenib, does not induce activation of wild-type BRAF. Our data support the development of targeted treatment paradigms for BRAF-altered pediatric astrocytomas and also demonstrate that therapies must be tailored to the specific mutational context and distinct mechanisms of action of the mutant kinase.

Topics: Animals; Astrocytoma; Cell Line, Tumor; Cell Transformation, Neoplastic; Child; Dimerization; Enzyme Inhibitors; Genetic Vectors; HEK293 Cells; Humans; Indoles; Mice; Mice, Inbred BALB C; Mutation; Neoplasm Transplantation; NIH 3T3 Cells; Oncogene Proteins, Fusion; Phenotype; Protein Interaction Mapping; Protein Structure, Tertiary; Proto-Oncogene Proteins B-raf; Signal Transduction; Sulfonamides; Vemurafenib

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.

Topics: Animals; Apoptosis; Astrocytoma; Blotting, Western; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Child; Cyclin-Dependent Kinase Inhibitor p16; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Immunohistochemistry; Indoles; Mice; Mice, Inbred BALB C; Mice, Knockout; Mice, Nude; Mice, SCID; Neural Stem Cells; Phosphorylation; Piperazines; Proto-Oncogene Proteins B-raf; Pyridines; Sulfonamides; 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