Despite advancement in treatment for childhood central nervous system (CNS) tumors, they remain the leading cause of death in pediatric oncology. One potential therapeutic intervention is targeting the autophagic pathway, a complex catabolic process that contributes to tumor cell survival. Recent data has shown BRAFV600E mutations in a range of these tumors and my own research finds that these tumors show autophagy-dependence not seen in BRAFWT tumors. There is also evidence that autophagy inhibition is a potential mechanism in preventing or reversing resistance to direct inhibition against activated BRAFV600E. The proposed study will examine the interaction of the BRAFV600E mutation and autophagy in brain tumors. I hypothesize that BRAFV600E identifies tumors that will respond to combination therapy with autophagy inhibition with enhanced tumor cell death, establishing a basis for future rational clinical trial design for pediatric brain tumo patients harboring the mutation. Aim 1 will determine how the BRAFV600E mutation results in sensitization of CNS tumor cells to autophagy inhibition by evaluating ERK activity using isogenic BRAF models. Aim 2 will evaluate the effectiveness of autophagy inhibition to improve chemosensitivity in BRAF mutant CNS tumors ex vivo and in vivo. Combination therapy with chloroquine (autophagy inhibition) and vemurafenib (Raf-inhibitor) will be assessed in orthotopic murine models as well as organotypic slice cultures. Aim 3 will evaluate the effectiveness of autophagy inhibition as a therapeutic strategy to overcome acquired small molecule inhibition of oncogenic BRAF in pediatric brain tumors and the role of PUMA and apoptosis in mediating these effects. A combination of in vitro, ex vivo and in vivo methods will be utilized to achieve the stated Aims and clarify the role of BRAFV600E and autophagy in pediatric CNS tumors. Autophagy will be studied using the latest methods including flow cytometry and fluorescent microscopy. Detailed analysis of the MAP kinase pathways will be included. Isogenic cell lines will be used to remove other confounding genetic interactions. Intracranial mouse tumor models and organotypic slice cultures will be used to validate in vitro findings. In vivo studies have bee designed to simulate clinical treatment as best as possible to provided pre-clinical data for clinical trial proposals. Understanding these differences is vital to identifying patients who will most likely benefit from autophagy inhibition as an added therapy, allowing patients who will not benefit to focus their treatment efforts on more potentially beneficial efforts. Data obtained from these studies will be influential in the design of future clinical trials specifically devised to optimize the manipulation of autophagy with the intent of improving the care and survival of pediatric brain tumor patients.

K08CA193982

2015-09-23

2020-08-31