LGG-16. NOVEL COMBINATION THERAPIES FOR PEDIATRIC LOW-GRADE GLIOMA AND CHARACTERIZATION OF THE UNDERLYING CELL DEATH MECHANISMS

Abstract

Abstract BACKGROUND The majority of pediatric low-grade gliomas (pLGGs) harbor a MAPK/ERK pathway overactivating alteration, making them suitable for MAPK-targeted therapies such as MEK inhibition. However, MEK inhibitor treatment alone is in most cases not sufficient to induce complete response, and a rapid rebound of the tumor is the consequence after withdrawal of the therapy. METHODS To identify novel cytotoxic drug combinations, we screened 89 clinically relevant drugs in combination with the MEK inhibitors trametinib and binimetinib. We assessed the regression of cultured 3D microtumors of patient-derived BT40 and BT314 cells, both harboring a BRAFV600E mutation. Depending on the inducible expression of SV40 large T antigen, the BT314 model can reflect two states, proliferation and oncogene-induced senescence. The area of spheroids was measured both in brightfield and with the fluorescent dye TMRE (mitochondrial polarization) at the start and the end of the treatment (6 days). Moreover, metabolic activity was determined through bulk ATP measurement. Drug hit combinations were further analyzed for the successful induction of cell death using high-content microscopy (HCM) imaging and enzymatic caspase activity assays. The microscopic readouts included Hoechst 33342 for the detection of nuclear morphology, BODIPY for sensitizing lipid peroxidation and CM-H2DCFDA as a general oxidative stress indicator. The images were analyzed with the automated image analysis program CellProfiler and the machine learning application CellProfiler Analyst. RESULTS The screening identified the following drug hits: navitoclax (BCL-2 family inhibitor), BRAF inhibitor dabrafenib, mTOR inhibitor everolimus and selinexor, a selective inhibitor of nuclear export. All drugs increased apoptotic cell death and ROS levels at clinically achievable drug concentrations, except the mTOR inhibitors. These results are currently validated in vivo with BT40 zebrafish embryo xenograft models. CONCLUSION Combinations of MEK inhibitors with navitoclax, dabrafenib or selinexor resulted in sufficient apoptosis to induce regression of pLGG microtumors in vitro.