ID #720 Macropinocytosis as a Target in Pediatric High-Grade Glioma: Insights from a CRISPR-Cas9 Metabolic Library Screen
Thien Nguyen, Sanjay Palta-Hill, Kaitlyn Spees, Cesar Garcia, Fudhail Sayed, Sophia Arana, Peter Du, Roni Konigsberg, Alun Vaughan-Jackson, Ella Nettnin, Michael Bassik, Laura ProloAbstract
Background
Childhood brain tumors are the leading cause of cancer-related deaths in children, particularly pediatric high-grade gliomas (pHGGs) given limited treatment options. pHGGs exhibit distinct biological and clinical features from adult gliomas, rendering conventional adult therapies ineffective. To evaluate additional novel vulnerability in pHGGs, we investigate a novel pathway macropinocytosis, a nonselective bulk endocytosis process that allows cells to internalize extracellular material and contributes to tumor growth and proliferation. The aims included: (1) Assessing ability of pHGGs undergo macropinocytosis, (2) Identifying the genetic drivers of macropinocytosis through a dedicated CRISPR screen to develop targeted therapies.
Methods
Hemispheric and midline patient-derived pHGGs (SF188, DIPG04, STN049, and STN128) were assessed for macropinocytosis, using a large 70kDa dextran-conjugated fluorophore. Intracellular localization of macropinosomes were quantified using immunocytochemistry. To determine underlying molecular contributors of macropinoctyosis, a CRISPR-Cas9 screen was done in pHGG with a library containing cellular uptake genes, including macropinocytosis related genes, and downstream lysosomal processing genes. Knock down cells were stained with the macropinocytosis assay for 1 hour and then flow-sorted to top versus bottom 10% of uptake. Sequencing analysis was done through our high-throughput sequencing pipeline. Individual candidates from the screen were generated using Cas9 and validated using the macropinocytosis assay.
Results
ICC staining demonstrates macropinocytosis uptake in the commercially available SF188 and 3 primary patient-derived cell lines. From our CRIPSR-knock down screen, we identified 3 significant drivers underlying macropinocytosis: SNAP23, PELP1, and AMBRA1. While knock down of AMBRA1 and SNAP23, decreased macropinocytosis, knock down of PELP1 increased the phenotype. Knock down cell lines of each of these genes recapitulates the expected macropinocytosis phenotype (P-value < 0.05), compared to safe-guide knock outs. Additionally, incubation with a PELP1 inhibitor, increases macropinocytosis uptake compared to DMSO controls (P= .004; 95%-Confidence interval: [9.7%,25.7%]; R2= 0.91).
Conclusions
PELP1, SNAP23 and AMBRA1 are drivers of macropinocytosis and targeting these genes may allow for potential metabolic vulnerability in pHGGs.