Abstract 2858: Phenotypic screens identify genetic regulators of nanoparticle delivery to pediatric brain tumors

Abstract Pediatric central nervous system tumors are the leading cause of cancer death in children. Diffuse midline glioma (DMG) is a universally fatal pediatric brain tumor and despite many clinical trials over the past decades, less than 10% of patients are long-term survivors. The clinical standard of care is radiotherapy, however it only provides limited symptom relief. There is a clear need to develop creative treatment strategies for patients with DMG. Nanomedicine has the potential to address this challenge, allowing the encapsulation of difficult to deliver therapeutic cargoes, and selectively targeting them to tumors while crossing innate biological barriers. However, a deep understanding of disease-specific nano-bio interactions is crucial to leverage the full potential of these therapies. To functionally validate the role of candidate biomarkers in nanoparticle delivery to DMGs, we performed pooled genetic perturbation screens using a flow based phenotypic output. We developed a lentiviral all-in-one CRISPR/Cas9 knock-out library paired with fluorescence-activated cell sorting to probe the functional impact of 700 genes on the delivery of nanoparticles to DMG. Layer-by-layer electrostatic assembly was used to generate liposomal nanoparticles with a polymer surface coating of hyaluronic acid. Two patient-derived neurosphere models of DMG were transduced with the pooled all-in-one CRISPR/Cas9 library. Following antibiotic selection, cells were incubated with hyaluronic acid or polymeric nanoparticles for 4 or 24 hours and sorted into populations based on nanoparticle affinity. A dynamic cell-sorting strategy was optimized to separate cells infected with inert DNA barcodes based on their degree of nanoparticle-association. DNA was isolated from sorted cells and subjected to next generation sequencing. The enrichment of each single guide was determined relative to matched controls. We found strong agreement between biologic replicates, and determined which genes, when knocked down, shifted nanoparticle uptake into DMG cells. Our pooled phenotypic screen revealed strong biologic regulators of nanoparticle uptake. These can be categorized as formulation dependent and independent, as well as time-dependent. We identified several negative regulators of nanoparticle uptake including members of the mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) pathways. The top target genes are currently being validated in DMG and human neural stem cell models. In ongoing work, we are leveraging genetic and chemical inhibition to sensitize DMG cells to nanoparticle therapeutics. We plan to leverage our screening technique to develop novel combination therapy approaches that could be implemented across multiple administration routes (intravenous, intraventricular, or intratumoral) to enhance the use of nanomedicine for DMG and other pediatric brain tumors. Citation Format: Marissa Coppola, Julianna Kenny-Serrano, John G. Doench, Paula Hammond, Pratiti Bandopadhayay, Jessica W. Tsai, Joelle Straehla. Phenotypic screens identify genetic regulators of nanoparticle delivery to pediatric brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2858.