Abstract
Diffuse midline gliomas (DMGs) are aggressive tumors characterized by infiltration into normal midline brain tissue, hindering surgical resection and contributing to overall morbidity and mortality. We established a novel two-step pooled whole genome CRISPR-Migration screen by using serum-free conditions and laminin-dependent cell attachment, mimicking cell migration on brain vasculature scaffolds, a common feature in glioblastoma (GBM). Comparing DMG and GBM primary cell lines (n=5), genes involved in focal adhesion (ITGB1, CRKL, PARVA, PTK2, FERMT2) were identified as significantly restricting migration in all cell lines. Multi-institutional transcriptomic data revealed an association of expression of these focal adhesion genes with survival primarily in K27M-DMG and pediatric high-grade glioma compared to lower-grade brain tumors, and ITGB1 expression increased with glioma grade. Single-cell RNA-seq and spatial transcriptomic data demonstrated higher expression of ITGB1 in OPC-like/Mesenchymal cells associated with leading edge and microvasculature in both K27M-DMG and GBM tumors. Unexpectedly, knockout of ITGB1 (ITGB1-KO), primarily responsible for cell adhesion to laminin, led to the decreased expression of multiple mitochondrial NADH dehydrogenase genes, and reduced levels of glycolysis and oxidative phosphorylation (OXPHOS) activity. Preliminary mechanistic data linked this phenotype to impaired mitochondrial fusion. Orthotopic implantation of control and ITGB1-KO models of H3K27M-DMG and adult GBM in mouse brains revealed two distinct phenotypes: (1) ITGB1-KO reducing proliferation and significantly increasing survival; and (2) ITGB1-KO leading to upregulation of ITGB3 and ITGB5 in vivo without impacting survival. Cilengitide (αvβ3 and αvβ5 inhibitor) ablated migration of all ITGB1-KO models on vitronectin (secreted by pericytes surrounding blood vessels) in vitro. Ongoing combinatorial therapy with an experimental anti-Integrin Beta 1 antibody (AIIB2, CNS delivered) and Cilengitide is underway with promising early results. In summary, multi-integrin inhibition provides a feasible avenue to curb glioma spread in the brain through impaired focal adhesion as well as blunting mitochondrial glucose metabolism.