TMOD-29. BETA INTEGRINS REGULATE MITOCHONDRIALLY-MEDIATED CELL MIGRATION IN H3K27M-DIFFUSE MIDLINE GLIOMA

Abstract Infiltration into the brain tissue is one of the main features of diffuse midline gliomas (DMG), also characterized by the histone mutation H3K27M and overall survival of 12-15 months from diagnosis. To study which genes are critical for H3K27M-DMG tumor cell migration in the brain, we established a novel two-step pooled whole genome CRISPR-Migration screen of metastatic H3K27M-DMG (n=3) and glioblastoma (GBM, n=2) stem cells by using serum-free conditions and laminin-dependent cell attachment, mimicking cell migration on brain vasculature scaffolds. Genes involved in focal adhesion (ITGB1, CRKL, PARVA, PTK2, FERMT2) were identified as significantly restricting migration in all H3K27M-DMG lines. From those, only knockout of ITGB1 (ITGB1-KO) fully ablated in vitro migration in all models. In human tumor RNAseq, expression of ITGB1 correlated with higher glioma grade and worse survival specifically in H3K27M-DMG across pediatric brain tumors. In H3K27M-DMG scRNAseq, increased ITGB1 expression was found in cells with OPC-like/Mesenchymal cell signature associated with leading edge and microvasculature. Unexpectedly, ITGB1-KO 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 increased mitochondrial fusion. Genetic (ITGB1-KO) and pharmacologic (anti-ITGB1 antibody AIIB2, CNS delivered) targeting of ITGB1 in a novel orthotopic model of metastatic H3K27M-DMG (UMPED83) led to reduced proliferation and significant extension of survival (p=0.0003 ITGB1-KO, and p=0.0006 AIIB2). Interestingly, ITGB1-KO in another orthotopic H3K27M-DMG model (SVZ) led to upregulation of other integrins (ITGB3 and ITGB5) without change in survival, resulting in susceptibility to treatment with the ITGB3/5 inhibitor Cilengitide (p=0.006). Treatment with AIIB2 and Cilengitide alone or in combination was ineffective in an H3WT adult GBM orthotopic model suggesting unique H3K27M-DMG targetability. In summary, targeting of integrin pathways offers untapped therapeutic opportunities in H3K27M-DMG by impairing tumor cell survival through altered mitochondrial dynamics.