DISRUPTING INTEGRIN SIGNALING IMPAIRS MIGRATION, MYC-DRIVEN PURINE BIOSYNTHESIS, AND STEMNESS IN H3K27M DIFFUSE MIDLINE GLIOMAS

Diffuse midline glioma (DMG) is an aggressive pediatric brain tumor driven by the H3K27M histone mutation and represents the leading cause of cancer-related death in children. These tumors are highly infiltrative and can occasionally migrate to distant CNS regions. To uncover migration dependencies, we developed a novel two-step pooled whole-genome CRISPR-migration screen in metastatic H3K27M-DMG stem cells (n=3). Genes involved in focal adhesion (ITGB1 [integrin beta-1], CRKL, PARVA, PTK2, FERMT2) significantly restricted migration across all models; notably, only ITGB1 knockout (ITGB1-KO) completely abrogated migration. In H3K27M-DMG patient samples, unlike other brain tumor types, expression of ITGB1 correlates with higher glioma grade and worse survival. ITGB1-KO models demonstrated a reduction in expression of MYC target genes, including MYC-regulated metabolic genes involved in purine biosynthesis (e.g., IMPDH2). Further integrated RNA/metabolomic analyses revealed that loss of ITGB1 downregulates purine metabolism and the citric acid (TCA) cycle. Importantly, in in vivo models, ITGB1 deficiency significantly prolonged survival (UMPED83: 100 vs. 163.5 days, p=0.0003; pSCG-SVZ: 49 vs. 68 days, p=0.0095). Spatial transcriptomic and proteomic analyses of ITGB1-KO orthotopic H3K27M tumors showed widespread reduction in MYC target genes and depletion of precursor, undifferentiated (OPC-like), and an increase in differentiated (OC-like) K27M cells in the infiltrating edge. Direct pharmacological targeting of ITGB1 (anti-ITGB1 antibody, CNS delivered) significantly extended survival in DMG models (UMPED83: 100 vs. 125.5 days, p=0.0169; pSCG-SVZ: 49 vs. 72 days, p=0.0384). However, ITGB1-deficient pSCG-SVZ tumors exhibited compensatory alternative integrins upregulation (i.e., ITGB3, ITGB5). Promisingly, co-treatment with anti-ITGB1 antibody and cilengitide (ITGB3/5 inhibitor) further improved survival and resulted in 75% long-term survivors, free of disease. Strikingly, this combinatorial strategy failed to confer any survival benefit in adult glioblastoma (H3WT) models. Overall, these findings highlight integrin targeting as a promising therapeutic avenue in H3K27M-DMG, capable of disrupting tumor-specific migration, MYC-driven purine biosynthesis, and stemness programs.