TMET-01 - Therapeutic targeting of crosstalk between mitochondrial fitness and cell adhesion/migration in diffuse midline glioma (DMG)

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. To study which genes are driving this invasive phenotype, we established a novel two-step pooled whole genome CRISPR-Migration screen for DMG primary cell cultures (n=3) derived from patients. After selecting for genes critical for cell survival, each cell line was seeded in the upper chamber of a transwell assay optimized for DMG cells to promote complete serum-free cell attachment and migration through laminin, one of the major components of the blood vessels basement membrane. Following sgRNA library sequencing of low and high-migratory populations, we found a strong positive correlation with genes directly involved in the focal adhesion machinery (ITGB1, CRKL, RAPGEF1, PARVA, PTK2, FERMT2). Stable CRISPR knockout of these genes validated significant reduction in adhesion/migration, without affecting proliferation. Interestingly, we also identified in the screen genes controlling different aspects of mitochondrial function (NDUFAF6, PITRM1, MINOS1, MICU3, LIPT1, MRPL38), especially affecting outer-inner mitochondrial membrane integrity. This finding is in line with the higher sensitivity of multiple DMG cell cultures to Bcl-2 family inhibitors, readily prompting apoptosis in combination with sub-lethal doses of various drugs. Agents that specifically modulate mitochondrial oxidative phosphorylation (OXPHOS) (Rotenone, Oligomycin A, Antimycin A) reduced DMG cell migration without affecting proliferation. Surprisingly, silencing of ITGB1 and CRKL led to a marked decrease in glycolysis and OXPHOS levels, again without any changes in proliferation. Additionally, silencing of ITGB1 (primarily responsible for cell adhesion to laminin) led to the decreased expression (RNA) of multiple mitochondrial NADH dehydrogenases, pointing to an intricate bi-directional mechanism connecting mitochondrial fitness to DMG cell migration. Studies are underway to validate these findings in vivo and to determine an optimal therapeutic disruption of this critical DMG malignant phenotype.