CDKN2A/B deletion activates c-MET dependent pro-angiogenic signaling in IDH-mutant astrocytoma

Diffuse gliomas are the most common primary malignant brain tumors and remain incurable, with standard therapy for high-grade disease unchanged for over two decades. The 2016 and 2021 WHO classifications distinguish IDH-mutant astrocytoma from IDH-wildtype glioblastoma and designate CDKN2A/B homozygous deletion (HD) as a criterion for grade 4 IDH-mutant astrocytoma irrespective of histopathologic features, underscoring the need for new therapeutic strategies tailored to molecularly defined subgroups. To identify pathways underlying molecular grade 4 behavior in IDH-mutant astrocytoma, IDH-mutant astrocytoma from The Cancer Genome Atlas (TCGA) were stratified by CDKN2A/B status for pathway enrichment analysis. In a separate cohort of 40 WHO grade 4 IDH-mutant astrocytomas, tumors were stratified by CDKN2A/B status and vascular density was quantified using available histopathologic sections. To model the functional consequences of CDKN2A/B -HD, molecular grade 4 IDH-mutant astrocytoma cell lines were engineered for Tet-inducible re-expression of CDKN2A/B -encoded proteins p14 ARF , p15 INK4B , and p16 INK4A . RNA sequencing, angiogenic factor secretion and signaling assays, endothelial migration and sprouting assays, and in vivo vascular recruitment models were used to characterize angiogenic behavior. Candidate mediators of the CDKN2A/B -HD-associated angiogenic phenotype were investigated using siRNA and pharmacological inhibition, alone and in combination with VEGF inhibition. TCGA analysis identified enrichment of angiogenesis pathways in CDKN2A/B -HD tumors and patient tumors harboring CDKN2A/B -HD demonstrated increased vascular formation. The in vitro IDH-mutant astrocytoma model recapitulated patient tumors in pathway enrichment analysis via RNA sequencing and in functional models of angiogenic behavior. Re-expression of CDKN2A/B gene products reduced VEGF and bFGF secretion, while conditioned media (CM) from re-expression cells impaired endothelial angiogenic signaling, migration, and sprouting in vitro and vascular recruitment in vivo compared with CM from CDKN2A/B -HD cells. We identified elevated c-MET expression and activity in CDKN2A/B -HD tumors and cell lines as a potential mediator of this phenotype. Inhibition of c-MET via siRNA or pharmacological inhibition reduced VEGF secretion in IDH-mutant astrocytoma cells. CM from IDH-mutant astrocytoma cells following c-MET inhibition, either alone or combined with VEGF inhibition, more effectively suppressed endothelial angiogenic function than CM from cells treated with inhibition of VEGF alone. These findings identify c-MET as a novel therapeutic target in molecularly defined IDH-mutant astrocytoma with CDKN2A/B -HD and suggest that c-MET inhibition may exert anti-angiogenic effects in this aggressive tumor subtype.