CDKN2A gene fusions in glioblastoma: validation and functional characterization

Glioblastoma, IDH-wildtype (GB), is the most aggressive primary brain tumor in adults, characterized by poor prognosis, elevated intratumoral heterogeneity, and limited therapeutic options. Despite extensive genomic profiling efforts, only a small number of GB patients currently benefit from precision oncology approaches. In this thesis, we investigated the landscape and functional relevance of gene fusions in GB, with a particular focus on CDKN2A-involving rearrangements as potential therapeutic target. Using RNA sequencing of patient-derived GB tissues and low-passage primary cell lines, we identified and molecularly validated two previously uncharacterized fusions, CDKN2A–CDKN2B and CDKN2A–IZUMO3. Functional assays revealed that these fusion-positive GB models exhibit enhanced sensitivity to the CDK4/6 inhibitor palbociclib, outperforming temozolomide (TMZ) in both 2D and 3D models. Further analyses showed that fusion-positive cells maintain p16/INK4A expression and undergo treatment-induced transcriptional reprogramming marked by stemness/EMT-associated markers (e.g., CD44, SNAI2). Moreover, co-culture models demonstrated that palbociclib selectively modulates the tumor–microglia axis in fusion-positive contexts, inducing TGFβ expression and increased microglial migration, suggestive of an early immunosuppressive M2-like shift. Jointly, these findings place CDKN2A-involving fusions as clinically actionable biomarkers with both cell-intrinsic and microenvironmental impact. Although future directions include assessing fusion prevalence in larger patient cohorts and validating therapeutic responses in orthotopic PDX systems, these findings demonstrate the feasibility of integrating fusion screening into routine GB molecular profiling, to enable more precise patient stratification and guide the rational application of targeted therapies.