Unmasking the NF-YC splicing program that fuels glioblastoma aggressiveness and challenges therapeutic response

Background Glioblastoma (GBM), the most aggressive primary brain tumor, poses significant challenges due to its rapid progression, limited treatment options, and median survival not exceeding 18 months. This devastating prognosis reflects a critical need to uncover novel molecular mechanisms driving GBM progression and identify novel key targets for more effective therapies. Recent studies highlight that Nuclear Factor-Y Subunit C (NF-YC), a core part of the trimeric NF-Y transcription factor, could serve as a prognostic biomarker and oncogene, promoting GBM aggressiveness by regulating pathways involved in cell proliferation, adhesion, and migration. NF-YC undergoes alternative splicing, generating distinct isoforms (37, 39, and 50 kDa), whose functional roles in GBM remain unexplored. Hypothesis Our preliminary data show differential expression of NF-YC isoforms in GBM patients and cell lines, correlating with distinct phenotypes. We hypothesize that isoform-specific expression and function of NF-YC are key drivers of GBM progression, influencing tumor aggressiveness and patient prognosis. Targeting specific NF-YC isoforms may offer new therapeutic opportunities to impair tumor progression and increase therapy sensitivity. Aims This study aims to: - Characterize NF-YC isoforms expression across glioma and GBM patient samples, correlating these patterns with clinical parameters such as tumor grade, subtypes, patient survival, and treatment response. - Dissect phenotypic and molecular functions of each NF-YC isoform in GBMs using gain- and loss-of-function strategies. - Assess in vivo oncogenic potential and therapeutic relevance of NF-YC isoforms in zebrafish and mouse models of GBM. Experimental Design In Aim 1, we will leverage large-scale RNA sequencing datasets from GBM patients to quantify NF-YC isoform expression and correlate these with survival, tumor grade, subtype, treatment response, and tumor microenvironment interactions. We will also examine the co-expression patterns of NF-YC with splice variants of NF-YA - another critical NF-Y subunit previously implicated in tumor progression across multiple cancer types - to uncover a potential coordinated splicing program linked to GBM aggressiveness. In Aim 2, we will stably overexpress (via lentiviral transduction) or selectively silence (via RNAi and CRISPRCas9) NF-YC isoforms in GBM cell lines. Phenotypic features such as proliferation, migration, invasion, therapy resistance, and interaction with tumor-associated macrophages will be evaluated. Functional assays in both 2D and 3D models will be coupled with DNA-binding (ChiP-seq) and transcriptomic (RNA-seq) profiling to identify isoform-specific downstream targets and pathways. In Aim 3, we will validate the in vivo tumorigenicity of GBM cells expressing individual NF-YC isoforms using zebrafish xenografts and orthotopic mouse models. Tumor growth, invasion, and therapeutic responsiveness will be monitored, laying the groundwork for isoform-specific RNA-targeted strategies. Expected Results We expect to define distinct NF-YC isoform expression signatures associated with poor prognosis and tumor aggressiveness. Functional studies will likely reveal divergent roles for each isoform, with at least one acting as a key oncogenic driver in GBM. In vivo validation will confirm their relevance as potential therapeutic targets. Impact On Cancer This study will elucidate the biological significance of NF-YC splicing in GBM and establish its isoforms as promising precision oncology targets. Results may support future development of personalized treatment strategies based on molecular patient stratification and the identification of novel therapeutic targets.