Abstract 1416: Defining the mechanisms of cancer specific transcription factor, tGLI1

Abstract Glioblastoma (GBM) is the most common malignant central nervous system tumor in adults with a five-year survival rate of less than 7%. First identified in GBM, truncated GLI1 (tGLI1), an alternative splicing isoform of GLI1, has emerged as a cancer specific GLI1 variant as tGLI1 is highly expressed in both GBM cell lines and primary specimens but not in normal tissue. Despite an in-frame loss of 41 amino acids, the tGLI1 protein has retained all known GLI1 functional domains and appears to respond to sonic hedgehog in a similar manner as GLI1. However, tGLI1 operates as a gain of function transcription factor with the ability to bind to and activate genes unique from GLI1 to promote invasion, migration, angiogenesis, and stemness in both GBM and breast cancer. Despite the advancements in our understanding of tGLI1, a genome wide DNA binding pattern of tGLI1 has not been established and the mechanism by which tGLI1 gains access to these genes is unknown. To establish a more robust understanding of the differential DNA binding patterns of GLI1 and tGLI1, we carried out ChIP-sequencing (ChIP-seq) and found only 14% of GLI1 and tGLI1 binding sites are shared. These results suggest GLI1 and tGLI1 have even more unique binding pattens across the entire genome that previously thought. However, the underlying mechanism for why GLI1 and tGLI1 bind different regions of the genome remains elusive. To determine whether variations in protein interactions of GLI1 and tGLI1 contribute to the unique binding pattern of tGLI1, we carried out immunoprecipitation (IP) followed by mass spectrometry (mass spec). Results identified 45 GLI1 associated proteins and 52 tGLI1 associated proteins with only a 29% overlap between the two. Furthermore, gene ontology analysis revealed 43% of the tGLI1-specific associated proteins are involved in processes related to RNA binding, processing, and metabolism. From these RNA-related proteins, we validated an interaction between tGLI1 and the protein NONO. NONO, which can bind both RNA and DNA, is involved in a range of molecular processes including paraspeckle formation and transcriptional regulation. In GBM, high NONO expression correlates with poor patient outcomes and the mesenchymal subtype. Interestingly, we observed a greater interaction between NONO and tGLI1 as compared to GLI1 and that co-expression of NONO and tGLI1 increased the nuclear localization of both proteins. Overall, these findings enhance our understanding of tGLI1’s distinct role in the cell and suggest a potential mechanism underlying its contribution to GBM. Future research will focus on further elucidating the NONO-tGLI1 interaction and investigating the role of NONO in tGLI1-driven phenotypes and GBM progression. Citation Format: Haddie DeHart, Nolan Gregg, Julie Heldman, Richard Carpenter. Defining the mechanisms of cancer specific transcription factor, tGLI1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1416.