tGLI1 is a Novel Targetable Transcription Factor that Promotes Resistance to CDK4/6 Inhibitors and Chemoradiation in Glioblastoma

Introduction: Glioblastoma (GBM) is an aggressive, treatment-resistant brain tumor. CDK4/6 inhibitors (CDK4/6i) show potential as targeted therapies, but clinical trials have been unsatisfactory, possibly due to resistant glioma stem cells(GSCs). tGLI1 is a transcription factor that promotes GSCs and mesenchymal GBM transition. Here, we investigated the role of tGLI1 in conferring CDK4/6i and chemoradiation resistance in GBM and evaluated the efficacy of combination therapies in overcoming treatment resistance by co-targeting tGLI1 and CDK4/6.

Methods: tGLI1-overexpressing GBM cell lines were used for in vitro and in vivo experiments, including cell viability assays, neurosphere formation, immunohistochemistry, Western blots, and flow cytometry. Two orthotopic GBM xenograft models in mice were used to assess treatment response.

Results: Genomic analysis from the TCGA(n=642) and patient samples(n=314) revealed CDK4/6 pathway alterations in 70% of tumors, correlating with reduced overall survival (TCGA:13.76 vs. 17.18 months,p < 0.001) and progression-free survival (TCGA:7.1 vs. 9.56 months, p=0.008). Immunohistochemistry confirmed elevated co-expression of tGLI1 and CDK4/CDK6 proteins in patient tumors (p < 0.001). tGLI1-overexpressing GBMs in vitro exhibited enhanced resistance to CDK4/6-inhibitors, temozolomide(TMZ) and ionizing radiation(IR) in neurosphere formation and cell death assays. Ketoconazole(KCZ), a tGLI1-specific inhibitor, resensitized these cells to CDK4/6i, TMZ and IR (p < 0.01). Moreover, the KCZ-CDK4/6i combination demonstrated synergistic effects: significantly increased cell death, apoptosis and significantly inhibited stemness in tGLI1-overexpressing GBMs. In vivo orthotopic GBM xenografts confirmed the superior efficacy of KCZ-CDK4/6i combination therapy than either monotherapy: tumors were 2.5 and 3-times smaller than those receiving monotherapy (p=0.03,KCZ-CDK4/6i combination versus KCZ only or CDK4/6i only).

Conclusion : We identified tGLI1 as a novel targetable regulator of CDK4/6i, TMZ and IR resistance in GBM. Moreover, we demonstrated the efficacy of tGLI1-CDK4/6 targeted combination therapy in GBM in overcoming CDK4/6i resistance. These results highlight the potential of co-targeting tGLI1 with CDK4/6 inhibitors in the clinic to overcome GBM treatment resistance and maximize therapeutic potential.