DDDR-29. A Novel BBB-Permeable tGLI1 Inhibitor Overcomes CDK4/6 Inhibitor Resistance in Glioblastoma

Glioblastoma (GBM) remains the most aggressive primary brain tumor, with treatment resistance largely driven by glioma stem cells (GSCs). Although CDK4/6 inhibitors (CDK4/6i) demonstrate therapeutic potential, their efficacy is hindered by resistance mediated by a transcription factor named truncated glioma-associated oncogene homolog 1 (tGLI1). Ketoconazole (KCZ) inhibits tGLI1 but has limited clinical utility due to potent CYP3A4 inhibition and associated toxicities. To address this, we developed WF-229A, a novel blood-brain barrier (BBB)-penetrant tGLI1 inhibitor without CYP3A4 inhibition. We evaluated WF-229A’s efficacy and safety in overcoming CDK4/6i resistance in GBM. Treatment of tGLI1-overexpressing GBM cells with WF-229A significantly restored sensitivity to CDK4/6i, resulting in synergistic apoptosis (2.3-fold increase, p<0.01), reduced neurosphere formation (68% decrease, p<0.001), diminished invasion and migration (50% and 40% decreases, respectively; p<0.001 and p<0.01), and enhanced cell rounding (form factor increase: 0.32 to 0.70, p<0.001). WF-229A demonstrated minimal CYP3A4 inhibition (IC50>100,000 nM) compared to KCZ (IC50=15.72 nM), with in vitro safety confirmed by negligible toxicity on astrocytes and hepatocytes (IC50>1,000 nM). Maximal tolerated dose (MTD) studies revealed WF-229A exhibited no significant toxicity at doses up to 300 mg/kg, while KCZ induced notable elevations in serum ALT, ACTH, and lipase at lower doses (100 mg/kg, p<0.01). These findings demonstrate that WF-229A effectively and safely targets tGLI1, re-sensitizing GBM cells to CDK4/6 inhibitors, and reducing stemness and invasiveness without the CYP3A4-associated toxicities observed with KCZ. Thus, WF-229A represents a promising therapeutic approach poised for clinical translation in combination therapies to enhance CDK4/6i treatment efficacy in GBM patients.