Single-nucleus transcriptomics, pharmacokinetics, and pharmacodynamics of CDK4/6 and mTOR inhibition in a Phase 0/1 trial of recurrent high-grade glioma

Background: Outcomes for adult patients with high-grade glioma (HGG) remain poor, necessitating new treatment strategies. Key challenges include poor drug penetration in the brain and malignant cell state plasticity. Phase 0 studies identify agents that achieve target modulation through pharmacologically relevant brain concentrations. Methods: A Phase 0/1 clinical trial combined the 2 targeted inhibitors ribociclib (CDK4/6 inhibitor) and everolimus (mTOR inhibitor) in recurrent HGG patients, aiming to identify brain-penetrant combinations and assess their impact on malignant cell states. We enrolled 24 patients with recurrent HGG, characterized by CDKN2A/B deletion or CDK4/6 amplification, PTEN loss or PIK3CA mutations, and wildtype retinoblastoma protein (Rb). Tumors were evaluated for pharmacokinetics, pharmacodynamics, and single nucleus transcriptomics. Results: Median unbound ribociclib concentrations in gadolinium non-enhancing tumor regions were significantly above the biochemical IC50 for CDK4/6 inhibition at 400 and 600 mg QD doses. Unbound everolimus concentrations were undetectable (<0.1 nM) in tumor regions across all dose levels. Ribociclib treatment was associated with significantly decreased Ki-67-positive cells. Single-nucleus RNA sequencing of 17 on-trial IDH-wildtype recurrences and 88 standard-of-care-treated recurrences showed a significantly lower fraction of cycling and neural progenitor-like malignant cell populations in ribociclib-everolimus-treated tumors. CDK4/6 inhibitor-directed malignant cell state shifts were validated using 3 patient-derived cell lines. Conclusions: This trial underscores the value of integrating pharmacokinetics, pharmacodynamics, and single-nucleus transcriptomics in Phase 0/1 surgical studies to assess treatment effects, including malignant cell state shifts. Clini-calTrials.gov identifier: NCT03834740.