Preclinical Evaluation of the Efficacy of a Cyclin-dependent Kinase Inhibitor Ribociclib in Combination with Letrozole Against Patient-Derived Glioblastoma Cells

Ongoing studies suggest that letrozole (LTZ), a drug used in the treatment of breast cancer, can potentially be repurposed as a novel therapeutic for Glioblastoma (GBM). In a phase 0/1 trial in recurrent GBM patients we observed that LTZ permeates into the GBM tissue and triggers dose-dependent changes in the expression of genes regulating cell cycle (e.g. CDKN2A/N2B, CDK4). Based on these observations, we hypothesized that a combination of cyclin dependent kinase (CDK) 4/6 inhibitors with LTZ may result in synergistic anti-GBM activity. Therefore, we assessed the anti-tumor effects of LTZ in combination with ribociclib, a third-generation CDK4/6 inhibitor and the brain pharmacokinetics of ribociclib. Using cell viability and neurosphere growth assay against a panel of patient-derived GBM lines both compounds were found to be cytotoxic when used as single agents and were strongly synergistic when used in combination. We then assessed the DNA damaging effects (ɣH2AX induction), cell cycle arrest and the induction of apoptosis (Annexin V-FITC/PI) of both compounds as single agents and when used in combination. LTZ potentiated ribociclib-induced DNA damage and cell cycle arrest leading to apoptosis. Systemic and brain pharmacokinetics analysis of ribociclib in Sprague-Dawley (SD) rats by serial blood and brain extracellular fluid (ECF) sampling showed that ribociclib penetrates the blood-brain barrier with a partitioning coefficient (Kpu, u, brain) of about 10%. Overall, our studies suggest that a combination of ribociclib with LTZ is likely to be strongly synergistic against GBM at concentrations of the drugs that can be achieved in the brain RNAseq analysis of tumor samples from recurrent GBM patients treated with LTZ in a phase 0/1 clinical study (NCT03122197). Formalin-Fixed, Paraffin-Embedded (FFPE) tumor samples LTZ dose cohorts (n = 3 each) in the 2.5 mg (Group 1), 5 mg (Group 2), 10 mg (Group 3) and in the 12.5 mg (Group 4), and (n = 6) in the 15 mg (Group 5) were processed for mRNA isolation via depletion RNA purification.