Preclinical Evaluation of the Efficacy of the Cyclin-Dependent Kinase Inhibitor Ribociclib in Combination with Letrozole against Patient-Derived Glioblastoma Cells

Abstract

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/I trial in patients with recurrent GBM, we observed that LTZ permeates into the GBM tissue and triggers dose-dependent changes in the expression of genes regulating the cell cycle [e.g., cyclin-dependent kinase (CDK) inhibitor 2A/2B, CDK4]. Based on these observations, we hypothesized that a combination of CDK4/6 inhibitors and LTZ may result in synergistic anti-GBM activity. Therefore, we assessed the antitumor effects of LTZ in combination with ribociclib, a third-generation CDK4/6inhibitor, and the brain pharmacokinetics of ribociclib. Using cell viability and neurosphere growth assays 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 (γH2AX induction), cell-cycle arrest, and the induction of apoptosis (Annexin V-FITC/propidium iodide) 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 pharmacokinetic analysis of ribociclib in Sprague-Dawley rats by serial blood and brain extracellular fluid 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 and LTZ is likely to be strongly synergistic against GBM at concentrations of the drugs that can be achieved in the brain.

Figure 1:

A, Oncoprint of CDKN2A, CDKN2B, CDK4, CDK6 and RB1 gene expression in GBM samples from the TCGA, Cell 2013 dataset (577), accessed via cBioPortal. This Oncoprint displays alterations (amplifications, deletions, and mutations) in these key cell cycle regulators across GBM samples, highlighting the frequency and distribution of expression changes within the CDK4/6 pathway in GBM. The methylation status of the samples from the selected dataset and the comparison of the overall survival between the CDKN2A, CDK4 (altered) vs the unaltered group. B, RNA-seq analysis of FFPE tumor samples from our Phase 0/1 clinical trial of LTZ in recurrent GBM patients (NCT03122197). Differential gene expression between the 2.5mg (d2p5) vs the 5 (d5),10 (d10),12.5 9d12.5p5) and 15mg (d15) LTZ-treated tumors. Genes upregulated after LTZ treatment, associated with cell cycle and DNA repair. C, Log2 Counts Per Million (log2CPM) represents normalized expression level of CDK4 and CDKN2A in each sample across the entire dose cohort D, Log2 Fold Change (log2FC) represents relative change in CDK4 and CDKN2A expression between the 2.5mg vs the 5,10,12.5 and 15mg LTZ treated cohort. E, Western blot analysis of Aromatase expression in the patient-derived GBM lines used in the study vs MCF7 (ER +) and MDA-MB-468 (ER +) cell lines.

Figure 2:

The effect of drug treatment on cell viability of patient-derived GBM lines was determined using CellTiter-Glo^® 3D Cell Viability Assay. A, Cells (5000 /well) were treated with the indicated drug concentrations for 72 h (N = 3 per treatment group) in G75, G43, JHH-136 and G59 cells. (data: mean ± SD). RM one-way ANOVA (Šídák’s multiple comparisons) were used to analyze statistical differences across the treatment groups (G75: ***, G43: *, JHH-136: ** and G59: *. The graphs and statistical analysis were performed using GraphPad^® Prism version 10.4.1. (* = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001 & **** P ≤ 0.0001). B, Comparison of the ability of single cells to form spheroids between vehicle control (Group A) vs ribociclib alone (1 µM) (Group B), LTZ alone (1 µM) (Group C) and in combination (1 µM) (Group D) treated cells. The calculated stem cell frequencies were (500, 250, 125, 60, 30, 15, 7 and 3 cells per well) (12 wells each). Accutase-treated single cells were able to form spheroids, and treatment with ribociclib and LTZ inhibited spheroid formation. Statistical significance was determined with Extreme Limiting Dilution Assay software. C, Representative immunofluorescence images showing expression of CD133 and SOX2 in G43 and JHH-136 cells plated at 125 cells/well and 60 cells/well, respectively Quantification of relative fluorescence intensity for both markers is shown as bar graphs positioned above the IF images on the right. Both dilutions demonstrated positive staining for these stem cell markers, indicating that stemness is maintained even after serial dilution to low cell densities. Nuclei were counterstained with DAPI (blue). Scale bars = 50 μm.

Figure 3:

Flow cytometry pseudo color gated plots exhibiting induction of γH2AX in G75, G43, JHH-136 and G59 cells treated with ribociclib, LTZ and in combination for 5 h. Percentage γH2AX induction vs treatment was plotted using GraphPad^® Prism version 10.4.1. One-way ANOVA and Šídák’s multiple comparisons test was used to analyze the statistical difference across treatment groups (* = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001 & **** P ≤ 0.0001).

Figure 4:

A, Cell cycle analysis was performed employing flow cytometry by fixing, permeabilizing and staining the cells with PI. The plots are gated as per corresponding histograms (PE-A/H vs count) to determine the % of cells in each stage. A grouped bar graph with % of cells in various stages of the cell cycle vs treatment is plotted using GraphPad^® Prism version 10.4.1. Two-way ANOVA and Dunnett’s multiple comparisons test was used to analyze the difference between treatment groups (* = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001 & **** P ≤ 0.0001). B, Annexin V-FITC Apoptosis Staining / Detection Kit (ab14085) was used to assess the induction of apoptosis in G75, G43, JHH-136 and G59 cells using flow cytometry. (1*10⁵) cells were treated with the above-mentioned concentration of ribociclib, LTZ and in combination, incubated with serum free medium for 48h, vehicle served as NegCtrl. Pseudo color gated plots represent live cells: lower left (Annexin V− / PI−), early apoptosis: lower right (Annexin V+ / PI−), late apoptosis / secondary necrosis: upper right (Annexin V+ / PI+) and necrosis: upper left (Annexin V− / PI+). Representative bar graphs of mean (%) ± SEM] early apoptotic (annexin V⁺/PI⁻) and late apoptotic (annexin V⁺/PI⁺) cells plotted using GraphPad^® Prism version 10.4.1 are shown Two-way ANOVA and Tukey’s multiple comparisons test was used to analyze the difference between treatment groups (* = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001 & **** P ≤ 0.0001).

Figure 5:

Annexin V-FITC Apoptosis Staining / Detection Kit (ab14085) was used to determine apoptosis (using both the Annexin V-FITC and PI) in G75, G43, JHH-136, and G59 cells. (1*10⁵) cells were treated with the above-mentioned concentration of ribociclib alone, LTZ alone and ribociclib + LTZ, for 48 h in serum free medium. The green label on the plasma membrane (Annexin V-FITC) and the absence of nuclear red (PI) staining indicates apoptosis rather than necrosis. Leica DMi8 widefield fluorescence was used to analyze the cells.

Figure 6:

Brain and Systemic Pharmacokinetics of ribociclib. A) Plasma levels of ribociclib in SD rats (N = 5) at single i.p. dose of 100 mg/kg. (Corrected with protein binding ~28%) and B) Brain extracellular fluid (ECF) concentration–time profile of ribociclib in SD rats after single i.p. (N = 5) dose of 100 mg/kg (corrected with in-vitro recovery 6.1%). Data are presented as mean ± SD. The best-fit regression curves are plotted using Phoenix WinNonLin^® (Certara)