(W0930-10-58) Translational Research to Facilitate Development of Novel Therapeutic Combination of Letrozole with a CDK 4/6 Inhibitor Ribociclib for the Treatment of Glioblastoma

Purpose: Our pre-clinical and translational studies have identified letrozole (LTZ), a third-generation aromatase inhibitor, as a promising therapeutic for glioblastoma (GBM). Collaborating with the UC Cancer Center, we conducted a phase 0/1 clinical trial of LTZ in recurrent GBM patients. RNA-seq analysis of LTZ-treated tumor samples from the clinical trial revealed dose-dependent downregulation of key genes involved in cell cycle progression and proliferation, including CDK1, CCNA2, E2F8, CHEK1, and CCND1. Additionally, genomic analysis of GBM patient data from cBioPortal highlighted frequent deep deletions of CDKN2A and CDKN2B, underscoring the therapeutic potential of CDK inhibitors in GBM.

Methods: We employed cell viability (Cell titer Glo®) and neurosphere growth assays to assess the effects of LTZ and ribociclib against genotypically diverse patient-derived G75, G43, JHH-136 and G59 GBM lines.  Quantitative assessment of the cytotoxic effects of the combination was performed using Combination Index analysis.  The DNA damaging effects of CDK inhibitors were evaluated by flow cytometric analysis of γH2A.X induction and the mechanism of cell death was assessed using Annexin V-FITC staining. The blood-brain barrier (BBB) permeability of ribociclib in jugular vein cannulated Sprague-Dawley rats was assessed using intra-cranial microdialysis which facilitated serial sampling and analysis of blood and brain extracellular fluid (ECF) pharmacokinetics (PK) of the compound.

Results: The treatment of GBM cells with the combination of LTZ and ribociclib resulted in synergistic cytotoxic effects. For instance, the IC₅₀ values of ribociclib alone ranged from 0.4- 3.6 µM and in the presence of non-cytotoxic concentrations of LTZ, ribociclib IC₅₀ values ranged from 0.2 – 1.9 µM. As such, an approximate 2-2.5-fold reduction in the IC₅₀ values was observed. Furthermore, the CI values ranged from 0.02 to 0.6 suggesting strong synergistic interaction. Similar potentiation of ribociclib by LTZ was observed in neurosphere growth assays. Flow cytometric analysis revealed that LTZ significantly enhanced the DNA damaging effects of ribociclib, as evident by increased γH2A.X induction. This combination induced cell death through apoptosis which was microscopically quantified using the Annexin V-FITC staining. In-vivo intra-cranial microdialysis studies in Sprague-Dawley rats revealed that ribociclib has a blood-brain ECF partitioning of 0.095. The unbound concentration of ribociclib achieved in the rat brain was about 0.45 µM which was higher the IC₅₀ values achieved in-vitro.

Conclusion: Our studies provide a strong foundation for pursuing further development of combination therapy of GBM with LTZ and ribociclib.  Further in vivo efficacy studies and insights into molecular markers to identify patients most likely to benefit from this combination are warranted.  Overall, our findings underscore the promise of LTZ and ribociclib combination therapy as a potential treatment option for GBM patients.

References: Desai PB, Karve A, Zawit M, et al. A Phase 0/1 Pharmacokinetic and Pharmacodynamics and Safety and Tolerability Study of Letrozole in Combination with Standard Therapy in Recurrent High-Grade Gliomas. Clin Cancer Res. Published online March 26, 2024. doi:10.1158/1078-0432.CCR-23-3341

Acknowledgements: The authors declare that they have no conflicts of interest regarding this research. This study was funded by NIH, grant number 5 R61 NS128232-02

Effect of drug treatment on cell viability of patient-derived GBM lines was determined using CellTiter-Glo® 3D Cell Viability Assay. Cells (5000 /well) were treated with the indicated drug concentrations for 72 h (N = 3 per treatment group). A G43, B G75, C G59 and D JHH-136 cells. (data: mean ± SD). Multi-parametric correlation matrix analysis and repeated measures one-way ANOVA were used to analyze statistical differences across the treatment groups. p value: * = < 0.05; ** < 0.01; *** < 0.001; n.s. > 0.05. The graphs and statistical analysis was performed using Graph Pad® Prism 10.2.2.

Flow cytometry pseudo color plots exhibiting induction of γH2A.X in G43 (A), G75 (B), G59 (C) and JHH-136 (D) cells treated with the above mentioned concentrations for 5 h. % Gamma-H2AX staining vs treatment with ribo and LTZ is plotted using Graph Pad® Prism 10.2.2 (E,F,G&H).

Annexin V-FITC Apoptosis Staining / Detection Kit (ab14085) was used to determine apoptosis (using both the Annexin V-FITC and PI) in A) G43, B) JHH-136 & C) G59 cells. (1*105) cells were treated with the above mentioned concentration of ribociclib in combination with LTZ, incubated with serum free medium for 48h. 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 analyse the cells.