Combination of palbociclib and radiotherapy for glioblastoma

Abstract

The cyclin-dependent kinase inhibitor, palbociclib has shown compelling efficacy in breast cancer patients. Several pre-clinical studies of glioblastoma (GBM) have also shown palbociclib to be efficacious. In this study, we investigated palbociclib in combination with radiation therapy (RT) for treating GBM. We tested palbociclib (with and without RT) on four patient-derived cell lines (PDCLs; RB1 retained; CDKN2A loss). We investigated the impact of therapy on the cell cycle and apoptosis using flow cytometry, in vitro. Balb/c nude mice were intracranially injected with the PDCL, GBM-L1 and treated orally with palbociclib (with and without RT). Overall survival was measured. Palbociclib treatment resulted in a significant increase in the percentage of cells in the G1 cell cycle phase. Apoptotic cell death, measured by Annexin V was induced. Palbociclib combined with RT acted synergistically with the significant impediment of colony formation. The oral treatment of mice with palbociclib did not show any significant survival advantage when compared to control mice, however when combined with RT, a survival advantage of 8 days was observed. Our results support the use of palbociclib as an adjuvant treatment to RT and warrant translation to the clinic.

Figure 1

Treatment of patient-derived cell lines (PDCLs) with palbociclib. (a) Expression of retinoblastoma protein (Rb1) pathway proteins in PDCLs without treatment; (b) Dose-response curves of the PDCLs treated with increasing concentrations of palbociclib; (c) Median IC₅₀ doses of palbociblib for each PDCL. All experiments were repeated three times. Error bars represent the standard deviation of the mean.

Figure 2

Distribution of cell cycle phase in HW1 and RN1 cell lines treated with palbociclib. (a, b) DNA histograms generated by flow cytometry showing the distribution of cells in various stages of the cell cycle for cell lines HW1 and RN1. Cells were treated with either DMSO vehicle (control) or 4 μM palbociclib (treated) for 48 h. Histograms are representative of n=3 experiments. (c, d) Graphical representation of DNA histograms for HW1 and RN1 cell lines showing the percentage of cells in the G0/G1, S-Phase and G2/M phases of the cell cycle (n=3 experiments).

Figure 3

Analysis of apoptosis in HW1 and RN1 cell lines treated with palbociclib. (a, c) Flow cytometric analysis of apoptosis using annexin V/PI staining of HW1 and RN1 cell lines treated with 0, 0.5, 1 and 1.5× their respective IC50 concentrations of palbociclib for 72 h. Dot-plot images are representative of n=3 experiments. (b, d) Graphical representation of flow cytometric data for HW1 and RN1 cell lines showing the percentage of live, early apoptotic, late apoptotic and necrotic cells (n=3 experiments).

Figure 4

Analysis of colony formation with or without palbociclib treatment of irradiated PDCLs. (a) Colony formation assay; (b) Colonies were counted from quadruplicate samples for each treatment condition and represented as a percentage of the control (DMSO treated cells). Mean and SD are shown.

Figure 5

Protein expression in response to treatment with palbociclib with or without RT. (a) Representative western blot and protein intensities determined using Image J software (NIH, Bethesda, MD, USA); (b) Rb1; (c) E2F; (d) CDK4; (e) CDK6; (f) H2AX; (g) cPARP. *P<0.05; **P<0.01; ***P<0.005; ****P<0.001.

Figure 6

Kaplan–Meier Survival curve for treatments of mice with intracranial RN1 xenografts. Mice were treated at day 55 (indicated by arrow) and received two weeks of palbociclib treatment. Mice were irradiated on days 55 and 56 to receive a total of 4 Gy. LogRank P=0.048.