Combined use of CDK4/6 and mTOR inhibitors induce synergistic growth arrest of diffuse intrinsic pontine glioma cells via mutual downregulation of mTORC1 activity

Abstract

Background

Diffuse intrinsic pontine glioma (DIPG) is a lethal type of pediatric brain tumor that is resistant to conventional chemotherapies. Palbociclib is a putative novel DIPG treatment that restricts the proliferation of rapidly dividing cancer cells via selective inhibition of cyclin-dependent kinase (CDK) 4 and CDK6. However, implementing palbociclib as a monotherapy for DIPG is unfeasible, as CDK4/6 inhibitor resistance is commonplace and palbociclib does not readily cross the blood–brain barrier (BBB) or persist in the central nervous system. To inhibit the growth of DIPG cells, we aimed to use palbociclib in combination with the rapamycin analog temsirolimus, which is known to ameliorate resistance to CDK4/6 inhibitors and inhibit BBB efflux.

Materials and methods

We tested palbociclib and temsirolimus in three patient-derived DIPG cell lines. The expression profiles of key proteins in the CDK4/6 and mammalian target of rapamycin (mTOR) signaling pathways were assessed, respectively, to determine feasibility against DIPG. Moreover, we investigated effects on cell viability and examined in vivo drug toxicity.

Results

Immunoblot analyses revealed palbociclib and temsirolimus inhibited CDK4/6 and mTOR signaling through canonical perturbation of phosphorylation of the retinoblastoma (RB) and mTOR proteins, respectively; however, we observed noncanonical downregulation of mTOR by palbociclib. We demonstrated that palbociclib and temsirolimus inhibited cell proliferation in all three DIPG cell lines, acting synergistically in combination to further restrict cell growth. Flow cytometric analyses revealed both drugs caused G₁ cell cycle arrest, and clonogenic assays showed irreversible effects on cell proliferation. Palbociclib did not elicit neurotoxicity in primary cultures of normal rat hippocampi or when infused into rat brains.

Conclusion

These data illustrate the in vitro antiproliferative effects of CDK4/6 and mTOR inhibitors in DIPG cells. Direct infusion of palbociclib into the brain, in combination with systemic delivery of temsirolimus, represents a promising new approach to developing a much-needed treatment for DIPG.

Keywords:

• palbociclib
• temsirolimus
• brain tumor
• DIPG
• retinoblastoma protein
• cyclin-dependent kinase

Supplementary materials

Figure S1 DIPG cells, ostensibly, do not express p16^INK4A.

Notes: Immunoblot using p16^INK4A-specific antibody against protein from SF7761, SF8628, and SU-DIPG IV cells treated with vehicle (−) or 10 μM palbociclib (+) for 24 hours. HeLa cell protein was used as a positive control for p16^INK4A expression. ®-actin was used as a loading control. Representative immunoblot of three determinations.

Abbreviation: p16^INK4A, cyclin-dependent kinase inhibitor 2A.

Figure S2 Logarithmic dose-response curves for palbociclib and temsirolimus single-agent and combination treatments.

Notes: (A) SF7761, (B) SF8628, and (C) SU-DIPG IV cells were treated for 24 hours with increasing concentrations of single-agent palbociclib (0.2–200 µM), single-agent temsirolimus (0.2–200 μM) or temsirolimus (0.2–200 μM) combined with a single fixed dose of palbociclib (2, 10, 12, 15 or 25 μM). Cell viability was assessed using calcein-AM staining and an IC₅₀ modeled in each instance. Data are the mean ± SEM of triplicate determinations.

Abbreviations: PD, palbociclib; TM, temsirolimus.

Figure S3 Representative cell cycle analysis histograms illustrating G₁-S arrest in DIPG cells in response to palbociclib and temsirolimus treatment compared to control cells.

Notes: SF7761 cells were treated with vehicle, 2 μM palbociclib or 10 μM temsirolimus for 0, 24, 48, or 72 hours, as shown. DRAQ5 fluorescent dye was used to conduct flow cytometric cell cycle analysis on cells following treatment. G₁ peak (left), G₂ peak (right), and S-phase cells (transitional central area) are shown in all instances. Percentage value (top right) indicates the percentage of total cells in G₁ phase. Each panel is a representative histogram of three determinations.

Figure S4 Palbociclib dose-dependently reduces clonogenicity in DIPG cells.

Notes: SU-DIPG IV cells were treated with different concentrations of palbociclib for 24–72 hours, and colonies were counted after 14 days. Data are the mean ± SEM of triplicate determinations.

Acknowledgments

The authors thank Abbie’s Army DIPG brain tumor research charity for their generous financial support. The authors also thank Dr Nalin Gupta for the SF7761 and SF8628 cell lines, Dr Michelle Monje for the SU-DIPG IV cell line, and Dr Alex Hoose for general advice and assistance. This study was funded by Abbie’s Army DIPG brain tumor research charity (to NU Barua and AS Bienemann), the Functional Neurosurgery Research Fund held by Southmead Hospital Charities (SS Gill), the Gatsby foundation (to WGB Singleton), and joint funding from the Medical Research Council and the Brain Tumour Charity (grant no. MR/N00130/1 to WGB Singleton).

Disclosure

SS Gill is a consultant for Renishaw PLC. NU Barua is a consultant for Renishaw PLC. The authors report no other conflicts of interest in this work.