Cancer Management and Research (Sep 2018)
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
Daniel J Asby,1 Clare L Killick-Cole,1 Lisa J Boulter,1 William GB Singleton,1,2 Claire A Asby,3 Marcella J Wyatt,1 Neil U Barua,1,2 Alison S Bienemann,1 Steven S Gill1,2 1Functional Neurosurgery Research Group, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, UK; 2Department of Neurosurgery, North Bristol NHS Trust, Southmead Hospital, Bristol, UK; 3Department of Neurology, North Bristol NHS Trust, Southmead Hospital, Bristol, UK 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
