(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. Cancer and

: 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 CNS. We aimed to use palbociclib to inhibit growth of DIPG cells, in combination with the rapamycin analogue 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. We also 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, while we also observed non-canonical 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 G 1 cell cycle arrest and clonogenic assays showed non-reversible 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. S4). The clonogenicity of SU-DIPG IV showed very high sensitivity to palbociclib in our preliminary combination experiments, and thus a dose of 0.5  M palbociclib (in line with our initial immunoblotting assays) was used in combination with temsirolimus, which was sufficient to considerably augment the ability of temsirolimus to reduce colony numbers. Taken together these data suggest the drugs provided sustained antiproliferative effects, via cell cycle arrest.