THE COMBINATION OF THE CDK4/6 INHIBITOR PALBOCICLIB WITH THE RAPALOGUE TEMSIROLIMUS INHIBITS DIPG CELL PROLIFERATION VIA SYNERGISTIC ATTENUATION OF CELL CYCLE REGULATORS

Current chemotherapy for diffuse intrinsic pontine glioma (DIPG) is ineffective and requires revitalization. DIPG accounts for up to 15% of all childhood central nervous system tumours and has a two-year survival rate of less than 10%. DIPG is highly chemotherapy-resistant and children show little response to conventional high-grade glioma chemotherapies. Palbociclib has the potential to treat DIPG by selectively inhibiting the proliferation of cancer cells via inhibition of cyclin-dependent kinase (CDK) 4 and CDK6, which triggers cell cycle arrest at the G1 checkpoint. However it has been found that CDK inhibitors alone are ineffective as long-term cancer treatments and resistance can be acquired. We aimed to use palbociclib to inhibit DIPG cell proliferation by inducing cell cycle arrest via targeted disruption of cyclin D-CDK4/6 complexes, in combination with the rapamycin analogue temsirolimus, used to attenuate mTOR-driven cyclin protein expression. Cell viability was assessed in two ex vivo DIPG cell lines treated with palbociclib, temsirolimus or both for 24 hours. We demonstrated that palbociclib and temsirolimus both inhibit proliferation in DIPG cells and that their effects were potentiated when used in combination. This was substantiated via flow cytometric and clonogenic analyses, the latter revealing longer-term dose-dependent reductions in colony formation. Quantitative analysis of the dose-effect relationship between the two drugs indicated a synergistic relationship when used in combination. Furthermore, immunoblot analyses showed inhibition of CDK4/6 and mTOR signalling using palbociclib and temsirolimus, respectively, each of which was enhanced in combination. This work illustrates the in vitro effectiveness of these drugs against DIPG when used individually, and the amplified effects when used concurrently. The combination of these two established FDA-approved therapeutics represents a putative fast-track approach to creating a much-needed treatment for DIPG.