CDKs as target structures for cancer therapy – an in vitro analysis on patient-derived glioblastoma cell lines

Background Glioblastoma (GBM) is the most common and lethal brain tumor. Despite radical therapy consisting of surgical resection, combined chemoradiotherapy and continuous development of treatment strategies survival prognosis remains poor. This underlines the urgent need for new (alternative) therapeutic strategies. Cyclin-dependent kinases (CDKs) play essential roles in regulation of cell cycle progression, transcription, DNA damage repair, stem cell self-renewal as well as nerve cell differentiation. Glioma cells frequently show genomic alterations involved in cell cycle control, especially in specific interphase CDKs essential for tumor cell proliferation. To counteract the CDK dysregulation in malignant patient-derived glioma cell cultures we used selective CDK inhibitors (CDKIs) like abemaciclib (AB), palbociclib (P) and the novel inhibitor dinaciclib (D). Since GBM are also known to be highly radioresistant we tested these inhibitors in combination with radiation therapy (RT).

Methods A panel of low passaged patient-derived glioblastoma cell lines with different molecular characteristics for 2D and 3D cultures (Spheroids and Glioma stem cell populations; GSCs) were included (N = 5). Impact of CDKIs on proliferation and cytotoxicity was examined on these cell lines in clinically relevant doses (1 – 10 µM = AB, P; 0.001– 0.1 µM = D) and different treatment durations (24 h, 48 h, 72 h, 2 × 72 h). Treatment was performed with single agents with and without RT. Read out was performed using Calcein AM and 3D-Glo-viability assays. By taking advantage of the Clariom^™ S Assay, a microarray analysis offered first insights into molecular mechanisms of D treatment.

Results D significantly affected viability of GBM cell lines in both 2D and 3D culture, respectively. Of note, antitumoral effects were visible shortly after low-dose treatment. AB and P had weaker effects, additionally being cell line specific, time- and dose-dependent. Interestingly, a radio-sensitizing effect was seen after treatment with AB and D, but not upon P exposure. Initial microarray analysis confirmed CDK inhibition by D (CDK2/4/5/6), but also high upregulation of genes inducing apoptosis (Casp8, Casp3), senescence (ASF1A, ID1), and autophagy (SOGA1, ATG4D, ATG16L1).

Conclusions Dinaciclib treatment was most effective against all tested GBM cell lines in a 2D and 3D model. Combined application of Dinaciclib or Abemaciclib and low-dose radiotherapy seems to be a promising strategy to kill tumor target cells. Deciphering the molecular mechanisms of CDKI mediated cell death along with in vivo proof-of-concept studies will help to move forward this treatment strategy.

 

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