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Glioblastoma (GBM) is one of the most common and aggressive forms of brain cancer. It is typically associated with poor survival rates, in part due to the characteristically infiltrative nature of GBM tumour cells. This allows GBM cells to disseminate through the brain via existing white matter tracts and perivascular spaces making complete surgical resection unachievable and contributing to high recurrence rates. For the majority of patients treatment of GBM includes radiotherapy. However, radiation resistance results in populations of surviving tumour cells with the potential for modified behaviour through radiation-induced alterations in signalling and gene expression profiles Myotonic dystrophy kinase-related CDC42- binding kinase (MRCK) is involved in regulating actin-myosin dynamics during cell migration through phosphorylation of its targets MLC2 and MYPT. Upon radiation of GBM cell lines in vivo we observed an increase in MRCK activity which was concomitant with a significant increase in GBM cell motility and invasion in in vitro and ex vivo assays. In support of these data we have observed increased MRCK activity at the invasive edges of both clinical GBM samples and an orthotopic GBM xenograft model that recapitulates key histological features of human GBM. Furthermore, therapeutic doses of irradiation were found to promote GBM invasion in vivo in this xenograft model. These observations suggest that MRCK activity may be required to promote invasion following irradiation and therefore may present a promising novel target for GBM treatment. To explore this further we performed MRCK RNAi on GBM cells and found we were able to reverse the radiation dependent promotion of GBM cell motility. We then used a novel MRCK selective inhibitor BDP-9066 to recapitulate this effect, observing dose dependent effects on both MRCK biomarkers, PMLC2 and PMYPT, and motility in vitro and ex vivo. These data suggest that MRCK activity is indeed required for GBM invasion and can be targeted effectively in multiple GBM cell lines. These observations provide evidence that glioblastoma cell migration may be enhanced following radiation therapy. As such, this might provide a substrate for therapy resistance and early recurrence in these tumours whilst offering a potential target for novel therapies in GBM management.

Background:\ud The myotonic dystrophy kinase-related CDC42-binding kinases MRCKα and MRCKβ regulate actin-myosin contractility and have been implicated in cancer metastasis. Along with the related ROCK1 and ROCK2 kinases, the MRCK proteins initiate signalling events that lead to contractile force generation which powers cancer cell motility and invasion. A potential strategy for cancer therapy is to reduce metastasis by blocking MRCK activity, either alone or in combination with ROCK inhibition. However, to date no potent small molecule inhibitors have been developed with selectivity towards MRCK.\ud \ud Results:\ud Screening a kinase-focused small molecule chemical library resulted in the identification of compounds with inhibitory activity towards MRCK. Medicinal chemistry combined with in vitro enzyme profiling led to the discovery of 4-chloro-1-(4-piperidyl)-N-[5-(2-pyridyl)-1H-pyrazol-4-yl]pyrazole-3-carboxamide (BDP00005290; abbreviated as BDP5290) as a potent MRCK inhibitor. X-ray crystallography of the MRCKβ kinase domain in complex with BDP5290 revealed how this ligand interacts with the nucleotide binding pocket. BDP5290 demonstrated marked selectivity for MRCKβ over ROCK1 or ROCK2 for inhibition of myosin II light chain (MLC) phosphorylation in cells. While BDP5290 was able to block MLC phosphorylation at both cytoplasmic actin stress fibres and peripheral cortical actin bundles, the ROCK selective inhibitor Y27632 primarily reduced MLC phosphorylation on stress fibres. BDP5290 was also more effective at reducing MDA-MB-231 breast cancer cell invasion through Matrigel than Y27632. Finally, the ability of human SCC12 squamous cell carcinoma cells to invade a three-dimensional collagen matrix was strongly inhibited by 2 μM BDP5290 but not the identical concentration of Y27632, despite equivalent inhibition of MLC phosphorylation.\ud \ud Conclusions:\ud BDP5290 is a potent MRCK inhibitor with activity in cells, resulting in reduced MLC phosphorylation, cell motility and tumour cell invasion. The discovery of this compound will enable further investigations into the biological activities of MRCK proteins and their contributions to cancer progression.