A Novel Small-Molecule Inhibitor of MRCK Prevents Radiation-Driven Invasion in Glioblastoma.

Glioblastoma (GBM) is an aggressive and incurable primary brain tumor that causes severe neurologic, cognitive, and psychologic symptoms. Symptoms are caused and exacerbated by the infiltrative properties of GBM cells, which enable them to pervade the healthy brain and disrupt normal function. Recent research has indicated that although radiotherapy (RT) remains the most effective component of multimodality therapy for patients with GBM, it can provoke a more infiltrative phenotype in GBM cells that survive treatment. Here, we demonstrate an essential role of the actin-myosin regulatory kinase myotonic dystrophy kinase-related CDC42-binding kinase (MRCK) in mediating the proinvasive effects of radiation. MRCK-mediated invasion occurred via downstream signaling to effector molecules MYPT1 and MLC2. MRCK was activated by clinically relevant doses per fraction of radiation, and this activation was concomitant with an increase in GBM cell motility and invasion. Furthermore, ablation of MRCK activity either by RNAi or by inhibition with the novel small-molecule inhibitor BDP-9066 prevented radiation-driven increases in motility both in vitro and in a clinically relevant orthotopic xenograft model of GBM. Crucially, treatment with BDP-9066 in combination with RT significantly increased survival in this model and markedly reduced infiltration of the contralateral cerebral hemisphere.Significance: An effective new strategy for the treatment of glioblastoma uses a novel, anti-invasive chemotherapeutic to prevent infiltration of the normal brain by glioblastoma cells.Cancer Res; 78(22); 6509-22. ©2018 AACR.

Discovery of Potent and Selective MRCK Inhibitors with Therapeutic Effect on Skin Cancer.

The myotonic dystrophy-related Cdc42-binding kinases MRCKα and MRCKß contribute to the regulation of actin-myosin cytoskeleton organization and dynamics, acting in concert with the Rho-associated coiled-coil kinases ROCK1 and ROCK2. The absence of highly potent and selective MRCK inhibitors has resulted in relatively little knowledge of the potential roles of these kinases in cancer. Here, we report the discovery of the azaindole compounds BDP8900 and BDP9066 as potent and selective MRCK inhibitors that reduce substrate phosphorylation, leading to morphologic changes in cancer cells along with inhibition of their motility and invasive character. In over 750 human cancer cell lines tested, BDP8900 and BDP9066 displayed consistent antiproliferative effects with greatest activity in hematologic cancer cells. Mass spectrometry identified MRCKα S1003 as an autophosphorylation site, enabling development of a phosphorylation-sensitive antibody tool to report on MRCKα status in tumor specimens. In a two-stage chemical carcinogenesis model of murine squamous cell carcinoma, topical treatments reduced MRCKα S1003 autophosphorylation and skin papilloma outgrowth. In parallel work, we validated a phospho-selective antibody with the capability to monitor drug pharmacodynamics. Taken together, our findings establish an important oncogenic role for MRCK in cancer, and they offer an initial preclinical proof of concept for MRCK inhibition as a valid therapeutic strategy.Significance: The development of selective small-molecule inhibitors of the Cdc42-binding MRCK kinases reveals their essential roles in cancer cell viability, migration, and invasive character. Cancer Res; 78(8); 2096-114. ©2018 AACR.

A novel small-molecule MRCK inhibitor blocks cancer cell invasion.

BACKGROUND: The myotonic dystrophy kinase-related CDC42-binding kinases MRCKα and MRCKß 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. RESULTS: 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 MRCKß kinase domain in complex with BDP5290 revealed how this ligand interacts with the nucleotide binding pocket. BDP5290 demonstrated marked selectivity for MRCKß 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. CONCLUSIONS: 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.

Three different approaches to Transversus abdominis planeblock: a cadaveric study.

AIM: The transversus abdominis plane (TAP) block is a new technique for providing analgesia to the anterior abdominal wall. There is ongoing debate regarding access point for TAP block. The aim of this cadaveric study was to compare the spread of 40 mL of dye using three different approaches to TAP: subcostal , via the mid-axillary and via the lumbar triangle of Petit (LTOP). METHODS: Injection of black dye into the TAP was performed for each hemi-abdominal wall of 13 embalmed human cadavers by using 3 different access points: subcostal (9 hemi-abdomens), mid-axillary (9) and LTOP (8). This was followed by dissection to determine the extent of dye spread and nerve involvement in the dye injection. The shapes of the dye were traced onto clear plastic, which was then photographed. These digital photographs were loaded into the mathematical software programme Matlab, and the outline of the dye spread was digitised using a piecewise cubic spline, enabling the shapes to be plotted on a graph and the areas to be calculated. RESULTS: The area of the dye spread for subcostal, mid-axillary and LTOP was 85.1 (T7-L1), 58.9 (T10-L1) and 77.9 cm² (T10-L1), respectively. There was statistically significant difference between area of dye spread between subcostal and mid-axillary approach (p<0.01). CONCLUSIONS: This dye injection study in a cadaver model indicates that subcostal approach is associated with a larger area of spread of dye than the mid-axillary approach. Dye injected through subcostal, mid-axillary and LTOP approaches demonstrated different nerve involvement.

An anatomical study of the transversus abdominis plane block: location of the lumbar triangle of Petit and adjacent nerves.

BACKGROUND: The transversus abdominis plane (TAP) block is a new technique for providing analgesia to the anterior abdominal wall. Most previous studies have used the lumbar triangle of Petit as a landmark for the block. In this cadaveric study, we determined the exact position and size of the lumbar triangle of Petit and identified the nerves affected by the TAP block. METHODS: The position of the lumbar triangle of Petit was assessed unilaterally in 26 cadaveric specimens relative to reliably palpable surface landmarks. In addition, a series of dissections were performed to explore the course of the nerves blocked by the TAP. RESULTS: The mean distance from the midaxillary line along the iliac crest to the center of the base of the lumbar triangle of Petit at the level of the subcutaneous tissue and over the skin surface was 6.9 cm (range, 4.5-9.2 cm) and 9.3 cm (range, 4-15.1 cm), respectively. The center of the lumbar triangle of Petit was 1.4 cm above the iliac crest. The depth of the TAP at the lumbar triangle of Petit position was 0.5-4 cm and at the midaxillary line it was 0.5-2 cm. The average size of the lumbar triangle of Petit was 2.3 cm x 3.3 cm x 2.2 cm, with an average area of 3.63 +/- 1.93 cm2. The three cadaveric specimens we explored showed the nerves blocked by TAP passed lateral to the triangle. An incidental finding was that in 66% of specimens the lumbar triangle of Petit contained small branches of the subcostal artery. CONCLUSIONS: The lumbar triangles of Petit found in the specimens in this study were more posterior than the literature suggests. The position of the lumbar triangle of Petit varies largely and the size is relatively small. The relevant nerves to be blocked had not entered the TAP in the specimens in this study at the point of the lumbar triangle of Petit. At the midaxillary line, however, all the nerves were in the TAP.