Targeting tousled-like kinase 1 signalling pathways in glioblastoma multiforme therapy

Abstract

Glioblastoma multiforme (GBM) is an aggressive and highly invasive brain tumour that commonly exhibits resistance to conventional chemotherapy and radiotherapy. It is a heterogeneous disease involving complex kinomic alterations causing aberrant dysregulation of signalling pathways. Although many specific kinase inhibitors have been developed, only 30% of patients’ survived due to extreme proliferation, invasion and highly vascularized nature of GBM cells. The aim of this study was to explore novel kinome pathways and to identify potential therapeutic targets for GBM. Meta analysis was performed by filtering significant genes having p-value <0.05 from the Oncomine database using a total of 415 glioblastoma versus normal samples. This analysis identified 113 significantly upregulated kinases. RNAi screening on identified 113 kinases was performed subsequently, using Dharmacon Custom siRNA SMARTpoolTM library on LN18 and U87MG cells. k-Median Absolute Deviation statistical analysis has identified Tousled-like kinase 1 (TLK1) as a potential molecular target. TLK1, a serine-threonine kinase, was selected for an in vitrofunctional validation as its role in GBM is still unknown. Silencing of TLK1 using 25nM siRNA and shRNA in GBM cells resulted in a significant reduction in cellviability, clonogenicity and proliferation by inducing S-phase cell cycle arrest. In addition, inhibition of TLK1 induced apoptosis signals in GBM cells. Silencing of TLK1 also chemosensitised GBM cells towards sub-lethal dose of temozolomide (250µM). Invasion and migration of GBM cells were also inhibited (p<0.05). Interestingly, in normal human astrocytes, silencing of TLK1 did not cause significant changes in cell viability, apoptosis, invasion as well as migration. The downstream pathways of TLK1 in U87MG cells were interrogated using Illumina HumanHT12-12 v4 BeadChip microarray. WebGestalt pathways analysis on 2,632 significant probes with more than 1.1 fold change difference identified key signalling pathways involved in GBM cancer pathways including DNA replication, cell cycle, focal adhesion, TGF beta, and integrin-mediated cell adhesion signalling pathways. Gene expression analysis using qPCR confirmed the differential expressions of Thrombospondin-2, Paxillin, Ras-related C3 botulinum toxin substrate 2 (RAC2), Collagen type IV α2, FYN proto-oncogene Src family tyrosine kinase and Rho associated coiled-coil containing protein kinase 2 which were involved in the focal adhesion pathway. TLK1 was postulated to regulate invasion and migration of GBM cells through signalling with a small GTP binding protein, RAC2. RAC2 signal was significantly suppressed in both TLK1 knockdown U87MG and LN18 cells using RAC2 activation assay. TLK1 knockdown GBM cells also significantly reduced phosphorylated p70S6 kinase expression which acted downstream of the PI3K/AKT/mTOR signalling pathway. In vivo subcutaneous GBM xenograft of stably transfected U87MG cells with sh-TLK1in balb/c female nude mice showed significant difference (p<0.05) in tumour growth potential. Subsequent in silico homology modelling of TLK1 and high throughput virtual screening of protein-ligand docking identified two potential compounds that bound to catalytic site of TLK1 modelled protein. These findings suggest that TLK1 and its related pathways are potential molecular targets for GBM therapy.