Small Molecule Protein Kinase Inhibitors Research Articles

Comparison of signal transduction drug response markers in immortalized cell lines and primary patient specimens

Abstract Background: The epidermal growth factor pathway is an important driver of cancer growth. Erlotinib is a small molecule protein-kinase inhibitor targeting the epidermal growth factor receptor (EGFR) that may benefit a select subtype of cancer patients. However, identification of the characteristics of patients who may benefit from the use of erlotinib is not easy. Previous publication explored the activation of signal transduction proteins in the EGFR pathway as a method of determining erlotinib response and demonstrated that lung cancer cell lines characterized by increased pEGFR/pAKT ratios are more resistant to erlotinib. The aim of this study was to compare the activation of signal transduction markers in the EGFR pathway between immortalized cell lines and primary cultures of patient-derived tumor cells. Predictive potential of pEGFR/pAKT ratios in both immortalized cell lines and primary cultures of lung cancer cells was also compared. Methods: Western blotting and In-Cell western analysis were used to determine the activation state of the EGFR signaling pathway. Signaling pathway components were compared in the presence and absence of erlotinib treatment in immortalized lung cancer cell lines and primary cultures of lung cancer specimens. Chemosensitivity of cell lines and specimens to erlotinib was determined using an in vitro drug response marker. Cells were treated with a 10 dose concentration range of erlotinib for 72 hours and the remaining post-treatment live cells were enumerated by nuclear staining. Results: The pEGFR/pAKT ratio was increased in erlotinib-responsive immortalized cell lines when compared to non-responsive cell lines (p<0.05). This difference in pEGFR/pAKT ratio was significant after treatment with erlotinib as well. The difference in pEGFR/pAKT ratios in response to erlotinib treatment between erlotinib-responsive and non-responsive primary cultures of lung cancer cells was not, however, statistically significant. Conclusion: These studies confirmed that pEGFR/pAKT ratios may be indicative of erlotinib response in immortalized lung cancer cell lines. However, when evaluating the signal transduction markers in patient-derived primary cultures, no correlation with in vitro erlotinib response was found. These results highlight the value of using in vitro drug response markers, which utilize primary patient tissue, in the development of predictive biomarkers.

New Test System for Serine/Threonine Protein Kinase Inhibitors Screening: E. coli APHVIII/ Pk25 design

An efficient test system for serine/threonine protein kinase inhibitors screening has been developed based on theE. coliprotein system APHVIII/Pk25. Phosphorylation of aminoglycoside phosphotransferase VIII (APHVIII) by protein kinases enhances resistance of the bacterial cell to aminoglycoside antibiotics, e.g. kanamycin. Addition of protein kinase inhibitors prevents phosphorylation and increases cell sensitivity to kanamycin. We have obtained modifications of APHVIII in which phosphorylatable Ser146 was encompassed into the canonical autophosphorylation sequence ofStreptomyces coelicolorPk25 protein kinase. Mutant and wild-typeaphVIII were cloned intoE. coliwith the catalytic domain ofpk25. As a result of the expression of these genes, accumulation of corresponding proteins was clearly observed. Extracted from bacterial lysates, Pk25 demonstrated its ability to autophosphorylate. It was shown that variants ofE. colicontaining bothaphVIIIand рк25were more resistant to kanamycin than those carrying onlyaphVIII. Protein kinase inhibitors of the indolylmaleimide class actively inhibited Pk25 and reduced cell resistance to kanamycin. Modeling of APHVIII and Pk25 3D structures showed that pSer146 is an analog of phosphoserine in the ribose pocket of protein kinase A. Pk25 conformation was similar to that of РknB ofMycobacterium tuberculosis. Potential indolylmaleimide inhibitors were docked into the ATP-binding pocket of Pk25. The designed test system can be used for the primary selection of ATP-competitive small molecule protein kinase inhibitors.

Correction: A Novel Small-Molecule Inhibitor of Protein Kinase D Blocks Pancreatic Cancer Growth In vitro and In vivo

Correction: A Novel Small-Molecule Inhibitor of Protein Kinase D Blocks Pancreatic Cancer Growth <i>In vitro</i> and <i>In vivo</i>

Epidermal Growth Factor Receptor-Mediated Regulation of Urokinase Plasminogen Activator Expression and Glioblastoma Invasion via C-SRC/MAPK/AP-1 Signaling Pathways

One of the major pathophysiological features of malignant astrocytomas is their ability to infiltrate surrounding brain tissue. The epidermal growth factor receptor (EGFR) and proteases are known to be overexpressed in glioblastomas (GBMs), but the interaction between the activation of the EGFR and urokinase plasminogen activator (uPA) in promoting astrocytic tumor invasion has not been fully elucidated. Here, we characterized the signal transduction pathway(s) by which EGF regulates uPA expression and promotes astrocytoma invasion. We show that EGFR activation and constitutively active EGFR vIII in GBM cell lines upregulate uPA expression. Small-molecule inhibitors of mitogen-activated protein kinase, tyrosine kinase, and small interfering RNA targeting c-Src blocked uPA upregulation. Similarly, mutations in the activator protein 1 binding site of the uPA promoter reduced EGF-induced increases in uPA promoter activity. Treatment of GBM cells with EGF increased in vitro cell invasion, and the invasive phenotype was attenuated by gene silencing of uPA using small interfering RNA and short hairpin RNA. In addition, uPA knockdown clones formed smaller well-circumscribed tumors than nontarget U1242 control cells in a xenograft GBM mouse model in vivo. In summary, these results suggest that c-Src, mitogen-activated protein kinase, and a composite activator protein 1 on the uPA promoter are responsible for EGF-induced uPA expression and GBM invasion.

Cyclin-Dependent Kinase Inhibitors as Anticancer Drugs

Poor therapeutic outcomes and serious side effects, together with acquired resistance to multiple drugs, are common problems of current cancer therapies. Therefore, there is an urgent need for new cancer-targeted drugs, which has led (inter alia) to the development of molecules that can specifically inhibit cyclin-dependent kinases (CDKs). In addition to their cell cycle regulatory functions, CDKs, especially CDK7 and CDK9, play important roles in the regulation of RNA polymerase II-mediated transcription. Here, we report on progress in the preclinical development of CDK inhibitors and their anticancer activities. Special attention is paid to the action mechanisms of the pan-specific CDK inhibitors flavopiridol and roscovitine, which have already entered phase II clinical trials as treatments for various tumours. The links between their ability to inhibit transcription and sensitisation of some types of cancer to apoptosis, mechanisms leading to p53 activation, and their synergistic cooperation with common DNA damaging drugs are also discussed. It has been demonstrated that drug-resistant cancer cells can arise during therapeutic application of small molecule protein kinase inhibitors. Clinical resistance to CDK inhibitors has not yet been described, but by comparing CDKs to other kinases, and CDK inhibitors to other clinically used protein kinase inhibitors, we also discuss possible mechanisms that could lead to resistance to CDK inhibitors.

Targeted therapies in epithelial ovarian cancer: Molecular mechanisms of action

Ovarian cancer is the leading cause of death in women with gynecological cancer. Most patients are diagnosed at an advanced stage and have a poor prognosis. Currently, surgical tumor debulking, followed by platinum- and taxane-based chemotherapy is the standard treatment for advanced ovarian cancer. However, these patients are at great risk of recurrence and emerging drug resistance. Therefore, novel treatment strategies are required to improve outcomes for women with advanced ovarian cancer. A variety of molecular targeted agents, the majority of which are monoclonal antibodies and small-molecule protein-kinase inhibitors, have been explored in the management of ovarian cancer. The targets of these agents include angiogenesis, the human epidermal growth factor receptor family, ubiquitin-proteasome pathway, epigenetic modulators, poly(ADP-ribose) polymerase (PARP), and mammalian target of rapamycin (mTOR) signaling pathway, which are aberrant in tumor tissue. The antiangiogenic agent, bevacizumab, has been reported as the most effective targeted agent and should be included in the standard chemotherapeutic regimen for advanced ovarian cancer. PARP inhibitors, which are mainly used in breast and ovarian cancer susceptibility gene-mutated patients, and mTOR inhibitors are also attractive treatment strategies, either alone or combination with chemotherapy, for ovarian cancer. Understanding the tumor molecular biology and identification of predictive biomarkers are essential steps for selection of the best treatment strategies. This article reviews the molecular mechanisms of the most promising targeted agents that are under early phase clinical evaluation for ovarian cancer.

Abstract C192: Characterization of the target profile of XL228, a multi-targeted protein kinase inhibitor in phase 1 clinical development

Abstract Background: XL228 is a small molecule protein kinase inhibitor currently in Phase 1 clinical development targeting Ph+ leukemias, multiple myeloma, and solid tumors. Cellular and in vivo data have demonstrated activity of XL228 against IGF1R, ABL (including the T315I variant) and SRC family kinases. XL228 was tested in a panel of biochemical protein kinase assays, and additional putative targets were identified, including Aurora family kinases, FGFR1-3, and ALK. As these potential targets are known to enhance cell proliferation and survival in certain malignancies, the validation of their inhibition by XL228 through in vitro studies and in clinical pharmacodynamic samples was performed. Results: A panel of kinase inhibitors including XL228 was profiled against a series of cancer cell lines with known alterations in major signaling pathways. Approximately 30% of the lines demonstrated XL228 IC50 values of &amp;lt;100nM in viability assays, including many lines with characterized ALK or FGFR mutations or amplifications. Cell lines showing the highest level of response to XL228 were often sensitive to other Aurora, ALK or FGFR inhibitors as well. Sub-20nM activity was demonstrated against FGFR2 in a cellular kinase ELISA assay, and confirmed by immunoblot analysis. Potency against ALK in the cell kinase assay was lower (approximately 200 nM). The Aurora inhibitory activity of XL228 was examined through the use of immunoblots, immunofluorescence, and substrate phosphorylation assays. XL228 eliminated the phosphorylation of Aurora A and B at concentrations above 10 nM. Short-term treatment of HeLa cells led to disruption of mitotic spindle formation, with the majority of mitotic cells exhibiting a unipolar spindle and disorganized chromosomes. After exposure to XL228 for 24 hours, there was a significant increase in cells with &amp;gt;4N DNA content, suggesting a failure of cytokinesis and consequent endoreduplication. Activity of XL228 against Aurora kinases was further verified by dose-dependent inhibition of histone H3 phosphorylation, where it showed approximately 5-fold higher potency than the control compound VX-680. To confirm the relevance of the in vitro observations to the clinical activity of XL228, tumor biopsy specimens obtained from the solid tumor Phase 1 clinical study were evaluated for changes in phosphorylation of pathway markers by immunofluorescence. Inhibition of Aurora, FGFR, IGF1R, and SRC family kinases was observed, as measured by decreases in kinase substrate phosphorylation in these specimens. Conclusions: XL228 shows a broad pattern of protein kinase inhibition, including the tyrosine kinases IGF1R, SRC, ABL, FGFR1-3, and ALK and the serine/threonine kinases Aurora A and Aurora B. The clinical impact of this target profile is currently being assessed in two Phase 1 studies. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C192.

Interaction of ABC Multidrug Transporters with Anticancer Protein Kinase Inhibitors: Substrates and/or Inhibitors?

Protein kinase inhibitors (PKI) are becoming key agents in modern cancer chemotherapy, and combination of PKIs with classical chemotherapeutic drugs may help to overcome currently untreatable metastatic cancers. Since chemotherapy resistance is a recurrent problem, mechanisms of resistance should be clarified in order to help further drug development. Here we suggest that in addition to PKI resistance based on altered target structures, the active removal of these therapeutic agents by the MDR-ABC transporters should also be considered as a major cause of clinical resistance. We discuss the occurring systemic and cellular mechanisms, which may hamper PKI efficiency, and document the role of selected MDR-ABC transporters in these phenomena through their interactions with these anticancer agents. Moreover, we suggest that PKI interactions with ABC transporters may modulate overall drug metabolism, including the fate of diverse, chemically or target-wise unrelated drugs. These effects are based on multiple forms of MDR-ABC transporter interaction with PKIs, as these compounds may be both substrates and/or inhibitors of an ABC transporter. We propose that these interactions should be carefully considered in clinical application, and a combined MDR-ABC transporter and PKI effect may bring a major advantage in future drug development.

Non-specific in vivo inhibition of CK1 by the pyridinyl imidazole p38 inhibitors SB 203580 and SB 202190

Small-molecule inhibitors of protein kinases have contributed immensely to our understanding of biological signaling pathways and have been exploited therapeutically for the treatment of cancers and other disease states. The pyridinyl imidazole compounds SB 203580 and SB 202190 were identified as ATP competitive antagonists of the p38 stress-activated protein kinases and have been widely used to elucidate p38-dependent cellular processes. Here, we identify SB 203580 and SB 202190 as potent inhibitors of stress-induced CREB phosphorylation on Serine 111 (Ser-111) in intact cells. Unexpectedly, we found that the inhibitory activity of SB 203580 and SB 202190 on CREB phosphorylation was independent of p38, but instead correlated with inhibition of casein kinase 1 (CK1) in vitro. The inhibition of CK1-mediated CREB phosphorylation by concentrations of pyridinyl imidazoles commonly employed to suppress p38, suggests that in some cases conclusions of p38-dependence derived solely from the use of these inhibitors may be invalid.

Targeted Small-Molecule Inhibitors of Protein Kinase B as Anticancer Agents

Protein kinase B (PKB or Akt) is a central component of the PI3K - PKB - mTOR signalling cascade and is firmly established as an attractive target for pharmacological intervention in cancer. A number of small molecule inhibitors with well-defined, direct molecular interactions with PKB are now known, covering a range of mechanisms from ATP- or substrate-competitive inhibition, through allosteric modulation of the kinase activity, to inhibition of the phosphatidylinositol-dependent activation process. The development of small molecule inhibitors of PKB has benefited greatly from the application of structural biology techniques, particularly for lead generation and the optimisation of compound potency and selectivity. The development of the major chemical series of PKB inhibitors will be outlined, with an emphasis on the application of structure-based design and the strategies used to optimise compound pharmacodynamics, efficacy and therapeutic window. The development of small molecules targeting PKB for anticancer therapy has reached an exciting stage, with the first selective inhibitors entering clinical trials, and several additional chemotypes demonstrating efficacy in preclinical models.

Kinase-likeness and Kinase-Privileged Fragments: Toward Virtual Polypharmacology

Small molecule protein kinase inhibitors are widely employed as biological reagents and as leads in the design of drugs for a variety of diseases. We investigated the phenomenon of kinase-likeness, i.e., the propensity of ligands to inhibit protein kinases, in the context of kinase-specific substructural fragments. The frequency of occurrence of multiple structural fragments in kinase inhibitor libraries relative to nonkinase compounds has been analyzed. A combination of structural fragment counts, termed the "2-0" kinase-likeness rule, provides approximately 5-fold enrichment in kinase active compounds. This rule has been validated using in-house kinase counterscreening data and applied prospectively to uncover kinase activities in marketed drugs. In addition, the role of discriminating fragments in kinase recognition was interrogated using available structural data, providing an insight into their effect on inhibitor potency and selectivity. One of these fragments, bisarylaniline, has been characterized as a kinase-privileged fragment with specific binding preferences and a link to increased activity within kinases.

The selectivity of protein kinase inhibitors: a further update

The specificities of 65 compounds reported to be relatively specific inhibitors of protein kinases have been profiled against a panel of 70-80 protein kinases. On the basis of this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small-molecule inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiological roles of p38 MAPK (mitogen-activated protein kinase) isoforms, PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-kinases, PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK kinase-1) or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB (protein kinase B/Akt), rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)-raptor (regulatory associated protein of mTOR) complex], CT 99021 to inhibit GSK3 (glycogen synthase kinase 3), BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 kinase), D4476 to inhibit CK1 (casein kinase 1), VX680 to inhibit Aurora kinases, and roscovitine as a pan-CDK (cyclin-dependent kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-molecule inhibitors of protein kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds that we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular protein kinases in cellular processes.

Protein Kinases as Small Molecule Inhibitor Targets in Inflammation

The human kinome describes a major group of intracellular signalling molecules. In the last few years, many molecules in the group have become targets for therapeutic interventions. Due to the conserved reaction mechanism of catalysis, protein kinases seem well "drugable" by small molecular weight inhibitors, thus opening the chance to novel oral bioavailable drug development. A large number of small molecule weight inhibitors for protein kinases have already been introduced into research and these molecules are extensively analysed in regard to their efficiency, potency and selectivity. Here we summarise the use of small molecule protein kinase inhibitors relevant for acute and chronic inflammation based on their essential role in cellular signaling mechanisms in immune cells such as macrophages, lymphoytes and granulocytes. We describe the progress made to develop inhibitors against Toll-like receptor associated kinases (IRAKs), against the MAPK kinase kinases Cot/Tpl-2 and TAK1, against Inhibitor-kappaB kinases (IKKs), against MAPK kinases (MEKs, MKKs), against MAPKs (ERK2, p38, JNKs) and against their downstream kinases MNK1 and MK2/3. This overview should help to keep up with the fast developing field and the continuously growing number of protein kinase inhibitors.

Duplexed, bead‐based competitive assay for inhibitors of protein kinases

Many cellular signal transduction cascades have protein kinases as critical components. Small molecule protein kinase inhibitors can be effective as laboratory probes and drugs. Methods that allow two or more kinases to be evaluated simultaneously for inhibition by a small molecule would allow unequivocal tests of specificity and selectivity of action of the small molecule. Two hexahistidine-tagged activin receptor-like kinases were expressed in E. coli, purified, and bound to nickel beads. A fluorescent kinase ligand (F-KL) that binds to the ATP-binding site of these kinases with nanomolar affinity was developed. Binding of F-KL with kinase on the bead made the beads bright, and inhibitors decreased the brightness. A test panel of 17 nonfluorescent kinase inhibitors, spanning two orders of magnitude affinity for the kinases, gave K(d) values for the kinases that correlated well with a fluorescence polarization assay. Results were obtained for the kinases in duplex, using an autosampler to send beads from a 96-well plate to a flow cytometer in a format suitable for high throughput screening. Inhibitors of kinases can be measured in duplex in a high throughput format by flow cytometry, if a suitable fluorescent ligand is available.

Spreading of mammalian DNA-damage response factors studied by ChIP-chip at damaged telomeres

Phosphorylated histone H2AX (gammaH2AX) is generated in nucleosomes flanking sites of DNA double-strand breaks, triggering the recruitment of DNA-damage response proteins such as MDC1 and 53BP1. Here, we study shortened telomeres in senescent human cells. We show that most telomeres trigger gammaH2AX formation, which spreads up to 570 kb into the subtelomeric regions. Furthermore, we reveal that the spreading patterns of 53BP1 and MDC1 are very similar to that of gammaH2AX, consistent with a structural link between these factors. Moreover, different subsets of telomeres signal in different cell lines, with those that signal tending to equate to the shortest telomeres of the corresponding cell line, thus linking telomere attrition with DNA-damage signalling. Notably, we find that, in some cases, gammaH2AX spreading is modulated in a manner suggesting that H2AX distribution or its ability to be phosphorylated is not uniform along the chromosome. Finally, we observe weak gammaH2AX signals at telomeres of proliferating cells, but not in hTERT immortalised cells, suggesting that low telomerase activity leads to telomere uncapping and senescence in proliferating primary cells.

Histone H2AX Is a Mediator of Gastrointestinal Stromal Tumor Cell Apoptosis following Treatment with Imatinib Mesylate

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract and are caused by activating mutations of the KIT or platelet-derived growth factor receptor alpha (PDGFRA) tyrosine kinases. GISTs can be successfully treated with imatinib mesylate, a selective small-molecule protein kinase inhibitor that was first clinically approved to target the oncogenic BCR-ABL fusion protein kinase in chronic myelogenous leukemia, but which also potently inhibits KIT and PDGFR family members. The mechanistic events by which KIT/PDGFRA kinase inhibition leads to clinical responses in GIST patients are not known in detail. We report here that imatinib triggers GIST cell apoptosis in part through the up-regulation of soluble histone H2AX, a core histone H2A variant. We found that untreated GIST cells down-regulate H2AX in a pathway that involves KIT, phosphoinositide-3-kinase, and the ubiquitin/proteasome machinery, and that the imatinib-mediated H2AX up-regulation correlates with imatinib sensitivity. Depletion of H2AX attenuated the apoptotic response of GIST cells to imatinib. Soluble H2AX was found to sensitize GIST cells to apoptosis by aberrant chromatin aggregation and a transcriptional block. Our results underscore the importance of H2AX as a human tumor suppressor protein, provide mechanistic insights into imatinib-induced tumor cell apoptosis and establish H2AX as a novel target in cancer therapy.

Small Molecule Inhibitors of Protein Kinases in Cancer- How to Overcome Resistance

Small molecule protein kinase inhibitors show great promise as anti-cancer agents, however, de novo and acquired resistance present problems. These are reviewed and illustrated using the receptor tyrosine kinase, KIT, as an example. Emerging solutions are presented, such as targeting active kinase conformations.

Recent Progress in Development of Non-ATP Competitive Small-Molecule Inhibitors of Protein Kinases

The majority of marketed and late stage development kinase inhibitors are reported to be ATP-competitive. As a result, many promising drug candidates display non-specific activity that results in undesired physiological effects. There is growing interest towards non-ATP competitive kinase inhibitors, as they are expected to yield highly specific and efficacious molecules devoid of non-mechanistic toxicity. Recent developments in this area are summarized in our review.

A Small‐Molecule–Protein Interaction System with Split‐Ubiquitin as Sensor

The identification of receptors for small molecules is of great pharmaceutical importance for drug-discovery research. Several systems for the identification of protein-small-molecule interactions have been developed in the past. These were modifications of the classical yeast two-hybrid system, relying on a transcriptional read-out following nuclear translocation of the complex. Here we present a novel three-hybrid technology based on the split-ubiquitin system for the analysis of protein-small-molecule interactions independently of a nuclear translocation of the complex. The performance of the system is compared to a method based on the classical yeast two-hybrid system by using a chemical inducer of dimerization (CID) comprised of methotrexate linked to dexamethasone. Steric issues are addressed by varying the linker length of the compounds, as well as by comparing the orientation of fusion proteins. The system is further extended to the analysis of a small-molecule inhibitor of human PCTAIRE protein kinase 3, which is related to cyclin-dependent kinases (CDKs), an important class of pharmaceutical targets.

Combined inhibition of the phosphatidylinositol 3-kinase/Akt and Ras/mitogen-activated protein kinase pathways results in synergistic effects in glioblastoma cells

The present study uses cell-based screening assays to assess the anticancer effects of targeting phosphatidylinositol 3-kinase-regulated integrin-linked kinase (ILK) in combination with small-molecule inhibitors of Raf-1 or mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (MEK). The objective was to determine if synergistic interactions are achievable through the use of agents targeting two key cell signaling pathways involved in regulating glioblastoma cancer. The phosphatidylinositol 3-kinase/protein kinase B (PKB)/Akt and the Ras/MAPK pathway were targeted for their involvement in cell survival and cell proliferation, respectively. The glioblastoma cell lines U87MG, SF-188, and U251MG were transiently transfected with an antisense oligonucleotide targeting ILK (ILKAS) alone or in combination with the Raf-1 inhibitor GW5074 or with the MEK inhibitor U0126. Dose and combination effects were analyzed by the Chou and Talalay median-effect method and indicated that combinations targeting ILK with either Raf-1 or MEK resulted in a synergistic interaction. Glioblastoma cells transfected with ILKAS exhibited reduced levels of ILK and phosphorylated PKB/Akt on Ser473 but not PKB/Akt on Thr308 as shown by immunoblot analysis. These results were confirmed using glioblastoma cells transfected with ILK small interfering RNA, which also suggested enhanced gene silencing when used in combination with U0126. U87MG glioblastoma cells showed a 90% (P < 0.05) reduction in colony formation in soft agar with exposure to ILKAS in combination with GW5074 compared with control colonies. A substantial increase in Annexin V-positive cells as determined by using fluorescence-activated cell sorting methods were seen in combinations that included ILKAS. Combinations targeting ILK and components of the Ras/MAPK pathway result in synergy and could potentially be more effective against glioblastoma cancer than monotherapy.