Supplementary data are available at Bioinformatics online.

Mutant K-ras activity leads to the activation of the RAS/RAF/MEK/ERK pathway in approximately 44% of colorectal cancer (CRC) tumors. Accordingly, several inhibitors of the MEK pathway are under clinical evaluation in several malignancies including CRC. The aim of this study was to develop and characterize predictive biomarkers of response to the MEK1/2 inhibitor AZD6244 in CRC in order to maximize the clinical utility of this agent. Twenty-seven human CRC cell lines were exposed to AZD6244 and classified according to the IC 50 value as sensitive ( 0.1 mmol/L) or resistant (>1 mmol/L). All cell lines were subjected to immunoblotting for effector proteins, K-ras/BRAF mutation status, and baseline gene array analysis. Further testing was done in cell line xenografts and K-ras mutant CRC human explants models to develop a predictive genomic classifier for AZD6244. The most sensitive and resistant cell lines were subjected to differential gene array and pathway analyses. Members of the Wnt signaling pathway were highly overexpressed in cell lines resistant to AZD6244 and seem to be functionally involved in mediating resistance by shRNA knockdown studies. Baseline gene array data from CRC cell lines and xenografts were used to develop a k-top scoring pair (k-TSP) classifier, which predicted with 71% accuracy which of a test set of patient-derived K-ras mutant CRC explants would respond to AZD6244, providing the basis for a patient-selective clinical trial. These results also indicate that resistance to AZD6244 may be mediated, in part, by the upregulation of the Wnt pathway, suggesting potential rational combination partners for AZD6244 in CRC. Mol Cancer Ther; 9(12); 3351-62. Ó2010 AACR.

Purpose The MAPK pathway is a crucial regulator of cell proliferation, survival, and resistance to apoptosis. MEK inhibitors are being explored as a treatment option for patients with KRAS mutant colorectal cancer (CRC) who are not candidates for EGFR-directed therapies. Initial clinical results of MEK inhibitors have yielded limited single-agent activity in CRC, indicating that rational combination strategies are needed. Experimental Design In this study, we performed unbiased GSEA and synthetic lethality screens with selumetinib, which identified the non-canonical Wnt/Ca++ signaling pathway as a potential mediator of resistance to the MEK1/2 inhibitor selumetinib. To test this, we utilized shRNA constructs against relevant WNT receptors and ligands resulting in increased responsiveness to selumetinib in CRC cell lines. Further, we evaluated the rational combination of selumetinib and WNT pathway modulators and demonstrated synergistic antiproliferative effects in in vitro and in vivo models of CRC. Results Importantly, this combination not only demonstrated tumor growth inhibition but also tumor regression in the more clinically relevant patient-derived tumor explant (PDTX) models of CRC. In mechanistic studies, we observed a trend towards increased markers of apoptosis in response to the combination of MEK and WntCa++ inhibitors, which may explain the observed synergistic antitumor effects. Conclusions These results strengthen the hypothesis that targeting both the MEK and Wnt pathways may be a clinically effective rational combination strategy for metastatic CRC patients.

Purpose The Aurora kinases are a family of conserved serine-threonine kinases with key roles in mitotic cell division. As with other promising anti-cancer targets, patient selection strategies to identify a responsive subtype will likely be required for successful clinical development of Aurora kinase inhibitors. The purpose of this study was to evaluate the antitumor activity of the Aurora and angiogenic kinase inhibitor ENMD-2076 against preclinical models of breast cancer with identification of candidate predictive biomarkers. Experimental Design Twenty-nine breast cancer cell lines were exposed to ENMD-2076 and the effects on proliferation, apoptosis, and cell cycle distribution were evaluated. In vitro activity was confirmed in in MDA-MB-468 and MDA-MB-231 triple-negative breast cancer xenografts. Systematic gene expression analysis was used to identify up- and down-regulated pathways in the sensitive and resistant cell lines, including within the triple-negative breast cancer subset. Results ENMD-2076 demonstrated anti-proliferative activity against breast cancer cell lines, with more robust activity against cell lines lacking estrogen receptor expression and those without increased HER2 expression. Within the triple-negative breast cancer subset, cell lines with a p53 mutation and increased p53 expression were more sensitive to the cytotoxic and pro-apoptotic effects of ENMD-2076 exposure than cell lines with decreased p53 expression. Conclusions ENMD-2076 exhibited robust anticancer activity against models of triple-negative breast cancer and the candidate predictive biomarkers identified in this study may be useful in selecting patients for Aurora kinase inhibitors in the future.

Purpose Results from clinical trials involving resistance to molecularly targeted therapies have revealed the importance of rational single agent and combination treatment strategies. In this study, we tested the efficacy of a type 1 insulin-like growth factor receptor (IGF1R)/insulin receptor (IR) tyrosine kinase inhibitor (TKI), OSI-906, in combination with a MEK 1/2 inhibitor based on evidence that the MAPK pathway was upregulated in colorectal cancer (CRC) cell lines that were resistant to OSI-906. Experimental Design The antiproliferative effects of OSI-906 and the MEK 1/2 inhibitor U0126, were analyzed both as single agents and in combination in 13 CRC cell lines in vitro. Apoptosis, downstream effector proteins, and cell cycle were also assessed. Additionally, the efficacy of OSI-906 combined with the MEK 1/2 inhibitor selumetinib (AZD6244, ARRY-142886), was evaluated in vivo using human CRC xenograft models. Results The combination of OSI-906 and U0126 resulted in synergistic effects in 11 out of 13 CRC cell lines tested. This synergy was variably associated with apoptosis or cell cycle arrest in addition to molecular effects on pro-survival pathways. The synergy was also reflected in the in vivo xenograft studies following treatment with the combination of OSI-906 and selumetinib. Conclusions Results from this study demonstrate synergistic antiproliferative effects in response to the combination of OSI-906 with a MEK 1/2 inhibitor in CRC cell line models both in vitro and in vivo, which supports the rational combination of OSI-906 with a MEK inhibitor in patients with CRC.

Sequencing and microarray samples often are collected or processed in multiple batches or at different times. This often produces technical biases that can lead to incorrect results in the downstream analysis. There are several existing batch adjustment tools for ‘-omics’ data, but they do not indicate a priori whether adjustment needs to be conducted or how correction should be applied. We present a software pipeline, BatchQC, which addresses these issues using interactive visualizations and statistics that evaluate the impact of batch effects in a genomic dataset. BatchQC can also apply existing adjustment tools and allow users to evaluate their benefits interactively. We used the BatchQC pipeline on both simulated and real data to demonstrate the effectiveness of this software toolkit.Availability and Implementation: BatchQC is available through Bioconductor: http://bioconductor.org/packages/BatchQC and GitHub: https://github.com/mani2012/BatchQC.Contact: wej@bu.eduSupplementary information: Supplementary data are available at Bioinformatics online.

High-throughput RNA sequencing is rapidly emerging as a favourite method for gene expression studies. We review three software packages -- edgeR, DEGseq and baySeq -- from Bioconductor http://bioconductor.org for analysing RNA-sequencing data. We focus on three aspects: normalisation, statistical models and the testing employed on these methods. We also discuss the advantages and limitations of these software packages.

The p21-activated kinase (PAK) family of serine/threonine kinases, which are overexpressed in several cancer types, are critical mediators of cell survival, motility, mitosis, transcription, and translation. In the study presented here, we utilized a panel of colorectal cancer (CRC) cell lines to identify potential biomarkers of sensitivity or resistance that may be used to individualize therapy to the PAK inhibitor PF-03758309. We observed a wide range of proliferative responses in the CRC cell lines exposed to PF-03758309, this response was recapitulated in other phenotypic assays such as anchorage-independent growth, three-dimensional (3D) tumor spheroid formation, and migration. Interestingly, we observed that cells most sensitive to PF-03758309 exhibited up-regulation of genes associated with a mesenchymal phenotype (CALD1, VIM, ZEB1) and cells more resistant had an up-regulation of genes associated with an epithelial phenotype (CLDN2, CDH1, CLDN3, CDH17) allowing us to derive an epithelial-to-mesenchymal transition (EMT) gene signature for this agent. We assessed the functional role of EMT-associated genes in mediating responsiveness to PF-3758309, by targeting known genes and transcriptional regulators of EMT. We observed that suppression of genes associated with the mesenchymal phenotype conferred resistance to PF-3758309, in vitro and in vivo. These results indicate that PAK inhibition is associated with a unique response phenotype in CRC and that further studies should be conducted to facilitate both patient selection and rational combination strategies with these agents.