Overcoming IGF1R/IR resistance through inhibition of MEK signaling in colorectal cancer models.

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, OSI-906, in combination with a mitogen-activated protein (MAP)-ERK kinase (MEK) 1/2 inhibitor based on evidence that the MAP kinase pathway was upregulated in colorectal cancer 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 colorectal cancer cell lines in vitro. Apoptosis, downstream effector proteins, and cell cycle were also assessed. In addition, the efficacy of OSI-906 combined with the MEK 1/2 inhibitor selumetinib (AZD6244, ARRY-142886) was evaluated in vivo using human colorectal cancer xenograft models. RESULTS: The combination of OSI-906 and U0126 resulted in synergistic effects in 11 of 13 colorectal cancer cell lines tested. This synergy was variably associated with apoptosis or cell-cycle arrest in addition to molecular effects on prosurvival 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 an MEK 1/2 inhibitor in colorectal cancer cell line models both in vitro and in vivo, which supports the rational combination of OSI-906 with an MEK inhibitor in patients with colorectal cancer. Clin Cancer Res; 19(22); 6219-29. ©2013 AACR.

Association of the epithelial-to-mesenchymal transition phenotype with responsiveness to the p21-activated kinase inhibitor, PF-3758309, in colon cancer models.

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.

Preclinical activity of the rational combination of selumetinib (AZD6244) in combination with vorinostat in KRAS-mutant colorectal cancer models.

PURPOSE: Despite the availability of several active combination regimens for advanced colorectal cancer (CRC), the 5-year survival rate remains poor at less than 10%, supporting the development of novel therapeutic approaches. In this study, we focused on the preclinical assessment of a rationally based combination against KRAS-mutated CRC by testing the combination of the MEK inhibitor, selumetinib, and vorinostat, a histone deacetylase (HDAC) inhibitor. EXPERIMENTAL DESIGN: Transcriptional profiling and gene set enrichment analysis (baseline and posttreatment) of CRC cell lines provided the rationale for the combination. The activity of selumetinib and vorinostat against the KRAS-mutant SW620 and SW480 CRC cell lines was studied in vitro and in vivo. The effects of this combination on tumor phenotype were assessed using monolayer and 3-dimensional cultures, flow cytometry, apoptosis, and cell migration. In vivo, tumor growth inhibition, (18)F-fluoro-deoxy-glucose positron emission tomography (FDG-PET), and proton nuclear magnetic resonance were carried out to evaluate the growth inhibitory and metabolic responses, respectively, in CRC xenografts. RESULTS: In vitro, treatment with selumetinib and vorinostat resulted in a synergistic inhibition of proliferation and spheroid formation in both CRC cell lines. This inhibition was associated with an increase in apoptosis, cell-cycle arrest in G(1), and reduced cellular migration and VEGF-A secretion. In vivo, the combination resulted in additive tumor growth inhibition. The metabolic response to selumetinib and vorinostat consisted of significant inhibition of membrane phospholipids; no significant changes in glucose uptake or metabolism were observed in any of the treatment groups. CONCLUSION: These data indicate that the rationally based combination of the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, selumetinib, with the HDAC inhibitor vorinostat results in synergistic antiproliferative activity against KRAS-mutant CRC cell lines in vitro. In vivo, the combination showed additive effects that were associated with metabolic changes in phospholipid turnover, but not on FDG-PET, indicating that the former is a more sensitive endpoint of the combination effects.

Phase I safety, pharmacokinetic, and pharmacodynamic study of ENMD-2076, a novel angiogenic and Aurora kinase inhibitor, in patients with advanced solid tumors.

PURPOSE: ENMD-2076 is a unique orally bioavailable Aurora kinase and VEGFR inhibitor. The purpose of this phase 1 study of ENMD-2076 was to determine the MTD, pharmacokinetic, and pharmacodynamic profiles and preliminary antitumor activity. EXPERIMENTAL DESIGN: Patients with refractory advanced solid malignancies were treated with ENMD-2076 orally with continuous once daily dosing. Doses from 60 to 200 mg/m(2) were evaluated using a standard 3 (to 4) + 3 design. Pharmacokinetic parameters were studied on days 1, 28, and 30 to 35 of cycle 1. Expanded MTD cohorts included patients with ovarian cancer, colorectal cancer, and refractory solid tumors. RESULTS: A total of 67 patients (46 F, 21M; ages 30-76) entered the study. Dose levels of 60, 80, 120, 200, and 160 mg/m(2) were evaluated. Two patients experienced grade 3 hypertension at 200 mg/m(2), and additional grade 3 neutropenia events limited tolerability at this dose. An intermediate dose of 160 mg/m(2) was determined to be the MTD. The most common drug-related adverse events included hypertension, nausea/vomiting, and fatigue. The pharmacokinetics of ENMD-2076 were characterized by a rapid absorption phase (T(max) 3-7.8 hours), a t(1/2) of 27.3 to 38.3 hours after a single dose, and dose proportional exposure. Decreased plasma sVEGFR2 was observed posttreatment. Two patients with platinum refractory/resistant ovarian cancer had RECIST partial responses. CONCLUSIONS: ENMD-2076 was well tolerated, had a linear pharmacokinetic profile, and showed promising antitumor activity, particularly in ovarian cancer. The recommended phase 2 dose of ENMD-2076 is 160 mg/m(2) administered orally once daily with continuous dosing.

Identification of predictive markers of response to the MEK1/2 inhibitor selumetinib (AZD6244) in K-ras-mutated colorectal cancer.

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 µmol/L) or resistant (>1 µmol/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.

Development of an integrated genomic classifier for a novel agent in colorectal cancer: approach to individualized therapy in early development.

BACKGROUND: A plethora of agents is in early stages of development for colorectal cancer (CRC), including those that target the insulin-like growth factor I receptor (IGFIR) pathway. In the current environment of numerous cancer targets, it is imperative that patient selection strategies be developed with the intent of preliminary testing in the latter stages of phase I trials. The goal of this study was to develop and characterize predictive biomarkers for an IGFIR tyrosine kinase inhibitor, OSI-906, that could be applied in CRC-specific studies of this agent. METHODS: Twenty-seven CRC cell lines were exposed to OSI-906 and classified according to IC(50) value as sensitive (5 micromol/L). Cell lines were subjected to immunoblotting and immunohistochemistry for effector proteins, IGFIR copy number by fluorescence in situ hybridization, KRAS/BRAF/phosphoinositide 3-kinase mutation status, and baseline gene array analysis. The most sensitive and resistant cell lines were used for gene array and pathway analyses, along with shRNA knockdown of highly ranked genes. The resulting integrated genomic classifier was then tested against eight human CRC explants in vivo. RESULTS: Baseline gene array data from cell lines and xenografts were used to develop a k-top scoring pair (k-TSP) classifier, which, in combination with IGFIR fluorescence in situ hybridization and KRAS mutational status, was able to predict with 100% accuracy a test set of patient-derived CRC xenografts. CONCLUSIONS: These results indicate that an integrated approach to the development of individualized therapy is feasible and should be applied early in the development of novel agents, ideally in conjunction with late-stage phase I trials.