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BRAF V600E Disrupts AZD6244-Induced Abrogation of Negative Feedbackpathways Between Extracellular Signal-Regulated Kinase and Raf Proteins

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BRAF V600E Disrupts AZD6244-Induced Abrogation of Negative Feedbackpathways Between Extracellular Signal-Regulated Kinase and Raf Proteins Research Article BRAF V600E Disrupts AZD6244-Induced Abrogation of Negative FeedbackPathways between Extracellular Signal-Regulated Kinase and Raf Proteins Bret B. Friday,1 Chunrong Yu,4 Grace K. Dy,4 Paul D. Smith,3 Liang Wang,2 Stephen N. Thibodeau,2 and Alex A. Adjei4 Departments of 1Oncology and 2Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota; 3AstraZeneca, Macclesfield, United Kingdom; and 4Department of Medicine, Roswell Park Cancer Institute, Buffalo, New York Abstract Raf phosphorylates and activates the dual specificity kinases AZD6244 (ARRY 142886) is a potent and selective mitogen- MEK1 and MEK2, which successively phosphorylate and activate activated protein kinase (MAPK)/extracellular signal-regulated the proline-directed serine/threonine kinases ERK1 and ERK2, kinase (ERK) kinase (MEK) inhibitor currently in early clinical which have multiple downstream targets, including Elk-1, c-Ets1, trials. We examined the activity of AZD6244 in a panel of c-Ets2, p90RSK1, MNK1, MNK2, and TOB (1–3). non–small cell lung cancer and a panel of cell lines represent- Hyperactivation of the MAPK signaling cascade resulting from ing many cancer types using in vitro growth assays. AZD6244 receptor tyrosine kinase overexpression/mutation or MAPK family member mutation has a well-established role in oncogenesis and induced G0-G1 cell cycle arrest in sensitive cell lines that pri- marily included cells containing the BRAF V600E mutation. tumor growth (4, 5). Activating mutations in Ras and BRAF are common in many types of cancer and result in constitutive In these cells, G0-G1 arrest is accompanied by the up-regulation of the cell cycle inhibitors p21WAF1 and p27Kip1 and down- activation of MAPK signaling. K-ras mutations are found in up to regulation of cyclin D1. In the majority of cell lines tested, 30% of all cancers and are particularly common in pancreatic including those with K-ras or non-V600E BRAF mutations, cancers (90%) and colon cancers (50%; ref. 6). BRAF mutations have AZD6244 induced the accumulation of phospho-MEK, an effect a more narrow distribution but are prevalent in a few specific not observed in the most sensitive BRAF V600E-containing malignancies, including melanoma (63%), papillary thyroid cancers cells. Accumulation of phospho-MEKin non–V600E-containing (45%), and low-grade ovarian cancers (36%; refs. 7–9). Of interest, cell lines is due to abrogation of negative feedback pathways. BRAF V600E has also been found in up to 70% of colon cancers BRAF V600E disrupts negative feedback signaling, which results characterized by the presence of defective DNA mismatch repair, in enhanced baseline phospho-MEKexpression. Exogenous specifically those resulting from MLH1 promoter hypermethylation expression of BRAF V600E disrupts feedback inhibition but (10). Although many BRAF mutations have been described, a single does not sensitize cells to AZD6244. Specific suppression of amino acid change, resulting in a valine to glutamic acid sub- f endogenous BRAF V600E does not confer resistance to AZD6244 stitution at position 600 (V600E), accounts for 80% of the but enhances sensitivity to AZD6244. Thus, our findings show mutations (11). The V600E mutation results in elevated constitutive that BRAF V600E marks cells with an in vitro requirement for kinase activity, which is thought to occur by disruption of the MAPKsignaling to support proliferation. These cells are inactive conformation by the mutation (12). Given its well-defined role in cancer growth, therapeutic exquisitely sensitive to AZD6244 (IC50, <100 nmol/L), have high baseline levels of phospho-MEK, and lack feedback inhibition targeting of the MAPK pathway has been an area of intense between ERKand Raf. These data suggest an approach to investigation, and MEK inhibitors are seen as a promising new identifying cells that may be sensitive to AZD6244 and other approach for cancer treatment (13). The first MEK inhibitor tested MEKinhibitors. [Cancer Res 2008;68(15):6145–53] clinically was PD184352 (CI-1040), but further development was discontinued due to concerns over bioavailability and pharmaco- Introduction dynamics (14). Clinical development of two additional MEK inhi- bitors, AZD6244 (ARRY-142886) and PD0325901, continues (15, 16). The Ras/Raf/mitogen-activated protein kinase (MAPK)/extracel- The biological characterization and chemical structure of this lular signal-regulated kinase (ERK) kinase (MEK)/ERK MAPK path- agent was recently published (16). AZD6244 is a potent and way plays an important role in regulating diverse cellular functions, selective noncompetitive inhibitor of MEK1 and MEK2, with an including cell proliferation, cell cycle regulation, cell survival, and in vitro IC50 of 10 to 14 nmol/L against purified enzyme. When cell migration. Initiation of MAPK signaling occurs through acti- tested against a broad range of other serine/threonine and tyro- vation of cell surface receptor tyrosine kinases that in turn induce sine kinases, little inhibition at concentrations up to 10 Amol/L is the G protein Ras to exchange GDP for GTP. The serine/threonine detected. This high degree of specificity is thought to occur as a kinase Raf proteins are recruited to the plasma membrane by GTP- result of binding to an allosteric inhibitory site adjacent to the bound Ras and are activated via a complex mechanism. Activated ATP-binding site (17). AZD6244 has excellent preclinical activity (16, 18, 19). Safety and tolerability have been shown in initial reports from a phase I clinical trial in patients with advanced solid Requests for reprints: Alex A. Adjei, Department of Medicine, Roswell Park malignancies, and phase II efficacy studies are now under way. Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Phone: 716-845-4101; Fax: Response to MEK inhibition in vitro is variable, although the MAPK 716-845-3423; E-mail: [email protected]. I2008 American Association for Cancer Research. pathway is activated in a large proportion of tumors. Genotype, doi:10.1158/0008-5472.CAN-08-1430 with respect to activating mutations in Ras and BRAF, seems to www.aacrjournals.org 6145 Cancer Res 2008; 68: (15). August 1, 2008 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2008 American Association for Cancer Research. Cancer Research play an important role in conferring sensitivity. Tumor cell lines Immunoprecipitation kinase assay. Kinase assays were performed containing the V600E BRAF mutation are exquisitely sensitive to using a nonradioactive assay kit for BRAF activity from Promega, and the MEK inhibition, whereas cells containing Ras mutations display manufacturer’s protocol was followed with the following exceptions. The variable sensitivity, and cells with wild-type (WT) Ras and BRAF are antibody for immunoprecipitation was a rabbit polyclonal anti-BRAF from Santa Cruz Biotechnology. For analysis, levels of phosphorylated gluta- relatively resistant (20). The molecular mechanisms conferring S in vitro thione -transferase-MEK were determined in the fraction not bound to sensitivity to MEK inhibitors are not well understood. Sepharose beads. Initial studies with AZD6244 suggest that the pattern of sensitivity Exogenous BRAF expression. Cells were plated at f80% confluence in with respect to BRAF and KRAS mutation status is similar to other a six-well culture dish. After 24 h, the cells were transfected with MEK inhibitors (16). To investigate the determinants of sensitivity hemagglutinin-tagged plasmids expressing either WT or V600E BRAF to AZD6244 and enhance the clinical development of this exciting (Biomyx Technology) using Lipofectamine 2000 (Invitrogen) following the class of compounds, we undertook the present study to further manufacturer’s protocol. characterize the in vitro effects of AZD6244 in a broad panel of Retroviral-mediated short hairpin RNA expression. Retroviral cell lines. plasmids containing control or BRAF-specific short hairpin RNAs (shRNA) were provided by Dr. David Tuveson (Cambridge Research Institute, Cambridge, United Kingdom). BRAF-specific constructs used in this study Materials and Methods include COM-4, which targets both WT and V600E BRAF, and MU-A, which Materials and cells. AZD6244 was provided by AstraZeneca. U0126 and targets only V600E BRAF (23). Retroviruses were prepared by transient PD98059 were purchased from Calbiochem. All non–small cell lung cancer transfection of Phoenix helper virus–free amphotropic producer cells (NSCLC) cell lines, except MV522, were obtained from the American Type (ATCC), and cell lines were infected with retroviral supernatants using Culture Collection (ATCC). MV522 cells were provided by Dr. Julian Molina previously described techniques (24). (Mayo Clinic, Rochester, MN). HT29, Colo205, HCT116, MiaPaCa-2, MDA- Allele-specific PCR. Total cellular RNA was prepared from cells using MB-231, CAKI-1, and BxPC3 were obtained from ATCC. SK-MEL 3 and SK- the RNeasy Mini kit (Qiagen) following the manufacturer’s protocol. cDNA MEL 28 cells were provided by Dr. Svetomir Markovic (Mayo Clinic). All cells was prepared with 5 Ag RNA and oligo(dT) primers using the SuperScript III were cultured in RPMI 1640 containing 10% fetal bovine serum, penicillin, First-Strand Synthesis System following the manufacturer’s protocol ¶ and streptomycin and incubated in a humidified incubator with 5% CO2 at (Invitrogen). Primers used in PCRs included 5 -GTGATTTTGGTCTAGCTA- 37jC. Genotypes for all cell lines, except MV522, have been previously CAGT-3¶ and 5¶-GTGATTTTGGTCTAGCTACAGA-3¶,
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