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 inhibitors, 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¶, which amplify the WT determined (11, 21). The genotype of MV522 relative to K-ras and BRAF was allele or BRAF V600E mutation, respectively (25), and the reverse primer determined by direct sequencing of genomic PCR products encompassing 5¶-GTCCCTGTTGTTGATGTTTGAATA-3¶, which produces a 212-bp PCR exons 2 and 3 of K-ras and exons 11 and 15 of BRAF using previously product. Amplification was carried out using FastStart Taq DNA polymerase described primers and techniques (22). (Roche) following the manufacturer’s guidelines. Annealing temperature Cell growth assays. For 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme- was 58jC for 29 or 31 cycles in WT or mutant samples, respectively. Control thoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) cell proliferation reactions using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) assays, cells were plated in 96-well culture dishes at a density of 500 to 1,000 primers were done as previously described for 23 cycles (26). PCR samples per well. The following day, AZD6244 or DMSO was added to the cells at were separated using agarose gel electrophoresis and the products were concentrations ranging between 1 nmol/L and 10 Amol/L depending on the visualized using ethidium bromide staining. For quantitation, gel images experiment. Cell proliferation was determined 4 to 5 d later using CellTiter were captured and analyzed using a Syngene bioimaging system. 96 AQueous One Solution (Promega). For clonogenic assays, cells were plated at 500 to 1,000 in 35-mm culture dishes. AZD6244 or DMSO was Results added the next day, and cells were grown for 7 to 10 d to allow colonies In vitro to form. The number of viable colonies was counted after staining with activity of AZD6244. Sensitivity to the MEK inhibitor Coomassie blue. CI-1040 seems to depend on the presence of the BRAF activating Cell cycle analysis. Cells were plated at f50% confluence in 100-mm mutation V600E (20); however, sensitivity among cell lines culture dishes. AZD6244 (1 Amol/L) or DMSO was added 24 h later and cells containing activating Ras mutations is variable and cells containing were collected after a 24-h incubation. Cells were rinsed in PBS, fixed in 95% WT MAPK pathway proteins are resistant. Sensitivity to AZD6244 ethanol, pelleted by centrifugation, and resuspended in RNase A at 1 mg/mL seems to follow the same general characteristics based on recently in 0.1% sodium citrate. Following incubation at 37jC for 15 min, propidium published data (16). To more fully characterize the in vitro activity A iodide was added to 100 g/mL in 0.1% sodium citrate. DNA content was of AZD6244, we used a panel of NSCLC cell lines as well as multiple determined by flow cytometry and the proportion of cells in each cell cycle additional cell lines that harbor a variety of Ras and BRAF phase was determined using ModFit software (Verity Software House). mutations (Table 1). For each cell line, the IC was determined in a Immunoblot analysis. Following drug treatment, cell lysates were 50 prepared and subjected to SDS-PAGE immunoblot analysis using standard MTS assay using a 4- to 6-day continuous exposure to AZD6244. All techniques. The following primary antibodies were used: rabbit polyclonal cell lines containing BRAF V600E mutation were exquisitely 202 204 antibodies to MEK1/2, p27, phospho-ERK (Thr /Tyr ), and phospho- sensitive to AZD6244 (IC50, <100 nmol/L), whereas cells containing MEK1/2 (Ser217/221) from Cell Signaling Technology; rabbit polyclonal non-V600E BRAF mutations or activating N-ras or K-ras mutations antibodies to ERK, actin, and BRAF and murine monoclonal antibodies to with WT BRAF were variably sensitive to AZD6244. Cells Raf-1, tubulin, and cyclin D1 from Santa Cruz Biotechnology; murine containing WT BRAF and Ras proteins were resistant to AZD6244. WAF1 monoclonal antibody to p21 from NeoMarkers; and rabbit polyclonal To examine the determinants of sensitivity to AZD6244, we antibody to sprouty 2 (Spry2) from Abcam. To ensure equivalent loading chose to examine two NSCLC cell lines that harbor mutations and transfer, blots were probed with anti-actin where indicated. For resulting in constitutively active ERK, MV522 (BRAF V600E), and quantitation, blots were exposed using a Syngene bioimaging system. A549 (K-ras G12S) in detail. Both cell lines show dose-dependent Short interfering RNA knockdown. Short interfering RNA (siRNA) against BRAF, Raf-1, ERK1, and ERK2 was purchased from Ambion. Cells growth inhibition with AZD6244 using either a colony-forming or A were transfected with siRNAs at a final concentration of 20 nmol/L using MTS proliferation assay (Table 1; Fig. 1 ). The IC50s in A549 versus siPORT NeoFX Transfection Agent (Ambion) following the manufacturer’s MV522 cells differed by f20-fold using the clonogenic assay protocol. Cells were retransfected 24 h later and cell lysates were harvested (1.3 versus 0.06 Amol/L, respectively) or by f80-fold using the MTS 72 h after initial transfection. assay (6.3 versus 0.08 Amol/L, respectively). Consistent with a

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Table 1. Effects of AZD6244 in cancer cell lines in relation to the presence or absence of Ras and BRAF mutations

Cell line Ras BRAF AZD6244 IC50 (Amol/L)

NSCLC cell lines H1755 WT Mut (G468A) 8.1 MV522 WT Mut (V600E) 0.08 H1666 WT Mut (G465V) 0.36 H727 K-ras (G12V) WT 0.01 SW900 K-ras (G12V) WT 0.28 H460 K-ras (Q61H) WT 9.6 A549 K-ras (G12S) WT 6.3 H157 K-ras (G12R) WT 9.3 H358 K-ras (G12C) WT >10 H1299 N-ras (Q61K) WT >10 H661 WT WT >10 H1703 WT WT >10 H650 WT WT >10 Additional cell lines HT29 (colon) WT Mut (V600E) 0.09 Colo205 (colon) WT Mut (V600E) 0.034 SK-MEL 3 (melanoma) WT Mut (V600E) 0.039 SK-MEL 28 (melanoma) WT Mut (V600E) 0.04 HCT116 (colon) K-ras (G13D) WT 1.6 MiaPaCa-2 (pancreas) K-ras (G12C) WT 0.7 MDA-MB-231 (breast) K-ras (G13D) WT 1.6 HepG2 (hepatocellular) N-ras (Q61L) WT 0.04 CAKI-1 (kidney) WT WT >10 BxPC3 (pancreas) WT NA 5.6 HeLa (cervical) WT WT >10 U2OS (osteosarcoma) WT WT >10

previous report on MEK inhibitors in NSCLC cell lines (27), significantly increased in A549 cells by 24 h. This result suggests AZD6244 induced a G0-G1 cell cycle arrest in the sensitive cell line that AZD6244 abrogates negative feedback between ERK and MV522 and to a lesser extent in A549 cells (Fig. 1B). In neither of upstream targets in A549 cells, thus resulting in increased these cell lines was AZD6244 able to induce apoptosis, as the phospho-MEK. The negative feedback loop does not seem to be percentage of sub-G0-G1 cells in these experiments was <1% in all active in MV522 cells, as there is no corresponding change in samples. In colony-forming assays, significantly fewer colonies phospho-MEK levels. To determine if phospho-MEK accumulation were seen in the presence of AZD6244, but many isolated cells and following treatment with AZD6244 is a class effect relative to MEK small groups of cells were observed, suggesting that our results inhibitors, A549 cells were treated with AZD6244 or the alternative were due to growth inhibition rather than induction of apoptosis. commercially available laboratory-grade MEK inhibitors U0126 Sensitivity to AZD6244 correlates with MEKinhibition and or PD98059 (Fig. 2A). In each case, MEK inhibitor treatment the lack of phospho-MEKaccumulation. To examine the substantially increased phospho-MEK levels. differential sensitivity of MV522 and A549 cells to AZD6244- Accumulation of phospho-MEK after treatment with a MEK induced growth arrest, immunoblot analysis was used to determine inhibitor may be due to abrogation of negative feedback between C if AZD6244 had similar target effects (Fig. 1 ). The IC50 for MEK ERK and upstream targets; alternatively, the effect could result inhibition in MV522 was approximately 10 to 20 nmol/L and 20 to from pharmacologic effects on the MEK protein itself that inhibit 50 nmol/L in A549 cells, a difference of f2-fold, which is unlikely phosphatase activity. To show the phospho-MEK accumulation to explain the 20- to 80-fold difference in IC50 determined in results from abrogation of negative feedback, ERK function was growth assays and is probably within the inherent variability of this directly inhibited using siRNAs to down-regulate ERK1 and ERK2 assay. Next, we examined target effects as well as downstream protein expression. Because knockdown of ERK was inefficient in effectors in the MAPK pathway that have previously been A549 cells, two additional cell lines, SW900 and HeLa, were used implicated in the response of NSCLC cell lines to MEK inhibition (Fig. 2B). Significant accumulation of phospho-MEK in these cell (Fig. 1D; ref. 27). AZD6244 effectively inhibited MEK, as detected by lines was seen after knockdown of both ERK proteins, whereas the absence of phospho-ERK, after only 2 h of treatment in both knockdown of either ERK1 or ERK2 alone resulted in intermediate cell types. In MV522 cells, phospho-ERK inhibition was accompa- phospho-MEK accumulation. These results confirm that phospho- nied by increased p21WAF1 and p27Kip1 protein expression and MEK accumulation following treatment with a MEK inhibitor is decreased cyclin D1 levels, consistent with the observed G0-G1 due to abrogation of ERK-dependent feedback inhibition. arrest. Limited changes in p27Kip1 and cyclin D1 levels were also BRAF V600E mutation disrupts ERK-directed negative feed- seen in A549 cells. Interestingly, levels of phospho-MEK remained back. Our initial studies showed that accumulation of phospho- constant in MV522 cells throughout the time course but were MEK in response to AZD6244 occurred in a cell line containing an www.aacrjournals.org 6147 Cancer Res 2008; 68: (15). August 1, 2008

Figure 1. Effects of AZD6244 in NSCLC cell lines. A, MV522 and A549 cells were treated with AZD6244 at varying concentrations and response was determined using a clonogenic colony-forming assay. After 7 d, colonies were counted and the data are presented as the percentage of colonies formed relative to vehicle-treated cells (% Control). B, MV522 and A549 cells were treated with the indicated concentrations of AZD6244 for 24 h. The percentage of cells in each phase of the cell cycle was determined by quantitation of DNA content by propidium iodide staining and flow cytometry. C, MV522 and A549 cells were treated with the indicated concentrations of AZD6244 for 2 h and cell extracts were analyzed by immunoblotting for ERK and phospho-ERK (P-ERK). D, MV522 and A549 cells were treated for the indicated times with 500 nmol/L AZD6244. Cell extracts were analyzed by immunoblotting for the indicated proteins. activating K-ras mutation but did not occur in a cell line containing AZD6244 abrogates negative feedback between ERKand the BRAF V600E mutation. At least one study has suggested that Raf. Raf proteins are a likely target of AZD6244-induced feedback the BRAF V600E mutation disrupts feedback by disrupting inhibition because direct feedback inhibition between and ERK and interactions between the MAPK inhibitory proteins Spry2 or Spry4 Raf-1 or BRAF has been described (29, 30). We used two approaches and BRAF (28). To determine if AZD6244-induced phospho-MEK to test this hypothesis. First, siRNAs were used to knock down accumulation was correlated to the precise mutation contained in Raf-1 and BRAF expression in HeLa cells treated with vehicle or each cell line, MEK, phospho-MEK, ERK, and phospho-ERK protein AZD6244 (Fig. 3A). In cells treated with AZD6244, phospho-MEK expression levels were determined using immunoblot analysis in accumulation was greatest in cells transfected with a control the presence and absence of AZD6244 for all cell lines described in siRNA. Knockdown of Raf-1 or BRAF reduced phospho-MEK Table 1. If a negative feedback loop was inactive in cells containing accumulation, and combined knockdown resulted in the greatest BRAF V600E, then these cells would be expected to have higher effect. These results show that ERK feedback inhibition targets baseline levels of phospho-MEK and there should be little change both Raf-1 and BRAF. Second, BRAF kinase activity was in phospho-MEK after treatment with AZD6244. Indeed, baseline determined using an immunoprecipitation kinase assay from levels of phospho-MEK are significantly higher in cell lines con- MV522 and A549 cell extracts treated with vehicle or AZD6244 taining BRAF V600E relative to cell lines containing non-V600E (Fig. 3B). As expected, levels of phospho-MEK increased substan- mutations or WT proteins, whereas baseline levels of phospho-ERK tially in whole-cell extracts from A549 cells treated with AZD6244 are similar (Fig. 2C). In response to AZD6244, phospho-MEK levels but remained relatively unchanged in MV522 cells. BRAF kinase are essentially unchanged in V600E-containing cell lines but activity was much greater in MV522 cells compared with A549 cells, increase in all other cell lines tested as a result of abrogation but the activity was unchanged by AZD6244. However, A549 BRAF of feedback inhibition (Fig. 2D). To determine if the effect on feed- activity increased substantially after AZD6244 treatment. We also back in cell lines containing non-V600E or WT proteins was only attempted to assay for changes in Raf-1 activity with AZD6244 a characteristic of cancer cell lines, we also examined the effect of treatment in A549 cells but were unable to detect a change in Raf-1 AZD6244 on phospho-MEK expression in mouse embryonic fibro- kinase activity after AZD6244 treatment (data not shown). blasts (MEF). MEFs were resistant to growth inhibition due to Nevertheless, our results show that AZD6244 induces increased A in vitro AZD6244 (IC50, >10 mol/L) and accumulated phospho- BRAF kinase activity in a non-V600E cell line. MEK similar to cancer cells containing WT BRAF (data not shown). Negative feedback in the MAPK pathway is partially mediated by Thus, BRAF V600E seems to disrupt a negative feedback loop in the ERK-directed positive regulation of the inhibitory protein Spry2. To MAPK pathway. determine if Spry2 expression is reduced with AZD6244 treatment,

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MV522 and A549 cells were treated with AZD6244 and the change target total BRAF expression (COM-4) or just the mutant protein in Spry2 expression was determined by immunoblotting (Fig. 3C). (MU-A), and the effects on growth and responsiveness to AZD6244 In both cell lines, Spry2 expression is decreased following AZD6244 were determined. COM-4 expression reduced the expression of treatment. both the mutant and WT RNA efficiently in MV522 and HT29 cells, Effect of exogenous BRAF V600E expression on feedback whereas MU-A only affected the mutant RNA (Fig. 5A). Effects on and sensitivity to AZD6244. Cell lines containing the BRAF V600E the RNA were confirmed at the protein level. Currently, a mutation- mutation are exquisitely sensitive to AZD6244 and do not show specific antibody is not available, but expression of COM-4 feedback inhibition. To determine if exogenous expression of BRAF significantly reduced total BRAF protein levels and MU-A had an V600E disrupts normal feedback inhibition and confers sensitivity intermediate effect as expected because both of theses cell lines are to AZD6244, cell lines containing WT proteins or an activating heterozygous (Fig. 5B). Downstream effects for COM-4 and MU-A Ras mutation were transiently transfected with either WT BRAF were similar in both cell lines. BRAF V600E knockdown or BRAF V600E. In both H1299 (N-ras Q61K, Fig. 4A) and U2OS significantly reduces both phospho-MEK and phospho-ERK (WT, Fig. 4C) cells, transfection with WT BRAF did not sub- expression, and knockdown of the WT protein does not enhance stantially change baseline phospho-MEK or AZD6244-induced the effect. Treating the cells with AZD6244 further reduced phospho-MEK accumulation, but transfection with BRAF V600E phospho-ERK but did not alter phospho-MEK expression. substantially increased baseline phospho-MEK and there was no To determine the effect of BRAF V600E suppression on growth, change in the phospho-MEK level after AZD6244 treatment. These cells were infected with COM-4, MU-A, or control retroviruses and results are consistent with the findings we have observed in cell the effects on cell growth were determined using a MTS assay. lines naturally containing BRAF V600E. In neither H1299 nor U2OS BRAF V600E suppression clearly inhibits growth in both MV522 did exogenous BRAF V600E expression alter the sensitivity to and HT29 cells by approximately 50% to 75% at 4 days (Fig. 5C). AZD6244 in proliferation assays (Fig. 4B and D). Thus, BRAF V600E Although growth is inhibited, the cells remain sensitive to AZD6244 disrupts negative feedback pathways but is not able to confer (Fig. 5D). Thus, cell lines containing BRAF V600E clearly depend on sensitivity to MEK inhibition in resistant cell lines. activation of phospho-ERK via the activating mutation that Mutation-specific suppression of V600E expression. Our data supports a model in which BRAF V600E confers sensitivity to as well as the work of other groups show that cell lines containing AZD6244 by marking cells that are ‘‘addicted’’ to MAPK signaling to BRAF V600E are exquisitely sensitive to MEK inhibitors (16, 20). support in vitro growth. In fact, the sensitivity to AZD6244 is Studies have also shown that suppression of BRAF V600E expres- enhanced by BRAF V600E suppression, which supports this model. sion results in growth suppression both in vitro and in vivo (31, 32). The effect of suppressing the WT BRAF allele on sensitivity to However, it has not been shown whether specific suppression AZD6244 is not clear because we did not test a construct that of the BRAF V600E allele in cell lines that are heterozygous for specifically suppresses on the WT allele. the BRAF mutation would render those cells resistant to MEK inhibition. Several shRNAs have been described that specifically suppress expression of the BRAF V600E protein but have no effect Discussion on WT BRAF expression (32). We used these constructs to AZD6244 is a potent and selective MEK inhibitor. Prior studies determine if BRAF V600E is required for the cytostatic effects of have suggested that cell lines harboring activating Ras and BRAF AZD6244 in cell lines containing the mutant protein. MV522 and mutations were more likely to be sensitive to AZD6244 or other HT29 were infected with retroviruses expressing shRNAs that MEK inhibitors (16, 20, 33, 34). In particular, cells containing the

Figure 2. MEK inhibitors disrupt negative feedback pathways. A, A549 cells were treated with vehicle, 500 nmol/L AZD6244 (AZD), 25 Amol/LU0126, or 30 Amol/L PD98059 (PD) for the indicated times. Cell extracts were analyzed by immunoblotting for the indicated proteins. B, SW900 or HeLa cells were transfected with control (Ctrl), ERK1, ERK2, or a combination of ERK1 and ERK2 siRNAs. After 72 h, protein extracts were analyzed by immunoblotting for the indicated proteins. C, baseline levels of phospho-MEK and phospho-ERK in the absence of AZD6244 were determined for each cell line in Table 1. Data are presented as the expression relative to phospho-MEK (P-MEK) and phospho-ERK (P-ERK) levels in MV522 cells. Columns, mean expression in cell lines containing BRAF V600E and cell lines containing WT proteins or non-V600E mutations in BRAF or Ras; bars, SE. Student’s t test revealed a significant difference between the expression of phospho-MEK in the two groups (*, P < 0.0001). D, phospho-MEK levels were determined for each cell line from immunoblots and expressed as the relative phospho-MEK level in AZD6244 versus vehicle-treated cells. Each data point represents an individual cell line and cell lines are separated as those containing BRAF V600E and those containing WT proteins or non-V600E mutations in BRAF or Ras. Student’s t test revealed a significant difference between the means of each group (P < 0.05).

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Figure 3. AZD6244-induced phospho-MEK accumulation requires both BRAF and Raf-1. A, HeLa cells were transfected with control, Raf-1, BRAF, or a combination of Raf-1 and BRAF (R/B) siRNAs. After 48 h, vehicle (DMSO) or 500 nmol/L AZD6244 was added to the cells. After 24 h of incubation, protein extracts were analyzed by immunoblotting for the indicated proteins. B, MV522 or A549 cells were treated with vehicle or 500 nmol/L AZD6244 for 24 h. Whole-cell lysates were analyzed by immunoblotting for MEK, phospho-MEK, and actin. BRAF activity was determined in cell extracts using an immunoprecipitation (IP) kinase assay. Background activity was determined in a control (Ctrl) sample that did not contain cell lysate. Immunoprecipitation and BRAF activity were shown by immunoblotting for the indicated proteins. GST, glutathione S-transferase. C, A549 and MV522 cells were treated with the indicated concentrations for 24 h. Cell extracts were analyzed by immunoblotting for the indicated proteins. Tubulin is included as a loading control.

BRAF V600E mutation are exquisitely sensitive to MEK inhibitors, the patterns of sensitivity observed for the various mutations is whereas the response in cell lines containing Ras mutations is not understood. Ras proteins have pleiotropic effects on multiple variable. In this study, we have confirmed these observations in signaling pathways, whereas the target effects of Raf proteins are a panel of NSCLC cell lines as well as a panel of cell lines more limited. Raf-1 seems to have specific targets in addition to representing a broad array of malignancies. Although all activating MEK, such as MST2 and ASK1 (35, 36), but no MEK-independent Ras and BRAF mutations are expected to result in constitutive targets of BRAF have been described. Therefore, the variable activation of the MAPK pathway, the mechanism responsible for resistance to MEK inhibitors in cell lines containing Ras mutations

Figure 4. Exogenous BRAF V600E expression disrupts negative feedback. H1299 (A and B) or U2OS (C and D) cells were transfected with control (Ctrl), WT BRAF, or V600E expression plasmids. A and C, after 24 h, 0.5 Amol/L AZD6244 or vehicle was added. After an additional 24-h incubation, cell extracts were analyzed by immunoblotting for the indicated proteins. HA, hemagglutinin. B and D, after 24 h, cells were plated on a 96-well dish and AZD6244 was added to the indicated concentrations. After 4 d, cell proliferation was determined using a MTS assay.

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Figure 5. Endogenous BRAF V600E suppression does not reverse AZD6244 sensitivity. A, MV522 and HT29 cells were infected with retroviral supernatants containing control (CT), COM-4 (C), or MU-A (M) shRNAs. Seventy-two hours after infection, RNA was isolated and analyzed for expression of WT and V600E BRAF by allele-specific PCR. H661 RNA was used as a control as it does not contain BRAF V600E. GAPDH was used as a control to show equivalent input RNA. Samples containing only 10% of the input RNA for MV522 (MV) and HT29 (HT) are shown to show that amplification occurred in a quantitative manner. Numbers below each gel represent the relative expression in each sample compared with cells infected with control shRNA. B, after 72 h, cells infected with the indicated retroviral shRNAs were treated for 24 h with vehicle or 0.5 Amol/L AZD6244. Cell extracts were analyzed for the indicated proteins by immunoblotting. C and D, cells infected with the indicated shRNA retroviruses were plated on 96-well dishes. C, relative cell proliferation was determined using a MTS assay on subsequent days. Data are presented as the absorbance determined after a 60-min incubation in MTS reagent in three independent experiments. D, responsiveness to AZD6244 was determined by incubating the cells in various concentrations of AZD6244 for 4 d and determining relative proliferation using a MTS assay. Points, mean compared with vehicle-treated cells; bars, SE. For each cell line, the differences observed between control and COM-4 or MU-A were significant based on a paired t test (P < 0.05).

may be due to the activation of proliferative MEK-independent heterodimerization with Raf-1 (12, 39). The NSCLC cell line H1755 pathways that are not activated in cell lines containing BRAF contains the high-activity BRAF mutation G468A and is resistant to mutations. However, cell lines containing BRAF mutations distinct AZD6244 (IC50,8.1Amol/L) relative to cell lines containing the from V600E, such as H1755 (G468A) and H1666 (G465V), do not V600E mutation. Therefore, solely the presence of a high-activity have the same degree of sensitivity as V600E-containing cell lines, mutant does not confer sensitivity to AZD6244, suggesting that suggesting that other mechanisms may also be involved. V600E may have additional effects. Thus, our results show that cell Specific suppression of BRAF V600E expression by RNA lines containing V600E are dependent on the mutant allele for interference abrogates growth of cell lines containing the mutation activation of MAPK and are dependent on MAPK for in vitro in vitro and in vivo (23, 31, 37). Our results in NSCLC (MV522) and growth, which explains their exquisite sensitivity to AZD6244. colon (HT29) are similar. Interestingly, in cell lines heterozygous for Feedback regulation is a critical component of intracellular the V600E allele, specific knockdown of the mutant protein almost signal transduction networks and may be involved in determining completely blocks phospho-MEK and phospho-ERK expression the specificity of cellular responses to upstream receptor activation. despite the presence of WT BRAF protein (Fig. 5). Other groups Within the MAPK signaling network, both positive and negative have shown similar findings (32, 37). Activation of WT BRAF regulatory feedback loops have been described that affect typically occurs through upstream activation of Ras by cell surface differential cellular responses (40). Here, we have observed the receptors (6, 38), pathways that are induced by the presence of high presence of a negative feedback loop between ERK and upstream concentrations of FCS. Yet, despite growth in medium containing components of the MAPK pathway, including Raf proteins, in a high serum concentrations, we observe near-complete abrogation broad range of cancer cell lines. Additionally, the presence of a of MAPK signaling by suppression of the BRAF V600E allele, which feedback loop was confirmed using specific down-regulation of suggests that the mutant allele or other pathways may suppress ERK1 and ERK2 expression by RNA interference, which also upstream signaling pathways in these cell lines. V600E-containing resulted in phospho-MEK accumulation. Phospho-MEK accumula- cell lines seem to rely solely on activation of MAPK by the tion after treatment with a MEK inhibitor has been described by activating mutation in BRAF. Whether other BRAF mutations have several groups (41, 42) and was presumed to be due to disruption of the same effect is not known. BRAF mutations have been separated negative feedback pathways, but the phenomenon has not been into two classes: those that produce high intrinsic kinase activity systematically examined. Both direct and indirect feedback path- toward MEK, such as V600E and G468A, and those that have inter- ways between ERK and Raf have been described. ERK activation mediate/low intrinsic kinase activity that activate MAPK through results in the hyperphosphorylation of multiple proline-directed www.aacrjournals.org 6151 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 target sites of Raf-1 that reduce Raf-1 kinase activity and disrupt tion. AZD6244 disrupts negative feedback regulation between ERK Ras/Raf interactions (30). Similar results have also been observed and upstream targets resulting in accumulation of phospho-MEK. for BRAF, whereby ERK-directed phosphorylation of a SPKTP motif The BRAF V600E mutation disrupts normal feedback inhibition in the COOH terminus of BRAF reduces activity (29). and results in elevated baseline phospho-MEK expression and Indirect pathways may also mediate the negative feedback interrupts the AZD6244-induced phospho-MEK accumulation. between ERK and Raf. Spry1 and Spry2 are growth factor Although exogenous expression of BRAF V600E disrupts feedback regulators, whose expression is positively regulated by ERK, which signaling, it does not sensitize cells to AZD6244-induced growth seem to be involved in the negative feedback regulation of both Ras arrest. In addition, specific down-regulation of endogenous BRAF and Raf proteins (28, 43–45). Interestingly, sprouty proteins are V600E does not reverse the impaired feedback pathway nor does it unable to bind to the V600E isoform of BRAF (44), and this may confer resistance to AZD6244. In fact, suppression of endogenous underlie the differences we have observed in AZD6244-induced BRAF sensitizes the cells to AZD6244. Thus, BRAF V600E is not phospho-MEK changes between cell lines having BRAF V600E and sufficient to confer MEK inhibitor sensitivity but does mark cells WT/non-V600E mutations. In cell lines heterozygous for BRAF that are dependent on MAPK for in vitro growth. Finally, feedback V600E, we expected to see intact negative feedback after V600E- regulation is an important component of multiple cellular signaling specific suppression, but in both HT29 and MV522, phospho-MEK cascades and our results highlight the possibility that sequential levels were significantly decreased after BRAF V600E suppression targeting of multiple proteins within a cascade may be an and there was no change with AZD6244 treatment. Although the important clinical goal to ensure sufficient target inhibition and reasons for this finding are not clear, it likely occurs because there to block upstream activation of additional pathways that may be is no upstream activation of MAPK other than that provided by up-regulated by abrogation of negative feedback. BRAF V600E. Although we observed a small amount of residual phospho-ERK in cells after BRAF V600E suppression, this likely Disclosure of Potential Conflicts of Interest occurs due to incomplete knockdown. Aside from the exon 11 A. A. Adjei: Honoraria, Array Biopharma. The other authors disclosed no potential BRAF mutations G468A and G465V, the effect of other BRAF or Raf- conflicts of interest. 1 mutations on AZD6244-induced phospho-MEK accumulation is not clear. Non-V600E BRAF exon 15 mutations may have a similar effect because prior studies have shown that they can also disrupt Acknowledgments binding to Spry2 and Spry4 (44). Received 7/23/2007; revised 4/23/2008; accepted 5/7/2008. In summary, we show that the MEK inhibitor AZD6244 displays The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance preclinical activity in a broad range of human cancer cell lines, with 18 U.S.C. Section 1734 solely to indicate this fact. particularly in lines containing the BRAF V600E activating muta- We thank Dr. Scott Kaufmann for his ongoing advice during this project.

References 12. Wan PT, Garnett MJ, Roe SM, et al. Mechanism of 22. McIntyre A, Summersgill B, Spendlove HE, Huddart activation of the RAF-ERK signaling pathway by R, Houlston R, Shipley J. Activating mutations and/or 1. Maekawa M, Nishida E, Tanoue T. Identification of the oncogenic mutations of B-RAF. Cell 2004;116:855–67. expression levels of tyrosine kinase receptors GRB7, anti-proliferative protein TOB as a MAPK substrate. 13. Wallace EM, Lyssikatos JP, Yeh T, Winkler JD, Koch K. RAS, and BRAF in testicular germ cell tumors. Neoplasia J Biol Chem 2002;277:37783–7. Progress towards therapeutic small molecule MEK 2005;7:1047–52. 2. Roux PP, Ballif BA, Anjum R, Gygi SP, Blenis J. Tumor- inhibitors for use in cancer therapy. Curr Top Med 23. Sharma A, Trivedi NR, Zimmerman MA, Tuveson DA, promoting phorbol esters and activated Ras inactivate Chem 2005;5:215–29. Smith CD, Robertson GP. Mutant V599EB-Raf regulates the tuberous sclerosis tumor suppressor complex via 14. Sebolt-Leopold JS, Dudley DT, Herrera R, et al. growth and vascular development of malignant mela- p90 ribosomal S6 kinase. Proc Natl Acad Sci U S A 2004; Blockade of the MAP kinase pathway suppresses growth noma tumors. Cancer Res 2005;65:2412–21. 101:13489–94. of colon tumors in vivo. Nat Med 1999;5:810–6. 24. Abbott KL, Friday BB, Thaloor D, Murphy TJ, Pavlath 3. Ledwith BJ, Manam S, Kraynak AR, Nichols WW, 15. BrownAP,CarlsonTC,LoiCM,GrazianoMJ. GK. Activation and cellular localization of the cyclo- Bradley MO. Antisense-fos RNA causes partial reversion Pharmacodynamic and toxicokinetic evaluation of the sporine A-sensitive transcription factor NF-AT in of the transformed phenotypes induced by the c-Ha-ras novel MEK inhibitor, PD0325901, in the rat following skeletal muscle cells. Mol Biol Cell 1998;9:2905–16. oncogene. Mol Cell Biol 1990;10:1545–55. oral and intravenous administration. Cancer Chemother 25. Sapio MR, Posca D, Troncone G, et al. Detection of 4. Porter AC, Vaillancourt RR. Tyrosine kinase receptor- Pharmacol 2007;59:671–9. BRAF mutation in thyroid papillary carcinomas by activated signal transduction pathways which lead to 16. Yeh TC, Marsh V, Bernat BA, et al. Biological mutant allele-specific PCR amplification (MASA). Eur J oncogenesis. Oncogene 1998;17:1343–52. characterization of ARRY-142886 (AZD6244), a potent, Endocrinol 2006;154:341–8. 5. Zebisch A, Czernilofsky AP, Keri G, Smigelskaite J, Sill highly selective mitogen-activated protein kinase kinase 26. Meng XW, Chandra J, Loegering D, et al. Central role H, Troppmair J. Signaling through RAS-RAF-MEK-ERK: 1/2 inhibitor. Clin Cancer Res 2007;13:1576–83. of Fas-associated death domain protein in apoptosis from basics to bedside. Curr Med Chem 2007;14:601–23. 17. Ohren JF, Chen H, Pavlovsky A, et al. Structures of induction by the mitogen-activated protein kinase 6. Friday BB, Adjei AA. K-ras as a target for cancer human MAP kinase kinase 1 (MEK1) and MEK2 kinase inhibitor CI-1040 (PD184352) in acute lympho- therapy. Biochim Biophys Acta 2005;1756:127–44. describe novel noncompetitive kinase inhibition. Nat cytic leukemia cells in vitro. J Biol Chem 2003;278: 7. Xing M. BRAF mutation in thyroid cancer. Endocr Struct Mol Biol 2004;11:1192–7. 47326–39. Relat Cancer 2005;12:245–62. 18. Tai YT, Fulciniti M, Hideshima T, et al. Targeting 27. Brognard J, Dennis PA. Variable apoptotic response 8. Sieben NL, Macropoulos P, Roemen GM, et al. In MEK induces myeloma cell cytotoxicity and inhibits of NSCLC cells to inhibition of the MEK/ERK pathway ovarian neoplasms, BRAF, but not KRAS, mutations are osteoclastogenesis. Blood 2007;110:1656–63. by small molecules or dominant negative mutants. Cell restricted to low-grade serous tumours. J Pathol 2004; 19. Huynh H, Soo KC, Chow PK, Tran E. Targeted Death Differ 2002;9:893–904. 202:336–40. inhibition of the extracellular signal-regulated kinase 28. Yusoff P, Lao D-H, Ong SH, et al. Sprouty2 inhibits the 9. Brose MS, Volpe P, Feldman M, et al. BRAF and RAS kinase pathway with AZD6244 (ARRY-142886) in the Ras/MAP kinase pathway by inhibiting the activation of mutations in human lung cancer and melanoma. Cancer treatment of hepatocellular carcinoma. Mol Cancer Raf. J Biol Chem 2002;277:3195–201. Res 2002;62:6997–7000. Ther 2007;6:138–46. 29. Brummer T, Naegele H, Reth M, Misawa Y. Identifi- 10. Wang L, Cunningham JM, Winters JL, et al. BRAF 20. Solit DB, Garraway LA, Pratilas CA, et al. BRAF cation of novel ERK-mediated feedback phosphorylation mutations in colon cancer are not likely attributable to mutation predicts sensitivity to MEK inhibition. Nature sites at the C-terminus of B-Raf. Oncogene 2003;22: defective DNA mismatch repair. Cancer Res 2003;63: 2006;439:358–62. 8823–34. 5209–12. 21. Valenzuela DM, Groffen J. Four human carcinoma 30. Dougherty MK, Muller J, Ritt DA, et al. Regulation of 11. Davies H, Bignell GR, Cox C, et al. Mutations of the cell lines with novel mutations in position 12 of c-K-ras Raf-1 by direct feedback phosphorylation. Mol Cell 2005; BRAF gene in human cancer. Nature 2002;417:949–54. oncogene. Nucleic Acids Res 1986;14:843–52. 17:215–24.

Cancer Res 2008; 68: (15). August 1, 2008 6152 www.aacrjournals.org

31. Hoeflich KP, Gray DC, Eby MT, et al. Oncogenic BRAF 36. Chen J, Fujii K, Zhang L, Roberts T, Fu H. Raf-1 kinase kinase activation domain recognized by the is required for tumor growth and maintenance in promotes cell survival by antagonizing apoptosis inhibitor PD 184352. Mol Cell Biol 2002;22:7593–602. melanoma models. Cancer Res 2006;66:999–1006. signal-regulating kinase 1 through a MEK-ERK indepen- 42. Eppstein AC, Sandoval JA, Klein PJ, et al. Differential 32. Hingorani SR, Jacobetz MA, Robertson GP, Herlyn M, dent mechanism. Proc Natl Acad Sci U S A 2001;98: sensitivity of chemoresistant neuroblastoma subtypes to Tuveson DA. Suppression of BRAFV599E in human 7783–8. MAPK-targeted treatment correlates with ERK, p53 melanoma abrogates transformation. Cancer Res 2003; 37. Salvatore G, De Falco V, Salerno P, et al. BRAF is a expression, and signaling response to U0126. J Pediatr 63:5198–202. therapeutic target in aggressive thyroid carcinoma. Clin Surg 2006;41:252–9. 33. Yeh T, Wallace E, Lyssikatos J, Winkler J. ARRY- Cancer Res 2006;12:1623–9. 43. Ozaki K, Kadomoto R, Asato K, Tanimura S, Itoh N, 142886, a potent and selective MEK inhibitor. II. Potency 38. Midgley RS, Kerr DJ. Ras as a target in cancer Kohno M. ERK pathway positively regulates the against cellular MEK leads to inhibition of cellular therapy. Crit Rev Oncol Hematol 2002;44:109–20. expression of Sprouty genes. Biochem Biophys Res proliferation and induction of apoptosis in cell lines 39. Garnett MJ, Rana S, Paterson H, Barford D, Marais R. Commun 2001;285:1084–8. with mutant Ras or B-Raf. Proc Am Assoc Cancer Res Wild-type and mutant B-RAF activate C-RAF through 44. Tsavachidou D, Coleman ML, Athanasiadis G, et al. 2004;45:3889. distinct mechanisms involving heterodimerization. Mol SPRY2 is an inhibitor of the Ras/extracellular signal- 34. Ball DW, Jin N, Rosen DM, et al. Selective growth Cell 2005;20:963–9. regulated kinase pathway in melanocytes and melano- inhibition in BRAF mutant thyroid cancer by the 40. SantosSD,VerveerPJ,BastiaensPI.Growth ma cells with wild-type BRAF but not with the V599E mitogen-activated protein kinase kinase 1/2 inhibitor factor-induced MAPK network topology shapes Erk mutant. Cancer Res 2004;64:5556–9. AZD6244. J Clin Endocrinol Metab 2007;92:4712–8. response determining PC-12 cell fate. Nat Cell Biol 2007; 45. Gross I, Bassit B, Benezra M, Licht JD. Mammalian 35. O’Neill E, Rushworth L, Baccarini M, Kolch W. Role of 9:324–30. sprouty proteins inhibit cell growth and differentiation the kinase MST2 in suppression of apoptosis by the proto- 41. Delaney AM, Printen JA, Chen H, Fauman EB, Dudley by preventing Ras activation. J Biol Chem 2001;276: oncogene product Raf-1. Science 2004;306:2267–70. DT. Identification of a novel mitogen-activated protein 46460–8.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2008 American Association for Cancer Research. BRAF V600E Disrupts AZD6244-Induced Abrogation of Negative Feedback Pathways between Extracellular Signal-Regulated Kinase and Raf Proteins

Bret B. Friday, Chunrong Yu, Grace K. Dy, et al.

Cancer Res 2008;68:6145-6153.

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