MEK-Independent Survival of B-RAFV600E Melanoma Cells Selected for Resistance to Apoptosis Induced by the RAF Inhibitor PLX4720

Published OnlineFirst November 18, 2010; DOI: 10.1158/1078-0432.CCR-10-2225

Clinical Cancer Cancer Therapy: Preclinical Research

Chen Chen Jiang, Fritz Lai, Rick F. Thorne, Fan Yang, Hao Liu, Peter Hersey, and Xu Dong Zhang

Abstract Purpose: To examine mechanisms that determine long-term responses of B-RAFV600E melanoma cells to B-RAF inhibitors. Experimental Design: B-RAFV600E melanoma cells were exposed to the B-RAF inhibitor PLX4720 for prolonged periods to select for cells resistant to apoptosis induced by the inhibitor. The resultant cells were analyzed for activation of extracellular signal regulated kinase (ERK), MAP/ERK kinase (MEK), and Akt, and related signals. Their roles in survival of the cells were also examined. Results: B-RAFV600E melanoma cells selected for resistant to PLX4720-induced apoptosis retained the V600E mutation in B-RAF, and proliferated steadily in the presence of the inhibitor, albeit with slow growth rate. These cells displayed high levels of ERK activation, that is, at least in part, independent of the conventional RAF/MEK/ERK pathway, as MEK activation was low and inhibition of MEK did not significantly block activation of ERK. In contrast, extracellular signals appeared involved. This was associated with elevated activation of the phosphoinositide 3-kinase (PI3k)/Akt pathway and could be inhibited by serum starvation and inhibition of PI3k/Akt. Inhibition of MEK did not impact on survival of these cells, whereas serum starvation, inhibition of PI3K/Akt, and inhibition of ERK1/2 reduced their viability. Conclusions: These results indicate that sensitivity to induction of apoptosis may be a major determinant of long-term responses of B-RAFV600E melanomas to specific inhibitors and suggest that rebound melanoma growth after initial treatment with the inhibitors may not be responsive to MEK inhibitors, but may be susceptible to inhibition of the PI3k/Akt pathway. Clin Cancer Res; 17(4); 1–10. 2010 AACR.

Introduction It is well established that blockade of the RAF/MEK/ERK pathway inhibits melanoma cell growth (5). Moreover, Results from clinical studies with small molecule inhi- induction of apoptosis has also been shown in varying bitors of the mutant B-RAFV600E have been very encoura- in vitro systems, in particular, in B-RAFV600E melanoma cells ging and promise to provide a much needed breakthrough (6–11). Apoptosis of such cells was clearly demonstrated in in the treatment of melanoma by targeting B-RAFV600E an ex vivo model after administration of the B-RAF inhibitor (1, 2). The latter is found in about 50% of melanomas, PLX4720 that is selective for the mutant B-RAFV600E (6). leading to constitutive activation of the RAF/MAP/ERK Consistently, regression of metastatic mutant B-RAF mel- kinase (MEK)/extracellular signal regulated kinase (ERK) anomas is a frequent sign of the response to administration pathway that is important for melanoma cell growth and of PLX4032/RG7204, an analogue to PLX4720, suggesting survival, and is involved in resistance to many therapeutic that induction of apoptosis may be a major biological agents (3, 4). However, a number of questions have already consequence of inhibition of mutant B-RAF that causes been raised from these studies, such as the durability of remission of melanoma (1, 2). responses and why some melanomas with mutant B-RAF Although molecular mechanisms that regulate sensitivity have not shown major responses. of B-RAFV600E melanoma cells to apoptosis induced by specific inhibitors are currently not well understood, it has been reported that acquired resistance of melanoma cells to the inhibitors was associated with rebound activa- Authors' Affiliation: Immunology and Oncology Unit, Newcastle, New tion of the RAF/MEK/ERK pathway (12, 13). Signaling to South Wales, Australia activate MEK/ERK has been shown to switch to C-RAF when Note: Chen Chen Jiang and Fritz Lai, equal contribution. mutant B-RAF was inhibited due to elevated C-RAF expres- Corresponding Authors: Peter Hersey or Xu Dong Zhang, Immunology sion (12). Moreover, most melanoma cells harboring the B- and Oncology Unit, Room 443, David Maddison Clinical Sciences Build- V600E ing, Cnr. King and Watt Streets, Newcastle, NSW 2300, Australia. Phone: RAF mutation retain the wild-type B-RAF allele (14). 61-2-49138174; Fax: 61-2-49138184; E-mails: These cells can be rescued by signals mediated by extra- [email protected] or [email protected] cellular growth factors when B-RAFV600E was knocked down doi: 10.1158/1078-0432.CCR-10-2225 (14). In addition, mutant B-RAF inhibitors can cause activa- 2010 American Association for Cancer Research. tion of ERK by transactivation of C-RAF in cells carrying

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Translational Relevance in Dimethyl sulfoxide (DMSO) and made up in stock solutions of 4 mmol/L. The MEK inhibitor U0126 and Results from clinical studies with small molecule the PI3k inhibitor LY294002 were purchased from Pro- inhibitors of mutant B-RAF have been very encouraging mega Corporation. in the treatment of melanoma, but frequent recurrence of tumor growth following initial remission remains a Generation of melanoma cells resistant to PLX4720- V600E major obstacle for more successful treatment of the induced apoptosis from B-RAF melanoma cell disease. We show in this report that B-RAFV600E mela- lines noma cells selected for resistance to apoptosis induced Mel-RMu and Mel-CV cells were seeded in wells of 24- by the RAF inhibitor PLX4720 by prolonged exposure to well plates or 6-well plates till they reached 70% to 80% the inhibitor can proliferate even in the presence of the confluence. Cells were then switched into medium con- m inhibitor. Survival of these cells is primarily mediated by taining PLX4720 (10 mol/L). The surviving cells were fed activation of ERK that is largely independent of the every 3 days with medium containing PLX4720 for 6 to conventional RAF/MEK/ERK pathway. In contrast, acti- 8 weeks until they reached 70% to 80% confluence. The vation of the PI3k/Akt pathway as a consequence of resulting cells were then passaged and cultured in the extracellular stimulation appears essential for activation presence of PLX4720 for at least 20 weeks. The resulting of ERK. These results suggest that regrowth of B-RAFV600E cell lines were designated as PLX-selected melanoma cells melanomas after initial responses to B-RAF inhibitors (Mel-RMu.S and Mel-CV.S, respectively). may not be responsive to MEK inhibitors, but may be susceptible to inhibitors against the PI3k/Akt pathway. Apoptosis Quantitation of apoptotic cells was carried out by mea- surement of sub-G1 DNA content using propidium iodide wild-type B-RAF allele (15–17). On the contrary, constitu- (PI) on a flow cytometer as described elsewhere (20, 21). tive activation of other survival signaling pathways such as the phosphoinositide 3-kinase (PI3k)/Akt pathway has Cell viability assays been reported to mediate resistance of mutant B-RAF mel- Cell viability assays (MTS assays) were performed as anoma cells to inhibition of B-RAF (18, 19). reported previously (20). In brief, cells were seeded at To understand the mechanism(s) that determines long- 5,000 cells/well onto flat-bottomed 96-well culture plates term responses of mutant B-RAF melanoma cells to specific and allowed to grow for 24 hours followed by the desired inhibitors, we generated B-RAFV600E melanoma cells resis- treatment. Cells were then labeled with the VisionBlue tant to PLX4720-induced apoptosis by prolonged exposure reagent and detected by Synergy 2 multidetection micro- to the inhibitor. We show in this report that these cells plate reader (BioTek) according to the manufacturer’s display high levels of activation of ERK that is important for instruction. survival of the cells. Unexpectedly, our results indicate that activation of ERK in the cells is largely independent of Flow cytometry C-RAF and MEK. Instead, extracellular signals appear are Immunostaining on intact and permeabilized cells was important in ERK activation. This is, at least in part, carried out as described previously (20, 21). mediated by increased activation of the PI3k/Akt pathway. Western blot analysis Materials and Methods Western blot analysis was carried out as described previously (20, 21). Labeled bands were detected by Immun- Cell culture and reagents Star HRP Chemiluminescent Kit, and images were captured Human melanoma cell lines Mel-RMu and Mel-CV have and the intensity of the bands was quantitated with the Bio- been described previously (20). They were cultured in Rad VersaDoc image system (Bio-Rad). Dulbecco’s Modified Eagle Medium (DMEM) containing 5% fetal calf serum (FCS) (Commonwealth Serum Labora- Small RNA interference tories). The mouse monoclonal antibody (mAbs) against The small RNA interference (siRNA) constructs used phospho-ERK (p-ERK) (Thr202/Tyr204), phospho-Akt (p- were obtained as the siGENOME SMARTpool reagents Akt) (Ser473), phospho-MEK (p-MEK), ERK, ERK1, ERK2, (Dharmacon), MEK1 siGENOME SMARTpool (M- Akt, Akt1, Akt2, Akt3, MEK1, C-RAF, and B-RAF were 003571–01-0005), C-RAF siGENOME SMARTpool (M- purchased from Cell Signaling Technology. The rabbit 003601–02-0010), ERK1 siGENOME SMARTpool polyclonal Ab against Caspase-3 was from Stressgen. (M-003592–03-0010), ERK2 siGENOME SMARTpool Isotype control Abs used were the ID4.5 (mouse IgG2a) (M-003555–04-0010), Akt1 siGENOME SMARTpool (M- mAb against Salmonella typhi supplied by Dr. L. Ashman 003000–03-0010), Akt2 siGENOME SMARTpool (Institute for Medical and Veterinary Science, Adelaide, (M-003001–02-0010), Akt3 siGENOME SMARTpool (M- Australia), the 107.3 mouse IgG1 mAb purchased from 003002–02-0010), and siGENOME Nontargeting siRNA PharMingen, and rabbit IgG from Sigma Chemical Co. pool (D-001206–13-20). Transfection of siRNA pools was PLX4720 was provided from Plexxikon Inc. It was dissolved carried out as described previously (20, 21).

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MEK-Independent Survival of PLX4720-Selected Melanoma Cells

Results any treatment (5%), indicative of successful selection of B-RAFV600E melanoma cells resistant to apoptosis induced Prolonged exposure to PLX4720 selects B-RAFV600E by PLX4720 (Fig. 1A). For standardization, only the cells melanoma cells resistant to PLX4720-induced that had been exposed to PLX4720 for at least 20 weeks apoptosis were designated as PLX-selected cells and used for sub- We exposed 2 melanoma cell lines, Mel-CV that carried sequent experiments. Notably, both PLX-selected Mel- B-RAFV600E and Mel-RMu that harbored B-RAFV600E and an RMu (Mel-RMu.S) and Mel-CV (Mel-CV.S) cells retained activating mutation in EGFR (L747_S752/P753S dele- the V600E mutation in B-RAF, whereas Mel-RMu.S cells tion), to the B-RAFV600E inhibitor PLX4720 at 10 mmol/ also retained the activating mutation in EGFR (data not L for prolonged periods. This concentration was chosen shown). because it induces high levels of apoptosis (>50% apop- Despite resistance to apoptosis, the PLX-selected cells totic cells) in most B-RAFV600E melanoma cell lines and proliferated significantly slower than their parental coun- fresh melanoma isolates (20). In addition, it is concei- terparts (Fig. 1B). Consistently, the cells displayed vably relevant to applications in vivo,asplasmaconcen- increased levels of p21 and p27, and decreased levels of trations of a closely related inhibitor PLX4032/RG7204 of cyclin D1 and CDK4 (Fig. 1C). Nevertheless, these cells around 60 mmol/L were not associated with significant were able to form stable populations and be passaged in the adverse effects in phase I clinical trials (1, 22). As presence of PLX4720 (Fig. 1D). expected, initial exposure to PLX4720 at 10 mmol/L for 3 days induced apoptosis in more than 60% of Mel-RMu PLX-selected B-RAFV600E melanoma cells exhibit high and Mel-CV cells. However, the levels of apoptosis in the levels of ERK activation that is largely independent of remaining cells exposed to fresh PLX4720 decreased pro- MEK gressively (Fig. 1A). At the end of 3 weeks, the levels of As reported before, initial exposure to PLX4720 at 10 apoptosis of the cells in the presence of PLX4720 were mmol/L resulted in marked inhibition of ERK activation, equivalent to those of the parental counterparts without with the inhibitory effect sustained up to 3 days in

Figure 1. Selection of B-RAFV600E melanoma cells resistant to apoptosis induced by PLX4720 by prolonged exposure to the inhibitor. A, B-RAFV600E Mel-RMu and Mel-CV cells seeded in wells of 24-well plates were exposed to PLX4720 (10 mmol/L). Culture medium containing PLX4720 was changed every 3 days. Apoptosis was measured in cells from representative wells at indicated time points by the PI method. Data shown are the mean Æ SE of results from 3 individual culture wells. B, Mel-RMu.S and Mel-CV. S cells and their parental counterparts were subjected to colony formation assays. The data shown are representative of 3 individual experiments. C, whole cell lysates from Mel-RMu.S and Mel-CV.S cells and their parental counterparts were subjected to Western blot analysis of p21, p27, CDK4, Cyclin D1, and glyceraldehyde 3 phosphate dehydrogenase (GAPDH) (as a loading control). The data shown are representative of 3 individual experiments. D, representative of microscopic photographs of Mel- RMu.S and Mel-CV.S cells and their parental counterparts grown in 75 mm2 culture flasks.

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Figure 2. Activation of ERK in the PLX-selected melanoma cells is largely independent of MEK and C-RAF. A, left panel: whole cell lysates from Mel-RMu and Mel-CV cells treated with PLX4720 (10 mmol/L) for indicated periods were subjected to Western blot analysis of p-ERK and ERK. Right panel: whole cell lysates from Mel-RMu.S and Mel-CV.S cells and their parental counterparts were subjected to Western blot analysis of p-ERK and ERK. The data shown are representative of 3 individual experiments. B, left panel: whole cell lysates from Mel-RMu.S and Mel-CV.S cells and their parental counterparts were subjected to Western blot analysis of p-MEK and MEK. Right panel: whole cell lysates from Mel-RMu.S and Mel-CV.S cells and their parental counterparts with or without treatment with U0126 (20 mmol/L) for 3 hours were subjected to Western blot analysis of p-ERK and ERK. The data shown are representative of 3 individual experiments. C, Mel-RMu.S and Mel-CV.S cells and their parental counterparts were transfected with the control and MEK1 siRNA, respectively. Twenty-four hours later, whole cell lysates were subjected to Western blot analysis of MEK1, p-ERK, and ERK. The data shown are representative of 3 individual experiments. D, left panel: whole cell lysates from Mel-RMu.S and Mel-CV.S cells and their parental counterparts were subjected to Western blot analysis of C-RAF and GAPDH (as a loading control). Right panel: Mel-RMu.S and Mel-CV.S cells and their parental counterparts were transfected with the control and C-RAF siRNA, respectively. Twenty-four hours later, whole cell lysates were subjected to Western blot analysis of C-RAF, p-ERK, and ERK. The data shown are representative of 3 individual experiments.

Mel-RMu and Mel-CV cells (ref. 20; Fig. 2A). However, exposure to PLX4720 (Fig. 2A). Notably, the levels activation of ERK in the remaining cells was steadily recov- appeared even higher in the PLX-selected cells (the cells ered in the presence of fresh PLX4720. By 9 days, the levels that had been exposed to PLX4720 for at least 20 weeks) of ERK activation were comparable to those in cells without than their parental counterparts.

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MEK-Independent Survival of PLX4720-Selected Melanoma Cells

To study the mechanism by which ERK is highly acti- increase in C-RAF in B-RAFV600E melanoma cells after vated in PLX-selected cells, we examined the activation prolonged inhibition of B-RAFV600E may not be responsible status of MEK that is directly upstream of ERK in the for rebound activation of ERK. RAF/MEK/ERK pathway. Figure 2B shows that while phosphorylated (activated) MEK was readily detected in the Activation of ERK contributes to survival of PLX- parental Mel-RMu and Mel-CV cells, it was expressed at selected melanoma cells markedly lower levels in Mel-RMu.S and Mel-CV.S cells. We examined the role of activation of ERK in survival of This suggests that activation of MEK may not play a major the PLX-selected melanoma cells by transfecting siRNA role in activation of ERK in the cells. Consistently, treat- pools for ERK1 and ERK2 into the Mel-RMu.S and Mel- ment with the MEK inhibitor U0126 caused only moderate CV.S cells, respectively (Fig. 3A). As shown in Figure 3B, inhibition of phosphorylation (activation) of ERK in these inhibition of ERK1 or ERK2 by siRNA induced moderate cells, whereas the levels of phosphorylated (activated) ERK levels of apoptosis (25–35%) in both lines. This was were reduced markedly in the parental Mel-RMu and Mel- associated with activation of caspase-3 (Fig. 3C). In con- CV cells (Fig. 2B). The similar effect was observed with trast, siRNA knockdown of ERK1 or ERK2 did not induce another MEK inhibitor AZD6244 (Data not shown). apoptosis in the parental counterparts (data not shown). Furthermore, siRNA knockdown of MEK1 significantly These results indicate that activation of ERK is responsible reduced the levels of ERK phosphorylation in the parental for survival of B-RAFV600E melanoma cells after long-term cells, but had no detectable effect on activation of ERK in exposure to PLX4720. Mel-RMu.S and Mel-CV.S cells (Fig. 2C). We studied whether signaling to activate ERK was Extracellular signaling is critical for activation of ERK switched to C-RAF in the PLX-selected cells. As shown in in the PLX4720-selected B-RAFV600E melanoma cells Figure 2D, the levels of C-RAF in Mel-RMu.S and Mel-CV.S Extracellular signals are essential for activation of ERK in cells were increased in comparison with their parental the absence of activating mutations of its upstream kinases counterparts. However, inhibition of C-RAF by siRNA (23, 24). We, therefore, examined if ERK activation in the did not reduce the levels of ERK activation in the PLX- PLX-selected cells is due to extracellular stimulation selected cells, nor did it inhibit activation of ERK in the afforded by FCS in culture medium by reducing its con- parental counterparts. These results suggest that the centration from 5% to 0.5%. Twenty-four hours after

Figure 3. Activation of ERK contributes to survival of the PLX-selected melanoma cells. A, Mel-RMu.S and Mel-CV.S cells were transfected with the control, ERK1, and ERK2 siRNA, respectively. Twenty-four hours later, whole cell lysates were subjected to Western blot analysis of ERK and GAPDH (as a loading control). The data shown are representative of 3 individual experiments. B, Mel-RMu.S and Mel-CV.S cells were transfected with the control, ERK1, and ERK2 siRNA, respectively. Forty-eight hours later, cells were subjected to quantitation of apoptosis using the PI method. The data shown are the mean Æ SE of results from 3 individual experiments. C, Mel-RMu.S and Mel-CV.S cells were transfected with the control, ERK1, and ERK2 siRNA, respectively. Forty- eight hours later, whole cell lysates were subjected to Western blot analysis of caspase-3 and GAPDH (as a loading control). The data shown are representative of 3 individual experiments.

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culturing in medium with 0.5% FCS, the levels of activation tion of MEK (Fig. 2B, 5B, and 5C). To test this, we treated of ERK were decreased in both the PLX-selected cells and Mel-RMu.S and Mel-CV.S cells with U0126 for 72 hours. the parental counterparts (Fig. 4A). Of note, while the The addition of U0126 did not cause significant apoptosis levels of ERK activation remained relatively stable in the of the selected cells (Fig. 6A). In contrast, cotreatment with parental counterparts, the levels in the PLX4720-selected U0126 and PLX4720 resulted in moderate enhancement in cells continued to decrease. By 72 hours, activation of ERK induction of apoptosis in the parental counterparts was hardly detectable in both Mel-RMu.S and Mel-CV.S (Fig. 6A). A similar effect on survival of PLX-selected cells (Fig. 4A). melanoma cells was observed with another MEK inhibitor We also examined whether the PLX4720-selected cells AZD6244 (data not shown). rely on extracellular signals for their survival. After switch- We also examined the effect of inhibition of the PI3k/Akt ing cells to medium with 0.5% FCS, the growth rate was pathway on survival of the PLX4720-selected B-RAFV600E reduced by approximately 30% in parental Mel-RMu and melanoma cells. As shown in Figure 6B, the addition of the Mel-CV cells at 48 hours, which remained stable thereafter PI3k inhibitor LY294002 induced apoptosis in 36% and (Fig. 4B). These cells otherwise appeared healthy with cell 45% of Mel-RMu.S and Mel-CV.S cells, respectively. Nota- volumes and morphology comparable to those grown in bly, LY294002 did not cause apoptotic cell death in the medium with 5% FCS (Fig. 4C). In contrast, the growth rate parental counterparts, but it enhanced PLX4720-induced of Mel-RMu.S and Mel-CV.S cell was reduced by approxi- apoptosis of the cells (Fig. 6B). The effect of the PI3k/Akt mately 60% and 75%, respectively, at 48 hours. This was pathway on survival of the PLX-selected melanoma cells associated with induction of apoptosis (Fig. 4D). Never- was also shown by siRNA knockdown of Akt3 that similarly theless, by the end of 2 weeks, sparse colonies were found induced apoptosis of Mel-CV.S cells (Fig. 5C and 6C). in the PLX-selected cell cultures (Fig. 4C). Discussion Activation of ERK is associated with increased activation of Akt in PLX-selected B-RAFV600E The above results show that B-RAFV600E melanoma cells melanoma cells selected for resistance to apoptosis induced by PLX4720 Another important survival signaling pathway in mela- can proliferate, albeit with reduced growth rate, in the noma is the PI3k/Akt pathway that can be activated in presence of the inhibitor. Survival of these cells is asso- response to extracellular signaling and is able to activate ciated with rebound activation of ERK that is largely inde- ERK independent of MEK (18, 25). As shown in Figure 5A, pendent of the conventional RAF/MEK/ERK pathway. the levels of Akt activation were elevated in both Mel-RMu.S Instead, elevated activation of the PI3k/Akt pathway as a and Mel-CV.S cells, but activation was decreased after the result of extracellular stimulation appears to be important. cells were switched into medium containing only 0.5% FCS. The results suggest that melanoma recurrence in patients While Akt activation was inhibited by the PI3k inhibitor treated with selective mutant B-RAF inhibitors may not LY294002, the levels of activation of ERK were also respond to MEK inhibitors, but may be susceptible to decreased in the PLX-selected cells treated with the inhibitor inhibitors against the PI3k/Akt pathway. (Fig. 5B). In contrast, LY294002 did not have any notable Although the RAF/MEK/ERK pathway was initially effect on ERK activation in the parental counterparts. thought to be mainly responsible for regulation of cell To further confirm the role of the PI3k/Akt pathway in proliferation (27, 28), it is now clear that this pathway also activation of ERK in PLX4720-selected cells, we knocked plays an important role in regulation of cell survival (27– down Akt1, Akt2, and Akt3 in Mel-CV and Mel-CV.S cells, 29). Blockade of this pathway can cause not only inhibition respectively, by siRNA. Consistent with previous reports of cell growth but also induction of apoptosis in melanoma that Akt3 is the major isoform of Akt in melanoma (19, 26), cells (6–9). Mutant B-RAF-specific inhibitors display the knockdown of Akt3, but not knockdown of Akt1 or Akt 2, dual effects on B-RAFV600E melanoma cells, but the relative resulted in marked reduction in the levels of phosphory- contributions of inhibition of cell growth and induction of lated Akt in both the PLX-selected and the parental cells apoptosis to the therapeutic effect remain ambiguous (6, (Fig. 5C). Similar to inhibition of PI3k by LY294002, 13). Despite the high rate of clinical responses in patients knockdown of Akt3 by siRNA partially inhibited ERK treated with mutant B-RAF-specific inhibitors such as activation in Mel-CV.S cells. In contrast, knockdown of PLX4032/RG7204 and GSK2118436, longer follow-up Akt1 or Akt2 did not reduce the levels of ERK activation in has shown frequent recurrence of tumor growth (13, the cells (Fig. 5C). Neither knockdown of Akt3 nor knock- 30). It is conceivable that induction of apoptosis may occur down of Akt1 or Akt2 had notable effect on activation of at early stages after inhibition of mutant B-RAF, but the ERK in the parental counterparts (Fig. 5C). remaining cells that are presumably resistant to apoptosis may retain low proliferation potential even in the presence The PLX4720-selected B-RAFV600E melanoma cells are of the inhibitors and regain high growth rate after the not sensitive to MEK inhibitors, but are susceptible to inhibition is removed. In corroboration with this, we inhibition of PI3k/Akt found in this study that although initial exposure to MEK-independent activation of ERK in PLX-selected cells PLX4720 induced apoptosis in most melanoma cells har- suggests that these cells may not be susceptible to inhibi- boring B-RAFV600E, the remaining cells could proliferate,

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Figure 4. Extracellular signals are critical for activation of ERK and survival of the PLX-selected melanoma cells. A, Mel-RMu.S and Mel-CV.S cells and their parental counterparts were cultured in medium containing 5% FCS till approximate 50% confluence. Cells were then switched into medium containing 0.5% FCS for indicated periods. Whole cell lysates were subjected to Western blot analysis of p-ERK and ERK. The data shown are representative of 3 individual experiments. B, Mel-RMu.S and Mel-CV.S cells and their parental counterparts were cultured in medium containing 5% FCS till approximate 50% confluence. Half of the cultures were then switched into medium with 0.5% FCS, and the rest were switched into fresh medium containing 5% FCS for the same periods. Cell viability was quantitated at indicated time points using MTS assays. Results from cells cultured in medium with 0.5% FCS were compared with those from cells cultured in medium with 5% FCS for the same periods and were expressed as the percentages of the latter. The data shown are the mean Æ SE of results from 3 individual culture wells. C, representative microscopic photographs of Mel-RMu.S and Mel-CV.S cells and their parental counterparts cultured in medium containing 0.5% FCS for indicated periods. D, Mel-RMu.S and Mel-CV.S cells and their parental counterparts were cultured in medium containing 5% FCS till approximate 50% confluence. Cells were then switched into medium with 0.5% FCS for 48 hours. Apoptosis was quantitated by the PI method. The data shown are the mean Æ SE of results from 3 individual experiments.

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Figure 5. Increased activation of Akt is responsible for activation of ERK in the PLX-selected melanoma cells. A, Mel-RMu.S and Mel-CV.S cells and their parental counterparts were cultured in medium containing 5% FCS till approximate 50% confluence. Half of the cultures were then switched into medium with 0.5% FCS, and the rest were switched into fresh medium containing 5% FCS for 24 hours. Whole cell lysates were subjected to Western blot analysis of p-Akt and Akt. The data shown are representative of 3 individual experiments. B, Mel-RMu.S and Mel-CV.S cells and their parental counterparts with or without exposure to LY294002 (20 mmol/L) for 6 hours were subjected to Western blot analysis of p-Akt, Akt, p-ERK, and ERK. The data shown are representative of 3 individual experiments. C, Mel-CV and Mel-CV.S cells were transfected with the control, Akt1, Akt2, and Akt3 siRNA, respectively. Twenty-four hours later, whole cell lysates were subjected to Western blot analysis of p-Akt, Akt1, Akt2, Akt3, p-ERK, and ERK. The data shown are representative of 3 individual experiments.

albeit with slow growth rate, even in the presence of a reason for this discrepancy is unknown, but it may be relatively high concentration of the inhibitor for prolonged related to different biochemical properties of the 2 inhi- periods. These results highlight the importance of induc- bitors used in the studies (12). tion of apoptosis in determining long-term responses of B- The present results show that reactivation of ERK appears RAFV600E melanoma cells to specific inhibitors and suggest largely independent of MEK. This was demonstrated by the that it is critical to administer the inhibitors at dosages that lack of MEK activation and the failure of MEK inhibitors efficiently induce apoptosis in melanomas that are naive to and siRNA knockdown of MEK to inhibit ERK activation in the inhibitors. the cells. The lack of activation of MEK may be due to Reactivation of ERK is a common finding after inhibition impaired upstream signaling, as the levels of MEK in the of the RAF/MEK/ERK pathway (30). In melanoma cells PLX-selected cells remained comparable to those in the harboring wild-type B-RAF, this is largely due to relief of parental counterparts. Another possible explanation is loss- negative feedback mediators such as the Sprouty proteins of-function mutations in MEK, which has been reported to and the mitogen-activated protein kinase phosphatases be responsible for the failure of melanoma cells to respond (31–33). However, the feedback is impaired in melanoma to the MEK inhibitor AZD6244 (36). Regardless of the cells carrying activating mutations in B-RAF (33, 34). This is mutational status of MEK in the PLX-selected melanoma associated with the inability of mutant B-RAF to bind to cells, our results clearly indicated that mechanisms other Sprouty family proteins (34, 35). Nevertheless, we found in than MEK can substitute its role in activation of ERK. this study that ERK was highly activated in PLX-selected B- Available evidence suggests that ERK activation was RAFV600E melanoma cells. Although elevated C-RAF has dependent on activation of the PI3k/Akt pathway in that been reported to be a mechanism of reactivation of MEK/ Akt activation was increased in the PLX-selected melanoma ERK and acquired resistance to the RAF inhibitor AZ628 in cells and that inhibition of the pathway by inhibitors of mutant B-RAF melanoma cells (12), the increase in C-RAF PI3k or knockdown of Akt3 inhibited activation of ERK in did not appear to play a major role in reactivation of ERK in the cells. Increased activation of the PI3k/Akt pathway in the PLX-selected B-RAFV600E cells in this study. The exact the PLX-selected cells appeared to result from extracellular

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MEK-Independent Survival of PLX4720-Selected Melanoma Cells

these 2 major survival pathways (18, 25, 37) and indicate that ERK can be activated by the PI3k/Akt pathway when its conventional upstream signaling is switched off in melanoma cells. The rebound activation of ERK in PLX-selected melanoma cells appeared necessary for survival of the cells because siRNA knockdown of either ERK1 or ERK2 caused reduction in cell viability. This was recapitulated by inhibition of the PI3k/Akt pathway and reduction in extracellular stimulation, consistent with their roles in activation of ERK in the PLX-selected melanoma cells. Notably, inhibition of ERK did not induce apoptosis in the parental counterparts, suggesting that the PLX-selected cells were more addicted to activation of ERK for their survival. It is of note that inhibition of MEK did not impinge on survival of PLX- selected melanoma cells, although it enhanced PLX- induced apoptosis in the parental counterparts (13). There- fore, activation of ERK is responsible for survival of the PLX-selected B-RAFV600E melanoma cells, which may not be susceptible to inhibition of MEK, but may be responsive to inhibition of PI3k/Akt. In conclusion, we have shown in this study that (1) B- RAFV600E melanoma cells resistant to apoptosis induced by PLX4720 selected by prolonged exposure to the inhibitor can proliferate even in the presence of the inhibitor; (2) the conventional RAF/MEK/ERK pathway is dispen- sable for survival of the PLX-selected melanoma cells; and (3) activation of ERK by the PI3k/Akt pathway resulting from extracellular stimulations is responsible for survival of B-RAFV600E melanoma cells after long-term exposure to PLX4720. These results suggest that (1) induction of apoptosis is a major determinant of long-term responses of melanoma cells to mutant B-RAF inhibitors; (2) regrowth of B-RAFV600E melanomas after discontinuation of BRAF inhibitors may not respond to single-agent MEK inhibitor; and (3) inhibition of the PI3k/Akt pathway may be a useful strategy in the treatment of recurrent B- RAFV600E melanomas after discontinuation of B-RAF inhi-

Figure 6. Inhibition of PI3K/Akt, but not inhibition of MEK, induces bitors. In addition, these results, along with others (12), apoptosis of the PLX-selected melanoma cells. A, Mel-RMu and Mel-CV suggest that combination of B-RAF inhibitors and those cells were treated with U0126 (20 mmol/L), PLX4720 (10 mmol/L), or the against MEK and/or PI3k/Akt should be considered at the combination of both for 72 hours. Mel-RMu.S and Mel-CV.S were also initial treatment of melanomas carrying activating muta- m exposed to U0126 (20 mol/L; the selected cells were routinely cultured in tions in B-RAF. the presence of PLX4720) for 72 hours. Apoptosis was quantitated by the PI method. The data shown are the mean Æ SE of results from 3 individual culture wells. B, Mel-RMu and Mel-CV cells were treated with LY294002 Disclosure of Potential Conflicts of Interest (20 mmol/L), PLX4720 (10 mmol/L), or the combination of both for 72 hours. Mel-RMu.S and Mel-CV.S were also exposed to LY294002 (20 mmol/L; the No potential conflicts of interest were disclosed. selected cells were routinely cultured in the presence of PLX4720) for 72 hours. Apoptosis was quantitated by the PI method. The data shown are the mean Æ SE of results from 3 individual culture wells. C, Mel-CV and Acknowledgments Mel-CV.S cells were transfected with the control and Akt3 siRNA, respectively. Forty-eight hours later, apoptosis was quantitated by the PI Æ This work was supported by the NSW State Cancer Council and National method. The data shown are the mean SE of results from 3 individual Health and Medical Research Council, Australia. X.D. Zhang and R.F. culture wells. Thorne are Cancer Institute NSW Fellows. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked signals as reduction in concentrations of FCS in culture advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate medium caused not only marked decreases in activation of this fact. ERK but also decreases in activation of Akt. These observa- Received August 19, 2010; revised November 2, 2010; accepted tions are in line with the existence of cross talk between November 8, 2010; published OnlineFirst November 18, 2010.

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References 1. Flaherty KT, Puzanov I, Sosman J, Kim K, Ribas A, McArthur G, et al. 19. Shao Y, Aplin AE. Akt3-mediated resistance to apoptosis in B-RAF- Phase I study of PLX4032: proof of concept for V600E BRAF mutation targeted melanoma cells. Cancer Res 2010;70:6670–81. as a therapeutic target in human cancer. J Clin Oncol 2009;27;461s, 20. Jiang CC, Lai F, Tay KH, Hersey P, Zhang XD. Apoptosis of human Abstract 9000. melanoma cells induced by inhibition of B-RAFV600E involves pre- 2. Chapman P, Puzanov I, Sosman J, Kim K, Ribas A, McArthur G, et al. ferential splicing of BimS. Cell Death Dis 2010;1:e69. Early efficacy signal demonstrated in advanced melanoma in a phase I 21. Yang F, Tay KH, Dong L, Thorne RF, Jiang CC, Yang E, et al. Cystatin trial of the oncogenic BRAF-selective inhibitor PLX4032. Eur J Cancer B inhibition of TRAIL-induced apoptosis is associated with the pro- Suppl 2009;7:5. tection of FLIP(L) from degradation by the E3 ligase itch in human 3. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al. melanoma cells. Cell Death Differ 2010;17:1354–67. Mutations of the BRAF gene in human cancer. Nature 2002;417:949– 22. Puzanov I, Nathanson KL, Chapman PB, Xu X, Sosman G, McArthur 54. GA, et al. Correlation of activity with pharmacokinetic and pharma- 4. Platz A, Egyhazi S, Ringborg U, Hansson J. Human cutaneous codynamic parameters in a phase I trial. J Clin Oncol 2009;27;p466s, melanoma; a review of NRAS and BRAF mutation frequencies in Abstract 9021. relation to histogenetic subclass and body site. Mol Oncol 23. Robinson MJ, Cobb MH. Mitogen-activated protein kinase pathways. 2008;1:395–405. Curr Opin Cell Biol 1997;9:180–6. 5. Wellbrock C, Rana S, Paterson H, Pickersgill H, Brummelkamp T, 24. Smalley KS. Understanding melanoma signaling networks as the Marais R. Oncogenic BRAF regulates melanoma proliferation through basis for molecular targeted therapy. J Invest Dermatol 2010; the lineage specific factor MITF. PLoS One 2008;3:e2734. 130:28–37. 6. Tsai J, Lee JT, Wang W, Zhang J, Cho H, Mamo S, et al. Discovery of a 25. Aksamitiene E, Kholodenko BN, Kolch W, Hoek JB, Kiyatkin A. PI3K/ selective inhibitor of oncogenic B-Raf kinase with potent antimela- Akt-sensitive MEK-independent compensatory circuit of ERK activa- noma activity. Proc Natl Acad Sci U S A 2008;105:3041–6. tion in ER-positive PI3K-mutant T47D breast cancer cells. Cell Signal 7. Cragg MS, Jansen ES, Cook M, Harris C, Strasser A, Scott CL. 2010;22:1369–78. Treatment of B-RAF mutant human tumor cells with a MEK inhibitor 26. Stahl JM, Sharma A, Cheung M, Zimmerman M, Cheng JQ, requires Bim and is enhanced by a BH3 mimetic. J Clin Invest Bosenberg MW, et al. Deregulated Akt3 activity promotes devel- 2008;118:3651–9. opment of malignant melanoma. Cancer Res 2004;64:7002– 8. Wang YF, Jiang CC, Kiejda KA, Gillespie S, Zhang XD, Hersey P. 10. Apoptosis induction in human melanoma cells by inhibition of MEK is 27. Balmanno K, Cook SJ. Tumour cell survival signalling by the ERK1/2 caspase-independent and mediated by the Bcl-2 family members pathway. Cell Death Differ 2009;16:368–77. PUMA, Bim, and Mcl-1. Clin Cancer Res 2007;13:4934–42. 28. Lavoie JN, Rivard N, L’Allemain G, Pouyssegur J. A temporal and 9. Panka DJ, Atkins MB, Mier JW. Targeting the mitogen-activated biochemical link between growth factor-activated MAP kinases, protein kinase pathway in the treatment of malignant melanoma. Clin cyclin D1 induction and cell cycle entry. Prog Cell Cycle Res 1996; Cancer Res 2006;12:2371s–5s. 2:49–58. 10. VanBrocklin MW, Verhaegen M, Soengas MS, Holmen SL. Mitogen- 29. Ballif BA, Blenis J. Molecular mechanisms mediating mammalian activated protein kinase inhibition induces translocation of Bmf to mitogen-activated protein kinase (MAPK) kinase (MEK)-MAPK cell promote apoptosis in melanoma. Cancer Res 2009;69:1985–94. survival signals. Cell Growth Differ 2001;12:397–408. 11. Sndergaard JN, Nazarian R, Wang Q, Guo D, Hsueh T, Mok S, et al. 30. Pratilas CA, Solit DB. Targeting the mitogen-activated protein kinase Differential sensitivity of melanoma cell lines with BRAFV600E muta- pathway: physiological feedback and drug response. Clin Cancer Res tion to the specific Raf inhibitor PLX4032. J Transl Med 2010;8:39. 2010;16:3329–34. 12. Montagut C, Sharma SV, Shioda T, McDermott U, Ulman M, Ulkus LE, 31. Kim HJ, Bar-Sagi D. Modulation of signalling by Sprouty: a developing et al. Elevated CRAF as a potential mechanism of acquired resistance story. Nat Rev Mol Cell Biol 2004;5:441–50. to BRAF inhibition in melanoma. Cancer Res 2008;68:4853–61. 32. Caunt CJ, Armstrong SP, Rivers CA, Norman MR, McArdle CA. 13. Paraiso KH, Fedorenko IV, Cantini LP. Recovery of phospho-ERK Spatiotemporal regulation of ERK2 by dual specificity phosphatases. activity allows melanoma cells to escape from BRAF inhibitor therapy. J Biol Chem 2008;283:26612–23. Br J Cancer 2010;102:1724–30. 33. Yeung K, Seitz T, Li S, Janosch P, McFerran B, Kaiser C, et al. 14. Christensen C, Guldberg P. Growth factors rescue cutaneous mela- Suppression of Raf-1 kinase activity and MAP kinase signalling by noma cells from apoptosis induced by knockdown of mutated (V 600 RKIP. Nature 1999;401:173–7. E) B-RAF. Oncogene 2005;24:6292–302. 34. Friday BB, Yu C, Dy GK, Smith PD, Wang L, Thibodeau SN, et al. 15. Heidorn SJ, Milagre C, Whittaker S, Nourry A, Niculescu-Duvas I, BRAF V600E disrupts AZD6244-induced abrogation of negative feed- Dhomen N, et al. Kinase-dead BRAF and oncogenic RAS cooperate to back pathways between extracellular signal-regulated kinase and Raf drive tumor progression through CRAF. Cell 2010;140:209–21. proteins. Cancer Res 2008;68:6145–53. 16. Hatzivassiliou G, Song K, Yen I, Brandhuber BJ, Anderson DJ, 35. Ritt DA, Monson DM, Specht SI, Morrison DK. Impact of feedback Alvarado R, et al. RAF inhibitors prime wild-type RAF to activate phosphorylation and Raf heterodimerization on normal and mutant B- the MAPK pathway and enhance growth. Nature 2010;464:431–5. Raf signaling. Mol Cell Biol 2010;30:806–19. 17. Poulikakos PI, Zhang C, Bollag G, Shokat KM, Rosen N. RAF inhi- 36. Emery CM, Vijayendran KG, Zipser MC, Sawyer AM, Niu L, Kim JJ, bitors transactivate RAF dimers and ERK signalling in cells with wild- et al. MEK1 mutations confer resistance to MEK and B-RAF type BRAF. Nature 2010;464:427–30. inhibition. Proc Natl Acad Sci U S A 2009;106:20411–6. 18. Grammer TC, Blenis J. Evidence for MEK-independent pathways 37. Sato S, Fujita N, Tsuruo T. Involvement of 3-phosphoinositide-depen- regulating the prolonged activation of the ERK-MAP kinases. Onco- dent protein kinase-1 in the MEK/MAPK signal transduction pathway. gene 1997;14:1635–42. J Biol Chem 2004;279:33759–67.

OF10 Clin Cancer Res; 17(4) February 15, 2011 Clinical Cancer Research

MEK-Independent Survival of B-RAFV600E Melanoma Cells Selected for Resistance to Apoptosis Induced by the RAF Inhibitor PLX4720

Chen Chen Jiang, Fritz Lai, Rick F. Thorne, et al.

Clin Cancer Res Published OnlineFirst November 18, 2010.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-10-2225

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