MEK Inhibition

ORIGINAL ARTICLE

Acquired Resistance to BRAF Inhibition Can Confer Cross-Resistance to Combined BRAF/MEK Inhibition Kavitha Gowrishankar1, Stephanie Snoyman1, Gulietta M. Pupo1, Therese M. Becker1, Richard F. Kefford1 and Helen Rizos1 Aberrant activation of the BRAF kinase occurs in B60% of melanomas, and although BRAF inhibitors have shown significant early clinical success, acquired resistance occurs in most patients. Resistance to chronic BRAF inhibition often involves reactivation of mitogen-activated protein kinase (MAPK) signaling, and the combined targeting of BRAF and its downstream target MAPK/ERK kinase (MEK) may delay or overcome resistance. To investigate the efficacy of combination BRAF and MEK inhibition, we generated melanoma cell clones resistant to the BRAF inhibitor GSK2118436. These BRAF inhibitor–resistant sublines acquired resistance through several distinct mechanisms, including the acquisition of activating N-RAS mutations and increased accumulation of COT1. These alterations uniformly promoted MAPK reactivation and most conferred resistance to MEK inhibition and to the concurrent inhibition of BRAF and MEK. These data indicate that melanoma tumors are likely to develop heterogeneous mechanisms of resistance, many of which will confer resistance to multiple MAPK inhibitory therapies. Journal of Investigative Dermatology (2012) 132, 1850–1859; doi:10.1038/jid.2012.63; published online 22 March 2012

INTRODUCTION Constitutively activating mutations in the serine/threonine kinase BRAF are found in a large proportion of metastatic melanomas (Davies et al., 2002; Long et al., 2011). The majority of these mutations result in a single amino-acid substitution of valine by glutamic acid at amino acid 600, and this leads to a 500-fold increase in the kinase activity of BRAF (Davies et al., 2002; Wan et al., 2004). As a result, most melanomas display a dependency on the RAF/MEK/ERK mitogen-activated protein kinase (MAPK) pathway, as shown by early clinical success with the RAF-targeted inhibitors Vemurafenib (PLX4032) and GSK2118436 (Flaherty et al., 2010; Kefford et al., 2010). Despite the marked initial responses to BRAF inhibitors, tumor regrowth occurs in most patients (Kefford et al., 2010), with a median progression-free survival of 5.3 months (Flaherty et al., 2010; Chapman et al., 2011). Resistance to 1

Westmead Institute for Cancer Research, University of Sydney at Westmead Millennium Institute and Melanoma Institute Australia, Westmead Hospital, Westmead, New South Wales, Australia Correspondence: Helen Rizos, Westmead Institute for Cancer Research, University of Sydney at Westmead Millennium Institute, Westmead Hospital, Westmead, New South Wales 2145, Australia. E-mail: [email protected] Abbreviations: DR, drug resistant; ERK, extracellular signal–regulated kinase; IC50 , half-maximal inhibitory concentration; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; mTOR, mammalian target of rapamycin; PDGFRb, platelet-derived growth factor receptor-b; p-ERK, phosphorylated extracellular signal–regulated kinase; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog; pRb, retinoblastoma protein; shRNA, short hairpin RNA Received 16 August 2011; revised 26 January 2012; accepted 30 January 2012; published online 22 March 2012

1850 Journal of Investigative Dermatology (2012), Volume 132

chronic BRAF inhibition involves reactivation of MAPK signaling and/or induction of prosurvival signals. For instance, a subset of BRAF inhibitor–resistant melanoma tumors restored MAPK activity by expressing truncated forms of BRAF or by elevating the expression of the serine/threonine kinases BRAF, CRAF, or COT1 (Montagut et al., 2008; Corcoran et al., 2010; Johannessen et al., 2010; Poulikakos et al., 2011). Similarly, activating mutations in N-RAS (Q61K/ R) and MEK1 (C121S) triggered phosphorylated extracellular signal–regulated kinase (p-ERK) signaling in BRAF inhibitor–resistant cell lines and clinical samples (Nazarian et al., 2010; Wagle et al., 2011). Moreover, the persistent expression of the receptor tyrosine kinases platelet-derived growth factor receptor-b (PDGFRb) and IGF-1R conferred BRAF inhibitor resistance in a MAPK-independent manner that may involve enhanced phosphatidylinositol 3-kinase (PI3K)/AKT pathway activation (Nazarian et al., 2010; Villanueva et al., 2010; Shi et al., 2011). In line with these data, PTEN (phosphatase and tensin homolog) loss also activated PI3K/AKT signaling and conferred partial resistance to BRAF inhibition in BRAFV600Epositive melanoma cell lines (Paraiso et al., 2011). Importantly, recent studies indicate that MAPK reactivation can modulate sensitivity to MAPK/ERK kinase (MEK) inhibition. For instance, melanoma cell lines harboring oncogenic N-RAS were sensitive to MEK inhibitors (Montagut et al., 2008; Nazarian et al., 2010), whereas tumor cells expressing mutant MEK1 or COT1 were refractory to MEK inhibition and maintained ERK phosphorylation in the presence of these inhibitors (Emery et al., 2009; Johannessen et al., 2010). There are currently limited data available on the activity of combining BRAF and MEK inhibitors, although the concurrent use of these inhibitors overcame resistance to & 2012 The Society for Investigative Dermatology

K Gowrishankar et al. MAPK Reactivation Dampens BRAF and MEK Inhibition

single agents in the setting of MEK mutations and COT1 overexpression (Emery et al., 2009; Johannessen et al., 2010). In this report, we demonstrate that distinct mechanisms of acquired resistance to BRAF inhibition can originate from a single melanoma tumor cell. These resistance drivers included the acquisition of activating N-RAS mutations and increased accumulation of COT1. These distinct alterations uniformly reactivated MAPK signaling and typically conferred cross-resistance to MEK inhibitors and the combination of MEK and BRAF inhibitors. These data indicate that melanoma tumors from a single patient are likely to develop heterogeneous mechanisms of resistance, many of which will confer resistance to multiple MAPK inhibitory therapies. RESULTS Generation of melanoma cell clones with acquired resistance to the RAF kinase inhibitor GSK2118436

To examine acquired resistance to the potent and highly selective BRAF kinase inhibitor, GSK2118436 (GSK-BRAFi), we generated resistance in the MelRMu human melanoma–derived cell line (Jiang et al., 2010). This cell line harbors activating mutations in BRAF (V600E), EGFR (P753S), and CDK4 (R24C), and is exquisitely sensitive to BRAF inhibition (half-maximal inhibitory concentration (IC50) 6 nM; Figure 1a). As expected, MelRMu cells responded to increasing concentrations of GSK-BRAFi by undergoing a G1 cell cycle arrest followed by potent apoptosis (Figure 1b and c). The BRAF inhibitor–induced proliferative arrest was associated with activation of the retinoblastoma protein (pRb; i.e, loss of

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Figure 1. Analysis of the BRAF inhibitor–sensitive MelRMu melanoma cell line. (a) Viability curve for MelRMu cell line treated with increasing concentrations of GSK-BRAFi for 72 hours (relative to DMSO-treated controls; mean±SD; n ¼ 3). (b) Cell cycle distribution of MelRMu cells treated with either DMSO (0 nM) or GSK-BRAFi (100 nM) for 72 hours. (c) Dual-color flow cytometric Annexin V analysis for apoptosis of MelRMu cells treated with DMSO (0 nM) or GSK-BRAFi (100 nM) for 72 hours. APC, allophycocyanin; PI, propidium iodide. (d) MelRMu cells were treated with 100 nM GSK-BRAFi for increasing duration. The effects on extracellular signal–regulated kinase (ERK) activation and cell cycle regulators were determined by immunoblotting.

hyperphosphorylated and total pRb), accumulation of the CDK inhibitor p27Kip1, and loss of cyclin D1 expression. Importantly, activation of pRb and cell cycle arrest was preceded by decreases in p-ERK that were detectable within 1 hour and up to 24 hours after exposure to GSK-BRAFi (Figure 1d). At 5 weeks after continuously exposing MelRMu cells to 100 nM GSK-BRAFi, single cell–derived drug-resistant clones emerged at a frequency of B1 in 105 cells. Five of these drugresistant (DR) clones (DR2, 4, 6, 8, and 9) were expanded and characterized. All five DR clones retained the activating BRAF, EGFR, and CDK4 mutations found in the parental line (data not shown) and showed strong resistance to GSK-BRAFi (Figure 2a) with IC50 not reached at 1,000 nM (data not shown). In all resistant DR clones, MAPK reactivation was confirmed using protein and gene expression analyses; p-ERK expression was maintained in the presence and absence of the drug (Figure 2b), and the resistant sublines demonstrated a MEK/ERK transcriptome signature indicative of persistent MEK/ERK activation (Dry et al., 2010; Nazarian et al., 2010; Figure 2c). This gene set included many known transcription targets of ERK signaling, including MYC, ETV5, and the negative feedback regulators DUSP4/6 and SPRY1/2. As a result, the DR clones responded to BRAF inhibition with a continued, although somewhat slower, rate of proliferation (data not shown), with little evidence of GSK-BRAFi-induced cell death (Figure 2d). Notably, although DR clones survived in response to BRAF inhibition, they all showed evidence of reduced S-phase entry that was associated with p27Kip1 accumulation and reduced levels of cyclin D1 (Figure 2b). Analyses for mutations in the candidate oncogenes H-RAS, K-RAS, N-RAS, BRAF, and KIT revealed that MelRMu DR2 and DR6 sublines carried the N-RASQ61H-activating mutation at a frequency of B30%. This mutation was not detected in the parental MelRMu cell line. Concordant with these data, unsupervised clustering of differential gene expression profiles showed that DR2 and DR6 share characteristics and group away from the parental and other DR clones (Supplementary Figure S1 online). We also noted that PTEN expression was retained, CRAF levels were not substantially elevated (Figure 3a), and truncated variant forms of BRAF were not detected in the DR sublines (Supplementary Figure S2 online). Furthermore, although AKT was activated (increased p-AKT) and PDGFRb expression was elevated in the DR8 and DR9 sublines (Figure 3a), these clones did not show activation-associated PDGFRb phosphorylation in a phospho-RTK array or western immunoblotting (detailed analyses of DR9 are shown in Figure 3b), and they did not display an increased PDGFRbspecific gene signature (Wu et al., 2008; Nazarian et al., 2010; Supplementary Figure S3 online). Finally, the concurrent inhibition of MAPK with PI3K/mammalian target of rapamycin (mTOR; BEZ235) or PDGFRb (imatinib mesylate) did not trigger DR9 cell death (Figure 3c). Collectively, these data suggest that neither PDGFRb nor the PI3K pathway mediate BRAF inhibitor resistance in the DR9 subline. The DR4 cell clone displayed elevated expression of the COT1 kinase (Figure 3a), and in accordance with a recent report www.jidonline.org 1851

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tion to suppress DR4 viability, without affecting the sensitivity of the MelRMu parental line (Figure 3d).

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Activating N-RAS reactivates MAPK and confers resistance to BRAF inhibition

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Activating N-RAS mutations (Q61K and Q61R) have previously been identified in two biopsy samples derived from BRAF inhibitor (PLX4032)–resistant melanoma metastases from a single patient (Nazarian et al., 2010). To thoroughly examine the role of oncogenic N-RAS in regulating tumor growth and conferring resistance to BRAF inhibition, we initially silenced the expression of N-RAS using highly specific shRNA molecules (Figure 4a). The suppression of N-RAS in the N-RAS wild-type DR4 and N-RAS mutant DR2 and DR6 sublines did not result in proliferative arrest

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We then compared the gene expression profiles of the MelRMu-derived resistant sublines with a signature predictive of MEK resistance, which appears to be driven upstream of RAF and independently of RAS or PI3K pathway mutations (Dry et al., 2010). Although only 6 of the 13 genes in this published resistance signature mapped to the Illumina platform, these genes showed significantly increased expression in the closely related DR8- and DR9-resistant sublines (Supplementary Figure S4 online). These data indicate that distinct mechanisms originating from a single melanoma cell line mediate MAPK reactivation and resistance to BRAF inhibition.

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Figure 3. BRAF inhibitor–resistant isogenic MelRMu sublines display distinct mechanisms of acquired resistance. (a) MelRMu parental and BRAF inhibitor–resistant sublines were treated with DMSO () or 100 nM GSK-BRAFi ( þ ) for 24 hours, and the effects on known drug-resistance regulators were determined by immunoblotting. (b) Phosphorylation of platelet-derived growth factor receptor-b (PDGFRb) was analyzed in the MelRMu parent and DR9 subline using phospho-RTK antibody arrays (left panel) and western immunoblotting (right panel). The positions of phosphorylated EGFR (positive in MelRMu and DR9) and phosphorylated PDGFRb (negative in MelRMu and DR9) are indicated. DR, drug resistant. (c) Impact of mitogen-activated protein kinase (MAPK) inhibition (100 nM GSK-BRAFi and 5 nM GSK-MEKi) either alone or in combination with receptor tyrosine kinase inhibition (100 nM imatinib mesylate) or phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibition (1 mM BEZ235) on cell survival at 72 hours after treatment. The inhibitory activity of imatinib and BEZ235 on c-Kit and AKT activation was confirmed in the c-Kit mutant (c-KitW557K558) melanoma cell line, MelMS. (d) MelRMu and DR4 were transfected with COT1 short hairpin RNA (shRNA; þ ) or control () shRNA. At 24 hours after transfection, cells were treated with DMSO () or 100 nM GSK-BRAFi ( þ ) for an additional 72 hours. COT1 silencing (right panel) and the effects on cell death (left panel) were measured 72 hours after drug addition.

Figure 2. Mitogen-activated protein kinase (MAPK) reactivation in cells with acquired resistance to GSK-BRAFi. (a) Viability curves of the parental and isogenic melanoma MelRMu sublines treated with the indicated GSK-BRAFi concentrations for 72 hours (relative to DMSO-treated controls; mean±SD; n ¼ 3). DR, drug resistant. (b) MelRMu parental cells and BRAF inhibitor–resistant sublines were treated with DMSO () or 100 nM GSK-BRAFi ( þ ) for 24 hours, and the effects on extracellular signal–regulated kinase (ERK) activation and cell cycle regulators were determined by immunoblotting. (c; left panel) Heat map for MEK/ERK activation signature in each of the cell lines treated with DMSO () or 100 nM GSK-BRAFi ( þ ) for 24 hours. Color scale, log2-transformed expression (red, high; green, low) for each gene (row) normalized by the mean of all samples. The probe ID is shown after each gene symbol. (c; right panel) Histograms of mean log2-transformed expression of all transcripts included in the MEK/ERK activation signature (see left panel). MEK, MAPK/ERK kinase. (d) GSK-BRAFi effects on cell cycle distribution and apoptosis. APC, allophycocyanin; PI, propidium iodide.

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Figure 4. Oncogenic N-RAS confers BRAF inhibitor resistance. (a) MelRMu-derived resistant cell lines were transduced with N-RAS no. 1 short hairpin RNA (shRNA; þ ) or control () shRNA. At 5 days after transduction, cells were treated with DMSO () or 100 nM GSK-BRAFi ( þ ) for an additional 72 hours. The effects on phosphorylated extracellular signal–regulated kinase (p-ERK) accumulation were detectable 24 hours after exposure to GSK-BRAFi (left panel), and the downstream effects on cell death were measured 72 hours after drug addition (right panel). DR, drug resistant. (b) MelRMu parental cell lines were lentivirally transduced with N-RASQ61K or copGFP control, and after 10 days cells were treated with DMSO () or 100 nM GSK-BRAFi ( þ ). The effects on p-ERK, cell cycle regulators, and N-RAS accumulation (left panel) and apoptosis (right panel) are shown.

(data not shown) or apoptosis in the absence of the BRAF inhibitor, indicating that these resistant clones did not depend on N-RAS signaling for growth (Figure 4a). In the presence of BRAF inhibitor, however, the stable suppression of N-RAS expression suppressed ERK activation, reinstated sensitivity to GSK-BRAFi, and resulted in potent cell death in the N-RAS mutant DR2 and DR6 sublines (Figure 4a and Supplementary Figure S5 online). This effect was highly specific, as suppression of N-RAS expression in the parental MelRMu and DR4 cells (both wild type for N-RAS) had no effect on GSK-BRAFi sensitivity (Figure 4a and Supplementary Figure S5 online). Conversely, the stable overexpression of MYCtagged N-RASQ61K promoted accumulation of p-ERK and conferred BRAF inhibitor resistance in the MelRMu parental cell line (Figure 4b). Oncogenic N-RAS dampens sensitivity to MEK inhibition

As shown in Figure 3a, activated N-RAS did not activate the PI3K/AKT pathway in the DR2 and DR6 sublines. Consequently, we predicted that melanoma cells expressing oncogenic N-RAS signaled predominantly via the reactivated MAPK pathway and would retain sensitivity to MEK inhibition. Surprisingly, we found that the pattern of sensitivity to MEK inhibition, using the allosteric MEK inhibitor GSK1120212 (GSK-MEKi; Gilmartin et al., 2011), was significantly diminished in most BRAF inhibitor–resistant MelRMu DR sublines: GSK-MEKi IC50 o2 nM for MelRMu 1854 Journal of Investigative Dermatology (2012), Volume 132

parental cells (Figure 5a). Critically, the level of resistance to MEK inhibition paralleled the unsupervised clustering of our DR clones in genome-wide, differential expression patterns (Supplementary Figure S1 online). Specifically, both N-RAS mutant DR2 and DR6 clones showed diminished MEK inhibitor sensitivity; the DR4 clone (expresses COT1 and clusters closely with the parental MelRMu line in gene profiling; Supplementary Figure S1 online) was sensitive to MEK inhibition and showed effective suppression of ERK phosphorylation (Figure 5b). The DR8 and DR9 clones, which are distinct from the other DR clones but share a common MEK resistance signature (Supplementary Figure S4 online), were highly resistant to MEK inhibition (Figure 5). Importantly, all resistant sublines showed minimal cell death in response to MEK inhibition but displayed a degree of S-phase inhibition that was associated with reduced cyclin D1 (Figure 5b and c). Finally, we also showed that the stable overexpression of MYC-tagged N-RASQ61K conferred MEK inhibitor resistance in the MelRMu parental cell line (Figure 5d). Dual inhibition of MEK and BRAF can restore partial sensitivity of melanoma cells with activating N-RAS and BRAF mutations

MelRMu parental cells were exquisitely sensitive to BRAF inhibition, and the combination of BRAF and MEK inhibition enhanced cell death in these cells (Figure 6). Similarly, in the majority of DR clones, targeting the MAPK pathway at both the BRAF and MEK nodes increased the percentage of cells

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Figure 5. Acquired resistance to BRAF inhibition can dampen MAPK/ERK kinase (MEK) inhibitor sensitivity. (a) Viability curves of the parental and isogenic melanoma MelRMu sublines treated with the indicated GSK-MEKi concentrations for 72 hours (relative to DMSO-treated controls; mean±SD; n ¼ 2). DR, drug resistant. (b) MelRMu parental cells and BRAF inhibitor–resistant sublines were treated with DMSO () or 5 nM GSK-MEKi ( þ ) for 24 hours, and the effects on extracellular signal–regulated kinase (ERK) activation and cell cycle regulators were determined by immunoblotting. (c) GSK-MEKi effects on cell cycle distribution and apoptosis. APC, allophycocyanin; PI, propidium iodide. (d) MelRMu parental cell lines were lentivirally transduced with N-RASQ61K or copGFP control, and after 10 days cells were treated with DMSO () or 5 nM GSK-MEKi ( þ ) for 72 hours. Percentage of cells in sub-G1 fraction is shown.

undergoing cell death, although this increase never reached the level of apoptosis seen in the parental cells (Figure 6b). For example, in response to the combined inhibition of BRAF and MEK, the N-RAS mutant DR2 and DR6 sublines

displayed a sub-G1 fraction of B20% compared with over 60% sub-G1 cells in the parent melanoma line (Figure 6a and b). Moreover, ectopic expression of N-RASQ61K in the MelRMu cells diminished the efficacy of the combination of www.jidonline.org 1855

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Figure 6. Combined BRAF and MAPK/ERK kinase (MEK) inhibition does not restore sensitivity to mitogen-activated protein kinase (MAPK) inhibition in BRAF inhibitor–resistant cells. (a) The effects of GSK-BRAFi (100 nM) and GSK-MEKi (5 nM) on cell cycle distribution and apoptosis of MelRMu parental and derived sublines at 72 hours after treatment. APC, allophycocyanin; PI, propidium iodide. (b) Activity of combination and single MAPK inhibitors in promoting cell death in MelRMu parental and drug resistant (DR) sublines at 72 hours after drug treatment. (c) MelRMu parental cell lines were lentivirally transduced with N-RASQ61K or copGFP control, and after 10 days cells were treated with DMSO () or 100 nM GSK-BRAFi and 5 nM GSK-MEKi ( þ ) for 72 hours. Percentage of cells in subG1 fraction is shown. (d) MelRMu parental cells and BRAF inhibitor–resistant sublines were treated with DMSO () or 100 nM GSK-BRAFi and 5 nM GSK-MEKi ( þ ) for 24 hours, and the effects on extracellular signal–regulated kinase (ERK) activation and cell cycle regulators were determined by immunoblotting.

MEK and BRAF inhibition (Figure 6c). Finally, the highly resistant DR9 subline remained refractory to the simultaneous inhibition of MEK and BRAF. In line with these data, the combination of BRAF and MEK inhibition did not significantly inhibit MAPK signaling in the DR clones, although the reduced levels of cyclin D1 and p-pRb (both markers of cell cycle inhibition) were comparable in the DR clones and MelRMu parent (Figure 6d). DISCUSSION The prevalent acquisition of resistance to BRAF inhibitors is a major problem in clinical oncology and reflects the capacity 1856 Journal of Investigative Dermatology (2012), Volume 132

of melanomas to either circumvent their dependence on MAPK activity or, more commonly, restore MAPK signaling. For instance, increased expression of the IGF-1R or PDGFRb receptor tyrosine kinases activates the compensatory PI3K/ AKT survival pathway (Nazarian et al., 2010; Villanueva et al., 2010). Similarly, alterations that facilitate ERK activation, such as increased abundance of CRAF or COT1, activating mutations in MEK or N-RAS, and amplification or truncation of BRAF, have been identified in vivo and in vitro (Montagut et al., 2008; Corcoran et al., 2010; Johannessen et al., 2010; Nazarian et al., 2010; Poulikakos et al., 2011; Wagle et al., 2011). In this report, we show that MAPK

K Gowrishankar et al. MAPK Reactivation Dampens BRAF and MEK Inhibition

reactivation is the predominant mechanism of acquired BRAF inhibitor resistance, with all resistant melanoma sublines displaying reactivation and often enhanced MAPK signaling in the presence of the BRAF inhibitor GSK2118436. The restoration of MAPK activity in the presence of BRAF inhibitor, and the observation that tumor regression requires near-complete cessation of ERK activity, suggests that the combination of inhibitors targeting multiple nodes in the MAPK pathway may provide a more durable and prolonged clinical response (Bollag et al., 2010). Consistent with this hypothesis, combined MEK and BRAF inhibition overcame resistance of colorectal cancer cells to MEK or BRAF inhibitors used singly and was more effective in parental cells compared with either agent alone (Corcoran et al., 2010). Moreover, drug trials testing concurrent therapy with BRAF and MEK inhibitors are underway in patients with mutant BRAF metastatic melanoma (Infante et al., 2011). In this report, the potency of combined MEK and BRAF inhibition was assessed in a series of melanoma sublines with resistance to BRAF inhibition. These DR sublines were generated from a single parental line; although they displayed distinct mechanisms of resistance, all showed increased MAPK signal pathway output in the presence of GSK-BRAFi. The majority of DR clones showed weaker sensitivity to MEK inhibition alone and to the combination of MEK and BRAF inhibitors when compared with the parental clone, indicating that acquiring resistance to BRAF inhibition commonly confers cross-resistance to MEK inhibitors. The activation of N-RAS conferred potent resistance to GSK-BRAFi, but significantly diminished the sensitivity of melanoma clones to GSK-MEKi alone, or the combination of MEK and BRAF inhibitors. These data strongly suggest that activation of N-RAS maintains MAPK signaling in the presence of BRAF inhibitor, and that additional MEKindependent N-RAS effectors contribute to MEK and BRAF inhibitor resistance. It is known that mutant N-RAS uses CRAF to signal to MEK and ERK, thus bypassing inhibited BRAF (Dumaz et al., 2006; Jaiswal et al., 2009), and that N-RAS can signal via multiple pathways including the PI3K/AKT cascade (Smalley, 2010). Nevertheless, there are discrepancies in the literature regarding the role of activated RAS in selectively sensitizing cancer cells to MEK inhibition. In one study, BRAF inhibitor–resistant melanoma cell lines with an acquired NRASQ61K mutation were sensitive to MEK inhibition in the presence of the RAF inhibitor, Vemurafenib/PLX4032 (Nazarian et al., 2010). Conversely, N-RAS mutation status did not predict MEK inhibitor sensitivity in melanoma cell lines (Solit et al., 2006), and MEK inhibitors show only modest clinical activity in patients with RAS-mutant tumors (Nissan and Solit, 2011). It seems likely that the impact of mutant RAS on MEK inhibitor responses reflects its expression and activity and ultimately the network of activated N-RAS-dependent effectors. This is in agreement with a recent report demonstrating that K-RAS13D-mutant HCT116 colorectal cancer cells became resistant to MEK inhibition upon amplification of the driving K-RAS13D oncogene (Little et al., 2011).

The DR8 and DR9 sublines showed constitutive reactivation of MAPK signaling, elevated levels of PDGFRb, and activation of AKT. There was no evidence of activationassociated PDGFRb phosphorylation, and thus this receptor tyrosine kinase did not contribute to AKT activity or BRAF inhibitor resistance in these clones. Signaling via AKT was also not mediating resistance, as the combined inhibition of MAPK (BRAF þ MEK), PI3K, and mTOR did not trigger apoptosis in the DR8 and DR9 clones. In contrast, melanoma cells with PDGFRb activation did not show significant reactivation of MAPK signaling (Nazarian et al., 2010), and the concurrent inhibition of the MAPK and PI3K/AKT/mTOR pathways overcame resistance to the BRAF inhibitor, Vemurafenib (Shi et al., 2011). The dual targeting of BRAF and the PI3K network appears effective when BRAF inhibition leads to increased AKT phosphorylation, a compensatory prosurvival mechanism not observed in any of our DR clones (Atefi et al., 2011; SanchezHernandez et al., 2012). The DR4 subline differed significantly in retaining some sensitivity to MEK inhibition, in the absence and presence of BRAF inhibitor. This presumably reflects the fact that DR4 accumulates elevated levels of the COT1 kinase, which activates ERK through MEK-dependent and -independent mechanisms (Johannessen et al., 2010). Our study confirms that many perturbations restoring MAPK activation have the potential to cause resistance to MEK inhibition and to the concurrent inhibition of BRAF and MEK. These data are in agreement with a recent report showing a high degree of cross-resistance to BRAF and MEK inhibitors in BRAF inhibitor–resistant melanoma cell lines generated in vitro (Ribas et al., 2010). Furthermore, experimental and computational modeling of the MAPK pathway showed that switching activated ERK from BRAF to CRAF is accompanied by increased resistance to MEK inhibition (Sturm et al., 2010). The clinical significance of these findings will require validation, and it remains unclear whether initial combination therapy with MEK and RAF inhibitors yields improved efficacy over single agents in terms of the frequency and duration of response. What is becoming increasingly evident, however, is that individualized combinations of highly targeted agents affecting multiple signaling pathways will be required to overcome the complex and heterogeneous resistance networks in melanoma metastases. MATERIALS AND METHODS Cell culture and compounds MelRMu and MelMS melanoma cells were obtained from Professor P Hersey (Newcastle, NSW, Australia). Cells were grown in DMEM with 10% fetal bovine serum and glutamine (Gibco BRL, Carlsbad, CA). Cells were cultured in a 37 1C incubator with 5% CO2. Stocks of GSK-BRAFi and GSK-MEKi (GlaxoSmithKline, Collegeville, PA), BEZ235, and imatinib mesylate (Selleck Chemicals, Houston, TX) were prepared in DMSO. Cell authentication was confirmed using the AmpFlSTR Identifiler PCR Amplification Kit from Applied Biosystems (Mulgrave, Victoria, Australia), and using the GeneMarker V1.91 software (SoftGenetics, State College, PA). www.jidonline.org 1857

K Gowrishankar et al. MAPK Reactivation Dampens BRAF and MEK Inhibition

Pharmacological growth inhibition assays Cultured cells were seeded into 96-well plates (1,000 cells per well) and 24 hours after seeding serial dilutions of the relevant compound prepared in media were added. Cells were incubated for 72 hours, and cell viability was measured using the Cell proliferation Aqueous MTS assay (Promega, Wisconsin, MD) on a VICTOR2 Multilabel counter (Perkin Elmer, Waltham, MA). Viability was calculated as a percentage of control (DMSO-treated cells) after background subtraction. A minimum of two independent viability assays each performed in triplicate was carried out for each cell line and drug combination.

The shRNA no.1 and 2 sequences targeting N-RAS correspond to nucleotides 567–586 and 741–759 (NM_002524.3), respectively. The COT1 shRNA sequence corresponds to nucleotides 1826–1846 (NM_005204.2), and the non-silencing negative control shRNA did not show complete homology to any known human transcript and had the following sequence: 50 -TTAGAGGCGAGCAAGACTA-30 . CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS

Genomic DNA was extracted from 1–2  106 cells using the Wizard SV Genomic DNA purification system (Promega). MelRMu cells were genotyped as part of the Sequenom OncoCarta Assay Panel v1.0 (San Diego, CA).

We are grateful to Tona Gilmer and Sylvie Laquerre (GlaxoSmithKline) for providing GSK-BRAFi and GSK-MEKi. We thank Suzanah Philipsz, Carina Fung, and Jason Todd for excellent technical assistance. This work is supported by program grant 633004 and project grants of the National Health and Medical Research Council of Australia (NHMRC) and an infrastructure grant to Westmead Millennium Institute by the Health Department of NSW through Sydney West Area Health Service. Westmead Institute for Cancer Research is the recipient of capital grant funding from the Australian Cancer Research Foundation. HR is a recipient of a Cancer Institute New South Wales Research Fellowship and an NHMRC Senior Research Fellowship. The support of the Melanoma Foundation of the University of Sydney and colleagues from the Melanoma Institute Australia (incorporating the Sydney Melanoma Unit) and the Department of Anatomical Pathology at the Royal Prince Alfred Hospital are also gratefully acknowledged.

RNA extraction and microarray gene expression analysis

SUPPLEMENTARY MATERIAL

Total RNA was extracted as described previously (Scurr et al., 2010). Gene expression analysis was performed using the Sentrix HumanRef-6 v.4.0 Expression BeadChip (Illumina, San Diego, CA) and BeadStation system from Illumina according to the manufacturer’s instructions.

Supplementary material is linked to the online version of the paper at http:// www.nature.com/jid

Cell cycle and apoptosis analysis Adherent and floating cells were combined and cell cycle and apoptosis analyses were performed as previously described (Gallagher et al., 2005).

DNA extraction and genotyping

Protein detection Total cellular proteins were extracted at 4 1C using RIPA lysis buffer containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). Proteins (40 mg) were resolved on 12% SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Bedford, MA). Phospho-RTK arrays were performed according to the manufacturer’s instructions (Human Phospho-RTK Array Kit; R&D Systems, Minneapolis, MN). Western blots were probed with antibodies against p-ERK (E4; Santa Cruz Biotechnology, Santa Cruz, CA), total ERK (137F5; Cell Signaling, Danvers, MA), BRAF (L12G7 and 55C6, Cell Signaling), pRbS807/811 (Cell Signaling), totalpRb (G3-245, Becton Dickinson, Franklin Lakes, NJ), b-actin (AC-74; Sigma-Aldrich), p27Kip1 (Becton Dickinson), p16INK4a (JC8, Santa Cruz Biotechnology), cyclin D1 (G124-326, Becton Dickinson), CRAF (E10, Santa Cruz Biotechnology), N-RAS (F155, Santa Cruz Biotechnology), pAKTS473 (736E11, Santa Cruz Biotechnology), total AKT (11E7, Cell Signaling), PTEN (A2B1, Santa Cruz Biotechnology), COT/Tpl2 (Cell Signaling), IGF-1R (Cell Signaling), pPDGFRbY751 (88H8, Cell Signaling), and PDGFRb (C82A3, Cell Signaling).

Lentiviral transductions Lentiviruses were produced in HEK293T cells as described previously (Haferkamp et al., 2009). Cells were infected using a multiplicity of infection of 5 to provide an efficiency of infection above 90%. For N-RAS silencing experiments, cells were transduced with control-pSIH-copGFP or N-RAS-pSIH-copGFP for 5 days. Inhibitors or DMSO were added and cells were harvested 72 hours after inhibitor treatment for cell cycle and western blot analyses. 1858 Journal of Investigative Dermatology (2012), Volume 132

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