Antitumor Activity of the Selective Pan-RAF Inhibitor TAK-632 in BRAF Inhibitor-Resistant Melanoma

Published OnlineFirst October 11, 2013; DOI: 10.1158/0008-5472.CAN-13-1825

Cancer Therapeutics, Targets, and Chemical Biology Research

Akito Nakamura, Takeo Arita, Shuntarou Tsuchiya, Jill Donelan, Jouhara Chouitar, Elizabeth Carideo, Katherine Galvin, Masanori Okaniwa, Tomoyasu Ishikawa, and Sei Yoshida

Abstract The mitogen-activated protein kinase (MAPK) pathway is particularly important for the survival and proliferation of melanoma cells. Somatic mutations in BRAF and NRAS are frequently observed in melanoma. Recently, the BRAF inhibitors vemurafenib and dabrafenib have emerged as promising agents for the treatment of melanoma patients with BRAF-activating mutations. However, as BRAF inhibitors induce RAF paradoxical activation via RAF dimerization in BRAF wild-type cells, rapid emergence of acquired resistance and secondary skin tumors as well as presence of few effective treatment options for melanoma bearing wild- type BRAF (including NRAS-mutant melanoma) are clinical concerns. Here, we demonstrate that the selective pan-RAF inhibitor TAK-632 suppresses RAF activity in BRAF wild-type cells with minimal RAF paradoxical activation. Our analysis using RNAi and TAK-632 in preclinical models reveals that the MAPK pathway of NRAS-mutated melanoma cells is highly dependent on RAF. We also show that TAK-632 induces RAF dimerization but inhibits the kinase activity of the RAF dimer, probably because of its slow dissociation from RAF. As a result, TAK-632 demonstrates potent antiproliferative effectsbothonNRAS-mutatedmelanoma cells and BRAF-mutated melanoma cells with acquired resistance to BRAF inhibitors through NRAS mutation or BRAF truncation. Furthermore, we demonstrate that the combination of TAK-632 and the MAPK kinase (MEK) inhibitor TAK-733 exhibits synergistic antiproliferative effects on these cells. Our ﬁndings characterize the unique features of TAK-632 as a pan-RAF inhibitor and provide rationale for its further investigation in NRAS-mutated melanoma and a subset of BRAF-mutated melanomas refractory to BRAF inhibitors. Cancer Res; 73(23); 1–13. 2013 AACR.

Introduction line) showed high response rates and improved overall survival – Melanoma is one of the deadliest and most aggressive forms in melanomas with BRAF-activating mutations (7 12). Thus, of skin cancer, arising from the malignant transformation of the rationale for targeting BRAF to treat BRAF-mutant mela- pigment-producing cells, melanocytes (1). The mitogen-acti- nomas is strongly demonstrated. However, several reports have vated protein kinase (MAPK) pathway is particularly impor- shown that BRAF inhibitors activate RAF in BRAF wild-type – tant for the survival and proliferation of melanoma cells (2, 3). cells by inducing RAF dimer formation (13 15). Since the Since oncogenic mutations frequently occur in components of paradoxical activation of RAF requires cooperation with RAS, the MAPK pathway in melanoma (BRAF, NRAS, and KRAS in it does not occur in BRAF-mutated melanoma cells in which approximately 45%, 18%, and 2%, respectively; ref. 4), research BRAF activates downstream components in a RAS-indepen- and development targeting the MAPK pathway has been dent manner (16, 17). Therefore, while vemurafenib showed in vitro extensively performed (2, 5, 6). comparable inhibitory activity to all RAF isoforms in Recently, it has been reported that the BRAF inhibitors kinase assays (7), it suppressed cellular RAF activity exclusively vemurafenib (Roche/Plexxicon) and dabrafenib (GlaxoSmithK- in BRAF-mutant cells (18, 19). Such a RAF inhibitor is catego- rized as a "BRAF inhibitor," which demonstrates preferential inhibition of the MAPK pathway and proliferation of BRAF- Authors' Afﬁliation: Oncology Drug Discovery Unit, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, mutated melanoma cells. On the other hand, a lack of such a Kanagawa, Japan treatment option for melanomas with wild-type BRAF remains Note: Supplementary data for this article are available at Cancer Research an unresolved clinical issue. A subset of these tumors contains Online (http://cancerres.aacrjournals.org/). activating mutations in NRAS, implying the relevance of the A. Nakamura and T. Arita contributed equally to this work. MAPK pathway. These observations warrant the development of a small-molecule inhibitor that suppresses RAF activity Corresponding Authors: Akito Nakamura, Takeda Pharmaceutical Com- pany Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251- without inducing RAF paradoxical activation for the treatment 8555, Japan. Phone: 81-466-32-2650; Fax: 81-466-29-4410; E-mail: of NRAS-mutated melanomas (here referred to as "pan-RAF [email protected]; and Sei Yoshida, [email protected] inhibitor"). doi: 10.1158/0008-5472.CAN-13-1825 Although the response of BRAF inhibitors to BRAF-mutant 2013 American Association for Cancer Research. melanomas is apparent, rapid development of acquired

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resistance has been observed (11, 12). Many resistance Kinase assay mechanisms have been identified to date (16, 20–27), and most Immunoprecipitated BRAF or CRAF was incubated with involve reactivation of the MAPK pathway, which suggests that recombinant inactive MEK (K97R) (Millipore) at 30C for 30 þ tumor survival and growth remains dependent on the MAPK minutes in kinase reaction buffer containing ATP/Mg2 . Addi- pathway. However, as many resistance mechanisms (e.g., tional details are provided in the Supplementary Methods. RAS mutation, CRAF overexpression, and BRAF truncation; refs. 16, 21, 25) involve RAF dimer signaling and thus the Animal study potential for RAF paradoxical activation by BRAF inhibitors, A solid dispersion (SD) formulated compound was dissolved a pan-RAF inhibitor may be useful to develop a new thera- in distilled water, and the resultant suspension was orally peutic option in these resistant settings. administered to xenograft-implanted nude mice. All the ani- In this preclinical study, we describe the biologic charac- mals were dosed with vehicle (SD powder formulated in water) terization of TAK-632 (28) as a potent and selective pan-RAF or compound suspension by oral gavages. Additional details inhibitor that overcomes paradoxical RAF activation. We are provided in the Supplementary Methods. used both genetic and chemical approaches to investigate the dependence of NRAS-mutated melanoma and BRAF inhibitor-resistant BRAF mutant melanoma cells on RAF. Results In addition, we explored the potential of TAK-632 as a TAK-632 suppresses the MAPK pathway without robust monotherapy and in combination with a MEK inhibitor to RAF paradoxical activation in multiple cell lines robustly suppress MAPK pathway activation in melanoma To screen for pan-RAF inhibitors, we evaluated MAPK cells. pathway regulation in BRAF-mutant melanoma cells and fibroblast cells. Since we observed that the MAPK pathway of fibroblast cells was activated by BRAF inhibitors via RAF Materials and Methods paradoxical activation, we explored RAF inhibitors that sup- Cell lines and culture pressed the MAPK pathway in BRAF-mutant melanoma cells Human fibroblast Cell System-Fb (CsFb) cells were obt- but did not activate that in fibroblast cells. Some of our (5,6)- ained from Cell Systems Corporation, GAK cells from the fused bicyclic class inhibitors achieved these criteria. Eventu- Health Science Research Resources Bank, HMV-II cells from ally, among the screened RAF inhibitors, TAK-632 was selected European Collection of Cell Cultures, and other cell lines from as a candidate for further development (28). Potent and American Type Culture Collection. The cell lines were cul- selective inhibition of RAF by TAK-632 was confirmed by a tured at 37 Cwith5%CO2 in the recommended medium panel of in vitro kinase assays (IC50 of BRAF ¼ 8.3 nmol/L, supplemented with 10% FBS (Invitrogen). The cell lines were CRAF ¼ 1.4 nmol/L, BRAF-V600E ¼ 2.4 nmol/L; ref. 28). We fi authenticated by the cell banks with short-tandem repeat also con rmed RAF kinase inhibition by vemurafenib (IC50 of profiling and used within 2 months after resuscitation. A375 BRAF ¼ 64 nmol/L, CRAF ¼ 90 nmol/L, BRAF-V600E ¼ 43 Q61K stable cells (AcGFP-mock, -NRAS ,and-DN-BRAF cells) nmol/L, comparable to the reported IC50 values; ref. 7) and were established by virus transduction and cultured in the used vemurafenib as a representative BRAF inhibitor in fol- presence of 1 mg/mL puromycin. lowing studies. Here, to formally characterize its cellular activity, we initially Reagents compared the effect of TAK-632 with vemurafenib on the TAK-632 (28) and TAK-733 (29) were synthesized by Takeda MAPK pathway in BRAF mutant A375 cells, which have dem- Pharmaceutical Company Limited. Vemurafenib was prepared onstrated sensitivity to BRAF inhibitors (27). Consistent with according to a published method (7). the inhibitory activities to purified RAF, TAK-632 exhibited relatively high inhibitory activities on MEK and ERK phos- Transfection, immunoprecipitation, and Western blot phorylation compared with vemurafenib, although the differ- analysis ence was not significant (Supplementary Fig. S1A; IC50 of TAK- Cells were transfected with plasmids or siRNAs using Lipo- 632 for pERK ¼ 25 nmol/L and IC50 of vemurafenib for pERK ¼ fectamine 2000 or RNAi MAX reagent (Invitrogen), respective- 75 nmol/L). To investigate the effect of TAK-632 on BRAF wild- ly. Details of immunoprecipitation and Western blot analysis type cells in detail, we tested multiple cell lines with wild-type are described in the Supplementary Methods. RAS/RAF, cell lines with KRAS mutations, and NRAS-mutated melanoma cell lines. We found that TAK-632 had a biphasic Cell viability assay effect on the MAPK pathway: MEK and ERK phosphorylation Cell viability was assessed (3 replicates) using the Sulforho- was modestly induced at low TAK-632 concentrations but damine B assay or by the CellTiter-Glo luminescent cell via- inhibited at higher concentrations in BRAF wild-type cells bility assay (Promega). The concentrations of chemical com- (Fig. 1A). Although many ATP-competitive RAF inhibitors pounds that produced 50% growth inhibition (GI50) were increase MEK and ERK phosphorylation in cells with wild- calculated using PCP software (SAS Japan). The combination type RAF (15), interestingly, paradoxical activation by TAK-632 index (CI) was calculated using CalcuSyn software (Biosoft). appeared to be weak and occurred at low concentrations. CI < 0.9, CI ¼ 0.9–1.1, and CI > 1.1 indicate synergism, additive Notably, this weak paradoxical activation occurred in a similar effect, and antagonism, respectively. pattern in the cells; however, the MAPK pathway of NRAS-

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A RAS/RAF wild-type KRAS mutant NRAS mutant

A431 A549 GAK 40 8 5,000 5,000 1,000 200 1,000 200 40 8 1.6 0 5,000 1,000 200 40 0 8 0 TAK-632 (2 h, nmol/L) 0.32 P-MEK

P-ERK

α-Tubulin

CsFb HCT-116 HMV-II

Figure 1. Effect of TAK-632 on 1,000 200 1,000 1.6 5,000 1,000 200 8 5,000 40 0 200 8 0.32 0.064 40 0 TAK-632 (2 h, nmol/L) 8 40 0 MAPK pathway. A, RAS/RAF wild- type (A431, CsFb, and HeLa), P-MEK KRAS-mutant (A549, HCT-116, P-ERK and MIA PaCa-2), and NRAS- α mutant melanoma (GAK, HMV-II, -Tubulin and SK-MEL-2) cells were treated with TAK-632 at the indicated HeLa MIA PaCa-2 SK-MEL-2 concentrations for 2 hours. Cell lysates were analyzed by Western 40 8 8 5,000 1,000 200 0 5,000 200 40 8 0 5,000 1,000 200 40 1.6 0.32 0 blot analysis. B, NRAS-mutant TAK-632 (2 h, nmol/L) 1,000 cells (GAK, HMV-II, and SK-MEL- P-MEK 2) were transfected with siRNA pools as indicated. Forty-eight P-ERK hours after transfection, cell α-Tubulin lysates were analyzed by Western blot analysis. B GAK HMV-II SK-MEL-2 ARAF BRAF A&B Control B&C A&B&C A&C CRAF B&C ARAF BRAF A&B&C CRAF A&B A&C Control Control A&B A&C B&C siRNA ARAF BRAF CRAF A&B&C P-MEK

P-ERK

ARAF

BRAF

CRAF

β-Actin

mutated melanoma cells was suppressed by TAK-632 at lower although the inhibitory activity was relatively weak compared concentrations than that of other cells (Fig. 1A). In contrast, as to that in BRAF-mutated melanoma cells. reported previously, vemurafenib signiﬁcantly induced RAF To investigate the relative sensitivity to TAK-632 in NRAS- paradoxical activation in NRAS-mutated melanoma cells in a mutated melanoma cells, we performed knockdown analysis biphasic manner (Supplementary Fig. S1B; ref. 18). We also using siRNA to each RAF isoform. We found that ARAF deple- compared the effect of TAK-632 on the MAPK signaling with tion by siRNA did not affect MEK and ERK phosphorylation but the potent MEK inhibitor TAK-733 (29). Western blot analysis BRAF or CRAF depletion decreased it in multiple NRAS-mutat- revealed that the inhibitory activity to ERK phosphorylation of ed melanoma cells (Fig. 1B). Furthermore, simultaneous BRAF TAK-733 in A375 cells was comparable to that in SK-MEL-2 and CRAF depletion suppressed MEK and ERK phosphorylation cells (Supplementary Fig. S1C). Meanwhile, the inhibition of more robustly than each siRNA alone (Fig. 1B). The data suggest ERK phosphorylation by TAK-632 was relatively weak in SK- that BRAF and CRAF but not ARAF dominantly regulate the MEL-2 cells compared to the inhibition in A375 cells (Fig. 1A MAPK pathway in NRAS-mutated melanoma cells. and Supplementary Fig. S1A), suggesting that the minimal RAF paradoxical activation by TAK-632 reduces its inhibitory activ- RAF dimer formation mediates RAF paradoxical ity at low concentrations. Thus, our data indicate that TAK-632 activation by TAK-632 displays potent inhibition of MAPK pathway in NRAS-mutated BRAF inhibitors reportedly induce RAF paradoxical activa- melanoma cells with minimal RAF paradoxical activation, tion through RAF dimer formation (13–15). Therefore, we

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investigated whether RAF dimer formation is responsible for To demonstrate the signiﬁcance of RAF in the paradoxical paradoxical activation by TAK-632. When SK-MEL-2 cells were activation of the MAPK pathway, we examined the effect of treated with TAK-632, the inhibitor induced the formation of TAK-632 in RAF-depleted cells. The paradoxical activation BRAF–CRAF dimers, detectable in coprecipitation experi- induced by TAK-632 and vemurafenib was suppressed by CRAF ments, in a concentration-dependent manner (Fig. 2A, top). depletion in SK-MEL-2 cells (Fig. 2C). Similar data was We also observed an increase in BRAF–CRAF dimers in obtained in KRAS-mutant cells (data not shown). In these vemurafenib-treated cells, albeit at higher concentrations than experiments, we used the most appropriate concentration of those in TAK-632 (Fig. 2A, top). Such modest RAF dimerization each inhibitor that induced the paradoxical activation of the was also observed in PLX4720, a precursor analog of vemur- MAPK pathway (TAK-632, 10 nmol/L and vemurafenib, 1,000 afenib (14, 30, 31). With regard to the effects of TAK-632 and nmol/L; Fig. 1A and Supplementary Fig. S1B). These results are vemurafenib on RAF dimerization, similar results were consistent with previous reports using BRAF inhibitors (13– obtained in KRAS-mutant A549 cells (Fig. 2B, top). Interest- 15), indicating that CRAF is particularly required for RAF ingly, the level of MEK and ERK phosphorylation induced by paradoxical activation induced by TAK-632. RAF inhibitors was the maximum at lower concentrations than Although we have shown that TAK-632 induced modest RAF those required to induce maximal RAF dimer formation (Fig. paradoxical activation and RAF dimerization, a direct link 2A and B). The data support the model that the paradoxical between them remains to be determined. To clarify whether activation of the MAPK pathway is mediated by RAF dimers in TAK-632–mediated paradoxical activation was attributed to which one protomer is occupied by the RAF inhibitor and RAF dimerization, we prepared plasmids encoding the dimer another is not (13, 15). At higher concentrations of RAF interface mutants E586K-BRAF and E478K-CRAF, which dimer- inhibitors, more RAF dimers are formed; however, both pro- ize easily (13, 32), and evaluated the contribution of RAF dimer tomers tend to be occupied by inhibitors, resulting in effective formation to paradoxical RAF activation. Unlike the biphasic inhibition of kinase activity (Supplementary Fig. S2). effects observed in control cells, the paradoxical activation of the

A C

TAK-632 Vemurafenib siRNA Control ARAF BRAF CRAF 50 Inhibitors (2 h, μmol/L) 0 0.01 0.1 1 10 0.01 0.1 1 10

CRAF IP: BRAF - - - - TAK-632, 1 Vem, 1 TAK-632, 1 TAK-632, 0.01 TAK-632, 1 TAK-632, 0.01 Vem, 1 TAK-632, 0.01 Vem, 1 TAK-632, 1 Vem, 1 BRAF Inhibitors (2 h, μmol/L) TAK-632, 0.01

P-MEK P-MEK P-ERK P-ERK ARAF Lysates BRAF BRAF CRAF

α-Tubulin CRAF α-Tubulin B D

TAK-632 Vemurafenib HA-FLAG- BRAF/CRAF Mock E586K/E478K

Inhibitors (2 h, μmol/L) 0 0.01 1 10 0.01 1 10 50 μ 0.04 0.0016 1 0.2 0.04 0.008 0.0016 0.2 0.008 0 0 CRAF TAK-632 (2 h, mol/L) 1 IP: BRAF Intensity (P-MEK) 1.0 2.6 4.4 5.1 4.7 3.6 1.0 2.6 4.8 6.3 6.7 6.3 BRAF P-MEK

P-MEK Intensity (P-ERK) 1.0 3.9 8.4 10.8 9.3 7.9 1.0 2.9 7.1 10.3 11.8 12.1

P-ERK P-ERK BRAF Lysates BRAF HA CRAF CRAF α-Tubulin α-Tubulin

Figure 2. Formation of RAF dimer mediates RAF paradoxical activation by TAK-632. A and B, SK-MEL-2 and A549 cells were treated with TAK-632 and vemurafenib at the indicated concentrations for 2 hours, respectively. Immunoprecipitated proteins (IP) and cell lysates were analyzed by Western blot analysis. C, SK-MEL-2 cells were transfected with siRNA as indicated. Forty-eight hours after transfection, cells were treated with TAK-632 or vemurafenib (Vem) at the indicated concentrations. Cell lysates were analyzed by Western blot analysis. Individual panels with dividing lines are combined from a single electrophoresis gel. D, HCT-116 cells were transfected with plasmids as indicated. Twenty-four hours after transfection, cells were treated with TAK-632 at indicated concentrations for 2 hours. Cell lysates were analyzed by Western blot analysis. Individual panels with dividing lines are combined from a single electrophoresis gel. Numbers in a rectangle represent densitometric analysis of phospho-MEK and phospho-ERK, normalizedto DMSO-treated control.

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MAPK pathway was not detected in response to TAK-632 activity in RAF immunoprecipitates with minimal paradoxical treatment of cells coexpressing E586K-BRAF and E478K-CRAF activation because slow off-rate compounds persistently occu- (Fig. 2D). Therefore, dimerization of RAF appears to be associ- py both protomers through prolonged binding. ated with minimal activation of the MAPK pathway by TAK-632. TAK-632 inhibits the kinase activity of RAF dimer Inhibitor off-rate affects RAF dimer activation/ To examine the effects of RAF inhibitors on the kinase inhibition activity of RAF dimers in cells, we expressed dimerization- To directly determine the modulation of cellular RAF kinase prone or dimerization-incompetent RAF mutants (13, 32) in activity by RAF inhibitors, we performed immunoprecipita- BRAF-mutated A375 cells, in which endogenous BRAF function–kinase assays. Consistent with the effect on MEK or ERK tions as a monomer (16, 17). In cells expressing dimerization- phosphorylation observed in SK-MEL-2 cell lysates (Fig. 2A, incompetent mutants (R509H-BRAF and R401H-CRAF), TAK- bottom), TAK-632 slightly activated kinase activity detected in 632 and vemurafenib inhibited MEK and ERK phosphorylation CRAF or BRAF immunoprecipitates at low concentrations and as seen in control cells (Fig. 3C, bottom). Notably, TAK-632 suppressed these kinase activities at higher concentrations suppressed MEK and ERK phosphorylation derived from (Fig. 3A and Supplementary Fig. S3A). On the other hand, dimerization-prone mutants (Fig. 3C, bottom). These data vemurafenib increased RAF activities in a concentration- revealed that in contrast to existing BRAF inhibitors (13), dependent manner (Fig. 3A and Supplementary Fig. S3A), TAK-632 suppresses activated BRAF–CRAF heterodimers (Fig. which was different from the effects on MEK and ERK phos- 3C). The interaction between dimerization-prone mutants was phorylation in cell lysates (Fig. 2A, bottom). Similar observa- hardly detected in dimethyl sulfoxide (DMSO)-treated cells but tions have been reported for other BRAF inhibitors (13). Taken was induced in RAF inhibitor-treated cells (Fig. 3C, top), together, RAF kinase activities in immunoprecipitates and suggesting that the endogenous BRAFV600E/MEK/ERK path- MEK and ERK phosphorylation in cell lysates were well cor- way stimulates a negative feedback mechanism to inhibit RAF related in TAK-632–treated cells but not in BRAF inhibitor- dimerization in BRAF-mutant A375 cells (17). treated cells, suggesting differences in the profile between pan- The formation of CRAF–CRAF homodimers by PLX4720 RAF and BRAF inhibitors. reportedly mediated paradoxical RAF activation (13). To exam- Since the kinase activity of RAF dimers is suppressed only ine whether CRAF homodimer formation is also induced by when both protomers are occupied by inhibitors, we suspected TAK-632, we performed immunoprecipitation assays using that a difference in inhibitor off-rate from RAF may contribute A375 cells expressing CRAF with a distinct size tag (HA- to the abovementioned gap. In fact, TAK-632 displayed a very FLAG-CRAF and GFP-CRAF). HA-FLAG-CRAF was coprecipi- long residence time to purified wild-type RAF (28) and tated with GFP-CRAF in a concentration-dependent manner PLX4720 demonstrated fast dissociation kinetics and a short by TAK-632 and vemurafenib (Fig. 3D, top). Furthermore, the residence time (13, 28). In addition to the data in cell-free RAF kinase assay revealed that TAK-632 slightly activated GFP- conditions, washout assay revealed that the inhibition of MEK CRAF at low concentrations and suppressed it at higher phosphorylation by TAK-632 was partially rescued but concentrations in cells coexpressing HA-FLAG-BRAF/GFP- remained suppressed 2 hours after medium replacement in CRAF and HA-FLAG-CRAF/GFP-CRAF, respectively. Mean- SK-MEL-2 cells, whereas the increased MEK phosphorylation while, vemurafenib increased these activities in a concentra- by vemurafenib mostly returned to the basal level in 2 hours tion-dependent manner (Fig. 3D, bottom bars). Similar results (Supplementary Fig. S4). Next, to investigate the importance of were obtained by using SK-MEL-2 cells expressing dimeriza- the off-rate in suppressing the kinase activity of RAF dimers, tion-prone mutants (Supplementary Fig. S5). Thus, our find- RAF inhibitors were added to the reaction mixture containing ings suggest that TAK-632 blocks the activity of both RAF immunoprecipitated RAF and kinase assay was performed heterodimers and homodimers by prolonged binding to RAF. (described here as "in vitro-treated"). In this analysis, we examined whether in vitro-treated BRAF inhibitor could sup- Antiproliferative effect of TAK-632 press immunoprecipitated RAF that had been preactivated by To investigate the antiproliferative activity of TAK-632, we treatment with the inhibitor in cells. Notably, in vitro-treated performed proliferation assays in various cell lines harboring vemurafenib suppressed paradoxically activated RAF, indicat- mutated BRAF, NRAS, or KRAS. Although TAK-632 exhibited ing that the inhibitor could inhibit the kinase activity of RAF antiproliferative activity in various types of cancer cells, BRAF- dimers at adequate concentrations (Fig. 3B and Supplementary and NRAS-mutated melanoma cells appeared to be more Fig. S3B). Thus, our data suggest two possibilities: First, a fast sensitive to TAK-632 (Table 1). These results are consistent off-rate RAF inhibitor partially dissociates from RAF dimers in with the observation that the MAPK pathway is easily sup- a standard immunoprecipitation–kinase assay procedure and pressed by TAK-632 in BRAF- or NRAS-mutated melanoma the residual inhibitor then reinduces allosteric transactivation. cells (Fig. 1A and Supplementary Fig. S1A). Meanwhile, TAK- Second, a fast off-rate RAF inhibitor dissociates from immu- 733 showed stronger antiproliferative effects than TAK-632 ¼ – noprecipitated RAF; however, another activating mechanism both in A375 and SK-MEL-2 cells (GI50 of TAK-632 40 190 contributes to paradoxical activation, such as relief from nmol/L, TAK-733 ¼ 6 nmol/L in A375 cells and GI50 of TAK-632 inhibitory autophosphorylation (33). Importantly, the slow ¼ 190–250 nmol/L, TAK-733 ¼ 11 nmol/L in SK-MEL-2 cells). off-rate of TAK-632 can also explain why TAK-632 suppresses The data are also consistent with the inhibitory activities to both MEK phosphorylation in cell lysates and RAF kinase ERK phosphorylation (Fig. 1A and Supplementary Fig. S1A and

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Characterization of a Novel Selective Pan-RAF Inhibitor

Table 1. In vitro GI50 of TAK-632 in various cell lines

Mutation status (from COSMIC database)

Cell line Origin BRAF NRAS KRAS Mean GI50 (nmol/L) A375 Melanoma V600E WT WT 160 HT-144 Melanoma V600E WT WT 110 Malme 3M Melanoma V600E WT WT 50 CHL-1 Melanoma WT WT WT 2,070 HMCB Melanoma WT Q61K WT 350 HMVII Melanoma G469V Q61K WT 60 SK-MEL-2 Melanoma WT Q61R WT 190 A427 Lung cancer WT WT G12D 2,490 A549 Lung cancer WT WT G12S 1,700 Calu-6 Lung cancer WT WT Q61K 770 NCI-H727 Lung cancer WT WT G12V 1,430 HCT-116 Colorectal cancer WT WT G13D 1,770 LS-174T Colorectal cancer WT WT G12D 1,040 HRT-18 Colorectal cancer WT WT G13D 1,760 MIA-PaCa-2 Pancreatic cancer WT WT G12C 3,210 HeLa Cervical cancer WT WT WT 5,240 293T Kidney epithlial WT WT WT 660 PC-3 Prostate cancer WT WT WT >10,000 MRC5 Lung ﬁbroblast WT WT WT 1,210

NOTE: Various cell lines were treated with TAK-632. The mean GI50 value was derived from the cell viability assay 72 hours after drug treatment.

S1C). Next, we examined the in vivo efficacy of TAK-632 in independent manner (20). Notably, dimer formation was NRAS-mutant melanoma using a SK-MEL-2 xenograft model. observed when NRASQ61K was expressed in A375 cells (Fig. TAK-632 exhibited potent antitumor efficacy when orally 5E). The observed BRAF–CRAF dimer may be induced by high administered at 60 mg/kg once daily (T/C ¼ 37%, P < 0.001) levels of GTP-bound NRAS, conferring insensitivity to vemur- or at 120 mg/kg once daily (T/C ¼ 29%, P < 0.001) for 21 days afenib in the cells (Fig. 5E) as reported previously (13–15, 17). In without severe toxicity (Fig. 4A). Western blot analysis in addition, aberrantly spliced BRAF has emerged as a mecha- parallel studies of mice treated for 3 days revealed significant nism of resistance to vemurafenib (16). The hyperactive spliced reductions of ERK phosphorylation in SK-MEL-2 tumors from variants lack exons 2–10, including the RAS-binding domain, animals treated with 60 or 120 mg/kg TAK-632 compared with but retain the kinase domain and constitutively dimerize in a that in vehicle-treated mice (Fig. 4B and C). The data indicate RAS-independent manner. Therefore, we expressed N-termi- that antitumor activity and MAPK pathway inhibition are well nally truncated BRAF (DN-BRAF) that lacks exons 1–10 and correlated in the model. examined the effect of TAK-632. Consistent with previous findings (16), MEK and ERK phosphorylation was increased TAK-632 suppresses the MAPK pathway in by DN-BRAF and vemurafenib had a little effect on them (Fig. vemurafenib-resistant melanoma cells 5F). In contrast, TAK-632 suppressed MEK and ERK phosphor- One of the major clinical challenges is to overcome the ylation in cells expressing DN-BRAF (Fig. 5F). Interestingly, acquired resistance to BRAF inhibitors for the treatment of TAK-632 and vemurafenib strongly induced RAF heterodimer patients with melanoma (11, 12). To preclinically examine the formation in cells expressing DN-BRAF compared with that in effects of TAK-632 on several known mechanisms of vemur- cells expressing wild-type BRAF (Fig. 5F), indicating that DN- afenib resistance (20, 21, 26), we expressed wild-type NRAS, BRAF easily forms RAF heterodimers both in a steady state and NRAS with activating mutation (G12D, G13D, Q61K, or Q61R), in the presence of RAF inhibitors. These observations indicate KRAS with activating mutation (G12V), and COT in A375 cells. that TAK-632 can suppress the MAPK pathway in vemurafe- Although the MAPK pathway in cells expressing wild-type nib-resistant melanoma cells bearing RAS mutation or BRAF NRAS, mutated NRASs, and KRASG12V is resistant to vemur- truncation. afenib as reported previously (15, 21), TAK-632 suppressed Subsequently, we examined the effects of RAF inhibitors and MEK and ERK phosphorylation in these cells as seen in control TAK-733 on cell proliferation of vemurafenib-resistant mela- cells (Fig. 5A–C). On the other hand, COT conferred resistance noma cells. TAK-632, TAK-733, and vemurafenib displayed to both TAK-632 and vemurafenib (Fig. 5D), consistent with a potent antiproliferative activities in parent or mock-trans- previous report that COT activates MEK and ERK in a RAF- fected A375 cells (Table 2). We showed that cells expressing

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A B TAK-632 / 60 mpk / QDx3 Vehicle TAK-632 20 mg/kg Vehicle 2 h 4 h 8 h 24 h TAK-632 60 mg/kg TAK-632 120 mg/kg P-ERK 1,000 ERK ) 3 800 TAK-632 / 120 mpk / QDx3 Vehicle2 h 4 h 8 h 16 h 24 h 600 P-ERK 400 * ERK 200 * C Tumor volume (mm 140 0 120 0 5 10 15 20 25 100 TAK-632_60 mpk TAK-632_120 mpk Days after treatment 80 60 40 20

P-ERK (% of vehicle) 0 Vehicle 2h 4h 8h 24h 2h 4h 8h 16h 24h

Figure 4. Effect of TAK-632 on xenograft proliferation. A, mice bearing SK-MEL-2 xenografts were treated once daily for 21 consecutive days with vehicle or TAK-632 SD at the indicated concentrations (10 mice per each treatment group). Day 0 indicates the beginning of treatment. Tumors were measured twice a week. Points, mean tumor volumes; bars, SE. P values were calculated using t test (, P < 0.001). B and C, mice bearing SK-MEL-2 xenografts were treated once daily (QD) for 3 days with vehicle, TAK-632 SD at 60 mg/kg (60 mpk), or TAK-632 SD at 120 mg/kg (120 mpk). Tumor xenografts were obtained at indicated time points after the ﬁnal treatment and analyzed by Western blot analysis. Individual blots with dividing lines are combined from a single electrophoresis gel. Bars represent densitometric analysis of phospho-ERK, normalized to vehicle-treated control (mean SD).

NRASQ61K and DN-BRAF demonstrated resistance to vemur- therefore, the accumulation of phosphorylated MEK does not afenib, as reported previously (16, 21), but retained sensitivity occur (37, 38). Notably, when HMV-II and SK-MEL-2 cells were to TAK-632 and TAK-733, albeit at higher doses than parent or cotreated with TAK-632 and TAK-733, the increase in MEK mock-transfected cells (Table 2). Consistent with these results, phosphorylation by a MEK inhibitor was prevented by TAK-632 Western blot analysis revealed that both TAK-632 and TAK-733 (Fig. 6B and C). To further examine the combination effects of significantly suppressed ERK phosphorylation not only in TAK-632 and TAK-733 in NRAS-mutated melanoma cells, we mock-transfected cells but also in NRASQ61K and DN-BRAF- investigated the expression level of several cell-cycle/trans- expressing cells (Supplementary Fig. S6A). Given that some lation/apoptosis markers in SK-MEL-2 cells. Western blot dabrafenib-resistant BRAF-mutated melanoma cells reported- analysis on the downstream markers shows the benefitofthe ly had NRAS mutation or BRAF truncation (34, 35), TAK-632 combination over the single agents in cell cycle as measured may also have the potential to show efficacy in such dabrafe- by cyclin D1, translation as measured by S6 phosphorylation, nib-resistant melanoma cells. Further investigation will clarify and apoptosis as measured by cleaved PARP and lamin the point in the future. (Fig. 6C). Consistent with the potent inhibition of ERK phosphor- Combination of TAK-632 with a MEK inhibitor enhances ylation and downstream markers, TAK-632 showed a stron- antiproliferative activities in BRAF- and NRAS-mutated ger inhibitory effect on cell viability when combined with melanoma cells TAK-733 in HMV-II and SK-MEL-2 cells (Fig. 6D and E). The Given that near complete inhibition of the MAPK pathway calculated CI value at EC50 was 0.64 in HMV-II cells and 0.46 may be required for significant antitumor effects in melanomas in SK-MEL-2 cells, indicating synergy between the drugs in (7), vertical combinations of agents targeting multiple steps blocking the proliferation of NRAS-mutant melanoma cells. along the MAPK pathway can be a reasonable therapeutic The combination displayed similar synergistic effects in strategy. Therefore, we examined combinational effects of other NRAS-mutant cells, HMCB and GAK cells (data not TAK-632 and TAK-733 on MAPK pathway activation and cell shown). On the other hand, additive antiproliferative effects proliferation in melanomas. Compared with single treatment, were observed in A375 cells, indicated by a CI value of 1.1 at the combination robustly suppressed ERK phosphorylation in the EC50 concentration (Fig. 6F). To determine whether the A375, HMV-II, and SK-MEL-2 (Fig. 6A–C). As shown in a combined effects preferentially occurred in these melano- previous study (36), TAK-733 induced the accumulation of mas, we tested 293T and HeLa cells, both of which bear wild- phosphorylated MEK in NRAS-mutant HMV-II and SK-MEL-2 type RAF and RAS. We found that the combination did not cells but not in BRAF-mutant A375 cells (Fig. 6A–C), because induce potent inhibition of their proliferation (Supplemen- treatment with a MEK inhibitor induces the accumulation of tary Fig. S7A and S7B). Collectively, our data indicate that phosphorylated MEK by relieving the negative feedback loop the combination of TAK-632 and a MEK inhibitor effectively from ERK in BRAF wild-type cells (37, 38). In contrast, in BRAF- inhibits the proliferation of melanoma cells with BRAF or mutant cells, the negative feedback mechanism is abrogated; NRAS mutation.

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A C Mock WT-NRAS NRASQ61K Mock KRASG12V

632 Vem 632 Vem 632 Vem 632 Vem 632 Vem

μ 0.01 0 0.01 1 0.1 1 0.01 0 0.01 0.1 0.1 1 0.01 0.1 1 0.1 1 0.01 0.1 1 0 0.01 0.1 1 0.01 0.1 1 0 0.1 1 0.01 0.1 0 0.01 Inhibitors (2 h, μmol/L) 1 Inhibitors (2 h, mol/L) P-MEK P-MEK

P-ERK P-ERK

FLAG FLAG

α-Tubulin α-Tubulin

B Q61R G12D G13D D NRAS NRAS NRAS COT WT-BRAF

632 Vem 632 Vem 632 Vem 632 Vem 632 Vem 0.1 1 1 0.01 0.1 0.01 1 0 0.01 0.1 0.01 0.1 1 0.1 1 0 0.01 0 1 0.01 0.1 0.01 1 0.1 1 0 0.1 0.01 0.1 0 0.01 0.01 0.1 Inhibitors (2 h, μmol/L) Inhibitors (2 h, μmol/L) 1 1 P-MEK P-MEK

P-ERK P-ERK

FLAG FLAG α-Tubulin α-Tubulin

E F WT-BRAF ΔN-BRAF Mock NRASQ61K

632 Vem 632 Vem TAK-632 Vem TAK-632 Vem 10 0 0.1 50 1 10 0.1 50 1 10 0.1 1 10 1 0.1 μ μ 0 1 1 0 10 1 10 Inhibitors (2 h, mol/L) 10 10 0 1 Inhibitors (2 h, mol/L) CRAF CRAF IP: BRAF CRAF BRAF (long ex.) IP: FLAG CRAF (BRAF) FLAG FLAG * Lysates P-MEK P-ERK P-MEK α-Tubulin P-ERK Lysates CRAF

α-Tubulin

Figure 5. TAK-632 suppresses MAPK pathway in vemurafenib-resistant melanoma cells. A–D, A375 cells were transfected with plasmids as indicated. Twenty- four hours after transfection, cells were treated with TAK-632 (632) or vemurafenib (vem) at the indicated concentrations for 2 hours. Cell lysates were analyzed by Western blot analysis. E, A375 cells were transfected with plasmids as indicated. Twenty-four hours after transfection, cells were treated with TAK-632 (632) or vemurafenib (Vem) at the indicated concentrations for 2 hours. Immunoprecipitated proteins (IP) and cell lysates were analyzed by Western blot analysis. F, A375 cells were transfected with plasmids as indicated. Twenty-four hours after transfection, cells were treated with TAK-632 or vemurafenib (Vem) at the indicated concentrations for 2 hours. Immunoprecipitated proteins (IP) and cell lysates were analyzed by Western blot analysis. Individual panels with dividing lines are combined from a single electrophoresis gel. , indicates bands derived from IgG, and "long ex." indicates longer exposure for detection of chemiluminescence.

TAK-632 shows synergy with a MEK inhibitor in (Supplementary Fig. S6B), reactivation of the MAPK pathway vemurafenib-resistant melanoma models is expected to warrant combined treatment with TAK-632 Since levels of MEK and ERK phosphorylation in A375 and a MEK inhibitor to further inhibit the MAPK pathway cells expressing NRASQ61K or DN-BRAF were increased com- and consequently suppress cell proliferation. The combi- paredwiththelevelsinparentormock-transfectedcells nation of TAK-632 and TAK-733 demonstrated highly

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Nakamura et al.

Table 2. In vitro GI (nmol/L) of RAF or MEK noprecipitates even at high concentrations that suppress MEK 50 and ERK phosphorylation in cell lysates (Figs. 2A and 3A). This inhibitor in A375 cells expressing DN-BRAF or Q61K discrepancy provides supporting evidence that vemurafenib NRAS has a rapid off-rate from RAF and thereby easily dissociates from RAF during immunoprecipitation and subsequent kinase TAK-632 Vemurafenib TAK-733 assays. The difference in dissociation rates between the pan- Parent 40 180 6 RAF inhibitor TAK-632 and BRAF inhibitors illustrates the Mock 60 250 6 following model: TAK-632 (pan-RAF inhibitors) continuously DN-BRAF 500 >10,000 240 binds to both protomers of RAF dimers and thereby inhibits NRAS-Q61K 1,710 >10,000 460 kinase activity of the RAF dimer so that minimal RAF paradoxical activation occurs only at low concentrations. Mean- Q61K NOTE: A375 cells expressing mock, NRAS , and DN- while, BRAF inhibitors induce RAF dimerization but do not BRAF were treated with TAK-632, vemurafenib, or TAK-733. persistently occupy both protomers, thereby robustly stimu- The mean GI50 value was derived from the cell viability assay lating paradoxical RAF activation. 72 hours after drug treatment. Notably, the RAF inhibitory activity of TAK-632 appears to be several times higher than that of vemurafenib, as shown in our data and existing findings from enzymatic assays and Q61K D synergistic antiproliferative effects in NRAS and N- cellular assays (Supplementary Fig. S1A; refs. 7, 15, 28). How- ¼ BRAF cells (Fig. 6G and H; CI 0.30 and 0.36, respectively). ever, such moderate differences in the potencies of these Consistent with these results, the combined treatment inhibitors may not be sufficient to explain the remarkable Q61K robustly suppressed ERK phosphorylation in NRAS and difference in the RAF dimer inhibitory activities (Fig. 3). We D N-BRAF cells (Supplementary Fig. S8A; 2 hours of treat- show that in vitro-treated vemurafenib suppresses paradoxi- ment and Fig. 6I and S8B; 24 hours of treatment). The 24- cally activated RAF (Fig. 3B and S3B), suggesting that the hour treatment assay also revealed that the cotreatment led varying RAF dimer inhibitory activity may be derived from to more drastic inhibition of cell-cycle progression, as indi- the difference in the dissociation rate from RAF. cated by decreased cyclin D1 expression in these cells (Fig. 6I The model complies with a recently reported model that and Supplementary Fig. S8B). Given the strong inhibitory RAF inhibitors mediate RAF paradoxical activation by reliev- effects of the combination of TAK-632 and TAK-733 on the ing inhibitory autophosphorylation (33). Loss of glycine-rich MAPK pathway and cell proliferation, reactivation of the phosphate-binding (P)-loop phosphorylation is considered to MAPK pathway appears to play a major role in the resistance destabilize the inactive DFG-out conformation and allow a to vemurafenib in the models. shift to the active DFG-in conformation (41, 42). Consistent with the report (33), we noted that vemurafenib did not Discussion induce significant paradoxical RAF activation in HMV-II NRAS is mutated in approximately 18% of melanomas and cells that bear not only NRAS mutation but also BRAF 10% of hematologic cancers (4). Our data using siRNA or the mutation at G469, which may disrupt P-loop autoinhibition pan-RAF inhibitor TAK-632 indicate that NRAS-mutated mel- (data not shown). As the formation of RAF dimers and anoma highly depends on the RAF/MEK/ERK pathway (Fig. 1). inhibition of P-loop autophosphorylation are not mutually Given that melanoma possesses outstanding mutation rates in exclusive, these mechanisms are expected to cooperatively components of the MAPK pathway, it is not surprising that this induce RAF paradoxical activation. pathway is particularly important for the initiation and/or Paradoxical RAF activation by BRAF inhibitors plays a sig- maintenance of melanoma. Although we have not examined nificant role in the development of squamous cell carcinoma the dependence of hematologic cancers on RAF, further inves- and keratoacanthoma in the clinic (8–11, 43). Several BRAF tigations will provide us with an insight into therapeutic inhibitors have paradoxically stimulated normal cell prolifera- interventions for the cancers. tion in mouse epithelial cells in vivo (13, 44). When we performed The inhibitor-induced conformational change in the acti- pharmacodynamic analysis using melanoma xenograft rat mod- vation loop of a kinase is observed in cocrystal structures of a els, we found that TAK-632 suppressed ERK phosphorylation in DFG-out (inactive conformation) type inhibitor and offers a xenograft tumors but did not induce RAF paradoxical activation slower dissociation rate than a DFG-in (active conformation) in normal skin tissues (data not shown). Our toxicologic studies type inhibitor (39). We have shown that TAK-632 binds to also showed no proliferative effects on skin epithelial cells in BRAF in the DFG-out state (28), whereas several BRAF inhi- animals (data not shown), suggesting that TAK-632 may avoid bitors, including vemurafenib, are known to associate with the risk of secondary skin tumors in the clinic. However, whereas BRAF in the DFG-in state (7, 13). In fact, TAK-632 had a much vemurafenib did not have skin proliferative effects in preclinical slower off-rate from RAF than BRAF inhibitors (13, 28). Con- animal models (7), skin tumor incidence was observed in sistent with these observations, it has been reported that an patients (8, 9). Moreover, sorafenib, which is a multi-kinase investigational RAF inhibitor AZ-628 binds to BRAF in the inhibitor with pan-RAF inhibitory effects (14, 45), is associated DFG-out state, has slow off-rate, and does not significantly with the emergence of skin tumors during treatment (46). induce RAF paradoxical activation (13, 40). Notably, our data Collectively, we should cautiously consider whether TAK-632 show that vemurafenib elevates RAF kinase activity in immu- has an effect on cutaneous cell proliferation.

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A C A375 TAK-632 TAK-632 TAK-632 SK-MEL-2 TAK-733 TAK-733 TAK-733 μ μ 0 μmol/L 0.1 μmol/L 1 μmol/L 0 μmol/L 0.001 mol/L 0.01 mol/L 0 0.001 0.01 0.1 0.01 0 0.001 0.1 0 0.001 0.1 TAK-733 (24 h, μmol/L) 0.01 0.005 0.005 0 0.5 0 0.05 0.005 0.05 0.5 0 TAK-632 (24 h, μmol/L) 0.05 0.5 P-MEK P-MEK P-ERK P-ERK MEK P-S6 ERK Cyclin D1 Cleaved PARP HMV-II TAK-632 TAK-632 B 0 μmol/L 1 μmol/L Cleaved Lamin A GAPDH 0 0.01 1 0.01 1 0 0.1 TAK-733 (24 h, μmol/L) 0.1 P-MEK P-ERK MEK ERK

D HMV-II E SK-MEL-2 F A375

120 CI=0.64 TAK-632 120 CI=0.46 TAK-632 120 CI=1.1 TAK-632 0 nmol/L 0 nmol/L 0 nmol/L 100 100 100 1 nmol/L 3 nmol/L 1 nmol/L 80 80 80 3 nmol/L 10 nmol/L 3 nmol/L 60 60 60 10 nmol/L 30 nmol/L 10 nmol/L 40 40 40 Cell viability Cell viability Cell viability (% of control) (% of control) 30 nmol/L 100 nmol/L (% of control) 30 nmol/L 20 20 20 100 nmol/L 300 nmol/L 100 nmol/L 0 0 0 0.01 0.1 1 10 100 0.01 0.1 1 10 100 0.01 0.1 1 10 100 Concentration (TAK-733, nmol/L) Concentration (TAK-733, nmol/L) Concentration (TAK-733, nmol/L)

NRASQ61K G I Mock NRASQ61K ΔN-BRAF TAK-632 120 CI=0.30 0 μmol/L

100 μ 632

0.02 mol/L - 80 μ Vemurafenib DMSO Vemurafenib DMSO Vemurafenib DMSO 0.06 mol/L Inhibitors (24 h, 1 μmol/L) TAK-632 TAK TAK-632 60 0.19 μmol/L 40 μ Cell viability 0 TAK-733 (24 h, mol/L) 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 0 0.1 0.1 (% of control) 0.56 μmol/L 20 1.67 μmol/L P-ERK 0 0.0001 0.01 1 5 μmol/L cyclin D1 Concentration (TAK-733, μmol/L)

Δ AcGFP H N-BRAF TAK-632 120 CI=0.36 0 μmol/L α-Tubulin 100 0.02 μmol/L 80 0.06 μmol/L 60 0.19 μmol/L 40 Cell viability (% of control) 0.56 μmol/L 20 1.67 μmol/L 0 0.0001 0.01 1 5 μmol/L Concentration (TAK-733, μmol/L)

Figure 6. TAK-632 shows synergy with a MEK inhibitor. A–C, A375, HMV-II, or SK-MEL-2 cells were cotreated with TAK-632 and TAK-733 at the indicated concentrations for 24 hours. Cell lysates were analyzed by Western blot analysis. Individual panels with dividing lines are combined from a single electrophoresis gel. D–F, HMV-II, SK-MEL-2, or A375 cells were cotreated with TAK-632 and TAK-733 at the indicated concentrations for 72 hours. Cell Q61K viability was measured. The CI value at EC50 was calculated. G and H, A375 cells stably expressing NRAS or DN-BRAF were cotreated with TAK-632 and TAK-733 at the indicated concentrations for 72 hours. Cell viability was measured. The CI value at EC50 was calculated. I, A375 cells stably expressing mock, NRASQ61K,orDN-BRAF were cotreated with TAK-632 and TAK-733 or vemurafenib and TAK-733 at the indicated concentrations for 24 h. Cell lysates were analyzed by Western blot analysis. Individual panels with dividing lines are combined from a single electrophoresis gel. www.aacrjournals.org Cancer Res; 73(23) December 1, 2013 OF11

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Since resistance to BRAF inhibitor treatment is associated TAK-632 potentially targets other tumors whose growth and with reactivation of the MAPK pathway, the combination of the survival are dependent on the MAPK pathway. Further inves- BRAF inhibitor dabrafenib and the MEK inhibitor trametinib in tigations will be required to identify such sensitive patient patients with BRAF-mutant melanomas has been clinically populations. tested. Phase I and II trials showed significant improvements in progression-free survival of the combination group compared Disclosure of Potential Conflicts of Interest with that of the monotherapy group (47). Notably, we showed No potential conflicts of interest were disclosed. that TAK-632 exhibits highly synergistic effects with TAK-733 in BRAF inhibitor-resistant melanoma cells (Fig. 6), suggesting Authors' Contributions Conception and design: A. Nakamura, T. Arita, K. Galvin, T. Ishikawa, the potential clinical benefit of the combination of pan-RAF S. Yoshida inhibitors and MEK inhibitors. In the abovementioned clinical Development of methodology: A. Nakamura Acquisition of data (provided animals, acquired and managed patients, trials, the rate of skin proliferative lesions was reduced by the provided facilities, etc.): S. Tsuchiya, J. Chouitar combination therapy (47). Although the potential risk of TAK- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, 632 inducing skin tumors in patients remains unknown, com- computational analysis): S. Tsuchiya, J. Donelan Writing, review, and/or revision of the manuscript: A. Nakamura, T. Arita, bined therapy with MEK inhibitors would be a reasonable J. Donelan, E. Carideo, K. Galvin, M. Okaniwa, T. Ishikawa, S. Yoshida strategy not only to enhance the antitumor activity but also to Administrative, technical, or material support (i.e., reporting or orga- possibly reduce the risk of skin tumor incidence. Given that nizing data, constructing databases): E. Carideo, M. Okaniwa Study supervision: T. Arita, J. Donelan, S. Yoshida amplification of the upstream oncogenic driver of the MAPK fi pathway has been identi ed as a mechanism for MEK inhibitor Acknowledgments resistance in colorectal cancer cell lines, the combination of The authors thank Masato Yabuki, Momoko Oohori, and Yuka Hasegawa for pan-RAF inhibitors and MEK inhibitors may also contribute to helpful discussion and technical assistance, Akihiro Ohashi and Hiromichi Kimura for critical reading of the manuscript, and Tomohiro Kawamoto, Akihiko overcoming the drug resistance in tumors besides melanoma Sumita, and Yoshitaka Inui for enzymatic, pharmacokinetic, and toxicological (48–50). evaluations of compounds, respectively. In this preclinical study, we demonstrated that TAK-632 has favorable characteristics in terms of killing NRAS-mutant Grant Support The study was funded by Takeda Pharmaceutical Company Limited. melanoma and BRAF inhibitor-resistant BRAF-mutant mela- The costs of publication of this article were defrayed in part by the noma cells. TAK-632 can prevent RAF paradoxical activation payment of page charges. This article must therefore be hereby marked by suppressing the kinase activity of the RAF dimer (Supple- advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this mentary Fig. S9). Furthermore, our findings suggest that TAK- fact. 632 has the potential to delay the emergence of drug resistance Received June 26, 2013; revised September 11, 2013; accepted September 24, caused by NRAS mutation and BRAF truncation. In addition, 2013; published OnlineFirst October 11, 2013.

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www.aacrjournals.org Cancer Res; 73(23) December 1, 2013 OF13

Antitumor Activity of the Selective Pan-RAF Inhibitor TAK-632 in BRAF Inhibitor-Resistant Melanoma

Akito Nakamura, Takeo Arita, Shuntarou Tsuchiya, et al.

Cancer Res Published OnlineFirst October 11, 2013.

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