Abstract
Alectinib/CH5424802 is a known inhibitor of anaplastic lymphoma kinase (ALK) and is being evaluated in clinical trials for the treatment of ALK fusion–positive non–small cell lung cancer (NSCLC). Recently, some RET and ROS1 fusion genes have been implicated as driver oncogenes in NSCLC and have become molecular targets for antitumor agents. This study aims to explore additional target indications of alectinib by testing its ability to inhibit the activity of kinases other than ALK. We newly verified that alectinib inhibited RET kinase activity and the growth of RET fusion–positive cells by suppressing RET phosphorylation. In contrast, alectinib hardly inhibited ROS1 kinase activity unlike other ALK/ROS1 inhibitors such as crizotinib and LDK378. It also showed antitumor activity in mouse models of tumors driven by the RET fusion. In addition, alectinib showed kinase inhibitory activity against RET gatekeeper mutations (RET V804L and V804M) and blocked cell growth driven by the KIF5B-RET V804L and V804M. Our results suggest that alectinib is effective against RET fusion–positive tumors. Thus, alectinib might be a therapeutic option for patients with RET fusion–positive NSCLC. Mol Cancer Ther; 13(12); 2910–8. ©2014 AACR.
Introduction
Rearranged during transfection (RET) is an oncogene that when genetically altered, is involved in the development of several human cancers. Activation of the RET point mutation is associated with medullary thyroid carcinoma (MTC; ref. 1), and RET rearrangement has been detected in papillary thyroid carcinoma (2). In addition, some RET fusion genes such as KIF5B-RET and CCDC6-RET have also recently been identified as driver oncogenes in non–small cell lung cancer (NSCLC), with the associated fusion genes present in approximately 1% to 2% of cases (3–5). Vandetanib and cabozantinib, which are multikinase inhibitors targeting RET, were approved by the FDA in 2011 and 2012, respectively, for the treatment of patients with MTC (6). In a prospective phase II trial that included RET fusion–positive NSCLC patients, 2 patients with RET fusion–positive NSCLC responded to cabozantinib (7). Thus, the genetic alterations of RET are considered to be promising targets for anticancer therapy.
Most of the clinically approved small-molecule inhibitors that are currently available are able to inhibit multiple kinases, and in fact, some inhibitors are approved or are currently in clinical trials for several indications. For example, imatinib, which has inhibitory activity against ABL and KIT, was approved for use in the treatment of Philadelphia chromosome (BCR-ABL fusion)–positive chronic myeloid leukemia and KIT-positive metastatic gastrointestinal stromal tumor (8). In addition, crizotinib, which was granted accelerated approval by the FDA in 2011 as the first anaplastic lymphoma kinase (ALK) inhibitor for advanced ALK-positive NSCLC patients (9), inhibits not only ALK but several other kinases including MET, ROS1, and RON (10). Clinical trials with crizotinib have recently demonstrated the drug's efficacy in the treatment of NSCLC with ROS1 translocations (11–13). In contrast, off-target toxicities are generally observed when therapeutic agents inhibit kinases that were not intended as targets (14). For example, anticancer therapies involving the use of multikinase inhibitors that target KDR are associated with the development of hypertension and proteinuria. These side effects commonly disappear upon drug withdrawal (15, 16).
Alectinib is a potent ALK inhibitor that exhibits antitumor activity against cancers with ALK gene alterations (17). A recent report on a phase I/II clinical study shows that alectinib is well tolerated and highly active in patients with advanced ALK-rearranged NSCLC (18). One ongoing clinical study is testing the activity of alectinib in patients in whom crizotinib treatment failed (Trial registration ID: NCT01588028). In this study, we explored indications for alectinib treatment in non–ALK-positive cancers, and provide evidence that alectinib is a potent RET inhibitor for RET fusion–driven NSCLC. Alectinib inhibited RET kinase activity and RET fusion–driven cell growth by suppressing phospho-RET. Alectinib also showed antitumor activity in mouse models of a RET fusion–positive tumor.
Materials and Methods
Compounds and cell lines
Alectinib was synthesized at Chugai Pharmaceutical Co. Ltd. according to the procedure described in patent publication WO2010143664. Crizotinib, cabozantinib, and vandetanib were purchased from Selleck Chemicals; gefitinib was purchased from Kemprotec Limited; and LDK378 was purchased from Active Biochemicals.
LC-2/ad cells were obtained from RIKEN in September 2011, NCI-H2228 cells were obtained from American Type Culture Collection (ATCC) in February 2008, NCI-H522 cells were obtained from ATCC in July 2001, and Ba/F3 cells were obtained from RIKEN in July 2008. Each cell line was cultured using the medium recommended by the suppliers. The cell lines have not been authenticated by the authors.
Kinase inhibitory assays
Recombinant human RET, mutated RET (G691S, Y791F, V804L, V804M, S891A, and M918T), ROS1, RON, EGFR, KDR, PDGFRβ, FGFR2, KIT, SRC, HER2, MET, RAF1, and MEK1 were purchased from Carna Biosciences or Merck. Inhibitory activity against each kinase was evaluated using the time-resolved fluorescence resonance energy transfer assay to examine each compound's ability to phosphorylate substrate peptide Biotin-EGPWLEEEEEAYGWMDF in the presence of the drug and 30 μmol/L ATP (17). The IC50 values were calculated using XLfit software (ID Business Solutions).
In silico modeling
The structure models of RET with alectinib, crizotinib, and LDK378 were modeled by Discovery Studio 3.5 (Accelrys). All figures were drawn using PyMol software (Schrödinger K.K.).
Generation of Ba/F3 cells expressing KIF5B-RET and mutated KIF5B-RET
The plasmids that express KIF5B-RET, KIF5B-RET V804L, and KIF5B-RET V804M, respectively, named CS-GS104J-M67, CS-GS104J-M67-m1, and CS-GS104J-M67-m2, were purchased from Genecopoeia. To generate Ba/F3 cells that stably expressed KIF5B-RET and mutated KIF5B-RET, Ba/F3 cells were transfected with the appropriate expression plasmids using a Nucleofector device (Amaxa). Stable transfectants were then isolated from the cultured medium without IL3.
Cell growth inhibition and caspase-3/7 assay
Cells were cultured in 96-well spheroid plates (Sumilon Celltight Spheroid 96U; Sumitomo Bakelite Inc.) overnight and incubated with various concentrations of compound for the indicated time. The viable cells were measured by the CellTiter-Glo luminescent cell viability assay (Promega). The IC50 values were calculated using XLfit software. The caspase-3/7 assay was evaluated using the Caspase-Glo 3/7 Assay Kit (Promega). Fluorescence was quantified using Envision (PerkinElmer).
Transcriptome profiling and qRT-PCR
Total RNA was extracted using the RNeasy mini-kit (Qiagen), and cDNA libraries were generated using the TruSeq RNA sample preparation kit (Illumina). The cDNA libraries were sequenced by an Illumina HiSeq 2000 instrument according to the manufacturer's instructions. RSEM software (19) was used to align reads against the RefSeq transcript and to calculate the expression value for each gene. For qRT-PCR, RNA was amplified by QuantiFast Multiplex RT-PCR (Qiagen) using a Universal probe library (Roche Applied Science) and the LightCycler System (Roche). GAPDH served as an internal control.
Immunoblotting
Cells were lysed in Cell Lysis Buffer (Cell Signaling Technology) containing 1 mmol/L PMSF, 1% (v/v) phosphate inhibitor cocktail 2 (Sigma), 1% (v/v) phosphate inhibitor cocktail 3 (Sigma), and Complete Mini, EDTA-Free (Roche). Cell lysates were subjected to SDS-PAGE, and the separated proteins were electrophoretically transferred to Immobilon-P membranes (Millipore). After blocking in Blocking One (Nacalai Tesque, Inc.), the membranes were incubated independently in primary antibody diluted with anti-RET (Santa Cruz Biotechnology, sc-167 or Cell Signaling Technology, #3341), anti- Phospho-RET (Tyr 905; Cell Signaling Technology, #3221), anti-STAT3 (Cell Signaling Technology, #9132), anti-Phospho-STAT3 (Tyr 705; Cell Signaling Technology, #9131), anti-AKT (Cell Signaling Technology, #9277), anti-Phospho-AKT (Ser 473; Cell Signaling Technology, #9271), anti-p44/42 MAP Kinase (ERK1/2; Cell Signaling Technology, #9102), anti-Phospho-ERK1/2 (Thr 202/Tyr 204; Cell Signaling Technology, #9101), anti-Cleaved PARP (Asp214; Cell Signaling Technology, #9546), anti-BIM (Cell Signaling Technology, #2819), anti-MCL-1 (Cell Signaling Technology, #4572), anti-BAX (Cell Signaling Technology, #2774), or anti-β-actin (Sigma, A5441). To detect phosphorylated RET, cell lysates were immunoprecipitated with anti-phosphotyrosine (PY-20) antibody (BD Biosciences). The membranes were incubated with an anti-rabbit or anti-mouse IgG, horseradish peroxidase-linked antibody (Cell Signaling Technology). The bands were detected using Chemi-Lumi One Super (Nacalai Tesque, Inc.) technology with LAS-4000 (Fujifilm).
In vivo studies
Male SCID mice (C.B-17/Icr-scid/scidJc, 5-week-old) were obtained from CLEA Japan, Inc. Cells (LC-2/ad cells at 8.4 × 106 or Ba/F3 cells expressing KIF5B-RET at 5.0 × 106) were grown as subcutaneous tumors in SCID mice. Mice were randomized to treatment groups to receive vehicle or alectinib (oral, qd) for the indicated duration of treatment. The final concentration of vehicle was 0.02N HCl, 10% DMSO, 10% Cremophor EL, 15% PEG400, and 15% HPCD (2-hydroxypropyl-β-cyclodextrin). The length (L) and width (W) of the tumor mass were measured, and tumor volume (TV) was calculated as: TV = (L × W2)/2. The rate of change in body weight (BW) was calculated using the following formula: BW = W/W0 × 100, where W and W0 are the body weight on a specific experimental day and on the first day of treatment, respectively. All animal experiments in this study were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Chugai Pharmaceutical Co., Ltd.
Results
Inhibition of RET kinase activity mediated by alectinib
The ALK inhibitor crizotinib inhibits several kinases including ALK, MET, ROS1, and RON (10). Several ROS1 fusion genes have recently been identified in NSCLC (4, 11, 12), and crizotinib has recently shown efficacy in the treatment of NSCLC with ROS1 translocations (13). Having previously reported that alectinib potently inhibited ALK (IC50 = 1.9 nmol/L) but not MET kinase (IC50, >5,000 nmol/L; ref. 17), we further checked the inhibitory activity of alectinib for ROS1 and RON. However, alectinib hardly inhibited ROS1 (IC50 = 3,700 nmol/L) and did not inhibit RON (IC50, >5,000 nmol/L; Fig. 1A; Table 1).
Structural model of alectinib with RET. A, chemical structure of alectinib. B, X-ray structure of alectinib with ALK at the ATP-binding site and 3D model of alectinib with RET. Amino acid residues, which form hydrogen bonds to the ligand directly or via water molecules, are depicted. Alectinib and amino acid residues are shown in stick form (C in yellow, white, and green; O in red; and N in blue). C, X-ray structure of alectinib with ALK (PDB ID: 3AOX), crizotinib with ALK (PDB ID: 2XP2), and LDK378 with ALK (PDB ID: 4MKC) and structural models of RET (PDB ID: 2K2K) with them. Alectinib (C in yellow), crizotinib (C in pink), and LDK378 (C in aqua) are shown in stick form.
Kinase inhibitory activity of alectinib against RET, ROS1, and RON
To examine the additional kinase inhibitory activity of alectinib, we evaluated 451 biochemical kinases, including several mutated kinases, using KINOMEscan technology (DiscoveRx). Using this technology, we had previously found that alectinib at 10 nmol/L bound to only three kinases: ALK, LTK, and GAK (17). In this study, we found that in addition to ALK and LTK, alectinib at 100 nmol/L bound to CHEK2, FLT3 (D835Y), PHKG2, RET, and RET (M918T) with greater than 95% inhibition (data not shown). Because several RET fusion genes have recently been identified as driver oncogenes in NSCLC (3–5), we focused on the potential of alectinib as a RET inhibitor. The results of a kinase inhibitory assay revealed that alectinib strongly inhibited RET kinase activity (IC50 = 4.8 nmol/L; Table 1) and RET kinases with point mutations that were identified in MTC (IC50 = 5.7−53 nmol/L, Table 1; refs. 20–22). In contrast, crizotinib hardly inhibited RET (IC50 = 3,200 nmol/L) and LDK378, a second-generation ALK inhibitor (23, 24), did not inhibit RET kinase activity (IC50, >5,000 nmol/L). In addition, ATP-competitive binding assay showed that alectinib bound to RET at a dissociation constant (KD) value of 7.6 nmol/L. The kinase domain of RET shares 36.6% amino acid sequence homology with ALK in the kinase domains, and a structure-based in silico analysis revealed that common hydrophobic interactions of alectinib bound to ALK (25) and RET (Fig. 1B). In addition, the differences among ALK inhibitors show that alectinib has a unique chemical scaffold (it is a benzo[b]carbazole derivative) that is unlike the scaffold used in ALK inhibitors of other chemical classes, such as crizotinib and LDK378 (17, 23, 26, 27). Moreover, in silico analysis suggested that RET would cause steric hindrance, thereby interfering with the action of crizotinib and LDK378 but not with that of alectinib (Fig. 1C).
Alectinib mediated inhibition of RET fusion–positive cell growth
Next, to investigate the sensitivity of RET fusion–positive cells to alectinib, we evaluated alectinib's capacity to inhibit cell growth in LC-2/ad NSCLC cells harboring the CCDC6-RET fusion gene (28). Alectinib, similarly to well-known RET inhibitors cabozantinib and vandetanib, inhibited the growth of LC-2/ad cells (Fig. 2A). However, other ALK inhibitors (crizotinib and LDK378) and the EGFR inhibitor gefitinib did not inhibit cell growth. To investigate the sensitivity of another line of RET fusion–positive cells to alectinib, we generated Ba/F3 cells expressing KIF5B-RET. An established Ba/F3 transfectant expressing KIF5B-RET shows IL3 independent growth and that the cells depend on KIF5B-RET. Alectinib, cabozantinib, and vandetanib were also effective against Ba/F3 cells expressing KIF5B-RET (Fig. 2B). From public information, ponatinib also inhibited Ba/F3 cells expressing KIF5B-RET (IC50 = 11 nmol/L; ref. 29), consistent with the potency of enzyme inhibition on RET (Supplementary Fig. S1). In contrast, gefitinib and crizotinib did not inhibit these cells and LDK378 hardly inhibited these cells (Fig. 2B and Supplementary Fig. S2). Moreover, alectinib induced caspase-3/7 activation in LC-2/ad cells as well as in NCI-H2228 cells harboring EML4-ALK (Fig. 2C), indicating that apoptosis is involved in the antitumor activity of alectinib. To understand the effects of phospho-RET suppression and the contribution of factors downstream to RET, we conducted a cellular phosphorylation assay using LC-2/ad cells that had been treated with alectinib. Alectinib suppressed the autophosphorylation of RET in a concentration-dependent manner (Fig. 2D). Alectinib also suppressed the phosphorylation of ERK1/2, but not of STAT3 and AKT (Fig. 2D), suggesting that the proliferation of LC-2/ad cells requires the MAPK signaling pathways. Taken together, these results indicate that alectinib exhibits potent inhibitory activity against RET fusion–positive cells by suppressing RET phosphorylation.
Inhibition of RET-driven tumors by alectinib. A, growth inhibition of LC-2/ad cells by alectinib. Cells were seeded in a spheroid culture plate and incubated overnight, and then treated with the indicated concentrations of alectinib, cabozantinib, vandetanib, crizotinib, LDK378, and gefitinib. The viable cells were measured using a CellTiter-Glo luminescent cell viability assay after 5 days of treatment. Data are shown as mean ± SD (n = 3 per group). B, effect of alectinib against Ba/F3 cells expressing KIF5B-RET. Cells were seeded and then treated with various concentrations of alectinib, cabozantinib, vandetanib, and gefitinib for 2 days. The viable cells were measured using the CellTiter-Glo luminescent cell viability assay. Data are shown as mean ± SD (n = 3). C, alectinib-mediated increase in caspase-3/7 activity. Cells were seeded in spheroid culture plate and incubated overnight, then treated with alectinib at the indicated concentrations. The viable cells were measured using a Caspase-Glo 3/7 assay kit after 2 days of treatment. Data are shown as mean ± SD (n = 3 per group). D, effects of alectinib on the phosphorylation of RET, STAT3, AKT, and ERK1/2 in LC-2/ad cells. LC-2/ad cells were treated with alectinib for 2 hours at the indicated concentrations. Cell lysates were subjected to immunoprecipitation with anti-phosphorylated tyrosine antibodies, and the level of RET phosphorylation was detected by immunoblot analysis using anti-RET antibody. The expression level of RET, phospho-STAT3 (Tyr 705), STAT3, phospho-AKT (Ser 473), AKT, phospho-ERK1/2 (Thr 202/Tyr 204), and ERK1/2 were detected by immunoblot analysis with the appropriate antibodies.
Downstream signaling pathway in LC-2/ad cells harboring CCDC6-RET
To understand the downstream signal pathway of CCDC6-RET in NSCLC, we performed comprehensive gene expression analysis of alectinib-treated LC-2/ad cells based on Illumina HiSeq 2000 next-generation sequencing. The majority of genes downregulated by alectinib were negative feedback regulators of ERK such as DUSP6 (30) and DUSP2 (31) and MAPK pathway downstream genes such as ETV1, ETV4, ETV5, FOS (32), and EREG (33). A previous report has demonstrated that certain MAPK-related genes, such as DUSP6 and EREG, are suppressed by a knockdown of RET using siRNA in LC-2/ad cells (34). To validate our data, we conducted qRT-PCR and confirmed significant decreases in the expression of these genes (e.g., DUSP2, EREG, ETV5, and FOS; Fig. 3A). We also examined the effects of alectinib on the expression of apoptosis-related proteins in LC-2/ad cells. Alectinib induced PARP cleavage in LC-2/ad cells and increased the abundance of BIM, a key proapoptotic member of the BCL-2 family of proteins, whereas the levels of BCL-2 family members MCL-1 and BAX remained unaffected (Fig. 3B), suggesting that alectinib induces apoptosis through the upregulation of BIM via inhibition of the MAPK signaling pathway. In Ba/F3 cells expressing KIF5B-RET, alectinib also suppressed phosphorylation of ERK and increased the abundance of BIM (Supplementary Fig. S3). These data were consistent with a previous report that inhibition of the MAPK signaling pathway contributes to EGFR inhibitor-induced BIM upregulation in EGFR-mutated NSCLC cells (35). However, we recognize that the full downstream signal pathway of RET fusion protein in NSCLC, including that in LC-2/ad cells, remains unknown. Further detailed studies are needed to elucidate the downstream signal pathway of RET fusion protein in NSCLC to explore options for combination therapy.
Downstream signaling pathway in LC-2/ad cells harboring CCDC6-RET. A, the levels of each transcript were measured by qRT-PCR. Data are shown as mean ± SD (n = 2). Student t test: *, P < 0.05, versus DMSO treatment. B, effects of alectinib on the expression levels of cleaved PARP, BIM, MCL-1, and BAX in LC-2/ad cells. LC-2/ad cells were treated with alectinib for 24 hours at the indicated concentrations. The expression level of cleaved PARP, BIM, MCL-1, BAX, and β-actin were detected by immunoblot analysis using the appropriate antibodies.
Antitumor activity of alectinib against RET fusion–positive tumors
To evaluate the in vivo antitumor activity of alectinib against RET fusion–positive tumors, we used a mouse xenograft model of LC-2/ad cells expressing CCDC6-RET. Although the engraftment of LC-2/ad cells using SCID mice was slow, RET break-apart FISH analysis using the RET Split Dual Color FISH Probe (ver. 1 SP018, GSP Lab., Inc.; ref. 3) showed that LC-2/ad xenograft tumors continued to harbor the RET rearrangement up to 141 days after the inoculation (data not shown). Using this xenograft model, we confirmed that once-daily oral administration of alectinib resulted in remarkable tumor regression at all doses without significant weight loss (Fig. 4A). Similar efficacy was also observed in the case of treatment with vandetanib at 50 mg/kg, but significant body weight loss was seen at this dose level (Fig. 4A).
Antitumor activity of alectinib against RET fusion–driven tumors. A, mice bearing LC-2/ad cells were administered alectinib or vandetanib orally at the indicated doses, once daily for 14 days. Tumor volume and changes in body weight were measured for each dose group. Data are shown as mean ± SD (n = 5 per group). Parametric Dunnett test: ***, P < 0.001; *, P < 0.05; N.S., not significant, versus treatment with vehicle of alectinib on the final day of the experiment. B, mice bearing Ba/F3 cells expressing KIF5B-RET were treated with vehicle, 60 mg/kg of alectinib orally once daily for 10 days. Tumor volume was measured for each dose group. Data are shown as mean ± SD (n = 5 per group). Parametric Dunnett' test: ***, P < 0.001, versus vehicle treatment on the final day of the experiment. C, mice bearing Ba/F3 cells expressing KIF5B-RET were orally administered a dose of 0 (vehicle) or 60 mg/kg alectinib, and the tumors were collected and lysed at 4 hours postdosing. The expression levels of phospho-RET and RET were detected by immunoblot analysis using the appropriate antibodies.
We next tested a subcutaneous mouse model of Ba/F3 cells expressing KIF5B-RET. We found that treatment with alectinib at 60 mg/kg significantly inhibited the growth of KIF5B-RET–driven tumors (Fig. 4B). In a phosphorylation assay of the Ba/F3 cells expressing KIF5B-RET tumors, alectinib suppressed autophosphorylation of RET in vivo (Fig. 4C). These in vitro and in vivo findings confirmed that alectinib is effective against RET fusion–positive tumors.
Alectinib is active in cancers driven by RET gatekeeper mutations
Point mutations in the kinase domain are known as a mechanism of acquired resistance to small-molecule kinase inhibitors. In particular, gatekeeper mutations, such as T790M in EGFR, T315I in ABL, and L1196M in ALK, are one of the most frequent causes of resistance (36–38). To evaluate the inhibitory effect of alectinib on the most predictable resistant RET mutations (V804L and V804M), which are known as gatekeeper mutations (20), we measured the kinase inhibitory activity using recombinant glutathione S-transferase (GST)-fused RET V804L and V804M. Alectinib had substantial inhibitory potency against both RET gatekeeper mutants and the IC50 values on RET V804L and V804M were 32 and 53 nmol/L, respectively (Table 1). Next, to investigate the sensitivity of these mutant-driven cells to alectinib, we generated Ba/F3 cells expressing KIF5B-RET V804L and V804M. Alectinib was effective against each mutant-driven cell, and the IC50 ratio of alectinib against the mutated KIF5B-RET as compared with the nonmutated KIF5B-RET (6.5- to 8.1-fold) was lower than that of cabozantinib (15- to 25-fold) and vandetanib (50- to 57-fold; Fig. 5A). The degree of cell sensitivity to these drugs was consistent with the suppression of phospho-RET (Fig. 5B).
Inhibition of RET gatekeeper mutants by alectinib, vandetanib, or cabozantinib. A, effect of alectinib, vandetanib, or cabozantinib against Ba/F3 cells expressing KIF5B-RET and gatekeeper mutations (V804L and V804M). Cells were seeded and then treated with various concentrations of alectinib, vandetanib, or cabozantinib for 2 days. The viable cells were measured using a CellTiter-Glo luminescent cell viability assay. IC50 values were determined by plotting the drug concentration versus the percentage of cell growth inhibition. Data are shown as mean ± SD (n = 3). B, effects of alectinib on the phosphorylation levels of RET in Ba/F3 cells expressing KIF5B-RET and gatekeeper mutations (V804L and V804M). Ba/F3 cells expressing KIF5B-RET were treated with alectinib, vandetanib, or cabozantinib for 2 hours at the indicated concentrations. The expression levels of phospho-RET, phospho-AKT, AKT, phospho-ERK1/2, ERK1/2, and β-actin were detected by immunoblot analysis using the corresponding antibodies.
To understand the difference in the inhibitory effects on RET V804L and V804M as exhibited by alectinib versus vandetanib, we examined an in silico model based on the crystal structures of alectinib with ALK (PDB ID: 3AOX). This model was then superimposed on the crystal structure of RET bound to vandetanib. Both V804L and V804M mutation would cause steric hindrance that interfered with the action of vandetanib but not with that of alectinib (Supplementary Fig. S4). These results indicated potential antitumor activity of alectinib against tumors harboring gatekeeper-mutated RET fusion genes.
Discussion
Discovery of the RET fusion gene in NSCLC has led to the development of RET inhibitors for patients with NSCLC with RET fusion–positive tumors. Clinical trials have been designed to investigate the therapeutic effects of multikinase inhibitors targeting RET, such as vandetanib (Trial registration ID: NCT01823068), cabozantinib (NCT01639508), sunitinib (NCT01829217), lenvatinib (NCT01877083), dovitinib (NCT01831726), and ponatinib (NCT01813734). In a recent study of cabozantinib in 3 patients with NSCLC with the RET fusion gene, cabozantinib achieved partial responses in 2 patients and stable disease in 1 patient (7). Of those patients, grade 3 toxicities of fatigue, proteinuria, and hypertension were observed, and 2 patients treated with cabozantinib required dose reduction of cabozantinib. To understand the differences between alectinib and other RET inhibitors, we confirmed the associated kinase selectivity profiles. Cabozantinib, vandetanib, sunitinib, sorafenib, ponatinib, lenvatinib, and dovitinib inhibited not only RET kinase, but also other kinases such as KDR, which is a receptor for VEGF (Supplementary Fig. S1; refs. 39, 40). In contrast, alectinib only slightly inhibited KDR kinase activity (Supplementary Fig. S1). The major toxicities of KDR tyrosine kinase inhibitors are associated with the development of hypertension and proteinuria and they are commonly regressive on drug withdrawal (15, 16). In a clinical trial of alectinib in patients with ALK-rearranged NSCLC, alectinib was generally tolerable with no dose-limiting toxicities observed and KDR-related toxicities such as hypertension observed rarely (18). Therefore, potent RET inhibitors with weak KDR inhibition may offer a clinical advantage in the treatment of RET fusion–positive NSCLC.
Although alectinib inhibited the growth of RET C634W mutation-positive MTC TT cells (data not shown; ref. 41), this effect was weak in a mouse xenograft model. Nevertheless, alectinib inhibited RET phosphorylation in these tumor samples (data not shown). In contrast, a multikinase inhibitor, sorafenib, showed tumor growth inhibition against this mouse model and inhibited RET phosphorylation in these tumor samples (data not shown). In a previous report, cabozantinib also inhibited tumor growth in a mouse xenograft model of TT cells (42). Thus, the growth of TT xenograft tumors might depend not only on RET, but also on other kinases such as KDR. Further investigation is needed to confirm the mechanisms underlying the effects of several RET inhibitors in RET-mutated MTC xenograft models.
In addition to the RET fusion gene, the ROS1 fusion gene has been identified as an oncogenic driver that is found in approximately 1% to 2% of NSCLC (11, 12). Alectinib hardly inhibited ROS1 kinase activity (Table 1) and did not inhibit the growth of HCC78 cells harboring the SLC34A2-ROS1 fusion gene (IC50, >1,000 nmol/L; refs. 11, 12, 43), although crizotinib strongly inhibited growth, and LDK378 weakly inhibited the growth of these cells under three-dimensional (3D) spheroid culture conditions. In addition, in silico analysis suggested that ROS1 would cause steric hindrance, thus interfering with the action of alectinib but not with that of crizotinib or LDK378 (Supplementary Fig. S5). In contrast, alectinib inhibited the growth of RET fusion–driven cells but crizotinib and LDK378 did not (Fig. 2A and B). Alectinib also showed antitumor activity in mouse models of RET fusion–driven tumors by suppressing phospho-RET (Fig. 4A and B). On the basis of our studies, in contrast with other ALK inhibitors, alectinib would not be useful for the treatment of ROS1 fusion-positive NSCLC but would be useful for the treatment of RET fusion–positive NSCLC.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: T. Kodama, H. Sakamoto
Development of methodology: Y. Watanabe
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T. Kodama, T. Tsukaguchi, Y. Satoh, M. Yoshida, Y. Watanabe
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): T. Kodama, Y. Watanabe
Writing, review, and/or revision of the manuscript: T. Kodama, H. Sakamoto
Study supervision: O. Kondoh, H. Sakamoto
Acknowledgments
The authors thank Y. Tachibana and M. Izawa for the biologic assay and A. Nakagawa for the in vivo study.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Footnotes
Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).
- Received March 31, 2014.
- Revision received September 19, 2014.
- Accepted September 25, 2014.
- ©2014 American Association for Cancer Research.
References
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