MAPK Pathway Alterations Correlate with Poor Survival and Drive Resistance to Therapy in Patients with Lung Cancers Driven by ROS1 Fusions Hiroki Sato1,2, Adam J

Published OnlineFirst March 2, 2020; DOI: 10.1158/1078-0432.CCR-19-3321

CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

MAPK Pathway Alterations Correlate with Poor Survival and Drive Resistance to Therapy in Patients with Lung Cancers Driven by ROS1 Fusions Hiroki Sato1,2, Adam J. Schoenfeld3, Evan Siau1, Yue Christine Lu1, Huichun Tai1,2, Ken Suzawa1,2, Daisuke Kubota1,2, Allan J.W. Lui1, Besnik Qeriqi4, Marissa Mattar4, Michael Ofﬁn3, Masakiyo Sakaguchi5, Shinichi Toyooka6, Alexander Drilon3, Neal X. Rosen4, Mark G. Kris3, David Solit4, Elisa De Stanchina4, Monika A. Davare7, Gregory J. Riely3, Marc Ladanyi1,2, and Romel Somwar1,2

ABSTRACT ◥ Purpose: ROS1 tyrosine kinase inhibitors (TKI) provide signif- activation of MEK/ERK signaling with minimal effects on AKT icant benefit in lung adenocarcinoma patients with ROS1 fusions. signaling, suggesting the importance of the MAPK pathway in driving However, as observed with all targeted therapies, resistance arises. ROS1 fusion-positive cancers. Of 8 patients, 2 patients harbored novel Detecting mechanisms of acquired resistance (AR) is crucial to in-frame deletions in MEK1 (MEK1delE41_L54) and MEKK1 finding novel therapies and improve patient outcomes. (MEKK1delH907_C916) that were acquired after ROS1 TKIs, and Experimental Design: ROS1 fusions were expressed in HBEC 2 patients harbored NF1 loss-of-function mutations. Expression of and NIH-3T3 cells either by cDNA overexpression (CD74/ROS1, MEK1del or MEKK1del, and knockdown of NF1 in ROS1 fusion- SLC34A2/ROS1) or CRISPR-Cas9–mediated genomic engineering positive cells activated MEK/ERK signaling and conferred resistance (EZR/ROS1). We reviewed targeted large-panel sequencing data to ROS1 TKIs. Combined targeting of ROS1 and MEK inhibited (using the MSK-IMPACT assay) patients treated with ROS1 TKIs, growth of cells expressing both ROS1 fusion and MEK1del. and genetic alterations hypothesized to confer AR were modeled in Conclusions: We demonstrate that downstream activation of the these cell lines. MAPK pathway can mediate of innate acquired resistance to ROS1 Results: Eight of the 75 patients with a ROS1 fusion had a TKIs and that patients harboring ROS1 fusion and concurrent concurrent MAPK pathway alteration and this correlated with shorter downstream MAPK pathway alterations have worse survival. Our overall survival. In addition, the induction of ROS1 fusions stimulated findings suggest a treatment strategy to target both aberrations.

Introduction vation of downstream pathways and malignant transformation, and occur in 1%–2% of lung adenocarcinomas (2–4). Although signaling ROS1 is a proto-oncogene that encodes a receptor tyrosine kinase activated by ROS1 fusion proteins involve multiple growth and involved in cell growth and differentiation, and belongs to the insulin survival pathways, the detailed mechanisms by which ROS1 fusions receptor subfamily (1). ROS1 gene rearrangements resulting from the promote an oncogenic phenotype remain unclear (5–7). The presence fusion of the ROS1 tyrosine kinase domain with various gene partners of a ROS1 fusion confers susceptibility of tumors to ROS1 tyrosine including CD74, EZR, SDC4, and SLC34A2, causes constitutive acti- kinase inhibitors (TKI) such as crizotinib, cabozantinib, and lorlatinib (8). However, several on-target mutations such as G2032R and D2033N kinase domain mutations have already been reported as 1Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, 2 mechanisms of acquired resistance (AR) to crizotinib and cabozanti- New York. Human Oncology and Pathogenesis Program, Memorial Sloan – Kettering Cancer Center, New York, New York. 3Thoracic Oncology Service, nib, respectively (9 11). To overcome the resistance to crizotinib Division of Solid Tumor Oncology, Department of Medicine, Memorial Sloan caused by on-target mutations, additional ROS1-targeted drugs Kettering Cancer Center, New York, New York. 4Molecular Pharmacology and including lorlatinib, a potent, oral, third-generation TKI directed at Chemistry Program, Memorial Sloan Kettering Cancer Center, New York, New ALK and ROS1, is currently being investigated in clinical trials. In a 5 York. Department of Cell Biology, Okayama University Graduate School of phase I study of patients with ALK or ROS1 fusion–positive lung Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan. 6Depart- adenocarcinoma, lorlatinib showed overall response rate of 46% for ment of Thoracic, Breast and Endocrinological Surgery, Okayama University ALK Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, -rearranged patients (19 of 41 including 19 patients who had Japan. 7Department of Pediatrics, Oregon Health & Science University, Portland, received prior ALK TKI) and 50% for ROS1-rearranged patients (6 of Oregon. 12 including 7 crizotinib-pretreated patients), and was effective for ALK ROS1 Note: Supplementary data for this article are available at Clinical Cancer patients harboring G1202R mutation, which analogous to Research Online (http://clincancerres.aacrjournals.org/). G2032R (12). On the other hand, little is known about speciﬁc H. Sato and A.J. Schoenfeld contributed equally to this article. off-target mechanisms of resistance to any ROS1 TKI. In this study, we analyzed our clinical and genomic data for Corresponding Author: Marc Ladanyi, Memorial Sloan Kettering Cancer Center, concurrent ROS1 and other pathway alterations that could potentially Molecular Diagnostics Service, 1275 York Ave., New York, NY 10065. Phone: 212- 639-6369; Fax: 212-717-3515; E-mail: [email protected] impinge upon response of tumor cells to therapy. We found several RAS–MAPK pathway alterations that were present at diagnosis or Clin Cancer Res 2020;XX:XX–XX acquired after resistance to therapy emerged. Using isogenic cells doi: 10.1158/1078-0432.CCR-19-3321 genetically engineered to express a ROS1 fusion, we demonstrate that 2020 American Association for Cancer Research. a novel in frame MEK1 or MEKK1 deletion and knockdown of NF1

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reagent and 72 hours later, a pool of transfected cells was harvested for Translational Relevance RNA extraction, cDNA synthesis, and RT-PCR to detect the fusion The identification of mechanisms of innate and acquired resis- mRNA. Primer sequences and PCR conditions are provided in Sup- tance to therapy in ROS1 fusion-driven lung cancers is essential to plementary Table S3. After gRNA validation, px458-gRNAs were improving outcomes for patients. Here we reviewed NGS data of transfected into HBECp53 cells using FuGENE HD (Promega). After MSKCC patients with ROS1 fusion-driven lung cancers to identify 48 hours, GFP-positive cells were isolated by FACS and seeded at a genomic alterations that affect responses to therapy. Eleven percent density of one cell/well into 96-well plates. Three days after plating, the (8/75) of patients with a ROS1 fusion had a concurrent MAPK growth media was changed from complete KSFM to DMEM/F12 pathway alteration and this correlated with shorter overall survival. supplemented with 5% FBS. Clones were serially moved to larger plates In-frame deletions in MEK1 (MEK1delE41_L54) and MEKK1 and then subjected to RT-PCR for fusion detection. The resulting (MEKK1delH907_C916) were acquired after ROS1 therapy. Two HBECp53 cell line with an EZR/ROS1 fusion is referred to as HBEC- patients had loss-of-function mutations in NF1 prior to receiving a ER cells, with numbers to indicate individual, distinct clones. ROS1 tyrosine kinase inhibitor. Expression of MEK1delE41_L54 or MEKK1delH907_C916, or knockdown of NF1 activated ERK Efficacy testing in allograft models signaling and conferred resistance to ROS1-specific therapies. Six-week-old female NOD/SCID gamma mice (Envigo) were used Combined targeting of ROS1 and MEK inhibited growth of cells for in vivo study. All mice were cared for in accordance with guidelines expressing both ROS1 fusions and MEK1delE41_L54 in vitro and approved by the Memorial Sloan Kettering Cancer Center Institutional in vivo. Animal Care and Use Committee and Research Animal Resource Center. Each cell line (5 106 cells) was mixed with Matrigel (50%) and injected into the subcutaneous flank of mice. Tumor volume was calculated using the empirical formula: V ¼ length width2 0.52. block sensitivity to ROS1 TKIs. A combination of lorlatinib and When tumors reached approximately 100 mm3 (7 days after injection), selumetinib was effective in blocking growth of allograft tumors. Our mice were randomly assigned to 6 groups (n ¼ 5 for each group) that results suggest that genetic alterations in the MAPK pathway represent received either vehicle control, 25 mg/kg crizotinib (once daily), novel molecular mechanisms mediating either primary or acquired 3 mg/kg lorlatinib (once daily), 40 mg/kg selumetinib (once daily), resistance to ROS1 TKIs in patients with ROS1-rearranged lung or a combination of 25 mg/kg crizotinib or 3 mg/kg lorlatinib plus adenocarcinoma. 40 mg/kg selumetinib (once daily). Crizotinib and lorlatinib were prepared in 2% DMSO, 30% PEG 300, and water. Selumetinib was prepared in 4% DMSO, 30% PEG 300, 5% Tween 80, and water. Materials and Methods Vehicles and drugs were administered orally as a suspension by gavage Patients once daily, on a 4-day-on and 3-day-off-schedule. Mice were observed In accordance with the Belmont report and following Institutional daily throughout the treatment period for signs of morbidity/mortal- Review Board/Privacy Board at Memorial Sloan Kettering (MSK) for ity. Data were analyzed by Student t test for significance. retrospective review of records and waiver of consent, we identified all patients with lung cancer who had ROS1 rearrangements with/without Immunofluorescence concurrent MAPK pathway alterations identified by targeted next- HEK-293T cells were directly cultured on 1-well glass slide generation sequencing. MSK-IMPACT (13) was the primary platform, (Lab-Tek II Chamber Slide System, Thermo Fisher Scientific) for but results from other methods such as FoundationOne (14) were also 24 hours, and then were transfected with cDNAs encoding MEK1 included if performed. MSK-Fusion, a custom targeted RNA sequenc- wild-type or MEK1delE41_L54. Forty-eight hours after transfection, ing (RNA-seq) panel, was used to confirm fusion status in cases with cells were fixed and permeabilized by methanol at 20C for 15 min- sufficient tissue (15). The frequency of ROS1 rearrangements with/ utes. Nonspecific binding was blocked using PBST (PBS with 0.1% without concurrent MAPK pathway among all patients was queried. Tween 20) supplemented with 1% BSA. Samples were incubated To assess the impact of concurrent alterations in advanced disease, with an anti-ERK1/2 mAb in a humidified chamber at 4C over- patient records with stage IV disease were reviewed to collect demo- night. After washing by PBST, the slides were counterstained with graphic, clinical outcome, and molecular data. Overall survival (OS) Hoechst 33342 (Thermo Fisher Scientific). Fluorescent microscopy was defined as date of metastatic diagnosis to date of last follow as of was used for visualization of the antigen–antibody complexes. March 15, 2019 or date of death. Fisher exact and log-rank tests were used to assess relationships between patients with/without MAPK Gene expression analysis alterations and Kaplan–Meier methodology was used to generate To better understand the transcriptional response evoked by ROS1 overall survival curves. fusion protein, we downloaded RNA-seq data, mutation profiles, and clinical information of 510 lung adenocarcinoma cases profiled by Generation of EZR/ROS1 fusion using CRISPR/Cas9 in human The Cancer Genome Atlas via the cBioPortal (http://www.cbioportal. bronchial epithelial cells org/). ROS1 fusion status for those cases were determined using the To generate a fusion linking EZR exons 1- 9 with ROS1 exons 34–43, cBioPortal, as a result, seven of 510 had ROS1 fusion. To reduce the two gRNAs were designed to target EZR intron 9 and three gRNAs statistical bias that may exist between ROS1 fusion–positive (n ¼ 7) were designed to target ROS1 intron 33 (http://crispr.mit.edu/). The and -negative (n ¼ 503) groups, a propensity score (PS)–matched sequence of gRNAs is shown in Supplementary Table S2. gRNAs were analysis was performed. The PS was calculated by logistic regression cloned into pSpCas9(BB)-2A-GFP (px458; Addgene, plasmid #48138) based on available factors that potentially confound the association as described previously (16, 17). To test the efficacy of fusion gener- between ROS1 fusion–positive and -negative groups. Six factors that ation, HEK-293T cells were transfected with each possible pair of were selected in the PS calculation were age, sex, histology, clinical gRNAs (one targeting EZR and one targeting ROS1) using Fugene HD stage, tumor size, and mutation status in RAS pathway. According to a

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Figure 1. Clinical and molecular characteristics of patients with ROS1-rearranged lung cancer and MAPK pathway alterations. A, Demographics of patients with metastatic ROS1-rearranged lung cancer with and without MAPK alterations. B, Spectrum of MAPK alterations identiﬁed among patients with metastatic ROS1-rearranged lung cancer. C, Survival of patients with or without concurrent MAPK alterations at metastatic diagnosis. D, The schema of MAPK pathway.

PS-matched analysis, seven cases were extracted from each group. ence or absence of concurrent MAPK alterations were generally Gene set enrichment analysis (GSEA) was then performed against the similar (Fig. 1A). There were no significant differences in the two Oncogenic Pathways (18). The GSEA software (GSEA ver. 3.0) down- groups by age, sex, or smoking status. The most common ROS1 loaded from the GSEA Website (http://software.broadinstitute.org/ fusion partner in samples with a concurrent MAPK alteration was gsea/index.jsp). CD74 (n ¼ 3) followed by SLC34A2 (n ¼ 2). In cases with sufficient tissue, targeted RNA-seq (n ¼ 3) confirmed the presence of the ROS1 rearrangement. MAPK pathway gene alterations included 2 Results MAP3K1 mutations, 1 MAP2K1 mutation, 1 MAP2K4 mutation, Clinical characteristics and molecular alterations 1 KRAS mutation, and 1 BRAF mutation (Fig. 1B; Supplementary Among 5,470 patients with non–small cell lung cancer who Table S1). In addition, we identified two neurofibromin 1 (NF1) underwent next-generation sequencing via MSK-IMPACT, we iden- truncating mutations. Among the patients with metastatic disease, 5 tified 75 (1%) patients with ROS1 fusions. Overall, 8 (11%) of the 75 patients (ID#1-5) had concurrent MAPK alterations and ROS1 patients and 7 (13%) of 53 patients with metastatic disease had fusions detected at the time of metastatic diagnosis and 2 concurrent MAPK pathway alterations in the same tumor specimen. patients (ID#6, 7) had MAPK alterations detected at the time of The initial clinical characteristics of the patients stratified by pres- resistance to ROS1 TKIs.

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Patient treatment and outcomes MAPK pathway is preferentially activated in cell lines with ROS1 The patients received several ROS1-directed therapies, including fusions crizotinib (n ¼ 3), entrectinib (n ¼ 3), lorlatinib (n ¼ 2), TPX-0005 To assess the changes in downstream signaling caused by expression (n ¼ 1), and cabozantinib (n ¼ 1), sequentially before and after of ROS1 fusion, the phosphorylation status of key signaling elements in standard cytotoxic chemotherapy and immunotherapy (Supplemen- the MAPK pathway and the PI3K/AKT pathway were examined by tary Table S1). Patients with advanced stage lung adenocarcinoma with Western blot analysis. As shown in Fig. 3A and Supplementary de novo concurrent MAPK alterations at diagnosis typically had Fig. S2A, phosphorylation of MEK1/2 and ERK1/2 were highly minimal or very short time to discontinuation on ROS1 TKIs (Sup- induced in ROS1 fusion–positive cells, compared with that of AKT plementary Table S1). When compared with other patients with ROS1- or 4EBP1. S6 phosphorylation more closely mirrored activation of rearranged advanced lung adenocarcinoma, patients with concurrent ERK1/2 than AKT in the ROS1 fusion–positive cells. p70 S6 Kinase MAPK alterations had shorter overall survival despite similar initial phosphorylation on the other hand, seems to be either cell line-specific clinical characteristics (Fig. 1C). Two patients had two novel MAPK or ROS1 fusion-specific. alterations (deletions of several nucleotides in MAP3K1 and MAP2K1 To gain insight into the relationship between ROS1 fusion proteins deletions) acquired at the time of resistance to TKIs. Case summaries and MAPK pathway, we performed coimmunoprecipitation analysis of the 2 patients with acquired MAPK alterations are provided to look at the association of upstream MAPK activators GRB2 and (Supplementary Case Report). MAP3K1 encodes the protein MEKK1, SOS1 with ROS1. GRB2 is well known to activate MAPK pathway via which has an N-terminal ubiquitin ligase domain and a C-terminal binding to SOS1, leading to cell proliferation and mitogenesis (20, 21). serine/threonine kinase domain, and can activate the ERK and JNK We found that ROS1 fusion proteins coimmunoprecipitated with pathways (19). MAP2K1 encodes MEK1, a well-characterized GRB2 and SOS1 (Fig. 3B; Supplementary Fig. S2B). In contrast, we upstream ERK kinase. The schema of the MAPK pathway is shown could not detect any association between the adaptor protein GAB1 in Fig. 1D. (which links PI3K to growth factor receptors) or the p85 regulatory subunit of PI3K, key upstream mediators of AKT signaling. Knock- Generation of TKI-responsive isogenic ROS1 fusion–positive down of GRB2 by siRNA caused a reduction in MEK1/2 and ERK1/2 cell line models phosphorylation, and inhibited growth of HBEC-ER1 cells (Fig. 3C To examine the influence of mutations in the MAPK pathway on and D). Furthermore, GSEA analysis revealed that gene sets related to sensitivity of ROS1 fusion–positive cells to ROS1 TKIs, we established the activation of KRAS signaling were upregulated in ROS1 fusion– stable isogenic NIH-3T3 cells expressing CD74/ROS1 or SLC34A2/ positive lung adenocarcinoma samples, compared with ROS1 fusion– ROS1 fusions using retroviral plasmids harboring the respective negative lung adenocarcinoma (NOM P ¼ 0.00; Fig. 3E). These results chimeric cDNAs. In addition, to faithfully model a ROS1 fusion in indicate that a direct interaction between ROS1 fusion protein with the lung adenocarcinoma, we also generated an endogenous EZR/ROS1 GRB2–SOS1 complex plays an important role in the activation of fusion in HBECp53 cells using CRISPR/Cas9 genome editing system as MAPK pathway and the tumorigenic properties of ROS1 fusions. Next, we have previously done for BRAF fusions (17). A schematic diagram to determine the sensitivity of ROS1 fusion–positive cells to MEK and of CRISPR/Cas9 genome engineering is illustrated in Fig. 2A. gRNAs RAF inhibitors, we examined the growth of cells in the presence of were transfected into HEK-293Ts and then the presence of EZR/ROS1 trametinib (MEK1/2 inhibitor), selumetinib (MEK1 inhibitor), cobi- fusion was examined by RT-PCR in the transfected population. metinib (MEK1 inhibitor), and a pan-RAF inhibitor, LY3009120. The validation of gRNAs revealed that the pool of cells containing ROS1 fusion–positive cells showed increased sensitivity to the three EZR gRNA #2 and ROS1 gRNA #3 generated more detectable fusion MEK inhibitors and the pan-RAF inhibitor (Fig. 3F; Supplementary mRNA (Supplementary Fig. S1A). We hence used this pairing of Fig. S2C). Taken together, these results indicate that introduction of gRNAs for the transfection. Three clones of HBECp53 cells that were ROS1 fusion proteins induces the preferential activation of MAPK positive for EZR/ROS1 fusion at the mRNA and protein level were pathway via the binding to GRB2–SOS1 complex, and this pathway is successfully isolated (Fig. 2B and C). Sanger sequencing of the cDNA essential for the growth of ROS1-driven cancer cells. using primers that cover the fusion junction confirmed that the translocation resulted from the pairing of EZR exon 9 with ROS1 Alterations in MAPK pathway drive resistance to ROS1 TKIs exon 34 (Fig. 2D). Among the three clones isolated, the clone with the To explore whether the novel in-frame deletions MEK1delE41_L54 highest EZR/ROS1 fusion expression was used for further analysis and MEKK1delH907_C916 that were acquired after ROS1 TKIs (HBEC-ER1). Subsequently, we examined the sensitivity of growth induce drug resistance in ROS1 fusion–positive lung adeno- and phosphorylation of downstream signaling proteins to ROS1 TKIs carcinoma, we generated isogenic cell line models by transduction in established isogenic ROS1 fusion–positive cells (3T3-CD74/ROS1, with lentiviral vectors. MEK1 wild-type and MEK1delE41_L54 3T3-SLC34A2/ROS1, and HBEC-ER1) to ROS1 TKIs. As shown were overexpressed in 3T3-SLC34A2/ROS1 cells, and MEKK1 in Fig. 2E and Supplementary Fig. S1B, growth of cells with a ROS1 wild-type and MEKK1delH907_C916 del were overexpressed in fusion was more sensitive to crizotinib and cabozantinib compared 3T3-CD74/ROS1 cells. In addition, HBEC-ER1 cells established by with the isogenic control lines. Western blot analysis showed that CRISPR/Cas9 genome editing, which maintain physiologic pro- phosphorylation of ROS1, MEK1/2 and ERK1/2 were inhibited by tein expression, were used to examine the effect of expression crizotinib in both 3T3-SLC34A2/ROS1 and HBEC-ER1 cells in a dose- of MEK1delE41_L54 and MEKK1delH907_C916 on sensitivity to dependent manner (Fig. 2F). On the other hand, phosphorylation of ROS1-TKI. Successful introduction of MEK1 delE41_L54 or AKT, p70 S6 kinase, and S6 were less inhibited by crizotinib in MEKK1delH907_C916 del was confirmed by Sanger sequencing comparison with ERK or MEK1/2, suggesting that the RAS–MEK– (Supplementary Fig. S3A). Western blot analysis revealed that ERK signaling axis is more dependent on ROS1 for activation than the expression of MEK1delE41_L54 or MEKK1delH907_C916 induced PI3K–AKT pathway (Fig. 2F; Supplementary Fig. S1C). These results remarkable phosphorylation of ERK, suggesting that these muta- prompted us to further explore the importance of MAPK pathway in tions are activating (Fig. 4A; Supplementary Fig. S3B). In addition, growth and survival of ROS1 fusion–positive cells. the expression of BIM, a proapoptotic protein, was more reduced

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Figure 2. Generation of a TKI-responsive ROS1 fusion cell line by CRISPR-Cas9–mediated genomic engineering. A, Schematic outline of CRISPR/Cas9 genome engineering to generate an EZR/ROS1 fusion. B and C, Expression of EZR/ROS1 fusion by RT-PCR or Western blotting. D, Sanger sequencing of cDNA conﬁrmed that the EZR/ROS1 rearrangement results in a fusion transcript of EZR exon 9 joined to ROS1 exon 34. E, Cells were treated with crizotinib or cabozantinib for 96 hours and then viability was determined. Data represent the mean SE of four independent experiments. F, Western blot analysis of cell extracts prepared after 4 hours of treatment with the indicated concentrations of crizotinib.

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Figure 3. MAPK pathway is preferentially activated in ROS1 fusion–positive cell lines. Isogenic stable 3T3-CD74/ROS1 or SLC34A2/ROS1 and HBEC-ER1 cell lines were serum-starved for 6 hours and then whole-cell extracts were prepared. Lysates were subjected to Western blot analysis (A) or coimmunoprecipitation followed by Western blot analysis (B). Rabbit IgG isotype antibody was used for negative control. C, HBEC-ER1 cells were transfected with nontargeting or GRB2 siRNAs; 72 hours after transfection, lysates prepared and then subjected to Western blot analysis. D, Twenty-four hours after transfection, cells were reseeded onto 6-well plates at a density of 100,000 cells/well, and the cells counted every 24 hours. Each condition was assayed in duplicate determinations, and data are representative of three independent experiments (mean SE). E, The top GSEA Oncogenic Signatures upregulated in ROS1 fusion–positive lung adenocarcinoma tumors, compared with ROS1 fusion–negative lung adenocarcinoma tumors. Gene sets related to KRAS signaling are indicated in bold. Representative GSEA enrichment plots for the KRAS-related signatures in TCGA are shown (bottom). F, Cells were treated with trametinib, selumetinib, cobimetinib, or LY3009120 for 96 hours and then viability was determined. Data represented the mean value of growth inhibition ratio at each concentration of the drugs in four independent experiments.

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Figure 4. Alterations in MAPK pathway drives resistance to ROS1 TKIs. A, Cells were serum-starved for 6 hours, whole-cell lysates prepared and then subjected to Western blot analysis. B and C, Isogenic cell lines were treated with each drug for 96 hours. Each condition was assayed in six replicate determinations and data are representative of three independent experiments (mean SE). D, HBEC-ER1 lines were treated with increasing concentrations of crizotinib for 4 hours and lysates were subjected to Western blot analysis. E, Cells were transfected for 48 hours then lysates were prepared and subjected to Western blot analysis. F, Twenty-four hours after transfection, cells were reseeded onto 96-well plates and treated with each drug for and viability determined 96 hours after treatment. Each condition was assayed in six replicate determinations, and data are representative of three independent experiments (mean SE).

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in cells expressing both ROS1 fusion protein and MEK1delE41_L54, cells. Collectively, our results indicate the possibility that the activa- compared with cells expressing ROS1 fusion alone (Supple- tion of MAPK pathway by multiple mutations is responsible for the mentary Fig. S3C). We next examined the effect that these mutant resistance to ROS1 TKIs. MAPKs have on the sensitivity of ROS1 fusion–positive cells to ROS1 TKIs that had been used for each patient: the patient with MEK1delE41_L54 is a novel oncogene MEK1delE41_L54 was treated with crizotinib, entrectinib, and Because little is known about the MEK1delE41_L54 mutation, we lorlatinib, and the patient with MEKK1delH907_C916 was treated assessed its transforming ability in Ba/F3 cells. Ba/F3 cells require IL3 with crizotinib and cabozantinib. Cell viability assay revealed for growth and survival; however, if they are transformed by intro- that ROS1 fusion–positive cells expressing MEK1delE41_L54 or duction of an oncogene, they become IL3-independent. We expressed MEKK1delH907_C916 were less sensitive to ROS1 TKIs, compared MEK1delE41_L54 or the wild-type MEK1 and cultured the cells in with control cells expressing an empty vector (Fig. 4B and C; growth media without IL3. Whereas Ba/F3 cells expressing MEK1 Supplementary Fig. S3D–S3G). Expression of wild-type MEK1 or wild-type were not able to survive without IL3, Ba/F3 cells expressing MEKK1 did not affect sensitivity to ROS1 inhibitors. To better MEK1delE41_L54 can grow without IL3, suggesting that Ba/F3 cells understand how MEK1delE41_L54- and MEKK1delH907_C916– were transformed by the MEK1delE41_L54 transduction (Fig. 5A). mediated TKI resistance, we examined their effects on ROS1 fusion To investigate the functional role of MEK1delE41_L54, we genera- and other downstream signaling proteins. Whereas phosphorylation ted NIH-3T3 and HBECp53 cell line models and examined the effects of ROS1 was inhibited by crizotinib in a dose-dependent fashion in of expression on growth and phosphorylation status of key signaling cells expressing either wild-type or the corresponding in-frame molecules. As shown in Supplementary Fig. S4A, induction of deletion, phosphorylation of ERK remained insensitive to crizotinib MEK1delE41_L54 promoted cell growth in NIH-3T3 cell line models. treatment only in MEK1delE41_L54- or MEKK1delH907_C916– In Western blot analysis, ERK was highly phosphorylated in 3T3 expressing cells (Fig. 4D). Similar results were obtained with the and HBEC cells expressing MEK1delE41_L54. However, the degree other ROS1 TKIs: entrectinib and lorlatinib for MEK1delE41_L54 of MEK1 phosphorylation was lower than that of ERK (Fig. 5B). and cabozantinib for MEKK1delH907_C916 (Supplementary Of note, phosphorylation of BRAF, which is upstream of MEK1, was Fig. S3H–S3J). As for the patient with MEK1delE41_L54, PIK3CA suppressed in cells expressing MEK1delE41_L54. We then performed E545K mutation was also detected in the post-TKI sample. To knockdown of BRAF by siRNA and examined the phosphorylation assess whether this mutation affects the drug sensitivity, we over- of MEK1 and ERK. In HBECp53 cells expressing MEK1 wild-type, expressed PIK3CA wild-type and E545K by retroviral transduction knockdown of BRAF suppressed the phosphorylation of MEK1 and using 3T3-SLC34A2/ROS1 and HBEC-ER1 cells. As shown in ERK (Fig. 5C). In contrast, despite knockdown of BRAF, the phos- Supplementary Fig. S3K–S3M, the expression of PIK3CA E545K phorylation of MEK1 and ERK were sustained at the same level as mutation induced the phosphorylation of AKT, but did not alter the negative control in HBECp53 cells expressing MEK1delE41_L54. sensitivity of growth of ROS1 fusion–positive cells to crizotinib and To better elucidate this phenomenon, we transiently transfected cabozantinib. MEK1 wild-type or MEK1delE41_L54 into HEK-293T cells, and As indicated in Supplementary Table S1, 2 patients had a ROS1 the lysates were subjected to coimmunoprecipitation Western blot fusion and a concurrent NF1 truncating mutation. One of these 2 cases analysis. As shown in Fig. 5D, whereas the binding of MEK1 to BRAF (case #2) also showed an intragenic deletion of NF1 exon 17. However, was observed in MEK1 wild-type–expressing cells, a physical asso- whether this intragenic deletion occurred in cis or in trans with the ciation between MEK1 and BRAF was not observed in cells expressing nonsense mutation cannot be determined. We could not identify a MEK1delE41_L54. These results indicate that the E41_L54 in-frame second hit in the remaining two cases, although one of these had low deletion in MEK1 resulted in a constitutively active kinase. We tumor content. Studies of NF1 truncating mutations in lung adeno- then examined the changes in drug sensitivity to MEK and RAF carcinoma have generally not documented biallelic events in most inhibitors caused by expression of MEK1delE41_L54. Introduction cases, even when the presence of the NF1 truncating mutation of MEK1delE41_L54 increased the sensitivity to MEK inhibitors appeared biologically significant (22), possibly due to technical limita- in Ba/F3 and HBECp53 cell line models (Fig. 5E; Supplementary tions in detecting single copy loss or small intragenic deletions. Fig. S4B and S4C). In contrast, LY3009120, a pan-RAF inhibitor, Likewise, NF1-mutant lung cancer cell lines show complete loss of did not affect growth of MEK1delE41_L54–expressing cells, reinfor- NF1 protein expression even when no second hit is obvious (23). cing that MEK1delE41_L54 can activate downstream pathways inde- Finally, even in the absence of a second hit, it is also possible that NF1 pendently of RAF signaling. haploinsufficiency may been significant, as it has been shown that To further characterize MEK1delE41_L54, we subsequently per- reduced NF1 expression can contribute to EGFR TKI resistance (24). formed comparisons with MEK1 harboring several well-known On the basis of these data and considerations, and given that NF1 is a activating mutations (F53L, Q56P, and K57N). MEK1 wild-type, well-characterized negative regulator of the RAS/MEK/ERK pathway MEK1delE41_L54, MEK1-F53L, MEK1-Q56P, and MEK1-K57N and the pathway is suggested to be highly activated in ROS1 fusion– were transiently transfected into HEK-293T cells, and the phosphor- positive lung adenocarcinoma, we sought to examine the effect of NF1 ylation status of ERK was compared. We found that ERK is highly loss on ROS1 fusion–positive cells. To model NF1 loss-of-function phosphorylated in MEKdelE41_L54–transfected cells in comparison mutation in lung adenocarcinoma with a ROS1 fusion, we performed with MEK1-F53L-, MEK1-Q56P-, and MEK1-K57N–transfected cells, knockdown of NF1 using siRNAs in HBEC-ER1 cells. Knockdown of suggesting the more potent activating ability of MEK1delE41_L54 NF1 was confirmed at mRNA and protein level by qRT-PCR and (Fig. 5F). The E41-L54 deletion overlaps the region encoding the Western blot analysis (Fig. 4E; Supplementary Fig. S3N). As expected, nuclear export signal (NES, residues 32– 51) of MEK1 (Supplementary ERK phosphorylation was increased in NF1-knockdown cells. We next Fig. S4D). Therefore, we examined the influence of this deletion on examined growth of cells in the presence of crizotinib and cabozanti- the subcellular localization of MEK1. Wild-type MEK1 or MEK1 nib. As shown in Fig. 4F and Supplementary Fig. S3O, knockdown of mutants (E41_L54 del, F53L, Q56P, and K57N) tagged with GFP NF1 conferred resistance to crizotinib and cabozantinib in HBEC-ER1 were transiently transfected into HEK-293T cells, and GFP-labeled

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cells were observed by fluorescence microscope. The results revealed MEK1E41_L54del when treated with a combination of crizotinib that in MEK1 wild-type or other mutations except for E41_L54 del– and selumetinib (Supplementary Fig. S5D). To validate these transfected cells, MEK1 is largely excluded from the nucleus, whereas in vitro findings, we performed in vivo experiments testing the in cells expressing MEK1delE41_L54, MEK1 remained in the nucleus efficacy of the combined inhibition of MEK and ROS1. We first without being exported (Fig. 5G). For further confirmation, we evaluated the in vivo tumorigenicity of MEK1 wild-type and compared expression of MEK1 in the nuclear fraction by Western MEK1delE41_L54. Whereas allografts of 3T3-SLC34A2/ROS1- blot analysis. Consequently, a remarkable enrichment of MEK1 was MEK1E41_L54del grew significantly faster than 3T3-SLC34A2/ observed in MEK1delE41_L54–expressing cells (Supplementary ROS1-empty or MEK1 wild-type allografts, the expression of MEK1 Fig. S4E). In contrast to MEK1 localization, ERK remained largely wild-type had no effect on tumorigenesis (Supplementary Fig. S5E). cytoplasmic in MEKdelE41_L54–expressing cells, suggesting the Next, mice bearing 3T3-SLC34A2/ROS1-Vector control or 3T3- acceleration of ERK dynamics (Fig. 5H). Finally, we searched for SLC34A2/ROS1-MEK1delE41_L54 allografts were treated with MEK1 deletion that overlaps the NES region across all cancers in either crizotinib alone (25 mg/kg once daily), lorlatinib alone MSK-IMPACT. As a result, we identified two patients (melanoma (3 mg/kg), selumetinib alone (40 mg/kg once daily), a combination and pancreatic cancer) with a similar deletion (E41_F53), suggesting of crizotinib and selumetinib, or a combination of lorlatinib and that in-frame deletion of the NES region is a recurrent mutation. selumetinib. The changes in volume of individual tumors upon treatment are shown in Fig. 6F and Supplementary Fig. S5F. Combined inhibition of ROS1 and MEK as potential therapy Crizotinib or lorlatinib treatment suppressed growth of To overcome the resistance to ROS1 TKIs caused by the activation 3T3-SLC34A2/ROS1-EV allograft tumors, but was less effective of the MAPK pathway, we first examined the efficacy of MEK at modulating growth of 3T3-SLC34A2/ROS1-MEK1delE41_L54 inhibitors. As shown in Fig. 6A and Supplementary Fig. S5A and allograft tumors, indicating that MEK1delE41_L54 can confer S5B, the induction of MEK1delE41_L54 or MEKK1delH907_C916 resistance to ROS1 TKIs in vivo.Consistentwithourin vitro increased sensitivity to MEK inhibitors in comparison with cells data, the combination of crizotiniborlorlatinibwithselumetinib expressing the wild-type proteins. In contrast, cells expressing successfully inhibited phosphorylation of ERK in 3T3-SLC34A2/ MEK1delE41_L54 showed decreased sensitivity to LY3009120. Con- ROS1-MEK1delE41_L54 allograft tumors (Supplementary Fig. S5G). sistent with this result, Western blot analysis revealed that phosphor- In addition, in agreement with our observations that the MEK–ERK ylation of ERK was sustained even at the high concentration of pathway is preferentially activated by ROS1 fusions, allograft tumors 1 mmol/L LY3009120 in MEK1delE41_L54–expressing cells (Fig. 6B). with ROS1 fusion alone were more sensitive to a combination of To better elucidate how MEK inhibitors affect growth, we examined selumetinib and lorlatinib, than to a monotherapy with lorlatinib their effect on phosphorylation of EGFR, HER2, ROS1, and other (Supplementary Fig. S5H). The drug combination was well tolerated downstream signaling proteins. Time-course experiments showed during the treatment course with no significant changes in animal that treatment with selumetinib completely suppressed phosphor- weight (Supplementary Fig. S5I). ylation of ERK by 48 hours, but reactivation of ERK was observed in the cells treated with trametinib (Fig. 6C). Although the feedback activation of RTKs and AKT was unclear in Fig. 6C,itiswell Discussion known that MEK inhibition causes the activation of several In this study, we observed that 11% of patients with a ROS1 upstream receptor tyrosine kinases by relieving physiologic feed- fusion had a concurrent MAPK pathway alteration and that this back suppression (25–27). Therefore, we tested the hypothesis that correlated with poor survival. Two patients acquired novel activa- coinhibition of MEK and ROS1 could be an efficient therapeutic ting mutations in the MAPK pathway (MEK1delE41_L54 and strategy to overcome resistance to ROS1 TKIs through MAPK MEKK1delH907_C916) following a ROS1 TKI, and we demonstrated activation. We examined the combined effect of ROS1 TKIs and that aberrant activation of MEK–ERK pathway caused by these MEK inhibitors in cell viability assays, and synergism was evaluated mutations can confer resistance to ROS1 TKIs. We also found that using the Chou–Talalay Method (28). In HBEC-ER1 and 3T3- loss-of-function mutations in the RASGAP NF1, which enhances SLC34A2/ROS1 isogenic models, combination of selumetinib and RAS signaling, thereby activating downstream MEK/ERK pathway, crizotinib showed better synergistic effect than trametinib and were present in 2 patients prior to ROS1 TKI. Deletion of NF1 in crizotinib (Fig. 6D; Supplementary Fig. S5C). We also confirmed cells with ROS1 fusion reduced sensitivity to ROS1 TKIs, suggesting by Western blot analysis that phosphorylation of both ERK and that loss-of-function NF1 mutations may also contribute to resistance AKT was effectively suppressed by the combination of crizotinib to ROS1 TKIs. Several studies support the notion that loss of NF1 and selumetinib (Fig. 6E). In addition, expression of cleaved PARP plays an important role in resistance to kinase inhibitors in cancers was prominently induced in cells expressing ROS1 fusion and with genetic alterations causing upstream activation of MAPK

Figure 5. MEK1 E41_L54 deletion is a novel oncogene. A, Ba/F3 cells stably expressing the indicated cDNAs were cultured with or without IL3 and cells counted every 24 hours. Each condition was assayed in duplicate determinations and data are representative of three independent experiments (mean SE). B, Isogenic stable cell lines were serum-starved for 6 hours and then lysates were prepared and subjected to Western blot analysis. Forty-eight hours after transfection, lysates were extracted and subjected to Western blot analysis. C, Forty-eight hours after transfection, lysates were extracted and subjected to Western blot analysis. D, Forty-eight hours after transfection, the lysates extracted from each cell were subjected to coimmunoprecipitation and Western blot analysis. Rabbit IgG isotype antibody was used for negative control. E, Isogenic cell lines were treated with each drug for 96 hours prior to determination of cell viability. Parental Ba/F3 cells and Ba/F3 cells expressing EV or MEK1 WT were cultured in the presence of IL3. Each condition was assayed in six replicate determinations and data are representative of three independent experiments (mean SE). F, Forty-eight hours after transfection, lysates were extracted and subjected to Western blot analysis (G); MEK1 tagged with GFP cDNA (green) was transfected into HEK-293T cells and 24 hours later cells were observed by fluorescence microscopy. Hoechst 33342 was used for nuclear staining (blue). H, HEK-293T cells were transfected with MEK1 or MEK1delE41_L54 and 48 hours later immunofluorescence was performed using mAbs against ERK1/2 labeled with the fluorescent dye Alexa488 (green). Nuclear counterstaining was performed using Hoechst 33342 (blue).

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pathway (24, 29, 30). Because we find that expression of a ROS1 therapy, a few studies have reported on PI3K–AKT and JAK–STAT3 fusion preferentially activates MAPK signaling over the AKT pathway gene mutations as mechanisms of TKI resistance in ALK- pathway, loss of NF1 is a candidate mechanism to be examined as rearranged and ROS1-rearranged lung adenocarcinoma (11, 38–40). part of the landscape of off-target alterations that can mediate resis- This may imply a less dominant role of these pathways in ROS1-or tance to ROS1-targeted TKI therapy. To overcome MAPK pathway– ALK-mediated proliferation. Indeed, our results show that the mediated resistance, we tested the efficacy of combined inhibition of PIK3CA E545K mutation has no effect on sensitivity to ROS1 TKIs. ROS1 and MEK with several inhibitors and found that the combina- Taken together, our results indicate the importance of MEK/ERK tion therapy successfully suppressed the growth of ROS1 fusion– signaling in primary or acquired resistance in ROS1-rearranged lung positive cells in vitro and in vivo. As for the optimal types of MEK adenocarcinoma. inhibitors for combination therapy, our results suggest the possibility Several studies have explored the specific signaling pathways that that selumetinib may be a better partner to be used with ROS1 TKIs are activated by ROS1 fusion protein; Jun and colleagues reported that because the more commonly used MEK1 inhibitor trametinib acti- CD74/ROS1, but not FIG/ROS1, induced phosphorylation of E-Syt1, vates ERK signaling, likely helping to counteract inhibition of resulting in more invasive properties (41). Neel and colleagues showed ROS1. Further investigation of the optimal types of MEK and ROS1 that ROS1 fusion proteins exhibit differential activation of MAPK inhibitors for combination therapy should be explored. signaling according to the subcellular localization of fusion pro- When considering a therapeutic strategy to overcome drug resis- teins (42). These studies suggested the possibility that downstream tance, on-target resistance mechanism and off-target resistance mech- signaling of ROS1 fusion may differ depending on the fusion partners. anism, such as activation of bypass signaling should be distinguished. In this study, we demonstrated that ROS1 fusion proteins can interact For the former, a more potent and structurally modified inhibitor with the GRB2–SOS1 complex by coimmunoprecipitation studies. targeting the oncogene itself can be used, whereas for the latter, This phenomenon was observed with three fusions (CD74, SLC34A2, combination therapy with targeted agents is likely to be more effec- and EZR), suggesting that binding of ROS1 fusion protein to GRB2 is a tive (11). In ROS1-rearranged lung adenocarcinoma, multiple on- common phenomenon that does not depend on the 50 fusion partner. target mechanisms for the resistance to crizotinib have been The tyrosine phosphatase, PTPN111 (SHP2) is associated with ROS1 reported (9, 10, 31, 32). However, there are few reports of drug via phosphorylated Y2274, and it has been shown to recruit GRB2 resistance caused by off-target mechanisms in ROS1-rearranged lung and SOS1, and activate the MAPK pathway (43, 44). Although we adenocarcinoma. Dziadziuszko and colleagues have reported that an did not investigate SHP2 in our study, it is possible that the activating KIT receptor mutation caused crizotinib resistance in a ROS1–GRB2–SOS1 complex includes SHP2. More than 10 ROS1 ROS1-rearranged lung adenocarcinoma patient and this was overcome fusion partner genes have been reported in lung adenocarcinoma by combined inhibition of ROS1 and KIT (33). All other studies and the detailed mechanisms of ROS1–GRB2 interaction are still describing off-target mechanisms of drug resistance to ROS1 TKIs unclear. Further investigation is important to clarify these potentially were conducted in cells in culture. Song and colleagues reported that important biochemical interactions. upregulation of the EGFR pathway conferred resistance to crizotinib in Our results suggest that MEK1_E41_L54 in-frame deletion is a the HCC78 cell line (harbors an SLC34A2/ROS1 fusion; ref. 32) and novel oncogenic driver mutation. Gao and colleges previously per- several studies have suggested the possibility that activation of RAS formed a broad survey of MEK1 mutations and defined three subsets of signaling pathway can render HCC78 cells resistant to ROS1- MEK1 mutations based on RAF dependency (RAF-dependent, RAF- TKIs (7, 34). Here we reported 2 cases of acquired resistance to ROS1 regulated, and RAF-independent groups; ref. 45). Among them, the TKI caused by recurrent activation of MAPK pathway in ROS1- RAF-independent group was shown to possess RAF-independent rearranged LUAD and demonstrate that other co-occurring MAPK kinase activity and to be insensitive to allosteric MEK inhibitors. pathway alterations such as loss-of-function NF1 mutations can be However, in cells expressing MEK1delE41_L54, RAF-independent consequential. Moreover, several studies have demonstrated that the ERK activation and increased sensitivity to allosteric MEK inhibitors activation of MAPK pathway was one of the most important off-target were simultaneously observed. One rationale for these contrasting mechanisms in the setting of resistance to EGFR TKIs (35–37). results may be explained by changes in ERK and MEK1 subcellular Considering these findings, the activation of MAPK pathway should localization. In general, it is known that MEK1/2 shuttles between the be noted as one of the crucial off-target mechanisms in ROS1-rear- cytoplasm and nucleus, and relocalization of nuclear MEK1/2 to the ranged lung adenocarcinoma. Considering these findings, activation of cytoplasm is regulated by the NES. MEK1/2 in the cytoplasm act as a MAPK pathway should be noted as one of the crucial off-target cytoplasmic anchor of ERK and in the nucleus, it regulates nuclear mechanisms in ROS1-rearranged lung adenocarcinoma. In contrast export of dephosphorylated ERK (46, 47). We demonstrated that to the evidence for MAPK pathway alteration as driving resistance to deletion of a segment overlapping the NES region leads to retention

Figure 6. Combined inhibition of ROS1 and MEK as potential therapy. A, Cells were treated with each drug for 96 hours and then viability determined. Each condition was assayed in six replicate determinations and data are representative of three independent experiments (mean SE). B, HBEC-ER1 lines were treated with increasing concentrations of LY3009120 for 4 hours and lysates were subjected to Western blot analysis. C, HBEC-ER1 lines were treated with trametinib (100 nmol/L) or selumetinib (200 nmol/L) for the indicated time and then lysates were extracted and subjected to Western blot analysis. D, Isogenic HBEC lines were treated with trametinib (0–50 nmol/L) or selumetinib (0–100 nmol/L) and crizotinib (0–1 mmol/L) for 96 hours and then viability was assessed. The combination index (CI) was calculated using CompuSyn software. Data represent the mean value of growth inhibition ratio at each concentration of the drugs in two independent experiments. Dot plot indicates the combination index and fraction affected (inhibition ratio) of various drug concentrations. E, Cells were treated with crizotinib (500 nmol/L), selumetinib (200 nmol/L), or combination for 4 hours. Lysates were then prepared and subjected to Western blot analysis. F, 3T3-SLC34A2/ROS1-EV and 3T3-SLC34A2/ROS1-MEK1delE41_L54 cells were implanted into a subcutaneous ﬂank of NSG mice. When tumors reached approximately 100 mm3, vehicle control, 25 mg/kg crizotinib (one daily), 3 mg/kg lorlatinib (once daily), 40 mg/kg selumetinib (once daily), or a combination of 25 mg/kg crizotinib or 3 mg/kg lorlatinib plus 40 mg/kg selumetinib (once daily). Tumor volume was determined on the indicated days after the onset of treatment. Data represent mean SE (n ¼ 3; , P < 0.05, compared with vehicle-treated group).

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of MEK1 in the nucleus and promotes export of dephosphorylated research grants from Pfizer, Novartis, Merck, Mirati, Roche, and Takeda, and is an ERK to the cytoplasm. Rapid relocalization of deactivated ERK to the unpaid consultant/advisory board member for Daiichi and Takeda. R. Somwar cytoplasm may accelerate the activation of the MAPK cascade, thereby reports receiving commercial research grants from Helsinn Health Care, Loxo Oncology, Merus, and 14Ner Oncology. No potential conflicts of interest were leading to higher phosphorylation level of ERK in comparison with disclosed by the other authors. other MEK1 mutations. Although there are some unknowns, such as how MEK redistribute to the cytoplasm, critical changes in the Authors’ Contributions intracellular dynamics of MEK and ERK are pivotal characteristics Conception and design: H. Sato, E. Siau, K. Suzawa, M. Offin, A. Drilon, M.G. Kris, of mutations occurring in NES region, and such specific MEK1 R. Somwar mutations may need to be considered as a new subset. Development of methodology: H. Sato, E. Siau, D. Kubota, D.B. Solit, R. Somwar In conclusion, our results indicate that the activation of MAPK Acquisition of data (provided animals, acquired and managed patients, provided pathway via the interaction with GRB2–SOS1 complex plays a key role facilities, etc.): H. Sato, A.J. Schoenfeld, E. Siau, Y.C. Lu, D. Kubota, A.J.W. Lui, ROS1 B. Qeriqi, M. Mattar, M.G. Kris, D.B. Solit, E. de Stanchina, G.J. Riely, M. Ladanyi, in the tumorigenic properties of -rearranged LUAD. Activation R. Somwar of the MAPK pathway by multiple genetic alterations can lead to high Analysis and interpretation of data (e.g., statistical analysis, biostatistics, proliferative ability in the presence of a ROS1 TKI manifested computational analysis): H. Sato, A.J. Schoenfeld, E. Siau, D. Kubota, M. Offin, clinically as drug resistance. Combined inhibition of ROS1 and M. Sakaguchi, A. Drilon, D.B. Solit, M.A. Davare, M. Ladanyi, R. Somwar MEK can be a promising therapeutic strategy that should be explored Writing, review, and/or revision of the manuscript: H. Sato, A.J. Schoenfeld, E. Siau, fi clinically in patients that have a ROS1 fusion and a MAPK pathway M. Of n, S. Toyooka, A. Drilon, N. Rosen, M.G. Kris, D.B. Solit, M.A. Davare, G.J. Riely, M. Ladanyi, R. Somwar alteration. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): H. Tai, D. Kubota, M. Offin, A. Drilon, M.G. Kris, D.B. Solit, Disclosure of Potential Conflicts of Interest G.J. Riely M. Offin reports receiving speakers bureau honoraria from PharmaMar, Novartis, Study supervision: M. Offin, M. Sakaguchi, M. Ladanyi, R. Somwar and Targeted Oncology. A. Drilon reports receiving speakers bureau honoraria from Ignyta/Roche/Genentech, Loxo/Bayer/Lilly, Takeda/Ariad/Millenium, TP Therapeu- Acknowledgments fi tics, AstraZeneca, P zer, Blueprint, Helsinn, Beigene, BergenBio, Hengrui, Exelixis, This work was supported by NIH grants U54 OD020355, P30 CA008748, and P01 Tyra, Verastem, MOREHealth, and Abbvie, and reports receiving other remuneration CA 129243. from GlaxoSmithKline, Teva, Taiho, PharmaMar, WoltersKluwer, Puma, Merus, and Boehringer Ingelheim. N. Rosen is a paid consultant for Tarveda, AstraZeneca, Ribon, The costs of publication of this article were defrayed in part by the payment of page Chugai, Beigene, MapCURE, Zai Labs, and Boehringer Ingelheim; reports receiving charges. This article must therefore be hereby marked advertisement in accordance commercial research grants from Chugai; and holds ownership interest (including with 18 U.S.C. Section 1734 solely to indicate this fact. patents) in Beigene, Zai Labs, Kura, and Fortress. M.G. Kris is a paid consultant for AstraZeneca, Regeneron, Pfizer, and Daiichi-Sankyo. D. B. Solit is a paid advisory board member for Loxo Oncology, Pfizer, Lilly Oncology, QED Therapeutics, Received October 9, 2019; revised January 21, 2020; accepted February 25, 2020; Illumina, and Vivideon Therapeutics. G. J. Riely reports receiving commercial published first March 2, 2020.

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OF14 Clin Cancer Res; 2020 CLINICAL CANCER RESEARCH

MAPK Pathway Alterations Correlate with Poor Survival and Drive Resistance to Therapy in Patients with Lung Cancers Driven by ROS1 Fusions

Hiroki Sato, Adam J. Schoenfeld, Evan Siau, et al.

Clin Cancer Res Published OnlineFirst March 2, 2020.

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