Abstract
Lung cancer is one of the most aggressive tumour types. Targeted therapies stratified by oncogenic drivers have substantially improved therapeutic outcomes in patients with non-small-cell lung cancer (NSCLC)1. However, such oncogenic drivers are not found in 25–40% of cases of lung adenocarcinoma, the most common histological subtype of NSCLC2. Here we identify a novel fusion transcript of CLIP1 and LTK using whole-transcriptome sequencing in a multi-institutional genome screening platform (LC-SCRUM-Asia, UMIN000036871). The CLIP1–LTK fusion was present in 0.4% of NSCLCs and was mutually exclusive with other known oncogenic drivers. We show that kinase activity of the CLIP1–LTK fusion protein is constitutively activated and has transformation potential. Treatment of Ba/F3 cells expressing CLIP1–LTK with lorlatinib, an ALK inhibitor, inhibited CLIP1–LTK kinase activity, suppressed proliferation and induced apoptosis. One patient with NSCLC harbouring the CLIP1–LTK fusion showed a good clinical response to lorlatinib treatment. To our knowledge, this is the first description of LTK alterations with oncogenic activity in cancers. These results identify the CLIP1–LTK fusion as a target in NSCLC that could be treated with lorlatinib.
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Data availability
The WTS data that support the findings of this study are not publicly available and restrictions apply to the availability of these data. Such WTS data are available through the corresponding authors for academic non-commercial research purposes upon reasonable request, and subject to review of a project proposal that will be evaluated by a LC-SCRUM-Asia data access committee, entering into an appropriate data access agreement and subject to any applicable ethical approvals. The presence of LTK fusion was explored in various types of cancer using publicly available data generated by TCGA consortium (https://gdc.cancer.gov), accessed through cBioPortal (https://www.cbioportal.org/datasets). Source data are provided with this paper.
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Acknowledgements
We thank Y. Murata and PREMIA for administrative assistance with managing clinical samples, molecular screening and the clinico-genomic database in LC-SCRUM-Asia; and T. Y. Morita for assistance with flow cytometry analyses. This work was supported by the Princess Takamatsu Cancer Research Fund 18-250 (S.S.K.), JSPS KAKENHI Grant Number JP20K17215 (H.I.), JP16K21746 (S.S.K.) and JP21K15541 (K. Tanaka.), the National Cancer Center Research and Development Fund 31-A-5 (A.O.), 31-A-6 (S.S.K) and 28-A-6 (K.G.), and AMED Grant Number JP21ck0106289 (K.G.), JP21ck0106294 (K.Y.), JP21ck0106483 (K. Nosaki), JP21ck0106568 (K.G.), JP20ck0106411 (S. Matsumoto), JP20ck0106449 (I.O.), JP20ck0106450 (S.N.), JP20ak0101050 (K. Tsuchihara), JP18Ik0201056 (A.O.), JP18kk0205004 (H. Nakagama), JP17Ack0106148 (K.G.), JP17Ack0106147 (S. Yano), and JP16ck0106041 (K.G.). Molecular screening in LC-SCRUM-Asia was supported by Amgen, Astellas, AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Chugai, Daiichi sankyo, Eisai, Janssen, Kyowa kirin, Merck, Medical & Biological Laboratories, MSD, Novartis, Ono, Pfizer, Sumitomo Dainippon, Taiho and Takeda. WTS was supported by Merus.
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H.I., S. Matsumoto, S.S.K. and K.G. conceived the idea and designed the experiments. H.I., S. Mori, S. Matsumoto, K.W., T.F., K.Y., A.O. and K.G. treated patients and interpreted data. H.I. performed cloning and mutagenesis of expression constructs for in vitro analysis, J.L., S. Kumagai and S.S.K. generated stable cell lines. H.I., J.L., K. Tanaka., S. Mori, T.H. and S.S.K. carried out biochemical analysis. K.H. performed FISH analysis. H.I., S. Matsumoto, Y.S., S. Mori, K.W., T.F., T.I., K.Y., T.K., K. Nishino, A.N., I.N., S. Kuyama, N.F., J.S.-K., I.O., K. Taima., N.E., H.D., A.Y., M.K., H.U., K.K., Y.Z., K. Nosaki, E.S., T.S., S.N. and K.G. planned and performed screening for the gene fusion in LC-SCRUM-Asia cohorts. T.N. and G.I. performed pathological evaluation. S.S.K. and K.G. supervised the project. H.I., S. Matsumoto, K.Y., S.S.K. and K.G. wrote the manuscript with input from all authors.
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H.I. reports research support from Amgen and personal fees (honoraria) from Ono. S. Matsumoto reports research support from Chugai, Novartis, Eli Lilly, Merck and MSD, and personal fees (honoraria) from AstraZeneca, Chugai, Novartis, Pfizer and Eli Lilly. Y.S. reports research support from MSD, and personal fees (honoraria) from Ono, Pfizer, Chugai, Novartis, Bristol-Myers Squibb, AstraZeneca and Taiho Pharmaceutical Co. T.F. reports research support from Pfizer, Chgai, Novartis and AstraZeneca. K.Y. reports research support from AstraZeneca, Eli Lilly, Pfizer, Daiichi sankyo, Abbvie, Taiho, Bayer, Takeda and MSD, and personal fees (honoraria) from AstraZeneca, Bristol-Myers Squibb, Chugai, Daiichi sankyo, Janssen, Eli Lilly, Taiho, Novartis, Kyowa kirin and Boehringer Ingelheim. T.K. reports research support from Pfizer, Chugai, Takeda, Novartis, Turning Point Therapeutics and AstraZeneca, and personal fees (honoraria) from Pfizer, Chugai, Takeda, Novartis and AstraZeneca. K. Nishino reports research support from Pfizer and personal fees (honoraria) from Pfizer, Chugai, Takeda, Novartis and AstraZeneca. A.N. reports personal fees (honoraria) from Chgai and Novartis. S. Kuyama reports personal fees (honoraria) from Pfizer, Chugai and AstraZeneca. N.F. reports personal fees (honoraria) from Pfizer, Chugai, Novartis and AstraZeneca. I.O. reports research support from Chugai, Takeda and Novartis, and personal fees (honoraria) from Pfizer, Chugai, Novartis and AstraZeneca. K. Taima. reports personal fees (honoraria) from Chugai, Novartis and AstraZeneca. H.D. reports research support from Pfizer, Chugai and Takeda, and personal fees from Chugai. A.Y. reports personal fees (honoraria) from Takeda. K.K. reports personal fees (honoraria) from Pfizer, Chugai, Novartis and AstraZeneca. Y.Z. reports research support from AstraZeneca and personal fees (honoraria) from Pfizer, Chugai, Takeda and AstraZeneca. K. Nosaki reports research support from Chugai and Takeda, and personal fees (honoraria) from Pfizer, Chugai, Takeda, Novartis and AstraZeneca. T.S. reports personal fees (honoraria) from Chugai and AstraZeneca. G.I. reports research support from Takeda and personal fees (honoraria) from Pfizer, Chugai, Takeda, Novartis and AstraZeneca. S.N. reports research support from Pfizer, Chugai, Takeda and AstraZeneca, and personal fees (honoraria) from Pfizer, Chugai, Takeda, Novartis and AstraZeneca. A.O. reports personal fees (honoraria) from Chugai. S.S.K. reports research support from Boehringer Ingelheim, MiNA Therapeutics and Taiho Therapeutics, as well as personal fees (honoraria) from Boehringer Ingelheim, Bristol Meyers Squibb, AstraZeneca, Chugai Pharmaceutical and Takeda Pharmaceuticals, all outside of the submitted work. K.G. reports research support from Boehringer Ingelheim, Bristol-Myers Squibb, Chugai, Daiichi sankyo, Eisai, Eli Lilly, Guardant Health, Janssen, Kyowa Kirin, Life Technologies Japan, MSD, Novartis, Ono, Otsuka, Pfizer, Taiho and Takeda, and personal fees (honoraria) from Bristol-Myers Squibb, Chugai, Daiichi sankyo, Eisai, Eli Lilly, Haihe Biopharma, Ignyta, Janssen, KISSEI, Kyowa Kirin, LOXO Oncology, Medical & Biological Laboratories, Merck Biopharma, Merus, MSD, Ono, Pfizer, Sumitomo Dainippon Pharma, Shanghai Haihe, Sysmex Corporation, Taiho, Takeda, and Xcoo. The authors report no other competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Electropherogram showing Sanger sequencing of the CLIP1-LTK fusion transcript.
cDNAs were generated from RNAs isolated from patient tumours and amplified by RT-PCR with CLIP1-LTK F3 and R3 primers. PCR products were directly sequenced using respective primers.
Extended Data Fig. 2 LTK break-apart FISH assay.
Non-tumour cells from Patient #1 showed orange (5′)-green (3′) fused signals (yellow arrows), while 82% (41/50) of scored tumour cells showed one fused (yellow arrows) and at least one green (3′) signals (white arrows), which indicates the presence of LTK rearrangement. Bars = 10 µm.
Extended Data Fig. 3 Histological findings of tumours positive for the CLIP1-LTK fusion.
a, Hematoxylin and eosin (H&E) stained images. Samples from Patient #1 and #3 were diagnosed with adenocarcinoma morphologically. Samples from Patient #2 was diagnosed with NSCLC. Bars = 100 µm. b, Immunohistochemical analysis of samples from Patient #2. TTF-1 positive (upper panel) and p40 negative (lower panel) staining supported the diagnosis of NSCLC favor adenocarcinoma. Bars = 100 µm.
Extended Data Fig. 4 Analysis of Ba/F3 (a) and NIH3T3 (b) cells stably transduced using MIGR1 IRES-GFP vectors harboring indicated constructs.
GFP-positive cells were sorted and expanded and then cell extracts were immunoblotted with antibodies indicated at the left side of each graph.
Extended Data Fig. 5 Cellular localization of LTK and CLIP1-LTK.
a, NIH3T3-Mock NIH3T3-LTK, or NIH3T3-CLIP1-LTK cells were stained with primary antibody specific for LTK, conjugated with Alexa Fluor 488, and subjected to flow cytometry analysis. Viable cells were gated as shown in Supplementary Information Figure 2. b, NIH3T3-Mock NIH3T3-LTK, or NIH3T3-CLIP1-LTK cells were fixed, permeabilized, and stained with anti-DDDDK-tag antibody conjugated to Alexa Fluor 594. Cells were subjected to immunofluorescence analysis.
Extended Data Fig. 6 Light microscopy images of indicated stably-transduced NIH3T3 cells.
a, Cells were plated in 10 cm plates at 2 × 105 cells/ml and cultured in DMEM supplemented with 10% FBS and P/S for 2–3 days until cells reached 100% confluency. Bars = 100 µm. b, Cells were plated in 6-well plates at 1 × 104 cells/ml and cultured in a soft agar medium for 14 days. Bars = 100 µm.
Extended Data Fig. 7 Analysis of viability of Ba/F3-CLIP1-LTK cells.
Ba/F3-CLIP1-LTK cells were treated with various concentrations of lorlatinib in the presence of 5% WEHI medium as a source of IL-3. Results are shown as an average ± standard deviation from three independent experiments.
Extended Data Fig. 8 Lorlatinib suppresses anchorage-independent growth of NIH3T3-CLIP1-LTK cell colonies.
Colony diameters were measured in lorlatinib- versus vehicle (DMSO)-treated cells and shown as average ± standard deviation from three independent experiments.
Supplementary information
Supplementary Information
This file contains Supplementary Figures 1 and 2 and their accompanying legends.
Supplementary Information Table 1
: Oligonucleotides used in this study.
Supplementary Information Table 2
: Antibodies used in this study
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Izumi, H., Matsumoto, S., Liu, J. et al. The CLIP1–LTK fusion is an oncogenic driver in non‐small‐cell lung cancer. Nature 600, 319–323 (2021). https://doi.org/10.1038/s41586-021-04135-5
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DOI: https://doi.org/10.1038/s41586-021-04135-5
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