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.
This is a preview of subscription content
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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.
Konig, D., Savic Prince, S. & Rothschild, S. I. Targeted therapy in advanced and metastatic non-small cell lung cancer. An update on treatment of the most important actionable oncogenic driver alterations. Cancers 13, 713 (2021).
Saito, M. et al. Gene aberrations for precision medicine against lung adenocarcinoma. Cancer Sci. 107, 713–720 (2016).
The Cancer Genome Atlas Research Network. Comprehensive molecular profiling of lung adenocarcinoma. Nature 511, 543–550 (2014).
Fernandez-Cuesta, L. et al. CD74–NRG1 fusions in lung adenocarcinoma. Cancer Discov. 4, 415–422 (2014).
Non-small cell lung cancer version 4.2021 National Comprehensive Cancer Network https://www.nccn.org/professionals/physician_gls/pdf/nscl.pdf (2021).
Roll, J. D. & Reuther, G. W. ALK-activating homologous mutations in LTK induce cellular transformation. PLoS ONE 7, e31733 (2012).
Scheel, J. et al. Purification and analysis of authentic CLIP-170 and recombinant fragments. J. Biol. Chem. 274, 25883–25891 (1999).
Grigoryan, G. & Keating, A. E. Structural specificity in coiled-coil interactions. Curr. Opin. Struct. Biol. 18, 477–483 (2008).
Varmus, H. E. The molecular genetics of cellular oncogenes. Annu. Rev. Genet. 18, 553–612 (1984).
Warmuth, M., Kim, S., Gu, X. J., Xia, G. & Adrián, F. Ba/F3 cells and their use in kinase drug discovery. Curr. Opin. Oncol. 19, 55–60 (2007).
Soda, M. et al. Identification of the transforming EML4–ALK fusion gene in non-small-cell lung cancer. Nature 448, 561–566 (2007).
Greulich, H. et al. Oncogenic transformation by inhibitor-sensitive and -resistant EGFR mutants. PLoS Med. 2, e313 (2005).
Kobayashi, S. et al. An alternative inhibitor overcomes resistance caused by a mutation of the epidermal growth factor receptor. Cancer Res. 65, 7096–7101 (2005).
Yasuda, H. et al. Structural, biochemical, and clinical characterization of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in lung cancer. Sci. Transl. Med. 5, 216ra177 (2013).
Zou, H. Y. et al. PF-06463922, an ALK/ROS1 inhibitor, overcomes resistance to first and second generation ALK inhibitors in preclinical models. Cancer Cell 28, 70–81 (2015).
Yamada, S. et al. Expression of a chimeric CSF1R–LTK mediates ligand-dependent neurite outgrowth. Neuroreport 19, 1733–1738 (2008).
Bruno, R. & Fontanini, G. Next generation sequencing for gene fusion analysis in lung cancer: a literature review. Diagnostics 10, 521 (2020).
Kohno, T. et al. Beyond ALK–RET, ROS1 and other oncogene fusions in lung cancer. Transl. Lung Cancer Res. 4, 156–164 (2015).
The Cancer Genome Atlas Research Network. Integrated genomic characterization of papillary thyroid carcinoma. Cell 159, 676–690 (2014).
Yoshihara, K. et al. The landscape and therapeutic relevance of cancer-associated transcript fusions. Oncogene 34, 4845–4854 (2015).
Muller-Tidow, C. et al. High-throughput analysis of genome-wide receptor tyrosine kinase expression in human cancers identifies potential novel drug targets. Clin. Cancer Res. 10, 1241–1249 (2004).
Carvalho, P., Gupta, M. L., Jr., Hoyt, M. A. & Pellman, D. Cell cycle control of kinesin-mediated transport of Bik1 (CLIP-170) regulates microtubule stability and dynein activation. Dev. Cell 6, 815–829 (2004).
Drilon, A. et al. Cabozantinib in patients with advanced RET-rearranged non-small-cell lung cancer: an open-label, single-centre, phase 2, single-arm trial. Lancet Oncol. 17, 1653–1660 (2016).
Kuroda, N. et al. ALK rearranged renal cell carcinoma (ALK-RCC): a multi-institutional study of twelve cases with identification of novel partner genes CLIP1, KIF5B and KIAA1217. Mod. Pathol. 33, 2564–2579 (2020).
Pinsolle, J. et al. A rare fusion of CLIP1 and ALK in a case of non-small-cell lung cancer with neuroendocrine features. Clin. Lung Cancer 20, e535–e540 (2019).
Yeh, I. et al. Clinical, histopathologic, and genomic features of Spitz tumors with ALK fusions. Am. J. Surg. Pathol. 39, 581–591 (2015).
Gainor, J. F. et al. Molecular mechanisms of resistance to first- and second-generation ALK inhibitors in ALK-rearranged lung cancer. Cancer Discov. 6, 1118–1133 (2016).
Haas, B. J. et al. Accuracy assessment of fusion transcript detection via read-mapping and de novo fusion transcript assembly-based methods. Genome Biol. 20, 213 (2019).
Pear, W. S. et al. Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow. Blood 92, 3780–3792 (1998).
Borowicz, S. et al. The soft agar colony formation assay. J. Vis. Exp. (92), e51998 (2014).
Tomayko, M. M. & Reynolds, C. P. Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother. Pharmacol. 24, 148–154 (1989).
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.
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.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
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.
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.
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.
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.
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.
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.
About this article
Cite this article
Izumi, H., Matsumoto, S., Liu, J. et al. The CLIP1–LTK fusion is an oncogenic driver in non‐small‐cell lung cancer. Nature (2021). https://doi.org/10.1038/s41586-021-04135-5