GCC2-ALK as a targetable fusion in lung adenocarcinoma and its enduring clinical responses to ALK inhibitors

Lung Cancer 115 (2018) 5–11

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Lung Cancer journal homepage: www.elsevier.com/locate/lungcan

Research paper

GCC2-ALK as a targetable fusion in lung adenocarcinoma and its enduring clinical responses to ALK inhibitors

MARK

Junhong Jianga,1, Xue Wub,1, Xiaoling Tongb, Wangzhi Weib, Anan Chenb,2, Xiaonan Wangc, ⁎ ⁎⁎ Yang W. Shaob,d, , Jianan Huanga, a

Department of Respiratory Medicine, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, 215006, China Translational Medicine Research Institute, Geneseeq Technology Inc., Toronto, Ontario, M5G 1L7, Canada c Nanjing Geneseeq Technology Inc., Nanjing, Jiangsu, 210032, China d School of Public Health, Nanjing Medical University, Nanjing, Jiangsu, 211166, China b

A R T I C L E I N F O

A B S T R A C T

Keywords: GCC2-ALK fusion Next generation sequencing Targeted therapy ALK inhibitor Non-small cell lung cancer

Objectives: ALK, RET and ROS1 fusions have been identified as treatable targets in 5%–15% of non-small-cell lung cancers, and thanks to the advanced sequencing technologies, their new partner genes have been steadily detected. Here we identified a rare fusion of ALK (GCC2-ALK) in a patient with advanced lung adenocarcinoma and monitored the treatment efficacy of ALK inhibitors on this patient. We further performed in vitro functional studies of this fusion protein for evaluating its oncogenic potential. Materials and methods: The GCC2-ALK fusion gene was identified by targeted next generation sequencing (NGS) from the tumor DNA samples, and its fusion product was confirmed by Sanger sequencing the cDNA product. The functional study of GCC2-ALK was performed in Ba/F3 cells with cell proliferation and viability assays. The activation of downstream signaling pathways of ALK and their responses to crizotinib inhibition were studied in HEK-293 and 293T cells with ectopic expression of GCC2-ALK. In parallel, disease progression in the patient was monitored by computed tomography scanning and targeted NGS of either liquid or tissue biopsy samples throughout and after crizotinib treatment. Results: Similarly to EML4-ALK, the GCC2-ALK fusion protein promotes IL-3-independent growth of Ba/F3 cells. Ectopic expression of GCC2-ALK leads to hyper-activation of ALK downstream signaling that can be inhibited by crizotinib. Crizotinib treatment of the patient resulted in 18 months of progression free survival without any trace of GCC2-ALK fusion in the liquid biopsies. Re-biopsy of a lung lesion at progression identified the reoccurrence of GCC2-ALK. The patient was then administrated with a second-generation ALK inhibitor, ceritinib, and received partial response until the last follow-up. Conclusion: We identified and functionally validated GCC2-ALK as a constitutively activated fusion in NSCLC. The patient was benefited from crizotinib treatment initially and then ceritinib after progression, suggesting GCC2-ALK as a novel therapeutic target for ALK inhibitors.

1. Introduction Lung cancer is the leading cause of cancer-related deaths worldwide [1], with 85–90% of diagnosed cases being non-small cell lung cancers (NSCLCs) [2]. Gene rearrangements of anaplastic lymphoma kinase (ALK) is identified in approximately 3–7% of NSCLCs, with the most common being the echinoderm microtubule-associated protein like-4 (EML4)-ALK fusion [3–6], and their occurrences appear to be mutually

exclusive with EGFR and KRAS mutations [7]. Structural characterization indicates that the amino terminal coiled-coil domain of EML4 causes ligand-independent oligomerization and consequently constitutive activation of the kinase domain of ALK [8]. ALK has also been found to partner with TFG, KIF5B, TPR, HIP1, DCTN1 and KLC1 genes in NSCLCs, although at a much lower frequency [9–14]. ALK fusion-positive NSCLC is clinically actionable because it can be targeted by several FDA-approved drugs, including the first generation

⁎ Corresponding author at: Translational Medicine Research Institute, Geneseeq Technology Inc., Suite 300, MaRS Centre, South Tower, 101 College Street, Toronto, ON, M5G 1L7, Canada. ⁎⁎ Corresponding author at: Department of Respiratory Medicine, The First Affiliated Hospital of Soochow University, 188 Shizi St., Cang Lang Qu, Suzhou, Jiangsu, 215006, China. E-mail addresses: [email protected] (Y.W. Shao), [email protected] (J. Huang). 1 These authors contributed equally to this work. 2 Present address: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.

https://doi.org/10.1016/j.lungcan.2017.10.011 Received 10 August 2017; Received in revised form 15 October 2017; Accepted 26 October 2017 0169-5002/ © 2017 Elsevier B.V. All rights reserved.

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TKI crizotinib, which is a dual inhibitor to MET and ALK [15], and the second generation inhibitors, alectinib and ceritinib, both of which are highly-selective ALK inhibitors. The latter two were not only approved as the first line treatment for ALK-positive NSCLC [16,17], but also in treating crizotinib-resistant or brain metastatic NSCLC [18,19]. Nowadays high-throughput sequencing technology increases the chance of identifying new fusion genes that have activities similar to classical ALK fusions [20,21], potentially matching more patients to existing drugs. Here, we identified a rare ALK fusion partner, GCC2 (GRIP and coiledcoil domain-containing protein 2), in a patient with NSCLC by targeted next generation sequencing (NGS). Our in vitro functional study and the clinical observations of the patient proved that GCC2-ALK has similar kinase activities as EML4-ALK, and that the patient with this variant can be treated with existing ALK inhibitors.

Cedar Creek, TX). Once ready, libraries were sequenced on Illumina HiSeq4000 platforms (Illumina, San Diego, CA) and representative parameters of sequencing quality for each sample were summarized in Supplementary Table S2. 2.4. Sequencing data processing Trimmomatic [22] software was used for FASTQ file quality control and adapter removal. Sequencing reads were mapped to Human Genome version 19 (hg19) using the Burrows-Wheeler Aligner (BWAmem, v0.7.12) [23]. Local realignment around indels and base quality score recalibration were applied with the Genome Analysis Toolkit (GATK 3.4.0) [24]. Single nucleotide polymorphisms (SNPs)/indels were identified using VarScan2 (http://dkoboldt.github.io/varscan/) for mutations with mutant allele frequency (MAF) < 10% and HaplotypeCaller/UnifiedGenotyper in GATK for mutations with MAF > 10%, and filtered with dbSNP and 1000 Genome data sets for common SNPs. Germline mutations were identified by comparing the tumor sample to its matched whole blood DNA sample. Copy number variations (CNVs) were detected using ADTEx (http://adtex.sourceforge.net) with default parameters. Genomic fusions were identified by FACTERA [25] with default parameters. The fusion reads were further manually reviewed and confirmed on Integrative Genomics Viewer (IGV) [26].

2. Materials and methods 2.1. Clinical sample collection The patient has given written consent for specimen collection and genetic testing. The study has been approved by the Ethics Committee of the First Affiliated Hospital of Soochow University. Formalin-fixed and paraffin-embedded (FFPE) sections that were prepared from the tissue biopsy of pleura pulmonalis at the time of diagnosis were used for genomic DNA extraction. During crizotinib treatment, 10 ml of peripheral blood was collected every 2–3 months using EDTA-coated blood collection tubes (BD Biosciences, Mississauga, ON), and plasma was prepared within 48 h of blood collection. When progressed on crizotinib, a re-biopsy of the lung lesion was obtained for cancer genomic testing.

2.5. Reverse transcriptase-polymerase chain reaction (RT-PCR) and sanger sequencing Total RNA from the patient's FFPE tissue sample and the whole blood sample was extracted using RNeasy FFPE kit and RNeasy mini kit, respectively (QIAGEN). Reverse transcription was performed with Superscript Vilo mastermix (Life Technologies). The GCC2-ALK fusion fragment was amplified with the primer pair (Forward: 5′-AGCTTCAG AAAACCATGCAAGAA-3′; Reverse: 5′-GCTCAGCTTGTACTCAGGGC-3′), and gel-purified for Sanger sequencing to confirm the fusion sequence in cDNA.

2.2. DNA extraction FFPE sections were de-paraffinized with xylene and DNA was extracted using QIAamp DNA FFPE Tissue Kit (QIAGEN, Louisville, KY). Genomic DNA of the whole blood sample was extracted by DNeasy Blood & Tissue kit (QIAGEN) as a germline control. Circulating tumor DNA (ctDNA) was extracted from plasma sample with QIAamp Circulating Nucleic Acid kit (QIAGEN). DNA samples were quantified by Qubit 3.0 using dsDNA HS Assay Kit (Life Technologies, Grand Island, NY).

2.6. Plasmid construction To generate human EML4-ALK variant 1 (E13;A20) fusion cDNA as the positive control of the experiment, the N-terminal and C-terminal DNA fragments with overlapping sequence were synthesized using gBlocks technology (Integrated DNA Technology). EML4-ALK fusion cDNA (E13;A20) was then generated using Gibson assembly method (NEB, Ipswich, MA), which was further amplified and subcloned into a pcDNA3 expression vector by HindIII and NotI sites. To generate human GCC2-ALK (G13;A20) fusion cDNA, total RNA from HEK-293 cell line was reverse-transcribed into cDNA library and used as template to amplify the 5′ GCC2 cDNA sequence. The GCC2ALK fusion fragment (c.3322-3613 of the GCC2 cDNA followed by c.3173-3289 of the ALK cDNA) was synthesized using gBlocks technology and then ligated to the 3′ ALK gBlocks above by the Gibson assembly method. The resulting 3′ fusion cDNA was then ligated to the 5′ GCC2 cDNA by stitching PCR [27]. Finally, the resulting full length GCC2-ALK fusion cDNA was subcloned into the pcDNA3 expression vector by HindIII and NotI sites. To subclone the cDNA of EML4-ALK and GCC2-ALK fusions into pBabe-puro retroviral vector, their original 3′ NotI restriction site was modified to EcoRI by PCR amplification, and then inserted into the vector by HindIII and EcoRI sites.

2.3. Library preparation and sequencing Sequencing libraries were prepared with KAPA Hyper Prep kit (KAPA Biosystems, Wilmington, MA). In brief, 1 μg of genomic DNA from the whole blood or the FFPE samples was fragmented by Covaris M220 instrument (Covaris, Woburn, MA), and subsequently subjected to end repair, A-Tailing and adapter ligation. Unligated adapters were removed by Agencourt AMPure XP beads (Beckman Coulter, Beverly, MA). The resulting libraries were PCR-amplified and purified with Agencourt AMPure XP beads. Sequencing libraries of the ctDNA were prepared from 10 to 50 ng DNA with the same above procedures except the DNA shearing because of their already fragmented properties. Indexed libraries were pooled together for target enrichment with customized IDT xGen lockdown probes (Integrated DNA Technologies, Coralville, IA) using NimbleGen SeqCap EZ Hybridization & Wash Kit (Roche, Madison, WI) and Dynabeads M-270 (Life Technologies). The customized target probes were designed and optimized to capture all the fusion-involved introns of 16 genes (ALK, BCL2, BCR, BIRC3, BRAF, ETV1, ETV5, EWSR1, KMT2A, MYC, PDGFB, RAF1, RARA, RET, ROS1, TMPRSS2), as well as the exons of 384 cancer-relevant genes. Captured libraries were PCR-amplified using Illumina p5 and p7 primers and purified with Agencourt AMPure XP beads. Library was quantified using KAPA Library Quantification kit (KAPA Biosystems), and its fragment size was analyzed by Bioanalyzer 2100 (Agilent Technologies,

2.7. Cell culture HEK-293, 293T and Ba/F3 cell lines were obtained from American Type Culture Collection (ATCC) and cultured in a 5% CO2 atmosphere at 37 °C. HEK-293 and 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies) supplemented with 10% 6

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Fig. 1. Identification and validation of the GCC2-ALK fusion in a lung cancer patient. (A) Sequencing reads of ALK and GCC2 were visualized by the Integrative Genomics Viewer (IGV). The sequencing reads in the ALK intron19 region aligned to a different gene locus are color-coded to identify the other locus, which are mated to GCC2 region. (B) A schematic map showing the structure of the GCC2-ALK fusion locus. Exons 1–13 of GCC2 (blue) were fused to exons 20–29 of ALK (green) through intron13 of GCC2 and intron19 of ALK. (C) RT-PCR of total tumor RNA with primers spanning the GCC2-ALK cDNA fusion junction amplified a product of 275 bp. This product was not detected in the negative control RNA from the patient’s whole blood. PCR amplification of the housekeeping gene β-actin was set as a positive control for reverse transcription. (D) Sanger sequencing chromatogram of the RT-PCR product in (C) identified the fusion point (dotted line) between GCC2 and ALK.

transfected into HEK-293 cells or 293T cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA). 24 h after transfection, the cells were serumstarved for 24 h, and then treated with 1 μM crizotinib (Cell Signaling Technology) in serum-free culture medium for indicated time points. To generate retroviruses, 1 × 106 293T packaging cells were cotransfected with 3 μg pBabe-puro retroviral and 3 μg EcoPac packaging plasmids using FuGENE HD Transfection Reagent (Promega, Madison,

fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (Wisent Inc., Quebec, CA). Ba/F3 cells were cultured in RPMI1640 (Life Technologies) supplemented with 10% FBS, 1% Penicillin/ Streptomycin, and 10 ng/ml mouse interleukin-3 (IL-3) (Cell Signaling Technology, Danvers, MA). For transient transfection, pcDNA3-EML4-ALK, pcDNA3-GCC2-ALK and the empty pcDNA3 control plasmids were independently 7

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Fig. 2. Transforming activity of GCC2-ALK and its response to crizotinib treatment. (A) Ba/F3 cells retrovirally expressing EML4-ALK, GCC2-ALK or pBabe-puro empty vector were cultured in the presence (+IL3) or absence of IL-3 (-IL3). Representative images after 4 days of incubation are shown. (B) Ba/F3 cells of different genotypes were grown in IL-3-deprived medium and the live cell numbers were counted every day. Data is presented as mean ± SD in three independent experiments, each performed in triplicate. (C) Ba/F3 cells expressing EML4-ALK or GCC2-ALK were cultured in the absence or presence of crizotinib at indicated concentrations. Cell viability was determined by the AlamarBlue cell viability assay (570 nm/ 600 nm). Experiments were performed in triplicate. Two-way ANOVA was used to statistically analyze growth rate (B) and cell viability (C) between groups. *, P < 0.05; ****, P < 0.0001; ns, not significant.

2.9. Cell proliferation and cell viability assays

WI). After overnight incubation, transfection media was replaced with fresh growth medium. Virus-containing supernatants were collected at 48 h post-transfection, and passed through a 0.45 μm filter to remove cell debris. To establish stable cell lines, Ba/F3 cells were transduced in 6 cm2 cell culture plates with 2 ml virus-containing medium in the presence of 8 μg/ml polybrene for 24 h, followed by selection with 2 μg/ml puromycin for 48–72 h. After puromycin selection, transduced cells were re-plated for further analysis.

For cell proliferation assay, Ba/F3 cell lines stably expressing EML4ALK, GCC2-ALK or empty pBabepuro control vector were seeded at 5 × 104 cells per well in a 6-well plate in the presence or absence of IL3. Cells were harvested at indicated time points and stained with trypan blue solution to count live cells with a hemocytometer under the microscope. For cell viability assay, Ba/F3 cells stably expressing EML4-ALK or GCC2-ALK were seeded at a density of 1.5 × 104 cells per well in a 96well plate in the absence or presence of crizotinib at various doses in IL3 free medium for 96 h. After treatment, cells were incubated with Alamar blue reagent (Life Technologies) for 4 h, and the cell viability was determined by absorbance signal at 570 nm/600 nm wavelengths on a Synergy H1 plate reader (BioTek, Winooski, VT).

2.8. Western blotting assay Cells were lysed in RIPA buffer (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 10% glycerol, 1% NP-40, 0.5% Na Deoxyclolate, 0.1% SDS and 2 mM EDTA) containing 1% Halt protease and phosphatase inhibitor cocktail (ThermoFisher, Rockford, IL). Homogenates were centrifuged at 16,000g for 15 min at 4 °C. Protein concentration was quantified using Coomassie Plus (Bradford) assay kit (ThermoFisher). 25 μg of total protein were separated with SDS-PAGE, transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA), and analyzed by Western Blot with primary antibodies listed in Supplementary Table S1. In brief, the membrane was incubated with the primary antibody overnight at 4 °C and subsequently with antimouse or anti-rabbit antibody (LI-COR Biosciences, Lincoln, NE) at a dilution of 1:2000 for 1 h at room temperature. Protein level was visualized using Licor WesternSure™ ECL Substrate on a C-DiGit blot scanner (LI-COR Biosciences).

2.10. Statistical analysis All results are based on three independent experiments, each was performed in triplicates, and the data was presented as mean ± SD. Significance of differences was assessed by two-way ANOVA using GraphPad Prism 6. Significance was assigned at p < 0.05.

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of the ALK kinase domain in the fusion protein. 3.2. GCC2-ALK fusion induces IL-3-independent cell growth and can be suppressed by crizotinib To assess the effect of the GCC2-ALK fusion protein on cell proliferation and compare it to the previously established functions of the EML4-ALK fusion, we generated stable Ba/F3 cell lines expressing either GCC2-ALK or EML4-ALK variant 1 (the most common subtype of EML4-ALK fusions) (Supplementary Fig. 2). Same as EML-ALK cells, GCC2-ALK cells grew in an IL-3-independent manner (Fig. 2A-B), and there was no significant difference between the proliferation rates of these two cell lines in the presence or absence of IL-3 (Fig. 2B and Supplementary Fig. 2B). Moreover, the viability of both EML4-ALK- and GCC2-ALK-expressing cells was highly susceptible to crizotinib treatment, with the IC50 for the GCC2-ALK cells (7.75 nM) being much lower than that of the EML4-ALK cells (68.81 nM) (p < 0.0001) (Fig. 2C), indicating a higher sensitivity of GCC2-ALK expressing cells to crizotinib in vitro. We next analyzed the downstream signaling pathways that could be influenced by GCC2-ALK by transiently expressing the GCC2-ALK fusion protein in HEK-293 cells. EML4-ALK expressing vector and the empty control vector were also transfected individually as controls. Activating phosphorylation of the ALK kinase domain on Tyr1604 was detected in both serum-starved GCC2-ALK and EML4-ALK expressing cells, suggesting the constitutively activation of the two ALK fusion proteins (Fig. 3A). It has been reported that ALK mediates downstream signals mainly via the RAS-MAPK, PI3K-AKT and JAK-STAT pathways [31]. In HEK293 cells overexpressed EML4-ALK or GCC2-ALK, we observed increased phosphorylation of MEK and ERK in the RAS-MAPK pathway, as well as elevated STAT3 phosphorylation in the JAK-STAT pathway, but not activated PI3K-AKT pathway as shown with unchanged phospho-AKT level (Fig. 3A–C). However, when we performed the experiments in 293T cells, elevated AKT activation was also observed with either EML4-ALK or GCC2-ALK overexpression (Supplementary Fig. 3), suggesting that PI3K-AKT pathway can also been activated by GCC2-ALK in a cell-context dependent manner. Meanwhile, crizotinib treatment suppressed ALK phosphorylation and the activation of its downstream effector molecules, MEK, ERK, STAT3 and AKT in the GCC2-ALK- and EML4-ALK-expressing cells (Fig. 3 and Supplementary Fig. 3).

Fig. 3. Activation of downstream signaling pathways by the GCC2-ALK fusion. HEK-293 cells expressing GCC2-ALK, EML4-ALK or empty vector were cultured in serum-free medium overnight, followed by treatment with DMSO (vehicle control) or 1 μM crizotinib for 2 and 5 h. Cell lysates were subjected to SDS-PAGE and immunoblotting analysis with specific antibodies as indicated (Supplementary Table S3). Results shown are representative of two independent experiments.

3. Results 3.1. Detection of a rare ALK fusion, GCC2-ALK in a NSCLC patient The subject in this study is a 28-year-old Chinese non-smoking female. Her first chest computed tomography (CT) scans revealed a large opacity in the upper lobe of the left lung with pleural effusion around the mass lesion. A subsequent histopathologic analysis of a pleural biopsy indicated poorly differentiated lung adenocarcinoma. To optimize her treatment, the FFPE tissue block generated from a pleural biopsy was submitted for genomic testing by targeted NGS using a pan-cancer gene panel. Mutation profiling revealed the presence of a rearrangement within the chromosome 2 with a mutant allele frequency (MAF) of 5% (Fig. 1A), generated by an inversion of the intron 13 of GCC2 on 2q12.3 to the intron 19 of ALK on 2p23. Based on the DNA sequencing data, the fusion transcript was proposed to contain the N-terminal exons 1–13 of GCC2 gene and the C-terminal exons 20–29 of ALK gene, the latter of which encodes the entire ALK intracellular kinase domain (Fig. 1B). This fusion product was further confirmed at mRNA level by RT-PCR and Sanger sequencing of the tumor FFPE RNA sample (Fig. 1C and D). Coiled-coil motifs in fusion partners often mediate ligand-independent oligomerization of the receptor tyrosine kinases, leading to the constitutive activation of their intracellular kinase domains in various malignancies [28–30]. Similar to other ALK fusions, the Coils server (http://embnet.vital-it.ch/software/COILS_form.html) estimates the N-terminus of GCC2 to be highly abundant with α-helical coiled-coil motifs (Supplementary Fig. 1), suggesting the possibility of GCC2 Nterminus in mediating the constitutively ligand-independent activation

3.3. GCC2-ALK represents a potential therapeutic target As soon as identifying GCC2-ALK fusion in the tumor and observing its structural similarity to other defined ALK fusions, the patient was orally administered crizotinib twice per day at a dose of 250 mg. After one month of treatment, her lung neoplastic mass and the pleural effusion were dramatically reduced (Fig. 4A). As treatment continued, her lung lesion became invisible in the CT scanning (Fig. 4A). The patient was released from the hospital, and follow-ups were performed with CT scans and ctDNA monitoring by targeted NGS every 2–3 months to monitor the disease status. Her response to crizotinib was maintained for 18 months with no signs of tumor recurrence from the CT images and no trace of GCC2-ALK fusion in the liquid biopsies (Fig. 4). A new lung lesion was detected by CT scanning after 18 months of treatment, and targeted NGS of the tissue biopsy sample from the lesion identified the recurrent of the GCC2-ALK fusion at a MAF of 20% as well as an ALK E1407K mutation with unknown significance at 1% MAF (Fig. 4B and Supplementary Table S1). Soon after, multiple metastatic lesions were observed in the brain by magnetic resonance imaging (MRI), further confirming the development of crizotinib resistance (Supplementary Fig. 4A). The patient was then subjected to cycles of chemo- and radiotherapy to treat her brain and lung lesions. After her brain lesions improved and her lung lesions were stable, she was switched to ceritinib treatment, a more robust second-generation 9

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Fig. 4. CT scans and dynamic monitoring of the tumor mutational status during disease progression. A) Chest CT scans at the time of diagnosis show the presence of a large opacity in the upper lobe of the left lung with malignant pleural effusion. Significant and consistent reduction of the tumor volume and pleural effusion was observed by the follow-up CT scans at 1 and 6 months post-crizotinib treatment. After 18 months of the therapy, a new lesion (orange arrow) was found in the left lung. The upper panel is the lung window and the lower panel is the bone window. B) Target NGS of the tumor tissues or the ctDNA samples from plasma to monitor the tumor burden and the genomic changes of the tumor during the treatment course. A FFPE sample (0 month) was obtained prior to crizotinib treatment, and multiple liquid biopsies were performed at 2.5, 5, 8 and 11 months post crizotinib treatment. After tumor relapse (18 months), a tissue biopsy was collected for DNA sequencing.

ALK mutations L1196M and G1269A, ALK amplification, cKIT amplification, EGFR or KRAS activation [40–43]. In this patient, we have not identified any newly acquired mutations after drug resistance except ALK E1407K at a MAF of 1%. However, it is unlikely that this mutation will influence the ALK activity or crizotinib binding since the mutation site is located outside of the kinase domain. We also observed a dramatic increase of GCC2-ALK MAF from 5% in the primary tumor to 20% in the recurrent lesion, and the new lesion responded well to the second generation of ALK inhibitor ceritinib, suggesting that the relapsed tumor is still ALK-driven. However, the mechanism for crizotinib-resistance in this patient is still unknown. In summary, our study identified a targetable ALK fusion, GCC2ALK, in an advance NSCLC patient. In vitro functional studies and clinical observations of the patient consistently show that GCC2-ALK is constitutively activated and can be inhibited by crizotinib, suggesting the benefit of including it into clinical practice for diagnosis and targeted treatment.

ALK inhibitor, and received an immediate size reduction of her lung lesion (Supplementary Fig. 3B), suggesting that GCC2-ALK was still the driving force in her lung tumor progression. 4. Discussion The ALK receptor tyrosine kinase undergoes genetic rearrangements in a variety of human cancers, including anaplastic large-cell lymphoma [32], diffuse large B-cell lymphoma [33], colon, breast and lung cancers [34]. ALK translocations drive tumorigenesis mainly by enabling constitutive, ligand-independent activation of the ALK kinase. The fusion of EML4-ALK is the most common ALK fusion in NSCLCs [35]. Given the well-established transforming activities of EML4-ALK and its sensitivity to crizotinib [36], attempts to identify more partner genes to ALK that have the same effects in tumorigenesis and clinical responses to ALK inhibitors have recently been made [37,38]. In this study, we identified a rare GCC2-ALK fusion (G13:A20) in NSCLC by targeted NGS. A similar GCC2-ALK fusion (G12:A20) has been reported recently in one of the 158 ALK-positive patients in a study of 3000 lung cancer patients from Korea [6] without any functional and clinical follow-ups. Herein, we found that introduction of GCC2-ALK (G13:A20) into Ba/F3 cells enabled their IL-3-independent cell growth that can be suppressed by crizotinib. Molecular pathway studies in both HEK-293 and 293T cells uncovered the enhanced ALK kinase activity of GCC2-ALK and the excess downstream RAS-MAPK, JAK-STAT and PI3K-AKT signals. In the absence of direct ligand binding, GCC2 is proposed to facilitate the oligomerization of the ALK kinase domain through its tandem coiled-coil domains similar to other fusion partners [39], and therefore leads to the constitutive activation of the ALK kinase and its downstream signaling pathways. As reported, resistant mechanisms to crizotinib include secondary

Conflict of interest Yang W. Shao, Xue Wu and Xiaoling Tong are the shareholders or employees of Geneseeq Technology Inc., Canada; Wangzhi Wei and Anan Chen are the former employees of Geneseeq Technology Inc., Canada; Xiaonan Wang is the employee of Nanjing Geneseeq Technology Inc. Financial Support The work is supported by research grants from Clinical Key Specialty Project of China and Clinical Medical Center of Suzhou, China (Szzx201502) and Jiangsu Province Special Program of Medical 10

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Science, China (BE2016672).

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