Structural Alterations of MET Trigger Response to MET Kinase Inhibition in Lung Adenocarcinoma Patients

Author Manuscript Published OnlineFirst on December 28, 2017; DOI: 10.1158/1078-0432.CCR-17-3001 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Structural alterations of MET trigger response to MET kinase inhibition in lung adenocarcinoma patients

Dennis Plenker1,2*, Miriam Bertrand3*, Adrianus J. de Langen4*, Richard Riedel5*, Carina Lorenz1,2*, Andreas H. Scheel6, Judith Müller3, Johannes Brägelmann1,2, Juliane Daßler- Plenker7, Carsten Kobe8, Thorsten Persigehl9, Alexander Kluge10, Thomas Wurdinger11,12,13, Pepijn Schellen14, Gunther Hartmann7, Tobias Zacherle3, Roopika Menon3, Erik Thunnissen15, Reinhard Büttner6, Frank Griesinger16, Jürgen Wolf5, Lukas Heukamp17, Martin L. Sos1,2,18*, Johannes M. Heuckmann3*

Correspondence

Martin L. Sos ([email protected]), Molecular Pathology, Institute of Pathology and Department of Translational Genomics, University of Cologne, Weyertal 115b,

50931 Cologne, Germany. Tel.: +49 221 478 96175 Fax: +49 221 478 97902

Johannes M. Heuckmann ([email protected]), NEO New Oncology GmbH,

Gottfried-Hagen-Str. 20, 51105 Cologne, Germany. Tel.: +49 221 8882380 Fax: +49 221

88823822

Affiliations 1 Molecular Pathology, Institute of Pathology, University of Cologne, Kerpener Str. 62, 50937 Cologne, Germany. 2 Department of Translational Genomics, Medical Faculty, University of Cologne, Weyertal 115b, 50931 Cologne, Germany 3 NEO New Oncology GmbH, Gottfried-Hagen-Str. 20, 51105 Köln, Germany. 4 Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, The Netherlands. 5 Department of Internal Medicine, Center for Integrated Oncology Köln Bonn, University Hospital Cologne, Cologne, 50931 Cologne, Germany. 6 Institute of Pathology, Center of Integrated Oncology, University Hospital Cologne, 50937 Cologne, Germany 7 Department of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany 8 Department of Nuclear Medicine, University Hospital of Cologne, Cologne, Germany. 9 Department of Radiology, University Hospital of Cologne, Germany. 10 Institute for diagnostic and interventional Radiology, Pius-Hospital, Medical Campus University of Oldenburg, Germany 11 Department of Neurosurgery, VU University Medical Center, Cancer Center Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands. 12 Brain Tumor Center Amsterdam, VU University Medical Center, Cancer Center Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands. 13 Department of Neurology Massachusetts General Hospital and Neuroscience Program, Harvard Medical School, 149 13(th) Street, Charlestown, MA 02129, USA.

Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 28, 2017; DOI: 10.1158/1078-0432.CCR-17-3001 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

14 Department of Neurosurgery, VU University Medical Center, Cancer Center Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands; Brain Tumor Center Amsterdam, VU University Medical Center, Cancer Center Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands. 15 Dept of Pathology, VU University Medical Center, Amsterdam, The Netherlands. 16 Lung Cancer Network NOWEL, Georgstrasse 12, 26121, Oldenburg, Germany; Department of Hematology and Oncology, Pius-Hospital, University Department Internal Medicine-Oncology, Medical Campus University of Oldenburg, Germany. 17 NEO New Oncology GmbH, Gottfried-Hagen-Str. 20, 51105 Köln, Germany; Institute for Hematopathology, Fangdieckstr. 75a, Hamburg, Germany; Lung Cancer Network NOWEL, Georgstrasse 12, 26121, Oldenburg, Germany. 18 Center for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany.

Current affiliation of D. Plenker: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA

Running title: MET alterations trigger response to MET inhibition

Disclosures of Potential Conflict of Interest

A.H.S. attended advisory boards of MSD, BMS, Roche and received sponsored travels from QuIP GmbH, NordiQC within the last 12 months. R.B. is an employee of Targos

Molecular Pathology. J.W. is a consultant/advisory board member for AstraZeneca,

Bristol-Myers Squibb, Boehringer-Ingelheim, Clovis, MSD, Novartis, Pfizer, and Roche.

M.L.S received a commercial research grant from Novartis. R.R. attended advisory boards of Boehringer Ingelheim and received sponsored travels from Lilly Oncology and

Boehringer Ingelheim within the last 12 months. M.B., J.M., R.M., T.Z., L.H. and J.M.H. are employees of NEO New Oncology GmbH. M.L.S received a commercial research grant from Novartis.

Abstract

Purpose: We sought to investigate the clinical response to MET inhibition in patients diagnosed with structural MET alterations and to characterize their functional relevance in cellular models.

Experimental Design: Patients were selected for treatment with crizotinib upon results of hybrid capture-based next generation sequencing. To confirm the clinical observations we analyzed cellular models that express these MET kinase alterations.

Results: Three individual patients were identified to harbor alterations within the

MET receptor. Two patients showed genomic rearrangements leading to a gene fusion of KIF5B or STARD3NL and MET. One patient diagnosed with an EML4-ALK rearrangement developed a MET kinase domain duplication as a resistance mechanism to ceritinib. All three patients showed a partial response to crizotinib that effectively inhibits MET and ALK among other kinases. The results were further confirmed using orthogonal cellular models.

Conclusions: Crizotinib leads to a clinical response in patients with MET rearrangements. Our functional analyses together with the clinical data suggest that these structural alterations may represent actionable targets in lung cancer patients.

Statement of translational relevance

Oncogenically activated MET kinases have been implicated in the tumorigenesis of several cancer subtypes. We identified three structurally unique MET kinase alterations in lung adenocarcinoma patients. More specifically we characterize two different MET kinase fusions as well as a MET kinase domain duplication that developed in an ALK-

rearranged tumor along with acquired resistance to ceritinib. Off-label use of the kinase inhibitor crizotinib led to a marked response of all patients. Together with these observations our cellular analyses provide a functional basis for the oncogenic role of these structural MET alterations. Our findings will have an immediate impact for both the diagnostic and the therapeutic routine of lung cancer patients.

Introduction

Over the past years we have witnessed a dramatic shift in the clinical routine of lung adenocarcinoma patients, driven by the identification of oncogenically activated and therapeutically actionable targets. Next to oncogenic mutations, structural rearrangements of receptor kinases involving ALK, ROS1 or RET represent an ever- increasing pool of druggable targets in lung cancer patients (1-6). Effective inhibition of these oncogenic drivers frequently results in dramatic clinical responses (1,4-6). Most common oncogenic alterations of MET kinase involve MET exon 14 skipping mutations but the therapeutic relevance of more complex structural rearrangements remains largely unknown (2,7-9). Here, we report the discovery of structural alterations of MET that may predict response to targeted MET inhibition in lung cancer patients.

Materials and Methods

Patient material

All three patients consented to testing, therapy and registration of data for future publication. Genetic testing was conducted in concordance with local ethical guidelines and reviewed by the institutional ethics committee. The patient material was sequenced using hybrid capture-based next-generation sequencing (NEO, New Oncology GmbH).

Hybrid-capture based sequencing

NEOplus

Genomic DNA was extracted from FFPE material, sheared (Covaris) and subjected to hybrid capture-based next-generation sequencing to detect point mutations, small insertions and deletions, copy number alterations and rearrangement/gene fusions in a single assay (NEO New Oncology GmbH, Cologne, Germany). In brief, after shearing, adapters were ligated and individual genomic regions of interest were enriched using complementary bait sequences (hybrid capture procedure). The selected baits ensure optimal coverage of all relevant genomic regions. After enrichment, targeted fragments were amplified (clonal amplification) and sequenced in parallel at high sequencing depth. Computational analysis was performed using NEO New Oncology’s proprietary computational biology analysis pipeline to detect relevant genomic alterations in a quantitative manner.

NEOliquid

Whole blood (18 ml) was collected in Streck tubes (Cell-Free DNA BCT, Streck, Ref.

#218997) and cell free DNA (cfDNA) was extracted using Qiagen’s QIAamp Circulating

Nucleic Acid kit (QIAGEN, Cat.-No. #19419). Fragmented DNA was subjected to hybrid capture-based next-generation sequencing to detect point mutations, small insertions and deletions, copy number alterations and genomic translocations in a single assay

(NEO New Oncology GmbH, Cologne, Germany). In brief, after DNA extraction, adapters were ligated and individual genomic regions of interest were enriched using complementary bait sequences (hybrid capture procedure). The selected baits ensure optimal coverage of all relevant genomic regions. After enrichment, targeted fragments were amplified (clonal amplification) and sequenced in parallel at ultra-high sequencing depth. Computational analysis was performed using NEO New Oncology’s proprietary computational biology analysis pipeline to remove sequencing artifacts and detect relevant genomic alterations in a quantitative manner.

DNA sequencing

Dideoxy sequencing was performed using manufacturers standard protocols at the

Cologne Center for Genomics or at GATC GmbH. Alignments and verification of sequences were performed using Geneious R8.

Immunohistochemistry

For KIF5B- and STARD3NL-MET cases immunohistochemistry was performed by using the Ventana CONFIRM SP44 rabbit monoclonal antibody on the Ventana Benchmark XT staining platform according to the manufacturer's instructions (Roche Diagnostics /

Ventana Medical Systems, Tucson, AZ, USA). In brief, antigene retrieval was done with buffer 'CC1' (high pH), detection was done with the OptiView kit that uses DAB as chromogen, counterstaining was done with hematoxylin. Phospho-MET was stained using D26 rabbit monoclonal antibody (Cell Signaling). For the MET kinase domain duplication case immunohistochemistry was performed using the following antibodies: p40 (Biocare, BC28, 1:100), TTF1 (Dako, 8G7G3-1,1:800), MET (SP44, Ventana, ready to use) and ALK (Monosan, 5A4, 1:10).

Fluorescence in situ hybridization (FISH).

ALK break-apart FISH was performed using the Vysis ALK Break Apart FISH Probe Kit

(Abbott, #06N38) according to manufacturer’s instructions.

Cellular analyses

Generation of cell lines, viability assays and immunoblotting was performed as described previously (5). A detailed description of all additional methods can be found in the Supplementary Materials section.

Statistical analyses

All statistical analyses were performed using Microsoft Excel 2011 or GraphPad Prism

6.0h for Mac or R (https://www.r-project.org/).

Results

MET-rearranged lung tumors respond to treatment with crizotinib.

To identify patients with structural MET alterations we used hybrid capture-based paired-end sequencing (NEOplus, NEO New Oncology) of patients that were negative for any common driver mutation for lung cancer and we were able to identify two patients with lung adenocarcinoma (LADC) (2/337, ~0.5%) from two different clinical centers.

We identified spanning reads involving the MET kinase (3`) and sequences matching to either KIF5B or STARD3NL (5´) (Fig. 1A and Supplementary Fig. S1A) (10). Both genes encode domains that may facilitate homodimerization (Coiled-coil domain KIF5B;

MENTAL domain STARD3NL) of the resulting fusion protein (Fig. 1A) (11,12). We next confirmed the expression of both fusion transcripts by dideoxy sequencing of the RT-

PCR products isolated from the FFPE material (Supplementary Fig. S1B). In addition, immunohistochemical staining revealed high MET protein expression and was positive for phospho-MET in both samples (Fig. 1B). While another variant of KIF5B-MET fusion has been observed previously, STARD3NL represents a novel fusion partner for MET (2).

Since our sequencing panel was negative for any other oncogenic driver in both of the patients, the MET kinase fusion seemed to be a potential druggable target.

Crizotinib, initially developed as a MET inhibitor, effectively inhibits ALK and MET among several other kinases (4,13). Therefore both patients underwent off-label treatment with crizotinib (8,14).

The first patient, identified with the KIF5B-MET gene fusion, was a 33-year-old female with stage IV LADC who is a former smoker (10 pack-years) (Supplementary Fig.

1C). Two weeks after off-label treatment with crizotinib, cough and dyspnea improved remarkably. A subsequent PET/CT showed a response with a decrease in tumor size and

FDG-uptake (74% SUVmax reduction based on PERCIST) (Fig. 1C, 1D and

Supplementary Fig. S1C) (15). The following PET/CT scans confirmed the response (up to SUVmax 77% reduction based on PERCIST) and the treatment is ongoing with minimal side effects and a clinical benefit of currently more than eight months after initiation (Fig. 1C, 1D and Supplementary Fig. S1C).

The STARD3NL-MET gene fusion was identified in a 62-year-old female with a stage IV LADC who is a never smoker. After initial diagnosis, off-label treatment with crizotinib was started leading to a partial response (69% reduction based on RECIST

1.1) of the primary tumor and the metastases (Fig. 1C, D). Thus, our genomic analyses together with clinical observations suggest that MET rearrangements represent a druggable target in lung cancer patients.

KIF5B-MET and STARD3NL-MET fusions represent oncogenic driver alterations in cellular models.

To characterize the oncogenicity and druggability of KIF5B-MET and STARD3NL-MET we stably transduced KIF5B-MET and STARD3NL-MET into NIH-3T3 cells (Fig. 2A). The overexpression resulted in the activation of MET signaling that was robustly inhibited with crizotinib along with partial inhibition of downstream MAPK and PI3K signaling

(Fig. 2A). In these cells, stable inhibition of oncogenic MET signaling was obtained during treatment with crizotinib and tepotinib, two structurally independent MET- targeting drugs (Supplementary Fig. S1D). As expected, we observed robust colony formation in soft agar assays only in cells expressing the MET fusions (Fig. 2B). Of note colony formation was robustly inhibited under crizotinib treatment in a dose-dependent manner suggesting each MET rearrangement being the oncogenic driver for anchorage independent growth (Fig. 2B). Furthermore, stable expression of KIF5B-MET or

STARD3NL-MET induced IL-3 independent growth in Ba/F3 cells, confirming the

oncogenic transformation potential of the MET rearrangements. Both gene fusions showed nanomolar sensitivity towards inhibition of MET either with crizotinib or more effectively with tepotinib in viability screening assays (Fig. 2C, D, E and Supplementary

Fig. S1E). Thus, the orthogonal results derived from our functional in vitro experiments clearly demonstrate the therapeutic relevance of MET fusions for lung cancer patients.

MET kinase domain duplication drives resistance against ALK targeted therapy.

With a similar sequencing approach we sought to identify a potential resistance mechanism in an ALK-rearranged LADC (stage IV, 60, male) that acquired resistance to ceritinib. The patient initially showed a partial response to the selective ALK inhibitor that lasted for three months (Fig. 3A). Here, hybrid capture-based sequencing of circulating tumor DNA (ctDNA, NEOliquid) revealed a duplication of the MET kinase domain (MET-KDD) that was confirmed in a FFPE re-biopsy sample but was not present in the pre-therapy sample (Fig. 3B and Supplementary Fig. S2A). We validated the expression of the MET-KDD transcript in the post-ceritinib FFPE sample. Sequencing of the cDNA showed a retained short intronic region between the kinase domains that may represent a linker function (Supplementary Fig. S2B).

We speculated that MET-KDD may override the activity of a specific ALK inhibitor such as ceritinib and that dual ALK/MET inhibition may overcome this resistance. To test this hypothesis we transiently overexpressed the wild type and the rearranged receptor kinase in NIH-3T3 cells (Supplementary Fig. S2C). We observed a protein size shift of the MET-KDD transfected cells compared to cells expressing wild type MET

(Supplementary Fig. S2C). Both cell lines displayed high phospho-MET levels and activation of downstream MAPK signaling indicating that MET-KDD retains its kinase activity (Supplementary Fig. S2C). To more precisely model the clinical situation in vitro

we stably transduced NIH-3T3 cells with EML4-ALK and transiently transfected the cells with MET-KKD. As expected, only crizotinib but not ceritinib was able to inhibit both

ALK- and MET-dependent signaling in cells expressing rearranged ALK and MET-KDD

(Fig. 3C).

A subsequent treatment of the patient expressing MET-KDD with crizotinib resulted in a partial response (51% reduction based on RECIST 1.1) that lasted for three months (Fig. 3D). Follow-up imaging showed oligo-progression of a thoracic wall lesion.

Hybrid capture-based sequencing was performed on ctDNA and on a re-biopsy sample of the oligo-progressive lesion, but did not reveal any additional mutations that may be associated with resistance. Both samples were positive for the ALK fusion and MET-

KDD. We also confirmed the presence of the initial ALK-rearrangement throughout the treatment lines (Supplementary Fig. S2D). Thus, it is conceivable that the MET-KDD rearrangement identified in the patient is associated with acquired resistance against ceritinib (Fig. 3A, D).

Of note, we were not able to identify a single MET-KDD in a re-analysis of the

TCGA dataset (n>11,000 patients) suggesting that MET-KDD might be specifically associated with acquired ALK-inhibitor resistance (Supplementary Fig. S2E). Overall,

MET-KDD represents a rare structural alteration that may emerge as a resistance mechanism against ceritinib in ALK-rearranged tumors.

Discussion

In summary, our results provide evidence, that structural rearrangements of MET, such as MET fusions and MET kinase duplications, represent immediate druggable targets in lung cancer patients. While the MET kinase fusions identified in two LADCs fulfill the

criteria of primary oncogenic drivers, the MET-KDD rearrangement may be specifically associated with the ceritinib-resistance phenotype in an ALK-rearranged background.

Oncogenic MET alterations are found in several cancers including lung, glioblastoma and papillary renal cell carcinomas (8,14,16,17). In lung cancer patients exon 14 skipping mutations have been identified as recurrent alterations that lead to a hyper activated MET signaling and that trigger response to MET inhibition (3,8). More recently, MET gene fusions have been shown to represent a potentially druggable target in pediatric gliablastoma (7). Our results provide evidence that these type of MET alterations may be rare but can lead to a therapeutically actionable dependency in lung cancer patients.

MET amplifications on the other hand are recurrently found in EGFR mutant patients that acquire resistance against first and third generation EGFR inhibitors

(18,19). Two reports suggest that MET signaling could also promote resistance in ALK- rearranged tumors to ALK kinase inhibition (20,21). Our data suggest that under selective pressure with selective ALK inhibitors, subclones harboring MET kinase duplications may arise and trigger resistance against these drugs. Interestingly, a similar link between the selection of kinase domain duplications and resistance to targeted therapy has been previously described for BRAF in vemurafenib resistant melanoma

(22).

Overall, our functional data provide important insight into the cellular signaling that is engaged through MET kinase fusions and MET kinase domain duplications. The availability of clinically active MET-directed drugs urges for the testing of these rare structural rearrangements within the diagnostic routine and future clinical trials.

Authors’ Contributions

D.P., M.B., J.M., C.L., J.B., J.D.P., A.H.S., G.H., T.Z., R.M. and L.H. performed and/or coordinated the experimental work. D.P., M.B., A.H.S., J.M., J.B., C.K., T.S., A.K., R.B., L.H.,

M.L.S. and J.M.H. performed data analysis. A.J.d.L., R.R., T.W., P.S., E.T., F.G. and J.W. collected data and provided patient materials. D.P., M.B., A.J.d.L., M.L.S. and J.M.H. prepared the initial manuscript and figures. D.P., J.M.H and M.L.S supervised the study.

Acknowledgments

This work was supported by the German federal state North Rhine Westphalia (NRW) as part of the EFRE initiative (EFRE-0800397 to R.B. and M.L.S) and by the German

Ministry of Science and Education (BMBF) as part of the e:Med program (grant no.

01ZX1303 to J.W., R.B and grant no. 01ZX1406 to M.L.S). Additional funding was provided by the Deutsche Krebshilfe as part of the Oncology Centers of Excellence funding program (to R.B., J.W. and G.H.) and by the DFG-funded Cluster of Excellence

ImmunoSensation (to G.H.).

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Figure legends

Figure 1. Oncogenic MET fusions in LADC. A) Schematic representation of wild type

KIF5B, STARD3NL and MET proteins and the corresponding rearrangements. The red bar indicates the breakpoint. CC, coiled-coil. TM, transmembrane. KD, kinase domain. B)

Immunohistochemistry stainings of FFPE patient tissue for H&E, MET and phospho-

MET. The scale bar is equal to 25 µm. C) Schematic presentation of clinical course for patients expressing either KIF5B-MET or STARD3NL-MET. Dotted line of KIF5B-MET graph marks 1 week break of crizotinib treatment. SD, stable disease. PR, partial response. D) PET/CT (KIF5B-MET) and CT (STARD3NL-MET) scans of patients prior and post crizotinib treatment. Arrows indicate regions of tumor growth or shrinkage.

Figure 2. Assessing the oncogenic potential of KIF5B- and STARD3NL-MET in vitro. A)

Immunoblot of NIH-3T3 cells transduced with empty vector, KIF5B-MET or STARD3NL-

MET treated for 4 h with various concentrations of crizotinib. HSP90 serves as loading control. A representative blot from n=3 independent experiments is shown. B) Colony formation assay of NIH-3T3 cells stably transduced with empty vector (e.v.), KIF5B-MET or STARD3NL-MET under crizotinib treatment. Pictures were taken after 10 days. Scale bar is equal to 100 µm. C) Dose-response curves (72 h) of Ba/F3 cells transduced with

KIF5B- or STARD3NL-MET and not supplemented with IL-3. Cells were treated with crizotinib, tepotinib or erlotinib (as control compound). (n=3) D) Immunoblot of Ba/F3 cells transduced with KIF5B-MET under treatment with crizotinib or tepotinib (4 h).

HSP90 serves as loading control. A representative blot from n=3 independent experiments is shown. E) Immunoblot of Ba/F3 cells transduced with STARD3NL-MET under treatment with crizotinib or tepotinib (4 h). HSP90 serves as loading control. A representative blot from n=3 independent experiments is shown.

Figure 3. MET kinase duplication (KDD) is a new resistance mechanism in EML4-ALK- rearranged LADC. A) CT scans of a patient under therapy with ceritinib. Arrows indicate regions of tumor growth or shrinkage. B) Schematic representation of wild type MET and MET kinase domain duplication. TM, transmembrane. KD, kinase domain. cDNA sequence and translation of duplication site is indicated below. KD1 is marked in black,

KD2 is marked in blue, intronic linker sequence is printed in bold. C) Immunoblot of transiently transfected NIH-3T3 cells (48 h) stably transduced with EML4-ALK v1. Cells were treated with crizotinib and ceritinib for 4 h. A representative blot from n=3 independent experiments is shown. D) Clinical information of MET KDD case prior and post acquired resistance. SD, stable disease. PR, partial response (upper panel) and CT scans of patient under therapy with crizotinib. Arrows indicate regions of tumor growth or shrinkage (lower panel).

Structural alterations of MET trigger response to MET kinase inhibition in lung adenocarcinoma patients

Dennis Plenker, Miriam Bertrand, Adrianus Johannes de Langen, et al.

Clin Cancer Res Published OnlineFirst December 28, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-17-3001

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