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1 Clinical response of the novel activating ALK-I1171T mutation in
2 neuroblastoma to the ALK inhibitor ceritinib.
3
4 Jikui Guan1*, Susanne Fransson2*, Joachim Tetteh Siaw1*, Diana Treis4*, Jimmy Van den 5 Eynden1, Damini Chand1, Ganesh Umapathy1, Kristina Ruuth9, Petter Svenberg4, Sandra 6 Wessman5, Alia Shamikh5, Hans Jacobsson6, Lena Gordon7, Jakob Stenman8, Pär-Johan 7 Svensson8, Magnus Hansson3, Erik Larsson1, Tommy Martinsson2#, Ruth H. Palmer1#, Per 8 Kogner5#, Bengt Hallberg1#.
9
10 Departments of 1Medical Biochemistry and Cell Biology, 2Pathology and Genetics, 3Pediatrics and 11 Pathology, Institute of Biomedicine, The Sahlgrenska Academy, University of Gothenburg, 12 Gothenburg, Sweden, 4Childhood Cancer Research Unit, Department of Women’s and Children’s 13 Health, and Pediatric Oncology Program Karolinska University Hospital, 5Department of Oncology- 14 Pathology, Karolinska Institutet, and Dept. of Clinical Pathology, Karolinska University Hospital, 15 6Department of Radiology, Karolinska University Hospital, 7Dept. of Pediatric Radiology, Astrid 16 Lindgren Children’s Hospital, Karolinska University Hospital, 8Department of Pediatric Surgery, 17 Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Stockholm, Sweden and 18 9Institute of Molecular Biology, Umeå University, Umeå, Sweden.
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21 Running title: Clinical response to ceritinib in ALK-positive neuroblastoma.
22
23 #Correspondence: Bengt Hallberg ([email protected]), Ruth. H. Palmer 24 ([email protected]), Tommy Martinsson ([email protected]) and Per Kogner 25 ([email protected]).
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1 Abstract
2
3 Tumors with Anaplastic Lymphoma Kinase (ALK) fusion rearrangements, including
4 non-small cell lung cancer and anaplastic large cell lymphoma, are highly sensitive to ALK
5 tyrosine kinase inhibitors (TKIs), underscoring the notion that such cancers are addicted to
6 ALK activity. While mutations in ALK are heavily implicated in childhood neuroblastoma,
7 response to the ALK TKI crizotinib has been disappointing. Embryonal tumors in patients
8 with DNA repair defects such as Fanconi anemia (FA) often have a poor prognosis, due to
9 lack of therapeutic options. Here we report a child with underlying FA and ALK mutant high-
10 risk neuroblastoma responding strongly to precision therapy with the ALK TKI ceritinib.
11 Conventional chemotherapy treatment caused severe, life-threatening toxicity. Genomic
12 analysis of the initial biopsy identified germ-line FANCA mutations as well as a novel ALK-
13 I1171T variant. ALK-I1171T generates a potent gain-of-function mutant, as measured in
14 PC12 cell neurite outgrowth and NIH3T3 transformation. Pharmacological inhibition
15 profiling of ALK-I1171T in response to various ALK TKIs identified an 11-fold improved
16 inhibition of ALK-I1171T with ceritinib when compared with crizotinib. Immunoaffinity-
17 coupled LC-MS/MS phosphoproteomics analysis indicated a decrease in ALK signaling in
18 response to ceritinib. Ceritinib was therefore selected for treatment in this child. Mono-
19 therapy with ceritinib was well tolerated and resulted in normalized catecholamine markers
20 and tumor shrinkage. After 7.5 months treatment, residual primary tumor shrunk, was
21 surgically removed and exhibited hallmarks of differentiation together with reduced Ki67
22 levels. Clinical follow-up after 21 months treatment revealed complete clinical remission
23 including all metastatic sites. Therefore, ceritinib presents a viable therapeutic option for
24 ALK-positive neuroblastoma.
25
26
2
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1 Introduction
2 Anaplastic Lymphoma Kinase (ALK) is a receptor tyrosine kinase (RTK) comprising an
3 extracellular ligand-binding domain, a transmembrane domain and an intracellular tyrosine
4 kinase domain (Iwahara, Fujimoto et al. 1997, Morris, Naeve et al. 1997). ALK was first
5 described in 1994 as a fusion partner in the t(2;5) chromosomal translocation in anaplastic
6 large cell lymphoma (ALCL) (Morris, Kirstein et al. 1994). Vertebrate ALK is activated by
7 the recently described small secreted ALKAL (FAM150/AUG) ligands, which potently
8 activate ALK signaling (Zhang, Pao et al. 2014, Guan, Umapathy et al. 2015, Reshetnyak,
9 Murray et al. 2015, Mo, Cheng et al. 2017, Fadeev, Mendoza-Garcia et al. 2018). ALK
10 activates multiple signaling pathways, such as the PI3K-AKT, CRKL-C3G, MEKK2/3-
11 MEK5-ERK5, JAK-STAT and MAPK pathways (Hallberg and Palmer 2016).
12 Mutation of full-length ALK has been well described in the neural crest derived
13 pediatric cancer neuroblastoma (Caren, Abel et al. 2008, Chen, Takita et al. 2008, George,
14 Sanda et al. 2008, Janoueix-Lerosey, Lequin et al. 2008, Mosse, Laudenslager et al. 2008,
15 Hallberg and Palmer 2013). Of the more than 35 mutations in ALK described, the majority
16 are point mutations in the kinase domain, although deletions in the extracellular domain and
17 translocations have also been reported (Okubo, Takita et al. 2012, Cazes, Louis-Brennetot et
18 al. 2013, Hallberg and Palmer 2013, Bresler, Weiser et al. 2014, Fransson, Hansson et al.
19 2015). Within the kinase domain three hotspot residues, F1174, F1245 and R1275, represent
20 most patient mutations (De Brouwer, De Preter et al. 2010, Hallberg and Palmer 2013,
21 Bresler, Weiser et al. 2014). In addition to ALK mutations in treatment-naïve neuroblastoma,
22 examination of ALK status in relapsed neuroblastoma samples has highlighted an increase in
23 activating ALK mutations appearing later in the disease course (Martinsson, Eriksson et al.
24 2011, Schleiermacher, Javanmardi et al. 2014, Eleveld, Oldridge et al. 2015).
3
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1 In neuroblastoma, ALK signaling has been shown to act synergistically with MYCN to
2 drive tumor development (Berry, Luther et al. 2012, Heukamp, Thor et al. 2012, Schonherr,
3 Ruuth et al. 2012, Zhu, Lee et al. 2012, Cazes, Lopez-Delisle et al. 2014, Ueda, Nakata et al.
4 2016), with combined occurrence of MYCN amplification and ALK mutations being
5 associated with particularly bad prognosis (De Brouwer, De Preter et al. 2010). The
6 importance of ALK has lead to the suggestion that inhibition of ALK with small molecule
7 tyrosine kinase inhibitors (TKIs) may offer clinical benefit in neuroblastoma. The ALK TKI
8 crizotinib was approved for clinical use in patients with ALK-positive non-small cell lung
9 cancer (NSCLC) in 2011 ((FDA) 26th of August, 2011, Kwak, Bang et al. 2010), based on a
10 robust response in this patient population. Although significant problems with resistance to
11 ALK TKIs occur, a trial comparing crizotinib with chemotherapy concluded that crizotinib is
12 superior in patients with previously treated, advanced ALK-positive NSCLC (Shaw, Kim et
13 al. 2013). Similarly, treatment with crizotinib resulted in a strong response in a phase I
14 crizotinib monotherapy trial of pediatric patients with ALK-fusion positive tumors,
15 although patient responses in pediatric ALK mutant neuroblastoma were less
16 encouraging (Mosse, Lim et al. 2013, Mosse, Voss et al. 2017). It is not clear whether this
17 relates to clinical factors unique to neuroblastoma or to issues of efficacy of inhibition of
18 ALK by crizotinib. The clinical data thus far motivates exploration of alternative strategies
19 in neuroblastoma, including ALK monotherapy with next-generation TKIs and combination
20 strategies (Berry, Luther et al. 2012, Moore, Azarova et al. 2014, Umapathy, El Wakil et al.
21 2014, Guan, Tucker et al. 2016, Infarinato, Park et al. 2016, Krytska, Ryles et al. 2016).
22 Other ALK TKIs include ceritinib, brigatinib, alectinib and lorlatinib (Christensen, Zou
23 et al. 2007, Katayama, Khan et al. 2011, Sakamoto, Tsukaguchi et al. 2011, Marsilje, Pei et al.
24 2013, Chia, Mitchell et al. 2014, Johnson, Richardson et al. 2014, Katayama, Lovly et al.
25 2015) (for latest details of clinical trials employing ALK TKIs, please see Clinicaltrials.gov).
4
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1 These ALK inhibitors bind slightly differently within the ATP-binding pocket of the ALK
2 kinase domain, show varying abilities to cross the blood brain barrier and have differing
3 profiles of inhibition for the wild-type ALK kinase domain compared with the various ALK
4 kinase mutants. These properties have important implications for potential treatment of ALK-
5 positive neuroblastoma in which ALK mutations are present in treatment-naïve tumors.
6 Ceritinib gained US Food and Drug Administration approval in 2014 following accelerated
7 review for the treatment of patients with ALK-positive (ALK+) metastatic NSCLC who have
8 progressed on, or are intolerant to, crizotinib ((FDA) 26th of May, 2017, (FDA) 29th of April,
9 2014, Shaw, Kim et al. 2014).
10 Here we report the robust response of an ALK-positive neuroblastoma patient to
11 ceritinib treatment. The patient received chemotherapy according to protocol after being
12 diagnosed with high-risk neuroblastoma but displayed severe hematological failure early after
13 initial treatment. This led to suspicion of Fanconi anemia (FA), that was confirmed by
14 chromosomal breakage assessment and identification of FANCA gene mutations. FA is a rare
15 recessive genetic disorder clinically characterized by congenital abnormalities and progressive
16 bone marrow failure (Kutler, Singh et al. 2003), although some patients may show only subtle
17 symptoms or no phenotype at all (Neveling, Endt et al. 2009). FA is caused by mutations in
18 one of at least 21 different FA genes encoding proteins that function in interstrand cross-link
19 and double strand DNA repair (Mamrak, Shimamura et al. 2017). Due to the impaired DNA
20 damage response, FA patients have significantly increased cancer susceptibility, particularly
21 to acute myeloid leukemia, head and neck squamous cell carcinoma (Kutler, Auerbach et al.
22 2003, Kutler, Singh et al. 2003) and more rarely, embryonic tumors such as Wilms tumor,
23 hepatoblastoma or neuroblastoma (Abbondanzo, Manz et al. 1986, Bissig, Staehelin et al.
24 2002, Berrebi, Lebras et al. 2006, Kopic, Eirich et al. 2011, Malric, Defachelles et al. 2015).
25 The management of cancer in FA patients is challenging as defective DNA repair leads to
5
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1 hypersensitivity to both radiation and crosslinking agents such as mitomycin C and cisplatin
2 with increased risk of developing severe toxicity (Walsh, Chang et al. 2017)
3 (http://fanconi.org/index.php/publicaionts/guidelines). In parallel to identification of FANCA
4 gene mutations in this neuroblastoma patient, we also noted the presence of an ALK mutation
5 at residue I1171.
6 Preclinical analysis showed that the mutation of ALK-I1171 to threonine (T), which has
7 not previously been reported in neuroblastoma, generates a potent gain-of-function mutant.
8 PC12 cells expressing ALK-I1171T display ligand-independent activation of ALK, neurite
9 outgrowth and activation of downstream signaling. In addition, ALK-I1171T drives foci
10 formation in NIH3T3 cells. We were able to identify ceritinib as a next-generation ALK TKI
11 that effectively inhibits ALK-I1171T in a preclinical setting, and based on these experimental
12 data, the patient was treated with ceritinib. A dramatic response was observed and 7.5 months
13 after initiation of treatment, tumor material was surgically removed, exhibiting reduced Ki67
14 levels and upregulation of markers of differentiation. Based on this case, we suggest that
15 ceritinib presents a viable therapeutic option for patients with ALK-positive neuroblastoma .
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1 Materials and methods
2
3 Patient
4 A 16-month-old boy presented at the pediatric emergency unit with neurologic symptoms
5 including facial palsy, impaired balance and strabismus. There was no past medical history of
6 note except for examination by a hand surgeon and a dysmelia team due to slightly deviant
7 thumbs which had been present since birth. At the emergency visit, exophthalmus and an
8 abdominal mass were also noted, and imaging using MIBG scintigraphy, CT scans and MRI
9 revealed a metastatic neuroblastoma with metastases in bone, lungs and intracranially (Fig. 1,
10 Fig. S1) but with no neuroblastoma detected in the bone marrow. Biological workup of the
11 diagnostic biopsy showed a number of unfavorable chromosomal aberrations, including 11q
12 loss, 17q gain, 2q loss, 4p loss,7q and 9q gain (Fig. 2). Treatment was initiated according to
13 the high-risk neuroblastoma protocol HR-NBL-1 SIOPEN (Ladenstein, Potschger et al. 2017)
14 (ethical permit 02-294). In response to this treatment, the patient developed protracted
15 neutropenia, anemia and thrombocytopenia necessitating repeated transfusions of erythrocytes
16 and platelets for almost 6 months (Fig. 3). Furthermore the patient experienced repeated
17 episodes of severe septicemia and typhlitis requiring intensive care. Based on the suspicion of
18 underlying FA (Walsh, Chang et al. 2017), chromosomal breakage assessment was perfomed
19 and found positive. Further genomic analysis revealed inherited germline mutations in the
20 FANCA gene, confirming FA diagnosis (Fig. 2B, Table S1). Detailed genomic analysis
21 further identified a somatic ALK-I1171T mutation in the patient, raising the possibility of
22 ALK TKI treatment (Fig. 2C, Table S1). Following preclinical investigation of the ALK-
23 I1171T mutation, a clinical multidisciplinary conference, an Institutional Ethical Board
24 meeting and a Clinical Ethical Board meeting endorsed application for a Medical Products
25 Agency (MPA) license (according to LVFS 2008:1). In addition, written informed consent
7
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1 from the patient’s guardians was obtained and the patient was included in the Novartis
2 compassionate use Individual Patient Program for ceritinib (CLDK378A2003M). Sampling
3 and analyses of tumor tissues and linkage to clinical information were performed according to
4 the ethical permit 2009/1369-31/1. Written informed consent was obtained from the
5 patient’s guardians allowing scientific research publication.
6
7 Immunohistochemistry
8 Staining for NB84 (Novocastra, NCL-NB84), NF (DAKO, 2F11) and Ki-67 (DAKO, MIB-1)
9 was performed using a DAKO Autostainer. Slides were rehydrated with Xylene followed by a
10 series of alcohol dilutions and a rinse in buffer (DAKO 8007). PTLINK (DAKO) was used
11 for enzyme antigen retrieval. Endogenous enzyme block was performed for 5 minutes with
12 EnVision FLEX Peroxidase-Blocking Reagent (DAKO). The antibodies (diluted at DAKO
13 Company) were incubated at room temperature for 20 minutes. After 5 minutes rinse in buffer
14 the labeled polymer, EnVision FLEX/HRP (DAKO), was applied and incubated for 20
15 minutes with two additional rinses before the slides were incubated with substrate-
16 chromogen, Substrate Working Solution (DAKO), for 10 minutes. Slides were ultimately
17 rinsed and counterstained in EnVision FLEX Hematoxylin prior to mounting.
18
19 Genomics profile with SNP array
20 Microarray analyses of DNA from the tumor samples were performed using Affymetrix
21 Human Cytoscan High Density arrays essentially as described earlier (Fransson, Ostensson et
22 al. 2016). For primary data analysis the GDAS software (Affymetrix) was used, while
23 genomic profiles were generated using CNAG (Copy Number Analyzer for
24 AffymetrixGeneChip Mapping arrays) version 3.3 (Genome Laboratory, Tokyo University;
25 http://www.genome.umin.jp; 11).
8
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1
2 Detection of ALK mutation with massive parallel DNA sequencing
3 DNA was extracted from tumor sample using standard procedures and evaluated through
4 fluorometric quantitation and DNA integrity assessment on Agilent Tapestation (Agilent,
5 Santa Clara, CA) prior to exome sequencing. Exome sequencing was performed at GATC
6 (GATC, Konstanz, Germany) through paired-end sequencing (101 bp read length) on
7 Illumina platforms after enrichment with Agilent SureSelect Human All Exon v5 enrichment
8 kit giving an average coverage of 85X (sequencing details are supplied in supplemental
9 information file: ‘Exome sequencing’). Alignment against hg19 was performed using BWA
10 with GATK local realignment followed by SNV calling using SNPeff and Annovar
11 annotation. Visualization of mapped reads and manual QC of called variants was done
12 through IGV (Robinson, Thorvaldsdottir et al. 2011). Only variants located at regions covered
13 by at least 10 unique reads were kept. A systematic filtering approach was used to identify
14 critical variants by removal of common variants (e.g. showing an allele frequency above
15 0.005 in 1000 genomes or exome variant server) as well as excluding all synonymous variants
16 or variants in non-coding regions except those affecting canonical splice sites. PolyPhen 2
17 (PP2) and SIFT were used for prediction of functional relevance of called SNVs. Sanger
18 sequencing of the patient’s constitutional and tumor DNA verified presence of a somatic ALK
19 mutation in the tumor. Sanger sequencing of ALK exons was performed as described
20 previously (Caren, Abel et al. 2008). All genomic positions are specified according
21 hg19/GRCh37.
22
23 Generation of human ALK mutant constructs in PC12 cells
24 The ALK-I1171T mutation was generated based on pcDNA3-ALK-WT (NM_004304.3) by
25 Eurofins Genomics (Ebersberg, Germany). ALK-F1174L has been described previously
9
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1 (Martinsson, Eriksson et al. 2011). All mutations generated in the kinase domain were
2 confirmed by sequencing from both directions.
3
4 Neurite outgrowth assay
5 PC12 (2 x 106) cells were co-transfected with 0.5 µg of empty pcDNA3 vector, pcDNA3-
6 ALK-WT, pcDNA3-ALK-I1171T or pcDNA3-ALK-F1174L respectively together with 0.5
7 µg of pEGFP-N1 (Clonetech) as indicated by electroporation using Amaxa electroporator
8 (Amaxa Biosystems) and Ingenio electroporation solution (Mirus Bio LCC). After
9 electroporation, cells were kept in minimum essential medium (MEM) supplemented with 7%
10 horse serum and 3% fetal bovine serum and seeded into 24-well plates. For samples
11 stimulated with ALK ligand, purified ALKAL1 (FAM150A) protein was added to the well
12 with a final concentration of 1µg/ml (Guan, Umapathy et al. 2015). After 48 hours of
13 incubation, the fraction of Green fluorescent protein (GFP)-positive and neurite carrying cells
14 versus GFP-positive cells was observed under a Zeiss Axiovert 40 CFL microscope. To be
15 judged as a neurite carrying cell, the neurite of the cell should be at least twice the diameter of
16 a normal cell body. Experiments were performed in triplicate, and each sample within an
17 experiment was performed in triplicate.
18
19 Transformation assay
20 NIH 3T3 cells (5 × 104) were seeded into collagen-coated 12-well plates and transfected with
21 0.55μg of pcDNA3 vector, pcDNA3-ALK-WT, pcDNA3-ALK-I1171T, or pcDNA3-ALK-
22 F1174L respectively using Lipofectamine 2000 according to the manufacturer’s protocol
23 (Invitrogen). Twenty-four hours later, three fifths of the cells from each well were transferred
24 to wells in 6-well plates and kept in DMEM supplemented with 10% FBS and 0.5 mg/ml
10
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1 G418 until the cells reached confluence. Thereafter, cells were kept in DMEM supplemented
2 with 5% FBS and 0.25 mg/ml G418 for another 10 days, with replacement of medium every
3 second day. Plates were washed with PBS and air-dried, fixed with methanol for 20 minutes,
4 and cells were stained with 0.2% crystal violet in 20% ethanol for 20 minutes, followed by
5 three short rinses in water.
6
7 Cell culture, viability, lysis, and immunoblotting
8 Cell viability was assessed as relative redox metabolic activity using aresazurin-based assay.
9 Cell lines employed were CLB-BAR, CLB-GE, CLB-GA, Kelly, SK-N-AS, SK-N-BE, SK-
10 N-DZ, IMR32 and CLB-PE neuroblastoma cells (Umapathy, Guan et al. 2017). Cells (4 ×104)
11 were plated on collagen-coated 48-well plates and treated with either ceritinib or crizotinib
12 with indicated concentrations. Cell viability was determined after 72 hours with 55μM
13 resazurin (Sigma, Stockholm, Sweden). After 3h at 37°C, the amount of metabolized
14 resazurin was analyzed as relative fluorescence with an Infinit200 plate reader (TEKAN,
15 Männedorf, Switzerland). Results were from one of three representative experiments, with
16 each experiment being performed in triplicate. GraphPad Prism 6.0 was used to calculate IC50
17 values by fitting data to a log (inhibitor concentration) versus normalized response (variable
18 slope) equation.
19 PC12 cells were transfected with 0.5 µg of empty pcDNA3 vector, pcDNA3-ALK-WT, or
20 pcDNA3-ALK variants as specified respectively by electroporation as described above. Cells
21 were serum-starved for 36 hours before lysis. For samples stimulated with ALK ligand, they
22 were treated with 1μg/ml of purified ALKAL1 protein for 30 minutes prior to lysis. Cells
23 were washed twice with ice-cold PBS prior to harvest in lysis buffer [25mmol/L Tris-Cl,
24 pH7.5, 150mmol/L NaCl, 1% (v/v) Triton X-100, 1mmol/L DTT, protease inhibitor cocktail
11
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1 tablet (Roche)]. Cell lysates were cleared by centrifugation at 14,000 rpm for 15 minutes at
2 4°C. Samples were boiled in 1x SDS sample buffer and analyzed by immunoblotting. Primary
3 antibodies used for immunoblotting were: anti-pan-ERK (1:10,000), purchased from BD
4 Transduction Laboratories (Franklin Lakes, NJ), anti-pALK (Y1604) and anti-pERK1/2
5 (T202/Y204) from Cell Signaling Technology (Danvers, MA). Monoclonal antibody 135
6 (anti-ALK) was produced in the Hallberg laboratory against the extracellular domain of ALK
7 as described (Moog-Lutz, Degoutin et al. 2005, Schonherr, Ruuth et al. 2012). Horseradish-
8 peroxidase-conjugated secondary antibodies goat anti-rabbit IgG and goat anti-mouse IgG
9 (1:5,000) were from Thermo Scientific (Waltham, MA).
10
11 ALK inhibitor profiles on ALK variants
12 PC12 cells (2×106) were transfected with 1.0 μg of pcDNA3-ALK-WT, or pcDNA3-ALK
13 variants as described (Guan, Tucker et al. 2016, Siaw, Wan et al. 2016). Cells from three
14 electroporations were pooled together, mixed and equally seeded into 9 wells of one 24-well
15 plate. After 24-36 hours culture, cells were treated with serial dilutions of the indicated
16 inhibitors for 1 hour before lysis. Cells were washed with cold 1X PBS and lysed with 1X
17 SDS sample buffer and samples were boiled at 95ºC for 5 minutes. Phospho-ALK (Y1604)
18 antibody was used to detect ALK phosphorylation and ALK mAb135 was used to detect total
19 ALK. The intensity of pALK (Y1604) and total ALK bands was quantified with Image Studio
20 Lite 3.1 software. Data were normalised to the 0 nM inhibitor samples. GraphPad Prism 6.0
21 was used to calculate IC50 values by fitting data to a log (inhibitor concentration) vs.
22 normalized response (variable slope) equation.
23
24 Phosphoproteomics
12
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1 Tyrosine and serine/threonine phosphorylation profiling of neuroblastoma cells (CLB-BAR,
2 CLB-GE and SK-N-AS) in the presence or absence of ceritinib (200 nM for 60 mins) was
3 undertaken by immunoaffinity purification with P-Tyr-100 or P-Ser/Thr-antibodies
4 accordingly (Cell Signaling Technology, Danvers, MA, USA). Cells were lysed in lysis buffer
5 (20 mM Hepes pH 8.0, 9M UREA, 1 mM sodium orthovanadate, 2.5 mM sodium
6 pyrophosphate and 1 mM β-glycerol-phosphate) and approximately 20 mg protein per sample
7 subjected to LC-MS/MS. This was performed as previously described (Sattu, Hochgrafe et al.
8 2013). Phosphorylation responses were quantified for each site (tyrosine, serine or threonine)
9 under analysis as log2 fold change (FC) values of ceritinib treated/untreated control counts. If
10 a specific site was measured in multiple peptides, its median log2 FC value was used for
11 analysis. Protein sites were considered differentially phosphorylated when their absolute log2
12 FC values were higher than 1.5. A gene set enrichment analysis (GSEA) was performed using
13 Fisher’s exact test and false discovery rate correction using the Benjamini Hochberg method
14 (Benjamini and Hochberg 1995). Reactome pathway information was downloaded from the
15 Molecular Signatures Database v5.2 (Subramanian, Tamayo et al. 2005). The differentially
16 phosphorylated proteins were mapped to the InWeb_InBioMap protein-protein interaction
17 (PPI) network (Li, Wernersson et al. 2017). For the analysis of related TRKs, a list of 40
18 different TRKs was downloaded from HGNC (Gray et al., 2015).
19
20
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1 Results
2
3 Patient presentation
4 A 16-month-old boy presenting with neurologic symptoms at the pediatric emergency
5 unit was found to have metastatic neuroblastoma stage M (INRGSS) (Fig. 1). There was no
6 previous family history of neuroblastoma. SNP-microarray analysis of genomic DNA from
7 initial biopsy showed no MYCN-amplification but revealed several unfavorable segmental
8 alterations (Fig. 2A). COJEC chemotherapy induction was started according to HR-NBL-1
9 SIOPEN (Ladenstein, Potschger et al. 2017) and the patient received one A course (day 0;
10 vincristine, carboplatin, etoposide), one B course (day 10; vincristine, cisplatin) and a partial
11 C course (day 20; vincristine, etoposide, cyclophosphamide), before chemotherapy had to be
12 discontinued because of severe bone marrow toxicity with anemia, neutropenia and
13 thrombocytopenia. For the ensuing four months, the patient was continuously hospitalized and
14 suffered repeated episodes of septicemia, intestinal hemorrhage and typhlitis as well as
15 respiratory failure requiring intensive care. Because of the unusual severity of the toxicity,
16 and also because the patient displayed moderate radial dysplasia with deviant thumbs
17 bilaterally, suspicion of Fanconi anemia was raised. A clinical chromosome breakage
18 assessment was found positive. A more detailed genomic analysis then revealed inherited
19 mutations in the FANCA gene; one novel missense variant at genomic position
20 chr16:89831348 (NM_000135.2; c.2728C>T, p.L910F) predicted to be deleterious and
21 probably damaging by SIFT and PP2 respectively and one splice site mutation at the
22 acceptor site of intron 42 at genomic position chr16:89805118, (NM_000135.2; c.4261-
23 2A>C) (Fig. 2B, Table S1). The L910F variant is not present in either ClinVar or
24 HGMD and to best of our knowledge, this is the first report of a L910F variant in
25 association with FA. A third rare missense variant at chr16:89871709 (c.688G>A; p.V230I),
14
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1 was detected in the patient although predicted to be benign by SIFT and PP2, indicating a
2 non-pathogenic function. This is supported by a recent study showing that the V230I variant
3 is localized to the nucleus and does not confer sensitivity to mitomycin C (Kimble, Lach et al.
4 2017). The fact that the child presents as a compound heterozygote for FANCA
5 (p.L910F/c.4261-2A>C) is a likely cause for the above described toxicity (Fig. 2B, Table S1).
6 Further use of chemotherapy, or irradiation, was advised against in this patient due to the
7 DNA repair defects in Fanconi anemia. Since initial genomic analysis also identified an ALK-
8 I1171T mutation in the patient (Fig. 2C, Table S1), ALK TKI therapy was considered and
9 experimentally investigated preclinically for this patient.
10
11 ALK-I1171T is a gain-of-function ALK mutation
12 Exome sequence analysis of a tumor biopsy sample revealed a heterozygous mutation
13 of ALK (chr2:29445213) in exon 22; (NM_004304.4; c.3512T>C) leading to a missense
14 mutation I117IT (Fig. 2C, Table S1). Sanger sequencing of tumor and patient’s
15 corresponding constitutional DNA indicated that the ALK variant was caused by a somatic
16 event. I1171 is located in the ALK kinase domain in close proximity to F1174, one of the
17 mutational hotspots of ALK in neuroblastoma that is localized in the αC-helix in the N-
18 terminal lobe of the kinase (Fig. 4A). Mutation of I1171 to either N/T or S has previously
19 been described in ALK TKI-treated patients with ALK fusion-positive NSCLC (in the EML4-
20 ALK and HIP1-ALK fusion proteins) (Katayama, Friboulet et al. 2014, Ou, Klempner et al.
21 2014, Toyokawa, Hirai et al. 2014), resulting in tumor resistance.
22 This is the first report of mutation of ALK-I1171T in neuroblastoma, although mutation
23 of ALK-I1171N has been reported (Mosse, Laudenslager et al. 2008, Bresler, Weiser et al.
24 2014). Intriguingly, the residue I1171 contributes to the regulatory spine (Kornev and Taylor
25 2015) (Fig. 4A), prompting us to experimentally validate its behavior in the context of the full
15
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1 length ALK RTK. To evaluate the ALK-I1171T mutant we employed a PC12 cell culture
2 system for expression of either wild type or ALK-I1171T. Wild type ALK can be activated
3 with the ALKAL1 ligand (FAM150A, AUGβ) (Guan, Umapathy et al. 2015, Reshetnyak,
4 Murray et al. 2015) leading to extensive tyrosine phosphorylation of the receptor and
5 activation of downstream targets, such as ERK1/2 (Fig. 4B). In contrast to wild type ALK we
6 observed ligand-independent auto-trans-phosphorylation of the ALK-I1171T mutant together
7 with activation of the downstream target ERK1/2 (Fig. 4B).
8 We next investigated whether the ALK-I1171T mutant was capable of stimulating
9 neurite outgrowth in the PC12 cell line. PC12 cells are a clonal rat adrenal
10 pheochromocytoma cell line with enteric cell origin, which differentiate and extend neurites
11 upon extended ERK1/2 stimulation (Cowley, Paterson et al. 1994). We and others have
12 previously shown that activation of ALK triggers differentiation of PC12 cells into
13 sympathetic-like neurons, a process characterized by extension of neurites (Fig. 4C) (Motegi,
14 Fujimoto et al. 2004, Schonherr, Ruuth et al. 2011). Expression of wild type ALK mediates
15 only a low level of neurite outgrowth in this assay (Fig. 4C). In contrast, expression of the
16 ALK-I1171T mutant results in robust neurite outgrowth to a level similar to that observed for
17 the well characterized ALK-F1174L mutant employed as positive control.
18 Finally, we investigated whether the human ALK-I1171T mutant displays transforming
19 potential in NIH3T3 cells. Expression of either the positive control human ALK-F1174L or
20 the ALK-I1171T mutant led to formation of foci of transformed cells over the background
21 monolayer (Fig. 4D), whereas expression of the wild type human ALK receptor was unable to
22 mediate foci formation. Thus, ALK-I1171T exhibits intrinsic transforming activity in a focus
23 formation assay, in addition to ligand-independent activation of downstream targets and
24 neurite outgrowth.
25
16
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1 Ceritinib abrogates growth in ALK-addicted neuroblastoma cell lines.
2 Ceritinib is a small molecule ALK TKI that has been extensively explored for ALK-
3 positive NSCLC and is FDA approved for clinical use in that setting. It has not yet been
4 reported as being employed clinically in the context of ALK mutations in neuroblastoma.
5 Previous analysis of the I1171T mutation in NSCLC (arising in response to sequential
6 crizotinib and alectinib treatment) noted that the I1171 mutation is relatively insensitive to
7 inhibition by crizotinib and alectinib but was still sensitive to other ALK TKIs such as
8 TAE684 and ceritinib (Katayama, Friboulet et al. 2014). We therefore assembled a panel of
9 neuroblastoma cell lines harboring either mutated or wildtype ALK in combination with other
10 genetic aberrations, CLB-GE, CLB-GA, CLB-BAR, CLB-PE, IMR32, Kelly, SK-N-AS, SK-
11 N-DZ and SK-N-BE (Table 1) to examine the effect of ceritinib on ALK activity and cell
12 proliferation in a neuroblastoma setting (Kohl, Gee et al. 1984, Combaret, Turc-Carel et al.
13 1995, Schleiermacher, Janoueix-Lerosey et al. 2003, Cazes, Louis-Brennetot et al. 2013,
14 Fransson, Hansson et al. 2015, Umapathy, Guan et al. 2017). In this analysis we also
15 compared the ability of ceritinib to inhibit proliferation of neuroblastoma cells with the well-
16 characterized clinical ALK TKI crizotinib (Christensen, Zou et al. 2007), treating cells with
17 increasing doses of either crizotinib or ceritinib. Proliferation of ALK-addicted cell lines such
18 as CLB-BAR, CLB-GE, CLB-GA and Kelly was inhibited in a dose-dependent manner by
19 both crizotinib and ceritinib (Table 1). Of the neuroblastoma cell lines tested, growth of CLB-
20 BAR and CLB-GE was inhibited in a manner similar to that previously shown for crizotinib
21 (Schonherr, Ruuth et al. 2012), although the observed IC50 values differ up to 2-fold. IC50
22 values were similar for both ceritinib and crizotinib in CLB-GA and Kelly cells with values of
23 approximately 110 nM and 330 nM, respectively (Table 1). Importantly, we did not observe
24 inhibition of proliferation in non-ALK-addicted cell lines such as CLB-PE, SK-N-AS SK-N-
25 BE, SK-N-DZ, which carry other driver mutations. IMR32 cells, which express wild type
17
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1 ALK and respond to stimulation with ALKAL ligands (Guan, Umapathy et al. 2015), are
2 more sensitive to crizotinib than ceritinib, although IC50 values are high, more than 350 nM
3 (Table 1). Thus, these preclinical results show that while activity of the ALK-dependent
4 neuroblastoma cells tested here is inhibited by crizotinib, treatment with ceritinib has greater
5 efficacy.
6
7 Ceritinib effectively inhibits the crizotinib resistant ALK-I1171T mutant in PC12 cells.
8 To determine which ALK TKI would be suitable in this clinical case we examined the
9 ability of different ALK inhibitors to abrogate ALK-I1171T activity. As readout for ALK
10 activity we employed phosphorylation of ALK Y1604, which reflects ALK activation, in
11 PC12 cells (Chand, Yamazaki et al. 2013). PC12 cells expressing ALK-I1171T were treated
12 with serial dilution of ALK inhibitors crizotinib, ceritinib, lorlatinib and brigatinib and Y1604
13 phosphorylation levels were quantified. We observed that full length ALK-I1171T was more
14 resistant to crizotinib than other inhibitors as measured by Y1604 phosphorylation (Fig. 4E).
15 The IC50 of crizotinib for ALK-I1171T in this analysis was 193 ± 57 nM, approximately 11
16 times that of ceritinib for ALK-I1171T (Fig. 4F). The next generation ALK TKIs also showed
17 strong anti-ALK-I1171T activity. The observed IC50 values of ALK-I1171T for brigatinib and
18 lorlatinib were in a similar range (6.8 nM to 7.5 nM), approximately 1/28th of the crizotinib
19 IC50 for ALK-I1171T (Fig. 4F). Taken together, our results indicate that ceritinib as well as
20 the third generation TKIs lorlatinib and brigatinib represent experimentally well supported
21 choices for treatment of tumor(s) harboring the ALK-I1171T mutation.
22 To extend our analysis we examined the activity of ceritinib to inhibit a range of gain-
23 of-function ALK variants expressed in PC12 cells, employing ALK Y1604 phosphorylation
24 as readout (Fig. S2; includes all loading ALK control blots for this experiment). In this
25 analysis crizotinib abrogates wild type ALK phosphorylation with an IC50 of approximately
18
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1 18 nM, which is similar to earlier reports (Schonherr, Ruuth et al. 2011, Siaw, Wan et al.
2 2016). In comparison, ceritinib blocks ALK Y1604 phosphorylation with a 3-fold lower IC50
3 level, of approximately 5 nM. The IC50 values for crizotinib inhibition of ALK Y1604
4 phosphorylation of the various ALK mutations tested here are similar to earlier reports,
5 highlighting a requirement for higher crizotinib dosage for effective inhibition of ALK-
6 I1171N (Table 2) (Schonherr, Ruuth et al. 2011, Guan, Tucker et al. 2016, Siaw, Wan et al.
7 2016). ALK-G1269A has only been described in EML4-ALK fusions arising in NSCLC
8 patients that have developed resistance to ALK TKI treatment (Doebele, Pilling et al.
9 2012), but is included here as reference in the context of full length ALK.
10 Our extensive examination of the inhibition profiles of both crizotinib and ceritinib on
11 the different ALK mutations reveals one important exception - ALK-I1171T. We observe that
12 inhibition of ALK-I1171T by ceritinib exhibits an IC50 value (17.4 ± 3.1, Fig. 4F) that is not
13 too far from that of wild type ALK (5.3 ± 0.2) (Table 2). This is in stark contrast to the ALK-
14 I1171N mutation at the same residue, which displays resistance to both ceritinib (46.6 ± 0.16)
15 and crizotinib (139.5 ± 13.8) when compared with wild type ALK (Table 2). In summary,
16 ceritinib was active in a range comparable to crizotinib as measured by inhibition of ALK
17 phosphorylation/activation in PC12 cells with one exception, the ALK-I1171T mutant which
18 was insensitive to crizotinib but efficiently inhibited by ceritinib.
19
20 Phosphoproteomic profiling of ceritinib in different neuroblastoma cell lines
21 Phosphoproteomic profiling has clinical value since it can be translated into prognostic
22 biomarkers, which can be employed to identify functional downstream targets as
23 combinatorial treatment target options together with ceritinib. Here we determined the
24 difference in the phosphoproteomic patterns upon treatment with ceritinib in ALK-addicted
25 (CLB-BAR and CLB-GE) neuroblastoma cell lines with that of a non-ALK-addicted (SK-N-
19
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1 AS) neuroblastoma cell line (Fig. 5A). The CLB-BAR and CLB-GE cell lines harbor
2 activating ALK mutations, are ALK/MYCN amplified, lack p53/RAS mutations and display
3 high levels of NF1 (Umapathy, Guan et al. 2017). The SK-N-AS cell line harbors an
4 activating mutation in the NRAS gene and exhibits a down-regulated NF1 expression leading
5 to an activation of the RAS-MAPK pathway. SK-N-AS has a 11q deletion, is non-MYCN
6 amplified, has no p53/ALK mutations and undetectable levels of ALK protein (Umapathy,
7 Guan et al. 2017). SK-N-AS was employed as a control cell line for off-target effects of the
8 inhibitors employed, since ALK protein is undetectable in this cell line with no MYCN
9 amplification.
10 To identify sites with altered phosphorylation associated with ALK activity, we utilized
11 an immunoaffinity-coupled LC-MS/MS approach by treating neuroblastoma cell lines with
12 ceritinib. Prior to MS, lysates were examined for ALK-, ERK- and AKT-activity, which have
13 previously been shown to be reduced upon ALK TKI treatment for 1 hour (Fig. S3). We
14 observed reduced phosphorylation levels of ALK and ERK in response to ceritinib, validating
15 response to inhibitor treatment (Fig. S3). Phosphoproteomic analysis of neuroblastoma cell
16 lines (CLB-BAR, CLB-GE and SK-N-AS) identified 2,223 phosphothreonine sites, 2,583
17 phosphotyrosine sites and 5,013 phosphoserine residue sites in 3,345 different proteins (Fig.
18 5A). All previously observed 11 ALK phosphotyrosine sites were identified and showed
19 drastically reduced phosphorylation signals in both ALK-addicted neuroblastoma cell lines
20 (i.e. CLB-BAR and CLB-GE) after ceritinib treatment. The strongest dephosphorylation
21 signals were measured at ALK tyrosine sites 1096, 1278, 1282 and 1283 (Fig. 5B). The latter
22 three sites were previously reported as important residues in the ALK activation loop
23 (Donella-Deana, Marin et al. 2005, Rush, Moritz et al. 2005, Rikova, Guo et al. 2007, Tartari,
24 Gunby et al. 2008, Wang, Wu et al. 2010, Sattu, Hochgrafe et al. 2013, Guan, Yamazaki et al.
25 2017). As expected, no phosphorylation signal was observed in the control SK-N-AS cell line.
20
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1 Furthermore, this strong dephosphorylation response was only observed for ALK and not for
2 any other RTK (Fig. S4). In CLB-BAR, 182 proteins were dephosphorylated and 52 proteins
3 were phosphorylated upon treatment with ceritinib (Fig. 5C). Similarly, in CLB-GE, 200
4 proteins were dephosphorylated and 120 proteins were phosphorylated (Fig. 5C). Treatment
5 with ceritinib in SK-N-AS revealed only 35 proteins that were dephosphorylated and 20
6 proteins that were phosphorylated. Few similarities were found between the RAS-addicted
7 SK-N-AS cell line and the ALK-addicted CLB-BAR/GE cell lines and, while phosphorylation
8 responses were specific between all cell lines with little overlap, 60 proteins were
9 dephosphorylated in both CLB-BAR and CLB-GE but not SK-N-AS (Fig. 5D and Table S2).
10 Assuming that these 60 proteins represent the most specific response to ceritinib, they were
11 employed for further analysis. To illustrate downstream phosphorylation targets of ALK
12 signaling activity that are inhibited upon treatment with ceritinib in ALK-addicted
13 neuroblastoma cells, we mapped the dephosphorylated proteins to a protein-protein
14 interaction network (Fig. 5E). This network analysis highlighted FRS2, IRS2, ERK1/2, AKT
15 family members, SHC family members, SOS1 and GAB1/2 as key components of the
16 underlying signaling pathways. Many of these proteins are known to be active in insulin, NGF
17 and FGFR receptor signaling pathways, as confirmed by a gene set enrichment analysis (Fig.
18 5F). Similar results were found when independently focusing on the 182 and 200
19 dephosphorylated proteins of the CLB-BAR and CLB-GE cell lines respectively (Figs. S5 and
20 S6).
21
22 Patient response to ceritinib.
23 Based on the experimental data presented here that indicated a potentially poor response
24 to crizotinib, as well as evidence for FA prohibiting use of conventional chemotherapy or
25 irradiation, the patient was started on ceritinib monotherapy as soon as severe acute toxicity
21
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1 had subsided (initiated 9.5 months after diagnosis, indicated by *; Fig. 3A). Initial dosage was
2 225 mg once daily (450 mg/m2 body surface area) for a period of 10.5 months, after which
3 dosage was increased to 540 mg/m2 body surface area. Hematological counts were stabilized
4 at normal levels (Fig. 3A). After the first 4 week cycle, elevated urinary catecholamines had
5 decreased to levels below the upper reference limit (Fig. 3B). Other than mild gastrointestinal
6 side effects, no toxicity was observed, and the child was able to lead a normal life (Kim,
7 Mehra et al. 2016). After 6.5 months of ceritinib treatment, the primary tumor had decreased
8 in size (-43.6% according to MRI with volumetric measurements, Fig. 1) and the remaining
9 CNS metastasis showed response by MRI (Fig. S1), although not as sensitive as CT scans
10 performed at diagnosis and after 21 months of treatment showing complete metastatic
11 remission (Fig. 1). The remaining primary was radically removed surgically together with the
12 left adrenal gland at 7.5 months from start of ceritinib treatment. The tumor was subjected to
13 immunohistochemical analysis with both proliferation and differentiation markers. The
14 treated tumor displayed a large proportion of calcification and non-malignant stroma, reduced
15 cell proliferation as measured by Ki-67 positivity (5% versus 27% before treatment) as well
16 as hallmarks of differentiation as assessed with NB84 and NFP (Fig. 6). The overall
17 morphology of the primary tumor indicated differentiation in response to treatment,
18 resembling a ganglioneuroblastoma, and was rich in schwannian stroma containing scattered
19 foci of tumor cells of varying degrees of maturation to ganglion cells. Complete clinical
20 evaluation with CT and MRI one year after surgery and 21 months after initiation of ceritinib
21 treatment showed no residual tumor in the abdomen, and complete resolution of metastases at
22 all sites (Fig. 1). MIBG-I123 scintigraphy showed normal findings, indicating completely
23 resolved tumor activity (Fig. 1B). After 34 months of continuous ceritinib treatment, the child
24 is in continous complete remission (Park, Bagatell et al. 2017).
22
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1 Discussion
2 Here we report the case of a metastatic high-risk neuroblastoma that was treated
3 according to HR_NBL-1 SIOPEN protocol and developed severe toxicity including protracted
4 neutropenia, anemia and thrombocytopenia that required intensive clinical care for 4 months.
5 During this time genetic analyses revealed inherited biallelic sequence variants in FANCA.
6 This confirmed the initial suspicion of FA as likely cause of the observed toxicity and a
7 potential contributing factor to tumor development. Genomic analysis also detected a somatic
8 ALK-I1171T mutation that has not previously been described in neuroblastoma. This
9 presented a clinical option to treat the child, whose FA status precluded further chemotherapy
10 or irradiation, with targeted inhibition of ALK. Therefore an extensive preclinical
11 experimental investigation of the ALK-I1171T mutant was performed.
12 ALK-I1171T exhibits robust gain-of-function activity in several different systems, such
13 as triggering neurite outgrowth in PC12 cells, activation of validated downstream ALK targets
14 and transformation competence in NIH 3T3 cells. A critical part of this investigation was
15 defining the sensitivity of ALK-I1171T to available ALK TKIs, including crizotinib, ceritinib,
16 brigatinib and lorlatinib. Of these, the next-generation inhibitors ceritinib, brigatinib and
17 lorlatinib, which have been explored in preclinical neuroblastoma models (Guan, Tucker et al.
18 2016, Wood, Krytska et al. 2016), were effective in inhibition. Most importantly, crizotinib
19 was a very poor inhibitor of ALK-I1171T activity and does not efficiently cross the blood
20 brain barrier. This is in agreement with data from non-small cell lung cancer patients, where
21 resistance mutations in ALK fusions have been reported at I1171, although in these cases to
22 Aspargine and Serine, and not as yet to Threonine (Ou, Klempner et al. 2014, Ou, Greenbowe
23 et al. 2015). We also performed a preclinical LC-MS/MS analysis of ceritinib activity in
24 neuroblastoma confirming a strong dephosphorylation response at all tyrosine residues that
25 are known to be involved in the ALK activation loop, but not in any other RTK, indicating the
23
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1 specificity of the response. The downstream signaling response included FRS2, ERK1/2,
2 AKT family members, SHC family members and GAB1/2 amoung others and was similar to
3 insulin, NGF and FGFR signaling pathways. Based on the collective preclinical information,
4 the child was included in the compassionate use individual patient program for ceritinib.
5 The patient responded strongly to ceritinib with minimal side effects allowing the child
6 to lead a normal life. To date, the patient is in complete clinical remission after continuous
7 ceritinib mono-therapy treatment for 34 months. It is unclear to what extent the efficacy of
8 ceritinib treatment may have been influenced by the initial chemotherapy treatment which
9 induced partial remission. One possibility that should be considered, is that the initial
10 chemotherapy treatment sensitized the tumor to the subsequent ALK TKI treatment. A
11 preclinical report investigating combination of crizotinib with commonly employed
12 chemotherapy showed synergistic cytotoxicity and increased apoptosis in neuroblastoma cell
13 lines with ALK aberrations, supporting this senario (Krytska, Ryles et al. 2016). However, it
14 is important to note that at initiation of ceritinib treatment the patient displayed significant
15 active disease, as shown by persistant elevated urine catecholamine markers, metastatic
16 disease and remaining primary tumor tissue, which all responded strongly and rapidly to ALK
17 TKI therapy with finally complete clinical remission at all sites.
18 In conclusion, we have shown the importance of comprehensive genetic profiling
19 combined with preclinical investigation, which was conducted during a forced drug holiday of
20 a neuroblastoma patient later discovered to present with FA due to FANCA mutations and an
21 ALK mutation. A compassionate use protocol with ceritinib has shown a clear benefit for the
22 patient who is in complete clinical remission with clearance of all metastatic sites 34 months
23 from start of targeted ceritinib treatment. After the intial 7.5 months of treatment with
24 ceritinib the primary tumor had decreased in size and was removed surgically. The removed
25 tumor displayed a large amount of calcification and non–malignant stroma, reduced
24
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1 proliferation index and hallmarks of differentiation. Here we show that mono-therapy with
2 next generation ALK TKIs can be effective against metastatic high-risk neuroblastoma
3 carrying an activating ALK mutation, confirming ceritinib as a viable therapeutic option for
4 ALK-positive neuroblastoma patients.
5
25
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1 Additional information
2
3 Data deposition and Access
4 The detected DNA variants in FANCA and ALK have been submitted to ClinVar
5 (http://www.ncbi.nlm.nih.gov/clinvar/) and can be found under accession numbers
6 SCV000777897 - SCV000777901.
7
8 Ethics Statement
9 Treatment was initiated according HR-NBL-1 SIOPEN protocol (permit 02-294). A Clinical
10 Ethical Board meeting and an endorsed license application from the MPA license (according
11 to LVFS 2008:1) supported LDK378 compassionate use. Written informed consent from the
12 patient's guardians was obtained and the patient was included in the Novartis compassionate
13 use Program for ceritinib (CLDK378A2003M). Tumor analyses and linkage to clinical
14 information were performed according to the ethical permit 2009/1369-31/1. Written
15 informed consent was obtained from the patient's guardians allowing the scientific research
16 publication and are filed with the case notes.
17
18 Acknowledgements
19 This work has been supported by grants from the Swedish Cancer Society
20 (TMCAN2015/794; BHCAN15/775; RHP CAN15/391; PK CAN16/3625), the Swedish
21 Childhood Cancer Foundation (TM PR2016-0147; BH 2015-80, and 2014-150; RHP 2015-
22 96; PK PR2014-0084; JG 2016-0011; SF 15-0061; DT 12-009 and 12-002), the Swedish
23 Research Council (RHP 2015-04466; BH 521-2012-2831; TM 521-2014-3031; PK 2014-
24 3036); the Swedish Foundation for Strategic Research (RB13-0204, www.nnbcr.se) and the
25 Göran Gustafsson Foundation (RHP2016). D.T was supported by a PhD-student program
26
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1 (NC2012-0026), J.G. was supported by a Postdoctoral Research Fellow Position (TJ2016-
2 0088) and S.F. was supported by a Research Assistant Fellowship (14-0064), by the Swedish
3 Childhood Cancer Foundation. The CLB-BAR, CLB-GE, CLB-GA, and CLB-PE cell lines
4 used in this study were kindly provided by Valérie Combaret under an MTA agreement with
5 the Center
6 Léon Bérard laboratory (Lyon, France). Novartis supplied ceritinib free of charge within the
7 compassionate use program “Ceritinib IPP for ALK-Positive IMT, ALCL and Neuroblastoma
8 Pediatric Patients”.
9
10
11
12
27
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1 Figure Legends
2
3 Figure 1. Patient tumor imaging. Disease at initial diagnosis and after targeted therapy
4 in full clinical remission (A-D). (A) Whole body MIBG-I123 scintigraphy showing extensive
5 metastatic disease at diagnosis of high-risk neuroblastoma (upper panels). Left panel –
6 anterior view; Right panel – posterior view. Lower panels display head and neck region. (B)
7 MIBG-scintigraphy after 21 months of ceritinib therapy showing complete remission at all
8 metastatic sites. (C) Brain CT scans at diagnosis showing a CNS metastasis (43 mm x 46.7
9 mm) posterior to the right orbit. (D) CT scans after 21 months of ceritinib therapy showing
10 complete metastatic response. (E-H) Primary tumor immediately before and after
11 targeted therapy (E-H). (E) Magnetic resonance imaging (MRI) of primary abdominal
12 tumor (indicated by arrows and measurements inserted) immediately before start of
13 ceritinib targeted therapy (approximately 18.8 mL tumor volume), (F) MRI after 3
14 months therapy (approximately 13.8 mL tumor volume) and (G) MRI after 6 months
15 therapy immediately prior to radical surgery (approximately 10.6 mL tumor volume).
16 (H) Decrease of evaluable tumor volume after 3 and 6 months of targeted therapy.
17
18
19 Figure 2. Genomic analysis of the tumor sample. (A) Copy number profiling using
20 Affymetrix HD SNP-microrray shows a genomic profile with 11q-deletion and 17q-gain
21 indicative of high-risk neuroblastoma. (B) Two different likely pathogenic variants in the
22 FANCA gene were detected through exome sequencing and confirmed in constitutional DNA
23 of the patient. Read mapping is visualized in IGV. (C) Exome sequencing also detected a
24 novel ALK I1171T mutation in the tumor that was confirmed to be somatic through Sanger
28
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1 sequencing. Electrophorogram of tumor in upper panel and from constitutional DNA in lower
2 panel.
3
4 Figure 3. Hematological counts and catecholamine metabolites in urine. (A) After
5 conventional high-risk neuroblastoma chemotherapy was started, the patient suffered severe
6 bone marrow toxicity with protracted anemia and thrombocytopenia requiring frequent
7 transfusions (each peak indicates post transfusion measurement). When hematological counts
8 stabilized, ceritinib treatment could be started (*). Hemoglobin and platelet counts remained
9 stable during TKI treatment, allowing for surgery (#) with radical removal of the
10 differentiated and calcified decreased primary tumor. (B) Catecholamine metabolites
11 (dopamine in black, homovanillic acid, HVA in red, and vanillylmandelic acid, VMA in
12 green) as neuroblastoma markers in the patient's urine (molar concentrations/creatinine
13 concentration). During ceritinib therapy (*), elevated urine catecholamine metabolites
14 returned to normal (below respective dashed line), indicating a biologically inactive residual
15 tumour. At surgery (#) normal levels had been reached.
16
17 Figure 4. Characterization of ALK-I1171T with cell culture systems. (A) Model of the
18 ALK kinase domain (PDB #3LCS) employing PyMol, which can be divided into the upper N-
19 terminal lobe and the lower C-terminal lobe by the hinge region and ceritinib binding/ATP
20 (red) binding pocket. Regulatory spine (R-spine) residues are shown in magenta. The spine is
21 anchored in the αF helix (D1311), and includes the DFG (F1271) and the HRD motif (H1247)
22 of the C-lobe. R-spine residues in the N-terminal lobe include the β4 strand (C1182) and the
23 residue mutated in this patient in the α-C-helix (I1171). Mutation of residue I1171 to
24 threonine in PyMol, results in a small shift, that potentially drives a dynamic allostery that
29
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1 results in a gain-of-function ALK activity (upper insert:wild type ALK kinase domain; lower
2 insert: ALK-I1171T kinase domain). (B) ALK kinase activity and activation of downstream
3 signaling pathways were visualized by western blot with antibodies against pALK (Y1604)
4 and pERK1/2. Total ALK and pan-ERK were used as loading controls. Blots are
5 representative of three independent experiments. (C) Neurite outgrowth of PC12 cells as a
6 readout for ALK kinase activity was performed with wild type ALK and ALK variants in the
7 absence or presence of ALKAL1 ligand. Bars represent mean percentage ± STD of neurite-
8 carrying cells among GFP-positive cells from three independent experiments. (D)
9 Representative focus formation assays for NIH 3T3 cells transfected with wild type ALK,
10 ALK variants (ALK-I1171T and ALK-F1174L) or empty vector. (E) Inhibition profiling of
11 ALK TKIs on ALK-I1171T. PC12 cells expressing ALK-I1171T were treated with serial
12 dilution of ALK inhibitors as indicated. Phosphorylation of ALK was detected with pALK
13 (Y1604) antibody and total ALK was used as loading control. (F) IC50 values of different
14 ALK inhibitors were calculated with GraphPad Prism 6.0 by fitting data to a log (inhibitor
15 concentration) vs. normalized response (variable slope) equation and shown in the
16 accompanying table. Values represent average ± STD from three independent experiments.
17
18 Figure 5. Ceritinib phosphoproteomic profile in neuroblastoma cell lines. (A) Three
19 neuroblastoma cell lines (CLB-BAR, CLB-GE and SK-N-AS) were cultured in control
20 conditions or in the presence of ceritinib. Cells were harvested for tyrosine and
21 serine/threonine phosphoproteomic analysis after 60 mins. Pie chart indicates the number of
22 targeted sites. (B) Overview of all ALK tyrosine phosphorylation sites with log2 fold change
23 values (FC; treated/untreated) in CLB-BAR and CLB-GE. Note that no signal was measured
24 in SK-N-AS cells reflecting known absent expression of ALK. (C) Log2 FC values of all
25 analyzed phosphosites indicating the number of proteins found to contain phosphorylated
30
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1 (red) or dephosphorylated (green) sites for the three cell lines analyzed. Dotted lines indicate
2 thresholds to determine differential phosphorylation. (D) Venn diagrams show the correlation
3 between phosphorylated (red) and dephosphorylated (green) proteins in the different cell
4 lines. Sixty proteins were found to be dephosphorylated upon ceritinib treatment in the ALK-
5 addicted CLB-BAR and CLB-GE lines but not in the SK-N-AS cell line. These proteins were
6 mapped to the InWeb_InBioMap protein protein interaction network (E). For visualization
7 purposes only direct interactions between the identified proteins are shown. Node sizes are
8 proportional to the number of connections while node colors indicate different log2 FC values
9 as indicated in the color scale legend on the top left. (F) Reactome gene set enrichment
10 analysis on all 60 proteins that were identified to be dephosphorylated. The 10 most
11 significantly enriched pathways are shown and ranked on FDR values.
12
13 Figure 6. Immunohistochemical analysis of tumor material. Tumor sample taken in
14 November 2014 was stroma-poor, comprising of small, round, primitive appearing cells
15 positive for the Ki-67 proliferation marker in up to 27%. Post ceritinib treatment resected
16 tumor displayed few Ki-67 positive cells, and increased expression of NB84 and NFP, that are
17 markers for differentiated neuroblastoma.
18
19
31
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1 Supplementary Figure Legends
2
3 Figure S1. Patient tumor imaging. (A-B) CNS metastasis immediately before and after
4 targeted therapy. (A) MRI of CNS with metastasis indicated by arrows immediately before
5 start of targeted therapy. (B) MRI of CNS after 6 months of targeted therapy showing
6 decreased metastasis indicated by arrows. (C-D) Imaging showing metastatic bone
7 involvement at diagnosis and after completed targeted therapy. (C) 3D bone visualization of
8 CT scan at diagnosis showing metastatic bone involvement of the skull. (D) CT scan after
9 completed 21 months of targeted therapy with no remaining metastatic bone involvement.
10
11 Figure S2. Ceritinib blocks phosphorylation of mutant ALK variants expressed in PC12
12 cells. (A) PC12 cells were transfected with ALK-mutant variants or ALK-wild type as
13 indicated. 48 hours post-transfection cells were treated with serial diluted amounts of either
14 ceritinib or crizotinib. Whole cell lysates were blotted for pALK (Y1604) to measure ALK
15 activation/phosphorylation inhibition. (B) Western blot analysis of ALK expression, in PC12
16 whole cell lysate. (C) Inhibition profiles for ceritinib and crizotinib were determined with
17 GraphPad Prism 6.0 by fitting data from A, to a log (inhibitor concentration) vs. normalised
18 response (variable slope) equation. IC50 values are shown in Table 1. Values represent
19 average ± STD from at least two independent experiments.
20
21 Figure S3. Ceritinib inhibits ALK activity and proliferation of ALK addicted
22 neuroblastoma cell lines. (A) CLB-BAR (ALK-∆4-11), and CLB-GE (ALK-F1174F), both
23 ALK addicted cell lines and SK-N-AS (Ras-Q61K), a RAS addicted cell line, were treated
32
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1 with increasing concentrations of ceritinib for 72 hours and cell viability assessed by resazurin
2 assay. Plotted values are means ± SE from growth curves from at least three independent
3 experiments preformed in triplicate. (B) CLB-BAR, CLB-GE and SK-N-AS were treated with
4 crizotinib (250 nM) and ceritinib (200 nM) for 1 hour. Non-treated cells was employed as a
5 negative control. Cell lysates were resolved by SDS/PAGE followed by immunoblotting as
6 indicated.
7
8 Figure S4. Ceritinib phosphorylation response of different tyrosine receptor kinases
9 (RTKs) in neuroblastoma cell lines. Log2 FC values of all analyzed phosphosites in
10 different RTKs and neuroblastoma cell lines as indicated. Median values are indicated by
11 horizontal lines.
12
13 Figure S5. Ceritinib phosphoproteomic profile in CLB-BAR. (A) Reactome gene set
14 enrichment analysis on all 182 proteins that were dephosphorylated upon ceritinib treatment.
15 The 10 most significantly enriched pathways are shown and ranked on FDR values. (B) All
16 dephosphorylated proteins were mapped to the InWeb_InBioMap protein protein interaction
17 network. For visualization purposes only direct interactions between the identified proteins
18 are shown. Node sizes are proportional to the number of connections while node colors
19 indicate different fold change values as indicated in the color scale legend on the top left.
20
21 Figure S6. Ceritinib phosphoproteomic profile in CLB-GE. (A) Reactome gene set
22 enrichment analysis on all 200 proteins that were dephosphorylated upon ceritinib treatment.
23 The 10 most significantly enriched pathways are shown and ranked on FDR values. (B) All
33
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1 dephosphorylated proteins were mapped to the InWeb_InBioMap protein protein interaction
2 network. For visualization purposes only direct interactions between the identified proteins
3 are shown. Node sizes are proportional to the number of connections while node colors
4 indicate different fold change values as indicated in the color scale legend on the top left.
5
6
7
8
34
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1 Table Legends
2
3 Table 1
4 Indicated neuroblastoma cell lines were treated with increasing doses of either ceritinib or
5 crizotinib for 72 hours and cell viability was assassed by resazurin assay. NA, non-amplified;
6 A - amplified; g-o-f ∆exon 4-11 – gain of function deletion of exons in the extracellular
7 domain of ALK; WT - wild type.
8 9
10 Table 2
11 IC50 values for inhibition of ALK Y1604 phosphorylation by ceritinib or crizotinib in PC12
12 cells. IC50 values were determined by quantification of Y1604 phosphorylation from the
13 immunoblots in Supplementary figure 1. Values represent mean ± SD from at least two
14 independent experiments.
15
35
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1 Supplementary Table Legends
2
3 Table S1. FANCA and ALK mutation details. Variant specific information including
4 position, transcript-ID, coverage and presence in publically available databases.
5
6 Table S2. Log2 FC values of 64 dephosphorylated proteins in ALK expressing
7 neuroblastoma cell lines. Sixty-four proteins were dephosphorylated upon ceritinib treatment
8 in the CLB-BAR and CLB-GE but not the SK-N-AS cell lines with indication of log2 FC
9 values for the three cell lines.
10
36
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TABLE 1
Cell line ALK status MYCN IC50 nM (ceritinib) IC50 nM (crizotinib) status CLB-GE F1174V, A A 131,3 ± 24 233,7 ± 18 CLB-BAR g-o-f ∆exon 4-12 A 36,1 ± 6.2 54,9 ± 20 KELLY F1174L, A A 328,9 ± 36 333,3 ± 78 CLB-GAR R1275Q NA 102,3 ± 16 119,3 ± 45 IMR32 Wt A ˃ 500 348,7 ± 45 SK-N-BE Wt, NA A ˃ 1500 ˃ 1000 SK-N-DZ Wt A ˃ 800 ˃ 4000 SKNAS Wt, NA NA ˃ 3000 353,0 ± 27 CLB-PE Wt A ˃ 15000 ˃ 4000
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TABLE 2
ALK mutation Ceritinib Normalised to Crizotinib Normalised to Fold (IC50 nM) IC50 of WT (IC50 nM) IC50 of WT change WT 5.3 ± 0.2 1.0 17.8 ± 0.2 1 3.4 G1128A 19.8 ± 3.5 3.7 22.2 ± 3.4 1.2 1.1 I1171N 46.6 ± 16 8.8 139.5 ± 13.8 7.8 3.0 F1174L 16.9 ± 8 3.2 29.9 ± 4.8 1.7 1.8 R1192P 26.1 ± 1.2 4.9 28.1 ± 7.5 1.6 1.1 F1245C 24.6 ± 6.4 4.6 37.5 ± 7.7 2.1 1.5 G1269A 28.9 ± 3.6 5.5 113.5 ± 8.5 6.4 3.9 R1275Q 10.8 ± 3.5 2.0 30.3 ± 0.9 1.7 2.8 Y1278S 20.5 ± 0.1 3.9 69.5 ± 0.03 3.9 3.4 Downloaded from molecularcasestudies.cshlp.org on October 9, 2021 - Published by Cold Spring Harbor Laboratory Press
Figure1 A B
C D
E F G H FigureDownloaded 2 from molecularcasestudies.cshlp.org on October 9, 2021 - Published by Cold Spring Harbor Laboratory Press Figure 3Downloaded from molecularcasestudies.cshlp.org on October 9, 2021 - Published by Cold Spring Harbor Laboratory Press
A
B Downloaded from molecularcasestudies.cshlp.org on October 9, 2021 - Published by Cold Spring Harbor Laboratory Press Figure 4
A B
pcDNA3 + + - - - - ALK-WT - - + + - - ALK-I1171T - - - - + - ALK-F1174L - - - - - + ALKAL1 - + - + - -
pALK
ALK
pERK1/2
panERK
C D
pcDNA3 ALK-WT
pcDNA3 + + - - - - ALK-WT - - + + - - ALK-I1171T - - - - + - ALK-F1174L - - - - - + ALK-I1171T ALK-F1174L ALKAL1 - + - + - -
E F
Inhibitor (nM): 0 5 10 25 50 100 250 500 1000
pALK crizotinib ALK
pALK ceritinib ALK
pALK lorlatinib ALK ALK TKI IC50 nM crizotinib 193 ± 57 pALK brigatinib ceritinib 17.4 ± 3.1 ALK lorlatinib 6.8 ± 1.3
brigatinib 7.5 ± 0.4 Downloaded from molecularcasestudies.cshlp.org on October 9, 2021 - Published by Cold Spring Harbor Laboratory Press Figure 5
A B Log2 FC CLB-BAR Ceritinib (LDK-378) 3,345 proteins Site CLB-BAR CLB-GE CLB-GE Y1078 -1.98 -2.56 S SK-N-AS Y1092 -1.52 -1.87 5,013 Y1096 -3.49 -3.56 P Y1131 -1.74 -1.82 2,223 2,583 Y1278 -3.63 -2.79 Y1282 -3.42 -2.94 T Y Phosphoproteomics Y1283 -2.98 -1.93 Y1401 -0.41 0h 1h Y1507 -0.82 -1.47 Y1584 -1.49 -2.15 Y1586 -1.70 -2.15 Y1604 -1.36 -2.15 C E Log2 FC >4.5 SHC3 3.5-4.5 NEK9 BICD2
2.5-3.5 SHC2 EIF4B 5 1-5-2.5 FNBP1L TNKS1BP1 <1.5 ALK RPS6 AP2B1
GAB1
20 proteins 20 52 proteins 52 120 proteins 120 AKT1 FRS2 MAPK1 SOS1 PDCD11
AKT3 0 PTPN11 MAPK3 CCT2 GAB2 SLC35E1 BCR
Log2 FC treated/untreated FC Log2 MAP1A HSF1 IRS2
-5 SLC35B3 ERF
35 proteins 35 200 proteins 200 182 proteins 182 RBM4 PDCD4
F CLB-GE CLB-BAR SK-N-AS Reactome GSEA (60 dephosphorylated proteins)
INSULIN_RECEPTOR_SIGNALLING_CASCADE SIGNALING_BY_INSULIN_RECEPTOR D SK-N-AS SK-N-AS NGF_SIGNALLING_VIA_TRKA_FROM_THE_PLASMA_MEMBRANE SIGNALING_BY_FGFR_IN_DISEASE SHC_MEDIATED_SIGNALLING 17 26 SIGNALING_SIGNAL_ATTENUATION 1 1 3 2 SHC_RELATED_EVENTS 1 4 SIGNALLING_TO_ERKS 44 6 112 115 60 134 SIGNALLING_BY_NGF PI3K_CASCADE
CLB-BAR CLB-GE CLB-BAR CLB-GE 0 2 4 6 8 -log10 (FDR) Figure 6 Downloaded from molecularcasestudies.cshlp.org
Ki-67 NFP NB84 Nov 2014 Nov onOctober9,2021-Publishedby Dec 2015 Dec Cold SpringHarborLaboratoryPress
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Clinical response of the novel activating ALK-I1171T mutation in neuroblastoma to the ALK inhibitor ceritinib.
Jikui Guan, Susanne Fransson, Joachim Tetteh T Siaw, et al.
Cold Spring Harb Mol Case Stud published online June 15, 2018 Access the most recent version at doi:10.1101/mcs.a002550
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Published online June 15, 2018 in advance of the full issue.
Accepted Peer-reviewed and accepted for publication but not copyedited or typeset; Manuscript accepted manuscript is likely to differ from the final, published version. Published onlineJune 15, 2018in advance of the full issue.
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