Downloaded from molecularcasestudies.cshlp.org on October 9, 2021 - Published by Cold Spring Harbor Laboratory Press 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. 19 20 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]). 26 27 1 Downloaded from molecularcasestudies.cshlp.org on October 9, 2021 - Published by Cold Spring Harbor Laboratory Press 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 Downloaded from molecularcasestudies.cshlp.org on October 9, 2021 - Published by Cold Spring Harbor Laboratory Press 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 Downloaded from molecularcasestudies.cshlp.org on October 9, 2021 - Published by Cold Spring Harbor Laboratory Press 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 Downloaded from molecularcasestudies.cshlp.org on October 9, 2021 - Published by Cold Spring Harbor Laboratory Press 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