The role of the ALK receptor in cancer biology

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Annals of Oncology 27 (Supplement 3): iii4–iii15, 2016 doi:10.1093/annonc/mdw301

The role of the ALK receptor in cancer biology B. Hallberg* & R. H. Palmer*

A vast array of oncogenic variants has been identified for anaplastic lymphoma kinase (ALK). Therefore, there is a need to better understand the role of ALK in cancer biology in order to optimise treatment strategies. This review summarises the latest research on the receptor tyrosine kinase ALK, and how this information can guide the management of patients with cancer that is ALK-positive. A variety of ALK gene alterations have been described across a range of tumour types, including point mutations, deletions and rearrangements. A wide variety of ALK fusions, in which the kinase domain of ALK and the amino-terminal portion of various protein partners are fused, occur in cancer, with echinoderm microtubule-associated protein-like 4 (EML4)-ALK being the most prevalent in non-small-cell lung cancer (NSCLC). Different ALK fusion proteins can mediate different signalling outputs, depending on properties such as subcellular localisation and protein stability. The ALK fusions found in tumours lack spatial and temporal regulation, which can also affect dimerisation and substrate specificity. Two ALK tyrosine kinase inhibitors (TKIs), crizotinib and ceritinib, are currently approved in Europe for use in ALK-positive NSCLC and several others are in development. These ALK TKIs bind slightly differently within the ATP-binding pocket of the ALK kinase domain and are associated with the emergence of different resistance mutation patterns during therapy. This emphasises the need to tailor the sequence of ALK TKIs according to the ALK signature of each patient. Research into the oncogenic functions of ALK, and fast paced development of ALK inhibitors, has substantially improved outcomes for patients with ALK-positive NSCLC. Limited data are available surrounding the physiological ligand-stimulated activation of ALK signalling and further research is needed. Understanding the role of ALK in tumour biology is key to further optimising therapeutic strategies for ALK-positive disease. Key words: anaplastic lymphoma kinase, non-small-cell lung cancer, neuroblastoma, tyrosine kinase inhibitor, crizotinib, ceritinib

introduction discovery of the NPM-ALK fusion in ALCL Anaplastic lymphoma kinase (ALK), discovered in 1994, was first described as a fusion partner in the t(2;5) chromosomal translocation in anaplastic large cell lymphoma (ALCL), from which ALK takes its name [1]. This report revealed the identity of p80, a highly tyrosine-phosphorylated protein that had previously been described in ALCL [1, 2]. The NPM-ALK/p80 protein comprises the kinase domain of ALK together with an amino-terminal fusion derived from the nucleophosmin (NPM) protein, which serves to dimerise and activate the ALK kinase domain [1] (Figure 1A). NPM-ALK would prove to be the first of many ALK fusion proteins to be described in a wide range of cancer types.

the full-length ALK receptor tyrosine kinase and its ligands Several years passed before the unveiling of the full-length ALK receptor tyrosine kinase (RTK), which consists of an extracellular ligand-binding domain, a transmembrane domain and an *Correspondence to: Prof. Ruth Palmer and Prof. Bengt Hallberg, Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, SE-40530 Gothenburg, Sweden. Tel: +46-317863906; E-mail: ruth. [email protected]; Prof. Bengt Hallberg, Tel: +46-317863815; E-mail: [email protected]

intracellular tyrosine kinase domain [3, 4]. While the tyrosine kinase domain of human ALK shares a high level of similarity with that of the insulin receptor (IR), the ALK extracellular domain (ECD) is unique among the RTK family, containing a glycine-rich region, a low-density lipoprotein receptor class A (LDLa) domain and meprin, A-5 protein and receptor proteintyrosine phosphatase mu (MAM) domains [4, 5] (Figure 1A). The ALK extracellular domain. Based on the similarity of the ECDs, ALK and the leucocyte tyrosine kinase (LTK) RTK, which also has a glycine-rich region in its ECD (Figure 1A), represent a unique subfamily within the IR superfamily. The human ALK/LTK subfamily is activated by the recently described small secreted peptide/protein ligands FAM150A (AUGβ) and FAM150B (AUGα), which potently activate ALK signalling [6–8]. Whether additional activators of human ALK exist, and how important a role the FAM150A/B (AUG) ligands play in ALK-dependent cancer biology, remain key questions. The ALK ECD can be divided into several domains with presumed functions for ligand binding, interaction with potential co-receptors and secreted regulatory proteins, and dimerisation, all of which may potentially relay conformational changes that initiate activation of the intracellular kinase domain. Another potential regulatory mechanism involves proteolytic cleavage of

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review

Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden

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Annals of Oncology

A

B

ALK gain-of-function (ligand independent)

Jeb

FAM150

ALK wild-type (ligand dependent)

ceAlk (Scd-2)

dAlk

Cell membrane FP

HEN-1

FP

downstream signalling

ALK fusions (ligand independent)

PLC-γ

p130CAS

SRC

Jak/STAT

CBL

GRB2

CRKL

Pl3K

Cyclin D2

FAK

SHP2

NIPA

IRS1

SHC

C3G

Akt

MEK5

JUN

NF-κB

PTPN1

JNK

FRS2

RAS/ MAPK

RAP1

mTOR GSK3β Foxo

ERK5

Nuclear membrane

MEKK2/3

ERK5 MYCN, JUNB, C/EBP, BCL-2A1, MMP9, INK4A, HIF1-2α, VEGF

Figure 1. The anaplastic lymphoma kinase (ALK) receptor tyrosine kinase. (A) ALK signalling in cancer. ALK was first described as the carboxy-terminal portion of the NPM-ALK fusion. In fusions such as NPM-ALK, the amino-terminal fusion partner (FP, in blue) is fused to the intracellular tyrosine kinase domain of ALK (in red), leading to activation of downstream signalling. The full-length ALK receptor is a classical receptor tyrosine kinase, comprising an amino-terminal ECD and an intracellular tyrosine kinase domain (in red), connected by a single transmembrane domain (in green). The ALK ECD contains two MAM domains (in pink), one LDLa domain (in yellow) and a glycine-rich region (in grey). Human ALK is activated by the small secreted FAM150A (AUGβ) and FAM150B (AUGα) ligands, which are the only two proteins encoded by the human genome identified as containing a FAM150 (AUG) domain [6, 7]. Mutations in the context of full-length ALK, e.g. in neuroblastoma (gold stars) result in activation. ALK signals via numerous downstream pathways. These include PI3K-Akt activation, leading to signalling via mTOR, GSK3β and FOXO proteins. Akt also activates the MEKK2/3/MEK5/ERK5 pathway, which regulates MYCN transcription. Other pathways reported to be activated by ALK are the RAS-MAPK, CRKL-C3G-RAP1, JAK-STAT and JUN pathways. Proteins such as insulin receptor substrate 1 (IRS1), SHC, growth factor receptor-bound protein 2 (GRB2), phospholipase C-γ (PLCγ), CBL, CRKL and fibroblast growth factor receptor substrate 2 (FRS2) interact with and are phosphorylated by ALK upon activation. ALK activity stimulates initiation of transcription of a number of genes, including MYCN, JUNB, C/EBP, BCL2A1, MMP9, INK4A, HIF1-2a, VEGF, CDC42, FOXO, GSK3β, JNK, NF-κB, NIPA and SHH. (B) C. elegans ALK (ceALK) and D. melanogaster Alk (dAlk) are activated upon binding of the HEN-1 and Jeb ligands, respectively. In humans and mice, no Jeb/HEN-1-like ligand has been reported to date, and there is no structural similarity between the FAM150 (AUG) and HEN-1/Jeb proteins. Furthermore, FAM150 (AUG) domain-containing proteins do not appear to be present in the C. elegans or D. melanogaster genomes [6].

the full-length ALK receptor ECD [9] that releases an ECD fragment of around 80 kDa, as well as a highly tyrosine-phosphorylated 140 kDa truncated receptor. The physiological significance and the underlying molecular mechanisms of this cleavage event are unclear; cleaved ALK may be more stable/active than intact ALK, and/or the cleavage event may play a part in ligandmediated activation. The ALK kinase domain. Similar to other kinases, the ALK kinase domain comprises a conserved amino-terminal lobe and a carboxy-terminal lobe [10–12] linked with a ‘hinge’ region to form a cleft that presents a binding pocket for ATP (Figure 2A), allowing the catalytic kinase reaction to proceed. Additional internal structures within the kinase domain mediate not only flexibility but also the dynamic infrastructure of the kinase, allowing important allosteric regulation within and between the

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lobes that promotes the switch between active and inactive kinase conformations [13]. ALK contains two hydrophobic non-contiguous motifs, termed the ‘regulatory’ and ‘catalytic’ spines (Figure 2A). These comprise hydrophobic amino acids that span both lobes and are conserved across all kinases [10–12]. The catalytic spine is completed by the adenine ring of bound ATP (Figure 2A). The regulatory spine (comprising I1171, C1182, H1247, F1271 and D1311 in ALK) is generally assumed to be assembled when the kinase is active, and then disconnected upon inactivation, clearly demonstrated by the active and inactive conformations of IR (Figure 2C and D). Inactive ALK displays a number of the features of an active kinase, adopting an active but dynamically uncommitted configuration. For instance, structures of ALK in the apo- and ADP-bound forms show assembled regulatory spines [10–12]. Furthermore, all published ALK structures in

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Figure 2. The anaplastic lymphoma kinase (ALK) kinase domain. (A) Global cartoon view of the ALK kinase domain, which can be divided into amino- and carboxy-terminal lobes. The two kinase spines are shown; the catalytic spine residues shown as lime green, the regulatory spine (R-spine) residues shown as light pink. The spines are anchored in the αF helix of the carboxy-lobe and assemble through both lobes of the kinase. ADP is shown in magenta. The αC helix is shown in dark green. Under the αC helix is the structured short unique helical segment (blue) included in the activation loop (A-loop) following the DFG motif (the F residue in the DFG is included in the R-spine). HRD is in yellow (the D residue in HRD is also included in the R-spine). Residues contributing to the hydrophobic pocket are shown in orange. The amino-terminal two-stranded antiparallel β-sheet is indicated as purple. (Inset) Under the αC helix (green), a close up of the helical segment of the A-loop including residue Y1278 of the Y’RAS’YY autophosphorylation motif (light blue, indicated as sticks with dots) and C1097 ( purple, indicated as sticks with dots). Y1278 is inaccessible for phosphorylation since it is engaged in the interaction interface through bonding with C1097 in the amino-terminal β-sheet. Also shown are the two other tyrosines, 1282 and 1283, of the Y’RAS’YY autophosphorylation motif in the A-loop (light blue, sticks, no dots). (B–D) The regulatory and catalytic spines of ALK. Comparison of the regulatory and catalytic spines of ALK with those of the insulin receptor (IR) which are shown in the active and inactive state. (B) The inactive ALK kinase domain (PDB: 3LCT); shown in green is the αC-helix, and highlighted are the two hydrophobic non-contiguous motifs, conserved in all kinases, that span both the amino- and carboxy-terminal lobes of the kinase, termed the regulatory (light pink) and catalytic spines (lime green). The ALK regulatory spine comprises I1171 (N-lobe, α-helix), C1182 (N-lobe, β4 strand), H1247 (C-lobe, HRD motif), F1271 (C-lobe, DFG motif ) and D1311 (C-lobe, αF-helix). The catalytic spine comprises V1130, A1148, L1204, C1255, L1256, L1257, L1318 and I1322. (C and D) The kinase domain of IR in inactive (C) and active (D) conformations, with regulatory spine (light pink) and catalytic spine (lime green), and hydrophobic pocket (orange) in between (PDB: 1IRK and 1IR3). The regulatory spine is generally assumed to be assembled when the kinase is active and then disconnected upon inactivation, clearly seen in the case of active IR (D) and inactive IR (C). Inactive ALK, however, displays features of an active kinase such as an assembled regulatory spine (B).

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ALK signalling The characterisation of downstream ALK signalling events has focused on fusion proteins such as NPM-ALK and EML4-ALK, where kinase activation and signalling is driven by oligomerisation. This has been complemented in recent years by an increasing body of work examining the ALK-dependent signalling events in neuroblastoma, where the focus has been on the fulllength ALK receptor and the impact of associated mutations. Similarities and differences in signalling output between these two scenarios exist, due to a range of factors. These include differences in ALK fusion partners, differing tumour cell types and also, particularly in neuroblastoma, differences in the genetic background of the tumour cell. Wild-type ALK is a membrane-bound receptor. Several ALK variants displaying defects in intracellular trafficking have been reported, although the significance of this is as yet unclear [17]. In general, ALK activates multiple signalling pathways, such as the PI3K-AKT, CRKL-C3G, MEKK2/3-MEK5-ERK5, JAKSTAT and MAPK pathways (Figure 1). Activation of adaptor proteins and other cellular proteins, such as PTPN11, Src, FRS2, Shc-GRB2, IRS2, GSK-3a and FAK, has been observed downstream of ALK, indicating roles for alternative pathways. Other reported downstream ALK targets include BIM, p27, cyclin D2, NIPA, RAC1, CDC42, p130CAS, SHP1 and FYVE finger containing phosphoinositide kinase (PIKFYVE) [18–27]. In neuroblastoma, ALK signalling acts synergistically with MYCN to drive tumour development [28–33]; whether ALK

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fusion proteins function synergistically with c-MYC in nonsmall-cell lung cancer (NSCLC) remains to be seen. NPM-ALK has been shown to phosphorylate PLCγ, PI3K-Akt and JAK2 [34–36]; different ALK fusion proteins can mediate different signalling outputs, depending on properties such as subcellular localisation and protein stability [37, 38]. Proteomic posttranslational modification and metabolic profiling analyses have documented targets downstream of ALK, many of which remain to be functionally characterised [39–44]. At a genomic level, MYCN, JUNB, CDKN2A, HIF-1α, MMP9, CEBPB and BCL2A1, among others, have been shown to be activated by ALK [18, 30, 45–48]. In addition, several microRNAs have been implicated in NPM-ALK signalling, such as miR96, miR-135b, mir-29a and miR-16 [49–52].

the biological function of ALK: lessons from model organisms ALK in worm, fly and fish development ALK gene disruption has been investigated in a number of model organisms, including the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans and the zebrafish Danio rerio. A single ALK family member is observed in worms (SCD-2) [53] and flies (Alk) [5]. In C. elegans, ALK (SCD-2) signalling, while not essential for nematode development, is required for the integration of sensory inputs [53, 54]. In D. melanogaster, Alk signalling is critical for embryonic development, and flies lacking Alk die due to lack of specification of a particular cell type—the founder cell—in the embryonic visceral muscle [55–57]. Throughout fly development, Alk signalling is highly regulated in a spatial and temporal fashion, and while not critical for viability after embryogenesis, important roles in the nervous system have been described [58–63]. Two ALK family members have been described in zebrafish— DrLtk and DrAlk. In this organism, loss or gain of gene function results in a loss or gain of iridophores (pigment cells in fish that arise from the neural crest) for DrLtk, and impaired neurogenesis for DrAlk [64–66]. ALK-activating ligands containing LDLa domains have been identified in model organisms: Jeb in D. melanogaster [55–57, 67] and HEN-1 in C. elegans [68, 69] (Figure 1B). To date, no such ligand has been reported to activate ALK signalling in either zebrafish or other vertebrate systems. Several FAM150 (AUG)-like ligands are present in the zebrafish genome and may represent ligands for DrAlk/DrLtk, although these have not yet been experimentally investigated [6].

ALK function during mouse development Several groups have published the results of ALK loss-of-function analyses in mice, including the analysis of ALK/LTK double mutants [70–72]. While defects in neurogenesis and testosterone production have been reported, these mice are viable, suggesting that ALK is not critical to developmental processes, making ALK an attractive candidate for long-term targeted therapy in paediatric patient populations. Gain-of-function knock-in ALK mice have also been generated, highlighting a role for ALK in neurogenesis and neuroblastoma progression in combination with MYCN [32, 33].

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the protein data bank are in a ‘DFG-in’ conformation, a common feature of active kinases. The catalytically important salt bridge observed between K1150:E1167 is also consistent with an active kinase conformation, although the lobe closure appears to be incomplete. The ALK field is still missing important structural clues that would explain the activation of the kinase domain. Biochemical data support an important role for the activation loop (A-loop). Like IR, ALK has a Y’XXX’YY autophosphorylation motif (Y’RAS’YY) in the A-loop [14–16] and in ALK fusions, the tyrosine at position Y1278 in ALK is the first residue in the motif to be phosphorylated. Within the ALK kinase domain, a unique inhibitory structural feature of the short proximal A-loop αhelix places it against the αC-helix, and a β-turn motif containing C1097 obstructs the substrate binding region (Figure 2A, inset). In this arrangement, Y1278 is inaccessible for phosphorylation, as it is engaged through bonding with C1097 in the amino-terminal β-sheet [10, 12]. These observations suggest that the initial activation of ALK may be mediated by the regulation of Y1278 phosphorylation, by releasing ALK from inactive conformational restraints [10, 12]. Future work should shed light on the role of ligands, activating mutations and fusion partner-induced dimerisation in ALK kinase activation, presenting a more complete picture. Recent trends in the field of kinase structure have explored cooperation between interconnected ‘communities’ within the kinase domain, including assembly of the spines and post-translational modifications, such as phosphorylation (reviewed in [13]). Understanding the conformational and allosteric changes that regulate ALK activation will be important in coming years.

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ALK in cancer The initial NPM-ALK fusion protein described in ALCL in 1994 has now been joined by a large number of additional fusion proteins spanning a wide range of tumour types, typically representing only a small proportion of cases within each tumour type (Figure 3) [1, 18]. In 2008, a report of ALK mutation in the paediatric cancer neuroblastoma added another mechanism by which ALK can be oncogenically activated, typically by point mutation in the full-length RTK (Figure 3) [18, 73–77]. Overexpression of ALK has been reported in many different human tumours and cell lines [18]; although to date there is little evidence of a role in tumour initiation or progression, investigation of the recently described FAM150A (AUGβ) and FAM150B (AUGα) ALK ligands [6–8] in this context may help to delineate the significance of ALK overexpression in human cancer.

Nearly 30 different ALK fusion protein partners have now been described (Figure 3), suggesting that the ALK locus may be prone to translocation for reasons that are not currently understood. Although a number of fusion proteins have been experimentally characterised, only a few have been extensively studied, in particular NPM-ALK in ALCL and EML4-ALK in NSCLC. Indeed, both EML4-ALK-driven transgenic NSCLC models [78–81] and NPM-ALK-driven ALCL models [82–84] have been developed. The clinical evidence base for ALK fusion

ALK fusions

ALK mutations

ALCL: NPM–ALK, ALO17–ALK, TFG–ALK, MSN–ALK, TPM3–ALK, TPM4–ALK, ATIC–ALK, MYH9–ALK, CLTC–ALK, TRAF1–ALK NSCLC: EML4– ALK, KIF5B– ALK, TFG– ALK, KLC1– ALK, PTPN3–ALK, HIP1–ALK, TPR–ALK, STRN–ALK IMT: TPM3–ALK, TPM4–ALK, CLTC–ALK, ATIC–ALK, SEC31A– ALK, RANBP2–ALK, PPFIBP1–ALK, CARS–ALK DLBCL: NPM–ALK, CLTC–ALK, SQSTM1–ALK, SEC31A–ALK Others: ESCC: TPM4–ALK; RMC/RCC: VCL–ALK, TPM3–ALK, EML4– ALK; Breast: EML4–ALK; Colon: EML4– ALK; C2orf44–ALK; SOC: FN1–ALK; ATC: STRN–ALK, EML4–ALK, GFPT1–ALK, TFG–ALK Neuroblastoma: Predominantly point mutations in the kinase domain, hotspots 1174, 1245 and 1275 (shown as red balls, left). • Point mutations: R1060H, K1062M, D1091N, T10871, D1091N, A1099T, G1128A, T1151M/R, M1166R, T1151R, I1170N/S/T, I1171N, F1174C/I/L/S/V, l1183T, R1192P, L1196M, A1200V, L1204F, R1231Q, A1234T, L1240V, F1245C/I/L/V, I1250T, D1270G, R1275L/Q, Y1278S, G1286R, T13431, R1464* • Deletions/translocations: ALKΔexon2–3, ALKΔexon4–11, ALKΔexon1–5 ATC: Activating point mutations, L1198F and G1201E, in the kinase domain.

Figure 3. Schematic summary of the different categories of anaplastic lymphoma kinase (ALK)-positive cancers. ALK fusion proteins, in which the kinase domain of ALK (red) is fused to the amino-terminal portion of various protein fusion partners (FP; blue), have been described in numerous cancers, such as anaplastic large cell lymphoma (ALCL), non-small-cell lung cancer (NSCLC), inflammatory myofibroblastic tumour (IMT), diffuse large B-cell lymphomas (DLBCL), oesophageal squamous cell carcinoma (ESCC), renal medulla carcinoma (RMC), renal cell carcinoma (RCC), breast cancer, colon carcinoma, serous ovarian carcinoma (SOC) and anaplastic thyroid carcinoma (ATC). ALK mutations comprise a second group found mainly in neuroblastoma as well as NSCLC and ATC; most are point mutations within the kinase domain. Germline mutations in neuroblastoma are highlighted in blue; *truncation. ALK overexpression, which has been reported in a variety of cancer types and cell lines is not shown here.

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ALK fusion oncogenes

proteins in cancer largely originates from cohorts of patients with ALK-positive NSCLC (as discussed in more detail in this edition of Annals of Oncology). Although ALK fusions occur in only around 5% of NSCLC cases, the large number of NSCLC patients makes EML4-ALK the most prevalent ALK gene rearrangement, accounting for ∼40 000 new occurrences per year worldwide [85–87]. Studies of ALK fusion proteins have highlighted some general principles, such as the importance of oligomerisation. This is mediated through the ALK fusion partners, for instance, the coiled-coil of the EML4 portion of EML4-ALK that serves to dimerise and activate the ALK kinase domain. The ALK fusions described to date involve the entire kinase domain of ALK, and translocation almost always occurs at a common breakpoint in exon 20 of the ALK locus. In addition, different ALK fusion proteins can exhibit differential stability, as well as differential sensitivity to ALK tyrosine kinase inhibitors (TKIs) [38]. To add a further level of complexity, some ALK fusions have multiple variants; for instance, more than 15 have been described for EML4-ALK in NSCLC (expertly reviewed in [88]). This may alter the properties of the resulting fusion protein, for example, its stability or activity. While a range of different ALK fusions can occur, typically one fusion may occur more frequently in a particular subtype of tumour. This can be illustrated in NSCLC, where ALK fusion proteins account for around 5% of NSCLC cases, often in younger, non-smoking patients with the adenocarcinoma-type of NSCLC. EML4-ALK is the main fusion in NSCLC,

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Anaplastic thyroid cancer. ALK mutations have been reported in anaplastic thyroid cancer (ATC), including gain-of-function mutations in L1198F and G1201E [110] (Figure 3).

representing the majority of ALK-positive cases [89], while KIF5B-ALK, TFG-ALK, KLC1-ALK, PTPN3-ALK, HIP1-ALK, STRN-ALK and TPR-ALK have also been reported [18, 40, 90– 98]. In ALCL, however, where ALK fusions are found in more than 50% of cases, NPM-ALK is the most common variant, while ALO17-ALK, TFG-ALK, MSN-ALK, TPM3-ALK, TPM4ALK, ATIC-ALK, MYH9-ALK, CLTC-ALK and TRAF1-ALK fusions have also been reported [1, 18, 99].

TKIs targeting ALK

ALK point mutations Neuroblastoma. Mutant variants in the full-length ALK receptor have been identified in both somatic and familial cases of neuroblastoma, a childhood tumour arising from the neural crest [73–77]. More than 35 mutations in ALK have been described, predominantly point mutations, although deletions in the ECD and translocations have also been reported [18, 100– 103] (Figure 3). The majority of these mutations are found in one of three hotspot residues in the kinase domain: F1174, F1245 and R1275 [18, 100] (Figure 3). F1174, F1245 and R1275, among others, have been reported to be activating ALK mutations [74, 75, 100, 104–106]. Transgenic and knock-in ALK mouse models highlight the synergy between ALK and MYCN in neuroblastoma progression [28, 29, 32, 33, 107]. Other reported ALK mutations show ligand-dependent activation or are kinase dead [100, 108, 109] and their significance in the development of neuroblastoma remains to be defined.

A

B

C

D

E

F

Figure 4. Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) in the ATP binding pocket of the ALK kinase domain. Visualisation of the ATP-binding pocket of the wild-type ALK kinase domain modelled with a selection of ALK TKIs, showing the variation in contact sites between different TKI molecules. (A) TAE684 (PDB: 2XB7) [111]. (B) Crizotinib (PF2341066, PDB: 2XP2) [112]. (C) Ceritinib (LDK378, PDB: 4MKC) [113]. (D) Alectinib (CH5424802, PDB: 3AOX) [114]. (E) Lorlatinib (PF06463922, PDB: 4CLI) [115]. (F). Brigatinib (AP26113, modelling kindly provided by Tianjau Zhou, ARIAD Pharmaceuticals) [116]. Figures were generated with PyMol showing the different inhibitors (red) interacting within the ATP binding pocket of ALK.

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In 2011, crizotinib, an ATP analogue inhibitor of ALK (Figure 4), was approved for use in patients with ALK-positive NSCLC based on strong clinical response data [117, 118]. It is now clear that long-term treatment with crizotinib and other TKIs is limited by the development of drug resistance [119, 120]. Around 30% of cases of ALK-targeted agent resistance in NSCLC are due to mutations within the ALK fusion gene; activation of bypass pathways and ALK copy number gain may also contribute to resistance [121]. Such dynamic events in the clinical course highlight the importance of tumour re-biopsy and repeated ALK evaluation during disease progression, as well as careful consideration of initial diagnostic testing. This is important not only for patients with NSCLC who are already known to be ALK-positive, since resistance mutations can emerge during treatment with ALK TKIs such as crizotinib [119, 122], but also for patients with neuroblastoma, where activating ALK mutations have been documented later in the disease course, without prior exposure to ALK TKIs [105, 123, 124]. Further examples of ALK TKIs include ceritinib, brigatinib, alectinib and lorlatinib [85, 112–116, 125] (for latest details of clinical trials employing ALK TKIs, see ClinicalTrials.gov). All bind slightly differently within the ATP-binding pocket of the

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Table 1. Resistance mutations reported in patients treated with anaplastic lymphoma kinase tyrosine kinase inhibitors

1151Ins L1152 L1156

G1128

ALK TKI R1192

L1198 G1123

M1166

G1202

Crizotinib PF-02341066

I1171

G1269

Clinically reported ALK TKI resistance mutations

TKI activity

ALK, MET, ROS-1

1151Tins, L1152R, C1156Y, F1174L/V, L1196M, G1202R, S1206Y, F1245C, G1269A [89, 119, 120, 126–130]

[112]

ALK, IGF1R, InsR, STK22D

G1123S, F1174C/V, G1202R [126, 131]

[113]

ALK, GAK, LTK

I1171N/T/S, G1202R [95, 127, 132]

[114]

ALK, ROS1

L1198F [133]

[115]

I1170

S1206

F1174 R1275 Y1278

Ceritinib LDK378

F1245 L1240

Figure 5. Mutations in the anaplastic lymphoma kinase (ALK) kinase domain: comparison of activating ALK mutations found in neuroblastoma with secondary resistance mutations reported from patients treated with ALK tyrosine kinase inhibitors (TKIs). Resistance mutations in ALK fusions from ALK TKI-treated patients (red) generally cluster around the inhibitor/ ATP-binding site (ADP in cyan). Strongly activating, neuroblastoma-associated ALK mutations (blue) occur in close proximity to residues thought to be important for initial activation of ALK activity (αC-helix in green, A-loop in teal). Four exceptions are the I1171, F1174, F1245 and L1196 mutations ( purple), which are reported in both neuroblastoma and ALK TKI-resistant NSCLC patients. The regulatory spine (light pink) and catalytic spine (lime green) are also shown.

ALK kinase domain (Figure 4) and show differing profiles of inhibition for the wild-type ALK kinase domain compared with the various ALK kinase mutants. The growing clinical evidence suggests that different resistance mutation patterns emerge during therapy with different ALK TKIs (Table 1). Furthermore, the effects of different ALK mutations on kinase activity may be compounded by differential effects in mediating signals to downstream targets, differences that may have implications for potential combination therapy strategies. Thus, a complex and dynamic picture is emerging as an increasing number of patients are treated sequentially with multiple ALK TKIs. This can be illustrated by a recent case report of a patient initially treated with crizotinib who developed a C1156Y crizotinib resistance kinase domain mutation. After several additional lines of therapy, this patient subsequently received the nextgeneration ALK TKI lorlatinib, and later developed a lorlatinib resistance mutation (L1198F) that then restored crizotinib sensitivity [133]. Such scenarios serve to highlight the challenges for clinicians in monitoring and interpreting ALK mutational data to allow a tailored, personalised ALK TKI treatment sequence.

ALK activation: lessons from kinase domain mutations in cancer One way to investigate the activation mechanisms important in the regulation of ALK activity is to examine the various disease mutations that have been reported. For the purposes of this

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Alectinib CH5424802/ RO5424802 Lorlatinib PF-06463922

The most frequently observed mutations observed in crizotinib-resistant tumours are the L1196M gatekeeper mutation and G1269A. Of those resistance mutations reported to date, the I1171, F1174, L1196 and F1245 residue mutations overlap with ALK mutations in neuroblastoma (italicised).

review, we will consider some selected subclasses of the known mutations: the point mutations described in neuroblastoma patients and resistance mutations that have been shown to arise within the kinase domain during ALK TKI therapy for ALKpositive NSCLC.

mutations in the full-length ALK RTK in neuroblastoma The majority of ALK mutations described in neuroblastoma patients are point mutations within the kinase domain, although deletions in the ECD and translocations have also been reported [18, 73–77, 100–103]. Point mutations within the kinase domain that confer constitutive activity tend to occur around the A-loop and the αC helix (Figure 5), with the exception of the G1128 and R1192 mutations. These two residues are important for coordination of ATP binding and formation of a salt bridge with the αC helix, respectively, and mutation results in ligand-independent activation of the ALK kinase domain. The three hotspot residues F1174, R1275 and F1245, all surround the αC-helix, and mutations in them transform ALK to an active kinase. Interconnected ‘communities’ within the kinase domain help explain this increased kinase activity, as F1174 and R1275 are central amino acids of a hydrophobic core between the αC helix and the catalytic loop, respectively, maintaining the kinase domain in an auto-inhibited conformation [10, 12, 13]. Mutation of these residues alters the dynamics within this region, releasing inhibition of kinase activity. The F1245 residue forms part of an additional auto-inhibitory region between the αC helix and the α-helix of the A-loop; mutations in this residue occur in both somatic and germline neuroblastomas [10,

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12, 13]. A third example is the Y1278S A-loop neuroblastoma mutation that results in ligand-independent ALK kinase activity. It has been proposed from structural studies that Y1278 interacts via a hydrogen bond with C1097 situated in the amino-terminal β-turn [10] (Figure 2). Mutation of Y1278, or indeed phosphorylation of Y1278 in the A-loop in response to ligand stimulation, is predicted to disrupt the bond between Y1278 and C1097, leading to ALK kinase activation.

resistance mutations in the kinase domain

summary and perspective ALK kinase signalling across different tumour types, such as NSCLC, ALCL and neuroblastoma, shares many features, but also exhibits certain differences. Most of our currently available data relates to oncogenic ALK, either activated through mutation or through dimerisation driven by a fusion partner, and information regarding physiological ligand-stimulated activation of ALK signalling in vivo is scarce. The ALK fusions found in tumours such as NSCLC and ALCL lack spatial and temporal regulation, with their expression regulated by the amino-terminal fusion partner, which can also affect dimerisation, substrate specificity and signalling. The transforming and

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acknowledgements Modelling of Brigatinib with the ALK kinase domain was kindly provided by Tianjau Zhou, ARIAD Pharmaceuticals. Medical writing support was provided by ACUMED® (Tytherington, UK), an Ashfield company, part of UDG Healthcare plc, and was funded by Pfizer.

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As discussed above, treatment with ALK TKIs leads to the emergence of resistance in the form of ALK kinase domain mutation (around 30%), amplification of the ALK locus or activation of ‘bypass’ signalling pathways. A number of ALK TKIs are currently approved worldwide for use in ALK-positive tumours, with others in development. Figure 4 illustrates a range of ALK TKIs bound in the ATP-binding pocket of ALK, where they all vary slightly in their contact point with ALK (shown are TAE684 [111], crizotinib/PF2341066 [112], ceritinib/LDK378 [113], alectinib/CH5424802 [114], lorlatinib/ PF06463922 [115] and brigatinib/AP26113 [116]). The differing interactions of the ALK TKIs with the ALK kinase domain explain in part the differing patterns of resistance observed in TKI-treated patients. As the first encouraging reports of crizotinib therapy were published (in patients with ALK-positive NSCLC and inflammatory myofibroblastic tumour [IMT]) [118, 134], the appearance of TKI-resistant ALK mutations within the target kinase domain was also reported [119]. The majority of ALK TKI resistance mutations reported to date cluster around the ATP/ TKI binding site in the kinase domain, conferring a steric interference for inhibitor binding that enables resumption of ALK fusion kinase activity even in the presence of TKI (Figure 5) [120]. Such resistance mutations may arise as a consequence of ALK TKI treatment, or may pre-exist as minority clones within the tumour, which expand under the selective pressure exerted by ALK TKI therapy. A number of ALK TKI resistance mutations in NSCLC patients overlap with point mutations in neuroblastoma patients (Figure 5). These kinase domain mutations, which include I1171 [95, 132], F1174 [120, 126, 127], F1245 [128] and the L1196M gatekeeper mutation [119, 129, 130], confer the full-length ALK receptor with constitutive activity, and are capable of driving oncogenic transformation.

tumorigenic potential of ALK fusion proteins can be expected to differ substantially from that of the full-length ALK receptor mutants observed in neuroblastoma, which are activated only in cells normally expressing the receptor; indeed, signalling also differs from that of the wild-type ALK receptor activated by natural ligands in a physiologically appropriate, temporal and spatial manner. From a clinical perspective, further understanding of the role of ALK in tumour biology is key to optimise future therapeutic options. As the range of TKIs available to treat ALK-positive tumours continues to expand, many questions remain: which ALK TKI should be employed in the first-, second- and third-line, and beyond? Can therapeutic efficacy be optimised by starting with crizotinib, which has proven efficacy against the wild-type ALK kinase domain, or by starting with second- or third-generation ALK TKIs which have higher affinities for ALK but for whom the pattern of resistance is currently less well defined (Table 1)? These clinical considerations are discussed in more detail in later articles of this edition of Annals of Oncology. Investigation of additional targets to provide an effective second hit that will enhance the effect of ALK inhibition of tumour growth is also important. A number of such molecular targets have been identified, such as Hsp90, mTOR, CDK4/6 and ERK5, as potential therapeutic partners for use in combination with ALK TKIs, as well as possible combinations with conventional chemotherapies [107, 135, 136]. These targets offer the opportunity to further weaken downstream pathways to amplify tumour growth inhibition and prevent feedback signalling scenarios. Such approaches are suggested to reduce the ability of the tumour to survive and adapt to TKI monotherapy through bypass signalling mechanisms and resistance mutations. In developing clinical combination regimens, increasing therapeutic efficacy while limiting additional toxicity will be a key consideration. A number of interesting studies have suggested the potential of immunotherapy in the form of DNA vaccination, as explored in mouse models of ALCL and NSCLC [137, 138]. In the case of tumours involving full-length ALK, such as neuroblastoma, preclinical observations support further evaluation of ALKtargeted immunotherapy directed against the ECD of the receptor [6, 9, 139]. Characterisation of the FAM150 (AUG) ALK ligands has opened a new avenue for exploration of potential therapeutic targets for tumours expressing activated full-length ALK. It is indeed rewarding to see that as clinical and preclinical investigators work side-by-side, the tremendous pace of ALK inhibitor development has translated into improved duration and quality of life for patients with ALKpositive tumours in, for example, NSCLC and ALCL [140– 142]. While the promise of ALK TKIs for these patients has surpassed its initial short-term goals, it is clear that many significant challenges remain to be met in the coming years to attain the longer term treatment goals for these patients.

review funding This work was supported by the Swedish Cancer Society (15/ 391 to RHP; 15/775 to BH), the Swedish Childhood Cancer Foundation (2015-0096 to RHP; 2015-80 and 2014-150 to BH), the Swedish Research Council (2015-04466 to RHP; 521-20122831 to BH) and the SSF Programme Grant (RB13-0204 to RHP and BH).

disclosure The authors have declared no conflicts of interest.

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Annals of Oncology

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Volume 27 | Supplement 3 | September 2016

doi:10.1093/annonc/mdw301 | iii