Structural basis for ligand reception by anaplastic lymphoma kinase.

The proto-oncogene ALK encodes anaplastic lymphoma kinase, a receptor tyrosine kinase that is expressed primarily in the developing nervous system. After development, ALK activity is associated with learning and memory1 and controls energy expenditure, and inhibition of ALK can prevent diet-induced obesity2. Aberrant ALK signalling causes numerous cancers3. In particular, full-length ALK is an important driver in paediatric neuroblastoma4,5, in which it is either mutated6 or activated by ligand7. Here we report crystal structures of the extracellular glycine-rich domain (GRD) of ALK, which regulates receptor activity by binding to activating peptides8,9. Fusing the ALK GRD to its ligand enabled us to capture a dimeric receptor complex that reveals how ALK responds to its regulatory ligands. We show that repetitive glycines in the GRD form rigid helices that separate the major ligand-binding site from a distal polyglycine extension loop (PXL) that mediates ALK dimerization. The PXL of one receptor acts as a sensor for the complex by interacting with a ligand-bound second receptor. ALK activation can be abolished through PXL mutation or with PXL-targeting antibodies. Together, these results explain how ALK uses its atypical architecture for its regulation, and suggest new therapeutic opportunities for ALK-expressing cancers such as paediatric neuroblastoma.

Mechanism for the activation of the anaplastic lymphoma kinase receptor.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that regulates important functions in the central nervous system1,2. The ALK gene is a hotspot for chromosomal translocation events that result in several fusion proteins that cause a variety of human malignancies3. Somatic and germline gain-of-function mutations in ALK were identified in paediatric neuroblastoma4-7. ALK is composed of an extracellular region (ECR), a single transmembrane helix and an intracellular tyrosine kinase domain8,9. ALK is activated by the binding of ALKAL1 and ALKAL2 ligands10-14 to its ECR, but the lack of structural information for the ALK-ECR or for ALKAL ligands has limited our understanding of ALK activation. Here we used cryo-electron microscopy, nuclear magnetic resonance and X-ray crystallography to determine the atomic details of human ALK dimerization and activation by ALKAL1 and ALKAL2. Our data reveal a mechanism of RTK activation that allows dimerization by either dimeric (ALKAL2) or monomeric (ALKAL1) ligands. This mechanism is underpinned by an unusual architecture of the receptor-ligand complex. The ALK-ECR undergoes a pronounced ligand-induced rearrangement and adopts an orientation parallel to the membrane surface. This orientation is further stabilized by an interaction between the ligand and the membrane. Our findings highlight the diversity in RTK oligomerization and activation mechanisms.

Structural basis of cytokine-mediated activation of ALK family receptors.

Anaplastic lymphoma kinase (ALK)1 and the related leukocyte tyrosine kinase (LTK)2 are recently deorphanized receptor tyrosine kinases3. Together with their activating cytokines, ALKAL1 and ALKAL24-6 (also called FAM150A and FAM150B or AUGß and AUGα, respectively), they are involved in neural development7, cancer7-9 and autoimmune diseases10. Furthermore, mammalian ALK recently emerged as a key regulator of energy expenditure and weight gain11, consistent with a metabolic role for Drosophila ALK12. Despite such functional pleiotropy and growing therapeutic relevance13,14, structural insights into ALK and LTK and their complexes with cognate cytokines have remained scarce. Here we show that the cytokine-binding segments of human ALK and LTK comprise a novel architectural chimera of a permuted TNF-like module that braces a glycine-rich subdomain featuring a hexagonal lattice of long polyglycine type II helices. The cognate cytokines ALKAL1 and ALKAL2 are monomeric three-helix bundles, yet their binding to ALK and LTK elicits similar dimeric assemblies with two-fold symmetry, that tent a single cytokine molecule proximal to the cell membrane. We show that the membrane-proximal EGF-like domain dictates the apparent cytokine preference of ALK. Assisted by these diverse structure-function findings, we propose a structural and mechanistic blueprint for complexes of ALK family receptors, and thereby extend the repertoire of ligand-mediated dimerization mechanisms adopted by receptor tyrosine kinases.

Extracellular domain shedding of the ALK receptor mediates neuroblastoma cell migration.

Although activating mutations of the anaplastic lymphoma kinase (ALK) membrane receptor occur in ∼10% of neuroblastoma (NB) tumors, the role of the wild-type (WT) receptor, which is aberrantly expressed in most non-mutated cases, is unclear. Both WT and mutant proteins undergo extracellular domain (ECD) cleavage. Here, we map the cleavage site to Asn654-Leu655 and demonstrate that cleavage inhibition of WT ALK significantly impedes NB cell migration with subsequent prolongation of survival in mouse models. Cleavage inhibition results in the downregulation of an epithelial-to-mesenchymal transition (EMT) gene signature, with decreased nuclear localization and occupancy of ß-catenin at EMT gene promoters. We further show that cleavage is mediated by matrix metalloproteinase 9, whose genetic and pharmacologic inactivation inhibits cleavage and decreases NB cell migration. Together, our results indicate a pivotal role for WT ALK ECD cleavage in NB pathogenesis, which may be harnessed for therapeutic benefit.

Computational studies of anaplastic lymphoma kinase mutations reveal common mechanisms of oncogenic activation.

Kinases play important roles in diverse cellular processes, including signaling, differentiation, proliferation, and metabolism. They are frequently mutated in cancer and are the targets of a large number of specific inhibitors. Surveys of cancer genome atlases reveal that kinase domains, which consist of 300 amino acids, can harbor numerous (150 to 200) single-point mutations across different patients in the same disease. This preponderance of mutations-some activating, some silent-in a known target protein make clinical decisions for enrolling patients in drug trials challenging since the relevance of the target and its drug sensitivity often depend on the mutational status in a given patient. We show through computational studies using molecular dynamics (MD) as well as enhanced sampling simulations that the experimentally determined activation status of a mutated kinase can be predicted effectively by identifying a hydrogen bonding fingerprint in the activation loop and the αC-helix regions, despite the fact that mutations in cancer patients occur throughout the kinase domain. In our study, we find that the predictive power of MD is superior to a purely data-driven machine learning model involving biochemical features that we implemented, even though MD utilized far fewer features (in fact, just one) in an unsupervised setting. Moreover, the MD results provide key insights into convergent mechanisms of activation, primarily involving differential stabilization of a hydrogen bond network that engages residues of the activation loop and αC-helix in the active-like conformation (in >70% of the mutations studied, regardless of the location of the mutation).

Mutation-mediated influences on binding of anaplastic lymphoma kinase to crizotinib decoded by multiple replica Gaussian accelerated molecular dynamics.

Anaplastic lymphoma kinase (ALK) has been thought to be a prospective target of anti-drug resistance design in treatment of tumors and specific neuron diseases. It is highly useful for the seeking of possible strategy alleviating drug resistance to probe the mutation-mediated effect on binding of inhibitors to ALK. In the current work, multiple replica Gaussian accelerated molecular dynamics (MR-GaMD) simulations, molecular mechanics generalized Born surface area (MM-GBSA) and free energy landscapes were coupled to explore influences of mutations L1198F, L1198F/C1156Y, and C1156Y on the binding of the first ALK inhibitor crizotinib to ALK. The results suggest that three mutations obviously affect structural flexibility, motion modes and conformational changes of ALKs. L1198F and L1198F/C1156Y strengthen the binding of crizotinib to the mutated ALKs but C1156Y induces evident drug resistance toward crizotinib. Analyses of free energy landscapes show that stability in the orientation and positions of crizotinib relative to ALK plays a vital role in alleviating drug resistance of mutations toward crizotinib. Residue-based free energy decomposition method was utilized to evaluate the contributions of separate residues to the binding of crizotinib. The results not only indicate that the tuning of point mutation L1198F on interaction networks of crizotinib with ALK can be regarded as a possible strategy to relieve drug resistance of the mutated ALK but also further verify that residues L1122, V1130, L1196, L1198, M1199, and L1256 can be used as efficient targets of anti-drug resistance design induced by mutations.

Preparation of a phosphotyrosine-protein standard for use in semiquantitative western blotting with enhanced chemiluminescence.

Protein tyrosine phosphorylation is key to activation of receptor tyrosine kinases (RTK) that drive development of some cancers. One challenge of RTK-targeted therapy is identification of those tumors that express non-mutated but activated RTKs. Phosphotyrosine (pTyr) RTK levels should be more predictive of the latter than expressed total protein. Western blotting (WB) with a pTyr antibody and enhanced chemiluminescence (ECL) detection is sufficiently sensitive to detect pTyr-RTKs in human tumor homogenates. Presentation of results by comparing WB images, however, is wanting. Here we describe the preparation of a new pTyr-protein standard, pTyr-ALK48-SB (pA), derived from a commercial anaplastic lymphoma kinase (ALK) recombinant fragment, and its use to quantify pTyr-epidermal growth factor receptor (pTyr-EGFR) in commercial A431 cell lysates. Linearity of one-dimensional (1D) WB plots of pA band density versus load as well as its lower level of detection (0.1 ng, 2 fmole) were determined for standardized conditions. Adding pA to two lots of A431 cell lysates with high and low pTyr-EGFR allowed normalization and quantification of the latter by expressing results as density ratios for both 1D and 2D WB. This approach is semi-quantitative because unknown RTKs may be outside the linear range of detection. Semiquantitative ratios are an improvement over comparisons of images without a reference standard and facilitate comparisons between samples.

Dual potent c-Met and ALK inhibitors: from common feature pharmacophore modeling to structure based virtual screening.

Everyday plenty of people succumb to various forms of cancer across the world and it stands as one of the main reasons of death in our today's life. Receptor tyrosine kinases (RTKs) are a class of receptors involved in cancer progression. Since aberrant signaling has critical roles in cancer, both c-Met and ALK enzymes are regarded as attractive oncology targets for therapeutic objects. A number of potent dual inhibitors of c-Met and ALK are reported in literature that in the present work we based them to construct multiple common feature pharmacophore models and then applied them for ligand-based virtual screening. The score values of the models ranged from 22.489 to 28.169. The retrieved compounds from virtual screening were subjected to the docking study and the interaction pattern of common hits between two enzymes with high predicted affinity has been investigated. To this end, common hit compound ZINC000223394281 (z1) was directed to the molecular dynamics study and the results indicated that the hydrogen bond interaction between this compound and Asp1222 was mostly stable during the equilibrium time range. The life time of hydrogen bond made between the complex of ALK and Met1199 was also stable in 63%.

Improvement in predicting drug sensitivity changes associated with protein mutations using a molecular dynamics based alchemical mutation method.

While molecular-targeted drugs have demonstrated strong therapeutic efficacy against diverse diseases such as cancer and infection, the appearance of drug resistance associated with genetic variations in individual patients or pathogens has severely limited their clinical efficacy. Therefore, precision medicine approaches based on the personal genomic background provide promising strategies to enhance the effectiveness of molecular-targeted therapies. However, identifying drug resistance mutations in individuals by combining DNA sequencing and in vitro analyses is generally time consuming and costly. In contrast, in silico computation of protein-drug binding free energies allows for the rapid prediction of drug sensitivity changes associated with specific genetic mutations. Although conventional alchemical free energy computation methods have been used to quantify mutation-induced drug sensitivity changes in some protein targets, these methods are often adversely affected by free energy convergence. In this paper, we demonstrate significant improvements in prediction performance and free energy convergence by employing an alchemical mutation protocol, MutationFEP, which directly estimates binding free energy differences associated with protein mutations in three types of a protein and drug system. The superior performance of MutationFEP appears to be attributable to its more-moderate perturbation scheme. Therefore, this study provides a deeper level of insight into computer-assisted precision medicine.

Targeted degradation of anaplastic lymphoma kinase by gold nanoparticle-based multi-headed proteolysis targeting chimeras.

Anaplastic lymphoma kinase (ALK) is a major target in treating non-small-cell lung cancer, and several ALK inhibitors have been developed to antagonize its kinase activity. However, patients treated with inhibitors ultimately develop drug resistance. Therefore, therapies with new mechanisms of action are needed. Proteolysis targeting chimeras (PROTACs) are molecules that comprise a ligand for binding a protein of interest (POI), a connecting linker and a ligand for recruiting E3 ligase, and cause degradation of the target POI. Here, the first multi-headed PROTAC, as a proof of concept, is developed as a gold nanoparticle (GNP)-based drug delivery system for delivering PROTACs to target ALK. Pegylated GNPs loaded with both ceritinib and pomalidomide molecules, termed Cer/Pom-PEG@GNPs, showed good stability in several media. The GNP conjugates potently decreased the levels of ALK fusion proteins in a dose- and time-dependent manner, and specifically inhibited the proliferation of NCI-H2228 cells. In comparison with small molecule PROTACs, the new multi-headed PROTAC promoted the formation of coacervates of POIs/multi-headed PROTAC/E3 ubiquitin ligases, and POI and E3 ubiquitin ligase interacted through multidirectional ligands and a flexible linker, thereby avoiding the need for complicated structure optimization of PROTACs. In conclusion, Cer/Pom-PEG@GNPs can degrade intracellular ALK fusion proteins with minor off-target toxicity and can be applied in patients resistant to ALK inhibitors. As a nano-based drug carrier, Cer/Pom-PEG@GNPs have the potential to enable prolonged circulation and specifically distribute drugs to tumor regions in vivo; thus, further investigation is warranted.

Exploring New Structural Features of the 18ß-Glycyrrhetinic Acid Scaffold for the Inhibition of Anaplastic Lymphoma Kinase.

Novel 18ß-glycyrrhetinic acid derivatives possessing a carbamate moiety and structurally similar ester derivatives were developed and evaluated for their efficacy as antitumor inhibitors. In the cellular assays, most of the N-substituted carbamate derivatives at the C3-position exhibited potent activities. The results of SAR investigation revealed that the introduction of the morpholine group at the C30-COOH led to a significant loss of the inhibitory potency. Among the ester derivatives, the ester group at C3-position also determined a noticeable reduction in the efficacy. Compound 3j exhibited the most prominent antiproliferative activity against six human cancer cells (A549, HT29, HepG2, MCF-7, PC-3, and Karpas299). Furthermore, compound 3j exerted a moderate inhibiting effect on the ALK. The results of molecular docking analyses suggested that it could bind well to the active site of the receptor ALK, which was consistent with the biological data. These results might inspire further structural optimization of 18ß-glycyrrhetinic acid aiming at the development of potent antitumor agents. The structures 4d, 4g, 4h, 4j, and 4n were studied by X-ray crystallographic analyses.

Covalent chemistry on nanostructured substrates enables noninvasive quantification of gene rearrangements in circulating tumor cells.

Well-preserved mRNA in circulating tumor cells (CTCs) offers an ideal material for conducting molecular profiling of tumors, thereby providing a noninvasive diagnostic solution for guiding treatment intervention and monitoring disease progression. However, it is technically challenging to purify CTCs while retaining high-quality mRNA.Here, we demonstrate a covalent chemistry-based nanostructured silicon substrate ("Click Chip") for CTC purification that leverages bioorthogonal ligation-mediated CTC capture and disulfide cleavage-driven CTC release. This platform is ideal for CTC mRNA assays because of its efficient, specific, and rapid purification of pooled CTCs, enabling downstream molecular quantification using reverse transcription Droplet Digital polymerase chain reaction. Rearrangements of ALK/ROS1 were quantified using CTC mRNA and matched with those identified in biopsy specimens from 12 patients with late-stage non-small cell lung cancer. Moreover, CTC counts and copy numbers of ALK/ROS1 rearrangements could be used together for evaluating treatment responses and disease progression.

Synthesis and anti-tumor efficacy of novel 2, 4-diarylaminopyrimidine derivatives bearing N-(3-pyridinylmethyl) urea moiety as anaplastic lymphoma kinase inhibitors.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase responsible for development of various tumor types. In this study, we synthesized a series of novel 2,4-diarylaminopyrimidine derivatives possessing a unique N-(3-pyridinylmethyl)urea moiety as ALK inhibitors. The most promising analog 5m bearing a 3-methoxy-4-morpholinophenyl substituent significantly inhibited proliferation of ALK positive H3122 and Karpas-299 cells with IC50 values about 10 nM, which were comparable with positive control LDK378. Compound 5m suppressed phosphorylation of ALK and its downstream proteins, and showed low cytotoxicity on normal human primary fibroblast cells (BJ cells). The binding mode of 5m was proposed by docking simulation, which explains the important role of N-(3-pyridinylmethyl)urea moiety. Furthermore, compound 5m exhibited favorable liver microsomal stability and significant efficacy in H3122 xenograft mice model. Interestingly, compound 5m also showed broader anti-proliferative activity on other human tumor cell lines, which was different from other ALK inhibitors.

Expression of C-terminal ALK, RET, or ROS1 in lung cancer cells with or without fusion.

BACKGROUND: Genetic alterations, including mutation of epidermal growth factor receptor or v-Ki-ras2 kirsten rat sarcoma viral oncogene homolog and fusion of anaplastic lymphoma kinase (ALK), RET proto-oncogene (RET), or v-ros UR2 sarcoma virus oncogene homolog 1 (ROS1), occur in non-small cell lung cancers, and these oncogenic drivers are important biomarkers for targeted therapies. A useful technique to screen for these fusions is the detection of native carboxy-terminal (C-terminal) protein by immunohistochemistry; however, the effects of other genetic alterations on C-terminal expression is not fully understood. In this study, we evaluated whether C-terminal expression is specifically elevated by fusion with or without typical genetic alterations of lung cancer. METHODS: In 37 human lung cancer cell lines and four tissue specimens, protein and mRNA levels were measured by capillary western blotting and reverse transcription-PCR, respectively. RESULTS: Compared with the median of all 37 cell lines, mRNA levels at the C-terminus of all five of the fusion-positive cell lines tested (three ALK, one RET, and one ROS1) were elevated at least 2000-, 300-, or 2000-fold, respectively, and high C-terminal protein expression was detected. In an ALK fusion-positive tissue specimen, the mRNA and protein levels of C-terminal ALK were also markedly elevated. Meanwhile, in one of 36 RET fusion-negative cell lines, RET mRNA levels at the C-terminus were elevated at least 500-fold compared with the median of all 37 cell lines, and high C-terminal protein expression was detected despite the absence of RET fusion. CONCLUSIONS: This study of 37 cell lines and four tissue specimens shows the detection of C-terminal ALK or ROS1 proteins could be a comprehensive method to determine ALK or ROS1 fusion, whereas not only the detection of C-terminal RET protein but also other methods would be needed to determine RET fusion.

Tyrosine kinase inhibitor acquired resistance mechanism alternates between EGFR and ALK in a lung adenocarcinoma patient.

Drive gene mutation positive non-small cell lung cancer achieves reliable clinical responses to subsequent target therapy. However, most patients will inevitably develop disease progression with multiple treatment failure. Next generation sequencing can identify clear resistance mechanisms. We report a case of a late stage, non-smoking, male non-small cell lung cancer patient that developed dual mutations and our attempts to determine the novel resistance mechanism. After systematic chemotherapy, he was administered multiple target therapy according to different genotypes. Larger panel gene detection was adapted after the failure of different treatments to investigate the resistance mechanism. Re-biopsy and large panel NGS revealed an EGFR mutant lung adenocarcinoma with alternating changes in acquired resistance between EGFR and ALK. The total survival time was 73 months. The genotypes and treatments in this patient provide new insight of target therapy resistance mechanisms. Re-biopsy and large panel gene detection should be performed for each driver gene mutation to provide precision treatment strategies.

Prediction of ALK mutations mediating ALK-TKIs resistance and drug re-purposing to overcome the resistance.

BACKGROUND: Alectinib has shown a greater efficacy to ALK-rearranged non-small-cell lung cancers in first-line setting; however, most patients relapse due to acquired resistance, such as secondary mutations in ALK including I1171N and G1202R. Although ceritinib or lorlatinib was shown to be effective to these resistant mutants, further resistance often emerges due to ALK-compound mutations in relapse patients following the use of ceritinib or lorlatinib. However, the drug for overcoming resistance has not been established yet. METHODS: We established lorlatinib-resistant cells harboring ALK-I1171N or -G1202R compound mutations by performing ENU mutagenesis screening or using an in vivo mouse model. We performed drug screening to overcome the lorlatinib-resistant ALK-compound mutations. To evaluate these resistances in silico, we developed a modified computational molecular dynamic simulation (MP-CAFEE). FINDINGS: We identified 14 lorlatinib-resistant ALK-compound mutants, including several mutants that were recently discovered in lorlatinib-resistant patients. Some of these compound mutants were found to be sensitive to early generation ALK-TKIs and several BCR-ABL inhibitors. Using our original computational simulation, we succeeded in demonstrating a clear linear correlation between binding free energy and in vitro experimental IC50 value of several ALK-TKIs to single- or compound-mutated EML4-ALK expressing Ba/F3 cells and in recapitulating the tendency of the binding affinity reduction by double mutations found in this study. Computational simulation revealed that ALK-L1256F single mutant conferred resistance to lorlatinib but increased the sensitivity to alectinib. INTERPRETATION: We discovered lorlatinib-resistant multiple ALK-compound mutations and an L1256F single mutation as well as the potential therapeutic strategies for these ALK mutations. Our original computational simulation to calculate the binding affinity may be applicable for predicting resistant mutations and for overcoming drug resistance in silico. FUND: This work was mainly supported by MEXT/JSPS KAKENHI Grants and AMED Grants.

Structural basis for drug resistance mechanisms against anaplastic lymphoma kinase.

Drug resistance to anaplastic lymphoma kinase (ALK) inhibitors (crizotinib and ceritinib) is caused by mutation in the region encoding kinase domain of ALK. Compounds with potential ability to inhibit all strains of ALK are a solution to tackle the problem of drug resistance. In this study, we delineated positions of residues possessing the ability to make ALK drug resistant upon mutation by assessing them using five parameters (conservation index, binding-site root-mean-square deviation, protein structure stability, change in ATP, and drug-binding affinity). Four residual positions (Leu 1122, Thr 1151, Phe 1245, and Gly 1269) were ascertained. This study will be beneficial for designing drugs with better proficiency against ALK and the issues of drug resistance. This study can be taken as a pipeline for investigating drug-resistant mutations in other diseases as well.

Molecular inhibitory mechanism study on the potent inhibitor brigatinib against four crizotinib-resistant ALK mutations.

As a potent and selective drug, brigatinib exhibits high efficacy against wild-type and mutant anaplastic lymphoma kinase (ALK) proteins to treat non-small cell lung cancer. In this work, the mechanisms of brigatinib binding to wild type and four mutant ALKs were investigated to gain insight into the dynamic energetic and structural information with respect to the design of novel inhibitors. Comparison between ALK-brigatinib and ALK-crizotinib suggests that the scaffold of brigatinib is well anchored to the residue Met1199 of hinge region by two hydrogen bonds, and the residue Lys1150 has the strong electrostatic interaction with the dimethylphosphine oxide moiety in brigatinib. These ALK mutations have significant influences on the flexibility of P-loop region and DFG sequences, but do not impair the hydrogen bonds between brigatinib and the residue Met1199 of hinge region. And mutations (L1196M, G1269A, F1174L, and R1275Q) induce diverse conformational changes of brigatinib and the obvious energy variation of residues Glu1167, Arg1209, Asp1270, and Asp1203. Together, the detailed explanation of mechanisms of those mutations with brigatinib further provide several guidelines for the development of more effective ALK inhibitors.

Anaplastic Lymphoma Kinase (ALK) Receptor Tyrosine Kinase: A Catalytic Receptor with Many Faces.

The anaplastic lymphoma kinase (ALK) receptor is a membrane-bound tyrosine kinase. The pathogenesis of several cancers is closely related to aberrant forms of ALK or aberrant ALK expression, including ALK fusion proteins, ALK-activated point mutations, and ALK amplification. Clinical applications of different ALK inhibitors represent significant progress in targeted therapy. Knowledge of different aspects of ALK biology can provide significant information to further the understanding of this receptor tyrosine kinase. In this mini-review, we briefly summarize different features of ALK. We also summarize some recent research advances on ALK fusion proteins in cancers.