Andrey V. Reshetnyak

Heparin is an activating ligand of the orphan receptor tyrosine kinase ALK

Heparin binds and activates the receptor tyrosine kinase ALK, which has no known ligand. De-Orphaning ALK Receptor tyrosine kinases (RTKs), including anaplastic lymphoma kinase (ALK), are important during development and regeneration and are often aberrantly activated in cancer. ALK is an orphan RTK with no known ligand. Murray et al. found that heparin bound directly and with high affinity to a specific region in the extracellular domain of ALK. Heparin stimulated ALK activation and signaling in NB1 cells through a mechanism that required heparin sulfation and the ability of heparin to promote dimerization of ALK. Moreover, an antibody that competed with heparin for binding to ALK prevented heparin-mediated activation of ALK. These results define heparin as an ALK-binding agonist, thereby providing potential avenues for therapeutic intervention. Anaplastic lymphoma kinase (ALK) is one of the few remaining “orphan” receptor tyrosine kinases (RTKs) in which the ligands are unknown. Ligand-mediated activation of RTKs is important throughout development. ALK is particularly relevant to the development of the nervous system. Increased activation of RTKs by mutation, genetic amplification, or signals from the stroma contributes to disease progression and acquired drug resistance in cancer. Aberrant activation of ALK occurs in subsets of lung adenocarcinoma, neuroblastoma, and other cancers. We found that heparin is a ligand that binds specifically to the ALK extracellular domain. Whereas heparins with short chain lengths bound to ALK in a monovalent manner and did not activate the receptor, longer heparin chains induced ALK dimerization and activation in cultured neuroblastoma cells. Heparin lacking N- and O-linked sulfate groups or other glycosaminoglycans with sulfation patterns different than heparin failed to activate ALK. Moreover, antibodies that bound to the extracellular domain of ALK interfered with heparin binding and prevented heparin-mediated activation of ALK. Thus, heparin and perhaps related glycosaminoglycans function as ligands for ALK, revealing a potential mechanism for the regulation of ALK activity in vivo and suggesting an approach for developing ALK-targeted therapies for cancer.

Reactibodies Generated by Kinetic Selection Couple Chemical Reactivity with Favorable Protein Dynamics.

Igs offer a versatile template for combinatorial and rational design approaches to the de novo creation of catalytically active proteins. We have used a covalent capture selection strategy to identify biocatalysts from within a human semisynthetic antibody variable fragment library that uses a nucleophilic mechanism. Specific phosphonylation at a single tyrosine within the variable light-chain framework was confirmed in a recombinant IgG construct. High-resolution crystallographic structures of unmodified and phosphonylated Fabs display a 15-Å-deep two-chamber cavity at the interface of variable light (VL) and variable heavy (VH) fragments having a nucleophilic tyrosine at the base of the site. The depth and structure of the pocket are atypical of antibodies in general but can be compared qualitatively with the catalytic site of cholinesterases. A structurally disordered heavy chain complementary determining region 3 loop, constituting a wall of the cleft, is stabilized after covalent modification by hydrogen bonding to the phosphonate tropinol moiety. These features and presteady state kinetics analysis indicate that an induced fit mechanism operates in this reaction. Mutations of residues located in this stabilized loop do not interfere with direct contacts to the organophosphate ligand but can interrogate second shell interactions, because the H3 loop has a conformation adjusted for binding. Kinetic and thermodynamic parameters along with computational docking support the active site model, including plasticity and simple catalytic components. Although relatively uncomplicated, this catalytic machinery displays both stereo- and chemical selectivity. The organophosphate pesticide paraoxon is hydrolyzed by covalent catalysis with rate-limiting dephosphorylation. This reactibody is, therefore, a kinetically selected protein template that has enzyme-like catalytic attributes.

Augmentor α and β (FAM150) are ligands of the receptor tyrosine kinases ALK and LTK: Hierarchy and specificity of ligand–receptor interactions

Significance Many cancers (e.g., subpopulations of lung cancer, anaplastic lymphoma, and neuroblastoma) are driven by mutations in the receptor tyrosine kinase ALK (for anaplastic lymphoma kinase). However, the extracellular protein signals that regulate ALK’s activity and its ligand-induced mechanism of activation remain elusive. Here we describe a cytokine designated augmentor-α that binds with high affinity and specificity to ALK’s extracellular glycine-rich region, resulting in robust receptor activation. Augmentor-α also potently activates the related leukocyte tyrosine kinase (LTK) receptor, whereas a previously identified LTK ligand (augmentor-β) only weakly activates ALK. These experiments reveal an important missing link necessary for the regulation of a known oncogenic RTK, providing important insights into its biology and offering new opportunities for therapeutic intervention. Receptor tyrosine kinases (RTKs) are a class of cell surface receptors that, upon ligand binding, stimulate a variety of critical cellular functions. The orphan receptor anaplastic lymphoma kinase (ALK) is one of very few RTKs that remain without a firmly established protein ligand. Here we present a novel cytokine, FAM150B, which we propose naming augmentor-α (AUG-α), as a ligand for ALK. AUG-α binds ALK with high affinity and activates ALK in cells with subnanomolar potency. Detailed binding experiments using cells expressing ALK or the related receptor leukocyte tyrosine kinase (LTK) demonstrate that AUG-α binds and robustly activates both ALK and LTK. We show that the previously established LTK ligand FAM150A (AUG-β) is specific for LTK and only weakly binds to ALK. Furthermore, expression of AUG-α stimulates transformation of NIH/3T3 cells expressing ALK, induces IL-3 independent growth of Ba/F3 cells expressing ALK, and is expressed in neuroblastoma, a cancer partly driven by ALK. These experiments reveal the hierarchy and specificity of two cytokines as ligands for ALK and LTK and set the stage for elucidating their roles in development and disease states.

Structural basis for KIT receptor tyrosine kinase inhibition by antibodies targeting the D4 membrane-proximal region

Significance The receptor tyrosine kinase KIT is aberrantly activated primarily by somatic mutations in gastrointestinal stromal tumors and in a subset of acute myeloid leukemia, melanoma, and other cancers. Treatment of these cancers with tyrosine kinase inhibitors shows durable clinical response, but drug resistance and disease progression eventually occur in all patients. Here we describe monoclonal antibodies that block the activity of KIT and its oncogenic mutant. Structural and biochemical analyses of anti-KIT antibodies in complex with a KIT fragment demonstrated that KIT antibodies bind to a critical Achilles heel region that is essential for receptor activation. These antibodies may provide a potentially unique therapeutic approach for the treatment of tumors driven by WT or oncogenically mutated KIT. Somatic oncogenic mutations in the receptor tyrosine kinase KIT function as major drivers of gastrointestinal stromal tumors and a subset of acute myeloid leukemia, melanoma, and other cancers. Although treatment of these cancers with tyrosine kinase inhibitors shows dramatic responses and durable disease control, drug resistance followed by clinical progression of disease eventually occurs in virtually all patients. In this report, we describe inhibitory KIT antibodies that bind to the membrane-proximal Ig-like D4 of KIT with significant overlap with an epitope in D4 that mediates homotypic interactions essential for KIT activation. Crystal structures of the anti-KIT antibody in complex with KIT D4 and D5 allowed design of affinity-matured libraries that were used to isolate variants with increased affinity and efficacy. Isolated antibodies showed KIT inhibition together with suppression of cell proliferation driven by ligand-stimulated WT or constitutively activated oncogenic KIT mutant. These antibodies represent a unique therapeutic approach and a step toward the development of “naked” or toxin-conjugated KIT antibodies for the treatment of KIT-driven cancers.

Catalytic transformations of supercoiled DNA as studied by flow linear dichroism technique

A catalytic turnover of supercoiled DNA (scDNA) transformation mediated by topoisomerases leads to changes in the linking number (Lk) of the polymeric substrate by 1 or 2 per cycle. As a substrate of the topoisomerization reaction it is chemically identical to its product; even a single catalytic event results in the quantum leap in the scDNA topology. Non‐intrusive continuous assay to measure the kinetics of the scDNA topoisomerization was performed. The development of such a technique was hindered because of multiple DNA species of intermediate topology present in the reaction mixture. The interrelation of DNA topology, its hydrodynamics, and optical anisotropy enable us to use the flow linear dichroism technique (FLD) for continuous monitoring of the scDNA topoisomerization reaction. This approach permits us to study the kinetics of DNA transformation catalyzed by eukaryotic topoisomerases I and II, as well as mechanistic characteristics of these enzymes and their interactions with anticancer drugs. Moreover, FLD assay can be applied to any enzymatic reaction that involves scDNA as a substrate. It also provides a new way of screening drugs dynamically and is likely to be potent in various biomedical applications.

Alk and Ltk ligands are essential for iridophore development in zebrafish mediated by the receptor tyrosine kinase Ltk

Significance Receptor tyrosine kinases (RTKs) and their stimulatory ligands regulate a variety of cellular processes. Many diseases, including cancer, are driven by mutations in or dysregulation of RTKs and their signaling pathways. The physiological ligands responsible for activating the RTKs, ALK and LTK, remained elusive. Recently, the ligands of each were identified and named “ALKAL1” and “ALKAL2” (“FAM150” or “augmentors”). Here, we demonstrate that zebrafish contain three distinct ligand molecules and describe their role in controlling pigment development in the embryo and adult zebrafish eye and body. These experiments show that these hormone-like molecules are critical factors influencing neural crest cell differentiation and progenitor/stem cell fates in zebrafish, suggesting a role for augmentors in control of similar processes in higher organisms. Anaplastic lymphoma kinase (Alk) and leucocyte tyrosine kinase (Ltk) were identified as “orphan” receptor tyrosine kinases (RTKs) with oncogenic potential. Recently ALKAL1 and ALKAL2 (also named “augmentor-β” and “augmentor-α” or “FAM150A” and “FAM150B,” respectively) were discovered as physiological ligands of Alk and Ltk. Here, we employ zebrafish as a model system to explore the physiological function and to characterize in vivo links between Alk and Ltk with their ligands. Unlike the two ligands encoded by mammalian genomes, the zebrafish genome contains three genes: aug-α1, aug-α2, and aug-β. Our experiments demonstrate that these ligands play an important role in zebrafish pigment development. Deficiency in aug-α1, aug-α2, and aug-β results in strong impairment in iridophore patterning of embryonic and adult zebrafish that is phenocopied in zebrafish deficient in Ltk. We show that aug-α1 and aug-α2 are essential for embryonic iridophore development and adult body coloration. In contrast, aug-α2 and aug-β are essential for iridophore formation in the adult eye. Importantly, these processes are entirely mediated by Ltk and not by Alk. These experiments establish a physiological link between augmentor ligands and Ltk and demonstrate that particular augmentors activate Ltk in a tissue-specific context to induce iridophore differentiation from neural crest-derived cells and pigment progenitor cells.

Distinct cellular properties of oncogenic KIT receptor tyrosine kinase mutants enable alternative courses of cancer cell inhibition

Significance A common practice in modern clinics is to identify a match between a mutated oncogenic protein that functions as a “driver” of a particular cancer with a known or new cancer drug from available targeted therapies. To understand mechanisms underlying the differential clinical impact of various targeted therapies on cancers driven by the receptor tyrosine kinase KIT, we analyzed a variety of biochemical and cellular properties of the most common KIT somatic mutations identified in human cancers. Surprisingly, each of the six major KIT oncogenic mutants exhibits distinct properties and responds differently to targeted therapies. These experiments show that detailed biochemical and cellular analyses of oncogenic mutations are required to optimize precision medicine for cancer treatment. Large genomic sequencing analysis as part of precision medicine efforts revealed numerous activating mutations in receptor tyrosine kinases, including KIT. Unfortunately, a single approach is not effective for inhibiting cancer cells or treating cancers driven by all known oncogenic KIT mutants. Here, we show that each of the six major KIT oncogenic mutants exhibits different enzymatic, cellular, and dynamic properties and responds distinctly to different KIT inhibitors. One class of KIT mutants responded well to anti-KIT antibody treatment alone or in combination with a low dose of tyrosine kinase inhibitors (TKIs). A second class of KIT mutants, including a mutant resistant to imatinib treatment, responded well to a combination of TKI with anti-KIT antibodies or to anti-KIT toxin conjugates, respectively. We conclude that the preferred choice of precision medicine treatments for cancers driven by activated KIT and other RTKs may rely on clear understanding of the dynamic properties of oncogenic mutants.

Dynamics of DNA Supercoiling

A catalytic turnover of supercoiled DNA (scDNA) transformation mediated by topoisomerases leads to the changes of the linking number (Lk) of the polymeric substrate by 1 or 2. While a substrate of the topoisomerisation reaction is chemically identical to its product, even single catalytic event results in the quantum leap in the scDNA topology. A continuous non-disturbing assay for measurement of kinetics of the scDNA topoisomerisation was lacking. The intrinsic connections of DNA topology, its hydrodynamics and optical anisotropy, studied in this chapter allowed the use of flow linear dichroism technique (FLD) for continuous monitoring of scDNA topoisomerisation reaction. This approach permits studying the kinetics of DNA transformation catalysed by eukaryotic topoisomerases I and II, mechanistic properties of these enzymes and their interactions with anti-cancer drugs.

Identification of a biologically active fragment of ALK and LTK-Ligand 2 (augmentor-α)

Significance The limited availability of the two ligands of the receptor tyrosine kinases ALK and LTK precluded the elucidation of their physiological roles and mechanism of action. In this report we describe two new approaches to produce full-length ALKAL2/AUG-α and an active variant lacking the N-terminal variable region. Detailed characterization of the primary structure and disulfide bridges using mass spectrometry demonstrates that the N-terminal variable region plays an important role in AUG-α dimerization and provides insight into the structural organization of a previously unknown augmentor fold. A variety of cellular experiments show that both AUG-α and its deletion mutant induce similar activation of ALK and LTK and stimulation neuronal differentiation of human neuroblastoma NB1 and rat pheochromocytoma PC12 cells. Elucidating the physiological roles and modes of action of the recently discovered ligands (designated ALKAL1,2 or AUG-α,β) of the receptor tyrosine kinases Anaplastic Lymphoma Kinase (ALK) and Leukocyte Tyrosine Kinase (LTK) has been limited by difficulties in producing sufficient amounts of the two ligands and their poor stability. Here we describe procedures for expression and purification of AUG-α and a deletion mutant lacking the N-terminal variable region. Detailed biochemical characterization of AUG-α by mass spectrometry shows that the four conserved cysteines located in the augmentor domain (AD) form two intramolecular disulfide bridges while a fifth, primate-specific cysteine located in the N-terminal variable region mediates dimerization through formation of a disulfide bridge between two AUG-α molecules. In contrast to AUG-α, the capacity of AUG-α AD to undergo dimerization is strongly compromised. However, full-length AUG-α and the AUG-α AD deletion mutant stimulate similar tyrosine phosphorylation of cells expressing either ALK or LTK. Both AUG-α and AUG-α AD also stimulate a similar profile of MAP kinase response in L6 cells and colony formation in soft agar by autocrine stimulation of NIH 3T3 cells expressing ALK. Moreover, both AUG-α and AUG-α AD stimulate neuronal differentiation of human neuroblastoma NB1 and PC12 cells in a similar dose-dependent manner. Taken together, these experiments show that deletion of the N-terminal variable region minimally affects the activity of AUG-α toward LTK or ALK stimulation in cultured cells. Reduced dimerization might be compensated by high local concentration of AUG-α AD bound to ALK at the cell membrane and by potential ligand-induced receptor–receptor interactions.