Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the ...Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of “conformational disruptors” to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.
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•Pseudokinases share autoinhibitory interactions of their closest kinase relatives•Structural dynamics of pseudokinases closely resemble those of canonical kinases•ROR1 ATP-binding site can be targeted with small molecules despite being occluded•Inhibitor binding disrupts pseudokinase conformation and may inhibit signaling
Sheetz et al. ask how Wnt-binding receptor tyrosine kinases can signal through intracellular “pseudokinase” domains. They find that these domains resemble autoinhibited kinases but are dynamic enough to regulate signaling through conformational switching. They find small molecules that can alter pseudokinase conformation, suggesting routes for modulating this receptor class therapeutically.
The nonreceptor tyrosine TEC kinases are key regulators of the immune system and play a crucial role in the pathogenesis of diverse hematological malignancies. In contrast to the substantial efforts ...in inhibitor development for Bruton's tyrosine kinase (BTK), specific inhibitors of the other TEC kinases, including the bone marrow tyrosine kinase on chromosome X (BMX), remain sparse. Here we present a novel class of dual BMX/BTK inhibitors, which were designed from irreversible inhibitors of Janus kinase (JAK) 3 targeting a cysteine located within the solvent-exposed front region of the ATP binding pocket. Structure-guided design exploiting the differences in the gatekeeper residues enabled the achievement of high selectivity over JAK3 and certain other kinases harboring a sterically demanding residue at this position. The most active compounds inhibited BMX and BTK with apparent IC
values in the single digit nanomolar range or below showing moderate selectivity within the TEC family and potent cellular target engagement. These compounds represent an important first step towards selective chemical probes for the protein kinase BMX.
Ibrutinib, a Bruton's tyrosine kinase inhibitor, is active in CLL, but resistance may emerge. The authors observed the emergence of a CLL clone with a cysteine-to-serine change in amino acid 481 of ...the target protein that substantially weakens drug binding and leads to resistance.
To the Editor:
Ibrutinib, an inhibitor that binds covalently to C481 of Bruton's tyrosine kinase (BTK), has produced remarkable responses in patients with relapsed and refractory chronic lymphocytic leukemia (CLL).
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However, 5.3% of patients have disease progression, and the mechanism of resistance is largely unknown. Herein we describe the mechanism of resistance in such a case.
A 49-year-old woman had a diagnosis of CLL established in 2000. After the failure of multiple treatments, she began receiving ibrutinib at a dose of 560 mg daily in 2010 as part of a phase 1, dose-escalation study of ibrutinib in B-cell cancers. . . .
Bruton's agammaglobulinemia tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase important in B-lymphocyte development, differentiation, and signaling. Btk is a member of the Tec family of kinases. ...Mutations in the Btk gene lead to X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Activation of Btk triggers a cascade of signaling events that culminates in the generation of calcium mobilization and fluxes, cytoskeletal rearrangements, and transcriptional regulation involving nuclear factor-κB (NF-κB) and nuclear factor of activated T cells (NFAT). In B cells, NF-κB was shown to bind to the Btk promoter and induce transcription, whereas the B-cell receptor-dependent NF-κB signaling pathway requires functional Btk. Moreover, Btk activation is tightly regulated by a plethora of other signaling proteins including protein kinase C (PKC), Sab/SH3BP5, and caveolin-1. For example, the prolyl isomerase Pin1 negatively regulates Btk by decreasing tyrosine phosphorylation and steady state levels of Btk. It is intriguing that PKC and Pin1, both of which are negative regulators, bind to the pleckstrin homology domain of Btk. To this end, we describe here novel mutations in the pleckstrin homology domain investigated for their transforming capacity. In particular, we show that the mutant D43R behaves similar to E41K, already known to possess such activity.
The pleckstrin homology (PH) domain is well known for its phospholipid targeting function. The PH-TEC homology (PHTH) domain within the TEC family of tyrosine kinases is also a crucial component of ...the autoinhibitory apparatus. The autoinhibitory surface on the PHTH domain has been previously defined, and biochemical investigations have shown that PHTH-mediated inhibition is mutually exclusive with phosphatidylinositol binding. Here we use hydrogen/deuterium exchange mass spectrometry, nuclear magnetic resonance (NMR), and evolutionary sequence comparisons to map where and how the PHTH domain affects the Bruton’s tyrosine kinase (BTK) domain. The data map a PHTH-binding site on the activation loop face of the kinase C lobe, suggesting that the PHTH domain masks the activation loop and the substrate-docking site. Moreover, localized NMR spectral changes are observed for non–surface-exposed residues in the active site and on the distal side of the kinase domain. These data suggest that the association of PHTH induces allosteric conformational shifts in regions of the kinase domain that are critical for catalysis. Through statistical comparisons of diverse tyrosine kinase sequences, we identify residues unique to BTK that coincide with the experimentally determined PHTH-binding surface on the kinase domain. Our data provide a more complete picture of the autoinhibitory conformation adopted by full-length TEC kinases, creating opportunities to target the regulatory domains to control the function of these kinases in a biological setting.
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.
RATIONALE:Clinical benefits of reperfusion after myocardial infarction are offset by maladaptive innate immune cell function, and therapeutic interventions are lacking.
OBJECTIVE:We sought to test ...the significance of phagocytic clearance by resident and recruited phagocytes after myocardial ischemia reperfusion.
METHODS AND RESULTS:In humans, we discovered that clinical reperfusion after myocardial infarction led to significant elevation of the soluble form of MerTK (myeloid-epithelial-reproductive tyrosine kinase; ie, soluble MER), a critical biomarker of compromised phagocytosis by innate macrophages. In reperfused mice, macrophage Mertk deficiency led to decreased cardiac wound debridement, increased infarct size, and depressed cardiac function, newly implicating MerTK in cardiac repair after myocardial ischemia reperfusion. More notably, Mertk(CR) mice, which are resistant to cleavage, showed significantly reduced infarct sizes and improved systolic function. In contrast to other cardiac phagocyte subsets, resident cardiac MHCIICCR2 (major histocompatibility complex II/C-C motif chemokine receptor type 2) macrophages expressed higher levels of MerTK and, when exposed to apoptotic cells, secreted proreparative cytokines, including transforming growth factor-β. Mertk deficiency compromised the accumulation of MHCII phagocytes, and this was rescued in Mertk(CR) mice. Interestingly, blockade of CCR2-dependent monocyte infiltration into the heart reduced soluble MER levels post-ischemia reperfusion.
CONCLUSIONS:Our data implicate monocyte-induced MerTK cleavage on proreparative MHCII cardiac macrophages as a novel contributor and therapeutic target of reperfusion injury.
In some patients with CLL, resistance to the BTK inhibitor ibrutinib develops. Two classes of resistance mutations have been found: the more common involves alteration of the drug-binding site to ...make binding reversible; the less common activates a downstream kinase that effectively bypasses BTK.
The development of B-cell–receptor antagonists has been a therapeutic advance in chronic lymphocytic leukemia (CLL). Although B-cell–receptor ligation in normal cells induces proliferation, apoptosis, or anergy,
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pathway dysregulation in CLL results in the propagation of proliferative and prosurvival signals.
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Several agents targeting the B-cell–receptor pathway are in development, including the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib. Although
BTK
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it is up-regulated at the transcript level and is constitutively active.
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Ibrutinib irreversibly binds BTK at the C481 residue, rendering it kinase-inactive, inducing modest CLL-cell apoptosis, and abolishing proliferation and B-cell–receptor signaling in . . .
Granulocyte-colony stimulating factor receptor (G-CSFR) controls myeloid progenitor proliferation and differentiation to neutrophils. Mutations in CSF3R (encoding G-CSFR) have been reported in ...patients with chronic neutrophilic leukemia (CNL) and acute myeloid leukemia (AML); however, despite years of research, the malignant downstream signaling of the mutated G-CSFRs is not well understood. Here, we used a quantitative phospho-tyrosine analysis to generate a comprehensive signaling map of G-CSF induced tyrosine phosphorylation in the normal versus mutated (proximal: T618I and truncated: Q741x) G-CSFRs. Unbiased clustering and kinase enrichment analysis identified rapid induction of phospho-proteins associated with endocytosis by the wild type G-CSFR only; while G-CSFR mutants showed abnormal kinetics of canonical Stat3, Stat5, and Mapk phosphorylation, and aberrant activation of Bruton's Tyrosine Kinase (Btk). Mutant-G-CSFR-expressing cells displayed enhanced sensitivity (3-5-fold lower IC50) for ibrutinib-based chemical inhibition of Btk. Primary murine progenitor cells from G-CSFR-Q741x knock-in mice validated activation of Btk by the mutant receptor and retrovirally transduced human CD34
umbilical cord blood cells expressing mutant receptors displayed enhanced sensitivity to Ibrutinib. A significantly lower clonogenic potential was displayed by both murine and human primary cells expressing mutated receptors upon ibrutinib treatment. Finally, a dramatic synergy was observed between ibrutinib and ruxolinitib at lower dose of the individual drug. Altogether, these data demonstrate the strength of unsupervised proteomics analyses in dissecting oncogenic pathways, and suggest repositioning Ibrutinib for therapy of myeloid leukemia bearing CSF3R mutations. Phospho-tyrosine data are available via ProteomeXchange with identifier PXD009662.
Background The Nod-like receptor NACHT, LRR, and PYD domain–containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK) are protagonists in innate and adaptive immunity, respectively. NLRP3 senses ...exogenous and endogenous insults, leading to inflammasome activation, which occurs spontaneously in patients with Muckle-Wells syndrome; BTK mutations cause the genetic immunodeficiency X-linked agammaglobulinemia (XLA). However, to date, few proteins that regulate NLRP3 inflammasome activity in human primary immune cells have been identified, and clinically promising pharmacologic targeting strategies remain elusive. Objective We sought to identify novel regulators of the NLRP3 inflammasome in human cells with a view to exploring interference with inflammasome activity at the level of such regulators. Methods After proteome-wide phosphoproteomics, the identified novel regulator BTK was studied in human and murine cells by using pharmacologic and genetic BTK ablation. Results Here we show that BTK is a critical regulator of NLRP3 inflammasome activation: pharmacologic (using the US Food and Drug Administration–approved inhibitor ibrutinib) and genetic (in patients with XLA and Btk knockout mice) BTK ablation in primary immune cells led to reduced IL-1β processing and secretion in response to nigericin and the Staphylococcus aureus toxin leukocidin AB (LukAB). BTK affected apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and caspase-1 cleavage and interacted with NLRP3 and ASC. S aureus infection control in vivo and IL-1β release from cells of patients with Muckle-Wells syndrome were impaired by ibrutinib. Notably, IL-1β processing and release from immune cells isolated from patients with cancer receiving ibrutinib therapy were reduced. Conclusion Our data suggest that XLA might result in part from genetic inflammasome deficiency and that NLRP3 inflammasome–linked inflammation could potentially be targeted pharmacologically through BTK.
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