RESUMO
Sarcomas are a heterogenous group of tumours that commonly carry poor prognosis with limited therapeutic options. Adolescents and young adults (AYAs) with sarcoma are a unique and understudied patient population that have only achieved modest survival gains compared to other groups. We present our institutional experience of AYAs with sarcoma who underwent comprehensive molecular profiling (CMP) via either large-panel targeted DNA sequencing or whole genome and transcriptome sequencing and evaluated the feasibility and clinical impact of this approach. Genomic variants detected were determined to be clinically relevant and actionable following evaluation by the Molecular Tumour Board. Clinicians provided feedback regarding the utility of testing three months after reporting. Twenty-five patients who were recruited for CMP are included in this analysis. The median time from consent to final molecular report was 45 days (interquartile range: 37-57). Potentially actionable variants were detected for 14 patients (56%), and new treatment recommendations were identified for 12 patients (48%). Pathogenic germline variants were identified in three patients (12%), and one patient had a change in diagnosis. The implementation of CMP for AYAs with sarcoma is clinically valuable, feasible, and should be increasingly integrated into routine clinical practice as technologies and turnaround times continue to improve.
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Congenital amegakaryocytic thrombocytopenia (CAMT) is a severe inherited thrombocytopenia due to loss-of-function mutations affecting the thrombopoietin (TPO) receptor, MPL. Here, we report a new homozygous MPL variant responsible for CAMT in 1 consanguineous family. The propositus and her sister presented with severe thrombocytopenia associated with mild anemia. Next-generation sequencing revealed the presence of a homozygous MPLR464G mutation resulting in a weak cell-surface expression of the receptor in platelets. In cell lines, we observed a defect in MPLR464G maturation associated with its retention in the endoplasmic reticulum. The low cell-surface expression of MPLR464G induced very limited signaling with TPO stimulation, leading to survival and reduced proliferation of cells. Overexpression of a myeloproliferative neoplasm-associated calreticulin (CALR) mutant did not rescue trafficking of MPLR464G to the cell surface and did not induce constitutive signaling. However, it unexpectedly restored a normal response to eltrombopag (ELT), but not to TPO. This effect was only partially mimicked by the purified recombinant CALR mutant protein. Finally, the endogenous CALR mutant was able to restore the megakaryocyte differentiation of patient CD34+ cells carrying MPLR464G in response to ELT.
Assuntos
Benzoatos/farmacologia , Calreticulina , Síndrome Congênita de Insuficiência da Medula Óssea , Hidrazinas/farmacologia , Mutação de Sentido Incorreto , Pirazóis/farmacologia , Receptores de Trombopoetina , Trombocitopenia , Adulto , Substituição de Aminoácidos , Calreticulina/genética , Calreticulina/metabolismo , Criança , Pré-Escolar , Síndrome Congênita de Insuficiência da Medula Óssea/tratamento farmacológico , Síndrome Congênita de Insuficiência da Medula Óssea/genética , Síndrome Congênita de Insuficiência da Medula Óssea/metabolismo , Síndrome Congênita de Insuficiência da Medula Óssea/patologia , Feminino , Células HEK293 , Homozigoto , Humanos , Lactente , Masculino , Receptores de Trombopoetina/genética , Receptores de Trombopoetina/metabolismo , Trombocitopenia/tratamento farmacológico , Trombocitopenia/genética , Trombocitopenia/metabolismo , Trombocitopenia/patologiaRESUMO
Mutations in the MPL gene encoding the human thrombopoietin receptor (TpoR) drive sporadic and familial essential thrombocythemias (ETs). We identified 2 ET patients harboring double mutations in cis in MPL, namely, L498W-H499C and H499Y-S505N. Using biochemical and signaling assays along with partial saturation mutagenesis, we showed that L498W is an activating mutation potentiated by H499C and that H499C and H499Y enhance the activity of the canonical S505N mutation. L498W and H499C can activate a truncated TpoR mutant, which lacks the extracellular domain, indicating these mutations act on the transmembrane (TM) cytosolic domain. Using a protein complementation assay, we showed that L498W and H499C strongly drive dimerization of TpoR. Activation by tryptophan substitution is exquisitely specific for position 498. Using structure-guided mutagenesis, we identified upstream amino acid W491 as a key residue required for activation by L498W or canonical activating mutations such as S505N and W515K, as well as by eltrombopag. Structural data point to a common dimerization and activation path for TpoR via its TM domain that is shared between the small-molecule agonist eltrombopag and canonical and novel activating TpoR mutations that all depend on W491, a potentially accessible extracellular residue that could become a target for therapeutic intervention.
Assuntos
Benzoatos/farmacologia , Predisposição Genética para Doença , Hidrazinas/farmacologia , Mutação , Pirazóis/farmacologia , Receptores de Trombopoetina/agonistas , Receptores de Trombopoetina/genética , Trombocitemia Essencial/genética , Alelos , Substituição de Aminoácidos , Linhagem Celular , Estudos de Associação Genética , Humanos , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Trombocitemia Essencial/diagnóstico , Trombocitemia Essencial/metabolismoRESUMO
Introduction: Classical Myeloproliferative Neoplasms (MPNs) include three disorders: Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). MPNs are associated with constitutive activation of JAK2 leading to persistent cell signaling downstream of the dimeric myeloid cytokine receptors due to mutations in three genes encoding JAK2, calreticulin (CALR) and the thrombopoietin (TPO) receptor (MPL or TPOR). CALR and MPL mutants induce JAK2 activation that depends on MPL expression, thus explaining why they induce megakaryocyte pathologies including ET and PMF, but not PV. In contrast, JAK2 V617F drives all three diseases as it induces persistent signaling via EPOR, G-CSFR (CSF3R) and MPL. Areas Covered: Here, we review how different pathogenic mutations of MPL are translated into active receptors by inducing stable dimerization. We focus on the unique role of MPL on the hematopoietic stem cell (HSC), explaining why MPL is indispensable for the development of all MPNs. Last but not least, we describe how CALR mutants are pathogenic via binding and activation of MPL. Expert Opinion: Altogether, we believe that MPL is an important, but challenging, therapeutic target in MPNs that requires novel strategies to interrupt the specific conformational changes induced by each mutation or pathologic interaction without compromising the key functions of wild type MPL.
Assuntos
Transtornos Mieloproliferativos/genética , Receptores de Trombopoetina/metabolismo , HumanosRESUMO
A well-functioning hematopoietic system requires a certain robustness and flexibility to maintain appropriate quantities of functional mature blood cells, such as red blood cells and platelets. This review focuses on the cytokine receptor that plays a significant role in thrombopoiesis: the receptor for thrombopoietin (TPO-R; also known as MPL). Here, we survey the work to date to understand how this receptor functions at a molecular level throughout its lifecycle, from traffic to the cell surface, dimerization and binding cognate cytokine via its extracellular domain, through to its subsequent activation of associated Janus kinases and initiation of downstream signaling pathways, as well as the regulation of these processes. Atomic level resolution structures of TPO-R have remained elusive. The identification of disease-causing mutations in the receptor has, however, offered some insight into structure and function relationships, as has artificial means of receptor activation, through TPO mimetics, transmembrane-targeting receptor agonists, and engineering in dimerization domains. More recently, a novel activation mechanism was identified whereby mutated forms of calreticulin form complexes with TPO-R via its extracellular N-glycosylated domain. Such complexes traffic pathologically in the cell and persistently activate JAK2, downstream signal transducers and activators of transcription (STATs), and other pathways. This pathologic TPO-R activation is associated with a large fraction of human myeloproliferative neoplasms.
RESUMO
The mechanisms behind the hereditary thrombocytosis induced by the thrombopoietin (THPO) receptor MPL P106L mutant remain unknown. A complete trafficking defect to the cell surface has been reported, suggesting either weak constitutive activity or nonconventional THPO-dependent mechanisms. Here, we report that the thrombocytosis phenotype induced by MPL P106L belongs to the paradoxical group, where low MPL levels on platelets and mature megakaryocytes (MKs) lead to high serum THPO levels, whereas weak but not absent MPL cell-surface localization in earlier MK progenitors allows response to THPO by signaling and amplification of the platelet lineage. MK progenitors from patients showed no spontaneous growth and responded to THPO, and MKs expressed MPL on their cell surface at low levels, whereas their platelets did not respond to THPO. Transduction of MPL P106L in CD34+ cells showed that this receptor was more efficiently localized at the cell surface on immature than on mature MKs, explaining a proliferative response to THPO of immature cells and a defect in THPO clearance in mature cells. In a retroviral mouse model performed in Mpl-/- mice, MPL P106L could induce a thrombocytosis phenotype with high circulating THPO levels. Furthermore, we could select THPO-dependent cell lines with more cell-surface MPL P106L localization that was detected by flow cytometry and [125I]-THPO binding. Altogether, these results demonstrate that MPL P106L is a receptor with an incomplete defect in trafficking, which induces a low but not absent localization of the receptor on cell surface and a response to THPO in immature MK cells.
Assuntos
Membrana Celular/metabolismo , Mutação/genética , Receptores de Trombopoetina/genética , Trombocitose/genética , Trombocitose/patologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Família , Feminino , Humanos , Masculino , Megacariócitos/metabolismo , Camundongos , Linhagem , Transporte Proteico , Receptores de Trombopoetina/metabolismo , Retroviridae/metabolismo , Transdução GenéticaRESUMO
The pseudokinase MLKL (mixed lineage kinase domain-like), has recently emerged as a critical component of the necroptosis cell death pathway. Although it is clear that phosphorylation of the activation loop in the MLKL pseudokinase domain by the upstream protein kinase RIPK3 (receptor-interacting protein kinase-3), is crucial to trigger MLKL activation, it has remained unclear whether other phosphorylation events modulate MLKL function. By reconstituting Mlkl(-/-), Ripk3(-/-) and Mlkl(-/-)Ripk3(-/-) cells with MLKL phospho-site mutants, we compared the function of known MLKL phosphorylation sites in regulating necroptosis with three phospho-sites that we identified by MS, Ser(158), Ser(228) and Ser(248). Expression of a phosphomimetic S345D MLKL activation loop mutant-induced stimulus-independent cell death in all knockout cells, demonstrating that RIPK3 phosphorylation of the activation loop of MLKL is sufficient to induce cell death. Cell death was also induced by S228A, S228E and S158A MLKL mutants in the absence of death stimuli, but was most profound in Mlkl(-/-)Ripk3(-/-) double knockout fibroblasts. These data reveal a potential role for RIPK3 as a suppressor of MLKL activation and indicate that phosphorylation can fine-tune the ability of MLKL to induce necroptosis.
Assuntos
Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Substituição de Aminoácidos , Animais , Ativação Enzimática/fisiologia , Técnicas de Inativação de Genes , Humanos , Camundongos , Mutação de Sentido Incorreto , Fosforilação/fisiologia , Proteínas Quinases/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Células U937RESUMO
The JAK (Janus kinase) family members serve essential roles as the intracellular signalling effectors of cytokine receptors. This family, comprising JAK1, JAK2, JAK3 and TYK2 (tyrosine kinase 2), was first described more than 20 years ago, but the complexities underlying their activation, regulation and pleiotropic signalling functions are still being explored. Here, we review the current knowledge of their physiological functions and the causative role of activating and inactivating JAK mutations in human diseases, including haemopoietic malignancies, immunodeficiency and inflammatory diseases. At the molecular level, recent studies have greatly advanced our knowledge of the structures and organization of the component FERM (4.1/ezrin/radixin/moesin)-SH2 (Src homology 2), pseudokinase and kinase domains within the JAKs, the mechanism of JAK activation and, in particular, the role of the pseudokinase domain as a suppressor of the adjacent tyrosine kinase domain's catalytic activity. We also review recent advances in our understanding of the mechanisms of negative regulation exerted by the SH2 domain-containing proteins, SOCS (suppressors of cytokine signalling) proteins and LNK. These recent studies highlight the diversity of regulatory mechanisms utilized by the JAK family to maintain signalling fidelity, and suggest alternative therapeutic strategies to complement existing ATP-competitive kinase inhibitors.
Assuntos
Janus Quinase 1/metabolismo , Janus Quinase 2/metabolismo , Janus Quinase 3/metabolismo , TYK2 Quinase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Ativação Enzimática , Humanos , Síndromes de Imunodeficiência/genética , Peptídeos e Proteínas de Sinalização Intracelular , Antígenos Comuns de Leucócito/metabolismo , Transtornos Mieloproliferativos/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Estrutura Terciária de Proteína/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas/metabolismo , Receptores de Citocinas/fisiologia , Transdução de Sinais , Domínios de Homologia de srcRESUMO
IL-6 a multi-functional cytokine with important effects in both inflammation and haematopoiesis. SOCS3 is the primary inhibitor of IL-6 signalling, interacting with gp130, the common shared chain of the IL-6 family of cytokines, and JAK1, JAK2 and TYK2 to control both the duration of signalling and the biological response. Recent biochemical and structural studies have shown SOCS3 binds to only these three JAKs, all of which are associated with IL-6 signalling, and not JAK3. This specificity is determined by a three residue "GQM" motif in the kinase domain of JAK1, JAK2 and TYK2. SOCS3 binds to JAK and gp130 simultaneously, and inhibits JAK activity in an ATP-independent manner by partially occluding the kinase's substrate binding groove with its kinase inhibitory region. We therefore propose a model in which each of gp130, JAK and SOCS3 are directly bound to the other two, allowing SOCS3 to inhibit IL6 signalling with high potency and specificity.
Assuntos
Interleucina-6/antagonistas & inibidores , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Catálise , Receptor gp130 de Citocina/metabolismo , Humanos , Interleucina-6/metabolismo , Janus Quinases/antagonistas & inibidores , Janus Quinases/metabolismo , Modelos Biológicos , Família Multigênica , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de Interleucina-6/metabolismo , Transdução de Sinais , Proteínas Supressoras da Sinalização de Citocina/química , Proteínas Supressoras da Sinalização de Citocina/genéticaRESUMO
Activation of the cell surface receptor, c-Mpl, by the cytokine, thrombopoietin (TPO), underpins megakaryocyte and platelet production in mammals. In humans, mutations in c-Mpl have been identified as the molecular basis of Congenital Amegakaryocytic Thrombocytopenia (CAMT). Here, we show that CAMT-associated mutations in c-Mpl principally lead to defective receptor presentation on the cell surface. In contrast, one CAMT mutant c-Mpl, F104S, was expressed on the cell surface, but showed defective TPO binding and receptor activation. Using mutational analyses, we examined which residues adjacent to F104 within the membrane-distal cytokine receptor homology module (CRM) of c-Mpl comprise the TPO-binding epitope, revealing residues within the predicted Domain 1 E-F and A-B loops and Domain 2 F'-G' loop as key TPO-binding determinants. These studies underscore the importance of the c-Mpl membrane-distal CRM to TPO-binding and suggest that mutations within this CRM that perturb TPO binding could give rise to CAMT.
Assuntos
Mutação/genética , Receptores de Trombopoetina/genética , Trombocitopenia/genética , Trombopoetina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Plaquetas/metabolismo , Células COS , Linhagem Celular , Proliferação de Células , Chlorocebus aethiops , Síndrome Congênita de Insuficiência da Medula Óssea , Megacariócitos/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Oncogênicas/genética , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Receptores da Prolactina/genética , Alinhamento de Sequência , Transdução de Sinais/genéticaRESUMO
JAK2 (Janus kinase 2) initiates the intracellular signalling cascade downstream of cell surface receptor activation by cognate haemopoietic cytokines, including erythropoietin and thrombopoietin. The pseudokinase domain (JH2) of JAK2 negatively regulates the catalytic activity of the adjacent tyrosine kinase domain (JH1) and mutations within the pseudokinase domain underlie human myeloproliferative neoplasms, including polycythaemia vera and essential thrombocytosis. To date, the mechanism of JH2-mediated inhibition of JH1 kinase activation as well as the susceptibility of pathological mutant JAK2 to inhibition by the physiological negative regulator SOCS3 (suppressor of cytokine signalling 3) have remained unclear. In the present study, using recombinant purified JAK2JH1-JH2 proteins, we demonstrate that, when activated, wild-type and myeloproliferative neoplasm-associated mutants of JAK2 exhibit comparable enzymatic activity and inhibition by SOCS3 in in vitro kinase assays. SAXS (small-angle X-ray scattering) showed that JAK2JH1-JH2 exists in an elongated configuration in solution with no evidence for interaction between JH1 and JH2 domains in cis. Collectively, these data are consistent with a model in which JAK2's pseudokinase domain does not influence the activity of JAK2 once it has been activated. Our data indicate that, in the absence of the N-terminal FERM domain and thus cytokine receptor association, the wild-type and pathological mutants of JAK2 are enzymatically equivalent and equally susceptible to inhibition by SOCS3.
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Neoplasias Hematológicas/prevenção & controle , Janus Quinase 2/antagonistas & inibidores , Mutação de Sentido Incorreto/genética , Transtornos Mieloproliferativos/prevenção & controle , Proteínas Supressoras da Sinalização de Citocina/fisiologia , Domínio Catalítico/genética , Predisposição Genética para Doença , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Janus Quinase 2/química , Janus Quinase 2/genética , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Estrutura Secundária de Proteína/genética , Proteínas Recombinantes/genética , Espalhamento a Baixo Ângulo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Difração de Raios XRESUMO
Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.
Assuntos
Janus Quinase 2/química , Janus Quinase 2/classificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Receptor ErbB-3/química , Receptor ErbB-3/classificação , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Insetos , Janus Quinase 2/genética , Dados de Sequência Molecular , Ligação Proteica/fisiologia , Receptor ErbB-3/genéticaRESUMO
Mixed lineage kinase domain-like (MLKL) is a component of the "necrosome," the multiprotein complex that triggers tumor necrosis factor (TNF)-induced cell death by necroptosis. To define the specific role and molecular mechanism of MLKL action, we generated MLKL-deficient mice and solved the crystal structure of MLKL. Although MLKL-deficient mice were viable and displayed no hematopoietic anomalies or other obvious pathology, cells derived from these animals were resistant to TNF-induced necroptosis unless MLKL expression was restored. Structurally, MLKL comprises a four-helical bundle tethered to the pseudokinase domain, which contains an unusual pseudoactive site. Although the pseudokinase domain binds ATP, it is catalytically inactive and its essential nonenzymatic role in necroptotic signaling is induced by receptor-interacting serine-threonine kinase 3 (RIPK3)-mediated phosphorylation. Structure-guided mutation of the MLKL pseudoactive site resulted in constitutive, RIPK3-independent necroptosis, demonstrating that modification of MLKL is essential for propagation of the necroptosis pathway downstream of RIPK3.
Assuntos
Apoptose , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fatores de Necrose Tumoral/metabolismo , Animais , Domínio Catalítico , Linhagem Celular , Cristalografia por Raios X , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necrose , Fosfoproteínas Fosfatases , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Proteínas Quinases/química , Proteínas Quinases/genética , Transdução de SinaisRESUMO
The inhibitory protein SOCS3 plays a key part in the immune and hematopoietic systems by regulating signaling induced by specific cytokines. SOCS3 functions by inhibiting the catalytic activity of Janus kinases (JAKs) that initiate signaling within the cell. We determined the crystal structure of a ternary complex between mouse SOCS3, JAK2 (kinase domain) and a fragment of the interleukin-6 receptor ß-chain. The structure shows that SOCS3 binds JAK2 and receptor simultaneously, using two opposing surfaces. While the phosphotyrosine-binding groove on the SOCS3 SH2 domain is occupied by receptor, JAK2 binds in a phosphoindependent manner to a noncanonical surface. The kinase-inhibitory region of SOCS3 occludes the substrate-binding groove on JAK2, and biochemical studies show that it blocks substrate association. These studies reveal that SOCS3 targets specific JAK-cytokine receptor pairs and explains the mechanism and specificity of SOCS action.
Assuntos
Citocinas/metabolismo , Janus Quinase 2/metabolismo , Transdução de Sinais , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/química , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Homologia de Sequência de Aminoácidos , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/químicaRESUMO
Janus kinases (JAKs) are key effectors in controlling immune responses and maintaining hematopoiesis. SOCS3 (suppressor of cytokine signaling-3) is a major regulator of JAK signaling and here we investigate the molecular basis of its mechanism of action. We found that SOCS3 bound and directly inhibited the catalytic domains of JAK1, JAK2, and TYK2 but not JAK3 via an evolutionarily conserved motif unique to JAKs. Mutation of this motif led to the formation of an active kinase that could not be inhibited by SOCS3. Surprisingly, we found that SOCS3 simultaneously bound JAK and the cytokine receptor to which it is attached, revealing how specificity is generated in SOCS action and explaining why SOCS3 inhibits only a subset of cytokines. Importantly, SOCS3 inhibited JAKs via a noncompetitive mechanism, making it a template for the development of specific and effective inhibitors to treat JAK-based immune and proliferative diseases.