RESUMO
Classical BCR-ABL-negative myeloproliferative neoplasms (MPNs) are clonal disorders of hematopoietic stem cells (HSCs) caused mainly by recurrent mutations in genes encoding JAK2 (JAK2), calreticulin (CALR), or the thrombopoietin receptor (MPL). Interferon α (IFNα) has demonstrated some efficacy in inducing molecular remission in MPNs. To determine factors that influence molecular response rate, we evaluated the long-term molecular efficacy of IFNα in patients with MPN by monitoring the fate of cells carrying driver mutations in a prospective observational and longitudinal study of 48 patients over more than 5 years. We measured the clonal architecture of early and late hematopoietic progenitors (84 845 measurements) and the global variant allele frequency in mature cells (409 measurements) several times per year. Using mathematical modeling and hierarchical Bayesian inference, we further inferred the dynamics of IFNα-targeted mutated HSCs. Our data support the hypothesis that IFNα targets JAK2V617F HSCs by inducing their exit from quiescence and differentiation into progenitors. Our observations indicate that treatment efficacy is higher in homozygous than heterozygous JAK2V617F HSCs and increases with high IFNα dose in heterozygous JAK2V617F HSCs. We also found that the molecular responses of CALRm HSCs to IFNα were heterogeneous, varying between type 1 and type 2 CALRm, and a high dose of IFNα correlates with worse outcomes. Our work indicates that the long-term molecular efficacy of IFNα implies an HSC exhaustion mechanism and depends on both the driver mutation type and IFNα dose.
Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Fatores Imunológicos/uso terapêutico , Interferon-alfa/uso terapêutico , Mutação/efeitos dos fármacos , Transtornos Mieloproliferativos/tratamento farmacológico , Calreticulina/genética , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Fatores Imunológicos/farmacologia , Interferon-alfa/farmacologia , Janus Quinase 2/genética , Estudos Longitudinais , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Estudos Prospectivos , Receptores de Trombopoetina/genética , Células Tumorais CultivadasRESUMO
An inhibitor for the thrombopoietin receptor (TpoR) would be more specific for the treatment of myeloproliferative neoplasms (MPNs) due to constitutively active mutant TpoR compared to the current treatment approach of inhibiting Janus kinase 2 (JAK2). We describe a cell-based high-throughput phenotypic screening approach to identify inhibitors for constitutively active mutant TpoR. A stepwise elimination process is used to differentiate generally cytotoxic compounds from compounds that specifically inhibit growth of cells expressing wild-type TpoR and/or mutant TpoR. We have systematically optimized the phenotypic screening assay and documented in this chapter critical parameters for a successful phenotypic screen, such as cell growth and seeding optimization, plate reproducibility and uniformity studies, and an assay robustness analysis with a pilot screen.
Assuntos
Descoberta de Drogas , Fenótipo , Receptores de Trombopoetina/antagonistas & inibidores , Receptores de Trombopoetina/genética , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Ligantes , Medições Luminescentes/métodos , Camundongos , Reprodutibilidade dos Testes , Bibliotecas de Moléculas PequenasRESUMO
The constitutively active JAK2 V617F mutant is the major determinant of human myeloproliferative neoplasms (MPNs). We show that coexpression of murine JAK2 V617F and the murine thrombopoietin (Tpo) receptor (TpoR, c-MPL) in hematopoietic cell lines or heterozygous knock-in of JAK2 V617F in mice leads to down-modulation of TpoR levels. Enhanced TpoR ubiquitinylation, proteasomal degradation, reduced recycling, and maturation are induced by the constitutive JAK2 V617F activity. These effects can be prevented in cell lines by JAK2 and proteasome inhibitors. Restoration of TpoR levels by inhibitors could be detected in platelets from JAK2 inhibitor-treated myelofibrosis patients that express the JAK2 V617F mutant, and in platelets from JAK2 V617F knock-in mice that were treated in vivo with JAK2 or proteasome inhibitors. In addition, we show that Tpo can induce both proliferative and antiproliferative effects via TpoR at low and high JAK2 activation levels, respectively, or on expression of JAK2 V617F. The antiproliferative signaling and receptor down-modulation by JAK2 V617F were dependent on signaling via TpoR cytosolic tyrosine 626. We propose that selection against TpoR antiproliferative signaling occurs by TpoR down-modulation and that restoration of down-modulated TpoR levels could become a biomarker for the treatment of MPNs.
Assuntos
Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/fisiologia , Inibidores de Proteassoma , Inibidores de Proteínas Quinases/farmacologia , Receptores de Trombopoetina/genética , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/fisiologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação de Sentido Incorreto/fisiologia , Fenilalanina/genética , Receptor Cross-Talk/efeitos dos fármacos , Receptor Cross-Talk/fisiologia , Receptores de Trombopoetina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Valina/genéticaRESUMO
Amyloid fibrils associated with Alzheimer's disease and a wide range of other neurodegenerative diseases have a cross beta-sheet structure, where main chain hydrogen bonding occurs between beta-strands in the direction of the fibril axis. The surface of the beta-sheet has pronounced ridges and grooves when the individual beta-strands have a parallel orientation and the amino acids are in-register with one another. Here we show that in Abeta amyloid fibrils, Met35 packs against Gly33 in the C-terminus of Abeta40 and against Gly37 in the C-terminus of Abeta42. These packing interactions suggest that the protofilament subunits are displaced relative to one another in the Abeta40 and Abeta42 fibril structures. We take advantage of this corrugated structure to design a new class of inhibitors that prevent fibril formation by placing alternating glycine and aromatic residues on one face of a beta-strand. We show that peptide inhibitors based on a GxFxGxF framework disrupt sheet-to-sheet packing and inhibit the formation of mature Abeta fibrils as assayed by thioflavin T fluorescence, electron microscopy, and solid-state NMR spectroscopy. The alternating large and small amino acids in the GxFxGxF sequence are complementary to the corresponding amino acids in the IxGxMxG motif found in the C-terminal sequence of Abeta40 and Abeta42. Importantly, the designed peptide inhibitors significantly reduce the toxicity induced by Abeta42 on cultured rat cortical neurons.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Amiloide/efeitos dos fármacos , Amiloide/ultraestrutura , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/farmacologia , Peptídeos beta-Amiloides/toxicidade , Animais , Benzotiazóis , Corantes Fluorescentes , Glicina/química , Humanos , Metionina/química , Microscopia Eletrônica , Dados de Sequência Molecular , Neurônios/efeitos dos fármacos , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Espectrometria de Fluorescência , TiazóisRESUMO
The erythropoietin receptor (EpoR) is crucial for erythrocyte formation. The x-ray crystal structures of the EpoR extracellular domain lack the juxtamembrane (JM) region and the junction to the transmembrane (TM) domain. Yet the JM-TM regions are important for transmitting the conformational change imposed on the receptor dimer by Epo binding. Cysteine-scanning mutagenesis of the JM-TM regions identified three novel constitutively active mutants, demonstrating close disulfide-bonded juxtapositioning of these residues in the JM (L223C) and N-terminal TM domain (L226C, I227C). Chemical cross-linking defined the interface of the active helical TM dimer and revealed that the JM-TM segment encompassing Leu(226)-Leu(230) is non-helical. Molecular dynamics and NMR studies indicated that the TM-JM junction forms an N-terminal helix cap. This structure is important for EpoR function because replacement of this motif by consecutive leucines rendered the receptor constitutively active.
Assuntos
Glicina/análogos & derivados , Receptores da Eritropoetina/química , Motivos de Aminoácidos , Animais , Western Blotting , Linhagem Celular , Membrana Celular/metabolismo , Proliferação de Células , Separação Celular , Reagentes de Ligações Cruzadas/farmacologia , Cristalografia por Raios X , Cisteína/química , Análise Mutacional de DNA , DNA Complementar/metabolismo , Dimerização , Dissulfetos/química , Citometria de Fluxo , Glicina/química , Substâncias de Crescimento/metabolismo , Humanos , Leucina/química , Luciferases/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Mutagênese , Mutagênese Sítio-Dirigida , Mutação , Peptídeos/química , Fosforilação , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores da Eritropoetina/metabolismo , TransfecçãoRESUMO
We have cloned a novel adapter protein containing Src homology 2 and Src homology 3 domains similar to the Src family of tyrosine kinases. This molecule lacks a catalytic tyrosine kinase domain and is related to a previously identified protein, Src-like adapter protein (SLAP), and is therefore designated SLAP-2. Northern blot analysis indicates that SLAP-2 is predominantly expressed in the immune system. Jurkat T cells express SLAP-2 protein and overexpression of SLAP-2 in these cells negatively regulates T cell receptor signaling as assessed by interleukin-2 promoter or NF-AT promoter reporter constructs. Mutational analysis revealed that an intact SH2 domain of SLAP-2 is essential for this inhibitory effect, whereas mutation of the SH3 domain alone has no effect. This inhibitory effect is upstream of the activation of Ras and increase of intracellular calcium levels, as no inhibition was observed when the cells were activated by phorbol ester plus ionomycin. SLAP-2 interacts with Cbl in vivo in a phosphorylation independent manner and with ZAP-70 and T cell receptor zeta chain upon T cell receptor activation. Finally, we show that the mutation of a predicted myristoylation site within the NH(2)-terminal of SLAP-2 is essential for its inhibitory effect. This report therefore implicates SLAP and SLAP-2 as a family of adapter proteins that negatively regulate T cell receptor signaling.