RESUMO
Cytokines signal through cell surface receptor dimers to initiate activation of intracellular Janus kinases (JAKs). We report the 3.6-angstrom-resolution cryo-electron microscopy structure of full-length JAK1 complexed with a cytokine receptor intracellular domain Box1 and Box2 regions captured as an activated homodimer bearing the valineâphenylalanine (VF) mutation prevalent in myeloproliferative neoplasms. The seven domains of JAK1 form an extended structural unit, the dimerization of which is mediated by close-packing of the pseudokinase (PK) domains from the monomeric subunits. The oncogenic VF mutation lies within the core of the JAK1 PK interdimer interface, enhancing packing complementarity to facilitate ligand-independent activation. The carboxy-terminal tyrosine kinase domains are poised for transactivation and to phosphorylate the receptor STAT (signal transducer and activator of transcription)-recruiting motifs projecting from the overhanging FERM (four-point-one, ezrin, radixin, moesin)-SH2 (Src homology 2)-domains. Mapping of constitutively active JAK mutants supports a two-step allosteric activation mechanism and reveals opportunities for selective therapeutic targeting of oncogenic JAK signaling.
Assuntos
Janus Quinase 1 , Receptores de Citocinas , Domínios de Homologia de src , Regulação Alostérica , Microscopia Crioeletrônica , Ativação Enzimática , Humanos , Janus Quinase 1/química , Janus Quinase 1/metabolismo , Mutação , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , Fosforilação , Multimerização Proteica , Receptores de Citocinas/química , Fatores de Transcrição STAT/metabolismoRESUMO
Mutations in the Janus Kinase 2 (JAK2) gene resulting in constitutive kinase activation represent the most common genetic event in myeloproliferative neoplasms (MPN), a group of diseases involving overproduction of one or more kinds of blood cells, including red cells, white cells, and platelets. JAK2 kinase inhibitors, such as ruxolitinib, provide clinical benefit, but inhibition of wild-type (wt) JAK2 limits their clinical utility due to toxicity to normal cells, and small molecule inhibition of mutated JAK2 kinase activity can lead to drug resistance. Here, we present a strategy to target mutated JAK2 for degradation, using the cell's intracellular degradation machinery, while sparing non-mutated JAK2. We employed a chemical genetics screen, followed by extensive selectivity profiling and genetic studies, to identify the deubiquitinase (DUB), JOSD1, as a novel regulator of mutant JAK2. JOSD1 interacts with and stabilizes JAK2-V617F, and inactivation of the DUB leads to JAK2-V617F protein degradation by increasing its ubiquitination levels, thereby shortening its protein half-life. Moreover, targeting of JOSD1 leads to the death of JAK2-V617F-positive primary acute myeloid leukemia (AML) cells. These studies provide a novel therapeutic approach to achieving selective targeting of mutated JAK2 signaling in MPN.
Assuntos
Enzimas Desubiquitinantes/antagonistas & inibidores , Janus Quinase 2/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Mutação , Transtornos Mieloproliferativos/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Idoso , Idoso de 80 Anos ou mais , Apoptose , Proliferação de Células , Humanos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Fosforilação , Prognóstico , Células Tumorais CultivadasAssuntos
Cromossomos Humanos Par 8/genética , Rearranjo Gênico , Linfoma , Transtornos Mieloproliferativos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Translocação Genética , Adulto , Humanos , Linfoma/enzimologia , Linfoma/genética , Linfoma/patologia , Masculino , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genéticaAssuntos
Células da Medula Óssea , Mutação , Transtornos Mieloproliferativos , Enzimas Ativadoras de Ubiquitina/genética , Idoso , Células da Medula Óssea/enzimologia , Células da Medula Óssea/patologia , Humanos , Masculino , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , SíndromeRESUMO
The chronic myeloproliferative neoplasms (MPNs) are clonal stem cell disorders. The hematopoietic stem/progenitor cell (HSPC) compartment in patients with MPNs is heterogeneous with the presence of both wild-type and JAK2V617F mutant cells. Mechanisms responsible for mutant stem cell expansion in MPNs are not fully understood. Vascular endothelial cells (ECs) are an essential component of the hematopoietic microenvironment. ECs carrying the JAK2V617F mutation can be detected in patients with MPNs. Utilizing an ex vivo EC-HSPC co-culture system with mixed wild-type and JAK2V617F mutant ECs, we show that even small numbers of JAK2V617F mutant ECs can promote the expansion of JAK2V617F mutant HSPCs in preference to wild-type HSPCs during irradiation or cytotoxic chemotherapy, the two treatments commonly used in the conditioning regimen for stem cell transplantation, the only curative treatment for patients with MPNs. Mechanistically, we found that both cell-cell interactions and secreted factors are important for JAK2V617F mutant EC-mediated neoplastic hematopoiesis. Further understanding of how the JAK2V617F mutation alters vascular niche function will help identify new strategies to not only control neoplastic cell expansion but also prevent disease relapse in patients with MPNs.
Assuntos
Células Endoteliais/enzimologia , Neoplasias Hematológicas , Hematopoese , Células-Tronco Hematopoéticas/enzimologia , Janus Quinase 2 , Mutação de Sentido Incorreto , Transtornos Mieloproliferativos , Células-Tronco Neoplásicas/enzimologia , Microambiente Tumoral , Substituição de Aminoácidos , Animais , Neoplasias Hematológicas/enzimologia , Neoplasias Hematológicas/genética , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Camundongos , Camundongos Transgênicos , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genéticaRESUMO
VEXAS syndrome (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is a monogenic disease of adulthood caused by somatic mutations in UBA1 in hematopoietic progenitor cells. Patients develop inflammatory and hematologic symptoms. Myeloid-driven autoinflammation and progressive bone marrow failure lead to substantial morbidity and mortality. Effective medical treatments need to be identified. Reports in the current issue of Blood describe novel UBA1 genetic variants, treatment options, and insight into disease pathophysiology. VEXAS syndrome represents a prototype for a new class of diseases.
Assuntos
Genes Ligados ao Cromossomo X , Doenças Genéticas Inatas , Mutação , Transtornos Mieloproliferativos , Enzimas Ativadoras de Ubiquitina/genética , Células Eritroides/enzimologia , Doenças Genéticas Inatas/diagnóstico por imagem , Doenças Genéticas Inatas/enzimologia , Doenças Genéticas Inatas/genética , Humanos , Masculino , Células Mieloides/enzimologia , Transtornos Mieloproliferativos/diagnóstico por imagem , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , SíndromeAssuntos
Células da Medula Óssea , Células Eritroides , Genes Ligados ao Cromossomo X , Mutação , Células Mieloides , Células Progenitoras Mieloides , Transtornos Mieloproliferativos , Enzimas Ativadoras de Ubiquitina/genética , Vacúolos , Adulto , Idoso , Idoso de 80 Anos ou mais , Células da Medula Óssea/enzimologia , Células da Medula Óssea/patologia , Criança , Células Eritroides/enzimologia , Células Eritroides/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Células Mieloides/enzimologia , Células Mieloides/patologia , Células Progenitoras Mieloides/enzimologia , Células Progenitoras Mieloides/patologia , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Síndrome , Vacúolos/enzimologia , Vacúolos/genética , Vacúolos/patologiaAssuntos
Doenças Genéticas Inatas , Mutação de Sentido Incorreto , Mutação , Transtornos Mieloproliferativos , Enzimas Ativadoras de Ubiquitina/genética , Doenças Genéticas Inatas/enzimologia , Doenças Genéticas Inatas/genética , Humanos , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , SíndromeRESUMO
DNA methylation regulates gene transcription and is involved in various physiological processes in mammals, including development and hematopoiesis. It is catalyzed by DNA methyltransferases including Dnmt1, Dnmt3a, and Dnmt3b. For Dnmt3b, its effects on transcription can result from its own DNA methylase activity, the recruitment of other Dnmts to mediate methylation, or transcription repression in a methylation-independent manner. Low-frequency mutations in human DNMT3B are found in hematologic malignancies including cutaneous T-cell lymphomas, hairy cell leukemia, and diffuse large B-cell lymphomas. Moreover, Dnmt3b is a tumor suppressor in oncogene-driven lymphoid and myeloid malignancies in mice. However, it is poorly understood how the different Dnmt3b activities contribute to these outcomes. We modulated Dnmt3b activity in vivo by generating Dnmt3b+/- mice expressing one wild-type allele as well as Dnmt3b+/CI and Dnmt3bCI/CI mice where one or both alleles express catalytically inactive Dnmt3bCI. We show that 43% of Dnmt3b+/- mice developed T-cell lymphomas, chronic lymphocytic leukemia, and myeloproliferation over 18 months, thus resembling phenotypes previously observed in Dnmt3a+/- mice, possibly through regulation of shared target genes. Interestingly, Dnmt3b+/CI and Dnmt3bCI/CI mice survived postnatal development and were affected by B-cell rather than T-cell malignancies with decreased penetrance. Genome-wide hypomethylation, increased expression of oncogenes such as Jdp2, STAT1, and Trip13, and p53 downregulation were major events contributing to Dnmt3b+/- lymphoma development. We conclude that Dnmt3b catalytic activity is critical to prevent B-cell transformation in vivo, whereas accessory and methylation-independent repressive functions are important to prevent T-cell transformation.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Leucemia Linfocítica Crônica de Células B/genética , Linfoma de Células B/genética , Linfoma de Células T/genética , Transtornos Mieloproliferativos/genética , Neoplasias Experimentais/genética , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA (Citosina-5-)-Metiltransferases/deficiência , Metilação de DNA , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Heterozigoto , Homozigoto , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma de Células B/enzimologia , Linfoma de Células B/patologia , Linfoma de Células T/enzimologia , Linfoma de Células T/patologia , Masculino , Camundongos , Camundongos Knockout , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/patologia , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/patologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , DNA Metiltransferase 3BAssuntos
Aminopiridinas/uso terapêutico , Isocitrato Desidrogenase/genética , Transtornos Mieloproliferativos/tratamento farmacológico , Triazinas/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , Resultado do TratamentoRESUMO
We report on 18 patients with myeloid neoplasms and associated tyrosine kinase (TK) fusion genes on treatment with the TK inhibitors (TKI) ruxolitinib (PCM1-JAK2, n = 8; BCR-JAK2, n = 1) and imatinib, nilotinib or dasatinib (ETV6-ABL1, n = 9). On ruxolitinib (median 24 months, range 2-36 months), a complete hematologic response (CHR) and complete cytogenetic response (CCR) was achieved by five of nine and two of nine patients, respectively. However, ruxolitinib was stopped in eight of nine patients because of primary resistance (n = 3), progression (n = 3) or planned allogeneic stem cell transplantation (allo SCT, n = 2). At a median of 36 months (range 4-78 months) from diagnosis, five of nine patients are alive: four of six patients after allo SCT and one patient who remains on ruxolitinib. In ETV6-ABL1 positive patients, a durable CHR was achieved by four of nine patients (imatinib with one of five, nilotinib with two of three, dasatinib with one of one). Because of inadequate efficacy (lack of hematological and/or cytogenetic/molecular response), six of nine patients (imatinib, n = 5; nilotinib, n = 1) were switched to nilotinib or dasatinib. At a median of 23 months (range 3-60 months) from diagnosis, five of nine patients are in CCR or complete molecular response (nilotinib, n = 2; dasatinib, n = 2; allo SCT, n = 1) while two of nine patients have died. We conclude that (a) responses on ruxolitinib may only be transient in the majority of JAK2 fusion gene positive patients with allo SCT being an important early treatment option, and (b) nilotinib or dasatinib may be more effective than imatinib to induce durable complete remissions in ETV6-ABL1 positive patients.
Assuntos
Neoplasias Hematológicas , Transtornos Mieloproliferativos , Proteínas de Fusão Oncogênica , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Tirosina Quinases , Adulto , Idoso , Intervalo Livre de Doença , Feminino , Seguimentos , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/enzimologia , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/mortalidade , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/mortalidade , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Inibidores de Proteínas Quinases/efeitos adversos , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Taxa de SobrevidaAssuntos
Transtornos Mieloproliferativos/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Regulação para Baixo , Camundongos , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/metabolismo , Transtornos Mieloproliferativos/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de SinaisRESUMO
Constitutive JAK2 signaling is central to myeloproliferative neoplasm (MPN) pathogenesis and results in activation of STAT, PI3K/AKT, and MEK/ERK signaling. However, the therapeutic efficacy of current JAK2 inhibitors is limited. We investigated the role of MEK/ERK signaling in MPN cell survival in the setting of JAK inhibition. Type I and II JAK2 inhibition suppressed MEK/ERK activation in MPN cell lines in vitro, but not in Jak2V617F and MPLW515L mouse models in vivo. JAK2 inhibition ex vivo inhibited MEK/ERK signaling, suggesting that cell-extrinsic factors maintain ERK activation in vivo. We identified PDGFRα as an activated kinase that remains activated upon JAK2 inhibition in vivo, and PDGF-AA/PDGF-BB production persisted in the setting of JAK inhibition. PDGF-BB maintained ERK activation in the presence of ruxolitinib, consistent with its function as a ligand-induced bypass for ERK activation. Combined JAK/MEK inhibition suppressed MEK/ERK activation in Jak2V617F and MPLW515L mice with increased efficacy and reversal of fibrosis to an extent not seen with JAK inhibitors. This demonstrates that compensatory ERK activation limits the efficacy of JAK2 inhibition and dual JAK/MEK inhibition provides an opportunity for improved therapeutic efficacy in MPNs and in other malignancies driven by aberrant JAK-STAT signaling.
Assuntos
Neoplasias Hematológicas/tratamento farmacológico , Janus Quinase 2/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mutação de Sentido Incorreto , Transtornos Mieloproliferativos/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Substituição de Aminoácidos , Animais , Becaplermina/genética , Becaplermina/metabolismo , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Neoplasias Hematológicas/enzimologia , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Trombopoetina/genética , Receptores de Trombopoetina/metabolismoAssuntos
Biomarcadores Tumorais/genética , Análise Mutacional de DNA , Janus Quinase 2/genética , Mutação , Transtornos Mieloproliferativos/genética , Neoplasias/genética , Idoso , Idoso de 80 Anos ou mais , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/enzimologia , Neoplasias/enzimologia , FenótipoAssuntos
Janus Quinase 2 , Mutação de Sentido Incorreto , Transtornos Mieloproliferativos , Transdução de Sinais/genética , Substituição de Aminoácidos , Feminino , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Masculino , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologiaRESUMO
The myeloproliferative neoplasms (MPNs) are stem cell disorders characterized by hematopoietic stem/progenitor cell (HSPC) expansion and overproduction of mature blood cells. The acquired kinase mutation JAK2V617F plays a central role in these disorders. The mechanisms responsible for HSPC expansion in MPNs are not fully understood, limiting the effectiveness of current treatments. One hallmark feature of the marrow in patients with MPNs is megakaryocyte (MK) hyperplasia. Previously, we reported that JAK2V617F-bearing MKs cause a murine myeloproliferative syndrome with HSPC expansion. Here we show that JAK2V617F MKs promote MPN stem cell function by inducing HSPC quiescence with increased repopulating capacity. In addition, we demonstrate that thrombopoietin and its receptor MPL are critical for the JAK2V617F-bearing MK-induced myeloproliferation, both by directly affecting the quantity and quality of MKs and by altering the MK-endothelial interaction and vascular niche function. Therefore, targeting HSPC niche-forming MKs and/or their interactions within the vascular niche could provide novel, more effective therapeutic strategies in patients with MPNs. Stem Cells 2018;36:1676-1684.
Assuntos
Células-Tronco Hematopoéticas/enzimologia , Janus Quinase 2/metabolismo , Megacariócitos/metabolismo , Transtornos Mieloproliferativos/metabolismo , Receptores de Trombopoetina/metabolismo , Trombopoetina/metabolismo , Animais , Proliferação de Células/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Janus Quinase 2/genética , Megacariócitos/enzimologia , Megacariócitos/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Transdução de Sinais , Trombopoetina/genéticaAssuntos
Neoplasias Hematológicas/genética , Mutação , Transtornos Mieloproliferativos/genética , Tirosina Quinase 3 Semelhante a fms/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Neoplasias Hematológicas/enzimologia , Neoplasias Hematológicas/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/patologia , Domínios ProteicosRESUMO
Truncating mutations in the terminal exon of protein phosphatase Mg2+/Mn2+ 1D (PPM1D) have been identified in clonal hematopoiesis and myeloid neoplasms, with a striking enrichment in patients previously exposed to chemotherapy. In this study, we demonstrate that truncating PPM1D mutations confer a chemoresistance phenotype, resulting in the selective expansion of PPM1D-mutant hematopoietic cells in the presence of chemotherapy in vitro and in vivo. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease mutational profiling of PPM1D in the presence of chemotherapy selected for the same exon 6 mutations identified in patient samples. These exon 6 mutations encode for a truncated protein that displays elevated expression and activity due to loss of a C-terminal degradation domain. Global phosphoproteomic profiling revealed altered phosphorylation of target proteins in the presence of the mutation, highlighting multiple pathways including the DNA damage response (DDR). In the presence of chemotherapy, PPM1D-mutant cells have an abrogated DDR resulting in altered cell cycle progression, decreased apoptosis, and reduced mitochondrial priming. We demonstrate that treatment with an allosteric, small molecule inhibitor of PPM1D reverts the phosphoproteomic, DDR, apoptotic, and mitochondrial priming changes observed in PPM1D-mutant cells. Finally, we show that the inhibitor preferentially kills PPM1D-mutant cells, sensitizes the cells to chemotherapy, and reverses the chemoresistance phenotype. These results provide an explanation for the enrichment of truncating PPM1D mutations in the blood of patients exposed to chemotherapy and in therapy-related myeloid neoplasms, and demonstrate that PPM1D can be a targeted in the prevention of clonal expansion of PPM1D-mutant cells and the treatment of PPM1D-mutant disease.
Assuntos
Sequência de Bases , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Neoplasias Hematológicas , Células-Tronco Hematopoéticas/enzimologia , Transtornos Mieloproliferativos , Proteínas de Neoplasias , Células-Tronco Neoplásicas/enzimologia , Proteína Fosfatase 2C , Deleção de Sequência , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/enzimologia , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Células-Tronco Hematopoéticas/patologia , Humanos , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/patologia , Proteína Fosfatase 2C/antagonistas & inibidores , Proteína Fosfatase 2C/genética , Proteína Fosfatase 2C/metabolismoRESUMO
Janus tyrosine kinase 2 (JAK2) mediates downstream signaling of cytokine receptors in all hematological lineages, constitutively active somatic JAK2 mutations play key roles in the pathology of myeloproliferative neoplasms (MPNs). Recently, germline JAK2 mutations are also associated with triple-negative MPNs. A novel germline mutation JAK2 V625F is reported to be involved in a subset of MPNs patients. However, the pathogenesis of this mutation caused MPN is still unclear. In this study, the homology models of JAK2 V625F showed that the newly formed interaction between F625 and Y613 disrupted the JAK2 JH1-JH2 domain interactions was responsible for its activation, when F625 and Y613 interaction was disrupted, its activity significantly decreased. While, when this interaction was repaired whether by forming hydrogen bond or salt bond, it would cause JAK2 activation. Biochemical studies also demonstrated that JAK2 V625F mutation led to JAK2-STAT5 pathway activation and promoted the proliferation of BaF3 cells. Thus, our results herein provide clues to understand the mechanism JAK2 V625F mutation caused MPNs and give information for the development of JAK2 mutation specific inhibitors.
Assuntos
Mutação em Linhagem Germinativa , Neoplasias Hematológicas , Janus Quinase 2 , Mutação de Sentido Incorreto , Transtornos Mieloproliferativos , Proteínas de Neoplasias , Transdução de Sinais , Substituição de Aminoácidos , Linhagem Celular Tumoral , Neoplasias Hematológicas/enzimologia , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Ligação de Hidrogênio , Janus Quinase 2/química , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismoRESUMO
OBJECTIVE: Philadelphia-negative myeloproliferative neoplasms (MPNs) commonly share hyperactive JAK-STAT signaling affecting hematopoietic stem cells (HSC) and their progeny. The JAK1/2 inhibitor Ruxolitinib has remarkable clinical efficacy, including spleen reduction, improvement of constitutional symptoms, and bone marrow (BM) fibrosis reversal. Whether this is due to inhibition of JAK2-mutated HSC only, or whether Ruxolitinib also affects BM stroma is not known. METHODS: This study investigated potential effects of Ruxolitinib on BM mesenchymal stromal cells (MSC), which are not only major regulators of hematopoiesis but also contribute to fibrosis, from 10 healthy donors and 7 JAK2V617F -positive MPN patients. RESULTS: Ruxolitinib moderately inhibited the growth of healthy donor MSC (HD-MSC) and MSC from JAK2V617F+ MPN patients (P-MSC) in short- and long-term assays. The clonogenic potential of HD-MSC was not affected by Ruxolitinib. JAK-STAT signaling, however, was markedly inhibited in both HD-MSC and P-MSC, the latter of which showed higher expression of fibrosis-associated and hematopoiesis-maintenance genes. Moreover, Ruxolitinib reduced MSC secretion of MCP-1 and IL-6. CONCLUSION: Ruxolitinib affected JAK2 signaling in MSC at clinically relevant doses, which is likely to contribute to the normalization of the inflammatory milieu in MPNs. Thus, combined HSC and stroma-directed interventions have the potential to improve constitutional symptoms and reduce stromal proliferation in MPNs.