RESUMO
BCR::ABL1-negative myeloproliferative neoplasms (MPNs) are clonal diseases originating from a single hematopoietic stem cell that cause excessive production of mature blood cells. The 3 subtypes, that is, polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are diagnosed according to the World Health Organization (WHO) and international consensus classification (ICC) criteria. Acquired gain-of-function mutations in 1 of 3 disease driver genes (JAK2, CALR, and MPL) are the causative events that can alone initiate and promote MPN disease without requiring additional cooperating mutations. JAK2-p.V617F is present in >95% of PV patients, and also in about half of the patients with ET or PMF. ET and PMF are also caused by mutations in CALR or MPL. In â¼10% of MPN patients, those referred to as being "triple negative," none of the known driver gene mutations can be detected. The common theme between the 3 driver gene mutations and triple-negative MPN is that the Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway is constitutively activated. We review the recent advances in our understanding of the early events after the acquisition of a driver gene mutation. The limiting factor that determines the frequency at which MPN disease develops with a long latency is not the acquisition of driver gene mutations, but rather the expansion of the clone. Factors that control the conversion from clonal hematopoiesis to MPN disease include inherited predisposition, presence of additional mutations, and inflammation. The full extent of knowledge of the mutational landscape in individual MPN patients is now increasingly being used to predict outcome and chose the optimal therapy.
Assuntos
Transtornos Mieloproliferativos , Policitemia Vera , Mielofibrose Primária , Trombocitemia Essencial , Humanos , Mielofibrose Primária/genética , Calreticulina/genética , Calreticulina/metabolismo , Receptores de Trombopoetina/genética , Receptores de Trombopoetina/metabolismo , Transtornos Mieloproliferativos/metabolismo , Policitemia Vera/genética , Trombocitemia Essencial/genética , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , MutaçãoRESUMO
Mutant calreticulin (CALR) proteins resulting from a -1/+2 frameshifting mutation of the CALR exon 9 carry a novel C-terminal amino acid sequence and drive the development of myeloproliferative neoplasms (MPNs). Mutant CALRs were shown to interact with and activate the thrombopoietin receptor (TpoR/MPL) in the same cell. We report that mutant CALR proteins are secreted and can be found in patient plasma at levels up to 160 ng/mL, with a mean of 25.64 ng/mL. Plasma mutant CALR is found in complex with soluble transferrin receptor 1 (sTFR1) that acts as a carrier protein and increases mutant CALR half-life. Recombinant mutant CALR proteins bound and activated the TpoR in cell lines and primary megakaryocytic progenitors from patients with mutated CALR in which they drive thrombopoietin-independent colony formation. Importantly, the CALR-sTFR1 complex remains functional for TpoR activation. By bioluminescence resonance energy transfer assay, we show that mutant CALR proteins produced in 1 cell can specifically interact in trans with the TpoR on a target cell. In comparison with cells that only carry TpoR, cells that carry both TpoR and mutant CALR are hypersensitive to exogenous mutant CALR proteins and respond to levels of mutant CALR proteins similar to those in patient plasma. This is consistent with CALR-mutated cells that expose TpoR carrying immature N-linked sugars at the cell surface. Thus, secreted mutant CALR proteins will act more specifically on the MPN clone. In conclusion, a chaperone, CALR, can turn into a rogue cytokine through somatic mutation of its encoding gene.
Assuntos
Transtornos Mieloproliferativos , Neoplasias , Humanos , Citocinas/metabolismo , Calreticulina/genética , Transtornos Mieloproliferativos/genética , Mutação , Fatores Imunológicos , Janus Quinase 2/genéticaRESUMO
Interferon-α (IFN-α)-based treatments can induce hematologic and molecular responses (HRs and MRs, respectively) in polycythemia vera (PV); however, patients do not respond equally. Germline genetic factors have been implicated in differential drug responses. We addressed the effect of common germline polymorphisms on HR and MR after treatment of PV in the PROUD-PV and CONTINUATION-PV studies in a total of 122 patients who received ropeginterferon alfa-2b. Genome-wide association studies using longitudinal data on HR and MR over a 36-month follow-up did not reveal any associations at the level of genome-wide statistical significance. Furthermore, we performed targeted association analyses at the interferon lambda 4 (IFNL4) locus, well known for its role in hepatitis C viral clearance and recently reported to influence HR during treatment of myeloproliferative neoplasms. We did not observe any association of IFNL4 polymorphisms with HR in our study cohort; however, we demonstrated a statistically significant effect of the functionally causative IFNL4 diplotype (haplotype pair, including the protein-coding variants rs368234815/rs117648444) on MR (P = 3.91 × 10-4; odds ratio, 10.80; 95% confidence interval, 2.39-69.97) as reflected in differential JAK2V617F mutational burden changes according to IFNL4 diplotype status. Stratification of patients with PV based on IFNL4 functionality may allow for optimizing patient management during IFN-α-based therapy.
Assuntos
Células Germinativas/metabolismo , Interferon-alfa/uso terapêutico , Policitemia Vera/tratamento farmacológico , Policitemia Vera/genética , Seguimentos , Predisposição Genética para Doença , Humanos , Interleucinas/genética , Fases de Leitura Aberta/genética , Polimorfismo de Nucleotídeo Único/genética , Resultado do TratamentoRESUMO
Somatic mutations of calreticulin (CALR) have been identified as a main disease driver of myeloproliferative neoplasms, suggesting that development of drugs targeting mutant CALR is of great significance. Site-directed mutagenesis in the N-glycan binding domain (GBD) abolishes the ability of mutant CALR to oncogenically activate the thrombopoietin receptor (MPL). We therefore hypothesized that a small molecule targeting the GBD might inhibit the oncogenicity of the mutant CALR. Using an in silico molecular docking study, we identified candidate binders to the GBD of CALR. Further experimental validation of the hits identified a group of catechols inducing a selective growth inhibitory effect on cells that depend on oncogenic CALR for survival and proliferation. Apoptosis-inducing effects by the compound were significantly higher in the CALR-mutated cells than in CALR wild-type cells. Additionally, knockout or C-terminal truncation of CALR eliminated drug hypersensitivity in CALR-mutated cells. We experimentally confirmed the direct binding of the selected compound to CALR, disruption of the mutant CALR-MPL interaction, inhibition of the JAK2-STAT5 pathway, and reduction at the intracellular level of mutant CALR upon drug treatment. Our data indicate that small molecules targeting the GBD of CALR can selectively kill CALR-mutated cells by disrupting the CALR-MPL interaction and inhibiting oncogenic signaling.
Assuntos
Calreticulina/metabolismo , Hematoxilina/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Receptores de Trombopoetina/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Calreticulina/química , Calreticulina/genética , Linhagem Celular , Descoberta de Drogas , Humanos , Camundongos , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Mutação , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Ligação Proteica/efeitos dos fármacos , Receptores de Trombopoetina/químicaRESUMO
Myeloproliferative neoplasms (MPN) are characterized by uncontrolled expansion of myeloid cells, disease-related mutations in certain driver-genes including JAK2, CALR, and MPL, and a substantial risk to progress to secondary acute myeloid leukemia (sAML). Although behaving as stem cell neoplasms, little is known about disease-initiating stem cells in MPN. We established the phenotype of putative CD34+ /CD38- stem cells and CD34+ /CD38+ progenitor cells in MPN. A total of 111 patients with MPN suffering from polycythemia vera, essential thrombocythemia, or primary myelofibrosis (PMF) were examined. In almost all patients tested, CD34+ /CD38- stem cells expressed CD33, CD44, CD47, CD52, CD97, CD99, CD105, CD117, CD123, CD133, CD184, CD243, and CD274 (PD-L1). In patients with PMF, MPN stem cells often expressed CD25 and sometimes also CD26 in an aberrant manner. MPN stem cells did not exhibit substantial amounts of CD90, CD273 (PD-L2), CD279 (PD-1), CD366 (TIM-3), CD371 (CLL-1), or IL-1RAP. The phenotype of CD34+ /CD38- stem cells did not change profoundly during progression to sAML. The disease-initiating capacity of putative MPN stem cells was confirmed in NSGS mice. Whereas CD34+ /CD38- MPN cells engrafted in NSGS mice, no substantial engraftment was produced by CD34+ /CD38+ or CD34- cells. The JAK2-targeting drug fedratinib and the BRD4 degrader dBET6 induced apoptosis and suppressed proliferation in MPN stem cells. Together, MPN stem cells display a unique phenotype, including cytokine receptors, immune checkpoint molecules, and other clinically relevant target antigens. Phenotypic characterization of neoplastic stem cells in MPN and sAML should facilitate their enrichment and the development of stem cell-eradicating (curative) therapies.
Assuntos
Leucemia Mieloide Aguda , Transtornos Mieloproliferativos , Policitemia Vera , Animais , Camundongos , Calreticulina/genética , Janus Quinase 2/genética , Mutação , Transtornos Mieloproliferativos/genética , Células-Tronco Neoplásicas , Proteínas Nucleares/genética , Fenótipo , Policitemia Vera/genética , Fatores de Transcrição/genética , HumanosRESUMO
Myeloproliferative neoplasms (MPN) are chronic stem cell disorders characterized by enhanced proliferation of myeloid cells, immune deregulation, and drug resistance. JAK2 somatic mutations drive the disease in 50-60% and CALR mutations in 25-30% of cases. Published data suggest that JAK2-V617F-mutated MPN cells express the resistance-related checkpoint PD-L1. By applying RNA-sequencing on granulocytes of 113 MPN patients, we demonstrate that PD-L1 expression is highest among polycythemia vera patients and that PD-L1 expression correlates with JAK2-V617F mutational burden (R = 0.52; p < .0001). Single nucleotide polymorphism (SNP) arrays showed that chromosome 9p uniparental disomy (UPD) covers both PD-L1 and JAK2 in all MPN patients examined. MPN cells in JAK2-V617F-positive patients expressed higher levels of PD-L1 if 9p UPD was present compared to when it was absent (p < .0001). Moreover, haplotype-based association analyses provided evidence for germline genetic factors at PD-L1 locus contributing to MPN susceptibility independently of the previously described GGCC risk haplotype. We also found that PD-L1 is highly expressed on putative CD34+ CD38- disease-initiating neoplastic stem cells (NSC) in both JAK2 and CALR-mutated MPN. PD-L1 overexpression decreased upon exposure to JAK2 blockers and BRD4-targeting agents, suggesting a role for JAK2-STAT5-signaling and BRD4 in PD-L1 expression. Whether targeting of PD-L1 can overcome NSC resistance in MPN remains to be elucidated in forthcoming studies.
Assuntos
Antígeno B7-H1 , Transtornos Mieloproliferativos , Policitemia Vera , Dissomia Uniparental , Antígeno B7-H1/genética , Proteínas de Ciclo Celular/genética , Humanos , Janus Quinase 2/genética , Mutação , Transtornos Mieloproliferativos/genética , Proteínas Nucleares/genética , Policitemia Vera/genética , Fatores de Transcrição , Dissomia Uniparental/genéticaRESUMO
Calreticulin (CALR) +1 frameshift mutations in exon 9 are prevalent in myeloproliferative neoplasms. Mutant CALRs possess a new C-terminal sequence rich in positively charged amino acids, leading to activation of the thrombopoietin receptor (TpoR/MPL). We show that the new sequence endows the mutant CALR with rogue chaperone activity, stabilizing a dimeric state and transporting TpoR and mutants thereof to the cell surface in states that would not pass quality control; this function is absolutely required for oncogenic transformation. Mutant CALRs determine traffic via the secretory pathway of partially immature TpoR, as they protect N117-linked glycans from further processing in the Golgi apparatus. A number of engineered or disease-associated TpoRs such as TpoR/MPL R102P, which causes congenital thrombocytopenia, are rescued for traffic and function by mutant CALRs, which can also overcome endoplasmic reticulum retention signals on TpoR. In addition to requiring N-glycosylation of TpoR, mutant CALRs require a hydrophobic patch located in the extracellular domain of TpoR to induce TpoR thermal stability and initial intracellular activation, whereas full activation requires cell surface localization of TpoR. Thus, mutant CALRs are rogue chaperones for TpoR and traffic-defective TpoR mutants, a function required for the oncogenic effects.
Assuntos
Calreticulina/genética , Calreticulina/metabolismo , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Receptores de Trombopoetina/metabolismo , Animais , Humanos , Camundongos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Transporte Proteico/fisiologiaRESUMO
Ph-negative myeloproliferative neoplasms (MPNs) are hematological cancers that can be subdivided into entities with distinct clinical features. Somatic mutations in JAK2, CALR, and MPL have been described as drivers of the disease, together with a variable landscape of nondriver mutations. Despite detailed knowledge of disease mechanisms, targeted therapies effective enough to eliminate MPN cells are still missing. In this study of 113 MPN patients, we aimed to comprehensively characterize the mutational landscape of the granulocyte transcriptome using RNA sequencing data and subsequently examine the applicability of immunotherapeutic strategies for MPN patients. Following implementation of customized workflows and data filtering, we identified a total of 13 (12/13 novel) gene fusions, 231 nonsynonymous single nucleotide variants, and 21 insertions and deletions in 106 of 113 patients. We found a high frequency of SF3B1-mutated primary myelofibrosis patients (14%) with distinct 3' splicing patterns, many of these with a protein-altering potential. Finally, from all mutations detected, we generated a virtual peptide library and used NetMHC to predict 149 unique neoantigens in 62% of MPN patients. Peptides from CALR and MPL mutations provide a rich source of neoantigens as a result of their unique ability to bind many common MHC class I molecules. Finally, we propose that mutations derived from splicing defects present in SF3B1-mutated patients may offer an unexplored neoantigen repertoire in MPNs. We validated 35 predicted peptides to be strong MHC class I binders through direct binding of predicted peptides to MHC proteins in vitro. Our results may serve as a resource for personalized vaccine or adoptive cell-based therapy development.
Assuntos
Antígenos de Neoplasias/genética , Transtornos Mieloproliferativos/genética , Idoso , Calreticulina/genética , Feminino , Humanos , Imunoterapia/métodos , Masculino , Pessoa de Meia-Idade , Mutação , Receptores de Trombopoetina/genética , Análise de Sequência de RNA/métodos , TranscriptomaRESUMO
Over 80% of patients with myeloproliferative neoplasms (MPNs) harbor the acquired somatic JAK2 V617F mutation. JAK inhibition is not curative and fails to induce a persistent response in most patients, illustrating the need for the development of novel therapeutic approaches. We describe a critical role for CDK6 in MPN evolution. The absence of Cdk6 ameliorates clinical symptoms and prolongs survival. The CDK6 protein interferes with 3 hallmarks of disease: besides regulating malignant stem cell quiescence, it promotes nuclear factor κB (NF-κB) signaling and contributes to cytokine production while inhibiting apoptosis. The effects are not mirrored by palbociclib, showing that the functions of CDK6 in MPN pathogenesis are largely kinase independent. Our findings thus provide a rationale for targeting CDK6 in MPN.
Assuntos
Apoptose , Quinase 6 Dependente de Ciclina/farmacologia , Janus Quinase 2/genética , Mutação , Transtornos Mieloproliferativos/etiologia , NF-kappa B/metabolismo , Humanos , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/mortalidade , Transtornos Mieloproliferativos/patologia , Neoplasias , Transdução de SinaisRESUMO
Myeloproliferative neoplasms (MPNs) are characterized by a pathologic expansion of myeloid lineages. Mutations in JAK2, CALR and MPL genes are known to be three prominent MPN disease drivers. Mutant CALR (mutCALR) is an oncoprotein that interacts with and activates the thrombopoietin receptor (MPL) and represents an attractive target for targeted therapy of CALR mutated MPN. We generated a transgenic murine model with conditional expression of the human mutant exon 9 (del52) from the murine endogenous Calr locus. These mice develop essential thrombocythemia like phenotype with marked thrombocytosis and megakaryocytosis. The disease exacerbates with age showing prominent signs of splenomegaly and anemia. The disease is transplantable and mutCALR stem cells show proliferative advantage when compared to wild type stem cells. Transcriptome profiling of hematopoietic stem cells revealed oncogenic and inflammatory gene expression signatures. To demonstrate the applicability of the transgenic animals for immunotherapy, we treated mice with monoclonal antibody raised against the human mutCALR. The antibody treatment lowered platelet and stem cell counts in mutant mice. Secretion of mutCALR did not constitute a significant antibody sink. This animal model not only recapitulates human MPN but also serves as a relevant model for testing immunotherapeutic strategies targeting epitopes of the human mutCALR.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Calreticulina/antagonistas & inibidores , Modelos Animais de Doenças , Células-Tronco Hematopoéticas/metabolismo , Terapia de Alvo Molecular , Trombocitemia Essencial/terapia , Animais , Anticorpos Monoclonais/imunologia , Plaquetas/imunologia , Plaquetas/metabolismo , Calreticulina/genética , Calreticulina/imunologia , Calreticulina/fisiologia , Éxons/genética , Mutação da Fase de Leitura , Técnicas de Introdução de Genes , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Quimera por Radiação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Esplenomegalia/etiologia , Trombocitemia Essencial/sangue , Trombocitemia Essencial/complicações , Trombocitemia Essencial/genética , TranscriptomaRESUMO
The data on the pharmacology of 4-thiazolidinones showed that 5-ene-2-(imino)amino-4-thiazolidinones are likely to comprise one of the most promising groups of compounds possessing anticancer properties. A series of 5-arylidene-2-(4-hydroxyphenyl)aminothiazol-4(5H)-ones was designed, synthesized, and studied against 10 leukemia cell lines, including the HL-60, Jurkat, K-562, Dami, KBM-7, and some Ba/F3 cell lines. The structure-activity relationship analysis shows that almost all tested 5-arylidene-2-(4-hydroxyphenyl)aminothiazol-4(5H)-ones were characterized by ÐС50 values lower or comparable to that of the control drug chlorambucil. Among the tested compounds, (5Z)-5-(2-methoxybenzylidene)- (12), (5Z)-(2-ethoxybenzylidene)- (21), (5Z)-5-(2-benzyloxybenzylidene)- (25), and (5Z)-5-(2-allyloxybenzylidene)-2-(4-hydroxyphenylamino)thiazol-4(5H)-ones (28) possessed the highest antileukemic activity at submicromolar concentrations (ÐС50 = 0.10-0.95 µM).
Assuntos
Antineoplásicos/farmacologia , Tiazóis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/químicaRESUMO
Inhibition of Janus-kinase 1/2 (JAK1/2) is a mainstay to treat myeloproliferative neoplasms (MPN). Sporadic observations reported the co-incidence of B-cell non-Hodgkin lymphomas during treatment of MPN with JAK1/2 inhibitors. We assessed 626 patients with MPN, including 69 with myelofibrosis receiving JAK1/2 inhibitors for lymphoma development. B-cell lymphomas evolved in 4 (5.8%) of 69 patients receiving JAK1/2 inhibition compared with 2 (0.36%) of 557 with conventional treatment (16-fold increased risk). A similar 15-fold increase was observed in an independent cohort of 929 patients with MPN. Considering primary myelofibrosis only (N = 216), 3 lymphomas were observed in 31 inhibitor-treated patients (9.7%) vs 1 (0.54%) of 185 control patients. Lymphomas were of aggressive B-cell type, extranodal, or leukemic with high MYC expression in the absence of JAK2 V617F or other MPN-associated mutations. Median time from initiation of inhibitor therapy to lymphoma diagnosis was 25 months. Clonal immunoglobulin gene rearrangements were already detected in the bone marrow during myelofibrosis in 16.3% of patients. Lymphomas occurring during JAK1/2 inhibitor treatment were preceded by a preexisting B-cell clone in all 3 patients tested. Sequencing verified clonal identity in 2 patients. The effects of JAK1/2 inhibition were mirrored in Stat1-/- mice: 16 of 24 mice developed a spontaneous myeloid hyperplasia with the concomitant presence of aberrant B cells. Transplantations of bone marrow from diseased mice unmasked the outgrowth of a malignant B-cell clone evolving into aggressive B-cell leukemia-lymphoma. We conclude that JAK/STAT1 pathway inhibition in myelofibrosis is associated with an elevated frequency of aggressive B-cell lymphomas. Detection of a preexisting B-cell clone may identify individuals at risk.
Assuntos
Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Linfoma de Células B/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Mielofibrose Primária/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Linfoma de Células B/enzimologia , Linfoma de Células B/genética , Linfoma de Células B/patologia , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Mielofibrose Primária/enzimologia , Mielofibrose Primária/genética , Mielofibrose Primária/patologia , Estudos RetrospectivosRESUMO
Recurrent gain-of-function mutations in the transcription factors STAT5A and much more in STAT5B were found in hematopoietic malignancies with the highest proportion in mature T- and natural killer-cell neoplasms (peripheral T-cell lymphoma, PTCL). No targeted therapy exists for these heterogeneous and often aggressive diseases. Given the shortage of models for PTCL, we mimicked graded STAT5A or STAT5B activity by expressing hyperactive Stat5a or STAT5B variants at low or high levels in the hematopoietic system of transgenic mice. Only mice with high activity levels developed a lethal disease resembling human PTCL. Neoplasia displayed massive expansion of CD8+ T cells and destructive organ infiltration. T cells were cytokine-hypersensitive with activated memory CD8+ T-lymphocyte characteristics. Histopathology and mRNA expression profiles revealed close correlation with distinct subtypes of PTCL. Pronounced STAT5 expression and activity in samples from patients with different subsets underline the relevance of JAK/STAT as a therapeutic target. JAK inhibitors or a selective STAT5 SH2 domain inhibitor induced cell death and ruxolitinib blocked T-cell neoplasia in vivo We conclude that enhanced STAT5A or STAT5B action both drive PTCL development, defining both STAT5 molecules as targets for therapeutic intervention.
Assuntos
Leucemia , Linfoma de Células T Periférico , Animais , Linfócitos T CD8-Positivos/metabolismo , Citocinas , Humanos , Linfoma de Células T Periférico/genética , Camundongos , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Proteínas Supressoras de TumorRESUMO
The genetic landscape of classical myeloproliferative neoplasm (MPN) is in large part elucidated. The MPN-restricted driver mutations, including those in JAK2, calreticulin (CALR), and myeloproliferative leukemia virus (MPL), abnormally activate the cytokine receptor/JAK2 pathway and their downstream effectors, more particularly the STATs. The most frequent mutation, JAK2V617F, activates the 3 main myeloid cytokine receptors (erythropoietin receptor, granulocyte colony-stimulating factor receptor, and MPL) whereas CALR or MPL mutants are restricted to MPL activation. This explains why JAK2V617F is associated with polycythemia vera, essential thrombocythemia (ET), and primary myelofibrosis (PMF) whereas CALR and MPL mutants are found in ET and PMF. Other mutations in genes involved in epigenetic regulation, splicing, and signaling cooperate with the 3 MPN drivers and play a key role in the PMF pathogenesis. Mutations in epigenetic regulators TET2 and DNMT3A are involved in disease initiation and may precede the acquisition of JAK2V617F. Other mutations in epigenetic regulators such as EZH2 and ASXL1 also play a role in disease initiation and disease progression. Mutations in the splicing machinery are predominantly found in PMF and are implicated in the development of anemia or pancytopenia. Both heterogeneity of classical MPNs and prognosis are determined by a specific genomic landscape, that is, type of MPN driver mutations, association with other mutations, and their order of acquisition. However, factors other than somatic mutations play an important role in disease initiation as well as disease progression such as germ line predisposition, inflammation, and aging. Delineation of these environmental factors will be important to better understand the precise pathogenesis of MPN.
Assuntos
Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Mutação , Policitemia Vera/genética , Mielofibrose Primária/genética , Trombocitemia Essencial/genética , Calreticulina/genética , Calreticulina/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Progressão da Doença , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Policitemia Vera/metabolismo , Policitemia Vera/fisiopatologia , Mielofibrose Primária/metabolismo , Mielofibrose Primária/fisiopatologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/metabolismo , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Receptores de Trombopoetina/genética , Receptores de Trombopoetina/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Trombocitemia Essencial/metabolismo , Trombocitemia Essencial/fisiopatologiaAssuntos
Proteínas Mutadas de Ataxia Telangiectasia , Mutação em Linhagem Germinativa , Transtornos Mieloproliferativos , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Mutadas de Ataxia Telangiectasia/genética , Predisposição Genética para Doença , Itália/epidemiologia , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/diagnóstico , LinhagemRESUMO
External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay set-up and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.
Assuntos
Janus Quinase 2/genética , Mutação de Sentido Incorreto , Patologia Molecular/normas , Garantia da Qualidade dos Cuidados de Saúde , Reação em Cadeia da Polimerase em Tempo Real/normas , Substituição de Aminoácidos , Feminino , Humanos , MasculinoRESUMO
Frameshift mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and myelofibrosis patients. To address the contribution of the CALR mutants to the pathogenesis of myeloproliferative neoplasms, we engrafted lethally irradiated recipient mice with bone marrow cells transduced with retroviruses expressing these mutants. In contrast to wild-type CALR, CALRdel52 (type I) and, to a lesser extent, CALRins5 (type II) induced thrombocytosis due to a megakaryocyte (MK) hyperplasia. Disease was transplantable into secondary recipients. After 6 months, CALRdel52-, in contrast to rare CALRins5-, transduced mice developed a myelofibrosis associated with a splenomegaly and a marked osteosclerosis. Monitoring of virus-transduced populations indicated that CALRdel52 leads to expansion at earlier stages of hematopoiesis than CALRins5. However, both mutants still specifically amplified the MK lineage and platelet production. Moreover, a mutant deleted of the entire exon 9 (CALRdelex9) did not induce a disease, suggesting that the oncogenic property of CALR mutants was related to the new C-terminus peptide. To understand how the CALR mutants target the MK lineage, we used a cell-line model and demonstrated that the CALR mutants, but not CALRdelex9, specifically activate the thrombopoietin (TPO) receptor (MPL) to induce constitutive activation of Janus kinase 2 and signal transducer and activator of transcription 5/3/1. We confirmed in c-mpl- and tpo-deficient mice that expression of Mpl, but not of Tpo, was essential for the CALR mutants to induce thrombocytosis in vivo, although Tpo contributes to disease penetrance. Thus, CALR mutants are sufficient to induce thrombocytosis through MPL activation.
Assuntos
Calreticulina/metabolismo , Mutação INDEL , Megacariócitos/metabolismo , Mielofibrose Primária/metabolismo , Receptores de Trombopoetina/metabolismo , Trombocitose/metabolismo , Animais , Calreticulina/genética , Mutação da Fase de Leitura , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Megacariócitos/patologia , Camundongos , Camundongos Mutantes , Mielofibrose Primária/etiologia , Mielofibrose Primária/genética , Mielofibrose Primária/patologia , Receptores de Trombopoetina/genética , Trombocitose/complicações , Trombocitose/genética , Trombocitose/patologiaRESUMO
The role of somatic JAK2 mutations in clonal myeloproliferative neoplasms (MPNs) is well established. Recently, germ line JAK2 mutations were associated with polyclonal hereditary thrombocytosis and triple-negative MPNs. We studied a patient who inherited 2 heterozygous JAK2 mutations, E846D from the mother and R1063H from the father, and exhibited erythrocytosis and megakaryocytic atypia but normal platelet number. Culture of erythroid progenitors from the patient and his parents revealed hypersensitivity to erythropoietin (EPO). Using cellular models, we show that both E846D and R1063H variants lead to constitutive signaling (albeit much weaker than JAK2 V617F), and both weakly hyperactivate JAK2/STAT5 signaling only in the specific context of the EPO receptor (EPOR). JAK2 E846D exhibited slightly stronger effects than JAK2 R1063H and caused prolonged EPO-induced phosphorylation of JAK2/STAT5 via EPOR. We propose that JAK2 E846D predominantly contributes to erythrocytosis, but is not sufficient for the full pathological phenotype to develop. JAK2 R1063H, with very weak effect on JAK2/STAT5 signaling, is necessary to augment JAK2 activity caused by E846D above a threshold level leading to erythrocytosis with megakaryocyte abnormalities. Both mutations were detected in the germ line of rare polycythemia vera, as well as certain leukemia patients, suggesting that they might predispose to hematological malignancy.
Assuntos
Mutação em Linhagem Germinativa/genética , Janus Quinase 2/genética , Megacariócitos/patologia , Policitemia/congênito , Adolescente , Adulto , Feminino , Humanos , Masculino , Megacariócitos/metabolismo , Pessoa de Meia-Idade , Fosforilação , Policitemia/genética , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Adulto JovemRESUMO
Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed "triple negative." We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders.
Assuntos
Exoma , Sequenciamento de Nucleotídeos em Larga Escala , Janus Quinase 2/genética , Mutação , Transtornos Mieloproliferativos/genética , Receptores de Trombopoetina/genética , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Genômica/métodos , Humanos , Janus Quinase 2/metabolismo , Transtornos Mieloproliferativos/metabolismo , Mielofibrose Primária/genética , Mielofibrose Primária/metabolismo , Receptores de Trombopoetina/metabolismo , Trombocitemia Essencial/genética , Trombocitemia Essencial/metabolismo , Inativação do Cromossomo X/genéticaRESUMO
Mutations in the calreticulin gene (CALR) represented by deletions and insertions in exon 9 inducing a -1/+2 frameshift are associated with a significant fraction of myeloproliferative neoplasms (MPNs). The mechanisms by which CALR mutants induce MPN are unknown. Here, we show by transcriptional, proliferation, biochemical, and primary cell assays that the pathogenic CALR mutants specifically activate the thrombopoietin receptor (TpoR/MPL). No activation is detected with a battery of type I and II cytokine receptors, except granulocyte colony-stimulating factor receptor, which supported only transient and weak activation. CALR mutants induce ligand-independent activation of JAK2/STAT/phosphatydylinositol-3'-kinase (PI3-K) and mitogen-activated protein (MAP) kinase pathways via TpoR, and autonomous growth in Ba/F3 cells. In these transformed cells, no synergy is observed between JAK2 and PI3-K inhibitors in inhibiting cytokine-independent proliferation, thus showing a major difference from JAK2V617F cells where such synergy is strong. TpoR activation was dependent on its extracellular domain and its N-glycosylation, especially at N117. The glycan binding site and the novel C-terminal tail of the mutant CALR proteins were required for TpoR activation. A soluble form of TpoR was able to prevent activation of full-length TpoR provided that it was N-glycosylated. By confocal microscopy and subcellular fractionation, CALR mutants exhibit different intracellular localization from that of wild-type CALR. Finally, knocking down either MPL/TpoR or JAK2 in megakaryocytic progenitors from patients carrying CALR mutations inhibited cytokine-independent megakaryocytic colony formation. Taken together, our study provides a novel signaling paradigm, whereby a mutated chaperone constitutively activates cytokine receptor signaling.