Myeloproliferative Neoplasms Transcriptome Reveals Pro-Inflammatory Signature and Enrichment in Peripheral Blood Monocyte-Related Genes.

Myeloproliferative neoplasms (MPN) are hematological diseases associated with genetic driver mutations in the JAK2, CALR, and MPL genes and exacerbated oncoinflammatory status. Analyzing public microarray data from polycythemia vera (n = 41), essential thrombocythemia (n = 21), and primary myelofibrosis (n = 9) patients' peripheral blood by in silico approaches, we found that pro-inflammatory and monocyte-related genes were differentially expressed in MPN patients' transcriptome. Genes related to cell activation, secretion of pro-inflammatory and pro-angiogenic mediators, activation of neutrophils and platelets, coagulation, and interferon pathway were upregulated in monocytes compared to controls. Together, our results suggest that molecular alterations in monocytes may contribute to oncoinflammation in MPN.

Exploring hematological alterations and genetics linked to SNV rs10974944 in myeloproliferative neoplasms among Amazon patients.

BCR::ABL1-negative myeloproliferative neoplasms are hematopoietic disorders characterized by panmyelosis. JAK2 V617F is a frequent variant in these diseases and often occurs in the 46/1 haplotype. The G allele of rs10974944 has been shown to be associated with this variant, specifically its acquisition, correlations with familial cases, and laboratory alterations. This study evaluated the association between the 46/1 haplotype and JAK2 V617F in patients with myeloproliferative neoplasms in a population from the Brazilian Amazon. Clinical, laboratory and molecular sequencing analyses were considered. Carriers of the G allele of rs10974944 with polycythemia vera showed an increase in mean corpuscular volume and mean corpuscular hemoglobin, while in those with essential thrombocythemia, there was an elevation in red blood cells, hematocrit, and hemoglobin. Associations were observed between rs10974944 and the JAK2 V617F, in which the G allele (OR 3.4; p < 0.0001) and GG genotype (OR 4.9; p = 0.0016) were associated with JAK2 V617F + and an increase in variant allele frequency (GG: OR 15.8; p = < 0.0001; G: OR 6.0; p = 0.0002). These results suggest an association between rs10974944 (G) and a status for JAK2 V617F, JAK2 V617F + _VAF ≥ 50%, and laboratory alterations in the erythroid lineage.

Updated threshold, renewed problems: should the diagnostic criteria of polycythemia vera be reconsidered? A retrospective cross-sectional cohort study.

OBJECTIVE: This aim of this study was to evaluate hemoglobin and hematocrit values of polycythemia vera and secondary polycythemia patients with updated World Health Organization thresholds. In addition, by determining our own threshold values, we aimed to demonstrate the necessity of bone marrow biopsy and genetic analysis to be used for further diagnosis in patients with high-normal hematocrit and hemoglobin values. METHODS: A cross-sectional and retrospective study was performed with the medical records of patients from Eskisehir City Hospital hematology clinics and outpatient clinics between July 1, 2019 and July 1, 2020. The study included patients with polycythemia, divided into two groups according to polycythemia vera and secondary polycythemia. A bone marrow biopsy was performed on patients with either Janus kinase mutation positivity and/or subnormal erythropoietin levels. Receiver operating characteristics analysis was used to find threshold values, and the diagnostic efficiency of these values in differentiating World Health Organization thresholds in 2008 and 2016 was evaluated. RESULTS: A total of 73 patients were included. The median age was 43.5 years (min: 18; max: 84). The hematocrit value of 54.1 was predicted to diagnose polycythemia vera with a sensitivity of 45% and a specificity of 80%. Subsequent analysis revealed that an hemoglobin value of 17.7 was indicative of diagnosing polycythemia vera with a sensitivity of 60% and a specificity of 63%. The mean follow-up length was 6.4 months (2-12). CONCLUSION: Our study demonstrated that modified World Health Organization criteria might lead to unnecessary additional tests for polycythemia vera patients with high-normal hemoglobin and hematocrit values.

Hsa_circ_0013561 promotes progression of nasopharyngeal carcinoma by activating JAK2/STAT3 signaling pathway.

OBJECTIVE: Nasopharyngeal Carcinoma (NPC) is a malignancy of epithelium of epithelium of the nasopharynx, with the highest incidence of otolaryngeal malignancies. A growing number of studies confirm that Circular RNA (circRNA) plays an important role in tumor development, including Hsa_circ_0013561. This study aims to elucidate the process and mechanism of NPC regulation hsa_circ_0013561. METHODS: In this study, circRNA expression nodes and subcellular localization in NPC tissues were analyzed by fluorescence in situ hybridization. The expression of hsa_circ_0013561 in NPC cells was further clarified by RT-qPCR. At the same time, the lentivirus vector interfered by hsa_circ_0013561 was constructed and transfected. The cell proliferation was detected by CCK-8 method, EdU assay and plate cloning assay. The cell cycle and apoptosis were detected by flow cytometry, and the cell migration ability was detected by wound healing assay and Transwell assay. Western blotting examined the expression of apoptosis, Epithelial-Mesenchymal Transition (EMT)-associated proteins, and Janus Kinase/Signal Transductor and Activator of Transcription (JAK/STAT) signaling pathway-related proteins. RESULTS: The results showed that the expression of hsa_circ_0013561 in NPC samples was significantly upregulated and hsa_circ_0013561 localized in the cytoplasm. After down-regulating hsa_circ_0013561 expression, it significantly inhibited the proliferation and metastasis ability of NPC, inhibited EMT progression, and promoted apoptosis. Further studies showed that interference hsa_circ_0013561 significantly inhibited JAK2/STAT3 signaling pathway activation and induced the expression of apoptosis-related proteins. CONCLUSION: In summary, we found that hsa_circ_0013561 is a pro-tumor circRNA in NPC, which can reduce the activation of JAK2/STAT3 pathway by knocking down hsa_circ_0013561, thereby slowing down the malignant progression of NPC. OXFORD CENTRE FOR EVIDENCE-BASED MEDICINE 2011 LEVELS OF EVIDENCE: Level 4.

JAK2, CALR, and MPL Mutation Profiles in Colombian patients with BCR-ABL Negative Myeloproliferative Neoplasms.

Background: Among the chronic myeloproliferative neoplasms (MPNs) not associated with BCR-ABL mutations are polycythemia vera, primary myelofibrosis, and essential thrombocythemia. These diseases are caused by mutations in genes, such as the JAK2, MPL, and CALR genes, which participate in regulating the JAK-STAT signaling pathway. Objective: This study aimed to establish the frequencies of mutations in the JAK2, MPL, and CALR genes in a group of Colombian patients with a negative clinical diagnosis of BCR-ABL chronic myeloproliferative neoplasms. Methods: The JAK2 V617F and MPL W515K mutations and deletions or insertions in exon 9 of the CALR gene were analyzed in 52 Colombian patients with polycythemia vera, primary myelofibrosis, and essential thrombocythemia. Results: The JAK2V617F mutation was carried by 51.9% of the patients, the CALR mutation by 23%, and the MPL mutation by 3.8%; 23% were triple-negative for the mutations analyzed. In these neoplasms, 6 mutation types in CALR were identified, one of which has not been previously reported. Additionally, one patient presented a double mutation in both the CALR and JAK2 genes. Regarding the hematological results for the mutations, significant differences were found in the hemoglobin level, hematocrit level, and platelet count among the three neoplasms. Conclusion: Thus, this study demonstrates the importance of the molecular characterization of the JAK2, CALR and MPL mutations in Colombian patients (the genetic context of which remains unclear in the abovementioned neoplasms) to achieve an accurate diagnosis, a good prognosis, adequate management, and patient survival.

Antecedentes: Entre las neoplasias mieloproliferativas crónicas no asociadas con mutaciones BCR-ABL se encuentran la policitemia vera, la mielofibrosis primaria y la trombocitemia esencial. Estas enfermedades están causadas por mutaciones en genes, como los genes JAK2, MPL y CALR, que participan en la regulación de la vía de señalización JAK-STAT. Objetivo: Establecer las frecuencias de mutaciones en los genes JAK2, MPL y CALR en un grupo de pacientes colombianos con diagnóstico clínico negativo de NMP BCR-ABL. Metodos: Se analizaron las mutaciones y deleciones o inserciones JAK2 V617F y MPL W515K en el exón 9 del gen CALR en 52 pacientes colombianos con policitemia vera, mielofibrosis primaria y trombocitemia esencial. Resultados: La mutación JAK2V617F la portaban el 51.9% de los pacientes, la mutación CALR el 23.0% y la mutación MPL el 3.8%; El 23.0% fueron triple negativos para las mutaciones analizadas. En estas neoplasias se identificaron seis tipos de mutación en CALR, uno de los cuales no ha sido reportado previamente. Además, un paciente presentó una doble mutación tanto en el gen CALR como en el JAK2. En cuanto a los resultados hematológicos para las mutaciones, se encontraron diferencias significativas en el nivel de hemoglobina, el nivel de hematocrito y el recuento de plaquetas entre las tres neoplasias. Conclusiones: Así, este estudio demuestra la importancia de la caracterización molecular de las mutaciones JAK2, CALR y MPL en pacientes colombianos (cuyo contexto genético aún no está claro en las neoplasias antes mencionadas) para lograr un diagnóstico certero, un buen pronóstico, un manejo adecuado y una mejoría del paciente. supervivencia.

Altered distribution and function of NK-cell subsets lead to impaired tumor surveillance in JAK2V617F myeloproliferative neoplasms.

In cancer, tumor cells and their neoplastic microenvironment can sculpt the immunogenic phenotype of a developing tumor. In this context, natural killer (NK) cells are subtypes of lymphocytes of the innate immune system recognized for their potential to eliminate neoplastic cells, not only through direct cytolytic activity but also by favoring the development of an adaptive antitumor immune response. Even though the protective effect against leukemia due to NK-cell alloreactivity mediated by the absence of the KIR-ligand has already been shown, and some data on the role of NK cells in myeloproliferative neoplasms (MPN) has been explored, their mechanisms of immune escape have not been fully investigated. It is still unclear whether NK cells can affect the biology of BCR-ABL1-negative MPN and which mechanisms are involved in the control of leukemic stem cell expansion. Aiming to investigate the potential contribution of NK cells to the pathogenesis of MPN, we characterized the frequency, receptor expression, maturation profile, and function of NK cells from a conditional Jak2V617F murine transgenic model, which faithfully resembles the main clinical and laboratory characteristics of human polycythemia vera, and MPN patients. Immunophenotypic analysis was performed to characterize NK frequency, their subtypes, and receptor expression in both mutated and wild-type samples. We observed a higher frequency of total NK cells in JAK2V617F mutated MPN and a maturation arrest that resulted in low-numbered mature CD11b+ NK cells and increased immature secretory CD27+ cells in both human and murine mutated samples. In agreement, inhibitory receptors were more expressed in MPN. NK cells from Jak2V617F mice presented a lower potential for proliferation and activation than wild-type NK cells. Colonies generated by murine hematopoietic stem cells (HSC) after mutated or wild-type NK co-culture exposure demonstrated that NK cells from Jak2V617F mice were deficient in regulating differentiation and clonogenic capacity. In conclusion, our findings suggest that NK cells have an immature profile with deficient cytotoxicity that may lead to impaired tumor surveillance in MPN. These data provide a new perspective on the behavior of NK cells in the context of myeloid malignancies and can contribute to the development of new therapeutic strategies, targeting onco-inflammatory pathways that can potentially control transformed HSCs.

The Contribution of JAK2 46/1 Haplotype in the Predisposition to Myeloproliferative Neoplasms.

Haplotype 46/1 (GGCC) consists of a set of genetic variations distributed along chromosome 9p.24.1, which extend from the Janus Kinase 2 gene to Insulin like 4. Marked by four jointly inherited variants (rs3780367, rs10974944, rs12343867, and rs1159782), this haplotype has a strong association with the development of BCR-ABL1-negative myeloproliferative neoplasms (MPNs) because it precedes the acquisition of the JAK2V617F variant, a common genetic alteration in individuals with these hematological malignancies. It is also described as one of the factors that increases the risk of familial MPNs by more than five times, 46/1 is associated with events related to inflammatory dysregulation, splenomegaly, splanchnic vein thrombosis, Budd-Chiari syndrome, increases in RBC count, platelets, leukocytes, hematocrit, and hemoglobin, which are characteristic of MPNs, as well as other findings that are still being elucidated and which are of great interest for the etiopathological understanding of these hematological neoplasms. Considering these factors, the present review aims to describe the main findings and discussions involving the 46/1 haplotype, and highlights the molecular and immunological aspects and their relevance as a tool for clinical practice and investigation of familial cases.

Splanchnic vein thrombosis as a manifestation of latent myeloproliferative neoplasm associated with sticky platelet syndrome / Trombosis esplácnica como presentación de neoplasia mieloproliferativa latente asociada con síndrome de plaqueta pegajosa

Abstract Vein thrombosis of unusual sites such as the splanchnic region continues to be not only a diagnostic but also a therapeutic challenge for the clinician due to its manifestation and associated pathologies. Latent JAK2 (Janus kinase 2) positive myeloproliferative neoplasm associated with sticky platelet syndrome is unusual. We present a clinical case of a 38-year-old female patient who presented with sudden onset abdominal pain of a possible vascular origin. Splanchnic thrombosis was diagnosed in latent myeloproliferative neoplasm by identifying the JAK2V617F mutation and sticky platelet syndrome via platelet aggregometry. Off-label anticoagulation with rivaroxaban 20 mg/day was administered. During her outpatient follow-up, she did not suffer any new thrombotic episodes.

Resumen La trombosis venosa de sitios inusuales como la esplácnica continúa siendo un reto no solo diagnóstico sino también terapéutico para el clínico debido a su forma de presentación y las patologías asociadas. La neoplasia mieloproliferativa latente JAK2 (cinasa de Janus 2) positiva asociada con síndrome de plaqueta pegajosa es inusual. Se presenta un caso clínico de una paciente de 38 años de edad que debutó con dolor abdominal de inicio súbito que sugirió un posible origen vascular. Se diagnosticó trombosis esplácnica en relación con neoplasia mieloproliferativa latente por la identificación de la mutación de la JAK2V617F y síndrome de plaqueta pegajosa mediante agregometría plaquetaria. Se administró de manera off-label anticoagulación con rivaroxabán 20 mg/día. Durante su seguimiento ambulatorio no ha presentado nuevos episodios trombóticos.

Potential limitations of diagnostic standard codes to distinguish polycythemia vera and secondary erythrocytosis.

Red cell overproduction is seen in polycythemia vera (PV), a bone marrow myeloproliferative neoplasm characterized by trilinear cell proliferation (WBC, platelets), as well as in secondary erythrocytosis (SE), a group of heterogeneous disorders characterized by elevated EPO gene transcription. We aimed to verify the concordance of the International Classification of Diseases (ICD) code-based diagnosis of "polycythemia" or "erythrocytosis" with the true clinical diagnosis of these conditions. We retrospectively reviewed the electronic medical records (January 1, 2005, to December 31, 2016) of adult patients with ICD codes of polycythemia and/or erythrocytosis who had testing done for the presence of the JAK2V617F mutation. We verified the accuracy of the ICD code-based diagnoses by meticulous chart review and established whether these patients fulfilled the criteria by the evaluating physician for PV or SE and according to the World Health Organization 2016 diagnostic guidelines. The reliability of ICD coding was calculated using Cohen's kappa. We identified and chart reviewed a total of 578 patient records. Remarkably, 11% of the patients had concurrent diagnosis codes for PV and SE and were unable to be classified appropriately without individual chart review. The ICD code-based diagnostic system led to misidentification in an important fraction of cases. This represents a problem for the detection of PV or SE cases by ICD-based registries and their derived studies. Research based exclusively on ICD codes could have a potential impact on patient care and public health, and limitations must be weighed when research findings are conveyed.

JAK2 Variant Signaling: Genetic, Hematologic and Immune Implication in Chronic Myeloproliferative Neoplasms.

The JAK2V617F variant constitutes a genetic alteration of higher frequency in BCR/ABL1 negative chronic myeloproliferative neoplasms, which is caused by a substitution of a G ˃ T at position 1849 and results in the substitution of valine with phenylalanine at codon 617 of the polypeptide chain. Clinical, morphological and molecular genetic features define the diagnosis criteria of polycythemia vera, essential thrombocythemia and primary myelofibrosis. Currently, JAK2V617F is associated with clonal hematopoiesis, genomic instability, dysregulations in hemostasis and immune response. JAK2V617F clones induce an inflammatory immune response and lead to a process of immunothrombosis. Recent research has shown great interest in trying to understand the mechanisms associated with JAK2V617F signaling and activation of cellular and molecular responses that progressively contribute to the development of inflammatory and vascular conditions in association with chronic myeloproliferative neoplasms. Thus, the aim of this review is to describe the main genetic, hematological and immunological findings that are linked to JAK2 variant signaling in chronic myeloproliferative neoplasms.

PI3K-AKT, JAK2-STAT3 pathways and cell-cell contact regulate maspin subcellular localization.

BACKGROUND: Maspin (SERPINB5) is a potential tumor suppressor gene with pleiotropic biological activities, including regulation of cell proliferation, death, adhesion, migration and gene expression. Several studies indicate that nuclear localization is essential for maspin tumor suppression activity. We have previously shown that the EGFR activation leads to maspin nuclear localization in MCF-10A cells. The present study investigated which EGFR downstream signaling molecules are involved in maspin nuclear localization and explored a possible role of cell-cell contact in this process. METHODS: MCF-10A cells were treated with pharmacological inhibitors against EGFR downstream pathways followed by EGF treatment. Maspin subcellular localization was determined by immunofluorescence. Proteomic and interactome analyses were conducted to identify maspin-binding proteins in EGF-treated cells only. To investigate the role of cell-cell contact these cells were either treated with chelating agents or plated on different cell densities. Maspin and E-cadherin subcellular localization was determined by immunofluorescence. RESULTS: We found that PI3K-Akt and JAK2-STAT3, but not MAP kinase pathway, regulate EGF-induced maspin nuclear accumulation in MCF-10A cells. We observed that maspin is predominantly nuclear in sparse cell culture, but it is redistributed to the cytoplasm in confluent cells even in the presence of EGF. Proteomic and interactome results suggest a role of maspin on post-transcriptional and translation regulation, protein folding and cell-cell adhesion. CONCLUSIONS: Maspin nuclear accumulation is determined by an interplay between EGFR (via PI3K-Akt and JAK2-STAT3 pathways) and cell-cell contact. Video Abstract.

Toll-like receptor gene polymorphisms in patients with myeloproliferative neoplasms.

Toll-like receptors (TLRs) are a family of transmembrane receptors whose signaling control cellular processes of cell proliferation, survival, apoptosis, angiogenesis, remodeling, and repair of tissues. Polymorphisms in TLR genes can change the balance between pro and anti-inflammatory cytokines, modulating the risk of infection, chronic inflammation, and cancer. Although many studies have demonstrated the direct involvement of TLR signaling in the benefit of tumor cells in certain cancers, little is known about the influence of these gene polymorphisms on myeloproliferative neoplasms (MPNs). In this context, the objective of the study was to investigate a possible association between the TLR polymorphisms and the development of MPNs. 167 patients diagnosed with MPN and 222 healthy controls from the same region were evaluated. Genomic DNA was extracted and the TLR2 (rs5743708), TLR4 (rs4986790, rs4986791), TLR9 (rs5743836, rs187084) and JAK2V617F polymorphisms were genotyped by PCR-RFLP. The statistical analysis was performed by OpenEpi and SNPstat software. The JAK2V617F mutation was found in 68.32% of patients. TLR9-1486C/T CT genotype was less frequent in patients with polycythemia vera (PV) (OR 0.39, 95% CI 0.20-0.78, P = 0.025). When haplotype frequencies were analyzed, -1237T/-1486C (TLR9) was also less frequent in men (OR 0.58, 95% CI 0.36-0.94) and JAK negative men patients (OR 0.43, 95% CI 0.21-0.88). We can infer that the TLR9-1486 CT genotype could be associated with protection for PV and the TLR9-1237T/-1486C haplotype, protection for men, as well as for JAK negative men patients with MPN. There were no associations between TLR2 and TLR4 gene polymorphisms and MPN.

[Clinical and epidemiological characteristics of the Philadelphia negative myeloproliferative neoplasms in Chile]. / Características clínicas y epidemiológicas de las neoplasias mieloproliferativas Philadelphia negativas en el sistema público de salud de Chile.

BACKGROUND: Philadelphia-negative myeloproliferative neoplasms (Ph-MPN) are chronic hematological disorders characterized by the overproduction of one or more mature myeloid blood cell lineages. Classical Ph-MPN are polycythemia vera (PV), essential thrombocytopenia (ET) and primary myelofibrosis (PMF). AIM: To assess the epidemiological, clinical and diagnostic characteristics of Ph-MPN in Chile. MATERIAL AND METHODS: Retrospective review of medical records of all patients referred as MPN from 2012 to 2017. Patients with (9;21) translocation were excluded. RESULTS: Data of 462 cases with a median age of 69 years from 10 public hospitals was reviewed. ET was the most frequently Ph-MNP found. The incidence of Ph-MPN was 1.5 x 100.000 cases. The JAK2 V617F mutation study was performed in 96% of patients and only 30% had a bone marrow biopsy. Thrombotic events were observed in 29% of patients. Bleeding events were observed in 7%. Five-year overall survival was 87%. CONCLUSIONS: ET is the most frequent Ph-MPN. The mean incidence was lower than reported in the literature, in part because of a sub diagnosis.

[Clinical and epidemiological characteristics of the Philadelphia negative myeloproliferative neoplasms in Chile]. / Características clínicas y epidemiológicas de las neoplasias mieloproliferativas Philadelphia negativas en el sistema público de salud de Chile.

BACKGROUND: Philadelphia-negative myeloproliferative neoplasms (Ph-MPN) are chronic hematological disorders characterized by the overproduction of one or more mature myeloid blood cell lineages. Classical Ph-MPN are polycythemia vera (PV), essential thrombocytopenia (ET) and primary myelofibrosis (PMF). AIM: To assess the epidemiological, clinical and diagnostic characteristics of Ph-MPN in Chile. MATERIAL AND METHODS: Retrospective review of medical records of all patients referred as MPN from 2012 to 2017. Patients with (9;21) translocation were excluded. RESULTS: Data of 462 cases with a median age of 69 years from 10 public hospitals was reviewed. ET was the most frequently Ph-MNP found. The incidence of Ph-MPN was 1.5 x 100.000 cases. The JAK2 V617F mutation study was performed in 96% of patients and only 30% had a bone marrow biopsy. Thrombotic events were observed in 29% of patients. Bleeding events were observed in 7%. Five-year overall survival was 87%. CONCLUSIONS: ET is the most frequent Ph-MPN. The mean incidence was lower than reported in the literature, in part because of a sub diagnosis.

Diagnostic Performance of Erythropoietin Levels in Polycythemia Vera: Experience at a Comprehensive Cancer Center.

INTRODUCTION: Considering the evolving diagnostic criteria of polycythemia vera (PV), we analyzed the utility of serum erythropoietin (EPO) as a predictive marker for differentiating polycythemia vera (PV) from other etiologies of erythrocytosis. PATIENTS AND METHODS: We conducted a retrospective study after a review of electronical medical records from January 2005 to December 2016 with diagnosis of erythrocytosis using International Classification of Disease-specific codes. To evaluate the diagnostic performance of EPO levels and JAK2-V617F mutation, we constructed a receiver-operated characteristic curve of sensitivity versus 1-specificity for serum EPO levels and JAK2-V617F mutation as predictive markers for differentiating PV from other causes of erythrocytosis. RESULTS: We surveyed 577 patients with erythrocytosis. Median patient age was 59.2 years, 57.72% (n = 329) were male, 86.3% (n = 491) were white, and only 3.3% (n = 19) were African American. A total of 80.88% (n = 351) of those diagnosed with PV had a JAK2-V617F mutation compared to only 1.47% (n = 2) whose primary diagnosis was secondary polycythemia. When comparing JAK2-V617 mutation to the EPO level, the area under the curve of JAK2-V617 (0.8970) was statistically larger than that of EPO test (0.6765). Therefore, the PV diagnostic methodology using JAK2-V617 is better than the EPO test. An EPO level of < 2 mIU/mL was > 99% specific to predict PV but was only 12% sensitive. CONCLUSION: In the appropriate clinical setting, cytogenetic and molecular studies such as JAK2 mutation status prevail as the most useful tools for PV case identification. The use of isolated EPO to screen patients with erythrocytosis is not a good diagnostic approach.

RAGE Mediates Cholesterol Efflux Impairment in Macrophages Caused by Human Advanced Glycated Albumin.

We addressed the involvement of the receptor for advanced glycation end products (RAGE) in the impairment of the cellular cholesterol efflux elicited by glycated albumin. Albumin was isolated from type 1 (DM1) and type 2 (DM2) diabetes mellitus (HbA1c > 9%) and non-DM subjects (C). Moreover, albumin was glycated in vitro (AGE-albumin). Macrophages from Ager null and wild-type (WT) mice, or THP-1 transfected with siRNA-AGER, were treated with C, DM1, DM2, non-glycated or AGE-albumin. The cholesterol efflux was reduced in WT cells exposed to DM1 or DM2 albumin as compared to C, and the intracellular lipid content was increased. These events were not observed in Ager null cells, in which the cholesterol efflux and lipid staining were, respectively, higher and lower when compared to WT cells. In WT, Ager, Nox4 and Nfkb1, mRNA increased and Scd1 and Abcg1 diminished after treatment with DM1 and DM2 albumin. In Ager null cells treated with DM-albumin, Nox4, Scd1 and Nfkb1 were reduced and Jak2 and Abcg1 increased. In AGER-silenced THP-1, NOX4 and SCD1 mRNA were reduced and JAK2 and ABCG1 were increased even after treatment with AGE or DM-albumin. RAGE mediates the deleterious effects of AGE-albumin in macrophage cholesterol efflux.

A proteomic study of myeloproliferative neoplasms using reverse-phase protein arrays.

Myeloproliferative neoplasms polycythemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis constitute a group of haematological diseases. The comprehensive assessment of signaling pathway activation in blood cells may aid the understanding of MPN pathophysiology. Thus, levels of post-translational protein modifications and total protein expression were determined in MPN patients and control leukocytes by using reverse-phase protein arrays (RPPA). Compared to control samples, p-SRC, p-CTNNB1, c-MYC, MCL-1, p-MDM2, BAX and CCNB1 showed higher expression in PV samples than controls. P-JAK2/JAK2 and pro-apoptotic BIM showed differential expression between JAK2V617F-positive and -negative ET patients. Apoptosis, cancer and PI3K/AKT pathways proteins showed differential expression among the studied groups. For most of the proteins analyzed using Western-Blot and RPPA, RPPA showed higher sensitivity to detect subtle differences. Taken together, our data indicate deregulated protein expression in MPN patients compared to controls. Thus, RPPA may be a useful method for broad proteome analysis in MPN patients´ leukocytes.

Genomic characterization and prognostication applied to a Brazilian cohort of patients with myelofibrosis.

Genomic characterization of patients with myeloproliferative neoplasms (MPN) may lead to better diagnostic classification, prognostic assessment, and treatment decisions. These goals are particularly important in myelofibrosis (MF). We performed target Next Generation Sequencing for a panel of 255 genes and Chromosome Microarray Analysis (CMA) in 27 patients with MF. Patients were classified according to genomic findings and we compared the performance of a personalized prognostication system with IPSS, MIPSS70 and MIPSS70 + v2. Twenty-six patients presented mutations: 11.1% had single driver mutations in either JAK2, CALR or MPL; 85.2% had mutations in non-restricted genes (median: 2 per patient). CMA was abnormal in 91.7% of the 24 cases with available data. Copy-Number-Neutral Loss-of-Heterozygosity was the most common finding (66.7%). Del13q was the most frequent copy number variation, and we could define a 2.4 Mb minimally affected region encompassing RB1, SUCLA2 and CLLS2 loci. The largest genomic subgroup consisted of patients with mutations in genes involved with chromatin organization and splicing control (40.7%) and the personalized system showed better concordance and accuracy than the other prognostic systems. Comprehensive genomic characterization reveals the striking genetic complexity of MF and, when combined with clinical data, led, in our cohort, to better prognostication performance.

NT157 has antineoplastic effects and inhibits IRS1/2 and STAT3/5 in JAK2V617F-positive myeloproliferative neoplasm cells.

Recent data indicate that IGF1R/IRS signaling is a potential therapeutic target in BCR-ABL1-negative myeloproliferative neoplasms (MPN); in this pathway, IRS2 is involved in the malignant transformation induced by JAK2V617F, and upregulation of IGF1R signaling induces the MPN phenotype. NT157, a synthetic compound designed as an IGF1R-IRS1/2 inhibitor, has been shown to induce antineoplastic effects in solid tumors. Herein, we aimed to characterize the molecular and cellular effects of NT157 in JAK2V617F-positive MPN cell lines (HEL and SET2) and primary patient hematopoietic cells. In JAK2V617F cell lines, NT157 decreased cell viability, clonogenicity, and cell proliferation, resulting in increases in apoptosis and cell cycle arrest in the G2/M phase (p < 0.05). NT157 treatment inhibited IRS1/2, JAK2/STAT, and NFκB signaling, and it activated the AP-1 complex, downregulated four oncogenes (CCND1, MYB, WT1, and NFKB1), and upregulated three apoptotic-related genes (CDKN1A, FOS, and JUN) (p < 0.05). NT157 induced genotoxic stress in a JAK2/STAT-independent manner. NT157 inhibited erythropoietin-independent colony formation in cells from polycythemia vera patients (p < 0.05). These findings further elucidate the mechanism of NT157 action in a MPN context and suggest that targeting IRS1/2 proteins may represent a promising therapeutic strategy for MPN.