Lrig1 regulates cell fate specification of glutamatergic neurons via FGF-driven Jak2/Stat3 signaling in cortical progenitors.

The cell-intrinsic mechanisms underlying the decision of a stem/progenitor cell to either proliferate or differentiate remain incompletely understood. Here, we identify the transmembrane protein Lrig1 as a physiological homeostatic regulator of FGF2-driven proliferation and self-renewal of neural progenitors at early-to-mid embryonic stages of cortical development. We show that Lrig1 is expressed in cortical progenitors (CPs), and its ablation caused expansion and increased proliferation of radial/apical progenitors and of neurogenic transit-amplifying Tbr2+ intermediate progenitors. Notably, our findings identify a previously unreported EGF-independent mechanism through which Lrig1 negatively regulates neural progenitor proliferation by modulating the FGF2-induced IL6/Jak2/Stat3 pathway, a molecular cascade that plays a pivotal role in the generation and maintenance of CPs. Consistently, Lrig1 knockout mice showed a significant increase in the density of pyramidal glutamatergic neurons placed in superficial layers 2 and 3 of the postnatal neocortex. Together, these results support a model in which Lrig1 regulates cortical neurogenesis by influencing the cycling activity of a set of progenitors that are temporally specified to produce upper layer glutamatergic neurons.

Biological Evaluations and Computer-Aided Approaches of Janus Kinases 2 and 3 Inhibitors for Cancer Treatment: A Review.

Cancer remains one of the leading diseases of mortality worldwide. Janus kinases 2/3 (JAK2/3) have been considered a drug target for the development of drugs to treat different types of cancer. JAK2/3 play a critical role in innate immunity, inflammation, and hematopoiesis by mediating the signaling of numerous cytokines, growth factors, and interferons. The current focus is to develop new selective inhibitors for each JAK type. In this review, the current strategies of computer-aided studies, and biological evaluations against JAK2/3 are addressed. We found that the new synthesized JAK2/3 inhibitors are prone to containing heterocyclic aromatic rings such as pyrimidine, pyridine, and pyrazolo [3,4-d]pyrimidine. Moreover, inhibitors of natural origin derived from plant extracts and insects have shown suitable inhibitory capacities. Computer-assisted studies have shown the important features of inhibitors for JAK2/3 binding. Biological evaluations showed that the inhibition of the JAK receptor affects its related signaling pathway. Although the reviewed compounds showed good inhibitory capacity in vitro and in vivo, more in-depth studies are needed to advance toward full approval of cancer treatments in humans.

Effect of 28 days treatment of baricitinib on mechanical allodynia, osteopenia, and loss of nerve fibers in an experimental model of type-1 diabetes mellitus.

BACKGROUND: Type-1 diabetes mellitus (T1DM) is associated with numerous health problems, including peripheral neuropathy, osteoporosis, and bone denervation, all of which diminish quality of life. However, there are relatively few therapies to treat these T1DM-related complications. Recent studies have shown that Janus kinase (JAK) inhibitors reverse aging- and rheumatoid arthritis-induced bone loss and reduce pain associated with peripheral nerve injuries, and rheumatoid arthritis. Thus, we assessed whether a JAK1/JAK2 inhibitor, baricitinib, ameliorates mechanical pain sensitivity (a measure of peripheral neuropathy), osteoporosis, and bone denervation in the femur of mice with T1DM. METHODS: Female ICR mice (13 weeks old) received five daily administrations of streptozotocin (ip, 50 mg/kg) to induce T1DM. At thirty-one weeks of age, mice were treated with baricitinib (po; 40 mg/kg/bid; for 28 days) or vehicle. Mechanical sensitivity was evaluated at 30, 33, and 35 weeks of age on the plantar surface of the right hind paw. At the end of the treatment, mice were sacrificed, and lower extremities were harvested for microcomputed tomography and immunohistochemistry analyses. RESULTS: Mice with T1DM exhibited greater blood glucose levels, hind paw mechanical hypersensitivity, trabecular bone loss, and decreased density of calcitonin gene-related peptide-positive and tyrosine hydroxylase-positive axons within the marrow of the femoral neck compared to control mice. Baricitinib treatment significantly reduced mechanical hypersensitivity and ameliorated sensory and sympathetic denervation at the femoral neck, but it did not reverse trabecular bone loss. CONCLUSIONS: Our findings suggest that baricitinib may represent a new therapeutic alternative to treat T1DM-induced peripheral neuropathy and bone denervation.

Sphingosine 1-phosphate protective effect on human proximal tubule cells submitted to an in vitro ischemia model: the role of JAK2/STAT3.

Acute kidney injury is a serious public health problem worldwide, being ischemia and reperfusion (I/R) the main lesion-aggravating factor that contributes to the evolution towards chronic kidney disease. Nonetheless, intervention approaches currently available are just considered palliative options. In order to offer an alternative treatment, it is important to understand key factors involved in the development of the disease including the rescue of the affected cells and/or the release of paracrine factors that are crucial for tissue repair. Bioactive lipids such as sphingosine 1-phosphate (S1P) have significant effects on the modulation of signaling pathways involved in tissue regeneration, such as cell survival, proliferation, differentiation, and migration. The main objective of this work was to explore the protective effect of S1P using human kidney proximal tubule cells submitted to a mimetic I/R lesion, via ATP depletion. We observed that the S1P pre-treatment increases cell survival by 50% and preserves the cell proliferation capacity of injured cells. We showed the presence of different bioactive lipids notably related to tissue repair but, more importantly, we noted that the pre-treatment with S1P attenuated the ischemia-induced effects in response to the injury, resulting in higher endogenous S1P production. All receptors but S1PR3 are present in these cells and the protective and proliferative effect of S1P/S1P receptors axis occur, at least in part, through the activation of the SAFE pathway. To our knowledge, this is the first time that S1PR4 and S1PR5 are referred in these cells and also the first indication of JAK2/STAT3 pathway involvement in S1P-mediated protection in an I/R renal model.

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.

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

Abstract 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.

CARL y MPL en pacientes cubanos con trombocitemia esencial y mielofibrosis primaria / CARL and MPL in Cuban patients with essential thrombocythemia and primary mielofibrosi

Introducción: La trombocitemia esencial y la mielofibrosis primaria comparten la presencia de las mutaciones JAK2, CALR y MPL. En total, están presentes en poco más del 90 % de los pacientes con estas enfermedades. Objetivos: Determinar el comportamiento de las mutaciones más frecuentes en los genes MPL y CALR en pacientes cubanos. Métodos: Se realizó un estudio ambispectivo, descriptivo y longitudinal en el Instituto de Hematología e Inmunología de Cuba, entre los años 2010 y 2020. Se incluyeron todos los pacientes con sospecha de trombocitemia esencial y de mielofibrosis primaria con muestras de ADN válidas. Se les identificaron las mutaciones CALR y MPL por PCR en tiempo real. Resultados: De los 53 pacientes estudiados, el 67,9 % fueron diagnosticados con trombocitemia esencial, el 22,6 % con mielofibrosis primaria. En el 90,6 % se pudo detectar alguna de las mutaciones conductoras; el 67,9 % fueron positivos a la mutación JAK2V617F, el 13,2 % a las mutaciones en el gen que codifica para la calreticulina y en el 9,4 % se identificaron mutaciones en el gen MPL. Conclusiones: El comportamiento de las mutaciones conductoras JAK2V617F, CALR y MPL en la muestra de pacientes cubanos con trombocitemia esencial y mielofibrosis primaria estuvo en correspondencia con lo descrito en la mayoría de las investigaciones.

Introduction: Essential thrombocythemia and primary myelofibrosis share the presence of JAK2, CALR and MPL mutations. In total, they comprise slightly more than 90 % of patients with these diseases. Objectives: To determine the behavior of the most frequent mutations in MPL and CALR genes in Cuban patients. Methods: An ambispective, descriptive and longitudinal study was performed at the Institute of Hematology and Immunology of Cuba, between 2010 and 2020. All patients with suspected essential thrombocythemia and primary myelofibrosis with valid DNA samples were included. CALR and MPL mutations were identified by real-time PCR. Results: Of the 53 patients studied, 67.9% were diagnosed with essential thrombocythemia, and 22.6% with primary myelofibrosis. In 90.6% it was possible to detect any of the driver mutations: 67.9% were positive for the JAK2V617F mutation, 13.2% for mutations in the gene coding for calreticulin and in 9.4% mutations in the MPL gene were identified. Conclusions: The behavior of the driver mutations JAK2V617F, CALR and MPL in the sample of Cuban patients with essential thrombocythemia and primary myelofibrosis was in correspondence with what is described in the majority of the investigations.

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.

High cell-free DNA is associated with disease progression, inflammasome activation and elevated levels of inflammasome-related cytokine IL-18 in patients with myelofibrosis.

Myelofibrosis (MF) is a clonal hematopoietic stem cell disorder classified among chronic myeloproliferative neoplasms, characterized by exacerbated myeloid and megakaryocytic proliferation and bone marrow fibrosis. It is induced by driver (JAK2/CALR/MPL) and high molecular risk mutations coupled to a sustained inflammatory state that contributes to disease pathogenesis. Patient outcome is determined by stratification into risk groups and refinement of current prognostic systems may help individualize treatment decisions. Circulating cell-free (cf)DNA comprises short fragments of double-stranded DNA, which promotes inflammation by stimulating several pathways, including inflammasome activation, which is responsible for IL-1ß and IL-18 maturation and release. In this work, we assessed the contribution of cfDNA as a marker of disease progression and mediator of inflammation in MF. cfDNA was increased in MF patients and higher levels were associated with adverse clinical outcome, a high-risk molecular profile, advanced disease stages and inferior overall survival, indicating its potential value as a prognostic marker. Cell-free DNA levels correlated with tumor burden parameters and markers of systemic inflammation. To mimic the effects of cfDNA, monocytes were stimulated with poly(dA:dT), a synthetic double-stranded DNA. Following stimulation, patient monocytes released higher amounts of inflammasome-processed cytokine, IL-18 to the culture supernatant, reflecting enhanced inflammasome function. Despite overexpression of cytosolic DNA inflammasome sensor AIM2, IL-18 release from MF monocytes was shown to rely mainly on the NLRP3 inflammasome, as it was prevented by NLRP3-specific inhibitor MCC950. Circulating IL-18 levels were increased in MF plasma, reflecting in vivo inflammasome activation, and highlighting the previously unrecognized involvement of this cytokine in MF cytokine network. Monocyte counts were higher in patients and showed a trend towards correlation with IL-18 levels, suggesting monocytes represent a source of circulating IL-18. The close correlation shown between IL-18 and cfDNA levels, together with the finding of enhanced DNA-triggered IL-18 release from monocytes, suggest that cfDNA promotes inflammation, at least in part, through inflammasome activation. This work highlights cfDNA, the inflammasome and IL-18 as additional players in the complex inflammatory circuit that fosters MF progression, potentially providing new therapeutic targets.

Molecular landscape of the JAK2 gene in chronic myeloproliferative neoplasm patients from the state of Amazonas, Brazil.

JAK2V617F (dbSNP: rs77375493) is the most frequent and most-studied variant in BCR::ABL1 negative myeloproliferative neoplasms and in the JAK2 gene. The present study aimed to molecularly characterize variants in the complete coding region of the JAK2 gene in patients with BCR::ABL1 negative chronic myeloproliferative neoplasms. The study included 97 patients with BCR::ABL1 negative myeloproliferative neoplasms, including polycythemia vera (n=38), essential thrombocythemia (n=55), and myelofibrosis (n=04). Molecular evaluation was performed using conventional PCR and Sanger sequencing to detect variants in the complete coding region of the JAK2 gene. The presence of missense variants in the JAK2 gene including rs907414891, rs2230723, rs77375493 (JAK2V617F), and rs41316003 were identified. The coexistence of variants was detected in polycythemia vera and essential thrombocythemia. Thus, individuals with high JAK2V617F variant allele frequency (≥50% VAF) presented more thrombo-hemorrhagic events and manifestations of splenomegaly compared with those with low JAK2V617F variant allele frequency (<50% VAF). In conclusion, individuals with BCR::ABL1 negative neoplasms can display >1 variant in the JAK2 gene, especially rs2230722, rs2230724, and rs77375493 variants, and those with high JAK2V617F VAF show alterations in the clinical-laboratory profile compared with those with low JAK2V617F VAF.

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.

Crosstalk between the JAK2 and TGF-β1 signaling pathways in scleroderma-related interstitial lung disease targeted by baricitinib

Abstract Background and objective Systemic sclerosis (SSc) is an immune-mediated rheumatic disease characterized by fibrosis and vascular lesions. Interstitial lung disease is an early complication of SSc and the main cause of death from SSc. Although baricitinib shows good efficacy in a variety of connective tissue diseases, its role in systemic sclerosis-related interstitial lung disease (SSc-ILD) is unclear. The objective of our study was to explore the effect and mechanism of baricitinib in SSc-ILD. Methods We explored crosstalk between the JAK2 and TGF-β1 pathways. In vivo experiments, SSc-ILD mice model were constructed by subcutaneous injection of PBS or bleomycin (7.5 mg/kg) and intragastric administration of 0.5% CMC-Na or baricitinib (5 mg/kg) once every two days. We used ELISA, qRT-PCR, western blot and immunofluorescence staining to evaluate the degree of fibrosis. In vitro experiments, we used TGF-β1 and baricitinib to stimulate human fetal lung fibroblasts (HFLs) and assessed protein expression by western blot. Results The vivo experiments showed that baricitinib notably alleviated skin and lung fibrosis, decreased the concentration of pro-inflammatory factors and increased those of the anti-inflammatory factors. Baricitinib affected the expression of TGF-β1 and TβRI/II inhibitiing JAK2. In the vitro experiments, following the culture of HFLs with baricitinib or a STAT3 inhibitor for 48 h, the expression levels of TβRI/II decreased. Conversely, with successful inhibition of TGF-β receptors in HFLs, JAK2 protein expression decreased. Conclusions Baricitinib attenuated bleomycin-induced skin and lung fibrosis in SSc-ILD mice model by targeting JAK2 and regulating of the crosstalk between the JAK2 and TGF-β1 signaling pathways.

Baricitinib improves pulmonary fibrosis in mice with rheumatoid arthritis-associated interstitial lung disease by inhibiting the Jak2/ Stat3 signaling pathway

Abstract Objective The study explored improvements in pulmonary inflammation and fibrosis in a bovine type II collagen-induced rheumatoid arthritis-associated interstitial lung disease mouse model after treatment with baricitinib and the possible mechanism of action. Methods A rheumatoid arthritis-associated interstitial lung disease mouse model was established, siRNA Jak2 and lentiviral vectors were transfected with human embryonic lung fibroblast cells. And the levels of relevant proteins in mouse lung tissue and human embryonic lung fibroblasts were detected by Western blotting. Results The levels of JAK2, p-JAK2, p-STAT3, p-SMAD3, SMA, TGFβR2, FN and COL4 were increased in the lung tissues of model mice (P < 0.5) and decreased after baricitinib intervention (P < 0.05). The expression levels of p-STAT3, p-SMAD3, SMA, TGFβR2, FN and COL4 were reduced after siRNA downregulation of the JAK2 gene (P < 0.01) and increased after lentiviral overexpression of the JAK2 gene (P < 0.01). Conclusion Baricitinib alleviated fibrosis in the lung tissue of rheumatoid arthritis-associated interstitial lung disease mice, and the mechanism of action may involve the downregulation of Smad3 expression via inhibition of the Jak2/Stat3 signaling pathway, with consequent inhibition of the profibrotic effect of transforming growth factor-β1.

Acute exercise reduces feeding by activating IL-6/Tubby axis in the mouse hypothalamus.

Background: Acute exercise contributes to decreased feeding through leptin and interleukin/Janus kinase 2/signal transducers and activators of transcription 3 (IL-6/JAK2/STAT3) signaling. Considering the pleiotropic use of substrates by JAK2 and that JAK2 can phosphorylate the Tubby protein (TUB) in CHO-IR cells, we speculated that acute exercise can activate the IL-6/JAK2/TUB pathway to decrease food intake. Aims: We investigated whether acute exercise induced tyrosine phosphorylation and the association of TUB and JAK2 in the hypothalamus and if IL-6 is involved in this response, whether acute exercise increases the IL-6/TUB axis to regulate feeding, and if leptin has an additive effect over this mechanism. Methods: We applied a combination of genetic, pharmacological, and molecular approaches. Key findings: The in vivo experiments showed that acute exercise increased the tyrosine phosphorylation and association of JAK2/TUB in the hypothalamus, which reduced feeding. This response was dependent on IL-6. Leptin had no additive effect on this mechanism. Significance: The results of this study suggest a novel hypothalamic pathway by which IL-6 released by exercise regulates feeding and reinforces the beneficial effects of exercise.

Influencia de la carga alélica del JAK2V617F en pacientes cubanos con trombocitemia esencial y mielofibrosis primaria / Influence of JAK2V617F allelic burden in Cuban patients with essential thrombocythemia and primary myelofibrosis

Introducción: La frecuencia de la mutación JAK2V617F se estima entre el 50 y 60 por ciento en pacientes con trombocitemia esencial y mielofibrosis primaria. El 30 por ciento de los pacientes con policitemia vera y mielofibrosis primaria. Entre 2-4 por ciento de los pacientes con trombocitemia esencial presentan pérdida de heterocigosidad. Objetivos: Evaluar la influencia de la carga alélica de la mutación JAK2V617F y su relación con variables clínico-hematológicas en el diagnóstico de estas enfermedades en pacientes cubanos. Métodos: Se realizó un estudio retrospectivo, descriptivo y longitudinal en el Instituto de Hematología e Inmunología entre 2010 y 2020. Se incluyeron todos los pacientes con sospecha de trombocitemia esencial y mielofibrosis primaria con muestras de ADN válidas. Se les cuantificó la carga alélica de la mutación por PCR en tiempo real. Resultados: Se detectó la mutación en 66,7 por ciento de los diagnosticados con trombocitemia esencial y mielofibrosis primaria. El 62,5 por ciento de los pacientes con mielofibrosis primaria fueron homocigotos a la mutación, mientras que en la trombocitemia esencial solo el 20,8 por ciento. La diferencia de medias de cargas alélicas entre ambas enfermedades fue estadísticamente significativa. No se encontraron diferencias significativas en la comparación de las variables clínicas y hematológicas en estas enfermedades ni asociación con la carga alélica con excepción de las plaquetas en la mielofibrosis primaria. Conclusiones: El estudio estuvo limitado por la escasa muestra de pacientes, pero se corresponde con otras investigaciones que sostienen el concepto de que la presentación fenotípica de las neoplasias mieloproliferativasestá influenciada por la carga mutacional del JAK2V617F(AU)

Introduction: The frequency of the JAK2V617F mutation is estimated to be between 50 percent and 60 percent in patients with essential thrombocythemia and primary myelofibrosis. 30 percent of patients with polycythemia vera and primary myelofibrosis and 2-4 percent of patients with essential thrombocythemia show loss of heterozygosity. Objectives: To evaluate the influence of the allelic load of the JAK2V617F mutation in the diagnosis of these diseases in Cuban patients and its relationship with clinical-hematological variables. Methodology: A retrospective, descriptive and longitudinal study was carried out at the Institute of Hematology and Immunology between 2010 and 2020. All patients with suspected essential thrombocythemia and primary myelofibrosis with valid DNA samples were included. The allelic load of the mutation was quantified by real-time PCR. Results: The mutation was detected in 66.7 percent of those diagnosed with essential thrombocythemia and primary myelofibrosis. 62.5 percent of the patients with primary myelofibrosis were homozygous for the mutation, while in essential thrombocythemia only 20.8 percent. The difference in mean allelic loads between both diseases was statistically significant. No significant differences were found in the comparison of clinical and hematological variables in these diseases or association with allelic load, with the exception of platelets in primary myelofibrosis. Conclusions: The study was limited by the small sample of patients, but it corresponds to other investigations that support the concept that the phenotypic presentation of myeloproliferative neoplasms is influenced by the mutational load of JAK2V617F(AU)

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.

Association of JAK/STAT genetic variants with cutaneous melanoma.

Background: The Janus-activated kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway regulates cutaneous melanoma (CM) development and progression. The JAK1, JAK2, and STAT3 proteins are encoded by polymorphic genes. This study aimed to verify whether single-nucleotide variants (SNVs) in JAK1 (c.1648+1272G>A, c.991-27C>T), JAK2 (c.-1132G>T, c.-139G>A), and STAT3 (c.*1671T>C, c.-1937C>G) altered the risk, clinicopathological aspects, and survival of CM patients as well as protein activity. Methods: CM patients (N = 248) and controls (N = 274) were enrolled in this study. Genotyping was performed by real-time polymerase chain reaction (PCR), and JAK1, JAK2, and STAT3 expression was assessed by quantitative PCR (qPCR). STAT3 c.-1937C>G SNV was investigated by luciferase, qPCR, western blot, apoptosis, and cell cycle assays in SKMEL-28 cells with CC or GG genotype. Results: Individuals with STAT3 c.*1671TT and c.-1937CC genotypes and TC haplotype of both SNVs were under about 2.0-fold increased risk of CM. Specific JAK1, JAK2, and STAT3 combined genotypes were associated with up to 4.0-fold increased risk of CM. Higher luciferase activity [4,013.34 vs. 2,463.32 arbitrary units (AU); p = 0.004], STAT3 expression by qPCR (649.20 vs. 0.03 AU; p = 0.003) and western blot (1.69 vs. 1.16 AU; p = 0.01), and percentage of cells in the S phase of the cell cycle (57.54 vs. 30.73%; p = 0.04) were more frequent in SKMEL-28 with STAT3 c.-1937CC than with GG genotype. CM cell line with CC genotype presented higher STAT3 protein levels than the one with GG genotype (1.93 versus 1.27 AU, p = 0.0027). Conclusion: Our data present preliminary evidence that inherited abnormalities in the JAK/STAT pathway can be used to identify individuals at a high risk of CM, who deserve additional attention for tumor prevention and early detection.

Differential cytotoxic activity of pharmacological inhibitors of IGF1R-related pathways in JAK2V617F driven cells.

Myeloproliferative neoplasms (MPN) belong to a group of clonal diseases of hematopoietic stem cells characterized by aberrant proliferation of mature myeloid lineages. The constitutive activation of the JAK2/STAT signaling pathway is now well established to play a central role in MPN pathogenesis; however, accumulating evidence now indicates that the IGF1R-mediated signaling pathway contributes to the maintenance of the malignant phenotype. Studies using inhibitors of IGF1-mediated signaling have reported cytotoxic effects in cellular and murine models of MPN, but no consensus has been reached regarding the potency and efficacy of inhibitors of the IGF1R-related pathway in this context. In the present study, we compared the potency and efficacy of three inhibitors of IGF1R-related pathways in a JAK2V617F-driven cellular model. These inhibitors (NT157, OSI-906, and NVP-AEW54) present antineoplastic activity with similar efficacy in Ba/F3 JAK2V617F cells, with NT157 showing the greatest potency. Both the induction of apoptosis and reduction in cell proliferation were associated with the observed reduction in cell viability. Downregulation of JAK2/STAT signaling was an advantageous off-target effect of all three inhibitors. These preclinical studies reinforce the potential of the IGF1R-related pathway as a therapeutic target in MPN.

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.