A novel cancer-germline gene DAZL promotes progression and cisplatin resistance of non-small cell lung cancer by upregulating JAK2 and MCM8.

Germline-specific genes are usually activated in cancer cells and drive cancer progression; such genes are called cancer-germline or cancer-testis genes. The RNA-binding protein DAZL is predominantly expressed in germ cells and plays a role in gametogenesis as a translational activator or repressor. However, its expression and role in non-small cell lung cancer (NSCLC) are unknown. Here, mining of RNA-sequencing data from public resources and immunohistochemical analysis of tissue microarrays showed that DAZL was expressed exclusively in testis among normal human tissues but ectopically expressed in NSCLC tissues. Testis and NSCLC cells expressed the shorter and longer transcript variants of the DAZL gene, respectively. Overexpression of the longer DAZL transcript promoted tumor growth in a mouse xenograft model. Silencing of DAZL suppressed cell proliferation, colony formation, migration, invasion, and cisplatin resistance in vitro and tumor growth in vivo. Quantitative proteomic analysis based on tandem mass tag and Western blot analysis showed that DAZL upregulated the expression of JAK2 and MCM8. RNA-binding protein immunoprecipitation assays showed that DAZL bound to the mRNA of JAK2 and MCM8. The JAK2 inhibitor fedratinib attenuated the oncogenic outcomes induced by DAZL overexpression, whereas silencing MCM8 counteracted the effects of DAZL overexpression on cisplatin-damaged DNA synthesis and half-maximal inhibitory concentration of cisplatin. In conclusion, DAZL was identified as a novel cancer-germline gene that enhances the translation of JAK2 and MCM8 to promote NSCLC progression and resistance to cisplatin, respectively. These findings suggest that DAZL is a potential therapeutic target in NSCLC.

Drug Repositioning via Graph Neural Networks: Identifying Novel JAK2 Inhibitors from FDA-Approved Drugs through Molecular Docking and Biological Validation.

The increasing utilization of artificial intelligence algorithms in drug development has proven to be highly efficient and effective. One area where deep learning-based approaches have made significant contributions is in drug repositioning, enabling the identification of new therapeutic applications for existing drugs. In the present study, a trained deep-learning model was employed to screen a library of FDA-approved drugs to discover novel inhibitors targeting JAK2. To accomplish this, reference datasets containing active and decoy compounds specific to JAK2 were obtained from the DUD-E database. RDKit, a cheminformatic toolkit, was utilized to extract molecular features from the compounds. The DeepChem framework's GraphConvMol, based on graph convolutional network models, was applied to build a predictive model using the DUD-E datasets. Subsequently, the trained deep-learning model was used to predict the JAK2 inhibitory potential of FDA-approved drugs. Based on these predictions, ribociclib, topiroxostat, amodiaquine, and gefitinib were identified as potential JAK2 inhibitors. Notably, several known JAK2 inhibitors demonstrated high potential according to the prediction results, validating the reliability of our prediction model. To further validate these findings and confirm their JAK2 inhibitory activity, molecular docking experiments were conducted using tofacitinib-an FDA-approved drug for JAK2 inhibition. Experimental validation successfully confirmed our computational analysis results by demonstrating that these novel drugs exhibited comparable inhibitory activity against JAK2 compared to tofacitinib. In conclusion, our study highlights how deep learning models can significantly enhance virtual screening efforts in drug discovery by efficiently identifying potential candidates for specific targets such as JAK2. These newly discovered drugs hold promises as novel JAK2 inhibitors deserving further exploration and investigation.

Insights into the role of JAK2-I724T variant in myeloproliferative neoplasms from a unique cohort of New Zealand patients.

OBJECTIVES: This study aimed to compile bioinformatic and experimental information for JAK2 missense variants previously reported in myeloproliferative neoplasms (MPN) and determine if germline JAK2-I724T, recently found to be common in New Zealand Polynesians, associates with MPN. METHODS: For all JAK2 variants found in the literature, gnomAD_exome allele frequencies were extracted and REVEL scores were calculated using the dbNSFP database. We investigated the prevalence of JAK2-I724T in a cohort of 111 New Zealand MPN patients using a TaqMan assay, examined its allelic co-occurrence with JAK2-V617F using Oxford Nanopore sequencing, and modelled the impact of I724T on JAK2 using I-Mutant and ChimeraX software. RESULTS: Several non-V617F JAK2 variants previously reported in MPN had REVEL scores greater than 0.5, suggesting pathogenicity. JAK2-I724T (REVEL score 0.753) was more common in New Zealand Polynesian MPN patients (n = 2/27; 7.4%) than in other New Zealand patients (n = 0/84; 0%) but less common than expected for healthy Polynesians (n = 56/377; 14.9%). Patients carrying I724T (n = 2), one with polycythaemia vera and one with essential thrombocythaemia, had high-risk MPN. Both patients with JAK2-I724T were also positive for JAK2-V617F, found on the same allele as I724T, as well as separately. In silico modelling did not identify noticeable structural changes that would give JAK2-I724T a gain-of-function. CONCLUSION: Several non-canonical JAK2 variants with high REVEL scores have been reported in MPN, highlighting the need to further understand their relationship with disease. The JAK2-I724T variant does not drive MPN, but additional investigations are required to exclude any potential modulatory effect on the MPN phenotype.

Neuropsychiatric disturbance detecting polycythemia vera myelofibrosis: a case report and literature review.

Background: Neuropsychiatric disturbances and chorea are less recognized consequences of polycythemia vera (PV), and their role in post-PV myelofibrosis (MF) has not been reported. Clinical features that predict post-PV MF lack specificity. Case presentation: We describe an elderly patient with PV who developed acute-onset reversible neuropsychiatric disturbances accompanied by generalized chorea and was finally diagnosed with post-PV MF after a bone marrow examination. We also reviewed four cases of late PV associated with neuropsychiatric symptoms since 1966 and analyzed their clinical characteristics and therapeutic effects. Conclusion: Our case indicates that Janus kinase 2 (JAK2)-related PV is a treatable cause of late-onset chorea and that chorea may herald the deterioration of hematological parameters. Our case provides a clinically specific representation of post-PV MF. Patients with a long course of PV are recommended to undergo bone marrow re-examinations when they present with neuropsychiatric symptoms to achieve an early diagnosis of post-PV MF.

Interleukin-6 in Skeletal Muscle Mass Regulation / 中国生物化学与分子生物学报

Interleukin-6 (IL-6) is a spreading pleiotropic cytokine, with both anti-inflammatory and proinflammatory effects. It not only participates in the body immune responses but also is involved in the biological regulative processes among different organs, tissues, and cells. IL-6 has both anti-inflammatory and pro-inflammatory effects. In the early stage of pathogen infection, IL-6 plays an anti-inflammatory role in the body, and its level is moderately increased in the body to resist inflammation and maintain internal homeostasis. However, a large amount of IL-6 release can cause excessive inflammation and trigger other pathological changes in the body. Il-6 also has the dual effect of stimulating the synthesis and degradation of skeletal muscle protein in regulating skeletal muscle mass. As an important locomotive organ, skeletal muscle is also one of the key targets of IL-6. IL-6 takes part in the biological control of skeletal muscle hypertrophy through regulating muscle satellite cell proliferation and differentiation under specific stresses. In addition IL-6 is also associated with skeletal muscle atrophy induced by aging and other pathological stresses. In addition, during exercise stress, skeletal muscle can also serve as an endocrine organ to secrete and release IL-6 that facilitates the "crosstalk" between skeletal muscle and other organs or tissues. As IL-6 plays as a versatile role in our body, this paper reviews the research progress of the mechanism of IL-6 in the regulation of skeletal muscle mass, which may provide theoretical support for revealing the molecular mechanism of skeletal muscle stresses and adaptations.

Mechanism of Hedysari Radix Polysaccharide on Diabetic Nephropathy in db/db Mice Based on JAK2/STAT3 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of Hedysari Radix polysaccharide （HRP） on the Janus kinase 2 （JAK2）/signal transducer and activator of transcription protein 3 （STAT3） signaling pathway in diabetic nephropathy db/db mice. MethodFifty db/db mice were randomly divided into model group， irbesartan group （irbesartan suspension， 22.75 mg·kg-1）， and high-， medium-， and low-dose HRP groups （HRP suspension， 200， 100， 50 mg·kg-1） according to the body weight， with 10 mice in each group. Another 10 C57BL/6 mice were assigned to the normal group. The mice were treated with corresponding drugs by gavage， while those in the normal group and the model group received distilled water at 5 mL·kg-1. The mice in the six groups were administered once a day by gavage for 12 consecutive weeks. The uric acid （UA）， triglycerides （TG）， and total cholesterol （TC） were detected. Periodic acid-Schiff （PAS） staining and Masson staining were used to observe the pathological changes in kidney tissues. Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） were used to detect the protein and mRNA expression levels of JAK2， STAT3， suppressor of cytokine signaling 3 （SOCS3）， and tumor necrosis factor-α （TNF-α） in the kidney. ResultAfter 12 weeks of treatment， compared with the normal group， the model group showed significant pathological ultrastructural changes in kidney tissues and increased UA， TG， and TC levels （P<0.01）. Compared with the model group， the high- and medium-dose HRP groups and the irbesartan group showed improvement in pathological ultrastructure of kidney tissues and reduced UA， TG， and TC levels （P<0.05， P<0.01）. Compared with the normal group， the model group showed a decrease in SOCS3 protein and mRNA expression levels and an increase in JAK2， STAT3， and TNF-α protein and mRNA expression levels （P<0.01）. Compared with the model group， the high- and medium-dose HRP groups and the irbesartan group showed an increase in SOCS3 protein and mRNA expression levels and a decrease in JAK2， STAT3， and TNF-α protein and mRNA expression levels （P<0.05， P<0.01）. ConclusionHRP can alleviate renal damage in diabetic nephropathy to a certain extent， and its mechanism may be related to the inhibition of the activation of the JAK2/STAT3 signaling pathway.

Curculigoside Represses the Proliferation and Metastasis of Osteosarcoma via the JAK/STAT and NF-κB Signaling Pathways.

Curculigoside (Cur) is a natural component from Curculigo orchioides Gaertn, with various bioactivities. The function of Cur in the nervous system and osteoarthritis has been reported. However, its role in osteosarcoma (OS) needs to be investigated. Hence, we focus on probing the impact of Cur on OS. In vitro, cell counting kit 8 (CCK-8), flow cytometry and Transwell assay were used to investigate the effects of Cur on OS cell proliferation, apoptosis, migration and invasion. In vivo, we developed a xenograft model to figure out the effect of Cur on tumor growth in nude mice. Western blotting (WB) was conducted to compare the levels of Cur on apoptosis-related proteins (C-caspase-3, Bax, and Bcl-2), epithelial-mesenchymal transition (EMT)-related proteins (N-cadherin, Snail, and E-cadherin) and the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and nuclear factor-κB (NF-κB) pathways in vitro and in vivo. In-vitro data testified that Cur treatment markedly hampered OS cells' growth, migration and invasion and intensified their apoptosis compared to that of the control group. In vivo, Cur treatment notably hampered the growth of OS tumors in mice. In addition, both in vitro and in vivo experiments demonstrated that the phosphorylation of JAK2, STAT3, and NF-κB were inhibited through Cur treatment. Furthermore, the inhibition of Cur in OS cells was demonstrated by up-regulating the expression of JAK/STAT and NF-κB pathways protein levels. In summary, the data suggest that Cur curbs OS growth by down-regulating the JAK/STAT and NF-κB pathways, which is an underlying therapeutic option for OS treatment.

Cell proliferation effects of S-allyl-L-cysteine are associated with phosphorylation of janus kinase 2, insulin-like growth factor type-I receptor tyrosine kinase, and extracellular signal-regulated kinase 2 in primary cultures of adult rat hepatocytes.

The cell proliferation effect of S-allyl-L-cysteine (SAC) and its mechanisms were examined in primary cultures of adult rat hepatocytes. In serum-free cultivation, SAC (10-6 M)-stimulated hepatocytes showed significant proliferation compared to control at 5-h culture; the effect was dependent on the culture time and the dose of SAC (EC50 value 8.58 × 10-8 M). In addition, SAC-stimulated hepatocytes significantly increased mRNA expression levels of c-Myc and c-Fos at 1 h and cyclin B1 at 3.5 and 4 h, respectively. In contrast, alliin and allicin, structural analogs of SAC, did not show these effects observed with SAC. The SAC-induced hepatocyte proliferation effects were completely suppressed by monoclonal antibodies against growth hormone receptor and insulin-like growth factor type-I (IGF-I) receptor, respectively. Furthermore, the Janus kinase 2 (JAK2) inhibitor TG101209, phospholipase C (PLC) inhibitor U-73122, IGF-I receptor tyrosine kinase (RTK) inhibitor AG538, PI3 kinase inhibitor LY294002, MEK inhibitor PD98059, and mTOR inhibitor rapamycin completely suppressed the SAC-induced hepatocyte proliferation. JAK2 (p125 kDa) phosphorylation in cultured hepatocytes peaked 5 min after SAC stimulation. SAC-induced IGF-I RTK (p95 kDa) and ERK2 (p42 kDa) phosphorylation had slower rises than JAK2, peaking at 20 and 30 min, respectively. These results indicate that SAC promoted cell proliferation by growth hormone receptor/JAK2/PLC pathway activation followed by activation of the IGF-I RTK/PI3K/ERK2/mTOR pathway in primary cultures of adult rat hepatocytes.

Study of CALR, MPL, and c-kit Gene Mutations in Thai Patients with JAK2 V617F Negative Myeloproliferative Neoplasms.

OBJECTIVE: The aim of this study to determine the prevalence of CALR, MPL and c-kit gene mutations in JAK2 V617F negative-MPN patients. METHODS: The retrospective study of CALR, MPL and c-kit mutations were analyzed in 113 samples collected from March 2010 to May 2017 and identified as JAK2 V617F-negative MPN Thai patients. The samples were analysis by gel electrophoresis and direct sequencing. RESULTS: 28.3% of JAK2 V617F-negative MPN patients showed CALR gene mutations. Within the MPN patients with CALR mutation, 46.9% were classified as essential thrombocythemia (ET) and 20.9% were classified as primary myelofibrosis (PMF). Previous studies classified CALR mutations into three types using negatively charged amino acid stretches at the C-terminal domain. Type 1-like mutations were observed in 12 of 49 (24.5%) ET patients and type 2-like mutations were observed in 10 of 49 (20.4%) patients. In addition, 8 of 43 (18.6%) PMF patients showed type 1-like mutations and 1 of 43 (2.3%) showed type 2-like CALR mutation. Interestingly, platelet counts were higher in patients with CALR gene mutation than in patients without CALR gene mutation. MPL mutations (W515K and W515L) were identified in 2 of 109 (1.8%) MPN patients; the MPL mutations were only found in ET patients, which was consistent with previous studies. We did not detect exon 17 c-kit mutation in JAK2-negative MPN patients but detected intronic single nucleotide polymorphisms at c.74,978 and c.75,255 in these samples. Approximately 66% of patients did not have mutations in CALR and MPL genes, in addition to lacking JAK2 gene mutation, and these cases are classified as triple-mutations. CONCLUSION: Our results showed that 66% of cases were triple-negative mutation MPN because they lacked mutations in JAK2, CALR and MPL genes. The frequencies of CALR and MPL mutation in this study are similar to other CALR and MPL patient data.

S-Allyl-L-cysteine Promotes Cell Proliferation by Stimulating Growth Hormone Receptor/Janus Kinase 2/Phospholipase C Pathways and Promoting Insulin-Like Growth Factor Type-I Secretion in Primary Cultures of Adult Rat Hepatocytes.

The mechanism of insulin-like growth factor type-I (IGF-I) secretion stimulated by S-allyl-L-cysteine (SAC) was investigated as part of a study of SAC-induced DNA synthesis and cell proliferation in primary cultures of adult rat hepatocytes. When 10-6 M SAC was added to the culture, the amount of IGF-I in the medium was significantly increased at 10 min. The peak IGF-I level (140 pg/mL) was observed 20 min after SAC stimulation. The SAC-induced IGF-I secretion was completely suppressed by a selective Janus kinase 2 (JAK2) inhibitor (TG101209), a selective phospholipase C (PLC) inhibitor (U-73122), an intracellular Ca2+ chelating agent (BAPTA-AM), and a granule secretion inhibitor (somatostatin). On the other hand, 10-6 M SAC-stimulated hepatocytes showed increased intracellular Ca2+ concentration in a time-dependent manner from 0 to 10 min. Phosphorylation of SAC-induced JAK2 and IGF-I receptor tyrosine kinase (RTK) was completely suppressed by TG101209. In addition, U-73122, BAPTA-AM, and somatostatin did not suppress SAC-induced JAK2 phosphorylation, but significantly suppressed SAC-induced IGF-I RTK phosphorylation. Furthermore, binding of the monoclonal antibody against growth hormone (GH) to GH receptor was dose-dependently suppressed by SAC on immunofluorescence. These results showed that SAC promotes cell proliferation by stimulating GH receptor/JAK2/phospholipase C pathways and promoting autocrine secretion of IGF-I in primary cultures of adult rat hepatocytes.

Association analysis of SOCS3, JAK2 and STAT3 gene polymorphisms and genetic susceptibility to type 2 diabetes mellitus in Chinese population.

AIM: The association of polymorphisms in the three genes of SOCS3, JAK2 and STAT3 with genetic susceptibility to type 2 diabetes mellitus (T2DM) was explored, and its interaction with environmental factors such as hypertension and triglycerides was analyzed. METHODS: The Hardy-Weinberg balance test was used to analyze the random balance of genes in the population. The analysis of the association of SNPs with T2DM was performed using Pearson's chi-square test. Haplotype frequency distribution, SNPs-SNPs interaction and environmental factors were analyzed by chi-square test and logistic regression. RESULTS: The genotype distribution of SNPs rs2280148 of the SOCS3 gene was statistically significant. The allele frequency distribution of SNPs (rs4969168/rs2280148) was statistically different. After covariate correction, the SOCS3 gene locus (rs4969168) showed an association with T2DM in additive model, while the rs2280148 locus showed an association with T2DM in all three models. The locus (rs10974914/rs10815157) allele and genotype frequency distribution of JAK2 were statistically significant. After covariate correction, two SNPs in the gene showed association with T2DM in both additive and recessive models. The distribution of genotype frequencies of SNPs rs1053005 locus in gene STAT3 was statistically significant between the two groups. In recessive genetic models, rs1053005 locus polymorphisms was associated with T2DM. Haplotype S3 (G G)/S 4 (G T) of the SOCS3 gene as well as haplotype J2 (A G)/J 3 (G C) of the JAK2 gene were closely associated with T2DM. There was an interaction between SNPs rs4969168 and SNPs rs2280148 in the SOCS3 gene. There was an interaction between the SOCS3, JAK2 and STAT3 genes and hypertension/triglycerides. CONCLUSION: The SOCS3 and JAK2 genes may be associated with T2DM in the Chinese population, in which SNPs carrying the A allele (rs4969168)/G allele (rs2280148)/C allele (rs10815157) have a reduced risk of T2DM. Haplotype S3 (G G)/S 4 (G T) of the SOCS3 gene and haplotype J2 (A G)/J 3 (G C) of the JAK2 gene may be influencing factor for T2DM. The interaction between SNPs rs4969168 and SNPs rs2280148 increases the risk of T2DM. Hypertension and triglycerides may interact with SNPs of T2DM susceptibility genes.

Pacritinib for myelofibrosis in adults with thrombocytopenia.

Myelofibrosis is a rare progressive cancer of the bone marrow that disrupts the normal production of healthy blood cells, leading to bone marrow failure. Patients with myelofibrosis and severe thrombocytopenia (platelet count below 50 × 109/L) have a wide range of unmet medical needs compared with those without thrombocytopenia. Usually, these patients have an increased disease burden, increased transfusion dependence, shorter overall survival, and limited treatment options. Pacritinib is a new oral kinase inhibitor specifically targeting Janus kinase 2 (JAK2), interleukin-1 receptor-associated kinase 1 (IRAK-1) and colony-stimulating factor 1 receptor (CSF-1R), and is indicated for the treatment of adults with moderate or high risk of primary or secondary myelofibrosis (post-polycythemia or post-primary thrombocytopenia) whose platelet count is less than 50 × 109/L. In this review, we introduce pacritinib and make a brief comparison of different JAK inhibitors in clinical application.

Mechanism of Xueniao Capsule in Treatment of Acute Pyelonephritis Based on Network Pharmacology / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Xueniao capsule in the treatment of acute pyelonephritis （APN） by network pharmacology and experimental verification. MethodThe effect of Xueniao capsule on APN was investigated based on the APN model in rats. The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）， Chemistryl Database， and SymMap were searched for the chemical components of Smilacis Chinae Rhizoma，Coicis Semen， and Trachycarpi Petiolus. The target information of the components was collected from PharmMapper and SwissTargetPrediction， and disease target information from Therapeutic Target Database （TTD）， DrugBank， DisGeNET， GeneCards， and Online Mendelian Inheritance in Man（OMIM）. The key genes of Xueniao capsule for APN underwent Gene Ontology（GO） and Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway enrichment analyses by Metascap. Real-time quantitative polymerase chain reaction （PCR） and Western blot were employed to verify the prediction results. ResultCompared with the blank group and the sham operation group， the model group showed an increased ratio of the left kidney to the right kidney and organ index（P<0.05， P<0.01），up-regulated white blood cells （WBC），neutrophils （NEUT），monocytes （MONO）， and lymphocytes （LY）（P<0.05， P<0.01）， and elevated levels of nuclear factor-κB（NF-κB）， interleukin-6 （IL-6）， and tumor necrosis factor-α （TNF-α）（P<0.05， P<0.01）. Compared with the model group， the norfloxacin group， the low- and high-dose Xueniao capsule groups showed a decreased ratio of the left kidney to the right kidney and organ index（P<0.05， P<0.01）， dwindled levels of WBC， NEUT， MONO， and LY（P<0.05， P<0.01）， and reduced levels of NF-κB， IL-6， and TNF-α（P<0.05， P<0.01）. The medium-dose Xueniao capsule group showed a decreased ratio of the left kidney to the right kidney and organ index（P<0.05， P<0.01）， reduced levels of WBC， NEUT， MONO， and LY（P<0.05， P<0.01）， and dwindled levels of IL-6 and TNF-α（P<0.05， P<0.01）. Network pharmacological analysis revealed 17 active compounds from Smilacis Chinae Rhizoma， 18 active compounds from Coicis Semen， six active compounds from Trachycarpi Petiolus， and 39 key genes for the treatment of APN in Xueniao capsule. GO enrichment analysis demonstrated 704 biological processes， 22 cellular components， and 59 molecular functions. Sixty-two pathways were enriched in KEGG enrichment analysis. The experimental verification results showed that compared with the blank group， the model group showed increased mRNA expression of prostaglandin-endoperoxide synthase 2 （PTGS2）， mitogen-activated protein kinase 1 （MAPK1）/extracellular signal-regulated protein kinase 2 （ERK2），phosphoinositide 3 kinase （PI3K），protein kinase B2（Akt2），Janus kinase 2 （JAK2），and signal transducer and activator of transcription 3 （STAT3）and protein expression of PI3K， Akt2， JAK2， and STAT3 （P<0.05， P<0.01）. Compared with the model group， the low-dose Xueniao capsule group showed decreased mRNA expression of MAPK1， PI3K， JAK2， and STAT3 and protein expression of PI3K， JAK2， and STAT3 （P<0.05， P<0.01）. The medium-dose Xueniao capsule group showed decreased mRNA expression of MAPK1， PTGS2， PI3K， JAK2， and STAT3， and protein expression of PI3K， JAK2， and STAT3 （P<0.05， P<0.01）. The high-dose Xueniao capsule group showed reduced mRNA expression of PTGS2， MAPK1， PI3K， Akt2， JAK2， and STAT3 and protein expression of PI3K， Akt2， JAK2， and STAT3 （P<0.05， P<0.01）. ConclusionXueniao capsule has a certain curative effect on APN via multiple targets and multiple pathways. The mechanism may be related to the inhibition of the PI3K/Akt signaling pathway and the JAK2/STAT3 signaling pathway.

ORF3a Protein of Severe Acute Respiratory Syndrome Coronavirus 2 Inhibits Interferon-Activated Janus Kinase/Signal Transducer and Activator of Transcription Signaling via Elevating Suppressor of Cytokine Signaling 1.

Coronavirus disease 2019 (COVID-19) has caused a crisis to global public health since its outbreak at the end of 2019. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen of COVID-19, appears to efficiently evade the host immune responses, including interferon (IFN) signaling. Several SARS-CoV-2 viral proteins are believed to involve in the inhibition of IFN signaling. In this study, we discovered that ORF3a, an accessory protein of SARS-CoV-2, inhibited IFN-activated Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling via upregulating suppressor of cytokine signaling 1 (SOCS1), a negative regulator of cytokine signaling. ORF3a induced SOCS1 elevation in a dose- and time-dependent manner. RNAi-mediated silencing of SOCS1 efficiently abolished ORF3a-induced blockage of JAK/STAT signaling. Interestingly, we found that ORF3a also promoted the ubiquitin-proteasomal degradation of Janus kinase 2 (JAK2), an important kinase in IFN signaling. Silencing of SOCS1 by siRNA distinctly blocked ORF3a-induced JAK2 ubiquitination and degradation. These results demonstrate that ORF3a dampens IFN signaling via upregulating SOCS1, which suppressed STAT1 phosphorylation and accelerated JAK2 ubiquitin-proteasomal degradation. Furthermore, analysis of ORF3a deletion constructs showed that the middle domain of ORF3a (amino acids 70-130) was responsible for SOCS1 upregulation. These findings contribute to our understanding of the mechanism of SARS-CoV-2 antagonizing host antiviral response.

Second-Generation Jak2 Inhibitors for Advanced Prostate Cancer: Are We Ready for Clinical Development?

Androgen deprivation therapy (ADT) for metastatic and high-risk prostate cancer (PC) inhibits growth pathways driven by the androgen receptor (AR). Over time, ADT leads to the emergence of lethal castrate-resistant PC (CRPC), which is consistently caused by an acquired ability of tumors to re-activate AR. This has led to the development of second-generation anti-androgens that more effectively antagonize AR, such as enzalutamide (ENZ). However, the resistance of CRPC to ENZ develops rapidly. Studies utilizing preclinical models of PC have established that inhibition of the Jak2-Stat5 signaling leads to extensive PC cell apoptosis and decreased tumor growth. In large clinical cohorts, Jak2-Stat5 activity predicts PC progression and recurrence. Recently, Jak2-Stat5 signaling was demonstrated to induce ENZ-resistant PC growth in preclinical PC models, further emphasizing the importance of Jak2-Stat5 for therapeutic targeting for advanced PC. The discovery of the Jak2V617F somatic mutation in myeloproliferative disorders triggered the rapid development of Jak1/2-specific inhibitors for a variety of myeloproliferative and auto-immune disorders as well as hematological malignancies. Here, we review Jak2 inhibitors targeting the mutated Jak2V617F vs. wild type (WT)-Jak2 that are currently in the development pipeline. Among these 35 compounds with documented Jak2 inhibitory activity, those with potency against WT-Jak2 hold strong potential for advanced PC therapy.

Identification of JAK2 and FOXM1 expression as novel candidate biomarkers for predicting the benefit of immunotherapy in lung squamous cell carcinoma.

BACKGROUND: Lung squamous cell carcinoma (LUSC) accounts for about 30% of all non-small cell lung cancers (NSCLC). However, only a small percentage of LUSC patients gain benefit from immune checkpoint inhibitors (ICIs). METHODS: This study analyzed LUSC patients from The Cancer Genome Atlas (TCGA), which were divided into 2 groups: PD-L1 high-expression/TMB-high (TPH) and PD-L1 low-expression/TMB-low (TPL) group based on programmed death-ligand 1 (PD-L1) expression and tumor mutational burden (TMB) status. The differences in tumor-infiltrating immune cells were estimated between the 2 groups. The overlap of differentially expressed genes and proteins (DEGs and DEPs) between 2 groups were used as candidate biomarkers. Kaplan-Meier curves were used to evaluate the association between risk score and overall survival (OS). RESULTS: More abundant immune infiltration fractions were found in TPH group. Janus kinase 2 (JAK2) and forkhead box protein M1 (FOXM1) were identified as DEGs between the TPH and TPL groups. Subsequently, we developed a risk score that combined the expression of JAK2 and FOXM1 in an effort to accurately determine the survival risk of LUSC patients. Patients with high-risk [hazard ratio (HR), median OS, 43.1 months 1.924; 95% confidence interval (CI): 1.256 to 2.945; P=0.002) had shorter survival than those with low-risk (median OS, 70.0 months). External data verification found that JAK2 and FOXM1 were significantly expressed at a higher level in the responders receiving immunotherapy (P=0.038 and P=0.009, respectively). CONCLUSIONS: The expressions of JAK2 and FOXM1 can be used as novel candidate biomarkers for predicting the benefit of immunotherapy in LUSC.

The Role of JAK/STAT Molecular Pathway in Vascular Remodeling Associated with Pulmonary Hypertension.

Pulmonary hypertension is defined as a group of diseases characterized by a progressive increase in pulmonary vascular resistance (PVR), which leads to right ventricular failure and premature death. There are multiple clinical manifestations that can be grouped into five different types. Pulmonary artery remodeling is a common feature in pulmonary hypertension (PH) characterized by endothelial dysfunction and smooth muscle pulmonary artery cell proliferation. The current treatments for PH are limited to vasodilatory agents that do not stop the progression of the disease. Therefore, there is a need for new agents that inhibit pulmonary artery remodeling targeting the main genetic, molecular, and cellular processes involved in PH. Chronic inflammation contributes to pulmonary artery remodeling and PH, among other vascular disorders, and many inflammatory mediators signal through the JAK/STAT pathway. Recent evidence indicates that the JAK/STAT pathway is overactivated in the pulmonary arteries of patients with PH of different types. In addition, different profibrotic cytokines such as IL-6, IL-13, and IL-11 and growth factors such as PDGF, VEGF, and TGFß1 are activators of the JAK/STAT pathway and inducers of pulmonary remodeling, thus participating in the development of PH. The understanding of the participation and modulation of the JAK/STAT pathway in PH could be an attractive strategy for developing future treatments. There have been no studies to date focused on the JAK/STAT pathway and PH. In this review, we focus on the analysis of the expression and distribution of different JAK/STAT isoforms in the pulmonary arteries of patients with different types of PH. Furthermore, molecular canonical and noncanonical JAK/STAT pathway transactivation will be discussed in the context of vascular remodeling and PH. The consequences of JAK/STAT activation for endothelial cells and pulmonary artery smooth muscle cells' proliferation, migration, senescence, and transformation into mesenchymal/myofibroblast cells will be described and discussed, together with different promising drugs targeting the JAK/STAT pathway in vitro and in vivo.

Discovery of imidazopyrrolopyridines derivatives as novel and selective inhibitors of JAK2.

Herein, we describe the design, synthesis, and structure-activity relationships of a series of imidazopyrrolopyridines derivatives that selectively inhibit Janus kinase 2 (JAK2). These screening cascades revealed that 6k was a preferred compound, with IC50 values of 10 nM for JAK2. Moreover, 6k was a selective JAK2 inhibitor with 19-fold, >30-fold and >30-fold selectivity over JAK1, JAK3 and TYK2 respectively. In cytokine-stimulated cell-based assays, 6k exhibited a higher JAK2 selectivity over JAK1 isoforms. Indeed, at a dose of 20 mg/kg compound 6k, pSTAT3 and pSTAT5 expression was reduced to levels comparable to those of control animals untreated with GM-CSF. Additionally, 6k showed a relatively good bioavailability (F = 38%), a suitable half-life time (T1/2 = 1.9 h), a satisfactory metabolic stability, suggesting that 6k might be a promising inhibitor of JAK2 for further development research for the treatment of MPNs.

Role of water in the determination of protonation states of titratable residues.

Water is the fundamental unit for living being, and its contribution in variety of crucial cellular functions is widely accepted. The presence of water molecules in protein's environment also accounts for structural optimization, in which highly conserved water molecules ensure structural stability of the biomolecule by providing protein-water (solute-solvent) hydrogen-bond interaction networks. Similarly, protonation states and pKa values of individual amino acid residues are also influenced by neighboring water molecules present in the protein's vicinity. In the present study, we have highlighted the role of water molecules in hydrogen-bond optimization, in determining pKa values and protonation states of titratable residues in JH2 domain of JAK2 apo protein. We found that inclusion or exclusion of water molecules while calculating pKa and assigning protonation states to amino acid residues during the molecular system build-up step resulted in slight differences in pKa values of few titratable residues and alternative protonation states of a certain residue. Accordingly, different protonation states of ionizable residues offer differing interaction patterns. Thus, we inferred that the presence of water optimizes the hydrogen-bond interactions by forming direct protein-water interactions and by linking via protein-protein bridging interactions. However, in the absence of water, the interaction pattern is somewhat disrupted. We assume that water molecules could modulate the plausibility of a particular protonation state of titratable residues on the basis of its fit with the local environment, by utilizing some particular hydrogen-bond contacts that would remain unexploited in the absence of water.

Concomitant Essential Thrombocythemia and Mature B -Lymphoproliferative Disorder in a Patient.

A-64-year old male presented with cough, weight loss, and maculopapular rash for 15-20 days. On examination, he was found to have cervical lymphadenopathy and splenomegaly. His leukocyte count was 62.1x109/L, platelets were 1169x109/L and LDH was 816 IU/L. Peripheral blood film showed a leukoerythroblastic picture with thrombocytosis. He was started on hydroxyurea and allopurinol. Subsequently, bone marrow evaluation was done which depicted increased lymphoid cells with an M:E ratio of 4:1. Cellular areas exhibited an increase in myeloid precursors along with prominent lymphoid cells and abundant megakaryocytes. Immunohistochemistry showed an increase in B-lymphocytes. Grade MF-2 reticulin fibrosis was noted. Overall findings suggested essential thrombocythemia (ET). On flow cytometry, CD45-positive lymphoid cells population was 31% and showed reactivity to Pan-B-markers with lambda light chain restriction. Janus Kinase 2 (JAK 2) mutation was detected while BCR-ABL1 translocation was negative. A diagnosis of ET progressing to myelofibrosis and mature B-lymphoproliferative disorder was made. Hydroxyurea and allopurinol were stopped while ruxolitinib was introduced and 2.5 years later he remains stable on this treatment.