JAK2 Kinase Activity Research Articles

Pterostilbene suppresses gastric cancer proliferation and metastasis by inhibiting oncogenic JAK2/STAT3 signaling: In vitro and in vivo therapeutic intervention

BackgroundGastric cancer (GC) represents a significant health burden with dire prognostic implications upon metastasis and recurrence. Pterostilbene (PTE) has been proven to have a strong ability to inhibit proliferation and metastasis in other cancers, while whether PTE exhibits anti-GC activity and its potential mechanism remain unclear. PurposeTo explore the efficacy and potential mechanism of PTE in treating GC. MethodsWe employed a comprehensive set of assays, including CCK-8, EdU staining, colony formation, flow cytometry, cell migration, and invasion assays, to detect the effect of PTE on the biological function of GC cells in vitro. The xenograft tumor model was established to evaluate the in vivo anti-GC activity of PTE. Network pharmacology was employed to predict PTE's potential targets and pathways within GC. Subsequently, Western blotting, immunofluorescence, and immunohistochemistry were utilized to analyze protein levels related to the cell cycle, EMT, and the JAK2/STAT3 pathway. ResultsOur study demonstrated strong inhibitory effects of PTE on GC cells both in vitro and in vivo. In vitro, PTE significantly induced cell cycle arrest at G0/G1 and S phases and suppressed proliferation, migration, and invasion of GC cells. In vivo, PTE led to a dose-dependent reduction in tumor volume and weight. Importantly, PTE exhibited notable safety, leaving mouse weight, liver function, and kidney function unaffected. The involvement of the JAK2/STAT3 pathway in PTE's anti-GC effect was predicted utilizing network pharmacology. PTE suppressed JAK2 kinase activity by binding to the JH1 kinase structural domain and inhibited the downstream STAT3 signaling pathway. Western blotting confirmed PTE's inhibition of the JAK2/STAT3 pathway and EMT-associated protein levels. The anti-GC effect was partially reversed upon STAT3 activation, validating the pivotal role of the JAK2/STAT3 signaling pathway in PTE's activity. ConclusionOur investigation validates the potent inhibitory effects of PTE on the proliferation and metastasis of GC cells. Importantly, we present novel evidence implicating the JAK2/STAT3 pathway as the key mechanism through which PTE exerts its anti-GC activity. These findings not only establish the basis for considering PTE as a promising lead compound for GC therapeutics but also contribute significantly to our comprehension of the intricate molecular mechanisms underlying its exceptional anti-cancer properties.

DUSP1 Specific Protac Selectively Targets the JAK2 V617F Expressing Cells

Constitutive JAK-STAT signaling resulting from mutations in JAK2 V617F and certain growth-factor receptors has provided a strong rationale for targeting JAK2 kinase activity in myeloproliferative neoplasms (MPNs). Although several JAK2 inhibitors have been approved for MPN treatment, their clinical efficacy is limited to alleviating the constitutional symptoms associated with inflammatory cytokine signaling. These inhibitors are not cytotoxic and only exert a cytostatic response, failing to eradicate the malignant clone, induce molecular remission, or prevent progression to acute myeloid leukemia (AML). The inability of JAK2 TKIs to fully suppress the inflammatory cytokine overproduction, a key feature of MPN development and transition to AML, contributes to clonal persistence and TKI refractoriness. Recently, we reported that the cytokine signaling, in the context of JAK2 V617F signaling induces the expression of DUSP1, which enhances P53 degradation that leads to TKI refractoriness. Enhanced DUSP1 expression blocks P53 phosphorylation at Serine 15 (S15 phosphorylation prevents its interaction with MDM2) by inhibiting the P38 kinase activity. Consequently, reduced phosphorylation under JAK2 V617F signaling enhances P53 turnover. Deletion of DUSP1 enhances P38 kinase activity, leading to greater P53 stabilization and expression, which selectively induce cell death in JAK2 V617F expressing MPN cells while sparing normal cells. However, chemical inhibition of DUSP1 using a small molecule inhibitor (BCI) was ineffective in achieving synthetic lethality to JAK2 V617F cells, mainly due to pERK1/2 rebound caused by the off-target inhibition of DUSP6. Reintroduction of DUSP6 and BCI treatment restored JAK2 V617F clonal selectivity. This study provided the first mechanistic basis of TKI refractoriness in JAK2 targeted MPNs and suggested that selectively targeting DUSP1 could offer a stable or curative response in JAK2 V617F -driven MPNs. To address this, we developed a DUSP1-protac, a novel and highly potent DUSP1 selective PROTAC. Treatment of BaF3 cells expressing JAK2 V617F with DUSP1-protac resulted in dose- and time-dependent degradation of DUSP1, accompanied by increased P38 activity and P53 stabilization. The maximal level of DUSP1 degradation (Dmax) achieved was 95% with a DC50 of 70 nM/L within 24 hours. Pre-treatment with the proteasome inhibitor MG132, but not the lysosomal inhibitor chloroquine, reversed DUSP1 degradation, confirming that degradation occurs via the 26S-proteasome. As expected, DUSP1-protac induced cytotoxicity specifically in JAK2 V617F expressing cells, with a cellular IC50 of 50 nM. No toxicity was observed in BaF3 cells or those expressing MPL or CALR variants at concentrations up to 1000 nM, confirming its selectivity. In conclusion, our data suggest that DUSP1-protac is an exceptionally potent first-in-class DUSP1 selective PROTAC. It exerts a cytotoxic response specifically in JAK2 V617F expressing cells by enhancing P53 expression. Cellular uptake experiments and efficacy studies in primary cells are currently underway and will be presented in subsequent reports. The development of DUSP1-protac represents a promising approach for targeting JAK2 V617F -driven MPNs, offering potential benefits in achieving a stable or curative therapeutic response.

Preclinical Evaluation of JAK2 Specific Investigational Oligonucleotide for the Treatment of MPNs

Background: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell malignancies with cytokine independency or hypersensitivity. Polycythemia vera (PV), an acquired MPN characterized by increased blood cell mass and hematocrit and leukocyte count, is associated with incidental myelofibrosis (MF). Myelofibrosis (MF) is a chronic and progressive disorder characterized by bone marrow fibrosis, extramedullary hematopoiesis, leukoerythroblastosis and splenomegaly. Essential Thrombocythemia (ET) is another type of MPN associated with increased number and size of circulating platelets. All MPNs leave patients at risk for progression to leukemia. Abnormal cytokine signaling due to an aberrant JAK2-STAT pathway has a vital role in pathogenesis of various MPNs. JAK2 mutations can result in hematologic malignancies, where hyperactive signaling of the JAK2-STAT pathway promotes tumor cell proliferation, invasion, and angiogenesis. Increased JAK2 kinase activity is observed in hematologic malignancies; somatic JAK2 V617F gain-of-function mutations are found in large numbers between different subtypes of MPNs patients (40-95%) and are also implicated in MDS-MPN cases. We hypothesize that preventing JAK2 transcriptionby antisense oligonucleotide (ASO) mediated exon masking of the JAK2 intron-exon junction will result in reduced JAK2 mRNA, and thus JAK2 protein, by providing nonsense mediated decay (NMD) in the reading frame. This may alleviate disease manifestations of many patients suffering from different forms of MPNs. Methods: This study utilized a HEL92.1.7 cell line harboring the V617F JAK2 gain-of-function mutation. Antisense Oligonucleotides (ASOs) are small fragments of DNA/RNA that have the ability to reduce production of target protein via interaction with RNA. We designed a series ASOs targeting JAK2 sequence and tested these at a range of concentrations. The ASOs were designed and prioritized based on in silico binding affinity and limited off-target activity. HEL92.1.7 cells underwent treatment with several ASO-prototypes and functional readout at the end of 72h of incubation. Results: We observed significant JAK2 protein decrease (~50%) in ASO-treated samples compared to untreated samples. JAK2 qPCR results confirmed reduction of target transcript in 40-60% range. STAT5 phosphorylation status further confirmed this effect, and we report a ~35% pSTAT5 reduction. Furthermore, this ASO showed limited off-target effects in silico. siRNA and CRISPR knockout protein lysates were used as internal controls. Our preclinical data support our ASO as a highly specific JAK2 agent, affecting direct levels of JAK2 as well as downstream STAT signaling. Conclusions: Around 50-60% of primary MF patients harbor the JAK2 V617F gain-of-function mutation. ASOs offer the capability to directly target JAK2-driven neoplasms with highest precision and effectively reduce JAK2 protein production, without off-target effects linked with small molecules targeting various kinase families. The ability to reduce JAK2 protein may alleviate the disease burden that patients with hematologic malignancies face, resulting in a higher quality of life through prevention and treatment.

Exploring the role of LIAS-related cuproptosis in systemic lupus erythematosus.

Cuproptosis is a novel mode of cell death, which is strongly related to energy metabolism in mitochondria and regulated by protein lipoylation. Currently, the molecular mechanisms of cuproptosis-related genes (CRGs) involved in systemic lupus erythematosus (SLE) largely remained unclear, our study is aimed to explore the mechanisms of cuproptosis and CRGs involved in SLE. Bulk RNA-seq datasets were collected to display the expressions of CRGs in peripheral blood mononuclear cells (PBMCs) of SLE and healthy individuals, and then ROC analysis was used to establish the diagnostic models of CRGs. Next, the immune infiltration analyses were applied to reveal the difference of immune cells infiltration in LIAS-low and LIAS-high group. Additionally, WGCNA analysis was performed to find the gene modules significantly correlated with the LIAS expression level. We also performed the functional enrichment analyses for LIAS-related gene modules to determine the potential pathways involved in the development of SLE. Finally, scRNA-seq dataset was used to cluster immune cell subsets, reveal the activated pathways, and study cell-cell interactions in LIAS-low and LIAS-high cells. We found CDKN2A was significantly increased and LIAS was significantly decreased in SLE patients compared with healthy individuals. The AUC score showed that LIAS had a great diagnostic value than other CRGs. Additionally, the results of immune infiltration analyses showed that immune cells proportion were diverse in LIAS-low and LIAS-high samples. The gene sets related to LIAS expression level were involved in dephosphorylation of JAK1 by SHP1, phosphorylation of STAT2, cytokine signaling in immune system, expression of interferon-alpha and beta, inhibition of JAK kinase activity by SOCS1/3, and so on. Finally, the results of cell-cell communication showed that CCL- (CCL5 + CCR1) and ANNEXIN- (ANXA1 + FPR1) might play an essential role in the communication network between LIAS-low and LIAS-high cells. Above findings inferred that LIAS-mediated cuproptosis might involve in a comprehensive cellular and molecular mechanism to cause the occurrence and development of SLE.

A novel germline hyperactivating JAK2 mutation L604F

Somatic JAK2 mutations are the main molecular cause of the vast majority of polycythemia vera (PV) cases. According to a recent structural model, the prevalent acquired V617F mutation improves the stability of the JAK2 dimer, thereby enhancing the constitutive JAK2 kinase activity. Germline JAK2 mutations usually do not largely alter JAK2 signaling, although they may modulate the impact of V617F. We found an unusual germline JAK2 mutation L604F in homozygous form in a young PV patient, along with a low allele burden JAK2 V617F mutation, and in her apparently healthy sister. Their father with a PV-like disease had L604F in a heterozygous state, without V617F. The functional consequences of JAK2 L604Fmutation were compared with those induced by V617F in two different in vitro model systems: (i) HEK293T cells were transfected with plasmids for exogenous JAK2-GFP expression, and (ii) endogenous JAK2 modifications were introduced into HeLa cells using CRISPR/Cas9. Both mutations significantly increased JAK2 constitutive activity in transfected HEK293T cells. In the second model, JAK2 modification resulted in reduced total JAK2 protein levels. An important difference was also detected: as described previously, the effect of V617F on JAK2 kinase activity was abrogated in the absence of the aromatic residue F595. In contrast, JAK2 hyperactivation by L604F was only partially inhibited by the F595 change to alanine. We propose that the L604F mutation increases the probability of spontaneous JAK2 dimer formation, which is physiologically mediated by F595. In addition, L604F may contribute to dimer stabilization similarly to V617F.

Differences in JAK Isoform Selectivity Among Different Types of JAK Inhibitors Evaluated for Rheumatic Diseases Through In Vitro Profiling.

The selectivity of JAK inhibitors (Jakinibs) forms the basis for understanding their clinical characteristics; however, evaluation of selectivity is hampered by the lack of comprehensive head-to-head studies. Our objective was to profile in parallel Jakinibs indicated or evaluated for rheumatic diseases for their JAK and cytokine selectivity in vitro. We analyzed 10 Jakinibs for JAK isoform selectivity by assaying their inhibition of JAK kinase activity, binding to kinase and pseudokinase domains, and inhibition of cytokine signaling using blood samples from healthy volunteers and using isolated peripheral blood mononuclear cells (PBMCs) from patients with rheumatoid arthritis and from healthy donors. Pan-Jakinibs effectively suppressed kinase activity of 2 to 3 JAK family members, whereas isoform-targeted Jakinibs possessed varying degrees of selectivity for 1 or 2 JAK family members. In human leukocytes, Jakinibs predominantly inhibited the JAK1-dependent cytokines interleukin-2 (IL-2), IL-6, and interferons (IFNs). In PBMCs from patients with rheumatoid arthritis compared with healthy controls, inhibition of these cytokines was more pronounced, and some cell-type and STAT isoform differences were observed. Novel Jakinibs demonstrated high selectivity: the covalent Jakinib ritlecitinib showed 900- to 2,500-fold selectivity for JAK3 over other JAKs and specific suppression of IL-2-signaling, whereas the allosteric TYK2 inhibitor deucravacitinib inhibited IFNα signaling with high specificity. Interestingly, deucravacitinib targeted the regulatory pseudokinase domain and did not affect JAK in vitro kinase activity. Inhibition of JAK kinase activity did not directly translate into cellular inhibition of JAK/STAT signaling. Despite differences in JAK selectivity, the cytokine inhibition profiles of currently approved Jakinibs were highly similar, with preference for JAK1-mediated cytokines. Novel types of Jakinibs showed narrow cytokine inhibition profile specific for JAK3- or TYK2-mediated signaling.

Preclinical evaluation of JAK2 specific investigational oligonucleotide for the treatment of MDS/PV.

122 Background: Myeloproliferative disorders (MPD) are clonal hematopoietic stem cell malignancies with cytokine independency or hypersensitivity. Polycythemia vera (PV), an acquired MPD characterized by increased blood cell mass and hematocrit and leukocyte count, is associated with incidental myelofibrosis (MF). MDS is characterized by cytopenia and the presence of morphological dysplasia of precursor and mature bone marrow blood cells. PV and MDS leave patients at risk for progression to acute myeloid leukemia. Abnormal cytokine signaling due to an aberrant JAK2-STAT pathway has a vital role in PV and MDS pathogenesis. JAK2 mutations can result in hematologic malignancies, where hyperactive signaling of the JAK2-STAT pathway promotes tumor cell proliferation, invasion, and angiogenesis. Increased JAK2 kinase activity is observed in hematologic malignancies; somatic JAK2 V617F gain-of-function mutations are found in at least 95% of PV patients and is implicated in MDS cases. We hypothesize that preventing JAK2 transcription by antisense oligonucleotide (ASO) mediated exon masking of the JAK2 intron-exon junction will result in reduced JAK2 mRNA, and thus JAK2 protein, by providing nonsense mediated decay (NMD) in the reading frame. This may alleviate disease manifestations of hematologic malignancies like PV and MDS. Methods: This study utilized a HEL cell line harboring the V617F JAK2 gain-of-function mutation. We designed a series 19mer ASO targeting JAK2 exon-intron junctions ( JAK2 intron-exon junction providing NMD in the reading frame, steric, non-RNAse H1) and tested these at a range of concentrations. The ASOs were designed with a phosphorothioate 2’-O-methoxyethyl backbone and prioritized based on in silico binding affinity and limited off-target binding. HEL cells underwent ASO treatment (1µM) and 72-hour incubation. Results: We observed significant JAK2 protein decrease (~50%) in ASO-treated samples compared to untreated samples. JAK2 qPCR results confirmed 40-60% of target transcript. STAT5 phosphorylation status further confirmed this effect, and we report a 35% pSTAT5 reduction. Furthermore, this ASO showed limited off-target effects in silico. siRNA and CRISPR knockout lysate were used as controls. Our preclinical data support this ASO as a highly specific JAK2 agent, affecting direct levels of JAK2 as well as downstream STAT signaling. Conclusions: Around 50-60% of primary MF patients harbor the JAK2 V617F gain-of-function mutation. ASOs offer the capability to directly target JAK2 mutations with high precision and effectively reduce JAK2 protein production, without off-target kinase effects. The ability to reduce JAK2 protein may alleviate the disease burden that patients with hematologic malignancies face, resulting in a higher quality of life through prevention and treatment.

Rational Polypharmacological Targeting of FLT3, JAK2, ABL and ERK1 By Pluripotin Suppresses the Adaptive Resistance to FLT3 Inhibitors in AML

Background: Despite significant advancement in developing FLT3 inhibitors, treatment failure is common due to emergence of resistance. Most patients developed resistance within short duration even on continued TKI therapy. Both type-I (gilteritinib and crenolanib) and type-II (quizartinib and sorafenib) FLT3 inhibitors have been evaluated in patients. Regardless of their selectivity and mode of inhibition, resistance emerged. Nonetheless, mechanisms driving resistance partly differ between type-I and type-II inhibitors. For instance, resistance to type-II inhibitors is predominately mediated by the on-target selection of resistant mutations in the FLT3 kinase domain, while resistance to type-I inhibitors is principally driven by off-target activation of RAS-MAPK, BCR-ABL, JAK2/JAK1 signaling. A minority of resistant patients showed the emergence of mutations in genes regulating metabolism and transcription. However, its relevance in conferring resistance has not been evaluated. Besides, like other TKI treated malignancies, gatekeeper mutation (F691L) conferred resistance to both type-I and type-II FLT3 inhibitors and poses a significant clinical challenge. It is not clear why on-target acquired resistance is more frequent with type-II inhibitors in comparison to type-I inhibitors where adaptive resistance to compensatory signaling is more common. It is likely possible that stabilization of active kinase conformation by type-I inhibitor is, nonetheless, enzymatically active but its non-enzymatic function (signaling scaffold) drives the adaptation to alternate survival pathways. Nevertheless, these studies informed that FLT3 resistant variants could be suppressed by switching to next-generation FLT3 inhibitors or dose escalation as demonstrated in managing the TKI resistance in chronic myeloid leukemia (CML). However, unlike CML, resistance conferred by off-target activation of MAPKs, BCR-ABL, and JAKs in AML remains a serious challenge. Results: The persistence of residual disease is the root cause of resistance. Strategies aimed at greater front-line disease eradication and suppression of resistance are needed, most of which depends on further research into combination chemotherapy or to develop polypharmacological agents targeting FLT3, its resistant variants, BCR-ABL, JAK2, and MAPKs. To address this, we performed a cell-based screening to identify the small molecule inhibitor active against FLT3, BCR-ABL, JAK2, and MAPKs. Here we show that pluripotin (SC-1), an inhibitor of RASGAP, and MAPK3, potently inhibits the kinase activity of FLT3, BCR-ABL, and JAK2. Structural modeling studies revealed that it binds with inactive conformations of FLT3, JAK2, and ABL. It is an equipotent inhibitor to both FLT3ITD and its most vexing resistant variant, the gatekeeper mutant F691L. Consequently, it potently suppressed the leukemic progression of TKI resistant primary AML in a preclinical mouse model, FLT3ITD/ITD/Tet2-/- mice develop robust leukemia and are resistant to FLT3 TKIs, e.g., Quizartinib and Gilteritinib, Fig 1. As expected, pluripotin treatment efficiently suppressed the adaptive resistance conferred by MAPK, BCR-ABL, and JAK2 signaling. As a proof of concept, we provide evidence that unique polypharmacology of pluripotin targeting FLT3, BCR-ABL, JAK2, and MAPK efficiently suppressed the leukemic progression in multiple preclinical mouse models as well as in mice engrafted with primary AML cells. Conclusions: Our preclinical data suggest that up-front targeting of key signaling nodes driving adaptive resistance by polypharmacological agents provides durable response, which is not achieved by currently used FLT3 inhibitors. Altogether, these studies warrant clinical evaluation of pluripotin to suppress the adaptive resistance for durable response. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Autosomal Dominant JAK2R715T Mutation Causes Polycythemia Vera in 3 Generations

Polycythemia vera (PV) has high hemoglobin (Hb) concentration caused by clonal proliferation of myeloid cells from a pluripotent stem cell (Prchal, N Engl J Med. 1974). Acquired somatic mutations of JAK2 gene, JAK2V617F and rarely exon 12, are present in virtually all PV patients (Ugo, Nature 2005, Scott N Engl J Med. 2007). The thromboses are the major causes of PV-related morbidity and mortality. A 38-year female suffered an ischemic stroke in 2017 at age 32 with negative thrombophilia work-up. She had elevated hemoglobin (Hb) 18 g/dL with intermittent elevation of leukocytes and normal platelets with borderline low erythropoietin (EPO) levels. JAK2V617F and exon 12 mutation analysis were negative. Her marrow was interpreted as compatible with PV and normal cytogenetics. Her hematologist treated her with phlebotomies. She was referred to us for an additional evaluation and management. Myeloid next generation sequencing (NGS) revealed a previously undescribed mutation JAK2R715T in the pseudokinase domain (JH2) with allelic burden ~50%. Some of her erythroid progenitors (BFU-Es) grew without extrinsic EPO, a hallmark of PV (Prchal, N Engl J Med. 1974). Phlebotomies were stopped; in two months, her Hb had risen to 17.4 g/dL and hematocrit had changed to 48.5% with normal leukocytes and platelets. We obtained JAK2 inhibitor fedratinib on compassionate basis from the manufacturer. On fedratinib, her Hb normalized to 15.2 g/dL with 42.8 % hematocrit. Her iron deficiency was corrected without increase of her Hb. The patient's germline mutation was confirmed by DNA sequencing of her nails. We encouraged her to have her only son and parents tested by NGS. The same JAK2R715T mutation was found in her 10-year-old son's blood sample. He also has elevated Hb at 16.2 g/dL (normal for age 11.5-15.5 g/dL), normal leukocytes and platelets were 400 K/µl. Her asymptomatic 63-year-old mother’ Hb was 17 g/dL, hematocrit 50.5%, and sequencing of her JAK2 also revealed JAK2R715T at ~50%. To assess how the R715T mutation may affect kinase activation, the JH1-JH2 domains of JAK2R715T and JAK2WT were modelled using AlphaFold 2.0 (Jumper, Nature 2021). The overall structure of JAK2R715T JH2 was more rigid than that of JAK2WT, in agreement with DynaMut in silico prediction (PMID: 29718330). -Helix C is shown in dark blue, Y570 and T555 in yellow, and ATP and T715 in red. Detailed comparison of JAK2R715T with JAK2WT JH2 (see Figure) indicated that the rigidification of the structure involved notably residues of the JH2-JH2 trans-dimerization domain centered around -Helix C. This has been suggested to mediate direct JH2-JH2 contacts (Wilmes, Science 2020) and a lengthening of the ß-sheet region surrounding the inhibitory phosphorylation site Tyr570 (Echarri, Mol Cell Biol. 2004). The R715 is in direct vicinity of the ATP binding pocket and promotes a closed lobe conformation through interaction with Thr555 of the nucleotide-binding loop (Bandaranayake, Nat Struct Mol Biol. 2012). We suggest that loss of this interaction may result in a more opened lobe conformation and disturbing of ATP binding to JH2, which regulates JAK2 kinase activity (Hammarén, Proc Natl Acad Sci USA 2015). To evaluate the effect of JAK2R715T in erythroid progenitor cells, the patient's erythroid progenitors derived from peripheral mononuclear cells were grown for 21 days with and without JAK inhibitor (ruxolitinib) following the previously published in vitro expansion protocol (Bruchova, Blood Cells Mol Dis, 2009). Compared to PVs and healthy controls, the patient cells showed increased proliferation. JAK inhibitor ruxolitinib treatment of patient cells induced decreased proliferation and differentiation compared to untreated cells. Assessment of STAT5 phosphorylation on patient's blood cells confirmed increased JAK2 activity to levels even higher than this of patients with confirmed JAK2V617F positive PV. X-chromosome based clonality studies of both females are ongoing. The EPO-independent colonies of her BFU-E is being evaluated for possible acquired conversion to homozygosity by uniparental disomy that is present in PV (Kralovics, Experimental Hematology, 2002). We conclude that this is the first example of dominantly inherited PV phenotype in 3 generations due to a novel germline JAK2R715T gain-of function mutation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

JAK1 inactivation promotes proliferation and migration of endometrial cancer cells via upregulating the hypoxia-inducible factor signaling pathway

BackgroundLoss-of-function (LOF) mutations of JAK1, a member of the JAK kinase family, were frequently observed in EC, indicating that JAK1 may act as a tumor suppressor, at least in EC. However, the mechanism of JAK1 mediated regulation of tumorigenesis remains poorly understood.MethodsThe genetic alterations of JAK1 in EC using latest sequencing dataset of EC deposited in TCGA database. The RNA-Seq dataset of EC and normal endometrial tissues from TCGA cohort was analyzed. The expression of JAK1 in EC and normal endometrial tissues were investigated using immunohistochemistry. The expression levels of genes in endometrial cancer cells were detected by quantitative reverse transcription-PCR (RT-qPCR) and western blotting. JAK1 protein was efficiently depleted by the two shRNAs. HIF1/2-α protein was efficiently depleted by siRNAs. JAK1 overexpressed EC cells were generated by an expressing plasmid. The proliferation and migration ability of cancer cells were evaluated by CCK8, colony formation assays and transwell assays. The global transcriptomic changes in JAK1-depleted KLE cells were investigated using RNA-Seq. Gene Ontology (GO) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to identify the most significant pathways that were altered in JAK1-depleted KLE cells. The physical association between HIF-1/2α and JAK1 using co-immunoprecipitation (co-IP) assays.ResultsIn the present study, we found that JAK1 was frequently mutated and downregulated in EC. JAK1 knockdown promotes EC cell proliferation and migration. JAK1 overexpression reduces EC cell proliferation and migration. We examined the transcriptional profiling changes in JAK1-depleted EC cells and unexpectedly found that the hypoxia inducible factor (HIF) pathway was activated. Mechanistically, JAK1 interacts with HIF-1/2α, and reduces HIF1/2-α protein expression under hypoxia. HIF-1/2α knockdown reverses the JAK1 knockdown–induced growth and migration of EC cells under hypoxia. JAK1 knockdown or pharmacological inhibition of JAK1 kinase activity by Ruxolitinib upregulates transcription of HIF target genes under hypoxia. JAK1 overexpression downregulates transcription of HIF target genes under hypoxia.ConclusionsThese findings provide novel insights into the functional link between JAK1 LOF mutations and abnormal HIF pathway activation in EC and suggest that pharmacological inhibition of HIF1/2 represents a promising therapeutic strategy targeting JAK1-mutated ECs.3W7_hN6trihWN8_decuxdBVideo

Itaconate and itaconate derivatives target JAK1 to suppress alternative activation of macrophages

The Krebs cycle-derived metabolite itaconate and its derivatives suppress the inflammatory response in pro-inflammatory "M1" macrophages. However, alternatively activated "M2" macrophages can take up itaconate. We therefore examined the effect of itaconate and 4-octyl itaconate (OI) on M2 macrophage activation. We demonstrate that itaconate and OI inhibit M2 polarization and metabolic remodeling. Examination of IL-4 signaling revealed inhibition of JAK1 and STAT6 phosphorylation by both itaconate and OI. JAK1 activation was also inhibited by OI in response to IL-13, interferon-β, and interferon-γ in macrophages and in T helper 2 (Th2) cells. Importantly, JAK1 was directly modified by itaconate derivatives at multiple residues, including cysteines 715, 816, 943, and 1130. Itaconate and OI also inhibited JAK1 kinase activity. Finally, OI treatment suppressed M2 macrophage polarization and JAK1 phosphorylation invivo. We therefore identify itaconate and OI as JAK1 inhibitors, suggesting a new strategy to inhibit JAK1 in M2 macrophage-driven diseases.

The SH2 domain and kinase activity of JAK2 target JAK2 to centrosome and regulate cell growth and centrosome amplification.

JAK2 is cytokine-activated non-receptor tyrosine kinase. Although JAK2 is mainly localized at the plasma membrane, it is also present on the centrosome. In this study, we demonstrated that JAK2 localization to the centrosome depends on the SH2 domain and intact kinase activity. We created JAK2 mutants deficient in centrosomal localization ΔSH2, K882E and (ΔSH2, K882E). We showed that JAK2 WT clone strongly enhances cell proliferation as compared to control cells while JAK2 clones ΔSH2, K882E and (ΔSH2, K882E) proliferate slower than JAK2 WT cells. These mutant clones also progress much slower through the cell cycle as compared to JAK2 WT clone and the enhanced proliferation of JAK2 WT cells is accompanied by increased S −> G2 progression. Both the SH2 domain and the kinase activity of JAK2 play a role in prolactin-dependent activation of JAK2 substrate STAT5. We showed that JAK2 is an important regulator of centrosome function as the SH2 domain of JAK2 regulates centrosome amplification. The cells overexpressing ΔSH2 and (ΔSH2, K-E) JAK2 have almost three-fold the amplified centrosomes of WT cells. In contrast, the kinase activity of JAK2 is dispensable for centrosome amplification. Our observations provide novel insight into the role of SH2 domain and kinase activity of JAK2 in centrosome localization of JAK2 and in the regulation of cell growth and centrosome biogenesis.

Design, synthesis and SAR study of 2-aminopyridine derivatives as potent and selective JAK2 inhibitors

The abnormal activation of JAK2 kinase is closely related to the occurrence and progression of myeloproliferative neoplasms (MPNs). At present, there is still an obvious unmet medical need for selective JAK2 inhibitors in clinic. In this paper, a class of 2-aminopyridine derivatives as potent and selective JAK2 inhibitors was obtained by combining drug design, synthesis and structure-activity relationship studies based on the previously identified lead Crizotinib. Among them, 21b exhibited high inhibitory activity against JAK2 with an IC50 of 9 nmol/L, moreover, it showed 276- and 184-fold selectivity over JAK1 and JAK3, respectively. Besides, 21b had a significant antiproliferative activity against HEL cells, and also inhibited the phosphorylation of JAK2 and its down-stream signaling pathway. These results indicated that 2-aminopyridine compound 21b had the potential to be developed as a selective JAK2 inhibitor for further study.

Eriocitrin, a dietary flavonoid suppressed cell proliferation, induced apoptosis through modulation of JAK2/STAT3 and JNK/p38 MAPKs signaling pathway in MCF-7 cells.

Eriocitrin, a lemons flavanone, exhibits several biological properties, antiproliferative, and proapoptotic effects. However,its molecular mechanical action is not entirely clarified. Oxidative stress causes abnormal stimulation of signal transducer and activator of transcription 3 (STAT3) and c-Jun NH2-terminal kinase (JNK), p38 mitogen-activated protein kinases (MAPKs) signaling has been strongly connected with the ruling of cell survival and apoptosis of cancer cells. Herein, we investigated an antiproliferative and proapoptotic effect that Eriocitrin modulates STAT3/MAPKs signaling activation in MCF-7 cells. We noticed that Eriocitrin strongly enhances reactive oxygen species (ROS) generation, alteration of mitochondrial outer membrane potential, and enhances apoptotic morphological changes. Furthermore, Eriocitrin suppressed STAT3 phosphorylation via inhibiting an upstream molecule of JAK2 and Src kinase activation, thereby blocking STAT3 nuclear translocation. Similarly, Eriocitrin causes oxidative stress-mediated JNK/p38 MAPK signaling activation. We confirmed that Eriocitrin induced ROS-mediated apoptosis inhibited by the antioxidant substance of N-acetylcysteine. Eriocitrin induced apoptosis via suppression of STAT3 signaling regulated proteins, activating proapoptotic factors Bax, caspase 7, 8, 9 and suppressing Bcl-2, Bcl-x expression in MCF-7 cells. Overall, these results evidenced that Eriocitrin can affect multiple signaling events associated with tumorigenesis. From this evidence, Eriocitrin, a novel chemotherapeutic agent, can be used to treat breast cancer.

Molecular Modeling, Interacting Residues and other Structural Analyses for Human SOCS3, Gp130 and JAK Proteins: A Detailed Computational Approach for Proteins Involved in Feedback Inhibition

Background: When STAT3 is activated only by the IL6 family of proteins, then gp130 (having a phosphopeptide motif) interacts with human SOCS3, which further binds to JAK and inhibits its protein kinase activity. Interaction of gp130 with SOCS3 targets only the IL-6 signaling cascade. The interaction occurs when SOCS3 binds to a particular motif on gp130 (centered upon pTyr759) after its phosphorylation. Previously, wet laboratory studies were done, but computational exploration for the participating residues remained unexplored. Methodology: The 3D structure of human SOCS3 protein was modeled, and its stereochemical parameters were satisfied. Crystallographic structures of gp130-phosphopeptide and JAK were studied. After protein docking, the complex underwent minimization and molecular dynamics simulation. Different stability parameters and binding patterns with residues were evaluated. Results and Discussion : The best-modeled structure of SOCS3 protein was selected and found that it had three helices and seven sheets interspersed with coils. Arg133, Tyr137 and Tyr98 from SOCS3 formed manifold binding patterns with gp130 (mainly with pTyr759 and Glu758). Lys62, Lys63 and Arg65 from SOCS3 were also found to interact with Val762 of gp130. Interactions with JAK were also studied. Residue 53, 62-65, 98, 133, 136 and 137 formed the predominant binding pockets in SOCS3. They can serve as important target sites as well. Altogether, it created electrostatically charged pockets to accommodate the partner proteins for each other. Gp130 phosphopeptide was observed to be tightly accommodated in the electrostatically positive zones on the SOCS3 surface. Net area for solvent accessibility was also found to get drastically reduced, implying high participation of residues. Earlier studies documented that the interaction of these three proteins occurs with affinity and have a satisfactory association with each other. Conclusion: Here in this study, the free energy of binding for the triple protein interaction through the ΔG values helped to infer that SOCS3 interacted spontaneously (in a thermodynamic sense). Many helical conformations formed coiled-coils providing high flexibility to interact spontaneously. Most of the interactions were through the responsible SH2 domain (46-127 residue length) of SOCS3. Residues 53, 62-64 and 98 formed coils while the residue number 137adopted sheet conformation from coils. Future Scope: This study shall instigate blocking the gp130-binding sites of SOCS3 through targeting of drugs, thereby preventing SOCS3-gp130 interaction. This would allow the JAK-STAT signaling cascade, which is paramount for several biological functions.

A Novel JAK2 Mutation Confers Cytokine-Independence in the Absence of a Type I Dimeric Cytokine Receptor

JAK2 fusions reported in hematologic malignancies are often associated with eosinophilia. In addition to JAK2 fusions such as PCM1-JAK2, BCR-JAK2 and ETV6-JAK2, the JAK2V617Fpoint mutant has been recurrently observed in patients with hypereosinophilic syndrome (HES), occurring in ~4% of patients with hypereosinophilia of unknown significance (Schwaab, Am J Hematol, 2015).Here, we describe a novel JAK2 mutation in a 69 year-old woman with a history of immune thrombocytopenia referred for steroid-refractory HES. Peripheral blood eosinophilia was present for at least five years, with absolute eosinophil counts ranging up to 17,500/μL. Bone marrow biopsy revealed hypercellular marrow with trilineage hematopoiesis, increased atypical megakaryocytes, and markedly increased eosinophils with abnormal morphology, consistent with myeloproliferative neoplasm. Results from an eosinophilia FISH panel for FIP1L1/CHIC2/PDGFRα, FGFR, PDGFRβ, and CBFβ fusions were negative, as were T-cell clonality studies by next-generation sequencing (NGS). An NGS-based myeloid malignancies panel revealed a novel mutation in JAK2 consisting of a ten base deletion and insertion of a single thymine (JAK2 c.1747_1756delinsT, p.Leu583_Ala586delinsSer;JAK2indel). Treatment with ruxolitinib improved the peripheral blood eosinophilia, but dose intensity was limited by thrombocytopenia (Table 1).For transformation of cells to cytokine-independence, JAK2V617F requires co-expression of a type I dimeric cytokine receptor, such as Epo-R, to enable downstream signaling in the absence of growth factors (Lu et al, Proc Natl Acad Sci, 2005). To characterize the signaling properties of the new JAK2indel mutant, we cloned JAK2WT, JAK2V617F, and JAK2indel into the MSCV-IRES-GFP (MIG) retroviral vector. IL-3-dependent Ba/F3 cells lacking type I cytokine receptors were infected with retrovirus to assess cytokine independence.Parental Ba/F3 cells and those transduced with JAK2WT, JAK2V617F, or JAK2indel proliferated at similar rates when grown in WEHI conditioned medium (CM). However, upon IL-3 (WEHI) withdrawal, only GFP-positive JAK2indel Ba/F3 cells demonstrated growth factor independence (n=3). Furthermore, parental and GFP-sorted JAK2WT- and JAK2V617F-expressingBa/F3 cells display acceleration of growth when cultured in graded dilutions of WEHI CM, while growth of JAK2indel Ba/F3 cells is not further enhanced by WEHI CM. JAK2indel-expressing Ba/F3 cells also demonstrated cytokine-independent growth in colony assays, whereas parental, JAK2WT-, and JAK2V617F-expressing cells displayed little to no colony formation in the absence of cytokines. Immunoblot analyses revealed that after 4 hours of culture ± IL-3, all cells grown in IL-3 demonstrated activation of STAT5 and ERK, but only JAK2indel Ba/F3 cells demonstrated cytokine-independent signaling activation.JAK2indel activates STAT5 and ERK without co-expression of a type I cytokine receptor, implicating a mechanism of JAK2 kinase activation that is distinct from JAK2V617F. Unexpectedly, preliminary structural analysis suggests that the conformational change induced by JAK2indel resembles that of JAK2V617F. Mutation of glutamic acid 596 to arginine (E596R) has been shown to abolish the constitutive activity of JAK2V617F via modulation of the C-terminal residues of the SH2-JH2 linker, thereby restoring pseudokinase domain autoinhibitory function (Leroy et al, Biochem J, 2016). To test whether this regulatory mechanism is preserved in JAK2indel, we introduced E596R into JAK2indel and are currently characterizing JAK2indel/E596R in cellular and biochemical assays. Additional modeling and biochemical work is underway to clarify whether JAK2indel confers a new functionality that supports signaling activation in the absence of a type I cytokine receptor.In summary we report a novel JAK2 mutation in a patient with myeloproliferative-variant HES that results in cytokine-independent growth, is associated with STAT5 and ERK phosphorylation and may be employing an alternative mechanism to achieve constitutive activation of signaling. The fact that the patient's eosinophilia improved with ruxolitinib treatment illustrates the importance of performing NGS in patients with HES to identify potentially targetable genetic lesions. [Display omitted] DisclosuresDeininger:Ariad Pharmaceuticals, Bristol Myers Squibb, CTI BioPharma Corp, Gilead, Incyte, Novartis, Pfizer, Celgene, Blue Print, Galena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Incyte: Consultancy; Novartis: Consultancy, Research Funding; Celgene: Research Funding; Pfizer: Consultancy; BMS: Consultancy, Research Funding; ARIAD: Consultancy.

Gestational zinc deficiency impairs brain astrogliogenesis in rats through multistep alterations of the JAK/STAT3 signaling pathway

We previously showed that zinc (Zn) deficiency affects the STAT3 signaling pathway in part through redox-regulated mechanisms. Given that STAT3 is central to the process of astrogliogenesis, this study investigated the consequences of maternal marginal Zn deficiency on the developmental timing and key mechanisms of STAT3 activation, and its consequences on astrogliogenesis in the offspring. This work characterized the temporal profile of cortical STAT3 activation from the mid embryonic stage up to young adulthood in the offspring from dams fed a marginal Zn deficient diet (MZD) throughout gestation and until postnatal day (P) 2. All rats were fed a Zn sufficient diet (control) from P2 until P56. Maternal zinc deficiency disrupted cortical STAT3 activation at E19 and P2. This was accompanied by altered activation of JAK2 kinase due to changes in PTP1B phosphatase activity. The underlying mechanisms mediating the adverse impact of a decreased Zn availability on STAT3 activation in the offspring brain include: (i) impaired PTP1B degradation via the ubiquitin/proteasome pathway; (ii) tubulin oxidation, associated decreased interactions with STAT3 and consequent impaired nuclear translocation; and (iii) decreased nuclear STAT3 acetylation. Zn deficiency-associated decreased STAT3 activation adversely impacted astrogliogenesis, leading to a lower astrocyte number in the early postnatal and adult brain cortex. Thus, a decreased availability of Zn during early development can have a major and irreversible adverse effect on astrogliogenesis, in part via multistep alterations in the STAT3 pathway.

Bioactive lipid-based therapeutic approach to COVID-19 and other similar infections.

COVID-19 is caused by SARS-CoV-2 infection. Epithelial and T, NK, and other immunocytes release bioactive lipids especially arachidonic acid (AA) in response to microbial infections to inactivate them and upregulate the immune system. COVID-19 (coronavirus) and other enveloped viruses including severe acute respiratory syndrome (SARS-CoV-1 of 2002-2003) and Middle East respiratory syndrome (MERS; 2012-ongoing) and hepatitis B and C (HBV and HCV) can be inactivated by AA, γ-linolenic acid (GLA, dihomo-GLA (DGLA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), which are precursors to several eicosanoids. Prostaglandin E1, lipoxin A4, resolvins, protectins and maresins enhance phagocytosis of macrophages and leukocytes to clear debris from the site(s) of infection and injury, enhance microbial clearance and wound healing to restore homeostasis. Bioactive lipids modulate the generation of M1 and M2 macrophages and the activity of other immunocytes. Mesenchymal and adipose tissue-derived stem cells secrete LXA4 and other bioactive lipids to bring about their beneficial actions in COVID-19. Bioactive lipids regulate vasomotor tone, inflammation, thrombosis, immune response, inactivate enveloped viruses, regulate T cell proliferation and secretion of cytokines, stem cell survival, proliferation and differentiation, and leukocyte and macrophage functions, JAK kinase activity and neutrophil extracellular traps and thus, have a critical role in COVID-19.

SOCS3 is a suppressor of γc cytokine signaling and constrains generation of murine Foxp3+ regulatory T cells

SOCS3 is a cytosolic inhibitor of cytokine signaling that suppresses the activation of cytokine receptor-associated JAK kinases. Mechanistically, SOCS3 is recruited to a site in the cytokine receptors known as the SOCS3-interaction motif, and then binds JAK molecules to inhibit their kinase activity. The SOCS3-interaction motif is found in receptors of the gp130 cytokine family but mostly absent from other cytokine receptors, including γc. Thus, SOCS3 has been considered a selective suppressor of gp130 family cytokines, but not γc cytokines. Considering that γc signaling induces SOCS3 expression in T cells, here we revisited the role of SOCS3 on γc signaling. Using SOCS3 transgenic mice, we found that increased abundance of SOCS3 not only suppressed signaling of the gp130 family cytokine IL-6, but also signaling of the γc family cytokine IL-7. Consequently, SOCS3 transgenic mice were impaired in IL-7-dependent T cell development in the thymus and the homeostasis of mature T cells in peripheral tissues. Moreover, enforced SOCS3 expression interfered with the generation of Foxp3+ regulatory T cells that requires signaling by the γc family cytokine IL-2. Collectively, we report an underappreciated role for SOCS3 in suppressing γc cytokine signaling, effectively expanding its scope of target cytokines in T cell immunity.

The SMAC mimetic LCL-161 selectively targets JAK2V617F mutant cells

BackgroundEvasion from programmed cell death is a hallmark of cancer and can be achieved in cancer cells by overexpression of inhibitor of apoptosis proteins (IAPs). Second mitochondria-derived activator of caspases (SMAC) directly bind to IAPs and promote apoptosis; thus, SMAC mimetics have been investigated in a variety of cancer types. particularly in diseases with high inflammation and NFĸB activation. Given that elevated TNFα levels and NFĸB activation is a characteristic feature of myeloproliferative neoplasms (MPN), we investigated the effect of the SMAC mimetic LCL-161 on MPN cell survival in vitro and disease development in vivo.MethodsTo investigate the effect of the SMAC mimetic LCL-161 in vitro, we utilized murine and human cell lines to perform cell viability assays as well as primary bone marrow from mice or humans with JAK2V617F–driven MPN to interrogate myeloid colony formation. To elucidate the effect of the SMAC mimetic LCL-161 in vivo, we treated a JAK2V617F–driven mouse model of MPN with LCL-161 then assessed blood counts, splenomegaly, and myelofibrosis.ResultsWe found that JAK2V617F-mutated cells are hypersensitive to the SMAC mimetic LCL-161 in the absence of exogenous TNFα. JAK2 kinase activity and NFĸB activation is required for JAK2V617F-mediated sensitivity to LCL-161, as JAK or NFĸB inhibitors diminished the differential sensitivity of JAK2V617F mutant cells to IAP inhibition. Finally, LCL-161 reduces splenomegaly and may reduce fibrosis in a mouse model of JAK2V617F-driven MPN.ConclusionLCL-161 may be therapeutically useful in MPN, in particular when exogenous TNFα signaling is blocked. NFĸB activation is a characteristic feature of JAK2V617F mutant cells and this sensitizes them to SMAC mimetic induced killing even in the absence of TNFα. However, when exogenous TNFα is added, NFĸB is activated in both mutant and wild-type cells, abolishing the differential sensitivity. Moreover, JAK kinase activity is required for the differential sensitivity of JAK2V617F mutant cells, suggesting that the addition of JAK2 inhibitors to SMAC mimetics would detract from the ability of SMAC mimetics to selectively target JAK2V617F mutant cells. Instead, combination therapy with other agents that reduce inflammatory cytokines but preserve JAK2 signaling in mutant cells may be a more beneficial combination therapy in MPN.