Activation Of JAK2 Research Articles

Open label safety and efficacy pilot to study mitigation of equine recurrent uveitis through topical suppressor of cytokine signaling-1 mimetic peptide

Equine recurrent uveitis (ERU) is a painful and debilitating autoimmune disease and represents the only spontaneous model of human recurrent uveitis (RU). Despite the efficacy of existing treatments, RU remains a leading cause of visual handicap in horses and humans. Cytokines, which utilize Janus kinase 2 (Jak2) for signaling, drive the inflammatory processes in ERU that promote blindness. Notably, suppressor of cytokine signaling 1 (SOCS1), which naturally limits the activation of Jak2 through binding interactions, is often deficient in autoimmune disease patients. Significantly, we previously showed that topical administration of a SOCS1 peptide mimic (SOCS1-KIR) mitigated induced rodent uveitis. In this pilot study, we test the potential to translate the therapeutic efficacy observed in experimental rodent uveitis to equine patient disease. Through bioinformatics and peptide binding assays we demonstrate putative binding of the SOCS1-KIR peptide to equine Jak2. We also show that topical, or intravitreal injection of SOCS1-KIR was well tolerated within the equine eye through physical and ophthalmic examinations. Finally, we show that topical SOCS1-KIR administration was associated with significant clinical ERU improvement. Together, these results provide a scientific rationale, and supporting experimental evidence for the therapeutic use of a SOCS1 mimetic peptide in RU.

Insulin‐like growth factor 1 induces a reparative neutrophil phenotype

Cardiovascular diseases are the leading cause of death in western societies and neutrophils were long thought to play a detrimental role in ischaemic heart disease. But recent data showed that neutrophils also play an important role in cardiac remodelling and can exhibit an anti‐inflammatory N2‐phenotype or a pro‐inflammatory N1 phenotype. Previous experiments showed that short‐term treatment with insulin‐like growth factor (IGF1) preserves cardiac function after myocardial infarction (MI). This effect was mediated by myeloid cells. Therefore, it was the aim to analyse to what extent and how IGF1 modulates neutrophil function.Murine bone marrow neutrophils were isolated by density gradient centrifugation and were left untreated or polarized for 4 hours in vitrowith IL‐4 (20 ng/ml) or IGF1 (10 ng/ml), Insulin (10 ng/ml) or LPS/IFN‐γ (10 ng/ml / 2 ng/ml).Transcript expression analysis and qRT‐PCR were performed to analyse to what extent transcriptional changes were triggered by growth factor and cytokine induced polarization after 4 hours. These analyses revealed that IL‐4, and interestingly also IGF1, induces a reparative N2‐like neutrophil phenotype, characterised by the induction of Arg1, Retnlα and Chi3l3.Surprisingly, Insulin, despite the high homology to IGF1, did not induce an antiinflammatory phenotype. LPS/IFN‐y treatment induced upregulation of proinflammatory genes (TNFα, IL12a, iNOS). As expected, RNAseq analysis showed, that IL‐4 treatment upregulated genes involved in the IL‐4 pathway. Surprisingly, IGF1 treatment induced almost the same pathways as IL‐4. Therefore, the canonical IL‐4 signaling pathway Jak‐Stat was analysed. This revealed that IL‐4, and also IGF1, induced phosphorylation of Stat6. Additionally, Jak1/2/3/Tyk2 was inhibited with pharmacological inhibitors (InSolution Jak Inhibitor I, Ruxolitinib, BMS‐911543). Inhibition of all Jaks prevented IL‐4, and also IGF1, induced upregulation of N2 marker genes. Specific inhibition of Jak2 showed that IL‐4‐ and IGF1‐induced polarization is most likely dependent on Jak2 activation, as N2 marker gene expression was reduced. Western blot analysis as well revealed that Stat6 phosphorylation was reduced after Jak2 inhibition and Proximity ligation assay proved an interaction of the IGF1R and Jak2. Regarding the missing effect of insulin also the canonical insulin/IGF1 pathway, as for example the Akt and Ras‐Raf‐Erk pathway, were analysed. By using Tie2 Akt1 and Tie2 Akt2 KO mice, we showed the independency of IL‐4 and IGF1 induced polarization on those Akt isoforms, as upregulation of antiinflammatory genes was not reduced.Additionally, the effect of different polarizers on the formation of neutrophil extracellular traps, an inflammatory mechanism often found in heart failure, was analysed. This revealed that both, IL‐4 and IGF1, are able to reduce the formation of NETs, whereas LPS/IFN‐y treatment induces the formation of NETs.In conclusion, these findings indicate that IGF1 is able to polarize neutrophils to a reparative N2‐like phenotype most likely via the non‐canonical Jak2‐Stat6 axis, comparable to that induced by the classical M2/N2 polarizer IL‐4. Additionally, IGF1 is able to reverse proinflammatory mechanisms.

Epigenetic downregulation of Socs2 contributes to mutant N-Ras-mediated hematopoietic dysregulation.

ABSTRACTRAS mutations occur in a broad spectrum of human hematopoietic malignancies. Activating Ras mutations in blood cells leads to hematopoietic malignancies in mice. In murine hematopoietic stem cells (HSCs), mutant N-RasG12D activates Stat5 to dysregulate stem cell function. However, the underlying mechanism remains elusive. In this study, we demonstrate that Stat5 activation induced by a hyperactive Nras mutant, G12D, is dependent on Jak2 activity. Jak2 is activated in Nras mutant HSCs and progenitors (HSPCs), and inhibiting Jak2 with ruxolitinib significantly decreases Stat5 activation and HSPC hyper-proliferation in vivo in NrasG12D mice. Activation of Jak2-Stat5 is associated with downregulation of Socs2, an inhibitory effector of Jak2/Stat5. Restoration of Socs2 blocks NrasG12D HSC reconstitution in bone marrow transplant recipients. SOCS2 downregulation is also observed in human acute myeloid leukemia (AML) cells that carry RAS mutations. RAS mutant AML cells exhibited suppression of the enhancer active marker H3K27ac at the SOCS2 locus. Finally, restoration of SOCS2 in RAS mutant AML cells mitigated leukemic growth. Thus, we discovered a novel signaling feedback loop whereby hyperactive Ras signaling activates Jak2/Stat5 via suppression of Socs2.

Philadelphia chromosome-negative myeloproliferative neoplasms: clinical aspects and treatment options.

Clinical studies of Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) have progressed greatly with the discovery of mutations in three driver genes: JAK2, MPL, and calreticulin. Other genes that may play important roles in pathogenesis and progression of MPN have also been identified. Several prognostic prediction systems based on various risk factors including these genetic factors have been developed and utilized in clinical practice. All mutations of the three driver genes result in JAK2 activation, and JAK inhibitors have indeed improved clinical outcomes for primary myelofibrosis and polycythemia vera. However, they have minimal ability to inhibit clonogenic growth, far below that of ABL tyrosine kinase inhibitors in chronic myeloid leukemia. Therefore, hematopoietic stem cell transplantation (HSCT), which still has a high mortality rate, remains the only curative treatment for MPN. Efforts are being made to advance the treatment of MPN by refining HSCT methods, combining JAK inhibitors with other molecularly targeted agents, and reviewing the safety and clonogenic inhibitory effects of interferon-alfa.

GH Action in Prostate Cancer Cells Promotes Proliferation, Limits Apoptosis, and Regulates Cancer-related Gene Expression.

Previous studies investigating the effects of blocking the growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis in prostate cancer found no effects of the growth hormone receptor (GHR) antagonist, pegvisomant, on the growth of grafted human prostate cancer cells in vivo. However, human GHR is not activated by mouse GH, so direct actions of GH on prostate cancer cells were not evaluated in this context. The present study addresses the species specificity of GH-GHR activity by investigating GH actions in prostate cancer cell lines derived from a mouse Pten-deletion model. In vitro cell growth was stimulated by GH and reduced by pegvisomant. These in vitro GH effects were mediated at least in part by the activation of JAK2 and STAT5. When Pten-mutant cells were grown as xenografts in mice, pegvisomant treatment dramatically reduced xenograft size, and this was accompanied by decreased proliferation and increased apoptosis. RNA sequencing of xenografts identified 1765 genes upregulated and 953 genes downregulated in response to pegvisomant, including many genes previously implicated as cancer drivers. Further evaluation of a selected subset of these genes via quantitative reverse transcription-polymerase chain reaction determined that some genes exhibited similar regulation by pegvisomant in prostate cancer cells whether treatment was in vivo or in vitro, indicating direct regulation by GH via GHR activation in prostate cancer cells, whereas other genes responded to pegvisomant only in vivo, suggesting indirect regulation by pegvisomant effects on the host endocrine environment. Similar results were observed for a prostate cancer cell line derived from the mouse transgenic adenocarcinoma of the mouse prostate (TRAMP) model.

Moxibustion regulates the polarization of macrophages through the IL-4/STAT6 pathway in rheumatoid arthritis.

To observe the effects of moxibustion on "Shenshu" and "Zusanli" on macrophage polarization and IL-4/STAT6 signaling pathway in rats with rheumatoid arthritis (RA). To further explore the possible anti-inflammatory mechanism of moxibustion in the treatment of RA. The rats' right hind paws were injected with freund's complete adjuvant (FCA) to establish the model of RA. Seven days after the injection of FCA, moxibustion therapy was performed on the acupoints of Shenshu (BL23) and Zusanli (ST36) once a day for three weeks. The researchers measured the thickness of the foot pad. ELISA and Histological Analysis were performed to observe the anti-inflammatory effect of moxibustion. Then researchers detected the expression of macrophage phenotype and the expression of IL-4/STAT6 signaling pathway related molecules. It was observed that after the injection of FCA, the rats' feet showed obvious symptoms of redness and swelling. But the symptoms were significantly improved when moxibustion was employed. The study found lower IL-23 and higher IL-4 level in the serum of FCA-injected rats after moxibustion treatment. HE staining showed that the synovium of the RA group was hyperemia and edema, with a large number of inflammatory cells infiltration and vascular dilatation. In the moxibustion group, the degree of synovial hyperemia and edema was improved, and the infiltration of inflammatory cells and vascular dilation were reduced. The study also found that there wer differences among the expressions of macrophage phenotypes in RA, and this was shown by the high expression of CD86 and low expression of CD206. However, the polarization of macrophages in the moxibustion group changed, and that was manifested by enhanced M2-polarized Mφs and inhibited M1-polarized Mφs. Meanwhile, moxibustion suppressed the activation of JAK1, JAK3 and STAT6 in the IL-4/STAT6 signaling pathway, which contributed to the polarization of M2 . The results demonstrate that moxibustion not only suppresses the polarization of M1, but also promotes the polarization of M1. The anti-inflammatory effect of moxibustion may be related to the regulation of macrophage polarization through IL-4/STAT6 signaling pathway.

Sphingomyelin maintains the cutaneous barrier via regulation of the STAT3 pathway.

Epidermal tissues play vital roles in maintaining homeostasis and preventing the dysregulation of the cutaneous barrier. Sphingomyelin (SM), a sphingolipid synthesized by sphingomyelin synthase (SMS) 1 and 2, is involved in signal transduction via modulation of lipid-raft functions. Though the implications of SMS on inflammatory diseases have been reported, its role in dermatitis has not been clarified. In this study, we investigated the role of SM in the cutaneous barrier using a dermatitis model established by employing Sgms1 and 2 deficient mice. SM deficiency impaired the cutaneous inflammation and upregulated signal transducer and activator of transcription 3 (STAT3) phosphorylation in epithelial tissues. Furthermore, using mouse embryonic fibroblast cells, the sensitivity of STAT3 to Interleukin-6 stimulation was increased in Sgms-deficient cells. Using tofacitinib, a clinical JAK inhibitor, the study showed that SM deficiency might participate in STAT3 phosphorylation via JAK activation. Overall, these results demonstrate that SM is essential for maintaining the cutaneous barrier via the STAT3 pathway, suggesting SM could be a potential therapeutic target for dermatitis treatment.

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.

A novel intramolecular negative regulation of mouse Jak3 activity by tyrosine 820.

Jak3, a member of the Janus kinase family, is essential for the cytokine receptor common gamma chain (γc)-mediated signaling. During activation of Jak3, tyrosine residues are phosphorylated and potentially regulate its kinase activity. We identified a novel tyrosine phosphorylation site within mouse Jak3, Y820, which is conserved in human Jak3, Y824. IL-2-induced tyrosine phosphorylation of Jak3 Y824 in human T cell line HuT78 cells was detected by using a phosphospecific, pY824, antibody. Mutation of mouse Jak3 Y820 to alanine (Y820A) showed increased autophosphorylation of Jak3 and enhanced signal transducer and activator of transcription 5 (STAT5) tyrosine phosphorylation and transcriptional activation. Stably expressed Jak3 Y820A in F7 cells, an IL-2 responsive mouse pro-B cell line Ba/F3, exhibited enhanced IL-2-dependent cell growth. Mechanistic studies demonstrated that interaction between Jak3 and STAT5 increased in Jak3 Y820A compared to wild-type Jak3. These data suggest that Jak3 Y820 plays a role in negative regulation of Jak3-mediated STAT5 signaling cascade upon IL-2-stimulation. We speculate that this occurs through an interaction promoted by the tyrosine phosphorylated Y820 or a conformational change by Y820 mutation with either the STAT directly or with the recruitment of molecules such as phosphatases via a SH2 interaction. Additional studies will focus on these interactions as Jak3 plays a crucial role in disease and health.

A55 INTERLEUKIN-10 ELICITS CYTOPROTECTION VIA MITOCHONDRIAL SIGNAL TRANSDUCER AND ACTIVATORS OF TRANSCRIPTION 3 (MTSTAT3) TO PREVENT BACTERIAL PATHOBIONT EVOKED MITOCHONDRIAL FRAGMENTATION IN GUT EPITHELIA

BackgroundAberrant mitochondrial function is increasingly reported in inflammatory bowel disease (IBD). We recently reported that the IBD-associated pathobiont, adherent-invasive E. coli (AIEC) (strain LF82)-evoked epithelial mitochondrial fragmentation and mitochondrial depolarization. Remarkably, the transcription factor, signal transducer and activator of transcription (STAT)-3, can migrate to mitochondria (mtSTAT3) to regulate functions such as reactive oxygen species and ATP production. This non-canonical activity is dependent on STAT3 phosphorylation at serine727. IL-10 is an important regulator of enteric homeostasis that signals via STAT-3, however whether IL-10 affects mitochondrial dynamics is unknown.AimsTo determine (1) if IL-10 preserves mitochondrial function in E. coli-LF82-infected epithelia through mtSTAT3, and if so, (2) to identify the mechanism by which mtSTAT3 restores mitochondrial functions.MethodsThe human colon-derived T84 epithelial cells or human organoids were exposed to E. coli LF82 (108 cfu/ml, 4h) ± co-treatment or an 18h pre-treatment with IL-10 (10 ng/ml). The effect of IL-10 on bacterial growth and its invasion of T84 cells were assessed by growth curve analysis and bacterial internalisation assays. We assessed mitochondrial network morphology using mito-tracker red and confocal microscopy, mitochondrial membrane potential with the fluorescent-dye TMRE and flow cytometry and fluorescence microscopy, and oxygen consumption rate (OCR) and ATP levels. STAT3 phosphorylation was analysed in whole cell protein extracts by western blot. Pharmacological inhibitors of JAK and Erk1/2 were used to examine the pathway of mtSTAT3 activation.ResultsIL-10 affected neither E. coli-LF82 growth nor invasion of T84 epithelia, but substantially reduced the AIEC-induced mitochondrial fragmentation in T84 cells and organoids. IL-10 preservation of the mitochondrial network was accompanied by increased mitochondrial membrane potential, OCR, and ATP levels in E. coli-LF82 infected T84 cells. IL-10 + E. coli-LF82-treated epithelia displayed increased phospho-S727 STAT3 that was reduced by inhibition of Erk1/2 but not JAK activity.ConclusionsMitochondria are a potential drug target in IBD, and the E. coli-LF82 pathobiont can be added to a growing list of microbial pathogens that evoke epithelial mitochondrial dysfunction. We have uncovered that IL-10 can stabilize mitochondrial function in E. coli-LF82 infected cells, possibly through mtSTAT-3. Our results underscore the role of IL-10/STAT3 signaling in preserving mitochondrial functions that could result in mitochondria-targeted therapeutics in IBD and other bacteria-driven enteropathies.Funding AgenciesCIHRAIHS

The PAX5‐JAK2 translocation acts as dual‐hit mutation that promotes aggressive B‐cell leukemia via nuclear STAT5 activation

While PAX5 is an important tumor suppressor gene in B‐cell acute lymphoblastic leukemia (B‐ALL), it is also involved in oncogenic translocations coding for diverse PAX5 fusion proteins. PAX5‐JAK2 encodes a protein consisting of the PAX5 DNA‐binding region fused to the constitutively active JAK2 kinase domain. Here, we studied the oncogenic function of the PAX5‐JAK2 fusion protein in a mouse model expressing it from the endogenous Pax5 locus, resulting in inactivation of one of the two Pax5 alleles. Pax5Jak2/+ mice rapidly developed an aggressive B‐ALL in the absence of another cooperating exogenous gene mutation. The DNA‐binding function and kinase activity of Pax5‐Jak2 as well as IL‐7 signaling contributed to leukemia development. Interestingly, all Pax5Jak2/+ tumors lost the remaining wild‐type Pax5 allele, allowing efficient DNA‐binding of Pax5‐Jak2. While we could not find evidence for a nuclear role of Pax5‐Jak2 as an epigenetic regulator, high levels of active phosphorylated STAT5 and increased expression of STAT5 target genes were seen in Pax5Jak2/+ B‐ALL tumors, implying that nuclear Pax5‐Jak2 phosphorylates STAT5. Together, these data reveal Pax5‐Jak2 as an important nuclear driver of leukemogenesis by maintaining phosphorylated STAT5 levels in the nucleus.

Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling

SummaryAsthma is a complex pulmonary disorder with multiple pathological mechanisms. A key pathological feature of chronic asthma is airway remodeling, which is largely attributed to airway smooth muscle (ASM) hyperplasia that contributes to thickening of the airway wall and further drives asthma pathology. The cellular processes that mediate ASM cell proliferation are not completely elucidated. Using multiple approaches, we demonstrate that the adapter protein Abi1 (Abelson interactor 1) is upregulated in ∼50% of ASM cell cultures derived from patients with asthma. Loss-of-function studies demonstrate that Abi1 regulates the activation of Jak2 (Janus kinase 2) and STAT3 (signal transducers and activators of transcription 3) as well as the proliferation of both nonasthmatic and asthmatic human ASM cell cultures. These findings identify Abi1 as a molecular switch that activates Jak2 kinase and STAT3 in ASM cells and demonstrate that a dysfunctional Abi1-associated pathway contributes to the progression of asthma.

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.

In silico Prediction on the PI3K/AKT/mTOR Pathway of the Antiproliferative Effect of O. joconostle in Breast Cancer Models.

The search for new cancer treatments from traditional medicine involves developing studies to understand at the molecular level different cell signaling pathways involved in cancer development. In this work, we present a model of the PI3K/Akt/mTOR pathway, which plays a key role in cell cycle regulation and is related to cell survival, proliferation, and growth in cancer, as well as resistance to antitumor therapies, so finding drugs that act on this pathway is ideal to propose a new adjuvant treatment. The aim of this work was to model, simulate and predict in silico using the Big Data-Cellulat platform the possible targets in the PI3K/Akt/mTOR pathway on which the Opuntia joconostle extract acts, as well as to indicate the concentration range to be used to find the mean lethal dose in in vitro experiments on breast cancer cells. The in silico results show that, in a cancer cell, the activation of JAK and STAT, as well as PI3K and Akt is related to the effect of cell proliferation, angiogenesis, and inhibition of apoptosis, and that the extract of O. joconostle has an antiproliferative effect on breast cancer cells by inhibiting cell proliferation, regulating the cell cycle and inhibiting apoptosis through this signaling pathway. In vitro it was demonstrated that the extract shows an antiproliferative effect, causing the arrest of cells in the G2/M phase of the cell cycle. Therefore, it is concluded that the use of in silico tools is a valuable method to perform virtual experiments and discover new treatments. The use of this type of model supports in vitro experimentation, reducing the costs and number of experiments in the real laboratory.

Colorectal cancer-associated fibroblasts promote metastasis by up-regulating LRG1 through stromal IL-6/STAT3 signaling

Cancer-associated fibroblasts (CAFs) have been shown to play a strong role in colorectal cancer metastasis, yet the underlying mechanism remains to be fully elucidated. Using CRC clinical samples together with ex vivo CAFs-CRC co-culture models, we found that CAFs induce expression of Leucine Rich Alpha-2-Glycoprotein 1(LRG1) in CRC, where it shows markedly higher expression in metastatic CRC tissues compared to primary tumors. We further show that CAFs-induced LRG1 promotes CRC migration and invasion that is concomitant with EMT (epithelial-mesenchymal transition) induction. In addition, this signaling axis has also been confirmed in the liver metastatic mouse model which displayed CAFs-induced LRG1 substantially accelerates metastasis. Mechanistically, we demonstrate that CAFs-secreted IL-6 (interleukin-6) is responsible for LRG1 up-regulation in CRC, which occurs through a direct transactivation by STAT3 following JAK2 activation. In clinical CRC tumor samples, LRG1 expression was positively correlated with CAFs-specific marker, α-SMA, and a higher LRG1 expression predicted poor clinical outcomes especially distant metastasis free survival, supporting the role of LRG1 in CRC progression. Collectively, this study provided a novel insight into CAFs-mediated metastasis in CRC and indicated that therapeutic targeting of CAFs-mediated IL-6-STAT3-LRG1 axis might be a potential strategy to mitigate metastasis in CRC.

JAK Inhibition Prevents DNA Damage and Apoptosis in Testicular Ischemia-Reperfusion Injury via Modulation of the ATM/ATR/Chk Pathway.

Testicular ischemia reperfusion injury (tIRI) causes oxidative stress-induced DNA damage leading to germ cell apoptosis (GCA). The aim of the study is to establish a direct link between JAK2 activation and the DNA damage response (DDR) signaling pathways and their role in tIRI-induced GCA using AG490, a JAK2 specific inhibitor. Male Sprague Dawley rats (n = 36) were divided into three groups: sham, unilateral tIRI and tIRI + AG490 (40 mg/kg). During tIRI, augmentation in the phosphorylation levels of the JAK2/STAT1/STAT3 was measured by immunohistochemistry. Observed spermatogenic arrest was explained by the presence of considerable levels of DSB, AP sites and 8OHdG and activation of caspase 9, caspase 3 and PARP, which were measured by colorimetric assays and TUNEL. The ATM/Chk2/H2AX and ATR/Chk1 pathways were also activated as judged by their increased phosphorylation using Western blot. These observations were all prevented by AG490 inhibition of JAK2 activity. Our findings demonstrate that JAK2 regulates tIRI-induced GCA, oxidative DNA damage and activation of the ATM/Chk2/H2AX and ATR/Chk1 DDR pathways, but the cell made the apoptosis decision despite DDR efforts.

Inside-out and outside-in organotypic normal human skin culture: JAK-STAT pathway is activated after pro-inflammatory psoriatic cytokine exposure

The Janus kinases–signal transducers and activators of transcription (JAK-STAT) signalling pathway are a pleiotropic cascade that involves ligands such as cytokines, hormones, and growth factors. Among cytokines, interleukin (IL)-17, IL-22, IL-23, and tumour necrosis factor (TNF)-alpha play a pivotal role in psoriasis. We aimed at investigating in an organotypic experimental model of normal human skin (n = 7 women between 20–40 years old, non-smokers) the early, direct, and specific effects of IL-17, IL-22, IL-23, TNF-alpha and a combination of the four cytokines (Mix) on the JAK-STAT/pathway. The expression of the psoriatic marker keratin (K) 17 was analyzed by immunofluorescence and molecular techniques after exposure to IL-23 or Mix. The Mix elicited a strong K17 up-regulation in keratinocytes at 72 h, reinforcing the hypothesis of a synergistic effect of different cytokines. High levels of JAK1 and STAT3 activation were detected, suggesting the involvement of JAK1/STAT3 pathway in the upregulation of K17. As the present study in an organotypic model of human skin reports a variable expression of JAK-STAT upon different cytokine stimuli and most of the JAK inhibitors for the psoriasis treatment have proven to have a clinical efficacy, these observations have a relevance to better understand the mechanisms of JAK-inhibitors in the skin.

A Novel CSF3R Activating Mutation Identified in a Patient with Chronic Neutrophilic Leukemia

Mutations in CSF3R, the growth factor receptor that signals to drive neutrophil production, are commonly found in Chronic Neutrophilic Leukemia (CNL). There are two main types of CSF3R mutations in CNL: membrane-proximal point mutations and cytoplasmic truncating mutations. The membrane proximal point mutations-notably T618I-are the most common in CNL. These point mutations lead to the loss of glycosylation sites, causing ligand-independent dimerization of the receptor and enhanced activation of downstream signaling pathways. Previously, these glycan-altering mutations were thought to reside in a relatively restricted region near the membrane spanning 610-618 that contains an N-linked glycosylation site and three putative O-linked glycosylation sites.A 55-year old man presented with a WBC of 50,000 and a predominant neutrophilia with 84% neutrophils and bands and no immature cells. A bone marrow examination showed a markedly hypercellular bone marrow exhibiting myeloid-predominant maturing trilineage hematopoiesis with left-shifted myeloid maturation, mild megakaryocytic atypia (small, hypolobated forms), mild reticulin fibrosis (grade 1 of 3). He had multi-station small volume lymphadenopathy and a spleen that measured 16.6 cm craniocaudally. The findings were most consistent with chronic neutrophilic leukemia.Targeted NGS analysis revealed three mutations in CSF3R: Q739* (VAF 26%), Q741* (VAF 24%) and N579Y (VAF 52%). ASXL1 G646fs (38% VAF) and ETNK1 H243Q (VAF 12%) mutations were also present. The truncating mutations in CSF3R were reported as pathogenic, but the N579Y mutation was listed as a variant of uncertain significance. When truncating mutations occur in CNL, they most frequently occur alongside an activating point mutation. This, combined with the knowledge that N579 was previously mapped as a site of receptor glycosylation prompted us to assess the oncogenicity of this variant.We undertook several parallel approaches to characterize this variant. First, a cytokine independent growth assay was performed in Ba/F3 cells. The CSF3R N579Y variant allowed these cells to proliferate in the absence of cytokine support, similarly to the well characterized CSF3R T618I variant. Furthermore, it enabled growth independent from the ligand for CSF3R (GCSF), indicating that this mutation likely also confers ligand-independent dimerization. In an orthogonal approach, we found that CSF3R N579Y enables primary murine hematopoietic cells to grow in cytokine-free methylcellulose to a similar extent as CSF3R T618I.To understand the downstream consequences of receptor activation we undertook immunoblot analysis and discovered that CSF3R N579Y robustly enhances JAK/STAT and MAPK signaling downstream of the receptor. Because of the know activation of JAK by CSF3R mutations, a JAK1/2 inhibitor (ruxolitinib) has been under investigation for treatment of CSF3R-mutant CNL. We find that CSF3R N579Y confers sensitivity to ruxolitinib in both Ba/F3 cells and murine colony forming unit assays. Furthermore, ruxolitinib inhibited colony formation of CD34 positive cells isolated from a bone marrow aspirate from the patient whose leukemia harbored CSF3R N579Y, CSF3R Q739* and CSF3R Q741* mutations. Collectively, our data suggests that N579Y is a pathogenic and potentially actionable variant and that N-linked glycosylation outside of the membrane-proximal region is necessary to maintain the receptor in an inactive state. [Display omitted] DisclosuresSanz-Altamira: Kura Oncology: Consultancy. DeAngelo: Autolus: Consultancy; Forty-Seven: Consultancy; Incyte: Consultancy; Jazz: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy; Servier: Consultancy; Takeda: Consultancy; Abbvie: Research Funding; Blueprint: Research Funding; Amgen: Consultancy; Agios: Consultancy; Glycomimetrics: Research Funding. Maxson: Ionis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Second Treatment Free Remission after Combination Therapy with Ruxolitinib Plus Tyrosine Kinase Inhibitors in Chronic Phase Chronic Myeloid Leukemia (CML)

Second Treatment Free Remission after Combination Therapy with Ruxolitinib Plus Tyrosine Kinase Inhibitors in Chronic Phase Chronic Myeloid Leukemia (CML)

Germline-Somatic Interactions in Myelofibrosis Susceptibility

▪Introduction: Myelofibrosis (MF) is a rare myeloproliferative neoplasm (MPN) characterized by bone marrow fibrosis, progressive bone marrow failure, and increased risk of acute myeloid leukemia. While MF arises from somatic driver mutations in JAK2, MPL, and CALR, some MPN patients may have a heritable component. To comprehensively examine the genetic etiology of MF, we performed the first integrative analysis of SNP array genotyping (using Infinium Global Screening Array), targeted long-read sequencing (using PacBio SMRT sequencing) and telomere length (TL, using qPCR assay).Methods: Our study included 937 MF patients who received an allogeneic hematopoietic cell transplant (HCT) between 2000 and 2016 and had an available pre-HCT blood sample at the Center for International Blood and Marrow Transplant Research Repository. Somatic mosaic chromosomal alterations (mCAs, including deletions, duplications, or copy-neutral losses-of-heterozygosity (CNLOH)) were called with the Mosaic Chromosomal Alteration (MoChA) algorithm using raw genotyping intensity data. A genome-wide association study (GWAS) was restricted to include 827 MF patients of European ancestry and utilized 4,135 genetically-matched healthy controls.Results: GWAS identified six independent MF susceptibility loci at genome-wide significance (P< 5×10 -8); four of which replicate prior MPN susceptibility loci [9p24.1(JAK2), 5p15.33(TERT), 3q25.33(IFT80), and 4q24(TET2)] and two novel MF loci [6p21.35(HLA-DQB1-AS1) and 17p13.1(TP53)] (Figure 1). A transcriptome-wide association analysis using whole blood GTEx data highlighted the 9p24.1 locus with increased JAK2 expression associated with elevated risk of MF (P= 2.18×10 -19). A strong colocalization statistic further indicated shared genetic component between eQTL and this JAK2 locus (HyPrColoc Posterior Probability= 0.6) (Figure 2).Based on the strong signal identified at TERT (Figure 1), we investigated the relationship between MF risk and genetically-inferred telomere length using a panel of 19 germline variants previously found to be associated with telomere length. Of the 19 telomere-length associated variants investigated, 7 were found to be associated with MF risk (binomial P= 2.31×10 -5, linear trend P= 5.48×10 -4) (Figure 3). Both Mendelian randomization and genome-wide genetic correlation analyses further indicated that increased risk of MF was associated with longer inherited telomere length.Utilizing available clinical mutation data on a subset of 185 patients, MF cases carrying the germline risk haplotype of the 9p24.1(JAK2) susceptibility locus were observed to more frequently have the JAK2 V617F mutation (71% vs 59%; P= 0.02). Targeted PacBio long-read sequencing around JAK2 provided further evidence of linkage between the germline risk allele and the JAK2 V617F mutation. Detectable autosomal mCAs were also abundant in MF cases with 67.4% having at least one mCA (compared to ~3% in the general population) and 27.6% having an mCA spanning JAK2 (mostly CNLOH) (Figure 4). In addition, using a binomial test for biased allelic imbalance, a cis relationship was identified at 9p24.1 in which the MF risk haplotype was predominantly duplicated by CNLOH (binomial P=1.36×10 -9). Regional sequencing of JAK2 further confirmed duplication of JAK2 V617F by CNLOH. Finally, we observed an inverse association between autosomal mCAs and qPCR measured telomere length (OR= 0.22, 95% CI= 0.07-0.65, P= 6.40×10 -3). These results were consistent by mCA chromosomal region and copy number state.Conclusion: Our results suggest a molecular framework for the genetic etiology of MF in which both genetically-inferred telomere length and germline variation at JAK2 are associated with increased MF risk. The 9p24.1 risk haplotype predisposes to the acquisition of a somatic JAK2 V617F mutation in cis and subsequent duplication of JAK2 V617F by mCAs (usually CNLOH). This process leads to aberrant JAK2 activity and increased clonal proliferation, accelerating telomere length shortening and increasing genomic instability in patients with MF. [Display omitted] DisclosuresGupta: AbbVie: Consultancy, Honoraria; Constellation Pharma: Consultancy, Honoraria; Roche: Consultancy; Pfizer: Consultancy; BMS-Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria, Research Funding. Lee: Janssen: Other; Incyte: Research Funding; AstraZeneca: Research Funding; Kadmon: Research Funding; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Syndax: Research Funding; Takeda: Research Funding; Amgen: Research Funding. Saber: Govt. COI: Other.