Activation Of JAK2 Research Articles

CYT387 Inhibits the Hyperproliferative Potential of Fibroblast-like Synoviocytes via Modulation of IL-6/JAK1/STAT3 Signaling in Rheumatoid Arthritis

ABSTRACT Fibroblast-like synoviocytes (FLS) are the critical effector cells primarily involved in rheumatoid arthritis (RA) disease pathogenesis. Interleukin (IL)-6, a proinflammatory cytokine most abundantly expressed in the rheumatoid synovium, promotes Janus kinase (JAK)/signal transducer and transcriptional activator (STAT) signaling cascade activation in RA-FLS, thus leading to its aggressive phenotype, invasiveness, and joint destruction. Momelotinib (CYT387) is a selective small-molecule inhibitor of JAK1/2 and is clinically approved to treat myelofibrosis. However, the therapeutic efficacy of CYT387 in FLS mediated RA pathogenesis is less known. In the present study, we investigated the modulatory effect of CYT387 on IL6/JAK/STAT signaling cascade in FLS induced RA pathogenesis. CYT387 treatment inhibited IL-6 induced high proliferative and migratory potential of FLS cells isolated from adjuvant-induced arthritic (AA) rats. CYT387 reduced the expression of PRMT5, survivin, and HIF-1α mediated by IL-6/sIL-6R in AA-FLS in a dose-dependent manner. The IL-6/sIL-6R induced expression of angiogenic factors such as VEGF and PIGF in AA-FLS cells was found downregulated by CYT387 treatment. Importantly, CYT387 significantly reduced IL-6/sIL-6R dependent activation of JAK1 and STAT3 and increased SOCS3 expression in AA-FLS cells. Next, the S3I-201 mediated blockade of STAT3 activation supported the inhibitory effect of CYT387 on IL-6/JAK1/STAT3 signaling cascade in AA-FLS. Overall, this study proves that CYT387 inhibits proliferation, migration, and pathogenic disease potential of FLS isolated from adjuvant-induced arthritic (AA) rats via targeting IL-6/JAK1/STAT3 signaling cascade.

The Synergistic Inhibitory Effect of Combining MK-2206 and AZD 6244 in MARIMO Cells Harboring a Calreticulin Gene Mutation

Introduction: Somatic mutations in the calreticulin (CALR) gene occur in most myeloproliferative neoplasm (MPN) patients who lack Janus kinase 2 or thrombopoietin receptor (MPL) mutations, but the molecular pathogenesis of MPN with mutated CALR is unclear, which limited the further treatment for CALR gene mutant patients. Objectives: Previous studies showed that CALR mutations not only activated serine/threonine protein kinase (AKT) in primary mouse bone marrow cells but also mitogen-activated protein kinases (MAPKs) in MARIMO cells harboring a heterozygous 61-bp deletion in CALR exon 9, which were responsible for mutant CALR cell survival, respectively. Hence, we aimed to initially explore the mechanism of AKT activation and observe the synergistic inhibitory effect of combining AKT (MK-2206) and MAPK kinase (AZD 6244) inhibitors in MARIMO cells. Methods: We detected the expression of phosphorylated AKT in MARIMO cells treated with inhibitors for 24 or 48 h by western blotting and analyzed cell proliferation, cell cycle, and apoptosis by flow cytometry. We further examined the synergistic inhibitory effect of combining MK-2206 and AZD 6244 in MARIMO cells using the median effect principle of Chou and Talalay. Results: We found that the AKT was activated in MARIMO cells, and blocking its activity significantly inhibited MARIMO cell growth with downregulation of cyclin D and E, and accelerated cell apoptosis by decreasing Bcl-2 but increasing Bax and cleaved caspase-3 levels in a dose-dependent manner. Further analysis showed that AKT activation was dependent on mammalian target of rapamycin but not on the JAK signaling pathway in MARIMO cells, displaying that inhibition of JAK activity by ruxolitinib (RUX) did not decrease the AKT phosphorylation. Furthermore, the combination of MK-2206 and AZD 6244 produced a significantly synergistic inhibitory effect on MARIMO cells. Conclusions: AKT activation is a feature of MARIMO cells and co-targeting of AKT and MAPKs signaling pathways synergistically inhibits MARIMO cell growth.

Blockade of JAK2 signaling produces immunomodulatory effect to preserve pancreatic homeostasis in severe acute pancreatitis

JAK/STAT plays an important role in cytokine signal transduction and it is potentially involved in the proinflammatory response during the early phase of severe acute pancreatitis (SAP). However, whether JAK2 activity is upregulated and whether JAK2 inhibition plays a role in the maintenance of pancreatic homeostasis during SAP is incompletely understood. Here we show that JAK2/STAT3 activity is highly elevated in SAP and blockade of JAK2 by AG-490 protects against SAP-induced pancreatic inflammation and injury. Gene expression and ELISA studies showed that JAK2 inhibition altered the cytokine profiles in both the circulation and pancreases. Further analysis revealed that JAK2 inhibition restored the level of cytokines critical for macrophage polarization towards M2 macrophage. Our findings suggest that pharmacological targeting at JAK2/STAT signalling may be an effective choice of therapeutic interventions against SAP.

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.

PHF6 and JAK3 mutations cooperate to drive T-cell acute lymphoblastic leukemia progression

T-cell acute lymphoblastic leukemia (T-ALL) is a malignant hematologic disease caused by gene mutations in T-cell progenitors. As an important epigenetic regulator, PHF6 mutations frequently coexist with JAK3 mutations in T-ALL patients. However, the role(s) of PHF6 mutations in JAK3-driven leukemia remain unclear. Here, the cooperation between JAK3 activation and PHF6 inactivation is examined in leukemia patients and in mice models. We found that the average survival time is shorter in patients with JAK/STAT and PHF6 comutation than that in other patients, suggesting a potential role of PHF6 in leukemia progression. We subsequently found that Phf6 deficiency promotes JAK3M511I-induced T-ALL progression in mice by inhibiting the Bai1-Mdm2-P53 signaling pathway, which is independent of the JAK3/STAT5 signaling pathway. Furthermore, combination therapy with a JAK3 inhibitor (tofacitinib) and a MDM2 inhibitor (idasanutlin) reduces the Phf6 KO and JAK3M511I leukemia burden in vivo. Taken together, our study suggests that combined treatment with JAK3 and MDM2 inhibitors may potentially increase the drug benefit for T-ALL patients with PHF6 and JAK3 comutation.

Requirement of Histone Deacetylase 6 for Interleukin-6 Induced Epithelial-Mesenchymal Transition, Proliferation, and Migration of Peritoneal Mesothelial Cells.

Aims: Influenced by microenvironment, human peritoneal mesothelial cells (HPMCs) acquired fibrotic phenotype, which was identified as the protagonist for peritoneal fibrosis. In this study, we examined the role of histone deacetylase 6 (HDAC6) for interleukin-6 (IL-6) induced epithelial-mesenchymal transition (EMT), proliferation, and migration of HPMCs.Methods: The role of HDAC6 in IL-6-elicited EMT of HPMCs was tested by morphological observation of light microscope, immunoblotting, and immune-fluorescence assay; and the function of HDAC6 in proliferation and migration of HPMCs was examined by CCK-8 assay, wound healing experiment, and immunoblotting.Results: IL-6 stimulation significantly increased the expression of HDAC6. Treatment with tubastatin A (TA), a highly selective HDAC6 inhibitor, or silencing of HDAC6 with siRNA decreased the expression of HDAC6. Moreover, TA or HDAC6 siRNA suppressed IL-6-induced EMT, as evidenced by decreased expressions of α-SMA, Fibronectin, and collagen I and the preserved expression of E-cadherin in cultured HPMCs. Mechanistically, HDAC6 inhibition suppressed the expression of transforming growth factor β (TGFβ) receptor I (TGFβRI), phosphorylation of Smad3, secretion of connective tissue growth factor (CTGF), and transcription factor Snail. On the other hand, the pharmacological inhibition or genetic target of HDAC6 suppressed HPMCs proliferation, as evidenced by the decreased optical density of CCK-8 and the expressions of PCNA and Cyclin E. The migratory rate of HPMCs also decreased. Mechanistically, HDAC6 inhibition blocked the activation of JAK2 and STAT3.Conclusion: Our study illustrated that IL-6-induced HDAC6 not only regulated IL-6 itself downstream JAK2/STAT3 signaling but also co-activated the TGF-β/Smad3 signaling, leading to the change of the phenotype and mobility of HPMCs. HDAC6 could be a potential therapeutic target for the prevention and treatment of peritoneal fibrosis.

Acquired JAK2 mutations confer resistance to JAK inhibitors in cell models of acute lymphoblastic leukemia

Ruxolitinib (rux) Phase II clinical trials are underway for the treatment of high-risk JAK2-rearranged (JAK2r) B-cell acute lymphoblastic leukemia (B-ALL). Treatment resistance to targeted inhibitors in other settings is common; elucidating potential mechanisms of rux resistance in JAK2r B-ALL will enable development of therapeutic strategies to overcome or avert resistance. We generated a murine pro-B cell model of ATF7IP-JAK2 with acquired resistance to multiple type-I JAK inhibitors. Resistance was associated with mutations within the JAK2 ATP/rux binding site, including a JAK2 p.G993A mutation. Using in vitro models of JAK2r B-ALL, JAK2 p.G993A conferred resistance to six type-I JAK inhibitors and the type-II JAK inhibitor, CHZ-868. Using computational modeling, we postulate that JAK2 p.G993A enabled JAK2 activation in the presence of drug binding through a unique resistance mechanism that modulates the mobility of the conserved JAK2 activation loop. This study highlights the importance of monitoring mutation emergence and may inform future drug design and the development of therapeutic strategies for this high-risk patient cohort.

Nitric Oxide-Releasing Drug Glyceryl Trinitrate Targets JAK2/STAT3 Signaling, Migration and Invasion of Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is a highly aggressive disease with invasive and metastasizing properties associated with a poor prognosis. The STAT3 signaling pathway has shown a pivotal role in cancer cell migration, invasion, metastasis and drug resistance of TNBC cells. IL-6 is a main upstream activator of the JAK2/STAT3 pathway. In the present study we examined the impact of the NO-donor glyceryl trinitrate (GTN) on the activation of the JAK2/STAT3 signaling pathway and subsequent migration, invasion and metastasis ability of TNBC cells through in vitro and in vivo experiments. We used a subtoxic dose of carboplatin and/or recombinant IL-6 to activate the JAK2/STAT3 signaling pathway and its functional outcomes. We found an inhibitory effect of GTN on the activation of the JAK2/STAT3 signaling, migration and invasion of TNBC cells. We discovered that GTN inhibits the activation of JAK2, the upstream activator of STAT3, and mediates the S-nitrosylation of JAK2. Finally, the effect of GTN (Nitronal) on lung metastasis was investigated to assess its antitumor activity in vivo.

Murine Norovirus Infection Results in Anti-inflammatory Response Downstream of Amino Acid Depletion in Macrophages.

ABSTRACTMurine norovirus (MNV) infection results in a late translation shutoff that is proposed to contribute to the attenuated and delayed innate immune response observed both in vitro and in vivo. Recently, we further demonstrated the activation of the α subunit of eukaryotic initiation factor 2 (eIF2α) kinase GCN2 during MNV infection, which has been previously linked to immunomodulation and resistance to inflammatory signaling during metabolic stress. While viral infection is usually associated with activation of double-stranded RNA (dsRNA) binding pattern recognition receptor PKR, we hypothesized that the establishment of a metabolic stress in infected cells is a proviral event, exploited by MNV to promote replication through weakening the activation of the innate immune response. In this study, we used multi-omics approaches to characterize cellular responses during MNV replication. We demonstrate the activation of pathways related to the integrated stress response, a known driver of anti-inflammatory phenotypes in macrophages. In particular, MNV infection causes an amino acid imbalance that is associated with GCN2 and ATF2 signaling. Importantly, this reprogramming lacks the features of a typical innate immune response, with the ATF/CHOP target GDF15 contributing to the lack of antiviral responses. We propose that MNV-induced metabolic stress supports the establishment of host tolerance to viral replication and propagation.IMPORTANCE During viral infection, host defenses are typically characterized by the secretion of proinflammatory autocrine and paracrine cytokines, potentiation of the interferon (IFN) response, and induction of the antiviral response via activation of JAK and Stat signaling. To avoid these and propagate, viruses have evolved strategies to evade or counteract host sensing. In this study, we demonstrate that murine norovirus controls the antiviral response by activating a metabolic stress response that activates the amino acid response and impairs inflammatory signaling. This highlights novel tools in the viral countermeasures arsenal and demonstrates the importance of the currently poorly understood metabolic reprogramming occurring during viral infections.

Curcumol may alleviate psoriasis-like inflammation by inhibiting keratinocyte proliferation and inflammatory gene expression via JAK1/STAT3 signaling

Psoriasis is a chronic inflammatory skin disease characterized by abnormal proliferation and differentiation of keratinocytes. Since curcumol exhibits anti-inflammatory properties in various diseases, we investigated its anti-inflammatory potential in stimulated human keratinocytes. Our data show that curcumol significantly inhibits proliferation and induces cell cycle arrest in NHEK cells stimulated with proinflammatory cytokines (IL-1α, IL-17A, IL-22, oncostatin M, and TNF-α; mix M5). In addition, curcumol markedly ameliorates inflammatory response and promotes differentiation of M5-stimulated NHEK cells. Curcumol inhibits activity of JAK1, resulting in the inhibition of STAT3, downregulation of cyclin D2, and cell cycle arrest in stimulated NHEK cells. Together, our data show that curcumol reduces proliferation and inflammatory gene expression in stimulated keratinocytes by inhibiting the JAK1/STAT3 signaling, suggesting that it might serve as a potential therapeutic option for the treatment of psoriasis.

Abstract 1454: Antitumor activity of a selective FAK/Pyk2 inhibitor, SJP1602 against TNBC as well as CRC

Abstract Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that serves as a key mediator in response to integrin and growth factor signaling to control multiple aspects of cellular behavior. High expression and/or aberrant activation of FAK is observed in a broad range of human cancers, contributing to malignant phenotype in cancer and development of tumor resistance. FAK, therefore, has been a prominent target in oncology and a small number of FAK inhibitors are currently in clinical development. However, selective FAK inhibitors have shown insufficient antitumor responses due to a potential compensatory role of proline-rich tyrosine kinase 2 (Pyk2), a homologue of FAK, and require dual specific FAK/Pyk2 inhibition. SJP1602 is a novel dual inhibitor of FAK and Pyk2 without multi-kinase inhibition. We have previously reported that SJP1602 is highly specific against FAK and Pyk2 and its antitumor efficacy in triple negative breast cancer (TNBC) is superior over the clinically active FAK inhibitors. In this study, we further assessed pharmacological efficacy of SJP1602 in another type of cancer, colorectal cancer (CRC). As evaluated by cell-based ELISA, SJP1602 has nanomolar potency against FAK and Pyk2 in a panel of CRC cell lines. Validation through immunoblotting analyses revealed significant reduction of FAK/Pyk2 activation by treatment of SJP1602 in CRC cell lines, consistent with previous observations in TNBC cell lines. We then investigated the inhibition efficiency of SJP1602 in three-dimensional (3D) spheroid models using the CRC cell lines. As determined by CellTiter-Glo 3D cell viability assay, SJP1602 showed the significantly lower IC50 values than other FAK inhibitors. Next, to investigate the mechanism of action (MOA) of SJP1602, we examined changes in activation of several FAK/Pyk2-mediated signaling pathways. As shown by immunoblotting analyses, SJP1602 decreased JAK2 and Akt activity and increased the expression levels of cleaved caspase 3, a critical marker of apoptosis. Further assessment of SJP1602 in vivo showed significant antitumor activities in different human CRC xenograft models containing BRAF-mutant or KRAS-mutant tumors. In addition, the specificity and MOA of SJP1602 were confirmed using xenograft tumors. In consistent with in vitro results, SJP1602 reduced JAK2 and Akt activity in tumors from mice with SJP1602 administration. Therefore, our results demonstrate that SJP1602 has the strong antitumor efficacy in vitro and in vivo via downregulation of JAK2 and PI3K/Akt signaling pathways. SJP1602 is currently undergoing preclinical toxicology studies and in vitro safety evaluation. Taken together, our data provides promising preclinical results for the further development of SJP1602 as a potent anti-cancer agent in TNBC, CRC and resistant cancer involving FAK activation. Citation Format: Kug Hwa Lee, Hyoung Min Cho, Yongbin Park, Sungpyo Hong, Soon Kil Ahn, Min-Hyo Ki. Antitumor activity of a selective FAK/Pyk2 inhibitor, SJP1602 against TNBC as well as CRC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1454.

Mechanism of Escape from LNK Negative Regulation: Constitutively Active JAK2V617F Retains Lysine63-Ubiquitin

IntroductionThe myeloproliferative neoplasms (MPN), polycythemia vera (PV), essential thrombocytosis and primary myelofibrosis are clonal hematopoietic stem cell disorders that arise from gain of function mutations that directly or indirectly activate JAK2, the cognate tyrosine kinase of the growth factor receptors for erythropoietin, thrombopoietin, and granulocyte colony-stimulating factor. Normally, JAK2 activation is modulated by the adapter protein LNK. However, this mechanism and how MPN mutations that activate JAK2 constitutively bypass LNK modulation are unknown. Clinically, loss of function LNK mutations mimic the MPN phenotype. LNK interacts with BRISC (BRCC36 isopeptidase complex) a lysine63-specific four member deubiquitinating complex. MERIT40 (Mediator of RAP80 Interactions and Targeting 40 kDa), the core subunit of BRISC, is necessary for both stability and activity of the BRISC deubiquitinating complex. This LNK-BRISC interaction suggested that deubiquitination might be a potential mechanism by which LNK could negatively regulate JAK2. We, therefore, examined the behavior of LNK, BRISC and MERIT40 in the context of JAK2 V617F.MethodsWe used a JAK2 V617F transgenic mouse PV model and a JAK2 V617F-expressing human cell line, DAMI, to examine JAK2 ubiquitination. The JAK2 V617F transgenic mouse, whether heterozygotic (VF) or homozygotic (VF/VF), authentically recapitulated human PV in a gene dosage-dependent manner: VF mice developed PV by 6 weeks, and splenomegaly and myelofibrosis by 30 weeks, while VF/VF mice demonstrated greater thrombocytosis, neutrophilia and erythrocytosis, with corresponding splenomegaly and fibrosis evident by 21 weeks.ResultsMERIT40 protein expression was found to be decreased in the JAK2 V617F transgenic mouse. Given the critical role of MERIT40 in BRISC protein stability and function, protein expression of both BRCC36 and LNK were assessed and also found to be decreased. Since we previously observed that BRISC activity is targeted to different proteins and cellular locations by interacting with different adaptor proteins (Patterson-Fortin et al. 2010; Zheng et al. 2013), we asked if LNK targeted BRISC deubiquitinating activity to JAK2. Lysates of unfractionated bone marrow from WT and VF mice were immunoprecipitated with a JAK2 antibody. The blots were probed with antibodies targeting lysine63-linked ubiquitin. JAK2 was robustly deubiquitinated in bone marrow samples from WT mice, whereas not only was this debubiquitination lost in in bone marrow samples from VF mice, there was also increased JAK2 phosphorylation. In the human DAMI cell model, MERIT40, LNK and BRISC protein levels were also all reduced, and JAK2 retained dramatic levels of lysine63-linked ubiquitin. Furthermore, we were able to rescue JAK2 deubiquitination in DAMI cells by transfecting MERIT40.We next performed immunohistochemistry on bone marrow samples from WT, VF and VF/VF age-matched mice (Figure). As expected, a JAK2 V617F gene dosage-dependent increase in megakaryocytes was observed with VF/VF mice having the greatest increase. Importantly, in contrast to WT mice, VF-positive megakaryocytes demonstrated cytoplasm localization of lysine63-linked ubiquitin and loss of this signal in the nucleus, most striking in VF/VF mice.ConclusionsWe employed a gene dosage-dependent JAK2 V617F transgenic mouse model of PV and human DAMI cells to demonstrate for the first time that activated JAK2 avoids LNK inhibition because both LNK and MERIT40, a core subunit of the BRISC deubiquitinating complex, are downregulated. This leads to increased JAK2 lysine63-linked ubiquitination and increased JAK2-mediated signaling as evidenced by increased JAK2 phosphorylation. Additionally, there is a marked translocation of the lysine63-linked ubiquitination complex from the nucleus to the cytoplasm in the JAK2 V617F context. These results establish that normally LNK negatively regulates JAK2 activity by targeting a lysine63-deubiquitinating complex to the JAK2 protein. This leads to deubiquitination and suppression of JAK2 activity. In contrast, in the constitutively-activated JAK2 V617F context, down regulation of LNK and MERIT40, and possibly lysine63-linked ubiquitin redistribution, results in loss of negative regulation, perpetuating JAK2 signaling. These data suggest that deubiquitinating agents could be a novel targeted therapy in MPN. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Reprogramming Cell-Signaling By Delivering the Catalytic Domain of Ptpgr Ameliorates Anemia of β-Thalassemia

β-thalassemia (β-thal) is one of the most common monogenic disorders worldwide, characterized by ineffective erythropoiesis leading to a chronic, debilitating anemia associated with high morbidity and mortality. Erythroid maturation is a dynamic process tightly regulated by complex signaling mechanisms, only partially described either in normal and diseased erythropoiesis. To investigate this issue, we carried out a high throughput kinome analysis by taking advantage of Kinexus array technology (http://www.kinexus.ca), in sorted erythroid precursors from a mouse model of β-thalassemia (Hbb3th/+) compared to wildtype animals. In β-thal mice, we found differential modulation of many protein kinases. Network computational analysis unveiled common as well as erythroid precursor-specific signaling mechanisms of altered erythrocyte differentiation in beta thalassemia, suggesting a selective perturbation in protein kinase/phosphatase balance in β-thal erythropoiesis.We reasoned that balancing kinome anomalies, by increasing phosphatome activity, could normalize kinome signaling pathways, thus ameliorating erythropoiesis. We explored the expression and function of different protein phosphatase and we found reduced expression and function of protein Tyr-phosphatase receptor type gamma (PTPRG). To investigate PTPRG role in erythropoiesis, we exploited a novel Trojan-fusion protein (TAT-ICD), we recently patented, that delivers intracellularly the catalytic domain of PTPRG and up-modulates its signaling cascade, as both a research tool to map dysfunctional pathways and as a potential therapeutic agent. In β-thal mice, TAT-ICD acted on multiple abnormally activated targets identified by computational analysis. TAT-ICD significantly reduced the activation of (i) Jak2- STAT5 pathway; (ii) Bruton tyrosine kinase (BTK), that has been reported to be part of the erythropoietin cascade; (iii) Akt, that is involved in TGF-β -SMAD signaling pathway. This was associated with down-regulation of Erfe and Gdf11 gene expression in sorted erythroblasts from TAT-ICD treated β-thal mice. Collectively, TAT-ICD treatment resulted in amelioration of β-thal ineffective erythropoiesis, evaluated by multiple approaches, including the profile of erythroid maturation and the amount of Annexin-V+ erythroid cells, reticulocyte count, circulating erythroblasts and hemolytic indices (U.S. Patent #62/109,555). The improvement of anemia was also associated with reduction in alpha aggregates and membrane bound hemichromes in circulating erythrocytes. We also found a reduction of liver and spleen iron accumulation in agreement with the beneficial effects on the hematologic phenotype. It is of note that TAT-ICD treatment did not affect either peripheral leukocyte counts or spleen lymphocyte pattern.Our data unveil abnormalities in signal transduction pathways as new mechanism involved in β-thal erythropoiesis, and validate a novel, breakthrough, therapeutic approach to reset back to homeostatic equilibrium altered kinome in diseased erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.

Beyond JAK-STAT: Contribution of Abi-1, STAT3, and NF-κB Signaling to Pathogenesis of Myeloproliferative Neoplasms

Activation of the JAK-STAT pathway is essential for the pathogenesis of primary myelofibrosis (PMF) and other myeloproliferative neoplasms (MPNs), but in the absence of curative treatment options, a major need remains to identify other targetable mechanisms in those diseases. We have previously shown that mice with conditional, inducible bone marrow-specific knockout of Abelson interactor 1 (Abi-1 KO) develop progressive myeloid hyperplasia, splenomegaly and bone marrow fibrosis leading to weight loss and decreased survival (Chorzalska, ASH 2016). Our data also indicated that hematopoietic progenitors from PMF patients show decreased levels of ABI1 transcript independent of the presence of JAK2 mutations. Abi-1 is one of several interactors of the Abelson kinase involved in Ras, PI3K and integrin signaling, and its deficiency in embryonic development leads to lethal defects of the heart and placenta. Despite suggestions that Abi-1 may act as a tumor suppressor, its involvement in hematopoiesis has not been elucidated.We have now characterized mechanisms that underlie the progressive PMF-like phenotype in our Abi-1 KO mice model. Colony-forming unit (CFU) assays indicated a significant decrease in the number of BFU-E colonies, a significant increase in CFU-GM colonies, and a nearly 3-fold increase in CFU-Mk colonies in the Abi-1 KO animals (Fig. 1A). Gene expression profiling studies showed a significant increase in the frequency of long-term hematopoietic stem cells (LT-HSCs) and HSCs enriched lineage- Sca-1+ cKit+ (LSK) cells in Abi-1 KO mice, associated with increased cell cycle activity in those cells: 3- to 4-fold higher expression of Mcm2 and Mki67 genes (specific to S phase and DNA replication), >6-fold upregulation of cMyb (specific for the G1/S phase transition), a 5-fold upregulation of M-phase specific Ccna1 and Ccnb1 and >15-fold upregulation of Cdk2, compared with wild-type (WT) animals. We also observed a 60 to 70% decrease in Cdkn1a, Cdkn1b, Cdkn2b (G1- or G2-phase-specific cell cycle inhibitors) transcripts in Abi-1 KO LT-HSCs (Fig. 1B). Investigation of cell cycle phases in HSC-enriched LSK cells using bromodeoxyuridine (BrdU) incorporation showed significantly more BrdU staining in cells from Abi-1 KO compared with WT mice, with a higher proportion of cells in S phase (Fig. 1C). Bone marrow transplantation assays done in C57BL/6 WT mice in the absence of competitor cells showed progressive loss of engraftment (decreased donor chimerism) of Abi-1 KO bone marrow cells over 6 months post transplant (Fig. 1D). Similarly, in competitive repopulation assays (C57BL/6 wild type mice engrafted with the whole bone marrow from Abi-1 KO or WT mice in 1:1 ratio with competitor, congenic CD45.2 WT cells), we observed a progressive decrease in donor chimerism with Abi1 inactivation (Fig. 1E).Using the Abi-1 KO model we then evaluated signaling pathways critical for the MPN pathophysiology, detecting only a modest increase in JAK2 and STAT5 activities (Fig. 1F). Immunoblotting analyses of the activities of Src Family Kinases (SFKs) showed increased phosphorylation on Tyr 416, consistent with activation of SFKs as well as increased activity of STAT3 and NF-κB (Fig. 1G). We confirmed these findings in CD34+ cell isolates from PMF patients (n=2), showing a decrease in Abi-1 protein levels and significant upregulation of SFKs, STAT3 and NF-κB activities (Fig. 1H).In summary, bone marrow-specific inactivation of Abi1 appears to be sufficient to induce a MPN-like phenotype, replicating features of human PMF. Results from gene expression profiling, immunoblotting, and proteomics consistently show that activation of SFKs, STAT3 and NF-κB may be involved in the genesis of this phenotype. We continue to explore the mechanisms responsible for the downregulation of ABI1 in hematopoietic progenitor cells from patients with PMF/MPN, as well as use of murine models with mutations in JAK2, CALR or MPL to determine mechanisms responsible for ABI1 loss and its association with classical MPN-associated pathways. Our work provides further evidence supporting a role for Abi-1 in pathogenesis of MPNs and a possible link to the SFK-STAT3-NF-κB signaling axis. Full elucidation of these mechanisms may couple the proliferative and pro-inflammatory phenomena that characterize PMF/MPNs, and provide a new therapeutic strategy for these diseases. [Display omitted] DisclosuresReagan:Teva: Membership on an entity's Board of Directors or advisory committees. Vannucchi:Shire: Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Monotcytes/Macrophages Are Involved in Erythrocytosis Caused By Both EPO-Dependent and -Independent Activation of EPOR

Excess activation of EPO receptor (EPOR) signaling leads to erythrocytosis, which is defined as an absolute increase of red cell mass. EPO-independent and erythrocytes-intrinsic EPOR activation is induced by gain-of-function EPOR or JAK2 mutations, resulting in “primary” erythrocytosis. Erythrocytes-extrinsic EPOR activation is called “secondary” erythrocytosis, which is caused by EPOR stimulation with high level of EPO.In order to investigate primary and secondary erythrocytosis in detail, we generated two unique mouse models. LysM -Cre; Jak2 V617F knock-in heterozygous mice express constitutively active JAK2 in LysM-expressing cells, which include a small portion of HSCs and their downstream progenitors. We also generated a Alb -Cre; Rosa26 LoxP-Stop-LoxP-rtTA (LSL-rtTA);human EPAS1 double-point-mutant (DPM) mice (Alb -Cre;DPM). EPAS1 is a master regulator of EPO gene. The EPAS1-DPM has mutations in P531 and N847 sites, which are critical sites for stability and transcriptional inactivation in normoxia. This mouse expresses the DPM mutant (P531A and N847A) specifically in hepatocytes in a doxycycline-inducible manner, resulting in high EPO production in the liver. We used LysM -Cre; Jak2 V617F as an erythrocytes-intrinsic “primary” erythrocytosis model, and Alb -Cre;DPM as an erythrocytes-extrinsic “secondary” erythrocytosis model. Interestingly, both of them developed comparable degree of erythrocytosis quickly. The plasma EPO level in LysM -Cre; Jak2 V617F was significantly decreased, while that in Alb -Cre/DPM was significantly increased in comparison to the control and also to LysM -Cre; Jak2 V617F. They both exhibited splenomegaly, but spleens of Lys M-Cre; Jak2 V617F mice were significantly larger than those of Alb -Cre;DPM mice. In Alb -Cre;DPM mice, CD45- CD71+ erythroblasts were detected in the liver, whereas there were almost no erythroblasts in the liver of the control mice, suggesting that extremely high level of EPO expression in the liver causes erythropoiesis in the liver of Alb -Cre;DPM mice.Increased monocyte count has been reported in patients with erythrocytosis. Consistent with the observation in the patients, total CD11b+ CD115+ monocytes were significantly increased in PB from both LysM -Cre; Jak2 V617F and Alb -Cre;DPM mice compared to those in PB from the wild-type mice. It is also well known that macrophages play a crucial role in erythropoiesis, supplying the iron and engulfing the protruded nuclei from erythroblasts in the terminal maturation process. The complex of erythroblasts and macrophages directly interacting each other is called as “erythroblastic island (EBI)”. Thus, we next analyzed macrophages in bone marrow (BM) and spleen (SP) in our erythrocytosis models. Macrophages in SP were dramatically increased in the both models, while those in BM were not significantly altered. The percentage of EBI (multiplet F4/80+ Ter119+) in BM and SP were increased in both models.EPOR expression on the cell surface is required for EPO-EPOR signaling. To confirm the expression of EPOR by genetic approach, we combined EPOR -Cre knock-in allele and Rosa26LSL-GFP reporter allele to generate EPOR -Cre;GFP reporter mice. In this model, pro-erythroblasts and basophilic erythroblasts in BM expressed GFP as expected. We did not find GFP expression on the Macrophage and dendritic cell precursors (MDPs), common monocyte progenitors (cMoPs), mature monocytes in BM and PB monocytes. In a single-cell-gated macrophage population, we found a small fraction expressing GFP. However, FACS sorting revealed that those GFP+ cells are not macrophages alone, but macrophages with erythroblasts adhered to them. Even after adding EDTA, some of those macrophages still retain bound erythroblasts. It is possible that they could acquire activated EPOR signaling from erythroblasts within EBI through direct interaction. We are currently investigating the mechanisms for expansion of monocytes/macrophages in erythrocytosis.Collectively, here we demonstrate that both Jak2V617F and high EPO could activate EPOR signaling and expand erythroblasts and monocytes/macrophages, which might contribute to erythrocytosis phenotype in our primary and secondary erythrocytosis models. Further exploration of the roles of monocytes/macrophages in erythrocytosis could lead to development of novel therapeutic strategy for erythrocytosis by modulating their functions in EBI. DisclosuresNo relevant conflicts of interest to declare.

STAT3 activation in thymic epithelial tumors: correlation with cyclin D1, JAK3, and clinical behavior.

Thymic epithelial tumors are the most common adult mediastinal tumors; however, their growth mechanism remains relatively unknown. Among the JAK/STAT pathway-related proteins, which control various intracellular events, STAT3 is deeply involved in cell proliferation. Constitutive activation of STAT3 and the resulting overexpression of cyclin D1 have been confirmed in various tumors, but have not been thoroughly investigated in thymic epithelial tumors. In this study, we immunohistochemically examined STAT3 activation, cyclin D1 expression, and JAK3 activation in thymic epithelial tumors and statistically analyzed their correlation with clinicopathological features. Formalin-fixed paraffin-embedded specimens of 94 thymic epithelial tumors surgically resected at Kyorin University Hospital between 2005 and 2018 were included in this study. pSTAT3, cyclin D1, and pJAK3 were immunohistochemically examined, and the correlation with histology, Masaoka stage, and survival time was statistically analyzed. Cyclin D1 was found to be significantly overexpressed in the STAT3-activated group. This phenomenon was associated with histology and Masaoka stage. JAK3 was also activated in thymic epithelial tumors; however, JAK3 and STAT3 activation were not always correlated. Using survival time analysis, the STAT3-activated group, cyclin D1-expressed group, and JAK3-activated group had significantly lower progression-free survival times than those for both the non-activated and non-expressed groups. STAT3 activation may promote cyclin D1 overexpression in thymic epithelial tumors, and intracellular signaling pathways other than JAK3 may be involved in STAT3 activation. STAT3 activation, cyclin D1 overexpression, and JAK3 activation are biomarker candidates that indicate clinically poor prognosis.

113-OR: Identification of Sorafenib for the Treatment of Type 1 Diabetes

Type 1 diabetes (T1D) is an immune-mediated disease characterized by impairment of pancreatic beta cells. To date, a comprehensive cure is not available. Th1 cell activation is considered a key mediator of the pathogenesis of T1D. IL-12-induced STAT4 activation is a paramount signaling pathway to instigate Th1 cell differentiation. Targeting IL-12-induced Th1 cell differentiation seems to be an effective way to block the development of T1D. However, given the critical function of Th1 in the immune system, the potential side effects hinder the application of anti-Th1 therapy in the treatment of T1D. To identify safe anti-Th1 treatment(s), we screened the FDA-approved tyrosine kinase inhibitor (TKI) drug library using an IL-12-induced Th1 differentiation cell model. We found that sorafenib, an oral anti-cancer drug, is a top contender for inhibition of Th1 differentiation relative to induced toxicity. Sorafenib inhibited IL-12-induced Th1 cell differentiation in a dose-dependent manner without any change of CD4+ T-cell viability. Treatment of NOD mice with sorafenib significantly impeded the development of T1D and ameliorated insulitis, which coincided with a specifically decreased aggregate of Th1 cell population in pancreas but not in peripheral immune organs. Inflammatory cytokines and Th1 signature transcription factor Tbet mRNA expression in pancreas were decreased in sorafenib-treated NOD mice compared to vehicle-treated NOD mice. Mechanistically, sorafenib directly inhibited JAK2 activity and blocked IL-12-induced phosphorylations of JAK2 and STAT4. In conclusion, sorafenib administration inhibits the development of T1D by suppressing IL-12b/JAK2/STAT4 signaling-mediated Th1 cell differentiation specifically in pancreas in NOD mice. Since sorafenib is classified as an FDA-approved drug, it serves as a preliminary lead point for additional experimentation and may be a promising therapeutic for T1D in humans. Disclosure Q. Zeng: None. J. Song: None. X. Sun: None. D. Wang: None. Y. Xiao: None. Z. Zhou: None. T. Deng: None.

Selected Articles from This Issue

Donor T cell costimulation and IL-6 cytokine activation mediate acute graft-versus-host disease (GVHD) via mTOR and JAK2 signaling pathways, respectively. In this first-in-human phase I trial, Pidala and colleagues show that the JAK2 inhibitor, pacritinib, plus sirolimus and low-dose tacrolimus (PAC/SIR/TAC) is safe and demonstrates preliminary efficacy in GVHD prevention after allogenic hematopoietic cell transplantation. PAC/SIR/TAC ablates aberrant JAK2 and mTOR activity in donor T cells, polarizes regulatory T-cell responses, and avoids cytopenia observed with pan JAK inhibitors. PAC/SIR/TAC is a promising GVHD prophylaxis regimen to achieve immune tolerance while maintaining anti-leukemia responses from NK cells and cytotoxic T lymphocytes.BAY1436032 is an inhibitor of mIDH1 that increases survival and induces markers of differentiation in animal models of mIDH1 glioma. Wick and colleagues describe the results of a first-in-human phase I clinical study of BAY1436032 in subjects with solid tumors. BAY1436032 was well-tolerated, demonstrated target inhibition, and showed evidence of clinical activity including durable objective responses in a small subset of heavily pretreated subjects with mIDH1 lower-grade glioma. These findings support the continued clinical evaluation of mIDH1 inhibitors in this patient population and indicate that some mIDH1 solid tumors may be susceptible to differentiation therapy.ATRT-MYC, ATRT-SHH, and ATRT-TYR are three molecular group. of atypical teratoid rhabdoid tumor (ATRT). However, the relationship between molecular group and outcome has not been studied prospectively. In this study, Upadhyaya and colleagues utilized two prospective multi-institutional clinical trials to assess the contribution of molecular group to outcome in children with newly diagnosed ATRT. Infants with ATRT-TYR tumors were likely to have localized disease and improved prognosis, while children with ATRT-SHH tumors were more likely to have metastatic disease and poor outcome. Germline mutations in SMARCB1 were associated with metastatic disease, but not progression-free survival. These results support the use of molecular grouping in future trials of children with ATRT.Total neoadjuvant therapy (TNT) is currently used for patients with locally advanced rectal cancer (LARC) and is often followed by a watch-and-wait approach. However, many patients will develop metastasis and succumb to their disease. Vidal and colleagues assess the role of circulating tumor (ct) DNA before rectal surgery to predict response to TNT, relapse, and survival in LARC patients in the GEMCAD 1402 trial. Using an ultrasensitive ctDNA assay, detectable presurgery ctDNA was significantly associated with systemic recurrence, shorter disease-free survival, and shorter overall survival. The authors propose future clinical trials using ctDNA to personalize treatment for patients with LARC.

Recent Advances in the Use of Molecular Analyses to Inform the Diagnosis and Prognosis of Patients with Polycythaemia Vera.

Genetic studies in the past decade have improved our understanding of the molecular basis of the BCR-ABL1-negative myeloproliferative neoplasm (MPN) polycythaemia vera (PV). Such breakthroughs include the discovery of the JAK2V617F driver mutation in approximately 95% of patients with PV, as well as some very rare cases of familial hereditary MPN caused by inherited germline mutations. Patients with PV often progress to fibrosis or acute myeloid leukaemia, both associated with very poor clinical outcome. Moreover, thrombosis and major bleeding are the principal causes of morbidity and mortality. As a result of increasingly available and economical next-generation sequencing technologies, mutational studies have revealed the prognostic relevance of a few somatic mutations in terms of thrombotic risk and risk of transformation, helping to improve the risk stratification of patients with PV. Finally, knowledge of the molecular basis of PV has helped identify targets for directed therapy. The constitutive activation of the tyrosine kinase JAK2 is targeted by ruxolitinib, a JAK1/JAK2 tyrosine kinase inhibitor for PV patients who are resistant or intolerant to cytoreductive treatment with hydroxyurea. Other molecular mechanisms have also been revealed, and numerous agents are in various stages of development. Here, we will provide an update of the recent published literature on how molecular testing can improve the diagnosis and prognosis of patients with PV and present recent advances that may have prognostic value in the near future.

JAK1: Number one in the family; number one in inflammation?

Several cytokines involved in inflammatory pathologies signal via the Janus kinase-signal transducer and activator of transcription pathway. Four JAKs are known: JAK1, JAK2, JAK3 and TYK2. The specific activation of JAKs and STATs determines the biological effects of each cytokine. JAK1 is involved in the signalling of 'γc' receptor cytokines (IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21), pro-inflammatory cytokines including IL-6, as well as IFN. The critical position of JAK1 downstream of these cytokines suggests that JAK1-selective inhibitors are comparable to non-selective ones, without the unwanted consequences of JAK2- or JAK3-blockade. JAK inhibition has led to a better understanding of the biology of synovial inflammation and bone homeostasis. Moreover, the efficacy of non-selective JAK inhibitors and novel JAK1-selective drugs in RA supports a role for JAK1 in its pathogenesis. JAK1-selective drugs are also showing promise in axial spondyloarthritis, suggesting that they may target additional regulatory pathways that impact cytokines such as TNF and IL-17A, which do not use JAKs. Additionally, evidence now supports a JAK1 predominance in the signalling of IL-6 and oncostatin M, and indirectly, of TNF in synovial fibroblasts, macrophages and endothelial cells. Notably, bone homeostasis is also dependent on cytokines relying on JAK1 signalling to promote receptor activator of NF-κB ligand expression in osteoblasts and T cells, contributing to osteoclastogenesis. Here, the contribution of JAK1 over other kinases is unclear. While beneficial effects of JAK inhibitors on bone erosion are supported by preclinical and clinical data, effects on new bone formation in axial spondyloarthritis requires additional study.