Cholinergic anti-inflammatory pathway ameliorates murine experimental Th2-type colitis by suppressing the migration of plasmacytoid dendritic cells

Ulcerative colitis (UC) is a chronic inflammatory bowel disease. Several studies have demonstrated that α7 nicotinic acetylcholine receptors (α7nAChRs) exert anti-inflammatory effects on immune cells and nicotine suppress UC onset and relapse. Plasmacytoid dendritic cells (pDCs) reportedly accumulate in the colon of UC patients. Therefore, we investigated the pathophysiological roles of α7nAChRs on pDCs in the pathology of UC using oxazolone (OXZ)-induced Th2-type colitis with BALB/c mice. 2-deoxy-D-glucose, a central vagal stimulant suppressed OXZ colitis, and nicotine also ameliorated OXZ colitis with suppressing Th2 cytokines, which was reversed by α7nAChR antagonist methyllycaconitine. Additionally, α7nAChRs were expressed on pDCs, which were located very close to cholinergic nerve fibers in the colon of OXZ mice. Furthermore, nicotine suppressed CCL21-induced bone marrow-derived pDC migration due to Rac 1 inactivation, which was reversed by methyllycaconitine, a JAK2 inhibitor AG490 or caspase-3 inhibitor AZ-10417808. CCL21 was mainly expressed in the isolated lymphoid follicles (ILFs) of the colon during OXZ colitis. The therapeutic effect of cholinergic pathway on OXZ colitis probably through α7nAChRs on pDCs were attributed to the suppression of pDC migration toward the ILFs. Therefore, the activation of α7nAChRs has innovative therapeutic potential for the treatment of UC.

Jak2 Inhibitor AG490 Improved Poststroke Central and Peripheral Inflammation and Metabolic Abnormalities in a Rat Model of Ischemic Stroke

Poststroke hyperglycemia and inflammation have been implicated in the pathogenesis of stroke. Janus Kinase 2 (Jak2), a catalytic signaling component for cytokine receptors such as Interleukin-6 (IL-6), has inflammatory and metabolic properties. This study aimed to investigate the roles of Jak2 in poststroke inflammation and metabolic abnormality in a rat model of permanent cerebral ischemia. Pretreatment with Jak2 inhibitor AG490 ameliorated neurological deficit, brain infarction, edema, oxidative stress, inflammation, caspase-3 activation, and Zonula Occludens-1 (ZO-1) reduction. Moreover, in injured cortical tissues, Tumor Necrosis Factor-α, IL-1β, and IL-6 levels were reduced with concurrent decreased NF-κB p65 phosphorylation, Signal Transducers and Activators of Transcription 3 phosphorylation, Ubiquitin Protein Ligase E3 Component N-Recognin 1 expression, and Matrix Metalloproteinase activity. In the in vitro study on bEnd.3 endothelial cells, AG490 diminished IL-6-induced endothelial barrier disruption by decreasing ZO-1 decline. Metabolically, administration of AG490 lowered fasting glucose, with improvements in glucose intolerance, plasma-free fatty acids, and plasma C Reactive Proteins. In conclusion, AG490 improved the inflammation and oxidative stress of neuronal, hepatic, and muscle tissues of stroke rats as well as impairing insulin signaling in the liver and skeletal muscles. Therefore, Jak2 blockades may have benefits for combating poststroke central and peripheral inflammation, and metabolic abnormalities.

A Novel ELISA-Based Peptide Biosensor Assay for Screening ABL1 Activity in vitro: A Challenge for Precision Therapy in BCR-ABL1 and BCR-ABL1 Like Leukemias

The pathogenic role of the overactivated ABL1 tyrosine kinase (TK) pathway is well recognized in some forms of BCR-ABL1 like acute lymphoblastic leukemia (ALL); TK inhibitors represent a useful therapeutic choice in these patients who respond poorly to conventional chemotherapy. Here we report a novel peptide biosensor (PABL)-ELISA assay to investigate ABL1 activity in four immortalized leukemic cell lines with different genetic background. The PABL sequence comprises an ABL1 tyrosine (Y) phosphorylation site and a targeting sequence that increases the specificity for ABL1; additional peptides (Y-site-mutated (PABL-F) and fully-phosphorylated (PPHOSPHO-ABL) biosensors) were included in the assay. After incubation with whole cell lysates, average PABL phosphorylation was significantly increased (basal vs. PABL phosphorylation: 6.84 ± 1.46% vs. 32.44 ± 3.25%, p-value < 0.0001, two-way ANOVA, Bonferroni post-test, percentages relative to PPHOSPHO-ABL in each cell line). Cell lines expressing ABL1-chimeric proteins (K562, ALL-SIL) presented the higher TK activity on PABL; a lower signal was instead observed for NALM6 and REH (p < 0.001 and p < 0.05 vs. K562, respectively). Phosphorylation was ABL1-mediated, as demonstrated by the specific inhibition of imatinib (p < 0.001 for K562, NALM6, ALL-SIL and p < 0.01 for REH) in contrast to ruxolitinib (JAK2-inhibitor), and occurred on the ABL1 Y-site, as demonstrated by PABL-F whose phosphorylation was comparable to basal levels. In order to validate this novel PABL-ELISA assay on leukemic cells isolated from patient’s bone marrow aspirates, preliminary analysis on blasts derived from an adult affected by chronic myeloid leukaemia (BCR-ABL1 positive) and a child affected by ALL (BCR-ABL1 negative) were performed. Phosphorylation of PABL was specifically inhibited after the incubation of BCR-ABL1 positive cell lysates with imatinib, but not with ruxolitinib. While requiring further optimization and validation in leukemic blasts to be of clinical interest, the PABL-based ELISA assay provides a novel in vitro tool for screening both the aberrant ABL1 activity in BCR-ABL1 like ALL leukemic cells and their potential response to TK inhibitors.

DUSP6 Mediates Resistance to JAK2 Inhibition and Drives Myeloproliferative Neoplasm Disease Progression

Myeloproliferative neoplasms (MPNs) are clonally derived from hematopoietic stem/progenitor cells (HSPCs) and typically harbor somatic mutations in one of three genes (JAK2, CALR, MPL) leading to aberrant activation of JAK-STAT signaling. While small molecule inhibitors of JAK2 provide symptomatic benefit for MPN patients, they do not eradicate the underlying malignant clone, nor do they prevent disease progression. Chronic MPNs exhibit a propensity for transformation to secondary acute myeloid leukemia (sAML), for which the underlying mechanisms remain poorly understood, resulting in limited treatment options and dismal clinical outcomes. To understand alterations to the transcriptional landscape underlying MPN disease progression, we performed bulk transcriptome profiling on myelofibrosis (MF) and sAML patient CD34+ HSPCs. Differential gene expression analysis revealed upregulation of dual-specificity phosphatase 6 (DUSP6), which encodes a MAPK phosphatase that regulates ERK signaling, in sAML CD34+ cells. Elevated DUSP6 protein expression accompanying disease progression was confirmed via MPN patient bone marrow immunofluorescence and imaging mass cytometry analysis. We performed further single cell RNA sequencing (scRNA-seq) in conjunction with TotalSeq surface protein marker detection on more than 50,000 sorted CD34+ cells of serial samples from three patients at chronic MPN and sAML stages, and two healthy controls, which revealed DUSP6 among the top 21 genes elevated in all three paired samples across disease progression. Subsequent differentiation trajectory pseudotime analysis demonstrated concomitant elevation of DUSP6 across state trajectories and disease progression. Genetic and pharmacologic targeting of DUSP6 followed by biochemical and mass cytometry analysis identified signaling inhibition through S6 and JAK/STAT, establishing them as novel, non-canonical effectors of DUSP6. DUSP6 inhibition also led to potent suppression of cell proliferation, induction of apoptosis and cell cycle arrest, and reduction of inflammatory cytokine production in primary MPN samples. Furthermore, ectopic DUSP6 expression augmented proliferation and mediated JAK2 inhibitor resistance, while DUSP6 inhibition reduced colony-forming potential of JAK2 inhibitor-persistent patient cells. Mechanistically, DUSP6 suppression dampened S6 signaling via inhibition of RSK1 (RPS6KA1), which we identified as a second indispensable candidate associated with poor clinical outcome via Kaplan-Meier overall survival (Log-rank p = 0.0005) and multivariate (RPS6KA1 expression hazard ratio = 1.60, 95% confidence interval: 1.10, 2.34) analyses of the TCGA LAML cohort. Strong correlation was observed (r = 0.68; p = 0.0009) between RPS6KA1 and DUSP6 expression in CD34+ HSPCs, and pharmacologic inhibition of RSK1 with BI-D1870 suppressed proliferation and colony formation across AML cell lines and primary samples. DUSP6 inhibition in vivo via small molecule inhibitor BCI resolved pathologically elevated hematocrit and white blood cell counts and reduced splenomegaly in Jak2 V617F knock-in mice. In the MPL W515L retroviral transplant model, BCI suppressed leukocytosis while reducing reticulin fibrosis and prolonging survival. In patient-derived xenograft (PDX) model of NSGS mice engrafted with sAML patient CD34+ cells, BCI treatment or DUSP6 knockdown reduced peripheral blood hCD45+ engraftment. Importantly, BCI treatment did not pathologically cause cytopenias or decrease spleen weights in wild-type mice, nor did it reduce hCD45+ engraftment in NSGS PDX mice engrafted with healthy donor CD34+ cells. Lastly, NSGS PDX mice engrafted with MF patient CD34+ cells ectopically expressing DUSP6 demonstrated marked leukocytosis, splenomegaly, and early lethality. These findings underscore DUSP6 in driving MPN disease progression and therapeutic resistance, and highlight the DUSP6-RSK1 axis as a novel, druggable pathway in myeloid malignancies. Figure 1 Figure 1. Disclosures Oh: Abbvie: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; Celgene Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Constellation: Membership on an entity's Board of Directors or advisory committees; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Membership on an entity's Board of Directors or advisory committees; Geron: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Kartos Therapeutics: Membership on an entity's Board of Directors or advisory committees; PharamaEssentia: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Membership on an entity's Board of Directors or advisory committees.

Targeting Metabolic Dysregulation for Ruxolitinib Failure in MPN

Background:Metabolic alterations are increasingly recognized as an important pathogenic process that underlies fibrosis and cancer cells. MPN models were demonstrated to exhibit metabolic vulnerabilities due to a high dependence on enhanced levels of glycolysis and oxidative phosphorylation. MPN patients often gain weight during ruxolitinib treatment effects on leptin signaling. Ruxolitinib provides symptomatic relief and can improve survival but generally fails to resolve the malignant clone,nealy 50% patients resistance to ruxolitinib.Here,we will study transcriptomic and metabolomic program in JAK2-mutant MPN who resistance to ruxolitinib. Methods:The collection of blood samples and clinical data from MPN patients, Mice used in this study were kept in accordance with Swiss federal regulations. Multi-omics detection by transcriptome sequencing and liquid chromatography . Results: We found that active lipid metabolism obviously in patients resistance to ruxolitinib in comparison with patients sensitive to ruxolitinib.Transcriptomic and metabolomic analyses identified numerous metabolic nodes in JAK2-mutant hematopoietic stem and progenitor cells that were altered in comparison with wild-type controls. We studied the consequences of key regulatory enzyme of Fatty acid metabolism gene(SREBP,FASN, ACC, SCD1) and found that pharmacological inhibition of SREBP(Betulin) reduced hematopoietic manifestations of MPNs. Conclusion: Our findings reveal the contribution of metabolic alterations to JAK2 inhibitor failure and suggest that abnormally active lipid metabolism of mutant cells represent vulnerabilities that can be targeted for treating MPNs. Keywords: metabolomic;resistance;resistance;Myeloproliferative Disclosures No relevant conflicts of interest to declare.

Recent Advances in Molecular Diagnostics and Targeted Therapy of Myeloproliferative Neoplasms

Somatic mutations in JAK2, calreticulin, and MPL genes drive myeloproliferative neoplasms (MPN), and recent technological advances have revealed a heterogeneous genomic landscape with additional mutations in MPN. These mainly affect genes involved in epigenetic regulation and splicing and are of diagnostic and prognostic value, predicting the risk of progression and informing decisions on therapeutic management. Thus, genetic testing has become an integral part of the current state-of-the-art laboratory work-up for MPN patients and has been implemented in current guidelines for disease classification, tools for prognostic risk assessment, and recommendations for therapy. The finding that JAK2, CALR, and MPL driver mutations activate JAK2 signaling has provided a rational basis for the development of targeted JAK2 inhibitor therapies and has fueled their translation into clinical practice. However, the disease-modifying potential of JAK2 inhibitors remains limited and is further impeded by loss of therapeutic responses in a substantial proportion of patients over time. Therefore, the investigation of additional molecular vulnerabilities involved in MPN pathogenesis is imperative to advance the development of new therapeutic options. Combination of novel compounds with JAK2 inhibitors are of specific interest to enhance therapeutic efficacy of molecularly targeted treatment approaches. Here, we summarize the current insights into the genetic basis of MPN, its use as a diagnostic and prognostic tool in clinical settings, and the most recent advances in targeted therapies for MPN.

The JAK2 inhibitor TG101209 exhibits anti-tumor and chemotherapeutic sensitizing effects on Burkitt lymphoma cells by inhibiting the JAK2/STAT3/c-MYB signaling axis

Constitutive activation of JAK2/STAT3 is a major oncogenic signaling event involved in the development of Burkitt lymphoma (BL). In the present study, we investigated the antilymphoma activity of TG101209, a specific JAK2 inhibitor, on EBV-positive and EBV-negative Burkitt lymphoma cell lines and primary BL cells. The results showed that TG101209 had a significant antilymphoma effect by inhibiting BL cell growth and inducing apoptosis along with cell differentiation toward mature B cells in vitro. We also found that TG101209 displayed significant synergistic action and a sensitizing effect on the anti-Burkitt lymphoma activity of doxorubicin. In vivo experiments indicated that TG101209 could suppress tumor growth and prolong the overall survival of BL cell-bearing mice. The mechanistic study indicated that TG101209, by suppressing the JAK2/STAT3/c-MYB signaling axis and crosstalk between the downstream signaling pathways, plays an antilymphoma role. These data suggested that TG101209 may be a promising agent or alternative choice for the treatment of BL.

Arbutin Ameliorates Murine Colitis by Inhibiting JAK2 Signaling Pathway

Background and objective: Abnormal activation of Janus kinase 2 (JAK2) promotes the pathogenesis and progress of inflammatory bowel disease (IBD) by stimulating the cytokine traffic. Based on docking studies, arbutin, a natural product extracted from a traditional medicinal plant bearberry, was found to bind to JAK2. The study aimed to investigate the effects and mechanisms of regulating JAK2 by arbutin on colitis in mice.Methods: A mice colitis model was established to mimic human IBD. The mice freely drank water containing dextran sulfate sodium. Inflammation in epithelial (IEC6) and immune (RAW264.7) cells was analyzed following treatment with lipopolysaccharides (LPS).Results: Colitis symptoms, including body weight loss, increased disease activity index, and increased colon weight/length ratio, were significantly alleviated by arbutin. Mediators of colonic pro-inflammatory cytokines as well as apoptosis markers in colitis were suppressed by the glycoside. High expression of phosphorylated JAK2 in colitis was significantly reversed by arbutin. The effects of arbutin treatment on colitis were considerably inhibited by the JAK2 inhibitor AG490. LPS-induced inflammatory responses were also suppressed by arbutin, which was notably inhibited by the JAK2 inhibitor AG490.Conclusion: The findings obtained herein suggest the protective role of arbutin and provide novel insights into alternative colitis treatments, which involve inhibition of the JAK2 signaling pathway.

CAMK2G is identified as a novel therapeutic target for myelofibrosis

Although JAK1/2 inhibition is effective into alleviating symptoms of myelofibrosis (MF), it does not result in the eradication of MF clones, which can lead to inhibitor-resistant clones emerging during the treatment. Here we established iPS cells derived from MF patient samples (MF-iPSCs) harboring JAK2 V617F, CALR type 1, or CALR type 2 mutations. We demonstrated that these cells faithfully recapitulate the drug sensitivity of the disease. These cells were utilized for chemical screening and calcium/calmodulin-dependent protein kinase 2 (CAMK2) was identified as a promising therapeutic target. MF model cells and mice induced by MPL W515L, another type of mutations recurrently detected in MF patients were used to elucidate the therapeutic potential of CAMK2 inhibition. CAMK2 inhibition was effective against JAK2 inhibitor-sensitive and JAK2 inhibitor-resistant cells. Further research revealed CAMK2 gamma subtype was important in MF model cells induced by MPL W515L. We showed that CAMK2G hetero knockout in the primary bone marrow cells expressing MPL W515Ldecreased colony-forming capacity. CAMK2G inhibition with berbamine, a CAMK2G inhibitor, significantly prolonged survival and reduced disease phenotypes such as splenomegaly and leukocytosis in a MF mouse model induced by MPL W515L. We investigated the molecular mechanisms underlying the therapeutic effect of CAMK2G inhibition and found that CAMK2G is activated by MPL signaling in MF model cells and is an effector in the MPL-JAK2 signaling pathway in these cells. These results indicate CAMK2G plays an important role in MF, and CAMK2G inhibition may be a novel therapeutic strategy that overcomes resistance to JAK1/2 inhibition.