JAK2 Signaling Research Articles

Fluid Shear Stress Ameliorates Prehypertension-Associated Decline in Endothelium-Reparative Potential of Early Endothelial Progenitor Cells.

This study investigated the effects of prehypertension and shear stress on the reendothelialization potential of human early EPCs and explored its potential mechanisms. Early EPCs from the prehypertensive patients showed reduced migration and adhesion in vitro and demonstrated a significantly impaired in vivo reendothelialization capacity. Shear stress pretreatment markedly promoted the in vivo reendothelialization capacity of EPCs. Although basal CXCR4 expression in early EPCs from prehypertensive donors was similar to that from healthy control, SDF-1-induced phosphorylation of CXCR4 was lower in prehypertensive EPCs. Shear stress up-regulated CXCR4 expression and increased CXCR4 phosphorylation, and restored the SDF-1/CXCR4-dependent JAK-2 phosphorylation in prehypertensive EPCs. CXCR4 knockdown or JAK-2 inhibitor treatment prevents against shear stress-induced increase in the migration, adhesion and reendothelialization capacity of the prehypertensive EPCs. Collectively, CXCR4 receptor profoundly modulates the reendothelialization potential of early EPCs. The abnormal CXCR4-mediated JAK-2 signaling may contribute to impaired functions of EPCs from patients with prehypertension.

Effect of hydroxychloroquine and chloroquine on syncytial differentiation and autophagy in primary human trophoblasts

During placentation, cytotrophoblasts differentiate and fuse to form multinucleated cells (syncytiotrophoblasts) in a process that involves autophagy. Appropriate syncytial differentiation is essential for establishing a healthy pregnancy. In this study, we evaluated the effect of two chloroquine compounds, hydroxychloroquine (HCQ) and chloroquine (CQ), on syncytial differentiation and autophagy in cultured primary human trophoblasts (PHTs). PHT cells were isolated from the human term placenta. Bafilomycin, a well-known autophagy inhibitor, was used as a positive control. Biochemical and morphological differentiation was assessed in syncytiotrophoblasts, and autophagy-related proteins and genes were evaluated. Affymetrix Human Gene 2.0 ST Array profiling was used to identify genes affected by HCQ during syncytial differentiation. Chloroquine compounds lowered the production of beta-human chorionic gonadotropin (β-hCG) and the fusion index in PHTs. Syncytial differentiation in PHT was associated with the increased expression of ATG4C mRNA (autophagy-related gene), and this expression was affected by CQ but not by HCQ. Microarray analysis revealed that HCQ or CQ affected several genes (MMP15, GPC3, CXCL10, TET-1, and S100A7) during syncytial differentiation, which were different from that of the syncytial differentiation suppression (Ham’s/Waymouth media) or autophagy inhibition (bafilomycin treatment). Using Kyoto Encyclopedia of Genes and Genomes analysis we identified that HCQ might affect JAK2 signaling in the syncytial differentiation of PHT. In conclusion, chloroquine compounds could mitigate biochemical and morphological syncytial trophoblast differentiation in cultured PHT cells through the JAK signaling pathway rather than the inhibition of autophagic activity.

SARS-CoV-2 NSP13 helicase suppresses interferon signaling by perturbing JAK1 phosphorylation of STAT1

BackgroundSARS-CoV-2 is the causative agent of COVID-19. Overproduction and release of proinflammatory cytokines are the underlying cause of severe COVID-19. Treatment of this condition with JAK inhibitors is a double-edged sword, which might result in the suppression of proinflammatory cytokine storm and the concurrent enhancement of viral infection, since JAK signaling is essential for host antiviral response. Improving the current JAK inhibitor therapy requires a detailed molecular analysis on how SARS-CoV-2 modulates interferon (IFN)-induced activation of JAK-STAT signaling.ResultsIn this study, we focused on the molecular mechanism by which SARS-CoV-2 NSP13 helicase suppresses IFN signaling. Expression of SARS-CoV-2 NSP13 alleviated transcriptional activity driven by type I and type II IFN-responsive enhancer elements. It also prevented nuclear translocation of STAT1 and STAT2. The suppression of NSP13 on IFN signaling occurred at the step of STAT1 phosphorylation. Nucleic acid binding-defective mutant K345A K347A and NTPase-deficient mutant E375A of NSP13 were found to have largely lost the ability to suppress IFN-β-induced STAT1 phosphorylation and transcriptional activation, indicating the requirement of the helicase activity for NSP13-mediated inhibition of STAT1 phosphorylation. NSP13 did not interact with JAK1 nor prevent STAT1-JAK1 complex formation. Mechanistically, NSP13 interacted with STAT1 to prevent JAK1 kinase from phosphorylating STAT1.ConclusionSARS-CoV-2 NSP13 helicase broadly suppresses IFN signaling by targeting JAK1 phosphorylation of STAT1.

JAK and STAT gene mutations and JAK-STAT pathway activation in lympho- and myeloproliferative neoplasms

Lympho- and myeloproliferative neoplasms are a very heterogeneous group of haematological malignancies originating from a haematopoietic stem cell (HSC). In most of them, the neoplastic transformation is a result of the acquisition of molecular defects by HSC or progenitor cells, impairing their proliferation, differentiation and maturation. Herein, the role of the Janus kinase-signal transduction and transcription activation (JAK-STAT) signalling pathway in the normal and neoplastic lympho- and myelopoiesis is presented. Particular attention is paid to the molecular aberrations of the JAK and STAT genes and their impact on JAK and STAT signalling pathway function and the mutation-driven mechanism of the lymphoid and myeloid cells neoplastic transformation. In the Authors’ opinion, its early identification allows to incorporate the molecularly targeted drugs, including JAK-STAT pathway signalling inhibitors, to the therapeutic algorithm used and to improve the treatment results of lymphoid- and myeloid neoplasms.

Understanding Aberrant Signaling to Elude Therapy Escape Mechanisms in Myeloproliferative Neoplasms.

Simple SummaryMyeloproliferative neoplasms are a group of rare disorders characterized by genetic mutations in hematopoietic stem cells and by the presence of systemic inflammation. The main driver mutations causing these diseases converge in activating the JAK2 signal transduction pathway, which plays a major role in disease onset and maintenance. Treatments based on JAK2 inhibitors ameliorate symptoms without suppressing the disease. This depends on the reactivation of JAK2 signaling and on the emergence of alternative pathways also sustained by inflammatory mediators. Molecular mechanisms at the basis of disease persistence and new therapeutic attempts to overcome them are discussed in the review.Aberrant signaling in myeloproliferative neoplasms may arise from alterations in genes coding for signal transduction proteins or epigenetic regulators. Both mutated and normal cells cooperate, altering fragile balances in bone marrow niches and fueling persistent inflammation through paracrine or systemic signals. Despite the hopes placed in targeted therapies, myeloid proliferative neoplasms remain incurable diseases in patients not eligible for stem cell transplantation. Due to the emergence of drug resistance, patient management is often very difficult in the long term. Unexpected connections among signal transduction pathways highlighted in neoplastic cells suggest new strategies to overcome neoplastic cell adaptation.

Glycerophospholipid metabolism is involved in rheumatoid arthritis pathogenesis by regulating the IL-6/JAK signaling pathway

To explore the metabolic mechanism of differential plasma interleukin (IL)-6 expression in patients with rheumatoid arthritis (RA). A total of 240 RA patients were enrolled in the non-target metabolomics study cohort and 69 healthy volunteers were included as healthy controls (HCs). Plasma IL-6 levels were detected by electrochemiluminescence assay. Plasma metabolites were detected by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Patients with active RA (n = 20) and remissive RA (n = 20) and 20 HCs were enrolled in the targeted validation cohort. Metabolites identified by non-target metabolomics were quantitatively analyzed by ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry. Effects of 1-oleoyl-sn-glycero-3-phosphocholine (OGPC) associated with IL-6 on MH7A cells were assessed. After 24-h or 48-h induction by TNF-α, the supernatants were collected for IL-6 quantification by enzyme-linked immunosorbent assay. Furthermore, Western blot was performed to investigate the relative JAK2 and p-JAK2 expressions. With an increasing IL-6 level, OGPC shown to be related to the glycerophospholipid metabolism pathway by Kyoto Encyclopedia of Genes and Genomes analysis displayed a significant decrease. In the validating RA cohort, the OGPC concentrations in remissive RA group and active RA group decreased compared with HC group. OGPC down-regulated IL-6 secretion and p-JAK2 expression in TNF-α-induced MH7A cells in vitro. In conclusion, glycerophospholipid metabolism is the main metabolic pathway associated with the differential IL-6 expression in RA patients. The down-regulated OGPC is a promoting factor for the increased IL-6 plasma level in RA patients, which further affects the downstream JAK signaling pathway.

Association of leptin with migration, invasion, and cancer stem-like properties in gastric cancer cells.

352 Background: Obesity is a risk factor for various types of cancer, including gastric cancer. Leptin, an adipocyte-derived hormone, may function in stimulating the proliferation of gastric cancer cell. The purpose of this study was to evaluate the role of leptin in gastric cancer. Methods: Leptin level was assessed by ELISA and was compared in obese patients with gastric cancer (n=23) and non-obese patients with gastric cancer (n=23) as a function of their clinicopathological characteristics. Gastric cancer cell lines were studied to investigate the effects of leptin on the Janus kinase/signal transducer and activator of transcription (JAK-STAT) and MEK signaling pathways using wound healing assay, inhibition study, and ELISA. Cancer-initiating cells derived from gastric cancer cell lines were studied to investigate the effects of leptin to the maintenance of stemness and epithelial-mesenchymal transition using invasion assay, RT- PCR, and immunoblotting. Results: Obesity was associated with elevated leptin level and BMI has positive correlation with the leptin level ( p=0.001 for both). Overall survival was not significantly different between the two groups ( p=0.098). Leptin induces the migration and invasion of gastric cancer cells by activating AKT and ERK and up-regulating the expression of vascular endothelial growth factor (VEGF). Pharmacological inhibitors of JAK and MEK signaling pathways decreased both leptin-induced migration and invasion and the leptin-induced expression of VEGF. Leptin increased the mRNA and protein levels of the markers for stemness (CD44) and epithelial-mesenchymal-transition (Snail and N-cadherin). Conclusions: Leptin plays a role in gastric cancer by stimulating the migration and invasion of gastric cancer cells via activating the JAK-STAT and MEK pathways and contributes to the maintenance of cancer stemness and metastatic potential. Leptin presents evidence supporting the adverse effect of obesity in gastric cancer. Treatment targeting leptin-associated signaling pathways could be a potential strategy for managing gastric cancer.

The Natural Compound Dehydrocrenatidine Attenuates Nicotine-Induced Stemness and Epithelial-Mesenchymal Transition in Hepatocellular Carcinoma by Regulating a7nAChR-Jak2 Signaling Pathways.

Background Exposure to nicotine has been observed associated with tumor progression, metastasis, and therapy resistance of many cancers. Hepatocellular carcinoma (HCC) is one major cancer related to the liver and the most difficult to treat malignancies worldwide. The underlying mechanism of nicotine in the stimulation of HCC tumorigenesis is still not studied well. Methods Classically, nicotine binds to nicotinic acetylcholine receptors (nAChRs) and induces many downstream cancer-associated signaling pathways. Big data analysis is used to explore the importance of a7nAChR-Jak2 axis in the progression of hepatocellular carcinoma. Bioinformatic analysis was performed to determine gene associated with a7nAChR-Jak2 axis of HCC patients. Biological importance of a7nAChR-Jak2 axis was investigated in vitro (Hun7 and HepG2 cell lines), and athymic nude mouse models bearing HepG2-HCC cells xenografts were established in vivo. Result We found that nicotine exposure stimulated the HCC tumorigenicity by inducing the expression of one of the key nAChRs subunit that is α7nAChR as well as the expression of Janus kinase (JAK)-2. In both the in vitro and in vivo studies, the reduced overexpression of α7nAChR and increased sensitization of HCC towards treatment is observed with dehydrocrenatidine (DHCT), a novel and potent JAK family kinase inhibitor. Interestingly, DHCT treatment results in the reduction of the epithelial-mesenchymal transition process which leads to a significant reduction of clonogenicity, migratory, and invasive ability of HCC cells. Moreover, DHCT treatment also inhibits the cancer stem cell phenotype by inhibiting the tumor-sphere formation and reducing the number of ALDH1+ cells population in nicotine-stimulated HCC cells. Conclusions Taken together, the presented results indicate the positive effect of inhibition of nicotine induced overexpression of α7nAChR and JAK2, unique to HCC. Thus, these findings suggest the nicotine effect on HCC progression via α7nAChR-mediated JAK2 signaling pathways, and DHCT treatment enhances the therapeutic potential of HCC patients via overcoming/reversing the effect of nicotine in HCC patients.

Identification of galangin as the bioactive compound from Alpinia calcarata (Haw.) Roscoe rhizomes to inhibit IRAK-1/ MAPK/ NF-κB p65 and JAK-1 signaling in LPS stimulated RAW 264.7 cells

Ethnopharmacological relevanceAlpinia calcarata (Haw.) Roscoe rhizomes are used to treat diabetes, rheumatism, gastrointestinal problems, inflammatory diseases, cough and respiratory problems in traditional practices. The primary objective of the study is to identify and isolate anti-inflammatory bioactive compounds from A.calcarata rhizomes and to assess its molecular mechanism. Materials and methodsThe bioassay-guided fractionation of methanolic extract of A. calcarata rhizomes yielded chloroform fraction as the effective fraction and galangin as the bioactive compound identified by NMR studies. The anti-inflammatory action of galangin was evaluated by determining NO and cytokine production in LPS stimulated RAW264.7 cells. Further, its mechanism was studied on the expression levels of mRNA and protein targets by qPCR and Western blot analysis. ResultsBased on the MTT assay, the concentration of 3.1–25 μM of galangin was selected for further studies. Galangin reduced the levels of NO and proinflammatory cytokines (TNF-α, IL-1β and IL-6) production in LPS induced RAW 264.7 cells in a dose-dependent manner. In addition, the qPCR analysis revealed a reduction in the mRNA expression levels of COX-2, IRAK 1 and JAK 1 in galangin treated LPS stimulated RAW 264.7 cells in a dose-dependent manner. Western blot analysis implicated that galangin has markedly reduced the protein expression levels of cell signaling regulators (JAK-1, IRAK-1, MyD88, MAPK (p38 and ERK) and NF-κB p65). ConclusionFrom the results, it is evident that the inhibition of these cell signaling regulators has contributed to the anti-inflammatory effects of galangin. To our knowledge, we are the first to report IRAK-1 and JAK-1 as therapeutic targets of galangin for its anti-inflammatory effect.

3080 – IL-3 SELECTIVELY RESCUES RUNX1-DEFICIENT HUMAN HSPCS WITH DYSREGULATED JAK/STAT SIGNALING

Both germline and somatic loss-of-function mutations in RUNX1 are commonly found in hematopoietic diseases, ranging from thrombocytopenia and autoimmunity to myeloid malignancies. However, our understanding of the underlying mechanisms is lacking. Here, we established a faithful CRISPR/AAV6 model of RUNX1 knockout (KO) in human CD34+ hematopoietic stem and progenitor cells (HSPCs), demonstrating monocytic skew at the expense of erythro-megakaryocytic potential and increased inflammatory signaling. Importantly, RUNX1 KO severely limited in vitro proliferation and in vivo competitive engraftment potential. Using ATAC-seq and RNA-seq, we determined that RUNX1 KO cells displayed decreased JAK/STAT signaling and elevated expression of the negative regulator SOCS3. Knockdown of SOCS3 rescued the RUNX1 KO proliferative defect and both RUNX1 KO HSPCs and RUNX1 mutant primary patient samples showed increased sensitivity to treatment with JAK inhibitors, suggesting that RUNX1 loss represses JAK signaling. Next, we investigated whether cytokines signaling through JAK/STAT can reverse the RUNX1 KO proliferative defect. Exposure to IL-3, but not other cytokines, rescued STAT5 phosphorylation, proliferation, and competitive engraftment defects, and both RUNX1 KO HSPCs and RUNX1 mutant primary AMLs upregulated the IL-3 receptor IL3RA (CD123) at the protein level. Thus, RUNX1 loss sensitizes cells to IL-3 and overcome the JAK/STAT signaling deficiency by regulating IL3RA receptor expression. In summary, we established a human model of RUNX1 loss and found dysregulated IL-3/JAK/STAT signaling, which is achieved by modulated expression of negative regulators and the IL-3 receptor. Importantly, we identify conditions under which RUNX1 KO cells regain proliferative competitiveness and anticipate that targeting these pathways may be beneficial for a range of RUNX1-mutant diseases. Both germline and somatic loss-of-function mutations in RUNX1 are commonly found in hematopoietic diseases, ranging from thrombocytopenia and autoimmunity to myeloid malignancies. However, our understanding of the underlying mechanisms is lacking. Here, we established a faithful CRISPR/AAV6 model of RUNX1 knockout (KO) in human CD34+ hematopoietic stem and progenitor cells (HSPCs), demonstrating monocytic skew at the expense of erythro-megakaryocytic potential and increased inflammatory signaling. Importantly, RUNX1 KO severely limited in vitro proliferation and in vivo competitive engraftment potential. Using ATAC-seq and RNA-seq, we determined that RUNX1 KO cells displayed decreased JAK/STAT signaling and elevated expression of the negative regulator SOCS3. Knockdown of SOCS3 rescued the RUNX1 KO proliferative defect and both RUNX1 KO HSPCs and RUNX1 mutant primary patient samples showed increased sensitivity to treatment with JAK inhibitors, suggesting that RUNX1 loss represses JAK signaling. Next, we investigated whether cytokines signaling through JAK/STAT can reverse the RUNX1 KO proliferative defect. Exposure to IL-3, but not other cytokines, rescued STAT5 phosphorylation, proliferation, and competitive engraftment defects, and both RUNX1 KO HSPCs and RUNX1 mutant primary AMLs upregulated the IL-3 receptor IL3RA (CD123) at the protein level. Thus, RUNX1 loss sensitizes cells to IL-3 and overcome the JAK/STAT signaling deficiency by regulating IL3RA receptor expression. In summary, we established a human model of RUNX1 loss and found dysregulated IL-3/JAK/STAT signaling, which is achieved by modulated expression of negative regulators and the IL-3 receptor. Importantly, we identify conditions under which RUNX1 KO cells regain proliferative competitiveness and anticipate that targeting these pathways may be beneficial for a range of RUNX1-mutant diseases.

Rotundic Acid Promotes Angiogenesis Potentially by Activating the STAT3 -JAK Signaling Pathway

Rotundic Acid Promotes Angiogenesis Potentially by Activating the STAT3 -JAK Signaling Pathway

Momelotinib is a highly potent inhibitor of FLT3-mutant AML

AbstractDespite the introduction of more selective FLT3 inhibitors to treat FLT3-mutated acute myeloid leukemia (AML), remissions are short lived, and patients show progressive disease after an initial response. Acquisition of resistance-conferring genetic mutations and growth factor signaling are 2 principal mechanisms that drive relapse. FLT3 inhibitors targeting both escape mechanisms could lead to a more profound and lasting clinical response. Here, we show that the JAK2 inhibitor momelotinib is an equipotent type 1 FLT3 inhibitor. Momelotinib showed potent inhibition of FLT3-internal tandem duplication in mouse and human primary cells and effectively suppressed its clinically relevant resistant variants within the activation loop at residues D835, D839, and Y842. Additionally, momelotinib efficiently suppressed the resistance mediated by growth factors and hematopoietic cytokine-activated JAK2 signaling. Consequently, concomitant inhibition of FLT3 and suppression of growth factor signaling by momelotinib treatment showed better efficacy in suppressing leukemia in a preclinical murine model of AML. Altogether, these data provide evidence that momelotinib is an effective type 1 dual JAK2/FLT3 inhibitor and may offer an alternative to gilteritinib. Its ability to impede the resistance conferred by growth factor signaling and activation loop mutants suggests that momelotinib treatment could provide a deeper and durable response and, thus, warrants its clinical evaluation.

CTNI-42. PHASE I STUDY OF RUXOLITINIB WITH RADIATION AND TEMOZOLOMIDE IN PATIENTS WITH NEWLY DIAGNOSED GRADE III GLIOMAS AND GLIOBLASTOMA

Abstract BACKGROUND Ruxolitinib is a novel, potent, selective inhibitor of JAK1 (Janus kinase 1) and JAK2 with modest to marked selectivity against TYK2 (tyrosine kinase 2) and JAK3. Ruxolitinib interferes with the signaling of a number of cytokines and growth factors that are important for hematopoiesis and immune function. JAK signaling involves recruitment of signal transducers and activators of transcription to cytokine receptors, activation, and subsequent localization of STATs to the nucleus leading to modulation of gene expression. METHODS Newly diagnosed patients with MGMT not hypermethylated, Glioblastoma or grade III glioma were recruited to Arm 1. Every patient received ruxolitinib and 60 Gy radiation for 6 weeks (2Gy x 30). The dose of Ruxolitinib was administered given the 3 + 3 design. Level 1 or starting dose was 10 mg twice daily, level 2 was 15 mg twice daily, level 3 was 20 mg twice daily. Patients with MGMT hypermethylated glioblastoma or grade III glioma were eligible for Arm 2. Every patient received ruxolitinib + radiation x 60 Gy + daily temozolomide at 75 mg/m2 for 6 weeks. RESULTS 45 patients had survival data, 25 patients were in Arm I and 20 arm in II. The median OS and PFS were 18.2 (95% CI: 3.6-NA) months for Arm 1 and were not reached for Arm 2. OS and PFS Rate at 1 year was 61% (95% CI: 43-85%) and 51% (35-76%) for Arm 1, and 95% (85-100%) for Arm 2 (p=0.01 and p= 0.002), respectively. 9 patients had partial response, 16 patients were stable, and 28 patients had progression. CONCLUSION Patients that received ruxolitinib + radiation x 60 Gy + daily temozolomide at 75 mg/m2 for 6 weeks over 6 weeks (Arm 2) had significantly better PFS and OS than those that received ruxolitinib + radiation x 60 Gy alone..

Ruxolitinib Plus Nivolumab in Patients with R/R Hodgkin Lymphoma after Failure of Check-Point Inhibitors: Preliminary Report on Safety and Efficacy

Background: Recent studies demonstrated that classical Hodgkin lymphoma (cHL) is characterized by chromosome 9p24.1 amplification with associated overexpression of PD-1 ligands as well as JAK2 tyrosine kinase activation. Immune evasion mediated by the PD-1/PD-1 ligand axis can be inhibited with IgG4 agonist antibody specific for human PD-1 nivolumab which has shown excellent overall response rate (ORR) of over 60% in relapsed cHL. JAK2 induces PD-1 ligand expression and augments tumor cell proliferation. JAK2 signaling can be inhibited with clinical grade small molecules (i.e. ruxolitinib). Given this biology, we designed a Phase I/II study to target both pathways in cHL patients who had failed PD-1 blocking agents.Methods: This is a Phase I/II, multicenter, open-label, dose escalation/dose-expansion study to evaluate the safety and tolerability of ruxolitinib when combined with nivolumab (fixed dose 3mg/kg IV every 2 weeks) in patients with relapsed or refractory (R/R) cHL (NCT03681561). Eligible patients had to fail prior check-point inhibitor (CPI; nivolumab or pembrolizumab). Three dose levels of ruxolitinib were tested: 10 mg orally twice a day (bid), 15 mg bid, and 20 mg bid. Planned duration of therapy was 2 years. The primary objective was to find the maximum tolerated dose (MTD) of ruxolitinib when given with nivolumab and characterize the safety and tolerability of this combination. The phase 2 objective was to evaluate the overall response rate (ORR).Results: We enrolled 19 patients. Median age was 38 years (range 22-76); 68% were males. Patients were a median of 3.4 years from the initial diagnosis (range 0.9-16.7 years), 89% had stage III-IV disease and all had experienced progressive disease following CPI. 17 (89%) had prior autologous HCT and 1 had prior allogeneic HCT. All patients received nivolumab combined with ruxolitinib, and the highest ruxolitinib dose 20mg BID (MTD) was reached without DLTs. The combination was well tolerated; most AEs were grade 1 and 2. Only 3 patients required hospitalization (2 had pneumonia, one for disease progression). Three patients experienced immune mediated adverse events (LFT elevation: 1 Gr 1 and 1 Gr 2; 1 Gr 3 pneumonitis) and all were reversible. Three subjects were not evaluable for response (2 patients who received < 1 month of therapy due to rapid disease progression and Gr 2 immune hepatitis and 1 patient with short follow-up is not yet evaluable for response). Median follow-up was 13 months (range 3-27 months).In 16 patients evaluable for response, best ORR was 75% (12 of 16); including 3 CRs (19%), 2 PRs (13%), 6 patients had stable disease (SD, 44%) with tumor bulk reduction ranging from 10-45%. One patient had indeterminate response (negative biopsy of new PET-avid site). Duration of responses were 12.5 months (ranged from 3.7 to 20.4 months); 5 responders continue study therapy and 2 completed 2 years; 1 went on to autologous HCT in PR, and 3 progressed while on therapy after 6, 6 and 23 months. One patient died after later experimental therapy. Progression-free survival at 1 year was 64% (95%CI 34-84%).Conclusions: Inhibition of JAK2 combined blockade of the PD-1 pathway represent complementary rational therapeutic targets in cHL. Therapy combining ruxolitinib with nivolumab is well tolerated and yield encouragingly high remission rates and durable responses in patients who had all failed previous CPI. Pharmacodynamic and correlative analyses on the mechanism of synergism are underway. DisclosuresBachanova: KaryoPharma: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; FATE: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gamida Cell: Membership on an entity's Board of Directors or advisory committees, Research Funding. Janakiram: ADC Therapeutics: Research Funding; FATE Therapeutics: Research Funding; Takeda Pharmaceuticals: Research Funding; Kyowa Kirin Therapeutics: Honoraria. Weisdorf: Fate Therapeutics: Research Funding; Incyte: Research Funding. Farooq: Kite, a Gilead Company: Honoraria. OffLabel Disclosure:ruxolitinib for Hodgkin lymphoma

Momelotinib Is a Highly Potent Inhibitor of FLT3-Mutant AML

Approximately one-third of AML patients harbor kinase activating mutations in FLT3. Several small-molecule first generation FLT3 tyrosine kinase inhibitors (TKIs) have been evaluated in the last two decades, but none could induce a durable response possibly due to poor pharmacokinetics and target selectivity. Second generation FLT3 inhibitors such as, quizartinib, gilteritinib, and crenolanib, were designed for greater selectivity with a narrow kinome-profile and better pharmacokinetic properties, but they failed to induce a durable response. Possibly due to intrinsic resistance conferred by growth factor signaling in the bone marrow resident leukemic cells, which serve as a reservoir to develop resistance resulting to disease relapse. Activation of Jak2 signaling by chemokines and cytokines from the stroma have been reported to confer TKI refractoriness. Recent studies from Martin Carroll's group showed that both GMCSF and IL-3 confer resistance by activating JAK2 signaling, which can be suppressed by combined FLT3 and JAK2 inhibition. However, so far, it is not established whether upfront FLT3/Jak2 inhibition will provide durable response.For instance, AML patients who achieved complete remission lacking FLT3-ITD clones showed better overall survival than patients with measurable MRD, suggesting that eradicating the FLT3-ITD clones will have a deeper response with better overall survival. Thus, an ideal FLT3 inhibitor should be able to suppress FLT3-ITD resistant mutants, and growth factor activated JAK2 signaling while sparing c-KIT receptor and hERG to avoid myelosuppression and cardiotoxicity, respectively. Here we show that Jak2 inhibitor, momelotinib, is an equipotent type-I FLT3 inhibitor (Fig 1A-D). Biochemical and structural modeling revealed that it binds to an active conformation of FLT3 kinase. Therefore, like gilteritinib, it efficiently suppresses the resistance conferred by activation loop mutations (Fig1 E-H). Moreover, its lack of activity against c-KIT and inhibition of ACVR1 provides additional benefit in alleviating myelosuppression and anemia, which is commonly observed with currently used JAK2 and FLT3 inhibitors (ruxolitinib, fedratinib, and quizartinib). Perhaps more interestingly, momelotinib efficiently suppressed the disease in a preclinical model of AML using NSGS mice which recapitulates cytokine induce refractoriness as usually observed in clinical setting (Fig1 I-L). Our preclinical data provide evidence that momelotinib is an equipotent dual JAK2/FLT3 inhibitor and suppresses resistance conferred by both activation-loop mutations and growth-factor signaling. These data provide evidence that momelotinib treatment will have clinical activity in FLT3-mutated AML. Thus, warrants its clinical evaluation. [Display omitted] DisclosuresStarczynowski: kurome Inc: Consultancy.

Targeting JAK2 Gene Rearrangements with a Novel Kinase Inhibitor in a Preclinical Model of Pediatric Acute Lymphoblastic Leukemia

Background. Although the Event Free Survival for Childhood Acute lymphoblastic leukemia (ALL) reaches 85%, the remaining 15% of patients relapse, and 25-40% of them die. Novel molecular targets may increase the efficacy of therapy and reduce treatment toxicity. Among B-other ALL patients, JAK/STAT pathway alterations represent about 7% of the ‘Philadelphia-like’ cases. JAK2 gene encodes for a non-receptor tyrosine kinase fundamental for hematopoiesis, cellular proliferation and survival. In last years, JAK2 mutations have been widely studied in leukemia and lymphoma, whereas JAK2 fusion genes are still poorly characterized.Aim. This study aims to identify JAK2 fusion genes among BCP-ALL pediatric patients and develop a target strategy in in vitro and in vivo preclinical models.Methods. A targeted capture RNA Next Generation Sequencing strategy was applied to discover JAK2 fusion genes in a large cohort of PCR-based MRD high risk (HR) BCP-ALL pediatric patients. Fusions were validated by RT-PCR and/or FISH. Primary patients' cells have been in vivo expanded in NSG mice. We performed ex vivo and in vivo drug treatments with JAK2 inhibitors; phosphoflow and apoptosis-viability assays were performed in patients' blasts in co-culture with human bone marrow stroma.Results. We identified 10 pediatric cases carrying a JAK2 fusion gene with different partners in single cases, such as ATF7IP, ZEB2, MPRIP, BCR, TLE4, GIT2 and RAB7, in addition to PAX5, which was the only recurrent in three cases. Cells were available from 3 cases, carrying PAX5-JAK2, ATF7IP-JAK2 and ZEB2-JAK2, respectively. After in vivo expansion, we demonstrated that the JAK2 signaling pathway was active at basal level, through phosphorylation on Y1007-1008 JAK2 residues inside the catalytic activation loop, compared to cases wild type for JAK2 and CRLF2 (+70%, two-tailed P value 0.0355); a positive trend was also shown compared to primary cells with P2RY8-CRLF2 rearrangements and JAK2 mutation, as positive controls (+40% two-tailed P value 0.158). The JAK2 downstream effectors pS727-STAT3 and pY694-STAT5 were also activated.We thus setup a JAK2 targeted drug treatment using CHZ868, a new class-II tyrosine kinase inhibitor (TKI) (Novartis, Basel, CH). After 30 minutes of treatment, we appreciated a mean inhibition of -62% of Y1007-1008 JAK2 residues in PAX5-JAK2, -22% in ATF7IP-JAK2 and -35% in ZEB2- JAK2. Contemporarily, we observed a decrease of pS727-STAT3 (-35-50%) and pY694-STAT5 (-15-50%).After 48h monotherapy treatment by CHZ868, we detected apoptosis induction and cell viability decrease between 20- 75% at IC50. In combination with dexamethasone, we assessed a further decrease of viability between 10 to 95%. A biological variability among the three different patients was appreciated, according to the different partner genes.Exclusively for the PAX5-JAK2 fusion, we also performed treatments with the kinase inhibitor BIBF1120/Nintedanib, targeting LCK, which is activated downstream PAX5 fusions and we observed a 20% reduction of cell viability. Importantly, combination of BIBF1120 and CHZ868 showed a synergistic effect (-45%, at IC50). Moreover, we found that ruxolitinib caused autophagy as observed by higher levels of LC3-II compared to untreated cells (+ 45%, p<0.01), with consequent reduction of apoptosis induction. Indeed, active caspase 3 increased when ruxolitinib was given in combination with chloroquine, an autophagy inhibitor (+20% vs ruxolitinib alone, p<0.01). CHZ868 alone or in combination with chloroquine instead does not induce autophagy as LC3-II and active caspase 3 levels are the same of untreated cells.Finally, we demonstrated the in vivo efficacy of CHZ868 in patient derived xenograft model in presence of PAX5-JAK2 fusion. After two weeks of 30mg/Kg daily treatment of CHZ868, we observed a significant reduction of leukemic CD10+/CD19+ cells both in bone marrow (p<0.01, -43%), spleen (p<0.001, -72%), central nervous system (-40%) and peripheral blood (p<0.05, -46%), compared to vehicle mice. Further in vivo experiments are ongoing in other JAK2 fusion settings.Conclusion. CHZ868 is a promising candidate for treatment of BCP-ALL carrying JAK2 fusions, showing high efficacy and specificity, both ex vivo and in vivo. Further studies will include combination with standard chemotherapy drugs with the aim to maintain its efficacy by reducing the intensity and toxicity of chemotherapy. DisclosuresBiondi: Novartis: Honoraria; Bluebird: Other: Advisory Board; Incyte: Consultancy, Other: Advisory Board; Colmmune: Honoraria; Amgen: Honoraria.

Integrative genomic and transcriptomic analysis in plasmablastic lymphoma identifies disruption of key regulatory pathways

AbstractPlasmablastic lymphoma (PBL) represents a clinically heterogeneous subtype of aggressive B-cell non-Hodgkin lymphoma. Targeted-sequencing studies and a single-center whole-exome sequencing (WES) study in HIV-positive patients recently revealed several genes associated with PBL pathogenesis; however, the global mutational landscape and transcriptional profile of PBL remain elusive. To inform on disease-associated mutational drivers, mutational patterns, and perturbed pathways in HIV-positive and HIV-negative PBL, we performed WES and transcriptome sequencing (RNA-sequencing) of 33 PBL tumors. Integrative analysis of somatic mutations and gene expression profiles was performed to acquire insights into the divergent genotype–phenotype correlation in Epstein-Barr virus–positive (EBV+) and EBV– PBL. We describe a significant accumulation of mutations in the JAK signal transducer and transcription activator (OSMR, STAT3, PIM1, and SOCS1), as well as receptor tyrosine-kinase RAS (ERBB3, NRAS, PDGFRB, and NTRK) pathways. We provide further evidence of frequent perturbances of NF-κB signaling (NFKB2 and BTK). Induced pathways, identified by RNA-sequencing, closely resemble the mutational profile regarding alterations accentuated in interleukin-6/JAK/STAT signaling, NF-κB activity, and MYC signaling. Moreover, class I major histocompatibility complex–mediated antigen processing and cell cycle regulation were significantly affected by EBV status. An almost exclusive upregulation of phosphatidylinositol 3-kinase/AKT/mTOR signaling in EBV+ PBL and a significantly induced expression of NTRK3 in concert with recurrent oncogenic mutations in EBV– PBL hint at a specific therapeutically targetable mechanism in PBL subgroups. Our characterization of a mutational and transcriptomic landscape in PBL, distinct from that of diffuse large B-cell lymphoma and multiple myeloma, substantiates the pathobiological independence of PBL in the spectrum of B-cell malignancies and thereby refines the taxonomy for aggressive lymphomas.

A miR-129-5P/ARID3A Negative Feedback Loop Modulates Diffuse Large B Cell Lymphoma Progression and Immune Evasion Through Regulating the PD-1/PD-L1 Checkpoint.

The activated B cell (ABC) and germinal center B cell (GCB) subtypes of diffuse large B cell lymphoma (DLBCL) have different gene expression profiles and clinical outcomes, and miRNAs have been reported to play important roles in tumorigenesis, progression, and metastasis. This study aimed to explore the differentially expressed miRNAs and target genes in the two main subtypes of DLBCL. Hub miRNAs were identified by constructing a regulatory network, and in vitro experiments and peripheral blood samples of DLBCL were used to explore the functions and mechanisms of differential miRNAs and mRNAs. Differentially expressed miRNAs and genes associated with the two DLBCL subtypes were identified using GEO datasets. Weighted gene co-expression network analysis shows that one gene module was associated with a better prognosis of patients with the GCB subtype. Through the construction of a regulatory network and qPCR verification of clinical samples and cell lines, miR-129-5p was identified as an important differential miRNA between the ABC and GCB subtypes. The negative relationship between miR-129-5p and ARID3A in DLBCL was confirmed using luciferase reporter assays. Overexpression of miR-129-5p and knockdown of ARID3A inhibited the proliferation of SU-DHL-2 (ABC-type) cells and promoted their apoptosis through the JAK and STAT6 signaling pathways. In addition, inhibition of miR-129-5p and overexpression of ARID3A promoted the proliferation and reduced apoptosis of DB and SU-DHL-6 (GCB-type) cells. Inhibition of miR-129-5p and overexpression of ARID3A in DB and SU-DHL-6 promoted immune escape by increasing PD-L1 expression, which was transcriptionally activated by ARID3A. In conclusion, we showed for the first time that the mir-129-5P/ARID3A negative feedback loop modulates DLBCL progression and immune evasion by regulating PD-1/PD-L1.

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.

Vitiligo Skin T Cells Are Prone to Produce Type 1 and Type 2 Cytokines to Induce Melanocyte Dysfunction and Epidermal Inflammatory Response Through Jak Signaling

Vitiligo is a T cell-mediated inflammatory skin disorder characterized by the loss of epidermal melanocytes. However, the contribution of melanocytes to the physiopathology of the disease in response to the T-cell microenvironment remains unclear. Here, using NanoString technology and multiplex ELISA, we show that active vitiligo perilesional skin is characterized by prominent type 1 and 2 associated immune responses. The vitiligo skin T-cell secretome downregulated melanocyte function and adhesion while increasing melanocyte mitochondrial metabolism and expression of inflammatory cytokines and chemokines by epidermal cells. The Jak1/2 inhibitor ruxolitinib strongly inhibited such effects on epidermal cells. Our data highlight that vitiligo is more complex than previously thought, with prominent combined activities of both T helper type 1- and T helper type 2-related cytokines inducing inflammatory responses of epidermal cells. Melanocytes do not appear only to be a target of T cells in vitiligo but could actively contribute to perpetuate inflammation. Jak inhibitors could prevent the impact of T cells on epidermal cells and pigmentation, highlighting their potential clinical benefit in vitiligo.