scholarly journals Thiol Isomerase Activity of β 3 Integrin Psi Domain and L33P Polymorphism: Implications in Blood Coagulation and Anti-Thrombotic Therapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1052-1052
Author(s):  
Eric Gaetano Cerenzia ◽  
Tyler W. Stratton ◽  
Daniel Mackeigan ◽  
Zhenze Liu ◽  
Guangheng Zhu ◽  
...  
Keyword(s):  
Blood Coagulation ◽  
High Speed ◽  
Platelet Adhesion ◽  
Conflicts Of Interest ◽  
Site Directed Mutagenesis ◽  
Isomerase Activity ◽  
Platelet Adhesion And Aggregation ◽  
Increased Risk ◽  
Β3 Integrin

Abstract Background: Integrins are α and β subunit heterodimeric receptors expressed ubiquitously on metazoan cells that play key roles in cell adhesion, movement, and signaling. The αIIbβ3 integrin on platelets is essential for thrombosis and hemostasis through its binding of fibrinogen and other ligands to mediate platelet adhesion and aggregation. The plexin-semaphorin-integrin (PSI) domain is an approximately 54 amino acid sequence at the N-terminus of the β3 subunit located in close proximity to the "knee" region. Our lab recently discovered that two "CXXC" motifs within PSI domains in the integrin family exert endogenous thiol isomerase activity, and blockade of this activity in β3 integrin with our novel anti-PSI domain monoclonal antibodies (mAbs) significantly attenuated platelet adhesion and aggregation without impairing hemostasis (Blood, 2017). Interestingly, intravital microscopy studies demonstrate that our anti-PSI antibodies inhibited thrombosis in vivo 10-30 fold more potent than their effects in vitro under anti-coagulant conditions, suggesting a possible inhibitory effect on blood coagulation. Notably, A L33P polymorphism (HPA-1b) within the β3 integrin PSI domain is linked to an approximately two-fold increased risk for cardiovascular disease (CVD), however, the underlying mechanism for this remains unclear. Methods/Results: To investigate the role of PSI domain in blood coagulation, we first employed thromboelastography (TEG) to compare our anti-PSI domain mAbs with other anti-β3 mAbs that do not directly bind to β3 PSI domain. Results show that anti-PSI domain mAbs inhibited blood coagulation in human and murine whole blood or platelet-rich plasma (PRP) significantly more than anti-αIIbβ3 antibodies JAN-D1, M1, and Abciximab precursor 7E3. To address whether the L33P polymorphism affects PSI domains thiol isomerase activity, we generated L33P PSI domain via site-directed mutagenesis in E. coli. Using a scrambled RNase assay, we found that L33P polymorphism enhanced thiol-isomerase activity relative to WT PSI domain. We further corroborated these findings through an insulin β chain reduction assay, and a MPB (N-Maleimidopropionyl-biocytin) western blot assay, which quantifies thiol isomerase activity through MPB binding to free thiols that have not been oxidized into RNase. Interestingly, TEG results show that recombinant human PSI domain enhanced blood coagulation in platelet-microparticle (PMP) free plasma, which was generated through high-speed centrifugation (17,000 x g) of platelet poor plasma (PPP)for 15 minutes that removed residual platelets and microparticles . Conclusion: We have discovered a novel role of integrin β3 PSI domain in blood coagulation, which is enhanced by the L33P polymorphism (HPA-1b). These data highlight the β3 PSI domain as a suitable therapeutic target for its roles in both platelet adhesion/aggregation, and blood coagulation. Furthermore, these data may explain the increased risk of CVD such as myocardial infarction and deep vein thrombosis for individuals with the L33P polymorphism. Disclosures No relevant conflicts of interest to declare.

Apolipoprotein AIV (ApoA-IV) Is a Novel Ligand of Platelet β3 Integrin That Negatively Regulates Platelet Adhesion, Aggregation, and Thrombosis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 156-156
Author(s):  
Christopher M. Spring ◽  
Wuxun Jin ◽  
Hong Yang ◽  
Adili Reheman ◽  
Guangheng Zhu ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Protein S ◽  
Vessel Occlusion ◽  
Platelet Adhesion And Aggregation ◽  
Β3 Integrin

Abstract Abstract 156 Platelet adhesion and aggregation at sites of vascular injury are key events required for haemostasis and thrombosis. It has been documented that von Willebrand factor (VWF) and fibrinogen (Fg) are required for platelet adhesion and aggregation. However, we previously showed that occlusive thrombi still form in mice deficient for both Fg and VWF (Fg/VWF−/−) via a β3 integrin-dependent pathway. Here, we have investigated novel, non-classical ligands of β3 integrin that may regulate platelet adhesion and aggregation. To identify potential ligand(s) of β3 integrin, latex beads were coated with purified human platelet β3 integrin and incubated with human plasma. Protein(s) specifically associated with β3 integrin were electrophoresed and apolipoprotein AIV (ApoA-IV) was identified by mass spectrometry. We found that ApoA-IV binds to the surface of stimulated platelets, but not to quiescent platelets or β3−/− platelets, and ApoA-IV/platelet association was blocked by the addition of a specific anti-β3 integrin monoclonal antibody. It appears that ApoA-IV binds to, but is not internalized by platelet β3 integrins. ApoA-IV-deficient (ApoA-IV−/−) mice exhibited enhanced platelet aggregation induced by ADP, Collagen, and TRAP in plasma (but not PIPES buffer) compared to wild type (WT) littermates. This enhancement was diminished when ApoA-IV−/− plasma was replaced by WT plasma, indicating that the reduction was due to plasma ApoA-IV and not an unrelated platelet effect. When platelets were incubated with FITC-Fg, ApoA-IV was able to reduce platelet/Fg association, indicating that ApoA-IV may act to displace pro-thrombotic β3 integrin ligand(s). In support of this, ApoA-IV reduced the number of adherent platelets on immobilized Fg in perfusion chamber assays and enhanced thrombus formation was observed when ApoA-IV−/− mouse blood was perfused over collagen. We found that addition of recombinant ApoA-IV inhibited platelet aggregation and thrombus formation in vitro, while the control apolipoprotein ApoA-I did not. Using intravital microscopy, we further demonstrated that early platelet deposition was increased, and the time for thrombus formation and vessel occlusion were shorter in ApoA-IV−/− mice, which can be corrected by recombinant ApoA-IV transfusion. Furthermore, recombinant ApoA-IV inhibited WT platelet aggregation, thrombus formation and enhanced thrombus dissolution both in vitro and in vivo. Our data demonstrate for the first time that ApoA-IV is a novel ligand of platelet β3 integrin that negatively regulates thrombosis. These new data are consistent with the reported association between ApoA-IV and reduced cardiovascular diseases, and establish the first link between ApoA-IV and thrombosis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Targeting β3 Integrin Psi Domain Inhibits Both Platelet Aggregation and Blood Coagulation: Two Birds with One Stone

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1246-1246
Author(s):  
Tyler W. Stratton ◽  
Guangheng Zhu ◽  
Yiming Wang ◽  
Pingguo Chen ◽  
Miguel A. D. Neves ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Blood Coagulation ◽  
Platelet Adhesion ◽  
Platelet Rich Plasma ◽  
Coagulation Factors ◽  
Inhibitory Effect ◽  
Clot Retraction ◽  
Isomerase Activity ◽  
Β3 Integrin ◽  
Human And Mouse

Abstract Integrin αIIbβ3 plays key roles in thrombosis and hemostasis primarily through mediating platelet adhesion and aggregation. We recently reported that the active site of thiol-isomerase enzymes, CXXC motif, is expressed twice within the plexin-semaphorin-integrin (PSI) domain across all integrins and species, and the PSI domain of β3 integrin possesses endogenous thiol-isomerase activity, which may be a novel target for anti-thrombotic therapy (Blood, 2017). We developed four mouse anti-mouse β3 integrin PSI domain monoclonal antibodies (mAbs). These mAbs cross-react with β3 PSI domains of human, mouse, pig, rat, and rabbit tested but not other regions of β3 integrin, other integrins or other thiol-isomerase enzymes. They inhibit the thiol-isomerase activity of β3 PSI domain, decrease platelet adhesion/aggregation and thrombosis without increasing bleeding. Interestingly, the inhibitory effect of these mAbs on thrombosis in vivo (no anti-coagulant) was 10-20 times greater than their inhibitory effect on platelet aggregation in anti-coagulated platelet-rich plasma in vitro. This motivated us to explore whether this PSI domain contributes to blood coagulation. To asses blood clot formation and retraction, blood was incubated in non-stick tubes for two hours at 37°C in clot retraction assays. These assays showed less clot retraction and significantly lower dry clot weight in human and mouse whole blood treated with these anti-PSI mAbs compared to controls (p<0.05). As a visual representation, laser scanning confocal microscopy of platelet-rich plasma samples with labelled fibrinogen/fibrin showed that fibrin network formation of anti-PSI mAb treated clots was decreased. To measure blood coagulation in a global assay in the presence of platelets and natural blood constituents, thromboelastography (TEG) was performed. Blinded TEG showed that anti-PSI mAb-treated whole blood (human and mouse) and platelet rich plasma (human) decreased in coagulation compared to controls (p<0.05). We further compared our anti-PSI mAbs with other anti-β3 integrin mAbs that inhibit αIIbβ3 ligand binding and platelet aggregation much stronger than the anti-PSI mAbs. In this TEG assay we found that the anti-PSI mAbs were still significantly decreasing platelet coagulation parameters, much more than other inhibitory anti-β3 mAbs including M1 and JAN D1 as well as the Abciximab precursor 7E3 (p<0.05). This suggests the decreased coagulation may result from the anti-thiol-isomerase activity of the anti-PSI mAbs. Disulfide bond exchanges are required for blood coagulation and thiol-isomerase enzymes play important roles in thrombosis. Given that blood coagulation is rapidly amplified on the platelet surface (i.e. cell-based coagulation), many coagulation factors are in close proximity to β3 integrin PSI domains (with thiol-isomerase activity). The study of interaction between β3 PSI domain and coagulation factors is currently being exploring. This is the first evidence of an allosteric therapeutic target of β3 integrin. This work suggests that a novel interaction exists between platelets and the coagulation cascade that requires the β3 integrin PSI domain and its thiol-isomerase activity. Importantly, the PSI domain can be targeted to partially block both platelet aggregation and coagulation, resulting in less bleeding while still providing a strong overall anti-thrombotic effect. Disclosures No relevant conflicts of interest to declare.

THE ROLE OF CONVECTION AND DIFFUSION ON PLATELET ADHESION AND AGGREGATION

1972 ◽  
Vol 201 (1) ◽  
pp. 329-342 ◽  
Author(s):  
Edward F. Leonard ◽  
Eric F. Grabowski ◽  
Vincent T. Turitto
Keyword(s):  
Platelet Adhesion ◽  
Platelet Adhesion And Aggregation ◽  
And Diffusion

Agonist-Induced Kindlin-3 Phsphorylation Regulates αIIbβ3 Integrin Activation In HEL Cells and Platelets

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 22-22
Author(s):  
Katarzyna Bialkowska ◽  
Eugene Podrez ◽  
Tatiana V. Byzova ◽  
Edward F. Plow
Keyword(s):  
Conflicts Of Interest ◽  
Focal Adhesions ◽  
Human Platelets ◽  
Phosphorylation Sites ◽  
Integrin Activation ◽  
Suspension Cells ◽  
Integrin Αiibβ3 ◽  
Hel Cells ◽  
Β3 Integrin

Abstract The contributions of integrins to platelet responses depend upon the dynamic regulation of their activation status, which in turn depends on engagement of binding partners by their cytoplasmic tails. It is well-established that not only talin but also kindlin family members are essential for integrin activation, and both must present for optimal integrin function. Recent studies in humans have specifically emphasized the vital role of kindlin-3 in integrin functions in hematopoietic cells, including platelets, where kindlin-3 deficiency can lead to episodic bleeding, frequent infections and osteopetrosis, consequences of an inability to activate β1, β2 and β3 integrins. Despite this evidence, little is known about kindlin-3 structure-function relationship. Here, we used human platelets and human erythroleukemic HEL cell line that expresses integrin αIIbβ3 to investigate whether posttranslational modification(s) of kindlin-3 occurs and can influence its integrin activity. Non-stimulated HEL cells are suspension cells, and they do not adhere to fibrinogen or bind soluble fibrinogen and PAC-1 antibody (specific for activated αIIbβ3) readily. Thrombopoietin or PMA stimulation activated αIIbβ3 such that the cells adhered and spread on fibrinogen and increased their binding of PAC-1 and soluble fibrinogen. β3 integrin and kindlin-3 colocalized in focal adhesions in the adherent cells, and there was enhanced β3 integrin-kindlin-3 association as detected by coimmunoprecipitation. Kindlin-3 knockdown impaired agonist-stimulated adhesion and spreading on fibrinogen. Since, as we have shown previously, β3 integrin phosphorylation regulates kindlin and integrin interaction, we sought to determine whether kindlin-3 is also phosphorylated. Human platelets were stimulated with thrombin and HEL cells with PMA, and kindlin-3 was immunoprecipitated from lysates of control and stimulated cells. A kindlin-3 peptide showing significant increase in phosphorylation upon agonist stimulation was identified in both platelets and HEL cells by mass spectrometry. T482 or S484 were identified as phosphorylation sites in sequence that resides in the kindlin-3 variable region, which is not present either in kindlin-1 or kindlin-2 but is conserved across all species in which kindlin-3 has been sequenced. When expressed in HEL cells, TS/AA kindlin-3 mutant displayed decreased soluble fibrinogen binding and cell spreading on immobilized fibrinogen when compared to wild-type kindlin-3. Membrane-permeable, poly-arginine tagged kindlin-3 peptide containing the candidate phosphorylation sites kindlin-3 was introduced into HEL cells and platelets. HEL cell adhesion and spreading was blunted by the kindlin-3 peptide when compared to a scramble poly-arginine control peptide. Moreover, thrombin-induced platelet aggregation was inhibited by kindlin-3 peptide but not by the scramble peptide. Thus, our data emphasizes a role of previously unknown, agonist-induced kindlin-3 phosphorylation, in integrin αIIbβ3 activation in HEL cells and platelets and provides a basis for functional differences between kindlin-3 and its other two paralogs, kindlin-1 and kindlin-2. Disclosures: No relevant conflicts of interest to declare.

Association of A313g Glutathione S-Transferase P1 (GSTP1) Inborn Polymorphism with Susceptibility to De Novo MDS

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1445-1445
Author(s):  
Sophia Zachaki ◽  
Chryssa Stavropoulou ◽  
Aggeliki Daraki ◽  
Marina Kalomoiraki ◽  
Panagoula Kollia ◽  
...  
Keyword(s):  
De Novo ◽  
Conflicts Of Interest ◽  
Type A ◽  
Common Mechanism ◽  
Genotype Distribution ◽  
Distribution Analysis ◽  
Wild Type ◽  
Increased Risk ◽  
Variant Genotype

Abstract Abstract 1445 Models for the pathogenesis of myelodysplastic syndromes (MDS) imply the role of individual genetic variations in genes involved in detoxification mechanisms. GSTP1 enzyme plays a key role in detoxification of a variety of electrophilic compounds, such as benzo [a]-pyrene and other polycyclic aromatic hydrocarbons (PAHs), chemotherapy drugs and products of oxidative stress. GSTP1 acts through a common mechanism of conjugating reactive oxygen species (ROS) with glutathione, enabling their detoxification and elimination and thus defending tissues against DNA damage. The corresponding gene is subject to a single-nucleotide polymorphism (A313G) leading to abolished enzyme activity. Thus, individuals homozygous for the variant G allele (G/G) have a lower conjugating activity than individuals homozygous for the wild type A allele (A/A), while heterozygotes (A/G) display intermediate activity. The aim of the present study was to evaluate whether the GSTP1 polymorphism influences susceptibility to MDS and/or promote specific chromosomal aberrations. We conducted a case-control study in 310 de novo MDS patients and 370 unrelated healthy controls using both a conventional PCR-RFLP assay and a novel Real-Time PCR genotyping method using hybridization probe technology. The GSTP1 gene status was also evaluated in relation to patients' characteristics and chromosomal abnormalities. Comparison of the genotype distribution between controls and MDS cases revealed a significantly higher frequency of the variant genotypes (heterozygotes A/A and homozygotes G/G) among MDS patients, as compared to controls (p<0.0001, χ2=31.167, df=2). The most marked statistical difference between MDS patients and controls was observed between the wild-type (A/A) and the homozygous variant genotype (G/G), since subjects carrying the G/G variant genotype showed a 4.1-fold increased risk of MDS prevalence than subjects carrying the wild-type A/A genotype (p=0.000, χ2=30.5, d.f.=1, OR=4.098, 95%CI=[2.433–6.897]). Allele frequencies distribution analysis between patients and controls, showed that MDS patients exhibited a 1.9-fold increased risk of carrying at least one variant G allele, as compared to the controls (p<0.0001, d.f.=1, OR =1.9, 95%CI=[1.48–2.34]). There was no association between the GSTP1 polymorphism and gender or any specific cytogenetic subgroup, while stratification of patients according to age showed a differential GSTP1 genotype distribution (p=0.007). Our results, derived from the larger series of primary MDS cases tested for the GSTP1 genetic background, reveal an increased incidence of the GSTP1 variant genotypes among MDS patients, providing evidence for a potential pathogenetic role of the GSTP1 polymorphism on de novo MDS risk. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Increased Adhesion and Aggregation of Platelets Lacking Cyclic Guanosine 3′,5′-Monophosphate Kinase I

1999 ◽  
Vol 189 (8) ◽  
pp. 1255-1264 ◽  
Author(s):  
Steffen Massberg ◽  
Matthias Sausbier ◽  
Peter Klatt ◽  
Markus Bauer ◽  
Alexander Pfeifer ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Platelet Adhesion ◽  
Ischemia Reperfusion ◽  
Vascular Lesions ◽  
Downstream Target ◽  
Platelet Adhesion And Aggregation ◽  
Smooth Muscle Proliferation ◽  
Aggregation Of Platelets

Atherosclerotic vascular lesions are considered to be a major cause of ischemic diseases, including myocardial infarction and stroke. Platelet adhesion and aggregation during ischemia–reperfusion are thought to be the initial steps leading to remodeling and reocclusion of the postischemic vasculature. Nitric oxide (NO) inhibits platelet aggregation and smooth muscle proliferation. A major downstream target of NO is cyclic guanosine 3′,5′-monophosphate kinase I (cGKI). To test the intravascular significance of the NO/cGKI signaling pathway in vivo, we have studied platelet–endothelial cell and platelet–platelet interactions during ischemia/reperfusion using cGKI-deficient (cGKI−/−) mice. Platelet cGKI but not endothelial or smooth muscle cGKI is essential to prevent intravascular adhesion and aggregation of platelets after ischemia. The defect in platelet cGKI is not compensated by the cAMP/cAMP kinase pathway supporting the essential role of cGKI in prevention of ischemia-induced platelet adhesion and aggregation.

scholarly journals In-Silico Transcriptome Analyses of Hemostasis Triggers in Inflamed Vs Normal Mucosa of IBD Patients

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 19-20
Author(s):  
Enrica Branca ◽  
Giovanna Carrà ◽  
Isabella Russo ◽  
Massimo Terzolo ◽  
Angelo Guerrasio ◽  
...  
Keyword(s):  
In Silico ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Normal Mucosa ◽  
Fibrinolytic System ◽  
Coagulation System ◽  
Increased Risk ◽  
Underlying Mechanisms

Background:inflammatory bowel disease (IBD) is characterized by chronic inflammation associated with an increased tendency to thrombosis and thromboembolic complications. The underlying mechanisms of VTE are not yet fully understood. Several studies reported different expressions of circulating procoagulant factors or fibrinolysis inhibitors in IBD. Others linked podoplanin overexpression with thrombosis, hypercoagulability, and increased risk of VTE. Here, we aimed to identify, among genes able to trigger thrombosis, those aberrantly expressed in IBD samples as compared with matched normal mucosa. Methods:we analyzed the transcriptome of matched normal and inflamed lesions in 168 patients with Adult Crohn Disease (CD) and 245 pediatric IBD using publicly available datasets. We intentionally assessed the expression levels of triggers and inhibitors of the coagulation system (Tissue Factor, TFI), the fibrinolytic system (PLAU, PLAT, PAI-1/2/3, TAFI, PN-1, SERPINF2, A2M, SNX1, SERPINC1) and platelets activation (podoplanin). Analyses were finally performed using additional datasets with 219 ulcerative colitis (UC) adult patients, 198 IBD patients and compared to normal colon transciptome. Results:in all datasets, when compared to matched normal mucosal transcriptome, CD and UC inflamed tissues over-expressed Podoplanin mRNA (p=2.46e-25). TF mRNA was also consistently up-regulated in inflamed mucosal of IBD patients, even if paralleled with up-regulation of TFPI as well, questioning on the functional role of TF in IBD. Among the regulators of the fibrinolytic system the urokinase-activator of the plasminogen is consistently deregulated in inflamed areas. Conclusion: our in-silico analyses on gene expression profiles, using matched normal and pathological mucosa of the same patients, suggested that IBD inflamed mucosa favors platelets activation due to podoplanin over-expression. These observations require further studies to assess the biological role of podoplanin in IBD patients and opens new insights on how to perform antithrombotic prophylaxis in IBD patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Increased Incidence of Infection in Patients with Myelofibrosis and Transfusion-Associated Iron Overload: A Monocentric Experience

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4186-4186
Author(s):  
Giovanni Caocci ◽  
Maria Pina Simula ◽  
Silvia Ghiani ◽  
Olga Mulas ◽  
Giorgia Mainas ◽  
...  
Keyword(s):  
Iron Overload ◽  
Conflicts Of Interest ◽  
Ferritin Level ◽  
Real Life ◽  
Observation Time ◽  
System Response ◽  
Anatomical Site ◽  
Increased Risk

Background. Transfusion-associated iron overload may increase the risk of infections both increasing bacterial or fungal growth and leading to the production of free iron that impairs immune system response. Anemia and transfusion dependency (TD) represent well known prognostic factors of survival in patients with myelofibrosis (MF). Compared to myelodysplastic syndrome (MDS), the role of iron overload on infection in MF has been scarcely explored. Methods. We identified consecutive adult patients diagnosed at our Centre with primary or secondary MF, between 1998 and 2018. Patients were stratified in 2 groups according to transfusion dependence defined as having received >2 unit of red blood cells (RBC) over 3 months. The total number of RBC units infused was also collected. All infections were recorded and graded according to Common Terminology Criteria for Adverse Events (CTCAE). Results. A total of 106 patients, median age 72 years (range 44-89) were retrospectively evaluated. A diagnosis of primary MF was performed in 75 of cases (71%), post Essential Thrombocythemia myelofibrosis (PET-MF) in 27 (25%) and post Polycythemia Vera myelofibrosis (PPV-MF) in 4 (4%). Splenomegaly was present in 83 patients (78%) and 30 (28%) reported constitutional symptoms.According to the IPSS, 43 patients (40 %) were classified at low or intermediate-1 risk, and 63 (60 %) at intermediate-2 or high risk. Molecular analysis was performed in 76 patients (72%): 54 of 76 patients (71%) were positive for the JAK2V617F mutation, 10 (13%) for CALR mutation, 4 (5%) for MPL, and 8 (11%) were negative for all tested mutations. Over time, 56 patients (53%) received hydroxyurea or other cytoreductive treatment, and 23 (22%) ruxolitinib. Overall, 39 patients (37%) were transfusion dependent with a median of 14 RBC units received during follow-up (range 4-199). The median serum ferritin level in the TD cohort was 840 ng/mL (range 130-12.129). Median observation time was 36 months (range 3-203). At last follow-up, 48 patients (45%) presented one or more infectious episodes. Total infectious events were 69 and 13 of them (19%) were defined as severe (grade 3-4 CTCAE). Anatomical site of infections was the respiratory tract in 28 (41%) of cases, gastro-intestinal in 22 (32%), gynecological-urological in 8 (11%), sepsis in 2 (3%), unspecified in the remaining cases. When detected, the etiological agents were bacterial in 8 (12%), viral in 5 (7%) and fungal in 4 (6%). The 60-month cumulative incidence of infection from diagnosis of MF was 64.1±6.5%. TD patients showed a higher incidence of infection (99±8.8% vs 53.3±8%, p=0.007) (Figure 1). In multivariate analysis no association was found between infection incidence and primary vs secondary MF, splenomegaly, age >65 years, ruxolitinib treatment; only transfusion dependencemaintained a significant association (p=0.019; HR=2.13; 95% C.I.=1.13-4.01). Among the 39 TD patients, the transfusion burden was significantly higher in those with infectious complication (median 24 RBC units vs 15 RBC units; p=0.012). The 60-month overall survival was 40±5.9%. Lower IPSS-risk (p<0.0001) and ruxolitinib treatment (p=0.027) were significantly associated with higher survival. Conclusions. This retrospective monocentric real-life study showed an increased risk of infections in MF patients with higher transfusion burden. The role of iron chelation in improving infection free survival in patients with MF and iron overload should be further investigated. Figure 1 Disclosures No relevant conflicts of interest to declare.

scholarly journals Brefeldin Α Reduces STAT3 Dependent Survival of Acute Leukemia and Neuroblastoma Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5762-5762
Author(s):  
Mitsuteru Hiwatari ◽  
Kentaro Watanabe ◽  
Yasuo Kubota ◽  
Masahiro Sekiguchi ◽  
Junko Takita
Keyword(s):  
High Risk ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
High Risk Group ◽  
Mycn Amplification ◽  
Alk Inhibitors ◽  
Anaplastic Lymphoma ◽  
Increased Risk ◽  
Clinical Resistance

Leukemic stem cells have several dysregulated pathways and the Janus kinases (JAKs) and signal transducer and activator of transcription (STAT) pathway are prominent among them. High STAT3 expression in leukemia has been associated with an increased risk for relapse and decreased overall survival. In addition, phosphorylated STAT3 can be found following prolonged activation of the anaplastic lymphoma kinase (ALK) ligand binding site, but the level of phospho-STAT3 engendered by ligand-independent activation of ALK was exponentially higher. In ALK positive tumors, ALK-STAT3 activation promotes tumor cell outgrowth and directly prevents apoptosis. The role of ALK in several types of ALK-expressing solid tumors as well as the therapeutic use of ALK inhibitors in treating these cancers also have been reviewed in a number of recent papers.However, clinical resistance to ALK inhibitors invariably occurs. Amplified ALK or mutated ALK was identified in ~14% of neuroblastomas (NB), the most common and aggressive childhood malignancy, and phase I trial of ALK inhibitor such as crizotinib showed a lack of response in patients harboring certain ALK mutations. In this study, we implemented a high throughput chemical screen in 4 NB-derived cell lines to identify compounds with the potential of inhibiting oncogenic activity of ALK in NB, using a curated library of ~450 compounds. Brefeldin A (BFA),an inhibitor of the transport of proteins from the endoplasmic reticulum (ER) to the Golgi, was identified as a suitable candidate. It was previously reported that BFA inhibited phosphorylation of ALK and its downstream molecule, STAT3. Since an increasing number of studies have found that STAT3 abnormal expression and activation are accompanied by leukemia development, which indicates the potential role of STAT3 in the pathogenesis of leukemia, we analyzed the cytotoxicity of BFA against 12 T-ALL and 6 AML cell lines. Assessment of BFA antitumor effect using an MTT assay revealed BFA to possess potent cytotoxic activity across a broad spectrum of hematopoietic malignancies, with 10 T-ALL and 5 AML being especially responsive. For example, T-ALL cell lines, as well as AML lines, were potently inhibited by BFA ( IC50 values: ICH-TALL-UK, 168.1 nM; MOLT-4, 23.2nM; Jurkat, 66.6 nM; MOLT14, 97.3 nM; ICH-TALL-SM, 54.9 nM). In an expanded panel of 20 NB cell lines, those with or without MYCN-amplification or 11q loss of heterozygousity which have been identified as two major oncogenic events in NB pathogenesis, especially in the high-risk group were the most sensitive to low nanomolar concentrations of BFA. In NB cell lines harboring F1174L or R1275Q-mutated ALK,BFAshowed cytotoxicity.BFA reduced the phosphorylation of ALK in NIH3T3 cells stably expressed F1174L ALK or TGW, cell line harboring R1275Q-mutated ALK. Of the molecules that are phosphorylated downstream of ALK that have been reported to be important for ALCL cells, the phosphorylation of STAT3, AKT and ERK was inhibited by BFA. These results suggested that BFA can be used to target STAT3 signaling pathways for leukemia treatment. Furthermore these results indicated that the effect of BFA on ALK mutated NB cell lines is accompanied by inhibition of the phosphorylation of ALK and its downstream key effector molecule STAT3.These findings provide the significance of ALK and STAT3 as they relate to NB or leukemia, explores the significance of STAT3, AKT and ERK downstream of ALK, and touches on the potential for new chemotherapeutics targeting ALK and STAT3 to improve high risk NB and leukemia patient prognosis. Disclosures No relevant conflicts of interest to declare.

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