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Integrins are transmembrane receptors that mediate cell-cell and cell-extracellular matrix adhesion. Although all integrins can undergo activation (affinity change for ligands), the degree of activation is most spectacular for integrins on blood cells. The β2 integrins are exclusively expressed on the surface of all leukocytes including neutrophils, lymphocytes, and monocytes. They are essential for many leukocyte functions and are strictly required for neutrophil arrest from rolling. The inside-out integrin activation process receives input from chemokine receptors and adhesion molecules. The integrin activation pathway involves many cytoplasmic signaling molecules such as spleen tyrosine kinase, other kinases like Bruton's tyrosine kinase, phosphoinositide 3-kinases, phospholipases, Rap1 GTPases, and the Rap1-GTP-interacting adapter molecule. These signaling events ultimately converge on talin-1 and kindlin-3, which bind to the integrin β cytoplasmic domain and induce integrin conformational changes: extension and high affinity for ligand. Here, we review recent structural and functional insights into how talin-1 and kindlin-3 enable integrin activation, with a focus on the distal signaling components that trigger β2 integrin conformational changes and leukocyte adhesion under flow.

A unique feature of Hodgkin lymphoma (HL) is the presence of CD4+ T cells that surround, protect, and promote survival of tumor cells. The adhesion molecules involved in this so-called T-cell rosetting are important components of the immunological synapse (IS). However, it is unknown whether this synapse is fully assembled and leads to T-cell activation by enabling interaction between the T-cell receptor (TCR) and human leukocyte antigen class II (HLA-II). We established a novel rosetting model by coculturing HLA-II–matched peripheral blood mononuclear cells with HL cell lines and showed IS formation with activation of rosetting T cells. HLA-II downregulation by class II transactivator knockout did not affect the extent of rosetting, but almost completely abrogated T-cell activation. Intriguingly, the level of CD58 expression correlated with the extent of rosette formation, and CD58 knockout or CD2 blockade reduced both rosette formation and T-cell activation. The extension of our findings to primary HL tissue by immunohistochemistry and proximity ligation assays showed interaction of CD2 with CD58 and of TCR-associated CD4 with HLA-II. In conclusion, T-cell rosetting in HL is established by formation of the IS, and activation of rosetting T cells critically depends on the interaction of both TCR-HLA-II and CD2-CD58.

Neutrophil adhesion and extravasation into tissue at sites of injury or infection depend on binding of the integrin lymphocyte function–associated antigen 1 (LFA-1) to ICAM-1 expressed on activated endothelial cells. The activation-dependent conformational change of LFA-1 to the high-affinity conformation (H+) requires kindlin-3 binding to the β2-integrin cytoplasmic domain. Here we show that genetic deletion of the known kindlin interactor integrin-linked kinase (ILK) impaired neutrophil adhesion and extravasation in the cremaster muscle and in a clinically relevant model of renal ischemia reperfusion injury. Using in vitro microfluidic adhesion chambers and conformation-specific antibodies, we show that knockdown of ILK in HL-60 cells reduced the conformational change of β2-integrins to the H+ conformation. Mechanistically, we found that ILK was required for protein kinase C (PKC) membrane targeting and chemokine-induced upregulation of its kinase activity. Moreover, PKC-α deficiency also resulted in impaired leukocyte adhesion in bone marrow chimeric mice. Mass spectrometric and western blot analyses revealed stimulation- and ILK-dependent phosphorylation of kindlin-3 upon activation. In summary, our data indicate an important role of ILK in kindlin-3–dependent conformational activation of LFA-1.

Coronavirus disease 2019 (COVID-19) is a heterogeneous clinical condition caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The initial stage of infection occurs at the time of respiratory viral invasion through angiotensin-converting enzyme 2 (ACE2) receptors in alveolar pneumocytes and macrophages. In the second stage of established pulmonary disease without and with hypoxia, viral multiplication and localized inflammation occurs. The third stage COVID-19 is the most severe, characterized by systemic hyperinflammation, acute respiratory distress syndrome (ARDS) and generalized microangiopathy, with significantly elevated biomarkers including IL-2, IL-6, IL-7, granulocyte-colony stimulating factor, macrophage inflammatory protein 1α, tumor necrosis factor-α, C-reactive protein, ferritin, D-dimer, von Willebrand factor (vWF) and thrombomodulin (TM). Evidence suggests that endothelial damage and activation play a key role in the pathobiology of the disease, and thus protecting endothelium and reversing endotheliitis may be a key therapeutic goal (Teuwen et al,Nat Rev Immunol2020). Defibrotide (DF), a complex mixture of poly-deoxyribonucleotides extracted from porcine mucosa, modulates expression of thrombogenic and inflammatory mediators produced in response to endothelial cell injury and/or leukocyte activation. DF increases tissue plasminogen activator (t-PA) and TM expression, thereby enhancing the enzymatic activity of plasmin to hydrolyze fibrin clot, while also decreasing vWF and plasminogen activator inhibitor-1 (PAI-1). Platelet adhesion is inhibited via increases in nitric oxide (NO), prostaglandin I2 (PGI2), and prostaglandin E2 (PGE2) (Falanga A et al,Leukemia 2003). Anti-inflammatory properties via inhibition of p38 MAPK pathway have been demonstrated with DF, attenuating the release of inflammatory mediators including IL-6, thromboxane A2, leukotriene B4, tumor necrosis factor-alpha, and reactive oxygen species (Mitsiades et al,Clin Cancer Res2009). Additionally, DF inhibits both the expression and activity of heparanase, which in turn modulates heparan sulfate, a vital moiety for viral infection (Richardson et al,Blood Adv2018; Koganti et al,Cell Mol Life Sci2020). DF also modulates endothelial cell injury in murine models by down-regulating the expression of endothelial cell adhesion molecules such as E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), that are critical to leukocyte adhesion and migration, and by increasing the endothelial cell release of anti-inflammatory cytokines, conferring survival benefit (Garcia-Bernal et al,J Cell Mol Med2020). DF is approved for the treatment of severe veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS) complicating stem cell transplantation and has demonstrated efficacy and safety in critically ill patients with multi-organ failure, underpinned by vasculopathy, endotheliitis, immune activation and hemorrhagic risk (Richardson et al,Blood2016). This multi-target and pleiotropic approach makes DF an attractive candidate for the treatment of advanced stage COVID 19 and the systemic endothelial complications underpinning both its pathobiology and ensuing mortality (Figure 1). Various clinical studies in Spain, Italy, Ireland, the UK and USA are underway, with the larger randomized placebo-controlled prospective phase 2 study in Spain (clinicaltrials.gov: NCT04348383) leading in accrual with promising early results, demonstrating safety to date with potential efficacy, as reflected by favorable outcome in a subset of intubated patients as part of an ongoing multi-center trial. Other studies within the group have focused on safety and particularly the therapeutic role of DF in advanced disease, including those patients requiring extracorporeal membrane oxygenation (ECMO) and full dose systemic anticoagulation. These trials include common in-depth biological correlatives, assessing biomarkers including heparanase, inflammatory cytokines, immune cell sub-populations, vWF and soluble TM, as well as other novel markers and pharmacokinetics. In aggregate, these studies will hopefully help demonstrate the effects of DF on the proposed targets in COVID-19 and successfully correlate these effects with improved clinical outcome. Disclosures Moraleda: Jazz Pharmaceuticals:Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding;Novartis, Sandoz, Takeda:Honoraria;Gilead:Honoraria, Other: Travel expenses.Carlo-Stella:Servier, Novartis, Genenta Science srl, ADC Therapeutics, F. Hoffmann-La Roche, Karyopharm, Jazz Pharmaceuticals:Membership on an entity's Board of Directors or advisory committees;Bristol-Myers Squibb, Merck Sharp & Dohme, Janssen Oncology, AstraZeneca:Honoraria;Boehringer Ingelheim and Sanofi:Consultancy;ADC Therapeutics and Rhizen Pharmaceuticals:Research Funding.Jara Rubio:Edwards Lifesciences, Getinge:Honoraria.Diaz-Ricart:Jazz Pharmaceuticals:Honoraria, Research Funding;German Jose Carreras Leukaemia Foundation:Research Funding.Carreras:Jazz Pharmaceuticals:Research Funding, Speakers Bureau;German Jose´ Carreras Leukaemia Foundation:Research Funding.Mo:Celgene/BMS:Membership on an entity's Board of Directors or advisory committees.Cooke:Jazz Pharmaceuticals:Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.Soiffer:Be the Match/ National Marrow Donor Program:Membership on an entity's Board of Directors or advisory committees;alexion:Consultancy;Celgene:Membership on an entity's Board of Directors or advisory committees;Juno:Membership on an entity's Board of Directors or advisory committees;Novartis:Consultancy;VOR Biopharma:Consultancy;Mana Therapeutics:Consultancy;Precision Bioscience:Consultancy;Cugene:Consultancy;Rheos Therapeutics:Consultancy;Gilead:Consultancy;Kiadis:Membership on an entity's Board of Directors or advisory committees.Baron:Genetech:Consultancy;Merck:Consultancy.Pagliuca:Jazz Pharmaceuticals:Membership on an entity's Board of Directors or advisory committees.Richardson:Celgene/BMS, Oncopeptides, Takeda, Karyopharm:Research Funding. OffLabel Disclosure: Defibrotide used for the treatment of endotheliitis complicating SARS-Cov-2 infection

Neutrophils represent the first line of cellular defense against invading microorganism by rapidly moving across the blood–endothelial cell (EC) barrier and exerting effector cell functions. The neutrophil recruitment cascade to inflamed tissues involves elements of neutrophil rolling, firm adhesion, and crawling onto the EC surface before extravasating by breaching the EC barrier. The interaction between neutrophils and ECs occurs via various adhesive modules and is a critical event determining the mode of neutrophil transmigration, either at the EC junction (paracellular) or directly through the EC body (transcellular). Once thought to be a homogenous entity, new evidence clearly points to the plasticity of neutrophil functions. This review will focus on recent advances in our understanding of the mechanism of the neutrophil transmigration process. It will discuss how neutrophil–EC interactions and the subsequent mode of diapedesis, junctional or nonjunctional, can be context dependent and how this plasticity may be exploited clinically.

Early stages of inflammation are characterized by extensive oxidative insult by recruited and activated neutrophils. Secretion of peroxidases, including the main enzyme, myeloperoxidase, leads to the generation of reactive oxygen species. We show that this oxidative insult leads to polyunsaturated fatty acid (eg, docosahexaenoate), oxidation, and accumulation of its product 2-(ω-carboxyethyl)pyrrole (CEP), which, in turn, is capable of protein modifications. In vivo CEP is generated predominantly at the inflammatory sites in macrophage-rich areas. During thioglycollate-induced inflammation, neutralization of CEP adducts dramatically reduced macrophage accumulation in the inflamed peritoneal cavity while exhibiting no effect on the early recruitment of neutrophils, suggesting a role in the second wave of inflammation. CEP modifications were abundantly deposited along the path of neutrophils migrating through the 3-dimensional fibrin matrix in vitro. Neutrophil-mediated CEP formation was markedly inhibited by the myeloperoxidase inhibitor, 4-ABH, and significantly reduced in myeloperoxidase-deficient mice. On macrophages, CEP adducts were recognized by cell adhesion receptors, integrin αMβ2 and αDβ2 Macrophage migration through CEP-fibrin gel was dramatically augmented when compared with fibrin alone, and was reduced by β2-integrin deficiency. Thus, neutrophil-mediated oxidation of abundant polyunsaturated fatty acids leads to the transformation of existing proteins into stronger adhesive ligands for αMβ2- and αDβ2-dependent macrophage migration. The presence of a carboxyl group rather than a pyrrole moiety on these adducts, resembling characteristics of bacterial and/or immobilized ligands, is critical for recognition by macrophages. Therefore, specific oxidation-dependent modification of extracellular matrix, aided by neutrophils, promotes subsequent αMβ2- and αDβ2-mediated migration/retention of macrophages during inflammation.

Introduction: Inappropriate activation of Wnt/β-catenin signaling plays a role in some cancers. β-catenin (β-cat) levels in the cell can be regulated by a cadherin-mediated sequestration into membrane-bound and free cytosolic pools, with the later translocating to the nucleus and driving TCF-mediated transcriptional activity following Wnt signal transduction. While sequencing has shown that MM lacks the mutations that typically lead to constitutive β-cat activation seen in other cancers, we and others have demonstrated that Wnt/β-catenin signaling is nonetheless activated in MM and can regulate MM growth. The mechanism driving β-cat stabilization and activation in MM is unclear. E- and N-cadherin (N-cad) expression is elevated in MM compared to plasma cells from healthy donors. We hypothesized that that cadherins can regulate Wnt/β-catenin signaling in MM. Materials and Methods: We detected different forms of β-cat expression in a panel of human MM cell lines (HMCLs) and CD138 PC from MM patients by several approaches. Cadherin gain- or loss-of-function MM models were produced by expressing wild-type N-cad in MMS1 and ARP1 (lack endogenous N-cadherin expression) using a lentiviral system to create stable cell lines (N-Cad/MMS1 and N-cad/ARP1) and empty vector control (EV/MMS1, and EV-ARP1). We knocked down N-cadherin in the JJN3 cell line expressing high level of endogenous N-cadherin using shRNA specific for N-cad (shNcad/JJN3) or scrambled control shRNA (shCont/JJN3) by lentiviral-mediated transfection. We used a TCF reporter system to evaluate β-cat transcriptional activity as previously described. Results: We surveyed 25 HMCLs and CD138-selected plasma cells from 72 newly diagnosed MM for active β-cat with an antibody that specifically recognizes the unphosphorylated active form of β-cat. Higher levels of cytosolic and/or nuclear β-cat protein were seen in 13 of 25 (52%) HMCLs and 36 of 72 (50%) primary MM PC. Correlation of β-cat protein levels with global mRNA expression levels in primary PC revealed significant correlation with only one gene, CDH2 (N-cad). Remarkably, those primary MM with high β-cat levels but low CDH2 levels expressed high levels of E-cadherin/CHD1 mRNA. This posed the question of whether CDH2 is a direct target of TCF/β-cat transcriptional activity or whether high levels of CDH2 lead to increased levels of β-cat protein via sequestration. Both CDH2 mRNA and protein were correlated with β-cat protein but not β-cat mRNA in 23/25 HMCLs. Co-immunoprecipitation revealed that N-cad and β-cat complexes could be identified in HMCLs and primary MM. Consistent with N-cad-mediated stabilization of β-cat both total and unphosphorylated β-cat levels and TCF activity were significantly elevated in N-cad/MMS1 and N-Cad/ARP1 cells relative to controls. In contrast, shRNA mediated knockdown of N-cad led to a loss of both N-cad and β-cat protein levels and TCF-dependent transcription activity relative to controls. These findings provide evidence that β-cat/TCF signaling can be regulated by N-cad in MM. CDH2 mRNA is significantly elevated in the MS and HY subgroups of MM. To search for a potential mechanism of CDH2 transcriptional regulation in MS MM, we compared TCF activity and β-cat protein levels in MS versus non-MS HMCLs. TCF activity and active β-cat were elevated in MS versus non-MS forms of MM and B-cell lymphoma lacking N-cadherin. To determine if MMSET is required to up-regulate N-cad expression, we depleted the full-length MMSET protein in KMS11 cells. The results revealed a dramatic loss of total and unphosphorylated β-cat protein, but not mRNA, and loss of both CDH2 mRNA and protein relative to controls. These data suggest that MMSET can regulate the transcription of the CDH2 gene. MMS1 and ARP1 cells stably expressing N-cad exhibited enhanced adhesion to bone marrow stromal cells and decreased sensitivity to bortezomib (Bzb). In contrast, blocking N-cadherin-mediated adhesion by CDH2 shRNA increased sensitivity to Bzb. These results suggests that N-cad/β-cat complexes can contribute to adhesion-mediated drug resistance in MM. Conclusion: Taken together, these findings establish that β-cat is stabilized by N-cadherin-, and likely E-cadherin-, adhesins junction formation in MM. This in turn leads to an increased pool of β-cat that can drive TCF transcriptional activation as well participate in cadherin-mediated cell adhesion and drug resistance. Disclosures Davies: Amgen: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Morgan: BMS: Membership on an entity's Board of Directors or advisory committees; Jansen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees. Walker: Bristol Myers Squibb: Research Funding; Sanofi: Speakers Bureau.

Sickle cell-related vaso-occlusive crises (VOCs) are among the primary clinical manifestations of sickle cell disease (SCD) and are associated with many acute and chronic complications that lead to significant morbidity and mortality. VOCs are caused by the adhesion of leukocytes and sickle erythrocytes to the endothelium, which results in vascular obstruction and tissue ischemia. By blocking the P-selectin- PSGL-1 (P-selectin glycoprotein ligand 1) mediated cell adhesion, crizanlizumab, a recently FDA approved humanized IgG2 anti-P-selectin antibody, reduced the frequency of VOCs in SCD patients and established the proof of principle for this approach (Ataga KI et al., N Engl J Med, 2017). Inclacumab is a novel, fully human IgG4 monoclonal antibody that selectively targets P-selectin and has safely demonstrated sustained anti-cell adhesion effects in over 700 participants including healthy volunteers and patients with cardiovascular disease (Schmitt C et al., J Cardiovasc Pharmacol. 2015; Tardif JC et al., J Am Coll Cardiol, 2013; Morrison M et al., Eur J Clin Pharmacol, 2015; Kling D et al., Thromb Res, 2013). A crystal structure of inclacumab and P-selectin reveals that inclacumab directly binds to an epitope in the PSGL-1 binding region on P-selectin and thus competitively inhibits P-selectin and its ligand interaction. In contrast, crizanlizumab binds to a more distant epitope to the PSGL-1 binding site on P-selectin. To further elucidate differences between the two antibodies, we characterized inclacumab and crizanlizumab in a series of in vitro functional assays including ligand binding affinity, competitive ligand binding by surface plasmon resonance (SPR), P-selectin mediated cell-based adhesion assay and cell-cell interaction with human whole blood samples. In vitro, inclacumab binds to human P-selectin with high affinity and potently suppresses the interaction of P-selectin with its main ligand PSGL-1. Both antibodies exhibited similar binding affinities to P-selectin (KD of 9.9 and 9.1 nM for inclacumab and crizanlizumab, respectively) and comparable potencies at preventing a PSGL-1 mimetic peptide from binding P-selectin (IC50 of 1.9 and 2.2 µg/mL for inclacumab and crizanlizumab, respectively) or blocking the adhesion of PSGL-1 expressing cells to an immobilized P-selectin (IC50 = 430 ng/mL for inclacumab and IC50 = 453 ng/mL for crizanlizumab). However, inclacumab demonstrated greater maximal platelet-leukocyte cell adhesion inhibition in response to thrombin receptor activating peptide (TRAP) in blood samples from both healthy volunteers and subjects with SCD in an in vitro efficacy assay (see figure). Inclacumab is differentiated from crizanlizumab as a fully human monoclonal antibody that directly blocks the PSGL-1 binding region of P-selectin and shows greater maximal inhibition of cell-cell interactions in vitro. At doses up to 20 mg/kg Q4W, which previous clinical trials have shown to be safe and well-tolerated, inclacumab has much greater drug exposure than the approved dose of crizanlizumab (5 mg/kg W0/W2/Q4W) (Ataga KI et al., N Engl J Med, 2017; Schmitt C et al., J Cardiovasc Pharmacol. 2015; Tardif JC et al., J Am Coll Cardiol, 2013). A single dose of inclacumab 20 mg/kg demonstrated full PLA inhibition for ≥84 days in healthy volunteers (Morrison M et al., Eur J Clin Pharmacol, 2015; Kling D et al., Thromb Res, 2013). Inclacumab may allow for a substantially longer and therefore more convenient dosing interval as compared with crizanlizumab. In aggregate, these data suggest that inclacumab has the potential to be a best-in-class P-selectin inhibitor to reduce VOCs in sickle cell disease. Clinical studies of inclacumab in patients with SCD are planned for the 1st half of 2021. Disclosures Geng: Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mihaila:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Yuan:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Strutt:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Benz:Roche Pharmaceuticals: Current Employment. Tang:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mayer:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Oksenberg:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Background: The pathogenesis of sickle cell disease (SCD) and its complications have been well characterized down to the molecular level. However, there remains a relative dearth of disease modifying therapies that reduce the frequency and number of vas-occlusive crises, hospitalizations, and deaths. Recent advancements in utilizing hydroxyurea and L-glutamine, which both impact unique disease pathways, should pave way for the identification of other molecular pathways as ideal drug targets. In this regard, our meta-analysis serves to identify key genes and associated pathways that are differentially expressed in SC patients. Methods: We employed our STARGEO platform to tag samples from the NCBI Gene Expression Omnibus and performed meta-analysis to compare SC and healthy control transcriptomes. For the meta-analysis, we tagged 285 peripheral blood samples from SC patients and 86 samples from healthy subjects as a control. We then analyzed the signature in Ingenuity Pathway Analysis to elucidate top disease functions from our analysis. Results: From our meta-analysis, we identified iron homeostasis signaling, NRF2-mediated oxidative stress response, cell senescence, and pyrimidine interconversion/biosynthesis as top canonical pathways that were upregulated in the peripheral blood samples from SC patients. Top upstream regulators included membrane associated protein and transporter ABCB6, non-coding RNY3, and erythroid maturation transcription factors GATA1, KLF1, and HIPK2 (with predicted activation). The most upregulated genes included inflammatory modulators RNF182 and IFI27, the latter of which has been shown to inhibit vascular endothelial growth and repair. Several membrane-associated protein coding genes such as GYPA, RAP1GAP, and PAQR9 were also upregulated in the SC samples. RAP1GAP is known to modulate neutrophil cell adhesion and homing while PAQR9 has roles in regulating protein quality control: a role also seen in similarly upregulated YOD1, a deubiquitinating enzyme involved in trafficking of misfolded proteins. Expectedly, also upregulated were HBBP1 and SOX6, which regulate globin genes and have been shown to silence γ-globin expression. Lastly, SLC6A19, the neutral amino acid transporter mutated in Hartnup disease, was also upregulated. Of the downregulated genes, WASF3, a member of the Wiskott-Aldrich syndrome protein family, has been linked to poor survival in many malignancies, including AML and CMML, but has not previously been linked to SCD pathogenesis. ENKUR was also downregulated and has been annotated as a tethering protein to cation channels as well as linked to pathways involving vascular leakage. SIGLEC10, which binds to vascular adhesion proteins, is a key suppressor of inflammatory responses to damage; it's downregulation along with ELAPOR1, a transmembrane protein involved in cellular response to stress, was also observed. Finally, based off the focus genes in our analysis we identified several networks with most being involved in amino acid metabolism, cellular assembly, function, and maintenance, hematological disease, and organismal injury. The top pathway is illustrated in Figure 1. Conclusions: Our study illustrates differentially expressed gene activity in SCD consistent with known pathophysiology such as immune response, endothelial damage and adherence, heme metabolism, and globin regulation. We also showed evidence of genes not previously studied in SCD, which may have novel roles such as those part of the ubiquitin-proteasome system like YOD1 and RNF182. Additionally, while some genes in our analysis like EKLF and GAT1 have been shown to enhance δ-globin expression, paving way for possible drug therapies for B-hemoglobinopathies, others like IFI27, PAQR9, RAP1GAP, ENKUR, SIGLEC10, WASF3, and SOX9 have yet to be studied as mediators of disease pathogenesis in SCD. A target to SOX9, a known suppressor of γ-globin, or ABCB6, a known modulator of erythroid cell shape and hydration, have particularly promising potential as disease modifying therapies. Finally, HIPK2, HBBP1, and SLC6A19 have previously been shown to have intriguing effects on hydroxyurea dosing and responsivity in SC patients and may also be candidate target molecules to enhance existing therapies. These data identify potential candidate pathways for mechanistic studies seeking to confirm a causative role in the pathogenesis of sickle cell disease. Disclosures No relevant conflicts of interest to declare.

Leukocyte adhesion to P-selectin on activated platelets and endothelial cells induces shedding of the P-selectin ectodomain into the circulation. Plasma soluble P-selectin (sP-selectin) is elevated threefold to fourfold in patients with cardiovascular disease. Circulating sP-selectin is thought to trigger signaling in leukocytes that directly contributes to inflammation and thrombosis. However, sP-selectin likely circulates as a monomer, and in vitro studies suggest that sP-selectin must dimerize to induce signaling in leukocytes. To address this discrepancy, we expressed the entire ectodomain of mouse P-selectin as a monomer (sP-selectin) or as a disulfide-linked dimer fused to the Fc portion of mouse immunoglobulin G (sP-selectin-Fc). Dimeric sP-selectin-Fc, but not monomeric sP-selectin, triggered integrin-dependent adhesion of mouse leukocytes in vitro. Antibody-induced oligomerization of sP-selectin or sP-selectin-Fc was required to trigger formation of neutrophil extracellular traps. Injecting sP-selectin-Fc, but not sP-selectin, into mice augmented integrin-dependent adhesion of neutrophils in venules, generated tissue factor-bearing microparticles, shortened plasma-clotting times, and increased thrombus frequency in the inferior vena cava. Furthermore, transgenic mice that overexpressed monomeric sP-selectin did not exhibit increased inflammation or thrombosis. We conclude that elevated plasma sP-selectin is a consequence rather than a cause of cardiovascular disease.

Integrins are a large family of heterodimeric cell surface receptors that bind prototypic ligands on neighboring cells or in the extracellular matrix. Numerous studies have revealed key roles for platelet and leukocyte integrins in adhesion and migration and, thereby, their significance for hemostasis and immunity. The clinical importance of these integrins has also become clear, because aberrant integrin expression and/or behavior are associated with bleeding disorders, immunodeficiency, or autoimmune diseases. Importantly, overwhelming evidence gathered over recent years shows that regulation of integrin function is far more complex than previously assumed; a picture has emerged of multiple cytoplasmic, cell surface, and extracellular regulators working together to ensure cell type-specific and integrin-specific control of integrin functions. Here, we discuss recent insights into the dynamic activation and suppression of hematopoietic integrins, as well as their implications for platelet and leukocyte function in health and disease.

Painful vaso-occlusive crisis (VOC) is the most common complication of sickle cell disease (SCD). Increasing evidence suggests that vaso-occlusion is initiated by increased adherence of sickle red blood cells (RBCs) to the vascular endothelium. Thus, the mechanisms that remove endothelial-attached sickle RBCs from the microvasculature are expected to be critical for optimal blood flow and prevention of VOC in SCD. We hypothesized that patrolling monocytes (PMos), which protect against vascular damage by scavenging cellular debris, could remove endothelial-adherent sickle RBCs and ameliorate VOC in SCD. We detected RBC (GPA+)-engulfed material in circulating PMos of patients with SCD, and their frequency was further increased during acute crisis. RBC uptake by PMos was specific to endothelial-attached sickle, but not control, RBCs and occurred mostly through ICAM-1, CD11a, and CD18. Heme oxygenase 1 induction, by counteracting the cytotoxic effects of engulfed RBC breakdown products, increased PMo viability. In addition, transfusions, by lowering sickle RBC uptake, improved PMo survival. Selective depletion of PMos in Townes sickle mice exacerbated vascular stasis and tissue damage, whereas treatment with muramyl dipeptide (NOD2 ligand), which increases PMo mass, reduced stasis and SCD associated organ damage. Altogether, these data demonstrate a novel mechanism for removal of endothelial attached sickle RBCs mediated by PMos that can protect against VOC pathogenesis, further supporting PMos as a promising therapeutic target in SCD VOC.

Background: In sickle cell disease (SCD), recurrent episodes of vaso-occlusive crisis (VOC) contribute to morbidity and accelerated mortality. Analysis of the RESET Phase 3 study demonstrated that administration of the pan-selectin inhibitor, rivipansel, early in the course of VOC was associated with efficacy outcomes in favor of rivipansel, suggesting that reversal of vaso-occlusion may be achieved by treatment early in VOC. While both P- and L-selectin likely contribute to the underlying inflammatory cascade leading to VOC, previous studies have shown that E-selectin expression on the vascular luminal surface appears to be crucial for adhesion of sickled (SS)-RBC and leukocytes to the endothelium. GMI-1687 is an innovative potent E-selectin specific antagonist that demonstrates high bioavailability following subcutaneous (SC) administration. In the current investigation, we report on in vitro and in vivo studies evaluating the activity of GMI-1687 in SCD mouse models of VOC. Methods: Townes transgenic mice and nude mice transfused with human SS-RBC were used as in vivo models of SCD. VOC was initiated by TNF injection in both models. Adhesion to inflamed venules and development of VOC was recorded using intravital microscopy at sites of window chamber implants. The in vivo activity of E-selectin inhibition with GMI-1687 was assessed following IV injection in the nude mouse and Townes transgenic models, and SC injection in the nude mouse model of SCD. Results: In an initial study, IV treatment with saline or GMI-1687 was initiated 30 min post infusion of SS-RBC (t30) into TNF-treated mice, a time during which sickle cells are adherent to inflamed venules and VOC has initiated, and repeated 30 minutes later (t60). As expected, mice treated with saline alone showed marked adhesion of human SS-RBC to venules (61% occlusion of the total vessels recorded at t30) with evident blood stasis. Under these conditions, GMI-1687 at 40 μg/kg significantly reversed SS-RBC adhesion and VOC at both t30 and t60 (p=0.002 and 0.0073, respectively, compared to saline treatment). This led to restoration of blood flow in 85% of the venules in GMI-1687 treated mice vs. 18% of vessels with normal blood flow in the control saline treated group. Because of reduced sickle cell adhesion and VOC, the number of circulating SS-RBC increased by approximately 6-fold in mice treated with GMI-1687. Similar anti-adhesive and anti-VOC activities of GMI-1687 treatment were obtained in the Townes transgenic model of SCD where following IV administration of the E-selectin antagonist, adhesion of sickled RBCs was significantly diminished and as a result, the percentage of the total occluded vessel segments was reduced by 80-90%. We next assessed the activity of SC administration with 40 μg/kg GMI-1687 on human SS-RBC adhesion and development of VOC in the nude mouse model. Similar to the IV experiments, GMI-1687 initiated at t30 and t60 post SS-RBC infusion significantly reduced SS-RBC adhesion in inflamed vessels (69% reduction at t30, and 63% at t60, p=0.0001). The reduction in SS-RBC adhesion led to a lower incidence of VOC (2% compared to 30% of vessels in mice injected with saline), and a 5.1-fold improvement (p=0.0315) in the number of circulating human SS-RBC. It is noteworthy that the effective dose of GMI-1687 used in these IV and SC studies resulting in the restoration of normal blood flow was approximately 500-fold less than that reported for rivipansel. Conclusions: These data demonstrate that the administration of the potent and E-selectin specific compound GMI-1687 was effective in restoring blood flow in two mouse models of SCD, and support the development of GMI-1687 formulated for SC use and self-administration with the potential for early intervention of VOC. Disclosures Thamilarasan: GlycoMimetics: Research Funding. Fogler:GlycoMimetics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Zennadi:GlycoMimetics: Research Funding.

Novel targeted therapies have substantially improved the prognosis of patients with B cell malignancies. However, a substantial fraction of patients still relapse, even after initially achieving deep remissions. Many studies have characterized the interactions between tumor cells and their microenvironment as integral to leukemia/ lymphoma homeostasis and for the provision of survival signals, also contributing to drug resistance (referred to as environment-mediated drug resistance (EMDR)). Therapeutic efforts to antagonize microenvironment-emanating survival cues have predominantly focused on perturbation of tumour cell adhesion enabling the physical displacement from protective niches (e.g. BCR-inhibitors). In an effort to address whether direct stromal targeting could more precisely mitigate EMDR, we recently characterised the molecular mechanisms underlying tumor-stroma interactions in B cell malignancies and identified a protein kinase C-β (PKC-β) as an essential kinase, required for activation of NF-κB in mesenchymal stromal cells (Lutzny et al Cancer Cell 2013). The dependency on stroma PKC-β was uniformly found for acute (ALL) and chronic (CLL, MCL) B cell malignancies. Importantly, our data further demonstrate that targeting stroma PKC-β is of key importance for multi-drug resistance of malignant B cells and can be used for therapeutic interventions (Park et al Science Trans Med 2020). Here we demonstrate novel mechanistic insights into stroma-mediated drug resistance in B cell malignancies. We identified that stroma PKC-β drives a transcriptional program in tumor cells, dependent on the activation of TGF-β and BMP-signaling, which ultimately leads to the stabilisation of BCL-XL. Our data show that BCL-XL expression in tumor cells is associated with SMAD1-induction by cytotoxic therapies, which simultaneously suppress SMAD4 expression. Importantly, SMAD1 expression was strictly dependent on stromal PKC-β activity. Antagonizing stroma signals with TGF-β inhibitors inhibits SMAD1 induction, abrogates the up-regulation of BCL-XL and overcomes stroma-dependent resistance to Venetoclax and conventional chemotherapy. The TGF-β pathway operates in parallel to the activation of the transcription factor EB (TFEB) as a down-stream target of PKC-β. Interference with these signaling pathways impairs plasma membrane integrity of stromal cells by down-regulation of numerous adhesion and signaling molecules, such as ADAM17, required for the reciprocal stabilization of BCL-XL in tumor cells. The significance of microenvironment PKC-β for drug resistance was demonstrated in vivo, using C57B/6 mice, diseased with EμTCL-1 driven B cell tumors and treated with Venetoclax in combination with or without PKC-β inhibitors. Combined treatment significantly prolonged survival, based on PKC-β mediated impairment of EMDR. Similarly, concurrent treatment of PKC-β inhibitors with chemotherapy also improved survival in an ALL-PDx model Our data demonstrate that mitigating EMDR with small molecule inhibitors of PKC-β or TGF-β signalling enhance the effectiveness of both targeted and non-targeted chemotherapies and moreover, has the ability to overcome Venetoclax resistance in B cell malignancies. Clinical trials with repurposed drugs inhibiting the here described pathways mediating EMDR are in planning. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Midostaurin as inhibitor of stroma PKC-β

Sickle cell disease (SCD) is an autosomal recessive genetic disease caused by the inheritance of a single point mutation, resulting in abnormal sickle hemoglobin (HbS). During hypoxia or dehydration, HbS polymerizes to form insoluble aggregates and induces sickling of red blood cells (RBCs). RBC sickling increases the adhesiveness of RBCs to alter the rheological properties of the blood and trigger inflammatory responses, leading to hemolysis, vasoocclusive crisis, pulmonary complications, plethora, and other pathological sequelae. Glycosaminoglycans, such as low molecular weight heparin (LMWH), have been suggested as treatments to relieve coagulation complications in SCD because of their ability to decrease thrombin generation and sickle cell adhesion. However, they are associated with bleeding complications after repeated dosing. An alternative, sulfated non-anticoagulant LMWH derivative (S-NACH) was previously reported to have none to low systemic anticoagulant activity and no bleeding side effects, and it interfered with P-selectin-dependent binding of sickle cells to endothelial cells, with concomitant decrease in the levels of adhesion biomarkers in SCD mice (1,2). S-NACH has been further engineered to possess an aldehyde moiety, which confers anti-sickling properties primarily due to specific interactions with HbS to increase its affinity for oxygen. Our in vitro sickling assay under hypoxic conditions using S-NACH at 0.5 - 2 mM demonstrated that S-NACH significantly reduced the sickling of SS cells and in a concentration-dependent manner, with comparable to that of 1 mM GBT440 (Figure 1 A-C). A similar concentration dependent effect on increasing HbS affinity for oxygen using oxygen equilibrium study was documented. In an vivo animal model using Townes' SCD animals plasma levels of pro-inflammatory cytokines IL-1β, IL-6, IFN-γ, MCP-1, TNF-α, M-CSF, and VEGF were increased in SCD untreated samples in contrast to a significant decrease (*P< 0.001) in S-NACH-treated animals, at both 2 and 6 h. In addition, S-NACH was able to increase the decreased levels of the endogenous anti-inflammatory IL-10 (Figure 1 D). The above set of novel findings about S-NACH established it to be effective for the management of SCD via the modulation of thromboinflammatory pathways, involved in thromboembolism and end organ damage, beside anti-sickling, anti-selectin and without causing any bleeding risk. Reference 1. Alshaiban A, Muralidharan-Chari V, Nepo A, Mousa SA. Modulation of Sickle Red Blood Cell Adhesion and its Associated Changes in Biomarkers by Sulfated Non-anticoagulant Heparin Derivative. Clin Appl Thromb Hemost. 2016 ;22(3):230-8. 2. Mousa SA: Compositions and method for anti-sickling of red blood cells in sickle cell disease. US Patent 9,822,190, November 2017. Disclosures Mousa: Vascular Vision Pharma Co.: Patents & Royalties.

Introduction The JAK2V617F+ mutation occurs in up to 95% of patients with polycythemia vera (PV) and increases the risk of thrombosis 6-fold. Recent studies demonstrate that JAK2V617F+ endothelial cells express pro-adhesive proteins, suggesting that the endothelium may contribute to increased thrombosis.1-3 The targeted JAK1/JAK2 inhibitor ruxolitinib is an approved second-line therapy for PV patients and is effective in alleviating constitutional symptoms, lowering hematocrit, and reducing cell number. However, there is limited data regarding efficacy of ruxolitinib in reducing thrombosis. Although recent work has demonstrated ruxolitinib reduces neutrophil extracellular trap formation,4 the vascular effects of ruxolitinib are unknown. Therefore, we hypothesize that ruxolitinib reduces endothelial cell pro-adhesive activation leading to decreased rolling and adhesion of JAK2V617F+ samples. Methods To mimic JAK2V617F activation, primary human umbilical vein endothelial cells (HUVEC, passage 1-5) were treated with TNF-α (10 ng/mL) +/- ruxolitinib (400 nM-4 µM, Selleckchem) and characterized 4h later. For confocal microscopy, cells were fixed with paraformaldehyde, permeabilized and stained for either VWF, VCAM-1, or P-selectin along with 2-(4-amidinophenyl)-1H-indole-carboxamide (DAPI, Thermo Fisher). Images were captured on Olympus FluoView FV1000 IX2 Inverted Confocal microscope. Secretion of VWF, VCAM-1 and P-selectin into conditioned media was assessed with ELISA (Molecular Innovations; BioLegend). Citrated normal and JAK2V617F+whole blood was obtained per IRB and labeled with calcein AM (ThermoFisher). To quantify leukocyte and platelet velocity and adhesion, an endothelialized poly-di-methyl-siloxane (PDMS) microchannel was prepared and treated with TNF-α (10 ng/mL) +/- ruxolitinib (400 nM-4 µM). Samples were perfused through the microfluidic at a shear stress of 0.35 dynes/cm2, and 10 s images were captured using a fluorescence microscope (Olympus). Cell velocity and adhesion were quantified using FIJI (NIH). Results Compared to TNF-α treated HUVEC alone, the combination of ruxolitinib + TNF-α reduced expression of VWF, VCAM-1 and P-selectin using both immunofluorescence and ELISA. In endothelialized microfluidic devices treated with TNF-α alone, both normal (n=3) and JAK2V617F+ (n=9) leukocyte and platelet velocity was significantly decreased and cell adhesion increased. This result was independent of hematocrit and platelet levels. In normal controls (n=2), TNF-α+ruxolitinib lead to a trend toward normal leukocyte and platelet velocity with decreased cell adhesion. Lastly, in a small subset of JAK2V617F+ patients (n=4), addition of ruxolitinib increased cell velocity. Conclusions In conclusion, in TNF-α-activated endothelial cells, treatment with ruxolitinib decreases pro-adhesive VWF, VCAM-1, and P-selectin expression. Using normal controls and JAK2V617F+ MPN blood samples, our TNF-α-activated endothelialized microfluidics model demonstrates significant reduction in leukocyte and platelet velocity and increased cell adhesion. In normal and JAK2V617F+ MPN whole blood, treatment of TNF-α-stimulated endothelium with ruxolitinib improves cell velocity. Further evaluation using JAK2V617F+ endothelial cells is planned. Collectively, these results suggest that ruxolitinib may reduce JAK2V617F+ thrombotic risk through reduction of pro-adhesive endothelial activation. Disclosures Vercellotti: CSL Behring: Research Funding.

Coagulation cascade activation and fibrin deposits have been implicated or observed in diverse forms of liver damage. Given that fibrin amplifies pathological inflammation in several diseases through the integrin receptor αMβ2, we tested the hypothesis that disruption of the fibrin(ogen)-αMβ2 interaction in Fibγ(390-396A) mice would reduce hepatic inflammation and fibrosis in an experimental setting of chemical liver injury. Contrary to our hypothesis, α-naphthylisothiocyanate (ANIT)-induced liver fibrosis increased in Fibγ(390-396A) mice, whereas inflammatory cytokine expression and hepatic necrosis were similar to ANIT-challenged wild-type (WT) mice. Increased fibrosis in Fibγ(390-396A) mice appeared to be independent of coagulation factor 13 (FXIII) transglutaminase, as ANIT challenge in FXIII-deficient mice resulted in a distinct pathological phenotype characterized by increased hepatic necrosis. Rather, bile duct proliferation underpinned the increased fibrosis in ANIT-exposed Fibγ(390-396A) mice. The mechanism of fibrin-mediated fibrosis was linked to interferon (IFN)γ induction of inducible nitric oxide synthase (iNOS), a gene linked to bile duct hyperplasia and liver fibrosis. Expression of iNOS messenger RNA was significantly increased in livers of ANIT-exposed Fibγ(390-396A) mice. Fibrin(ogen)-αMβ2 interaction inhibited iNOS induction in macrophages stimulated with IFNγ in vitro and ANIT-challenged IFNγ-deficient mice had reduced iNOS induction, bile duct hyperplasia, and liver fibrosis. Further, ANIT-induced iNOS expression, liver fibrosis, and bile duct hyperplasia were significantly reduced in WT mice administered leukadherin-1, a small molecule that allosterically enhances αMβ2-dependent cell adhesion to fibrin. These studies characterize a novel mechanism whereby the fibrin(ogen)-integrin-αMβ2 interaction reduces biliary fibrosis and suggests a novel putative therapeutic target for this difficult-to-treat fibrotic disease.