E-selectin Ligand Research Articles

Binding kinetics of liposome conjugated E-selectin and P-selectin glycoprotein ligand-1 measured with atomic force microscopy

Various ligand-functionalized liposomes have been developed for targeted therapies. Typically, the binding properties of the ligands and targeted proteins are measured with surface plasmon resonance (SPR), where the proteins are immobilized on a rigid surface. However, the difference of protein-ligand binding kinetics between liposome-conjugated protein and rigid surface-conjugated protein is not fully understood. In this work, the binding kinetics of P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin conjugated on liposome and on rigid surfaces are investigated with Atomic Force Microscopy (AFM). The results suggest that protein orientation and diffusion on liposomal membrane can alter the binding kinetics of the protein-ligand interaction. Specifically, the association and dissociation rate constant of AFM probe-conjugated E-selectin and glass-conjugated PSGL-1 are measured as 9.32 × 104 M−1s−1 and 1.54 s−1, respectively. While for the liposome-conjugated E-selectin and glass-conjugated PSGL-1, the kinetic constants are measured as 5.00 × 107 M−1s-1 and 2.76 s−1, respectively. Thus, there is an order’s magnitude increase of binding affinity (from kd = 16.51 μM to kd = 0.06 μM) when protein is attached to liposome compared to attached to a rigid surface. The results might provide better understanding and pave the way for the future design of the ligand-targeted liposomes.

Abstract CT144: Development of GMI-1359, a novel agent targeting tumor-microenvironment cross-talk in bone metastatic cancer

Abstract Pre-clinical and clinical data suggest that the bone marrow (BM) environment provides breast cancer (BC) cells a protective haven against chemotherapeutic insult, endocrine therapies, and immune recognition. Novel agents that intercept cross-talk between BC cells and the host could therefore improve the depth of response to therapies and potentially increase overall survival in metastatic BC. We have previously shown that bone metastatic BC cells reside in perivascular niches expressing high levels of SDF-1α (CXCL12) and E-selectin, two molecules for which emerging data have demonstrated a significant role in metastatic BC progression. Our prior pre-clinical studies have shown that E-selectin inhibition specifically prevents circulating BC cells from homing to the BM, whereas CXCR4 blockade mobilizes micrometastases from the marrow into circulation, where they may be sensitized to chemotherapeutic cell kill. Given the normal role of these molecules in host immune responses, E-selectin/CXCR4 blockade has additional potential to impact the tumor immune microenvironment. GMI-1359 is a small molecule, glycomimetic compound with dual inhibitory activity against E-selectin and CXCR4. We hypothesize that GMI-1359 can block E-selectin and CXCR4 binding to E-selectin ligands and SDF-1, disrupting the protective effects of the bone microenvironment on tumor cells. A single-center, phase 1b, open-label, single and multiple ascending dose study (NCT04197999) is therefore evaluating GMI-1359 in patients with HR+ metastatic breast cancer with bony metastases who are stable or minimally progressive on endocrine therapy, with or without a CDK4/6 inhibitor. To date, 2 patients have been enrolled and received single monthly doses of 3.5, 5.0 and 7.0 mg/kg followed by 3 daily doses of 7 mg/kg. Both patients have completed treatment with no dose limiting toxicities observed. Coincident with GMI-1359 administration at all dose levels, we observed mobilization of CD34+ cells coupled with a reduction in elevated serum sE-selectin levels, demonstrating the dual functionality of the compound. Initial peripheral blood immunophenotype data on one patient revealed a redistribution of myeloid derived suppressor cells and a shift in macrophage polarization from M2 to M1. Notably, we observed similar immune alterations in ongoing preclinical studies in the E0771 syngeneic BC mouse model. We found that orthotopically-engrafted mice treated with single agent 1359 had a significant increase in the ratio of CD8/Tregulatory cells isolated from the primary tumor and BM. We also observed a decrease in primary tumor M-MDSCs and lung metastasis-associated macrophages. In summary, our preliminary clinical data suggest that GMI-1359 is well-tolerated and elicits on-target effects expected from disruption of the host microenvironment. Ongoing clinical and preclinical work will define the efficacy of this strategy to enhance responses to chemo and immune therapies. Citation Format: P. Kelly Marcom, Trevor T. Price, Andrew S. Murray, William E. Fogler, John L. Magnani, Helen Thackray, Eric Feldman, Dorothy Sipkins. Development of GMI-1359, a novel agent targeting tumor-microenvironment cross-talk in bone metastatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT144.

CD44 fucosylation on bone marrow-derived mesenchymal stem cells enhances homing and promotes enteric nervous system remodeling in diabetic mice

BackgroundDiabetes can cause extensive enteric nervous system (ENS) injuries and gastrointestinal motility disorder. In developing possible treatments, researchers have engaged in tissue regeneration engineering with the very promising bone marrow-derived mesenchymal stem cells (BMSCs). However, BMSCs have poor homing ability to the targeted tissues after intravenous injection. Thus, we aimed to investigate whether enhancing the expression of E-selectin ligand on BMSCs could improve their homing ability and subsequently influence their role in ENS remodeling in diabetic mice.MethodsFirst, we constructed the fucosylation modification of CD44 on BMSCs through a fucosyltransferase VII (FTVII) system to generate a Hematopoietic Cell E-/L-selectin Ligand (HCELL) property, a fucosylated sialyllactosaminyl glycovariant of CD44 that potently binds E-selectin. Next, FTVII-modified and unmodified BMSCs labeled with green fluorescent protein (GFP) were injected into diabetic mice through the tail vein to compare their homing ability to the gastrointestinal tract and their effect on ENS remodeling, respectively. A bioluminescent imaging system was used to evaluate the homing ability of GFP-labeled BMSCs with and without FTVII modification, to the gastrointestinal tract. Gastrointestinal motility was assessed by gastrointestinal transient time, defecation frequency, stool water content and colon strips contractility. Immunofluorescence staining and western blotting were used to assess the expression levels of protein gene product 9.5 (PGP9.5), glial fibrillary acidic protein (GFAP) and glial cell line-derived neurotrophic factor (GDNF).ResultsThe FTVII-mediated α(1,3)-fucosylation modification of CD44 on BMSCs generated a HCELL property. Bioluminescent imaging assays showed that FTVII-modified BMSCs had enhanced homing ability to gastrointestinal tract, mainly to the colon, 24 h after injection through the tail vein. Compared with diabetic mice, FTVII-modified BMSCs significantly promoted the gastrointestinal motility and the ENS remodeling, including intestinal peristalsis (P < 0.05), increased feces excretion (P < 0.05) and the water content of the feces (P < 0.05), restored the spontaneous contraction of the colon (P < 0.05), and upregulated the protein expression levels of PGP9.5 (P < 0.01), GFAP (P < 0.001), and GDNF (P < 0.05), while unmodified BMSCs did not (P > 0.05).ConclusionsCD44 fucosylation modification on murine BMSCs promotes homing ability to the gastrointestinal tract and ENS remodeling in diabetic mice.

Glycomimetic Antagonist of E-Selectin, GMI-1271, Enhances Therapeutic Activity of the Hypomethylating Agent, 5-Azacitidine, in the KG1 Model of AML

Components of the bone marrow stroma afford a permissive environment for the malignant progression of AML. These stromal components include E-selectin, an adhesion molecule expressed in the bone marrow vasculature. Through the recognition and binding of E-selectin by specific ligands on AML blasts, survival pathways are activated within the blasts leading to the development of/increase in chemoresistance. GMI-1271 is a novel E-selectin antagonist that disrupts this process, down-regulating survival pathways and enhancing chemotherapy response with improved survival in mouse xenograft and syngeneic AML tumor models. GMI-1271 is currently in clinical trials for the treatment of AML in combination with intensive chemotherapy; the FDA has granted Breakthrough Therapy Designation to GMI-1271 for the treatment of adults with relapse/refractory AML. Azacitidine (5-AZC) is a DNA-methyltransferase inhibiting cytosine nucleoside analog that at low doses induces DNA hypomethylation and transcriptional activation, while at higher doses is directly cytotoxic to neoplastic cells including AML blasts. Vidaza (5-AZC, Celgene) is approved in Europe for the treatment of limited populations with AML. In these studies we evaluated the anti-tumor activity of GMI-1271 in combination with 5-AZC in the KG1 AML tumor model to identify if there was potential for therapeutic benefit of the combination.Seven days post i.v. injection of KG1 cells, female NSG mice (n=10/group) were treated with saline; GMI-1271 (40 mg/kg IP QDx14) alone; 5-AZC (5 mg/kg IP Q3Dx5) alone, or the combination of GMI-1271 and 5-AZC. All treatments were well tolerated. The median survival time (MST) of mice treated with 5-AZC was 88 days and statistically different (P<0.005) to groups treated with saline (MST=69.5 days) or GMI-1271 alone (MST=69 days). At study conclusion (Day 110 post tumor injection), 20% of mice treated with 5-AZC remained alive; as expected all mice treated with saline or GMI-1271 alone succumbed to progressive tumor growth. Importantly, the therapeutic activity of 5-AZC was significantly enhanced when combined with GMI-1271 (MST>110 days, P=0.0217 compared to 5-AZC alone) with 70% of mice surviving to study conclusion. These results indicate that E-selectin/AML blast interaction in the KG1 model protects from the anti-tumor activity of 5-AZC and that GMI-1271 can attenuate this protection.To investigate the nature of the observed in vivo activity of GM-1271 and 5-AZC in the KG1 model, in vitro assays of cytotoxicity and E-selectin ligand expression in KG1 and the KG1a variant were assessed. The IC50 of 5-AZC alone or in combination with a saturating dose of GMI-1271 on KG1 cells was 518 and 528 nM, respectively. The IC50 of 5-AZC alone or in combination with GMI-1271 on KG1a cells was 637 and 711 nM, respectively. These results imply that the enhanced anti-tumor activity observed in vivo was not due to a simple shift in the cytotoxicity profile of 5-AZC. KGI and KG1a cells were subsequently cultured for 96 h in a noncytotoxic concentration (100 nM) of 5-AZC and the reactivity of the cells to HECA-452, a surrogate marker of E-selectin ligand, or binding E-selectin was determined by flow cytometry. Treatment with 5-AZC resulted in an increased reactivity of KG1 and KG1a cells to HECA-452 (29 and 27%, respectively compared to nontreated cells) and an increased binding of E-selectin to KG1 cells (32% compared to nontreated cells). Collectively, these results demonstrate that noncytotoxic concentrations of 5-AZC lead to an increased expression of E-selectin ligands on KG1 and KG1a cells.In summary, these studies show that even though the hypomethylating agent 5-AZC improves outcomes with AML, therapeutic potential could be improved. In this regard, 5-AZC at noncytotoxic concentrations promotes the increased expression of E-selectin ligands on AML blasts which may dampen 5-AZC activity. However, attenuation or disruption of E-selectin ligand/E-selectin interactions with GMI-1271 in combination with 5-AZC overcomes this tumor survival pathway with the potential to maximize therapeutic outcome. This work has relevance to patients with AML who do not achieve optimal or long-term response with 5-AZC and other hypomethylating agents. Clinical assessment of GMI-1271 with hypomethylating agents in AML is planned. DisclosuresFogler:GlycoMimetics: Employment, Equity Ownership. Smith:GlycoMimetics: Employment, Equity Ownership. Lee:GlycoMimetics: Employment, Equity Ownership. Magnani:GlycoMimetics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Expression of functional E-selectin ligands on the plasma membrane of carcinoma cells correlates with poor prognosis in clear cell renal cell carcinoma

ObjectivesGiven the relatively high frequency of metastatic recurrence of clear cell renal cell carcinoma (ccRCC), reliable prognostic markers of ccRCC, particularly those associated with metastasis, are needed. Here, in search of those factors, we assessed the contribution of sialyl Lewis x (sLex) and sialyl Lewis a (sLea), as well as functional E-selectin ligand carbohydrates expressed on carcinoma cells, to metastasis and consequent poor prognosis in ccRCC. Materials and methodsPatients who underwent surgical resection (curative nephrectomy) for RCC, and whose post-operative pathological diagnosis was ccRCC (n = 117) were enrolled in this study. Expression of sLex/sLea carbohydrate antigens in ccRCC was evaluated by immunohistochemistry with an anti-sLex/sLea monoclonal antibody HECA-452. To evaluate membrane expression of sLex/sLea carbohydrate antigens quantitatively, we employed a histological scoring system used to evaluate membrane expression of human epidermal growth factor receptor 2 (HER2) in breast cancer. We also conducted an E-selectin•IgM chimera in situ binding assay to assess expression of functional E-selectin ligand carbohydrates in ccRCC. We then carried out statistical analysis to determine whether membrane expression of HECA-452-reactive sLex/sLea glycans as well as of E-selectin•IgM-binding functional E-selectin ligand carbohydrates correlates with progression-free, overall, or cancer-specific survival. ResultsBased on HECA-452 immunochemistry, 106 of 117 ccRCC specimens expressed detectable levels of sLex/sLea glycans, primarily on the plasma membrane, and of those, 31 that showed robust membrane expression were judged as HECA-452-positive. Membrane expression of HECA-452-positive sLex/sLea glycans correlated with shortened progression-free and overall survival. Moreover, in in situ analysis, these HECA-452-positive ccRCC tissues were decorated with E-selectin•IgM chimeric proteins, calcium-dependently. Comparable analysis in normal kidney showed both HECA-452 positivity and chimera binding almost exclusively in epithelial cells that constitute proximal tubules. Membrane expression of functional E-selectin ligand carbohydrates, as detected by the E-selectin•IgM chimera, correlated more significantly with poor prognosis of patients, namely, shortened progression-free, overall and cancer-specific survival, than did HECA-452 positivity. ConclusionsExpression of E-selectin•IgM-binding functional E-selectin ligand carbohydrates can serve as a reliable and potentially superior prognostic biomarker of patients with ccRCC.

Abstract PO038: Circulating cancer associated fibroblasts (cCAFs) enhance CTC extravasation and establishment of metastases

Abstract Background: Cancer associated fibroblasts (CAFs) are the most abundant non-cancerous cell component of the breast cancer (BC) TME and have been shown to participate in most all steps of metastasis. We have previously shown that CTCs cluster with circulating CAFs (cCAFs) in BC patients at all disease stages, and this observation is recapitulated in the mouse PYMT BC model. Our central hypothesis is that cCAF/CTC co-clusters comprise the functional metastatic unit, and here we sought to determine the role of cCAFs in co-clusters on CTC extravasation and metastatic seeding. Methods: Xenografts: In vitro co-clusters were produced by culturing human CAF23 cells and MDA231(Ffluc) cells in ULA plates for 18 hrs. Clusters were injected via tail vein into NSG mice and metastatic growth was monitored by BLI. Endothelial binding assays: In vitro clusters, single CAFs or single BC cells were labeled with Cell Tracker dyes, pipetted onto confluent HUVEC monolayers, and allowed to bind for 30 minutes before gentle washing. Bound cells were counted on a fluorescent microscope. Ex vivo extravasation assays: Cell Tracker labeled co-clusters were injected via tail vein into FVB mice. One hour before sacrifice, FITC-lectin was injected via tail vein to label lung endothelia. Lungs were then extracted and imaged by confocal microscopy. Results/Discussion: Xenograft experiments demonstrate that within the first week, CAF23/MDA231 co-clusters produce durable, proliferative lung metastases faster than MDA231 monoclusters. This early difference in metastatic growth suggests that CAFs help BC cells establish metastases faster, possibly by aiding extravasation into the lung parenchyma. Using our ex vivo model of lung extravasation, we saw that significantly more CAF23/MDA231 co-clusters than MDA231 monoclusters were in the process of and/or had extravasated into the lung parenchyma at 8 and 24 hrs post-injection. In vitro binding experiments revealed that HUVEC endothelial cells bind more CAF23/BC co-clusters than BC monoclusters. Similarly, HUVEC cells bound significantly more single CAF23 or CAF19 cells than BC cells. CAF binding increased when endothelial e-selectin expression was induced with IL1-b; binding to e-selectin being the first step in cancer cell rolling and extravasation. Immunofluorescence and flow cytometry showed that CAFs express the CA19-9 antigen, sialyl-Lewis A (sLeA), which acts as an e-selectin ligand. Tannic acid, an inhibitor of the chemokine SDF1/CXCL12, decreased binding, as did 5mM EDTA. CD44 siRNA had no effect on binding whereas depletion of hyaluronin, a ligand of CD44, had a significant effect on CAFs but not on BC cells. Together, these experiments point to CAFs in co-clusters as critical enhancers of endothelial binding and extravasation, which may ultimately result in faster establishment of metastases and enhanced survival of CTCs. Future investigations will focus on therapeutic targeting of the mechanisms we have identified to block cCAF/CTC extravasation and metastasis. Citation Format: Angela Spartz, Benjamin Troness, Utsav Sharma, Geneva Taylor, Dorraya El Ashry. Circulating cancer associated fibroblasts (cCAFs) enhance CTC extravasation and establishment of metastases [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO038.

Enforced sialyl-Lewis-X (sLeX) display in E-selectin ligands by exofucosylation is dispensable for CD19-CAR T-cell activity and bone marrow homing.

CD19‐directed chimeric antigen receptors (CAR) T cells induce impressive rates of complete response in advanced B‐cell malignancies, specially in B‐cell acute lymphoblastic leukemia (B‐ALL). However, CAR T‐cell‐treated patients eventually progress due to poor CAR T‐cell persistence and/or disease relapse. The bone marrow (BM) is the primary location for acute leukemia. The rapid/efficient colonization of the BM by systemically infused CD19‐CAR T cells might enhance CAR T‐cell activity and persistence, thus, offering clinical benefits. Circulating cells traffic to BM upon binding of tetrasaccharide sialyl‐Lewis X (sLeX)‐decorated E‐selectin ligands (sialofucosylated) to the E‐selectin receptor expressed in the vascular endothelium. sLeX‐installation in E‐selectin ligands is achieved through an ex vivo fucosylation reaction. Here, we sought to characterize the basal and cell‐autonomous display of sLeX in CAR T‐cells activated using different cytokines, and to assess whether exofucosylation of E‐selectin ligands improves CD19‐CAR T‐cell activity and BM homing. We report that cell‐autonomous sialofucosylation (sLeX display) steadily increases in culture‐ and in vivo‐expanded CAR T cells, and that, the cytokines used during T‐cell activation influence both the degree of such endogenous sialofucosylation and the CD19‐CAR T‐cell efficacy and persistence in vivo. However, glycoengineered enforced sialofucosylation of E‐selectin ligands was dispensable for CD19‐CAR T‐cell activity and BM homing in multiple xenograft models regardless the cytokines employed for T‐cell expansion, thus, representing a dispensable strategy for CD19‐CAR T‐cell therapy.

Atheroprotective Roles of Adiponectin via CCL2 Inhibition.

Aim: Adiponectin (APN) exhibits different atheroprotective effects, and we have previously reported that APN function is modulated by its binding proteins, E-selectin ligand 1, Mac-2 binding protein, and cystatin C. In the present study, we aimed to identify a novel atheroprotective mechanism of APN via C–C motif chemokine 2 (CCL2). Methods: We conducted iMAP ® -intravascular ultrasound (IVUS) in 111 Japanese male patients with stable angina. The plaque characteristics were determined where “plaque burden” [(EEM CSA − lumen CSA)/(EEM CSA)×100 (%)] ＞50%, and their correlation with serum CCL2 and APN levels was analyzed. Using western blot analysis, the effects of APN on the biological effects of CCL2 were examined in their mutual binding by co-immunoprecipitation assay, the monocyte migration, and the phosphorylation of MAP kinases. Results: In a clinical study, we found that the percentage of plaque in the culprit lesion was correlated positively with serum CCL2 and negatively with serum APN levels, with significance. We identified CCL2 as a novel APN-binding serum protein using immunoprecipitation and western blot analysis. CCL2-induced phosphorylation of MAP kinases and monocyte migration was significantly attenuated by APN in vitro . Conclusion: The opposite association of APN and CCL2 on the percentage of coronary plaque might be caused by their direct interaction and competitive functions on monocyte migration.

L1CAM as an E-selectin Ligand in Colon Cancer.

Metastasis is the main cause of death among colorectal cancer (CRC) patients. E-selectin and its carbohydrate ligands, including sialyl Lewis X (sLeX) antigen, are key players in the binding of circulating tumor cells to the endothelium, which is one of the major events leading to organ invasion. Nevertheless, the identity of the glycoprotein scaffolds presenting these glycans in CRC remains unclear. In this study, we firstly have characterized the glycoengineered cell line SW620 transfected with the fucosyltransferase 6 (FUT6) coding for the α1,3-fucosyltransferase 6 (FUT6), which is the main enzyme responsible for the synthesis of sLeX in CRC. The SW620FUT6 cell line expressed high levels of sLeX antigen and E-selectin ligands. Moreover, it displayed increased migration ability. E-selectin ligand glycoproteins were isolated from the SW620FUT6 cell line, identified by mass spectrometry, and validated by flow cytometry and Western blot (WB). The most prominent E-selectin ligand we identified was the neural cell adhesion molecule L1 (L1CAM). Previous studies have shown association of L1CAM with metastasis in cancer, thus the novel role as E-selectin counter-receptor contributes to understand the molecular mechanism involving L1CAM in metastasis formation.

Combined Blockage of E-Selectin and CXCR4 (GMI-1359) Enhances Anti-Leukemia Effect of FLT3 Inhibition (Sorafenib) and Protects Hematopoiesis in Pre-Clinical AML Models

Acute myelogenous leukemia (AML) is characterized by an accumulation of abnormal white blood cells. Internal tandem duplications in the fms-like tyrosine kinase 3 (FLT3-ITD) account for 30% of adult AML cases and confer poor prognosis (Nakao et al., Leukemia 1996). FLT3 inhibitors like sorafenib efficiently eliminate circulating leukemia blasts, but frequently not in the bone marrow (BM), which suggests a protective effect of the BM niche for leukemic stem cell survival (Zhang et al., JNCI 2008). The homing of AML cells in BM is mediated chiefly by the adhesion to E-selectin on endothelial cells (ECs) and by CXCR4-directed cellular migration to stromal CXCL12 (SDF1) sources (Chien et al., Blood 2013; Peled and Tavor, Theranostics 2013). In many respects, BM homing signals are shared between leukemia and hematopoietic stem cells (HSCs). Our previous study demonstrated that targeting E-selectin/CXCR4 with the dual E-selectin/CXCR4 antagonist GMI-1359 markedly reduced leukemia cell adhesion to ECs and mesenchymal stem cells, reduced the BM-mediated protection of leukemic cells during FLT3-targeted therapy in vitro, and effectively reduced leukemia cellularity in the BM in vivo (Zhang et al., Can Res suppl 2016). Further, GMI-1359 combined with cytarabine/daunorubicin provided a profound survival benefit in mice with FLT3-mutated leukemia (Zhang et al., Blood suppl 2015). In the present study, we sought to evaluate dual E-selectin/CXCR4 blockage in the context of FLT3 inhibition by sorafenib in vivo, and to better understand the underlying mechanism. We compared expression levels of E-selectin ligands and CXCR4 in FLT3 inhibitor-sensitive Ba/F3-FLT3-ITD cells and their inhibitor-resistant counterparts Ba/F3-FLT3-ITD+D835Y and Ba/F3-FLT3-ITD+F691L. Resistant cells expressed 1.7 to ~5.6-fold higher levels of total E-selectin ligand detected by a soluble E-selectin reagent, and 10-fold higher levels of CXCR4. In addition, BM-mimetic hypoxia culture profoundly upregulated the cell surface expression of E-selectin ligands and CXCR4 on leukemia cells. We evaluated anti-leukemia effects of co-targeting E-selectin/CXCR4 and FLT3 with GMI-1359 and sorafenib in a patient-derived AML xenograft (PDX) model harboring FLT3-ITD and WT1 mutations. We observed that addition of GMI-1359 to sorafenib greatly reduced leukemia cellularity compared to sorafenib alone, and as much as by 92%, 82%, 69% and 45% in, respectively, liver, lung, spleen and BM (Fig. 1) as compared with vehicle-treated mice (p &amp;lt; 0.05). As expected, the number of circulating leukemia cells transiently increased. The GMI-1359/sorafenib combination improved mouse survival (median survival 138.5 versus 109, 87 and 126 days for the GMI-1359/sorafenib versus vehicle, GMI-1359 and sorafenib, respectively, p &amp;lt; 0.001). Using intravital 2-photon microscopy, we observed AML cell behavior in calvarial BM and their response to acute GMI-1359 bolus infusion. Remarkably, AML cell mobility began to increase in the BM microenvironment as soon as 20 min after treatment (Fig. 2), followed by intravasation and cellular outflow through the BM capillary vasculature over the next 2-4 hours. Moreover, although BM homing signals are thought to be shared between leukemia and HSCs, the combination therapy improved hematopoiesis parameters compared to sorafenib alone. In particular, this important effect was associated with increased numbers of megakaryocytes (2.1-fold), myelocytes (2.1-fold), and erythrocytes (7.1-fold) in BM (p &amp;lt; 0.01). The underlying mechanism(s) of hematopoiesis protection by GMI-1359 are under investigation. Conclusion: Co-inhibition of E-selectin/CXCR4 enhances the anti-leukemia efficacy of FLT3 inhibition and preserves hematopoiesis in the BM in a PDX model of AML. Disclosures Fogler: GlycoMimetics: Current Employment, Current equity holder in publicly-traded company, Patents &amp; 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 &amp; Royalties. Zal:Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding. Andreeff:Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research &amp; Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

An inflammatory vascular endothelium-mimicking microfluidic device to enable leukocyte rolling and adhesion for rapid infection diagnosis

Recruitment of circulating leukocytes to sites of infection is of utmost importance in the development, propagation, and outcome of sepsis. These multi-step processes are mediated by interactions between adhesion receptors of leukocytes and cell adhesion molecules (CAMs) of endothelial cells, such as P-selectin, E-selectin and ICAM-1. However, the potential utility of the CAMs-facilitated leukocyte capture has not been thoroughly investigated as an index of the host response to infection for diagnostic purposes. Here, we report that the systemic infection affects the expression of CAMs ligands on leukocytes, upregulating the expression of P-selectin ligand-1 (PSGL-1) and increasing the number of PSGL-1- and E-selectin ligand-1 (ESL-1)-expressing leukocyte levels in septic blood. We leveraged this finding to determine infection by measuring the increased adhesion of leukocytes to an inflammatory vascular endothelium-mimicking microchannel coated with CAMs. We successfully validated that the proposed method can significantly differentiate infection in bacteremia and endotoxemia models in rats as early as an hour post-infection using a finger-prick volume of blood (50 μL), which were unachievable with the conventional diagnostic methods.

Abstract 2626: Exosomes derived from the BT-20 human breast cancer cell line express E-selectin ligands

Abstract Exosomes are small extracellular vesicles (40-150 nm in diameter) secreted by cells and initially proposed as a mechanism to eliminate cellular waste. More recently, exosomes have been reported to participate in cancer metastasis because of their ability to mediate local and distant cell-to-cell communication. Furthermore, exosomes create cancer-favored microenvironments, potentially enhancing cancer metastasis. However, it is unknown whether exosomes interact with E-selectin, an adhesion molecule expressed by vascular endothelium and shown to be involved in metastasis. Our lab has previously shown that BT-20 human breast cancer cells adhesively interact with endothelial E-selectin via several E-selectin ligands such as sialofucosylated CD44 variants, Mac-2BP, and gangliosides. Therefore, we hypothesize that exosomes secreted by the BT-20 human breast cancer cell line interact with E-selectin. To investigate this hypothesis, exosomes were generated by culturing cells in serum-depleted media and isolated by a series of ultracentrifugation steps. Molecular size analysis using dynamic light scattering revealed that the size range of the isolated particles was similar to the expected size of exosomes. To detect CD9 (an exosome marker) and E-selectin ligands, both breast cancer whole cell lysate and exosome lysate were tested by Western blot. The higher expression of CD9 in isolated particles than the whole cell lysates confirmed that the isolated particles were exosomes. When Western blots were labeled with recombinant E-selectin/Fc chimera with Ca2+, bands were detected at approximately 130 kDa, indicating the presence of E-selectin ligands. As a negative control, the divalent cation chelator EDTA was added to the primary labeling incubation. Bands were not detected in these negative control experiments, indicating no E-selectin interactions in the absence of Ca2+. Human IgG (hIgG) was also used as a negative control in Western blots and showed no signal with exosome lysates, as expected. In comparison, experiments were also performed with Hs578T breast cancer cells, a cell line that our lab has shown does not express E-selectin ligands. Exosomes isolated from Hs578T cells did not express E-selectin ligands, consistent with whole cells. Altogether, these results suggest that BT-20 human breast cancer exosomes express E-selectin ligands. Future work will focus on identifying and characterizing the exosome E-selectin ligands, as well as investigating other cancer cells that express E-selectin ligands. Ultimately, understanding the interaction between exosomes and E-selectin can lead to new diagnostic and therapeutic strategies for cancer metastasis. Citation Format: Yinan Huang, Christian A. Showalter, Monica M. Burdick. Exosomes derived from the BT-20 human breast cancer cell line express E-selectin ligands [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2626.

Abstract 5867: Transcriptome profiling of ST3GAL4 and FUT7 in multiple tumor types and prognostic value in adult acute myeloid leukemia

Abstract E-selectin is a cell adhesion glycoprotein expressed on endothelial cells and participates in the development of environmental-mediated drug resistance and poor clinical outcome in cancer. The E-selectin antagonist, uproleselan, interrupts leukemic cell homing to the vascular niche and increases susceptibility to cytotoxic therapies. Recent clinical data demonstrate a correlation between leukemic cell surface E-selectin ligands and response to uproleselan, linking E-selectin ligand expression to response. Multiple genes involved in the glycan synthesis of E-selectin ligands are highly expressed in pediatric AML. Expression levels of two of these genes, ST3GAL4 and FUT7 are associated with poor outcome and are associated with cell surface E-selectin ligand expression. In the current studies we extend transcriptome profiling of E-selectin ligand forming glycosylation genes with an emphasis on ST3GAL4 and FUT7 in different cancers. Initially, we investigated expression levels of ST3GAL4 and FUT7 in 10,258 samples covering 33 cancer types from the TCGA PanCanAtlas. ST3GAL4 and FUT7 were consistently expressed in the majority of cancers evaluated. The cancer types that expressed ST3GAL4 most highly were melanoma (uveal and skin), kidney chromophobe, adrenocortical carcinoma and bladder urothelial carcinoma, while for FUT7, it was AML, diffuse large B-cell lymphoma, thymoma, testicular germ cell tumors and head and neck squamous cell carcinoma. Of particular interest was the identification of adult AML for the highest expression of FUT7 and high expression of ST3GAL4 (mean log2 gene expression = 8.1 and 9.4, respectively). Augmented expression of FUT7 and ST3GAL4 were also observed in an analysis of 39 AML cell lines among the 1,457 cell lines comprising the Cancer Cell Line Encyclopedia RNAseq data set. The prognostic significance of FUT7 and ST3GAL4 in adult AML was further assessed using the TCGA-LAML RNAseq dataset for differential expression and associations with overall survival (OS). The data set included 151 RNAseq profiles of bone marrow samples from adult patients with AML, and within this data set the status of the FMS-like tyrosine kinase 3 (FLT3) proto-oncogene was considered. Mutational alterations of FLT3 are associated with higher risk of relapse and shorter OS compared with wild-type FLT3. ST3GAL4 and FUT7 were both identified as being upregulated (fold-change = 1.73 and 1.40, respectively) in the mutated FLT3 subset (n=46) as compared to wild type FLT-3 (p=0.000033 and 0.046, respectively). Notably in the mutated FLT-3 subset expression of FUT7 was significantly associated with a poor prognosis and decreased OS (HR = 4.56, p= 0.015). Collectively, these studies extend the prognostic importance of the E-selectin ligand glycosylation genes, ST3GAL4 and FUT7, to adult AML where these genes may be useful as predictive biomarkers. In addition, these studies suggest potential additional tumor types beyond AML where treatment protocols with uproleselan may have therapeutic benefits. Citation Format: William E. Fogler, Vince Deng, David Stewart, Michael Jarman, Simone Daminelli, Adrian Carr, John L. Magnani. Transcriptome profiling of ST3GAL4 and FUT7 in multiple tumor types and prognostic value in adult acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5867.

Abstract 6038: Combined targeting of E-selectin/CXCR4 and FLT3 by GMI-1359 and sorafenib effectively reduces leukemia cell burden and protects normal hematopoiesis in a patient-derived AML xenograft model

Abstract Acute myelogenous leukemia (AML) is characterized by an accumulation of abnormal white blood cells. Internal tandem duplications in the Fms-like tyrosine kinase 3 (FLT3-ITD) account for 30% of adult AML cases and confer poor prognosis (Kottaridis et al., 2003; Thiede et al., 2002). FLT3 inhibitors like sorafenib efficiently eliminate leukemia blast in the peripheral blood (PB), but frequently not in the bone marrow (BM) (Zhang et al., 2008). This suggests a protective effect of the BM on leukemic stem cells, which is mediated by E-selectin and CXCL12 expression in endothelial cells (ECs) and mesenchymal stem cells (MSCs) in the BM vascular and endosteal niches (Horacek et al., 2013; Peled and Tavor, 2013). Our previous study demonstrated that targeting E-selectin/CXCR4 with the dual E-selectin/CXCR4 antagonist GMI-1359 markedly reduced leukemia cell adhesion to ECs and MSCs and eliminated the BM-mediated protection of leukemic cells during FLT3-targeted therapy in vitro, and effectively reduced leukemia cell in the BM in vivo (Zhang et al., 2016). Further, GMI-1359 combined with cytarabine/daunorubicin provided a profound survival benefit in FLT3-mutated leukemia cell MV4-11-bearing mice (Zhang et al., 2015). In the preset study, we compared expression levels of E-selectin ligands and CXCR4 in FLT3 inhibitor-sensitive Ba/F3-FLT3-ITD cells and their inhibitor-resistant counterparts Ba/F3-FLT3-ITD+D835Y and Ba/F3-FLT3-ITD+F691L. The resistant cells expressed 1.7 to ~5.6-fold higher E-selectin ligand and 10-fold higher CXCR4 levels. In addition, BM-mimetic hypoxia culture profoundly upregulated P-selectin glycoprotein ligand-1 (PSGL-1) and CXCR4 levels on leukemia cells. PSGL-1 is also a high-efficiency ligand for E-selectin. We then evaluated anti-leukemia effects of co-targeting E-selectin/CXCR4 and FLT3 with GMI-1359 and sorafenib in a patient-derived AML xenograft model (harboring FLT3-ITD and WT1 mutations). Leukemia cell engraftment was evaluated using FACS and immunohistochemistry by hCD45 staining. We observed that GMI-1359 efficiently mobilized leukemic cells into PB and in combination with sorafenib enhanced the anti-leukemia activity and significantly reduced leukemia cell infiltration by 92%, 82%, 69% and 45% in liver, lung, spleen and BM, respectively, as compared with vehicle-treated mice (p &amp;lt; 0.05). The GMI-1359/sorafenib combination improved mouse survival (median survival were 109, 87, 126 and 138.5 days for the vehicle, GMI-1359, sorafenib and combination groups, respectively). Importantly, we observed that the combination protected normal hematopoiesis by increasing the number of BM megakaryocytes, myelocytes, and erythrocytes. The underlying mechanism(s) for this effect is under investigation. Our findings suggest that co-targeting E-selectin/CXCR4/FLT3 exerts remarkable protection of normal hematopoiesis in addition to more efficiently reducing leukemia cells. Citation Format: Weiguo Zhang, Kyung Hee Chang, Mahesh Basyal, Yannan Jia, Lauren Ostermann, William E. Fogler, John L. Magnani, Michael Andreeff. Combined targeting of E-selectin/CXCR4 and FLT3 by GMI-1359 and sorafenib effectively reduces leukemia cell burden and protects normal hematopoiesis in a patient-derived AML xenograft model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6038.

Abstract 486: Enhanced survival of lethally-irradiated mice with HSC reconstitution in combination with the E-selectin antagonist, GMI-1271 (uproleselan)

Abstract The involvement of the adhesion molecule E-selectin and its interactions with E-selectin ligands as pertains to hematopoietic stem cells (HSC) and transplantation has been investigated (Winkler et al. 2012; Winkler et al. 2014). These preclinical studies focused on the role of E-selectin and the use of uproleselan, an E-selectin antagonist, during HSC mobilization in harvesting procedures of donors to accelerate recovery in transplant recipients. However, the impact of E-selectin and uproleselan administration on transplant recipients was less clear. In the current investigations we assessed the survival outcome of bone marrow depleted mice when reconstituted with HSC in combination with uproleselan. Lethally-irradiated, bone marrow depleted C57BL/6 mice were reconstituted with bone-marrow harvested from a congenic strain. Twenty-four hours post irradiation (6Gy x2), cohorts of mice (n=10/group) were injected i.v. with 1 × 106 cells (study day 0) from congenic donors using three i.p. dosing regimens with 40 mg/kg uproleselan. These regimens were: (a) bid on study days 0 and1; (b) bid on study days 1 and 2; and (c) bid on study day 1 only. Control groups in this study included irradiated mice alone (expected survival = 0%), non-irradiated mice alone (expected survival = 100%), and irradiated, reconstituted mice (no uproleselan). The survival of mice was determined over the course of the study (Day 0 to 30). Additional parameters of evaluation included sinusoidal obstructive syndrome such as hepatic veno-occlusive disease known to be E-selectin dependent and a complication of HSC transplantation. Treatment with uproleselan as part of the transplant regimen significantly increased the median survival time (MST) of mice compared with the control group – the MST of mice treated with uproleselan and HSC was &amp;gt;30 days with 80-90% of mice alive at study completion. In contrast, the MST of irradiated mice (no transplant) was 11.5 days with no survivors at study conclusion. The MST of mice irradiated and transplanted with congenic HSC was 9 days with 40% survival on day 30. The impact of uproleselan on survival represented a &amp;gt;233.3% increase in life span. Flow cytometric analysis in all surviving mice on day 30 showed that the mean percentage of CD45.1+ cells from donor congenic mice was approximately 90% (blood and bone marrow) indicating that all surviving mice were successfully reconstituted. In summary, we report on a novel therapeutic use of inhibitors of E-selectin, such as uproleselan, which results in the increased survival of mice when combined with HSC transplantation for reconstitution of depleted and compromised bone marrow. The impact on increased host survival could extend to the use of peripheral blood and stem cell transplantations as a therapeutic option in various malignancies where curative intent is intended. Citation Format: William E. Fogler, Dylan Daniel, Sheri Barnes, Alden Wong, David Draper, John L. Magnani. Enhanced survival of lethally-irradiated mice with HSC reconstitution in combination with the E-selectin antagonist, GMI-1271 (uproleselan) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 486.

Nucleolin-Sle A Glycoforms as E-Selectin Ligands and Potentially Targetable Biomarkers at the Cell Surface of Gastric Cancer Cells.

Background: Gastric cancer (GC) is a major health burden worldwide, with half of patients developing metastases within 5 years after treatment, urging novel biomarkers for diagnosis and efficient therapeutic targeting. Sialyl-Lewis A (SLeA), a terminal glycoepitope of glycoproteins and glycolipids, offers tremendous potential towards this objective. It is rarely expressed in healthy tissues and blood cells, while it is present in highly metastatic cell lines and metastases. SLeA is also involved in E-selectin mediated metastasis, making it an ideal target to control disease dissemination. Methods and Results: To improve cancer specificity, we have explored the SLeA-glycoproteome of six GC cell models, with emphasis on glycoproteins showing affinity for E-selectin. A novel bioinformatics-assisted algorithm identified nucleolin (NCL), a nuclear protein, as a potential targetable biomarker potentially involved in metastasis. Several immunoassays, including Western blot and in situ proximity ligation reinforced the existence of cell surface NCL-SLeA glycoforms in GC. The NCL-SLeA glycophenotype was associated with decreased survival and was not reflected in relevant healthy tissues. Conclusions: NCL-SLeA is a biomarker of poor prognosis in GC holding potential for precise cancer targeting. This is the first report describing SLeA in preferentially nuclear protein, setting a new paradigm for cancer biomarkers discovery and targeted therapies.

Functional binding of E-selectin to its ligands is enhanced by structural features beyond its lectin domain

Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation. Selectins are composed of a lectin domain, an epidermal growth factor (EGF)-like domain, multiple short consensus repeats (SCRs), a transmembrane domain, and a cytoplasmic tail. It is well-established that the lectin and EGF domains are required to mediate interactions with ligands; however, the contributions of the other domains in mediating these interactions remain obscure. Using various E-selectin constructs produced in a newly developed silkworm-based expression system and several assays performed under both static and physiological flow conditions, including flow cytometry, glycan array analysis, surface plasmon resonance, and cell-rolling assays, we show here that a reduction in the number of SCR domains is correlated with a decline in functional E-selectin binding to hematopoietic cell E- and/or L-selectin ligand (HCELL) and P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, the binding was significantly improved through E-selectin dimerization and by a substitution (A28H) that mimics an extended conformation of the lectin and EGF domains. Analyses of the association and dissociation rates indicated that the SCR domains, conformational extension, and dimerization collectively contribute to the association rate of E-selectin–ligand binding, whereas just the lectin and EGF domains contribute to the dissociation rate. These findings provide the first evidence of the critical role of the association rate in functional E-selectin–ligand interactions, and they highlight that the SCR domains have an important role that goes beyond the structural extension of the lectin and EGF domains.

Abstract P3-01-28: Circulating cancer associated fibroblasts (cCAFs) enhance CTC extravasation and establishment of metastases

Abstract Introduction: Breast cancer metastasis is a multi-step process that involves cellular migration, invasion, travel through the circulation, extravasation and secondary site proliferation. Circulating tumor cell (CTC) travel is thought to be via rolling on vascular endothelium, and CTC clusters have been shown to have a shorter circulation half-life than single CTCs due to increased entrapment within capillaries. We have previously shown that CTCs cluster with circulating cancer associated fibroblasts (cCAFs) in breast cancer patients and this observation is recapitulated in the mouse PYMT breast cancer model. Circulating CAF/CTC clusters are seen in patients of all stages and in all PyMT mice with tumors, before overt metastases. Our central hypothesis is that cCAF/CTC co-clusters comprise the functional metastatic unit and here we sought to determine the role of cCAFs in co-clusters on metastatic seeding. Methods: Xenografts: In vitro co-clusters were produced by culturing 75,000 human CAF23 cells and 75,000 MDA231(Ffluc) cells in ultra-low attachment plates for 18 hrs. Clusters were injected via tail vein into NSG mice and metastatic growth was monitored by bioluminescent imaging. Endothelial binding assays: In vitro clusters were made as for xenografts except that CAFs and BC cells were labeled with Cell Tracker dyes. Clusters were pipetted onto confluent HUVEC monolayers and allowed to bind for 30 minutes before gentle washing. Bound cells were counted on a fluorescent microscope. Ex vivo extravasation assays: Co-clusters were made as above with Cell Tracker labeled MDA231 and CAF23 cells and injected via tail vein into FVB mice. Lung endothelia was labeled by tail vein injection of FITC-lectin an hour before lungs were extracted and imaged by confocal microscopy. Results: Xenograft experiments demonstrate that within the first week, CAF23/MDA231(Ffluc) co-clusters produce durable, proliferative lung metastases faster than MDA231(Ffluc) monoclusters. This early difference in metastatic growth suggests that CAFs help cancer cells establish metastases faster, possibly by enhancing the binding and rolling of CTCs along the vascular endothelium and/or by aiding extravasation into the lung parenchyma. In vitro binding experiments revealed that CAF23/MDA231 co-clusters bind to HUVEC endothelial cells better than MDA231 monoclusters under static conditions. Similar results were observed with the aggressive basal cell line, DT28. To determine the effect of shear stress, we performed endothelial binding assays in an orbital shaker at 100 rpm (used to mimic oscillatory and continuous flow shear stress), and under these conditions, CAFs continue to bind to HUVECs at significantly higher numbers than MDA231 cells. CAF binding increased two-fold when endothelial e-selectin expression was induced with IL1-β. Immunofluorescence and flow cytometry showed that CAFs express the CA19-9 antigen, sialyl-Lewis A (sLeA), which acts as an e-selectin ligand; binding to e-selectin being the first step in cancer cell rolling and extravasation. Using our ex-vivo extravasation imaging protocol, we observed that significantly more CAF23/MDA231 co-clusters than MDA231 monoclusters were in the process of extravasation and/or had extravasated into the lung parenchyma at 8 and 24 hrs post-injection. Conclusions: Together, these experiments point to CAFs in co-clusters as critical enhancers of endothelial binding and extravasation, which may ultimately result in quicker establishment of metastases and enhanced survival of CTCs in clusters. Future studies will focus on identifying the molecular mechanisms in these processes facilitated by cCAFs that could be amenable to therapeutic targeting. Citation Format: Angela Spartz, Benjamin Troness, Utsav Sharma, Dorraya El-Ashry. Circulating cancer associated fibroblasts (cCAFs) enhance CTC extravasation and establishment of metastases [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-28.

Nucleolin-SLe &lt;sup&gt;A&lt;/sup&gt; Glycoforms as E-Selectin Ligands and Potentially Targetable Biomarkers at the Cell Surface of Gastric Cancer Cells

Gastric cancer (GC) is the third leading cause of cancer-related death worldwide, with half of patients developing metastases within 5 years after treatment, urging the need for novel biomarkers for diagnosis and more efficient therapeutic targeting. The sialyl-Lewis A (SLeA) antigen, a terminal glycoepitope of glycoproteins and glycolipids, offers tremendous potential towards this objective. This glycan is rarely expressed by healthy tissues and blood cells but is often present in cancer cells of high metastatic potential, as well as in the metastases. The SLeA antigen is also directly involved in E-selectin mediated metastasis, making it an ideal target to control disease dissemination. To improve further on cancer specificity, we have zoomed in on the SLeA-glycoproteome of six distinct GC cell models. Emphasis was set on glycoproteins showing affinity for E-selectin. A bioinformatics-assisted algorithm, used for the first time in this study, identified nucleolin (NCL), a nuclear protein frequently observed at the cell-surface in cancer cells, as a potential targetable biomarker potentially involved in metastasis development. Several immunoassays, including western blot and in situ proximity ligation reinforced the existence of NCL-SLeA glycoforms at the cancer cells surface and in GC. The NCL-SLeA glycophenotype was associated with decreased survival and was not reflected in relevant healthy tissues, supporting its tremendous potential for precise cancer targeting with limited off-target effects. To our knowledge, this is also the first report describing SLeA in a protein preferentially found in the nucleus, setting a new paradigm for cancer biomarkers discovery and targeted therapies. Funding Statement: Portuguese Foundation for Science and Technology (FCT) for the human resources grants: PhD grant BD/103571/2014 (EF), SFRH/BD/111242/2015 (AP), SFRH/BD/146500/2019 (MRS), SFRH/BD/142479/2018 (JS), SFRH/BD/105355/2014 (RA), SFRH/BD/127327/2016 (CG); and FCT assistant researcher grant CEECIND/03186/2017 (JAF). FCT is co-financed by European Social Fund (ESF) under Human Potential Operation Programme (POPH) from National Strategic Reference Framework (NSRF). The authors also acknowledge FCT the funding for CI-IPOP research unit (PEst-OE/SAU/UI0776/201), the Portuguese Oncology of Porto Research Centre (CI-IPOP-29-2014; CI-IPOP-58-2015; CI-IPOPProj.70-bolsa2019-GPTE) and PhD Programs in Biomedical and Pathology and Molecular Genetics of ICBAS-University of Porto. The authors EF, DF and MRS also acknowledge the financial support of the Research Group of Digestive Cancers (GICD) and the “Early stage cancer treatment, driven by context of molecular imaging (ESTIMA)” framework (NORTE-01-0145-FEDER-000027), IPO-Score (DSAIPA/DS/0042/2018) and “Towards a single therapy with a synergistic combination against triple negative breast cancer and neuroblastoma by nucleolin mediated multicellular targeting” (EU joint call ENMed/0006/2015) for financial support. This article is also a result of the project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundacao para a Ciencia e a Tecnologia/Ministerio da Ciencia, Tecnologia e Ensino Superior in the framework of the project Institute for Research and Innovation in Health Sciences (POCI-01-0145-FEDER-007274). Declaration of Interests: The authors declare no conflict of interests. Ethics Approval Statement: All procedures concerning the inclusion of patients were approved by the institutional Ethics Committee after patient’s informed consent.

Synergistic Targeting of BTK and E-Selectin/CXCR4 in the Microenvironment of Mantle Cell Lymphomas

Synergistic Targeting of BTK and E-Selectin/CXCR4 in the Microenvironment of Mantle Cell Lymphomas