Targeting Selectins Mediated Biological Activities With Multivalent Probes

Selectins are type-I transmembrane glycoproteins that are ubiquitously expressed on activated platelets, endothelial cells, and leukocytes. They bind to cell surface glycoproteins and extracellular matrix ligands, regulate the rolling of leukocytes in the blood capillaries, and recruit them to inflammatory sites. Hence, they are potential markers for the early detection and inhibition of inflammatory diseases, thrombosis, cardiovascular disorders, and tumor metastasis. Fucosylated and sialylated glycans, such as sialyl Lewisx, its isoform sialyl Lewisa, and heparan sulfate, are primary selectin ligands. Functionalization of these selectin-binding ligands on multivalent probes, such as nanoparticles, liposomes, and polymers, not only inhibits selectin-mediated biological activity but is also involved in direct imaging of the inflammation site. This review briefly summarizes the selectin-mediated various diseases such as thrombosis, cancer and recent progress in the different types of multivalent probes used to target selectins.

FLT3 Inhibitors Upregulate CXCR4 and E-Selectin Ligands and CD44 Via ERK Suppression in AML Cells, and Blockade of CXCR4 and E-Selectin Signaling with GMI-1359 Overcomes AML Resistance to Quizartinib in Vitro and In Vivo

FLT3 inhibitors (FLT3i) have had transient success treating FLT3-mutant acute myeloid leukemia (AML) patients, especially those with FLT3 internal tandem duplication (ITD) mutations that account for one-third of adult AML cases (Daver et al., 2021). However, FLT3i are typically ineffective in eliminating leukemia stem cells in the protective bone marrow (BM) microenvironmental "niche" (Borthakur et al., 2011; Cortes et al., 2013; Zhang et al., 2008). Cytokines and chemokines such as CXCR4 and E-selectin ligands play a critical role in leukemia cell protection in the BM niche. Indeed, interactions of leukemic cells with their vicinal support cells, including mesenchymal stem cells (MSCs) and endothelial cells (ECs), in the BM niche is mediated mainly through the CXCR4/SDF-1 and E-selectin/HECA-452/CD44 axes (Erbani et al., 2020; Peled and Tavor, 2013). Therefore, evaluation of the effects of FLT3i on CXCR4 and E-selectin signaling in leukemia cells could enhance our understanding of AML FLT3i resistance mechanisms. To this end, we investigated the levels of CXCR4 and E-selectin ligands on FLT3-ITD-mutated AML cells in vitro and in vivo during FLT3i treatment (e.g., quizartinib or sorafenib), and evaluated the anti-leukemia effects of CXCR4/E-selectin blockade with the dual inhibitor GMI-1359. We first checked the effects of FLT3i on the levels of CXCR4 and E-selectin ligands, as well as CD44, in vitro in human MOLM14 AML cells, which harbor FLT3-ITD mutations. All of these were upregulated, as measured by flow cytometry, following exposure to quizartinib (p < 0.001) or sorafenib (p < 0.01) for 96 h. The mRNA levels were also increased roughly 2-fold, as measured by qPCR, suggesting transcriptional regulation was involved in the upregulation. Further, the upregulation of CXCR4 and E-selectin ligands and CD44 was time dependent (from 2 to 96 h). FLT3i profoundly suppresses activation of ERK, AKT, and Stat5 (Zhang et al., 2008). Therefore, we tested if the suppression of each signaling pathways individually could upregulate of CXCR4. Unexpectedly, 72-h suppression of MEK/ERK signaling with selumetinib or pimasertib also upregulated CXCR4 in MOLM14 cells. No effects in this regard were observed by suppressing AKT/mTOR or Stat5 with AZD8055 or STAT5-IN-1, respectively. Additionally, in Dox-inducible NRAS (G12D)-mutated MOLM13 AML cells which also harbor FLT3 ITD mutations, ERK activation by doxycycline downregulated CXCR4 levels implying the MEK/ERK signaling pathway was associated with the suppression of CXCR4. Furthermore, under BM microenvironment-mimicking, co-culture using human MSCs/ECs and MOLM14 cells, blockade of CXCR4 and/or E-selectin signaling using the CXCR4 antagonist plerixafor, the E-selectin antagonist GMI-1271, or the CXCR4 and E-selectin dual inhibitor GMI-1359 showed that GMI-1359 markedly abrogated BM protection and sensitized MOLM14 cells to quizartinib-induced apoptosis. We further validated the effect of GMI-1359 in a PDX model of AML which were from a patient who relapsed from sorafenib+E6201+DAC in clinic and showed resistant to quizartinib ex vivo. The combination of GMI-1359 with quizartinib profoundly reduced leukemia burden and extended survival of the PDX mice compared to the vehicle or the single-agent treatments (median survival was 158 days vs. 82.5, 79 and 128 days, respectively, in combination group vs. vehicle, quizartinib and GMI-1359; p < 0.0001) [Figure 1,2]. Our results suggest that FLT3i can upregulate CXCR4 and E-selectin ligands and CD44 in FLT3-ITD leukemia cells, which is mediated, at least in part, via suppression of MEK/ERK signaling. GMI-1359 sensitized AML cells to quizartinib-induced apoptosis in vitro and statistically significantly extended AML PDX mouse survival in vivo. These findings provide a pre-clinical rational for using GMI-1359 to prevent or overcome FLT3i resistance when treating FLT3-mutant AML patients. Figure 1 Figure 1. Disclosures Fogler: GlycoMimetics Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani: GlycoMimetics Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Konopleva: Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Forty Seven: Other: grant support, Research Funding; KisoJi: Research Funding; AstraZeneca: Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; Ablynx: Other: grant support, Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Rafael Pharmaceuticals: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Calithera: Other: grant support, Research Funding; Sanofi: Other: grant support, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Agios: Other: grant support, Research Funding; Cellectis: Other: grant support; Stemline Therapeutics: Research Funding; Ascentage: Other: grant support, Research Funding. Andreeff: Senti-Bio: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; AstraZeneca: Research Funding; Karyopharm: Research Funding; Glycomimetics: Consultancy; Aptose: Consultancy; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; ONO Pharmaceuticals: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Medicxi: Consultancy; Oxford Biomedica UK: Research Funding; Breast Cancer Research Foundation: Research Funding; Syndax: Consultancy.

Fra-2 overexpression upregulates pro-metastatic cell-adhesion molecules, promotes pulmonary metastasis, and reduces survival in a spontaneous xenograft model of human breast cancer

Purpose The transcription factor Fra-2 affects the invasive potential of breast cancer cells by dysregulating adhesion molecules in vitro. Previous results suggested that it upregulates the expression of E- and P-selectin ligands. Such selectin ligands are important members of the leukocyte adhesion cascade, which govern the adhesion and transmigration of cancer cells into the stroma of the host organ of metastasis. As so far, no in vivo data are available, this study was designed to elucidate the role of Fra-2 expression in a spontaneous breast cancer metastasis xenograft model. Methods The effect of Fra-2 overexpression in two stable Fra-2 overexpressing clones of the human breast cancer cell line MDA MB231 on survival and metastatic load was studied after subcutaneous injection into scid and E- and P-selectin-deficient scid mice. Results Fra-2 overexpression leads to a significantly shorter overall survival and a higher amount of spontaneous lung metastases not only in scid mice, but also in E- and P-deficient mice, indicating that it regulates not only selectin ligands, but also selectin-independent adhesion processes. Conclusion Thus, Fra-2 expression influences the metastatic potential of breast cancer cells by changing the expression of adhesion molecules, resulting in increased adherence to endothelial cells in a breast cancer xenograft model.

Single-molecule imaging and microfluidic platform reveal molecular mechanisms of leukemic cell rolling

Hematopoietic stem/progenitor cell (HSPC) and leukemic cell homing is an important biological phenomenon that occurs through key interactions between adhesion molecules. Tethering and rolling of the cells on endothelium, the crucial initial step of the adhesion cascade, is mediated by interactions between selectins expressed on endothelium to their ligands expressed on HSPCs/leukemic cells in flow. Although multiple factors that affect the rolling behavior of the cells have been identified, molecular mechanisms that enable the essential slow and stable cell rolling remain elusive. Here, using a microfluidics-based single-molecule live cell fluorescence imaging, we reveal that unique spatiotemporal dynamics of selectin ligands on the membrane tethers and slings, which are distinct from that on the cell body, play an essential role in the rolling of the cell. Our results suggest that the spatial confinement of the selectin ligands to the tethers and slings together with the rapid scanning of a large area by the selectin ligands, increases the efficiency of selectin-ligand interactions during cell rolling, resulting in slow and stable rolling of the cell on the selectins. Our findings provide novel insights and contribute significantly to the molecular-level understanding of the initial and essential step of the homing process.

Fra-2 Overexpression Upregulates Pro-metastatic Cell-adhesion Molecules, Promotes Pulmonary Metastasis and Reduces Survival in a Spontaneous Xenograft Model of Human Breast Cancer

Purpose:The transcription factor Fra-2 affects the invasive potential of breast cancer cells by dysregulating adhesion molecules in vitro. Previous results suggested that it upregulates the expression of E- and P-selectin ligands. Such selectin ligands are important members of the leukocyte adhesion cascade, which govern the adhesion and transmigration of cancer cells into the stroma of the host organ of metastasis. As so far, no in vivo data are available, this study was designed to elucidate the role of Fra-2 expression in a spontaneous breast cancer metastasis xenograft model. Methods:The effect of Fra-2 overexpression in two stable Fra-2 overexpressing clones of the human breast cancer cell line MDA MB231 on survival and metastatic load was studied after subcutaneous injection into scid and E- and P-selectin deficient scid mice.Results:Fra-2 overexpression lead to a significantly shorter overall survival and a higher amount of spontaneous lung metastases not only in scid mice, but also in E- and P-deficient mice, indicating that it regulates not only selectin ligands, but also selectin-independent adhesion processes. Conclusion:Thus, Fra-2 expression influences the metastatic potential of breast cancer cells by changing the expression of adhesion molecules, resulting in increased adherence to endothelial cells in a breast cancer xenograft model.

Supramolecular polymerization of sulfated dendritic peptide amphiphiles into multivalent L-selectin binders

The synthesis of a sulfate-modified dendritic peptide amphiphile and its self-assembly into one-dimensional rod-like architectures in aqueous medium is reported. The influence of the ionic strength on the supramolecular polymerization was probed via circular dichroism spectroscopy and cryogenic transmission electron microscopy. Physiological salt concentrations efficiently screen the charges of the dendritic building block equipped with eight sulfate groups and trigger the formation of rigid supramolecular polymers. Since multivalent sulfated supramolecular structures mimic naturally occurring L-selectin ligands, the corresponding affinity was evaluated using a competitive SPR binding assay and benchmarked to an ethylene glycol-decorated supramolecular polymer.

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 < 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 < 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 < 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 & 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. 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 & 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.