A vital sugar code for ricin toxicity.

Ricin is one of the most feared bioweapons in the world due to its extreme toxicity and easy access. Since no antidote exists, it is of paramount importance to identify the pathways underlying ricin toxicity. Here, we demonstrate that the Golgi GDP-fucose transporter Slc35c1 and fucosyltransferase Fut9 are key regulators of ricin toxicity. Genetic and pharmacological inhibition of fucosylation renders diverse cell types resistant to ricin via deregulated intracellular trafficking. Importantly, cells from a patient with SLC35C1 deficiency are also resistant to ricin. Mechanistically, we confirm that reduced fucosylation leads to increased sialylation of Lewis X structures and thus masking of ricin-binding sites. Inactivation of the sialyltransferase responsible for modifications of Lewis X (St3Gal4) increases the sensitivity of cells to ricin, whereas its overexpression renders cells more resistant to the toxin. Thus, we have provided unprecedented insights into an evolutionary conserved modular sugar code that can be manipulated to control ricin toxicity.

Deletions linked to TP53 loss drive cancer through p53-independent mechanisms.

Mutations disabling the TP53 tumour suppressor gene represent the most frequent events in human cancer and typically occur through a two-hit mechanism involving a missense mutation in one allele and a 'loss of heterozygosity' deletion encompassing the other. While TP53 missense mutations can also contribute gain-of-function activities that impact tumour progression, it remains unclear whether the deletion event, which frequently includes many genes, impacts tumorigenesis beyond TP53 loss alone. Here we show that somatic heterozygous deletion of mouse chromosome 11B3, a 4-megabase region syntenic to human 17p13.1, produces a greater effect on lymphoma and leukaemia development than Trp53 deletion. Mechanistically, the effect of 11B3 loss on tumorigenesis involves co-deleted genes such as Eif5a and Alox15b (also known as Alox8), the suppression of which cooperates with Trp53 loss to produce more aggressive disease. Our results imply that the selective advantage produced by human chromosome 17p deletion reflects the combined impact of TP53 loss and the reduced dosage of linked tumour suppressor genes.

Cooperative loss of RAS feedback regulation drives myeloid leukemogenesis.

RAS network activation is common in human cancers, and in acute myeloid leukemia (AML) this activation is achieved mainly through gain-of-function mutations in KRAS, NRAS or the receptor tyrosine kinase FLT3. We show that in mice, premalignant myeloid cells harboring a Kras(G12D) allele retained low levels of Ras signaling owing to negative feedback involving Spry4 that prevented transformation. In humans, SPRY4 is located on chromosome 5q, a region affected by large heterozygous deletions that are associated with aggressive disease in which gain-of-function mutations in the RAS pathway are rare. These 5q deletions often co-occur with chromosome 17 alterations involving the deletion of NF1 (another RAS negative regulator) and TP53. Accordingly, combined suppression of Spry4, Nf1 and p53 produces high levels of Ras signaling and drives AML in mice. Thus, SPRY4 is a tumor suppressor at 5q whose disruption contributes to a lethal AML subtype that appears to acquire RAS pathway activation through a loss of negative regulators.

Janus kinase inhibition by ruxolitinib extends dasatinib- and dexamethasone-induced remissions in a mouse model of Ph+ ALL.

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is initiated and driven by the oncogenic fusion protein BCR-ABL, a constitutively active tyrosine kinase. Despite major advances in the treatment of this highly aggressive disease with potent inhibitors of the BCR-ABL kinase such as dasatinib, patients in remission frequently relapse due to persistent minimal residual disease possibly supported, at least in part, by salutary cytokine-driven signaling within the hematopoietic microenvironment. Using a mouse model of Ph+ ALL that accurately mimics the genetics, clinical behavior, and therapeutic response of the human disease, we show that a combination of 2 agents approved by the US Food and Drug Administration (dasatinib and ruxolitinib, which inhibit BCR-ABL and Janus kinases, respectively), significantly extends survival by targeting parallel signaling pathways. Although the BCR-ABL kinase cancels the cytokine requirement of immature leukemic B cells, dasatinib therapy restores cytokine dependency and sensitizes leukemic cells to ruxolitinib. As predicted, ruxolitinib alone had no significant antileukemic effect in this model, but it prevented relapse when administered with dasatinib. The combination of dasatinib, ruxolitinib, and the corticosteroid dexamethasone yielded more durable remissions, in some cases after completion of therapy, avoiding the potential toxicity of other cytotoxic chemotherapeutic agents.

Systemic blockade of sialylation in mice with a global inhibitor of sialyltransferases.

Sialic acid terminates glycans of glycoproteins and glycolipids that play numerous biological roles in health and disease. Although genetic tools are available for interrogating the effects of decreased or abolished sialoside expression in mice, pharmacological inhibition of the sialyltransferase family has, to date, not been possible. We have recently shown that a sialic acid analog, 2,4,7,8,9-pentaacetyl-3Fax-Neu5Ac-CO2Me (3F-NeuAc), added to the media of cultured cells shuts down sialylation by a mechanism involving its intracellular conversion to CMP-3F-NeuAc, a competitive inhibitor of all sialyltransferases. Here we show that administering 3F-NeuAc to mice dramatically decreases sialylated glycans in cells of all tissues tested, including blood, spleen, liver, brain, lung, heart, kidney, and testes. A single dose results in greatly decreased sialoside expression for over 7 weeks in some tissues. Although blockade of sialylation with 3F-NeuAc does not affect viability of cultured cells, its use in vivo has a deleterious "on target" effect on liver and kidney function. After administration of 3F-NeuAc, liver enzymes in the blood are dramatically altered, and mice develop proteinuria concomitant with dramatic loss of sialic acid in the glomeruli within 4 days, leading to irreversible kidney dysfunction and failure to thrive. These results confirm a critical role for sialosides in liver and kidney function and document the feasibility of pharmacological inhibition of sialyltransferases for in vivo modulation of sialoside expression.

Targeted delivery of mycobacterial antigens to human dendritic cells via Siglec-7 induces robust T cell activation.

Lipids from mycobacteria can be presented to human T cells by group 1 CD1 Ag-presenting molecules (CD1a, CD1b, and CD1c). Group 1 CD1-restricted T cells are activated by lipid Ags presented by myeloid dendritic cells (DCs), after which they generate antibacterial effector functions, including IFN-γ secretion and cytolysis. Thus, mycobacterial lipids are being investigated as components of novel vaccines for mycobacterial infections. In this study we show that the mycobacterial lipid Ag C80 glucose-6-monomycolate can be delivered to human CD1b(+) DCs via targeted liposomal nanoparticles, leading to robust group 1 CD1-restricted activation of T cells. Targeting was achieved by decorating the liposomes with a high-affinity glycan ligand of sialic acid-binding Ig-like lectin (Siglec)-7, a siglec receptor expressed on DCs that mediates rapid endocytosis and transport of its cargo to lysosomes. An Ab to Siglec-7 completely blocked the binding of targeted liposomes to human monocyte-derived DCs (Mo-DCs), demonstrating their targeting specificity. Mo-DCs pulsed with targeted liposomes containing C80 glucose-6-monomycolate more potently activated a CD1b-restricted T cell line relative to Mo-DCs pulsed with free lipid Ag or antigenic liposomes without Siglec-7 ligand. These data suggest that the endocytic function of Siglec-7 can be exploited to deliver glycolipid Ags to their target cell and increase the efficiency of display to T cells.

Disubstituted Sialic Acid Ligands Targeting Siglecs CD33 and CD22 Associated with Myeloid Leukaemias and B Cell Lymphomas.

The siglec family of sialic acid-binding proteins are endocytic immune cell receptors that are recognized as potential targets for cell directed therapies. CD33 and CD22 are prototypical members and are validated candidates for targeting acute myeloid leukaemia and non-Hodgkin's lymphomas due to their restricted expression on myeloid cells and B-cells, respectively. While nanoparticles decorated with high affinity siglec ligands represent an attractive platform for delivery of therapeutic agents to these cells, a lack of ligands with suitable affinity and/or selectivity has hampered progress. Herein we describe selective ligands for both of these siglecs, which when displayed on liposomal nanoparticles, can efficiently target the cells expressing them in peripheral human blood. Key to their identification was the development of a facile method for chemo-enzymatic synthesis of disubstituted sialic acid analogues, combined with iterative rounds of synthesis and rapid functional analysis using glycan microarrays.

On-chip synthesis and screening of a sialoside library yields a high affinity ligand for Siglec-7.

The Siglec family of sialic acid-binding proteins are differentially expressed on white blood cells of the immune system and represent an attractive class of targets for cell-directed therapy. Nanoparticles decorated with high-affinity Siglec ligands show promise for delivering cargo to Siglec-bearing cells, but this approach has been limited by a lack of ligands with suitable affinity and selectivity. Building on previous work employing solution-phase sialoside library synthesis and subsequent microarray screening, we herein report a more streamlined 'on-chip' synthetic approach. By printing a small library of alkyne sialosides and subjecting these to 'on-chip' click reactions, the largest sialoside analogue library to date was generated. Siglec-screening identified a selective Siglec-7 ligand, which when displayed on liposomal nanoparticles, allows for targeting of Siglec-7(+) cells in peripheral human blood. In silico docking to the crystal structure of Siglec-7 provides a rationale for the affinity gains observed for this novel sialic acid analogue.

Click and pick: identification of sialoside analogues for siglec-based cell targeting.

Click 'n' chips: Azide and alkyne-bearing sialic acids (purple diamond; see picture) were subjected to high-throughput click chemistry to generate a library of sialic acid analogues. Microarray printing of the library and screening with the siglec family of sialic-acid-binding proteins, led to the identification of high-affinity ligands for siglec-9 and siglec-10.

Global metabolic inhibitors of sialyl- and fucosyltransferases remodel the glycome.

Despite the fundamental roles of sialyl- and fucosyltransferases in mammalian physiology, there are few pharmacological tools to manipulate their function in a cellular setting. Although fluorinated analogs of the donor substrates are well-established transition state inhibitors of these enzymes, they are not membrane permeable. By exploiting promiscuous monosaccharide salvage pathways, we show that fluorinated analogs of sialic acid and fucose can be taken up and metabolized to the desired donor substrate-based inhibitors inside the cell. Because of the existence of metabolic feedback loops, they also act to prevent the de novo synthesis of the natural substrates, resulting in a global, family-wide shutdown of sialyl- and/or fucosyltransferases and remodeling of cell-surface glycans. As an example of the functional consequences, the inhibitors substantially reduce expression of the sialylated and fucosylated ligand sialyl Lewis X on myeloid cells, resulting in loss of selectin binding and impaired leukocyte rolling.

High-throughput screening for inhibitors of sialyl- and fucosyltransferases.

Sweet screens: A high-throughput screening platform for identification of inhibitors of sialyl- and fucosyltransferases based on fluorescence polarization (FP) has been developed. An analogue of the natural donor substrate carrying a fluorescent label (green star) is transferred to a glycoprotein acceptor, which results in robust FP. The screening of 16,000 compounds against different glycosyltransferases has identified various interesting inhibitors.

Glycan microarrays for decoding the glycome.

In the last decade, glycan microarrays have revolutionized the analysis of the specificity of glycan-binding proteins (GBPs), providing information that simultaneously illuminates the biology mediated by them and decodes the informational content of the glycome. Numerous methods have emerged for arraying glycans in a "chip" format, and glycan libraries have been assembled that address the diversity of the human glycome. Such arrays have been successfully used for analysis of GBPs, which mediate mammalian biology, host-pathogen interactions, and immune recognition of glycans relevant to vaccine production and cancer antigens. This review covers the development of glycan microarrays and applications that have provided insights into the roles of mammalian and microbial GBPs.