Emerging role of sialylation in cancer therapy resistance: Mechanisms and therapeutic implications.

Meningiomas are the most common primary intracranial tumors and are often curable with gross resection. However, surgery is not entirely effective, as recurrences are reported more frequently in patients with meningiomas with higher-grade tumors, despite the extent of surgical resection. Therefore, elucidating tumor biology at the molecular level is needed. Aberrant sialylation, resulting from altered expression of sialyl-transferases (STs), plays an important role in cancer development and progression. However, the role of altered sialylation in meningioma progression remains unclear. In the present study, downregulation of β-galactoside α2,3-ST (ST3Gals) and β-galactoside α2,6-ST (ST6Gals) genes was found in malignant meningioma tissues from four different Gene Expression Omnibus (GEO) datasets (GEO entries: GSE16581, GSE43290, GSE74385 and GSE136661). Moreover, suppression of sialylation using a pan-sialylation inhibitor (3Fax-peracetyl-Neu5Ac, 3Fax) reduced the activity of STs in a malignant meningioma cell line, leading to an increase in cell migration and invasion capacities. Further investigation of epithelial-mesenchymal transition (EMT) markers, AKT, and ERK signaling in the 3Fax-treated cell lines revealed that high expression of EMT-related transcription factors (Snail) and EMT-related proteins (MMP9) were regulated via phosphorylation of AKT and ERK. Therefore, the present findings suggested that suppression of sialylation by 3Fax in malignant meningioma increases migration and invasion abilities by enhancing the EMT process.

Molecular hybridization, an emerging strategy for the discovery of new anticancer therapeutics, shows promise as a powerful tool for the development of new sialyltransferase (ST) inhibitors for cancer treatment. This concept inspired the design of novel ST inhibitors through the hybridization of lithocholic acid and diindolylmethane, leading to the discovery of LCA-DIM hybrids as potential chemical entities targeting STs. Preliminary screening revealed the significance of the DIM moiety and incorporation of Asp linker on enhancing the inhibitory activity and selectivity of the hybrids towards ST6GAL1, inhibiting up to 100% of ST6GAL1 activity at 25 μM with no ST3GAL1 inhibition even at 500 μM. Incorporation of various 5,5'-substituents enhanced the inherent antimigration properties of the hybrids, with IAN-5B (R = Cl) and IAN-15B (R = N3) presenting the highest antimigration activity across several triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, BT549, Hs578T) and considerable antiangiogenetic effect by suppressing HUVEC tube formation. This could be attributed to the excellent ST6GAL1 inhibitory activities of the two hybrids with IC50 values of 6.6 ± 0.2 μM and 3.3 ± 0.1 μM, respectively. Overall, this study highlights LCA-DIM hybrids as novel, potent, and N-glycan-selective ST inhibitors with promising antimigration properties against aggressive TNBC.

Abstract BACKROUND Glioblastoma (GBM) is the most prevalent and aggressive brain tumour, and is associated with an unfavourable prognosis. Consequently, there is an imperative for the identification of novel therapeutic targets. The attachment of sialic acids (Sia) to the cell surface represents a potential target for therapeutic intervention, as this process is already employed clinically and prognostically in other tumour entities. The sialyltransferases (ST) ST8SIA2 and ST8SIA4, which influence the interaction of cells via long sugar chains, are mainly responsible for polysialylation (PolySia) in tumours. The project investigates the expression of STs in different GBMs, and functionally characterises the influence of PolySia on malignancy. MATERIALS AND METHODS For the expression analysis of STs in primary tumours (PT) and primary cultures (PC), quantitative PCR (qPCR) and bulk RNAseq were utilised. The knockout of ST8SIA2 and ST8SIA4 is performed using CRISPR/Cas9. Following the transfection of cell lines and primary cultures, the influence of the knockout of both STs alone and the double knockout will be characterised using Real-Time Cell Analysis (RTCA). The effect on cellular properties will be measured by changes in proliferation, invasion, resistance to therapy and co-culture experiments. RESULTS The qPCR revealed a heterogeneous ST profile, with the ST3 group showing notable expressions in PCs. The data indicate changes in ST expression between PTs and corresponding PCs. The presence of poly-STs has been detected, while the predominance of either ST8SIA2 or ST8SIA4 in PTs is maintained in 2D culture. ST8SIA2 was stronger expressed in some PCs and ST8SIA4 in others. However, higher expressions were observed in 2D cultures than in PTs. With regard to gene editing, sgRNA was cloned into a plasmid, and the transfection protocol could be tested. In addition, the formation of spheroids from cell lines and PCs was achieved. CONCLUSION GBM is distinguished by intertumoural heterogeneity, a property that extends to the ST expression. The elevated expression of ST8SIA2 or ST8SIA4 in 2D cultures when compared to PTs may be attributed to the selection of cells, suggesting that these cells possess a growth advantage. Consequently, 3D GBM models have been developed to achieve maximum comparability with PTs. Given the divergent expression of ST8SIA2 to ST8SIA4 between PCs, the double knockout of both STs represents a promising prospect, with the potential to be used therapeutically in future.

The multifaceted roles of ST3GAL family in cancer: Mechanistic insights and therapeutic implications.

Neutrophils are the most abundant circulating leukocyte population that play critical roles in neuroinflammation following central nervous system (CNS) injury. CD177, a glycoprotein on neutrophils, is emerging as an important immune regulator which can fundamentally affect multiple human inflammatory diseases. However, the role and regulatory mechanism of CD177 glycobiology of neutrophils in neuroinflammation remain elusive. Here, we show that CD177+ neutrophils expand significantly and infiltrate the injured brain following CNS injury both in the human and mouse. Using single-cell RNA sequencing and genetic approaches, we find CD177+ neutrophils as an anti-inflammatory subset that is critical for modulating neuroinflammation after CNS injury. We further identify St3gal5, a sialyltransferase (ST), that can mediate the sialylation and cell surface presentation of glyco-CD177 on neutrophils. Glycoproteomics reveal downregulated sialylation levels in St3gal5-deficient neutrophils. Neutrophil-specific depletion of St3gal5 prevents the cell surface presentation of CD177 on brain-infiltrated neutrophils and exacerbates neuroinflammation. Administration of the FDA-approved anticonvulsant valproic acid (VPA), an St3gal5 upregulator, promotes the glycosylation of neutrophils and attenuates neuroinflammation following CNS injury. Our study reveals a glycoimmuno-regulatory effect of neutrophils and suggests VPA as a neutrophil glycobiology targeting approach to combat neuroinflammation following CNS injury.

Sialyltransferases (ST) are key enzymes found in, among others, mammals and bacteria that are responsible for producing sialylated glycans, which play critical roles in human health and disease. However, chemical tools to study sialyltransferases have been limited to non-covalent inhibitors and probes that do not allow isolation and profiling of these important enzymes. Here we report a new class of covalent affinity-based probes (AfBP) for ST by using ligand-directed chemistry (LDchem). Our affinity-based probes are armed with a simple to synthesise but robust O-nitrobenzoxadiazole (O-NBD) warhead, which is a lysine-specific SNAr electrophilic warhead with an advantageous turn-on fluorescence property. We chemoenzymatically synthesised a series of CMP-Neu5Ac based probes and demonstrated their high specificity in labelling a range of recombinant STs with submicromolar sensitivity. Importantly, with our LDchem ST probe, we successfully labelled the endogenous lipooligosaccharide ST (Lst) in live Neisseria gonorrhoeae, a clinically relevant human pathogen. Our results demonstrated that this new class of covalent ST probes offer a robust platform for ST profiling and future studies of STs in their native environments.

CD24, a highly sialylated glycosyl-phosphatidyl-inositol (GPI) cell surface protein that interacts with sialic acid-binding immunoglobulin-like lectins (Siglecs), serves as an innate immune checkpoint and plays a crucial role in inflammatory diseases and tumor progression. Recently, cytoplasmic CD24 has been observed in samples from patients with cancer. However, whether sialylation governs the subcellular localization of CD24 in cancer remains unclear, and the impact of CD24 expression and localization on the clinical prognosis of cancer remains controversial. Here, we performed a systematic pan-cancer analysis of the gene expression levels and clinical correlation of CD24. Our analysis revealed that CD24 was highly expressed in breast tumor tissues and tumor cells, significantly shortening patient survival time. However, this correlation was not evident in other types of cancer. Additionally, a correlation analysis of CD24 levels with sialyltransferases (STs) revealed that ST8SIA6 is the key ST affecting CD24 sialylation. Further investigation demonstrated that ST8SIA6 directly modified CD24, promoting its localization to the cell membrane. Taken together, these findings elucidate, for the first time, the mechanisms by which ST8SIA6 regulates CD24 subcellular localization, providing new insights into the biological functions and applications of CD24.

Preventing human influenza and coronaviral mono or coinfection by blocking virus-induced sialylation

Sialic acid (Neu5Ac) is installed onto glycoconjugates by sialyltransferases (STs) using cytidine monophosphate-Neu5Ac (CMP-β-d-Neu5Ac) as their donor. The only class of cell-active ST inhibitors are those based on a 3FaxNeu5Ac scaffold, which is metabolically converted into CMP-3FaxNeu5Ac within cells. It is essential for the fluorine to be axial, yet stereoselective installation of fluorine in this specific orientation is challenging. Sialic acid aldolase can convert 3-fluoropyruvate and 2-acetamido-2-deoxy-d-mannopyranose (ManNAc) to 3FNeu5Ac, but stereocontrol of the fluorine in the product has not been possible. We hypothesized that the 3Fax kinetic product of a sialic acid aldolase reaction could be trapped by coupling with CMP-sialic acid synthetase to yield CMP-3FaxNeu5Ac. Here, we report that highly active CMP-sialic acid synthetase and short reaction times produce exclusively CMP-3FaxNeu5Ac. Removal of CMP from CMP-3FaxNeu5Ac under acidic conditions unexpectedly led to 3-fluoro-β-d-Neu5Ac 2-phosphate (3FaxNeu5Ac-2P). Alkaline phosphatase successfully converted 3FaxNeu5Ac-2P to 3FaxNeu5Ac, enabling stereochemically controlled access to 3FaxNeu5Ac, which is effective in lowering the sialoglycan ligands for Siglecs on cells. Moreover, our kinetic trapping approach could be used to access CMP-3FaxNeu5Ac with modifications at the C5, C9, or both positions, which enabled the chemoenzymatic synthesis of a photo-cross-linkable version of CMP-3FaxNeu5Ac that selectively photo-cross-linked to ST6GAL1 over two other STs.

ST3GAL1 Promotes Malignant Phenotypes in Intrahepatic Cholangiocarcinoma

Tumour-derived sialoglycans, bearing the charged nonulosonic sugar sialic acid at their termini, play a critical role in tumour cell adhesion and invasion, as well as evading cell death and immune surveillance. Sialyltransferases (ST), the enzymes responsible for the biosynthesis of sialylated glycans, are highly upregulated in cancer, with tumour hypersialylation strongly correlated with tumour growth, metastasis and drug resistance. As a result, desialylation of the tumour cell surface using either targeted delivery of a pan-ST inhibitor (or sialidase) or systemic delivery of a non-toxic selective ST inhibitors are being pursued as potential new anti-metastatic strategies against multiple cancers including pancreatic, ovarian, breast, melanoma and lung cancer. Herein, we have employed molecular modelling to give insights into the selectivity observed in a series of selective ST inhibitors that incorporate a uridyl ring in place of the cytidine of the natural donor (CMP-Neu5Ac) and replace the charged phosphodiester linker of classical ST inhibitors with a neutral α-hydroxy-1,2,3-triazole linker. The inhibitory activities of the nascent compounds were determined against recombinant human ST enzymes (ST3GAL1, ST6GAL1, ST8SIA2) showing promising activity and selectivity towards specific ST sub-types. Our ST inhibitors are non-toxic and show improved synthetic accessibility and drug-likeness compared to earlier nucleoside-based ST inhibitors.

Single-Cell Transcriptomics Points to a Role of Stemness and Bone Marrow Niche Interactions in the Poor Response to Induction Therapy in T-Cell Acute Lymphoblastic Leukemia

The role of sialylation in gynecologic cancers.

Upregulation of surface expressed sialoglycans on tumor cells is one of the mechanisms which promote tumor growth and progression. Specifically, the interactions of sialic acids with sialic acid-binding immunoglobulin-like lectins (Siglecs) on lymphoid or myeloid cells transmit inhibitory signals and lead to suppression of anti-tumor responses. Here, we show that neutrophils express among others Siglec-9, and that EGFR and HER2 positive breast tumor cells express ligands for Siglec-9. Treatment of tumor cells with neuraminidases or a sialyl transferase inhibitor significantly reduced binding of a soluble recombinant Siglec-9-Fc fusion protein, while EGFR and HER2 expression remained unchanged. Importantly, the cytotoxic activity of neutrophils driven by therapeutic EGFR or HER2 antibodies in vitro was increased by blocking the sialic acid/Siglec interaction, either by reducing tumor cell sialylation or by a Siglec-9 blocking antibody containing an effector silenced Fc domain. In vivo a short-term xenograft mouse model confirmed the improved therapeutic efficacy of EGFR antibodies against sialic acid depleted, by a sialyltransferase inhibitor, tumor cells compared to untreated cells. Our studies demonstrate that sialic acid/Siglec interactions between tumor cells and myeloid cells can impair antibody dependent tumor cell killing, and that Siglec-9 on polymorphonuclear cells (PMN) is critically involved. Considering that PMN are often a highly abundant cell population in the tumor microenvironment, Siglec-9 constitutes a promising target for myeloid checkpoint blockade to improve antibody-based tumor immunotherapy.

Despite decades of research, glycosaminoglycans (GAGs) have not been known to interact with sialyl transferases (STs). Using our in-house combinatorial virtual library screening (CVLS) technology, we studied seven human isoforms, including ST6GAL1, ST6GAL2, ST3GAL1, ST3GAL3, ST3GAL4, ST3GAL5, and ST3GAL6, and predicted that GAGs, especially heparan sulfate (HS), are likely to differentially bind to STs. Exhaustive CVLS and molecular dynamics studies suggested that the common hexasaccharide sequence of HS preferentially recognized ST6GAL1 in a site overlapping the binding site of the donor substrate CMP-Sia. Interestingly, CVLS did not ascribe any special role for the rare 3-O-sulfate modification of HS in ST6GAL1 recognition. The computational predictions were tested using spectrofluorimetric studies, which confirmed preferential recognition of HS over other GAGs. A classic chain length-dependent binding of GAGs to ST6GAL1 was observed with polymeric HS displaying a tight affinity of ~65nM. Biophysical studies also confirmed a direct competition between CMP-Sia and an HS oligosaccharide and CS polysaccharide for binding to ST6GAL1. Overall, our novel observation that GAGs bind to ST6GAL1 with high affinity and compete with the donor substrate is likely to be important because modulation of sialylation of glycan substrates on cells has considerable physiological/pathological consequences. Our work also brings forth the possibility of developing GAG-based chemical probes of ST6GAL1.

Despite hypersialylation of cancer cells together with a significant upregulation of sialyltransferase (ST) activity contributes to the metastatic cascade at multiple levels, there are few dedicated tools to interfere with their expression. Although transition state-based ST inhibitors are well-established, they are not membrane permeable. To tackle this problem, herein, we design and construct long-circulating, self-assembled core-shell nanoscale coordination polymer (NCP) nanoparticles carrying a transition state-based ST inhibitor, which make the inhibitor transmembrane and potently strip diverse sialoglycans from various cancer cells. In the experimental lung metastasis and metastasis prevention models, the nanoparticle device (NCP/STI) significantly inhibits metastases formation without systemic toxicity. This strategy enables ST inhibitors to be applied to cells and animals by providing them with a well-designed nanodelivery system. Our work opens a new avenue to the development of transition state-based ST inhibitors and demonstrates that NCP/STI holds great promise in achieving metastases inhibition for multiple cancers.

Gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide. Despite progress in the last decades, there are still no reliable biomarkers for the diagnosis of and prognosis for GC. Aberrant sialylation is a widespread critical event in the development of GC. Neuraminidases (Neu) and sialyltransferases (STs) regulate the ablation and addition of sialic acid during glycoconjugates biosynthesis, and they are a considerable source of biomarkers in various cancers. This study retrospectively characterized Neu3 and ST3Gal3 expression by immunohistochemistry in 71 paraffin-embedded GC tissue specimens and analyzed the relationship between their expression and the clinicopathological parameters. Neu3 expression was markedly increased in GC tissues compared with non-tumoral tissues (p<0.0001). Intratumoral ST3Gal3 staining was significantly associated with intestinal subtype (p=0.0042) and was negatively associated with angiolymphatic invasion (p=0.0002) and higher histological grade G3 (p=0.0066). Multivariate analysis revealed that ST3Gal3 positivity is able to predict Lauren's classification. No associations were found between Neu3 staining and clinical parameters. The in silico analysis of mRNA expression in GC validation cohorts corroborates the significant ST3Gal3 association with higher histological grade observed in our study. These findings suggest that ST3Gal3 expression may be an indicator for aggressiveness of primary GC.

Cleavage of erythrocyte surface sialic acid (SA) by the sialidase produced by trypanosomes is implicated in the pathogenesis of anaemia in African animal trypanosomiasis (AAT). Sialyltransferase (ST) mediates the attachment of SA to cell surface glycoproteins and glycolipids of desialylated erythrocytes. ST activities in Trypanosoma congolense-infected sheep and control groups were investigated and variations in their physio-biochemical properties were evaluated. Six (6) apparently healthy Nigerian Yankassa breed of sheep comprising of T. congolense-infected (n=3) and non-infected (n=3) groups were used for the experiment. Parasitaemia and packed cell volume (PCV) were determined daily over a 5-week period. Enzyme kinetics of partially purified ST from the thyroid gland were also evaluated. Anaemia (mean PCV= 18.83±0.71%) was observed in the T. congolense-infected sheep when compared with the non-infected control group (mean PCV 39.75±0.35%) and the observed differences were significant (p &lt; 0.05) after five weeks post infection. Variations were also observed in the physio-biochemical properties (pH, temperature, activation energy) of the ST isolated from the T. congolense-infected and control sheep. This finding is believed to have been an induced response by the host to parasite’s activity and could be exploited further as a possible target in the control of AAT.

Upregulation of sialyltransferases ST3Gal1 and ST6Gal1 promotes stabilization of erythrocyte mass and recovery of anemia in Trypanosoma brucei brucei-infected pigs