Anticancer Properties of Hexosamine Analogs Designed to Attenuate Metabolic Flux through the Hexosamine Biosynthetic Pathway.

Altered cellular metabolism is a hallmark of cancer pathogenesis and progression; for example, a near-universal feature of cancer is increased metabolic flux through the hexosamine biosynthetic pathway (HBP). This pathway produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a potent oncometabolite that drives multiple facets of cancer progression. In this study, we synthesized and evaluated peracetylated hexosamine analogs designed to reduce flux through the HBP. By screening a panel of analogs in pancreatic cancer and glioblastoma multiform (GBM) cells, we identified Ac4Glc2Bz─a benzyl-modified GlcNAc mimetic─as an antiproliferative cancer drug candidate that down-regulated oncogenic metabolites and reduced GBM cell motility at concentrations non-toxic to non-neoplastic cells. More specifically, the growth inhibitory effects of Ac4Glc2Bz were linked to reduced levels of UDP-GlcNAc and concomitant decreases in protein O-GlcNAc modification in both pancreatic cancer and GBM cells. Targeted metabolomics analysis in GBM cells showed that Ac4Glc2Bz disturbed glucose metabolism, amino acid pools, and nucleotide precursor biosynthesis, consistent with reduced proliferation and other anti-oncogenic properties of this analog. Furthermore, Ac4Glc2Bz reduced the invasion, migration, and stemness of GBM cells. Importantly, normal metabolic functions mediated by UDP-GlcNAc were not disrupted in non-neoplastic cells, including maintenance of endogenous levels of O-GlcNAcylation with no global disruption of N-glycan production. Finally, a pilot in vivo study showed that a potential therapeutic window exists where animals tolerated 5- to 10-fold higher levels of Ac4Glc2Bz than projected for in vivo efficacy. Together, these results establish GlcNAc analogs targeting the HBP through salvage mechanisms as a new therapeutic approach to safely normalize an important facet of aberrant glucose metabolism associated with cancer.

Cell Line-, Protein-, and Sialoglycosite-Specific Control of Flux-Based Sialylation in Human Breast Cells: Implications for Cancer Progression.

Sialylation, a post-translational modification that impacts the structure, activity, and longevity of glycoproteins has been thought to be controlled primarily by the expression of sialyltransferases (STs). In this report we explore the complementary impact of metabolic flux on sialylation using a glycoengineering approach. Specifically, we treated three human breast cell lines (MCF10A, T-47D, and MDA-MB-231) with 1,3,4-O-Bu3ManNAc, a "high flux" metabolic precursor for the sialic acid biosynthetic pathway. We then analyzed N-glycan sialylation using solid phase extraction of glycopeptides (SPEG) mass spectrometry-based proteomics under conditions that selectively captured sialic acid-containing glycopeptides, referred to as "sialoglycosites." Gene ontology (GO) analysis showed that flux-based changes to sialylation were broadly distributed across classes of proteins in 1,3,4-O-Bu3ManNAc-treated cells. Only three categories of proteins, however, were "highly responsive" to flux (defined as two or more sialylation changes of 10-fold or greater). Two of these categories were cell signaling and cell adhesion, which reflect well-known roles of sialic acid in oncogenesis. A third category-protein folding chaperones-was unexpected because little precedent exists for the role of glycosylation in the activity of these proteins. The highly flux-responsive proteins were all linked to cancer but sometimes as tumor suppressors, other times as proto-oncogenes, or sometimes both depending on sialylation status. A notable aspect of our analysis of metabolically glycoengineered breast cells was decreased sialylation of a subset of glycosites, which was unexpected because of the increased intracellular levels of sialometabolite "building blocks" in the 1,3,4-O-Bu3ManNAc-treated cells. Sites of decreased sialylation were minor in the MCF10A (<25% of all glycosites) and T-47D (<15%) cells but dominated in the MDA-MB-231 line (~60%) suggesting that excess sialic acid could be detrimental in advanced cancer and cancer cells can evolve mechanisms to guard against hypersialylation. In summary, flux-driven changes to sialylation offer an intriguing and novel mechanism to switch between context-dependent pro- or anti-cancer activities of the several oncoproteins identified in this study. These findings illustrate how metabolic glycoengineering can uncover novel roles of sialic acid in oncogenesis.

Exploiting metabolic glycoengineering to advance healthcare.

Metabolic glycoengineering (MGE) is a technique for manipulating cellular metabolism to modulate glycosylation. MGE is used to increase the levels of natural glycans and, more importantly, to install non-natural monosaccharides into glycoconjugates. In this Review, we summarize the chemistry underlying MGE that has been developed over the past three decades and highlight several recent advances that have set the stage for clinical translation. In anticipation of near-term application to human healthcare, we describe emerging efforts to deploy MGE in diverse applications, ranging from the glycoengineering of biotherapeutic proteins and the diagnosis and treatment of complex diseases such as cancer to the development of new immunotherapies.

The Triticum Mosaic Virus Internal Ribosome Entry Site Relies on a Picornavirus-Like YX-AUG Motif To Designate the Preferred Translation Initiation Site and To Likely Target the 18S rRNA.

Several viruses encode an internal ribosome entry site (IRES) at the 5' end of their RNA, which, unlike most cellular mRNAs, initiates translation in the absence of a 5' m7GpppG cap. Here, we report a uniquely regulated translation enhancer found in the 739-nucelotide (nt) sequence of the Triticum mosaic virus (TriMV) leader sequence that distinguishes the preferred initiation site from a plethora of IRES-encoded AUG triplets. Through deletion mutations of the TriMV 5' untranslated region (UTR), we show that the TriMV 5' UTR encodes a cis-acting picornaviral Y16-X11-AUG-like motif with a 16-nt polypyrimidine CU-tract (Y16), at a precise, 11-nt distance (X11) from the preferred 13th AUG. Phylogenetic analyses indicate that this motif is conserved among potyviral leader sequences with multiple AUGs. Consistent with a broadly conserved mechanism, the motif could be functionally replaced with known picornavirus YX-AUG motifs and is predicted to function as target sites for the 18S rRNA by direct base pairing. Accordingly, mutations that disrupted overall complementarity to the 18S rRNA markedly reduced TriMV IRES activity, as did the delivery of antisense oligonucleotides designed to block YX-AUG accessibility. To our knowledge, this is the first report of a plant viral IRES YX-AUG motif, and our findings suggest that a conserved mechanism regulates translation for multiple economically important plant and animal positive single-stranded RNA viruses.IMPORTANCE Uncapped viral RNAs often rely on their 5' leader sequences to initiate translation, and the Triticum mosaic virus (TriMV) devotes an astonishing 7% of its genome to directing ribosomes to the correct AUG. Here we uncover a novel mechanism by which a TriMV cis-regulatory element controls cap-independent translation. The upstream region of the functional AUG contains a 16-nt polypyrimidine tract located 11 nt from the initiation site. Based on functional redundancy with similar motifs derived from human picornaviruses, the motif is likely to operate by directing ribosome targeting through base pairing with 18S rRNA. Our results provide the first report of a broad-spectrum mechanism regulating translation initiation for both plant- and animal-hosted picornaviruses.

Improving Immunotherapy Through Glycodesign.

Immunotherapy is revolutionizing health care, with the majority of high impact "drugs" approved in the past decade falling into this category of therapy. Despite considerable success, glycosylation-a key design parameter that ensures safety, optimizes biological response, and influences the pharmacokinetic properties of an immunotherapeutic-has slowed the development of this class of drugs in the past and remains challenging at present. This article describes how optimizing glycosylation through a variety of glycoengineering strategies provides enticing opportunities to not only avoid past pitfalls, but also to substantially improve immunotherapies including antibodies and recombinant proteins, and cell-based therapies. We cover design principles important for early stage pre-clinical development and also discuss how various glycoengineering strategies can augment the biomanufacturing process to ensure the overall effectiveness of immunotherapeutics.