Kevin J. Yarema

Ossification of the Posterior Longitudinal Ligament (OPLL) and Diffuse Idiopathic Skeletal Hyperostosis (DISH) are disorders in which patients suffer from pain and stiffness due to progressive spinal fractures and enthesospathy. Myelopathy and impaired mobility worsen with age and may progress to hemiplegia; the mechanisms driving disease progression are unknown, and effective treatments are lacking. To investigate OPLL disease pathogenesis we prospectively quantiated plasma PPi in consecutive OPLL and cervical osteoarthritic (OA) patients who presented for surgical correction, and further sequenced the OPLL patients for genes associated with phosphate wasting and spinal enthesopathy, and to evaluate novel OPLL therapeutics we explored the efficacy of soluble and bone targeted ENPP1 biologics on the plasma biomarkers and the skeletal phenotype of OPLL mice. We found that a significant proportion of OPLL patients harbored monoallelic pathogenic ENPP1 loss of function variants and that those OPLL patients requiring surgical intervention had significantly reduced plasma PPi levels in comparison to cervical OA patients with comparably severe disease. In murine OPLL, bone targeted ENPP1-Fc corrected plasma biomarkers and spinal hyperostosis, significantly improved or normalized spinal and long bone fragility, and ameliorated Achilles tendon enthesopathy. Our findings support the notion that OPLL patients are ENPP1 deficient, and that treatment with ENPP1 biologics may ameliorate the spinal hyperostosis, fracture risk, and enthesopathies present in OPLL.

Schwann cells (SCs) are myelinating cells that promote peripheral nerve regeneration. When nerve lesions form, SCs are destroyed, ultimately hindering nerve repair. The difficulty in treating nerve repair is exacerbated due to SC’s limited and slow expansion capacity. Therapeutic use of adipose-derived stem cells (ASCs) is emerging in combating peripheral nerve injury due to these cells’ SC differentiation capability and can be harvested easily in large numbers. Despite ASC’s therapeutic potential, their transdifferentiation period typically takes more than two weeks. In this study, we demonstrate that metabolic glycoengineering (MGE) technology enhances ASC differentiation into SCs. Specifically, the sugar analog Ac₅ManNTProp (TProp), which modulates cell surface sialylation, significantly improved ASC differentiation with upregulated SC protein S100β and p75NGFR expression and elevated the neurotrophic factors nerve growth factor beta (NGFβ) and glial cell-line-derived neurotrophic factor (GDNF). TProp treatment remarkably reduced the SC transdifferentiation period from about two weeks to two days in vitro, which has the potential to improve neuronal regeneration and facilitate future use of ASCs in regenerative medicine.

Altered cellula r 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 path w a y (HBP). This path w a y 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 mot i l i t y at concentrations non-toxic to non-neoplastic cells. More specifically, the gro w t h 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 anima l s 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.

Almost all therapeutic proteins are glycosylated, with the carbohydrate component playing a long-established, substantial role in the safety and pharmacokinetic properties of this dominant category of drugs. In the past few years and moving forward, glycosylation is increasingly being implicated in the pharmacodynamics and therapeutic efficacy of therapeutic proteins. This article provides illustrative examples of drugs that have already been improved through glycoengineering including cytokines exemplified by erythropoietin (EPO), enzymes (ectonucleotide pyrophosphatase 1, ENPP1), and IgG antibodies (e.g., afucosylated Gazyva^®, Poteligeo^®, Fasenra™, and Uplizna^®). In the future, the deliberate modification of therapeutic protein glycosylation will become more prevalent as glycoengineering strategies, including sophisticated computer-aided tools for “building in” glycans sites, acceptance of a broad range of production systems with various glycosylation capabilities, and supplementation methods for introducing non-natural metabolites into glycosylation pathways further develop and become more accessible.

This report describes novel thiol-modified N-acetylmannosamine (ManNAc) analogs that extend metabolic glycoengineering (MGE) applications of Ac₅ManNTGc, a non-natural monosaccharide that metabolically installs the thio-glycolyl of sialic acid into human glycoconjugates. We previously found that Ac₅ManNTGc elicited non-canonical activation of Wnt signaling in human embryoid body derived (hEBD) cells but only in the presence of a high affinity, chemically compatible scaffold. Our new analogs Ac₅ManNTProp and Ac₅ManNTBut overcome the requirement for a complementary scaffold by displaying thiol groups on longer, N-acyl linker arms, thereby presumably increasing their ability to interact and crosslink with surrounding thiols. These new analogs showed increased potency in human neural stem cells (hNSCs) and human adipose stem cells (hASCs). In the hNSCs, Ac₅ManNTProp upregulated biochemical endpoints consistent with Wnt signaling in the absence of a thiol-reactive scaffold. In the hASCs, both Ac₅ManNTProp and Ac₅ManNTBut suppressed adipogenic differentiation, with Ac₅ManNTBut providing a more potent response, and they did not interfere with differentiation to a glial lineage (Schwann cells). These results expand the horizon for using MGE in regenerative medicine by providing new tools (Ac₅ManNTProp and Ac₅ManNTBut) for manipulating human stem cells.