Targeting PGM3 as a Novel Therapeutic Strategy in KRAS/LKB1 Co-Mutant Lung Cancer

In non-small-cell lung cancer (NSCLC), concurrent mutations in the oncogene KRAS and tumor suppressor STK11 (also known as LKB1) confer an aggressive malignant phenotype, an unfavourability towards immunotherapy, and overall poor prognoses in patients. In a previous study, we showed that murine KRAS/LKB1 co-mutant tumors and human co-mutant cancer cells have an enhanced dependence on glutamine-fructose-6-phosphate transaminase 2 (GFPT2), a rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP), which could be targeted to reduce survival of KRAS/LKB1 co-mutants. Here, we found that KRAS/LKB1 co-mutant cells also exhibit an increased dependence on N-acetylglucosamine-phosphate mutase 3 (PGM3), an enzyme downstream of GFPT2. Genetic or pharmacologic suppression of PGM3 reduced KRAS/LKB1 co-mutant tumor growth in both in vitro and in vivo settings. Our results define an additional metabolic vulnerability in KRAS/LKB1 co-mutant tumors to the HBP and provide a rationale for targeting PGM3 in this aggressive subtype of NSCLC.

CNS GNPDA2 Does Not Control Appetite, but Regulates Glucose Homeostasis

GNPDA2 has been associated with human obesity and type-2 diabetes by using a GWAS approach. GNPDA2 is an enzyme involved in the hexosamine biosynthesis pathway, which is known to be important for nutrient sensing in various organism. Its counter enzyme, GFAT, has previously been shown to be important to the development of insulin resistance in diabetes. The implication of GNPDA2 and GFAT in metabolism is scarce and the effect of both enzymes over appetite and glucose homeostasis is unknown.Aim: Identify the role of GNPDA2 and GFAT in nutrient sensing circuits of the CNS that are important for the regulation of both appetite and glucose homeostasis.Methods: Using Long Evans rats, we administered either a GNPDA2 or GFAT antagonist or vehicle in i3vt.Key Findings:GNPDA2 is highly expressed in hypothalamus and adipose tissue, followed by muscle and liver. GNPDA2 is expressed in different hypothalamic nuclei (ARC, DMH, LHA, PVN). GNPDA2 is downregulated in hypothalamus under diet-induced obesity (as previously described), but GFAT expression does not change. Moreover, i3vt infusion of GNPDA2 or GFAT inhibitor resulted in increased c-Fos in areas related to appetite and glucose homeostasis control as PVN and DMH and to a lesser extent in the LHA and ARC. Central inhibition of GNPDA2 does not alter either acute food intake or body weight; however, GFAT inhibition diminished appetite and body weight due to visceral illness. In addition, central administration of the GNPDA2 antagonist, prior to an intraperitoneal glucose tolerance test, resulted in glucose intolerance in comparison to vehicle without altering insulin levels.Significance: These results suggest that central GNPDA2 does not control appetite, but regulates glucose homeostasis.

Glutamine deprivation triggers NAGK-dependent hexosamine salvage

Tumors frequently exhibit aberrant glycosylation, which can impact cancer progression and therapeutic responses. The hexosamine biosynthesis pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a major substrate for glycosylation in the cell. Prior studies have identified the HBP as a promising therapeutic target in pancreatic ductal adenocarcinoma (PDA). The HBP requires both glucose and glutamine for its initiation. The PDA tumor microenvironment is nutrient poor, however, prompting us to investigate how nutrient limitation impacts hexosamine synthesis. Here, we identify that glutamine limitation in PDA cells suppresses de novo hexosamine synthesis but results in increased free GlcNAc abundance. GlcNAc salvage via N-acetylglucosamine kinase (NAGK) is engaged to feed UDP-GlcNAc pools. NAGK expression is elevated in human PDA, and NAGK deletion from PDA cells impairs tumor growth in mice. Together, these data identify an important role for NAGK-dependent hexosamine salvage in supporting PDA tumor growth.

Oxidative phosphorylation safeguards pluripotency via UDP-N-acetylglucosamine

The roles of mitochondrial respiration in pluripotency remain largely unknown. We show here that mouse ESC mitochondria possess superior respiration capacity compared to somatic cell mitochondria, and oxidative phosphorylation (OXPHOS) generates the majority of cellular ATP in ESCs. Inhibition of OXPHOS results in extensive pluripotency and metabolic gene expression reprogram, leading to disruption of self-renewal and pluripotency. Metabolomics profiling identifies UDP-N-acetylglucosamine (UDP-GlcNAc) as one of the most significantly decreased metabolites in response to OXPHOS inhibition. The loss of ESC identity induced by OXPHOS inhibition can be ameliorated by directly adding GlcNAc both in vitro and in vivo. This work demonstrates that mitochondrial respiration, but not glycolysis, produces the majority of ATP in ESCs, and uncovers a novel mechanism whereby mitochondrial respiration is coupled with the hexosamine biosynthesis pathway to generate UDP-GlcNAc for ESC identity maintenance.

Glucose regulates expression of pro-inflammatory genes IL-1β and IL-12 through a mechanism involving hexosamine biosynthesis pathway dependent regulation of αEcatenin

High glucose levels are associated with changes in macrophage polarization and evidence indicates that the sustained or even short-term high glucose levels modulate inflammatory responses in macrophages. However, the mechanism by which macrophages can sense the changes in glucose levels are not clearly understood. We find that high glucose levels rapidly increase the α-E catenin protein level in RAW264.7 macrophages. We also find an attenuation of glucose induced increase of α-E catenin when hexosamine biosynthesis pathway is inhibited either with glutamine depletion or with the drugs azaserine and tunicamycin. This indicates the involvement of hexosamine biosynthesis pathway in this process. Then, we investigated the potential role of α-E catenin in glucose induced macrophage polarization. We find that the reduction of α-E catenin level using siRNA attenuates the glucose induced changes of both IL-1β and IL-12 mRNA levels under LPS stimulated condition but does not affect TNF-α expression. Together this indicates that α-E catenin can sense the changes in glucose levels in macrophages via hexosamine biosynthesis pathway and also can modulate the glucose induced gene expression of inflammatory markers such as IL-1β and IL-12. This identifies a new part of the mechanism by which macrophages are able to respond to changes in glucose levels.

Prognostic relevance of the hexosamine biosynthesis pathway activation in leiomyosarcoma

Metabolic reprogramming of tumor cells and the increase of glucose uptake is one of the hallmarks of cancer. In order to identify metabolic pathways activated in leiomyosarcoma (LMS), we analyzed transcriptomic profiles of distinct subtypes of LMS in several datasets. Primary, recurrent and metastatic tumors in the subtype 2 of LMS showed consistent enrichment of genes involved in hexosamine biosynthesis pathway (HBP). We demonstrated that glutamine-fructose-6-phosphate transaminase 2 (GFPT2), the rate-limiting enzyme in HBP, is expressed on protein level in a subset of LMS and the expression of this enzyme is frequently retained in patient-matched primary and metastatic tumors. In a new independent cohort of 327 patients, we showed that GFPT2 is associated with poor outcome of uterine LMS but not extra-uterine LMS. Based on the analysis of a small group of patients studied by 18F-FDG-PET imaging, we propose that strong expression of GFPT2 in primary LMS may be associated with high metabolic activity. Our data suggest that HBP is a potential new therapeutic target in one of the subtypes of LMS.

Glucose regulates expression of pro-inflammatory genes IL-1β and IL-12 through a mechanism involving hexosamine biosynthesis pathway dependent regulation of α-E catenin in the RAW 264.7 macrophage cell line

High glucose levels are associated with changes in macrophage polarization and evidence indicates that the sustained or even short-term high glucose levels modulate inflammatory responses in macrophages. However, the mechanism by which macrophages can sense the changes in glucose levels are not clearly understood. We find that high glucose levels rapidly increase the α-E catenin protein level in RAW264.7 macrophages. We also find an attenuation of glucose induced increase of α-E catenin when hexosamine biosynthesis pathway is inhibited either with glutamine depletion or with the drugs azaserine and tunicamycin. This indicates the involvement of hexosamine biosynthesis pathway in this process. Then, we investigated the potential role of α-E catenin in glucose induced macrophage polarization. We find that the reduction of α-E catenin level using siRNA attenuates the glucose induced change of IL-1β mRNA level under LPS stimulated condition. Further, we identified that the depletion of α-E catenin also decreases the IL-12 gene expression in basal glucose conditions leading to a reduction of glucose induced changes in IL-12. Together this indicates that α-E catenin can sense the changes in glucose levels in macrophages via hexosamine biosynthesis pathway and also can modulate the glucose induced gene expression of inflammatory markers such as IL-1-β and IL-12. This identifies a new part of the mechanism by which macrophages are able to respond to changes in glucose levels.

Evidence and manipulation of O-GlcNAcylation in granulosa cells of bovine antral follicles†

Glucose is a preferred energy substrate for metabolism by bovine granulosa cells (GCs). O-linked N-acetylglucosaminylation (O-GlcNAcylation), is a product of glucose metabolism that occurs as the hexosamine biosynthesis pathway (HBP) shunts O-GlcNAc sugars to serine and threonine residues of proteins. O-GlcNAcylation through the HBP is considered a nutrient sensing mechanism that regulates many cellular processes. Yet little is known of its importance in GCs. Here, O-GlcNAcylation in GCs and its effects on GC proliferation were determined. Bovine ovaries from a slaughterhouse, staged to the mid-to-late estrous period were used. Follicular fluid and GCs were aspirated from small (3–5 mm) and large (>10 mm) antral follicles. Freshly isolated GCs of small follicles exhibited greater expression of O-GlcNAcylation and O-GlcNAc transferase (OGT) than large follicles. Less glucose and more lactate was detectable in the follicular fluid of small versus large follicles. Culture of GCs revealed that inhibition of the HBP via the glutamine fructose-6-phosphate aminotransferase inhibitor, DON (50 μM), impaired O-GlcNAcylation and GC proliferation, regardless of follicle size. Direct inhibition of O-GlcNAcylation via the OGT inhibitor, OSMI-1 (50 μM), also prevented proliferation, but only in GCs of small follicles. Augmentation of O-GlcNAcylation via the O-GlcNAcase inhibitor, Thiamet-G (2.5 μM), had no effect on GC proliferation, regardless of follicle size. The results indicate GCs of bovine antral follicles undergo O-GlcNAcylation, and O-GlcNAcylation is associated with alterations of glucose and lactate in follicular fluid. Disruption of O-GlcNAcylation impairs GC proliferation. Thus, the HBP via O-GlcNAcylation constitutes a plausible nutrient-sensing pathway influencing bovine GC function and follicular growth.

Metabolomics analysis reveals metabolic changes associated with trans-resveratrol treatment in experimental cryptorchidism mice

This study aimed to analyse global metabolomic changes associated with trans-resveratrol (RSV) treatment in mice with cryptorchidism using untargeted metabolomics. Cryptorchidism was established surgically in Kunming mice, which were then treated with 20µg g–1 day–1, s.c., RSV for 35 consecutive days. Typical manifestations of spermatogenesis arrest were seen in mice with cryptorchidism, and RSV treatment for 35 days restored spermatogenesis. Liquid chromatography–tandem mass spectrometry was used to profile the metabolome of testes from mice in the control (non-cryptorchid, untreated), cryptorchid and RSV-treated cryptorchid groups. In all, 1386 and 179 differential metabolites were detected in the positive and negative modes respectively. Seven and six potential biomarkers were screened for spermatogenesis arrest and restoration respectively. Pathway analysis showed changes in 197 metabolic pathways. The hexosamine biosynthesis pathway was inhibited in the cryptorchid group, which probably resulted in a decrease in the end product, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Immunoblot analysis showed that total testicular protein O-linked β-N-acetylglucosamine glycosylation was related to spermatogenesis arrest, further indicating a decrease in UDP-GlcNAc in the cryptorchid group. Thus, untargeted metabolomics revealed the biochemical pathways associated with the restoration of metabolic status in the cryptorchid group following RSV treatment and the findings could be used to monitor the response to RSV treatment. This study provides a meaningful foundation for the future clinical application of RSV in the treatment of spermatogenesis dysfunction.