Kinetics of glucose-6-phosphate dehydrogenase (G6PD) activity during Plasmodium vivax infection: implications for early radical malaria treatment.

BACKGROUND: Plasmodium vivax relapses due to dormant liver hypnozoites can be prevented with primaquine. However, the dose must be adjusted in individuals with glucose-6-phosphate-dehydrogenase (G6PD) deficiency. In French Guiana, assessment of G6PD activity is typically delayed until day (D)14 to avoid the risk if misclassification. This study assessed the kinetics of G6PD activity throughout P. vivax infection to inform the timing of treatment. METHODS: For this retrospective monocentric study, data on G6PD activity between D1 and D28 after treatment initiation with chloroquine or artemisinin-based combination therapy were collected for patients followed at Cayenne Hospital, French Guiana, between January 2018 and December 2020. Patients were divided into three groups based on the number of available G6PD activity assessments: (i) at least two measurements during the P. vivax malaria infection; (ii) two measurements: one during the current infection and one previously; (iii) only one measurement during the malaria infection. RESULTS: In total, 210 patients were included (80, 20 and 110 in groups 1, 2 and 3, respectively). Data from group 1 showed that G6PD activity remained stable in each patient over time (D1, D3, D7, D14, D21, D28). None of the patients with normal G6PD activity during the initial phase (D1-D3) of the malaria episode (n = 44) was categorized as G6PD-deficient at D14. Patients with G6PD activity < 80% at D1 or D3 showed normal activity at D14. Sex and reticulocyte count were statistically associated with G6PD activity variation. In the whole sample (n = 210), no patient had severe G6PD deficiency (< 10%) and only three between 10 and 30%, giving a G6PD deficiency prevalence of 1.4%. Among the 100 patients from group 1 and 2, 30 patients (26.5%) were lost to follow-up before primaquine initiation. CONCLUSIONS: In patients treated for P. vivax infection, G6PD activity did not vary over time. Therefore, G6PD activity on D1 instead of D14 could be used for primaquine dose-adjustment. This could allow earlier radical treatment with primaquine, that could have a public health impact by decreasing early recurrences and patients lost to follow-up before primaquine initiation. This hypothesis needs to be confirmed in larger prospective studies.

Prolactin receptor potentiates chemotherapy through miRNAs-induced G6PD/TKT inhibition in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with a notoriously dismal prognosis. As a competitive inhibitor of DNA synthesis, gemcitabine is the cornerstone drug for treating PDAC at all stages. The therapeutic effect of gemcitabine, however, is often hindered by drug resistance, and the underlying mechanisms remain largely unknown. It is unclear whether their response to chemotherapeutics is regulated by endocrine regulators, despite the association between PDAC risk and endocrine deregulation. Here, we show that prolactin receptor (PRLR) synergizes with gemcitabine in both in vitro and in vivo treatment of PDAC. Interestingly, PRLR promotes the expression of miR-4763-3p and miR-3663-5p, two novel miRNAs whose functions are unknown. Furthermore, the analysis of transcriptome sequencing data of tumors from lactating mouse models enriches the PPP pathway, a multifunctional metabolic pathway. In addition to providing energy, the PPP pathway mainly provides a variety of raw materials for anabolism. We demonstrate that two key enzymes of the pentose phosphate pathway (PPP), G6PD and TKT, are directly targeted by miR-4763-3p and miR-3663-5p. Notably, miR-4763-3p and miR-3663-5p diminish the nucleotide synthesis of the PPP pathway, thereby increasing gemcitabine sensitivity. As a result, PRLR harnesses these two miRNAs to suppress PPP and nucleotide synthesis, subsequently elevating the gemcitabine sensitivity of PDAC cells. Also, PDAC tissues and tumors from LSL-KrasG12D/+, LSL-Trp53R172H/+, and PDX1-cre (KPC) mice exhibit downregulation of PRLR. Bisulfite sequencing of PDAC tissues revealed that PRLR downregulation is due to epigenetic methylation. In this study, we show for the first time that the endocrine receptor PRLR improves the effects of gemcitabine by boosting two new miRNAs that block the PPP pathway and nucleotide synthesis by inhibiting two essential enzymes concurrently. The PRLR-miRNAs-PPP axis may serve as a possible therapeutic target to supplement chemotherapy advantages in PDAC.

The PreQuine Platform: A novel diagnostic tool for measuring glucose-6-phosphate dehydrogenase (G6PD) activity and hemoglobin concentration.

Quantitative diagnosis of glucose-6-phosphate dehydrogenase (G6PD) deficiency is essential for the safe administration of 8-aminoquinoline based radical cure for the treatment of Plasmodium vivax infections. Here, we present the PreQuine Platform (IVDS, USA), a quantitative biosensor that uses a dual-analyte assay for the simultaneous measurement of Hemoglobin (Hgb) levels and G6PD enzyme activity within the same sample. The platform relies on a downloadable mobile application. The device requires 10µl of whole blood and works with a reflectance-based meter. Comparing the G6PD measurement normalized by Hgb of 12 samples from the PreQuine Platform with reference measurements methods (spectrophotometry, Pointe Scientific, USA and hemoglobin meter, HemoCue, Sweden) showed a positive and significant agreement with a slope of 1.0091 and an intercept of -0.0379 under laboratory conditions. Next steps will be to conduct field trials in Bangladesh, Cambodia, and the USA to assess diagnostic performance, user friendliness and acceptance.

G6PD maintains the VSMC synthetic phenotype and accelerates vascular neointimal hyperplasia by inhibiting the VDAC1-Bax-mediated mitochondrial apoptosis pathway.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) plays an important role in vascular smooth muscle cell (VSMC) phenotypic switching, which is an early pathogenic event in various vascular remodeling diseases (VRDs). However, the underlying mechanism is not fully understood. METHODS: An IP‒LC‒MS/MS assay was conducted to identify new binding partners of G6PD involved in the regulation of VSMC phenotypic switching under platelet-derived growth factor-BB (PDGF-BB) stimulation. Co-IP, GST pull-down, and immunofluorescence colocalization were employed to clarify the interaction between G6PD and voltage-dependent anion-selective channel protein 1 (VDAC1). The molecular mechanisms involved were elucidated by examining the interaction between VDAC1 and apoptosis-related biomarkers, as well as the oligomerization state of VDAC1. RESULTS: The G6PD level was significantly elevated and positively correlated with the synthetic characteristics of VSMCs induced by PDGF-BB. We identified VDAC1 as a novel G6PD-interacting molecule essential for apoptosis. Specifically, the G6PD-NTD region was found to predominantly contribute to this interaction. G6PD promotes VSMC survival and accelerates vascular neointimal hyperplasia by inhibiting VSMC apoptosis. Mechanistically, G6PD interacts with VDAC1 upon stimulation with PDGF-BB. By competing with Bax for VDAC1 binding, G6PD reduces VDAC1 oligomerization and counteracts VDAC1-Bax-mediated apoptosis, thereby accelerating neointimal hyperplasia. CONCLUSION: Our study showed that the G6PD-VDAC1-Bax axis is a vital switch in VSMC apoptosis and is essential for VSMC phenotypic switching and neointimal hyperplasia, providing mechanistic insight into early VRDs.

Field evaluation of a novel semi-quantitative point-of-care diagnostic for G6PD deficiency in Indonesia.

BACKGROUND: The WHO recommends routine testing of G6PD activity to guide radical cure in patients with Plasmodium vivax malaria. Females may have intermediate G6PD enzyme activity and to date, only complex diagnostics are able to reliably identify them. The semi-quantitative G6PD diagnostic "One Step G6PD Test" (Humasis, RoK; "RDT") is a lateral flow assay that can distinguish deficient, intermediate, and normal G6PD status and offers a simpler diagnostic alternative. METHODS: G6PD status of participants enrolled in Malinau and Nunukan Regencies and the capital Jakarta was assessed with the RDT, and G6PD activity was measured in duplicate by reference spectrophotometry. The adjusted male median (AMM) of the spectrophotometry measurements was defined as 100% activity; 70% and 30% of the AMM were defined as thresholds for intermediate and deficient G6PD status, respectively. Results were compared to those derived from spectrophotometry at the clinically relevant G6PD activity thresholds of 30% and 70%. RESULTS: Of the 161 participants enrolled, 10 (6.2%) were G6PD deficient and 12 (7.5%) had intermediate G6PD activity by spectrophotometry. At the 30% threshold, the sensitivity of the RDT was 10.0% (95%CI: 0.3-44.5%) with a specificity of 99.3% (95%CI: 96.4-100.0%); the positive predictive value was 50.0% (95%CI: 1.3-98.7%) and the negative predictive value 94.3% (95%CI: 89.5-97.4%). The corresponding figures at the 70% threshold were 22.7% (95%CI: 7.8-45.4%), 100.0% (95%CI: 97.4-100.0%), 100.0% (95%CI: 47.8-100.0%) and 89.1% (95%CI: 83.1-93.5%), respectively. CONCLUSION: While there is a dire need for an easy-to-use, economical, semi-quantitative diagnostic for the point of care, the observed performance of the "One Step G6PD Test" in its current form was insufficient to guide antimalarial treatment.

Associations between COVID-19 and Glucose-6-Phosphate Dehydrogenase Activity in Brazil.

Glucose-6 phosphate dehydrogenase deficiency (G6PDd) was suggested as a risk factor for severe disease in patients with COVID-19. We evaluated clinical outcomes and glucose-6 phosphate dehydrogenase (G6PD) activity during and after illness in patients with COVID-19. This prospective cohort study included adult participants (≥ 18 years old) who had clinical and/or radiological COVID-19 findings or positive reverse transcription-polymerase chain reaction results. Epidemiological and clinical data were extracted from electronic medical records. Glucose-6 phosphate dehydrogenase activity was measured using SD Biosensor STANDARD G6PD® equipment on admission and 1 year after discharge. Samples were genotyped for the three most common single nucleotide polymorphisms for G6PDd in the Brazilian Amazon. Seven hundred fifty-three patients were included, of whom 123 (16.3%) were G6PD deficient. There was no difference between groups regarding the risks of hospitalization (P = 0.740) or invasive mechanical ventilation (P = 0.31), but the risk of death was greater in patients with normal G6PD levels (P = 0.022). Only 29 of 116 participants (25%) carried the African G6PDd genotype. Of 30 participants tested as G6PD deficient during disease, only 11 (36.7%) results agreed 1 year after discharge. In conclusion, this study does not demonstrate an association of G6PDd with severity of COVID-19. Limitations of the test for detecting enzyme levels during COVID-19 illness were demonstrated by genotyping and retesting after the disease period. Care must be taken when screening for G6PDd in patients with acute COVID-19.

Human papillomavirus-16 E6 activates the pentose phosphate pathway to promote cervical cancer cell proliferation by inhibiting G6PD lactylation.

High-risk human papillomaviruses (HPVs) are the causative agents of cervical cancer. Here, we report that HPV16 E6E7 promotes cervical cancer cell proliferation by activating the pentose phosphate pathway (PPP). We found that HPV16 E6 activates the PPP primarily by increasing glucose-6-phosphate dehydrogenase (G6PD) enzyme activity. Mechanistically, HPV16 E6 promoted G6PD dimer formation by inhibiting its lactylation. Importantly, we suggest that G6PD K45 was lactylated during G6PD-mediated antioxidant stress. In primary human keratinocytes and an HPV-negative cervical cancer C33A cells line ectopically expressing HPV16 E6, the transduction of G6PD K45A (unable to be lactylated) increased GSH and NADPH levels and, correspondingly, decreasing ROS levels. Conversely, the re-expression of G6PD K45T (mimicking constitutive lactylation) in HPV16-positive SiHa cells line inhibited cell proliferation. In vivo, the inhibition of G6PD enzyme activity with 6-aminonicotinamide (6-An) or the re-expression of G6PD K45T inhibited tumor proliferation. In conclusion, we have revealed a novel mechanism of HPV oncoprotein-mediated malignant transformation. These findings might provide effective strategies for treating cervical and HPV-associated cancers.

Enabling High Activation of Glucose-6-Phosphate Dehydrogenase Activity Through Liquid Condensate Formation and Compression.

Droplet formation via liquid-liquid phase separation is thought to be involved in the regulation of various biological processes, including enzymatic reactions. We investigated a glycolytic enzymatic reaction, the conversion of glucose-6-phosphate to 6-phospho-D-glucono-1,5-lactone with concomitant reduction of NADP+ to NADPH both in the absence and presence of dynamically controlled liquid droplet formation. Here, the nucleotide serves as substrate as well as the scaffold required for the formation of liquid droplets. To further expand the process parameter space, temperature and pressure dependent measurements were performed. Incorporation of the reactants in the liquid droplet phase led to a boost in enzymatic activity, which was most pronounced at medium-high pressures. The crowded environment of the droplet phase induced a marked increase of the affinity of the enzyme and substrate. An increase in turnover number in the droplet phase at high pressure contributed to a further strong increase in catalytic efficiency. Enzyme systems that are dynamically coupled to liquid condensate formation may be the key to deciphering many biochemical reactions. Expanding the process parameter space by adjusting temperature and pressure conditions can be a means to further increase the efficiency of industrial enzyme utilization and help uncover regulatory mechanisms adopted by extremophiles.

Functional effects of an African glucose-6-phosphate dehydrogenase (G6PD) polymorphism (Val68Met) on red blood cell hemolytic propensity and post-transfusion recovery.

BACKGROUND: Donor genetic variation is associated with red blood cell (RBC) storage integrity and post-transfusion recovery. Our previous large-scale genome-wide association study demonstrated that the African G6PD deficient A- variant (rs1050828, Val68Met) is associated with higher oxidative hemolysis after cold storage. Despite a high prevalence of X-linked G6PD mutation in African American population (>10%), blood donors are not routinely screened for G6PD status and its importance in transfusion medicine is relatively understudied. STUDY DESIGN AND METHODS: To further evaluate the functional effects of the G6PD A- mutation, we created a novel mouse model carrying this genetic variant using CRISPR-Cas9. We hypothesize that this humanized G6PD A- variant is associated with reduced G6PD activity with a consequent effect on RBC hemolytic propensity and post-transfusion recovery. RESULTS: G6PD A- RBCs had reduced G6PD protein with ~5% residual enzymatic activity. Significantly increased in vitro hemolysis induced by oxidative stressors was observed in fresh and stored G6PD A- RBCs, along with a lower GSH:GSSG ratio. However, no differences were observed in storage hemolysis, osmotic fragility, mechanical fragility, reticulocytes, and post-transfusion recovery. Interestingly, a 14% reduction of 24-h survival following irradiation was observed in G6PD A- RBCs compared to WT RBCs. Metabolomic assessment of stored G6PD A- RBCs revealed an impaired pentose phosphate pathway (PPP) with increased glycolytic flux, decreasing cellular antioxidant capacity. DISCUSSION: This novel mouse model of the common G6PD A- variant has impaired antioxidant capacity like humans and low G6PD activity may reduce survival of transfused RBCs when irradiation is performed.

Glucose-6-Phosphatase-Dehydrogenase activity as modulative association between Parkinson's disease and periodontitis.

The association between periodontitis (PD) and Parkinson's disease (PK) is discussed due to the inflammatory component of neurodegenerative processes. PK severity and affected areas were determined using the following neuropsychological tests: Unified Parkinson's Disease Rating Score (UPDRS) and Hoehn and Yahr; non-motoric symptoms by Non-Motor Symptoms Scale (NMSS), and cognitive involvement by Mini-Mental State Examination (MMSE). Neuroinflammation and the resulting Glucose-6-Phosphatase-Dehydrogenase (G6PD) dysfunction are part of the pathophysiology of PK. This study aimed to evaluate these associations in periodontal inflammation. Clinical data and saliva-, serum-, and RNA-biobank samples of 50 well-characterized diametric patients with PK and five age- and sex-matched neurologically healthy participants were analyzed for G6PD function, periodontal pathogens (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Prevotella intermedia, Campylobacter rectus, Fusobacterium nucleatum, and Filifactor alocis), monocyte chemoattractant protein (MCP) 1, and interleukin (IL) 1-beta. Regression analysis was used to identify associations between clinical and behavioral data, and t-tests were used to compare health and disease. Compared with PK, no pathogens and lower inflammatory markers (p < 0.001) were detectible in healthy saliva and serum, PK-severity/UPDRS interrelated with the occurrence of Prevotella intermedia in serum as well as IL1-beta levels in serum and saliva (p = 0.006, 0.019, 0.034), Hoehn and Yahr correlated with Porphyromonas gingivalis, Prevotella intermedia, RNA IL1-beta regulation, serum, and saliva IL1-beta levels, with p-values of 0.038, 0.011, 0.008, <0.001, and 0.010, while MMSE was associated with Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, serum MCP 1 levels, RNA IL1-beta regulation and G6PD serum activity (p = 0.036, 0.003, 0.045, <0.001, and 0.021). Cognitive and motor skills seem to be important as representative tests are associated with periodontal pathogens and oral/general inflammation, wherein G6PD-saliva dysfunction might be involved. Clinical trial registration: https://www.bfarm.de/DE/Das-BfArM/Aufgaben/Deutsches-Register-Klinischer-Studien/_node.html, identifier DRKS00005388.

The Role of Glucose-6-phosphate Dehydrogenase in the Wine Yeast Hanseniaspora uvarum.

Hanseniaspora uvarum is the predominant yeast species in the majority of wine fermentations, which has only recently become amenable to directed genetic manipulation. The genetics and metabolism of H. uvarum have been poorly studied as compared to other yeasts of biotechnological importance. This work describes the construction and characterization of homozygous deletion mutants in the HuZWF1 gene, encoding glucose-6-phosphate dehydrogenase (G6PDH), which provides the entrance into the oxidative part of the pentose phosphate pathway (PPP) and serves as a major source of NADPH for anabolic reactions and oxidative stress response. Huzwf1 deletion mutants grow more slowly on glucose medium than wild-type and are hypersensitive both to hydrogen peroxide and potassium bisulfite, indicating that G6PDH activity is required to cope with these stresses. The mutant also requires methionine for growth. Enzyme activity can be restored by the expression of heterologous G6PDH genes from other yeasts and humans under the control of a strong endogenous promoter. These findings provide the basis for a better adaptation of H. uvarum to conditions used in wine fermentations, as well as its use for other biotechnological purposes and as an expression organism for studying G6PDH functions in patients with hemolytic anemia.

Analysis of the Effects of Pentose Phosphate Pathway Inhibition on the Generation of Reactive Oxygen Species and Epileptiform Activity in Hippocampal Slices.

The pentose phosphate pathway (PPP) is one of three major pathways involved in glucose metabolism, which is regulated by glucose-6-phosphate dehydrogenase (G6PD) controls NADPH formation. NADPH, in turn, regulates the balance of oxidative stress and reactive oxygen species (ROS) levels. G6PD dysfunction, affecting the PPP, is implicated in neurological disorders, including epilepsy. However, PPP's role in epileptogenesis and ROS production during epileptic activity remains unclear. To clarify these points, we conducted electrophysiological and imaging analyses on mouse hippocampal brain slices. Using the specific G6PD inhibitor G6PDi-1, we assessed its effects on mouse hippocampal slices, examining intracellular ROS, glucose/oxygen consumption, the NAD(P)H level and ROS production during synaptic stimulation and in the 4AP epilepsy model. G6PDi-1 increased basal intracellular ROS levels and reduced synaptically induced glucose consumption but had no impact on baselevel of NAD(P)H and ROS production from synaptic stimulation. In the 4AP model, G6PDi-1 did not significantly alter spontaneous seizure frequency or H2O2 release amplitude but increased the frequency and peak amplitude of interictal events. These findings suggest that short-term PPP inhibition has a minimal impact on synaptic circuit activity.

Metabolic remodeling of visceral and subcutaneous white adipose tissue during reacclimation of rats after cold.

Deciphering lipid metabolism in white adipose tissue (WAT) depots during weight gain is important to understand the heterogeneity of WAT and its roles in obesity. Here, we examined the expression of key enzymes of lipid metabolism and changes in the morphology of representative visceral (epididymal) and subcutaneous (inguinal) WAT (eWAT and iWAT, respectively)-in adult male rats acclimated to cold (4 ± 1 °C) for 45 days and reacclimated to room temperature (RT, 22 ± 1 °C) for 1, 3, 7, 12, 21, or 45 days. The relative mass of both depots decreased to a similar extent after cold acclimation. However, fatty acid synthase (FAS), glucose-6-phosphate dehydrogenase (G6PDH), and medium-chain acyl-CoA dehydrogenase (ACADM) protein level increased only in eWAT, whereas adipose triglyceride lipase (ATGL) expression increased only in iWAT. During reacclimation, the relative mass of eWAT reached control values on day 12 and that of iWAT on day 45 of reacclimation. The faster recovery of eWAT mass is associated with higher expression of FAS, acetyl-CoA carboxylase (ACC), G6PDH, and ACADM during reacclimation and a delayed increase in ATGL. The absence of an increase in proliferating cell nuclear antigen suggests that the observed depot-specific mass increase is predominantly due to metabolic adjustments. In summary, this study shows a differential rate of visceral and subcutaneous adipose tissue weight regain during post-cold reacclimation of rats at RT. Faster recovery of the visceral WAT as compared to subcutaneous WAT during reacclimation at RT could be attributed to observed differences in the expression patterns of lipid metabolic enzymes.

Butyrate induces oxidative burst mediated apoptosis via Glucose-6-Phosphate Dehydrogenase (G6PDH) in macrophages during mycobacterial infection.

Microorganisms present in the gut modulate host defence responses against infections in order to maintain immune homeostasis. This host-microbe crosstalk is regulated by gut metabolites. Butyrate is one such small chain fatty acid produced by gut microbes upon fermentation that has the potential to influence immune responses. Here we investigated the role of butyrate in macrophages during mycobacterial infection. Results demonstrate that butyrate significantly suppresses the growth kinetics of mycobacteria in culture medium as well as inhibits mycobacterial survival inside macrophages. Interestingly, butyrate alters the pentose phosphate pathway by inducing higher expression of Glucose-6-Phosphate Dehydrogenase (G6PDH) resulting in a higher oxidative burst via decreased Sod-2 and increased Nox-2 (NADPH oxidase-2) expression. Butyrate-induced G6PDH also mediated a decrease in mitochondrial membrane potential. This in turn lead to an induction of apoptosis as measured by lower expression of the anti-apoptotic protein Bcl-2 and a higher release of Cytochrome C as a result of induction of apoptosis. These results indicate that butyrate alters the metabolic status of macrophages and induces protective immune responses against mycobacterial infection.

Substitution of arginine 219 by glycine compromises stability, dimerization, and catalytic activity in a G6PD mutant.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common enzymopathies in humans, present in approximately half a billion people worldwide. More than 230 clinically relevant G6PD mutations of different classes have been reported to date. We hereby describe a patient with chronic hemolysis who presents a substitution of arginine by glycine at position 219 in G6PD protein. The variant was never described in an original publication or characterized on a molecular level. In the present study, we provide structural and biochemical evidence for the molecular basis of its pathogenicity. When compared to the wild-type enzyme, the Arg219Gly mutation markedly reduces the catalytic activity by 50-fold while having a negligible effect on substrate binding affinity. The mutation preserves secondary protein structure, but greatly decreases stability at higher temperatures and to trypsin digestion. Size exclusion chromatography elution profiles show monomeric and dimeric forms for the mutant, but only the latter for the wild-type form, suggesting a critical role of arginine 219 in G6PD dimer formation. Our findings have implications in the development of small molecule activators, with the goal of rescuing the phenotype observed in this and possibly other related mutants.

The Emerging Roles of the Metabolic Regulator G6PD in Human Cancers.

Metabolic reprogramming, especially reprogrammed glucose metabolism, is a well-known cancer hallmark related to various characteristics of tumor cells, including proliferation, survival, metastasis, and drug resistance. Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the pentose phosphate pathway (PPP), a branch of glycolysis, that converts glucose-6-phosphate (G6P) into 6-phosphogluconolactone (6PGL). Furthermore, PPP produces ribose-5-phosphate (R5P), which provides sugar-phosphate backbones for nucleotide synthesis as well as nicotinamide adenine dinucleotide phosphate (NADPH), an important cellular reductant. Several studies have shown enhanced G6PD expression and PPP flux in various tumor cells, as well as their correlation with tumor progression through cancer hallmark regulation, especially reprogramming cellular metabolism, sustaining proliferative signaling, resisting cell death, and activating invasion and metastasis. Inhibiting G6PD could suppress tumor cell proliferation, promote cell death, reverse chemoresistance, and inhibit metastasis, suggesting the potential of G6PD as a target for anti-tumor therapeutic strategies. Indeed, while challenges-including side effects-still remain, small-molecule G6PD inhibitors showing potential anti-tumor effect either when used alone or in combination with other anti-tumor drugs have been developed. This review provides an overview of the structural significance of G6PD, its role in and regulation of tumor development and progression, and the strategies explored in relation to G6PD-targeted therapy.

Molecular characterization of G6PD mutations identifies new mutations and a high frequency of intronic variants in Thai females.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked enzymopathy caused by mutations in the G6PD gene. A medical concern associated with G6PD deficiency is acute hemolytic anemia induced by certain foods, drugs, and infections. Although phenotypic tests can correctly identify hemizygous males, as well as homozygous and compound heterozygous females, heterozygous females with a wide range of G6PD activity may be misclassified as normal. This study aimed to develop multiplex high-resolution melting (HRM) analyses to enable the accurate detection of G6PD mutations, especially among females with heterozygous deficiency. Multiplex HRM assays were developed to detect six G6PD variants, i.e., G6PD Gaohe (c.95A>G), G6PD Chinese-4 (c.392G>T), G6PD Mahidol (c.487G>A), G6PD Viangchan (c.871G>A), G6PD Chinese-5 (c.1024C>T), and G6PD Union (c.1360C>T) in two reactions. The assays were validated and then applied to genotype G6PD mutations in 248 Thai females. The sensitivity of the HRM assays developed was 100% [95% confidence interval (CI): 94.40%-100%] with a specificity of 100% (95% CI: 88.78%-100%) for detecting these six mutations. The prevalence of G6PD deficiency was estimated as 3.63% (9/248) for G6PD deficiency and 31.05% (77/248) for intermediate deficiency by phenotypic assay. The developed HRM assays identified three participants with normal enzyme activity as heterozygous for G6PD Viangchan. Interestingly, a deletion in intron 5 nucleotide position 637/638 (c.486-34delT) was also detected by the developed HRM assays. G6PD genotyping revealed a total of 12 G6PD genotypes, with a high prevalence of intronic variants. Our results suggested that HRM analysis-based genotyping is a simple and reliable approach for detecting G6PD mutations, and could be used to prevent the misdiagnosis of heterozygous females by phenotypic assay. This study also sheds light on the possibility of overlooking intronic variants, which could affect G6PD expression and contribute to enzyme deficiency.

PBX3 promotes pentose phosphate pathway and colorectal cancer progression by enhancing G6PD expression.

Metabolic reprogramming is a hallmark of cancers crucial for fulfilling the needs of energy, building blocks, and antioxidants to support tumor cells' rapid proliferation and to cope with the harsh microenvironment. Pre-B-cell leukemia transcription factor 3 (PBX3) is a member of the PBX family whose expression is up-regulated in various tumors, however, whether it is involved in tumor cell metabolic reprogramming remains unclear. Herein, we report that PBX3 is a positive regulator of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway (PPP). PBX3 promoted G6PD transcriptional activity in tumor cells by binding directly to its promoter, leading to PPP stimulation and enhancing the production of nucleotides and NADPH, a crucial reductant, thereby promoting nucleic acid and lipid biosynthesis while decreasing intracellular reactive oxygen species levels. The PBX3/G6PD axis also promoted tumorigenic potential in vitro and in vivo. Collectively, these findings reveal a novel function of PBX3 as a regulator of G6PD, linking its oncogenic activity with tumor cell metabolic reprogramming, especially PPP. Furthermore, our results suggested that PBX3 is a potential target for metabolic-based anti-tumor therapeutic strategies.

Musashi-2 (MSI2) promotes neuroblastoma tumorigenesis through targeting MYC-mediated glucose-6-phosphate dehydrogenase (G6PD) transcriptional activation.

Neuroblastoma (NB) is the deadliest pediatric solid tumor due to its rapid proliferation. Aberrant expression of MYCN is deemed as the most remarkable feature for the predictive hallmark of NB progression and recurrence. However, the phenomenon that only detection of MYCN in the nearly 20% of NB patients hints that there should be other vital oncogenes in the progression of NB. Here, we firstly show that MSI2 mRNA is augmented by analyzing public GEO datasets in the malignant stage according to International Neuroblastoma Staging System (INSS) stages. Although accumulating evidences uncover the emerging roles of MSI2 in several cancers, the regulatory functions and underlying mechanisms of MSI2 in NB remain under-investigated. Herein, we identified that high-expressed MSI2 and low-expressed n-Myc group account for 43.1% of total NB clinical samples (n = 65). Meanwhile, MSI2 expression is profoundly upregulated along with NB malignancy and negatively associated with the survival outcome of NB patients in the NB tissue microarray (NB: n = 65; Ganglioneuroblastoma: n = 31; Ganglioneuroma: n = 27). In vitro, our results revealed that MSI2 promoted migration, invasion, and proliferation of NB cells via enhancing pentose phosphate pathway. Mechanistically, MSI2 upregulated the key enzyme glucose-6-phosphate dehydrogenase (G6PD) via directly binding to 3'-untranslated regions of c-Myc mRNA to facilitate its stability, resulting in enhancing pentose phosphate pathway. Our findings reveal that MSI2 promotes pentose phosphate pathway via activating c-Myc-G6PD signaling, suggesting that MSI2 exhibits a novel and powerful target for the diagnosis and treatment of NB.

Glucose-6-phosphate dehydrogenase: a therapeutic target for ovarian cancer.

INTRODUCTION: Ovarian cancer (OC) is a gynecological tumor disease, which is usually diagnosed at an advanced stage and has a poor prognosis. It has been established that the glucose metabolism rate of cancer cells is significantly higher than that of normal cells, and the pentose phosphate pathway (PPP) is an important branch pathway for glucose metabolism. Glucose-6-phosphate dehydrogenase (G6PD) is the key rate-limiting enzyme in the PPP, which plays an important role in the initiation and development of cancer (such as OC), and has been considered as a promisinganti-cancer target. AREAS COVERED: In this review, based on the structure and biological function of G6PD, recent research on the roles of G6PD in the progression, metastasis, and chemoresistance of OC are summarized and accompanied by proposed molecular mechanisms, which may provide a systematic understanding of targeting G6PD for the treatment of patients with OC. EXPERT OPINION: Accumulating evidence demonstrates that G6PD is a promising target of cancer. The development of G6PD inhibitors for cancer treatment merits broad application prospects.