Glycolytic Enzyme Enolase Research Articles

Abstract C029: Surface localization and targeting of enolase-1 (ENO1) as a novel theranostic approach for docetaxel-resistant neuroendocrine prostate cancer

Abstract Prostate cancer (PCa) is American men's second most common cancer. This cancer exhibits the most significant racial disparities among all cancer types, with elevated incidence and mortality in men of African ancestry. Although taxane-based chemotherapy with docetaxel (DTX) and cabazitaxel is the last line of defense for metastatic Castration Resistant Prostate Cancer (mCRPC), it invariably fails due to the development of chemoresistance. The protein-specific membrane antigen (PSMA) is currently an effective target for the imaging and therapy of mCRPC. However, although PSMA radioligand therapy (PSMA-RLT) is a theranostic option for men with advanced PCa, about 30% have a limited response to this treatment, particularly those that lack PSMA. In addition, some reports indicate that PSMA is suppressed in neuroendocrine PCa (NEPC), making it an unreliable theranostic target for this aggressive subtype. The glycolytic enzyme Enolase (ENO) is a promising theranostic alternative due to its cell surface localization in many human tumors. We hypothesized that ENO1 is abundant on the surface of NEPC-like cell lines and can be targeted with small molecule inhibitors (SMIs) that could potentially be used as theranostic agents. Western blotting (WB) analysis revealed that DTX-sensitive PCa cell lines expressing NEPC markers express both ENO1 and ENO2. By contrast, DTX-resistant NEPC-like cells only express ENO1 and therefore have a metabolic vulnerability due to the loss of ENO2. Additionally, we observed by WB and confocal microscopy changes in the expression and localization of ENO1 in NEPC-like cell lines under different glucose concentrations. Our results showed that under high glucose conditions, found on metabolically active metastatic tumors, ENO1 is highly abundant on the cell surface, making it a promising candidate target for theranostics. However, standard glucose conditions resulted in ENO1 downregulation, most likely due to inhibition of the activity of the c-MYC oncogene and upregulation of MBP1, the small splice variant of ENO1 that blocks the transcriptional activity of c-MYC. We also evaluated the cytotoxicity of SMIs designed to target ENO1 in chemoresistant NEPC-like cell lines using MTT viability assays, clonogenic assays, and Hoffman Modulation microscopy imaging. We observed that these inhibitors can significantly impair, with IC50 values at low micromolar concentrations, the proliferation and clonogenicity of NEPC cell lines. These results support our long-term goal to identify an alternative theranostic option for patients with NEPC by establishing ENO1 as a novel theranostic target. Citation Format: Krystal R. Santiago Torres, Kristen Whitley, Alfonso Duran, Bhaskar Das, Carlos Casiano, Frankis Almaguel. Surface localization and targeting of enolase-1 (ENO1) as a novel theranostic approach for docetaxel-resistant neuroendocrine prostate cancer [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C029.

Interference with ENO2 promotes ferroptosis and inhibits glycolysis in clear cell renal cell carcinoma by regulating Hippo‑YAP1 signaling.

Glycolytic enzyme enolase 2 (ENO2) is dysregulated in various cancer types. Nevertheless, the role and underlying mechanism of ENO2 in clear cell renal cell carcinoma (ccRCC) remain unclear. Therefore, the current study investigated the effect and mechanism of ENO2 in ccRCC. ENO2 expression in a ccRCC cell line was assessed using reverse transcription-quantitative PCR and western blotting. Analysis of glycolysis was performed by estimating the extracellular acidification rate, lactic acid concentration, glucose uptake and the expression of glucose transporter 1, pyruvate kinase muscle isozyme M2 and hexokinase 2. Moreover, ferroptosis was assessed by detecting the level of total iron, lipid peroxide, reactive oxygen species and the expression of ferroptosis-related protein. In addition, mitochondrial function was assessed using JC-1 staining and detection kits. The results indicated that ENO2 is expressed at high levels in ccRCC cell lines, and interference with ENO2 expression inhibits glycolysis, promotes ferroptosis and affects mitochondrial function in ccRCC cells. Further investigation demonstrated that interference with ENO2 expression affected ferroptosis levels in ccRCC cells by inhibiting the glycolysis process. Mechanistically, the present results indicated that ENO2 may affect ferroptosis, glycolysis and mitochondrial functions by regulating Hippo-yes-associated protein 1 (YAP1) signaling in ccRCC cells. In conclusion, the present study showed that ENO2 affects ferroptosis, glycolysis and mitochondrial functions in ccRCC cells by regulating Hippo-YAP1 signaling, hence demonstrating its potential as a therapeutic target in ccRCC.

KRAS-mutant non-small cell lung cancer (NSCLC) therapy based on tepotinib and omeprazole combination

BackgroundKRAS-mutant non-small cell lung cancer (NSCLC) shows a relatively low response rate to chemotherapy, immunotherapy and KRAS-G12C selective inhibitors, leading to short median progression-free survival, and overall survival. The MET receptor tyrosine kinase (c-MET), the cognate receptor of hepatocyte growth factor (HGF), was reported to be overexpressed in KRAS-mutant lung cancer cells leading to tumor-growth in anchorage-independent conditions.MethodsCell viability assay and synergy analysis were carried out in native, sotorasib and trametinib-resistant KRAS-mutant NSCLC cell lines. Colony formation assays and Western blot analysis were also performed. RNA isolation from tumors of KRAS-mutant NSCLC patients was performed and KRAS and MET mRNA expression was determined by real-time RT-qPCR. In vivo studies were conducted in NSCLC (NCI-H358) cell-derived tumor xenograft model.ResultsOur research has shown promising activity of omeprazole, a V-ATPase-driven proton pump inhibitor with potential anti-cancer properties, in combination with the MET inhibitor tepotinib in KRAS-mutant G12C and non-G12C NSCLC cell lines, as well as in G12C inhibitor (AMG510, sotorasib) and MEK inhibitor (trametinib)-resistant cell lines. Moreover, in a xenograft mouse model, combination of omeprazole plus tepotinib caused tumor growth regression. We observed that the combination of these two drugs downregulates phosphorylation of the glycolytic enzyme enolase 1 (ENO1) and the low-density lipoprotein receptor-related protein (LRP) 5/6 in the H358 KRAS G12C cell line, but not in the H358 sotorasib resistant, indicating that the effect of the combination could be independent of ENO1. In addition, we examined the probability of recurrence-free survival and overall survival in 40 early lung adenocarcinoma patients with KRAS G12C mutation stratified by KRAS and MET mRNA levels. Significant differences were observed in recurrence-free survival according to high levels of KRAS mRNA expression. Hazard ratio (HR) of recurrence-free survival was 7.291 (p = 0.014) for high levels of KRAS mRNA expression and 3.742 (p = 0.052) for high MET mRNA expression.ConclusionsWe posit that the combination of the V-ATPase inhibitor omeprazole plus tepotinib warrants further assessment in KRAS-mutant G12C and non G12C cell lines, including those resistant to the covalent KRAS G12C inhibitors.

BCAT proteins: A metabolic link between Type II Diabetes and Alzheimer’s Disease

AbstractBackgroundAberrant energy pathways compounded by dysregulated cellular redox plays a pivotal role in the pathogenesis of Alzheimer’s disease (AD) but the underlying mechanisms are not clear. Clinical and epidemiological data show that those with Type II Diabetes Mellitus (T2DM) have a 65% increased risk of developing AD (Arvanitakis et al., 2004). Our group and others have shown that serum branched chain amino acids and the branched chain aminotransferase proteins (BCAT) are increased in subjects with AD and T2DM, which has been linked with dysregulated autophagy and metabolic reprogramming. Here, we explore how changes in cellular redox regulate and influence metabolic‐related pathology changes associated with AD.MethodsSHSY‐5Y cells were used as a neuronal model to observe the effects of BCATc dysregulation in neuronal metabolism. Cells were transfected with our BCATc overexpression constructs and metabolite load assessed using LC‐MS/MS. Activity of glycolytic enzymes GAPDH and enolase were assessed, and overall glycolytic rate characterised using a Seahorse XF analyser. Oxidative stress was measured using the HyPer‐7 molecular probe (Bousolov et al, 2004) and cells were visualised using confocal microscopy. Finally, Western blot analysis was performed to assess levels of BCATc in the presence of increasing glucose (+/‐ 100 nM insulin) and hydrogen peroxide treatments and to demonstrate changes in levels of β‐amyloid and hyperphosphorylated tau.ResultsHere we show that BCATc overexpression increases the level of glucose and fructose, whilst decreasing GAPDH, enolase activity and glycolytic rate (p < 0.05). There was an observable increase in hydrogen peroxide generation (p<0.001), while levels of amyloid precursor protein (P<0.01), β‐amyloid (p>0.05) and hyperphosphorylated tau (p<0.05) were significantly increased. In addition, levels of BCATc increased dose‐dependently following hydrogen peroxide treatments (p < 0.001) and decreased in the presence of excess glucose (+ 100 nM insulin) (p<0.05).ConclusionOur results demonstrate that the reprogramming of BCAT as a result of hallmarks of T2DM such as excess dietary‐derived nutrient signals and a concurrent increase in oxidative stress, are associated with aberrant processing and aggregation of β‐amyloid and tau protein and are likely to contribute to the pathophysiology of AD.

Immunisation with the glycolytic enzyme enolase inhibits dissemination of Treponema pallidum in C57BL/6 mice

Treponema pallidum (T. pallidum), an obligate extracellular bacterium, is the causative agent of sexually transmitted bacterial diseases. In this study, the glycolytic enzyme enolase (Tp Eno) of T. pallidum were injected intramuscularly into C57BL/6 mice, resulting in higher levels of specific anti-Tp Eno antibodies and Tp Eno-specific splenocyte proliferation than those in the mice immunized with recombinant protein Tp Eno. Cytokine (IL-4, IL-6, IL-10, IFN-γ, and TNF-α) analysis of splenocytes showed that the Tp Eno could slightly trigger the Th1-biased immune response. Furthermore, immunization of mice with Tp Eno elicited a significant production of IFN-γ by CD4+ T-cells in the spleen. Subsequently, mice were inoculated intradermally (between the scapulae), intraperitoneally, intrarectally and via the corpora cavernosa with 2.5 × 106 organisms per site (1 × 107 total organisms). The bacterial organ burden detected in the blood, spleen, liver, testes or brain of immunized mice suggested that Tp Eno enhances protective immunity to inhibit T. pallidum colonization in distal tissues. Therefore, Tp Eno vaccination enhances Tp Eno-specific immunogenicity and provides protection against T. pallidum dissemination.

ENO2-derived phosphoenolpyruvate functions as an endogenous inhibitor of HDAC1 and confers resistance to antiangiogenic therapy.

Metabolic reprogramming is associated with resistance to antiangiogenic therapy in cancer. However, its molecular mechanisms have not been clearly elucidated. Here, we identify the glycolytic enzyme enolase 2 (ENO2) as a driver of resistance to antiangiogenic therapy in colorectal cancer (CRC) mouse models and human participants. ENO2 overexpression induces neuroendocrine differentiation, promotes malignant behaviour in CRC and desensitizes CRC to antiangiogenic drugs. Mechanistically, the ENO2-derived metabolite phosphoenolpyruvate (PEP) selectively inhibits histone deacetylase 1 (HDAC1) activity, which increases the acetylation of β-catenin and activates the β-catenin pathway in CRC. Inhibition of ENO2 with enolase inhibitors AP-III-a4 or POMHEX synergizes the efficacy of antiangiogenic drugs in vitro and in mice bearing drug-resistant CRC xenograft tumours. Together, our findings reveal that ENO2 constitutes a useful predictive biomarker and therapeutic target for resistance to antiangiogenic therapy in CRC, and uncover a previously undefined and metabolism-independent role of PEP in regulating resistance to antiangiogenic therapy by functioning as an endogenous HDAC1 inhibitor.

A variant in sperm-specific glycolytic enzyme enolase 4 (ENO4) causes human male infertility.

Although defects in sperm morphology and physiology lead to male infertility, in many instances, the exact disruption of molecular pathways in a given patient is often unknown. The glycolytic pathway is an essential process to supply energy in sperm cell motility. Enolase 4 (ENO4) is crucial for the glycolytic process, which provides the energy for sperm cells in motility. ENO4 is located in the sperm principal piece and is essential for the motility and organization of the sperm flagellum. In the present study, we characterized a family with asthenozoospermia and abnormal sperm morphology as a result of a variant in the enolase 4 (ENO4) gene. Computer-assisted semen analysis, papanicolaou smear staining and scanning electron microscopy were used to examine sperm motility and morphology for semen analysis in patients. For genetic analysis, whole-exome sequencing followed by Sanger sequencing was performed. Two brothers in a consanguineous family were being clinically investigated for sperm motility and morphology issues. Genetic analysis by whole-exome sequencing revealed a homozygous variant [c.293A>G, p.(Lys98Arg)] in the ENO4 gene that segregated with infertility in the family, shared by affected but not controls. In view of the association of asthenozoospermia and abnormal sperm morphology in Eno4 knockout mice, we consider this to be the first report describing the involvement of ENO4 gene in human male infertility. We also explore the possible involvement of another variant in explaining other phenotypic features in this family.

Early Events after Herpes Simplex Virus-Type 1 Entry Are Necessary for the Release of Gamma-Hydroxybutyrate upon Acute Infection.

We reported that gamma-hydroxybutyrate (GHB) is released upon Herpes Simplex Virus Type-1 (HSV-1) acute infection. However, the cellular biochemical processes involved in the production of GHB in infected cells are unclear. This study aims to shed light on the biochemical pathway and the stage within the viral life cycle responsible for the release of GHB in infected cells. UV-inactivation, acyclovir (ACV), and cycloheximide (CHX) treatments were used to inhibit HSV-1 replication at various stages. Vero cells treated with UV-inactivated HSV-1 significantly decreased GHB production. However, ACV or CHX treatments did not affect GHB production. We also showed that inhibition of glycolytic enzyme enolase by sodium fluoride (NaF) significantly reduces GHB production upon infection. This finding suggests that suppression of glycolytic activity negatively affects cellular GHB production. Our data also indicated that succinic semialdehyde dehydrogenase, an enzyme involved in the shunt of the tricarboxylic acid (TCA) cycle to generate succinic acid, was decreased upon infection, suggesting that infection may trigger the accumulation of succinic semialdehyde, causing the production of GHB. Although the precise mechanism has yet to be defined, our results suggest that early events following infection modulates the release of GHB, which is generated through the metabolic pathways of glycolysis and TCA cycle.

Axin2 coupled excessive Wnt‐glycolysis signaling mediates social defect in autism spectrum disorders

Social dysfunction is the core syndrome of autism spectrum disorder (ASD) and lacks effective medicine. Although numerous risk genes and relevant environmental factors have been identified, the convergent molecular mechanism underlying ASD‐associated social dysfunction remains largely elusive. Here, we report aberrant activation of canonical Wnt signaling and increased glycolysis in the anterior cingulate cortex (ACC, a key brain region of social function) of two ASD mouse models (Shank3−/− and valproic acid‐treated mice) and their corresponding human neurons. Overexpressing β‐catenin in the ACC of wild‐type mice induces both glycolysis and social deficits. Suppressing glycolysis in ASD mice partially rescued synaptic and social phenotype. Axin2, a key inhibitory molecule in Wnt signaling, interacts with the glycolytic enzyme enolase 1 (ENO1) in ASD neurons. Surprisingly, an Axin2 stabilizer, XAV939, effectively blocked Axin2/ENO1 interaction, switched glycolysis/oxidative phosphorylation balance, promoted synaptic maturation, and rescued social function. These data revealed excessive neuronal Wnt‐glycolysis signaling as an important underlying mechanism for ASD synaptic deficiency, indicating Axin2 as a potential therapeutic target for social dysfunction.

Abstract 387: Enolase-1 as a candidate theranostics target for neuroendocrine prostate cancer

Abstract Prostate cancer (PCa) is the second most common cancer in American men, with men of African ancestry (AA) having twice the mortality of men of European ancestry (EA). Although taxane-based chemotherapy is the last line of defense in men with advanced PCa, it ultimately fails due to chemoresistance. The protein-specific membrane antigen (PSMA) has been an effective target for the imaging and therapy of advanced PCa. Specifically, PSMA radioligand therapy (PSMA-RLT) is a theranostics (therapy + diagnostics) option for men with advanced PCa. However, about 30% of men with advanced PCa have limited response to PSMA-RLT due to the presence of neuroendocrine-differentiated PCa (NEPC), which lacks PSMA expression. A promising alternative to PSMA targeting is the glycolytic enzyme enolase (ENO), which localizes to the cell surface in advanced tumors. There are three enolases with similar functions, with ENO-1 and ENO-2 implicated in PCa. Here, we demonstrate, using immunoblotting, confocal microscopy, and cell fractionation studies with specific monoclonal antibodies, that chemosensitive NEPC cells express both ENO-1 and ENO-2; however, docetaxel (DTX)-resistant PC3-DR and DU145-DR cells only express ENO-1 with clear loss of ENO-2. Loss of ENO-2 in chemoresistant PCa cells generates a metabolic vulnerability due to loss of ENO redundancy. We also initiated the testing of small molecule inhibitors (SMIs) targeting ENO-1 in chemoresistant and chemosensitive NEPC cells, showing that these inhibitors have cytotoxic activity against these cells, with IC50 values in the low micromolar range. These results support the hypothesis that ENO-1 is expressed in the surface of NEPC cell lines and can be targeted with SMIs that could be modified as potential theranostics agents. Our long-term goal is to identify an alternative treatment for patients with NEPC by establishing ENO-1 as a novel cell surface theranostics target. Citation Format: Krystal Rubi Santiago Torres, Alfonso Duran. Enolase-1 as a candidate theranostics target for neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 387.

Abstract 3097: Prodrugs of a 1-hydroxy-2-oxopiperidin-3-yl phosphonate enolase inhibitor for the treatment of ENO1-deleted cancers

Abstract Cancers harboring homozygous deletion of the glycolytic enzyme enolase 1 (ENO1) are selectively vulnerable to inhibition of the paralogous isoform, enolase 2 (ENO2). We previously identified and characterized a competitive, small molecule phosphonate inhibitor of ENO2, 1-hydroxy-2-oxopiperidin-3-yl phosphonate (HEX), and its lipophilic bis-ester prodrug (POMHEX) in an ENO1-deleted intracranial orthotopic xenograft model of glioblastoma. Treatment with either HEX (150 mg/kg IV/IP) or POMHEX (20 mg/kg IV/IP) yielded tumor regression even after drug discontinuation. However, due to the poor pharmacokinetics of esterase-labile POMHEX, we synthesized a library of novel phosphonate prodrugs with distinct mechanisms of bioactivation and assessed their potency in D423 (ENO1-/-) cells. By conducting a prodrug structure activity relationship (SAR) study, we found that phosphonoamidate esters were efficiently bioactivated in ENO1-deleted glioma cells, while canonical McGuigan (ProTide) prodrugs were not. Other strategies, including salicylic alcohol (cycloSal) and lipid prodrugs of HEX, exhibited low micromolar IC50 values in ENO1-deleted glioma cells and improved stability in human serum over POMHEX. En route, we developed a novel class of aliphatic amine/ester prodrugs that can be broadly applied to efficiently deliver phosph(on)ate pharmacophores in cells. The activity of select prodrugs was also probed using the NCI-60 cell line screen, supporting its use to examine the relationship between prodrugs and cell line-dependent bioactivation. In sum, we have developed a novel class of phosph(on)ate prodrugs that is efficiently bioactivated in cells in vitro. Our prodrug SAR study disputes the common notion that ProTides are universally advantageous promoieties on phosph(on)ate pharmacophores and we provide mechanistic rationale for this observation with HEX. Finally, we show that the cycloSal prodrug yields efficient intracellular delivery of HEX in vitro, with a mechanism of bioactivation consistent with the GBM microenvironment, making this promoiety promising for further evaluation in vivo. Citation Format: Victoria C. Yan, Cong-Dat Pham, Elliot S. Ballato, Kristine L. Yang, Sunada Khadka, Yasaman Barektain, Prakriti Shrestha, Theresa Tran, Anton H. Poral, Mykia Washington, Sudhir Raghavan, Barbara Czako, Federica Pisaneschi, Yu-Hsi Lin, Nikunj Satani, Naima Hammoudi, Jeffrey J. Ackroyd, Dimitra K. Georgiou, Steven W. Millward, Florian L. Muller. Prodrugs of a 1-hydroxy-2-oxopiperidin-3-yl phosphonate enolase inhibitor for the treatment of ENO1-deleted cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3097.

Abstract C063: Enolase-1 as a candidate theranostics target for neuroendocrine prostate cancer

Abstract Prostate cancer (PCa) is the second most common cancer in American men, with men of African ancestry (AA) having twice the mortality of men of European ancestry (AA). Although taxane-based chemotherapy is the last line of defense in men with advanced PCa, it ultimately fails due to chemoresistance. The protein-specific membrane antigen (PSMA) has been an effective target for the imaging and therapy of advanced PCa. Specifically, PSMA radioligand therapy (PSMA-RLT) is a theranostics (therapy + diagnostics) option for men with advanced PCa. However, about 30% of men with advanced PCa have limited response to PSMA-RLT due to the presence of neuroendocrine-like PCa (NEPC), which lacks PSMA expression. A promising alternative to PSMA targeting is the glycolytic enzyme enolase (ENO), which localizes to the cell surface in advanced tumors. There are three enolases with similar functions, with ENO-1 and ENO-2 implicated in PCa. Previously, we showed that AA and EA men with PCa show differential serum antibody reactivity to ENO. While circulating serum antibodies from EA-PCa patients recognized ENO in both docetaxel (DTX)-sensitive and -resistant PCa cell lines, sera from AA-PCa men only recognized ENO in DTX-sensitive cells, with loss of immunoreactivity in the resistant cells. Here, we demonstrate, using immunoblotting and specific monoclonal antibodies, that chemosensitive NEPC cells express both ENO-1 and ENO-2; however, DTX-resistant cells only express ENO-1 with a clear loss of ENO-2. This pattern is identical to that produced by the AA-PCa serum antibodies, suggesting the possibility that AA-PCa patients may generate a preferential antibody response to ENO-2, with EA-PCa patients preferentially targeting ENO-1. This differential immunoreactivity may reflect differences between AA and EA PCa patients in tumor immunobiology, consistent with emerging literature. The loss of ENO-2 in chemoresistant PCa cells also generates a metabolic vulnerability due to the loss of ENO redundancy. We hypothesize that ENO-1 is expressed on the surface of NEPC cell lines and can be targeted with small molecule inhibitors (SMIs) that could be used as potential theranostics agents. Current studies are identifying ENO-1 surface expression on NEPC cell lines using monoclonal antibodies as well as AA and EA patient antibodies, via immunofluorescence microscopy, membrane fractionation analysis, and flow cytometry. We also initiated the testing of small molecule inhibitors (SMIs) targeting ENO-1 in chemoresistant NEPC cells. Our long-term goal is to identify an alternative treatment for patients with NEPC by establishing ENO-1 as a novel theranostics target. This could benefit AA-PCa patients, which may not mount a strong antitumor ENO-1 immune response. Citation Format: Krystal R. Santiago, Alfonso M. Duran, Carlos A. Casiano, Frankis G. Almaguel, Bhaskar Das. Enolase-1 as a candidate theranostics target for neuroendocrine prostate cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C063.

Prodrugs of a 1-Hydroxy-2-oxopiperidin-3-yl Phosphonate Enolase Inhibitor for the Treatment of ENO1-Deleted Cancers.

Cancers harboring homozygous deletion of the glycolytic enzyme enolase 1 (ENO1) are selectively vulnerable to inhibition of the paralogous isoform, enolase 2 (ENO2). A previous work described the sustained tumor regression activities of a substrate-competitive phosphonate inhibitor of ENO2, 1-hydroxy-2-oxopiperidin-3-yl phosphonate (HEX) (5), and its bis-pivaloyoxymethyl prodrug, POMHEX (6), in an ENO1-deleted intracranial orthotopic xenograft model of glioblastoma [Nature Metabolism 2020, 2, 1423-1426]. Due to poor pharmacokinetics of bis-ester prodrugs, this study was undertaken to identify potential non-esterase prodrugs for further development. Whereas phosphonoamidate esters were efficiently bioactivated in ENO1-deleted glioma cells, McGuigan prodrugs were not. Other strategies, including cycloSal and lipid prodrugs of 5, exhibited low micromolar IC50 values in ENO1-deleted glioma cells and improved stability in human serum over 6. The activity of select prodrugs was also probed using the NCI-60 cell line screen, supporting its use to examine the relationship between prodrugs and cell line-dependent bioactivation.

ENO2 Promotes Colorectal Cancer Metastasis by Interacting with the LncRNA CYTOR and Activating YAP1-Induced EMT.

The glycolytic enzyme enolase 2 (ENO2) is dysregulated in many types of cancer. However, the roles and detailed molecular mechanism of ENO2 in colorectal cancer (CRC) metastasis remain unclear. Here, we performed a comprehensive analysis of ENO2 expression in 184 local CRC samples and samples from the TCGA and GEO databases and found that ENO2 upregulation in CRC samples was negatively associated with prognosis. By knocking down and overexpressing ENO2, we found that ENO2 promoted CRC cell migration and invasion, which is dependent on its interaction with the long noncoding RNA (lncRNA) CYTOR, but did not depend on glycolysis regulation. Furthermore, CYTOR mediated ENO2 binding to large tumor suppressor 1 (LATS1) and competitively inhibited the phosphorylation of Yes-associated protein 1 (YAP1), which ultimately triggered epithelial–mesenchymal transition (EMT). Collectively, these findings highlight the molecular mechanism of the ENO2–CYTOR interaction, and ENO2 could be considered a potential therapeutic target for CRC.

Long non-coding RNA ANRIL promotes chemoresistance in triple-negative breast cancer via enhancing aerobic glycolysis

AimslncRNA ANRIL expression is dysregulated in many human cancers and is thus a useful prognostic marker for cancer patients. However, whether ANRIL is involved in drug resistance in triple-negative breast cancer (TNBC) has not yet been investigated. Main methodsA luciferase reporter assay was conducted to verify the binding between miR-125a and ANRIL. RT-PCR and western blotting were performed to detect the expression of miR-125a, ANRIL, and ENO1. Glycolysis stress was assessed using the Seahorse extracellular flux analyzer. Functional studies were performed using both in vitro and in vivo xenograft models. Key findingsANRIL was markedly upregulated in both patients with TNBC and TNBC cell lines. Knockdown of ANRIL increased the cytotoxic effect of ADR and repressed cellular glycolytic activity in TNBC cells. Mechanistic analysis showed that ANRIL may act as a competing endogenous RNA of miR-125a to relieve the repressive effect of miR-125a on its target glycolytic enzyme enolase (ENO1). Notably, 2-deoxy-glucose attenuated ANRIL-induced increase in drug resistance in TNBC cells. SignificanceThese results indicate that knockdown of ANRIL plays an active role in overcoming drug resistance in TNBC by inhibiting glycolysis through the miR-125a/ENO1 pathway, which may be useful for the development of novel therapeutic targets for treating patients with TNBC, especially those with drug resistance.

Investigation of the location and secretion features of Candida albicans enolase with monoclonal antibodies

PurposeThe glycolytic enzyme enolase plays important role in the pathogenesis of Candida albicans infection and has been also considered as a promising molecular marker for the diagnosis of invasive candidiasis. This study aimed to investigate the location and secretion features of Candida albicans enolase (CaEno) with a couple of specific monoclonal antibodies (mAbs).MethodsTwo mAbs named 9H8 and 10H8 against CaEno were generated by fusing SP2/0 myeloma cell with the spleen lymphocytes from CaEno immunized mice. The specificity of the mAbs was then validated by Western blot and liquid chromatography-mass spectrometry (LC–MS/MS). A diverse set of experiments were conducted based on the pair of mAbs which involved immunohistochemical staining analysis, whole cell enzyme-linked immunosorbent assay (ELISA), double antibody sandwich ELISA, and confocal microscopy to analyze the possible location and secretion features of CaEno.ResultsCaEno is abundantly expressed in the cytoplasm of C. albicans blastospores and is distributed in a ring-shaped pattern along the cell wall. CaEno appeared in the hyphal C. albicans as just a “mushroom” form. CaEno was found to be weakly expressed on the surface of blastospores but constantly expressed at various stages of growth. CaEno concentrations in C. albicans blastospores culture supernatant are considerably higher than in C. albicans hyphae culture supernatant. The dynamic changes of supernatant CaEno concentration in blastospores and hyphal C. albicans exhibit distinct features, although both appear to be associated with the C. albicans growth state. When cultivated under normal circumstances, however, no apparent CaEno degradation was seen in the cell-free supernatant.ConclusionOur results implied that CaEno was constantly expressed on the cell surface and its secretion features varied according to the growth stage of C. albicans. However, further experimental and theoretical studies are needed in future to identify the specific mechanisms by which this phenomenon can arise.

Riboregulation of Enolase 1 activity controls glycolysis and embryonic stem cell differentiation

Differentiating stem cells must coordinate their metabolism and fate trajectories. Here, we report that the catalytic activity of the glycolytic enzyme Enolase 1 (ENO1) is directly regulated by RNAs leading to metabolic rewiring in mouse embryonic stem cells (mESCs). We identify RNA ligands that specifically inhibit ENO1's enzymatic activity invitro and diminish glycolysis in cultured human cells and mESCs. Pharmacological inhibition or RNAi-mediated depletion of the protein deacetylase SIRT2 increases ENO1's acetylation and enhances its RNA binding. Similarly, induction of mESC differentiation leads to increased ENO1 acetylation, enhanced RNA binding, and inhibition of glycolysis. Stem cells expressing mutant forms of ENO1 that escape or hyper-activate this regulation display impaired germ layer differentiation. Our findings uncover acetylation-driven riboregulation of ENO1 as a physiological mechanism of glycolytic control and of the regulation of stem cell differentiation. Riboregulation may represent a more widespread principle of biological control.

The Dynamics of Mycoplasma gallisepticum Nucleoid Structure at the Exponential and Stationary Growth Phases.

The structure and dynamics of bacterial nucleoids play important roles in regulating gene expression. Bacteria of class Mollicutes and, in particular, mycoplasmas feature extremely reduced genomes. They lack multiple structural proteins of the nucleoid, as well as regulators of gene expression. We studied the organization of Mycoplasma gallisepticum nucleoids in the stationary and exponential growth phases at the structural and protein levels. The growth phase transition results in the structural reorganization of M. gallisepticum nucleoid. In particular, it undergoes condensation and changes in the protein content. The observed changes corroborate with the previously identified global rearrangement of the transcriptional landscape in this bacterium during the growth phase transition. In addition, we identified that the glycolytic enzyme enolase functions as a nucleoid structural protein in this bacterium. It is capable of non-specific DNA binding and can form fibril-like complexes with DNA.

Reduction of the canonical function of a glycolytic enzyme enolase triggers immune responses that further affect metabolism and growth in Arabidopsis.

Primary metabolism provides energy for growth and development as well as secondary metabolites for diverse environmental responses. Here we describe an unexpected consequence of disruption of a glycolytic enzyme enolase named LOW EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 2 (LOS2) in causing constitutive defense responses or autoimmunity in Arabidopsis thaliana. The autoimmunity in the los2 mutant is accompanied by a higher expression of about one-quarter of intracellular immune receptor nucleotide-binding leucine-rich repeat (NLR) genes in the genome and is partially dependent on one of these NLR genes. The LOS2 gene was hypothesized to produce an alternatively translated protein c-Myc Binding Protein (MBP-1) that functions as a transcriptional repressor. Complementation tests show that LOS2 executes its function in growth and immunity regulation through the canonical enolase activity but not the production of MBP-1. In addition, the autoimmunity in the los2 mutants leads to a higher accumulation of sugars and organic acids and a depletion of glycolytic metabolites. These findings indicate that LOS2 does not exert its function in immune responses through an alternatively translated protein MBP-1. Rather, they show that a perturbation of glycolysis from the reduction of the enolase activity results in activation of NLR-involved immune responses which further influences primary metabolism and plant growth, highlighting the complex interaction between primary metabolism and plant immunity.

Mining nematode protein secretomes to explain lifestyle and host specificity.

Parasitic nematodes are highly successful pathogens, inflicting disease on humans, animals and plants. Despite great differences in their life cycles, host preference and transmission modes, these parasites share a common capacity to manipulate their host’s immune system. This is at least partly achieved through the release of excretory/secretory proteins, the most well-characterized component of nematode secretomes, that are comprised of functionally diverse molecules. In this work, we analyzed published protein secretomes of parasitic nematodes to identify common patterns as well as species-specific traits. The 20 selected organisms span 4 nematode clades, including plant pathogens, animal parasites, and the free-living species Caenorhabditis elegans. Transthyretin-like proteins were the only component common to all adult secretomes; many other protein classes overlapped across multiple datasets. The glycolytic enzymes aldolase and enolase were present in all parasitic species, but missing from C. elegans. Secretomes from larval stages showed less overlap between species. Although comparison of secretome composition across species and life-cycle stages is challenged by the use of different methods and depths of sequencing among studies, our workflow enabled the identification of conserved protein families and pinpointed elements that may have evolved as to enable parasitism. This strategy, extended to more secretomes, may be exploited to prioritize therapeutic targets in the future.