Alpha-enolase Research Articles

TMET-14. ENOLASE 2 IS A UNIQUE METABOLIC VULNERABILITY INDUCED BY THE 1P19Q CODELETION IN OLIGODENDROGLIOMAS

Abstract Gliomas are devastating primary brain tumors in adults. The presence of an isocitrate dehydrogenase mutation combined with a chromosomal 1p/19q codeletion discriminates oligodendrogliomas from astrocytomas. The goal of this study was to leverage metabolic vulnerabilities induced by the 1p/19q codeletion for oligodendroglioma therapy. The 1p19q codeletion results in loss of enolase 1 (ENO1), which converts 2-phosphoglycerate to phosphoenolpyruvate during glycolysis. Here, we show that enolase 2 (ENO2) is upregulated in patient-derived oligodendroglioma (SF10417, BT88, BT54) models relative to astrocytoma (BT142, SF10602) and normal human astrocytes. ENO2 is also elevated in oligodendroglioma patient biopsies relative to astrocytoma or gliosis. Mechanistic studies indicate that inhibiting MEK1 or ERK1 abrogates ENO2 expression, suggesting that the MAPK pathway upregulates ENO2 in oligodendrogliomas. Next, we examined the therapeutic potential of targeting ENO2 in oligodendrogliomas. Genetic ablation of ENO2 or pharmacological inhibition using POMHEX inhibited the viability of SF10417 and BT88 cells with IC50 values of ~50 nM. In contrast, the IC50 for BT142 and SF10602 astrocytoma cells was ~5-8 uM, highlighting the exquisite dependence of oligodendrogliomas on ENO2. However, ENO2 inhibition did not lead to cytotoxicity in patient-derived oligodendroglioma cells or intracranial tumor xenografts. To assess the metabolic consequences of ENO2 inhibition and identify potential compensatory alterations, we traced [U-13C]-glucose metabolism in vivo in mice bearing intracranial SF10417 tumors. Although POMHEX abrogated glycolytic metabolism downstream of 2-phosphoglycerate, it shunted glucose towards biosynthesis of serine, glycine, methionine and purine nucleotides, an effect that was driven by phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme for serine biosynthesis. Importantly, the novel PHGDH inhibitor PHGDH-D8 was synthetically lethal in combination with POMHEX in oligodendroglioma cells and the combination induced apoptosis in mice bearing intracranial SF10417 tumors. Collectively, we exploit a mechanistic understanding of metabolic vulnerabilities induced by the 1p19q codeletion to identify combined inhibition of ENO2 and PHGDH as a novel therapeutic opportunity for oligodendrogliomas.

CircFndc3b Mediates Exercise-Induced Neuroprotection by Mitigating Microglial/Macrophage Pyroptosis via the ENO1/KLF2 Axis in Stroke Mice.

Circular RNA (circRNA) plays a pivotal role in regulating neurological damage post-ischemic stroke. Previous researches demonstrated that exercise mitigates neurological dysfunction after ischemic stroke, yet the specific contributions of circRNAs to exercise-induced neuroprotection remain unclear. This study reveals that mmu_circ_0001113 (circFndc3b) is markedly downregulated in the penumbral cortex of a mouse model subjected to middle cerebral artery occlusion (MCAO). However, exercise increased circFndc3b expression in microglia/macrophages, alleviating pyroptosis, reducing infarct volume, and enhancing neurological recovery in MCAO mice. Mechanistically, circFndc3b interacted with Enolase 1 (ENO1), facilitating ENO1's binding to the 3' Untranslated Region (3'UTR) of Krüppel-like Factor 2 (Klf2) mRNA, thereby stabilizing Klf2 mRNA and increasing its protein expression, which suppressed NOD-like Receptor Family Pyrin Domain Containing 3 (NLRP3) inflammasome-mediated microglial/macrophage pyroptosis. Additionally, circFndc3b enhanced ENO1's interaction with the 3'UTR of Fused in Sarcoma (FUS) mRNA, leading to increased FUS protein levels and promoting circFndc3b cyclization. These results suggest that circFndc3b mediates exercise-induced anti-pyroptotic effects via the ENO1/Klf2 axis, and a circFndc3b/ENO1/FUS positive feedback loop may potentiate exercise's neuroprotective effects. This study unveils a novel mechanism underlying exercise-induced neuroprotection in ischemic stroke and positions circFndc3b as a promising therapeutic target for stroke management, mimicking the beneficial effects of exercise.

EPIGENETIC MODULATION OF STEMNESS AND DIFFERENTIATION BY VALPROIC ACID IN MEDULLOBLASTOMA

Abstract AIMS Changes in epigenetic processes, including histone acetylation, are proposed as key events affecting tumor cell function and the initiation and progression of pediatric brain cancers. Valproic acid (VPA) is an antiepileptic drug that acts partially by inhibiting histone deacetylases (HDACs) and could be repurposed as an epigenetic anticancer therapy. Here, we report VPA-induced alterations in features related to stemness and differentiation in medulloblastoma (MB) cells. METHOD Human MB cells were treated with different concentrations of VPA and cell viability was measured with a trypan exclusion assay. Cell cycle was evaluated with flow cytometry. Expansion of MB cancer stem cells (CSCs) was evaluated by a neurosphere formation assay. Reverse transcriptase polymerase chain reaction (RT-qPCR) was used to analyze mRNA expression and Western Blot to analyze protein levels. H3K9 histone acetylation (H3K9ac) was measured with immunofluorescence, and chromatin Immunoprecipitation (ChIP) was used to evaluate the occupancy of gene promoter regions by H3K9ac. RESULTS VPA reduced MB cell viability and expansion of MB CSCs, induced morphological changes consistent with neuronal differentiation, and increased expression of differentiation markers tubulin beta 3 class III (TUBB3) and enolase 2 (ENO2). Expression of stemness markers SOX2 and Nestin was differentially affected by VPA in cells representative of SHH or Group 3/4 MB molecular subgroups. VPA increased H3K9ac and its occupancy of promoter regions of TP53 and CDKN1A genes. CONCLUSION Our results indicate that VPA may exert antitumor effects in MB by influencing histone acetylation, which may result in modulation of stemness and stimulation of neuronal differentiation.

ENO2 in progression and treatment of colon adenocarcinoma: integrative bioinformatics analysis on non-apoptotic cell death

Colon adenocarcinoma (COAD) is one of the most common types of cancer. The interconnection between non-apoptotic cell death and COAD has not been adequately addressed. In our study, an integrative bioinformatics analysis was performed to explore non-apoptotic cell death-related biomarkers in COAD. ENO2 was determined as a potent biomarker for prognosis, drug response, immunity, and immunotherapy prediction. We used EdU and RT-qPCR assays to test our hypothesis and investigate how the ENO2 gene may influence or regulate cancer-related processes. ENO2 was expected to be a potential target in COAD.

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.

Key genes and regulatory networks for diabetic retinopathy based on hypoxia-related genes: a bioinformatics analysis.

To prevent neovascularization in diabetic retinopathy (DR) patients and partially control disease progression. Hypoxia-related differentially expressed genes (DEGs) were identified from the GSE60436 and GSE102485 datasets, followed by gene ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Potential candidate drugs were screened using the CMap database. Subsequently, a protein-protein interaction (PPI) network was constructed to identify hypoxia-related hub genes. A nomogram was generated using the rms R package, and the correlation of hub genes was analyzed using the Hmisc R package. The clinical significance of hub genes was validated by comparing their expression levels between disease and normal groups and constructing receiver operating characteristic curve (ROC) curves. Finally, a hypoxia-related miRNA-transcription factor (TF)-Hub gene network was constructed using the NetworkAnalyst online tool. Totally 48 hypoxia-related DEGs and screened 10 potential candidate drugs with interaction relationships to upregulated hypoxia-related genes were identified, such as ruxolitinib, meprylcaine, and deferiprone. In addition, 8 hub genes were also identified: glycogen phosphorylase muscle associated (PYGM), glyceraldehyde-3-phosphate dehydrogenase spermatogenic (GAPDHS), enolase 3 (ENO3), aldolase fructose-bisphosphate C (ALDOC), phosphoglucomutase 2 (PGM2), enolase 2 (ENO2), phosphoglycerate mutase 2 (PGAM2), and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Based on hub gene predictions, the miRNA-TF-Hub gene network revealed complex interactions between 163 miRNAs, 77 TFs, and hub genes. The results of ROC showed that the except for GAPDHS, the area under curve (AUC) values of the other 7 hub genes were greater than 0.758, indicating their favorable diagnostic performance. PYGM, GAPDHS, ENO3, ALDOC, PGM2, ENO2, PGAM2, and PFKFB3 are hub genes in DR, and hypoxia-related hub genes exhibited favorable diagnostic performance.

ARNTL2 facilitates bladder cancer progression through potentiating ENO1-mediated glycolysis in a SLC31A1-independent and -dependent manner

BackgroundBasic helix-loop-helix ARNT like 2 (ARNTL2) is a transcription factor that controls the circadian rhythm. Amounts of studies have demonstrated the carcinogenic function of ARNTL2 in human malignant tumors albeit the underlying mechanisms remain poorly understood. We aimed to study the significance of ARNTL2 in bladder cancer (BLCA). MethodsImmunohistochemical staining, immunoblotting and the database from TCGA were used to analyze the clinical relevance of ARNTL2, enolase 1 (ENO1) and solute carrier family 31 member 1 (SLC31A1) in BLCA. The function of ARNTL2 was explored by cell proliferation assay, apoptosis, colony formation and xenografted tumorigenesis. The molecular mechanisms of ARNTL2-driving BLCA development were investigated by RT-qPCR, immunoblotting and luciferase assays. Glycolysis was checked by measuring glucose consumption and lactate production. ENO1 activity was assessed by using indicated assay kit. ResultsOverexpression of ARNTL2 facilitates the proliferation and tumorigenesis of BLCA cells through suppression of apoptosis and enhancement of glycolysis. Up-regulation of SLC31A1, ENO1, and enhancement of SLC31A1-mediated ENO1 activity were critical for ARNTL2-triggered glycolysis and malignant growth in BLCA cells. ARNTL2 was positively correlated with SLC31A1 and ENO1 in BLCA patients. High expression of ARNTL2, SLC31A1 or ENO1 predicted the poor prognosis of BLCA patients. Depletion of SLC31A1 and inhibition of glycolysis completely blunted the growth ability of BLCA cells. ConclusionIn summary, ARNTL2 facilitates the progression of BLCA via activating ENO1-mediated glycolysis in a SLC31A1-independent and -dependent manner. Inhibiting SLC31A1 and glycolysis may be an aspirational approach for the treatment of BLCA patients with overexpression of ARNTL2.

Circular RNA circESYT2 serves as a microRNA-665 sponge to promote the progression of hepatocellular carcinoma through ENO2.

Circular RNAs (circRNAs) have emerged as crucial regulators in tumor progression, yet their specific role in hepatocellular carcinoma (HCC) remains largely uncharacterized. In this study, we utilized high-transcriptome sequencing to identify the upregulation of circESYT2 (hsa_circ_002142) in HCC tissues. Functional experiments carried out invivo and invitro revealed that circESYT2 played a significant role in maintaining the growth and metastatic behaviors of HCC. Through integrative analysis, we identified enolase 2 (ENO2) as a potential target regulated by circESYT2 through the competitive endogenous RNA sponge mechanism. Additional gain- or loss-of-function experiments indicated that overexpression of circESYT2 led to a tumor-promoting effect, which could be reversed by transfection of microRNA-665 (miR-665) mimic or ENO2 knockdown in HCC cells. Furthermore, the direct interaction between miR-665 and circESYT2 and between miR-665 and ENO2 was confirmed using RNA immunoprecipitation, FISH, RNA pull-down, and dual-luciferase reporter assays, highlighting the involvement of the circESYT2/miR-665/ENO2 axis in promoting HCC progression. These findings shed light on the molecular characteristics of circESYT2 in HCC tissues and suggest its potential as a biomarker or therapeutic target for HCC treatment.

Enolase inhibitors as therapeutic leads for Naegleria fowleri infection.

Infections with the pathogenic free-living amoebae Naegleria fowleri can lead to life-threatening illnesses including catastrophic primary amoebic meningoencephalitis (PAM). Efficacious treatment options for these infections are lacking and the mortality rate remains >95% in the US. Glycolysis is very important for the infectious trophozoite lifecycle stage and inhibitors of glucose metabolism have been found to be toxic to the pathogen. Recently, human enolase 2 (ENO2) phosphonate inhibitors have been developed as lead agents to treat glioblastoma multiforme (GBM). These compounds, which cure GBM in a rodent model, are well-tolerated in mammals because enolase 1 (ENO1) is the predominant isoform used systemically. Here, we describe findings that demonstrate these agents are potent inhibitors of N. fowleri ENO (NfENO) and are lethal to amoebae. In particular, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) was a potent enzyme inhibitor (IC50 = 0.14 ± 0.04 μM) that was toxic to trophozoites (EC50 = 0.21 ± 0.02 μM) while the reported CC50 was >300 μM. Molecular docking simulation revealed that HEX binds strongly to the active site of NfENO with a binding affinity of -8.6 kcal/mol. Metabolomic studies of parasites treated with HEX revealed a 4.5 to 78-fold accumulation of glycolytic intermediates upstream of NfENO. Last, nasal instillation of HEX increased longevity of amoebae-infected rodents. Two days after infection, animals were treated for 10 days with 3 mg/kg HEX, followed by one week of observation. At the end of the one-week observation, eight of 12 HEX-treated animals remained alive (resulting in an indeterminable median survival time) while one of 12 vehicle-treated rodents remained, yielding a median survival time of 10.9 days. However, intranasal HEX delivery was not curative as brains of six of the eight survivors were positive for amoebae. These findings suggest that HEX requires further evaluation to develop as a lead for treatment of PAM.

ENO1 regulates IL-1β-induced chondrocyte inflammation, apoptosis and matrix degradation possibly through the potential binding to CRLF1

In this study, we aim to investigate the role of enolase 1 (ENO1) in osteoarthritis (OA) pathogenic process and to uncover the underlying mechanism. To this end, we used IL-1β to induce an in vitro OA‑like chondrocyte model in human immortalized chondrocyte C-28/I2 cells. We manipulated the expression of ENO1 and cytokine receptor-like factor 1 (CRLF1) in IL-1β-induced C-28/I2 cells using siRNA and/or overexpression and tested their effects on IL-1β-induced pathologies including cell viability, apoptosis and inflammatory cytokine levels (IL-6 and TNF-α), and the expression of extracellular matrix-related enzymes and major mediators in the NF-κB signaling pathway (p-p65, p65, p-IκBα and IκBα). We used co-immunoprecipitation and immunofluorescence imaging to study a possible binding between ENO1 and CRLF1. Our data showed that IL-1β induction elevated ENO1 and CRLF1 expression in C-28/I2 cells. Silencing ENO1 or CRLF1 inhibited the IL-1β-induced cell viability damage, apoptosis, inflammation, and extracellular matrix degradation. The inhibitory effect of silencing ENO1 was reversed by CRLF1 overexpression, suggesting a functional connection between ENO1 and CRLF1, which could be attributed to a binding between these two partners. Our study could help validate the role of ENO1 in OA pathogenies and identify novel therapeutic targets for OA treatment.

Exploring Canine Mammary Cancer through Liquid Biopsy: Proteomic Profiling of Small Extracellular Vesicles.

(Background). Canine mammary tumors (CMTs) have emerged as an important model for understanding pathophysiological aspects of human disease. Liquid biopsy (LB), which relies on blood-borne biomarkers and offers minimal invasiveness, holds promise for reflecting the disease status of patients. Small extracellular vesicles (SEVs) and their protein cargo have recently gained attention as potential tools for disease screening and monitoring. (Objectives). This study aimed to isolate SEVs from canine patients and analyze their proteomic profile to assess their diagnostic and prognostic potential. (Methods). Plasma samples were collected from female dogs grouped into CMT (malignant and benign), healthy controls, relapse, and remission groups. SEVs were isolated and characterized using ultracentrifugation (UC), nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Proteomic analysis of circulating SEVs was conducted using liquid chromatography-mass spectrometry (LC-MS). (Results). While no significant differences were observed in the concentration and size of exosomes among the studied groups, proteomic profiling revealed important variations. Mass spectrometry identified exclusive proteins that could serve as potential biomarkers for mammary cancer. These included Inter-alpha-trypsin inhibitor heavy chain (ITIH2 and ITI4), phosphopyruvate hydratase or alpha enolase (ENO1), eukaryotic translation elongation factor 2 (eEF2), actin (ACTB), transthyretin (TTR), beta-2-glycoprotein 1 (APOH) and gelsolin (GSN) found in female dogs with malignant tumors. Additionally, vitamin D-binding protein (VDBP), also known as group-specific component (GC), was identified as a protein present during remission. (Conclusions). The results underscore the potential of proteins found in SEVs as valuable biomarkers in CMTs. Despite the lack of differences in vesicle concentration and size between the groups, the analysis of protein content revealed promising markers with potential applications in CMT diagnosis and monitoring. These findings suggest a novel approach in the development of more precise and effective diagnostic tools for this challenging clinical condition.

ENO2, a Glycolytic Enzyme, Contributes to Prostate Cancer Metastasis: A Systematic Review of Literature.

Prostate cancer (PCa) is the second leading cause of male cancer deaths in the UK and the fifth worldwide. The presence of distant PCa metastasis can reduce the 5-year survival rate from 100% to approximately 30%. Enolase 2 (ENO2), a crucial glycolytic enzyme in cancer metabolism, is associated with the metastasis of multiple cancers and is also used as a marker for neuroendocrine tumours. However, its role in PCa metastasis remains unclear. In this study, we systematically reviewed the current literature to determine the association between ENO2 and metastatic PCa. Medline, Web of Science, and PubMed were searched for eligible studies. The search yielded five studies assessing ENO2 expression in PCa patients or cell lines. The three human studies suggested that ENO2 expression is correlated with late-stage, aggressive PCa, including castrate-resistant PCa (CRPC), metastatic CRPC, and neuroendocrine PCa (NEPC). This was further supported by two in vitro studies indicating that ENO2 expression can be regulated by the tumour microenvironment, such as androgen deprived conditions and the presence of bone-forming osteoblasts. Therefore, ENO2 may functionally contribute to PCa metastasis, possibly due to the unique metabolic features of PCa, which are glycolysis dependent only at the advanced metastatic stage.

BNIP3-mediated mitophagy boosts the competitive growth of Lenvatinib-resistant cells via energy metabolism reprogramming in HCC

An increasing evidence supports that cell competition, a vital selection and quality control mechanism in multicellular organisms, is involved in tumorigenesis and development; however, the mechanistic contributions to the association between cell competition and tumor drug resistance remain ill-defined. In our study, based on a contructed lenvitinib-resistant hepatocellular carcinoma (HCC) cells display obvious competitive growth dominance over sensitive cells through reprogramming energy metabolism. Mechanistically, the hyperactivation of BCL2 interacting protein3 (BNIP3) -mediated mitophagy in lenvatinib-resistant HCC cells promotes glycolytic flux via shifting energy production from mitochondrial oxidative phosphorylation to glycolysis, by regulating AMP-activated protein kinase (AMPK) -enolase 2 (ENO2) signaling, which perpetually maintaining lenvatinib-resistant HCC cells’ competitive advantage over sensitive HCC cells. Of note, BNIP3 inhibition significantly sensitized the anti-tumor efficacy of lenvatinib in HCC. Our findings emphasize a vital role for BNIP3-AMPK-ENO2 signaling in maintaining the competitive outcome of lenvitinib-resistant HCC cells via regulating energy metabolism reprogramming; meanwhile, this work recognizes BNIP3 as a promising target to overcome HCC drug resistance.

Evaluation of secretome biomarkers in glioblastoma cancer stem cells: A bioinformatics analysis.

Glioblastoma (GBM) is a malignant brain tumor that frequently occurs alongside other central nervous system (CNS) conditions. The secretome of GBM cells contains a diverse array of proteins released into the extracellular space, influencing the tumor microenvironment. These proteins can serve as potential biomarkers for GBM due to their involvement in key biological processes, exploring the secretome biomarkers in GBM research represents a cutting-edge strategy with significant potential for advancing diagnostic precision, treatment monitoring, and ultimately improving outcomes for patients with this challenging brain cancer. This study was aimed to investigate the roles of secretome biomarkers and their pathwayes in GBM through bioinformatics analysis. Using data from the Gene Expression Omnibus and the Cancer Genome Atlas datasets-where both healthy and cancerous samples were analyzed-we used a quantitative analytical framework to identify differentially expressed genes (DEGs) and cell signaling pathways that might be related to GBM. Then, we performed gene ontology studies and hub protein identifications to estimate the roles of these DEGs after finding disease-gene connection networks and signaling pathways. Using the GEPIA Proportional Hazard Model and the Kaplan-Meier estimator, we widened our analysis to identify the important genes that may play a role in both progression and the survival of patients with GBM. In total, 890 DEGs, including 475 and 415 upregulated and downregulated were identified, respectively. Our results revealed that SQLE, DHCR7, delta-1 phospholipase C (PLCD1), and MINPP1 genes are highly expressed, and the Enolase 2 (ENO2) and hexokinase-1 (HK1) genes are low expressions. Hence, our findings suggest novel mechanisms that affect the occurrence of GBM development, growth, and/or establishment and may also serve as secretory biomarkers for GBM prognosis and possible targets for therapy. So, continued research in this field may uncover new avenues for therapeutic interventions and contribute to the ongoing efforts to combat GBM effectively.

Ciwujianoside E inhibits Burkitt lymphoma cell proliferation and invasion by blocking ENO1-plasminogen interaction and TGF-β1 activation

Burkitt's lymphoma (BL) is a rare and highly aggressive B-cell non-Hodgkin lymphoma. Although the outcomes of patients with BL have greatly improved, options for patients with relapsed and refractory BL are limited. Therefore, there is an urgent need to improve BL therapeutics and to develop novel drugs with reduced toxicity. In this study, we demonstrated that enolase 1 (ENO1) is a potential novel drug target for BL treatment. We determined that ENO1 was aberrantly upregulated in BL, which was closely related to its invasiveness and poor clinical outcomes. Furthermore, using RNA interference, we demonstrated that ENO1 depletion significantly inhibited cell proliferation and invasion both in vitro and in vivo. Mechanistically, we established that ENO1 knockdown suppressed the PI3K-AKT and epithelial-mesenchymal transition (EMT) signaling pathways by reducing plasminogen (PLG) recruitment, plasmin (PL) generation, and TGF-β1 activation. Addition of activated TGF-β1 protein to the culture medium of shENO1 cells reversed the inhibitory effects on cell proliferation and invasion, as well as those on the PI3K-AKT and EMT signaling pathways. Notably, our research led to the discovery of a novel ENO1-PLG interaction inhibitor, Ciwujianoside E (L-06). L-06 effectively disrupts the interaction between ENO1 and PLG, consequently reducing PL generation and suppressing TGF-β1 activation. In both in vitro and in vivo experiments, L-06 exerted impressive antitumor effects. In summary, our study elucidated the critical role of ENO1 in BL cell proliferation and invasion and introduced a novel ENO1 inhibitor, which holds promise for improving the treatment of patients with BL in the future.

Integrative proteome analysis of bone marrow interstitial fluid and serum reveals candidate signature for acute myeloid leukemia

Acute myeloid leukemia (AML) is an aggressive form of blood cancer and clinically highly heterogeneous characterized by the accumulation of clonally proliferative immature precursors of myeloid lineage leading to bone marrow failure. Although, the current diagnostic methods for AML consist of cytogenetic and molecular assessment, there is a need for new markers that can serve as useful candidates in diagnosis, prognosis and understanding the pathophysiology of the disease. This study involves the investigation of alterations in the bone marrow interstitial fluid and serum proteome of AML patients compared to controls using label-free quantitative proteomic approach. A total of 201 differentially abundant proteins were identified in AML BMIF, while in the case of serum 123 differentially abundant proteins were identified. The bioinformatics analysis performed using IPA revealed several altered pathways including FAK signalling, IL-12 signalling and production of macrophages etc. Verification experiments were performed in a fresh independent cohort of samples using MRM assays led to the identification of a panel of three proteins viz., PPBP, APOH, ENOA which were further validated in a new cohort of serum samples by ELISA. The three-protein panel could be helpful in the diagnosis, prognosis and understanding of the pathophysiology of AML in the future. Biological SignificanceAcute Myeloid Leukemia (AML) is a type haematological malignancy which constitute one third of total leukemias and it is the most common acute leukemia in adults. In the current clinical practice, the evaluation of diagnosis and progression of AML is largely based on morphologic, immunophenotypic, cytogenetic and molecular assessment. There is a need for new markers/signatures which can serve as useful candidates in diagnosis and prognosis. The present study aims to identify and validate candidate biosignature for AML which can be useful in diagnosis, prognosis and understand the pathophysiology of the disease. Here, we identified 201 altered proteins in AML BMIF and 123 in serum. Among these altered proteins, a set of three proteins viz., pro-platelet basic protein (CXCL7), enolase 1 (ENO1) and beta-2-glycoprotein 1 (APOH) were significantly increased in AML BMIF and serum suggest that this panel of proteins could help in future AML disease management and thereby improving the survival expectancy of AML patients.

Inhibition of ALDOA‐ and ENO1‐Mediated Glycolysis of Oridonin as a Novel Antitumor Strategy of Non‐Small‐Cell Lung Cancer

AbstractNon‐small‐cell lung cancer (NSCLC) is the most common type of lung cancer. Oridonin (ORI), the main pharmacodynamic component of the Chinese herb Herba Rabdosiae with anticancer activity is reported to inhibit various types of cancer including NSCLC. However, its direct target proteins acting on NSCLC still require further investigation. Here, the direct protein profile is analyzed and the key targets of ORI are identified by combining activity‐based protein profiling (ABPP) and the cellular thermal shift assay (CETSA). As a result, ORI can significantly promote Lewis Lung Carcinoma (LLC) cell apoptosis, decrease mitochondrial membrane potential, and increase reactive oxygen species accumulation. In addition, both ABPP and CETSA indicate that ORI directly binds to key proteins of the glycolysis pathway, including Aldolase A (ALDOA), Enolase 1 (ENO1), pyruvate kinase M2, and lactate dehydrogenase. Based on the LLC mouse model, ORI markedly inhibits the growth of lung tumors, increases the serum levels of antioxidant enzymes catalase, superoxide dismutase, and glutathione peroxidase, and reduces the production of malondialdehyde. To sum up, ORI can be regarded as a novel antitumor strategy of NSCLC by inhibiting ALDOA‐ and ENO1‐mediated glycolysis.

The Role of α-Enolase on the Production of Interleukin (IL)-32 in Con A-Mediated Inflammation and Rheumatoid Arthritis (RA).

Interleukin (IL)-32 is produced by T lymphocytes, natural killer cells, monocytes, and epithelial cells. IL-32 induces the production of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, IL-1β, IL-6, and IL-8, and IL-32 expression is highly increased in rheumatoid arthritis (RA) patients. Enolase-1 (ENO1) is a glycolytic enzyme and the stimulation of ENO1 induces high levels of pro-inflammatory cytokines in concanavalin A (Con A)-activated peripheral blood mononuclear cells (PBMCs) and macrophages in RA patients. In addition, there are many reports that anti-ENO1 antibody is correlated with the disease progression of RA. It implies that ENO1 could regulate IL-32 production during inflammation related to the pathogenesis of RA. Therefore, we investigated the role of ENO1 in IL-32 production using Con A-activated PBMCs and RA PBMCs. IL-32 expression is increased by ENO1 stimulation using real-time PCR and ELISA. In addition, we confirmed that IL-32 production was decreased in Con A-activated PBMCs and RA PBMCs pre-treated with NF-κB or p38 MAPK pathway inhibitors. Taken together, these results suggest that ENO1 plays an important role in inflammation through the induction of IL-32 production by the activation of the NF-κB and p38 MAPK pathways.

Effect of Shoutai Pills on expression of key glycolytic proteins and apoptosis related factors at maternal fetal interface in mouse model of threatened abortion with syndrome of kidney deficiency

This study aims to explore the mechanism of Shoutai Pills in treating threatened abortion. According to the random number table method, ICR female mice were randomized into a normal group, a model group, a dydrogesterone group, and a Shoutai Pills group, with 15 mice in each group. Mice were administrated with normal saline(normal and model groups) or the suspension of Shoutai Pills or dydrogesterone by gavage at 9:00 am every day. At 16:00 every day, mice in the normal group were administrated with an equal volume of distilled water, while those in the model, Shoutai Pills, and dydrogesterone groups were administrated with hydrocortisone solution by gavage for 4 consecutive days. ICR female and male mice were caged in a ratio of 2∶1 during the pre-estrous or estrous period. From the first day of pregnancy, drug administration was continued for 5 consecutive days. On day 6, mice were administrated with mifepristone by gavage to establish the model of kidney deficiency-induced abortion. On day 6 of pregnancy, 10 female ICR mice were randomly selected from each group, and the uterus was collected for observation of the pathological changes of trophoblasts at the maternal-fetal interface by hematoxylin-eosin(HE) staining. The protein levels of key enzymes of glycolysis, hexokinase 2(HK2), enolase 1(ENO1), pyruvate kinase M2(PKM2), and lactate dehydrogenase A(LDHA), were determined by Western blot and immunofluorescence. The expression of apoptosis-related proteins including B cell lymphoma-2(Bcl-2), Bcl-2-associated protein X(Bax), and cysteinyl aspartate-specific proteinase-3(caspase-3) was determined by Western blot and real-time PCR. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling was employed to examine apoptosis. The embryo loss rate of the remaining five female mice was calculated by trypan blue staining method on day 14 of pregnancy. On day 14 of pregnancy, the embryo loss rate of the normal group was 5.00%, which was lower than that(27.78%) in the model group(P<0.05). Dydrogesterone and Shoutai Pills groups showed reduced embryo loss rates(10.26% and 7.50%, respectively) compared with the model group. On day 6 of pregnancy, compared with the normal group, the model group showed down-regulated expression of HK2, ENO1, PKM2, LDHA, and Bcl-2 and up-regulated expression of Bax and caspase-3(P<0.05). Compared with the model group, dydrogesterone and Shoutai Pills up-regulated the expression of HK2, ENO1, PKM2, LDHA, and Bcl-2 and down-regulated the expression of Bax and caspase-3(P<0.05). Compared with that in the normal group, the apoptosis rate in the model group increased(P<0.05). Compared with the model group, dydrogesterone and Shoutai Pills reduced the apoptosis rate(P<0.05). In conclusion, Shoutai Pills can reduce the embryo loss rate and protect embryos by promoting aerobic glycolysis at the maternal-fetal interface and inhibiting the apoptosis of trophoblasts in mice.

Abstract 4697: Surface localization of enolase-1 (ENO-1) as an attractive theranostic target in docetaxel-resistant neuroendocrine-like prostate cancer cells

Abstract Prostate cancer (PCa) is American men's second most common cancer. Although taxane-based chemotherapy is the last line of defense for Metastatic Castration Resistant Prostate Cancer (mCRPC), it invariably fails due to chemoresistance. The protein-specific membrane antigen (PSMA) has been an effective target for the imaging and therapy of mCRPC. Although PSMA radioligand therapy (PSMA-RLT) is a theranostics option for men with advanced PCa, about 30% have a limited response due to neuroendocrine-like PCa (NEPC), which lacks PSMA expression. Enolase (ENO) is a promising theranostic alternative due to its cell surface localization in many human tumors. We observed that chemosensitive PCa cell lines expressing NEPC markers express both ENO-1 and ENO-2; however, docetaxel-resistant NEPC-like cells only express ENO-1 and have a metabolic vulnerability due to the loss of ENO-2. We hypothesize that ENO-1 is expressed on the surface of NEPC-like cell lines and can be targeted with small molecule inhibitors (SMIs) that could potentially be used as theranostics agents. Additionally, we have observed changes in the expression and localization of ENO-1 in NEPC-like cell lines under different glucose concentrations. Our data show that under high glucose conditions, found on metabolically active metastatic tumors, ENO-1 is highly expressed on the cell surface, making it a promising candidate target for theranostics. However, low glucose conditions inhibit the activity of the c-MYC oncogene resulting in ENO-1 downregulation and upregulation of MBP1, the small splice variant of ENO1 that blocks the transcriptional activity of c-MYC. Our efforts to confirm ENO-1 surface expression on NEPC-like cell lines entailed using confocal microscopy, cell fractionation analysis followed by Western blotting, and Flow cytometric cell surface staining. We also evaluated the cytotoxicity of SMIs designed to target ENO-1 in chemoresistant NEPC-like cell lines using MTT viability assays, clonogenic assays, and Hoffman Modulation microscopy imaging. Our long-term goal is to identify an alternative treatment for patients with NEPC by establishing ENO-1 as a novel theranostics target. Citation Format: Krystal Rubi Santiago Torres, Carlos A. Casiano, Frankis Almaguel, Alfonso Duran, Kristen Whitley. Surface localization of enolase-1 (ENO-1) as an attractive theranostic target in docetaxel-resistant neuroendocrine-like prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4697.