Pyruvate kinase M2 sustains cardiac mitochondrial integrity in septic cardiomyopathy by regulating PHB2-dependent mitochondrial biogenesis.

Previous studies have highlighted the protective effects of pyruvate kinase M2 (PKM2) overexpression in septic cardiomyopathy. In our study, we utilized cardiomyocyte-specific PKM2 knockout mice to further investigate the role of PKM2 in attenuating LPS-induced myocardial dysfunction, focusing on mitochondrial biogenesis and prohibitin 2 (PHB2). Our findings confirmed that the deletion of PKM2 in cardiomyocytes significantly exacerbated LPS-induced myocardial dysfunction, as evidenced by impaired contractile function and relaxation. Additionally, the deletion of PKM2 intensified LPS-induced myocardial inflammation. At the molecular level, LPS triggered mitochondrial dysfunction, characterized by reduced ATP production, compromised mitochondrial respiratory complex I/III activities, and increased ROS production. Intriguingly, the absence of PKM2 further worsened LPS-induced mitochondrial damage. Our molecular investigations revealed that LPS disrupted mitochondrial biogenesis in cardiomyocytes, a disruption that was exacerbated by the absence of PKM2. Given that PHB2 is known as a downstream effector of PKM2, we employed PHB2 adenovirus to restore PHB2 levels. The overexpression of PHB2 normalized mitochondrial biogenesis, restored mitochondrial integrity, and promoted mitochondrial function. Overall, our results underscore the critical role of PKM2 in regulating the progression of septic cardiomyopathy. PKM2 deficiency impeded mitochondrial biogenesis, leading to compromised mitochondrial integrity, increased myocardial inflammation, and impaired cardiac function. The overexpression of PHB2 mitigated the deleterious effects of PKM2 deletion. This discovery offers a novel insight into the molecular mechanisms underlying septic cardiomyopathy and suggests potential therapeutic targets for intervention.

Pyruvate kinase 2 from Synechocystis sp. PCC 6803 increased substrate affinity via glucose-6-phosphate and ribose-5-phosphate for phosphoenolpyruvate consumption.

Pyruvate kinase (Pyk, EC 2.7.1.40) is a glycolytic enzyme that generates pyruvate and adenosine triphosphate (ATP) from phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP), respectively. Pyk couples pyruvate and tricarboxylic acid metabolisms. Synechocystis sp. PCC 6803 possesses two pyk genes (encoded pyk1, sll0587 and pyk2, sll1275). A previous study suggested that pyk2 and not pyk1 is essential for cell viability; however, its biochemical analysis is yet to be performed. Herein, we biochemically analyzed Synechocystis Pyk2 (hereafter, SyPyk2). The optimum pH and temperature of SyPyk2 were 7.0 and 55 °C, respectively, and the Km values for PEP and ADP under optimal conditions were 1.5 and 0.053 mM, respectively. SyPyk2 is activated in the presence of glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P); however, it remains unaltered in the presence of adenosine monophosphate (AMP) or fructose-1,6-bisphosphate. These results indicate that SyPyk2 is classified as PykA type rather than PykF, stimulated by sugar monophosphates, such as G6P and R5P, but not by AMP. SyPyk2, considering substrate affinity and effectors, can play pivotal roles in sugar catabolism under nonphotosynthetic conditions.

PKM2 promotes myoblast growth and inosine monophosphate-specific deposition in Jingyuan chicken.

Inosine monophosphate (IMP) is widely regarded as an important indicator for evaluating the flavour of poultry meat. However, little is known about the molecular mechanisms affecting the specific deposition of IMP. In this study, we functionally verified PKM2 (Pyruvate kinase M2), a candidate gene related to IMP synthesis, in order to reveal the important role of PKM2 in meat flavour and muscle development of Jingyuan chickens. The results showed that the IMP content in breast muscle of Jingyuan chickens was negatively correlated with PKM2 mRNA expression (r = -0.1710), while the IMP content in leg muscle was significantly positively correlated with PKM2 mRNA expression (r = 0.7350) (P < 0.05). During myogenesis, PKM2 promoted the proliferation rate of myoblasts and the expression of proliferation marker genes, inhibited the apoptosis rate and the expression of apoptosis marker genes, and decreased the expression of differentiation marker genes. Up-regulation of PKM2 enhanced the expression of key genes in the purine metabolic pathway and the de novo synthesis pathway of IMP, and suppressed the expression of key genes in the salvage pathway. ELISA assays showed that PKM2 decreased IMP and hypoxanthine (HX) contents, while adenosine triphosphate (ATP) and uric acid (UA) contents were clearly elevated. In summary, these studies revealed that PKM2 regulates myogenesis and specific deposition of IMP, which can be used to improve the quality of Jingyuan chicken meat.

Lung-specific interleukin 6 mediated transglutaminase 2 activation and cardiopulmonary fibrogenesis.

Pulmonary hypertension (PH) pathogenesis is driven by inflammatory and metabolic derangements as well as glycolytic reprogramming. Induction of both interleukin 6 (IL6) and transglutaminase 2 (TG2) expression participates in human and experimental cardiovascular diseases. However, little is known about the role of TG2 in these pathologic processes. The current study aimed to investigate the molecular interactions between TG2 and IL6 in mediation of tissue remodeling in PH. A lung-specific IL6 over-expressing transgenic mouse strain showed elevated right ventricular (RV) systolic pressure as well as increased wet and dry tissue weights and tissue fibrosis in both lungs and RVs compared to age-matched wild-type littermates. In addition, IL6 over-expression induced the glycolytic and fibrogenic markers, hypoxia-inducible factor 1α, pyruvate kinase M2 (PKM2), and TG2. Consistent with these findings, IL6 induced the expression of both glycolytic and pro-fibrogenic markers in cultured lung fibroblasts. IL6 also induced TG2 activation and the accumulation of TG2 in the extracellular matrix. Pharmacologic inhibition of the glycolytic enzyme, PKM2 significantly attenuated IL6-induced TG2 activity and fibrogenesis. Thus, we conclude that IL6-induced TG2 activity and cardiopulmonary remodeling associated with tissue fibrosis are under regulatory control of the glycolytic enzyme, PKM2.

CircPHGDH downregulation decreases papillary thyroid cancer progression through miR-122-5p/PKM2 axis.

BACKGROUND: Although papillary thyroid carcinoma (PTC) has a favorable prognosis, it could affect patient life quality and become a serious threat because of invasion and metastasis. Many investigations have suggested that circular RNAs (circRNAs) are involved in different cancer regulations. Nevertheless, circRNAs role in invasive PTC remains unclear. METHODS: In the present investigation, next-generation sequencing was applied to explore abnormal circRNA expression. The expression of circRNA phosphoglycerate dehydrogenase (circPHGDH) in PTC cell lines and tissues were examined. Then, we investigated regulatory mechanism and circPHGDH downstream targets using bioinformatics analysis and luciferase reporting analysis. Then transwell migration, Cell Counting Kit-8 (CCK8) and 5-ethynyl-2'-deoxyuridine (EdU) assays were used for cells migration and proliferation analysis. In vivo metastasis and tumorigenesis assays were also employed to evaluate the circPHGDH role in PTC. RESULTS: The data showcased that circPHGDH expression increased in both PTC cell lines and tissues, which suggested that circPHGDH functions in PTC progression. circPHGDH downregulation suppressed PTC invasion and proliferation in both in vivo and in vitro experiments. Bioinformatics and luciferase reporter results confirmed that both microRNA (miR)-122-5p and pyruvate kinase M2 subtype (PKM2) were downstream targets of circPHGDH. PKM2 overexpression or miR-122-5p suppression reversed PTC cell invasion and proliferation post silencing circPHGDH by restoring aerobic glycolysis. CONCLUSION: Taken together, our research found that circPHGDH downregulation reduced PTC progression via miR-122-5p/PKM2 axis regulation mediated by aerobic glycolysis.

Exploring the diverse role of pyruvate kinase M2 in cancer: Navigating beyond glycolysis and the Warburg effect.

Pyruvate Kinase M2, a key enzyme in glycolysis, has garnered significant attention in cancer research due to its pivotal role in the metabolic reprogramming of cancer cells. Originally identified for its association with the Warburg effect, PKM2 has emerged as a multifaceted player in cancer biology. The functioning of PKM2 is intricately regulated at multiple levels, including controlling the gene expression via various transcription factors and non-coding RNAs, as well as adding post-translational modifications that confer distinct functions to the protein. Here, we explore the diverse functions of PKM2, encompassing newly emerging roles in non-glycolytic metabolic regulation, immunomodulation, inflammation, DNA repair and mRNA processing, beyond its canonical role in glycolysis. The ever-expanding list of its functions has recently grown to include roles in subcellular compartments such as the mitochondria and extracellular milieu as well, all of which make PKM2 an attractive drug target in the pursuit of therapeutics for cancer.

Restricted glycolysis is a primary cause of the reduced growth rate of zinc-deficient yeast cells.

Zinc is required for many critical processes, including intermediary metabolism. In Saccharomyces cerevisiae, the Zap1 activator regulates the transcription of ∼80 genes in response to Zn supply. Some Zap1-regulated genes are Zn transporters that maintain Zn homeostasis, while others mediate adaptive responses that enhance fitness. One adaptive response gene encodes the 2-cysteine peroxiredoxin Tsa1, which is critical to Zn-deficient (ZnD) growth. Depending on its redox state, Tsa1 can function as a peroxidase, a protein chaperone, or a regulatory redox sensor. In a screen for possible Tsa1 regulatory targets, we identified a mutation (cdc19S492A) that partially suppressed the tsa1Δ growth defect. The cdc19S492A mutation reduced activity of its protein product, pyruvate kinase isozyme 1 (Pyk1), implicating Tsa1 in adapting glycolysis to ZnD conditions. Glycolysis requires activity of the Zn-dependent enzyme fructose-bisphosphate aldolase 1, which was substantially decreased in ZnD cells. We hypothesized that in ZnD tsa1Δ cells, the loss of a compensatory Tsa1 regulatory function causes depletion of glycolytic intermediates and restricts dependent amino acid synthesis pathways, and that the decreased activity of Pyk1S492A counteracted this depletion by slowing the irreversible conversion of phosphoenolpyruvate to pyruvate. In support of this model, supplementing ZnD tsa1Δ cells with aromatic amino acids improved their growth. Phosphoenolpyruvate supplementation, in contrast, had a much greater effect on growth rate of WT and tsa1Δ ZnD cells, indicating that inefficient glycolysis is a major factor limiting yeast growth. Surprisingly however, this restriction was not primarily due to low fructose-bisphosphate aldolase 1 activity, but instead occurs earlier in glycolysis.

The role of PKM2 in cancer progression and its structural and biological basis.

Pyruvate kinase M2 (PKM2), a subtype of pyruvate kinase (PK), has been shown to play an important role in the development of cancer. It regulates the last step of glycolytic pathway. PKM2 has both pyruvate kinase and protein kinase activity, and the conversion of these two functions of PKM2 depends on the mutual change of dimer and tetramer. The dimerization of PKM2 can promote the proliferation and growth of tumor cells, so inhibiting the dimerization of PKM2 is essential to curing cancer. The aggregation of PKM2 is regulated by both endogenous and exogenous cofactors as well as post-translational modification (PTM). Although there are many studies on the different aggregation of PKM2 in the process of tumor development, there are few summaries in recent years. In this review, we first introduce the role of PKM2 in various biological processes of tumor growth. Then, we summarize the aggregation regulation mechanism of PKM2 by various endogenous cofactors such as Fructose-1, 6-diphosphate (FBP), various amino acids, and post-translational modification (PTMs). Finally, the related inhibitors and agonists of PKM2 are summarized to provide reference for regulating PKM2 aggregation in the treatment of cancer in the future.

The HIF-1α/PKM2 Feedback Loop in Relation to EGFR Mutational Status in Lung Adenocarcinoma.

OBJECTIVE: Gene mutations in tumor cells can lead to several unique metabolic phenotypes, which are crucial for the proliferation of cancer cells. EGFR mutation (EGFR-mt) is the main oncogenic driving mutation in lung adenocarcinoma (LUAD). HIF-1 α and PKM2 are two key metabolic regulatory proteins that can form a feedback loop and promote cancer growth by promoting glycolysis. Here, the linkage between EGFR mutational status and HIF-1α/PKM2 feedback loop in LUAD were evaluated. METHODS: Retrospective study were performed on LUAD patients (n = 89) undergoing first-time therapeutic surgical resection. EGFR mutation was analyzed by real-time PCR. Immunohistochemistry was used to measure the expressions of HIF-1α and PKM2. RESULTS: We found that the protein expressions of HIF-1α and PKM2 were significantly higher in LUAD than normal lung tissues. In adenocarcinomas, the two protein expressions were both correlated with worse pTNM stage. Moreover, the correlation between the proteins of HIF-1α/PKM2 feedback loop and the EGFR mutational status were also analyzed. We found that EGFR-mt tumors showed higher HIF-1α and PKM2 proteins compared to tumors with EGFR wild-type. Meanwhile, HIF-1α expression was significantly correlated with higher pTNM stage, and PKM2 showed a similar trend, only in EGFR-mutated tumors. The expression of HIF-1α was positively correlated with PKM2 in LUAD, furthermore, this correlation was mainly in patients with EGFR-mt. CONCLUSION: Different expression and clinical features of HIF-1α/PKM2 feedback loop was existed between LUAD and normal lung tissues, especially in EGFR mutational tumors, supporting the relationship between EGFR mutation and the key related proteins of aerobic glycolysis (HIF-1α and PKM2) in lung adenocarcinomas.

[RBMX overexpression inhibits proliferation, migration, invasion and glycolysis of human bladder cancer cells by downregulating PKM2].

OBJECTIVE: To investigate the role of RNA-binding motif protein X-linked (RBMX) in regulating the proliferation, migration, invasion and glycolysis in human bladder cancer cells. METHODS: A lentivirus vectors system and RNA interference technique were used to construct bladder cancer 1376 and UC-3 cell models with RBMX overexpression and knockdown, respectively, and successful cell modeling was verified using RT-qPCR and Western blotting. Proliferation and colony forming ability of the cells were evaluated using EdU assay and colony-forming assay, and cell migration and invasion abilities were determined using Transwell experiment. The expressions of glycolysis-related proteins M1 pyruvate kinase (PKM1) and M2 pyruvate kinase (PKM2) were detected using Western blotting. The effects of RBMX overexpression and knockdown on glycolysis in the bladder cancer cells were assessed using glucose and lactic acid detection kits. RESULTS: RT-qPCR and Western blotting confirmed successful construction of 1376 and UC-3 cell models with RBMX overexpression and knockdown. RBMX overexpression significantly inhibited the proliferation, clone formation, migration and invasion of bladder cancer cells, while RBMX knockdown produced the opposite effects. Western blotting results showed that RBMX overexpression increased the expression of PKM1 and decreased the expression of PKM2, while RBMX knockdown produced the opposite effects. Glucose consumption and lactate production levels were significantly lowered in the cells with RBMX overexpression (P < 0.05) but increased significantly following RBMX knockdown (P < 0.05). CONCLUSION: RBMX overexpression inhibits bladder cancer progression and lowers glycolysis level in bladder cancer cells by downregulating PKM2 expression, suggesting the potential of RBMX as a molecular target for diagnosis and treatment of bladder cancer.

LncRNA PWRN1 inhibits the progression of hepatocellular carcinoma by activating PKM2 activity.

Hepatocellular carcinoma (HCC) is one of the most prevalent and leading causes of cancer-related mortality worldwide. Long non-coding RNAs (lncRNAs) have been demonstrated to play vital roles in cancer development and progression. The lncRNA PWRN1 (PWRN1), acts as a tumor suppressor factor, which is low expressed in some cancers. However, the molecular mechanisms underlying the effects of PWRN1, especially the regulatory relationship with RNA binding protein in HCC remain largely unknown. In the present study, we demonstrated that PWRN1 was significantly down-regulated in HCC and correlated with better prognosis; furthermore, gain-of-function experiments showed that PWRN1 inhibited the proliferation of HCC cells. We further found that PWRN1 up-regulated pyruvate kinase activity and thus hinders the proliferation of HCC in vitro and in vivo. Mechanistically, pyruvate kinase M2 (PKM2) was bound to it and maintained the high activity state of PKM2, thereby hindering PKM2 from entering the nucleus in the form of low-activity dimers, reducing the expression of c-Myc downstream gene LDHA, leading to a decrease in lactate levels, and inhibiting the growth of tumor cells. In addition, PWRN1 was found to inhibit aerobic glycolysis. Finally, TEPP-46, a pyruvate kinase activator, appeared to inhibit HCC proliferation by maintaining tetramer stability and increasing pyruvate kinase activity. Taken together, our results provide new insights into the biology hindering HCC proliferation and indicate that PWRN1 in combination with PKM2 activators might represent a novel therapeutic target for HCC.

MEK1/2-PKM2 Pathway Modulates the Immunometabolic Reprogramming of Proinflammatory Allograft-infiltrating Macrophages During Heart Transplant Rejection.

BACKGROUND: Emerging evidence has highlighted the role of macrophages in heart transplant rejection (HTR). However, the molecular signals modulating the immunometabolic phenotype of allograft-infiltrating macrophages (AIMs) during HTR remain unknown. METHODS: We analyzed single-cell RNA sequencing data from cardiac graft-infiltrating immunocytes to characterize the activation patterns and metabolic features of AIMs. We used flow cytometry to determine iNOS and PKM2 expression and MEK/ERK signaling activation levels in AIMs. We then generated macrophage-specific Mek1/2 knockout mice to determine the role of the MEK1/2-PKM2 pathway in the proinflammatory phenotype and glycolytic capacity of AIMs during HTR. RESULTS: Single-cell RNA sequencing analysis showed that AIMs had a significantly elevated proinflammatory and glycolytic phenotype. Flow cytometry analysis verified that iNOS and PKM2 expressions were significantly upregulated in AIMs. Moreover, MEK/ERK signaling was activated in AIMs and positively correlated with proinflammatory and glycolytic signatures. Macrophage-specific Mek1/2 deletion significantly protected chronic cardiac allograft rejection and inhibited the proinflammatory phenotype and glycolytic capacity of AIMs. Mek1/2 ablation also reduced the proinflammatory phenotype and glycolytic capacity of lipopolysaccharides + interferon-γ-stimulated macrophages. Mek1/2 ablation impaired nuclear translocation and PKM2 expression in macrophages. PKM2 overexpression partially restored the proinflammatory phenotype and glycolytic capacity of Mek1/2 -deficient macrophages. Moreover, trametinib, an Food and Drug Administration-approved MEK1/2 inhibitor, ameliorated chronic cardiac allograft rejection. CONCLUSIONS: These findings suggest that the MEK1/2-PKM2 pathway is essential for immunometabolic reprogramming of proinflammatory AIMs, implying that it may be a promising therapeutic target in clinical heart transplantation.

Pyruvate Kinase Differentially Alters Metabolic Signatures during Head and Neck Carcinogenesis.

During glycolysis, the muscle isoform of pyruvate kinase PKM2 produces ATP in exchange for dephosphorylation of phosphoenolpyruvate (PEP) into pyruvate. PKM2 has been considered as a tumor-promoting factor in most cancers, whereas the regulatory role of PKM2 during head and neck carcinogenesis remained to be delineated. PKM2 mRNA and protein expression was examined in head and neck tumorous specimens. The role of PKM2 in controlling cellular malignancy was determined in shRNA-mediated PKM2-deficient head and neck squamous cell carcinoma (HNSC) cells. In agreement with the results in other cancers, PKM2 expression is enriched in both mouse and human HNSC tissues. Nevertheless, PKM2 mRNA expression reversely correlated with tumor stage, and greater recurrence-free survival rates are evident in the PKM2high HNSC population, arguing that PKM2 may be tumor-suppressive. Multifaceted analyses showed a greater in vivo xenografic tumor growth and an enhanced cisplatin resistance in response to PKM2 loss, whereas PKM2 silencing led to reduced cell motility. At the molecular level, metabolic shifts towards mitochondrial metabolism and activation of oncogenic Protein kinase B (PKB/Akt) and extracellular signal-regulated kinase (ERK) signals were detected in PKM2-silencing HNSC cells. In sum, our findings demonstrated that PKM2 differentially modulated head and neck tumorigenicity via metabolic reprogramming.

Pyruvate kinase activators: targeting red cell metabolism in thalassemia.

Thalassemia is an inherited red blood cell disorder whereby the qualitative and/or quantitative imbalance in α- to ß-globin ratio results in hemolysis and ineffective erythropoiesis. Oxidative stress, from the precipitated excess globin and free iron, is a major factor that drives hemolysis and ineffective erythropoiesis. Pyruvate kinase activity and adenosine triphosphate availability are reduced due to the overwhelmed cellular antioxidant system from the excessive oxidative stress. Mitapivat, a pyruvate kinase activator in development as a treatment for thalassemia, was shown to increase hemoglobin and reduce hemolysis in a small phase 2 single-arm trial of patients with α- and ß-thalassemia. The ongoing phase 3 studies with mitapivat and the phase 2 study with etavopivat will examine the role of pyruvate kinase activators as disease modifying agents in thalassemia.

Role in post-translational modification of M2-type pyruvate kinase in tumorigenesis and development. / M2åž‹ä¸™é ®é ¸æ¿€é ¶ç¿»è¯‘åŽä¿®é¥°åœ¨è‚¿ç˜¤å‘ç”Ÿå’Œå‘å±•ä¸­çš„ä½œç”¨.

PKM2, also known as M2-type pyruvate kinase, has attracted significant attention due to its crucial role in glycolysis and its abnormal expression in various tumors. With the discovery of PKM2's non-metabolic functions, the transition between its pyruvate kinase activity (in the tetrameric form in the cytoplasm) and protein kinase activity (in the dimeric form in the nucleus) has once again made PKM2 a target of interest in cancer research. Studies have shown that PKM2 is a protein susceptible to various post-translational modifications, and different post-translational modifications play important regulatory roles in processes such as PKM2 cellular localization, structure, and enzyme activity conversion. In this review, we focused on the recent progress of multiple post-translational modifications of PKM2 and their important roles in tumor initiation and development. For example, phosphorylation and acetylation promote nuclear translocation by altering PKM2 cell localization; glycosylation and ubiquitination can promote the formation of dimer structure by affecting the structural transformation of PKM2; succinylation and redox modification promoted the enhancement of PKM2 kinase activity by affecting the transformation of kinase activity. Both changes affect the structure and cell localization of PKM2 and they play a role in promoting or inhibiting tumor development via altering its kinase activity.

PKM2 is a potential prognostic biomarker and related to immune infiltration in lung cancer.

Pyruvate kinase M2 (PKM2), a subtype of pyruvate kinase, plays a crucial role as a key enzyme in the final step of glycolysis. It is involved in regulating the tumor microenvironment and accelerating tumor progression. However, the relationship between PKM2 expression and the prognosis and immune infiltration remains unclear in lung cancer. In this study, we analyzed PKM2 expression in pan-cancer, and investigated its association with prognosis and immune cell infiltration of lung cancer by using multiple online databases, including Gent2, Tumor Immune Estimation Resource (TIMER), Gene Expression Profiling Interactive Analysis (GEPIA), PrognoScan, Kaplan-Meier plotter, and The Human Protein Atlas (HPA). The results showed that PKM2 expression is elevated in tumor tissues compared with the adjacent normal tissues of most cancers, including lung cancer. Prognostic analysis indicated that high expression of PKM2 was associated with poorer prognosis in overall lung cancer patients, especially in lung adenocarcinoma (LUAD). Notably, PKM2 exhibited a strong correlation with B cells and CD4+ T cells in LUAD; and with B cells, CD8+ T cells, CD4+ cells, and macrophages in lung squamous cell carcinoma (LUSC). Furthermore, PKM2 expression displayed a significant negative correlation with the expression of immune cell markers in both LUAD and LUSC. These findings suggested that PKM2 could serve as a promising prognostic biomarker for lung cancer and provided insights into its essential role in modulating the immune cell infiltration.

Transcription factor LHX9 (LIM Homeobox 9) enhances pyruvate kinase PKM2 activity to induce glycolytic metabolic reprogramming in cancer stem cells, promoting gastric cancer progression.

BACKGROUND: Glycolytic metabolic reprogramming is a phenomenon in which cells undergo altered metabolic patterns during malignant transformation, mainly involving various aspects of glycolysis, electron transport chain, oxidative phosphorylation, and pentose phosphate pathway. This reprogramming phenomenon can be used as one of the markers of tumorigenesis and development. Pyruvate kinase is the third rate-limiting enzyme in the sugar metabolism process by specifically catalyzing the irreversible conversion of PEP to pyruvate. PURPOSE: This study aimed to reveal the critical mediator(s) that regulate glycolytic metabolism reprogramming in gastric cancer and their underlying molecular mechanism and then explore the molecular mechanisms by which LHX9 may be involved in regulating gastric cancer (GC) progression. METHODS: Firstly, we downloaded the GC and glycolysis-related microarray datasets from TCGA and MSigDB databases and took the intersection to screen out the transcription factor LHX9 that regulates GC glycolytic metabolic reprogramming. Software packages were used for differential analysis, single gene predictive analysis, and Venn diagram. In addition, an enrichment analysis of the glycolytic pathway was performed. Immunohistochemical staining was performed for LHX9 and PKM2 protein expression in 90 GC patients, and the association between their expressions was evaluated by Spearman's correlation coefficient method. Three human GC cell lines (AGS, NCI-N87, HGC-27) were selected for in vitro experimental validation. Flow cytometry was utilized to determine the stem cell marker CD44 expression status in GCSCs. A sphere formation assay was performed to evaluate the sphere-forming capabilities of GCSCs. In addition, RT-qPCR and Western blot experiments were employed to investigate the tumor stem cell markers OCT4 and SOX2 expression levels in GCSCs. Furthermore, a lentiviral expression vector was constructed to assess the impact of downregulating LHX9 or PKM2 on the glycolytic metabolic reprogramming of GCSCs. The proliferation, migration, and invasion of GCSCs were then detected by CCK-8, EdU, and Transwell assays. Subsequently, the mutual binding of LHX9 and PKM2 was verified using chromatin immunoprecipitation and dual luciferase reporter genes. In vivo experiments were verified by establishing a subcutaneous transplantation tumor model in nude mice, observing the size and volume of tumors in vivo in nude mice, and obtaining fresh tissues for subsequent experiments. RESULTS: Bioinformatics analysis revealed that LHX9 might be involved in the occurrence and development of GC through regulating glycolytic metabolism. High LHX9 expression could be used as a reference marker for prognosis prediction of GC patients. Clinical tissue assays revealed that LHX9 and PKM2 were highly expressed in GC tissues. Meanwhile, GC tissues also highly expressed glycolysis-associated protein GLUT1 and tumor cell stemness marker CD44. In vitro cellular assays showed that LHX9 could enhance its activity and induce glycolytic metabolic reprogramming in GCSCs through direct binding to PKM2. In addition, the knockdown of LHX9 inhibited PKM2 activity and glycolytic metabolic reprogramming and suppressed the proliferation, migration, and invasive ability of GCSCs. In vivo animal experiments further confirmed that the knockdown of LHX9 could reduce the tumorigenic ability of GCSCs in nude mice by inhibiting PKM2 activity and glycolytic metabolic reprogramming. CONCLUSION: The findings suggest that both LHX9 and PKM2 are highly expressed in GCs, and LHX9 may induce the reprogramming of glycolytic metabolism through transcriptional activation of PKM2, enhancing the malignant biological properties of GCSCs and ultimately promoting GC progression.

Mitapivat: A Quinolone Sulfonamide to Manage Hemolytic Anemia in Adults With Pyruvate Kinase Deficiency.

BACKGROUND: Pyruvate kinase (PK) deficiency is a rare enzyme-linked glycolytic defect resulting in mild-to-severe chronic persistent erythrocyte hemolysis. The disease is an autosomal recessive trait caused by mutations in the PK liver and red blood cell gene characterized by insufficient erythrocyte PK activity. PK deficiency is most diagnosed in persons of northern European descent and managed with packed red blood cell transfusions, chelation, and splenectomy with cholecystectomy. Mitapivat is the first approved therapy indicated for hemolytic anemia in adults with PK deficiency with the potential for delaying splenectomy in mild-moderate disease. MECHANISM OF ACTION, PHARMACODYNAMICS, AND PHARMACOKINETICS: Mitapivat is a PK activator that acts by allosterically binding to the PK tetramer and increases PK activity. The red blood cell form of PK is mutated in PK deficiency, which leads to reduced adenosine triphosphate, shortened red blood cell lifespan, and chronic hemolysis. The half-life of elimination is 3-5 hours, with 73% bioavailability, 98% plasma protein binding, and a median duration of response of 7 months. CLINICAL TRIALS: Mitapivat has been investigated through various clinical trials for different therapeutic indications. Pivotal trials that serve the primary focus throughout this article are ACTIVATE, ACTIVATE-T, and RISE. ACTIVATE is a phase 3, randomized, double-blind, placebo-controlled study that evaluated the efficacy and safety of mitapivat in adult patients who were not receiving regular blood transfusions. Contrarily, ACTIVATE-T explored the safety and efficacy of mitapivat in adults with PK deficiency who received regular blood transfusions. Both trials demonstrated favorable use of mitapivat in PK deficiency. Focusing on another indication, the ongoing RISE trial investigates the optimal dosage of mitapivat in sickle cell disease. THERAPEUTIC ADVANCE: Mitapivat is an appropriate treatment for adults with PK deficiency requiring transfusions and may be considered for patients with symptomatic anemia who do not require transfusions and/or PK deficiency with compensated hemolysis without overt anemia.

Extracellular pyruvate kinase M2 induces cell migration through p-Tyr42 RhoA-mediated superoxide generation and epithelial-mesenchymal transition.

In the tumor microenvironment (TME), communication between cancer cells and tumor-associated macrophages (TAMs) through secreted extracellular proteins promotes cancer progression. Here, we observed that co-culturing cancer cells (4T1) and macrophage cells (Raw264.7) significantly enhanced superoxide production in both cell types. Using MALDI-TOF, we identified PKM2 as a highly secreted protein by Raw264.7 cells and bone marrow-derived monocytes. The extracellular recombinant PKM2 protein not only enhanced cancer cell migration and invasion but also increased superoxide production. Additionally, PKM2 was found to associate with the cell surface, and its binding to integrin α5/ß1 receptor was inhibited by antibodies specifically targeting it. Furthermore, we investigated downstream signaling pathways involved in PKM2-induced superoxide production. We found that knock-down of RhoA and p47phox using siRNAs effectively abolished superoxide generation in response to extracellular PKM2. Notably, extracellular PKM2 triggered the phosphorylation of p47phox at Ser345 residue and RhoA at Tyr42 residue (p-Tyr42 RhoA). Moreover, extracellular PKM2 exerted regulatory control over the expression of key epithelial-mesenchymal transition (EMT) markers, including ZEB1, Snail1, vimentin, and E-cadherin. Interestingly, p-Tyr42 RhoA translocated to the nucleus, where it bound to the ZEB1 promoter region. In light of these findings, we propose that extracellular PKM2 within the TME plays a critical role in tumorigenesis by promoting cancer cell migration and invasion through RhoA/p47phox signaling pathway.

The interaction between apigenin and PKM2 restrains progression of colorectal cancer.

Apigenin, a flavonoid that widely existed in vegetables and fruits, possesses anticarcinogenic, low toxicity, and no mutagenic properties, suggesting that apigenin is a potential therapeutic agent for tumors. However, the underlying anti-cancer molecular target of apigenin is still unclear. Therefore, to reveal the direct target and amino acid site of apigenin against colorectal cancer is the focus of this study. In the present study, the results proved that the anti-CRC activity of apigenin was positively correlated with pyruvate kinase M2 (PKM2) expression, characterized by the inhibition of cell proliferation and increase of apoptotic effects induced by apigenin in LS-174T cells of knock down PKM2. Next, pull-down and MALDI-TOF/TOF analysis determined that apigenin might interact directly with PKM2 in HCT-8 cells. Further, the study confirmed that lysine residue 433 (K433) was a key amino acid site for PKM2 binding to apigenin. Apigenin restricted the glycolysis of LS-174T and HCT-8 cells by targeting the K433 site of PKM2, thereby playing an anti-CRC role in vivo and in vitro. Meanwhile, apigenin markedly attenuated tumor growth without any adverse effects. Taken together, these findings reveal that apigenin is worthy of consideration as a promising PKM2 inhibitor for the prevention of CRC.