Pyruvate Kinase M2 Research Articles

SIRT3 suppression resulting from the enhanced β-catenin signaling drives glycolysis and promotes hypoxia-induced cell growth in hepatocellular carcinoma cells

ABSTRACT The precise mechanisms underlying the inhibitory effects of SIRT3, a mitochondrial sirtuin protein, on hepatocellular carcinoma (HCC) development, as well as its impact on mitochondrial respiration, remain poorly understood. We assessed sirtuins 3 (SIRT3) levels in HCC tissues and Huh7 cells cultured under hypoxic condition. We investigated the effects of SIRT3 on cell proliferation, glycolytic metabolism, mitochondrial respiration, mitophagy, and mitochondrial biogenesis in Huh7 cells. Besides, we explored the potential mechanisms regulating SIRT3 expression in hypoxically cultured Huh7 cells. Gradual reduction in SIRT3 expressions were observed in both adjacent tumor tissues and tumor tissues. Similarly, SIRT3 expressions were diminished in Huh7 cells cultured under hypoxic condition. Forced expression of SIRT3 attenuated the growth of hypoxically cultured Huh7 cells. SIRT3 overexpression led to a decrease in extracellular acidification rate while increasing oxygen consumption rate. SIRT3 downregulated the levels of hexokinase 2 and pyruvate kinase M2. Moreover, SIRT3 enhanced mitophagy signaling, as indicated by mtKeima, and upregulated key proteins involved in various mitophagic pathways while reducing intracellular reactive oxygen species levels. Furthermore, SIRT3 increased proxisome proliferator-activated receptor-gamma coactivator 1α levels and the amount of mitochondrial DNA in Huh7 cells. Notably, β-catenin expressions were elevated in Huh7 cells cultured under hypoxic condition. Antagonists and agonists of β-catenin respectively upregulated and downregulated SIRT3 expressions in hypoxically cultured Huh7 cells. The modulationsof glycolysis and mitochondrial respiration represent the primary mechanism through which SIRT3, suppressed by β-catenin, inhibits HCC cell proliferation.

ULK1-dependent phosphorylation of PKM2 antagonizes O-GlcNAcylation and regulates the Warburg effect in breast cancer.

Pyruvate kinase M2 (PKM2) is a central metabolic enzyme driving the Warburg effect in tumor growth. Previous investigations have demonstrated that PKM2 is subject to O-linked β-N-acetylglucosamine (O-GlcNAc) modification, which is a nutrient-sensitive post-translational modification. Here we found that unc-51 like autophagy activating kinase 1 (ULK1), a glucose-sensitive kinase, interacts with PKM2 and phosphorylates PKM2 at Ser333. Ser333 phosphorylation antagonizes PKM2 O-GlcNAcylation, promotes its tetramer formation and enzymatic activity, and decreases its nuclear localization. As PKM2 is known to have a nuclear role in regulating c-Myc, we also show that PKM2-S333 phosphorylation inhibits c-Myc expression. By downregulating glucose consumption and lactate production, PKM2 pS333 attenuates the Warburg effect. Through mouse xenograft assays, we demonstrate that the phospho-deficient PKM2-S333A mutant promotes tumor growth in vivo. In conclusion, we identified a ULK1-PKM2-c-Myc axis in inhibiting breast cancer, and a glucose-sensitive phosphorylation of PKM2 in modulating the Warburg effect.

Integrated metabolome, proteome, and transcriptome analysis explored the molecular mechanism of phosphoglycerate kinase 1 and pyruvate kinase M2 characterizing the postmortem meat quality

AbstractPhosphoglycerate kinase 1 (PGK1) and pyruvate kinase M2 (PKM2) have been identified as the postmortem meat quality biomarkers. However, the precise molecular mechanism through which they affect and regulate the development of meat quality remains unclear. In this work, the high‐ and low‐activity groups (n = 10) were selected from 60 lamb muscles at 24 h postmortem based on the activity levels of PGK1 and PKM2. The metabolomic, proteomic, and transcriptomic analyses combined with deeply integrated multi‐omics analysis were used to elucidate the mechanisms by which PGK1 and PKM2 characterize meat quality. The results indicated that glycolysis played a crucial role in regulating PGK1 and PKM2 activity at the metabolome, proteome, and transcriptome levels. In glycolysis pathway, we identified several key components closely related to PGK1 and PKM2 activity, including differential metabolites (adenosine triphosphate, adenosine diphosphate, glucose‐6‐phosphate, nicotinamide adenine dinucleotide phosphate, fructose‐6‐phosphate, dihydroxyacetone phosphate, 3‐phosphoglycerate, NAD+ nicotinamide adenine dinucleotide, lactate, and pyruvate), different abundance proteins (lactate dehydrogenase B and fructose bisphosphate aldolase B), and differentially expressed genes (hexokinase and fructose‐1,6‐bisphosphatase 1). It was concluded that PGK1 and PKM2 may affect the formation of meat quality by regulating these critical substrates. Additionally, PGK1 and PKM2 could also affect the tricarboxylic acid cycle, oxidative phosphorylation, and muscle contraction in postmortem and then influence meat quality. This integrative omics study offers valuable insight into unraveling the molecular mechanisms underlying postmortem meat quality development.

Transcriptional regulation and post-translational modifications in the glycolytic pathway for targeted cancer therapy.

Cancer cells largely rely on aerobic glycolysis or the Warburg effect to generate essential biomolecules and energy for their rapid growth. The key modulators in glycolysis including glucose transporters and enzymes, e.g. hexokinase 2, enolase 1, pyruvate kinase M2, lactate dehydrogenase A, play indispensable roles in glucose uptake, glucose consumption, ATP generation, lactate production, etc. Transcriptional regulation and post-translational modifications (PTMs) of these critical modulators are important for signal transduction and metabolic reprogramming in the glycolytic pathway, which can provide energy advantages to cancer cell growth. In this review we recapitulate the recent advances in research on glycolytic modulators of cancer cells and analyze the strategies targeting these vital modulators including small-molecule inhibitors and microRNAs (miRNAs) for targeted cancer therapy. We focus on the regulation of the glycolytic pathway at the transcription level (e.g., hypoxia-inducible factor 1, c-MYC, p53, sine oculis homeobox homolog 1, N6-methyladenosine modification) and PTMs (including phosphorylation, methylation, acetylation, ubiquitination, etc.) of the key regulators in these processes. This review will provide a comprehensive understanding of the regulation of the key modulators in the glycolytic pathway and might shed light on the targeted cancer therapy at different molecular levels.

Hypoxic microenvironment-induced exosomes confer temozolomide resistance in glioma through transfer of pyruvate kinase M2

ObjectiveGlioma, a malignant primary brain tumor, is notorious for its high incidence rate. However, the clinical application of temozolomide (TMZ) as a treatment option for glioma is often limited due to resistance, which has been linked to hypoxic glioma cell-released exosomes. In light of this, the present study aimed to investigate the role of exosomal pyruvate kinase M2 (PKM2) in glioma cells that exhibit resistance to TMZ.MethodsSensitive and TMZ-resistant glioma cells were subjected to either a normoxic or hypoxic environment, and the growth patterns and enzymatic activity of glycolysis enzymes were subsequently measured. From these cells, exosomal PKM2 was isolated and the subsequent effect on TMZ resistance was examined and characterized, with a particular focus on understanding the relevant mechanisms. Furthermore, the intercellular communication between hypoxic resistant cells and tumor-associated macrophages (TAMs) via exosomal PKM2 was also assessed.ResultsThe adverse impact of hypoxic microenvironments on TMZ resistance in glioma cells was identified and characterized. Among the three glycolysis enzymes that were examined, PKM2 was found to be a critical mediator in hypoxia-triggered TMZ resistance. Upregulation of PKM2 was found to exacerbate the hypoxia-mediated TMZ resistance. Exosomal PKM2 were identified and isolated from hypoxic TMZ-resistant glioma cells, and were found to be responsible for transmitting TMZ resistance to sensitive glioma cells. The exosomal PKM2 also contributed towards mitigating TMZ-induced apoptosis in sensitive glioma cells, while also causing intracellular ROS accumulation. Additionally, hypoxic resistant cells also released exosomal PKM2, which facilitated TMZ resistance in tumor-associated macrophages.ConclusionIn the hypoxic microenvironment, glioma cells become resistant to TMZ due to the delivery of PKM2 by exosomes. Targeted modulation of exosomal PKM2 may be a promising strategy for overcoming TMZ resistance in glioma.

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.

Apoptin Inhibits Glycolysis and Regulates Autophagy by Targeting Pyruvate Kinase M2 (PKM2) in Lung Cancer A549 Cells.

Pyruvate kinase M2 (PKM2) is a key enzyme in aerobic glycolysis and plays an important role in tumor energy metabolism and tumor growth. Ad-apoptin, a recombinant oncolytic adenovirus, can stably express apoptin in tumor cells and selectively causes cell death in tumor cells. The relationship between the anti-tumor function of apoptin, including apoptosis and autophagy activation, and the energy metabolism of tumor cells has not been clarified. In this study, we used the A549 lung cancer cell line to analyze the mechanism of PKM2 involvement in apoptin-mediated cell death in tumor cells. PKM2 expression in lung cancer cells was detected by Western blot and qRT-PCR. In the PKM2 knockdown and over-expression experiments, A549 lung cancer cells were treated with Ad-apoptin, and cell viability was determined by the CCK-8 assay and crystal violet staining. Glycolysis was investigated using glucose consumption and lactate production experiments. Moreover, the effects of Ad-apoptin on autophagy and apoptosis were analyzed by immunofluorescence using the Annexin v-mCherry staining and by western blot for c-PARP, p62, and LC3-II proteins. Immunoprecipitation analysis was used to investigate the interaction between apoptin and PKM2. In addition, following PKM2 knockdown and overexpression, the expression levels of p-AMPK, p-mTOR, p-ULK1, and p-4E-BP1 proteins in Ad-apoptin treated tumor cells were analyzed by western blot to investigate the mechanism of apoptin effect on the energy metabolism of tumor cells. The in vivo antitumor mechanism of apoptin was analyzed by xenograft tumor inhibition experiment in nude mice and immunohistochemistry of tumors' tissue. As a result, apoptin could target PKM2, inhibit glycolysis and cell proliferation in A549 cells, and promote autophagy and apoptosis in A549 cells by regulating the PKM2/AMPK/mTOR pathway. This study confirmed the necessary role of Ad-apoptin in the energy metabolism of A549 cells.

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.

Evaluation of Serum Pyruvate Kinase M2 and Key Glycolytic Signatures and Its Correlation with Phosphatidylinositol-3-kinase/Akt/Mammalian Target of Rapamycin Pathway in Women with Breast Carcinoma

Abstract Background: A desired target for cancer therapy is a cancer-specific metabolism which is associated with innate or acquired therapeutic resistance, malignancies, and aggressive cancer growth. Nevertheless, these cells utilize manifold metabolic routes to prolong their mass explosion. Human hexokinase 2 (HK2), phosphofructokinase, muscle (PFKM), pyruvate kinase M2 (PKM2), and lactate dehydrogenase A (LDHA) are fundamental signature players of cancer glycolysis playing a role in cancer cell regulation. Reflecting their metabolic roles may be possible with a better grasp of the metabolism transition in the early onset and spread of cancer. This study was designed to assess the serum protein levels of HK2, PFKM, PKM2, and LDHA in women with breast carcinoma and its correlation with phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) pathway. Methods: In the current research, case–control investigation was undertaken with breast cancer women (n = 30) and compatible healthy women (n = 30) visiting Justice K. S. Hegde Charitable Hospital, Deralakatte, Mangaluru, who were selected on the basis of inclusion and exclusion criteria. Baseline characteristics for the study population were marked down. The HK2, PFKM, PKM2, and LDHA levels within serum were estimated by means of sandwich enzyme-linked immunosorbent assay. Results: We observed a difference that was significant among the two groups with regard to age and postmenopausal status and also a difference in first-degree family history. The levels of PKM2 and key glycolytic enzymes in serum varied between the case and control groups. There was a positive correlation that was observed in breast carcinoma subjects with glycolytic proteins and signaling pathway (PI3K/Akt/mTOR). Conclusions: Nevertheless, assured processes that persuade the target enzymes remain vague and require further in-depth exploration. Overall, discovering anticancer therapeutics that target glycolytic enzymes involved in glucose metabolism remains a dilemma.

Tyrosine phosphatase SHP2 aggravates tumor progression and glycolysis by dephosphorylating PKM2 in gastric cancer.

Gastric cancer (GC) is among the most lethal human malignancies, yet it remains hampered by challenges in fronter of molecular-guided targeted therapy to direct clinical treatment strategies. The protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 2 (SHP2) is involved in the malignant progression of GC. However, the detailed mechanisms of the posttranslational modifications of SHP2 remain poorly understood. Herein, we demonstrated that an allosteric SHP2 inhibitor, SHP099, was able to block tumor proliferation and migration of GC by dephosphorylating the pyruvate kinase M2 type (PKM2) protein. Mechanistically, we found that PKM2 is a bona fide target of SHP2. The dephosphorylation and activation of PKM2 by SHP2 are necessary to exacerbate tumor progression and GC glycolysis. Moreover, we demonstrated a strong correlation between the phosphorylation level of PKM2 and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) in GC cells. Notably, the low phosphorylation expression of AMPK was negatively correlated with activated SHP2. Besides, we proved that cisplatin could activate SHP2 and SHP099 increased sensitivity to cisplatin in GC. Taken together, our results provide evidence that the SHP2/PKM2/AMPK axis exerts a key role in GC progression and glycolysis and could be a viable therapeutic approach for the therapy of GC.

Hypoxia-Challenged Pancreatic Adenocarcinoma Cell-Derived Exosomal circR3HCC1L Drives Tumor Growth Via Upregulating PKM2 Through Sequestering miR-873-5p.

Pancreatic adenocarcinoma (PAAD) is a fatal disease with poor survival. Increasing evidence show that hypoxia-induced exosomes are associated with cancer progression. Here, we aimed to investigate the function of hsa_circ_0007678 (circR3HCC1L) and hypoxic PAAD cell-derived exosomal circR3HCC1L in PAAD progression. Through the exoRBase 2.0 database, we screened for a circular RNA circR3HCC1L related to PAAD. Changes of circR3HCC1L in PAAD samples and cells were analyzed with real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation, migration, invasion were analyzed by colony formation, cell counting, and transwell assays. Measurements of glucose uptake and lactate production were done using corresponding kits. Several protein levels were detected by western blotting. The regulation mechanism of circR3HCC1L was verified by dual-luciferase reporter, RNA immunoprecipitation, and RNA pull-down assays. Exosomes were separated by differential ultracentrifugation. Animal experiments were used to verify the function of hypoxia-derived exosomal circR3HCC1L. CircR3HCC1L was upregulated in PAAD samples and hypoxic PAAD cells. Knockdown of circR3HCC1L decreased hypoxia-driven PAAD cell proliferation, migration, invasion, and glycolysis. Hypoxic PAAD cell-derived exosomes had higher levels of circR3HCC1L, hypoxic PAAD cell-derived exosomal circR3HCC1L promoted normoxic cancer cell malignant transformation and glycolysis in vitro and xenograft tumor growth in mouse models in vivo. Mechanistically, circR3HCC1L regulated pyruvate kinase M2 (PKM2) expression via sponging miR-873-5p. Also, PKM2 overexpression or miR-873-5p silencing offset circR3HCC1L knockdown-mediated effects on hypoxia-challenged PAAD cell malignant transformation and glycolysis. Hypoxic PAAD cell-derived exosomal circR3HCC1L facilitated PAAD progression through the miR-873-5p/PKM2 axis, highlighting the contribution of hypoxic PAAD cell-derived exosomal circR3HCC1L in PAAD.

In Vitro Antibacterial, DPPH Radical Scavenging Activities, and In Silico Molecular Modeling of Isolated Compounds from the Roots of Clematis hirsuta.

Clematis hirsuta is one of the traditional medicinal plants used in Ethiopia to treat different ailments, such as cancer and diseases related to the respiratory system. This study aimed to isolate the phytochemical components of the root of C. hirsuta and evaluate their in vitro and in silico biological activities. Oleic acid (1), palmitic acid (2), sterols (3 and 4), boehmenan (5), and carolignans E (6 and 7) were isolated by silica gel column chromatography and preparative thin layer chromatography and characterized by NMR spectroscopy. Compounds 5-7 were isolated from the plant for the first time. At 5 mg/mL, the inhibition zone of evaluated compounds ranged from 8.80 to 11.10 mm against all selected bacteria. The MIC of the MeOH and n-hexane: EtOAc (1 : 1) extracts was greater than or equal to 50 mg/mL against all selected bacteria. At 62.5 μg/mL, the % DPPH radical scavenging activity of tested compounds ranged from 30.3% to 92.1% with an IC50 value of 19.4 to 2.1 μg/mL. The results of molecular docking studies indicated that the docking scores of compounds 3-7 ranged from -6.4 to -7.9 kcal/mol against E. coli DNA gyrase B, -8.3 to -9.0 kcal/mol against the Pseudomonas quinolone signal A, -7.1 to -8.5 kcal/mol against pyruvate kinase M2, and -7.9 to -8.5 kcal/mol against human topoisomerase IIβ. The results of the in silico antibacterial activity of compounds 3, 5, and 6 supported the in vitro antibacterial test results. Compound 5 had a better docking score against human topoisomerase IIβ than the other test samples demonstrating its potential as an anticancer agent. Therefore, compounds 3-7 could be considered as a lead for developing antibacterial and anticancer drugs. Moreover, the presence of these active phytochemicals supports the traditional use of this plant against cancer and bacteria.

Ubiquitinome Analysis Uncovers Alterations in Synaptic Proteins and Glucose Metabolism Enzymes in the Hippocampi of Adolescent Mice Following Cold Exposure.

Cold exposure exerts negative effects on hippocampal nerve development in adolescent mice, but the underlying mechanisms are not fully understood. Given that ubiquitination is essential for neurodevelopmental processes, we attempted to investigate the effects of cold exposure on the hippocampus from the perspective of ubiquitination. By conducting a ubiquitinome analysis, we found that cold exposure caused changes in the ubiquitination levels of a variety of synaptic-associated proteins. We validated changes in postsynaptic density-95 (PSD-95) ubiquitination levels by immunoprecipitation, revealing reductions in both the K48 and K63 polyubiquitination levels of PSD-95. Golgi staining further demonstrated that cold exposure decreased the dendritic-spine density in the CA1 and CA3 regions of the hippocampus. Additionally, bioinformatics analysis revealed that differentially ubiquitinated proteins were enriched in the glycolytic, hypoxia-inducible factor-1 (HIF-1), and 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathways. Protein expression analysis confirmed that cold exposure activated the mammalian target of rapamycin (mTOR)/HIF-1α pathway. We also observed suppression of pyruvate kinase M2 (PKM2) protein levels and the pyruvate kinase (PK) activity induced by cold exposure. Regarding oxidative phosphorylation, a dramatic decrease in mitochondrial respiratory-complex I activity was observed, along with reduced gene expression of the key subunits NADH: ubiquinone oxidoreductase core subunit V1 (Ndufv1) and Ndufv2. In summary, cold exposure negatively affects hippocampal neurodevelopment and causes abnormalities in energy homeostasis within the hippocampus.

Abstract 4713: Chlorpromazine affects glioblastoma bioenergetics by interfering with pyruvate kinase M2: A route for drug repurposing in glioblastoma

Abstract Glioblastoma, the most common and deadly brain tumor, remains a critical unmet medical need due to the limited effectiveness of current treatments. Drug repurposing has recently emerged as a promising strategy to improve glioblastoma outcomes. Antipsychotic drugs, with their established safety profile and potential to disrupt tumor-neuron interactions, have drawn particular attention. Among these, chlorpromazine, a well-tolerated medication included in the 2021 WHO Model List of Essential Medicines, holds promise due to its therapeutic effects in psychiatric disorders stemming from its non-specific interference with various CNS neurotransmitter receptors. Our recent studies have demonstrated chlorpromazine's ability to inhibit several molecular and cellular processes in glioblastoma cells, suggesting its potential as a novel treatment option for this challenging disease.To elucidate chlorpromazine's mechanism of action as a potential anticancer drug, we employed two proteomics approaches: Reverse-Phase Protein microArrays to evaluate its impact on signal transduction pathways and Activity-Based Protein Profiling followed by mass spectrometry to identify novel molecular targets.Our data revealed that chlorpromazine significantly modulates major signal transduction pathways and implicates pyruvate kinase (PK) M2 as a drug target. PKM2, a PK variant characteristic of many cancers, plays a crucial role in orchestrating metabolic alterations, exemplified by the Warburg effect – the high glucose consumption and lactate production by cancer cells even under oxygen-rich conditions. PKM2 functions as a tetramer in the glycolytic pathway, while its dimeric form acquires nuclear localization, protein kinase activity, and interacts with various transcription factors, contributing to its pro-tumorigenic activity.Consistent with its ability to target PKM2, chlorpromazine promoted PKM2 tetramerization in glioblastoma cells, leading to significant alterations in glioblastoma energy metabolism. Notably, RPE-1 non-cancer neuroepithelial cells showed a reduced response to the drug. Additionally, silencing PKM2 diminished the effects of chlorpromazine. 3D modeling revealed that chlorpromazine interacts with the PKM2 tetramer at the same site involved in binding other small-molecule activators that stabilize the PKM2 tetramer.These findings suggest that chlorpromazine counteracts the Warburg effect and, consequently, malignancy in glioblastoma cells, while sparing non-cancerous RPE-1 cells. This preclinical evidence supports the rationale behind our recently completed multicenter Phase II clinical trial investigating the role of chlorpromazine in glioblastoma treatment. The study is registered as EudraCT #2019-001988-75 and ClinicalTrials.gov Identifier #NCT0422444. Citation Format: Claudia Abbruzzese, Silvia Matteoni, Paola Matarrese, Michele Signore, Barbara Ascione, Elisabetta Iessi, Aymone Gurtner, Andrea Sacconi, Andrea Pace, Veronica Villani, Andrea Polo, Susan Costantini, Alfredo Budillon, Gennaro Ciliberto, Marco G. Paggi. Chlorpromazine affects glioblastoma bioenergetics by interfering with pyruvate kinase M2: A route for drug repurposing in glioblastoma [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 4713.

Abstract 437: Hydrogen sulfide switches the glucose metabolism through sulfhydration on pyruvate kinase M2

Abstract Cancer cells reprogram their glucose metabolism from oxidative phosphorylation to aerobic glycolysis. This metabolic transformation is partly based on the activity alterations of a rate limiting enzyme known as the pyruvate kinase M2 (PKM2), which is responsible for the conversion of phosphoenolpyruvate (PEP) into pyruvate. Attributed to its critical regulatory role. PKM2 is recognized as the pivotal enzyme in cancer glucose metabolism By reducing the enzyme activity of PKM2, cancer cells attain a greater fraction of glycolytic metabolites for macromolecule synthesis needed for rapid proliferation. Hydrogen sulfide (H2S), an endogenously produced gasotransmitter that acts as a critical mediator in multiple physiological processes, modifies proteins mainly through the persulfide (-SSH) bond formation, which is called sulfhydration. Our preliminary study demonstrates that H2S stimulates PKM2 sulfhydration at multiple cysteine residues, including cysteine 326, leading to the destabilization of active tetrameric PKM2 form into dimers or monomers. The PKM2 dimer/monomer further translocates into the nucleus to simulate the activation of glycolytic related genes. Blocking PKM2 sulfhydration at cysteine 326 through amino acid mutation stabilizes PKM2 tetramer and crystal structure further indicates that the tetramer organization of PKM2C326S is different from the currently known T or R states, revealing PKM2C326S as a newly identified intermediate form. Blocking PKM2 sulfhydration at cysteine 326 inhibited cell proliferation and tumor growth in xenograft mouse model. In summary, our current study illustrates that H2S-mediated sulfhydration induces the dissociation of the PKM2 tetramer, resulting in the reduced PKM2 activity and subsequently inhibits breast cancer cell proliferation and tumor growth. Targeting the sulfhydration site of PKM2 emerges as a promising therapeutic target specific for cancer metabolism. Citation Format: Po-Chen Chien, Rong-Hsuan Wang, Pin-Ru Chen, Yue-Ting Chen, Yi-Chang Chen, Yu-Hsin Chu, Chia-Chen Chien, Shao-Yun Lo, Zhong-Liang Wang, Min-Chen Tsou, Ssu-Yu Chen, Guang-Shen Chiu, Wen-Ling Chen, Yi-Hsuan Wu, Hui-Ching Wang, Shu-Yi Lin, Wen-Ching Wang, Hsing-Jien Kung, Lu-Hai Wang, Hui-Chun Cheng, Kai-Ti Lin. Hydrogen sulfide switches the glucose metabolism through sulfhydration on pyruvate kinase M2 [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 437.

Cardioprotective potential of compound 3K, a selective PKM2 inhibitor in isoproterenol-induced acute myocardial infarction: A mechanistic study

Myocardial infarction (MI) or heart attack arises from acute or chronic prolonged ischemic conditions in the myocardium. Although several risk factors are associated with MI pathophysiology, one of the risk factors is an imbalance in the oxygen supply. The current available MI therapies are still inadequate due to the complexity of MI pathophysiology. Pyruvate kinase M2 (PKM2) has been implicated in numerous CVDs pathologies. However, the effect of specific pharmacological intervention targeting PKM2 has not been studied in MI. Therefore, in this study, we explored the effect of compound 3K, a PKM2-specific inhibitor, in isoproterenol-induced acute MI model. In this study, in order to induce MI in rats, isoproterenol (ISO) was administered at a dose of 100 mg/kg over two days at an interval of 24 h. Specific PKM2 inhibitor, compound 3K (2 and 4 mg/kg), was administered in MI rats to investigate its cardioprotective potential. After the last administration of compound 3K, ECG and hemodynamic parameters were recorded using a PV-loop system. Cardiac histology, western blotting, and plasmatic cardiac damage markers were evaluated to elucidate the underlying mechanisms. Treatment of compound 3K significantly reduced ISO-induced alterations in ECG, ventricular functions, cardiac damage, infarct size, and cardiac fibrosis. Compound 3K treatment produced significant increase in PKM1 expression and decrease in PKM2 expression. In addition, HIF-1α, caspase-3, c-Myc, and PTBP1 expression were also reduced after compound 3K treatment. This study demonstrates the cardioprotective potential of compound 3K in MI, and its mechanisms of cardioprotective action.

Abstract 3859: HSP90 blockade activates antitumor immunity by suppressing tumor glycolytic flux in head and neck squamous cell carcinoma

Abstract Accumulating evidence suggests that failure of antitumor immunity may be due to dysregulation of energy metabolism. Head and neck squamous cell carcinoma (HNSCC) exhibits increased activity of two key enzymes, pyruvate kinase M2 (PKM2) and platelet phosphofructokinase (PFKP), which are central to the glycolytic pathway responsible for the conversion of glucose to ATP energy. HSP90 is a molecular chaperone that plays a critical role in the folding, stabilization, and regulation of a wide range of client proteins involved in various cellular processes, including metabolism. However, whether HSP90 signaling contributes to tumor metabolism rewiring and antitumor immune response remains unknown. Using ganetespib as a drug model for HSP90 inhibition and combining results from clinical trials and animal treatment, our study demonstrated that HSP90 inhibition causes an obstruction of glycolytic flux in HNSCC cells by simultaneously suppressing both PKM2 and PFKP at the transcriptional and post-translational levels. In addition, ganetespib enhances T cell-driven antitumor immune responses by promoting CD8+ T cell infiltration into the tumor. Mechanistically, ganetespib downregulates IL8 signaling by inhibiting PKM2 and PFKP-mediated glycolysis in HNSCC cells, which in turn contributes to ganetespib-mediated T cell tumor infiltration. These findings provide a novel molecular basis for the use of effective HSP90 inhibitors to improve the outcome of HNSCC patients. Citation Format: Fanghui Chen, Chris Tang, Fan Yang, Asari Ekpenyong, Richard Qin, Nabil F. Saba, Yong Teng. HSP90 blockade activates antitumor immunity by suppressing tumor glycolytic flux in head and neck squamous cell carcinoma [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 3859.

N6-isopentenyladenosine inhibits aerobic glycolysis in glioblastoma cells by targeting PKM2 expression and activity.

Glioblastoma (GBM) is a primary tumor in the central nervous system with poor prognosis. It exhibits elevated glucose uptake and lactate production. This metabolic state of aerobic glycolysis is known as the Warburg effect. N6-isopentenyladenosine (iPA), a natural cytokine modified with an isopentenyl moiety derived from the mevalonate pathway, has well-established anti-tumor activity. It inhibits cell proliferation in glioma cells, inducing cell death by apoptosis and/or necroptosis. In the present study, we found that iPA inhibits aerobic glycolysis in unmodified U87MG cells and in the same cell line engineered to over-express wild-type epidermal growth factor receptor (EGFR) or EGFR variant III (vIII), as well as in a primary GBM4 patient-derived cell line. The detection of glycolysis showed that iPA treatment suppressed ATP and lactate production. We also evaluated the response of iPA treatment in normal human astrocyte primary cells, healthy counterpart cells of the brain. Aerobic glycolysis in treated normal human astrocyte cells did not show significant changes compared to GBM cells. To determine the mechanism of iPA action on aerobic glycolysis, we investigated the expression of certain enzymes involved in this metabolic pathway. We observed that iPA reduced the expression of pyruvate kinase M2 (PKM2), which plays a key role in the regulation of aerobic glycolysis, promoting tumor cell proliferation. The reduction of PKM2 expression is a result of the inhibition of the inhibitor of nuclear factor kappa-B kinase subunit, beta/nuclear factor-kappa B pathway upon iPA treatment. In conclusion, these experimental results show that iPA may inhibit aerobic glycolysis of GBM in stabilized cell lines and primary GBM cells by targeting the expression and activity of PKM2.

Pyruvate Kinase M2 Nuclear Translocation Regulate Ferroptosis-Associated Acute Lung Injury in Cytokine Storm

Cytokine storm (CS) is linked with macrophage dysfunction and acute lung injury (ALI), which can lead to patient mortality. Glycolysis is preferentially exploited by the pro-inflammatory macrophages, in which pyruvate kinase M2 (PKM2) is a critical enzyme. The mechanism underlying the link between CS and ALI involves cell death, with the recently discovered programmed cell death known as ferroptosis being involved. However, the relationship between the glycolysis and ferroptosis in the context of CS-related ALI remains unclear. CS-associated ALI induced by poly I:C (10 mg/kg, i.v) and LPS (5 mg/kg, i.p) (IC: LPS) exhibit significant ferroptosis. Ferrostatin-1 (ferroptosis inhibitor) treatment attenuated IC:LPS‑induced mortality and lung injury. Moreover, Alveolar macrophage (AM) from IC:LPS model exhibited enhanced glycolysis and PKM2 translocation. The administration of ML-265(PKM2 monomer/dimer inhibitor) resulted in the formation of a highly active tetrameric PKM2, leading to improved survival and attenuation of ALI. Furthermore, ML-265 treatment decreased ferroptosis and restored the balance between anaerobic glycolysis and oxidative phosphorylation. Notably, in patients with lung infection, intracellular expression level of PKM2 were correlated with circulating inflammation. Enhanced ferroptosis and PKM2 nuclear translocation was noticed in CD14+ blood monocytes of lung infection patients with CS. In conclusion, PKM2 is a key regulatory node integrating metabolic reprograming with intra-nuclear function for the regulation of ferroptosis. Targeting PKM2 could be explored as a potential means in the future to prevent or alleviate hyper-inflammatory state or cytokines storm syndrome with aberrant ferroptotic cell death.

Search progress of pyruvate kinase M2 (PKM2) in organ fibrosis.

Fibrosis is characterized by abnormal deposition of the extracellular matrix (ECM), leading to organ structural remodeling and loss of function. The principal cellular effector in fibrosis is activated myofibroblasts, which serve as the main source of matrix proteins. Metabolic reprogramming, transitioning from mitochondrial oxidative phosphorylation to aerobic glycolysis, is widely observed in rapidly dividing cells such as tumor cells and activated myofibroblasts and is increasingly recognized as a fundamental pathogenic basis in organ fibrosis. Targeting metabolism represents a promising strategy to mitigate fibrosis. PKM2, a key enzyme in glycolysis, plays a pivotal role in metabolic reprogramming through allosteric regulation, impacting both metabolic and non-metabolic pathways. Therefore, metabolic reprogramming induced by PKM2 activation is involved in the occurrence and development of fibrosis in various organs. A comprehensive understanding of the role of PKM2 in fibrotic diseases is crucial for seeking new anti-fibrotic therapeutic targets. In this context, we summarize PKM2's role in glycolysis, mediating the intricate mechanisms underlying fibrosis in multiple organs, and discuss the potential value of PKM2 inhibitors and allosteric activators in future clinical treatments, aiming to identify novel therapeutic targets for proliferative fibrotic diseases.