Discovery of PKM2 activators from Aconiti Lateralis Radix Praeparata via computer-aided drug design and experimental validation.

Upon growth factor stimulation or in some EGFR mutant cancer cells, PKM2 translocates into the nucleus to induce glycolysis and cell growth. Here, we report that nuclear PKM2 binds directly to poly-ADP ribose, and this PAR-binding capability is critical for its nuclear localization. Accordingly, PARP inhibition prevents nuclear retention of PKM2 and therefore suppresses cell proliferation and tumor growth. In addition, we found that PAR level correlates with nuclear localization of PKM2 in EGFR mutant brain and lung cancers, suggesting that PAR-dependent nuclear localization of PKM2 likely contributes to tumor progression in EGFR mutant glioblastoma and lung cancers. In addition, some EGFR-inhibitor-resistant lung cancer cells are sensitive to PARP inhibitors. Taken together, our data indicate that suppression of PKM2 nuclear function by PARP inhibitors represents a treatment strategy for EGFR-inhibitor-resistant cancers.

Objectives:Esophageal squamous cell carcinoma is a highly prevalent cancer withpoor survival rate and prognosis. Increasing evidence suggests an important role for metabolic regulation in treating esophageal squamous cell carcinoma, but the underlying mechanism remains unclear. The pyruvate kinase M2 isoform is a key enzyme in the energy production process, and the upregulation of pyruvate kinase M2 isoform also plays a crucial role in gene transcription and tumorigenesis. The mammalian target of rapamycin pathway regulates an array of cellular functions, including protein synthesis, metabolism, and cell proliferation. The pyruvate kinase M2 isoform and mammalian target of rapamycin pathways both affect metabolism in cancers, and evidence also suggests that the mammalian target of rapamycin downstream transcription factor hypoxia-inducible factor-1α regulates pyruvate kinase M2 isoform. We therefore investigated the regulatory mechanism among pyruvate kinase M2 isoform, mammalian target of rapamycin, and aerobic glycolysis in esophageal squamous cell carcinoma, hoping to prove that mammalian target of rapamycin pathway regulates pyruvate kinase M2 isoform to affect glycolysis in esophageal squamous cell carcinoma.Methods:Immunohistochemical staining was used to compare pyruvate kinase M2 isoform and phospho-mammalian target of rapamycin expression in 30 human pathological esophageal squamous cell carcinoma sections and 30 nontumoral esophageal tissues. Short hairpin RNA was used to inhibit pyruvate kinase M2 isoform and activate mammalian target of rapamycin, after which we monitored changes in glucose consumption and lactate production. Finally, we determined the expression of pyruvate kinase M2 isoform and the mammalian target of rapamycin downstream transcription factor hypoxia-inducible factor-1α, as well as glucose consumption and lactate production, following the modification of mammalian target of rapamycin expression.Results:Immunohistochemical staining showed that both phospho-mammalian target of rapamycin and pyruvate kinase M2 isoform expression were higher in esophageal squamous cell carcinoma than in nontumor tissues. Glucose consumption and lactate production measurements demonstrated that altering mammalian target of rapamycin and pyruvate kinase M2 isoform levels caused corresponding changes in glycolysis in esophageal squamous cell carcinoma cells. When mammalian target of rapamycin was activated or inhibited, expression of pyruvate kinase M2 isoform and hypoxia-inducible factor-1α as well as glycolysis were altered, indicating that mammalian target of rapamycin regulates pyruvate kinase M2 isoform via the downstream transcription factor hypoxia-inducible factor-1α, thereby affecting glycolysis in esophageal squamous cell carcinoma.Conclusion:Mammalian target of rapamycin pathway promotes aerobic glycolysis in esophageal squamous cell carcinoma by upregulating pyruvate kinase M2 isoform. Both proteins can serve as molecular targets for novel therapeutic strategies.

Role of, 29-non-synonymous, 15-intronic, 3-close to UTR, single nucleotide polymorphisms (SNPs) and 2 mutations of Human Pyruvate Kinase (PK) M2 were investigated by in-silico and in-vitro functional studies. Prediction of deleterious substitutions based on sequence homology and structure based servers, SIFT, PANTHER, SNPs&GO, PhD-SNP, SNAP and PolyPhen, depicted that 19% emerged common between all the mentioned programs. SNPeffect and HOPE showed three substitutions (C31F, Q310P and S437Y) in-silico as deleterious and functionally important. In-vitro activity assays showed C31F and S437Y variants of PKM2 with reduced activity, while Q310P variant was catalytically inactive. The allosteric activation due to binding of fructose 1-6 bisphosphate (FBP) was compromised in case of S437Y nsSNP variant protein. This was corroborated through molecular dynamics (MD) simulation study, which was also carried out in other two variant proteins. The 5 intronic SNPs of PKM2, associated with sporadic breast cancer in a case-control study, when subjected to different computational analyses, indicated that 3 SNPs (rs2856929, rs8192381 and rs8192431) could generate an alternative transcript by influencing splicing factor binding to PKM2. We propose that these, potentially functional and important variations, both within exons and introns, could have a bearing on cancer metabolism, since PKM2 has been implicated in cancer in the recent past.

AB0471 ELEVATED EXPRESSION OF PYRUVATE KINASE M2 IN GIANT CELL ARTERITIS

Tamoxifen has been reported to be associated with antagonism of estrogen-mediated cell growth signaling and activation of estrogen receptor-independent apoptosis events. It has been demonstrated that mammalian sterile 20-like kinase 1 is a direct target of Caspases to amplify the apoptotic signaling pathway. Here, we presented that breast cancer MCF-7 and SKBR3 cells under treatment with 4-hydroxytamoxifen displayed decreased level of pyruvate kinase M2. Western blot results also showed that 4-hydroxytamoxifen induced the activity of pro-apoptotic protein Caspase-3 in MCF-7 and SKBR3 cells, as evidenced by the cleavage of mammalian sterile 20-like kinase 1 substrate in a dose-dependent manner. Co-immunoprecipitation and immunofluorescence experiments were performed to clarify the relationship between pyruvate kinase M2 and mammalian sterile 20-like kinase 1. The results indicated that mammalian sterile 20-like kinase 1 was associated with pyruvate kinase M2 in cultured mammalian cells, and the interaction between mammalian sterile 20-like kinase 1 and pyruvate kinase M2 was decreased in response to 4-hydroxytamoxifen treatment. In addition, knockdown of pyruvate kinase M2 upregulated the level of cleaved Caspase-3 and subsequently facilitated the nuclear translocation of mammalian sterile 20-like kinase 1. Our data further supplemented the extensive functions of pyruvate kinase M2 in mediating breast cancer cell viability by substantially abating the mammalian sterile 20-like kinase 1-mediated apoptosis. In summary, our results identified that mammalian sterile 20-like kinase 1 is a novel downstream target of pyruvate kinase M2, and knockdown of pyruvate kinase M2 contributes apoptosis via promoting nuclear translocation of mammalian sterile 20-like kinase 1 by enhancing Caspase-3-dependent cleavage.

PKM2 Mediates Chronic Myeloid Leukemia Imatinib Resistance By Regulating Glycolysis Energy Metabolism

Pyruvate kinase (PK) M2 (PKM2), a glycolytic enzyme, is a hallmark of different types of tumors and plays a significant role in the Warburg effect. However, there is no fluorescent probe for PKM2 that has been reported yet. In this study, TEPC466, a novel TEPP-46-based aggregation-induced emission (AIE) probe for the detection of PKM2, was designed, synthesized, and fully characterized by 1H NMR, 13C NMR, and high-resolution mass spectrometry. When the fluorescent agent, coumarine, was conjugated to TEPP-46, the bioprobe TEPC466 showed a high degree of selectivity and sensitivity for the detection of PKM2 protein via the AIE effect. TEPC466 was then successfully applied in imaging the PKM2 protein in colorectal cancer cells with low toxicity. Moreover, structure-based modeling and the PK activity assay confirmed that TEPC466 has a better binding with PKM2 than TEPP-46, which suggests that TEPC466 could also be a good agonist of PKM2. Taken together, the bioprobe shows potential in selective detection of PKM2 and provides a useful tool for cancer diagnosis and therapy.

Aims/IntroductionTubulointerstitial fibrosis is a hallmark of diabetic nephropathy and is associated with an epithelial‐to‐mesenchymal transition (EMT) program and aberrant glycolysis. Dimeric pyruvate kinase (PK) M2 (PKM2) acts as a key protein kinase in aberrant glycolysis by promoting the accumulation of hypoxia‐inducible factor (HIF)‐1α, while tetrameric PKM2 functions as a pyruvate kinase in oxidative phosphorylation. The aim of the research is to study the effect of PKM2 tetramer activation on preventing kidney fibrosis via suppression of aberrant glycolysis and the EMT program.Materials and methods In vivo: Streptozotocin (STZ) was utilized to induce diabetes in 8‐week‐old CD‐1 mice; 4 weeks after diabetes induction, proteinuria‐induced kidney fibrosis was developed by intraperitoneal injection of bovine serum albumin (BSA: 0.3 g/30 g BW) for 14 days; The PKM2 activator TEPP‐46 was also administered orally simultaneously. In vitro: HK2 cells were co‐treated with high‐glucose media or/and TGF‐β1 and TEPP46 for 48 h, cellular protein was extracted for evaluation.ResultsDiabetic mice developed kidney fibrosis associated with aberrant glycolysis and EMT; BSA injection accelerated kidney fibrosis in both the control and diabetic mice; TEPP‐46 rescued the kidney fibrosis. In HK2 cells, TEPP‐46 suppressed the EMT program induced by TGF‐β1 and/or high‐glucose incubation. TEPP‐46‐induced PKM2 tetramer formation and PK activity resulted in suppression of HIF‐1α and lactate accumulation. Specific siRNA‐mediated knockdown of HIF‐1α expression diminished high glucose‐induced mesenchymal protein levels.ConclusionPKM2 activation could restore the tubular phenotype via suppression of the EMT program and aberrant glycolysis, providing an alternative target to mitigate fibrosis in diabetic kidneys.

Pyruvate Kinase M2 Coordinates Metabolism Switch between Glycolysis and Glutaminolysis in Cancer Cells.

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.

Pyruvate kinase M2 phosphorylates the transcription factor STAT3.

Introduction: Several factors released from activated platelets via exocytosis contribute to venous thrombosis (VT) by potentiating neutrophil extracellular traps formation (NETosis) at the sites of inflammation. The metabolic enzyme pyruvate kinase M2 (PKM2) contributes to platelet activation and arterial thrombosis. PKM2 possess protein kinase activity. The regulatory role of PKM2 in platelet granule exocytosis, NETosis, and VT has not been investigated yet. Aim: To test the hypothesis that dimeric PKM2 regulates SNAP23-mediated platelet granule exocytosis, and thereby contributes to NETosis, and VT. Methods: We utilized platelet-specific PKM2 –/– (PKM2 fl/fl PF4Cre + ), littermate (PKM2 fl/fl ) mice, and wild-type (WT) mice treated with small molecule ML265 (limits PKM2 dimerization) or vehicle to test the susceptibility to VT in inferior vena cava (IVC)-stenosis model. In vitro NETs formation was evaluated by co-incubating WT neutrophils with agonist (thrombin or convulxin)-stimulated platelets. The SNAP23 phosphorylation and PF4 release were measured using Western blot. Results: PKM2 fl/fl PF4Cre + or WT mice pre-treated with ML265 exhibited reduced susceptibility to VT in the 48 hours IVC-stenosis model as evaluated by less thrombus burden (% incidence, length, and weight, n=11-12/group, P<0.05 vs. controls). Myography revealed that limiting PKM2 dimerization improves the venous wall function post-VT. Activated PKM2 –/– or ML265 pre-treated WT platelets exhibited decreased SNAP23 phosphorylation and PF4 secretion suggesting a regulatory role for PKM2 in SNAP23 exocytosis and PF4 secretion. Furthermore, neutrophils stimulated with activated platelets from either PKM2 fl/fl PF4Cre + or WT mice pre-treated with ML265 showed reduced NETosis. Finally, we demonstrate that the % of the area covered by the thrombus is profoundly reduced in the ML265-treated human whole blood perfused over a tissue factor-coated surface at the venous shear. Conclusions: Dimeric PKM2 is a novel regulator of SNAP23-mediated platelet granule exocytosis. Targeting dimeric PKM2 in platelets inhibits NETosis in neutrophils stimulated with activated platelets and reduces susceptibility to experimental VT.

Development of colorectal cancer has been considered as a result of imbalance of pro- and anti-inflammatory intestinal microenvironment accompanied by macrophage recruitment. Despite macrophages are implicated in remodeling tumor microenvironment, the mechanism of macrophage recruitment is not fully elucidated yet. In this study, we reported clinical association of highly expressed pyruvate kinase M2 in colorectal cancer with macrophage attraction. The conditioned medium from Caco-2 and HT-29 cells with depleted pyruvate kinase M2 dramatically reduced macrophage recruitment, which is reversed by addition of, a critical chemotaxis factor to macrophage migration, rCCL2. Silencing of endogenous pyruvate kinase M2 markedly decreased CCL2 expression and secretion by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Endogenous pyruvate kinase M2 interacted with p65 and mediated nuclear factor-κB signaling pathway and mainly regulated phosphorylation of Ser276 on p65 nuclear factor-κB. In addition, inhibition of macrophage recruitment caused by pyruvate kinase M2 silencing was rescued by ectopic expression of p65. Interestingly, pyruvate kinase M2 highly expressed in colorectal cancer tissue, which is correction with macrophage distribution. Taken together, we revealed a novel mechanism of pyruvate kinase M2 in promoting colorectal cancer progression by recruitment of macrophages through p65 nuclear factor-κB-mediated expression of CCL2.

Background: PKM2 (M2 isoform of pyruvate kinase) was identified as a driver of aerobic glycolysis, leading to cell growth and tumor development. PKM2 is usually known to overexpress in cancer, however, there are still remaining questions about the function and the potential as anti-cancer treatment target. We investigated the expression status of PKM2 in gastric cancer tissues and evaluated the possibility of biomarker and anticancer target. Methods: Paraffin-embedded tissue microarray blocks of gastric adenocarcinoma tissue specimens were obtained from 363 gastric cancer patients. All patients underwent curative gastric resection from 1999 to 2007 and distributed from stage I to IV and their clinical characteristics were collected. IHC (Immunohistochemical) assay was performed to evaluate PKM2 expression levels. The correlation of PKM2 expression level with gastric cancer prognosis / stage / histology was evaluated. Results: In our previous microarray study using 60 gastric cancer tissues, we identified PKM2 mRNA overexpressed in cancer tissue than non-cancer tissue (P<0.01). In IHC assays, PKM2 expression level was classified to score 0 (negative, n=185), 1 (weak positive, n=136), and 2 (strong positive, n=42). 49% of gastric cancer tissues presented PKM2 expression (weak or strong positive). PKM2 expression had no relation with prognosis (p=0.913, recurrence free survival) and TNM stage (p=0.825). Intestinal and diffuse type cancer manifested different PKM2 expression pattern and signet ring cell carcinoma manifested 26.6% PKM2 expression. Within same Lauren classification, there was no significant difference of prognosis according to PKM2 expression except signet ring cell carcinoma. In signet ring cell cancer (n=78), PKM2 expression was related to poor overall survival (p=0.029) Conclusion: PKM2 protein expression was not correlated with gastric cancer prognosis. Only in signet ring cell gastric cancer, overexpressed PKM2 was associated with poor overall survival. The role of PKM2 should be further verified in signet ring cell gastric cancer model to find clinical significance in this subtype of gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4540. doi:1538-7445.AM2012-4540

Pyruvate kinase M2 (PKM2), playing a central role in regulating aerobic glycolysis, was considered as a promising target for cancer therapy. However, its role in cancer metastasis is rarely known. Here, we found a tight relationship between PKM2 and breast cancer metastasis, demonstrated by the findings that beta‐elemene (β‐elemene), an approved drug for complementary cancer therapy, exerted distinct anti‐metastatic activity dependent on PKM2. The results indicated that β‐elemene inhibited breast cancer cell migration, invasion in vitro as well as metastases in vivo. β‐Elemene further inhibited the process of aerobic glycolysis and decreased the utilization of glucose and the production of pyruvate and lactate through suppressing pyruvate kinase activity by modulating the transformation of dimeric and tetrameric forms of PKM2. Further analysis revealed that β‐elemene suppressed aerobic glycolysis by blocking PKM2 nuclear translocation and the expression of EGFR, GLUT1 and LDHA by influencing the expression of importin α5. Furthermore, the effect of β‐elemene on migration, invasion, PKM2 transformation, and nuclear translocation could be reversed in part by fructose‐1,6‐bisphosphate (FBP) and L‐cysteine. Taken together, tetrameric transformation and nuclear translocation of PKM2 are essential for cancer metastasis, and β‐elemene inhibited breast cancer metastasis via blocking aerobic glycolysis mediated by dimeric PKM2 transformation and nuclear translocation, being a promising anti‐metastatic agent from natural compounds.