PKM2 Inhibition Research Articles

Inhibition of abnormally activated HIF-1α-GLUT1/3-glycolysis pathway enhances the sensitivity of hepatocellular carcinoma to 5-caffeoylquinic acid and its derivatives

Over the past few years, the antitumor activity exhibited by 5-caffeylquinic acid (5-CQA), especially its inhibitory effect on hepatocellular carcinoma (HCC) proliferation and metastasis, has been recognized as a new research hotspot. However, impacted by the weak antitumor toxicity of 5-CQA, its clinical application has been limited. In this study, the antitumor effect arising from 5-CQA on HCC cells was evaluated through cell viability assay. In addition, proteomics, flow cytometry, qRT-PCR and western blotting were adopted to investigate the drug resistance mechanism of HCC cells to 5-CQA. As indicated by the results, 5-CQA significantly inhibited the proliferation of HCC cell lines MHCC97H and HCCLM3 with IC5048 h of 546.8 μM and 452 μM, respectively. According to the in-depth studies, the abnormal activation of HIF-1α/glucose transporters/glycolysis pathway of 5-CQA could be a key molecular mechanism leading to drug resistance of HCC cells. Thus, this study found that glucose starvation, glucose analogue 2-DG, hexokinase inhibitor bromopyruvic acid and PKM2 inhibitor compound 3k inhibited HCC cell proliferation in synergy with 5-CQA. Furthermore, though the 5-CQA derivatives methyl chlorogenate (MCGA) and 3,5-dicaffeoylquinic acid (3,5-diCQA) exhibited more potent antiproliferation activity in HCC cells than 5-CQA, they also up-regulated the expression of GLUT1/3, whereas they had no effect on hepatocytes. To be specific, under low-glucose culture conditions, the order of sensitivity of HCC cells to CQAs was 3,5-diCQA > MCGA > 5-CQA. In brief, the above results revealed that intervention in glucose metabolism can facilitate the effects of 5-CQA and its derivatives for treating HCC.

Inhibiting pyruvate kinase muscle isoform 2 regresses group 2 pulmonary hypertension induced by supra-coronary aortic banding.

Group 2 pulmonary hypertension (PH) has no approved PH-targeted therapy. Metabolic remodelling, specifically a biventricular increase in pyruvate kinase muscle (PKM) isozyme 2 to 1 ratio, occurs in rats with group 2 PH induced by supra-coronary aortic banding (SAB). We hypothesize that increased PKM2/PKM1 is maladaptive and inhibiting PKM2 would improve right ventricular (RV) function. Male, Sprague-Dawley SAB rats were confirmed to have PH by echocardiography and then randomized to treatment with a PKM2 inhibitor (intraperitoneal shikonin, 2mg/kg/day) versus 5% DMSO (n=5/group) or small interfering RNA-targeting PKM2 (siPKM2) versus siRNA controls (n=7/group) by airway nebulization. Shikonin-treated SAB rats had milder PH (PAAT 32.1±1.3 vs 22.1±1.2ms, P=.0009) and lower RV systolic pressure (RVSP) (31.5±0.9 vs 55.7±1.9mmHg, P<.0001) versus DMSO-SAB rats. siPKM2 nebulization reduced PKM2 expression in the RV, increased PAAT (31.7±0.7 vs 28.0±1.3ms, P=.025), lowered RVSP (30.6±2.6 vs 42.0±4.0mm Hg, P=.032) and reduced diastolic RVFW thickness (0.69±0.04 vs 0.85±0.06mm, P=.046). Both shikonin and siPKM2 regressed PH-induced medial hypertrophy of small pulmonary arteries. Increases in PKM2/PKM1 in the RV contribute to RV dysfunction in group 2 PH. Chemical or molecular inhibition of PKM2 restores the normal PKM2/PKM1 ratio, reduces PH, RVSP and RVH and regresses adverse PA remodelling. PKM2 merits consideration as a therapeutic cardiac target for group 2 PH.

Integrin β3-PKM2 pathway-mediated aerobic glycolysis contributes to mechanical ventilation-induced pulmonary fibrosis.

Background: Mechanical ventilation (MV) can induce pulmonary fibrosis. This study aims to investigate whether MV-induced pulmonary fibrosis is associated with aerobic glycolysis and seeks to uncover the underlying mechanisms mediated by integrin β3-pyruvate kinase M2 (PKM2) pathway.Methods: PKM2 knockdown or inhibition, integrin β3 knockout or inhibition and wild-type mice were exposed to MV (20 mL/kg) for 2 h.Results: Mice exposed to MV exhibited increased expression of collagen deposition, and upregulation of α-smooth muscle actin and collagen I in lung tissues. Single cells analysis showed that MV-induced pulmonary fibrosis was associated with increased gene expression of integrin and glycolysis in pulmonary fibroblasts, as well as upregulation of glycolytic products tested by metabolomics. Meanwhile, increased protein level of integrin β3 and PKM2 was confirmed by western blot and immunohistochemistry. Double immunofluorescence staining and flow cytometric analysis showed increased number of fibronectin+/integrin β3+ and fibronectin+/PKM2+ fibroblasts in lung tissues. Furthermore, MV-induced aerobic glycolysis and pulmonary fibrosis were ameliorated after treatment with PKM2 knockdown-AAV and inhibition, or in integrin β3 knockout and inhibition mice.Conclusions: Integrin β3-PKM2 pathway-mediated aerobic glycolysis contributes to MV-induced pulmonary fibrosis. The inhibition of aerobic glycolysis targeting integrin β3-PKM2 pathway may be a promising treatment for MV-induced pulmonary fibrosis.

PKM2 Is Essential for Bladder Cancer Growth and Maintenance.

Genetic manipulation and pharmacologic inhibition of PKM2 in mouse urothelial lesions highlight its essential role in promoting angiogenesis and metabolic addiction, events indispensable for tumor growth and maintenance.

PKM2 Is a Potential Diagnostic and Therapeutic Target for Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a major cause of blindness that is difficult to diagnose and treat. PKM2, a subtype of pyruvate kinase, is strongly associated with oxidative stress and is expressed in photoreceptors. We investigated whether PKM2 reduces photoreceptor cell apoptosis and evaluated possible antiapoptotic mechanisms in RP. We established RP models by exposing 661W cells to blue light and modulated PKM2 activity using a PKM2 inhibitor. We measured the apoptosis rates using calcein-acetoxymethyl ester/propidium iodide double staining and Cell Counting Kit-8, the oxidative stress levels using a reactive oxygen species assay, and the changes in protein expression by western blotting. Photodamage increased PKM2 expression, cellular oxidative stress, and apoptosis of 661W cells. PKM2 inhibition significantly reduced the levels of apoptosis and oxidative stress induced by photodamage. Our data suggest that PKM2 is a potential disease marker and therapeutic target for RP.

Pyruvate Kinase M2 Mediates Glycolysis Contributes to Psoriasis by Promoting Keratinocyte Proliferation.

Psoriasis is characterized by keratinocyte proliferation and immune cell infiltration. M2 isoform of pyruvate kinase (PKM2) was reported to have an important role in cell proliferation, which is a rate-limiting enzyme that regulates the final step of glycolysis. However, how PKM2 regulates cell metabolism and proliferation in psoriatic keratinocytes is still poorly understood. Interestingly, we found that PKM2 was highly expressed in psoriatic epidermis from patients and mouse models. PKM2 overexpression promoted keratinocyte glycolytic metabolism while knockdown inhibited keratinocyte proliferation and glycolysis. Mice lacking PKM2 specifically in keratinocytes, pharmacological inhibition of PKM2 or glycolysis inhibited keratinocyte proliferation and showed obvious remission in an imiquimod-induced psoriatic mouse model. Moreover, the inhibitor of the EGF-receptor blocked EGF-stimulated PKM2 expression and glycolysis in keratinocytes. We identify PKM2 as an upregulated gene in psoriasis. PKM2 is essential in keratinocyte over-proliferation and may represent a therapeutic target for psoriasis.

Kinetic Characterization and Inhibitor Screening of Pyruvate Kinase I From Babesia microti.

The apicomplexan Babesia microti is a main pathogenic parasite causing human babesiosis, which is one of the most widely distributed tick-borne diseases in humans. Pyruvate kinase (PYK) plays a central metabolic regulatory role in most living organisms and catalyzes the essentially irreversible step in glycolysis that converts phosphoenolpyruvate (PEP) to pyruvate. Hence, PYK is recognized as an attractive therapeutic target in cancer and human pathogens such as apicomplexans. In this study, we cloned, expressed, and purified B. microti PYK I (BmPYKI). Western blotting illustrated that anti-rBmPYKI antibody could specifically recognize the native BmPYKI protein in the lysate of B. microti with a 54-kDa band, which is consistent with the predicted size. In addition, the enzymatic activity of the purified recombinant PYKI (rPYKI) was tested under a range of pH values. The results showed that the maximum catalytic activity could be achieved at pH 7.0. The saturation curves for substrates demonstrated that the Km value for PEP was 0.655 ± 0.117 mM and that for ADP was 0.388 ± 0.087 mM. We further investigated the effect of 13 compounds on rBmPYKI. Kinetic analysis indicated that six inhibitors (tannic acid, shikonin, apigenin, PKM2 inhibitor, rosiglitazone, and pioglitazone) could significantly inhibit the catalytic activity of PYKI, among which tannic acid is the most efficient inhibitor with an IC50 value 0.49 μM. Besides, four inhibitors (tannic acid, apigenin, shikonin, and PKM2 inhibitor) could significantly decrease the growth of in vitro-cultured B. microti with IC50 values of 0.77, 2.10, 1.73, and 1.15 μM. Overall, the present study provides a theoretical basis for the design and development of new anti-Babesia drugs.

Shikonin contributes to intestinal epithelial cell differentiation through PKM2/NRF2-mediated Polyol pathway

BackgroundAlteration of polyol pathway is a critical event in serials of disease, including inflammatory bowel disease (IBD). Shikonin, an inhibitor of PKM2, has been reported to exert a wide range of biological and pharmacological properties. However, the function of shikonin-mediated polyol pathway in IBD remained to be addressed. MethodsWe conducted WB and qPCR to analyze the effect of shikonin on intestinal epithelial cell differentiation. IF, IP, luciferase assay and ChIP were employed to demonstrate the mechanism that shikonin regulated cell differentiation through polyol pathway. Elisa was used to measure the serum sorbitol in subjects enrolled in this study, and the clinical sample was collected to detect PKM2, AR and NRF2 expression. ResultsHerein, we showed that shikonin treatment in HT-29 and CaCO2 cells not only contributed to CDX2 and its targets expression, including MUC2 and villin, leading to cell differentiation, but also induced NRF2-mediated AKR1B1 and AKR1B10 expression, resulting in increasing AR activity. A synergistic effect on CDX2-mediated cell differentiation was observed in CaCO2 cells in response to cotreatment with shikonin and AR agonist bortezomib for 24 hours, while aldose reductase inhibitor fidarestat treatment in CaCO2 cells reversed the enhanced CDX2 expression caused by shikonin. Mechanistically, PKM2 interacted with NRF2, and this interaction was enhanced in response to shikonin stimulation, resulting in NRF2 phosphorylation and nuclear translocation, which further increased AKR1B1 and AKR1B10 transactivation. Moreover, both AKR1B1 and AKR1B10 expression were decreased, while PKM2 was increased in intestinal epithelial cells labeled with E-cadherin in patients with IBD, and a negative clinical relationship was observed between PKM2 and AKR1B1/AKR1B10, respectively. What's more, the serum sorbitol level was decreased in patients with IBD. ConclusionThese results suggested that shikonin could promote intestinal cell differentiation through NRF2-mediated polyol pathway, implying that shikonin may be a promising therapeutic drug for the treatment of IBD.

Cinnamic acid inhibits cell viability, invasion, and glycolysis in primary endometrial stromal cells by suppressing NF-κB-induced transcription of PKM2.

Endometriosis is a painful disorder characterized by the growth of endometrial tissue outside the uterine cavity. Here, we investigated the effects of the cinnamic acid isolated from the Chinese medicinal plant Cinnamomum cassia Presl on primary endometrial stromal cells. Immunohistochemistry was used to examine protein expression and cell purity. Quantitative RT-PCR was conducted to assess mRNA expression, and Western blot was performed to determine protein level. Cell viability was assessed using cell counting kit-8 (CCK-8) assay. Glycolysis and mitochondrial function were evaluated by measuring the extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR) of cells, respectively. Lastly, plasmid transfection and inhibitor treatment were used for overexpression and inhibition studies. Cinnamic acid inhibited cell viability and cell invasion, as well as decreased ECAR and OCR, in primary endometrial stromal cells. Cinnamic acid suppressed the effects of PKM2 overexpression, and inhibition of PKM2 using Compound 3k mimicked the effects of cinnamic acid. Treatment with Compound 3k and cinnamic acid did not lead to additive effects, but rather displayed effects similar to those of Compound 3k alone, suggesting that cinnamic acid elicits its effects on primary endometrial stromal cells by targeting PKM2. Our study identified cinnamic acid as a novel compound from Cinnamomum cassia Presl that displays potent effects on primary endometrial stromal cell viability, invasion, and glycolysis, suggesting its potential use for endometriosis treatment.

Mechanism of PKM2 affecting cancer immunity and metabolism in Tumor Microenvironment.

PKM2 is the enzyme that regulates the final rate-limiting step of glycolysis. PKM2 expression can reinforce the utilization of oxygen and synthesis of growth substances in cancer cells by enhancing OXPHOS and the Warburg effect. In cancer immunity, PKM2 can modulate the expression of PD-L1 in M2 macrophage and decrease the amount and activity of CD8+ T cells. This affects cancer cell killing and immune escape sequentially. How PKM2 regulates PD-L1 expression through immunometabolism is summarized. PKM2 builds a bridge between energy metabolism and cancer immunity. The activator and inhibitor of PKM2 both promote the anti-cancer immune response and inhibit cancer growth and metastasis by regulating the metabolism of cancer cells and immune cells in the tumor microenvironment through HIF-1α/PKM2 pathway. This review focuses on the precise role of PKM2 modulating immunometabolism, providing valuable suggestions for further study in this field.

Hypoxia facilitates the proliferation of colorectal cancer cells by inducing cancer-associated fibroblast-derived IL6

Colorectal cancer (CRC) is the most common malignancy worldwide, and its underlying molecular mechanisms remain largely unexplored. Accumulating evidences indicate cancer-associated fibroblasts (CAFs), abundant stromal cell population in the tumor microenvironment, play a key role in tumor development. Herein, we have successfully isolated CAFs and paired normal fibroblasts (NFs) from colorectal cancer tissues (n=10). By using a multiplex cytokine profiling assay, we have identified IL-6 as a major cytokine released by CAFs. Co-culturing of CAFs with CRC cell lines HCT116 or SW480 increases IL-6 release, and the secretion by CAFs can be further enhanced under hypoxia. By using the CCK-8 assay, we have found that HCT116 or SW480 cells treated with culture medium from CAFs, IL-6, or hypoxia showed a significant cell growth compared to control cells (p<0.01). Mechanistically, we have found that hypoxia could enhance the effect of the IL-6/STAT3 signaling on CRC cells, in part, through HIF-1a targeting PKM2. In conclusion, our data clearly proposes the interconnected mechanisms for constitutive activation of STAT3 signal by CAFs-derived IL-6 under hypoxia in colorectal cancer. The pharmacological inhibition of STAT3, PKM2, or HIF-1α can significantly reduce the oncogenic effect of IL-6, providing a potential therapeutic target for CRC patients.

SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing.

Background: A metabolic “switch” from oxidative phosphorylation to glycolysis provides tumor cells with energy and biosynthetic substrates, thereby promoting tumorigenesis and malignant progression. However, the mechanisms controlling this metabolic switch in tumors is not entirely clear.Methods: Clinical specimens were used to determine the effect of SAM68 on lung adenocarcinoma (LUAD) tumorigenesis and metastasis, and mouse models and molecular biology assays were performed to elucidate the function and underlying mechanisms in vitro and in vivo.Results: SAM68 mRNA levels were higher in LUAD tissue than in normal lung tissue, indicating that SAM68 expression is upregulated in LUAD. Patients with LUAD with SAM68high (n = 257) had a higher frequency of tumor recurrence (p = 0.025) and recurrence-free survival (p = 0.013) than did those with SAM68low (n = 257). Patients with SAM68high mRNA levels (n = 257) were at a higher risk for cancer-related death (p = 0.006), and had shorter overall survival (p = 0.044) than did those with SAM68low. SAM68 promotes tumorigenesis and metastasis of LUAD cells in vitro and in vivo by regulating the cancer metabolic switch. SAM68 drives cancer metabolism by mediating alternative splicing of pyruvate kinase (PKM) pre-mRNAs, and promoting the formation of PKM2. Mechanistically, SAM68 increased the binding of the splicing repressor hnRNP A1 to exon 9 of PKM, thereby enhancing PKM2 isoform formation and PKM2-dependent aerobic glycolysis and tumorigenesis.Conclusions: SAM68 promotes LUAD cell tumorigenesis and cancer metabolic programming via binding of the 351-443 aa region of SAM68 to the RGG motif of hnRNP A1, driving hnRNP A1-dependent PKM splicing, contributing to increased oncogene PKM2 isoform formation and inhibition of PKM1 isoform formation. SAM68 is therefore a promising therapeutic target for the treatment of LUAD.

Positive Feedback Regulation of Microglial Glucose Metabolism by Histone H4 Lysine 12 Lactylation in Alzheimer's Disease

The pro-inflammatory activation of microglia is a hallmark of Alzheimer’s disease (AD), and this process is known to involve a switch in energy metabolism from oxidative phosphorylation (OXPHOS) towards glycolysis. Here, we show how a positive feedback loop in microglia—comprising metabolic, histone lactylation, and transcriptional layers—drives AD pathogenesis, and we demonstrate that inhibiting this vicious cycle in microglia can ameliorate Aβ burden and cognitive deficits in AD model mice. After first detecting elevated histone lactylation in both AD model (5XFAD) mice and AD patient brains, we observed that H4K12la levels are elevated specifically in Aβ plaque-adjacent microglia. We subsequently found that this lactate-dependent histone modification is enriched at the promoters of glycolytic genes (e.g., Pkm). We confirmed that this enrichment activates transcription and thereby increases glycolysis activity, and ultimately demonstrate that a glycolysis/H4K12la/PKM2 positive feedback loop exacerbates microglial dysfunction in AD. Pharmacologic inhibition of PKM2 attenuated microglial activation, and microglia-specific ablation of Pkm2 improved spatial learning and memory in AD mice. Thus, beyond demonstrating a role for histone lactylation in a neurodegenerative disease and showing how multi-layered regulatory impacts attend altered glucose metabolism in microglia, our study illustrates that disrupting a positive feedback loop may support the development of innovative AD therapies.

&lt;p&gt;Simultaneous Inhibition of Ornithine Decarboxylase 1 and Pyruvate Kinase M2 Exerts Synergistic Effects Against Hepatocellular Carcinoma Cells&lt;/p&gt;

PurposePreviously, we showed that lactate promoted the proliferation and mobility of hepatocellular carcinoma (HCC) cells by increasing the expression of ornithine decarboxylase 1 (ODC1). In this study, we determined the relationship between ODC1 and pyruvate kinase M2 (PKM2, a key lactate metabolism enzyme), and determined the combined effects of difluoromethylornithine (DFMO; an ODC1 inhibitor) and compound 3k (a PKM2 inhibitor) on HCC cells.MethodsFirst, the relationship between PKM2 and ODC1 was analyzed using Western blotting, Cell Counting Kit (CCK)-8 assays, transwell assays, bioinformatics, quantitative real-time fluorescent PCR (qRT-PCR), and immunohistochemical staining. Thereafter, the ODC1 inhibitor DFMO and the PKM2 inhibitor compound 3k were employed. Their combined effects on HCC cell proliferation and mobility were evaluated via CCK-8 assay, flow cytometry, a subcutaneous xenograft tumor model in mice, wound healing assays, and transwell assays. Additionally, the effects of DFMO and compound 3k on the epithelial–mesenchymal transition phenotype and the AKT/GSK-3β/β-catenin pathway were explored using Western blotting and immunofluorescence.ResultsPKM2 knockdown significantly decreased the ODC1 expression, and the proliferation and invasion of HCC cells, while ODC1 overexpression reversed the inhibitory effects of PKM2 knockdown. Similarly, inhibition of ODC1 also decreased the expression of PKM2 via reducing the c-myc-induced transcription. PKM2 was co-expressed with ODC1 in HCC samples, while simultaneously upregulated PKM2 and ODC1 led to the poorest survival outcome. DFMO and compound 3k synergistically inhibited HCC cell proliferation, induced apoptosis, and suppressed cell mobility, as well as the EMT phenotype and the AKT/GSK-3β/β-catenin pathway. The AKT activator SC79 reversed the inhibitory effects.ConclusionPKM2/ODC1 are involved in a positive feedback loop. The simultaneous inhibition of ODC1 and PKM2 using DFMO and compound 3k exerts synergistic effects against HCC cells via the AKT/GSK-3β/β-catenin pathway. Thus, DFMO combined with compound 3k may be a novel effective strategy for treating HCC.

PKM2-c-Myc-Survivin Cascade Regulates the Cell Proliferation, Migration, and Tamoxifen Resistance in Breast Cancer.

The M2 isoform of pyruvate kinase (PKM2), as a key glycolytic enzyme, plays important roles in tumorigenesis and chemotherapeutic drug resistance. However, the intricate mechanism of PKM2 as a protein kinase regulating breast cancer progression and tamoxifen resistance needs to be further clarified. Here, we reported that PKM2 controls the expression of survivin by phosphorylating c-Myc at Ser-62. Functionally, PKM2 knockdown suppressed breast cancer cell proliferation and migration, which could be rescued by overexpression of survivin. Interestingly, we found that the level of PKM2 expression was upregulated in the tamoxifen resistant breast cancer cells MCF-7/TAMR, and knockdown of PKM2 sensitized the cells to 4-hydroxytamoxifen (4OH-T). In addition, the elevated level of PKM2 correlates with poor relapse-free survival in breast cancer patients treated with tamoxifen. Overall, our findings demonstrated that PKM2–c-Myc–survivin cascade regulated the proliferation, migration and tamoxifen resistance of breast cancer cells, suggesting that PKM2 represents a novel prognostic marker and an attractive target for breast cancer therapeutics, and that PKM2 inhibitor combined with tamoxifen may be a promising strategy to reverse tamoxifen resistance in breast cancer patients.

PKM2 inhibition may reverse therapeutic resistance to transarterial chemoembolization in hepatocellular carcinoma

BackgroundTherapeutic options for patients with hepatocellular carcinoma (HCC) are limited. Transarterial chemoembolization (TACE) is an interventional procedure used to deliver chemotherapy and embolizing agents directly to the tumor and is the procedure of choice for patients with intermediate stage HCC. While effective, more than 40% of patients do not respond to therapy, highlighting the need to investigate possible mechanisms of resistance. We sought to evaluate mechanisms of TACE resistance and evaluate a potential therapeutic target to overcome this resistance.MethodsUsing a prognostic gene signature which predicts TACE response (TACE Navigator) in a cohort of HCC patients who received TACE, patients were classified as responders and non-responders. Transcriptomic and gene pathway analysis were used to identify potential drivers of TACE resistance. Knockdown of the gene encoding rate limiting enzyme PKM2 using shRNA in HCC cell lines, as well as pharmacologic inhibition of PKM2 with shikonin using an in vitro TACE model measured response to chemotherapy under hypoxia. Finally, we replicated the TACE model with shikonin using patient derived cell line organoids (PDC). Functional studies were performed in vitro using immunoblotting, quantitative polymerase chain reaction, glycolysis and hypoxia assays.ResultsIn patient non-responders, we identified enrichment of the glycolysis pathway, specifically of the gene encoding the rate-limiting enzyme PKM2. We identified four HCC cell lines which recapitulated a TACE responder-like and non-responder-like phenotype. PKM2 knockdown in HCC cell lines demonstrated a less proliferative and aggressive phenotype as well as improved drug sensitivity to both doxorubicin and cisplatin. In vitro TACE model demonstrated that TACE non-responder-like cells overcame therapeutic resistance and rendered them susceptible to therapy through PKM2 knockdown. Lastly, we obtained similar results using a pharmacologic PKM2 inhibitor, shikonin in both cell lines, and PDC organoids.ConclusionElevated PKM2 is associated with treatment resistance and abbreviated survival in patients receiving TACE. Elevated PKM2 in vitro is associated with increased utilization of the glycolysis pathway, resulting in oxygen independent cell metabolism. Through PKM2 knockdown as well as with pharmacologic inhibition with shikonin, non-responder cells can be reprogrammed to act as responders and could improve TACE efficacy in patients.

Synthesis and Target Identification of Benzoxepane Derivatives as Potential Anti-Neuroinflammatory Agents for Ischemic Stroke.

Benzoxepane derivatives were designed and synthesized, and one hit compound emerged as being effective in vitro with low toxicity. In vivo, this hit compound ameliorated both sickness behavior through anti-inflammation in LPS-induced neuroinflammatory mice model and cerebral ischemic injury through anti-neuroinflammation in rats subjected to transient middle cerebral artery occlusion. Target fishing for the hit compound using photoaffinity probes led to identification of PKM2 as the target protein responsible for anti-inflammatory effect of the hit compound. Furthermore, the hit exhibited an anti-neuroinflammatory effect in vitro and in vivo by inhibiting PKM2-mediated glycolysis and NLRP3 activation, indicating PKM2 as a novel target for neuroinflammation and its related brain disorders. This hit compound has a better safety profile compared to shikonin, a reported PKM2 inhibitor, identifying it as a lead compound in targeting PKM2 for the treatment of inflammation-related diseases.

Synthesis and Target Identification of Benzoxepane Derivatives as Potential Anti‐Neuroinflammatory Agents for Ischemic Stroke

AbstractBenzoxepane derivatives were designed and synthesized, and one hit compound emerged as being effective in vitro with low toxicity. In vivo, this hit compound ameliorated both sickness behavior through anti‐inflammation in LPS‐induced neuroinflammatory mice model and cerebral ischemic injury through anti‐neuroinflammation in rats subjected to transient middle cerebral artery occlusion. Target fishing for the hit compound using photoaffinity probes led to identification of PKM2 as the target protein responsible for anti‐inflammatory effect of the hit compound. Furthermore, the hit exhibited an anti‐neuroinflammatory effect in vitro and in vivo by inhibiting PKM2‐mediated glycolysis and NLRP3 activation, indicating PKM2 as a novel target for neuroinflammation and its related brain disorders. This hit compound has a better safety profile compared to shikonin, a reported PKM2 inhibitor, identifying it as a lead compound in targeting PKM2 for the treatment of inflammation‐related diseases.

Targeting Metabolic Enzyme Pyruvate Kinase M2: A Novel Strategy to Inhibit Platelet Function and Arterial Thrombosis

Background: The cellular responses initiated upon platelet activation are energy consuming. Activated platelets, in comparison to their resting state, exhibit a high level of aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen) relative to oxidative phosphorylation (OXPHOS), suggesting that metabolic plasticity exists in platelets. Although aerobic glycolysis yields less total ATP when compared to OXPHOS, the rate of ATP generation is faster in aerobic glycolysis compared to OXPHOS, which we hypothesize is well suited for high-energy requirement during platelet activation. The glycolytic enzyme pyruvate kinases (PKs) catalyzes the final step of glycolysis and contributes to net ATP production. Four PK isoforms (L, R, M1 and M2) exist in mammals: L and R isoforms are expressed in the liver and red blood cells; the M1 isoform is expressed in most adult tissues that have high catabolic demands including muscle and brain; M2 is expressed in cells including activated platelets and leukocytes. Unlike other isoforms of PK that function only as tetramers, PKM2 can exist in either a tetrameric state or a dimeric state. PKM2 is allosterically regulated by the upstream metabolite fructose-1, 6 biphosphate. While PKM1 and tetrameric PKM2 favor ATP production from OXPHOS through the TCA cycle, dimeric PKM2 drives aerobic glycolysis. The glycolytic and non-glycolytic functions of PKM2 in platelets have not investigated yet. Objective: We tested an innovative concept that whether targeting metabolic enzyme PKM2 will inhibit platelet function and arterial thrombosis. Methods: Using a specific inhibitor of PKM2 (that prevents PKM2 dimerization and stabilizes tetramers) and a range of standardized platelet in vitro assays, we determined the mechanistic role of PKM2 in modulating platelet function in human and mice. To provide definitive evidence, we generated a megakaryocyte or platelet-specific PKM2-/- mouse (PKM2fl/flPF4Cre). Susceptibility to thrombosis was evaluated in vitro (microfluidics flow chamber) and in vivo (FeCl3-induced carotid and laser-injury induced mesenteric artery thrombosis models) by utilizing intravital microscopy. Susceptibility to hemostasis was evaluated in tail bleeding assay. Results: Human and mouse platelets pretreated with PKM2 inhibitor significantly decreased platelet aggregation to sub-optimal doses of collagen, convulxin, thrombin, and ADP. Consistent with this, inhibiting PKM2 dimerization reduced αIIbβ3 activation, alpha and dense granule secretion, clot retraction that was concomitant with decreased glucose uptake. Furthermore, treatment with PKM2 inhibitor reduced Akt and GSK3β phosphorylation, that are predominantly involved in PI3K/Akt signaling, suggesting a non-glycolytic role of the PKM2 in regulating platelet function. In microfluidics flow chamber assay, human and whole mouse blood pretreated with PKM2 inhibitor formed small thrombi when perfused over collagen for 5 minutes at an arterial shear rate of 1500s-1 (P&lt;0.05 vs. vehicle). In agreement with PKM2 inhibitor studies, platelets from PKM2fl/flPF4Cre mice exhibited decreased agonist-induced platelet aggregation, which was in agreement with decreased alpha and dense granule secretion, αIIbβ3 activation, clot retraction, lactate production, and Akt and GSK3β phosphorylation (P&lt;0.05 vs. PKM2fl/fl littermate controls). Wild-type mice-treated with PKM2 inhibitor and/or PKM2fl/flPF4Cre were less susceptible to thrombosis in the FeCl3-induced carotid and laser injury-induced mesenteric artery thrombosis models. Lack of effect on tail bleeding time suggested normal hemostasis in PKM2fl/flPF4Cre mice and PKM2 inhibitor-treated wild-type mice. No sex-based differences were observed. Currently, we are performing platelet metabolomics to determine the effect of targeting PKM2 on metabolic pathways. Conclusions: Our results suggest that manipulating metabolic plasticity by targeting dimeric PKM2 may be explored as a novel strategy to inhibit platelet function and arterial thrombosis. Disclosures Lentz: Novo Nordisk Inc.: Consultancy, Honoraria, Research Funding.

Activation of AMPK promotes thyroid cancer cell migration through its interaction with PKM2 and β-catenin

AMP-activated protein kinase (AMPK) is induced by the exhaustion of cellular energy and activates adaptive alterations in cellular metabolism, which is the basis for cell survival during different environmental stresses. We aimed to investigate the biological functions of AMPK and its molecular mechanism in regulating thyroid cancer (TC) progression. In current study, we found that activation of AMPK by multiple agonists suppresses TC cell proliferation. However, AMPK activation also led to TC cell migration at the same time. Depletion of AMPK abolished the effect of its agonist on cell multiplication and migration. Mechanistic investigations revealed that the impact of AMPK in terms of cell migration is dependent on its nuclear translocation, since site mutation of AMPK in its nuclear translocation domain (K244A) abolished TC cell migration but did not affect the inhibition of cell proliferation by AMPK agonist. Moreover, the nuclear AMPK recruits PKM2 and β-catenin by their interaction, which promotes the transcription of cell migration related genes, including MMP7 and c-Myc. Furthermore, depletion of PKM2/β-catenin abolished the migration effect of AMPK agonists, but did not affect their effects on suppression of cell proliferation. Our results provided a novel function of AMPK in cancer migration, and suggested that a combination of AMPK activation and PKM2 depletion or inhibition can be a new strategy to achieve better therapeutic effects for TC patients.