Inhibition Of LDHA Research Articles

Lactate Dehydrogenase A as a Target of Cancer Therapy

Lactate dehydrogenase (LDH) is an enzyme that catalyzes the conversion of pyruvate to lactate and on the contrary lactate to pyruvate. LDH is a tetramer consisting of two types of subunits M (LDHA) and H (LDHB). LDHA catalyzes the conversion of pyruvate to lactate, while LDHB converts lactate to pyruvate. LDHA increases its expression in cancer cells and is associated with cancer aggressiveness. Lactate that continues to increase gives the effect of an acidic microenvironment so that there is sufficient availability of oxygen for metabolism, which increases anaerobic glycolysis and decreases oxidative phosphorylation. Inhibition of LDHA can provide hope in cancer therapy because many studies have shown that inhibition of LDHA reduces cancer growth. This review will describe several cancer treatment approaches using LDH as a cancer therapy target until now.

Lactate dehydrogenases promote glioblastoma growth and invasion via a metabolic symbiosis

Lactate is a central metabolite in brain physiology but also contributes to tumor development. Glioblastoma (GB) is the most common and malignant primary brain tumor in adults, recognized by angiogenic and invasive growth, in addition to its altered metabolism. We show herein that lactate fuels GB anaplerosis by replenishing the tricarboxylic acid (TCA) cycle in absence of glucose. Lactate dehydrogenases (LDHA and LDHB), which we found spatially expressed in GB tissues, catalyze the interconversion of pyruvate and lactate. However, ablation of both LDH isoforms, but not only one, led to a reduction in tumor growth and an increase in mouse survival. Comparative transcriptomics and metabolomics revealed metabolic rewiring involving high oxidative phosphorylation (OXPHOS) in the LDHA/B KO group which sensitized tumors to cranial irradiation, thus improving mouse survival. When mice were treated with the antiepileptic drug stiripentol, which targets LDH activity, tumor growth decreased. Our findings unveil the complex metabolic network in which both LDHA and LDHB are integrated and show that the combined inhibition of LDHA and LDHB strongly sensitizes GB to therapy.

Lactate-induced lactylation in skeletal muscle is associated with insulin resistance in humans.

Elevated circulating lactate has been associated with obesity and insulin resistance. The aim of the current study was to determine if lactate-induced lysine lactylation (kla), a post-translational modification, was present in human skeletal muscle and related to insulin resistance. Fifteen lean (Body Mass Index: 22.1 ± 0.5 kg/m2) and fourteen obese (40.6 ± 1.4 kg/m2) adults underwent a muscle biopsy and 2-h oral glucose tolerance test. Skeletal muscle lactylation was increased in obese compared to lean females (19%, p < 0.05) and associated with insulin resistance (r = 0.37, p < 0.05) in the whole group. Skeletal muscle lactylation levels were significantly associated with markers of anaerobic metabolism (plasma lactate and skeletal muscle lactate dehydrogenase [LDH], p < 0.05) and negatively associated with markers of oxidative metabolism (skeletal muscle cytochrome c oxidase subunit 4 and Complex I [pyruvate] OXPHOS capacity, p < 0.05). Treatment of primary human skeletal muscle cells (HSkMC) with sodium lactate for 24 h increased protein lactylation and IRS-1 serine 636 phosphorylation in a similar dose-dependent manner (p < 0.05). Inhibition of glycolysis (with 2-deoxy-d-glucose) or LDH-A (with sodium oxamate or LDH-A siRNA) for 24 h reduced HSkMC lactylation which paralleled reductions in culture media lactate accumulation. This study identified the existence of a lactate-derived post-translational modification in human skeletal muscle and suggests skeletal muscle lactylation could provide additional insight into the regulation of skeletal muscle metabolism, including insulin resistance.

Genetic and Drug Inhibition of LDH-A: Effects on Murine Gliomas.

Simple SummaryThree different murine glioma cell lines were modified to downregulate expression of the murine LDH-A gene using shRNA knockdown (KD) and compared to pharmacologic (GNE-R-140) inhibition of the LDH enzyme complex, and to shRNA scrambled control (NC) cell lines. The effects of shRNA LDH-A knockdown and LDH drug-targeted inhibition (GNE-R-140) on tumor-cell metabolism, tumor growth, and animal survival were similar in each of the cell lines. However, an unexpected increase in the aggressiveness was observed in LDH-A KD and GNE-R-140 treated GL261 intracranial gliomas, but not in CT2A and ALTS1C1 i.c. gliomas. Our results show that LDH-A KD and GNE-R-140 treated GL261 cells are better able to metabolize lactate as a primary carbon source through the TCA cycle, and are a net consumer of lactate. These results suggest that inhibition of LDH-A/glycolysis may not be a general strategy to inhibit the i.c. growth of all gliomas, and that metabolic-inhibition treatment strategies need to be carefully assessed, since the inhibition of glycolysis may lead to the unexpected development and activation of alternative metabolic pathways resulting in enhanced tumor-cell survival in a nutrient-limited environment, leading to increased tumor aggressiveness.The effects of the LDH-A depletion via shRNA knockdown on three murine glioma cell lines and corresponding intracranial (i.c.) tumors were studied and compared to pharmacologic (GNE-R-140) inhibition of the LDH enzyme complex, and to shRNA scrambled control (NC) cell lines. The effects of genetic-shRNA LDH-A knockdown and LDH drug-targeted inhibition (GNE-R-140) on tumor-cell metabolism, tumor growth, and animal survival were similar. LDH-A KD and GNE-R-140 unexpectedly increased the aggressiveness of GL261 intracranial gliomas, but not CT2A and ALTS1C1 i.c. gliomas. Furthermore, the bioenergetic profiles (ECAR and OCR) of GL261 NC and LDH-A KD cells under different nutrient limitations showed that (a) exogenous pyruvate is not a major carbon source for metabolism through the TCA cycle of native GL261 cells; and (b) the unique upregulation of LDH-B that occurs in GL261 LDH-A KD cells results in these cells being better able to: (i) metabolize lactate as a primary carbon source through the TCA cycle, (ii) be a net consumer of lactate, and (iii) showed a significant increase in the proliferation rate following the addition of 10 mM lactate to the glucose-free media (only seen in GL261 KD cells). Our study suggests that inhibition of LDH-A/glycolysis may not be a general strategy to inhibit the i.c. growth of all gliomas, since the level of LDH-A expression and its interplay with LDH-B can lead to complex metabolic interactions between tumor cells and their environment. Metabolic-inhibition treatment strategies need to be carefully assessed, since the inhibition of glycolysis (e.g., inhibition of LDH-A) may lead to the unexpected development and activation of alternative metabolic pathways (e.g., upregulation of lipid metabolism and fatty-acid oxidation pathways), resulting in enhanced tumor-cell survival in a nutrient-limited environment and leading to increased tumor aggressiveness.

H3K9me3 represses G6PD expression to suppress the pentose phosphate pathway and ROS production to promote human mesothelioma growth.

The role of glucose-6-phosphate dehydrogenase (G6PD) in human cancer is incompletely understood. In a metabolite screening, we observed that inhibition of H3K9 methylation suppressed aerobic glycolysis and enhances the PPP in human mesothelioma cells. Genome-wide screening identified G6PD as an H3K9me3 target gene whose expression is correlated with increased tumor cell apoptosis. Inhibition of aerobic glycolysis enzyme LDHA and G6PD had no significant effects on tumor cell survival. Ablation of G6PD had no significant effect on human mesothelioma and colon carcinoma xenograft growth in athymic mice. However, activation of G6PD with the G6PD-selective activator AG1 induced tumor cell death. AG1 increased tumor cell ROS production and the resultant extrinsic and intrinsic death pathway, mitochondrial processes, and unfolded protein response in tumor cells. Consistent with increased tumor cell death in vitro, AG1 suppressed human mesothelioma xenograft growth in a dose-dependent manner in vivo. Furthermore, AG1 treatment significantly increased tumor-bearing mouse survival in an intra-peritoneum xenograft athymic mouse model. Therefore, in human mesothelioma and colon carcinoma, G6PD is not essential for tumor growth. G6PD acts as a metabolic checkpoint to control metabolic flux towards the PPP to promote tumor cell apoptosis, and its expression is repressed by its promotor H3K9me3 deposition.

Oxamate Attenuates Glycolysis and ER Stress in Silicotic Mice.

Glycolysis and ER stress have been considered important drivers of pulmonary fibrosis. However, it is not clear whether glycolysis and ER stress are interconnected and if those interconnections regulate the development of pulmonary fibrosis. Our previous studies found that the expression of LDHA, a key enzyme involved in glycolysis, was increased in silica-induced macrophages and silicotic models, and it was closely related to silicosis fibrosis by participating in inflammatory response. However, whether pharmacological inhibition of LDHA is beneficial to the amelioration of silicosis fibrosis remains unclear. In this study, we investigated the effects of oxamate, a potent inhibitor of LDHA, on the regulation of glycolysis and ER stress in alveolar macrophages and silicotic mice. We found that silica induced the upregulation of glycolysis and the expression of key enzymes directly involved in ER stress in NR8383 macrophages. However, treatment of the macrophages and silicotic mice with oxamate attenuated glycolysis and ER stress by inhibiting LDHA, causing a decrease in the production of lactate. Therefore, oxamate demonstrated an anti-fibrotic role by reducing glycolysis and ER stress in silicotic mice.

Inhibition of LDHA to induce eEF2 release enhances thrombocytopoiesis

AbstractTranslation is essential for megakaryocyte (MK) maturation and platelet production. However, how the translational pathways are regulated in this process remains unknown. In this study, we found that MK/platelet–specific lactate dehydrogenase A (LdhA) knockout mice exhibited an increased number of platelets with remarkably accelerated MK maturation and proplatelet formation. Interestingly, the role of LDHA in MK maturation and platelet formation did not depend on lactate content, which was the major product of LDHA. Mechanism studies revealed that LDHA interacted with eukaryotic elongation factor 2 (eEF2) in the cytoplasm, controlling the participation of eEF2 in translation at the ribosome. Furthermore, the interaction of LDHA and eEF2 was dependent on nicotinamide adenine dinucleotide (NADH), a coenzyme of LDHA. NADH-competitive inhibitors of LDHA could release eEF2 from the LDHA pool, upregulate translation, and enhance MK maturation in vitro. Among LDHA inhibitors, stiripentol significantly promoted the production of platelets in vivo under a physiological state and in the immune thrombocytopenia model. Moreover, stiripentol could promote platelet production from human cord blood mononuclear cell–derived MKs and also have a superposed effect with romiplostim. In short, this study shows a novel nonclassical function of LDHA in translation that may serve as a potential target for thrombocytopenia therapy.

LDH-A inhibitors as remedies to enhance the anticancer effects of PARP inhibitors in ovarian cancer cells.

Ovarian cancer is one of the most lethal gynecologic malignancies. It has been shown that PARP inhibitors can selectively target BRCA-mutated ovarian cancer and exert some effects on ovarian cancer without BRCA mutations. However, the mechanism is still unclear. In this study, wild-type BRCA ovarian cancer cells (A2780 and SKOV3) were used. Our results showed that using a PARP inhibitor (olaparib or AG14361) alone significantly inhibited the proliferation of A2780 cells but negligibly inhibited the proliferation of SKOV3 cells. We used RNA sequencing to explore differentially expressed genes and found that PARP inhibitors increased LDH-A in SKOV3 cells, which was confirmed by RT-PCR. Oxamate (a specific inhibitor of LDH-A) was used to investigate whether LDH-A inhibition enhances the suppressive effects of PARP inhibitors on ovarian cancer without BRCA mutations. CCK-8 assays, scratch assays and Transwell assays were used to determine cell proliferation, cell migration ability and invasion ability, respectively. Both olaparib and AG14361 significantly inhibited the proliferation/invasion ability of A2780 cells but not SKOV3 cells. Inhibition of LDH-A can remarkably promote the inhibitory effects of PARP inhibitors on both A2780 and SKOV3 cells. Thus, high expression level of LDH-A influenced the suppressive effects of PARP inhibitors on ovarian cancer with wild-type BRCA, and LDH-A inhibition notably enhanced this effect.

Melittin inhibits the expression of key genes involved in tumor microenvironment formation by suppressing HIF-1α signaling in breast cancer cells.

HIF-1α has critical roles in the formation of tumor microenvironment by regulating genes involved in angiogenesis and anaerobic respiration. TME fuels tumors' growth and metastasis and presents therapy with several challenges. Therefore, we aimed to investigate if Melittin disrupts HIF-1α signaling pathway in breast adenocarcinoma cell line MDA-MB-231. Breast adenocarcinoma cell line MDA-MB-231 was cultured in the presence of different doses of Melittin, and MTT assay was carried out to measure Melittin's cytotoxic effects. Cells were exposed to 5% O2 to mimic hypoxic conditions and Melittin. Western blot was used to measure HIF-1α protein levels. Gene expression analysis was performed using real-time PCR to measure relative mRNA abundance of genes involved in tumor microenvironment formation. Our results revealed that Melittin effectively inhibits HIF-1α at transcriptional and translational/post-translational level. HIF-1α protein and mRNA level were significantly decreased in Melittin-treated groups. It is found that inhibition of HIF-1α by Melittin is through downregulation of NFκB gene expression. Furthermore, gene expression analysis showed a downregulation in VEGFA and LDHA expression due to inhibition of HIF-1α protein by Melittin. In addition, cell toxicity assay showed that Melittin inhibits the growth of MDA-MB-231 cell line through activation of extrinsic and intrinsic apoptotic pathways by upregulating TNFA and BAX expression. Melittin suppresses the expression of genes responsible for formation of TME physiological hallmarks by suppressing HIF-1α signaling pathway. Our results suggest that Melittin can modulate tumor microenvironment by inhibition of VEGFA and LDHA.

Targeting lactate dehydrogenase a improves radiotherapy efficacy in non-small cell lung cancer: from bedside to bench

BackgroundLactate dehydrogenase A (LDHA) is overexpressed and associated with poor prognosis in many kinds of cancer. In the current study, we evaluated the prognostic value of LDHA expression in non-small cell lung cancer (NSCLC), and tested whether LDHA inhibition might improve radiotherapy efficacy in NSCLC.MethodsLDHA expression was investigated in NSCLC patients, using online database and further verified by immunohistochemistry. The prognostic value of LDHA was evaluated using Kaplan–Meier plotter database. In vitro, two NSCLC cell lines were pretreated with oxamate, an inhibitor of LDHA, and colony formation method was performed to determine cellular radiosensitivity. Comet assay was used to detect DNA damage after irradiation. Flow cytometry was applied to test cell cycle progression and apoptosis, and monodansylcadaverine (MDC) staining was used to examine cell autophagy.ResultsBoth mRNA and protein levels of LDHA expression were up-regulated in NSCLC tissues. High LDHA expression was a poor prognostic factor and associated with radioresistance in NSCLC patients. LDHA inhibition by oxamate remarkably increased radiosensitivity in both A549 and H1975 cancer cells, and enhanced ionizing radiation (IR)-induced apoptosis and autophagy, accompanied by cell cycle distribution alternations. Furthermore, LDHA inhibition induced reactive oxygen species (ROS) accumulation and cellular ATP depletion, which might increase DNA injury and hinder DNA repair activity.ConclusionsOur study suggests that inhibition of LDHA may be a potential strategy to improve radiotherapy efficacy in NSCLC patients, which needs to be further tested by clinical trials.

Inhibition of LDH-A by Oxamate Enhances the Efficacy of Anti-PD-1 Treatment in an NSCLC Humanized Mouse Model.

Immunotherapy is a curable treatment for certain cancers, but it is still only effective in a small subset of patients, partly because of the lack of sufficient immune cells in the tumor. It is reported that targeted lactate dehydrogenase (LDH) to reduce lactic acid production can promote the infiltration and activity of immune cells and turn tumors into hot tumors. Therefore, we constructed a humanized mouse model to evaluate the efficacy of using classical LDH inhibitor oxamate and pembrolizumab alone or in combination in non-small cell lung cancer (NSCLC). We found that both oxamate and pembrolizumab monotherapy significantly delayed tumor growth; moreover, combination therapy showed better results. Immunofluorescence analysis showed that oxamate treatment increased the infiltration of activated CD8+ T cells in the tumor, which might have enhanced the therapeutic effects of pembrolizumab. Treatment of the humanized mice with anti-CD8 abrogated the therapeutic effects of oxamate, indicating CD8+ T cells as the main force mediating the effect of oxamate. In conclusion, Our preclinical findings position that oxamate not only inhibits tumor growth at a high safe dose but also enhances the efficacy of pembrolizumab in Hu-PBMC-CDX mice. Our study also provides a preclinical model for exploring the efficacy of other immune-based combination therapies for NSCLC.

FSMP-13. HYPOXIC REGULATION OF LACTATE DEHYDROGENASE GENES (LDHA/B) IN T98 GLIOBLASTOMA MULTIFORME CELLS

Glioblastoma multiforme (GBM) is the most common primary brain cancer and carries a poor prognosis. GBM cells exhibit extensive metabolic alterations that enhance survival and proliferation in the mixed normoxic-hypoxic tumor microenvironment. Lactate dehydrogenase (LDH) enzymes are critical mediators of the normoxic to hypoxic transition in cells. Two LDH genes (A/B) encode monomers that combine to form five isoenzymes (LDH1-5) with different properties for pyruvate to lactate interconversion. Hypoxic induction of LDHA in all cells appears to occur via HIF-1 mediated transcription. However, little is known about hypoxic regulation of LDHB in cancer. We report on hypoxic regulation of LDHA/B in T98G, a rare cell line that has both normal and neoplastic features. Human T98 GBM cell lines were cultured in a humidified incubator at 37° C and 5% CO2 and were grown in normoxia (21% O2) or hypoxia (95% N2, 5% C02) for 72 hours. Relative expression of LDH isoforms 1-5 was assessed using native gel electrophoresis. Expression of the LDHA and LDHB genes was measured using qRT-PCR. LDHA-dominant isoforms (4/5) were detected in T98G cells subjected to normoxia and hypoxia via gel electrophoresis, however, LDHB-dominant isoforms (1/2) were not. The LDHA/B-equimolar isoform (3) was decreased in T98G cells subjected to hypoxia. LDHA gene expression was over two-fold greater than LDHB in normoxia (p = .00256 by one-tailed Mann-Whitney U test), and over nine-fold greater in hypoxia (p = .00256). LDHA:LDHB expression in hypoxia compared to normoxia was significantly different (p = .00256). LDHA expression increased three-fold in hypoxia (p = .00256), while LDHB expression decreased 0.3-fold in hypoxia (p = .03288). We document LDHB dysregulation in T98G cells as the gene is minimally responsive to oxygen. Therapeutic strategies aimed at promoting LDHB expression may complement inhibition of LDHA and reduce GBM survival in hypoxia.

Upregulation of lactate dehydrogenase A in a chronic model of temporal lobe epilepsy

The ketogenic diet treatment is effective for drug-resistant epilepsy. Because its antiepileptic effect is associated with lactate dehydrogenase (LDH), drug development is possible by targeting LDH enzymes. Seizures in rodent models are suppressed by inhibiting LDH; however, it remains unclear whether LDH in the brain is changed by seizures. In the present study, we examined the expression of LDH subunits (LDHA and LDHB) in a chronic model of temporal lobe epilepsy, in which seizures were induced by the microinjection of kainate into the mouse hippocampus. Using Western blot analyses, we found that LDHA expression was increased in the hippocampus of the chronic seizure model, whereas LDHB expression was not. Lactate levels in the hippocampus were also increased in this seizure model, suggesting elevated LDH enzymatic activities. Furthermore, the inhibition of LDHA suppressed spontaneous paroxysmal discharges in vivo in the chronic seizure model. In conclusion, our results show that chronic seizures increase LDHA, and conversely, the inhibition of LDHA suppresses seizures, which supports LDHA as a molecular target for the development of new antiepileptic drugs.

Lactate dehydrogenase A regulates autophagy and tamoxifen resistance in breast cancer

Estrogen receptor (ER) antagonist, tamoxifen has been universally used for the treatment of the ER-positive breast cancer; however, the inevitable emergence of resistance to tamoxifen obstructs the successful treatment of this cancer. So, there is an immediate requirement for the search of a novel therapeutic target for treatment of this cancer. Acquired tamoxifen-resistant breast cancer cell lines MCF-7 (MCF-7/TAM-R) and T47D (T47D/TAM-R) showed higher apoptotic resistance accompanied by induction of pro-survival autophagy compared to their parental cells. Besides, tamoxifen resistance was associated with reduced production of ATP and with overexpression of glycolytic pathways, leading to induced autophagy to meet the energy demand. Further, our study revealed that LDHA; one of the key molecules of glycolysis in association with Beclin-1 induced pro-survival autophagy in tamoxifen-resistant breast cancer. Mechanistically, pharmacological and genetic inhibition of LDHA reduced the pro-survival autophagy, with the restoration of apoptosis and reverting back the EMT like phenomena noticed in tamoxifen-resistant breast cancer. In total, targeting LDHA opened a novel strategy to interrupt autophagy and tamoxifen resistance in breast cancer.

ATM deficiency promotes progression of CRPC by enhancing Warburg effect

ATM is a well-known master regulator of double strand break (DSB) DNA repair and the defective DNA repair has been therapeutically exploited to develop PARP inhibitors based on the synthetic lethality strategy. ATM mutation is found with increased prevalence in advanced metastatic castration-resistant prostate cancer (mCRPC). However, the molecular mechanisms underlying ATM mutation-driving disease progression are still largely unknown. Here, we report that ATM mutation contributes to the CRPC progression through a metabolic rather than DNA repair mechanism. We showed that ATM deficiency generated by CRISPR/Cas9 editing promoted CRPC cell proliferation and xenograft tumor growth. ATM deficiency altered cellular metabolism and enhanced Warburg effect in CRPC cells. We demonstrated that ATM deficiency shunted the glucose flux to aerobic glycolysis by upregulating LDHA expression, which generated more lactate and produced less mitochondrial ROS to promote CRPC cell growth. Inhibition of LDHA by siRNA or inhibitor FX11 generated less lactate and accumulated more ROS in ATM-deficient CRPC cells and therefore potentiated the cell death of ATM-deficient CRPC cells. These findings suggest a new therapeutic strategy for ATM-mutant CRPC patients by targeting LDHA-mediated glycolysis metabolism, which might be effective for the PARP inhibitor resistant mCRPC tumors.

Metabolic Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes by Inhibition of HIF1α and LDHA

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are a readily available, robustly reproducible, and physiologically appropriate human cell source for cardiac disease modeling, drug discovery, and toxicity screenings in vitro. However, unlike adult myocardial cells in vivo, hPSC-CMs cultured in vitro maintain an immature metabolic phenotype, where majority of ATP is produced through aerobic glycolysis instead of oxidative phosphorylation in the mitochondria. Little is known about the underlying signaling pathways controlling hPSC-CMs' metabolic and functional maturation. To define the molecular pathways controlling cardiomyocytes' metabolic pathway selections and improve cardiomyocyte metabolic and functional maturation. We cultured hPSC-CMs in different media compositions including glucose-containing media, glucose-containing media supplemented with fatty acids, and glucose-free media with fatty acids as the primary carbon source. We found that cardiomyocytes cultured in the presence of glucose used primarily aerobic glycolysis and aberrantly upregulated HIF1α (hypoxia-inducible factor 1α) and its downstream target lactate dehydrogenase A. Conversely, glucose deprivation promoted oxidative phosphorylation and repressed HIF1α. Small molecule inhibition of HIF1α or lactate dehydrogenase A resulted in a switch from aerobic glycolysis to oxidative phosphorylation. Likewise, siRNA inhibition of HIF1α stimulated oxidative phosphorylation while inhibiting aerobic glycolysis. This metabolic shift was accompanied by an increase in mitochondrial content and cellular ATP levels. Furthermore, functional gene expressions, sarcomere length, and contractility were improved by HIF1α/lactate dehydrogenase A inhibition. We show that under standard culture conditions, the HIF1α-lactate dehydrogenase A axis is aberrantly upregulated in hPSC-CMs, preventing their metabolic maturation. Chemical or siRNA inhibition of this pathway results in an appropriate metabolic shift from aerobic glycolysis to oxidative phosphorylation. This in turn improves metabolic and functional maturation of hPSC-CMs. These findings provide key insight into molecular control of hPSC-CMs' metabolism and may be used to generate more physiologically mature cardiomyocytes for drug screening, disease modeling, and therapeutic purposes.

Abstract 21312: Lactate Dehydrogenase A Affects Neutrophil Transcellular Extravasation by Upregulating Invadopodia Signaling

During inflammation, such as ischemia reperfusion injury or atherosclerosis, excessive neutrophil infiltration drives pathogenesis. Yet, the relationship between neutrophil transmigration and inflammation is ill-defined. Neutrophils migrate through endothelial cells (EC) via para (between EC) and transcellular (through an EC, TCM) mechanisms. During TCM, neutrophils interact with EC via invadopodia-like structures to form a ‘pore’ into EC membrane, thus facilitating neutrophil migration through EC body. We recently reported that deficiency in Rap1b, a member of RasGTPase superfamily, enhanced neutrophil recruitment to inflamed lungs and susceptibility to endotoxin shock. Rap1b-/- neutrophils exhibit increased TCM, invadopodia-like formation and metalloproteinase (MMP) release, suggesting mode of migration may influence inflammation outcome. Here, we used Rap1b-/- neutrophils to further investigate the link between neutrophil migration and inflammation. Using mass spectrometry, we found 2-fold increase in glycolytic enzymes hexokinase1, LDHA and PGK1, in Rap1b-/- protrusions compared to WT. Immunofluorescent staining and western blotting confirmed this observation. Inhibition of LDHA with a specific inhibitor FX11, inhibited Rap1b-/- invadopodia protrusions, MMP release and TCM. We then tested effects of LDHA inhibition on neutrophil transendothelial migration in vivo. Rap1b-/- and WT neutrophils were tagged with cell tracker dyes and transferred to recipient mice, treated with FX11 or DMSO control. Ear microvasculature was stimulated with FMLP and labeled with PECAM antibody to visualize EC junctions. More Rap1b-/- cells migrated out of vessels compared to WT cells at the same vascular environment. FX11 treatment significantly abrogated this increase. This suggests that LDHA and enhanced glycolytic metabolism play critical roles during transmigration in vivo perhaps by changing neutrophil migratory behavior and inflammation outcome. Since lactate, the glycolytic byproduct, induces milieu acidification and acidity play a major role in ischemic damage to the heart, our findings may be important for our understanding of cardiovascular injury and for the development of approaches for effective treatment.

Discovery and Optimization of Potent, Cell-Active Pyrazole-Based Inhibitors of Lactate Dehydrogenase (LDH).

We report the discovery and medicinal chemistry optimization of a novel series of pyrazole-based inhibitors of human lactate dehydrogenase (LDH). Utilization of a quantitative high-throughput screening paradigm facilitated hit identification, while structure-based design and multiparameter optimization enabled the development of compounds with potent enzymatic and cell-based inhibition of LDH enzymatic activity. Lead compounds such as 63 exhibit low nM inhibition of both LDHA and LDHB, submicromolar inhibition of lactate production, and inhibition of glycolysis in MiaPaCa2 pancreatic cancer and A673 sarcoma cells. Moreover, robust target engagement of LDHA by lead compounds was demonstrated using the cellular thermal shift assay (CETSA), and drug-target residence time was determined via SPR. Analysis of these data suggests that drug-target residence time (off-rate) may be an important attribute to consider for obtaining potent cell-based inhibition of this cancer metabolism target.

NFκB mediated elevation of KCNJ11 promotes tumor progression of hepatocellular carcinoma through interaction of lactate dehydrogenase A

It has been well documented that changes in ion fluxes across cellular membranes is fundamental in maintaining cellular homeostasis. Dysregulation and/or malfunction of ion channels are critical events in the pathogenesis of diverse diseases, including cancers. In this study, we focused on the study of K+ channels in hepatocellular carcinoma (HCC). By data mining TCGA cohort, the expression of 27 K+ channels was investigated and KCNJ11 was identified as a key dysregulated K+ channels in HCC. KCNJ11 was differentially expressed in HCC and predicted a poor prognosis in HCC patients. Inhibition of NFκB signaling suppressed KCNJ11 expression in HCC cells. Knockdown of KCNJ11 expression inhibited cell proliferation, promoted cell apoptosis, and reduced cell invasive capacity. Mechanistically, we found that KCNJ11 promotes tumor progression through interaction with LDHA and enhancing its enzymatic activity. Pharmacological inhibition of LDHA largely compromised the oncogenic function of KCNJ11 in cell proliferation, cell apoptosis, and cell invasion. Collectively, our data, as a proof of principle, demonstrate that KCNJ11 acts as an oncogene in HCC though forming a complex with LDHA and suggest that targeting KCNJ11 can be developed as a candidate tool to dampen HCC.

Abstract 5932: Targeting tumor acidity with the LDHA inhibitor (FX11) and CAIX inhibitor (DH348) overcomes resistance to PD-1 blockade and inhibits metastasis in a pancreatic cancer model

Abstract Prior work by us and others has demonstrated that the extracellular pH (pHe) of solid tumors is acidic, due to a combination of increased fermentative metabolism, resulting in lactic acid production and poor perfusion. This acidity promotes tumor progression and metastasis formation. Recently we have shown in melanoma and pancreatic cancer models that acidity inhibits antitumor immunity by preventing T-cell activation. Reversal of acidity with buffer therapy (200mM NaHCO3) synergized with checkpoint blockade (anti-CTLA4 and anti-PD1) and adoptive T-cell therapy has resulted in cures. While this is promising, concerns are high regarding the ingestion of such large amounts of sodium bicarbonate, which makes clinical translation a challenge. Hence, we hypothesize that alternative pharmacological interventions can neutralize the pHe of tumors and remove this immunosuppressive effect. To study this we first investigated a series of agents for their ability to inhibit metastasis in the PC3 prostate cancer model, which is exquisitely sensitive to inhibition with buffer therapy. In this study, male SCID mice were grouped in to 6 groups (n=5) and treated with tap water, 200 mM bicarbonate ad lib, 30 mg/kg daily (q.d.) intraperitoneal (i.p.) Acetazolamide (CA inhibitor), 1.2 mg/kg q.d.i.p. Furosemide (diuretic), 10 mg/kg q.d.i.p. DH348 (selective CAIX inhibitor) or 2.1 mg/kg q.d.i.p. FX-11 (LDHA inhibitor). Mice were intravenously injected with 5*10^6 PC3M-luc cells and ventral bioluminescence images were acquired at time 0 and weekly thereafter. Our results showed that FX-11, acetazolamide, DH348 and bicarbonate were able to effectively (p&amp;lt;0.004) suppress metastasis formation in this system, whereas furosemide was not. Based on these results, a subsequent study investigated the combination of bicarbonate, FX-11 or DH348 with immune checkpoint blockade in a Panc02 mouse model of pancreatic cancer. Animals were inoculated orthotopically with Panc02 cells and randomized into 8 groups (n=10). Once tumors reached 0.3 cc in size, mice were treated with either 15 mg/kg anti-PD-1 antibody or normal rat IgG controls twice weekly alone or in combination with bicarbonate; FX-11 or DH348 and tumor response was evaluated using US 3D-Mode imaging and volume analysis (FUJIFILM VisualSonics) and histopathology. Anti-PD-1 antibody was ineffective as a monotherapy in this system. Both bicarbonate and DH348 neutralized tumor acidity and reduced tumor growth as monotherapies. Notably, despite the fact that FX-11 was shown to inhibit LDH-A in pancreatic cancer models, it did not affect pH and had no effect on tumor growth as monotherapy. However, FX-11 significantly reduced tumor growth (p &amp;lt;0.01) and metastasis formation in combination with anti-PD-1 antibody, suggesting that inhibition of LDH-A might be effective in combination with checkpoint blockade. Citation Format: Arig A. Ibrahim Hashim, Dominique Abrahams, Liping Xu, Barbra Centeno, Enakshi Sunassee, Rasha Abddelgader, Ludwig Dubois, Philippe Lambin, Robert A. Gatenby, Robert J. Gillies. Targeting tumor acidity with the LDHA inhibitor (FX11) and CAIX inhibitor (DH348) overcomes resistance to PD-1 blockade and inhibits metastasis in a pancreatic cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5932. doi:10.1158/1538-7445.AM2017-5932