Inhibitor AZD3965 Research Articles

Emodin regulated lactate metabolism by inhibiting MCT1 to delay non-small cell lung cancer progression.

Lung cancer is one of the most common malignant tumors in the world, with high incidence rate and mortality. Monocarboxylate transporter (MCT) 1 has been found to be widely expressed in various tumors and plays a crucial role in regulating energy metabolism. Emodin, as an important traditional Chinese medicine in China, has been reported to inhibit the progression of lung cancer. However, its potential mechanism has not been fully elucidated. The effects of emodin and MCT1 inhibitor AZD3965 on the proliferation, migration, and invasion of lung cancer cells were detected using cell counting kit-8 (CCK-8) assay, wound-healing assay, and transwell small chamber assay. The content of glucose, lactate, and pyruvate in the cell culture medium was detected using a glucose, lactate, and pyruvate detection kit, and also detected protein expression using western blotting. In addition, to investigate the effects of emodin and AZD3965 on lung cancer in vivo, we constructed nude mice subcutaneous transplant tumor model by subcutaneous injection of lung cancer cells. The results showed that emodin and AZD3965 could inhibit the proliferation, migration, and invasion of lung cancer cells. At the same time, they could inhibit the expression of MCT1 in lung cancer cells and promote the release of lactate, but did not affect the content of glucose and pyruvate. In vivo experiments had shown that emodin and AZD3965 could effectively inhibit the growth of lung cancer and inhibit the expression of MCT1. All in all, our data suggested that emodin inhibited the proliferation, migration, and invasion of lung cancer cells, possibly by inhibiting MCT1, providing important theoretical basis for elucidating the mechanism of emodin in treating lung cancer.

Carrier-Free Photodynamic Bioregulators Inhibiting Lactic Acid Efflux Combined with Immune Checkpoint Blockade for Triple-Negative Breast Cancer Immunotherapy.

Abnormal tumor metabolism creates a complex tumor immune microenvironment that plays a dominant role in the metastasis of triple-negative breast cancer (TNBC). TNBC is insensitive to immune checkpoint blockade (ICB) therapy because of insufficient cytotoxic T lymphocyte (CTL) infiltration and a hyper-lactic acid-suppressive immune microenvironment caused by abnormal glycolysis. Herein, we propose an amplified strategy based on lactic acid regulation to reprogram the immunosuppressive tumor microenvironment (ITM) and combine it with ICB therapy to achieve enhanced antitumor immunotherapy effects. Specifically, we constructed CASN, a carrier-free photodynamic bioregulator, through the self-assembly of the photosensitizer Chlorin e6 and monocarboxylate transporter 1 (MCT1) inhibitor AZD3965. CASN exhibited a uniform structure, good stability, and drug accumulation at the tumor site. CASN-mediated photodynamic therapy following laser irradiation inhibited primary tumor growth and induced immunogenic cell death. Furthermore, CASN reduced lactic acid-mediated regulatory T cell generation and M2 tumor-associated macrophage polarization by blocking MCT1-mediated lactic acid efflux to attenuate immune suppression, inducing the recruitment and activation of CTLs. Ultimately, CASN-mediated immunopotentiation combined with ICB therapy considerably strengthened tumor immunotherapy and effectively inhibited tumor growth and metastasis of TNBC. This synergistic amplification strategy overcomes the limitations of an acidic ITM and presents a potential clinical treatment option for metastatic tumors.

Deciphering metabolic heterogeneity in retinoblastoma unravels the role of monocarboxylate transporter 1 in tumor progression

BackgroundTumors exhibit metabolic heterogeneity, influencing cancer progression. However, understanding metabolic diversity in retinoblastoma (RB), the primary intraocular malignancy in children, remains limited.MethodsThe metabolic landscape of RB was constructed based on single-cell transcriptomic sequencing from 11 RB and 5 retina samples. Various analyses were conducted, including assessing overall metabolic activity, metabolic heterogeneity, and the correlation between hypoxia and metabolic pathways. Additionally, the expression pattern of the monocarboxylate transporter (MCT) family in different cell clusters was examined. Validation assays of MCT1 expression and function in RB cell lines were performed. The therapeutic potential of targeting MCT1 was evaluated using an orthotopic xenograft model. A cohort of 47 RB patients was analyzed to evaluate the relationship between MCT1 expression and tumor invasion.ResultsDistinct metabolic patterns in RB cells, notably increased glycolysis, were identified. This metabolic heterogeneity correlated closely with hypoxia. MCT1 emerged as the primary monocarboxylate transporter in RB cells. Disrupting MCT1 altered cell viability and energy metabolism. In vivo studies using the MCT1 inhibitor AZD3965 effectively suppressed RB tumor growth. Additionally, a correlation between MCT1 expression and optic nerve invasion in RB samples suggested prognostic implications.ConclusionsThis study enhances our understanding of RB metabolic characteristics at the single-cell level, highlighting the significance of MCT1 in RB pathogenesis. Targeting MCT1 holds promise as a therapeutic strategy for combating RB, with potential prognostic implications.

Transcriptome analysis of anaerobic glycolysis effects on Jurkat T cell proliferation.

To explore the effects of anaerobic glycolysis on Jurkat T cell proliferation and clarify the possible mechanism via transcriptomic analysis. The monocarboxylate transporter 1 inhibitor AZD3965 was used to target and block the transmembrane transport of lactate, thereby inhibiting anaerobic glycolysis in Jurkat T cells. Then, genes with differential expression between treated and untreated cells were detected by transcriptomic analysis, and constructs were generated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses as well as protein-protein interaction (PPI) network analysis were performed to explore the potential mechanism. Inhibition of anaerobic glycolysis reduced Jurkat T-cell proliferation. RNA sequencing identified 1723 transcripts that were differentially expressed, including 1460 upregulated genes and 263 downregulated genes. GO functional enrichment analysis showed that the differentially expressed genes were mainly involved in the biological processes of response to unfolded protein, response to topologically incorrect protein, and protein folding. KEGG pathway analysis of differentially expressed genes or hub genes from the PPI network analysis revealed enrichment in the estrogen signaling and PI3K-Akt pathways. Anaerobic glycolysis contributes to the regulation of Jurkat T-cell proliferation. The underlying mechanism may involve the estrogen signaling pathway or PI3K-Akt signaling pathway as well as protein metabolism.

Effects of Lactate Transport Inhibition by AZD3965 in Muscle-Invasive Urothelial Bladder Cancer.

The Warburg Effect is characterized by high rates of glucose uptake and lactate production. Monocarboxylate transporters (MCTs) are crucial to avoid cellular acidosis by internal lactate accumulation, being largely overexpressed by cancer cells and associated with cancer aggressiveness. The MCT1-specific inhibitor AZD3965 has shown encouraging results in different cancer models. However, it has not been tested in urothelial bladder cancer (UBC), a neoplasm where rates of recurrence, progression and platinum-based resistance are generally elevated. We used two muscle-invasive UBC cell lines to study AZD3965 activity regarding lactate production, UBC cells' viability and proliferation, cell cycle profile, and migration and invasion properties. An "in vivo" assay with the chick chorioallantoic membrane model was additionally performed, as well as the combination of the compound with cisplatin. AZD3965 demonstrated anticancer activity upon low levels of MCT4, while a general lack of sensitivity was observed under MCT4 high expression. Cell viability, proliferation and migration were reduced, cell cycle was arrested, and tumor growth "in vivo" was inhibited. The compound sensitized these MCT4-low-expressing cells to cisplatin. Thus, AZD3965 seems to display anticancer properties in UBC under a low MCT4-expression setting, but additional studies are necessary to confirm AZD3965 activity in this cancer model.

MCT4 blockade increases the efficacy of immune checkpoint blockade

Background & AimsIntratumoral lactate accumulation and acidosis impair T-cell function and antitumor immunity. Interestingly, expression of the lactate transporter monocarboxylate transporter (MCT) 4, but not MCT1, turned out to be...

Abstract 3930: MCT1 as a potential target in the progression of oral premalignant dysplasia to invasive squamous cell carcinoma

Abstract Introduction: Most oral squamous cell carcinomas (OSCC) progress through clinically identifiable stages, starting from premalignant epithelial dysplasia to early invasive OSCC. How cells transform from premalignant dysplasia to early invasive OSCC is unknown. High monocarboxylate transporter 1 (MCT1; SLC16A1) expression is correlated with poor outcomes in OSCC patients, where it is thought to promote tumor growth by supporting metabolic coupling between cancer cells and tumor stroma. Because expression of MCT1 is markedly increased in OSCC patients, we sought to determine if MCT1 played a role in the progression of oral dysplasia in early invasive OSCC. Methods: Immunohistochemistry (IHC) was used to evaluate changes in the levels of MCT1 expression across adjacent normal, dysplasia, and OSCC tissues to identify expression patterns during cancer progression. Genetic knockout (KO) of SLC16A1 via CRISPR/Cas9 and pharmacological blockade by the MCT1 inhibitor AZD3965 were used on OSCC (TR146) and dysplastic (DOK) cell lines. KO was confirmed by qRT-PCR and immunoblotting. MCT1-KO and AZD3965-treated cells were assayed for changes in cell invasion using Matrigel-coated transwell assays. Correlative changes in gene expression based on MCT1 staining and disease progression were also observed in adjacent normal, dysplasia, and OSCC regions in each tissue section using Nanostring GeoMx Digital Spatial Profiler. Results: Expression of MCT1 was directly correlated to the progression of disease from dysplasia to early superficially invasive OSCC relative to normal adjacent epithelium. KO of MCT1 significantly decreased the invasion of dysplastic and OSCC cell lines compared to the wild-type (parental) controls. AZD3965 also decreased invasion in the parental dysplastic and OSCC lines. To identify progressive changes in gene expression across normal, dysplasia, and early invasive OSCC, transcripts for cancer-related genes were quantified in the epithelial and stromal compartments from patient samples. Compared to the normal epithelium, upregulation in gene expression, including SLC16A1, was observed in the cell cycle and metabolic pathways in OSCC tissues. Conclusions: Our data suggest that MCT1 plays a key role in breaching of the basement membrane and facilitating tumor cell migration and invasion. Targeting MCT1 in oral dysplasia may prevent progression to OSCC in oral cancer patients. Citation Format: Ethan J. Wong, Ali Khammanivong, Ravi Maisuria, Erin B. Dickerson, Raj Gopalakrishnan. MCT1 as a potential target in the progression of oral premalignant dysplasia to invasive squamous cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3930.

PD04-11 MCT INHIBITION RESENSITIZES ENZALUTAMIDE-RESISTANT PROSTATE CANCER CELLS TO ENZALUTAMIDE

You have accessJournal of UrologyCME1 Apr 2023PD04-11 MCT INHIBITION RESENSITIZES ENZALUTAMIDE-RESISTANT PROSTATE CANCER CELLS TO ENZALUTAMIDE Sayani Bhattacharjee, Rebecca Wynn, Puneet Sindhwani, Firas Petros, and Nagalakshmi Nadiminty Sayani BhattacharjeeSayani Bhattacharjee More articles by this author , Rebecca WynnRebecca Wynn More articles by this author , Puneet SindhwaniPuneet Sindhwani More articles by this author , Firas PetrosFiras Petros More articles by this author , and Nagalakshmi NadimintyNagalakshmi Nadiminty More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003228.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate cancers (PCa) exhibit a unique metabolic profile with reliance on different forms of glucose metabolism at different stages of disease progression. Early stage PCa cells use the more efficient TCA cycle, while metastatic PCa cells switch to glycolysis (Warburg effect), leading to the accumulation of lactate. Monocarboxylate transporters (MCTs) play a major role in the export/import of lactate to maintain redox balance. Such metabolic reprogramming can lead to gain-of-function mutations and affect drug sensitivity. MCT expression levels are high in enzalutamide-resistant PCa cells. Hence, we hypothesized that MCT inhibition may be an attractive therapeutic strategy to overcome enzalutamide resistance in PCa cells. METHODS: We analyzed the expression levels of MCTs in PCa tissues using public datasets from Oncomine. We treated C4-2B and 22Rv1 parental and enzalutamide-resistant cells with varying concentrations of MCT inhibitors AR-C155858, AZD3965, or syrosingopine either singly or in combination with enzalutamide and assessed cell survival, cell viability, proliferation, clonogenic ability, survival in 3-D models, and glycolytic activity. We treated mouse xenografts of PCa cells with MCT inhibitors or enzalutamide either singly or in combination and assessed tumor growth. RESULTS: We found that the suppression of MCT activity using the MCT antagonists AR-C155858, AZD3965, or syrosingopine significantly diminished the proliferation, survival, and clonogenic ability of enzalutamide-resistant PCa cells compared with parental cells. MCT inhibition in combination with enzalutamide inhibited the metabolic adaptation of PCa cells. Tumor growth of enzalutamide-resistant PCa cell xenografts was suppressed significantly when treated with a combination of MCT inhibitors with enzalutamide. These findings imply that MCT inhibition may be an effective strategy to counter enzalutamide resistance in PCa. CONCLUSIONS: MCT activation may underlie the development of enzalutamide resistance in PCa and targeting it may represent a viable combinatorial option. Source of Funding: Department of Defense Prostate Prostate Cancer Research Program W81XWH2010794 (PC190332) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e146 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sayani Bhattacharjee More articles by this author Rebecca Wynn More articles by this author Puneet Sindhwani More articles by this author Firas Petros More articles by this author Nagalakshmi Nadiminty More articles by this author Expand All Advertisement PDF downloadLoading ...

Targeting cancer lactate metabolism with synergistic combinations of synthetic catalysts and monocarboxylate transporter inhibitors

Synthetic anticancer catalysts offer potential for low-dose therapy and the targeting of biochemical pathways in novel ways. Chiral organo-osmium complexes, for example, can catalyse the asymmetric transfer hydrogenation of pyruvate, a key substrate for energy generation, in cells. However, small-molecule synthetic catalysts are readily poisoned and there is a need to optimise their activity before this occurs, or to avoid this occurring. We show that the activity of the synthetic organometallic redox catalyst [Os(p-cymene)(TsDPEN)] (1), which can reduce pyruvate to un-natural d-lactate in MCF7 breast cancer cells using formate as a hydride source, is significantly increased in combination with the monocarboxylate transporter (MCT) inhibitor AZD3965. AZD3965, a drug currently in clinical trials, also significantly lowers the intracellular level of glutathione and increases mitochondrial metabolism. These synergistic mechanisms of reductive stress induced by 1, blockade of lactate efflux, and oxidative stress induced by AZD3965 provide a strategy for low-dose combination therapy with novel mechanisms of action.Graphical abstract

Effects of lactate on metabolism and differentiation of CD4+T cells

BackgroundLactate accumulation caused by abnormal tumor metabolism can induce the formation of an inhibitory immune microenvironment through a variety of pathways, which is characterized by regulatory T cells (Treg) infiltration and effector T cells (Teff) depletion. Studies have found that the key reason why Treg cells can survive in harsh environments lies in their flexible metabolic mode, which can use lactate in tumor microenvironment (TME) as an alternative energy substance to maintain their inhibitory activity. In addition, lactate could also promote the differentiation of CD4+T cells into Treg, but the mechanism was not completely clear. The purpose of this study was to investigate the possible mechanism by which lactate is utilized by CD4+T cells to influence Th17/Treg ratio. MethodsBasal cytokines (anti-CD3, anti-CD28, TGF-β) and 10 mM lactate was added into Naïve CD4+T cells basal medium for 3 days. After TCR stimulation, Naïve CD4+T converted to CD4+T. Flow cytometry was used to detect the proportion of Treg cells; ELISA was used to detect the activity of LDHA, LDHB and NADH and the amount of α -Ketoglutaric Acid (α-KG) and 2-Hydroxyglutaric Acid (2HG) after lactate entered the cells; Western Blot and RT-PCR were used to detect the protein and gene expression of Foxp3, RORγt, LDHA and LDHB. In the validation experiment, lactate uptake inhibitor AZD3965, LDHA inhibitor GSK2837808A and NADH conversion inhibitor Rotenone were added respectively to observe the differentiation ratio of Treg cells and confirm the key points of metabolism; the degradation of Treg cell transcription factor Foxp3 was interfered with ubiquitination inhibitors to observe whether it co-ubiquitinated with HIF-1α; the expression and activity of LDHA, LDHB and NADH in mitochondria and cytoplasm were detected to confirm cell localization. ResultsWhen basal cytokines (anti-CD3, anti-CD28, TGF-β) stimulated, lactate was added to the culture medium, and CD4+T cells absorbed a large amount of lactate not only through MCT1 (monocarboxylic acid transporter), but also increased the expression of lactate dehydrogenase and accelerated the intracellular metabolism of lactate. LDHB in cytoplasm mainly catalyzed the dehydrogenation of lactate to pyruvate, accompanied by the transformation reaction between NAD+ and NADH. The latter further entered the mitochondria and participates in the tricarboxylic acid cycle metabolism. In addition, lactate could significantly increase the level of LDHA in mitochondria and promote the transformation of α-KG to 2HG, accompanied by the transformation of NADH to NAD+. These metabolic changes eventually led to an increase in the intracellular 2HG/α-KG ratio. Abnormal 2HG increased the proportion of Treg by inhibiting ATP5B-mediated phosphorylation of mTOR and the synthesis of HIF-1α, causing it not be enough to ubiquitinate and degrade with Foxp3. ConclusionsLactate plays an important role in regulating the differentiation of Treg cells, inducing the expression and function of LDHA and promoting the transformation of α-KG to 2HG may be an important mechanism.

Antitumor effects of lactate transport inhibition on esophageal adenocarcinoma cells

As a consequence of altered glucose metabolism, cancer cell intake is increased, producing large amounts of lactate which is pumped out the cytosol by monocarboxylate transporters (MCTs). MCT 1 and MCT4 are frequently overexpressed in tumors, and recently, MCT inhibition has been reported to exert antineoplastic effects. In the present study, MCT1 and MCT4 levels were assessed in esophageal adenocarcinoma (EAC) cells and the effects of the MCT-1 selective inhibitor AZD3965, hypoxia, and a glucose overload were evaluated in vitro. Two EAC cell lines (OE33 and OACM5.1C) were treated with AZD3965 (10–100 nM) under different conditions (normoxia/hypoxia) and also different glucose concentrations, and parameters of cytotoxicity, oxidative stress, intracellular pH (pHi), and lactate levels were evaluated. MCT1 was present in both cell lines whereas MCT4 was expressed in OE33 cells and only in a small proportion of OACM5.1C cells. Glucose addition did not have any effect on apoptosis nor cell proliferation. AZD3965 increased apoptosis and reduced proliferation of OACM5.1C cells, effects which were abrogated when cells were growing in hypoxia. MCT1 inhibition increased intracellular lactate levels in all the cells evaluated, but this increase was higher in cells expressing only MCT1 and did not affect oxidative stress. AZD3965 induced a decrease in pHi of cells displaying low levels of MCT4 and also increased the sodium/hydrogen exchanger 1 (NHE-1) expression on these cells. These data provide in vitro evidence supporting the potential of MCT inhibitors as novel antineoplastic drugs for EAC and highlight the importance of achieving a complete MCT inhibition.

Simultaneous targeting of glycolysis and oxidative phosphorylation as a therapeutic strategy to treat diffuse large B-cell lymphoma

BackgroundWe evaluated the therapeutic potential of combining the monocarboxylate transporter 1 (MCT1) inhibitor AZD3965 with the mitochondrial respiratory Complex I inhibitor IACS-010759, for the treatment of diffuse large B-cell lymphoma (DLBCL), a potential clinically actionable strategy to target tumour metabolism.MethodsAZD3965 and IACS-010759 sensitivity were determined in DLBCL cell lines and tumour xenograft models. Lactate concentrations, oxygen consumption rate and metabolomics were examined as mechanistic endpoints. In vivo plasma concentrations of IACS-010759 in mice were determined by LC-MS to select a dose that reflected clinically attainable concentrations.ResultsIn vitro, the combination of AZD3965 and IACS-010759 is synergistic and induces DLBCL cell death, whereas monotherapy treatments induce a cytostatic response. Significant anti-tumour activity was evident in Toledo and Farage models when the two inhibitors were administered concurrently despite limited or no effect on the growth of DLBCL xenografts as monotherapies.ConclusionsThis is the first study to examine a combination of two distinct approaches to targeting tumour metabolism in DLBCL xenografts. Whilst nanomolar concentrations of either AZD3965 or IACS-010759 monotherapy demonstrate anti-proliferative activity against DLBCL cell lines in vitro, appreciable clinical activity in DLBCL patients may only be realised through their combined use.

PD43-03 MCT INHIBITION SYNERGIZES WITH TYROSINE KINASE AND MTOR INHIBITOR TREATMENT IN RENAL CELL CARCINOMA

You have accessJournal of UrologyCME1 May 2022PD43-03 MCT INHIBITION SYNERGIZES WITH TYROSINE KINASE AND MTOR INHIBITOR TREATMENT IN RENAL CELL CARCINOMA Bhaghyasree Jambunathan, Abdo Dergham, Sayani Bhattacharjee, Rebecca Wynn, Puneet Sindhwani, Firas Petros, and Nagalakshmi Nadiminty Bhaghyasree JambunathanBhaghyasree Jambunathan More articles by this author , Abdo DerghamAbdo Dergham More articles by this author , Sayani BhattacharjeeSayani Bhattacharjee More articles by this author , Rebecca WynnRebecca Wynn More articles by this author , Puneet SindhwaniPuneet Sindhwani More articles by this author , Firas PetrosFiras Petros More articles by this author , and Nagalakshmi NadimintyNagalakshmi Nadiminty More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002604.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: RCC has been recognized in recent years as a uniquely metabolic disease. All histologic types of RCC exhibit increase in aerobic glycolysis with disease progression. The TCGA-RCC study showed that high grade, high stage, and low survival are associated with a shift to aerobic glycolysis and a decrease in oxidative phosphorylation. Approaches targeting glucose metabolism in RCC such as GLUT1 inhibitors, hexokinase inhibitors, GAPDH inhibitor, and PKM2 inhibitor showed limited clinical benefit. Monocarboxylate transporters (MCTs) play a major role in the export/import of the glycolytic end-product lactate. Different MCT isoforms are differentially expressed in RCC tissues with an increase in the expression of MCT1 and MCT4 in clear-cell RCC and papillary RCC. Here we hypothesized that MCT inhibition may be an attractive therapeutic strategy against RCC cells. METHODS: We analyzed the expression levels of MCTs in RCC tissues using public datasets from Oncomine. We treated normal renal epithelial and renal cell carcinoma cells with varying concentrations of MCT inhibitors AR-C155858, AZD3965, or syrosingopine either singly or in combination with sunitinib or everolimus and assessed cell survival, cell viability, proliferation, clonogenic ability, survival in 3-D models, and glycolytic activity. We treated xenografts of ccRCC cells in mice with MCT inhibitors, sunitinib, or everolimus singly or in combination and assessed tumor growth. RESULTS: We found that the suppression of MCT activity using the MCT antagonists AR-C155858, AZD3965, or syrosingopine not only diminished the proliferation, survival, and clonogenic ability of RCC cells, but also potentiated the response of RCC cells to treatment with sunitinib or everolimus. MCT inhibitors inhibited the metabolic adaptation of ccRCC cells when treated with sunitinib or everolimus. Tumor growth of ccRCC xenografts was suppressed significantly when treated with a combination of MCT inhibitors with sunitinib or everolimus. These findings imply that MCT inhibition may be an effective strategy to develop combinatorial therapies in renal cell carcinoma. CONCLUSIONS: MCT inhibition may improve the anti-tumor effects of tyrosine kinase or mTOR inhibitors in renal cell carcinoma. Source of Funding: DoD-PCRP PC190332 (W81XWH2010794), University of Toledo College of Medicine and Life Sciences, NIH/NCI 1R21CA202404, NIH/NCI 1R03CA198696 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e702 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Bhaghyasree Jambunathan More articles by this author Abdo Dergham More articles by this author Sayani Bhattacharjee More articles by this author Rebecca Wynn More articles by this author Puneet Sindhwani More articles by this author Firas Petros More articles by this author Nagalakshmi Nadiminty More articles by this author Expand All Advertisement PDF DownloadLoading ...

Effect of Lactate Export Inhibition on Anaplastic Thyroid Cancer Growth and Metabolism.

Anaplastic thyroid cancer (ATC) is an aggressive malignancy without effective treatments. ATC cells demonstrate upregulated glycolysis (Warburg effect), generating lactate that is subsequently exported by monocarboxylate transporter 4 (MCT4). This study aims to determine whether MCT4 inhibition can suppress ATC growth. ATC cell lines 8505C, JL30, and TCO1 were grown in low (3 mmol/L; LG) or high (25 mmol/L; HG) glucose medium containing the lactate shuttle inhibitors acriflavine (10-25 μmol/L; ACF), syrosingopine (100 µmol/L; SYR), or AZD3965 (20 µmol/L; AZD). Lactate level and cell proliferation were measured with standard assays. Seahorse analysis was performed to determine glycolytic response. Compared with HG, addition of ACF to LG decreased lactate secretion for both 8505C (p < 10-5) and JL30 (p < 10-4) cells, whereas proliferation was also reduced (p < 10-4 and 10-5, respectively). During Seahorse analysis, addition of oligomycin increased acidification by 84 mpH/min in HG vs 10 mpH/min in LG containing ACF (p < 10-5). Treatment with LG and SYR drastically diminished 8505C and TCO1 growth vs HG (p < 0.01 for both). LG and AZD treatment also led to reduced proliferation in tested cell lines (p ≤ 0.01 for all) that was further decreased by addition of ACF (p < 10-4 vs HG, p ≤ 0.01 vs LG and AZD). Inhibition of lactate shuttles significantly reduced proliferation and glycolytic capacity of ATC cells in a low-glucose environment. Targeting suppression of glycolytic and lactate processing pathways may represent an effective treatment strategy for ATC.

Monocarboxylate Transporter 1 (MCT1) Mediates Succinate Export in the Retina.

PurposeSuccinate is exported by the retina and imported by eyecup tissue. The transporters mediating this process have not yet been identified. Recent studies showed that monocarboxylate transporter 1 (MCT1) can transport succinate across plasma membranes in cardiac and skeletal muscle. Retina and retinal pigment epithelium (RPE) both express multiple MCT isoforms including MCT1. We tested the hypothesis that MCTs facilitate retinal succinate export and RPE succinate import.MethodsWe assessed retinal succinate export and eyecup succinate import in short-term ex vivo culture using gas chromatography–mass spectrometry. We tested the dependence of succinate export and import on pH, proton ionophores, conventional MCT substrates, and the MCT inhibitors AZD3965, AR-C155858, and diclofenac.ResultsSuccinate exits retinal tissue through MCT1 but does not enter the RPE through MCT1 or any other MCT. Intracellular succinate levels are a contributing factor that determines if an MCT1-expressing tissue will export succinate.ConclusionsMCT1 facilitates export of succinate from retinas. An unidentified, non-MCT transporter facilitates import of succinate into RPE.

Metabolic drug survey highlights cancer cell dependencies and vulnerabilities

Interrogation of cellular metabolism with high-throughput screening approaches can unravel contextual biology and identify cancer-specific metabolic vulnerabilities. To systematically study the consequences of distinct metabolic perturbations, we assemble a comprehensive metabolic drug library (CeMM Library of Metabolic Drugs; CLIMET) covering 243 compounds. We, next, characterize it phenotypically in a diverse panel of myeloid leukemia cell lines and primary patient cells. Analysis of the drug response profiles reveals that 77 drugs affect cell viability, with the top effective compounds targeting nucleic acid synthesis, oxidative stress, and the PI3K/mTOR pathway. Clustering of individual drug response profiles stratifies the cell lines into five functional groups, which link to specific molecular and metabolic features. Mechanistic characterization of selective responses to the PI3K inhibitor pictilisib, the fatty acid synthase inhibitor GSK2194069, and the SLC16A1 inhibitor AZD3965, bring forth biomarkers of drug response. Phenotypic screening using CLIMET represents a valuable tool to probe cellular metabolism and identify metabolic dependencies at large.

Disrupting the Reverse Warburg Effect As a Therapeutic Strategy in Multiple Myeloma

Introduction: Altered cellular metabolism is a hallmark of every cancer cell. Aerobic glycolysis (“The Warburg Effect”) is one of the earliest recognized metabolic abnormalities in cancer cells whereby extracellular glucose is preferentially metabolized and eventually processed to generate lactate and energy in the form of ATP before the former is released extracellularly, irrespective of oxygen availability. While extracellular lactate produced and released from cancer cells has traditionally been considered a waste metabolic by-product, recent understanding of cell metabolism suggests that it can also serve as a primary metabolic fuel for cancer cells via uptake by monocarboxylate transporters (MCTs). Our goal was to evaluate this “Reverse Warburg Effect” phenomenon in multiple myeloma (MM) cells and determine if it can be exploited for therapeutic purposes.Methods: All HMCLs, MM1S, RPMI-8226 and U266, were grown in RPMI-1640 cell culture medium containing 11 mM glucose and supplemented with 10% dialyzed fetal bovine serum (FBS) and 2 mM Glutamine. Primary MM cells were extracted using magnetic bead CD138 positive selection from MM patient bone marrow aspirates. For 13C-labeling experiments, HMCLs and primary MM cells were suspended in RPMI-1640 cell culture media containing 13C-labeled isotopes. Isotopomer analysis of glycolytic and tricarboxylic acid (TCA) cycle metabolites from HMCL and primary MM cell pellets was performed using Agilent Technologies 5975C gas chromatography-mass spectrometry. Small molecule inhibitors, AZD3965 and syrosingopine, were purchased from Selleck Chemicals and Sigma respectively. Cellular viability and proliferation were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrasodium bromide (MTT) and CCK-8 assays respectively. MCT-1 and MCT-4 antibodies for western blotting were utilized to evaluate their cell membrane expression on HMCLs.Results: The HMCLs, MM1S and RPMI-8226 as well as primary CD138+ cells from MM patient bone marrow were cultured in cell culture media containing physiological levels (1 mM) of U-13C-Lactate. The incorporation of extracellular 13C into the intracellular glycolytic and TCA cycle metabolite pool was observed (Fig 1) based on the expected isotopomeric patterns, demonstrating the Reverse Warburg Effect in MM cells. The relative contribution of carbon substrate by extracellular lactate compared to extracellular glucose was assessed in the following HMCLs: MM1S, RPMI-8226 and U266 cells by culturing in cell culture media containing 3-13C-Lactate and U-13C-Glucose. Extracellular lactate (yellow bar) contribution to the formation of TCA metabolites equaled that of glucose (red bar) based on the expected isotopomer patterns, suggesting the relative importance of extracellular lactate as an essential nutrient like glucose (Fig 2). Since MCT-1 and MCT-4 are key bidirectional cell membrane transporters of lactate in and out of cells, we explored the clinical significance of their gene expression level on clinical outcomes using the COMMPASS dataset provided by the Multiple Myeloma Research Foundation (MMRF). When MM patients were dichotomized at above or below the median of the expression levels of fragments per kilobase of transcript per million (FPKM), MCT-1 and MCT-4 overexpression conferred a worse progression free survival and overall survival (Fig 3). The MCT-1/MCT-4 protein expression was detectable across the various HMCLs: MM1S, U266 and RPMI-8226 (Fig 4). Inhibition of MCT-1 by specific inhibitor AZD3965 was able to reduce proliferation but not affect viability of HMCLs at 48 hours (Fig 5). However, dual inhibition of MCT-1/MCT-4 using syrosingopine was able to significantly reduce proliferation and decrease viability of HMCLs in a dose dependent fashion (Fig 6). Finally, dual inhibition of MCT-1/MCT-4 using syrosingopine reduced the utilization of extracellular lactate into the TCA cycle pool by HMCLs in media containing 3-13C-Lactate (Fig 7).Conclusion: Utilization of extracellular lactate via Reverse Warburg Effect phenomenon appears highly active in MM cells. Disrupting the utilization of extracellular lactate by dual inhibition of both MCT-1 and MCT-4 appears therapeutic. In the future, dual inhibition of MCT-1/MCT-4 in combination with other anti-MM therapies should be evaluated to determine synergistic therapeutic potential. [Display omitted] DisclosuresKumar: Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Carsgen: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Antengene: Consultancy, Honoraria; Beigene: Consultancy; Bluebird Bio: Consultancy; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Consultancy; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Roche-Genentech: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding.

Phase I expansion study of the first-in-class monocarboxylate transporter 1 (MCT1) inhibitor AZD3965 in patients with diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL).

3115 Background: Tumours rely on lactate transporters (MCT1-4) to maintain glycolytic flux and avoid intracellular acidification. In haematological tumours the MCT1 transporter acts as a lactate and pyruvate exporter. AZD3965 is a potent and specific inhibitor of MCT1 causing intracellular lactate accumulation. In vivo efficacy was observed in xenografts of DLBCL and BL, expressing high levels of MCT1 and no or low levels of MCT4. In the AZD3965 phase I (NCT01791595) dose-escalation an oral recommended phase 2 dose (RP2D) of 10mg twice-daily (bd) was determined. Pharmacokinetic (PK) showed exposure estimated to produce a minimum MCT1 occupancy of 90% (based on modelling). DLTs were primarily on-target dose-dependent, reversible, asymptomatic alterations in retinal function seen on ERG. Methods: This expansion cohort enrolled patients with relapsed/refractory DLBCL and BL. Expression of MCT1/MCT4 was assessed by immunohistochemistry. Pharmacokinetic (PK) sampling was performed and pharmacodynamic assessments included [18F]FDG-PET/CT imaging and plasma/urine metabolomics. Results: 11 DLBCL patients were treated with AZD3965 10mg bd. PK showed exposure to be broadly in line with the escalation cohort. No significant ERG changes were observed. One patient experienced a dose-limiting SUSAR of Troponin I increase. MCT1 is expressed in erythrocytes, however no serious events of anaemia were seen, with one non-clinically significant episode of grade 3 anaemia reported. Urine analysis showed increased excretion of lactate and ketone bodies post AZD3965 treatment consistent with renal target engagement. No alteration was detected in plasma. Ongoing stable disease at cycle 5 was observed in one patient and an additional patient had a confirmed complete response (CR) lasting 15 months, with no significant toxicity. In the patient showing CR a reduction in tumour FDG uptake was observed on day 3 of cycle 1. The other four patients who consented to research imaging did not have a clinical response and no changes were observed on FDG-PET. Conclusions: AZD3965 can be safely administered at 10mg bd. In one DLBCL patient monotherapy activity was observed with changes in FDG-PET providing evidence indicative of proof of mechanism. These changes in FDG uptake as early as on day 3 in the responding patient warrant further investigation of FDG-PET as a predictive biomarker. Further biomarker analysis and preclinical studies are ongoing to understand the biology and explore effective combinations with other agents targeting tumour cell metabolism Clinical trial information: NCT01791595.

γ-Hydroxybutyric Acid-Ethanol Drug-Drug Interaction: Reversal of Toxicity with Monocarboxylate Transporter 1 Inhibitors.

The drug of abuse, γ-hydroxybutyric acid (GHB), is commonly co-ingested with ethanol, resulting in a high incidence of toxicity and death. Our laboratory has previously reported that GHB is a substrate for the monocarboxylate transporters (MCTs), necessary for its absorption, renal clearance, and tissue distribution, including across the blood-brain barrier. Our goal was to investigate the drug-drug interaction (DDI) between GHB and ethanol and to evaluate MCT1 inhibition as a strategy to reverse toxicity. The toxicokinetics of this DDI were investigated, including brain-to-plasma concentration ratios, in the presence and absence of ethanol. The toxicodynamic parameters examined were respiratory depression (breathing frequency, tidal volume) and sedation (time of return-of-righting reflex). Ethanol was administered (2 g/kg i.v.) 5 minutes before the intravenous or oral administration of GHB, and MCT1 inhibitors AZD-3965 and AR-C155858 (5 mg/kg i.v.) were administered 60 minutes after GHB administration. Ethanol administration did not alter the toxicokinetics or respiratory depression caused by GHB after intravenous or oral administration; however, it significantly increased the sedation effect, measured by return-to-righting time. AZD-3965 or AR-C155858 significantly decreased the effects of the co-administration of GHB and ethanol on respiratory depression and sedation of this DDI and decreased brain concentrations and the brain-to-plasma concentration ratio of GHB. The results indicate that ethanol co-administered with GHB increases toxicity and that MCT1 inhibition is effective in reversing toxicity by inhibiting GHB brain uptake when given after GHB-ethanol administration. SIGNIFICANCE STATEMENT: These studies investigated the enhanced toxicity observed clinically when γ-hydroxybutyric acid (GHB) is co-ingested with alcohol and evaluated strategies to reverse this toxicity. The effects of the novel monocarboxylate transporter 1 (MCT1) inhibitors AR-C155858 and AZD-3965 on this drug-drug interaction have not been studied before, and these preclinical studies indicate that MCT1 inhibitors can decrease brain concentrations of GHB by inhibiting brain uptake, even when administered at times after GHB-ethanol. AZD-3965 represents a potential treatment strategy for GHB-ethanol overdoses.

In Vitro and In Vivo Characterization of MCT1 Inhibitor AZD3965 Confirms Preclinical Safety Compatible with Breast Cancer Treatment.

Simple SummaryThe vast majority of tumors originate in tissues that use different substrates and oxygen to produce energy. However, tumors are disorganized structurally and functionally, which creates areas where oxygen and nutrients are poorly available. To survive and proliferate, cancer cells adapt by switching their metabolism to lactic fermentation. Their fate is further optimized by intercellular cooperation, but this creates a weakness that can be exploited therapeutically. Indeed, AZD3965 is a new drug currently tested in clinical trials that inhibits a cooperation based on lactate swapping for glucose between fermenting and respiring cells. It inhibits lactate transporter monocarboxylate transporter 1. Here, using malignant and nonmalignant cells representative of the breast tissue and several behavioral tests in mice, we establish that AZD3965 is safe for therapeutic use against cancer. The only side effect that we detected was a short-term memory retention defect that transiently perturbed the orientation of mice in space.To survive and proliferate in solid tumors, cancer cells adapt and evolve rapidly in microenvironments where oxygen and substrate bioavailability fluctuates over time and space. This creates metabolic heterogeneity. Cancer cells can further cooperate metabolically, for example by swapping glycolytic end-product lactate for blood-borne glucose. This type of cooperation can be targeted therapeutically, since transmembrane lactate exchanges are facilitated by lactate-proton symporters of the monocarboxylate (MCT) family. Among new drugs, AZD3965 is a first-in-class selective MCT1 inhibitor currently tested in Phase I/II clinical trials for patients with different types of cancers. Because MCT1 can function bidirectionally, we tested here whether and how malignant and nonmalignant cells adapt their metabolism and MCT repertoire when AZD3965 inhibits either lactate import or export. Using breast-associated malignant and nonmalignant cell lines as models, we report that AZD3965 is not directly cytotoxic. In the presence of glucose and glutamine, oxidative cells can survive when lactate uptake is blocked, and proliferating cells compensate MCT1 inhibition by overexpressing MCT4, a specialized facilitator of lactate export. Phenotypic characterization of mice focusing on metabolism, muscle and brain physiology found partial and transient memory retention defect as sole consequence of MCT1 inhibition by AZD3965. We therefore conclude that AZD3965 is compatible with anticancer therapy.