Inhibition Of Oxidative Phosphorylation Research Articles

Abstract 4183: Differential modulation of tumor versus T cell oxidative phosphorylation potentiates anti-tumor immunity

Abstract We sought to determine whether various inhibitors of Oxidative Phosphorylation (OxPhos) could restore tumor infiltrating T cell functionality and longevity while compromising metabolic fitness of tumor cells. Using a combination of in vitro metabolic profiling with the Seahorse XFe96 Analyzer and in vivo tumor immunotherapy in the immunocompetent TRAMP-C2 prostate adenocarcinoma model, we determined the differential impact of multiple OxPhos inhibitors on both tumor immune metabolic fitness and functional capacity. Impact on the tumor immune microenvironment of TRAMP-C2 tumors was assessed through high parameter flow cytometry on a BD X-30. While OxPhos inhibition compromised initial T cell activation, we found that two of the inhibitors increased the glucose uptake, proliferation, and activation status of effector T cells. In contrast, the same two inhibitors decreased the mitochondrial fitness, proliferation and OxPhos metabolic capacity of TRAMP-C2 tumor cells themselves. To determine whether this selective inhibition of tumor versus T cell metabolic function could improve immunotherapy responses in vivo, we implanted TRAMP-C2 tumors and treated them with these OxPhos inhibitors with or without concomitant immune checkpoint blockade (ICB). The combination of ICB and OxPhos inhibition achieved highly significant and durable rates of tumor control and regression suggesting that these approaches are potentially synergistic. Analysis of the tumor microenvironment has identified the cellular mechanisms underlying these therapeutic effects which may provide useful biomarkers as this novel combination is translated to the clinic. Citation Format: Krithikaa Rajkumar Bhanu, Priyamvada Jayaprakash, Meghan Rice, Brittany Morrow, Joseph R. Marszalek, Jason P. Gay, Christopher P. Vellano, Benjamin R. Cowen, Dean J. Welsh, Michael A. Curran. Differential modulation of tumor versus T cell oxidative phosphorylation potentiates anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4183.

Abstract 6222: Comprehensive monitoring of pyruvate metabolism in cancer cells and tumors reveals vulnerability to metabolic inhibition therapy with small molecules

Abstract Introduction: Pyruvate(Pyr) metabolism is a keystone in cancer metabolism, as well as normal cells, and has two major metabolic fluxes; Glycolysis and Oxidative Phosphorylation (OXPHOS). Especially in cancer cells, increased Pyr metabolism meets the demand of higher energy consumption during rapid cell growing state. Therefore, monitoring tumor Pyr metabolic flux in vitro and in vivo should be necessary to develop a therapeutic strategy using small molecules which can inhibit the fluxes.Aim: We performed in vitro metabolic analysis on pancreatic cancer cells (MiaPaCa2) and applied a novel in vivo imaging technology “hyperpolarized 13C-pyruvate magnetic resonance spectroscopic imaging (HP-MRSI)” to dynamic monitoring of the impact of Glycolysis and OXPHOS inhibitors on the MiaPaCa2 xenografts. Methods: We used a novel LDH inhibitor “NCI-006” for Glycolysis inhibition and Mitochondrial Complex 1(MC1) Inhibitor "Metformin(Met)", which is already clinically available, and “IACS-010759(IACS)” for OXPHOS inhibition, in vitro and in vivo. Extracellular flux (EXF) analysis and HP-MRSI were performed before and after administration of each or both to assess inhibitor impact on metabolic flux in vitro and in vivo, respectively. Results: HP-MRSI confirmed that LDH activity in the tumor was suppressed by NCI-006 (83.3±4.4% decrease) and accelerated by Met (69.9±6.6% increase) and IACS (88.6±25.1% increase). We confirmed these in vivo observations are fully consistent with the effect of those inhibitors in vitro on the energy profile of MiaPaCa2, and also the close correlation of these data with the results of the ex vivo LDH activity assay, suggesting HP-MRSI can reliably monitor in vivo on target effects of the inhibitors without need for tissue sampling. In addition, combined treatment with the NCI-006 and MC1 inhibitor significantly suppressed in vitro cell growth and tumor growth in an efficacy study of MiaPaCa2 xenografts, compared to each single administration. Apoptosis assay showed the combined treatment induced apoptosis in MIA Paca2 cells. Conclusion: Using EXF analyzer and HP-MRSI revealed that Glycolysis inhibition redirects tumor Pyr toward OXPHOS and also MC1 inhibition redirects tumor Pyr toward Glycolysis. Combination therapy suppresses metabolic plasticity, causing metabolic quiescence in vitro and tumor growth inhibition in vivo. HP-MRSI is thought to be useful for pancreatic cancer patients to establish the current treatment model because of no need to obtain clinical samples, such as patients-derived cancer cells or spheroid from the patients. The current proof of concept can be of great value in developing new therapeutic strategies using metabolic inhibitors to treat cancers. Citation Format: Nobu Oshima, Yuki Aisu, Shigeo Hisamori, Shigeru Tsunoda, Hiroki Hashida, Kenji Uryuhara, Hiroyuki Kobayashi, Masato Kondo, koji Kitamura, Satoshi Kaihara, Kazutaka Obama, Krishna Murali, Len Neckers. Comprehensive monitoring of pyruvate metabolism in cancer cells and tumors reveals vulnerability to metabolic inhibition therapy with small molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6222.

Therapeutic Targeting of Tumor Cells and Tumor Immune Microenvironment Vulnerabilities.

Therapeutic targeting of tumor vulnerabilities is emerging as a key area of research. This review is focused on exploiting the vulnerabilities of tumor cells and the immune cells in the tumor immune microenvironment (TIME), including tumor hypoxia, tumor acidity, the bidirectional proton-coupled monocarboxylate transporters (MCTs) of lactate, mitochondrial oxidative phosphorylation (OXPHOS), and redox enzymes in the tricarboxylic acid cycle. Cancer cells use glucose for energy even under normoxic conditions. Although cancer cells predominantly rely on glycolysis, many have fully functional mitochondria, suggesting that mitochondria are a vulnerable target organelle in cancer cells. Thus, one key distinction between cancer and normal cell metabolism is metabolic reprogramming. Mitochondria-targeted small molecule inhibitors of OXPHOS inhibit tumor proliferation and growth. Another hallmark of cancer is extracellular acidification due lactate accumulation. Emerging results show that lactate acts as a fuel for mitochondrial metabolism and supports tumor proliferation and growth. Metabolic reprogramming occurs in glycolysis-deficient tumor phenotypes and in kinase-targeted, drug-resistant cancers overexpressing OXPHOS genes. Glycolytic cancer cells located away from the vasculature overexpress MCT4 transporter to prevent overacidification by exporting lactate, and the oxidative cancer cells located near the vasculature express MCT1 transporter to provide energy through incorporation of lactate into the tricarboxylic acid cycle. MCTs are, therefore, a vulnerable target in cancer metabolism. MCT inhibitors exert synthetic lethality in combination with metformin, a weak inhibitor of OXPHOS, in cancer cells. Simultaneously targeting multiple vulnerabilities within mitochondria shows synergistic antiproliferative and antitumor effects. Developing tumor-selective, small molecule inhibitors of OXPHOS with a high therapeutic index is critical to fully exploiting the mitochondrial vulnerabilities. We and others developed small-molecule inhibitors containing triphenylphosphonium cation that potently inhibit OXPHOS in tumor cells and tissues. Factors affecting tumor cell vulnerabilities also impact immune cells in the TIME. Glycolytic tumor cells supply lactate to the tumor-suppressing regulatory T cells overexpressing MCTs. Therapeutic opportunities for targeting vulnerabilities in tumor cells and the TIME, as well as the implications on cancer health disparities and cancer treatment, are addressed.

Inhibition of Rho-associated protein kinase activity enhances oxidative phosphorylation to support corneal endothelial cell migration.

Corneal endothelial cell (CEC) dysfunction causes corneal edema and severe visual impairment that require transplantation to restore vision. To address the unmet need of organ shortage, descemetorhexis without endothelial keratoplasty has been specifically employed to treat early stage Fuchs endothelial corneal dystrophy, which is pathophysiologically related to oxidative stress and exhibits centrally located corneal guttae. After stripping off central Descemet's membrane, rho-associated protein kinase (ROCK) inhibitor has been found to facilitate CEC migration, an energy-demanding task, thereby achieving wound closure. However, the correlation between ROCK inhibition and the change in bioenergetic status of CECs remained to be elucidated. Through transcriptomic profiling, we found that the inhibition of ROCK activity by the selective inhibitor, ripasudil or Y27632, promoted enrichment of oxidative phosphorylation (OXPHOS) gene set in bovine CECs (BCECs). Functional analysis revealed that ripasudil, a clinically approved anti-glaucoma agent, enhanced mitochondrial respiration, increased spare respiratory capacity, and induced overexpression of electron transport chain components through upregulation of AMP-activated protein kinase (AMPK) pathway. Accelerated BCEC migration and in vitro wound healing by ripasudil were diminished by OXPHOS and AMPK inhibition, but not by glycolysis inhibition. Correspondingly, lamellipodial protrusion and actin assembly that were augmented by ripasudil became reduced with additional OXPHOS or AMPK inhibition. These results indicate that ROCK inhibition induces metabolic reprogramming toward OXPHOS to support migration of CECs.

IMG-16. Non-invasive metabolic imaging of response to therapy in diffuse midline gliomas

Abstract Diffuse midline gliomas (DMGs) are universally lethal pediatric tumors that are defined by the presence of histone H3K27 alterations. The infiltrative nature and anatomical location of these tumors prohibit surgical resection. Radiotherapy, which is standard of care, does not significantly enhance long-term outcome. Novel therapies are sorely needed for DMG patients. Imipridone drugs ONC201 and ONC206 have demonstrated anti-tumor activity in preclinical cancer models, including DMGs and have shown promise in pilot studies in DMG patients. Successful clinical deployment of imipridones requires the identification of companion imaging biomarkers that report on response to therapy. Magnetic resonance spectroscopy (MRS) is a safe, non-radioactive, non-invasive method of imaging metabolism in vivo. 1H-MRS assesses steady-state metabolite levels and is used in clinics. 2H-MRS following administration of 2H-labeled substrates is a novel, clinically translatable method of imaging metabolic pathway activity. Our results indicate that treatment of SF7761 DMG cells with ONC206 causes a significant reduction in 1H-MRS-detectable lactate, glutamate, glutathione and phosphocholine, pointing to inhibition of glycolysis, oxidative phosphorylation, redox and phosphatidylcholine biosynthesis respectively. Examination of [6,6’-2H]-glucose metabolism using 2H-MRS indicates that lactate production from [6,6’-2H]-glucose is significantly reduced in ONC206-treated SF7761 cells relative to controls. We then investigated the effect of ONC206 on mice bearing orthotopic SF8628 DMG tumors. At day 7 following the treatment onset, at a timepoint when no change in tumor volume can be observed by anatomical imaging, in vivo1H-MRS-detectable lactate and total choline are reduced relative to day 0. Collectively, our studies indicate that imipridones induce alterations in DMG metabolism that can be leveraged for non-invasive 1H- and 2H-MRS-based imaging of response to therapy. By providing clinicians with an early readout of treatment response prior to anatomical changes, our biomarkers will enable early assessment of treatment response and, thereby, clinical translation of these promising therapeutics.

The impact of berberine on the energy metabolism of cancer cells and CAFs in non-small lung cancer.

e15097 Background: The energy metabolism of tumor cells and infiltrating stromal cells is a promising target in anticancer therapy. One of the mechanisms of antitumor activity of berberine is its ability to suppress oxidative phosphorylation in cancer cells. However, there is relatively little data on how berberine affects stromal cells compared to cancer cells. This study assessed the effect of berberine on the energy metabolism in non-small cell lung cancer (NSCLC) cell, as well as cancer-associated fibroblasts (CAFs) of the same localization. Methods: Cells of CAFs, NSCLC primary cell culture and permanent lung cancer culture H1299 were seeded in an amount of 2*104 per well in a Seahorse XFp Analyzer plate (Agilent, USA) in DMEM medium (Gibco, USA) supplemented with 10% FBS (HyClone, USA). After cell adhesion berberine at a concentration of 2.5 µM or an equal amount of medium without berberine was added. For each cell culture experimental and control wells were set up in 3 repetitions. After 24 hours of cultivation, the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Seahorse XFp Analyzer (Agilent, USA) using the Seahorse XFp Cell Energy Phenotype Test (Agilent, USA), with 2 µM FCCP and 1 µM oligomycin. Results: Cultivation with berberine resulted in a significant (α = 0.05, df = 4) decrease in baseline OCR in all cultures, which was more pronounced in CAFs. The decrease in the baseline OCR in CAFs was 20.65±5%, in the primary culture 17.56±2.1% and in the permanent culture of H1299 8.29±1.1% compared to the control samples. At the same time, the maximum level of OCR also significantly decreased in CAFs compared to the control by 47.13±6.2% and in the H1299 culture by 14.9±3.1% (α = 0.05, df = 4). However, in the primary culture of NSCLC, the decrease in maximum OCR compared to the control was only 3.87±1.2% and was not significant. Both primary NSCLC culture and CAFs exhibited an increase in baseline glycolysis in response to berberine addition. In the primary cell culture ECAR increased by 19.7±2.3%, and in the culture of fibroblasts by 18.9±3.8% compared to the control. On the contrary, the baseline level of glycolysis in the permanent cell line H1299 decreased, which can be judged by a decrease in ECAR by 32.8±5.9% compared with the control. ECAR level changes were significant in all cultures at the accepted level of significance (α = 0.05, df = 4). Conclusions: Berberine causes oxidative phosphorylation inhibition in both cancerous cells and CAFs. Nevertheless, a compensatory increase in the level of glycolysis is only observed in primary cell cultures of NSCLC and CAFs.

Chronic inhibition of the mitochondrial ATP synthase in skeletal muscle triggers sarcoplasmic reticulum distress and tubular aggregates

Tubular aggregates (TA) are honeycomb-like arrays of sarcoplasmic-reticulum (SR) tubules affecting aged glycolytic fibers of male individuals and inducing severe sarcomere disorganization and muscular pain. TA develop in skeletal muscle from Tubular Aggregate Myopathy (TAM) patients as well as in other disorders including endocrine syndromes, diabetes, and ageing, being their primary cause unknown. Nowadays, there is no cure for TA. Intriguingly, both hypoxia and calcium dyshomeostasis prompt TA formation, pointing to a possible role for mitochondria in their setting. However, a functional link between mitochondrial dysfunctions and TA remains unknown. Herein, we investigate the alteration in muscle-proteome of TAM patients, the molecular mechanism of TA onset and a potential therapy in a preclinical mouse model of the disease. We show that in vivo chronic inhibition of the mitochondrial ATP synthase in muscle causes TA. Upon long-term restrained oxidative phosphorylation (OXPHOS), oxidative soleus experiments a metabolic and structural switch towards glycolytic fibers, increases mitochondrial fission, and activates mitophagy to recycle damaged mitochondria. TA result from the overresponse of the fission controller DRP1, that upregulates the Store-Operate-Calcium-Entry and increases the mitochondria-SR interaction in a futile attempt to buffer calcium overloads upon prolonged OXPHOS inhibition. Accordingly, hypoxic muscles cultured ex vivo show an increase in mitochondria/SR contact sites and autophagic/mitophagic zones, where TA clusters grow around defective mitochondria. Moreover, hypoxia triggered a stronger TA formation upon ATP synthase inhibition, and this effect was reduced by the DRP1 inhibitor mDIVI. Remarkably, the muscle proteome of TAM patients displays similar alterations in mitochondrial dynamics and in ATP synthase contents. In vivo edaravone treatment in mice with restrained OXPHOS restored a healthy phenotype by prompting mitogenesis and mitochondrial fusion. Altogether, our data provide a functional link between the ATP synthase/DRP1 axis and the setting of TA, and repurpose edaravone as a possible treatment for TA-associated disorders.

Platinum-induced mitochondrial OXPHOS contributes to cancer stem cell enrichment in ovarian cancer

BackgroundPlatinum based agents—cisplatin and carboplatin in combination with taxanes are used for the treatment of ovarian cancer (OC) patients. However, the majority of OC patients develop recurrent, platinum resistant disease that is uniformly fatal. Platinum treatment enriches for chemoresistant aldehyde dehydrogenase (ALDH) + ovarian cancer stem cells (OCSCs), which contribute to tumor recurrence and disease relapse. Acquired platinum resistance also includes metabolic reprograming and switching to oxidative phosphorylation (OXPHOS). Chemosensitive cells rely on glycolysis while chemoresistant cells have the ability to switch between glycolysis and OXPHOS, depending on which pathway drives a selective advantage for growth and chemoresistance. High expression of genes involved in OXPHOS and high production of mitochondrial ROS are characteristics of OCSCs, suggesting that OCSCs favor OXPHOS over glycolysis. Based on connections between OCSCs, chemoresistance and OXPHOS, we hypothesize that platinum treatment induces changes in metabolism that contribute to platinum-induced enrichment of OCSCs.MethodsThe effect of cisplatin on mitochondrial activity was assessed by JC1 staining and expression of OXPHOS genes by RT-qPCR. Cisplatin-induced changes in Sirtuin 1 (SIRT1) levels and activity were assessed by western blot. Small molecule inhibitors of mitochondrial complex I and SIRT1 were used to determine if their enzymatic activity contributes to the platinum-induced enrichment of OCSCs. The percentage of ALDH + OCSCs in OC cells and tumor tissue from xenograft models across different treatment conditions was analyzed using ALDEFLUOR assay and flow cytometry.ResultsWe demonstrate that platinum treatment increases mitochondrial activity. Combined treatment of platinum agents and OXPHOS inhibitors blocks the platinum-induced enrichment of ALDH + OCSCs in vitro and in vivo. Furthermore, platinum treatment increases SIRT1 levels and subsequent deacetylase activity, which likely contributes to the increase in platinum-induced mitochondrial activity.ConclusionsThese findings on metabolic pathways altered by platinum-based chemotherapy have uncovered key targets that can be exploited therapeutically to block the platinum-induced enrichment of OCSCs, ultimately improving the survival of OC patients.

Sorafenib and nitazoxanide disrupt mitochondrial function and inhibit regrowth capacity in three-dimensional models of hepatocellular and colorectal carcinoma

Quiescent cancer cells in malignant tumors can withstand cell-cycle active treatment and cause cancer spread and recurrence. Three-dimensional (3D) cancer cell models have led to the identification of oxidative phosphorylation (OXPHOS) as a context-dependent vulnerability. The limited treatment options for advanced hepatocellular carcinoma (HCC) and colorectal carcinoma (CRC) metastatic to the liver include the multikinase inhibitors sorafenib and regorafenib. Off-target effects of sorafenib and regorafenib are related to OXPHOS inhibition; however the importance of this feature to the effect on tumor cells has not been investigated in 3D models. We began by assessing global transcriptional responses in monolayer cell cultures, then moved on to multicellular tumor spheroids (MCTS) and tumoroids generated from a CRC patient. Cells were treated with chemotherapeutics, kinase inhibitors, and the OXPHOS inhibitors. Cells grown in 3D cultures were sensitive to the OXPHOS inhibitor nitazoxanide, sorafenib, and regorafenib and resistant to other multikinase inhibitors and chemotherapeutic drugs. Furthermore, nitazoxanide and sorafenib reduced viability, regrowth potential and inhibited mitochondrial membrane potential in an additive manner at clinically relevant concentrations. This study demonstrates that the OXPHOS inhibition caused by sorafenib and regorafenib parallels 3D activity and can be further investigated for new combination strategies.

Hydropersulfides (RSSH) Outperform Post-Conditioning and Other Reactive Sulfur Species in Limiting Ischemia-Reperfusion Injury in the Isolated Mouse Heart.

Hydrogen sulfide (H2S) exhibits protective effects in cardiovascular disease such as myocardial ischemia/reperfusion (I/R) injury, cardiac hypertrophy, and atherosclerosis. Despite these findings, its mechanism of action remains elusive. Recent studies suggest that H2S can modulate protein activity through redox-based post-translational modifications of protein cysteine residues forming hydropersulfides (RSSH). Furthermore, emerging evidence indicates that reactive sulfur species, including RSSH and polysulfides, exhibit cardioprotective action. However, it is not clear yet whether there are any pharmacological differences in the use of H2S vs. RSSH and/or polysulfides. This study aims to examine the differing cardioprotective effects of distinct reactive sulfur species (RSS) such as H2S, RSSH, and dialkyl trisulfides (RSSSR) compared with canonical ischemic post-conditioning in the context of a Langendorff ex-vivo myocardial I/R injury model. For the first time, a side-by-side study has revealed that exogenous RSSH donation is a superior approach to maintain post-ischemic function and limit infarct size when compared with other RSS and mechanical post-conditioning. Our results also suggest that RSSH preserves mitochondrial respiration in H9c2 cardiomyocytes exposed to hypoxia-reoxygenation via inhibition of oxidative phosphorylation while preserving cell viability.

Analysis of the metabolic proteome of lung adenocarcinomas by reverse-phase protein arrays (RPPA) emphasizes mitochondria as targets for therapy

Lung cancer is the leading cause of cancer-related death worldwide despite the success of therapies targeting oncogenic drivers and immune-checkpoint inhibitors. Although metabolic enzymes offer additional targets for therapy, the precise metabolic proteome of lung adenocarcinomas is unknown, hampering its clinical translation. Herein, we used Reverse Phase Protein Arrays to quantify the changes in enzymes of glycolysis, oxidation of pyruvate, fatty acid metabolism, oxidative phosphorylation, antioxidant response and protein oxidative damage in 128 tumors and paired non-tumor adjacent tissue of lung adenocarcinomas to profile the proteome of metabolism. Steady-state levels of mitochondrial proteins of fatty acid oxidation, oxidative phosphorylation and of the antioxidant response are independent predictors of survival and/or of disease recurrence in lung adenocarcinoma patients. Next, we addressed the mechanisms by which the overexpression of ATPase Inhibitory Factor 1, the physiological inhibitor of oxidative phosphorylation, which is an independent predictor of disease recurrence, prevents metastatic disease. We highlight that IF1 overexpression promotes a more vulnerable and less invasive phenotype in lung adenocarcinoma cells. Finally, and as proof of concept, the therapeutic potential of targeting fatty acid assimilation or oxidation in combination with an inhibitor of oxidative phosphorylation was studied in mice bearing lung adenocarcinomas. The results revealed that this therapeutic approach significantly extended the lifespan and provided better welfare to mice than cisplatin treatments, supporting mitochondrial activities as targets of therapy in lung adenocarcinoma patients.

Atovaquone: An Inhibitor of Oxidative Phosphorylation as Studied in Gynecologic Cancers.

Simple SummaryThe Warburg effect in cancer cells (high glucose update and lactate release) is an adaptation that is considered a hallmark of most neoplasms. Blocking oxidative phosphorylation is one way to combat carcinogenesis. Atovaquone is a mitochondrial complex III inhibitor. It is currently FDA-approved for the treatment of malaria, and is a well-tolerated, orally available medication. Our laboratory studied the anti-cancer properties of atovaquone in gynecologic cancers. We found that atovaquone slowed ovarian cancer growth in both cell lines and mouse models. Additional anti-cancer effects were seen, such as the reduced proliferation of cancer stem cells and spheroids implanted in mice. Atovaquone inhibited oxygen consumption and ATP production. Metabolic studies showed that atovaquone shifted glycolysis, electronic transport and the citric acid cycle. Our studies provided the mechanistic understanding and preclinical data to support the further investigation of atovaquone’s potential as a cancer therapy for gynecologic cancers.Oxidative phosphorylation is an active metabolic pathway in cancer. Atovaquone is an oral medication that inhibits oxidative phosphorylation and is FDA-approved for the treatment of malaria. We investigated its potential anti-cancer properties by measuring cell proliferation in 2D culture. The clinical formulation of atovaquone, Mepron, was given to mice with ovarian cancers to monitor its effects on tumor and ascites. Patient-derived cancer stem-like cells and spheroids implanted in NSG mice were treated with atovaquone. Atovaquone inhibited the proliferation of cancer cells and ovarian cancer growth in vitro and in vivo. The effect of atovaquone on oxygen radicals was determined using flow and imaging cytometry. The oxygen consumption rate (OCR) in adherent cells was measured using a Seahorse XFe96 Extracellular Flux Analyzer. Oxygen consumption and ATP production were inhibited by atovaquone. Imaging cytometry indicated that the majority of the oxygen radical flux triggered by atovaquone occurred in the mitochondria. Atovaquone decreased the viability of patient-derived cancer stem-like cells and spheroids implanted in NSG mice. NMR metabolomics showed shifts in glycolysis, citric acid cycle, electron transport chain, phosphotransfer, and metabolism following atovaquone treatment. Our studies provide the mechanistic understanding and preclinical data to support the further investigation of atovaquone’s potential as a gynecologic cancer therapeutic.

Genome-wide CRISPR screens reveal metabolic and transcriptional regulation of BTN3A and cancer susceptibility to Vγ9Vδ2 T cell targeting

Abstract γδ T cells are potent anti-cancer effectors with potential to target tumors broadly, independent of neoantigens or HLA-background. γδ T cells can sense conserved cell stress signals prevalent in transformed cells, although the mechanisms governing how γδ T cells sense and kill stressed target cells remain poorly characterized. Vγ9Vδ2 T cells – the most abundant subset of human γδ T cells – recognize a protein complex containing butyrophilin 3A1 (BTN3A1), a ubiquitously expressed cell surface protein that is activated by phosphoantigens abundantly produced by tumor cells. Here we performed genome-wide CRISPR screens in target cancer cells to identify pathways that regulate: (1) γδ T cell activity with a functional co-culture killing screen, and (2) BTN3A1 cell surface expression in a phenotypic screen. Multilayered regulation of BTN3A1 expression was uncovered: transcriptional regulation (IRF1, CTBP1, ZNF217, RUNX1), intracellular trafficking, sialylation, oxidative phosphorylation (OXPHOS), purine metabolism, and other metabolic pathways. Consistent with these results, we found upregulated BTN3A1 on cells undergoing an energy crisis due to glucose deprivation, glycolysis inhibition, or OXPHOS inhibition. Furthermore, we discovered that this BTN3A1 upregulation was induced by activation of the AMP-activated protein kinase (AMPK). Also, CRISPR screen-derived gene expression signatures correlated with higher survival in low-grade glioma patients whose tumors had high Vγ9Vδ2 T cell infiltration. Uncovering this AMPK-dependent mechanism of metabolic stress-induced ligand activation deepens our understanding of γδ T cell stress surveillance and suggests new avenues to enhance γδ T cell anti-cancer activity. M.R.M. is a Cancer Research Institute (CRI) Irvington Fellow supported by CRI and was funded by the Human Vaccines Project Michelson Prize for Human Immunology.

MICRORNA-10A NEGATIVELY REGULATES INTESTINAL IGA RESPONSE THROUGH SUPPRESSING REGULATORY T CELLS CONVERSION TO T FOLLICULAR REGULATOR CELLS

Abstract Intestinal IgA response is a key immune regulation between host and gut microbiota. Regulatory T cells (Tregs) have been recently shown to promote IgA responses through conversion to T follicular regulator cells (TFRs). However, how Treg converts to TFR to promote intestinal IgA response remain unclear. Tregs express microRNA (miR)-10a at high levels, however, whether it regulates TFR generation and function is unknown. We found that Treg cell-specific miR-10a deficient Foxp3YFP-cre miR-10af/f (Foxp3ΔmiR-10a) mice displayed higher levels of gut IgA compared to wild type (WT) Foxp3YFP-cre miR-10af/+ mice. TFRs in Peyer’s patches were also significantly increased while there were no differences in germinal center B cells, Tregs, and TFH cells. When orally immunized with cholera toxin (CT), Foxp3ΔmiR-10a mice showed more CT-specific fecal IgA and TFRs. miR-10a deficient Tregs induced more IgA compared to WT Tregs when co-cultured with naïve B cells in vitro. When transferred to TCRβxδ−/− mice, miR-10a deficient Tregs induced more gut IgA and higher levels of TFRs compared to WT Tregs in vivo. However, gut microbiota composition in Foxp3ΔmiR-10a mice was not altered by 16S rRNA gene sequencing. RNA-sequencing analysis showed that miR-10a was enriched in the oxidative phosphorylation (OXPHOS) process in Tregs. Consistently, miR-10a deficient Tregs displayed higher OXPHOS metabolic levels by seahorse analysis. Pretreatment of Tregs with oligomycin, which inhibits OXPHOS, inhibited Treg induction of IgA production in vitro, indicating miR-10a suppressed Tregs induction of IgA ability by inhibiting OXPHOS. Thus, miR-10a negatively regulates TFRs conversion through inhibition of OXPHOS to control intestinal IgA response.

Drug self-delivery nanorods enhance photodynamic therapy of triple-negative breast cancer by inhibiting oxidative phosphorylation

Photodynamic therapy (PDT) shows very high potential for the clinical treatment of triple-negative breast cancer. However, the efficacy of PDT is significantly weakened by tumor hypoxia, the relatively high intracellular glutathione levels and the active proliferation of cancer cells. To address these issues, we developed a novel drug self-delivery nanorod (defined as AINRs) through the hydrophobic interaction among the mitochondrial complex III inhibitor (atovaquone, ATO), the photosensitizer (indocyanine green, ICG) and the dispersion stabilizer (distearoyl phosphoethanolamine-polyethylene glycol 2000, DSPE-PEG 2000). The AINRs showed a rod-like morphology with a mean diameter of 120.6 ± 5.4 nm, a zeta potential of −26.35 ± 1.63 mV and a significantly high drug loading rate of 93.48%. The results of in vitro cell experiments involving triple-negative breast cancer cell lines (4T1 cells and MDA-MB-231 cells) indicated that the AINRs could effectively block the oxidative phosphorylation of cancer cells through the inhibition of mitochondrial complex III, which results in the reduction of endogenous oxygen consumption and the decrease of the intracellular ATP level. The reduction of ATP content further inhibited the glutathione synthesis and arrested the cell cycle at the S-phase, which results in enhanced in vitro PDT efficacy of ICG. The results of in vivo antitumor activity in 4T1-bearing mice showed that the tumor growth inhibition rate of the AINRs with near-infrared laser irradiation (NIR) was greater than 90%, whereas the tumor growth inhibition rates of the AINRs without NIR, ICG with NIR and doxorubicin (3 mg/kg) were only 31.68%, 61.15% and 24.59%, respectively. In addition, the results of safety studies, including body weights, biochemical indicators and H&E staining images of the main organs demonstrated the security of the AINRs. In summary, this study showed that the oxidative phosphorylation inhibition of triple-negative breast cancer was a safe and effective method to enhance its PDT efficacy.

Dual inhibition of glycolysis and oxidative phosphorylation by aptamer-based artificial enzyme for synergistic cancer therapy

Dual inhibition of glycolysis and oxidative phosphorylation by aptamer-based artificial enzyme for synergistic cancer therapy

Macrophage programming is regulated by a cooperative interaction between fatty acid binding protein 5 and peroxisome proliferator-activated receptor γ.

Resolution of inflammation is an active process that is tightly regulated to achieve repair and tissue homeostasis. In the absence of resolution, persistent inflammation underlies the pathogenesis of chronic lung disease such as chronic obstructive pulmonary disease (COPD) with recurrent exacerbations. Over the course of inflammation, macrophage programming transitions from pro‐inflammatory to pro‐resolving, which is in part regulated by the nuclear receptor Peroxisome Proliferator‐Activated Receptor γ (PPARγ). Our previous work demonstrated an association between Fatty Acid Binding Protein 5 (FABP5) expression and PPARγ activity in peripheral blood mononuclear cells of healthy and COPD patients. However, a role for FABP5 in macrophage programming has not been examined. Here, using a combination of in vitro and in vivo approaches, we demonstrate that FABP5 is necessary for PPARγ activation. In turn, PPARγ acts directly to increase FABP5 expression in primary human alveolar macrophages. We further illustrate that lack of FABP5 expression promotes a pro‐inflammatory macrophage programming with increased secretion of pro‐inflammatory cytokines and increased chromatin accessibility for pro‐inflammatory transcription factors (e.g., NF‐κB and MAPK). And finally, real‐time cell metabolic analysis using the Seahorse technology shows an inhibition of oxidative phosphorylation in FABP5‐deficient macrophages. Taken together, our data indicate that FABP5 and PPARγ reciprocally regulate each other's expression and function, consistent with a novel positive feedback loop between the two factors that mediates macrophage pro‐resolving programming. Our studies highlight the importance of defining targets and regulatory mechanisms that control the resolution of inflammation and may serve to inform novel interventional strategies directed towards COPD.

Local anesthetics elicit immune-dependent anticancer effects

BackgroundRetrospective clinical trials reported a reduced local relapse rate, as well as improved overall survival after injection of local anesthetics during cancer surgery. Here, we investigated the anticancer effects of...

Bladder cancer cells shift rapidly and spontaneously to cisplatin-resistant oxidative phosphorylation that is trackable in real time

Genetic mutations have long been recognized as drivers of cancer drug resistance, but recent work has defined additional non-genetic mechanisms of plasticity, wherein cancer cells assume a drug resistant phenotype marked by altered epigenetic and transcriptional states. Currently, little is known about the real-time, dynamic nature of this phenotypic shift. Using a bladder cancer model of nongenetic plasticity, we discovered that rapid transition to drug resistance entails upregulation of mitochondrial gene expression and a corresponding metabolic shift towards the tricarboxylic acid cycle and oxidative phosphorylation. Based on this distinction, we were able to track cancer cell metabolic profiles in real time using fluorescence lifetime microscopy (FLIM). We observed single cells transitioning spontaneously to an oxidative phosphorylation state over hours to days, a trend that intensified with exposure to cisplatin chemotherapy. Conversely, pharmacological inhibition of oxidative phosphorylation significantly reversed the FLIM metabolic signature and reduced cisplatin resistance. These rapid, spontaneous metabolic shifts offer a new means of tracking nongenetic cancer plasticity and forestalling the emergence of drug resistance.

Mitochondrial oxidative phosphorylation is dispensable for survival of CD34+ chronic myeloid leukemia stem and progenitor cells

Chronic myeloid leukemia (CML) are initiated and sustained by self-renewing malignant CD34+ stem cells. Extensive efforts have been made to reveal the metabolic signature of the leukemia stem/progenitor cells in genomic, transcriptomic, and metabolomic studies. However, very little proteomic investigation has been conducted and the mechanism regarding at what level the metabolic program was rewired remains poorly understood. Here, using label-free quantitative proteomic profiling, we compared the signature of CD34+ stem/progenitor cells collected from CML individuals with that of healthy donors and observed significant changes in the abundance of enzymes associated with aerobic central carbonate metabolic pathways. Specifically, CML stem/progenitor cells expressed increased tricarboxylic acid cycle (TCA) with decreased glycolytic proteins, accompanying by increased oxidative phosphorylation (OXPHOS) and decreased glycolysis activity. Administration of the well-known OXPHOS inhibitor metformin eradicated CML stem/progenitor cells and re-sensitized CD34+ CML cells to imatinib in vitro and in patient-derived tumor xenograft murine model. However, different from normal CD34+ cells, the abundance and activity of OXPHOS protein were both unexpectedly elevated with endoplasmic reticulum stress induced by metformin in CML CD34+ cells. The four major aberrantly expressed protein sets, in contrast, were downregulated by metformin in CML CD34+ cells. These data challenged the dependency of OXPHOS for CML CD34+ cell survival and underlined the novel mechanism of metformin. More importantly, it suggested a strong rationale for the use of tyrosine kinase inhibitors in combination with metformin in treating CML.