Inhibition Of Oxidative Phosphorylation Research Articles

First-in-Class NADH/Ubiquinone Oxidoreductase Core Subunit S7 (NDUFS7) Antagonist for the Treatment of Pancreatic Cancer.

Pancreatic cancer cells adapt to nutrient-scarce metabolic conditions by increasing their oxidative phosphorylation reserve to survive. Here, we present a first-in-class small-molecule NDUFS7 antagonist that inhibits oxidative phosphorylation (OXPHOS) for the treatment of pancreatic cancer. The lead compound, DX2-201, suppresses the proliferation of a panel of cell lines, and a metabolically stable analogue, DX3-213B, shows significant efficacy in a syngeneic model of pancreatic cancer. Exome sequencing of six out of six clones resistant to DX2-201 revealed a pV91M mutation in NDUFS7, providing direct evidence of its drug-binding site. In combination studies, DX2-201 showed synergy with multiple metabolic modulators, select OXPHOS inhibitors, and PARP inhibitors. Importantly, a combination with 2-deoxyglucose overcomes drug resistance in vivo. This study demonstrates that an efficacious treatment for pancreatic cancer can be achieved through inhibition of OXPHOS and direct binding to NDUFS7, providing a novel therapeutic strategy for this hard-to-treat cancer.

Redox-crippled MitoQ potently inhibits breast cancer and glioma cell proliferation: A negative control for verifying the antioxidant mechanism of MitoQ in cancer and other oxidative pathologies

Mitochondria-targeted coenzyme Q10 (Mito-ubiquinone, Mito-quinone mesylate, or MitoQ) was shown to be an effective antimetastatic drug in patients with triple-negative breast cancer. MitoQ, sold as a nutritional supplement, prevents breast cancer recurrence. It potently inhibited tumor growth and tumor cell proliferation in preclinical xenograft models and in vitro breast cancer cells. The proposed mechanism of action involves the inhibition of reactive oxygen species by MitoQ via a redox-cycling mechanism between the oxidized form, MitoQ, and the fully reduced form, MitoQH2 (also called Mito-ubiquinol). To fully corroborate this antioxidant mechanism, we substituted the hydroquinone group (-OH) with the methoxy group (-OCH3). Unlike MitoQ, the modified form, dimethoxy MitoQ (DM-MitoQ), lacks redox-cycling between the quinone and hydroquinone forms. DM-MitoQ was not converted to MitoQ in MDA-MB-231 cells. We tested the antiproliferative effects of both MitoQ and DM-MitoQ in human breast cancer (MDA-MB-231), brain-homing cancer (MDA-MB-231BR), and glioma (U87MG) cells. Surprisingly, DM-MitoQ was slightly more potent than MitoQ (IC50 = 0.26 μM versus 0.38 μM) at inhibiting proliferation of these cells. Both MitoQ and DM-MitoQ potently inhibited mitochondrial complex I-dependent oxygen consumption (IC50 = 0.52 μM and 0.17 μM, respectively). This study also suggests that DM-MitoQ, which is a more hydrophobic analog of MitoQ (logP: 10.1 and 8.7) devoid of antioxidant function and reactive oxygen species scavenging ability, can inhibit cancer cell proliferation. We conclude that inhibition of mitochondrial oxidative phosphorylation by MitoQ is responsible for inhibition of breast cancer and glioma proliferation and metastasis. Blunting the antioxidant effect using the redox-crippled DM-MitoQ can serve as a useful negative control in corroborating the involvement of free radical-mediated processes (e.g., ferroptosis, protein oxidation/nitration) using MitoQ in other oxidative pathologies.

Activation of TRPV4 channels promotes the loss of cellular ATP in organotypic slices of the mouse neocortex exposed to chemical ischemia.

The vertebrate brain has an exceptionally high energy need. During ischemia, intracellular ATP concentrations decline rapidly, resulting in the breakdown of ion gradients and cellular damage. Here, we employed the nanosensor ATeam1.03YEMK to analyse the pathways driving the loss of ATP upon transient metabolic inhibition in neurons and astrocytes of the mouse neocortex. We demonstrate that brief chemical ischemia, induced by combined inhibition of glycolysis and oxidative phosphorylation, results in a transient decrease in intracellular ATP. Neurons experienced a larger relative decline and showed less ability to recover from prolonged (>5min) metabolic inhibition than astrocytes. Blocking voltage-gated Na+ channels or NMDA receptors ameliorated the ATP decline in neurons and astrocytes, while blocking glutamate uptake aggravated the overall reduction in neuronal ATP, confirming the central role of excitatory neuronal activity in the cellular energy loss. Unexpectedly, pharmacological inhibition of transient receptor potential vanilloid 4 (TRPV4) channels significantly reduced the ischemia-induced decline in ATP in both cell types. Imaging with Na+ -sensitive indicator dye ING-2 furthermore showed that TRPV4 inhibition also reduced ischemia-induced increases in intracellular Na+ . Altogether, our results demonstrate that neurons exhibit a higher vulnerability to brief metabolic inhibition than astrocytes. Moreover, they reveal an unexpected strong contribution of TRPV4 channels to the loss of cellular ATP and suggest that the demonstrated TRPV4-related ATP consumption is most likely a direct consequence of Na+ influx. Activation of TRPV4 channels thus provides a hitherto unacknowledged contribution to the cellular energy loss during energy failure, generating a significant metabolic cost in ischemic conditions. KEY POINTS: In the ischemic brain, cellular ATP concentrations decline rapidly, which results in the collapse of ion gradients and promotes cellular damage and death. We analysed the pathways driving the loss of ATP upon transient metabolic inhibition in neurons and astrocytes of the mouse neocortex. Our results confirm the central role of excitatory neuronal activity in the cellular energy loss and demonstrate that neurons experience a larger decline in ATP and are more vulnerable to brief metabolic stress than astrocytes. Our study also reveals a new, previously unknown involvement of osmotically activated transient receptor potential vanilloid 4 (TRPV4) channels to the reduction in cellular ATP in both cell types and indicates that this is a consequence of TRPV4-mediated Na+ influx. We conclude that activation of TRPV4 channels provides a considerable contribution to the cellular energy loss, thereby generating a significant metabolic cost in ischemic conditions.

Lactic acidosis switches cancer cells from dependence on glycolysis to OXPHOS and renders them highly sensitive to OXPHOS inhibitors

Targeting oxidative phosphorylation (OXPHOS) has emerged as a strategy for cancer treatment. However, most tumor cells exhibit Warburg effect, they primarily rely on glycolysis to generate ATP, and hence they are resistant to OXPHOS inhibitors. Here, we report that lactic acidosis, a ubiquitous factor in the tumor microenvironment, increases the sensitivity of glycolysis-dependent cancer cells to OXPHOS inhibitors by 2–4 orders of magnitude. Lactic acidosis reduces glycolysis by 79–86% and increases OXPHOS by 177–218%, making the latter the main production pathway of ATP. In conclusion, we revealed that lactic acidosis renders cancer cells with typical Warburg effect phenotype highly sensitive to OXPHOS inhibitors, thereby greatly expanding the anti-cancer spectrum of OXPHOS inhibitors. In addition, as lactic acidosis is a ubiquitous factor of TME, it is a potential indicator to predict the efficacy of OXPHOS inhibitors in cancer treatment.

Various Energetic Metabolism of Microglia in Response to Different Stimulations.

The activation of the microglia plays an important role in the neuroinflammation induced by different stimulations associated with Alzheimer's disease (AD). Different stimulations, such as pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs) and cytokines, trigger a consequence of activation in the microglia with diverse changes of the microglial cell type response in AD. The activation of the microglia is often accompanied by metabolic changes in response to PAMPs, DAMPs and cytokines in AD. Actually, we do not know the distinct differences on the energetic metabolism of microglia when subject to these stimuli. This research assessed the changes of the cell type response and energetic metabolism in mouse-derived immortalized cells (BV-2 cells) induced by a PAMP (LPS), DAMPs (Aβ and ATP) and a cytokine (IL-4) in mouse-derived immortalized cells (BV-2 cells) and whether the microglial cell type response was improved by targeting the metabolism. We uncovered that LPS, a proinflammatory stimulation of PAMPs, modified the morphology from irregular to fusiform, with stronger cell viability, fusion rates and phagocytosis in the microglia accompanied by a metabolic shift to the promotion of glycolysis and the inhibition of oxidative phosphorylation (OXPHOS). Aβ and ATP, which are two known kinds of DAMPs that trigger microglial sterile activation, induced the morphology from irregular to amoebic, and significantly decreased others in the microglia, accompanied by boosting or reducing both glycolysis and OXPHOS. Monotonous pathological changes and energetic metabolism of microglia were observed under IL-4 exposure. Further, the inhibition of glycolysis transformed the LPS-induced proinflammatory morphology and decreased the enhancement of LPS-induced cell viability, the fusion rate and phagocytosis. However, the promotion of glycolysis exerted a minimal effect on the changes of morphology, the fusion rate, cell viability and phagocytosis induced by ATP. Our study reveals that microglia induced diverse pathological changes accompanied by various changes in the energetic metabolism in response to PAMPs, DAMPs and cytokines, and it may be a potential application of targeting the cellular metabolism to interfere with the microglia-mediated pathological changes in AD.

Endogenous PP2A inhibitor CIP2A degradation by chaperone-mediated autophagy contributes to the antitumor effect of mitochondrial complex I inhibition

Combined inhibition of oxidative phosphorylation (OXPHOS) and glycolysis has been shown to activate a PP2A-dependent signaling pathway, leading to tumor cell death. Here, we analyze highly selective mitochondrial complex I or III inhibitors invitro and invivo to elucidate the molecular mechanisms leading to cell death following OXPHOS inhibition. We show that IACS-010759 treatment (complex I inhibitor) induces a reactive oxygen species (ROS)-dependent dissociation of CIP2A from PP2A, leading to its destabilization and degradation through chaperone-mediated autophagy. Mitochondrial complex III inhibition has analogous effects. We establish that activation of the PP2A holoenzyme containing B56δ regulatory subunit selectively mediates tumor cell death, while the arrest in proliferation that is observed upon IACS-010759 treatment does not depend on the PP2A-B56δ complex. These studies provide a molecular characterization of the events subsequent to the alteration of critical bioenergetic pathways and help to refine clinical studies aimed to exploit metabolic vulnerabilities of tumor cells.

Atovaquone exerts its anticancer effect by inhibiting Na+/K+-ATPase ion transport in canine cancer cells.

New anticancer drugs are being developed to avoid the toxicity and chemoresistance of the currently available drugs. The Food and Drug Administration-approved anti-malarial drug atovaquone is known to act as a selective oxidative phosphorylation inhibitor in the mitochondria by competing with CO Q10 (mitochondrial complex II and III). This study aimed to investigate the effect of atovaquone by examining the Na+/K+-ATPase (NKA) activity in various canine cell lines. Canine cell lines were treated with various concentrations (2.5, 5, 10, 15, and 20 μM) of atovaquone for 24, 48, and 72 h. Human cell lines were used as a control to validate the canine cancer cell lines. The activities of the drugs against the cancer cell lines were measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromideassay. The cell metabolic activity was determined by measuring the activities of the nicotinamide adenine dinucleotide phosphate-dependent cellular oxidoreductase enzymes. The NKA activity was measured using the single-cell patch clamping assay. Atovaquone-induced apoptosis by elevating the concentration of reactive oxygen species (ROS) in the tumor cells, leading to cell death. Treatment of canine cancer cells with N-acetylcysteine (ROS inhibitor) reduced the activity of the drug. Furthermore, atovaquone inhibited more than 45% of the NKA ion current. This study demonstrated effects of atovaquone against canine cancer cell lines. The data may prove beneficial in repurposing the drug as a new anticancer agent in canine clinical trials, which might aid in fighting human cancer.

Targeting Mitochondrial DNA Transcription by POLRMT Inhibition or Depletion as a Potential Strategy for Cancer Treatment.

Transcription of the mitochondrial genome is essential for the maintenance of oxidative phosphorylation (OXPHOS) and other functions directly related to this unique genome. Considerable evidence suggests that mitochondrial transcription is dysregulated in cancer and cancer metastasis and contributes significantly to cancer cell metabolism. Recently, inhibitors of the mitochondrial DNA-dependent RNA polymerase (POLRMT) were identified as potentially attractive new anti-cancer compounds. These molecules (IMT1, IMT1B) inactivate cancer cell metabolism through reduced transcription of mitochondrially-encoded OXPHOS subunits such as ND1-5 (Complex I) and COI-IV (Complex IV). Studies from our lab have discovered small molecule regulators of the mitochondrial matrix caseinolytic protease (ClpP) as probable inhibitors of mitochondrial transcription. These compounds activate ClpP proteolysis and lead to the rapid depletion of POLRMT and other matrix proteins, resulting in inhibition of mitochondrial transcription and growth arrest. Herein we present a comparison of POLRMT inhibition and ClpP activation, both conceptually and experimentally, and evaluate the results of these treatments on mitochondrial transcription, inhibition of OXPHOS, and ultimately cancer cell growth. We discuss the potential for targeting mitochondrial transcription as a cancer cell vulnerability.

The deubiquitinase OTUB1 governs lung cancer cell fitness by modulating proteostasis of OXPHOS proteins

Aerobic glycolysis is a hallmark of cancer development, but this dogma has been challenged by reports showing a key role of oxidative phosphorylation (OXPHOS) in cancer cell survival. It has been proposed that increased levels of intramitochondrial proteins in cancer cells are associated with high OXPHOS activity and increased sensitivity to OXPHOS inhibitors. However, the molecular mechanisms leading to the high expression of OXPHOS proteins in cancer cells remain unknown. Multiple proteomics studies have detected the ubiquitination of intramitochondrial proteins, suggesting the contribution of the ubiquitin system to the proteostatic regulation of OXPHOS proteins. Here, we identified the ubiquitin hydrolase OTUB1 as a regulator of the mitochondrial metabolic machinery essential for lung cancer cell survival. Mitochondria-localized OTUB1 modulates respiration by inhibiting K48-linked ubiquitination and turnover of OXPHOS proteins. An increase in OTUB1 expression is commonly observed in one-third of non-small-cell lung carcinomas and is associated with high OXPHOS signatures. Moreover, OTUB1 expression highly correlates with the sensitivity of lung cancer cells to mitochondrial inhibitors.

Metabolic adaptation to tyrosine kinase inhibition in leukemia stem cells

AbstractTyrosine kinase inhibitors (TKIs) are very effective in treating chronic myelogenous leukemia (CML), but primitive, quiescent leukemia stem cells persist as a barrier to the cure. We performed a comprehensive evaluation of metabolic adaptation to TKI treatment and its role in CML hematopoietic stem and progenitor cell persistence. Using a CML mouse model, we found that glycolysis, glutaminolysis, the tricarboxylic acid cycle, and oxidative phosphorylation (OXPHOS) were initially inhibited by TKI treatment in CML-committed progenitors but were restored with continued treatment, reflecting both selection and metabolic reprogramming of specific subpopulations. TKI treatment selectively enriched primitive CML stem cells with reduced metabolic gene expression. Persistent CML stem cells also showed metabolic adaptation to TKI treatment through altered substrate use and mitochondrial respiration maintenance. Evaluation of transcription factors underlying these changes helped detect increased HIF-1 protein levels and activity in TKI-treated stem cells. Treatment with an HIF-1 inhibitor in combination with TKI treatment depleted murine and human CML stem cells. HIF-1 inhibition increased mitochondrial activity and reactive oxygen species (ROS) levels, reduced quiescence, increased cycling, and reduced the self-renewal and regenerating potential of dormant CML stem cells. We, therefore, identified the HIF-1–mediated inhibition of OXPHOS and ROS and maintenance of CML stem cell dormancy and repopulating potential as a key mechanism of CML stem cell adaptation to TKI treatment. Our results identify a key metabolic dependency in CML stem cells persisting after TKI treatment that can be targeted to enhance their elimination.

GPAM mediated lysophosphatidic acid synthesis regulates mitochondrial dynamics in acute myeloid leukemia.

Metabolic alterations, especially in the mitochondria, play important roles in several kinds of cancers, including acute myeloid leukemia (AML). However, AML-specific molecular mechanisms that regulate mitochondrial dynamics remain elusive. Through the metabolite screening comparing CD34+ AML cells and healthy hematopoietic stem/progenitor cells, we identified enhanced lysophosphatidic acid (LPA) synthesis activity in AML. LPA is synthesized from glycerol-3-phosphate by glycerol-3-phosphate acyltransferases (GPATs), rate-limiting enzymes of the LPA synthesis pathway. Among the four isozymes of GPATs, glycerol-3-phosphate acyltransferases, mitochondrial (GPAM) was highly expressed in AML cells, and the inhibition of LPA synthesis by silencing GPAM or FSG67 (a GPAM-inhibitor) significantly impaired AML propagation through the induction of mitochondrial fission, resulting in the suppression of oxidative phosphorylation and the elevation of reactive oxygen species. Notably, inhibition of this metabolic synthesis pathway by FSG67 administration did not affect normal human hematopoiesis in vivo. Therefore, the GPAM-mediated LPA synthesis pathway from G3P represents a critical metabolic mechanism that specifically regulates mitochondrial dynamics in human AML, and GPAM is a promising potential therapeutic target.

Discovery of a Potent and Oral Available Complex I OXPHOS Inhibitor That Abrogates Tumor Growth and Circumvents MEKi Resistance.

Targeting oxidative phosphorylation (OXPHOS) has emerged as a promising therapeutic strategy for cancer therapy. Here, we discovered a 1H-1,2,3-triazole derivative HP661 as a highly potent and orally available OXPHOS inhibitor that effectively blocked the activity of mitochondrial complex I. HP661 specifically compromised the mitochondrial oxygen consumption of high-OXPHOS lung cancer cells but not that of low-OXPHOS lung cancer cells or normal cells in the low nanomolar range. Notably, mitogen-activated protein kinase kinase (MEK) inhibitor (trametinib)-resistant lung cancer cells with high levels of OXPHOS also showed marked sensitivity to HP661, as indicated by decreased clonogenic growth and increased cell apoptosis upon treatment. In a mouse model of high-OXPHOS lung cancer, HP661 treatment not only significantly suppressed tumor growth but also augmented the therapeutic efficacy of trametinib by impairing tumor mitochondrial respiration. In summary, we identified HP661 as a highly effective OXPHOS inhibitor to abrogate the growth of high OXPHOS-dependent tumors and conquer high OXPHOS-mediated drug resistance.

Abstract A24: Targeting mitochondrial metabolism following Induction Chemotherapy eradicates Acute Myeloid Leukemia at the level of Minimal Residual Disease

Abstract Acute myeloid leukemia (AML) stem cells (AMLSCs) are thought to be responsible for disease initiation, chemoresistance and treatment failure. AMLSCs and residual cytarabine (AraC)-resistant AML cells (constituting minimal residual disease, MRD) were shown to be highly dependent on mitochondrial function for survival and thus are vulnerable to pharmacological blockade of the oxidative phosphorylation (OXPHOS) (Farge et al. Cancer Discov, 2017). Efficacy of OXPHOS inhibitor IACS-010759 was previously reported, demonstrating potent inhibition of mitochondrial complex I, OXPHOS suppression and growth inhibition of AML cells (Molina, et al. Nat Med, 2018). Here we evaluated OXPHOS dependency of AML MRD cells and determined impact of OXPHOS blockade on residual AML cells surviving standard chemotherapy (Doxorubicin/AraC, DA). Our results demonstrated that AML cell lines treated with AraC or DA induced accumulation of reactive oxygen species, mitochondrial superoxides, increased mitochondrial mass and mitochondrial membrane potential. AraC- and DA-based therapies in vitro were significantly potentiated by IACS-010759. OxPhos dependency shown as a significantly increased basal and maximal oxygen consumption rate after AraC and DA treatment, was fully inhibited by OxPhos inhibitor, leading to a total mitochondrial collapse. OxPhos inhibition in combination with DA consolidated reduction of viable cell number, induction of apoptosis and differentiation in cell lines and in AraC-resistant primary patient samples. Next, the efficacy of IACS-010759 together with DA chemotherapy was evaluated in several chemotherapy-sensitive and -resistant animal models in vivo. DA/IACS-010759 combination significantly reduced leukemia burden and significantly extended survival in OCI-AML3/Luc/GFP model and in FLT3-ITD+ AML PDX model. In the latter, IACS-010759 led to reduction of leukemia burden, and delayed leukemia recurrence when administered post completion of DA. At the single-cell level, CyTOF analysis demonstrated that this combination reduced frequency of CD34+CD38lowCD123+AML LSCs and has superior efficacy to facilitate differentiation of immature subpopulations. Furthermore, addition of IACS-010759 to DA extended survival of mice inoculated with chemoresistant PDX AML models. Finally, IACS-010759 administered during consolidation phase significantly extended mice survival compared to standard of care arm, supporting clinical utility of OXPHOS inhibitors in AML. In conclusion, our findings indicate that chemotherapy fosters mitochondrial respiration in AML, which could be abrogated by OXPHOS inhibitor at the LSCs and MRD level, in vitro and in vivo. Our data advocate for combining mitochondrial targeting strategies with chemotherapy as a part of induction and consolidation treatment for improved control of MRD, eradication of AMLSC and extended response duration. Citation Format: Natalia Baran, Lina Han, Lucille Stuani, Antonio Cavazos, Laurie M. Cooper, Cassandra L. Ramage, Vinitha M. Kuruvilla, Qi Zhang, Marie Sabatier, Emeline Boet, Jason P. Gay, Ningping Feng, Lokesh Battula, Emeline Chu-Van, Florence Castelli, Martin Carrol, Sergej Konoplev, Beenu Thakral, Hagop M. Kantarjian, Naval Daver, Joseph Marszalek, Michael Andreeff, Jean-Emmanuel Sarry, Marina Konopleva. Targeting mitochondrial metabolism following Induction Chemotherapy eradicates Acute Myeloid Leukemia at the level of Minimal Residual Disease [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A24.

Assays for profiling changes in T cell metabolism

Abstract Immune cell therapy, including engineered T cells, engineered NK cells, and treatments to enhance native T cell response, is a rapidly advancing field of research with great promise to treat multiple diseases. However, multiple challenges, from isolating the correct cell type, determining its mode of action, manufacturing and establishing appropriate quality control criteria, must be overcome to reach its potential. In recent years, cellular energy metabolism has emerged as an important consideration for efficacy and persistence when developing cell therapies. Here we evaluate a set of bioluminescent cell-based assays for measuring rate of metabolite uptake, monitoring activity for key metabolic pathways and determining changes in intracellular metabolite levels during T cell transition from naïve to activated to memory phenotypes. Metabolite levels measured include ATP, glucose uptake, malate, branched chain amino acids, and glycogen levels. Depending on the activation state of the cells, the cells respond differently to glycolysis and oxidative phosphorylation inhibitors, as demonstrated by changes to metabolite levels. Energy storage as measured by intracellular glycogen levels also differs depending on the T cell phenotype (naïve, activated, or memory). Understanding the processes governing dynamic metabolic changes during cell modification and expansion is required for next-generation cell therapy development. The developed bioluminescent cell-based assays provide the sensitivity, throughput and robustness required for rapid evaluation of such changes.

Mitochondria targeted metabolic inhibitors promote lymphocyte infiltration into murine pancreatic tumors

Abstract Pancreatic ductal adenocarcinoma is a malignant carcinoma with a 5-year survival rate of 11% characterized with poor prognosis, rising incidence, and resistance to chemo- and immune-therapies. Cancer cell evasion and suppression of anti-tumor immune responses in pancreas cancer are regulated in part by metabolic reprogramming within cancer and tumor accessory cells. We sought to determine if targeted inhibition of mitochondrial oxidative phosphorylation by mito-metformin (MMe) and newly developed variant pCF3-MMe influence the tumor immune microenvironment. Our results showed that MMe strongly inhibited tumor growth in murine models of pancreas cancer. Flow cytometry revealed a significant increase in total CD45+ leukocytes, with elevated cytolytic granzyme B+ and CD44+ CD8+ T cells. Immunodepleting CD8+ T cells, reversed the anti-tumor effects of MMe. Immunofluorescence microscopy and immunoblot analysis indicated that MMe stimulated oligomerization of mitochondrial antiviral-signaling protein (MAVS) and phosphorylation of IRF3 and IRF7. Further, levels of the IRF3/7 target chemokines CXCL9, CXCL10, and CXCL11 were increased in MMe-treated tumor cells, consistent with a role in CD8+ T cell infiltration. These data indicate OXPHOS inhibition stimulated virus-independent MAVS oligomerization, activation of downstream proinflammatory signaling, and reignition of the suppressed pancreatic tumor immune microenvironment. Funding Provided by NIH (2211376.1-5 R01 CA226279-01.04) and Center for Immunology MCW

Cell painting assays for profiling responses in Mycobacterium tuberculosis-infected macrophages after metabolic perturbation

Abstract Tuberculosis (TB) is the leading infectious disease killer in the world, and a lack of accessible, easy treatments contributes to its growing burden. Repurposing approved treatments for other conditions, including metabolic inhibitors under consideration for use in cancer therapy, as host-directed therapies may improve TB treatments. Detailed preliminary studies to identify mechanisms that underly drug function need to be performed before these drugs can be used in patients but there is a lack of tools to investigate metabolic perturbations in target cells. This problem is particularly acute for treatments that have uncharacterized effects on host cells or host-pathogen interactions, or for adherent cells where cell morphology and migration patterns may be factors that are modulated by treatments. Cell painting assays are microscopy-based analyses that use quantitative imaging data to generate insight into how drugs and infections perturb host cells. Here, we sought to optimize a cell painting assay to investigate changes in macrophage morphology and antimicrobial activity after metabolic perturbation and Mycobacterium tuberculosis(Mtb) infection. Our objective was to apply the quantification and analysis pipelines to macrophages that were infected with fluorescent Mtb reporter strains to identify cellular responses after inhibition of fatty acid oxidation, oxidative phosphorylation, or glycolysis. Moreover, we compared inhibitors that target different points in the glycolysis pathway to define how these points differentially affect macrophage antimicrobial activity. Our work provides a framework for using cell painting assays as tools for dissecting host-pathogen interactions in metabolically perturbed cells. Supported by grants from NIH grants AI167710 and #LINK#AI164970#ENDLINK#

NanoBeacon.AI: AI-Enhanced Nanodiamond Biosensor for Automated Sensitivity Prediction to Oxidative Phosphorylation Inhibitors.

Spalt-like transcription factor 4 (SALL4) is an oncofetal protein that has been identified to drive cancer progression in hepatocellular carcinoma (HCC) and hematological malignancies. Furthermore, a high SALL4 expression level is correlated to poor prognosis in these cancers. However, SALL4 lacks well-structured small-molecule binding pockets, making it difficult to design targeted inhibitors. SALL4-induced expression of oxidative phosphorylation (OXPHOS) genes may serve as a therapeutically targetable vulnerability in HCC through OXPHOS inhibition. Because OXPHOS functions through a set of genes with intertumoral heterogeneous expression, identifying therapeutic sensitivity to OXPHOS inhibitors may not rely on a single clear biomarker. Here, we developed a workflow that utilized molecular beacons, nucleic-acid-based, activatable sensors with high specificity to the target mRNA, delivered by nanodiamonds, to establish an artificial intelligence (AI)-assisted platform for rapid evaluation of patient-specific drug sensitivity. Specifically, when the HCC cells were treated with the nanodiamond-medicated OXPHOS biosensor, high sensitivity and specificity of the sensor allowed for improved identification of OXPHOS expression in cells. Assisted by a trained convolutional neural network, drug sensitivity of cells toward an OXPHOS inhibitor, IACS-010759, could be accurately predicted. AI-assisted OXPHOS drug sensitivity assessment could be accomplished within 1 day, enabling rapid and efficient clinical decision support for HCC treatment. The work proposed here serves as a foundation for the patient-based subtype-specific therapeutic research platform and is well suited for precision medicine.

The role and molecular mechanism of metabolic reprogramming of colorectal cancer by UBR5 through PYK2 regulation of OXPHOS expression study.

Colorectal carcinoma (CRC) is the third most malignant tumor in the world, but the key mechanisms of CRC progression have not been confirmed.UBR5 and PYK2 expression levels were detected by RT-qPCR. The levels of UBR5, PYK2, and mitochondrial oxidative phosphorylation(OXPHOS) complexes were detected by western blot analysis. Flow cytometry was used to detect ROS activity. The CCK-8 assay was used to assess cell proliferation and viability. The interaction between UBR5 and PYK2 was detected by immunoprecipitation. A clone formation assay was used to determine the cell clone formation rate. The ATP level and lactate production of each group of cells were detected by the kit. EdU staining was performed for cell proliferation.Transwell assay was performed for cell migration ability. For the CRC nude mouse model, we also observed and recorded the volume and mass of tumor-forming tumors.The expression of UBR5 and PYK2 was elevated in both CRC and human colonic mucosal epithelial cell lines, and knockdown of UBR5 had inhibitory effects on cancer cell proliferation and cloning and other behaviors in the CRC process by knockdown of UBR5 to downregulate the expression of PYK2, thus inhibiting the OXPHOS process in CRC; rotenone (OXPHOS inhibitor) treatment enhanced all these inhibitory effects.Knockdown of UBR5 can reduce the expression level of PYK2, thus downregulating the OXPHOS process in CRC cell lines and inhibiting the CRC metabolic reprogramming process.

Metabolic Intervention Liposome Boosted Lung Cancer Radio-Immunotherapy via Hypoxia Amelioration and PD-L1 Restraint.

At present, radiotherapy (RT) still acquires limited success in clinical due to the lessened DNA damage under hypoxia and acquired immune tolerance owing to the amplified programmed death ligand-1 (PD-L1) expression. Incredibly, intracellular PD-L1 expression depression is proven to better sensitize RT by inhibiting DNA damage repair. However, the disability of the clinically used antibodies in disrupting the extracellular PD-L1function still limits the effectiveness of radio-immunotherapy. Therefore, better PD-L1 regulation strategies are still urgently needed to better sensitize radio-immunotherapy. Hence, for this purpose, TPP-LND is synthesized by linking mitochondrial-targeted triphenylphosphine cations (TPP+ ) to the antineoplastic agent lonidamine (LND), which significantly reduces the dose needed for LND to induce effective oxidative phosphorylation inhibition (2 vs 300µM). Then, TPP-LND is wrapped with liposomes to form TPP-LND@Lip nanoparticles. By doing this, TPP-LND@Lip nanoparticles can sensitize RT by reversing the hypoxic microenvironment of tumors to generate more DNA damage and reducing the expression of PD-L1 via enhancing the adenosine 5'-monophosphate-activated protein kinase activation.As expected, these well-designed economical TPP-LND@Lip nanoparticles are more effective than conventional anti-PD-L1 antibodies to some extent.

Oxidative phosphorylation inhibitors inhibit proliferation of endometriosis cells.

Developing novel therapies to cure and manage endometriosis is a major unmet need that will benefit over 180 million women worldwide. Results from the current study suggest that inhibitors of oxidative phosphorylation may serve as novel agents for the treatment of endometriosis. Current therapeutic strategies for endometriosis focus on symptom management and are not curative. Here, we provide evidence supporting the inhibition of oxidative phosphorylation (OXPHOS) as a novel treatment strategy for endometriosis. Additionally, we report an organotypic organ-on-a-chip luminal model for endometriosis. The OXPHOS inhibitors, curcumin, plumbagin, and the FDA-approved anti-malarial agent, atovaquone, were tested against the endometriosis cell line, 12Z, in conventional as well as the new organotypic model. The results suggest that all three compounds inhibit proliferation and cause cell death of the endometriotic cells by inhibiting OXPHOS and causing an increase in intracellular oxygen radicals. The oxidative stress mediated by curcumin, plumbagin, and atovaquone causes DNA double-strand breaks as indicated by the elevation of phospho-γH2Ax. Mitochondrial energetics shows a significant decrease in oxygen consumption in 12Z cells. These experiments also highlight differences in the mechanism of action as curcumin and plumbagin inhibit complex I whereas atovaquone blocks complexes I, II, and III. Real-time assessment of cells in the lumen model showed inhibition of migration in response to the test compounds. Additionally, using two-photon lifetime imaging, we demonstrate that the 12Z cells in the lumen show decreased redox ratio (NAD(P)H/FAD) and lower fluorescence lifetime of NAD(P)H in the treated cells confirming major metabolic changes in response to inhibition of mitochondrial electron transport. The robust chemotoxic responses observed with atovaquone suggest that this anti-malarial agent may be repurposed for the effective treatment of endometriosis.