Inhibition Of Oxidative Phosphorylation Research Articles

A strategy for poisoning cancer cell metabolism: Inhibition of oxidative phosphorylation coupled to anaplerotic saturation.

The combination of inhibitor of oxidative phosphorylation (OXPHOS) with dimethyl-α-ketoglutarate, a cell-permeable precursor of α-ketoglutarate, is highly efficient in killing human cancer cells in vitro or in vivo, in xenotransplanted mice. This effect involves excessive anaplerosis, as demonstrated by the fact that inhibition of isocitrate dehydrogenase-1, IDH1, reduced the efficacy of cancer cell killing by the combination treatment. However, the signal transduction pathway leading to cell death turned out to be complex because it involved numerous atypical cell death effectors (such as AIF, APEX, MDM2, PARP1), as well as a profound remodeling of the transcriptome resulting in reduced expression of glycolytic enzymes. The combined inhibition of OXPHOS and glycolytic ATP generation culminated in a lethal bioenergetic catastrophe.

Protein Arginine Methyltransferase 3-Induced Metabolic Reprogramming Is a Vulnerable Target of Pancreatic Cancer

Background: The biological function of protein arginine methyltransferase 3 (PRMT3) is not well known because very few physiological substrates of PRMT3 have been identified to date. Methods: The clinical significance of PRMT3 in pancreatic cancer was studied by database analysis. The PRMT3 protein level in human pancreatic tumors was detected by immunoblotting. PRMT3-associated proteins and the methylation sites on the proteins were investigated using mass spectrometry. Capillary electrophoresis time-of-flight mass spectrometry and seahorse bioscience were performed to analyze metabolic reprogramming. Combination index analysis and xenograft animal model were conducted to explore the effects of combination of inhibitors of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and oxidative phosphorylation on tumor growth. Findings: We find the expression of PRMT3 is upregulated in pancreatic cancer, and its expression is associated with poor survival. We further demonstrate that GAPDH is methylated at R248 by PRMT3 in vivo. The methylation of GAPDH by PRMT3 enhances its catalytic activity while the mutation of R248 abolishes the effect. In cells, PRMT3 overexpression triggers metabolic reprogramming and enhances glycolysis and mitochondrial respiration simultaneously in a GAPDH-dependent manner. PRMT3-overexpressing cancer cells are addicted to GAPDH-mediated metabolism and sensitive to the inhibition of GAPDH and mitochondrial respiration. The combination of inhibitors of GAPDH and oxidative phosphorylation induces a synergistic inhibition on cellular growth in vitro and in vivo. Interpretation: Our results suggest PRMT3-induced metabolic reprogramming is a vulnerable target of pancreatic cancer. Funding Statement: This work was supported by the grants from the Ministry of Science and Technology (MOST-103-2314-B-037-075; MOST-104-2314-B-037-004; MOST-105-2314-B-037-001; MOST-107-2320-B-400-006-MY3 to M.-R.P. and W.-C.H. and MOST-106-2321-B-400-007 to L.-T.C.). This study was also supported by the grant from National Research Institutes (NHRI-107A1-CACO-02181811 to Y.-S.S.) and the Yen-Hsu Education Foundation to W.-C.H. Declaration of Interests: The authors have no conflicts of interest to declare. Ethics Approval Statement: Human pancreatic tumor tissues were obtained from patients undergoing surgical resection at Koo Foundation Sun Yat-Sen Cancer Center (Taipei, Taiwan) and National Cheng Kung University Hospital (Tainan, Taiwan) under the guidelines approved by the Institution Review Board at National Health Research Institutes. Written informed consent was obtained from each patient. All animal experiments were approved by Animal Care Committee of National Health Research Institutes.

Metabolic reprogramming of oncogene-addicted cancer cells to OXPHOS as a mechanism of drug resistance

The ability to selectively eradicate oncogene-addicted tumors while reducing systemic toxicity has endeared targeted therapies as a treatment strategy. Nevertheless, development of acquired resistance limits the benefits and durability of such a regime. Here we report evidence of enhanced reliance on mitochondrial oxidative phosphorylation (OXPHOS) in oncogene-addicted cancers manifesting acquired resistance to targeted therapies. To that effect, we describe a novel OXPHOS targeting activity of the small molecule compound, OPB-51602 (OPB). Of note, a priori treatment with OPB restored sensitivity to targeted therapies. Furthermore, cancer cells exhibiting stemness markers also showed selective reliance on OXPHOS and enhanced sensitivity to OPB. Importantly, in a subset of patients who developed secondary resistance to EGFR tyrosine kinase inhibitor (TKI), OPB treatment resulted in decrease in metabolic activity and reduction in tumor size. Collectively, we show here a switch to mitochondrial OXPHOS as a key driver of targeted drug resistance in oncogene-addicted cancers. This metabolic vulnerability is exploited by a novel OXPHOS inhibitor, which also shows promise in the clinical setting.

A Novel Patient-Derived Xenograft Model of Secondary CD20- CNS Lymphoma for Mechanistic and Therapeutic Exploration

IntroductionThe majority of secondary central nervous system (CNS) lymphoma cases are derived from histologically high-grade systemic lymphomas such as diffuse-large B cell lymphoma (DLBCL) and Burkitt's lymphoma. These CNS lymphomas are associated with poor prognosis and a low likelihood of long-term survival, especially rituximab-ineffective CD20- lymphoma. To investigate the biological features and potential therapeutic targets of CNS-involved CD20- lymphoma, we established the first known patient-derived xenograft (PDX) model using cerebrospinal fluid (CSF).MethodsSix- to 8-week-old male NSG mice (Jackson Laboratory) were housed and monitored in our animal research facility. All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee of The University of Texas MD Anderson Cancer Center. A total of 0.08 million cells were isolated from 2 mL of CSF obtained from a CD20- CD79A- DLBCL patient with Bcl-2 and Myc rearrangements (double-hit). The isolated cells were directly injected into a human fetal bone chip implanted into NSG (NSG-hu) mice. After passage, the mice were administered several small molecules, and tumor burden was measured via tumor volume and human β2M levels over the course of treatment. Kaplan-Meier survival analysis was also performed. PDX tissue was collected for histological analysis, immunophenotyping, genotyping, and proteomics analysis.ResultsThe PDX tumors possessed the same histological, genetic and immunophenotypical features as the primary patient tumor cells. The PDXs had Bcl-2 and Myc rearrangements and were positive for CD10, CD19, CD22, and CD45 and were negative for CD5, CD20, and CD79A. After tumor passage, the mice were treated with vehicle, 50 mg/kg ibrutinib (Bruton's tyrosine kinase (BTK) inhibitor) oral gavage daily, 50 mg/kg ABT199 (Bcl-2 inhibitor) oral gavage daily, 1 mg/kg carfilzomib (proteasome inhibitor) i.v. twice per week, or 7.5 mg/kg IACS-010759 (oxidative phosphorylation (OXPHOS) inhibitor) oral gavage daily. As shown in Figure 1, the PDX tumor was resistant to the BTK inhibitor ibrutinib and moderately responsive to the proteasome inhibitor carfilzomib. Although the Bcl-2 gene was rearranged, the PDX model only had a partial response to the Bcl-2 inhibitor ABT-199 (ABT-199 vs control, p = 0.0004). In contrast, inhibition of the OXPHOS pathway with IACS-010759 significantly inhibited 96% tumor growth (IACS-010759 vs control, p < 0.00001) and prolonged mouse survival by approximately 25 days compared with the vehicle control and the other drugs (IACS-010759 vs control, ABT-199, or ibrutinib, p < 0.0001). Moreover, proteomics analysis demonstrated that IACS-010759 not only inhibited OXPHOS but also upregulated the pro-apoptotic protein Bim.ConclusionTo our knowledge, this is the first PDX model generated from the CSF of a CD20- double-hit DLBCL patient. With this PDX model, we determined that the OXPHOS inhibitor IACS-010759 is a promising therapeutic agent for this aggressive cancer. [Display omitted] DisclosuresWang:Acerta Pharma: Honoraria, Research Funding; Kite Pharma: Research Funding; Novartis: Research Funding; Pharmacyclics: Honoraria, Research Funding; Dava Oncology: Honoraria; Juno: Research Funding; MoreHealth: Consultancy; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

EVI1 in Acute Myeloid Leukemia Triggers Metabolic Reprograming Associated with Leukemogenesis and Increases Sensitivity to L-Aspalaginase

Leukemia cells survive and proliferate under conditions of metabolic stress by acquiring mutations that increase energy metabolism. Here, we aimed to identify a specific metabolic inhibitor and examine transcription factor-enhanced changes in energy metabolism by refractory leukemia cells. Overexpression of Ecotropic Virus Integration site 1 protein homolog (EVI1) in adults and children with mixed lineage leukemia-rearrangement acute myeloid leukemia (MLL-r AML) has a very poor prognosis. We focused on metabolic reprograming of MLL leukemia cells expressing EVI1, since the metabolic relationship between MLL and EVI1 is unclear. We used an extracellular flux analyze to examine metabolic changes during leukemia development in a mouse model of MLL-r AML expressing high levels of EVI1 (EVI1+).To examine whether EVI1 regulates energy metabolism in MLL-rearranged leukemia cells, we used transgenic mice expressing EVI1 (TG) in LSK and GMP cells model in which AML is driven by the MLL-AF9 oncogene. We measured oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using a flux analyzer. TG MLL-AF9 mice showed a significantly higher basal and capacity of OCR than WT MLL-AF9 mice ex vivo. EVI1+ cells showed accelerated oxidative phosphorylation (OXPHOS) prior to activation of glycolysis, and higher dependency on glutamine as an energy source. To identify the metabolic pathways regulated by EVI1, we performed capillary electrophoresis time-of-fight mass spectrometry-based metabolome profiling of WT and TG MLL-AF9 leukemia cells. We found significant differences between the cells in terms of the amounts of metabolites derived from the glycolytic and TCA cycles. Fructose 1,6-bisphosphate and lactate were up-regulated in TG MLL-AF9 cells, implying activation of glycolysis. Moreover, the amounts of fumarate and malate (metabolites of the TCA cycle) were significantly higher in TG MLL-AF9 cells. EVI1 played a role in glycolysis as well as driving expression of genes engaged in the tricarboxylic acid cycle. Next, we tested whether pharmacological inhibition of glycolysis and glutaminolysis suppresses MLL-AF9. L-asparaginase (ASP) [which catalyzes hydrolysis of asparagine (Asn) and glutamine (Gln) to asparatic acid or glutamic acid, respectively] markedly suppressed proliferation of TG MLL-AF9 cells, EVI1highAML cell lines. To examine the therapeutic potential of ASP in vivo, we treated secondary recipients of TG MLL-AF9 AML cells with ASP or control (vehicle), beginning 5 days post-transplantation. Mice then received intraperitoneal injections (five times per week) of distilled water or ASP (1000 U/kg). ASP led to a significant reduction in the number of GFP+ AML cells in the peripheral blood and increased the survival of recipient mice. Next, we examined an AML xenograft model. Two groups of NOG mice were injected subcutaneously with UCSD/AML1 cells and then treated with ASP or control. ASP -treated mice showed a significant reduction in the growth of AML tumors. Overall, these findings indicate that ASP -mediated inhibition of OXPHOS is a potential treatment for AML.We clarified that increased glutamine dependency by MLL-r AML cells showing high EVI1 expression makes them sensitive to ASP. We found that the energy advantage of AML cells is acquired via transcription factor-mediated activation of mitochondrial metabolism, leading to a poor prognosis. Furthermore, we show that new therapeutic options can be identified by examining the energy-based metabolic characteristics of leukemia cells. DisclosuresNo relevant conflicts of interest to declare.

Venetoclax Synergistically Enhances the Antileukemic Activity of Imipridone ONC213, a Novel Imipridone ONC201 Analog, in Acute Myeloid Leukemia

Although standard induction therapy initially elicits a promising response in the majority of acute myeloid leukemia (AML) patients, the majority relapse. Leukemia stem cells (LSCs) that survive chemotherapy are believed to be responsible for AML relapse. Therefore, new therapies that eliminate LSCs are desperately needed.ONC201 is a TRAIL inducer and the founding member of the imipridone family. It has been shown to induce apoptosis in LSCs (Ishizawa et al, Science Signaling. 2016; 9:ra17). ONC201 was chemically modified to increase the potency and selectivity against cancer cells, resulting in the new analog ONC213. In this study, we investigated the antileukemic activity and the underlying molecular mechanism of ONC213 in preclinical AML models.ONC213 activity in AML cell lines and primary AML patient samples was first tested in vitro. MTT assay results revealed that ONC213 IC50s ranged from 91.7 nM to 2.4 µM in AML cell lines and primary AML patient samples, which are achievable in vivo based on results from a PK study in mice (a single dose of 50 and 100 mg/kg ONC213 resulted in peak plasma concentrations of 3.7 μM and 8 μM, respectively). Annexin V/propidium iodide staining and flow cytometry analysis results showed variable responses for the AML cell lines tested. After 48 h treatment with 500 nM ONC213, striking induction of cell death in MOLM-13 and MV4-11 cells was detected (at least 72% Annexin V+ cells), while THP-1 and U937 cells showed little to no increase in Annexin V+ cells (6-11%). Similar results were obtained in primary AML patient samples. In contrast to the 48 h treatment of THP-1 and U937 cells, increasing the treatment duration to 120 h resulted in greater than 50% Annexin V+ cells, suggesting that a longer exposure time is necessary in some cell lines. In MV4-11 and MOLM-13 cells, initiation of cell death was detected 8 to 12 h post ONC213 treatment. Colony formation assays revealed that ONC213 treatment significantly reduced colony formation capacity of primary AML patient samples to less than 5% compared to vehicle control, while having no significant effect on normal hematopoietic progenitor cells. A primary AML patient sample was treated with or without ONC213 for 48 h, transplanted into NSG mice, and ten weeks later bone marrow was harvested and human CD45+ cells were measured. ONC213 treatment significantly reduced human AML engraftment compared to vehicle control (0.6% vs. 21.3%; p<0.05), demonstrating that ONC213 kills LSCs in vitro. Next, we examined in vivo efficacy of ONC213 against an AML cell line derived xenograft mouse model. MV4-11 cells were injected into NSGS mice through the tail vein. Three days post-injection, the mice were randomized into vehicle control or 125 mg/kg ONC213 cohorts (5 mice per cohort) and treated daily for 8 days. Modest weight loss was noted but was entirely manageable. ONC213 treatment extended the survival of mice by 88% (median survival 62 vs 33 days).Unlike ONC201, ONC213 treatment of AML cells did not increase the expression of TRAIL. Interestingly, RNAseq results showed that 500 nM ONC213 treatment for 48 h downregulated 33 mRNAs in the oxidative phosphorylation (OXPHOS) pathway, suggesting that ONC213 treatment decreases OXPHOS in AML cells. Thus far, six of the downregulated mRNAs (UQCRQ, SDHA, COX6C, NDUFS5, ATP5D, and NDUFB1) were verified by real-time RT-PCR after both 8 h and 48 h ONC213 treatment. LSCs have been shown to be highly reliant on OXPHOS, while normal hematopoietic stem cells and some bulk AML cells can switch to glycolysis for ATP production during times of OXPHOS inhibition. Thus, ONC213 may kill LSCs through inhibition of OXPHOS.In addition to downregulation of OXPHOS related genes, we found that ONC213 treatment downregulates Mcl-1. Since Mcl-1 mediates resistance to the promising Bcl-2-selective inhibitor ABT-199 (Venetoclax) and inhibition of Bcl-2 impairs OXPHOS, ONC213 would likely synergize with ABT-199 in AML cells. Indeed, combined treatment resulted in striking synergistic induction of apoptosis in both AML cell lines and primary patient samples. Enhanced cell death was detected 8 h post combination treatment in both MOLM-13 and MV4-11 cells. Results from colony formation assays revealed that the combination spares normal hematopoietic progenitor cells. Taken together, ONC213 is active as a single agent and in combination with ABT-199 in AML. DisclosuresAllen:Oncoceutics: Employment. Stogniew:Oncoceutics: Employment. Prabhu:Oncoceutics: Employment. Ge:MEI Pharma: Research Funding.

Collagen density modulates triple-negative breast cancer cell metabolism through adhesion-mediated contractility

Extracellular matrix (ECM) mechanical properties upregulate cancer invasion, cell contractility, and focal adhesion formation. Alteration in energy metabolism is a known characteristic of cancer cells (i.e., Warburg effect) and modulates cell invasion. There is little evidence to show if collagen density can alter cancer cell metabolism. We investigated changes in energy metabolism due to collagen density in five breast cell lines by measuring the fluorescence lifetime of NADH. We found that only triple-negative breast cancer cells, MDA-MB231 and MDA-MB468 cells, had an increased population of bound NADH, indicating an oxidative phosphorylation (OXPHOS) signature, as collagen density decreased. When inhibiting ROCK and cell contractility, MDA-MB231 cells on glass shifted from glycolysis (GLY) to OXPHOS, confirming the intricate relationship between mechanosensing and metabolism. MCF10A cells showed less significant changes in metabolism, shifting towards GLY as collagen density decreased. The MCF-7 and T-47D, less invasive breast cancer cells, compared to the MDA-MB231 and MDA-MB468 cells, showed no changes regardless of substrate. In addition, OXPHOS or GLY inhibitors in MDA-MB231 cells showed dramatic shifts from OXPHOS to GLY or vice versa. These results provide an important link between cellular metabolism, contractility, and collagen density in human breast cancer.

Abstract 324: An Engineered Protein as an Antidote for Carbon Monoxide Poisoning That Can Reverse Cardiovascular Collapse Through Scavenging of Carbon Monoxide and Rescue of Mitochondrial Respiration

Background: Carbon monoxide (CO) is the most common human poisoning. There is no antidote. One-third of hospitalized patients will have cardiac dysfunction and increased mortality. Little is known about the nature of cardiovascular dysfunction during acute CO poisoning. CO toxicity results from: (1) global hypoxia and decreased oxygen delivery through CO binding to hemoglobin, (2) inhibition of oxidative phosphorylation from CO binding to cytochrome c oxidase, and (3) ensuing superoxide injury. We have developed a recombinant neuroglobin molecule (rNgb) with a high affinity for CO, that can chelate CO from intrinsic heme proteins. Hypothesis: The addition of rNgb will scavenge CO from hemoglobin and cytochrome c oxidase, reversing CO-induced cardiovascular dysfunction. Methods: We inserted micromanometer-tip catheters into the left ventricle of ventilated, severely CO poisoned mice to study the mechanisms of CO-induced cardiovascular collapse. In a similar model, we measured blood pressure and heart rate of severely CO poisoned, ventilated mice through a carotid arterial catheter and treated with phosphate buffered saline (PBS) or rNgb. Hearts were isolated from these mice and we measured tissue respiration using a Clark-type oxygen electrode. We compared the respiration rates of these animals and compared with non-poisoned, sedated control mice. We also compared the enzymatic activity of electron transport chain complexes. Results and Conclusions: When compared to control mice (exposed to 21% oxygen/no CO), CO poisoned mice develop a decreased contractility index (-32.3% baseline vs. -10.8% in control, t-test, p=0.002), hypotension and death, indicating primary cardiac toxicity. CO-poisoned mice treated with PBS had heart tissue respiration that was 62.3% (+/- 7.5%) of sedated control mice (t-test, P=0.015). Treatment of mice with rNgb restored tissue respiration (P=0.037 versus PBS treated mice) similar to control values. CO poisoning reduced the activity level of complex IV in PBS treated mice (P=0.011), but levels were similar to control in rNgb treated mice. These molecular changes were associated with reversal of cardiovascular collapse in rNgb treated mice versus PBS, demonstrating the potential of rNgb as a CO poisoning antidote.

Human Dendritic Cell Subsets Undergo Distinct Metabolic Reprogramming for Immune Response.

Toll-like receptor (TLR) agonists induce metabolic reprogramming, which is required for immune activation. We have investigated mechanisms that regulate metabolic adaptation upon TLR-stimulation in human blood DC subsets, CD1c+ myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). We show that TLR-stimulation changes expression of genes regulating oxidative phosphorylation (OXPHOS) and glutamine metabolism in pDC. TLR-stimulation increases mitochondrial content and intracellular glutamine in an autophagy-dependent manner in pDC. TLR-induced glutaminolysis fuels OXPHOS in pDCs. Notably, inhibition of glutaminolysis and OXPHOS prevents pDC activation. Conversely, TLR-stimulation reduces mitochondrial content, OXPHOS activity and induces glycolysis in CD1c+ mDC. Inhibition of mitochondrial fragmentation or promotion of mitochondrial fusion impairs TLR-stimulation induced glycolysis and activation of CD1c+ mDCs. TLR-stimulation triggers BNIP3-dependent mitophagy, which regulates transcriptional activity of AMPKα1. BNIP3-dependent mitophagy is required for induction of glycolysis and activation of CD1c+ mDCs. Our findings reveal that TLR stimulation differentially regulates mitochondrial dynamics in distinct human DC subsets, which contributes to their activation.

209 - Inhibition of respiratory chain Complex III irreversibly changes the mitochondria proteomic landscape

The role of mitochondria dysfunction in the pathogenesis of pulmonary hypertension (PH) is currently well-recognized. However, the particular mechanisms and the type of mitochondrial dysfunction are still being debated. We have recently shown that chronic inhibition of oxidative phosphorylation by Antimycin A (AA) results in increased pulmonary pressure and remodeled pulmonary arteries. AA (0.35mg/kg) was given to rats three times during first six days and lungs were analyzed 30 minutes (acute effect), 12 and 24 days (chronic effect) after the first AA injection. Mitochondria isolated from lungs were subjected to mass spectrometry and quantitative proteomic analysis to estimate changes in the isolated mitochondrial proteome over the course of disease development. Using a 4-fold change cutoff value, 48 mitochondrial proteins were discovered to be altered upon AA treatment, with 13 proteins exhibiting a significant difference. Our data indicate that proteomic data with applied 4-fold cutoff can completely distinguish between the control and 24-day groups using principal component analysis. Functional analysis of the proteome data revealed major downregulation of enzymes involved in fatty acids oxidation(ACDSB,HADH) and fatty acids transport(CPT1, ACSM5). Electron transport chain proteins showed mixed results with downregulation of Complex IV (COX1/2), altered Complex I subunits(NU4/5M) and assembly protein expression(NDUF4), and upregulation of ubiquinone biosynthesis (COQ3/7). Activation of ubiquinone biosynthesis could be explained as compensation for inhibition of mitochondrial respiration by AA. Mitochondrial machinery for importing proteins into the matrix(TIM9/10/13) was upregulated, but peptidases(LON,MPPA) that control the quality of matrix proteins were downregulated. Finally, proteins that regulate mitochondria morphology(MIRO2, PGAM5,TMM11) and degradation pathways (MIEAP,BCL2) were altered. Collectively, our data indicate that chronic inhibition of the respiratory chain for six days leads to irreversible changes in the proteomic landscape of mitochondria. These changes reprogram mitochondria to a different metabolic state and altered morphology that have been found in various models of PH and PH patients.

Mitochondria-driven elimination of cancer and senescent cells.

Mitochondria and oxidative phosphorylation (OXPHOS) are emerging as intriguing targets for the efficient elimination of cancer cells. The specificity of this approach is aided by the capacity of non-proliferating non-cancerous cells to withstand oxidative insult induced by OXPHOS inhibition. Recently we discovered that mitochondrial targeting can also be employed to eliminate senescent cells, where it breaks the interplay between OXPHOS and ATP transporters that appear important for the maintenance of mitochondrial morphology and viability in the senescent setting. Hence, mitochondria/OXPHOS directed pharmacological interventions show promise in several clinically-relevant scenarios that call for selective removal of cancer and senescent cells.

Low-Temperature EPR Spectroscopy as a Probe-Free Technique for Monitoring Oxidants Formed in Tumor Cells and Tissues: Implications in Drug Resistance and OXPHOS-Targeted Therapies.

Oxidants formed from oxidative and nitrative metabolism include reactive oxygen species (ROS) such as superoxide, hydrogen peroxide/lipid hydroperoxides and reactive nitrogen species (RNS) (e.g., peroxynitrite [ONOO-] and nitrogen dioxide), and reactive halogenated species (e.g., hypochlorous acid [HOCl]). Increasingly, ROS and RNS are implicated in tumorigenesis as well as tumor growth, progression, and metastasis. Recently, ROS were implicated in drug resistance, metabolic reprogramming, and T-cell metabolism in immunotherapy. Mostly, fluorescent probes have been used in cell culture systems. The identity of species is obtained by LC-MS analyses of diagnostic marker products. However, extrapolation of these assays to cancer xenografts is difficult if not impossible. Thus, development of a probe-free assay for monitoring and assessing oxidant formation in tumor cells and tumor xenografts is critical and timely. Here, we describe the use of ex vivo electron paramagnetic resonance (EPR) spectroscopy at cryogenic temperatures as a uniquely useful probe-free technique for assessing intracellular oxidation and oxidants via EPR signals from redox centers, particularly iron-sulfur clusters, in mitochondrial and cytosolic redox proteins. Examples of cancer cells subjected to inhibition of mitochondrial oxidative phosphorylation are presented. This ex vivo methodology can be readily extended to monitor oxidant formation in tumor tissues isolated from mice and humans.

Oxidative Phosphorylation: A Target for Novel Therapeutic Strategies Against Ovarian Cancer

Aerobic glycolysis is an important metabolic adaptation of cancer cells. There is growing evidence that oxidative phosphorylation is also an active metabolic pathway in many tumors, including in high grade serous ovarian cancer. Metastasized ovarian tumors use fatty acids for their energy needs. There is also evidence of ovarian cancer stem cells privileging oxidative phosphorylation (OXPHOS) for their metabolic needs. Metformin and thiazolidinediones such as rosiglitazone restrict tumor growth by inhibiting specific steps in the mitochondrial electron transport chain. These observations suggest that strategies to interfere with oxidative phosphorylation should be considered for the treatment of ovarian tumors. Here, we review the literature that supports this hypothesis and describe potential agents and critical control points in the oxidative phosphorylation pathway that can be targeted using small molecule agents. In this review, we also discuss potential barriers that can reduce the efficacy of the inhibitors of oxidative phosphorylation.

High-fat diet affects pregestational adiposity and glucose tolerance perturbing gestational placental macronutrient transporters culminating in an obese offspring in wild-type and glucose transporter isoform 3 heterozygous null mice

We examined the effect of a high-fat diet (HFD) vs. control diet (CD) upon pregestational and gestational wild-type (wt) and glucose transporter (glut)3 heterozygous (glut3+/−) female mice and observed an increase in pregestational body weights, white adiposity (wt > glut3+/−), circulating cholesterol, and high-density lipoproteins, with glucose intolerance in both genotypes. The HFD-exposed offspring displayed reduced birth weight with catch up to CD-fed in wt vs. an increased birth weight persisting as such at weaning by day 21 in glut3+/− mice. To decipher the mechanism behind this genotype-specific difference in the HFD offspring's phenotype, we first examined placental macronutrient transporters and noted HFD-induced increase in CD36 in wt with no change in other FATPs, sodium-coupled neutral amino acid transporters and system L amino acid transporter in both genotypes. In contrast, while placental Glut1 increased in both the genotypes, only Glut3 increased in the glut3+/− genotype in response to HFD. Hence, we next assessed glut3+/− embryonic (ES) cells under differing stressors of low glucose, hypoxia and inhibition of oxidative phosphorylation. Reduced Glut3-mediated glucose uptake in glut3+/− vs. wt ES cells culminated in deficient growth. We conclude that maternal HFD affects the in utero growth potential of the offspring by altering placental CD36 and Glut1 concentrations. In contrast, a differential effect on placental Glut3 concentrations between glut3+/− and wt genotypes is evident, with an increase occurring in the glut3+/− genotype alone. Deficient Glut3 in ES cells interferes with glucose uptake, cell survival and growth being further exaggerated with low glucose, hypoxia and inhibition of oxidative phosphorylation.

Insulin down-regulates cardioprotective SUR2A in the heart-derived H9c2 cells: A possible explanation for some adverse effects of insulin therapy

Some recent studies associated insulin therapy with negative cardiovascular events and shorter lifespan. SUR2A, a KATP channel subunit, regulate cardioprotection and cardiac ageing. Here, we have tested whether glucose and insulin regulate expression of SUR2A/KATP channel subunits and resistance to metabolic stress in heart H9c2 cells. Absence of glucose in culture media decreased SUR2A mRNA, while mRNAs of Kir6.2, Kir6.1, SUR1 and IES SUR2B were increased. 2-deoxyglucose (50 mM) decreased mRNAs of SUR2A, SUR2B and SUR1, did not affect IES SUR2A and IES SUR2B mRNAs and increased Kir6.2 mRNA. No glucose and 2-deoxyglucose (50 mM) decreased resistance to an inhibitor of oxidative phosphorylation, DNP (10 mM). 50 mM glucose did not alter KATP channel subunits nor cellular resistance to DNP (10 mM). Insulin (20 ng/ml) in both physiological and high glucose (50 mM) down-regulated SUR2A while upregulating Kir6.1 and Kir6.2 (in high glucose only). Insulin (20 ng/ml) in physiological and high glucose decreased cell survival in DNP (10 mM). As opposed to Kir6.2, infection with SUR2A resulted in titre-dependent cytoprotection. We conclude that insulin decreases resistance to metabolic stress in H9c2 cells by decreasing SUR2A expression. Lower cardiac SUR2A levels underlie increased myocardial susceptibility to metabolic stress and shorter lifespan.

Interplay between ShcA Signaling and PGC-1α Triggers Targetable Metabolic Vulnerabilities in Breast Cancer.

The ShcA adaptor protein transduces oncogenic signals downstream of receptor tyrosine kinases. We show here that breast tumors engage the ShcA pathway to increase their metabolism. ShcA signaling enhanced glucose catabolism through glycolysis and oxidative phosphorylation, rendering breast cancer cells critically dependent on glucose. ShcA signaling simultaneously increased the metabolic rate and flexibility of breast cancer cells by inducing the PGC-1α transcriptional coactivator, a central regulator of mitochondrial metabolism. Breast tumors that engaged ShcA signaling were critically dependent on PGC-1α to support their increased metabolic rate. PGC-1α deletion drastically delayed breast tumor onset in an orthotopic mouse model, highlighting a key role for PGC-1α in tumor initiation. Conversely, reduced ShcA signaling impaired both the metabolic rate and flexibility of breast cancer cells, rendering them reliant on mitochondrial oxidative phosphorylation. This metabolic reprogramming exposed a targetable metabolic vulnerability, leading to a sensitization of breast tumors to inhibitors of mitochondrial complex I (biguanides). Genetic inhibition of ShcA signaling in the Polyoma virus middle T (MT) breast cancer mouse model sensitized mammary tumors to biguanides during the earliest stages of breast cancer progression. Tumor initiation and growth were selectively and severely impaired in MT/ShcA-deficient animals. These data demonstrate that metabolic reprogramming is a key component of ShcA signaling and serves an unappreciated yet vital role during breast cancer initiation and progression. These data further unravel a novel interplay between ShcA and PGC-1α in the coordination of metabolic reprogramming and demonstrate the sensitivity of breast tumors to drugs targeting oxidative phosphorylation.Significance: This study uncovers a previously unrecognized mechanism that links aberrant RTK signaling with metabolic perturbations in breast cancer and exposes metabolic vulnerabilities that can be targeted by inhibitors of oxidative phosphorylation. Cancer Res; 78(17); 4826-38. ©2018 AACR.

Mycobacterial Cell Wall Synthesis Inhibitors Cause Lethal ATP Burst.

Mycobacterial cell wall inhibitors interfere with targets involved in synthesis of mycolic acids, arabinogalactan and peptidoglycan. These antibiotics corrupt structural integrity of the cell envelope and this is believed to be the cause of drug mediated cell death. Here, we show that treatment of Mycobacterium bovis BCG with these mechanistically different classes of cell wall inhibitors at MIC caused a 4 to 5-fold increase in intrabacterial ATP concentration. This effect on ATP homeostasis was specific to inhibitors of cell wall synthesis and not observed for other anti-tuberculosis drugs. Treating M. bovis BCG with sub-MIC concentrations of the ATP synthase inhibitor bedaquiline or the uncoupler carbonyl cyanide 3-chlorophenylhydrazone suppressed drug induced ATP surge, suggesting that the increase in ATP concentration was due to increased oxidative phosphorylation. Pharmacological suppression of the ATP burst attenuated bactericidal activity of the cell wall-targeting drugs up to 100-fold, suggesting that increased ATP levels are associated with the lethal effect of these antibiotics. Interestingly, inhibition of the ATP burst also suppressed induction of the promoter of the cell envelope stress response operon iniBAC by cell wall inhibitors suggesting a link between ATP surge and iniBAC expression. In conclusion, we show that treatment of M. bovis BCG with inhibitors of cell wall synthesis causes a burst of intrabacterial ATP by increasing oxidative phosphorylation. This ATP surge appears to be required for induction of the iniBAC cell envelope stress response operon and to contribute to drug induced cell death. Hence, this work revealed links between inhibition of cell wall synthesis, oxidative phosphorylation, iniBAC induction and cell death. The identification of the molecular mechanisms linking these processes may reveal novel targets for the discovery of bactericidal antibiotics.

Metformin Targets Mitochondrial Glycerophosphate Dehydrogenase to Control Rate of Oxidative Phosphorylation and Growth of Thyroid Cancer In Vitro and In Vivo.

Purpose: Mitochondrial glycerophosphate dehydrogenase (MGPDH) is the key enzyme connecting oxidative phosphorylation (OXPHOS) and glycolysis as well as a target of the antidiabetic drug metformin in the liver. There are no data on the expression and role of MGPDH as a metformin target in cancer. In this study, we evaluated MGPDH as a potential target of metformin in thyroid cancer and investigated its contribution in thyroid cancer metabolism.Experimental Design: We analyzed MGPDH expression in 253 thyroid cancer and normal tissues by immunostaining and examined its expression and localization in thyroid cancer-derived cell lines (FTC133, BCPAP) by confocal microscopy. The effects of metformin on MGPDH expression were determined by qRT-PCR and Western blot analysis. Seahorse analyzer was utilized to assess the effects of metformin on OXPHOS and glycolysis in thyroid cancer cells. We analyzed the effects of metformin on tumor growth and MGPDH expression in metastatic thyroid cancer mouse models.Results: We show for the first time that MGPDH is overexpressed in thyroid cancer compared with normal thyroid. We demonstrate that MGPDH regulates human thyroid cancer cell growth and OXPHOS rate in vitro Metformin treatment is associated with downregulation of MGPDH expression and inhibition of OXPHOS in thyroid cancer in vitro Cells characterized by high MGPDH expression are more sensitive to OXPHOS-inhibitory effects of metformin in vitro and growth-inhibitory effects of metformin in vitro and in vivoConclusions: Our study established MGPDH as a novel regulator of thyroid cancer growth and metabolism that can be effectively targeted by metformin. Clin Cancer Res; 24(16); 4030-43. ©2018 AACR.

Metabolic Mechanisms and a Rational Combinational Application of Carboxyamidotriazole in Fighting Pancreatic Cancer Progression after Chemotherapy.

The anticancer and anti-inflammatory effects of carboxyamidotriazole (CAI) have been demonstrated in several studies, but the underlying mechanisms remain to be elucidated. This study showed that CAI caused metabolic reprogramming of pancreatic cancer cells. The inhibition of mitochondrial oxidative metabolism by CAI led to increased glutamine-dependent reductive carboxylation and enhanced glycolytic metabolism. The presence of environmental substances that affect cellular metabolism, such as glutamine and pyruvate, attenuated the anticancer efficacy of CAI. Based on the action of CAI: 1) when glutamine was removed, the NAD+/NADH ratio was decreased, the synthesis of cellular aspartate was reduced, and autophagy flux was blocked; and 2) when glycolysis was pharmacologically inhibited, the ATP level was significantly decreased, the cell viability was greatly inhibited, and the compensatory rescue effect of glutamine was eliminated. When combined with chemotherapy, cotreatment with CAI and the glycolysis inhibitor 2-deoxyglucose (2-DG) inhibited the pancreatic cancer progression after chemotherapy. As the inhibition of mitochondrial oxidative metabolism can explain several anticancer activities of CAI reported previously, including inhibition of calcium entry and induction of reactive oxygen species, we demonstrate that inhibition of mitochondrial oxidative phosphorylation may be the fundamental mechanism of CAI. The combination of CAI and 2-DG causes energy depletion in cancer cells, eliminating the rescue effect of the metabolic environment. Inhibiting pancreatic cancer progression after chemotherapy is a rational application of this metabolism-disturbing combination strategy.

Abstract 1655: Discovery and development of IACS-010759, a novel inhibitor of Complex I currently in phase I studies to exploit oxidative phosphorylation dependency in acute myeloid leukemia and solid tumors

Abstract Over the past few years we and others reported that specific populations of tumor cells including AML, subsets of lymphoma, glioblastoma, triple negative breast cancer (TNBC), melanoma and pancreatic ductal adenocarcinoma (PDAC) are highly dependent upon oxidative phosphorylation (OXPHOS) to meet their energy and biomass needs. Inhibition of OXPHOS in the context of these dependent tumor populations represents therefore an exciting therapeutic opportunity. Through an extensive medicinal chemistry campaign we discovered IACS-010759, a potent, selective small molecule inhibitor of complex I of the mitochondria electron transport chain that possesses excellent pharmacokinetic (PK) and pharmacologic properties, making it suitable for clinical development. We advanced IACS-010759 through IND studies and have recently initiated Phase I studies in patients with relapsed/refractory acute myeloid leukemia (AML) and advanced solid tumors and lymphomas (NCT02882321 and NCT03291938). In this presentation we will describe the identification of a novel series of Complex I inhibitors and their optimization into the clinical candidate compound IACS-010759. Several challenges were successfully overcome, including the optimization of the pharmacokinetic profile and the identification of inhibitors with minimal activity shift across preclinical species, thus enabling a thorough evaluation of the efficacy and toxicology profile. Aspects of the extensive translational research conducted to elucidate the mechanism of action of IACS-010759 and to position it into the clinic will be discussed, including the compelling pharmacological response observed in multiple PDX models of primary AML, and PDX xenograft models of lymphoma, TNBC, glioblastoma, melanoma and PDAC. The observed response was associated with robust pharmacodynamic read-out as assessed by modulation of oxygen consumption rate (OCR), aspartate and specific transcriptional changes. The presentation will also cover the preclinical development activities which resulted in IACS-010759 advancing into on-going phase 1 evaluation in AML and solid tumors. Citation Format: Maria Emilia Di Francesco, Joseph R. Marszalek, Timothy McAfoos, Christopher L. Carroll, Zhijun Kang, Gang Liu, Jay P. Theroff, Jennifer P. Bardenhager, Madhavi L. Bandi, Jennifer R. Molina, Sonal Gera, Marina Protopopova, Yuting Sun, Mary K. Geck Do, Ningping Feng, Jason P. Gay, Florian Muller, Marina Konopleva, Funda Meric-Bernstam, Carlo Toniatti, Timothy P. Heffernan, Giulio F. Draetta, Philip Jones. Discovery and development of IACS-010759, a novel inhibitor of Complex I currently in phase I studies to exploit oxidative phosphorylation dependency in acute myeloid leukemia and solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1655.