Inhibition Of Oxidative Phosphorylation Research Articles

Initial B Cell Activation Induces Metabolic Reprogramming and Mitochondrial Remodeling

SummaryB lymphocytes provide adaptive immunity by generating antigen-specific antibodies and supporting the activation of T cells. Little is known about how global metabolism supports naive B cell activation to enable an effective immune response. By coupling RNA sequencing (RNA-seq) data with glucose isotopomer tracing, we show that stimulated B cells increase programs for oxidative phosphorylation (OXPHOS), the tricarboxylic acid (TCA) cycle, and nucleotide biosynthesis, but not glycolysis. Isotopomer tracing uncovered increases in TCA cycle intermediates with almost no contribution from glucose. Instead, glucose mainly supported the biosynthesis of ribonucleotides. Glucose restriction did not affect B cell functions, yet the inhibition of OXPHOS or glutamine restriction markedly impaired B cell growth and differentiation. Increased OXPHOS prompted studies of mitochondrial dynamics, which revealed extensive mitochondria remodeling during activation. Our results show how B cell metabolism adapts with stimulation and reveals unexpected details for carbon utilization and mitochondrial dynamics at the start of a humoral immune response.

Abstract 4953: Metabolic targeting of chemoresistance perturbs clonal complexity in pancreatic cancer

Abstract A major barrier to achieving durable remission and definitive cure in oncology patients is the emergence of tumor resistance, a common outcome of different disease types independent from the therapeutic approach undertaken. Patients with pancreatic ductal adenocarcinoma (PDAC) continue to have a poor prognosis despite concerted efforts to advance new drugs to the clinic. One reason for this, in PDAC and other tumors, is that tumors are constantly adapting and evolving in response to external perturbations. To better investigate tumor evolution in response to therapy we developed a new clonal tracking platform that enables the in vivo study of long term self-renewing compartments and the generation of cohorts of patient-derived xenografts in which tumors are virtually identical and maintained by the same clones (clonal replica tumors), representing a unique tool to address fundamental questions about clonal dynamics in response to pharmacological treatment. Using this novel approach we demonstrate that standard of care in pancreatic cancer, despite inducing tumor regression, has minimal effect on the clonal composition of tumors that eventually relapse. Transcriptomic and metabolic characterization of residual tumor cells in patient derived xenograft models as well as in patients after chemoradiation shows that resistant cells that contribute to tumor relapse are metabolically rewired to upregulate mitochondrial respiration (OXPHOS). Combining a novel inhibitor of oxidative phosphorylation (IACS-010759), developed at the MD Anderson Institute for Applied Cancer Science and currently in phase I clinical trial in relapsed/refractory acute myelogenous leukemia and advanced solid tumors, with standard of care drugs drastically reduces tumor clonal complexity, underscoring the promise of inhibiting mitochondrial respiration as a new therapeutic strategy to prolong patient survival by eradicating resistant clones that survive chemoradiation. Citation Format: Sara Loponte, Denise Corti, Sahil Seth, Edoardo Del Poggetto, I-Lin Ho, Chieh-Yuan Li, Shan Jiang, Joseph R. Marszalek, Maria Emilia Di Francesco, Giannicola Genovese, Giulio Draetta, Alessandro Carugo, Andrea Viale. Metabolic targeting of chemoresistance perturbs clonal complexity in pancreatic cancer [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 4953.

Abstract 5031: Organotypic brain explant culture as a drug evaluation system for malignant brain tumors

Abstract Background: Inhibition of major metabolic pathways is being assessed for several intractable tumors, including glioblastoma. Following screening in 2D-systems, evaluating metabolic inhibitors in assays that include both tumor and normal brain tissue is indispensable for selecting compounds that can affect gliomas within their microenvironment. Here we asked whether organotypic brain slices from adult mice can be used to evaluate the effects of metabolic inhibitors on malignant glioma cells. Methods: Retroviral expression of H-RasV12 and dsRed in Ink4a/Arf-/- neural stem / progenitor cells was adopted to establish glioma initiating cells (GICs). GICs with different extracellular acidification potential were derived by single cell cloning and used after one in vivo passage. GICs or their clones were implanted into the forebrain of 6-weeks-old wild-type mice. Tumor-bearing brain explants were established as 200 µm coronal brain slices, treated with cisplatin, dichloroacetate or phenformin. The effects of the drugs were assessed by fluorescence microscopy of the live explants, as well as immunohistochemical staining at the end of the treatment. Results: The feasibility of assessing drug effects in brain tumor explants from adult mice was confirmed with the use of the DNA-damaging agent cisplatin. Treatment of brain slices carrying tumors formed by GICs with 50 μM cisplatin for 4 days effectively decreased the tumor burden, as shown by a significant reduction of the tumor area on fluorescence imaging and a marked increase in tumor cells positive for cleaved caspase 3 on immunohistochemical analysis. The action of metabolic inhibitors was assessed in explants with tumors formed by GIC-derived populations that predominantly use either glycolysis or mitochondrial respiration. Treatment of the brain slices with dichloroacetate, an inhibitor of pyruvate dehydrogenase kinase, resulted in a marked increase in cleaved caspase 3-positive cells in glycolytic tumors. In contrast, phenformin, which inhibits respiratory complex I, had a pronounced pro-apoptotic effect in tumors formed by cells with a high mitochondrial activity. Conclusion: Inhibitors of both glycolysis and oxidative phosphorylation effectively and selectively induced apoptosis of the targeted tumor cells in cultured brain slices. Our results suggest that brain explants from adult mice can be used to assess the effects of anti-metabolic drugs on the survival of glioma initiating cells within a syngeneic microenvironment. Explants could thus be useful in the evaluation of new metabolic inhibitors. Citation Format: Noriaki Minami. Organotypic brain explant culture as a drug evaluation system for malignant brain 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 5031.

Abstract 2856: Targeting OXPHOS with IACS-010759 to eliminate standard of care resistant tumor cells

Abstract Tumors are comprised of heterogenous populations of tumor cells that rely on both glycolysis and oxidative phosphorylation (OXPHOS) for bioenergy and synthetic processes in support of cell proliferation. Over the past few years, we and others have reported that there is a subpopulation of tumors cells that are resistant to standard of care treatment or targeted therapies, and that these so-called persistent tumor cells possess stem cell like properties. Of note, these cells have elevated levels of mitochondria and are dependent on OXPHOS for survival. We have previously disclosed the discovery of IACS-010759, a potent, selective inhibitor of complex I of the electron transport chain, which is orally bioavailable and has excellent PK and physicochemical properties in preclinical species. IACS-010759 is currently in phase I clinical trials in relapsed/refractory AML and solid tumors where initial safety, pharmacokinetics, efficacy and pharmacodynamic impacts on tumor cell biology are being evaluated. As part of the development of IACS-010759, we were interested to explore the impact of the compound to target the persistent tumor cells, in particular by treating AML, TNBC and PDAC PDX models post-chemotherapy with IACS-010759. For all three contexts, IACS-010759 extended progression free survival, consistent with IACS-010759 targeting the recently described metabolically adapted residual tumor cells. For solid tumor indications, we have utilized innovative barcoding and clonal tracking strategies to confirm dependency of a specific subpopulation of tumor cells on OXPHOS. We show that OXPHOS inhibition extends survival and limits AML growth in secondary transplantation by stimulating terminal differentiation of putative stem cells. Taken together, these data provide rationale for multiple Phase II/III clinical trials where IACS-010759 will be used to target persistent tumor cell population and extend survival. Citation Format: Joseph R. Marszalek, Sahil Seth, Denise Corti, Qi Zhang, Gloria V. Echeverria, Lina Han, Yuting Sun, Jennifer Molina, Sonal Gera, Edward Chang, Tin O. Khor, Mikhila Mahendra, Ningping Feng, Jason P. Gay, Timothy McAfoos, Virginia Giuliani, Xi Shi, Sabrina Jeter-Jones, Sarah Loponte, Chieh-Yuan Li, Christopher A. Bristow, Maria Emilia Di Francesco, Helen Piwnica-Worms, Marina Konopleva, Alessandro Carugo, Andrea Viale, Philip Jones, Timothy P. Heffernan, Giulio F. Draetta. Targeting OXPHOS with IACS-010759 to eliminate standard of care resistant tumor cells [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 2856.

Abstract LB-397: SMIP004-7: A novel oxidative phosphorylation inhibitor selectively kills Breast Cancer Stem-Like Cells with metabolic vulnerabilities

Abstract Background The manifestation of metastases from <1% of the leftover subset of cells after apparently successful initial therapy is predictive of stem cell like properties such as self-renewal capacity, slow proliferation, and drug resistance. These “cancer stem cells” (CSCs) appear to drive relapse and form clinically relevant macro-metastases by establishing themselves in the microenvironment of distal sites. The aggressive nature of Triple Negative Breast Cancer (TNBC) may be explained by the presence of breast cancer stem cells (BCSCs) within TNBC tumors that are resistant to conventional therapies. Recent studies have shown an unexpected common denominator of these scenarios of therapy-selected quiescence is metabolic reprogramming to upregulate OXPHOS genes and increase oxygen consumption and ATP production. Efforts to target BCSCs have therefore focused on specific vulnerabilities of these cells. Multicellular tumor spheroid (MCTS) is one such 3D model that has been suggested to provide a more clinically relevant in vivo model. MCTS not only simulate the harsh conditions present in poorly vascularized tumors but also closely simulate the microenvironment with respect to glucose, oxygen, and lactate distribution thus enriched cancer cell phenotype. To counter this aggressive aspect of MCTS, we employed SMIP004-7 (N-(4-butyl-2-methyl-phenylaniline), a novel OXPHOS inhibitor in this study. Methods and Results Mammosphere assay (3D culture) for the enrichment of CD133+ CSCs in established breast cancer cell lines was performed. Overexpression of CD133+, as determined by flow cytometric analysis and immunoblot, correlated with an increased colonogenic, chemo-resistant, and invasive potential in vitro. This phenotype is concordant to that of CSCs in vivo. Our novel compound SMIP004-7, showed enhanced anti-cancer activity in 3D culture compared to its respective 2D monolayer cells in cytotoxicity assay. More specifically, we demonstrated that SMIP004-7 treatment of breast cancer cells inhibited oxygen-consumption and metabolically induced aerobic glycolysis, as well as oxidative stress determined through DCFH-DA, Glucose starvation experiment and OCR and ECAR assay. Remarkably, SMIP004-7 potently inhibited the propagation of TNBC-derived CSCs, with an IC50 of 1 μM, as measured using the mammosphere assay. Importantly, these results indicated that glycolysis itself is not sufficient to maintain the proliferation of CSCs, which is instead strictly dependent on mitochondrial function. Conclusions/Significance In conclusion, our mechanistic and pharmacokinetic insights are relevant to understanding and developing the role of SMIP04-7 using mammosphere cultures, we propose that mitochondria are new therapeutic targets for eradicating cancer stem cells, to prevent tumor recurrence, metastasis and poor clinical outcome in breast cancer patients. Our findings are in accordance with the hypothesis on dormant cells being CSCs. Citation Format: SHASHI JAIN, Dieter A. Wolf. SMIP004-7: A novel oxidative phosphorylation inhibitor selectively kills Breast Cancer Stem-Like Cells with metabolic vulnerabilities [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 LB-397.

Mutations in the SWI/SNF complex induce a targetable dependence on oxidative phosphorylation in lung cancer.

Lung cancer is a devastating disease that remains a top cause of cancer mortality. Despite improvements with targeted and immunotherapies, the majority of patients with lung cancer lack effective therapies, underscoring the need for additional treatment approaches. Genomic studies have identified frequent alterations in components of the SWI/SNF chromatin remodeling complex including SMARCA4 and ARID1A. To understand the mechanisms of tumorigenesis driven by mutations in this complex, we developed a genetically engineered mouse model of lung adenocarcinoma by ablating Smarca4 in the lung epithelium. We demonstrate that Smarca4 acts as a bona fide tumor suppressor and cooperates with p53 loss and Kras activation. Gene expression analyses revealed the signature of enhanced oxidative phosphorylation (OXPHOS) in SMARCA4 mutant tumors. We further show that SMARCA4 mutant cells have enhanced oxygen consumption and increased respiratory capacity. Importantly, SMARCA4 mutant lung cancer cell lines and xenograft tumors have marked sensitivity to inhibition of OXPHOS by a novel small molecule, IACS-010759, that is under clinical development. Mechanistically, we show that SMARCA4-deficient cells have a blunted transcriptional response to energy stress creating a therapeutically exploitable synthetic lethal interaction. These findings provide the mechanistic basis for further development of OXPHOS inhibitors as therapeutics against SWI/SNF mutant tumors.

An inhibitor of oxidative phosphorylation exploits cancer vulnerability.

Metabolic reprograming is an emerging hallmark of tumor biology and an actively pursued opportunity in discovery of oncology drugs. Extensive efforts have focused on therapeutic targeting of glycolysis, whereas drugging mitochondrial oxidative phosphorylation (OXPHOS) has remained largely unexplored, partly owing to an incomplete understanding of tumor contexts in which OXPHOS is essential. Here, we report the discovery of IACS-010759, a clinical-grade small-molecule inhibitor of complex I of the mitochondrial electron transport chain. Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in models of brain cancer and acute myeloid leukemia (AML) reliant on OXPHOS, likely owing to a combination of energy depletion and reduced aspartate production that leads to impaired nucleotide biosynthesis. In models of brain cancer and AML, tumor growth was potently inhibited in vivo following IACS-010759 treatment at well-tolerated doses. IACS-010759 is currently being evaluated in phase 1 clinical trials in relapsed/refractory AML and solid tumors.

Expression of glycolytic enzymes in ovarian cancers and evaluation of the glycolytic pathway as a strategy for ovarian cancer treatment

BackgroundNovel therapeutic approaches are required to treat ovarian cancer and dependency on glycolysis may provide new targets for treatment. This study sought to investigate the variation of expression of molecular components (GLUT1, HKII, PKM2, LDHA) of the glycolytic pathway in ovarian cancers and the effectiveness of targeting this pathway in ovarian cancer cell lines with inhibitors.MethodsExpression of GLUT1, HKII, PKM2, LDHA were analysed by quantitative immunofluorescence in a tissue microarray (TMA) analysis of 380 ovarian cancers and associations with clinicopathological features were sought. The effect of glycolysis pathway inhibitors on the growth of a panel of ovarian cancer cell lines was assessed by use of the SRB proliferation assay. Combination studies were undertaken combining these inhibitors with cytotoxic agents.ResultsMean expression levels of GLUT1 and HKII were higher in high grade serous ovarian cancer (HGSOC), the most frequently occurring subtype, than in non-HGSOC. GLUT1 expression was also significantly higher in advanced stage (III/IV) ovarian cancer than early stage (I/II) disease. Growth dependency of ovarian cancer cells on glucose was demonstrated in a panel of ovarian cancer cell lines. Inhibitors of the glycolytic pathway (STF31, IOM-1190, 3PO and oxamic acid) attenuated cell proliferation in platinum-sensitive and platinum-resistant HGSOC cell line models in a concentration dependent manner. In combination with either cisplatin or paclitaxel, 3PO (a novel PFKFB3 inhibitor) enhanced the cytotoxic effect in both platinum sensitive and platinum resistant ovarian cancer cells. Furthermore, synergy was identified between STF31 (a novel GLUT1 inhibitor) or oxamic acid (an LDH inhibitor) when combined with metformin, an inhibitor of oxidative phosphorylation, resulting in marked inhibition of ovarian cancer cell growth.ConclusionsThe findings of this study provide further support for targeting the glycolytic pathway in ovarian cancer and several useful combinations were identified.

PO-017 Androgen deprivation and oxidative phosporylation inhibition pose synergistic antitumor effects in subsets of prostate tumours in vitro

Introduction Cancer cells are able to survive oxidative phosphorylation (OXPHOS) inhibition by up-regulation of glycolysis. Androgens stimulate glycolysis in prostate cancer cells through activation of the androgen receptor (AR). Here we hypothesise that androgens might help subsets of prostate cancer cells to survive OXPHOS inhibition. In addition, the combined androgen signalling and OXPHOS inhibition might pose synergistic anti-tumour effects in vitro. Material and methods The effects of suppressing OXPHOS with 100 ng/ml oligomycin in charcoal stripped serum media (CSS), before and after the addition of 40 ng/dl testosterone in VCAP, LNCaP, and LNCaP-C4-2B cells in vitro were evaluated. Tumour cell viability after 72 hours was estimated using MTT assay. Results and discussions The addition of 100 ng/ml oligomycin to VCAP cells in a steroid-depleted environment (CSS) dramatically decreased cell viability compared to CSS alone (p Conclusion The combined androgen depletion and OXPHOS inhibition shows promising synergistic anti-tumour effects in vitro.

Oxidative Phosphorylation as an Emerging Target in Cancer Therapy.

Cancer cells have upregulated glycolysis compared with normal cells, which has led many to the assumption that oxidative phosphorylation (OXPHOS) is downregulated in all cancers. However, recent studies have shown that OXPHOS can be also upregulated in certain cancers, including leukemias, lymphomas, pancreatic ductal adenocarcinoma, high OXPHOS subtype melanoma, and endometrial carcinoma, and that this can occur even in the face of active glycolysis. OXPHOS inhibitors could therefore be used to target cancer subtypes in which OXPHOS is upregulated and to alleviate therapeutically adverse tumor hypoxia. Several drugs including metformin, atovaquone, and arsenic trioxide are used clinically for non-oncologic indications, but emerging data demonstrate their potential use as OXPHOS inhibitors. We highlight novel applications of OXPHOS inhibitors with a suitable therapeutic index to target cancer cell metabolism. Clin Cancer Res; 24(11); 2482-90. ©2018 AACR.

Redox mechanism of levobupivacaine cytostatic effect on human prostate cancer cells

Anti-cancer effects of local anesthetics have been reported but the mode of action remains elusive. Here, we examined the bioenergetic and REDOX impact of levobupivacaine on human prostate cancer cells (DU145) and corresponding non-cancer primary human prostate cells (BHP). Levobupivacaine induced a combined inhibition of glycolysis and oxidative phosphorylation in cancer cells, resulting in a reduced cellular ATP production and consecutive bioenergetic crisis, along with reactive oxygen species generation. The dose-dependent inhibition of respiratory chain complex I activity by levobupivacaine explained the alteration of mitochondrial energy fluxes. Furthermore, the potency of levobupivacaine varied with glucose and oxygen availability as well as the cellular energy demand, in accordance with a bioenergetic anti-cancer mechanism. The levobupivacaine-induced bioenergetic crisis triggered cytostasis in prostate cancer cells as evidenced by a S-phase cell cycle arrest, without apoptosis induction. In DU145 cells, levobupivacaine also triggered the induction of autophagy and blockade of this process potentialized the anti-cancer effect of the local anesthetic. Therefore, our findings provide a better characterization of the REDOX mechanisms underpinning the anti-effect of levobupivacaine against human prostate cancer cells.

Phase 1 study of an oxidative phosphorylation inhibitor IM156 in patients with advanced solid tumors.

TPS2620Background: IM156 is a novel oral agent with a biguanide structure, which has anticancer activity through AMPK activation and reduction of oxidative phosphorylation. Inhibition of oxidative ...

990 Targeting metabolism reprogramming of keratinocytes improves psoriasis-like skin inflammation

Keratinocyte-intrinsic metabolic reprogramming is the major fuel to epidermal hyperplasia in psoriasis, yet the underlying cause is poorly understood. We report that in psoriatic lesions, Protein Phosphatase 6 (PP6) is diminished in the epidermis, and its levels negatively correlate with the disease severity. Mice with genetic deficiency of Pp6 in keratinocytes spontaneously develop psoriasis-like skin phenotype resembling psoriasis clinically, histologically, in its gene expression profile and in its response to therapy. Mechanistically, Pp6-/- keratinocytes rely on inordinate ornithine cycle and enhanced oxidative phosphorylation (OXPHOS) to hyper-proliferate, mediated by increased Arginase-1 (Arg1) production resulting from the activation of CCAAT/enhancer-binding protein beta (C/EBPβ) and elevated levels of mitochondrial respiration chain components. Treating imiquimod-induced mouse model of psoriasis with an arginase inhibitor or (and) an OXPHOS inhibitor markedly improves the skin inflammation. Thus, our data reveal for the first time the molecular basis and the functional significance of keratinocyte metabolic adaptations in psoriasis, bringing forth novel therapeutic strategies targeting keratinocyte metabolism to treat psoriasis.

Phenformin inhibits proliferation of acquired gefitinib resistant non‐small‐cell lung cancer(NSCLC) through the reduction of NAD+/NADH ratio

Epidermal growth factor receptor(EGFR) tyrosine kinase inhibitors(TKI) including gefitinib and erlotinib are now recommended as a first‐line chemo therapy for NSCLC. Unfortunately majority of NSCLC patients administered with gefitinib showed acquired resistance within 1 yr. To overcome it, second‐ and third‐generation of EGFR inhibitors were developed, but eve0n in the third‐generation of EGFR inhibitors, NSCLCs were found to be resistant and new therapeutic strategies have become necessary.HCC827, a NSCLC cell line were cultured with stepwise escalation of gefitinib over 1 yrs to establish acquired gefitinib resistance HCC827(HCC827‐GR) cells. EGFR exons from 18 to 21(tyrosine kinase domain) in HCC827‐GR were not mutated, and the activities of EGFR tyrosine kinase and its downstream kinases(Akt and Erk) were all downregulated in HCC827‐GR cells compared to HCC827 cells. Because EGFR signaling is critical to the regulation of Warburg effect and oxidative phosphorylation(OXPHOS) in cancer cells, we assessed glucose metabolism in both HCC827 and HCC827‐GR cells. Determination of oxygen consumption rate(OCR) and extracellular acidification rate(ECAR) showed that HCC827‐GR cells mainly use OXPHOS in glucose utilization compared to HCC827 cells. Inhibition of OXPHOS by phenformin, known as a strong complex 1 inhibitor, exhibited significant anti‐proliferative effect in HCC827‐GR cells. Phenformin reduced the NAD+/NADH ratio and decreased cellular aspartate level. Moreover treatment of HCC827‐GR cells with α‐ketobutyrate (AKB) which is an electron acceptor or aspartate significantly reversed inhibition of HCC827‐GR cells proliferation. In conclusion, we propose that phenformin is effective to inhibit cell growth of acquired gefitinib resistant NSCLC and suggest OXPHOS targeting as a potential therapeutic target.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modeling Calcium‐Induced Inhibition of Oxidative Phosphorylation

Ischemic myocardium is characterized by a chronic elevation in cellular calcium concentration. In this setting, mitochondria accumulate calcium to levels that compromise their ability to synthesize ATP. Although total mitochondrial calcium can reach up to 1 M, the vast majority is presumed to be present as insoluble calcium phosphate granules. We recently reported calcium levels below thresholds that trigger mitochondrial permeability transition significantly lowers the rate of ADP‐stimulated respiration. To explain these data, we tested the following hypotheses using a novel mathematical model of cardiac mitochondrial energetics and calcium handling: i) depressed membrane potential caused by sodium/calcium cycling lowering the driving force for ATP production; ii) direct inhibition of calcium on mitochondrial processes critical for ATP production; iii) calpain activation leading to a decrease in ATP synthesis capacity; iv) depletion/reduction of available ADP for phosphorylation caused by calcium biding, incorporation into calcium phosphate granules, or net loss of adenine nucleotide. The model includes mechanistic descriptions of mitochondrial metabolism and energetics in addition to calcium uptake, sequestration, and efflux pathways. Based on the model analysis, sodium/calcium cycling lowers mitochondrial membrane potential, but does not lead to lower rates of oxidative phosphorylation. However, direct inhibition of calcium on ATP production did agree with these data. We also found that calpain activation is a possible explanation, but we experimentally ruled this mechanism out. Finally, ADP complexation with calcium did not affect the rate of oxidative phosphorylation, but net loss of nucleotides via incorporation into calcium phosphate granules or efflux out of mitochondria can explain these data. The model will be used to design further experiments to determine the most likely mechanism capable of explaining the calcium‐induced inhibition of oxidative phosphorylation.Support or Funding InformationR00‐HL121160This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cellular Respiration is Reduced in Ventricular Fibroblasts from Human Failing Heart under Statin Therapy

BackgroundCardiac fibrosis (CF) is a major factor underlying heart failure (HF) progression. HMG‐CoA (3‐hydroxy‐3‐methylglutaryl‐CoenzymeA) reductase inhibition by statins (indicated for dyslipidemia management) is reported to attenuate CF in various HF animal models but its effect in human failing heart ventricular fibroblasts (hVF), responsible for fibrosis, is not known. Also, statin‐induced mitochondrial inhibition in muscle is suggested to cause statin‐associated muscle disease, but its effect on hVF respiration is unknown. Hence, we compared the cellular respiration of hVF isolated from HF patients under statin therapy (HF+Statin) for at least 1 year to that not treated (HF‐No_Statin) and to hVFs from non‐failing hearts.MethodsPrimary cultures of hVFs from HF patients undergoing left‐ventricle assist device implantation either under‐ or no‐ statin therapy and from non‐diseased trauma victims (control) (n=6 each) were used at passage 3 or 4. After overnight culture in FM‐2 media at 25000 cells/well in collagen‐coated 96‐well Seahorse cell culture plate, cellular respiration was quantified using the Seahorse Extracellular Flux Analyzer XF‐96, as oxygen consumption rate (OCR) at baseline and following application of each well with mitochondrial modulators: Oligomycin (1μg/ml), FCCP (0.1μM) and antimycin A (1μg/ml), normalized to total protein, analyzed by unpaired t test or one‐Way Analysis of Variance.ResultsBasal or resting state respiration was significantly decreased (p=0.02) in hVFs from HF+statin group vs HF‐No_Statin, and control (0.14±0.06, 0.23±0.02 and 0.20±0.04 pmol O2/min/μg protein, respectively). Baseline respirations of hVFs from all three groups were suppressed by oligomycin, a specific inhibitor of mitochondrial ATP synthase and oxidative phosphorylation, however, was significantly reduced in HF+statin group compared to HF‐No_statin group. There was no significant difference in basal respiration between control and HF+No_statin groups (p=0.25). The maximal OCR of hVFs from control, HF‐No_Statin and HF+statin groups, determined after uncoupling of mitochondria with FCCP were 0.44±0.04, 0.52±0.06 and 0.26±0.10 pmol O2/min/μg protein, respectively. Non‐mitochondrial cellular OCR in hVFs, following treatment with antimycin A, a selective mitochondrial respiratory chain inhibitor, were 0.05±0.009, 0.06±0.0004 and 0.02±0.03 pmol O2/min/μg protein, respectively, control, HF‐No_Statin and HF+statin groups. In HF+statin group, the maximal OCR was significantly reduced compared to control (p=0.02) and HF+No statin (p=0.01). The maximal OCR (p=0.02) and the non‐mitochondrial OCR (p=0.03) were significantly higher in the HF+No_statin group compared to control hVFs, suggesting a compensatory O2 consumption in HF. Moreover, the extracellular acidification rate (ECAR), a pointer of glycolytic activity, did not significantly differ among the groups.ConclusionStatin therapy in human failing heart is associated with an energetic remodeling of ventricular fibroblasts with reduced mitochondrial respiration without any change in the glycolytic activity.Support or Funding InformationAurora Cardiovascular Surgical Research Award (CVSRA: 570‐5028) to G.R.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolite accumulation in VLCAD deficiency markedly disrupts mitochondrial bioenergetics and Ca2+ homeostasis in the heart.

We studied the effects of the major long-chain fatty acids accumulating in very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, namely cis-5-tetradecenoic acid (Cis-5) and myristic acid (Myr), on important mitochondrial functions in isolated mitochondria from cardiac fibers and cardiomyocytes of juvenile rats. Cis-5 and Myr at pathological concentrations markedly reduced mitochondrial membrane potential (ΔΨm ), matrix NAD(P)H pool, Ca2+ retention capacity, ADP- (state 3) and carbonyl cyanide 3-chlorophenyl hydrazine-stimulated (uncoupled) respiration, and ATP generation. By contrast, these fatty acids increased resting (state 4) respiration (uncoupling effect) with the involvement of the adenine nucleotide translocator because carboxyatractyloside significantly attenuated the increased state 4 respiration provoked by Cis-5 and Myr. Furthermore, the classical inhibitors of mitochondrial permeability transition (MPT) pore cyclosporin A plus ADP, as well as the Ca2+ uptake blocker ruthenium red, fully prevented the Cis-5- and Myr-induced decrease in ΔΨm in Ca2+ -loaded mitochondria, suggesting, respectively, the induction of MPT pore opening and the contribution of Ca2+ toward these effects. The findings of the present study indicate that the major long-chain fatty acids that accumulate in VLCAD deficiency disrupt mitochondrial bioenergetics and Ca2+ homeostasis, acting as uncouplers and metabolic inhibitors of oxidative phosphorylation, as well as inducers of MPT pore opening, in the heart at pathological relevant concentrations. It is therefore presumed that a disturbance of bioenergetics and Ca2+ homeostasis may contribute to the cardiac manifestations observed in VLCAD deficiency.

Bioenergetics of Mycobacterium: An Emerging Landscape for Drug Discovery.

Mycobacterium tuberculosis (Mtb) exhibits remarkable metabolic flexibility that enables it to survive a plethora of host environments during its life cycle. With the advent of bedaquiline for treatment of multidrug-resistant tuberculosis, oxidative phosphorylation has been validated as an important target and a vulnerable component of mycobacterial metabolism. Exploiting the dependence of Mtb on oxidative phosphorylation for energy production, several components of this pathway have been targeted for the development of new antimycobacterial agents. This includes targeting NADH dehydrogenase by phenothiazine derivatives, menaquinone biosynthesis by DG70 and other compounds, terminal oxidase by imidazopyridine amides and ATP synthase by diarylquinolines. Importantly, oxidative phosphorylation also plays a critical role in the survival of persisters. Thus, inhibitors of oxidative phosphorylation can synergize with frontline TB drugs to shorten the course of treatment. In this review, we discuss the oxidative phosphorylation pathway and development of its inhibitors in detail.

Abstract P4-03-02: Characterizing and targeting chemoresistant subclones in patient-derived xenograft models of triple negative breast cancer

Abstract Fifty percent of all triple negative breast cancer (TNBC) patients harbor significant residual tumor burden following treatment with standard neoadjuvant chemotherapy (NACT), resulting in poor prognosis. Recent studies in TNBC have revealed extensive intra-tumoral heterogeneity at the time of diagnosis and throughout disease progression, but the relative contributions of these heterogeneous populations of tumor cells to chemoresistance are not well understood. The primary tumor, dermal metastasis, and germline reference were obtained from a patient with untreated metastatic TNBC. Tumor cells were engrafted into the humanized mammary fat pads of NOD/SCID mice to establish PDX models of the primary (PIM001-P) and metastatic (PIM001-M) tumors. RNA sequencing and whole-exome sequencing (WES), performed on the patient's primary and metastatic tumors and the first- and third- passage PDX models revealed transcriptomic profiles and subclonal heterogeneity of the patient's tumors were recapitulated in the PDX models. Treatment of mice engrafted with PIM001-P tumors with NACT (Adriamycin plus cyclophosphamide, AC) resulted in partial response, the magnitude of which was diminished in mice bearing PIM001-M tumors. Tumor subclones were tracked during chemotherapy treatment in mice engrafted with PIM001-P tumors using lentiviral non-targeting DNA barcodes. Residual tumors maintained the clonal architecture of untreated tumors, and deep WES revealed stable maintenance of somatic mutant allele frequencies throughout treatment. Therefore, selection of pre-existing resistant clones did not lead to AC resistance in this model. Interestingly, only 25% of residual tumor clones contributed to primary relapse once treatment was halted, suggesting only a subpopulation of tumor cells was able to reconstitute the tumor. RNA sequencing and reverse phase protein array revealed that while vehicle-treated and regrown tumors were highly similar, residual tumors harbored a unique profile characterized by numerous significant alterations in RNA and protein levels. Together, these results suggest that residual tumors enter into a transient drug-resistant state that is reversible. Residual tumors were enriched for alterations in pathways such as metabolism, extracellular matrix remodeling, and cell-cell communication. Pharmacologic targeting of the residual tumor state with an inhibitor of mitochondrial oxidative phosphorylation led to significant inhibition of tumor regrowth following AC treatment. Additional vulnerabilities identified in residual tumors are being targeted therapeutically with the goal of eradicating residual tumor cells. Citation Format: Echeverria GV, Seth S, Ge Z, Sun Y, DiFrancesco E, Lau R, Marszalek J, Moulder S, Symmans F, Heffernan TP, Chang JT, Piwnica-Worms H. Characterizing and targeting chemoresistant subclones in patient-derived xenograft models of triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-03-02.

Mitochondrial Inhibition Augments the Efficacy of Imatinib by Resetting the Metabolic Phenotype of Gastrointestinal Stromal Tumor.

Purpose: Imatinib dramatically reduces gastrointestinal stromal tumor (GIST) 18F-FDG uptake, providing an early indicator of treatment response. Despite decreased glucose internalization, many GIST cells persist, suggesting that alternative metabolic pathways are used for survival. The role of mitochondria in imatinib-treated GIST is largely unknown.Experimental Design: We quantified the metabolic activity of several human GIST cell lines. We treated human GIST xenografts and genetically engineered KitV558del/+ mice with the mitochondrial oxidative phosphorylation inhibitor VLX600 in combination with imatinib and analyzed tumor volume, weight, histology, molecular signaling, and cell cycle activity. In vitro assays on human GIST cell lines were also performed.Results: Imatinib therapy decreased glucose uptake and downstream glycolytic activity in GIST-T1 and HG129 cells by approximately half and upregulated mitochondrial enzymes and improved mitochondrial respiratory capacity. Mitochondrial inhibition with VLX600 had a direct antitumor effect in vitro while appearing to promote glycolysis through increased AKT signaling and glucose transporter expression. When combined with imatinib, VLX600 prevented imatinib-induced cell cycle escape and reduced p27 expression, leading to increased apoptosis when compared to imatinib alone. In KitV558del/+ mice, VLX600 alone did not induce tumor cell death, but had a profound antitumor effect when combined with imatinib.Conclusions: Our findings show that imatinib alters the metabolic phenotype of GIST, and this may contribute to imatinib resistance. Our work offers preclinical proof of concept of metabolic targeting as an effective strategy for the treatment of GIST. Clin Cancer Res; 24(4); 972-84. ©2017 AACR.