Inhibition Of Oxidative Phosphorylation Research Articles

Protein arginine methyltransferase 3-induced metabolic reprogramming is a vulnerable target of pancreatic cancer

BackgroundThe biological function of protein arginine methyltransferase 3 (PRMT3) is not well known because very few physiological substrates of this methyltransferase have been identified to date.MethodsThe clinical significance of PRMT3 in pancreatic cancer was studied by database analysis. The PRMT3 protein level of human pancreatic tumors was detected by immunoblotting and immunohistochemical staining. PRMT3-associated proteins and the methylation sites on the proteins were investigated using mass spectrometry. Seahorse Bioscience analyzed the 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.ResultsWe found that the expression of PRMT3 is upregulated in pancreatic cancer, and its expression is associated with poor survival. We identified GAPDH as a PRMT3-binding protein and demonstrated that GAPDH is methylated at R248 by PRMT3 in vivo. The methylation of GAPDH by PRMT3 enhanced its catalytic activity while the mutation of R248 abolished the effect. In cells, PRMT3 overexpression triggered metabolic reprogramming and enhanced glycolysis and mitochondrial respiration simultaneously in a GAPDH-dependent manner. PRMT3-overexpressing cancer cells were addicted to GAPDH-mediated metabolism and sensitive to the inhibition of GAPDH and mitochondrial respiration. The combination of inhibitors of GAPDH and oxidative phosphorylation induced a synergistic inhibition on cellular growth in vitro and in vivo.ConclusionOur results suggest that PRMT3 mediates metabolic reprogramming and cellular proliferation through methylating R248 of GAPDH, and double blockade of GAPDH and mitochondrial respiration could be a novel strategy for the treatment of PRMT3-overexpressing pancreatic cancer.

Metformin Inhibits the Type 1 IFN Response in Human CD4+ T Cells.

In systemic lupus erythematosus, defective clearance of apoptotic debris and activation of innate cells result in a chronically activated type 1 IFN response, which can be measured in PBMCs of most patients. Metformin, a widely used prescription drug for Type 2 diabetes, has a therapeutic effect in several mouse models of lupus through mechanisms involving inhibition of oxidative phosphorylation and a decrease in CD4+ T cell activation. In this study, we report that in CD4+ T cells from human healthy controls and human systemic lupus erythematosus patients, metformin inhibits the transcription of IFN-stimulated genes (ISGs) after IFN-α treatment. Accordingly, metformin inhibited the phosphorylation of pSTAT1 (Y701) and its binding to IFN-stimulated response elements that control ISG expression. These effects were independent of AMPK activation or mTORC1 inhibition but were replicated using inhibitors of the electron transport chain respiratory complexes I, III, and IV. This indicates that mitochondrial respiration is required for ISG expression in CD4+ T cells and provides a novel mechanism by which metformin may exert a therapeutic effect in autoimmune diseases.

Abstract 3865: Discovery of novel DHODH and OXPHOS inhibitors

Abstract Inhibition of mitochondrial metabolism for treatment-resistant tumors has attracted renewed attention. Pharmacologic inhibition of mitochondrial oxidative phosphorylation (OXPHOS) is efficacious in preclinical models of chemo-resistant AML, glycolysis-deficient glioma, and Swi/Snf mutant lung cancer, and is associated with an apparent robust safety index (Molina et al., 2018, Nat Med 24: 1036-1046; Lissanu Deribe et al., 2018, Nat Med 24: 1047-1057). In addition, immune-suppressive cell types in the tumor micro-environment depend on OXPHOS metabolism, including CD4+ regulatory T-cells (Tregs), whereas tumor-infiltrating effector T-cells instead rely on glycolytic metabolism (Angelin et al., 2017, Cell Metab. 25: 1282-1293). Here, we have used a phenotypic drug discovery approach to identify selective inhibitors of mitochondrial -but not glycolytic- tumor cell metabolism. Based on initial hits derived from a high-throughput screening campaign, a chemical series was optimized to achieve single digit nM potency (best IC50= 2 nM) in targeting OXPHOS-dependent cancer cells (grown on lactate as sole carbon source) but not glycolytic cells (grown on glucose; IC50 > 10 microM). Target deconvolution within this chemical series revealed 2 distinct mechanisms. One chemical subseries are direct inhibitors of the mitochondrial enzyme, dihydroorotate dehydrogenase (DHODH). Consistent with previous reports on DHODH inhibitors, these compounds potently impair AML cell proliferation (best IC50= 10nM) through induction of myeloid cell differentiation -a trait that can be rescued by providing exogenous uridine to the cell cultures (IC50 > 10 microM). A second chemically distinct subset of compounds inhibit mitochondrial OXPHOS, but does not inhibit DHODH. As expected, these compounds selectively target OXPHOS-dependent cancer cell lines, and display a robust selectivity window (determined in glycolysis-dependent cell lines). Moreover, these inhibitors do not affect a mixed-lymphocyte reaction (MLR) assay. In-depth metabolomic profiling in cancer cells fueled by glucose, lactate or glutamine, the precise molecular target of these compounds, and further in vivo characterization of these compounds will be presented. Finally, the unique opportunity to simultaneously inhibit both DHODH and OXPHOS using dual inhibitors will be evaluated in chemo-resistant AML models. Citation Format: Matthias Versele, Philippe Selhorst, Kristine Metzger, Marnik Nijs, Hugo Klaassen, Amuri Kilonda, Damien Marchand, Philippe Arzel, Dominique Lambin, Jean-Christophe Vanherck, Arnaud Marchand, Patrick Chaltin, Cyril Corbet, Olivier Feron. Discovery of novel DHODH and OXPHOS inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3865.

Abstract 3664: Hypoxia-induced phenotypic and metabolic changes in Ewing sarcoma cells trigger bone metastasis

Abstract Ewing Sarcoma (ES) is an aggressive malignancy that arises in children and young adults. Although the survival for patients with localized tumors is relatively high, the metastatic form carries a dismal prognosis, particularly when bone metastases are present. The frequency of metastases and osseous dissemination increases in patients with necrotic ES tumors. In line with this, hypoxia, the major cause of tumor necrosis, has been shown to increase metastatic potential of ES cells. Previous data from our laboratory demonstrated that in an ES orthotopic xenograft model, primary tumor hypoxia specifically promotes bone metastasis, which is associated with accumulation of cells with enlarged nuclei and frequent chromosome gains in bone invasion areas. We have also shown that the progeny of hypoxia-induced polyploid ES cells (ES≈4n cells) preferentially metastasized to bone. Thus, the goal of our study was to determine the mechanisms enabling osseous dissemination of ES≈4n cells. To this end, we used FUCCI Cell Cycle Sensor followed by DNA staining with Hoechst 33342 and cell sorting to isolate tetraploid cells (4n) from hypoxia-exposed SK-ES1 ES cells. Subsequently, we compared the metastatic properties of their progeny (H-SK-ES1≈4n) with a diploid cell population selected from normoxic SK-ES1 cells (N-SK-ES1-2n cells). We have found that H-SK-ES1≈4n cells have increased motility and invasiveness, as compared to the N-SK-ES1-2n cells. H-SK-ES1≈4n cells had also an increased ability to grow in hypoxia in 2D culture and in soft agar. In line with this, H-SK-ES1≈4n cells were highly sensitive to a glycolysis inhibitor, 2D-glucose, but not an inhibitor of oxidative phosphorylation, metformin. Moreover, H-SK-ES1≈4n cells were more sensitive than controls to a growth-inhibitory effect of AICAR, a blocker of anabolic metabolism. In contrary, the tetraploid cell progeny were highly resistant to doxorubicin, particularly under hypoxic conditions, which in contrast sensitized N-SK-ES1-2n and wild type SK-ES1 cells to chemotherapy. Altogether, our data implicate the following mechanisms underlying osseous dissemination of the hypoxia-induced poplyploid cell progeny: 1) increased motility and invasiveness facilitating escape from the primary tumor; 2) ability to survive under low oxygen tension characteristic for bone tissue. Moreover, we have shown that the cells initiating bone dissemination are highly resistant to conventional chemotherapy, while strategies targeting their metabolic dependencies may be more successful in treatment of patients with bone metastases. Citation Format: Shiya Zhu, Akanksha Mahajan, Sung-Hyeok Hong, Susana Galli, Congyi Lu, You-Shin Chen, Sara Misiukiewicz, Stacey Chung, Jason Tilan, Joanna B. Kitlinska. Hypoxia-induced phenotypic and metabolic changes in Ewing sarcoma cells trigger bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3664.

Abstract 2900: Dissection of clonal heterogeneity unmasks pre-existing chemoresistance and new metabolic vulnerabilities in pancreatic cancer

Abstract Adaptive drug-resistance mechanisms allow human tumors to evade treatment through selection and expansion of treatment-resistant clones. Modeling the functional heterogeneity of tumors can unmask critical contributions of distinct tumor cell sub-populations toward identifying rational drug combinations. Here, studying clonal evolution of tumor cells derived from human pancreatic tumors, we demonstrate that in vitro adherent cultures and in vivo tumors are maintained by a common set of long term self-renewing tumorigenic cells that can be used to establish clonal replica tumors (CRTs), large cohorts of animals bearing human tumors with identical clonal composition. Using CRTs to conduct quantitative assessments of clonal dynamics and adaptive responses to therapeutic challenge over time, we uncovered that the tumorigenic compartment of pancreatic tumors maintains a multitude of functionally heterogeneous subpopulations of cells with differential degrees of sensitivity to therapeutics. High-throughput isolation and deep characterization of unique clonal lineages showed genetic and transcriptomic diversity underlying the functionally diverse subpopulations, positioning the origins of tumor heterogeneity within the long-term self-renewing compartment. Molecular annotation of gemcitabine-naïve clonal lineages with distinct responses to treatment in the context of CRTs generated signatures that can predict the response to chemotherapy and exposed pre-existing functional mechanisms of clonal resistance, primarily associated to DNA damage tolerance and mitochondrial respiration (OXPHOS). Further transcriptomic and metabolic characterization of residual tumor cells in patient derived xenograft models as well as in patients after chemoradiation showed that resistant cells that contribute to tumor relapse are metabolically rewired to upregulate OXPHOS. Combining a novel inhibitor of oxidative phosphorylation (IACS-10759) developed at the MD Anderson Institute for Applied Cancer Science, and currently in phase I clinical trial in acute myeloid leukemia and 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. Our study, correlating genomic and transcriptomic traits with specific functional phenotypes, uncovered new mechanisms that underlie intra-tumor sub-clonal heterogeneity, influence treatment response to drugs and sustain tumor relapse. Citation Format: Sahil Seth, Chieh-Yuan Li, Sara Loponte, I-Lin Ho, Denise Corti, Luigi Sapio, Edoardo Del Poggetto, Michael Peoples, Tatiana Karpinets, Frederick S. Robinson, Shan Jiang, Prasanta Dutta, Joseph Marszalek, Maria E. Di Francesco, Timothy P. Heffernan, Virginia Giuliani, Pratip K. Bhattacharya, Giannicola Genovese, Andrew Futreal, Giulio Draetta, Andrea Viale, Alessandro Carugo. Dissection of clonal heterogeneity unmasks pre-existing chemoresistance and new metabolic vulnerabilities in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2900.

Abstract 2901: Clonal dynamics and phenotypic evolution during chemoresistance and metastasis revealed by patient-derived xenograft models of triple negative breast cancer

Abstract Half of all triple negative breast cancer (TNBC) patients harbor significant residual cancer burden following standard neoadjuvant chemotherapy treatment, resulting in distant metastasis and death for most of these patients. Intra-tumor heterogeneity (ITH) is pervasive in TNBC as is a barrier to development of effective therapeutic strategies, but the relative contributions of heterogeneous tumor cell populations to chemoresistance and metastasis are not well understood. To investigate the clonal dynamics that accompany chemotherapy treatment and metastasis, we employed orthotopic patient-derived xenograft (PDX) models of treatment-naïve TNBC, thus enabling experimentation with heterogeneous populations of human tumor cells that have undergone minimal manipulation. To monitor the fates of PDX tumor cell lineages as they metastasized, we introduced a pooled lentiviral barcode library (Cellecta) into freshly dissociated PDX tumor cells which were orthotopically engrafted into recipient mice. Genomic analyses, including barcode enumeration, whole-exome sequencing, custom targeted DNA sequencing, and transcriptome sequencing, were conducted to characterize the clonal dynamics that accompanied metastasis. Of the thousands of diverse primary tumor clones, only ~2% harbored metastatic capacity. Of those, a rare population of the exact same clones predominated metastases in lung, liver, and brain, the three most common sites of human TNBC metastasis. These studies provide a quantitative map of the clonal architecture of multi-organ metastasis in TNBC and reveal that identical subclones can thrive in diverse secondary organ microenvironments. NACT resistance leads to metastasis and death for most patients, yet the origins of chemoresistance in TNBC are unclear. We modeled NACT resistance in an array of PDX models derived from treatment-naïve TNBC biopsies in alignment with an ongoing neoadjuvant clinical trial (NCT02276443). Upon partial response to NACT, tumors entered a transient drug-tolerant state characterized by distinct histologic, proteomic, and transcriptomic features that were reverted as tumors regrew after cessation of treatment. Barcode-mediated lineage tracking and whole-exome sequencing revealed that the drug-tolerant state was not mediated by clonal selection. Based on transcriptomic and metabolic features of the drug-tolerant state, we conducted preclinical trials with an inhibitor of mitochondrial oxidative phosphorylation (IACS-010759), which significantly delayed the regrowth of residual tumors in PDX models. Together, these studies revealed that TNBCs can resist NACT through non-selective adaptation of a reversible phenotypic state, and that inhibition of oxidative phosphorylation may be a promising therapy in the neoadjuvant setting for TNBC. Citation Format: Gloria V. Echeverria, Zhongqi Ge, Sahil Seth, Emily Powell, Xiaomei Zhang, Sabrina Jeter-Jones, Xinhui Zhou, Yan Jiang, Aaron McCoy, Shirong Cai, Yizheng Tu, Michael Peoples, Yuting Sun, Huan Qiu, Christopher Bristow, Alessandro Carugo, Jiansu Shao, Stacy L. Moulder, William F. Symmans, Timothy P. Heffernan, Jeffrey T. Chang, Helen M. Piwnica-Worms. Clonal dynamics and phenotypic evolution during chemoresistance and metastasis revealed by patient-derived xenograft models of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2901.

Abstract CT021: Phase I study of an oxidative phosphorylation inhibitor IM156 in patients with advanced solid tumors

Abstract Background: IM156 is a novel oral potent biguanide, which has anticancer activity through AMPK activation and reduction of oxidative phosphorylation. Inhibition of oxidative phosphorylation (OXPHOS) is detrimental to OXPHOS dependent cancer cells, which are prone to energy stress. Preclinical experiments demonstrated that IM156 can be effective in glioblastoma, gastric cancer and other solid tumors. Methods: This is an open label, first-in-human, single center, dose-escalation study (NCT03272256) using the 3+3 design to determine the maximum tolerated dose and/or recommended Phase II dose, dose limiting toxicities (DLT), safety, pharmacokinetics, pharmacodynamics and preliminary signals of anticancer efficacy of IM156 in patients with advanced solid tumors. Eligible patients are adults with advanced solid tumors refractory to standard therapies with adequate performance status (ECOG <2) and organ function with measurable disease per RECIST 1.1 (or RANO for gliomas). IM156 is administered orally every other day starting on day 1 of each 28 days cycle. Results: To date, 12 patients (gastric cancer, 5; endometrial cancer, 2; ovarian cancer, 2; other, 3) were enrolled to 4 dose levels (100mg to 800 mg orally every other day). There have been no DLTs or >other grade 3 treatment-related adverse events (AEs). Most frequent AEs included nausea (67% of patients), vomiting (17%), diarrhea (23%), insomnia (17%), epigastric and abdominal pain (17%), weakness (8%), neuropathy (8%) and fatigue (8%); however, there were all grade 1 except for four grade 2 episodes (nausea, 2; insomnia, 1; diarrhea, 1; epigastric pain, 1). Mild elevations (<5 mmol/L) in lactate levels were noted anecdotally. The best response was stable disease in 5 patients (gastric cancer, 2; ovarian cancer, 1; endometrial cancer, 1; colorectal, 1). Pharmacokinetic studies demonstrated dose proportional increase in Cmax and AUC, which were approaching expected efficacious range. Observed terminal half-life supported possible change of the schedule to daily oral administration. Pharmacodynamic marker studies are on-going. Conclusion: IM156 has been well tolerated with manageable AE profile. The dose escalation continues with a planned change from every other day administration to daily dosing. Citation Format: Sun Young Rha, Seung Hoon Beom Seung Hoon Beom, Gun Min Kim, Young Geun Shin, Dong-Seok Yim, Hyo Song Kim, Jong Hee Chang, Jae-Ho Cheong, Young Woo Lee, Yun Seon Chong, Vincent O’Neill, Sang Hee Yoo, Filip Janku. Phase I study of an oxidative phosphorylation inhibitor IM156 in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT021.

Abstract 1832: Ligand-dependent Wnt signaling directs metabolic adaptations to promote pancreatic tumorigenesis

Abstract Wnt/β-catenin signaling plays important roles in pancreatic adenocarcinoma (PDA) tumor initiation and progression via broad-ranging effects on proliferation, differentiation, survival, and stemness. Although Wnt has been shown to promote glycolysis in colon cancer, its role in regulating metabolism in PDA has not been explored in any detail. For this study, the AsPC-1 cell line was selected as an exemplar of ligand-dependent autocrine Wnt signaling in PDA. AsPC-1 harbors an inactivating mutation in RNF43 that confers Wnt growth-dependency and response to LGK974, a PORCN inhibitor that blocks Wnt ligand secretion and signaling. Global transcriptomic, proteomic, and metabolomic analysis revealed several metabolites and putative mediators of metabolism that are directly or indirectly linked to autocrine Wnt signaling in AsPC-1. In contrast to colon cancer, pharmacologic inhibition of Wnt by LGK974 in AsPC-1 led to the accumulation of pyruvate and lactate, as well as reduced tricarboxylic acid cycle metabolites and decreased mitochondrial membrane potential as measured by tetramethylrhodamine assay. These metabolic changes were accompanied by cell cycle arrest and downregulation of transcriptional programs involved in cell cycle progression, DNA replication, and nucleotide metabolism. Pyruvate analogs dichloroacetate and oxamate, that respectively target pyruvate dehydrogenase kinase and lactate dehydrogenase, restored mitochondrial membrane potential in the context of LGK974. Importantly, LGK974 results were phenocopied by siRNA-mediated knockdown of WNT7B. Thus, inhibition of mitochondrial oxidative phosphorylation by LGK974 in RNF43-mutant PDA appears mechanistically linked to its inhibition of canonical Wnt signaling, an adaptation and potential metabolic vulnerability that could be leveraged to therapeutic advantage via combinatorial strategies. Altogether, these results suggest tissue context and differing mechanisms of Wnt pathway activation or signal strength may profoundly influence its role in regulating cancer metabolism. Citation Format: Kristina Y. Aguilera, Rana Riahi, Edris A. Saadat, Anna R. Lay, Thuc Le, Anthony Cabebe, Timothy R. Donahue, Caius Radu, David W. Dawson. Ligand-dependent Wnt signaling directs metabolic adaptations to promote pancreatic tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1832.

Abstract 801: Dual inhibition of distinct metabolic features targets osteosarcoma stem cells

Abstract [Objective] Mitochondria are the places for the energy production of the cells, while reactive oxygen species (ROS) are also produced alongside. In recent years, it has been reported that cancer stem cells metabolize predominantly through oxidative phosphorylation (OXPHOS) rather than glycolysis in certain cancer cells. Targeting OXPHOS achieved by suppression of ATP synthesis through mitochondrial ATP synthase could be a potential therapeutic option against cancer stem cells. In the current study, we have identified the mitochondria metabolism as the potential therapeutic target in osteosarcoma (OS) stem cells, presenting the synergistic effects of combination of OXPHOS inhibition by pterostilebene (PTE) with c-Myc inhibitor, which target both OXPHOS-dominant cancer stem cells and glycolysis-dominant non-cancer stem cells as a ‘two hit’ or ‘dual inhibition’ of metabolic pathways, OXPHOS and glycolysis. [Materials & Methods] Using human OS cell lines of SaOS2, U2OS and MG63 cells, cell survival and the ability of sphere formation was assessed with or without PTE, and the expression of stem cell markers mRNA such as Oct3, NS, CD44 was examined by RT-PCR. Next, the activity of mitochondrial ATP synthase, mitochondrial respiration capacity of oxygen consumption rate, and the amount of ATP as well as ROS production were measured under the treatment of PTE. Furthermore, we examined the synergistic effect of PTE with cMyc transcription inhibitors of JQ1 or Honokiol (HNK). [Results] PTE treatment on human OS cell lines reduced the viabilities of all cell lines in dose-dependent manner and expression of stem cell marker and the ability of sphere formation were also decrease in terms of sphere number and size. PTE reduced the activity of F0F1-ATP synthase; Complex V predominantly, and the mitochondrial oxygen consumption rates and synthetic amount of ATP were also decreased in spheroid condition. These results suggest that PTE possibly targets stem cell population which preferably relies on OXPHOS, suppressing ATP synthesis via F0F1-ATP synthase inhibition as well as increased ROS production in OS cells and changes metabolic flax to glycolysis dependent feature. The dual inhibition of OXPHOS by PTE and c-Myc inhibition by HNK or JQ1 showed the synergistically inhibition of OS cell growth in a dose-dependent manner. [Discussion] Prognosis of the patients with osteosarcoma has reached to plateau without any breakthroughs over the last quarter century, and nearly 30% of patients still have to face very severe poor prognosis, especially with metastatic disease. Current study suggests that modulation of metabolic flux by c-Myc and OXPHOS inhibitors showed a greater synergistic effect with ‘two metabolic hit’ or ‘dual metabolic inhibition’ of distinct metabolic features and it could be a novel therapeutic strategy against osteosarcoma, possibly targeting both stem-like cell population and general tumor cell population. Citation Format: Shingo Kishi, Kanya Honoki, Shinji Tsukamoto, Hiromasa Fujii, Yumiko Kondo, Yasuhito Tanaka, Hiroki Kuniyasu. Dual inhibition of distinct metabolic features targets osteosarcoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 801.

Acute Iron Deprivation Reprograms Human Macrophage Metabolism and Reduces Inflammation In Vivo

SummaryIron is an essential metal that fine-tunes the innate immune response by regulating macrophage function, but an integrative view of transcriptional and metabolic responses to iron perturbation in macrophages is lacking. Here, we induced acute iron chelation in primary human macrophages and measured their transcriptional and metabolic responses. Acute iron deprivation causes an anti-proliferative Warburg transcriptome, characterized by an ATF4-dependent signature. Iron-deprived human macrophages show an inhibition of oxidative phosphorylation and a concomitant increase in glycolysis, a large increase in glucose-derived citrate pools associated with lipid droplet accumulation, and modest levels of itaconate production. LPS polarization increases the itaconate:succinate ratio and decreases pro-inflammatory cytokine production. In rats, acute iron deprivation reduces the severity of macrophage-dependent crescentic glomerulonephritis by limiting glomerular cell proliferation and inducing lipid accumulation in the renal cortex. These results suggest that acute iron deprivation has in vivo protective effects mediated by an anti-inflammatory immunometabolic switch in macrophages.

Abstract 3589: Targeting metabolic reprogramming and OXPHOS as a viable anti-melanoma strategy

Abstract Invasive melanoma is an aggressive form of skin cancer with poor clinical outcomes. Nearly 50% of melanoma patients exhibit oncogenic BRAFV600E mutations. Existing BRAF kinase inhibitor drugs provide a short-term benefit prior to the rapid onset of drug resistance. Emerging results suggest that BRAF and other kinase inhibitors induce metabolic reprogramming from glycolytic energy production to oxidative phosphorylation (OXPHOS) in a subset of malignant melanomas. Metabolic reprogramming to OXPHOS is emerging as an important mechanism of resistance to BRAF kinase inhibitors and is associated with worse prognosis. Consequently, there is a significant opportunity for multimodal therapeutic approaches that selectively target the metabolic reprogramming associated with resistance to BRAF kinase inhibitors and promote immune-targeted therapy. We have previously developed mitochondria-targeted drugs by exploiting the enhanced negative mitochondrial membrane potential of cancer cells to selectively target and disrupt tumor growth. We hypothesized that metabolic reprogramming from glycolysis to OXPHOS in BRAF inhibitor-resistant melanoma cells would make them susceptible to OXPHOS inhibitors. In this study, we have modified the structure of a naturally occurring polyphenol, magnolol into a mitochondria-targeted-magnolol (Mito-MGN). We tested the effects of Mito-MGN on mitochondrial respiration and bioenergetic function, cellular redox status, and mitophagy in wild type (WT) and BRAF-resistant (metabolically reprogrammed) melanoma cells. Our data show that Mito-MGN inhibits mitochondrial respiration, melanoma cell proliferation and invasion at concentrations more than 200-fold lower than that of magnolol. Furthermore, Mito-MGN increases ROS formation and oxidation of mitochondrial peroxiredoxin. Preliminary results also indicate that BRAF inhibitor resistant melanoma cells exhibit enhanced OXPHOS as a consequence of metabolic reprogramming and that Mito-MGN effectively inhibits proliferation and mitochondrial respiration in the resistant cells. Mito-MGN inhibited mitochondrial complex I activity in both WT and resistant cells. The IC50 values to inhibit complex I-mediated respiration was consistent with the values calculated for inhibition of cell respiration. We conclude that inhibition of complex I is a key initial event that is responsible for the anti-proliferative effects observed in the presence of Mito-MGN against wild-type as well as drug-resistant melanoma cells. Citation Format: Gang Cheng, Jacek Zielonka, Micael Hardy, Michael B. Dwinell, Balaraman Kalyanaraman. Targeting metabolic reprogramming and OXPHOS as a viable anti-melanoma strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3589.

Abstract 3589: Targeting metabolic reprogramming and OXPHOS as a viable anti-melanoma strategy

Invasive melanoma is an aggressive form of skin cancer with poor clinical outcomes. Nearly 50% of melanoma patients exhibit oncogenic BRAFV600E mutations. Existing BRAF kinase inhibitor drugs provide a short-term benefit prior to the rapid onset of drug resistance. Emerging results suggest that BRAF and other kinase inhibitors induce metabolic reprogramming from glycolytic energy production to oxidative phosphorylation (OXPHOS) in a subset of malignant melanomas. Metabolic reprogramming to OXPHOS is emerging as an important mechanism of resistance to BRAF kinase inhibitors and is associated with worse prognosis. Consequently, there is a significant opportunity for multimodal therapeutic approaches that selectively target the metabolic reprogramming associated with resistance to BRAF kinase inhibitors and promote immune-targeted therapy. We have previously developed mitochondria-targeted drugs by exploiting the enhanced negative mitochondrial membrane potential of cancer cells to selectively target and disrupt tumor growth. We hypothesized that metabolic reprogramming from glycolysis to OXPHOS in BRAF inhibitor-resistant melanoma cells would make them susceptible to OXPHOS inhibitors. In this study, we have modified the structure of a naturally occurring polyphenol, magnolol into a mitochondria-targeted-magnolol (Mito-MGN). We tested the effects of Mito-MGN on mitochondrial respiration and bioenergetic function, cellular redox status, and mitophagy in wild type (WT) and BRAF-resistant (metabolically reprogrammed) melanoma cells. Our data show that Mito-MGN inhibits mitochondrial respiration, melanoma cell proliferation and invasion at concentrations more than 200-fold lower than that of magnolol. Furthermore, Mito-MGN increases ROS formation and oxidation of mitochondrial peroxiredoxin. Preliminary results also indicate that BRAF inhibitor resistant melanoma cells exhibit enhanced OXPHOS as a consequence of metabolic reprogramming and that Mito-MGN effectively inhibits proliferation and mitochondrial respiration in the resistant cells. Mito-MGN inhibited mitochondrial complex I activity in both WT and resistant cells. The IC 50 values to inhibit complex I-mediated respiration was consistent with the values calculated for inhibition of cell respiration. We conclude that inhibition of complex I is a key initial event that is responsible for the anti-proliferative effects observed in the presence of Mito-MGN against wild-type as well as drug-resistant melanoma cells. Citation Format: Gang Cheng, Jacek Zielonka, Micael Hardy, Michael B. Dwinell, Balaraman Kalyanaraman. Targeting metabolic reprogramming and OXPHOS as a viable anti-melanoma strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3589.

Targeted metabolome analysis of the dog brain exposed to PCBs suggests inhibition of oxidative phosphorylation by hydroxylated PCBs

Canis lupus familiaris (domestic dog) possess a high capacity to metabolize higher-chlorinated polychlorinated biphenyls (PCBs) to thyroid hormone (TH)-like hydroxylated PCB metabolites (OH-PCBs). As a result, the brain could be at high risk of toxicity caused by OH-PCBs. To evaluate the effect of OH-PCBs on dog brain, we analyzed OH-PCB levels in the brain and the metabolome of the frontal cortex following exposure to a mixture of PCBs (CB18, 28, 70, 77, 99, 101, 118, 138, 153, 180, 187, and 202). 4-OH-CB202 and 4-OH-CB107 were major OH-PCBs in the brain of PCB-exposed dogs. These OH-PCBs were associated with metabolites involved in urea cycle, proline-related compounds, and purine, pyrimidine, glutathione, and amino-acid metabolism in dog brain. Moreover, adenosine triphosphate levels in the PCBs exposure group were significantly lower than in the control group. These results suggest that OH-PCB exposure is associated with a disruption in TH homeostasis, generation of reactive oxygen species, and/or disruption of oxidative phosphorylation (OXPHOS) in brain cells. Among them, OXPHOS disturbance could be associated with both disruptions in cellular amino-acid metabolism and urea cycle. Therefore, an OXPHOS activity assay was performed to evaluate the disruption of OXPHOS by OH-PCBs. The results indicated that 4-OH-CB107 inhibits the function of Complexes III, IV, and V of the electron transport chain, suggesting that 4-OH-CB107 inhibit these complexes in OXPHOS. The neurotoxic effects of PCB exposure may be mediated through mitochondrial toxicity of OH-PCBs in the brain.

Transcriptomic analysis reveals that BMP4 sensitizes glioblastoma tumor-initiating cells to mechanical cues

The poor prognosis of glioblastoma (GBM) is associated with a highly invasive stem-like subpopulation of tumor-initiating cells (TICs), which drive recurrence and contribute to intra-tumoral heterogeneity through differentiation. These TICs are better able to escape extracellular matrix-imposed mechanical restrictions on invasion than their more differentiated progeny, and sensitization of TICs to extracellular matrix mechanics extends survival in preclinical models of GBM. However, little is known about the molecular basis of the relationship between TIC differentiation and mechanotransduction. Here we explore this relationship through a combination of transcriptomic analysis and studies with defined-stiffness matrices. We show that TIC differentiation induced by bone morphogenetic protein 4 (BMP4) suppresses expression of proteins relevant to extracellular matrix signaling and sensitizes TIC spreading to matrix stiffness. Moreover, our findings point towards a previously unappreciated connection between BMP4-induced differentiation, mechanotransduction, and metabolism. Notably, stiffness and differentiation modulate oxygen consumption, and inhibition of oxidative phosphorylation influences cell spreading in a stiffness- and differentiation-dependent manner. Our work integrates bioinformatic analysis with targeted molecular measurements and perturbations to yield new insight into how morphogen-induced differentiation influences how GBM TICs process mechanical inputs.

PS1014 GLYCOLYSIS VERSUS OXIDATIVE PHOSPHORYLATION – POTENTIAL THERAPEUTIC TARGETS IN AML

Background:Cancer cells alter their metabolism to sustain high levels of growth and proliferation. One of the most common alterations is the metabolic shift of cancer cells from oxidative phosphorylation (OXPHOS) to glycolysis, regardless of oxygen presence (aerobic glycolysis), resulting in lower ATP production. This alteration is associated with up‐regulation of glucose transporters (GLUTs) and glycolytic enzymes, such as hexokinases, and enhanced lactate fermentation. This shift is mainly regulated by HIF‐1, p53 and MYC. Hence, metabolism constitute a new target in acute myeloid leukemia (AML), where mutations in metabolism [isocitrate dehydrogenase 1/2 (IDH1/2)] were already describe. AML is a heterogeneous hematologic neoplasia characterized by myeloid differentiation arrest and uncontrolled proliferation.Aims:This study aimed to metabolically characterize AML cell lines and assess the potential of glycolysis and OXPHOS metabolism as a therapeutic target in AML using in vitro models.Methods:Six AML cell lines of different subtypes and with different genetic backgrounds were used (HEL, HL‐60, K‐562, KG‐1, NB‐4 and THP‐1). The presence of IDH1/2 exon 4 mutations was assessed using Sanger sequencing. Intracellular peroxides, superoxide anion and mitochondrial membrane potential levels were measured by fluorimetry using DCFH2‐DA, DHE and JC‐1 probes, respectively. Cells were incubated in presence or absence of 2‐deoxy‐D‐glucose (2‐DG; glycolysis inhibitor) or oligomycin complex (OXPHOS inhibitor). Metabolic activity was assessed by resazurin assay and cell death by flow cytometry (FC) using Annexin V and 7‐AAD double staining. Cell cycle was also assessed by FC, using propidium iodide/RNase. Glucose intake was measured by 18F‐FDG uptake and expression levels of 8 SLC2A, 2 hexokinases and HIF‐1α were assessed using RT‐PCR.Results:NB‐4 cell line has the highest 18F‐FDG uptake, whereas HL‐60 and THP‐1 cells showed the lowest. HEL, K‐562 and KG‐1 had similar 18F‐FDG uptake. THP‐1 cells expressed the 8 tested SLC2A genes. NB‐4 cell line also had the highest peroxide and superoxide anion levels. K‐562 and KG‐1 cells had similar ROS levels and IDH1 genotype (single nucleotide variant rs11554137 in heterozygosity). Glycolysis inhibition reduced metabolic activity in a dose‐ and cell line dependent manner and HEL and KG‐1 were the most sensitive cell lines to 2‐DG. OXPHOS inhibition led to a decrease in metabolic activity in a cell line‐dependent manner. K‐562 cells were the most sensitive to this inhibition and THP‐1 cells were the most resistant to both compounds (2‐DG and oligomycim). These inhibitors induce cell death by apoptosis and cell cycle arrest in G0/G1 phase. 2‐DG induced a more evident cytotoxic effect on K‐562 and NB‐4 and a cytostatic effect on KG‐1 and THP‐1 cells. Conversely, oligomycin had a cytotoxic effect on K‐562 and KG‐1 cells and a cytostatic effect on NB‐4, THP‐1 and K 562. Moreover, 2‐DG decreased 18F‐FDG uptake, mainly in KG‐1 and NB‐4 cell lines, inducing up‐regulation of SLC2A11 and down‐regulation of HK1 gene expression. Lastly, oligomycin increased 18F‐FDG uptake, particularly in K‐562 cells, with up‐regulation of SLC2A1 and HK2 gene expression.Summary/Conclusion:AML cell lines have different metabolic preferences, with HEL, KG‐1 and NB‐4 cells appearing to be more glycolytic, whereas K‐562 cells more dependent on OXPHOS. Contrary, THP‐1 cells seems to be OXPHOS independent. These cell lines seem to reprogram their metabolism in order to overcome OXPHOS inhibition, suggesting that glycolysis could be a better therapeutic target in AML.

583-P: Increase in Mitochondrial Ceramide Contributes to Diabetes-Induced Retinal Endothelial Cell Damage

Mitochondrial damage precedes histopathological abnormalities in diabetic retinopathy (DR). Retinal endothelial cells (REC) have the highest level of Acid Sphingomyelinase (ASM) expression in the retina and the increase in ASM activity and ceramide production were shown to promote diabetes-induced pro-inflammatory and pro-apoptotic changes in the retina and REC. The role of diabetes-induced increase in ASM and ceramide production in mitochondrial damage in the retina and REC is not known. Retinal mitochondria from control, STZ-induced diabetic and ASM-/- rodent models, as well as Bovine REC (BREC) treated with diabetogenic conditions were isolated by differential centrifugation. Sphingolipids were analyzed by eSI-MS. Respiration was measured using a novel custom designed microrespirometer. Mitochondrial sphingomyelin (mSM) decreased (62 ± 1% compared to control 72.5 ± 7%, P<;0.05, n=3) and mitochondrial ceramide (mCer) increased (38 ± 4% compared to control 26.5 ± 6.5%, P<0.05, n=3 in 7 weeks diabetic rat retina. ASM-/- mouse model showed increased mSM (85.0 ± 6.7%) compared to wild type (77.0 ± 2.8%) and decreased mCer (11.5 ± 3.6%) compared to wild type (17.6 ± 3.6%, P<0.05, n=3), confirming that ASM is an essential contributor to mitochondrial ceramide regulation. Pre-treatment of BREC with an ASM inhibitor Desipramine revealed a 1.75-fold increase in maximal respiratory capacity compared to control cells. Diabetes results in decreased mitochondrial sphingomyelin and increased mitochondrial ceramide consistent with ASM-dependent ceramide production. Inhibition of ASM in BREC results in increased maximal, but not basal, respiratory rates. These results support the hypothesis that ASM activation and mitochondrial ceramide accumulation leads to inhibition of oxidative phosphorylation and REC damage in diabetic environment. Disclosure D. Pegouske: None. Y. Levitsky: None. S. Hammer: None. T.A. Lydic: None. A. Muchnik: None. D.A. Proshlyakov: None. J.V. Busik: None. Funding National Institutes of Health (R01EY028049, R01EY016077)

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Targeting lonidamine to mitochondria mitigates lung tumorigenesis and brain metastasis

Lung cancer often has a poor prognosis, with brain metastases a major reason for mortality. We modified lonidamine (LND), an antiglycolytic drug with limited efficacy, to mitochondria-targeted mito-lonidamine (Mito-LND) which is 100-fold more potent. Mito-LND, a tumor-selective inhibitor of oxidative phosphorylation, inhibits mitochondrial bioenergetics in lung cancer cells and mitigates lung cancer cell viability, growth, progression, and metastasis of lung cancer xenografts in mice. Mito-LND blocks lung tumor development and brain metastasis by inhibiting mitochondrial bioenergetics, stimulating the formation of reactive oxygen species, oxidizing mitochondrial peroxiredoxin, inactivating AKT/mTOR/p70S6K signaling, and inducing autophagic cell death in lung cancer cells. Mito-LND causes no toxicity in mice even when administered for eight weeks at 50 times the effective cancer inhibitory dose. Collectively, these findings show that mitochondrial targeting of LND is a promising therapeutic approach for investigating the role of autophagy in mitigating lung cancer development and brain metastasis.

An HK2 Antisense Oligonucleotide Induces Synthetic Lethality in HK1-HK2+ Multiple Myeloma.

Although the majority of adult tissues express only hexokinase 1 (HK1) for glycolysis, most cancers express hexokinase 2 (HK2) and many coexpress HK1 and HK2. In contrast to HK1+HK2+ cancers, HK1-HK2+ cancer subsets are sensitive to cytostasis induced by HK2shRNA knockdown and are also sensitive to synthetic lethality in response to the combination of HK2shRNA knockdown, an oxidative phosphorylation (OXPHOS) inhibitor diphenyleneiodonium (DPI), and a fatty acid oxidation (FAO) inhibitor perhexiline (PER). The majority of human multiple myeloma cell lines are HK1-HK2+. Here we describe an antisense oligonucleotide (ASO) directed against human HK2 (HK2-ASO1), which suppressed HK2 expression in human multiple myeloma cell cultures and human multiple myeloma mouse xenograft models. The HK2-ASO1/DPI/PER triple-combination achieved synthetic lethality in multiple myeloma cells in culture and prevented HK1-HK2+ multiple myeloma tumor xenograft progression. DPI was replaceable by the FDA-approved OXPHOS inhibitor metformin (MET), both for synthetic lethality in culture and for inhibition of tumor xenograft progression. In addition, we used an ASO targeting murine HK2 (mHK2-ASO1) to validate the safety of mHK2-ASO1/MET/PER combination therapy in mice bearing murine multiple myeloma tumors. HK2-ASO1 is the first agent that shows selective HK2 inhibition and therapeutic efficacy in cell culture and in animal models, supporting clinical development of this synthetically lethal combination as a therapy for HK1-HK2+ multiple myeloma. SIGNIFICANCE: A first-in-class HK2 antisense oligonucleotide suppresses HK2 expression in cell culture and in in vivo, presenting an effective, tolerated combination therapy for preventing progression of HK1-HK2+ multiple myeloma tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/10/2748/F1.large.jpg.

Antiproliferative effects of cimicifuga racemosa root extract Ze 450 mediated by inhibition of oxidative phosphorylation and indirect AMPK activation

Antiproliferative effects of cimicifuga racemosa root extract Ze 450 mediated by inhibition of oxidative phosphorylation and indirect AMPK activation