Mitochondrial Spare Respiratory Capacity Research Articles

P3‐061: ALLOPREGNANOLONE POTENTIATES BIOENERGETIC CAPACITY AND RESTRUCTURES MITOCHONDRIAL RETICULUM IN NEURONS AND ASTROCYTES

We reported previously that the neurosteroid allopregnanolone (Allo) promotes neural stem cell regeneration, reverses neurogenic, metabolic and cognitive deficits and reduces Alzheimer's disease (AD) pathology in a mouse model of AD. To compare and contrast the cell-type specific mechanisms of Allo in regulating brain energy metabolism, we assessed the effect of Allo on mitochondrial bioenergetic profile and their morphological changes in rat hippocampal neurons and astrocytes. E18 rat hippocampal neurons were cultured for 10 days in neurobasal medium (NBM) with 2% B27 and then starved in 0.2% B27 / NBM for 4 hours before treatment with either 100nM Allo or 0.001% Vehicle overnight. E18 rat hippocampal astrocyte were cultured for 10 days in DMEM:F12(1:1) with 10% FBS and then starved in 10% Charcoal stripped-FBS / DMEM:F12 for 24 hours before treatment with 100nM Allo or 0.001% Vehicle overnight. Upon completion of treatment, both cell types were subject to morphological, biochemical and metabolic characterization of their mitochondrial phenotypes. In primary hippocampal neurons, Allo treatment significantly reversed nutrient deprivation-induced deficits in mitochondrial maximal- and spare respiratory capacity and dendritic morphology. In parallel, in primary hippocampal astrocytes, Allo rescued serum depletion-induced decline in mitochondrial spare respiratory capacity. Allo treatment reduced the population of less efficient swollen globule mitochondria in both neurons and astrocytes. Specifically in astrocytes, Allo decreased the number of hyperfused tubule mitochondria and increased small globule mitochondria. The effect of Allo on re-structuring nutrient or serum depletion-induced mitochondrial reticulum in both neurons and astrocytes was further supported by the restoration of the balance between Drp1 and Opa1 expression, which are key regulators for mitochondrial fission and fusion, respectively. Outcomes of our findings further support the promising therapeutic effects of Allo against bioenergetic deficits and mitochondrial inefficiency that emerge in early phases of AD, with mitochondrial dynamics being a potentially key targeted mechanism.

219-OR: Hydroxychloroquine and Metabolic Outcomes in Patients Undergoing Total Pancreatectomy and Autologous Islet Transplantation

Rationale: Although autologous islet transplantation aims to prevent surgically-induced insulin-dependent diabetes in patients undergoing total pancreatectomy, only approximately one third of patients achieve insulin independence. To test the hypothesis that reducing nonspecific inflammation improves graft function post-AIT, we conducted a pilot clinical study using oral hydroxychloroquine (HCQ), an FDA-approved anti-malarial that inhibits thrombosis and a broad spectrum of inflammatory cytokines. Methods: We used a phase II, randomized, double-blinded, placebo-controlled trial to determine the effect of peri-transplant HCQ on individuals (n=8) undergoing total pancreatectomy with autologous islet transplantation (TPAIT). In vivo islet function was assessed via Mixed Meal Tolerance Test (MMTT) before and 6-12 months after surgery. We found that islet grafts were viable for at least 24 hours in culture and we optimized conditions for in vitro assessment of islet graft metabolic function via Seahorse Extracellular Flux. Results and Conclusions: Islet grafts from HCQ-treated patients showed a 4.7-fold greater mitochondrial spare respiratory capacity compared with placebo-treated patients (64.9 ± 20.7 vs. 13.7 ± 7.1% basal respiration, p = 0.058), suggesting that HCQ may improve metabolic function in the islet graft. We also found that the insulin incremental area under the curve after MMTT was 8.2-fold greater in HCQ-treated patients 6-12 months after surgery compared to that of placebo-treated patients (1154 ± 290 vs. 185 ± 180 mU/L, p = 0.026), suggesting that HCQ may also improve islet engraftment and/or function. Our results warrant further investigation into HCQ using a larger-scale clinical trial with the goal of reducing inflammatory islet destruction and enhancing mitochondrial function to improve metabolic outcomes in TPAIT. Disclosure R. McDowell: None. K.F. Ali: None. V.T. San Martin: None. R. Bottino: Research Support; Self; Imagine Pharma. J.P. Kirwan: None. B. Hatipoglu: Advisory Panel; Self; Novo Nordisk Inc. Speaker's Bureau; Self; Merck & Co., Inc. Funding Cleveland Clinic Foundation

IL-2 Signals Program the Fate of Exhausted CD8 T cells

Abstract The high affinity IL-2 receptor (IL-2Ra, CD25) is a key activation marker, which is transiently and heterogeneously expressed during the priming . While IL-2 signals are deemed critical for expansion of antigen-specific CD8 T cells, the role of IL-2 in programing the fate of exhausted CD8 T cells in vivo is poorly understood. Exhausted CD8 T cells are broadly divided into 2 subsets: one with brightest levels of inhibitory receptors such as PD-1, and lower mitochondrial spare respiratory capacity (SRC) – these cells are terminally exhausted and are largely unresponsive to PD-1 checkpoint blockade immunotherapy. The other subset has intermediate levels of inhibitory receptors and retains higher mitochondrial SRC – these are the clinically relevant cells that are highly responsive to PD-1 blockade. Based on the ability of IL-2 signals to drive glycolytic burnout in T cells, we hypothesized that the stochastic heterogeneity in in vivo assimilation of IL-2 signals during priming drives distinct CD8 T cell fates late during exhaustion. We sorted in vivo primed CD8 T cells based on their heterogeneity in CD25 expression, and adoptively transferred them into infection-matched chronically infected recipients. We observed that the exhausted CD8 T cells generated from CD25Hi donor cells phenotypically and functionally resembled terminally exhausted cells. Furthermore, knocking down the expression of CD25 or Special AT-rich sequence binding protein 1 (SATB1), which is known to regulate CD25 expression, resulted in better survival and function of exhausted CD8 T cells. Our data underscores the critical role of IL-2 signals in the exhaustion of CD8 T cells and has clinical significance in current combination checkpoint blockade immunotherapies.

Bok regulates mitochondrial fusion and morphology.

Bok (Bcl-2-related ovarian killer) is a member of the Bcl-2 protein family that governs the intrinsic apoptosis pathway, but the cellular role that Bok plays is controversial. Remarkably, endogenous Bok is constitutively bound to inositol 1,4,5-trisphosphate receptors (IP3Rs) and is stabilized by this interaction. Here we report that despite the strong association with IP3Rs, deletion of Bok expression by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease)-mediated gene editing does not alter calcium mobilization via IP3Rs or calcium influx into the mitochondria. Rather, Bok deletion significantly reduces mitochondrial fusion rate, resulting in mitochondrial fragmentation. This phenotype is reversed by exogenous wild-type Bok and by an IP3R binding-deficient Bok mutant, and may result from a decrease in mitochondrial motility. Bok deletion also enhances mitochondrial spare respiratory capacity and membrane potential. Finally, Bok does not play a major role in apoptotic signaling, since Bok deletion does not alter responsiveness to various apoptotic stimuli. Overall, despite binding to IP3Rs, Bok does not alter IP3R-mediated Ca2+ signaling, but is required to maintain normal mitochondrial fusion, morphology, and bioenergetics.

Mitochondrial Impairment Upregulates MICOS Expression in a Human Microglial Cell Model

Mitochondrial dysfunction and oxidative stress have been implicated as key pathological mechanisms underlying the dopaminergic neurodegenerative process of Parkinson's disease. The mitochondrial contact site and cristae‐organizing system (MICOS) complex, formed by two subcomplexes with a total of 7 subunits, is associated with the formation and maintenance of mitochondrial cristae structure and respiratory complexes as well as in the regulation of mitochondrial import machinery. Alterations of MICOS subunit protein levels, mutations, and protein modifications have been associated with distinct human diseases, including Parkinson's disease, diabetic cardiomyopathy, epilepsy, and cancer. Previous studies using the classic mitochondrial complex‐1 inhibitor rotenone have shown that overexpression of Mic60 attenuates the rotenone‐induced cell death and increased mitochondrial respiration and spare respiratory capacity in PC12 and SH‐SY5Y dopaminergic cell models. We recently reported that mitochondrial impairment in microglia amplifies the neuroinflammatory response in cell and animal models of rotenone‐induced neurotoxicity. To further understand the cellular mechanism underlying mitochondrial impairment and neuroinflammation, we investigated the effect of rotenone on MICOS subunits, specifically Mic27 and Mic60, in the novel C20 human microglia cell model. Our immunocytochemical, qPCR and Western blot analyses revealed that exposing C20 cells to rotenone for 24 hours induced the upregulation of Mic27 and Mic60. Microglia are known to be extremely resilient cells whose metabolic states differ greatly in comparison to neurons. Therefore, our future studies will determine if the upregulation of MICOS subunits in microglia represents a compensatory mechanism to relieve mitochondrial stress.Support or Funding Information(NIH grants NS100090, NS088206 and ES027245)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract A056: Incessant ER stress responses promote dendritic cell dysfunction in ovarian cancer

Abstract Harnessing the intrinsic ability of our immune system to recognize and eliminate malignanT-cells represents the most promising anticancer strategy since the development of chemotherapy. However, hostile microenvironmental conditions within aggressive solid tumors inhibit the optimal activity of protective immune cells. Targeting immunosuppression and re-programming immune cell function in the tumor microenvironment are thus fundamental requirements for developing successful cancer immunotherapies. Our CRI postdoctoral project aims at identifying, understanding and disabling the molecular mechanisms by which endoplasmic reticulum (ER) stress responses inhibit the natural function of dendritic cells (DCs) in the ovarian cancer microenvironment. The central hypothesis of this study is that ovarian tumors trigger ER stress and aberrant activation of the IRE1-XBP1 pathway in infiltrating DCs to cripple key immuno-metabolic processes and impede the development of protective T-cell responses. To determine how IRE1α-XBP1 overactivation defines regulatory DC phenotypes in the ovarian cancer microenvironment, metastatic ovarian tumors were developed in ER stress-Activated Indicator (ERAI) reporter mice. We found that DCs demonstrating IRE1 activation in the tumor microenvironment overexpress tolerogenic and immunosuppressive molecules. Next, to determine whether tumor-derived factors may affect DC metabolism, we optimized an ex vivo culture system that recreates the tumor microenvironment using malignant ascites samples from ovarian cancer patients. We treated human monocyte-derived DCs from healthy donors with patient-derived ovarian cancer malignant ascites supernatants and assessed the bioenergetic profile of DCs. Oxygen consumption rate (OCR), which measures mitochondrial respiration, ATP production and spare respiratory capacity, was increased in DCs exposed to ascites supernatants. We found that these metabolic changes upon ascites treatment relied on IRE1a-XBP1 pathway. Further, since therapeutic DC-based vaccines have shown limited effects in ovarian cancer patients, we tested the novel translational hypothesis that XBP1-deficient bone marrow-derived DCs (BMDCs) would be better equipped to endure and function in the tumor microenvironment when used as therapeutic vaccines. While adoptive transfer of WT BMDCs did not induce any therapeutic effect, treatment with XBP1-deficient BMDCs elicited a marked increase in overall host survival. This result suggest that compared with WT BMDCs, their XBP1-deficient counterparts were resistant to the immunosuppressive effects of the tumor microenvironment. Our results provide a unique mechanistic rationale for targeting the IRE1-XBP1 arm of the ER stress response as a potent approach to reprogram and enhance antitumor immune cell function in cancer. These findings should also pave the way for devising a new generation of cancer immunotherapies that may improve the dismal survival of >21,000 American women affected by ovarian cancer each year. Citation Format: Chang-Suk Chae. Incessant ER stress responses promote dendritic cell dysfunction in ovarian cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A056.

HMGA2 as a functional antagonist of PARP1 inhibitors in tumor cells.

Poly(ADP‐ribose) polymerase 1 inhibitors alone or in combination with DNA damaging agents are promising clinical drugs in the treatment of cancer. However, there is a need to understand the molecular mechanisms of resistance to PARP1 inhibitors. Expression of HMGA2 in cancer is associated with poor prognosis for patients. Here, we investigated the novel relationship between HMGA2 and PARP1 in DNA damage‐induced PARP1 activity. We used human triple‐negative breast cancer and fibrosarcoma cell lines to demonstrate that HMGA2 colocalizes and interacts with PARP1. High cellular HMGA2 levels correlated with increased DNA damage‐induced PARP1 activity, which was dependent on functional DNA‐binding AT‐hook domains of HMGA2. HMGA2 inhibited PARP1 trapping to DNA and counteracted the cytotoxic effect of PARP inhibitors. Consequently, HMGA2 decreased caspase 3/7 induction and increased cell survival upon treatment with the alkylating methyl methanesulfonate alone or in combination with the PARP inhibitor AZD2281 (olaparib). HMGA2 increased mitochondrial oxygen consumption rate and spare respiratory capacity and increased NAMPT levels, suggesting metabolic support for enhanced PARP1 activity upon DNA damage. Our data showed that expression of HMGA2 in cancer cells reduces sensitivity to PARP inhibitors and suggests that targeting HMGA2 in combination with PARP inhibition may be a promising new therapeutic approach.

Abstract 382: Inhibition of mitochondrial spare respiratory capacity by new securinine derivatives as a novel differentiation therapy for acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is characterized by a differentiation block in myeloid progenitors resulting in aberrant proliferation of immature myeloid cells. Therapeutics that induce differentiation of leukemic cells would overcome the high toxicity and low potency of conventional chemotherapy. Securinine, a plant-derived alkaloid from Securinega suffruticosa, has been shown to be a promising AML differentiation agent through an unclear mechanism, but is limited by its low potency (~20μM) and toxicity. We performed lead optimization chemistry on securinine that led to derivatives 250 and 317, with nanomolar efficacy and reduced toxicity. These derivatives effectively inhibit growth of AML cell lines and patient samples. Treatment of AML cell lines with compounds 250 and 317 induces the expression of differentiation markers CD11b and CD14c. Compounds 250 and 317 significantly reduced leukemic burden in a circulating model of AML in NSG mice. Additionally, these compounds were effective in inhibiting cell growth of HL60 cells that are resistant to a conventional chemotherapeutic, doxorubicin. By using the drug-affinity responsive target sensitivity (DARTS) assay, we identified that the redox mediator thioredoxin reductase (TrxR) is the primary target of these derivatives. 250 and 317 inhibit TrxR activity in vitro and in cells, and the resultant alterations in redox status are overcome by addition of a scavenger of reactive oxygen species (ROS), N-acetylcysteine. The ensuing oxidative metabolic stress rapidly alters the metabolite profile in AML cells including depletion of malate. Securinine derivatives decreased basal oxygen consumption rate and spare respiratory capacity, resulting in differentiation and cell death. Downregulating TrxR1 expression increased the sensitivity of OCI-AML3 cells to compounds 250 and 317. Together, we show here that these new derivatives of securinine exhibit preclinical efficacy in AML by decreasing mitochondrial spare respiratory capacity through the inhibition of TrxR. Citation Format: Sheela Karunanithi, Ruifu Liu, Anne Roe, Stephen Moreton, Yongchun Hou, Natasha Oldford, Juan Valentin Goyco, David Wald. Inhibition of mitochondrial spare respiratory capacity by new securinine derivatives as a novel differentiation therapy for acute myeloid leukemia [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 382.

Abstract 892: Doxorubicin-induced cardiotoxicity in iPSC-cardiomyocytes: Altered mitochondrial gene expression and function

Abstract Introduction: Cardiomyocytes are highly vulnerable to anthracycline-induced toxicity, which may lead to heart failure. This includes doxorubicin, which is a commonly used chemotherapeutic agent. Although mitochondrial function has been implicated as a mechanism of anthracycline-induced toxicity in rodent heart cells, the precise genes that regulate this response in humans remain to be elucidated. We hypothesized that doxorubicin significantly alters expression of mitochondrial genes in human cardiomyocytes, which impairs mitochondrial function. Methods: Human inducible pluripotent stem cell (iPSC)-derived cardiomyocytes were treated with doxorubicin or left untreated for control to assess changes in gene expression using RNAseq. A total of 169 genes involved in mitochondrial function, as defined by Ingenuity Pathway Analysis and KEGG, were analyzed for significant differences between untreated and treated conditions using DESeq2 and GenePattern 2.0. Mitochondrial respiration was measured in control and doxorubicin-treated cells using the Seahorse Bioscience XFe96 Cell Mito Stress Test kit. We used a Spearman's partial correlation coefficient analysis to correlate gene expression levels with mitochondrial basal respiration, ATP production, maximal respiration, and spare respiratory capacity for untreated and doxorubicin-treated iPSC-cardiomyocytes. Results: Of the 169 mitochondrial genes analyzed, we identified 25 genes that were significant in our global differential expression analysis across all conditions (P<0.05). Seven of these genes (ATP5D, COX5A, CYC1, HSD17B10, NDUFB10, NDUFS8, UQCRC1) remained significant in pairwise analyses between control and doxorubicin treated cells. We observed a decrease in mitochondrial respiration following treatment with doxorubicin. Maximal respiration (r=-0.929; P=0.022) and spare respiratory capacity (r=-0.98;P=0.0025) negatively correlated with NDUFS8 expression in doxorubicin-treated iPSC-cardiomyocytes. Conclusion: Our findings underscore a role for mitochondrial function in the development of doxorubicin-induced cardiotoxicity and implicate specific genes in this process. Doxorubicin-altered gene expression in cardiomyocytes may provide insight into how impaired mitochondrial function leads to heart failure in cancer survivors. Citation Format: Monica E. Reyes, Rashida Callender, Jianzhong Ma, Megan L. Grove, Alanna C. Morrison, Michelle A. Hildebrandt. Doxorubicin-induced cardiotoxicity in iPSC-cardiomyocytes: Altered mitochondrial gene expression and function [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 892.

Yerba Mate Stimulates Mitochondrial Biogenesis and Thermogenesis in High-Fat-Diet-Induced Obese Mice.

The potential effects of yerba mate (YM) on mitochondrial biogenesis and thermogenesis are evaluated. The in vitro effects of YM on mitochondrial respiration are assessed in C2C12 cells. The expression of genes related to mitochondrial biogenesis and thermogenesis are analyzed by quantitative PCR. The in vivo experiments are performed on mice fed a high-fat diet (HFD) and treated with YM extract. Indirect calorimetry was performed, and the expression of genes and proteins related to mitochondrial biogenesis, thermogenesis, and de novo lipogenesis is determined by quantitative PCR and western blot. Our in vitro data indicate that YM increases mtDNA copy number as well as mitochondrial spare respiratory capacity and coupling efficiency. The gene expression profile reinforces this evidence, indicating a modulation of genes downstream of Ampk. In vivo, it is found that YM partially prevents diet-induced obesity by increasing energy expenditure and enhancing mitochondrial biogenesis via the AMPK/SIRT1/PGC1α pathway. YM stimulates mitochondriogenesis and Ucp expression, leading to an increase in the spare respiratory capacity and energy dissipation. These effects may help to better understand the potential use of YM for obesity treatment.

Oxidative stress alters mitochondrial bioenergetics and modifies pancreatic cell death independently of cyclophilin D, resulting in an apoptosis-to-necrosis shift

Mitochondrial dysfunction lies at the core of acute pancreatitis (AP). Diverse AP stimuli induce Ca2+-dependent formation of the mitochondrial permeability transition pore (MPTP), a solute channel modulated by cyclophilin D (CypD), the formation of which causes ATP depletion and necrosis. Oxidative stress reportedly triggers MPTP formation and is elevated in clinical AP, but how reactive oxygen species influence cell death is unclear. Here, we assessed potential MPTP involvement in oxidant-induced effects on pancreatic acinar cell bioenergetics and fate. H2O2 application promoted acinar cell apoptosis at low concentrations (1–10 μm), whereas higher levels (0.5–1 mm) elicited rapid necrosis. H2O2 also decreased the mitochondrial NADH/FAD+ redox ratio and ΔΨm in a concentration-dependent manner (10 μm to 1 mm H2O2), with maximal effects at 500 μm H2O2. H2O2 decreased the basal O2 consumption rate of acinar cells, with no alteration of ATP turnover at <50 μm H2O2. However, higher H2O2 levels (≥50 μm) diminished spare respiratory capacity and ATP turnover, and bioenergetic collapse, ATP depletion, and cell death ensued. Menadione exerted detrimental bioenergetic effects similar to those of H2O2, which were inhibited by the antioxidant N-acetylcysteine. Oxidant-induced bioenergetic changes, loss of ΔΨm, and cell death were not ameliorated by genetic deletion of CypD or by its acute inhibition with cyclosporine A. These results indicate that oxidative stress alters mitochondrial bioenergetics and modifies pancreatic acinar cell death. A shift from apoptosis to necrosis appears to be associated with decreased mitochondrial spare respiratory capacity and ATP production, effects that are independent of CypD-sensitive MPTP formation.

Sorafenib promotes graft-versus-leukemia activity in mice and humans through IL-15 production in FLT3-ITD-mutant leukemia cells.

Individuals with acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) in the gene encoding Fms-related tyrosine kinase 3 (FLT3) who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) have a 1-year survival rate below 20%. We observed that sorafenib, a multitargeted tyrosine kinase inhibitor, increased IL-15 production by FLT3-ITD+ leukemia cells. This synergized with the allogeneic CD8+ T cell response, leading to long-term survival in six mouse models of FLT3-ITD+ AML. Sorafenib-related IL-15 production caused an increase in CD8+CD107a+IFN-γ+ T cells with features of longevity (high levels of Bcl-2 and reduced PD-1 levels), which eradicated leukemia in secondary recipients. Mechanistically, sorafenib reduced expression of the transcription factor ATF4, thereby blocking negative regulation of interferon regulatory factor 7 (IRF7) activation, which enhanced IL-15 transcription. Both IRF7 knockdown and ATF4 overexpression in leukemia cells antagonized sorafenib-induced IL-15 production in vitro. Human FLT3-ITD+ AML cells obtained from sorafenib responders following sorafenib therapy showed increased levels of IL-15, phosphorylated IRF7, and a transcriptionally active IRF7 chromatin state. The mitochondrial spare respiratory capacity and glycolytic capacity of CD8+ T cells increased upon sorafenib treatment in sorafenib responders but not in nonresponders. Our findings indicate that the synergism of T cells and sorafenib is mediated via reduced ATF4 expression, causing activation of the IRF7-IL-15 axis in leukemia cells and thereby leading to metabolic reprogramming of leukemia-reactive T cells in humans. Therefore, sorafenib treatment has the potential to contribute to an immune-mediated cure of FLT3-ITD-mutant AML relapse, an otherwise fatal complication after allo-HCT.

In Vitro-Generated Tc17 Cells Present a Memory Phenotype and Serve As a Reservoir of Tc1 Cells In Vivo.

Memory CD8+ T cells are ideal candidates for cancer immunotherapy because they can mediate long-term protection against tumors. However, the therapeutic potential of different in vitro-generated CD8+ T cell effector subsets to persist and become memory cells has not been fully characterized. Type 1 CD8+ T (Tc1) cells produce interferon-γ and are endowed with high cytotoxic capacity, whereas IL-17-producing CD8+ T (Tc17) cells are less cytotoxic but display enhanced self-renewal capacity. We sought to evaluate the functional properties of in vitro-generated Tc17 cells and elucidate their potential to become long lasting memory cells. Our results show that in vitro-generated Tc17 cells display a greater in vivo persistence and expansion in response to secondary antigen stimulation compared to Tc1 cells. When transferred into recipient mice, Tc17 cells persist in secondary lymphoid organs, present a recirculation behavior consistent with central memory T cells, and can shift to a Tc1 phenotype. Accordingly, Tc17 cells are endowed with a higher mitochondrial spare respiratory capacity than Tc1 cells and express higher levels of memory-related molecules than Tc1 cells. Together, these results demonstrate that in vitro-generated Tc17 cells acquire a central memory program and provide a lasting reservoir of Tc1 cells in vivo, thus supporting the use of Tc17 lymphocytes in the design of novel and more effective therapies.

The high-production volume fungicide pyraclostrobin induces triglyceride accumulation associated with mitochondrial dysfunction, and promotes adipocyte differentiation independent of PPARγ activation, in 3T3-L1 cells

Pyraclostrobin is one of the most heavily used fungicides, and has been detected on a variety of produce, suggesting human exposure occurs regularly. Recently, pyraclostrobin exposure has been linked to a variety of toxic effects, including neurodegeneration and triglyceride (TG) accumulation. As pyraclostrobin inhibits electron transport chain complex III, and as mitochondrial dysfunction is associated with metabolic syndrome (cardiovascular disease, type II diabetes, obesity), we designed experiments to test the hypothesis that mitochondrial dysfunction underlies its adipogenic activity. 3T3-L1 cells were differentiated according to standard protocols in the presence of pyraclostrobin, resulting in TG accumulation. However, TG accumulation occurred without activation of the peroxisome proliferator activated nuclear receptor gamma (PPARγ), the canonical pathway mediating adipogenesis. Furthermore, cells failed to express many markers of adipogenesis (PPARγ, lpl, CEBPα), while co-exposure to pyraclostrobin and two different PPARγ antagonists (GW9662, T0070907) failed to mitigate TG accumulation, suggesting TG accumulation occurred through a PPARγ-independent mechanism. Instead, pyraclostrobin reduced steady-state ATP, mitochondrial membrane potential, basal mitochondrial respiration, ATP-linked respiration, and spare respiratory capacity, demonstrating mitochondrial dysfunction, while reduced expression of genes involved in glucose transport (Glut-4), glycolysis (Pkm, Pfkl, Pfkm), fatty acid oxidation (Cpt-1b), and lipogenesis (Fasn, Acacα, Acacβ) further suggested a disruption of metabolism. Finally, inhibition of cAMP responsive element binding protein (CREB), a PPARγ coactivator, partially mitigated pyraclostrobin-induced TG accumulation, suggesting TG accumulation is occurring through a CREB-driven mechanism. In contrast, rosiglitazone, a known PPARγ agonist, induced TG accumulation in a PPARγ-dependent manner and enhanced mitochondrial function. Collectively, these results suggest pyraclostrobin-induced mitochondrial dysfunction inhibits lipid homeostasis, resulting in TG accumulation. Exposures that disrupt mitochondrial function may have the potential to contribute to the rising incidence of metabolic syndrome, and thus more research is needed to understand the human health impact of pyraclostrobin exposure.

41 - Lipid Hydroperoxides as Mediators of Ozone-Induced Oxidative Changes in Human Airway Epithelial Cells

Human exposure to tropospheric ozone (O3) is a significant public health concern. O3 inhalation induces airway inflammation and pulmonary function decrements, and may also lead to cardiovascular morbidity. The carbon-carbon double bonds in polyunsaturated fatty acids are primary targets for oxidation by O3, leading to formation of free radicals, hydrogen peroxide (H2O2), and lipid hydroperoxides. However, the role of these intermediates in mediating downstream cellular responses to O3 inhalation is not well understood. Previous studies have observed an O3-induced increase in the glutathione redox potential (EGSH) in human airway epithelial cells (HAEC). Impairment of mitochondrial respiration has also been previously observed in mice and rats exposed to O3. We hypothesized that lipid hydroperoxides contribute to these O3-induced oxidative changes in HAEC. In the present study we monitored the EGSH using the fluorogenic sensor roGFP and mitochondrial respiration with the Seahorse Extracellular Flux Analyzer in BEAS-2B cells exposed to lipid hydroperoxides. Overexpression of catalase was used to evaluate the role of H2O2 in the observed hydroperoxide-induced changes. We found that exposure to the linoleic acid-derived hydroperoxide 9-HpODE induces dose-dependent increases in EGSH that are independent of H2O2 production. Exposure to 9-HpODE also caused a decrease in mitochondrial basal oxygen consumption, maximal respiration, and spare respiratory capacity. These results suggest that lipid hydroperoxides are primary effectors of oxidative changes induced by O3 inhalation. This abstract of a proposed presentation does not necessarily reflect EPA policy.

Central nervous system-penetrating antiretrovirals impair energetic reserve in striatal nerve terminals.

The use of antiretroviral (ARV) drugs with central nervous system (CNS) penetration effectiveness (CPE) may be useful in the treatment of HIV-associated neurocognitive disorder (HAND) as well as targeting a CNS reservoir in strategies to achieve a functional cure for HIV. However, increased cognitive deficits are linked to at least one of these drugs (efavirenz). As mitochondrial dysfunction has been found with a number of ARVs, and as such can affect neuronal function, the objective of this study was to assess the effects of ARV with high CPE for toxicological profiles on presynaptic nerve terminal energy metabolism. This subcellular region is especially vulnerable in that a constant supply of ATP is required for the proper maintenance of neurotransmitter release and uptake supporting proper neuronal function. We evaluated the effects of acute treatment with ten different high CPE ARVs from five different drug classes on rat cortical and striatal nerve terminal bioenergetic function. While cortical nerve terminal bioenergetics were not altered, striatal nerve terminals exposed to efavirenz, nevirapine, abacavir, emtricitabine, zidovudine, darunavir, lopinavir, raltegravir, or maraviroc (but not indinavir) exhibit reduced mitochondrial spare respiratory capacity (SRC). Further examination of efavirenz and maraviroc revealed a concentration-dependent impairment of striatal nerve terminal maximal mitochondrial respiration and SRC as well as a reduction of intraterminal ATP levels. Depletion of ATP at the synapse may underlie its dysfunction and contribute to neuronal dysfunction in treated HIV infection.

P 100 - Isothiocyanates trigger early disruption of mitochondrial function in cells overexpressing Bcl-2

Epidemiological evidence indicates that increased consumption of isothiocyanates is associated with reduced incidence of cancer. One mechanism to explain this is the ability of isothiocyanates to trigger apoptosis, including in cells that overexpress the anti-apoptotic protein Bcl-2, which is overexpressed in a number of cancers. This study aimed to examine the mechanisms by which phenethyl isothiocyanate (PEITC) induces apoptosis in Bcl-2 overexpressing (Bcl-2 OE) cells. Bcl-2 OE cells were more sensitive than wild-type cells to PEITC (LC50=8 μM vs. 12.5 μM). Bax-/-Bak-/-and Bim-/-Bid-/- cells were resistant to PEITC, indicating a classical apoptotic cell death. Cell death occurred more rapidly in the Bcl-2 OE cells, and increased levels of caspase activity and earlier activation were observed. PEITC had a greater effect on mitochondrial metabolic activity in the Bcl-2 OE cells; they had higher basal mitochondrial respiration, however, this was susceptible to inhibition by PEITC. There was also a significant decline in mitochondrial spare respiratory capacity. These effects occurred prior to caspase activation and cell death. Together, these results suggest that isothiocyanates bypass the action of Bcl-2 by acting directly on mitochondria to promote Bax/Bak-dependent apoptosis. This information may be valuable for designing novel isothiocyanates to target Bcl-2 OE cancer cells.

Temperature induces significant changes in both glycolytic reserve and mitochondrial spare respiratory capacity in colorectal cancer cell lines

Thermotherapy, as a method of treating cancer, has recently attracted considerable attention from basic and clinical investigators. A number of studies and clinical trials have shown that thermotherapy can be successfully used as a therapeutic approach for various cancers. However, the effects of temperature on cancer bioenergetics have not been studied in detail with a real time, microplate based, label-free detection approach.This study investigates how changes in temperature affect the bioenergetics characteristics (mitochondrial function and glycolysis) of three colorectal cancer (CRC) cell lines utilizing the Seahorse XF96 technology. Experiments were performed at 32°C, 37°C and 42°C using assay medium conditions and equipment settings adjusted to produce equal oxygen and pH levels ubiquitously at the beginning of all experiments. The results suggest that temperature significantly changes multiple components of glycolytic and mitochondrial function of all cell lines tested. Under hypothermia conditions (32°C), the extracellular acidification rates (ECAR) of CRC cells were significantly lower compared to the same basal ECAR levels measured at 37°C. Mitochondrial stress test for SW480 cells at 37°C vs 42°C demonstrated increased proton leak while all other OCR components remained unchanged (similar results were detected also for the patient-derived xenograft cells Pt.93). Interestingly, the FCCP dose response at 37°C vs 42°C show significant shifts in profiles, suggesting that single dose FCCP experiments might not be sufficient to characterize the mitochondrial metabolic potential when comparing groups, conditions or treatments.These findings provide valuable insights for the metabolic and bioenergetic changes of CRC cells under hypo- and hyperthermia conditions that could potentially lead to development of better targeted and personalized strategies for patients undergoing combined thermotherapy with chemotherapy.

Continuous IL-15 Signaling Leads to Functional Exhaustion of Human Natural Killer Cells through Metabolic Changes That Alters Their In Vivo Anti-Tumor Activity

Natural Killer (NK) cells represent an exciting immunotherapeutic approach to treat cancer. We have shown that in vivo expansion and activation of donor NK cells supported by administration of IL-2 induces remission in patients with refractory AML. Recent clinical studies by our group have shown that IL-15 is superior to IL-2 to support NK cell persistence 14 days after adoptive transfer. However, only 36% of patients treated with 12 consecutive days of IL-15 had NK cell expansion to the level of ≥100 donor derived NK cells/µL blood compared to 10% in subjects treated with IL-2 (p=0.02). This leads us to conclude that we might not know the optimal route and interval to administer in vivo IL-15. We hypothesized that daily uninterrupted IL-15 dosing could lead to exhaustion or NK cellular stress. Therefore we designed an in vitro model system in which enriched NK cells are treated with three 3-day cycles of continuous IL-15 (IL-15cont) or were rested with a "gap" (skipping the middle cycle [IL-15gap]) before returning to the last cycle of IL-15. IL-15cont treatment yielded more proliferation and higher cell numbers compared to IL-15gap (4.8±0.44 vs. 1.9±0.26 million cells/ml, p < 0.0001) when cells were analyzed at the end of the three cycles (on day 9, where all in vitro measurements were taken). However, NK cell death, measured by flow cytometry, in the IL-15cont group was higher (18.9±2.2 vs 14.9±1.7 % cell death, p = 0.035) and this group also had an enrichment in genes involved in cell cycle checkpoint/ arrest, perhaps indicating more cellular stress in the IL-15cont. In an in vitro flow cytometric functional assay, the IL-15cont group had decreased activation when compared to the IL-15 gap group against K562 targets (43.6±2.1 vs 55.6±2.7 % CD107a [degranulation], p < 0.0001; 1.9±0.41 vs 7.1±0.93 % IFNg [inflammatory cytokine production], p = 0.0055). The decrease in NK cell activation correlated with a strong decrease in tumor target killing in an in vitro chromium release assay (Figure 1A) measuring killing of acute promyelocytic leukemia (HL-60) cell targets, in which the IL-15cont NK cells were potently outperformed by the IL-15gap cells (6.4±2.6 vs 51.5±4.8 % killing at 2.5:1 effector:target ratio, p < 0.0001). We used an in vivo xenogeneic model of AML, where conditioned NSG (NOD scid gamma) mice are engrafted with HL-60luc tumor targets 3 days prior to infusion with nothing, IL-15cont or IL-15gap human NK cells prepared within our 9 day culture system. Only the IL-15gap NK group mediated statistically significant tumor control when compared to tumor alone at two weeks following NK cell infusion (Figure 1B). To probe deeper into the functional defect we evaluated signaling after these treatments and noted decreased phosphorylation of several proteins in the IL-15cont group. These data led us to explore proteins involved in metabolism and we noted that CPT1A, a critical enzyme involved in fatty acid oxidation (FAO), was strongly increased in the IL-15gap treated NK cells (protein MFI of 15,759±2,603 [IL-15gap] vs 5,273±744 [IL-15cont], p = 0.009). Metabolic analysis using a Seahorse XFe24 analyzer showed an increased mitochondrial spare respiratory capacity (SRC) in the IL-15gap group, denoting better capability of the IL-15gap NK cells to respond to energetic demands (Figure 1C). In a separate experiment the groups were treated with etomoxir to inhibit CPT1A, and the SRC phenotype was reversed, with the IL-15gap group containing lower SRC than the IL-15cont group. To test these findings in a functional assay we repeated the IL-15cont treatment in combination with rapamycin, which can induce CPT1A through inhibition of mTORC1, and saw restoration of function to levels similar to IL-15gap (40.8±2.0 vs 49.3±2.9 % CD107a in the IL-15cont vs IL-15cont + rapamycin, p = 0.005; 2.4±0.47 vs 4.8±1.0 % IFNg in the IL-15cont vs IL-15cont + rapamycin, p = 0.03). These data indicate that NK cell functional exhaustion via continuous IL-15 signaling is mediated by a decrease in FAO. Intermittent IL-15 dosing or altering metabolism through other mechanisms may overcome this competition. These findings could impact ongoing clinical trials through simple alterations in dosing strategies in order to minimize NK cell exhaustion in the immunotherapeutic setting. [Display omitted] DisclosuresCooley:Fate Therapeutics: Research Funding. Miller:Oxis Biotech: Consultancy, Other: SAB; Fate Therapeutics: Consultancy, Research Funding.

Waldenstrom Macroglobulinemia Cells Modulate Mitochondrial Bioenergetics and Induce a Respiratory Hyper-Drive State upon Acquisition of Ibrutinib-Resistance

Background: Mitochondrial activity is critical to maintain a high cellular proliferation especially in drug-resistant clones. Ibrutinib-resistance (IR) in CLL is associated with highly aggressive clinical behavior, carrying a median survival of ~3 months. While these outcomes are not yet reported in WM, resistance to ibrutinib is foreseeable considering no WM patients achieve a complete remission with BTK inhibitors (reported series). The transition to a highly proliferative drive in the IR clone remains a biological dilemma and understanding this will allow rationale development of therapeutic strategies. It is established that increased cell proliferation is highly dependent on the functional capabilities of the mitochondria. Yet the respiratory proficiencies and adaptation of mitochondrial machinery in the IR state remains unknown. Here we interrogated mitochondrial function and its relevance in IR cells using our WM models.Materials: WM cell lines (BCWM.1, non-IR) and its isogenic IR clone (BCWM.1/IR; 20-fold resistant, without BTK or CXCR4WHIM-like mutations) were used. Mitochondrial respirometry was measured by Seahorse XF96 flux analyzer (Fig. 1a). Drug sensitivity and cytotoxicity were determined by Cell-Titer Glo cell viability assay. Whole exome sequencing (WES) was performed on an Illumina HiSeq2000.Results: IR WM cells were noted to be in G1 arrest with altered morphologic features and growth kinetics. Despite growth arrest, IR cells showed increased basal oxygen consumption rate (OCR) (>30% increase) vs. parental (non-IR) cells. Consistent with this we observed enhanced mitochondrial ATP production suggesting increased mitochondrial utilization by the IR clones. We then measured maximal respiratory potential of WM cells with FCCP pre-treatment, which evaluates cellular potential to respond to increased ATP demand. We observed upregulation of mitochondrial spare respiratory capacity in BCWM.1/IR cells vs. parental BCWM.1 cell. In addition, a >50% increase in non-mitochondrial oxygen consumption/respiration was also noted (Fig.1b, c). To identify potential genomic changes that could explain mitochondrial overdrive, WES of the non-IR and IR cells was performed. Interestingly, this analysis did not uncover any mutational alterations in genes directing cellular oxidative phosphorylation (NDUF, COX1, COQ7, SURF1, etc.), glycolysis (PDK1, HK2, PKM2, ALDOA, etc.) or ATP production (ATP5, ND1, UCP1 and others). We then asked if mitochondrial function impacts therapeutic potential of a specific drug (or a class of drug), focusing specifically on proteasome disruption. First, we observed that proteasome inhibition- independent of the site (20S vs. 19S), retained high cytotoxic potential, wherein IR cells showed exquisite sensitivity to proteasome disruption. Interestingly, VLX1570 (targets 19S proteasome) had a 2-fold lower IC50 in IR clones than its non-IR counterpart. Cell respirometry demonstrated that compared to parental cells, VLX1570 (500nM) treatment significantly (p<0.002) and rapidly (2hr) attenuated basal respiration in IR clones at concentrations half of those needed to achieve similar effects in BCWM.1 cells. A concomitant decrease in ATP production as well as spare respiratory capacity was observed at these time points/drug concentrations (significantly more evident than in BCWM.1 cells). While our focus was mitochondrial bioenergetics and its therapeutic relevance in drug-resistant WM, unexpectedly we observed for the first time, evidence that VLX1570 induces changes in mitochondrial bioenergetics as early as 2hrs leading to increased mitochondrial membrane permeability and cell apoptosis in 6-12hrs. Comparable observations were made with bortezomib (targeting 20S).Conclusions: This is the first report identifying the role of mitochondrial hyper-drive in WM cells with resistance to ibrutinib. Of note, enhancedmitochondrial bioenergetics in the IR state increased sensitivity to proteasome inhibition and this was independent of the site of disruption in the proteasome pathway. Although, WES findings alone were not sufficient in explaining mitochondrial overdrive noted in our system, ongoing analysis of various mitochondrial functions (Fig. 1d) at the gene expression, copy number and epigenetic level may elucidate the underlying biomechanics of mitochondrial disposition in IR WM cells. [Display omitted] DisclosuresAnsell:BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding. Linder:Vivolux, Ab: Other: Shareholder. Ailawadhi:Pharmacyclics: Consultancy; Novartis: Consultancy; Amgen Inc: Consultancy; Takeda Oncology: Consultancy.