Inhibition Of Oxidative Phosphorylation Research Articles

Targeting Residual Chemotherapy-Resistant Acute Myeloid Leukemia Cells By a Novel Oxphos Inhibitor IACS010759

Acute myeloid leukemia (AML) is initiated and maintained by a relatively rare leukemia stem cells (LSCs) that are capable of self-renewing, proliferation and differentiation into leukemia blasts. Recent data showed that LSCs (Lagadinou et al. Cell Stem Cell 2013) and residual cytarabine (Ara-C)-resistant AML cells (Farge et al. Cancer Discovery 2017) are highly dependent on mitochondrial function for survival. This unique metabolic biology makes LSCs and AML cells resistant to chemotherapy vulnerable to oxidative phosphorylation (OXPHOS) inhibition. We have identified a novel potent inhibitor, IACS-010759, which blocks mitochondrial complex I, suppresses OXPHOS and selectively inhibits the growth of AML cells (Molina et al. AACR 2016). In this study, we aimed to determine the effects of OXPHOS inhibition with IACS-010759 on residual AML cells and AML LSCs surviving standard chemotherapy (Doxorubicin/Ara-C, DA) in patient-derived xenograft (PDX) AML models.Consistent with Farge et al study, OCI-AML3 cells treated with DA in vitro produced elevated levels of reactive oxygen species, displayed increased mitochondrial membrane potential and higher mitochondrial mass (data not shown), indicating reliance on mitochondrial metabolism. Further, Ara-C facilitated statistically significant increase in the basal and maximal oxygen consumption rates (OCR) (36%±8%, p=0.03; 36%±3%, p=0.003, respectively) compared to control. In turn, OXPHOS inhibition with IACS-010759 at 30 nM fully inhibited basal and Ara-C-induced OCR (Fig. 1A). These findings indicate that chemotherapy fosters mitochondrial respiration in AML, which could be abrogated by OXPHOS inhibitor.To examine the efficacy of IACS-010759 on residual leukemia cells surviving chemotherapy in vivo, we first established a chemosensitive PDX AML model of minimal residual disease. Treatment of mice inoculated with a human AML PDX harboring FLT3-ITD mutation with DA (D: 1.5 mg/kg; A: 50 mg/kg) reduced leukemia burden (PB, 0.8 ± 0.6% vs 45.8 ± 8.2% in vehicle-treated mice, p=0.001). The residual AML cells in DA-treated mice expanded and caused rapidly progressing leukemia (78.2 ± 6.2% vs 95.3 ± 1.0% in vehicle-treated mice, p=0.047) on week 6 post DA. Daily oral treatment of mice with IACS-010759 (7.5 mg/kg) as a single agent reduced leukemia burden, and caused further reduction when administered post completion of DA (Fig. 1B,C). The CyTOF mass cytometry, which enables measurement of up to 40 parameters per cell, demonstrated reduced frequency of CD34+CD38lowCD123+ AML LSCs and resurgence of CD11c+ differentiated cells in both IACS and IACS/DA groups (Fig. 1D). On the contrary, chemotherapy alone failed to significantly reduce fractions of LSCs or induce differentiation (Fig. 1D). Proliferation measured by Ki67 was greatly reduced by IACS/DA combination in all populations (1.4 ± 0.3% vs 5.5 ± 0.4% in vehicle group, p<0.01). The expression of hypoxia-inducible factor 1α was downregulated, consistent with the decreased oxygen consumption by IACS-010759 (not shown).We next investigated the efficacy of IACS-010759 in a chemoresistant PDX model inoculated with chemorefractory MLL-rearranged AML. CyTOF analysis demonstrated that DA chemotherapy failed to reduce the primitive leukemia stem/progenitor populations, which comprised 84.7% of total cells in DA versus 71.2% in vehicle groups, while the combination of IACS-010759 and DA efficiently reduced this population down to 30.2%. Additionally, the combination increased the proportion of mature cells characterized by CD34-CD64+CD11b+ (43.1% versus 12.8% in vehicle), which remained low in the DA-only group (5.6%). DA/OXPHOS inhibitor combination significantly extended survival, compared to the vehicle or single-agent treatment arms (Fig. 1E).In conclusion, AML cells surviving chemotherapy depend on OXPHOS for survival, and OXPHOS inhibition with complex I inhibitor IACS-010759 reduces AML LSC pool, induces differentiation and extends survival in AML PDX models in vivo . Our data advocate for combining IACS-010759 with standard chemotherapy, upon identification of a recommended Phase II dose in an ongoing clinical trial of IACS-010759 in AML (NCT02882321). [Display omitted] DisclosuresBattula:United Therapeutics Corporation: Research Funding. Andreeff:Daiichi Sankyo: Consultancy. Kantarjian:ARIAD: Research Funding; Delta-Fly Pharma: Research Funding; Amgen: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Bristol-Meyers Squibb: Research Funding. Daver:Karyopharm: Consultancy, Research Funding; Incyte Corporation: Honoraria, Research Funding; Bristol-Myers Squibb Company: Consultancy, Research Funding; Daiichi-Sankyo: Research Funding; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Jazz: Consultancy; Otsuka America Pharmaceutical, Inc.: Consultancy; Pfizer Inc.: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Kiromic: Research Funding; Immunogen: Research Funding.

Novel Oxidative Phosphorylation Inhibitor IACS-010759 Induces AMPK-Dependent Apoptosis of AML Cells

Acute myeloid leukemia (AML) cells frequently adjust to increased energy/substrate demand under stress conditions in the bone marrow microenvironment. Recent studies indicate that AML cells including leukemia-initiating cells are highly dependent on oxidative phosphorylation (OxPhos) for survival, while normal hematopoietic stem cells predominantly utilize glycolysis for energy homeostasis.The aim of this study is to assess the biomarkers of anti-leukemia efficacy of the first-in-class oxidative phosphorylation inhibitor (OxPhosi) IACS-010759. IACS-010759 at nanomolar concentrations blocks cellular respiration through inhibition of complex I of the electron transport chain (Molina et al., AACR 2016), is orally bioavailable and is currently in Phase 1 clinical trial in AML (NCT #02882321).We utilized primary AML samples from patients with newly diagnosed or relapsed/refractory AML (11 sensitive and 3 resistant to IACS-010759) as well as OxPhosi-sensitive OCI-AML3 and -resistant MOLM13 AML cell lines . To identify transcript abundances for the transcriptome profiling, Cap Analysis of Gene Expression (CAGE)-Seq analysis has been conducted. CAGE-Seq identifies and quantifies the 5' ends of capped mRNA transcripts, which enables the identification of transcription start sites (TSS) and allows investigating promoter structures necessary for gene expression. CAGE-Seq analysis identified recurrent baseline differences of TSS gene expression between IACS-010759-sensitive and -resistant primary AML samples; 4 genes were higher and 124 genes were lower in sensitive samples compared to resistant samples (false positive rate; FDR<0.05). In IACS-010759 sensitive OCI-AML3 cells the expression levels of 1,623 genes were higher and 1,821 were lower than in resistant MOLM13 cells (triplicate, FDR<0.05). Based on these transcriptome differences, the Ingenuity Pathway Analysis (IPA) bioinformatics platform highlighted consistent overexpression of 4 factors in IACS-010759-resistant AML primary samples and cell line compared to the sensitive AML. These include mammalian target of rapamycin (mTOR) kinase, a positive regulator of cell metabolism; Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2) that enhances kinase-mediated activation; erythropoietin (EPO), a positive regulator of Jak-Stat pathway; and IL3, a potent growth-promoting cytokine. These findings indicate that the basal metabolic energetic capacity might affect the sensitivity of AML cells to IACS-010759. AMPK is a crucial cellular energy sensor and regulates energy metabolism through mTOR inhibition. We therefore studied the role of energy metabolism regulator AMP-activated protein kinase (AMPK) and of mTOR in anti-leukemia efficacy. To validate the role of AMPK, we utilized AMPK knockdown (sh AMPK) OCI-AML3 cells, and examined the sensitivity against IACS-010759, comparing to parental shC OCI-AML3 cells. By immunoblotting, IACS-010759 induced significant increase of p-AMPK in parental OCI-AML3 cells along with decrease of mTOR downstream target phospho- (p-) 4EBP1. OCI-AML3 stably infected with sh AMPK predictably overexpressed p-4EBP1 which was not modulated by IACS-010759. sh AMPK OCI-AML3 cells were more than 10-fold less sensitive against IACS-010759 compared to shC OCI-AML3 (IC50; 8.6±4.2 nM vs 0.5±0.1 nM, respectively), albeit remained in low nM concentrations. Interestingly, baseline level of p-AMPK expression in parental OCI-AML3 cells was markedly suppressed under physiologic hypoxic (1% oxygen) conditions, which caused activation of mTOR signaling (upregulation of p-4EBP1) and decreased sensitivity to IACS-010759 (IC50; 14.5±7.1 nM).Taken together, these results indicate that the responsiveness of AML to IACS-010759 depend at least in part on the AMPK-mTOR signaling, the gauge of cellular energy metabolic capacity. In sensitive AML cells IACS-010759 induces AMPK activation leading to mTOR suppression which results in cell growth inhibition in AML cells. In contrast, in cells with lower levels of AMPK, or in highly proliferating cells (such as stimulated by IL-3), mTOR is constitutively activated conferring resistance to Oxphosi, possibly through glycolytic adaptation. Altogether, these findings highlight AMPK and mTOR as putative biomarkers of anti-leukemia activity of the novel OxPhosi IACS-010759. DisclosuresAndreeff:Daiichi Sankyo: Consultancy.

Clinical Investigation of Mitochondrial Biogenesis in Multiple Myeloma

Background: Many cancers including multiple myeloma (MM) retain more cytosolic iron to promote tumor cell growth and drug resistance. Higher cytosolic iron promotes oxidative damages due to its interaction with reactive oxygen species generated by mitochondria. Though many other pre-clinical studies for targeting mitochondria have been reported in MM, it remains unclear whether the mitochondrial biogenesis is beneficial or detrimental for tumor cells is not clear. A better understanding of the genetic makeup of mitochondria in MM cells is required if we want to use mitochondria as specific targets.Materials and Methods: Gene expression profiles (GEP) of highly purified bone marrow plasma cells were analyzed from 559 newly diagnosed patients with MM including 351 from total therapy 2 (TT2) and 208 from TT3 cohorts. Kaplan-Meier analyses of event-free survival (EFS) and overall survival (OS) about patients in the TT2 and TT3 cohorts were performed using methods implemented in the SurvExpress website. We performed GSEA and ssGSEA by using the applications available at the GenePattern platform of the Broad Institute of MIT and Harvard. Raw enrichment scores were subjected to Hierarchiacal Clustering analysis and heatmaps were generated by Hierachical Clustering Image module from Genepattern platform. For the MM murine model, NOD.Cγ-Rag1 mice 6-8 weeks old were injected intravenously or intraperitoneal injection with ARP1 MM cells expressing luciferase. After one-week injection of ARP1 cells, mice were treated with either PAA injected intraperitoneal once a day, 5 days every week for 3 weeks. The mice were euthanized when a humane endpoint was reached.Results: The variation of mitochondrial biogenesis in different stages of MM disease was evaluated using gene expression profiles in a large clinical dataset. Sixteen of 18mitochondrial biogenesis related gene sets, including mitochondrial biogenesis signature and oxidative phosphorylation, were increased in myeloma cells compared with normal plasma cells and high expression was associated with an inferior outcome. Relapsed and drug resistant myeloma samples had higher expression of mitochondrial biogenesis signatures than newly diagnosed. The expression of mitochondrial biogenesis genes was regulated by the cellular iron content, which showed a synergistic effect in patient outcome in MM. Pharmacological ascorbic acid induced myeloma cell death by inhibition of mitochondria oxidative phosphorylation in an in vivo model.Conclusion: Here, we identify that dysregulated mitochondrial biogenesis and iron homeostasis play a major role in myeloma progression and patient outcome and pharmacological ascorbic acid through cellular iron content and mitochondrial oxidative species should be considered as a novel treatment in myeloma including drug-resistant and relapsed patients. DisclosuresNo relevant conflicts of interest to declare.

Analysis of Mitochondrial Metabolic Profile As a Predictor of Clinical Aggressiveness of Acute Myeloid Leukemia

Introduction:Acute myeloid leukemia (AML) is a clonal hematopoietic malignancy characterized by dysregulated differentiation, unrestricted growth, and inhibition of apoptosis of immature myeloid progenitors. A feature in AML is increased metabolic demand despite genetic heterogeneity. Mitochondria regulate ATP generation, metabolism, apoptosis and are central to proliferation and differentiation in normal and cancer cells. The Translocase of the Outer Mitochondrial Membrane Member 20 (TOMM20), a member of the outer membrane translocator complex, plays a critical role in import of precursor proteins like nuclear encoded oxidative phosphorylation (OXPHOS) subunits from cytosol to mitochondria, required for ATP generation. TOMM20 expression and uptake of the mitotracker (a fluorescent dye taken up by mitochondria with intact membrane potential), correlate with functional mitochondrial mass and OXPHOS activity. We aim to characterize the metabolic profile of AML, using TOMM20 as a surrogate marker of mitochondrial OXPHOS, and to assess in a subgroup of subjects, differences in mitotracker uptake between AML and non-cancer cells.Methods:This is a retrospective evaluation of 67 patients with AML treated at Thomas Jefferson University between 2005 and 2015. The patients were divided into 3 cohorts with (a) relapsed disease post-hematopoietic stem cell transplantation (HSCT) [n=20], (b) remission post-HSCT [n=27] and (c) refractory disease post induction therapy [n=20].The study hypotheses were: (1) Mitochondrial metabolism measured by TOMM20 expression is increased in AML compared to normal bone marrow biopsy specimens and there is higher mitotracker uptake in AML cells than non-cancer bone marrow cells (2) Biologic behavior of AML can be predicted by TOMM20 expression with patients who relapse having higher expression than those that maintain a remission and (3) At relapse, there will be an increase in TOMM20 expression from initial diagnosis with the patient serving as his/her own control. Immunostaining of TOMM20 in bone marrow specimens was graded by two pathologists (blinded to clinical characteristics) as 1+: faint fine granular staining in areas of maximum staining, 2+: coarse granular staining with distinctly visible granules in areas of maximum staining and 3+: coarse coalesced granular staining in areas of maximum staining. Overall survival in relation to TOMM20 expression was measured.The association of TOMM20 expression with unpaired categorical data was performed using chi-square or Fisher's exact test and paired data using the non-parametric Wilcoxon rank-sum test. Kaplan-Meier test was used to evaluate survival rates and compared using the log-rank test. Mitotracker deep red mean fluorescent intensity in CD34+ and CD34 bone marrow cells in AML were compared using a paired t-test.Results:The median age at AML diagnosis was 58 (range: 20-78) years and 63% were female. Per the Revised Disease Risk Index, patients fell into low (13%), intermediate (64%) and high (23%) risk groups. In the control bone marrow specimens analyzed (n=6), only scattered myeloid precursors showed 1+ TOMM20 expression compared to AML blasts that showed uniform 1+ to 3+ staining [figure 1]. Mitotracker uptake was 7.7 fold higher (p<0.05) in AML cells than in non-cancer bone marrow cells in 5 subjects with AML whose cancer cells expressed CD34.There was no significant difference in TOMM20 expression between the 3 patient groups at diagnosis (p=0.26). In the patient group relapsing post HSCT, there was a statistically significant increase in median TOMM20 expression compared to baseline (Z=-3.2, p=0.001). Patients with high TOMM20 expression (3+) had the lowest overall survival compared to those with 1+ or 2+ expression, although not statistically significant (Log rank p value=0.217) [figure 2].Discussion:We demonstrate that AML blasts have increased TOMM20 expression and mitotracker uptake, markers of mitochondrial metabolism, compared to normal myeloid precursors. We did not observe a significant difference in TOMM20 expression at diagnosis between patient cohorts. In AML patients who relapse post HSCT, there is an increase in functional mitochondrial mass as demonstrated by increased expression of TOMM20. Mitochondrial OXPHOS inhibitors are promising oncotherapeutic agents warranting prospective evaluation in treatment strategies for relapsed and/or refractory AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The therapeutic potential of mitochondrial toxins.

When screening active compounds by phenotypic assays, we often encounter mitochondrial toxins, which are compounds that can affect mitochondrial functions. In normal cells, these toxins may have relatively low toxicity but can nonetheless show measurable effects even at low concentrations. On the other hand, in animals, mitochondrial toxins can exert severe toxicity. Mitochondrial toxins that act as inhibitors of respiratory chain complexes in oxidative phosphorylation (OXPHOS) are typically avoided during drug discovery efforts, as such compounds can directly promote lethal inhibition of pulmonary respiration. However, mitochondrial toxins could in fact have beneficial therapeutic effects. Anti-cancer strategies that target mitochondrial functions, particularly OXPHOS, have received increasing attention in recent years. In this review article we examine the significance of OXPHOS inhibitors as anti-cancer drug candidates and discuss compounds having microbial origins.

Characterization of the liver proteome in dairy cows experiencing negative energy balance at early lactation

Negative energy balance (NEB) is associated with metabolic disorders in early lactation dairy cows. Therefore, our objective was to characterize the liver proteome in cows experiencing either NEB or positive energy balance (PEB). Forty-two multiparous Holstein dairy cows were milked either 2 or 3 times daily for the first 30 days in milk (DIM) to alter EB, and were classified retrospectively as NEB (n = 18) or PEB (n = 22). Liver biopsies were collected from 10 cows (n = 5 from each milking frequency) at 17 ± 3 DIM (NEB, n = 6; PEB, n = 4). The liver proteome was characterized using label-free quantitative shotgun proteomics and Ingenuity Pathway Analysis used to identify key affected canonical pathways. Overall, 2741 proteins were identified, and 68 of those were differentially abundant (P ≤ 0.05 and FC ± 1.5). ENO3 (FC = 10.3, P < 0.01) and FABP5 (FC = -12.5, P = 0.045) were the most dramatically upregulated and downregulated proteins, respectively, in NEB cows. Numerous mitochondrial proteins (NDUFA5, NDUFS3, NDUFA6, COX7A2L, COX6C, and COA5) were differentially abundant. Canonical pathways associated with NEB were LPS/IL-1 mediated inhibition of RXR function, oxidative phosphorylation, and mitochondrial dysfunction. Additionally, cows experiencing NEB had less hepatic IL10 transcript abundance than PEB. Together, NEB was associated with altered hepatic inflammatory status, likely due to oxidative stress from mitochondrial dysfunction. SignificanceOur manuscript describes the associations of negative energy balance with the liver proteome in early lactation dairy cows, when metabolic stress and the incidence of diseases is increased. Specifically, we found associations of negative energy balance with shifts in hepatic protein abundance involved in fatty acid uptake, impaired anti-inflammatory responses, and mitochondrial dysfunction. Moving forward, differentially abundant proteins found in this study may be useful as either biological markers for disease or therapeutic targets to improve metabolic adaptations to lactation in postpartum dairy cattle.

Involvement of RSK phosphorylation in PTTH-stimulated ecdysone secretion in prothoracic glands of the silkworm, Bombyx mori.

It is well known that phosphorylation of extracellular signal-regulated kinase (ERK) is involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in insect prothoracic glands (PGs). In the present study, we further investigated the downstream signalling pathways. Our results showed that PTTH stimulated p90 ribosomal S6 kinase (RSK) phosphorylation at Thr573 in Bombyx mori PGs both in vitro and in vivo. The in vitro PTTH stimulation was stage- and dose-dependent. The absence of Ca2+ reduced PTTH-stimulated RSK phosphorylation. Stimulation of RSK phosphorylation was also observed after treatment with either A23187 or thapsigargin. A phospholipase C (PLC) inhibitor, U73122, blocked PTTH-stimulated RSK phosphorylation. These results indicate the involvement of Ca2+ and PLC. Treatment with diphenylene iodonium (DPI), a mitochondrial oxidative phosphorylation inhibitor, blocked PTTH-regulated RSK phosphorylation, indicating its redox regulation. A mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor, U0126, but not a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, decreased PTTH-stimulated RSK phosphorylation, indicating that ERK is an upstream signalling. A protein kinase C (PKC) inhibitor, chelerythrine C, inhibited PTTH-stimulated RSK phosphorylation, and a PKC activator, phorbol 12-myristate acetate (PMA) stimulated RSK phosphorylation, indicating the involvement of PKC. BI-D1870, a specific RSK inhibitor, partly prevented PTTH-stimulated RSK phosphorylation and significantly inhibited PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated RSK phosphorylation is involved in ecdysteroidogenesis. Taken together, these data indicate that PTTH activates RSK phosphorylation which plays important roles in PTTH-stimulated ecdysteroidogenesis.

Elucidating the contribution of mitochondrial glutathione to ferroptosis in cardiomyocytes

Ferroptosis is a programmed iron-dependent cell death associated with peroxidation of lipids particularly, phospholipids. Several studies suggested a possible contribution of mitochondria to ferroptosis although the mechanisms underlying mitochondria-mediated ferroptotic pathways remain elusive. Reduced glutathione (GSH) is a central player in ferroptosis that is required for glutathione peroxidase 4 to eliminate oxidized phospholipids. Mitochondria do not produce GSH, and although the transport of GSH to mitochondria is not fully understood, two carrier proteins, the dicarboxylate carrier (DIC, SLC25A10) and the oxoglutarate carrier (OGC, SLC25A11) have been suggested to participate in GSH transport. Here, we elucidated the role of DIC and OGC as well as mitochondrial bioenergetics in ferroptosis in H9c2 cardioblasts. Results showed that mitochondria are highly sensitive to ferroptotic stimuli displaying fragmentation, and lipid peroxidation shortly after the onset of ferroptotic stimulus. Inhibition of electron transport chain complexes and oxidative phosphorylation worsened RSL3-induced ferroptosis. LC-MS/MS analysis revealed a dramatic increase in the levels of pro-ferroptotic oxygenated phosphatidylethanolamine species in mitochondria in response to RSL3 (ferroptosis inducer) and cardiac ischemia-reperfusion. Inhibition of DIC and OGC aggravated ferroptosis and increased mitochondrial ROS, membrane depolarization, and GSH depletion. Dihydrolipoic acid, an essential cofactor for several mitochondrial multienzyme complexes, attenuated ferroptosis and induced direct reduction of pro-ferroptotic peroxidized phospholipids to hydroxy-phospholipids in vitro. In conclusion, we suggest that ferroptotic stimuli diminishes mitochondrial bioenergetics and stimulates GSH depletion and glutathione peroxidase 4 inactivation leading to ferroptosis.

Mitochondria-targeted hydroxyurea inhibits OXPHOS and induces antiproliferative and immunomodulatory effects

SummaryHydroxyurea (HU), an FDA-approved drug for treating sickle cell disease, is used as an antitumor drug alone and together with conventional chemotherapeutics or radiation therapy. HU is used primarily to treat myeloproliferative diseases because it inhibits the enzyme ribonucleotide reductase involved in DNA synthesis. The hydroxyl group in HU is considered critical for its antiproliferative and chemotherapeutic effects. Here, we substituted the hydroxyl group in HU with a triphenylphosphonium cation attached to an alkyl group with different chain lengths, forming a new class of mitochondria-targeted HU (Mito-HU). Elongating the alkyl side chain length increased the hydrophobicity of Mito-HUs, inhibition of oxidative phosphorylation, and antiproliferative effects in tumor cells. Both mitochondrial complex I- and complex III-induced oxygen consumption decreased with the increasing hydrophobicity of Mito-HUs. The more hydrophobic Mito-HUs also potently inhibited the monocytic myeloid-derived suppressor cells and suppressive neutrophils, and stimulated T cell response, implicating their potential antitumor immunomodulatory mechanism.

Survival upon Staphylococcus aureus mediated wound infection in Caenorhabditis elegans and the mechanism entailed

Infection following injury is one of the major threats which causes huge economic burden in wound care management all over the world. Injury often results with poor healing when coupled by following infection. In contrast to this, we observed enhanced survival of wound infected worms compared to wounded worms in Caenorhabditis elegans wound model while infecting with Staphylococcus aureus. Hence, the study was intended to identify the mechanism for the enhanced survival of wound infected worms through LCMS/MS based high throughput proteomic analysis. Bioinformatics analyses of the identified protein players indicated differential enrichment of several pathways including MAPK signaling, oxidative phosphorylation and phosphatidylinositol signaling. Inhibition of oxidative phosphorylation and phosphatidylinositol signaling through chemical treatment showed complete reversal of the enhanced survival during wound infection nevertheless mutant of MAPK pathway did not reverse the same. Consequently, it was delineated that oxidative phosphorylation and phosphatidylinositol signaling are crucial for the survival. In this regard, elevated calcium signals and ROS including O− and H2O2 were observed in wounded and wound infected worms. Consequently, it was insinuated that presence of pathogen stress could have incited survival in wound infected worms with the aid of elevated ROS and calcium signals.

Utility and Mechanism of SHetA2 and Paclitaxel for Treatment of Endometrial Cancer

Simple SummaryIncidence and death rates for endometrial cancer are steadily rising world-wide. Endometrial cancer patients at high risk for recurrence are treated with chemotherapy, which causes significant toxicity. Molecularly targeted drugs have been found to cause less toxicity than chemotherapy. We studied a low-toxicity drug, called SHetA2, which targets three heat shock A proteins that are highly mutated in endometrial cancers. Our results demonstrated that SHetA2 inhibits endometrial cancer cells and tumors, and enhances therapeutic effects of paclitaxel without increasing toxicity. This information supports development of clinical trials to test if combining SHetA2 with paclitaxel can increase the paclitaxel therapeutic effect without increasing toxicity, or allows a lowered paclitaxel dose to achieve the same level of therapeutic effect, but with reduced toxicity. Our new knowledge about how SHetA2 works can be translated into development of biomarkers to predict with patients would most likely benefit from SHetA2-based therapy.Endometrial cancer patients with advanced disease or high recurrence risk are treated with chemotherapy. Our objective was to evaluate the utility and mechanism of a novel drug, SHetA2, alone and in combination with paclitaxel, in endometrial cancer. SHetA2 targets the HSPA chaperone proteins, Grp78, hsc70, and mortalin, which have high mutation rates in endometrial cancer. SHetA2 effects on cancerous phenotypes, mitochondria, metabolism, protein expression, mortalin/client protein complexes, and cell death were evaluated in AN3CA, Hec13b, and Ishikawa endometrial cancer cell lines, and on growth of Ishikawa xenografts. In all three cell lines, SHetA2 inhibited anchorage-independent growth, migration, invasion, and ATP production, and induced G1 cell cycle arrest, mitochondrial damage, and caspase- and apoptosis inducing factor (AIF)-mediated apoptosis. These effects were associated with altered levels of proteins involved in cell cycle regulation, mitochondrial function, protein synthesis, endoplasmic reticulum stress, and metabolism; disruption of mortalin complexes with mitochondrial and metabolism proteins; and inhibition of oxidative phosphorylation and glycolysis. SHetA2 and paclitaxel exhibited synergistic combination indices in all cell lines and exerted greater xenograft tumor growth inhibition than either drug alone. SHetA2 is active against endometrial cancer cell lines in culture and in vivo and acts synergistically with paclitaxel.

Rapamycin facilitates differentiation of regulatory T cells via enhancement of oxidative phosphorylation

We explored the interplay between energy metabolism and the impact of rapamycin (Rapa) on regulatory T cell (Treg) differentiation. Naïve CD4+ T cells were stimulated under Treg-polarizing conditions with or without Rapa. Rapa promoted Treg induction, as the expression of Foxp3 and Treg phenotypic markers were enhanced. Rapa disrupts glycolysis while favoring mitochondrial metabolism in induced Tregs (iTregs). Metabolic profiling showed reduced glycolytic metabolites in Rapa-treated iTregs, in line with the downregulation of glucose uptake and the expression of glycolytic enzymes. Conversely, Rapa increased the ratios of ATP/ADP and ATP/AMP, the production of mitochondrial ATP, and the expression of ATP5A. Treatment with oxidative phosphorylation inhibitors suppressed Foxp3 expression in Rapa-treated cells. Moreover, Rapa decreased oleic acid and palmitoleic acid levels and increased l-carnitine and acetylcarnitine levels and CPT1A expression in iTregs, indicative of augmented fatty acid oxidation. In conclusion, Rapa induces metabolic reprogramming in Tregs, affecting their differentiation.

Mitochondria Targeted O2 Economizer to Alleviate Tumor Hypoxia for Enhanced Photodynamic Therapy.

Photodynamic therapy (PDT) often suffers from the exacerbated tumor hypoxia and the heterogeneous distribution of photosensitizers, leading to an inefficient ROS productivity and availability. In this work, a mitochondria targeted O2 economizer (designated as Mito-OxE) is developed to improve PDT efficiency by alleviating tumor hypoxia and enhancing the subcellular localization of photosensitizers. Specifically, the photosensitizer of protoporphyrin IX (PpIX) is modified with the hydrophilic polyethylene glycol and the lipophilic cation of triphenylphosphine (TPP) to fabricate the biocompatible mitochondria targeted photosensitizers (designated as Mito-PSs). And Mito-OxE is prepared by using Mito-PSs to load the mitochondrial oxidative phosphorylation inhibitors of atovaquone (ATO). Benefiting from the targeting capability of TPP, Mito-OxE can selectively accumulate in mitochondria after cellular uptake. Subsequently, the mitochondrial respiration would be suppressed to with the participation of ATO, resulting in a local hypoxia mitigation for enhanced PDT. Compared with Mito-PSs, Mito-OxE maximizes the therapeutic effect against hypoxic tumors under light irradiation. This design of mitochondria targeted O2 economizer would advance the development of targeted drug delivery system for effective PDT regardless of hypoxic microenvironment.

Mechanisms of K ATP channel Activation by Metabolic Stress in Mediating Lymphatic Contractile Dysfunction

Lymphatic contractile dysfunction has been implicated in the sub-clinical edema observed in multiple disease states, including obesity, metabolic syndrome and inflammatory disease. The strong positive correlation between the upregulation or hyperactivation of KATP channels in lymphatic muscle and lymphatic contractile dysfunction suggests that KATP channel activation be a common cause of the contractile dysfunction. We investigated the role of KATP channels in lymphatic muscle in response to decreases in the intracellular ATP/ADP ratio and the increased generation of reactive oxygen species (ROS), both of which occur in metabolic diseases. We analyzed spontaneous lymphatic contractions and smooth muscle cell membrane potentials in isolated, pressurized popliteal lymphatic vessels from mice. Exposure of lymphatic vessels from WT mice to the mitochondrial complex inhibitor (antimycin, 30 nM) or the oxidative phosphorylation inhibitor (CCCP, 1 µM) led to an attenuated or complete block of lymphatic pumping, which was rescued by the KATP channel inhibitor glibenclamide (GLIB, 1 µM). In contrast, lymphatic vessels from Kir6.1-/- mice were resistant to antimycin and CCCP. Antimycin inhibited spontaneous action potentials in lymphatic muscle from WT vessels and treatment of GLIB rescued action potential generation. Antimycin increased DHR fluorescence in WT lymphatic vessels, confirming the production of ROS. The ROS scavengers, tiron (1 mM) and catalase (250 U/ml) blocked the increase in DHR fluorescence to antimycin. After pretreatment with tiron (1 mM), WT lymphatic vessels were resistant to the effects of antimycin, and there were no further changes in response to GLIB, suggesting that KATP channels were activated by ROS production. Similar results were found for catalase (250 U/ml) treatment. However, neither tiron nor catalase blocked the inhibitory effect of CCCP on lymphatic pumping. Based on these results, we propose that KATP channels in lymphatic muscle are activated by directly by metabolic stress or indirectly by ROS generation, leading to the inhibition of spontaneous lymphatic contractions and lymphatic contractile dysfunction.

Proper timing of a quiescence period in precursor prospermatogonia is required for stem cell pool establishment in the male germline.

The stem cell-containing undifferentiated spermatogonial population in mammals, which ensures continual sperm production, arises during development from prospermatogonial precursors. Although a period of quiescence is known to occur in prospermatogonia prior to postnatal spermatogonial transition, the importance of this has not been defined. Here, using mouse models with conditional knockout of the master cell cycle regulator Rb1 to disrupt normal timing of the quiescence period, we found that failure to initiate mitotic arrest during fetal development leads to prospermatogonial apoptosis and germline ablation. Outcomes of single-cell RNA-sequencing analysis indicate that oxidative phosphorylation activity and inhibition of meiotic initiation are disrupted in prospermatogonia that fail to enter quiescence on a normal timeline. Taken together, these findings suggest that key layers of programming are laid down during the quiescent period in prospermatogonia to ensure proper fate specification and fitness in postnatal life.

CCCP‐induced mitochondrial dysfunction – characterization and analysis of integrated stress response to cellular signaling and homeostasis

Mitochondrial dysfunction mediated by CCCP (carbonyl cyanide m-chlorophenyl hydrazone), an inhibitor of mitochondrial oxidative phosphorylation, evokes the integrated stress response (ISR), which is analyzed here by eIF2α phosphorylation and expression profiles of ATF4 and CHOP proteins. Our findings suggest that the CCCP-induced ISR pathway is mediated by activation of HRI kinase, but not by GCN2, PERK, or PKR. Also, CCCP activates AMPK, a cellular energy sensor, and AKT, a regulator implicated in cell survival, and suppresses phosphorylation of mTORC1 substrates eIF4E-BP1 and S6K. CCCP also downregulates translation and promotes autophagy, leading to noncaspase-mediated cell death in HepG2 cells. All these events are neutralized by NAC, an anti-ROS, suggesting that CCCP-induced mitochondrial dysfunction promotes oxidative stress. ISRIB, an inhibitor of the ISR pathway, mitigates CCCP-induced expression of ATF4 and CHOP, activation of AKT, and autophagy, similar to NAC. However, it fails to reverse CCCP-induced AMPK activation, suggesting that CCCP-induced autophagy is dependent on ISR and independent of AMPK activation. ISRIB restores partly, inhibition in eIF4E-BP1 phosphorylation, promotes eIF2α phosphorylation, albeit slowly, and mitigates suppression of translation accordingly, in CCCP-treated cells. These findings are consistent with the idea that CCCP-induced oxidative stress leading to eIF2α phosphorylation and ATF4 expression, which is known to stimulate genes involved in autophagy, play a pro-survival role together with AKT activation and regulate mTOR-mediated eIF4E-BP1 phosphorylation.

Pharmaceutical Drug Metformin and MCL1 Inhibitor S63845 Exhibit Anticancer Activity in Myeloid Leukemia Cells via Redox Remodeling.

Metabolic landscape and sensitivity to apoptosis induction play a crucial role in acute myeloid leukemia (AML) resistance. Therefore, we investigated the effect of metformin, a medication that also acts as an inhibitor of oxidative phosphorylation (OXPHOS), and MCL-1 inhibitor S63845 in AML cell lines NB4, KG1 and chemoresistant KG1A cells. The impact of compounds was evaluated using fluorescence-based metabolic flux analysis, assessment of mitochondrial Δψ and cellular ROS, trypan blue exclusion, Annexin V-PI and XTT tests for cell death and cytotoxicity estimations, also RT-qPCR and Western blot for gene and protein expression. Treatment with metformin resulted in significant downregulation of OXPHOS; however, increase in glycolysis was observed in NB4 and KG1A cells. In contrast, treatment with S63845 slightly increased the rate of OXPHOS in KG1 and KG1A cells, although it profoundly diminished the rate of glycolysis. Generally, combined treatment had stronger inhibitory effects on cellular metabolism and ATP levels. Furthermore, results revealed that treatment with metformin, S63845 and their combinations induced apoptosis in AML cells. In addition, level of apoptotic cell death correlated with cellular ROS induction, as well as with downregulation of tumor suppressor protein MYC. In summary, we show that modulation of redox-stress could have a potential anticancer activity in AML cells.

Saccharomyces cerevisiae as a model to study synthetic cannabinoids: the impact of using different carbon sources

Introduction Synthetic cannabinoids (SC) are potent agonists of cannabinoid receptors, that mime the psychoactive effect of Δ9-tetrahydrocannabinol (Δ9-THC), the principal psychoactive component of cannabis [1]. JWH-018 was the first novel psychoactive SC found in the recreational drug marketplace in 2008. A few years later, JWH-018 started to be controlled by authorities, so alternative molecules started to emerge. THJ-018 was designed to replace JWH-018, having a similar structural skeleton and also a naphthalene and pentyl chain connected via a middle core substructure. Eventually, THJ-018 was scheduled and alternatives emerged, such as EG-018 [2]. This practice makes almost impossible to characterise SC toxicological profiles on an acceptable time scale, mostly due to the time-consuming experiments that must be held in animal models or human cells by standard methods. The yeast Saccharomyces cerevisiae shares highly conserved molecular and cellular mechanisms with human cells and has been used before for synthetic cathinones [3]. The present work has studied the best carbon source (glucose or galactose) to measure the impact of synthetic cannabinoids on S. cerevisiae growth, aiming to develop a method able to profile synthetic cannabinoids toxicity in a short time scale. The difference between carbon sources is that in Crabtree-positive yeast strains, glucose induce a strong inhibition of mitochondrial oxidative phosphorylation [4]. Materials and methods The effect of EG-018 was evaluated by its impact on S. cerevisiae BY4741 (WT) growth on minimal medium with 2% Glucose or 1% Galactose as sole carbon sources, in the presence and absence of the SC, followed at OD600nm. Growth rates at each condition was fitted to a logistic equation. Results Figure 1 shows OD600max obtained from the non-linear regression to the logistic equation in the presence of different concentrations of EG-018. While in galactose there is no effect, in 2% Glucose the results points to a growth decrease with EG-018. Discussion and conclusions: Comparing the two carbon sources, yeast growth on glucose is more susceptible to EG-018. Recent studies points that JWH-018, a similar SC, exerts an effect on glycolytic and pentose phosphate pathway at high concentrations of glucose (personal communication). As yeast growth in galactose proceeds simultaneously via respiration and fermentation due to the “Crabtree effect” [4] an impact of EG-018 on glycolysis might be less effective. These results, suggest that glucose is the best carbon source to develop a yeast based toxicity sensor for SC. Figure 1. OD600max results in S. cerevisiae BY4741 in the presence of 0, 25 and 50 µM of EG-018 in glucose and galactose.

Multi-omics analyses reveal that HIV-1 alters CD4+ T cell immunometabolism to fuel virus replication

Human Immunodeficiency Virus type-1 (HIV-1)-infected individuals show metabolic alterations of CD4 T cells through unclear mechanisms with undefined consequences. We analyzed the transcriptome of CD4 T cells from HIV-1 patients and revealed that elevated oxidative phosphorylation (OXPHOS) pathway is associated with poor outcomes. Inhibition of OXPHOS by the FDA-approved drug metformin, which targets mitochondrial respiratory chain complex I, suppresses HIV-1 replication in human CD4 T cells and humanized mice. In patients, HIV-1 peak viremia positively correlates with the expression of NLRX1, a mitochondrial innate immune receptor. Quantitative proteomics and metabolic analyses reveal that NLRX1 enhances OXPHOS and glycolysis during HIV-1-infection of CD4 T cells to promote viral replication. At the mechanistic level, HIV infection induces the association of NLRX1 with the mitochondrial protein, FASTKD5, to promote the expression of mitochondrial respiratory complex components. This study uncovers the OXPHOS pathway in CD4 T cells as a target for HIV-1 therapy.

ETMM-05. LACTIC ACID FACILITATES GLIOBLASTOMA GROWTH THROUGH MODULATION OF THE EPIGENOME

Glioblastoma (GBM) is the most common primary malignant brain tumor with an unfavorable prognosis. While GBMs utilize glucose, there are other carbon sources at their disposal. Lactate accumulates to a significant amount in the infiltrative margin of GBMs. In the current study, we demonstrated that lactate rescued patient-derived xenograft (PDX) GBM cells from nutrient deprivation mediated cell death and inhibition of growth. Transcriptome analysis, ATAC-seq and CHIP-seq. showed that lactic acid exposure entertained a signature of cell cycle progression and oxidative phosphorylation (OXPHOS) /tricarboxylic acid (TCA)-cycle. LC/MS analysis demonstrated that U-13C-Lactate elicited substantial labeling of TCA-cycle metabolites, acetyl-CoA and histone protein acetyl-residues in PDX derived GBM cells. Given that acetyl-CoA is pivotal for histone acetylation we observed a dose-dependent elevation of histone marks (e.g. H3K27ac), which was rescued by genetic and pharmacological inhibition of lactic acid-uptake, ATP-citrate lyase, p300 histone-acetyl-transferase and OXPHOS, resulting in reversal of lactate mediated protection from cell death. CHIP-seq. analysis demonstrated that lactic acid facilitated enhanced binding of H3K27ac to gene promoters and cis-regulatory elements. Consistently, ATAC-seq. analysis highlighted enhanced accessibility of the chromatin by lactic acid. In a combined tracer experiment (U-13C-glucose and 3-C13-lactate), we made the fundamental observation that lactic acid carbons were predominantly labeling the TCA cycle metabolites over glucose, implying a critical role of lactic acid in GBMs. Finally, pharmacological blockage of the TCA-cycle, using a clinically validated drug, extended overall survival in an orthotopic PDX model in mice without induction of toxicity, implying a critical role of lactic acid in GBMs and establishing lactic acid metabolism as a novel drug target for GBM.