High Concentrations Of ATP Research Articles

Abstract 2718: Preclinical development of humanized CD39 and CD73 blocking antibodies targeting the ATP/adenosine immune checkpoint pathway for cancer immunotherapy

Abstract Within the tumor microenvironment, adenosine causes immune suppression and dysregulation of immune cell infiltrates resulting in tumor spreading. Adenosine accumulation results from the hydrolysis of extracellular immunoactivating ATP and ADP into AMP by the CD39 (ENTPD1) ectonucleotidase. AMP can be further hydrolyzed into immunosuppressive Adenosine (Ado) by CD73 (NT5E), a cell membrane ectonucleotidase of the NTPDase family.The immunosuppressive role of CD39 expressed on both Tregs and tumor cells has been demonstrated in several reports. Conversely, CD73 expression in the tumor environment has been associated with poor disease outcome and/or with a pro-metastatic phenotype. Blockade of CD39 and CD73 may promote anti-tumor immunity directly by accumulating immunostimulating ATP for CD39 and indirectly by reducing adenosine accumulation for both targets.In cancer tissue, using IHC and flow cytometry analyses, we observed that while CD73 is often expressed by tumor cells, CD39 is more frequently up-regulated on tumor infiltrating cells compared to PBMC or adjacent non-tumor tissue. We next describe the discovery and preclinical development of a unique anti-huCD39 blocking antibody and of an anti-human CD73 antibody for cancer immunotherapy. These anti-CD39 and anti-CD73 antibodies specifically bind with high affinity to huCD39 and huCD73 proteins, respectively. They potently inhibit enzyme activity of their respective targets under their soluble and membrane-associated forms, without inducing down-modulation of these enzymes expressed at the cell surface. Innate's antibodies efficiently reverse Ado-mediated T cell suppression in vitro in presence of ATP and both CD39- and CD73-expressing immune cells and additionally exhibit unique features. The anti-CD39 Ab maintains high concentration of ATP in the extracellular compartment that enhances DC activation and subsequent T cell proliferation in vitro. The anti-CD73 blocking Ab exhibits a more potent ability to block soluble and membrane-associated CD73 enzyme activity than benchmark Abs currently in clinical development. Finally, in vivo blockade of ATP/Ado pathway in CD39ko mice resulted in improved anti-tumor efficacy of immunogenic cell death inducer chemotherapy and of immune checkpoint therapies, including PD1 and CTLA4.Taken together, these data support the clinical development of anti-CD39 and anti-CD73 neutralizing Abs for cancer immunotherapy, potentially in combination with chemotherapy or Immune Checkpoint therapy. The humanized anti-huCD39 and anti-huCD73 monoclonal antibodies are currently in preclinical development.The research leading to CD73 results were obtained within the TumAdoR collaborative consortium that received funding from the European Community's Seventh Framework Program (FP7/2007-2013) under grant agreement n°602200. Citation Format: Ivan Perrot, Marc Giraudon Paoli, Séverine Augier, Marilyne Royannez Blemont, Marion Gaudin, Frédéric Bosco, Rachel Courtois, Stephane Delahaye, Diana Jecko, Nicolas Gourdin, Maryline Salin Agu, Cyril Perrier, Paul Ricaut, Aurélie Docquier, Stéphanie Chanteux, Benjamin Rossi, Agnès Représa, Caroline Denis, Romain Remark, Cécile Bonnafous, Laurent Gauthier, Ariane Morel, Nathalie Bonnefoy, Jérémy Bastid, Yannis Morel, Carine Paturel. Preclinical development of humanized CD39 and CD73 blocking antibodies targeting the ATP/adenosine immune checkpoint pathway for cancer immunotherapy [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 2718.

Abstract 3520: Targeting lipid metabolism eradicates peritoneal metastases of ovarian cancer

Abstract Ovarian cancer is one of the deadliest women malignancies in the world. The chemoresistance accompanied with metastasis lead to high mortality of this disease. Different from other solid tumors, the advanced ovarian cancers prefer transcoelomic route in metastasis. Clinical observation indicates ovarian cancer patients with intraperitoneal metastases are closely association with poor prognosis. Emerging evidence has indicated that ascites fluids which acts as a reservoir providing plenty of growth factors, chemokines and importantly, free fatty acids etc. However, the significance of ascites microenvironment in governing ovarian cancer metastatic progression remain unclear. Herein, we report that the ascites microenvironment alters metabolism that in turn, enhances ovarian cancer cell aggressiveness. By mimicking the ascites microenvironment, we established omental conditioned medium (OCM). Ovarian cancer cells co-cultured in OCM showed enhanced ovarian cancer oncogenic capacities as observed in ascites. By RNA-Seq and proteomics analyses, we found the activities of lipid metabolism were elevated in ovarian cancer cells cultured in OCM. Depletion of Glut-1/-3/-4 by shRNAi approach or treatment of STF31, a glycolysis inhibitor, showed no or slight effect on ATP production and cell growth of ovarian cancer cells. In contrast, knockdown of Acetyl-CoA Carboxylase-α or -β (ACC1/2) or treatment of FASN inhibitor, Orlistat, significantly inhibited the ATP production and cell growth, suggesting ovarian cancer cells undergone a metabolic shift from aerobic glycolysis to β-oxidation when co-cultured in fatty acid enriched OCM. On the other hand, AMP-activated protein kinase (AMPK) is a key energy sensor and affects oncogenic capacities of many cancer cells. We found that its activity was induced initially for triggering fatty acid oxidation in ATP production. But the AMPK activity was gradually reduced due to the negative feedback loop of high ATP content. The lowered AMPK activity led to mTOR and TAK1/NF-κB signaling activation that result in the increased oncogenic capacities of ovarian cancer cells. Notably, inhibition of the above signaling pathways by a combined cocktails of AMPK activator, TAK1 and FASN inhibitors could remarkably impair OCM-mediated oncogenic properties of ovarian cancer cells in vitro and in vivo. Taken together, these findings suggest that the ascites microenvironment induces metabolic reprogramming for ovarian cancer cells in production of energy supporting their aggressiveness, while targeting the lipid metabolism and associated signalings could impede peritoneal metastases of ovarian cancer. Citation Format: Run Chen, David Chan. Targeting lipid metabolism eradicates peritoneal metastases of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3520.

The type IV pilus assembly motor PilB is a robust hexameric ATPase with complex kinetics.

The bacterial type IV pilus (T4P) is a versatile nanomachine that functions in pathogenesis, biofilm formation, motility, and horizontal gene transfer. T4P assembly is powered by the motor ATPase PilB which is proposed to hydrolyze ATP by a symmetrical rotary mechanism. This mechanism, which is deduced from the structure of PilB, is untested. Here, we report the first kinetic studies of the PilB ATPase, supporting co-ordination among the protomers of this hexameric enzyme. Analysis of the genome sequence of Chloracidobacterium thermophilum identified a pilB gene whose protein we then heterologously expressed. This PilB formed a hexamer in solution and exhibited highly robust ATPase activity. It displays complex steady-state kinetics with an incline followed by a decline over an ATP concentration range of physiological relevance. The incline is multiphasic and the decline signifies substrate inhibition. These observations suggest that variations in intracellular ATP concentrations may regulate T4P assembly and T4P-mediated functions in vivo in accordance with the physiological state of bacteria with unanticipated complexity. We also identified a mutant pilB gene in the genomic DNA of C. thermophilum from an enrichment culture. The mutant PilB variant, which is significantly less active, exhibited similar inhibition of its ATPase activity by high concentrations of ATP. Our findings here with the PilB ATPase from C. thermophilum provide the first line of biochemical evidence for the co-ordination among PilB protomers consistent with the symmetrical rotary model of catalysis based on structural studies.

The changes of bacterial communities and antibiotic resistance genes in microbial fuel cells during long-term oxytetracycline processing

Microbial fuel cell (MFC) is regarded as a promising alternative for enhancing the removal of antibiotic pollutants. In this study, oxytetracycline served as an electron donor in the anode chamber of MFCs, and after continuous operation for 330 days, the efficiency of removal of 10 mg/L oxytetracycline in MFCs increased to 99.00% in 78 h, whereas removal efficiency of only 58.26% was achieved in microbial controls. Compared to microbial controls, higher ATP concentration and persistent electrical stimulation mainly contributed to bioelectrochemical reactions more rapidly to enhance oxytetracycline removal in MFCs. In addition, the analysis of bacterial communities revealed that Eubacterium spp.—as the main functional bacterial genus responsible for oxytetracycline biodegradation—flourished starting from merely 0.00%–91.69% ± 0.27% (mean ± SD) in MFCs. High-throughput quantitative PCR showed that the normalized copy numbers of total antibiotic resistance genes (ARGs) and mobile genetic elements in MFCs were 1.7364 and 0.0065 copies/cell respectively, which were markedly lower than those in the microbial controls. Furthermore, there was no significant correlation between oxytetracycline concentration in the influent and abundance of ARGs in effluent from MFCs. Nevertheless, Tp614, a transposase gene, was found to be enriched in both MFCs and microbial reactors, suggesting that it may be a common challenge for different biological processes for wastewater treatment. This study therefore showed a lower probability of upregulation and transmission of ARGs in MFCs when compared to a traditional anaerobic microbial treatment.

Cytokines Stimulate the Release of Microvesicles from Myeloid Cells Independently from the P2X7 Receptor/Acid Sphingomyelinase Pathway.

Microvesicles (MVs) are membrane particles of 200–500 nm released by all cell types constitutively. MVs of myeloid origin are found increased in the cerebrospinal fluid (CSF) of patients suffering from neuroinflammatory disorders, although the factors triggering their production have never been defined. Here, we report that both pro- and anti-inflammatory cytokines, specifically interferon-γ and interleukin-4, are equally able to stimulate the production of MVs from microglia cells and monocytes. Additionally, we found this process to be independent from the best characterized molecular pathway so far described for membrane shedding, which is centered on the purinergic receptor P2X7, whose activation by high concentrations of extracellular ATP (exATP) results in membrane blebbing operated by the secreted enzyme acid sphingomyelinase (ASMase). Moreover, a potent inhibitor of ASMase, injected in a mouse model of multiple sclerosis, failed to reduce the number of MVs in their CSF. This suggests that cytokines, rather than exATP, may exert a long-term control of MV production in the context of chronic inflammation, where both pro- and anti-inflammatory factors play coordinated roles.

Palmitic acid induces ceramide accumulation, mitochondrial protein hyperacetylation, and mitochondrial dysfunction in porcine oocytes.

Low oocyte quality is a possible causal factor of obesity-induced infertility. High palmitic acid (PA) concentration in follicular fluid is a crucial feature noted in obese women. This study examined how high PA concentration reduced mitochondrial quality in oocytes and investigated a possible countermeasure against mitochondrial dysfunction. Cumulus cell-oocyte complexes were obtained from the ovaries of gilts, and incubated in medium containing PA (0.5 mM) or vehicle (BSA) for 44 h. Culturing oocytes at high PA concentration induced mitochondrial dysfunction determined by high reactive oxygen species and low ATP content in oocytes. Furthermore, high PA levels increased mitochondrial acetylation levels determined by a high degree of co-localization of TOMM20 and acetylated-lysine. In addition, high PA levels reduced the expression of Sirtuin 3 (SIRT3) and phosphorylated AMP-activated protein kinase (AMPK), while the AMPK activator, AICAR, restored mitochondrial function as well as oocyte ability and reduced the acetylation of mitochondrial protein. Supplementation of culture medium with dorsomorphin dihydrochloride (an AMPK inhibitor) reduced mitochondrial function and increased mitochondrial protein acetylation. Treatment of oocytes with LB100 (an inhibitor of AMPK dephosphorylation) reduced mitochondrial acetylation levels and restored mitochondrial function. Furthermore, high PA levels increased ceramide accumulation in oocytes, and addition of ceramide to the culture medium also induced mitochondrial dysfunction and increased mitochondrial acetylation. This detrimental effect of ceramide was diminished by AICAR treatment of oocytes. Our results indicated that PA induces ceramide accumulation and downregulates the AMPK/SIRT3 pathway causing mitochondrial protein hyperacetylation and dysfunction in oocytes.

Structure-Based Design of Selective Noncovalent CDK12 Inhibitors.

Cyclin-dependent kinase (CDK) 12 knockdown via siRNA decreases the transcription of DNA-damage-response genes and sensitizes BRCA wild-type cells to poly(ADP-ribose) polymerase (PARP) inhibition. To recapitulate this effect with a small molecule, we sought a potent, selective CDK12 inhibitor. Crystal structures and modeling informed hybridization between dinaciclib and SR-3029, resulting in lead compound 5 [(S)-2-(1-(6-(((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)amino)-9-ethyl-9H-purin-2-yl)piperidin-2-yl)ethan-1-ol]. Further structure-guided optimization delivered a series of selective CDK12 inhibitors, including compound 7 [(S)-2-(1-(6-(((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)amino)-9-isopropyl-9H-purin-2-yl)piperidin-2-yl)ethan-1-ol]. Profiling of this compound across CDK9, 7, 2, and 1 at high ATP concentration, single-point kinase panel screening against 352 targets at 0.1 μm, and proteomics via kinase affinity matrix technology demonstrated the selectivity. This series of compounds inhibits phosphorylation of Ser2 on the C-terminal repeat domain of RNA polymerase II, consistent with CDK12 inhibition. These selective compounds were also acutely toxic to OV90 as well as THP1 cells.

Dietary l-arginine supplementation improves semen quality and libido of boars under high ambient temperature

l-Arginine is a nutritionally essential amino acid for spermatogenesis and plays versatile roles in animal health and can be utilized as a potential agent to improve reproductive performance of boars under high ambient temperature. The present study aimed to determine whether dietary l-arginine could alleviate heat stress-induced infertility in boars. In all, 20 boars (PIC 1040; 248.59±3.84 kg BW and 407.65±6.40 days of age) were selected and randomly assigned to four groups (group 0.0%, basal diet; group 0.6%, 0.8% or 1.0%, basal diet added with 0.6%, 0.8% or 1.0% l-arginine (wt:wt), respectively.) The four diets were made isonitrogenous by addition of appropriate amounts of l-alanine. Boars were pre-fed the corresponding experimental diet for 42 days. Then, the semen characteristics and libido were accessed for 6 weeks during the hot summer period (25.5° to 33.0°C). Results show that dietary l-arginine remarkably improved sperm motility, normality, total sperm number and effective total sperm number. Also, dietary l-arginine improved semen antioxidant capacity, such as decrease of malondialdehyde and 8-Hydroxy-2'-deoxyguanosine content in sperm (P<0.05), increase of the ratio of glutathione and oxidized glutathione, total antioxidant capacity, glutathione peroxidase and catalase activities in seminal plasma (P<0.05). Most of mitochondria contained intact ultrastructure in l-arginine-supplemented group which also accompany with higher ATP content than the 0.0% group. The boars fed 0.8% l-arginine show increased levels of estradiol-17β and testosterone and exhibit improved libido performance than boars in the 0.0% group. Adding dietary l-arginine linearly increased (P=0.002) nitric oxide content (as l-arginine increased). The scrotal surface temperature in the 0.6%, 0.8% and 1.0% group were decreased by 0.9°C, 0.9°C and 0.4°C, respectively, compared with the 0.0% group. l-Arginine levels caused linear effect on semen quality and antioxidant capacity, also caused quadratic effect on libido performance. During the hot summer months, the predicted optimal l-arginine levels for best semen quality and antioxidant capacity was 0.8% to 1.0% and for best libido performance was 0.8%. It can be concluded that l-arginine can be used as an effective agent to alleviate heat stress-induced infertility of boar, and that 0.8% to 1.0% can be considered as the optimum dosage.

Abstract B173: Preclinical characterization and antitumor efficacy of DS-5010, a highly potent and selective RET inhibitor

Abstract Background: RET gene rearrangement has been detected in several cancers including 1-2% and 50% of non-small cell lung cancer (NSCLC) and papillary thyroid carcinoma, respectively, and it is known as a driver mutation. Several US Food and Drug Administration (FDA)-approved multi-tyrosine kinase inhibitors (MTKIs) have shown inhibitory effects on RET kinase activity and have been tested in several clinical studies. However, MTKIs do not appear potent enough to show clinical benefits. The FDA-approved MTKIs have been reported to exhibit dose-limiting toxicity (DLT) at doses below those suppressing RET kinase activity. Kinase insert domain receptor (KDR) inhibition especially leads to anti-angiogenesis-related DLT such as hypertension. Thus, the development of highly potent and selective second-generation RET inhibitors is desired. DS-5010 is an orally available small-molecule RET inhibitor that shows a specific and highly potent activity against RET and gatekeeper-mutated RET (RET-GKm) and slight KDR activity. In this study, we characterized the in vitro and in vivo activities of DS-5010. Results: In biochemical assays of 106 kinases, RET and platelet-derived growth factor receptor (PDGFR) alpha/beta were inhibited more than 80% by 193 nM DS-5010. The half-maximal inhibitory concentration (IC50) values of DS-5010 against RET, RET-GKm (V804L) were single digit nano-molar even under a condition of high concentration of ATP; besides it against KDR was more than 1000 nM. In a Ba/F3-RET subcutaneous tumor model, DS-5010 dosing at 10 mg/kg twice daily (bid) induced tumor regression. Moreover, DS-5010 exhibited a similar antitumor effect in a Ba/F3-RET-GKm (V804L) subcutaneous tumor model. In contrast, the FDA-approved MTKIs (cabozantinib, vandetanib, and alectinib) showed no significant antitumor effect on a Ba/F3-RET-GKm (V804L) subcutaneous tumor model. In an LC2/ad NSCLC xenograft model, which has the RET-CCDC6 fusion gene, DS-5010 dosing at 1 mg/kg thrice daily (tid) induced tumor regression. To predict acquired mutations against FDA-approved MTKIs, resistant clones were established by prolonged incubation of Ba/F3-RET cells with cabozantinib. A sequence analysis revealed that all the resistant clones possessed V804E mutation in the RET kinase domain and DS-5010 inhibited cell proliferation of Ba/F3-RET (V804E) mutation in the low nano-molar range. However, the FDA-approved MTKIs failed to show strong growth inhibitory effects (half-maximal growth inhibition [GI50]: 1584-5381 nM). Conclusion: These results indicate that DS-5010 has potent in vitro and in vivo activities against RET and RET-GKm, suggesting the potential usefulness of the compound for targeted therapy of cancers with RET gene rearrangements and mutations. Moreover, its potential effectiveness against acquired MTKI-resistant cells was also demonstrated. We are currently performing investigational new drug-enabling studies of DS-5010. Citation Format: Yasuyuki Kaneta, Takahiro Komatsu, Masashi Miyamoto, Megumi Goto, Hidenori Namiki, Yoshihiro Shibata, Hideaki Kageji, Hiroaki Inagaki, Kiyoshi Nakayama, Yuichi Tominaga, Takeshi Isoyama. Preclinical characterization and antitumor efficacy of DS-5010, a highly potent and selective RET inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B173.

PhoU2 but Not PhoU1 as an Important Regulator of Biofilm Formation and Tolerance to Multiple Stresses by Participating in Various Fundamental Metabolic Processes in Staphylococcus epidermidis.

ABSTRACTPhoU, a conserved protein that has been proposed to coordinate phosphate import, is a negative regulator of drug tolerance in most bacteria. In Staphylococcus epidermidis, the role of PhoU in biofilm formation and drug tolerance has not yet been investigated. Two PhoU homologs in the genome of S. epidermidis have been identified by the presence of the conserved motif E(D)XXXD of PhoU. We separately constructed ΔphoU1 and ΔphoU2 mutants of S. epidermidis strain 1457. The ΔphoU2 mutant displayed growth retardation, a weakened biofilm formation capacity, a higher sensitivity to H2O2, and reduced tolerance to multiple antibiotics. However, deletion of phoU1 had no effect on those. We compared the transcriptome profiles of the ΔphoU2 and ΔphoU1 mutants with that of the parent strain. In the ΔphoU2 mutant, expression of genes related to inorganic phosphate uptake was significantly upregulated (pst operon) and the levels of intracellular inorganic polyphosphate (polyP) were increased. In the ΔphoU2 mutant, expression of enzymes in the pentose phosphate pathway (PPP) was downregulated and less NADP (NADPH) was detected, consistent with the high sensitivity to H2O2 and the growth retardation of the ΔphoU2 mutant. The upregulated expression of ATP synthase was consistent with the high intracellular ATP content in the ΔphoU2 mutant, which may have been related to the lower drug tolerance of the ΔphoU2 mutant. This study demonstrates that PhoU2, but not PhoU1, in S. epidermidis regulates bacterial growth, biofilm formation, oxidative stress, and drug tolerance in association with alterations to inorganic phosphate metabolism, the pentose phosphate pathway, galactose metabolism, the tricarboxylic acid (TCA) or citric cycle, glycolysis and gluconeogenesis, and respiratory reactions.IMPORTANCE PhoU is widely conserved throughout the bacterial kingdom and plays an important role in response to stress and metabolic maintenance. In our study, two PhoU homologs were found in S. epidermidis. The function of phoU2, but not phoU1, in S. epidermidis is related to growth, drug tolerance, the oxidative stress response, polyP levels, and ATP accumulation. In addition, phoU2 regulates biofilm formation. Hence, phoU2 is a regulator of both drug tolerance and biofilm formation, which are two bacterial properties that present major challenges to the clinical treatment of infections. Analysis of differential gene expression revealed that phoU2 is involved in fundamental metabolic processes, such as the PPP pathway. These findings indicate that phoU2 is a crucial regulator in S. epidermidis.

Co-opting ATP-generating glycolytic enzyme PGK1 phosphoglycerate kinase facilitates the assembly of viral replicase complexes.

The intricate interactions between viruses and hosts include exploitation of host cells for viral replication by using many cellular resources, metabolites and energy. Tomato bushy stunt virus (TBSV), similar to other (+)RNA viruses, induces major changes in infected cells that lead to the formation of large replication compartments consisting of aggregated peroxisomal and ER membranes. Yet, it is not known how TBSV obtains the energy to fuel these energy-consuming processes. In the current work, the authors discovered that TBSV co-opts the glycolytic ATP-generating Pgk1 phosphoglycerate kinase to facilitate the assembly of new viral replicase complexes. The recruitment of Pgk1 into the viral replication compartment is through direct interaction with the viral replication proteins. Altogether, we provide evidence that the ATP generated locally within the replication compartment by the co-opted Pgk1 is used to fuel the ATP-requirement of the co-opted heat shock protein 70 (Hsp70) chaperone, which is essential for the assembly of new viral replicase complexes and the activation of functional viral RNA-dependent RNA polymerase. The advantage of direct recruitment of Pgk1 into the virus replication compartment could be that the virus replicase assembly does not need to intensively compete with cellular processes for access to ATP. In addition, local production of ATP within the replication compartment could greatly facilitate the efficiency of Hsp70-driven replicase assembly by providing high ATP concentration within the replication compartment.

Myosin V: Chemomechanical-coupling ratchet with load-induced mechanical slip

A chemomechanical-network model for myosin V is presented on the basis of both the nucleotide-dependent binding affinity of the head to an actin filament (AF) and asymmetries and similarity relations among the chemical transitions due to an intramolecular strain of the leading and trailing heads. The model allows for branched chemomechanical cycles and takes into account not only two different force-generating mechanical transitions between states wherein the leading head is strongly bound and the trailing head is weakly bound to the AF but also load-induced mechanical-slip transitions between states in which both heads are strongly bound. The latter is supported by the fact that ATP-independent high-speed backward stepping has been observed for myosin V, although such motility has never been for kinesin. The network model appears as follows: (1) the high chemomechanical-coupling ratio between forward step and ATP hydrolysis is achieved even at low ATP concentrations by the dual mechanical transitions; (2) the forward stepping at high ATP concentrations is explained by the front head-gating mechanism wherein the power stroke is triggered by the inorganic-phosphate (Pi) release from the leading head; (3) the ATP-binding or hydrolyzed ADP.Pi-binding leading head produces a stable binding to the AF, especially against backward loading.

The Warburg effect as an adaptation of cancer cells to rapid fluctuations in energy demand.

To maintain optimal fitness, a cell must balance the risk of inadequate energy reserve for response to a potentially fatal perturbation against the long-term cost of maintaining high concentrations of ATP to meet occasional spikes in demand. Here we apply a game theoretic approach to address the dynamics of energy production and expenditure in eukaryotic cells. Conventionally, glucose metabolism is viewed as a function of oxygen concentrations in which the more efficient oxidation of glucose to CO2 and H2O produces all or nearly all ATP except under hypoxic conditions when less efficient (2 ATP/ glucose vs. about 36ATP/glucose) anaerobic metabolism of glucose to lactic acid provides an emergency backup. We propose an alternative in which energy production is governed by the complex temporal and spatial dynamics of intracellular ATP demand. In the short term, a cell must provide energy for constant baseline needs but also maintain capacity to rapidly respond to fluxes in demand particularly due to external perturbations on the cell membrane. Similarly, longer-term dynamics require a trade-off between the cost of maintaining high metabolic capacity to meet uncommon spikes in demand versus the risk of unsuccessfully responding to threats or opportunities. Here we develop a model and computationally explore the cell’s optimal mix of glycolytic and oxidative capacity. We find the Warburg effect, high glycolytic metabolism even under normoxic conditions, is represents a metabolic strategy that allow cancer cells to optimally meet energy demands posed by stochastic or fluctuating tumor environments.

Effects of Catalytic Action and Ligand Binding on Conformational Ensembles of Adenylate Kinase.

Crystal structures of adenylate kinase (AdK) from Escherichia coli capture two states: an "open" conformation (apo) obtained in the absence of ligands and a "closed" conformation in which ligands are bound. Other AdK crystal structures suggest intermediate conformations that may lie on the transition pathway between these two states. To characterize the transition from open to closed states in solution, X-ray solution scattering data were collected from AdK in the apo form and with progressively increasing concentrations of five different ligands. Scattering data from apo AdK are consistent with scattering predicted from the crystal structure of AdK in the open conformation. In contrast, data from AdK samples saturated with Ap5A do not agree with that calculated from AdK in the closed conformation. Using cluster analysis of available structures, we selected representative structures in five conformational states: open, partially open, intermediate, partially closed, and closed. We used these structures to estimate the relative abundances of these states for each experimental condition. X-ray solution scattering data obtained from AdK with AMP are dominated by scattering from AdK in the open conformation. For AdK in the presence of high concentrations of ATP and ADP, the conformational ensemble shifts to a mixture of partially open and closed states. Even when AdK is saturated with Ap5A, a significant proportion of AdK remains in a partially open conformation. These results are consistent with an induced-fit model in which the transition of AdK from an open state to a closed state is initiated by ATP binding.

Neuronal P2X7 Receptors Revisited: Do They Really Exist?

P2X7 receptors (Rs) constitute a subclass of ATP-sensitive ionotropic receptors (P2X1-P2X7). P2X7Rs have many distinguishing features, mostly based on their long intracellular C terminus regulating trafficking to the cell membrane, protein-protein interactions, and post-translational modification. Their C-terminal tail is especially important in enabling the transition from the nonselective ion channel mode to a membrane pore allowing the passage of large molecules. There is an ongoing dispute on the existence of neuronal P2X7Rs with consequences for our knowledge on their involvement in neuroinflammation, aggravating stroke, temporal lobe epilepsy, neuropathic pain, and various neurodegenerative diseases. Whereas early results appeared to support the operation of P2X7Rs at neurons, more recently glial P2X7Rs are increasingly considered as indirect causes of neuronal effects. Specific tools for P2X7Rs are of limited value because of the poor selectivity of agonists, and the inherent failure of antibodies to differentiate between the large number of active and inactive splice variants, or gain-of-function and loss-of-function small nucleotide polymorphisms of the receptor. Unfortunately, the available P2RX7 knock-out mice generated by pharmaceutical companies possess certain splice variants, which evade inactivation. In view of the recently discovered bidirectional dialogue between astrocytes and neurons (and even microglia and neurons), we offer an alternative explanation for previous data, which assumedly support the existence of P2X7Rs at neurons. We think that the unbiased reader will follow our argumentation on astrocytic or microglial P2X7Rs being the primary targets of pathologically high extracellular ATP concentrations, although a neuronal localization of these receptors cannot be fully excluded either.

Coordinated force generation of skeletal myosins in myofilaments through motor coupling

In contrast to processive molecular motors, skeletal myosins form a large motor ensemble for contraction of muscles against high loads. Despite numerous information on the molecular properties of skeletal myosin, its ensemble effects on collective force generation have not been rigorously clarified. Here we show 4 nm stepwise actin displacements generated by synthetic myofilaments beyond a load of 30 pN, implying that steps cannot be driven exclusively by single myosins, but potentially by coordinated force generations among multiple myosins. The simulation model shows that stepwise actin displacements are primarily caused by coordinated force generation among myosin molecules. Moreover, the probability of coordinated force generation can be enhanced against high loads by utilizing three factors: strain-dependent kinetics between force-generating states; multiple power stroke steps; and high ATP concentrations. Compared with other molecular motors, our findings reveal how the properties of skeletal myosin are tuned to perform cooperative force generation for efficient muscle contraction.

Abstract 1968: A novel mechanism of metastasis: Extracellular ATP promotes invasion and metastasis independent of purinergic receptor signaling

Abstract Cancer is one of the top deadliest diseases in the US and worldwide. Metastasis, the dissemination of cancer cells from the primary tumors to distant organs, is responsible for 90% of solid tumor-related deaths. Tumor invasion and metastasis is a multistep process in which loss of cell-cell adhesion, increased proteolysis, and cell motility has been shown to be critical steps. Extracellular ATP is potentially a very important factor involved in tumor invasion and metastasis because both cell detachment and migration require energy, which is known to be mainly provided by ATP. Recent studies have indicated that the extracellular microenvironment of tumors contains much higher concentrations of ATP than normal tissues of the same cell origins. Our recent studies demonstrated that extracellular ATP is taken up by cancer cells through macropinocytosis and other endocytosis and promotes cancer cell growth, survival, and drug resistance (1-3). Based on all these, we hypothesized that extracellular ATP (eATP) plays critical roles in the regulation of tumor cell detachment, motility, invasion and tumor metastasis initiation. It was previously shown that ATP-induced purinergic receptor (PR) signaling is involved in metastasis. Our hypothesis is different in that eATP also mediates invasion and metastasis independent of PR signaling. Various bioassays were used in human non-small cell lung cancer (NSCLC) A549 cells to test our hypothesis. Our results show that eATP treatment led to a substantially increased number of floating cancer cells, and these cells were viable and formed clones in a clonogenic assay. This indicates that eATP induces cancer cell detachment. Moreover, treatment of eATP also induces cell migration in cell wounding and Transwell migration assays. In vitro invasion assay showed that eATP induced a dose-and time-dependent increase in the invasive capacities of the A549 cells. Western blot analysis indicates that eATP treatment reduced the expression of cell-cell adhesion molecule E-cadherin. PR inhibitors only slightly attenuated these effects. All these suggest novel ATP mechanisms independent of the PR signaling that are unreported before and imply novel targets for inhibiting/preventing metastasis.

Abstract 4186: A biochemical approach to discriminate between ATP-competitive and non-ATP competitive protein kinase inhibitors

Abstract Protein kinases are central to cellular signal transduction and regulation of cellular processes, and are one of the most attractive target classes in modern drug discovery. Multiple kinase inhibitors have already been approved for treatment of various diseases including such severe conditions as cancer. The first and up to today largest group of drugs that effectively inhibits their respective kinase targets belong to the class of ATP competitive compounds. They bind into or near the ATP binding site of the enzymes and inhibit kinase activity by blocking access of ATP to the active site. Although there are numerous examples of highly specific ATP competitive compounds this mode of action is limited by several factors: The ATP binding pocket structures of kinases show a high degree of similarity, which makes finding highly selective compounds challenging. Furthermore, competing with ATP for binding to the same target site, compounds have to be of very high affinity due to the high intracellular ATP concentrations. Therefore, the interest to develop non-ATP competitive inhibitors has risen considerably over the last years. Such inhibitors bind to kinases at sites apart from the ATP binding site, inhibiting their activity e.g. by stabilizing an inactive conformation (like DFG-out state binders), displacing essential cofactors (like cyclins for CDKs) or by blocking activating modifications (like phosphorylation by upstream kinases). We present data of an in-vitro biochemical kinase activity assay setup which is suited to discriminate between ATP-competitive and non-ATP competitive inhibitors. The IC50 of an ATP-competitive inhibitor will increase with increasing ATP concentrations and the IC50 value at a given ATP concentration may be calculated using the equation of Cheng and Prussof: IC50=Ki+(Ki*[ATP]/KM[ATP] (Cheng Y., Prusoff W. H. (1973) Biochem. Pharmacol. 22: 3099-3108). By determining IC50 values for an inhibitor of a specific kinase at different ATP concentrations we examined whether the IC50 value changed according to the Cheng-Prusoff equation, indicating an ATP-competitive mode of action, or if the IC50 values remained unchanged in presence of elevated ATP indicating a non-ATP competitive or mixed type mode of action. In our assay setup we determined the IC50 values at ATP concentrations in a range of 0.1 to 10 fold the ATP KM of the kinase of interest. By comparing the results obtained for the non-ATP competitive MEK1 inhibitor selumetinib and the ATP competitive inhibitor staurosporine we could verify that our assay setup is well suited to discriminate between these different types of kinase inhibitors. Citation Format: Daniel Mueller, Frank Totzke, Thomas Weber, Carolin Heidemann-Dinger, Constance Ketterer, Diane Krämer, Marcel Pathe, Michael H. Kubbutat. A biochemical approach to discriminate between ATP-competitive and non-ATP competitive protein kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4186. doi:10.1158/1538-7445.AM2017-4186

Abstract 3194: A new mechanism of drug resistance in cancer: extracellular ATP-induced resistance through ATP internalization and upregulation of protein phosphorylation in Akt and ERK pathways

Abstract The opportunistic uptake of extracellular molecules has been named as a key emerging hallmark of cancer metabolism[1]. Extracellular ATP (eATP) levels of various cancer types are 103 to 104 times higher than those in their corresponding normal tissues[2]. However, the biological significance of the high ATP concentrations is not clear. We recently reported that cancer cells uptake extracellular ATP via different endocytoses to enhance growth, survival, and drug resistance to tyrosine kinase inhibitors (TKIs)[3 ,4]. We hypothesized that eATP induces resistance to TKIs by endocytoses-mediated internalization of eATP, which competes with TKIs for the ATP-binding site of receptor TKs (RTKs), phosphorylating and activating RTKs and RTK-mediated signaling pathways. In contrast to previous reports that synthesized intracellular ATP (iATP) elevation contributed acquired drug resistance, here we report a novel intrinsic drug resistance in which eATP was internalized and substantially increased iATP levels and promoted cancer cell drug resistance to the TKI sunitinib in human NSCLC A549 cells. The iATP level elevation and drug resistance were mediated primarily by macropinocytosis. The resistance was reduced when macropinocytosis was suppressed by inhibitors or an siRNA knockdown of a key micropinocytosis protein PAK1. Intracellularly, the elevated iATP upregulated phosphorylation of PDGFR and proteins/enzymes in the PDGFR-mediated Akt-mTOR and Raf-ERK signaling pathways, resulting in reduced apoptosis triggered by sunitinib. Furthermore, both in vitro and in A549 tumors, eATP partially restored phosphorylation levels of PDGFR and PDGFR-mediated proteins/enzymes suppressed by sunitinib. The resistance cannot be accounted for by the overall purinergic receptor-mediated signaling, glycolysis, or mitochondrial OXPHOS. These results strongly suggest that the eATP-elevated intracellular ATP levels reversed the inhibition of TKIs by using the mechanism we hypothesized, linking for the first time the ATP-rich tumor microenvironment with cancer drug resistance. All these findings significantly expand our understanding of the roles of extracellular ATP in cancer, and offer new anti-drug resistance targets. 1. Pavlova N, Thompson C. Cell Metab. (2016) 23:27-47. 2. Falzoni et al., Interface Focus.(2013) 3: 20120101. 3. Qian Y, Wang X, et al., Cancer Lett. (2014)351: 242-5. 4. Qian Y, Wang X et al., Mol Cancer Res (2016) 14:1087-96 Citation Format: Xuan Wang, Yunsheng Li, Yanrong Qian, Yanyang Cao, Xiaozhuo Chen. A new mechanism of drug resistance in cancer: extracellular ATP-induced resistance through ATP internalization and upregulation of protein phosphorylation in Akt and ERK pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3194. doi:10.1158/1538-7445.AM2017-3194

Abstract 979: Discovery of the JAK1 selective kinase inhibitor AZD4205

Abstract Janus kinases are a family of four enzymes; JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) that are critical in cytokine signalling, with constitutive activation of JAK/STAT pathways associated with a wide variety of malignancies. Elevated JAK/STAT signalling leading to increased activation of STAT3 is reported in a wide variety of cancers, including breast, liver, prostate, colorectal, head and neck, oesophageal, pancreatic, bladder, and non-small cell lung, and is implicated in the pathogenesis of diffuse large B-cell lymphoma and nasopharyngeal carcinomas. Overall, up to 70% of human tumours are linked to persistent elevated STAT3 activity which can be associated with poorer prognosis in many of these settings. In addition, elevated pSTAT3 is observed in response to chemotherapy treatment, and also in response to treatment with inhibitors of oncogenic signalling pathways such as EGFR, MAPK and AKT, and is associated with resistance or poorer response to agents targeting these pathways. In many of these cases, JAK1 is believed to be a primary driver of STAT3 phosphorylation and signalling, suggesting inhibition of JAKs as a therapeutic approach to treat these potential resistance mechanisms. The mixed JAK1/2 kinase inhibitor ruxolitinib is approved for the treatment of myeloproliferative neoplasms including intermediate or high risk myelofibrosis and polycythemia vera and has been tested in a variety of tumor settings. Since JAK2 is essential for the signal transduction downstream of erythropoietin, thrombopoietin and related receptors that control erythrocyte and megakaryocyte expansion, dosing of inhibitors that target JAK2 can be limited by toxicities such as thrombocytopenia and anaemia. Starting from a non-kinome selective screening hit, structure-based design was used to optimise a series of aminopyrimidines that led to JAK1-selective candidate drug AZD4205. This compound demonstrates ATP competitive binding with IC50’s in a high ATP concentration enzyme assay against JAK1 of 73 nM (Ki = 2.8 nM), with high selectivity against JAK2 and JAK3 with IC50’s of 13,233 nM and &amp;gt;30,000 nM respectively. In addition it showed potent inhibition of p-STAT3 in a cell based assay of JAK1 activity with an IC50 of 128 nM and excellent selectivity across the kinome. In summary, AZD4205 is a highly potent JAK1-selective kinase inhibitor with excellent preclinical pharmacokinetics with potential for further clinical development. The optimization from screening hit to first disclosure of this candidate drug will be presented. Citation Format: Jason G. Kettle, Qibin Su, Neil Grimster, Sameer Kawatkar, Scott Throner, Richard Woessner, Huawei Chen, Geraldine Bebernitz, Kristen Bell, Erica Anderson, Linette Ruston, Jon Winter-Holt, Paul Lyne, Melissa Vasbinder, Claudio Chuaqui. Discovery of the JAK1 selective kinase inhibitor AZD4205 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 979. doi:10.1158/1538-7445.AM2017-979