Cell-specific Activity Research Articles

Frequency-Specific Optogenetic Deep Brain Stimulation of Subthalamic Nucleus Improves Parkinsonian Motor Behaviors.

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is an effective therapy for the motor symptoms of Parkinson's disease (PD). However, the neural elements mediating symptom relief are unclear. A previous study concluded that direct optogenetic activation of STN neurons was neither necessary nor sufficient for relief of parkinsonian symptoms. However, the kinetics of the channelrhodopsin-2 (ChR2) used for cell-specific activation are too slow to follow the high rates required for effective DBS, and thus the contribution of activation of STN neurons to the therapeutic effects of DBS remains unclear. We quantified the behavioral and neuronal effects of optogenetic STN DBS in female rats following unilateral 6-hydroxydopamine (6-OHDA) lesion using an ultrafast opsin (Chronos). Optogenetic STN DBS at 130 pulses per second (pps) reduced pathologic circling and ameliorated deficits in forelimb stepping similarly to electrical DBS, while optogenetic STN DBS with ChR2 did not produce behavioral effects. As with electrical DBS, optogenetic STN DBS exhibited a strong dependence on stimulation rate; high rates produced symptom relief while low rates were ineffective. High-rate optogenetic DBS generated both increases and decreases in firing rates of single neurons in STN, globus pallidus externa (GPe), and substantia nigra pars reticular (SNr), and disrupted β band oscillatory activity in STN and SNr. High-rate optogenetic STN DBS can indeed ameliorate parkinsonian motor symptoms through reduction of abnormal oscillatory activity in the STN-associated neural circuit, and these results highlight that the kinetic properties of opsins have a strong influence on the effects of optogenetic stimulation.SIGNIFICANCE STATEMENT Whether STN local cells contribute to the therapeutic effects of subthalamic nucleus (STN) deep brain stimulation (DBS) in Parkinson's disease (PD) remains unclear. We re-examined the role of STN local cells in mediating the symptom-relieving effects of STN DBS using cell type-specific optogenetic stimulation with a much faster opsin, Chronos. Direct optogenetic stimulation of STN neurons was effective in treating the symptoms of parkinsonism in the 6-hydroxydopamine (6-OHDA) lesion rat. These results highlight that the kinetic properties of opsins can have a strong influence on the effects of optogenetic activation/inhibition and must be considered when employing optogenetic to study high-rate neural stimulation.

B Cell-Specific Transcription Activator PAX5 Recruits p300 To Support EBNA1-Driven Transcription.

The binding of Epstein-Barr Virus (EBV) nuclear antigen 1 (EBNA1) to the latent replication origin (oriP) triggers multiple downstream events to support virus-induced pathogenesis and tumorigenesis. Although EBV is widely recognized as a B-lymphotropic infectious agent, little is known about how tissue-specific factors are involved in the establishment of latency. Here, we showed that EBNA1 binds B cell activator PAX5 to promote EBNA1/oriP-dependent binding and transcription. In addition to showing that short hairpin RNA (shRNA)-mediated PAX5 knockdown substantially abrogated the above EBNA1-dependent functions, two mini-EBV reporter plasmids were used to perform nonlytic nano-luciferase (nLuc) activity and chromatin immunoprecipitation (ChIP) assays to show how EBNA1 cooperates with PAX5 to activate the transcription at the oriP site. The expression plasmids of two PAX5 mutants, V26G (EBNA1 binding mutant) and P80R (which remained EBNA1 associated), were used to assess their capability to restore the defects caused by PAX5 depletion in EBNA1/oriP-mediated binding, transcription, and maintenance of the genome copy number of the mini-EBV episome reporter in BJAB cells stably expressing EBNA1 or that of the EBV genome in EBV-infected BJAB cells. Since p300 is known to be associated with PAX5, we showed that the loss of function of the P80R mutant in support of EBNA1/oriP-mediated transcription under PAX5 depletion conditions was linked to its defective binding to p300. ChIP-quantitative PCR (qPCR) confirmed that P80R indeed failed to recruit p300 to the oriP DNA. Our discovery suggests that EBV has evolved an exquisite strategy to take advantage of tissue-specific factors to enable the establishment of viral latency.IMPORTANCE Although B cells are known to be the primary target for EBV infection, there is limited knowledge regarding the mechanism that determines this preferable tissue tropism. An in-depth understanding of the potential link of tissue-specific factors with the viral genes and their functioning is key to deciphering how EBV induces persistent infection in the distinct types of host cells. In this study, a substantial protein-protein interaction mediated by the B cell-specific activator PAX5 and EBNA1 was identified as the general requirement for the binding of EBNA1 to the latent replication origin and for downstream events. Of importance, the EBNA1-PAX5-p300 network is directly linked to EBNA1-dependent transcription. These findings suggest that targeting the viral gene-associated tissue-specific factors may lead to new therapeutic strategies for EBV-associated malignancies.

Weekly variations of viruses and heterotrophic nanoflagellates and their potential impact on bacterioplankton in shallow waters of the central Red Sea.

ABSTRACTBacterioplankton play a pivotal role in marine ecosystems. However, their temporal dynamics and underlying control mechanisms are poorly understood in tropical regions such as the Red Sea. Here, we assessed the impact of bottom-up (resource availability) and top-down (viruses and heterotrophic nanoflagellates) controls on bacterioplankton abundances by weekly sampling a coastal central Red Sea site in 2017. We monitored microbial abundances by flow cytometry together with a set of environmental variables including temperature, salinity, dissolved organic and inorganic nutrients and chlorophyll a. We distinguished five groups of heterotrophic bacteria depending on their physiological properties relative nucleic acid content, membrane integrity and cell-specific respiratory activity, two groups of Synechococcus cyanobacteria and three groups of viruses. Viruses controlled heterotrophic bacteria for most of the year, as supported by a negative correlation between their respective abundances and a positive one between bacterial mortality rates and mean viral abundances. On the contrary, heterotrophic nanoflagellates abundance covaried with that of heterotrophic bacteria. Heterotrophic nanoflagellates showed preference for larger bacteria from both the high and low nucleic acid content groups. Our results demonstrate that top-down control is fundamental in keeping heterotrophic bacterioplankton abundances low (< 5 × 10 5 cells mL−1) in Red Sea coastal waters.

Wireless, battery-free subdermally implantable photometry systems for chronic recording of neural dynamics

Recording cell-specific neuronal activity while monitoring behaviors of freely moving subjects can provide some of the most significant insights into brain function. Current means for monitoring calcium dynamics in genetically targeted populations of neurons rely on delivery of light and recording of fluorescent signals through optical fibers that can reduce subject mobility, induce motion artifacts, and limit experimental paradigms to isolated subjects in open, two-dimensional (2D) spaces. Wireless alternatives eliminate constraints associated with optical fibers, but their use of head stages with batteries adds bulk and weight that can affect behaviors, with limited operational lifetimes. The systems introduced here avoid drawbacks of both types of technologies, by combining highly miniaturized electronics and energy harvesters with injectable photometric modules in a class of fully wireless, battery-free photometer that is fully implantable subdermally to allow for the interrogation of neural dynamics in freely behaving subjects, without limitations set by fiber optic tethers or operational lifetimes constrained by traditional power supplies. The unique capabilities of these systems, their compatibility with magnetic resonant imaging and computed tomography and the ability to manufacture them with techniques in widespread use for consumer electronics, suggest a potential for broad adoption in neuroscience research.

Identification of an Embryonic Cell-Specific Region within the Pineapple SERK1 Promoter.

Plant tissue culture methods, such as somatic embryogenesis, are attractive alternatives to traditional breeding methods for plant propagation. However, they often suffer from limited efficiency. Somatic embryogenesis receptor kinase (SERK)1 is a marker gene of early somatic embryogenesis in several plants, including pineapple. It can be selectively induced and promotes a key step in somatic embryogenesis. We investigated the embryonic cell-specific transcriptional regulation of AcSERK1 by constructing a series of vectors carrying the GUS (Beta-glucuronidase) reporter gene under the control of different candidate cis-regulatory sequences. These vectors were transfected into both embryonic and non-embryonic callus, and three immature embryo stages and the embryonic-specific activity of the promoter fragments was analyzed. We found that the activity of the regulatory sequence of AcSERK1 lacking −983 nt ~−880 nt, which included the transcription initiation site, was significantly reduced in the embryonic callus of pineapple, accompanied by the loss of embryonic cell-specific promoter activity. Thus, this fragment is an essential functional segment with highly specific promoter activity for embryonic cells, and it is active only from the early stages of somatic embryo development to the globular embryo stage. This study lays the foundation for identifying mechanisms that enhance the efficiency of somatic embryogenesis in pineapple and other plants.

Aryl hydrocarbon receptor modulates stroke-induced astrogliosis and neurogenesis in the adult mouse brain

BackgroundThe aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor activated by environmental agonists and dietary tryptophan metabolites for the immune response and cell cycle regulation. Emerging evidence suggests that AHR activation after acute stroke may play a role in brain ischemic injury. However, whether AHR activation alters poststroke astrogliosis and neurogenesis remains unknown.MethodsWe adopted conditional knockout of AHR from nestin-expressing neural stem/progenitor cells (AHRcKO) and wild-type (WT) mice in the permanent middle cerebral artery occlusion (MCAO) model. WT mice were treated with either vehicle or the AHR antagonist 6,2′,4′-trimethoxyflavone (TMF, 5 mg/kg/day) intraperitoneally. The animals were examined at 2 and 7 days after MCAO.ResultsThe AHR signaling pathway was significantly upregulated after stroke. Both TMF-treated WT and AHRcKO mice showed significantly decreased infarct volume, improved sensorimotor, and nonspatial working memory functions compared with their respective controls. AHR immunoreactivities were increased predominantly in activated microglia and astrocytes after MCAO compared with the normal WT controls. The TMF-treated WT and AHRcKO mice demonstrated significant amelioration of astrogliosis and microgliosis. Interestingly, these mice also showed augmentation of neural progenitor cell proliferation at the ipsilesional neurogenic subventricular zone (SVZ) and the hippocampal subgranular zone. At the peri-infarct cortex, the ipsilesional SVZ/striatum, and the hippocampus, both the TMF-treated and AHRcKO mice demonstrated downregulated IL-1β, IL-6, IFN-γ, CXCL1, and S100β, and concomitantly upregulated Neurogenin 2 and Neurogenin 1.ConclusionNeural cell-specific AHR activation following acute ischemic stroke increased astrogliosis and suppressed neurogenesis in adult mice. AHR inhibition in acute stroke may potentially benefit functional outcomes likely through reducing proinflammatory gliosis and preserving neurogenesis.

In Vitro Cytotoxicity of Oleanolic/Ursolic Acids-Loaded in PLGA Nanoparticles in Different Cell Lines.

Oleanolic (OA) and ursolic (UA) acids are recognized triterpenoids with anti-cancer properties, showing cell-specific activity that can be enhanced when loaded into polymeric nanoparticles. The cytotoxic activity of OA and UA was assessed by Alamar Blue assay in three different cell lines, i.e., HepG2 (Human hepatoma cell line), Caco-2 (Human epithelial colorectal adenocarcinoma cell line) and Y-79 (Human retinoblastoma cell line). The natural and synthetic mixtures of these compounds were tested as free and loaded in polymeric nanoparticles in a concentration range from 2 to 32 µmol/L. The highest tested concentrations of the free triterpene mixtures produced statistically significant cell viability reduction in HepG2 and Caco-2 cells, compared to the control (untreated cells). When loaded in the developed PLGA nanoparticles, no differences were recorded for the tested concentrations in the same cell lines. However, in the Y-79 cell line, a decrease on cell viability was observed when testing the lowest concentration of both free triterpene mixtures, and after their loading into PLGA nanoparticles.

Generation of Amelx-iCre Mice Supports Ameloblast-Specific Role for Stim1

The identification and targeting of the molecular pathways regulating amelogenesis is an ongoing challenge in dental research, and progress has been restricted by the limited number of genetic tools available to study gene function in ameloblasts. Here, we generated 4 transgenic Cre-driver mouse lines that express improved Cre (iCre)–recombinase from the locus of the mouse ameloblast-specific gene amelogenin X (Amelx-iCre) with a large (250-kb) bacterial artificial chromosome DNA vector. All 4 Amelx-iCre transgenic lines were bred with ROSA26 reporter mice to characterize the iCre developmental pattern with the LacZ gene encoding β-galactosidase enzyme activity assay and Cre protein immunohistochemistry. From the 4 generated transgenic lines, 2 were selected for further analysis because they expressed a high amount of Cre recombinase exclusively in ameloblasts and showed developmental stage- and cell-specific β-galactosidase activity mimicking the endogenous amelogenin expression. To test the functionality of the selected transgenic models, we bred the 2 Amelx-iCre mice lines with stromal interaction molecule 1 (Stim1) floxed mice to generate ameloblast-specific Stim1 conditional knockout mice (Stim1 cKO). STIM1 protein serves as one of the main calcium sensors in ameloblasts and plays a major role in enamel mineralization and ameloblast differentiation. Amelx-iCre mice displayed exclusive CRE-mediated recombination in incisor and molar ameloblasts. Stim1 cKO mice showed a severely defected enamel phenotype, including reduced structural integrity concomitant with increased attrition and smaller teeth. The phenotype and genotype of the Amelx-iCre/Stim1 cKO showed significant differences with the previously reported Ker14-Cre/Stim1 cKO, highlighting the need for cell- and stage-specific Cre lines for an accurate phenotype-genotype comparison. Furthermore, our model has the advantage of carrying the entire Amelx gene locus rather than being limited to an Amelx partial promoter construct, which greatly enhances the stability and the specificity of our Cre expression. As such, the Amelx-iCre transgenic lines that we developed may serve as a powerful tool for targeting ameloblast-specific gene expression in future investigations.

Cell-Type-Specific CRISPR Activation with MicroRNA-Responsive AcrllA4 Switch.

Anti-CRISPR proteins have the potential to regulate CRISPR-Cas systems in a cell-type-specific manner. To selectively edit the genome in target cells, we controlled the expression of AcrllA4, a Streptococcus pyogenes Cas9 inhibitor, based on endogenous microRNA (miRNA) activity. We designed a miRNA-responsive AcrllA4 switch, which is a synthetic mRNA that contains a completely complementary sequence to an arbitrary miRNA at the 5'-UTR region and encodes AcrllA4. Together with the Cas9- or dCas9-VPR-guide RNA complex, this switch functions as a cell-specific Cas9 or dCas9-VPR activator that induces gene knockout or activation depending on the target miRNA. By sensing intracellular miRNAs, the conditional CRISPR-Cas9 ON system that we report could provide a powerful tool for future therapeutic applications and genome engineering.

Abstract 2244: DeepIL-15 primed T cells synergize with PD-L1 blockade to overcome resistance to checkpoint immunotherapy

Abstract Introduction Interleukin-15 (IL-15) activates and expands both CD8+T cells and NK cells but not immunosuppressive Tregcells. While IL-15 is an attractive asset for cancer immunotherapy, its systemic administration is limited by toxicities. To limit IL-15 systemic exposure, we have developed DeepTM IL-15, a multimer of chemically crosslinked IL-15/IL-15 Rα/Fc heterodimers (IL15-Fc). Deep IL-15 is surface anchored to tumor reactive T cells prior to adoptive cell transfer (ACT). This novel therapeutic approach enables Deep IL-15 loading onto cells at concentrations unachievable with systemic IL15-Fc, causes autocrine T cell activation and expansion, yet limits systemic exposure and associated toxicities. The anti-tumor activity of T cell therapies has been limited by insufficient T cell expansion and by checkpoint immunosuppression. Here, we combined Deep IL-15 primed T cells with PD-L1 blockade to overcome these limitations. Methods Deep IL-15 was synthesized by incubation of IL15-Fc with a crosslinker. PMEL CD8+T cells (PMEL) were isolated from B6.Cg-Thy1a/Cy Tg(TcraTcrb)8Rest/J mice,activated, expanded, and Deep IL-15 was anchored to PMEL to generate Deep IL-15 Primed PMEL (Deep-15 PMEL). B16-F10 tumor-bearing mice were treated with cyclophosphamide (4 mg/mouse) one day prior to injection of 107Deep-15 PMEL (carrying ~ 58 ug of IL15-Fc)± anti-PD-L1 antibody (10 mg/kg q2w). We monitored mouse weight, tumor volumes, transferred and endogenous T cells, IL15-Fc systemic exposure, and cytokine release. Results Deep-15 PMEL showed significantly improved expansion and anti-tumor activity compared to PMEL, and provided long-term protection against tumor re-challenge. Importantly, the toxicity profile of Deep-15 PMEL was better than that of IL15-Fc co-injection with PMEL, yielding lower systemic exposure to IL15-Fc, less IFN-γinduction and no expansion of endogenous CD8+T and NK cells. Deep-15 PMEL resulted in 2/10 partial regressions and a statistically significant tumor growth inhibition compared to both vehicle and PD-L1 blockade alone. Co-administration of anti-PD-L1 further improved the anti-tumor activity of Deep-15 PMEL, resulting in 10/10 Complete Regressions (CR) and 6/10 Tumor Free Survivors. Conclusions By loading tumor-reactive T cells with Deep IL-15 before ACT, Torque's proprietary Deep Priming technology leverages the immune-stimulating power of IL-15 while avoiding immunotoxicity. Deep IL-15 Priming promotes engrafted T cell-specific activation and proliferation, and a favorable toxicity profile. In vivo transfer of Deep-15 PMEL combined with checkpoint inhibition was well tolerated and elicited remarkable anti-tumor activity in the PD-L1 refractory B16-F10 model, with 10/10 CRs. Torque is initiating a Phase I trial for TRQ15-01 (Deep IL-15 Primed multi-targeted human T cells) and planning a combination arm with checkpoint blockade in solid tumors. Citation Format: Elena Geretti, Xiaoyan Liang, Jesse Lyons, Philip Bardwell, Santina Caruso, Becker Hewes, Jonathan Fitzgerald, Karsten Sauer, Thomas Andresen. DeepIL-15 primed T cells synergize with PD-L1 blockade to overcome resistance to checkpoint immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2244.

Abstract 2267: Therapeutic synergy of the chimeric, oncolytic virus VSV-GP with immune modulators

Abstract Introduction: Oncolytic viruses (OVs), which selectively replicate in and destroy tumor cells, represent a highly promising class of cancer therapeutics. In recent years, the focus in the OV field has significantly shifted from a pure oncolytic mode of action to their strong immune stimulatory potential. Tumor cells lysed by the viruses release pro-inflammatory signals, tumor associated antigens as well as display hallmarks of immunogenic cell death. VSV-GP, a chimeric VSV pseudotyped with the glycoprotein of the lymphocytic choriomeningitis virus (LCMV) is a potent tumor cell-lysing agent and capable of jump-starting α-tumor immunity. The current study explores the immune promoting properties of VSV-GP and investigates clinically relevant combination therapies with checkpoint inhibition (α-PD-1) as well as a “second mitochondria-derived activator of caspases“ mimetic (SMACm). Experimental Procedures: Immune competent mice bearing established LLC1-IFNARKO, B16-F1-OVA or CT26.CL25-IFNARKO tumors were subject to treatments comprising VSV-GP, α-PD-1, a SMACm or combinations of VSV-GP with either of the latter therapies. α-tumor effects were determined by tumor growth inhibition and/or changes in overall survival. VSV-GP's tumor cell selective replication, resulting in immune activation, was assessed using FFPE based IHC staining for the viral N-protein, PD-L1 and different immune cell subsets as well as the NanoString platform (nCounter Pan Cancer Immune Profiling Panel). Data Summary: Here we present data on VSV-GP, which displays potent α-tumor efficacy and upon systemic delivery increases the local T-cell infiltration, expression of chemokines, T-cell and myeloid cell specific activation markers as well as expression of immune checkpoints, such as PD-1 and PD-L1 within the tumor microenvironment. Building on the observed therapeutic potential of VSV-GP as monotherapy, its immune promoting properties and the delineated compensatory mechanisms, such as PD-1/-L1 upregulation, we explored therapeutic combinations with a PD-1 blocking antibody, which resulted in a strong improvement of VSV-GP-mediated α-tumor efficacy as well as overall survival. Cured, tumor free animals were protected from tumor rechallange indicating immunological memory formation. We further explored the therapeutic combination of VSV-GP with a SMACm, which also resulted in a strongly improved therapeutic benefit and increased overall survival. Conclusion: In summary VSV-GP induces a pro-inflammatory microenvironment within infected tumors, increases immune cell infiltration and synergizes with α-PD-1 as well as a SMACm. VSV-GP is a potent OV with a strong α-tumor immunity promoting potential. Preclinical data justify further testing of VSV-GP and the combinations with PD-1 blockade or a SMACm in clinical trials. Citation Format: Philipp Mueller, Patrik Erlmann, Monika Petersson, Tobias Nolden, Carles Urbiola, Liesa-Marie Schreiber, Bart Spiesschaert, Klaus Erb, Birgit Stierstorfer, Maria Antonietta Impagnatiello, Eric Borges, Knut Elbers, Guido Wollmann, John Park. Therapeutic synergy of the chimeric, oncolytic virus VSV-GP with immune modulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2267.

Abstract 3257: Activation of CD137 using multivalent and tumor targeted Bicyclic peptides

Abstract CD137 (4-1BB/TNFRSF9) is a costimulatory T-cell receptor belonging to the TNF receptor superfamily. Agonistic anti-CD137 antibodies have shown potent, often curative anti-tumor activity in preclinical models. Two human anti-CD137 antibodies, urelumab and utomilumab are currently undergoing clinical testing. Urelumab has shown several single-agent, partial responses, but its use has been hampered by hepatoxicity, while utomilumab has shown little or no single agent activity. Bicycles® are a new class of drugs - fully synthetic, constrained bicyclic peptides that combine the attributes of antibodies, small molecules, and peptides by delivering high affinity, selectivity, and rapid clearance. Their small size (1.5-2 kDa) delivers advantages in tumor penetration, and renal elimination may avoid the liver and GI toxicity often associated with other drug modalities, including certain antibodies. We hypothesized that fully synthetic Bicycle CD137 agonists with rapid clearance, minimal liver exposure and no Fc receptor interaction may induce CD137 mediated anti-tumor activity while avoiding liver toxicity. A high affinity lead BCY3814 (KD ~30 nM) that binds to the CD137 ligand-binding site was identified. CD137 activation requires receptor crosslinking, thus multivalent binding would be expected to recapitulate the action of the natural trimeric ligand. We envisioned that CD137 agonism could be achieved directly by using multimeric CD137 Bicycles or in a tumor targeted fashion with bispecific or “heterotandem” Bicycles. The synthetic simplicity and highly modular nature of the Bicycle® platform enabled us to rapidly explore both formats. To generate a “pure” CD137 agonist we synthesized &amp;gt;50 different multimeric variants of BCY3814 with chemical linkers of various lengths and rigidity and using different sites of attachments, while maintaining a compact size (&amp;lt;15 kDa). Tumor targeted CD137 agonists were generated as heterotandems, whereby BCY3814 is conjugated to a tumor antigen targeting Bicycle. In this design, the CD137 Bicycle only induces CD137 agonism after the molecule binds to a tumor cell with high receptor expression. We discovered Bicycle multimers that exhibit a range of potencies in a cell-based CD137-dependent reporter assay, activate human T cells in vitro as indicated by increased cytokine release, and show biological activity in vivo. Bicycle heterotandems targeting Nectin-4 and EphA2 exhibited highly potent and tumor cell specific activity in both the cell-based reporter assay and the human T cell assay. Selected CD137 multimers and heterotandems are being tested further in humanized mouse models for T cell activation, anti-tumor activity, and liver safety. These molecules are promising, novel cancer immunotherapy candidates and importantly, they pave the way for development of synthetic agonists of other TNF receptors that can be targeted to the local tumor microenvironment. Citation Format: Kristen E. Hurov, Punit Upadhyaya, Jessica Kublin, Xueyuan Zhou, Julia Kristensson, Rachid Lani, Gemma Mudd, Katerine van Rietschoten, Frank An, Johanna Lahdenranta, Liuhong Chen, Gavin Bennett, Kevin McDonnell, Peter Park, Nicholas Keen. Activation of CD137 using multivalent and tumor targeted Bicyclic peptides [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3257.

Effects of Carbon and Nitrogen Sources on Activity of Cell Envelope Proteinase Produced by Lactobacillus plantarum LP69

Abstract In present study, the effects of carbon sources (glucose, lactose, sucrose, galactose, maltose and soluble starch) and nitrogen sources (casein peptone, whey protein, soy peptone, yeast, tryptone, beef extract and peptone) on activity of cell envelope proteinases (CEP), specific activity, protein content, OD600 value and pH in MRS broth fermented by Lactobacillus plantarum LP69 were investigated by individual factor experiment. The results indicated that carbon and nitrogen sources have significant influence on the activity of CEP and specific activitys of L. plantarum LP69, glucose, maltose, casein peptone and peptone are superior to other selected carbon and nitrogen sources. The optimum concentrations of glucose, maltose, casein peptone and peptone for L. plantarum LP69 are 2%, 2%, 1% and 1%; the activity of CEP are 19.52U/mL, 21.13U/mL, 13.49U/mL and 20.61U/mL, respectively.

2173-P: Effects of AMP-Activated Protein Kinase Activation on Insulin Secretion in Mice

AMP-activated protein kinase (AMPK) is an important cellular energy sensor in regulation of metabolic activity. AMPK activation improves glucose homeostasis by increasing muscle glucose uptake and reducing hepatic glucose production. Although the effects on glucose-stimulated insulin secretion in the pancreatic β cells are still controversial. Our aim was to investigate the regulation of insulin secretion in pancreatic β cells using specific pharmacological AMPK activators and in mouse models of constitutive activation. We used a chronic global AMPK (γ1D316A) and a pancreatic β cell specific (β-D316A) activation mouse model that express the AMPK γamma1 isoform with a D316A mutation under the control of β-actin and ins1 promoter respectively. We tested how chronic AMPK activation impacted insulin secretion on C57BL6 mouse isolated islets using direct activators PF-06409577 (β1 isoform-selective) and RA089 (binds both β1 and β2 isoforms). Global activation of AMPK reduced insulin secretion from islets isolated from γ1D316A mice on chow and high-fat diet (2.4-fold, p&amp;lt;0.05 and 1.9-fold p&amp;lt;0.01, respectively). Dual AMPK activation of β1 and β2 (RA089) on control mouse islets enhanced in a dose-response manner insulin secretion in response to high glucose (17mM) and KCl after 30 minutes of incubation (1.7-fold p&amp;lt;0.005 and 1.7-fold p&amp;lt;0.005, respectively). The same effects were observed by activating AMPK β1 isoform with PF-06409577, increasing insulin release in response to high glucose and KCl (2-fold, p&amp;lt;0.005 and 1.7-fold, p&amp;lt;0.05). These findings suggest that the effects of AMPK on insulin secretion depends on its mode of activation. Acute pharmacological activation targetting β1 and/or β2 subunit increased insulin secretion whereas chronic global activation in mice reduced insulin release. Investigating our second model of chronic activation β-D316A and further studies of our models will help us decipher the role of AMPK in the pancreas. Disclosure M. Nguyen-Tu: None. J.P. Harris: None. A. Martinez-Sanchez: None. I. Leclerc: None. G.A. Rutter: Consultant; Self; Sun Pharma.

Anti-Cancer Activity of Novel Dihydrotestosterone-Derived Ring A-Condensed Pyrazoles on Androgen Non-Responsive Prostate Cancer Cell Lines.

Regioselective synthesis of novel ring A-fused arylpyrazoles of dihydrotestosterone (DHT) was carried out in two steps under facile reaction conditions. Aldol condensation of DHT with acetaldehyde afforded a 2-ethylidene derivative regio- and stereo-selectively, which was reacted with different arylhydrazines in the presence of iodine via microwave-assisted oxidative cyclization reactions. The 17-keto analogs of steroidal pyrazoles were also synthesized by simple oxidation in order to enlarge the compound library available for pharmacological studies and to obtain structure–activity relationship. The antiproliferative activities of the structurally related heteroaromatic compounds were tested in vitro on human cervical and breast adenocarcinoma cell lines (HeLa, MCF-7 and MDA-MB-231) and on two androgen-independent malignant prostate carcinoma cell lines (PC-3 and DU 145). Based on primary cytotoxicity screens and IC50 assessment, a structure-function relationship was identified, as derivatives carrying a hydroxyl group on C-17 exhibit stronger activity compared to the 17-one counterparts. Cancer cell selectivity of the derivatives was also determined using non-cancerous MRC-5 cells. Furthermore, the proapoptotic effects of some selected derivatives were verified on androgen therapy refractive p53-deficient PC-3 cells. The present study concludes that novel DHT-derived arylpyrazoles exert cancer cell specific antiproliferative activity and activate apoptosis in PC-3 cells.

Cell-specific CRISPR-Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins.

The rapid development of CRISPR–Cas technologies brought a personalized and targeted treatment of genetic disorders into closer reach. To render CRISPR-based therapies precise and safe, strategies to confine the activity of Cas(9) to selected cells and tissues are highly desired. Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins. We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and cardiac muscle cells, respectively, into the 3′UTR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and released Cas9 activity solely in hepatocytes or cardiomyocytes, while Cas9 was efficiently inhibited in off-target cells. We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes (Spy)Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64). Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme)Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3. Our Cas-ON switch should facilitate cell-specific activity of any CRISPR–Cas orthologue, for which a potent anti-CRISPR protein is known.

Cancer Gene Therapy by NF-κB-Activated Cancer Cell-Specific Expression of CRISPR/Cas9 Targeting to Telomere

NF-κB is an attractive target for cancer therapy due to its over activation in all tumors. However, the tranditional NF-κB inhibitor is difficult to become drugs due to their side effects. In this study, we testified a new cancer gene therapy that based on NF-κB overactivity in cancer cells and telomere-targeting CRISPR. In this gene therapy, a NFκB-specific promoter activated expression of Cas9 protein, which then damaged the telomeric DNA by guiding a telomere-targeting sgRNA. In this study, we firstly demonstrated the cancer cell-specific activation of NFκB. We then demonstrated the cancer cell specificity of this cancer gene therapy in various cell lines using a reporter gene ZsGreen under the control of NF-κB-specific promoter. We next demonstrated that this cancer gene therapy could specifically induce death of various cancer cells by expressing Cas9 protein under the control of NF-κB-specific promoter and a telomere-targeting sgRNA. We finally showed that the whole gene expression cassettes of this cancer gene therapy could be packaged in adeno-associated virus (AAV), which could be used to inhibit the growth of xenografted tumors in mouse by intravenously injecting recombinant AAV. Funding Statement: This work was supported by the National Natural Science Foundation of China (61571119) and the National Key Research and Development Program of China (2017YFA0205502) Declaration of Interests: None of the authors declared a conflict of interest. Ethics Approval Statement: All animal experiments in this study followed the guidelines and ethics of the Animal Care and Use Committee of Southeast University (Nanjing, China).

The Opposite Effect of c-Jun Transcription Factor on Apolipoprotein E Gene Regulation in Hepatocytes and Macrophages.

Apolipoprotein E (apoE) is mainly secreted by hepatocytes and incorporated into most plasma lipoproteins. Macrophages, which accumulate cholesterol and are critical for the development of the atherosclerotic plaque, are also an important, albeit smaller, apoE source. Distal regulatory elements control cell-specific activity of the apoE promoter: multienhancers (ME.1/2) in macrophages and hepatic control regions (HCR-1/2) in hepatocytes. A member of AP-1 cell growth regulator, c-Jun regulates the transcription of various apolipoproteins and proinflammatory molecules implicated in atherosclerosis. We aimed to investigate the effect of c-Jun on apoE expression in macrophages versus hepatocytes and to reveal the underlying molecular mechanisms. Herein we show that c-Jun had an opposite, cell-specific effect on apoE expression: downregulation in macrophages but upregulation in hepatocytes. Transient transfections using ME.2 deletion mutants and DNA pull-down (DNAP) assays showed that the inhibitory effect of c-Jun on the apoE promoter in macrophages was mediated by a functional c-Jun binding site located at 301/311 on ME.2. In hepatocytes, c-Jun overexpression strongly increased apoE expression, and this effect was due to c-Jun binding at the canonical site located at −94/−84 on the apoE proximal promoter, identified by transient transfections using apoE deletion mutants, DNAP, and chromatin immunoprecipitation assays. Overall, the dual effect of c-Jun on apoE gene expression led to decreased cholesterol efflux in macrophages resident in the atherosclerotic plaque synergized with an increased level of systemic apoE secreted by the liver to exacerbate atherogenesis.

The deep water of Eastern Mediterranean Sea is a hotspot for bacterial activity

We investigated the abundance and production of mesopelagic (150–650 m) and bathypelagic (>650 m) bacteria from the poorly-studied Levantine basin (Eastern Mediterranean Sea) during the summers of 2014–2017. We show that the mesopelagic and bathypelagic microbial populations have relatively high cell specific activity, amongst the highest worldwide, likely due to the relatively warm temperatures characterizing these depths in comparison to other oceanic regions. Furthermore, in order to investigate if mesopelagic and bathypelagic microbial populations are limited by the scarce organic carbon substrates in the area, we performed microcosm addition assays of pure glucose (Glu) and/or a mixture of dissolved free amino acids (DFAA). Both the mesopelagic and bathypelagic bacterial populations responded positively to the additions, with bacterial production rapidly increasing within a few hours following the Glu or DFAA amendments. Our results demonstrate that deep-water microbes in the Levantine Basin may be important nutrient recyclers, especially when labile organic carbon is introduced. Our observations may have implications on carbon cycling and sequestration in the oligotrophic Eastern Mediterranean Sea.

Abstract A188: T-cell priming with Deep IL-15 improves preclinical safety compared to systemic IL-15, and increases in vivo persistence and activity

Abstract Introduction: Interleukin-15 (IL-15), provides strong activation of both CD8+ T-cells and NK cells, without regulatory T-cells activation, making it an attractive immune modulator in cancer therapy. Systemic delivery of IL-15 to patients has revealed dose-limited toxicities resulting primarily in expansion of NK cells. Preclinical data suggest that IL-15 immunotoxicity is mediated by hyperproliferation and activation of NK cells (Guo Y, J Immunol 2015). In this study, we investigate safety and efficacy of T-cells loaded with Deep IL-15 (Deep IL-15 Primed T-cells), in a syngeneic mouse model. Deep IL-15 is a multimer of chemically crosslinked IL-15/IL-15 Rα/Fc heterodimers (IL15-Fc) that is designed for T-cell loading prior to adoptive cell transfer with the aim of improving the therapeutic window by autocrine signaling to the primed cells without causing the immunotoxicologic effects normally associated with IL-15. Deep IL-15 is loaded on the T-cells and, upon crosslinker cleavage, releases IL15-Fc to stimulate the primed cells. This novel T-cell-based therapeutic approach enables autocrine T-cell activation and expansion, and limits systemic exposure to IL15-Fc, thus reducing associated toxicities. Methods: Deep IL-15 was synthesized by incubation of IL15-Fc with a crosslinking reagent. PMEL CD8+ T (PMEL) cells were isolated from B6.Cg-Thy1a/Cy Tg(TcraTcrb)8Rest/J mice. PMEL cells carry a transgenic T-cell receptor specific for gp100, a protein expressed by B16-F10 melanoma cells. PMEL cells were activated, expanded, and loaded with Deep IL-15 to generate Deep IL-15 Primed PMEL (Deep-15 PMEL) cells. Deep-15 PMEL cells were transferred into naïve or B16-F10 tumor-bearing mice (10 x 106; 15 ug Deep IL-15/106 cells), and the toxicity of Deep-15 PMEL was compared with PMEL (10 x 106) co-injected with soluble IL15-Fc at the maximum tolerated dose of 10 μg/mouse (PMEL + IL15-Fc). Readouts included IL15-Fc systemic exposure (ELISA), cytokine release (Luminex), and changes in endogenous T-cells (complete blood counts, CBC; flow cytometry). In addition, the biodistribution and the antitumor activity of Deep-15 PMEL was evaluated in B16-F10 tumor-bearing mice. Results: Deep-15 PMEL cells, carrying 15-fold more IL15-Fc than PMEL + IL15-Fc, resulted in &amp;gt;300-fold lower systemic exposure to IL15-Fc and 30-fold lower circulating IFN-γ. Deep-15 PMEL did not affect CBCs and did not expand endogenous CD8+ nor NK cells. Conversely, IL15-Fc induced significant changes in CBCs and promoted expansion of both transferred and endogenous CD8+ (6.7-fold) and NK (18.2-fold) cells. Similar results were observed in both naïve and tumor-bearing mice. Deep-15 PMEL improved persistence of transferred cells across multiple tissues (15-44-fold rel. to PMEL co-injected with IL15-Fc; day 16): blood, spleen, lymph nodes (tumor draining and non-draining) and tumor. The tumor presence affected the biodistribution of Deep-15 PMEL cells, resulting in lower (0.5-fold) accumulation of cells in the spleen, and increased (2.1-fold) cell numbers in the tumor-draining lymph node. Furthermore, Deep-15 PMEL showed significantly improved anti-tumor activity compared to PMEL in the B16-F10 model. Conclusions: The Deep Priming technology developed by Torque offers the advantage of loading Deep IL-15 prior to cell infusion in ACT, at concentrations unachievable with systemic injection of IL15-Fc, thus resulting in controlled, local delivery and cell-specific activation and proliferation. In vivo transfer of Deep-15 PMEL cells was well tolerated. Importantly, Deep-15 PMEL cells persisted longer across multiple organs compared to PMEL cells exposed to systemic IL15-Fc, while not inducing expansion of endogenous cells, including NK cells, which are reported to be the primary mediators of IL-15 toxicity. Torque is preparing to initiate clinical trials for Deep IL-15 Primed multitargeted human T-cells, TRQ15-01, in both hematologic and solid tumors. Citation Format: Elena Geretti, Philip Bardwell, Xiaoyan Liang, Santina Caruso, De-Kuan Chang, Jesse Lyons, Austin Boesch, Aaron Handler, Carlos Tassa, Sanela Bilic, Janice Lancita, Becker Hawes, Jonathan Fitzgerald, Thomas Andresen. T-cell priming with Deep IL-15 improves preclinical safety compared to systemic IL-15, and increases in vivo persistence and activity [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 A188.