Late gastrointestinal tissue effects after hypofractionated radiation therapy of the pancreas

Background To consolidate literature reports of serious late gastrointestinal toxicities after hypofractionated radiation treatment of pancreatic cancer and attempt to derive normal tissue complication probability (NTCP) parameters using the Lyman-Kutcher-Burman model. Methods Published reports of late grade 3 or greater gastrointestinal toxicity after hypofractionated treatment of pancreatic cancer were reviewed. The biologically equivalent dose in 1.8 Gy fractions was calculated using the EQD model. NTCP parameters were calculated using the LKB model assuming 1–5 % of the normal tissue volume was exposed to the prescription dose with α/β ratios of 3 or 4. Results A total of 16 human studies were examined encompassing a total of 1160 patients. Toxicities consisted of ulcers, hemorrhages, obstructions, strictures, and perforations. Non-hemorrhagic and non-perforated ulcers occurred at a rate of 9.1 % and were the most commonly reported toxicity. Derived NTCP parameter ranges were as follows: n = 0.38–0.63, m = 0.48–0.49, and TD50 = 35–95 Gy. Regression analysis showed that among various study characteristics, dose was the only significant predictor of toxicity. Conclusions Published gastrointestinal toxicity reports after hypofractionated radiotherapy for pancreatic cancer were compiled. Median dose was predictive of late grade ≥ 3 gastrointestinal toxicity. Preliminary NTCP parameters were derived for multiple volume constraints

High-throughput 2-hydroxyglutarate Assays for Biological Screening in IDH Mutant Cells

Cancer cells display altered metabolic profiles that can be exploited with targeted therapeutics. The amino acid glutamine is shunted toward anabolic reactions and contributes to the production of 2-hydroxyglutarate (2HG), an onco-metabolite aberrantly elevated in several cancers. 2HG is structurally similar to α-ketoglutarate (αKG) and can modulate the activity of several αKG dependent enzymes including histone and DNA demethylases, as a well as prolyl hydroxylases. Modulation of these enzymes leads to transformation and blocked differentiation. Thus, inhibition of 2HG production could be therapeutic in patients with IDH mutant malignancies. A facile and high-throughput method for quantification of 2HG is needed to allow for large scale compound and siRNA screens to identify new strategies for reducing 2HG levels. Herein, we developed novel fluorimetric microplate assay for quantitation of 2HG and performed an unbiased small molecule screen in live cells to identify compounds capable of perturbing 2HG production. Zaprinast, a known PDE5 inhibitor, was identified as a modulator of 2HG production and confirmed to lower 2HG levels in vivo. The mechanism of action was not due to cGMP stabilization, but rather, profiling of metabolites upstream of mutant IDH1 pointed to targeted inhibition of the enzyme glutaminase (GLS1). With purified GLS1 in vitro, Zaprinast showed xi classic noncompetitive inhibition kinetics (Ki = 220 M). In human astrocytes expressing mutant IDH1, Zaprinast-mediated inhibition of GLS1reversed histone hypermethylation and cell growth in soft agar. Additionally, treatment of glutamine-addicted pancreatic ductal adenocarcinoma cells with Zaprinast reduced growth, increased ROS levels, and sensitized cells to oxidative damage, mimicking the effects of glutamine deprivation; metabolic rescue with glutamate reversed the Zaprinast-mediated blockade of GLS1. Thus, Zaprinast possesses efficacious off-target effects against glutamine metabolism, providing a convenient tool compound for further investigation of the linkages between GLS1 and 2HG-mediated oncogenesis as well as therapeutic development against IDH mutant or glutamine-addicted cancers

Abstract 4879: Real-time imaging of differential ligand-induced IKK activation and impact on the IκBα:NF-κB negative feedback loop within living cells and mice

Abstract The IKK (IκB Kinase) complex is a key regulator of the NF-κB signaling cascade and is a pharmacological target for cancer, autoimmune, and inflammatory therapies. IKK is a direct regulator of the IκBα:NF-κB negative feedback loop, a critical regulatory node within the NF-κB pathway. We have developed a systems-level reporter that utilizes bioluminescence imaging for real-time read-out of the dynamics of this negative feedback loop, enabling quantitative characterization of cell signaling in populations of live cells in vitro and in vivo. This novel transcriptionally-coupled IκBα-firefly luciferase fusion reporter (κB5→IκBα-FLuc), expressed in HepG2 cells and murine livers, monitors both IKK-induced degradation of IκBα and the subsequent NF-κB-driven re-synthesis of IκBα. Of particular interest was systematically evaluating the response of this negative feedback loop to subtle and/or drastic changes in ligand type, concentration, and exposure duration.To probe the effect of ligand type and concentration, HepG2 cells expressing κB5→IκBα-FLuc were stimulated with TNFα (0.57 – 570 pM) or IL-1β (0.06 – 60 pM) and the luciferase photon flux was followed for six hours. Increasing the concentration of either ligand increased the degree of degradation (EC50 = 6.7 pM for TNFα and 1.4 pM for IL-1β) and decreased the time required to achieve maximum degradation (from 53 to 29 min and 60 to 30 min, respectively). While higher concentrations of either ligand resulted in faster re-synthesis kinetics, IL-1β elicited a biphasic increase in IκBα re-synthesis amplitude and TNFα elicited increasing levels of IκBα re-synthesis up to a threshold (57 pM) beyond which higher amounts of TNFα actually elicited lower levels of re-synthesis (i.e. a “rollover” back down to 74 ± 3% of maximum levels). Concomitant modulation of TNFα concentration and duration revealed a prominent re-synthesis rollover when TNFα is given as a long pulse (&amp;gt; 15 min) that is less prominent or non-existent for short (30 sec to 10 min) pulses. Re-synthesis rollover was also observed for endogenous IκBα by Western blot analysis (although this technique reveals smaller changes in the IκBα levels) and within a computational model of the NF-κB pathway. Furthermore, in vivo somatic gene transfer of the κB5→IκBα-FLuc reporter into murine livers demonstrated TNFα dose-dependent increases in IκBα degradation and to a lesser extent re-synthesis, both with significantly faster kinetics than seen in vitro.In summation, a κB5→IκBα-FLuc reporter has revealed significant dynamic differences in the response of the IκBα:NF-κB negative feedback loop within populations of cells in vitro and in vivo to a variety of TNFα and IL-1β stimulation regimens, and represents a novel tool to qualitatively and quantitatively analyze regulation of negative feedback loops within complex biological signaling cascades. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4879. doi:10.1158/1538-7445.AM2011-4879</jats:p

Abstract LB-419: Multi-scalar approaches to interrogate the IκBα:NF-κB negative feedback loop: Quantitative dynamics in single cells, cell populations, and live animals <i>in vivo</i>

Abstract Cells have evolved complex molecular networks to sense environmental signals, transmit this information through the cell, and elicit appropriate biological responses. In particular, negative feedback loops represent a widely-utilized network motif capable of eliciting transient responses. To truly understand the biological significance of negative feedback processes, it is critical to study them at multiple scales: in single cells, in cell populations, and in animals. The IκBα:NF-κB negative feedback loop, a pivotal regulatory node of innate immunity and inflammation active in both immune cells and non-immune tissues, represents a model system for the use of multi-scalar reporter systems. To this end, we have utilized the κB5→IκBα-FLuc bioluminescent reporter to study dynamics of this transcriptionally-coupled negative feedback loop in response to diverse modes of stimulation which may be particularly relevant during cellular responses to inflammatory cytokines, such as TNFα. The κB5→IκBα-FLuc reporter enabled rigorous evaluation of the stimulus-specific dynamics of βκγα degradation and the downstream consequences of NF-κΔ nuclear translocation (i.e., NF-κΔ transcriptional activity) in single cells, cell populations and live animals in vivo. In response to modulation of TNFα concentration and pulse duration, complex, differential patterns in βκγα degradation and re-synthesis were discovered in both cell populations and single cells. Furthermore, IκBα dynamics observed in live animals in vivo upon modulation of TNFα dose strongly resembled those observed in single cells and cell populations upon modulating TNFα pulse duration, suggesting that increased doses of circulating TNFα were perceived by hepatocytes in vivo as pulses of increasing duration. Thus, a single bioluminescent reporter strategy enabled correlative quantitation of dynamic NF-κα:βκβα negative feedback loop responses in live single cells, cell populations, and tissues in vivo with a variety of rapid, low-cost, high-throughput approaches. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-419. doi:1538-7445.AM2012-LB-419</jats:p

Late gastrointestinal tissue effects after hypofractionated radiation therapy of the pancreas

Background: To consolidate literature reports of serious late gastrointestinal toxicities after hypofractionatedradiation treatment of pancreatic cancer and attempt to derive normal tissue complication probability (NTCP)parameters using the Lyman-Kutcher-Burman model.Methods: Published reports of late grade 3 or greater gastrointestinal toxicity after hypofractionated treatment ofpancreatic cancer were reviewed. The biologically equivalent dose in 1.8 Gy fractions was calculated using the EQDmodel. NTCP parameters were calculated using the LKB model assuming 1--5 % of the normal tissue volume wasexposed to the prescription dose with a/β ratios of 3 or 4.Results: A total of 16 human studies were examined encompassing a total of 1160 patients. Toxicities consisted ofulcers, hemorrhages, obstructions, strictures, and perforations. Non-hemorrhagic and non-perforated ulcers occurredat a rate of 9.1 % and were the most commonly reported toxicity. Derived NTCP parameter ranges were as follows:n = 0.38--0.63, m = 0.48--0.49, and TD50 = 35--95 Gy. Regression analysis showed that among various studycharacteristics, dose was the only significant predictor of toxicity.Conclusions: Published gastrointestinal toxicity reports after hypofractionated radiotherapy for pancreatic cancerwere compiled. Median dose was predictive of late grade ≥ 3 gastrointestinal toxicity. Preliminary NTCP parameterswere derived for multiple volume constraints

Figure S3 from A High-Throughput Fluorimetric Assay for 2-Hydroxyglutarate Identifies Zaprinast as a Glutaminase Inhibitor

PDF file 20K, cGMP does not inhibit GLS1 activity PDF. Enzyme activity of purified, full length, human glutaminase treated with increasing amounts of cGMP in the presence of 10 mM glutamine</p

Dual-Color Click Beetle Luciferase Heteroprotein Fragment Complementation Assays

SummaryUnderstanding the functional complexity of protein interactions requires mapping biomolecular complexes within the cellular environment over biologically relevant time scales. Herein, we describe a set of reversible multicolored heteroprotein complementation fragments based on various firefly and click beetle luciferases that utilize the same substrate, D-luciferin. Luciferase heteroprotein fragment complementation systems enabled dual-color quantification of two discrete pairs of interacting proteins simultaneously or two distinct proteins interacting with a third shared protein in live cells. Using real-time analysis of click beetle green and click beetle red luciferase heteroprotein fragment complementation applied to β-TrCP, an E3-ligase common to the regulation of both β-catenin and IκBα, GSK3β was identified as a candidate kinase regulating IκBα processing. These dual-color protein interaction switches may enable directed dynamic analysis of a variety of protein interactions in living cells

Figure S4 from A High-Throughput Fluorimetric Assay for 2-Hydroxyglutarate Identifies Zaprinast as a Glutaminase Inhibitor

PDF file 132K, Effect of Zaprinast on histone methylatio Immortalized NHA cells were transduced with vector, wild type IDH1, or IDH1 R132H plasmids and treated with vehicle or 300 microM Zaprinast. Histone extraction was then performed. Western blot was performed to assess the levels of histone lysine residues H3K9Me2 (A) and H3K9Me3 (B) (top) with band quantification (bottom)</p