p53-Mediated Radiosensitization of 177Lu-DOTATATE in Neuroblastoma Tumor Spheroids

p53 is involved in DNA damage response and is an exciting target for radiosensitization in cancer. Targeted radionuclide therapy against somatostatin receptors with 177Lu-DOTATATE is currently being explored as a treatment for neuroblastoma. The aim of this study was to investigate the novel p53-stabilizing peptide VIP116 in neuroblastoma, both as monotherapy and together with 177Lu-DOTATATE. Five neuroblastoma cell lines, including two patient-derived xenograft (PDX) lines, were characterized in monolayer cultures. Four out of five were positive for 177Lu-DOTATATE uptake. IC50 values after VIP116 treatments correlated with p53 status, ranging between 2.8–238.2 μM. IMR-32 and PDX lines LU-NB-1 and LU-NB-2 were then cultured as multicellular tumor spheroids and treated with 177Lu-DOTATATE and/or VIP116. Spheroid growth was inhibited in all spheroid models for all treatment modalities. The most pronounced effects were observed for combination treatments, mediating synergistic effects in the IMR-32 model. VIP116 and combination treatment increased p53 levels with subsequent induction of p21, Bax and cleaved caspase 3. Combination treatment resulted in a 14-fold and 1.6-fold induction of MDM2 in LU-NB-2 and IMR-32 spheroids, respectively. This, together with differential MYCN signaling, may explain the varying degree of synergy. In conclusion, VIP116 inhibited neuroblastoma cell growth, potentiated 177Lu-DOTATATE treatment and could, therefore, be a feasible treatment option for neuroblastoma.

Development of a Predictive Model for Cytokine Release Syndrome to Inform Risk Stratification and CRS Management Following Immunotherapy

Background: Cytokine release syndrome (CRS) is a potentially life-threatening toxicity caused by immune activation. CRS can be triggered non-specifically by T-cell engaging therapies. Risk factors for CRS are expected to be disease-specific and prediction of an individual patient's CRS risk is not currently possible. Glofitamab is a T-cell engaging bispecific antibody targeting CD20 and CD3 with a novel 2:1 format that has shown promising efficacy with a manageable safety profile in NP30179 (NCT03075696), an ongoing Phase I/II dose finding study evaluating glofitamab in patients with relapsed or refractory non-Hodgkin lymphoma (NHL). While CRS is observed with glofitamab, cases typically occur with grade 1/2 severity (per ASTCT, Lee et al 2019) with most occurrences confined to the first cycle of therapy. Data from NP30179 were used to develop a model to predict the occurrence of Grade ≥ 2 CRS after the first glofitamab dose to enable stratification of patients according to risk of CRS with possible future implications for intensity of monitoring for those at low risk. Methods: Glofitamab was administered intravenously over 4 to 8 hours as a fixed or step-up dosing regimen, as previously described (Hutchings, et al JCO 2021). Non-overlapping training and validation data sets were defined; the training data set included patients with aggressive (n=165) or indolent NHL (n=31) who received a first dose of 0.6-25 mg, and the model validation data set (n=51; 35 aggressive NHL) included patients who received a 2.5mg first dose. The primary outcome was defined as Grade ≥2 CRS in the week after the first glofitamab dose, and included 65 events (n=58 training, n=7 validation). In the training data set we evaluated the association between the dose, putative risk factors (including demographics, medical history, disease characteristic variables, baseline laboratory values) and the occurrence of CRS. Univariate and multivariate models were applied in a stratified cross-validation setting to assess the most predictive and stable combination of risk factors. Baseline and on-treatment cytokine levels were analyzed via ELISA in a subset of patients (n=89). Results: The temporal pattern of CRS occurrences revealed that the vast majority of first CRS events occur after the first dose of glofitamab. To predict the occurrence of Grade ≥ 2 CRS after the first glofitamab dose, a multivariate model was developed to include glofitamab first dose and a combined risk score, termed the "CRS risk score" (CRSRS), which is the weighted sum of binarized risk factor values at baseline (Figure). The predictive ability was tested in the validation data set in which the incidence of Grade ≥ 2 CRS was 14% (7/51). A low risk group (CRSRS <5.0) was identified to be 60% of the test cohort with patients within this group having only 5% chance (Negative Predictive Value=0.95, SE=0.03) of experiencing a Grade ≥2 CRS. Induction of cytokines, including IL-6 and TNFα, was observed upon treatment with glofitamab and peak magnitude of cytokine induction was associated with CRS incidence and severity. Cytokine induction was evident by end of glofitamab infusion and peak levels of TNFα were observed by mid-infusion. CRSRS and TNFα induction were evaluated together for the subset of patients with cytokine data available to determine whether risk classification incorporating both metrics could refine the risk stratification of patients. Early TNFα changes were layered on top of CRSRS (at the cutoff of 5.0) such that patients with less than 1.5-fold induction were classified as low risk, and patients with more than 8-fold induction were classified as high risk. In the training data set, this decision tree approach improved the performance of prediction compared to CRSRS alone. Conclusions: A model based on 8 baseline factors allowed an accurate classification of risk for Grade ≥2 CRS upon treatment with glofitamab. Addition of TNFα induction may improve the predictive value. The predictive performance was confirmed in a separate validation data set with additional analyses in independent cohorts ongoing. The CRSRS, alone or in combination with cytokine induction, represents a tool to predict the occurrence of Grade ≥2 CRS after the first glofitamab dose to enable stratification of patients according to risk of CRS with possible future implications for the intensity of monitoring for those at low risk. K.V.K. and A.B. have contributed equally. Disclosures Belousov: F. Hoffmann-La Roche Ltd: Current Employment. Byrtek: Genentech, Inc.: Current Employment; F. Hoffmann-La Roche Ltd: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Kwan: Genentech, Inc.: Current Employment, Current equity holder in publicly-traded company. Perez-Callejo: F. Hoffmann-La Roche Ltd: Current Employment, Current equity holder in publicly-traded company. Li: Genentech, Inc.: Current Employment, Current holder of individual stocks in a privately-held company. Carlile: AstraZeneca: Current equity holder in publicly-traded company, Ended employment in the past 24 months; F. Hoffmann-La Roche Ltd: Current Employment, Current equity holder in publicly-traded company. McCall: Genentech, Inc.: Current Employment; F. Hoffmann-La Roche Ltd: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Nielsen: F. Hoffmann-La Roche Ltd: Current Employment, Current equity holder in publicly-traded company. Piccione: Genentech, Inc.: Current Employment; F. Hoffmann-La Roche Ltd: Current equity holder in publicly-traded company. OffLabel Disclosure: Glofitamab is a full-length, humanized, immunoglobulin G1 bispecific antibody with a 2:1 molecular format that facilitates bivalent binding to CD20 on B-cells, and monovalent binding to CD3 on T-cells. Glofitamab redirects T cells to engage and eliminate malignant B cells. Glofitamab is an investigational agent.

Potent Nrf2-inducing, antioxidant, and anti-inflammatory effects and identification of constituents validate the anti-cancer use of Uvaria chamae and Olax subscorpioidea

Background Uvaria chamae (UC) and Olax subscorpioidea (OS) roots are included in traditional anti-cancer remedies and some studies have identified their chemopreventive/chemotherapeutic potential. This study aimed to identify some cellular/molecular mechanisms underlying such potential and the associated chemical constituents. Methods Effect on the viability of cancer cells was assessed using the Alamar Blue assay; ability to modulate oxidative stress was assessed using the 2′,7′-dichlorofluorescein diacetate (DCFDA) assay; potential to modulate Nuclear factor erythroid 2-related factor like-2 (Nrf2) activity was assessed in the AREc32 luciferase reporter cell line; and anti-inflammatory effect was assessed using lipopolysaccharide-induced nitric oxide release model in the RAW264.7 cells (Griess Assay). Chemical constituents were identified through liquid chromatography-mass spectrometry (LC-MS). Results Extracts up to 100 μg/ml were non-toxic or mildly toxic to HeLa, AREc32, PC3 and A549 cells (IC50 > 200 μg/ml). Each extract reduced basal and peroxide-induced levels of reactive oxygen species (ROS) in HeLa cells. OS and UC activated Nrf2, with UC producing nearly four-fold induction. Both extracts demonstrated anti-inflammatory effects. Chamanetin, isochamanetin, isouvaretin, uvaricin I and other compounds were found in U. chamae root extract. Conclusion As Nrf-2 induction, antioxidant and anti-inflammatory activities are closely linked with chemoprevention and chemotherapy of cancers, the roles of these plants in traditional anti-cancer remedies are further highlighted, as is their potential as sources of drug leads.

Highly tunable TetR-dependent target gene expression in the acetic acid bacterium Gluconobacter oxydans

For the acetic acid bacterium (AAB) Gluconobacter oxydans only recently the first tight system for regulatable target gene expression became available based on the heterologous repressor-activator protein AraC from Escherichia coli and the target promoter ParaBAD. In this study, we tested pure repressor-based TetR- and LacI-dependent target gene expression in G. oxydans by applying the same plasmid backbone and construction principles that we have used successfully for the araC-ParaBAD system. When using a pBBR1MCS-5-based plasmid, the non-induced basal expression of the Tn10-based TetR-dependent expression system was extremely low. This allowed calculated induction ratios of up to more than 3500-fold with the fluorescence reporter protein mNeonGreen (mNG). The induction was highly homogeneous and tunable by varying the anhydrotetracycline concentration from 10 to 200 ng/mL. The already strong reporter gene expression could be doubled by inserting the ribosome binding site AGGAGA into the 3’ region of the Ptet sequence upstream from mNG. Alternative plasmid constructs used as controls revealed a strong influence of transcription terminators and antibiotics resistance gene of the plasmid backbone on the resulting expression performance. In contrast to the TetR-Ptet-system, pBBR1MCS-5-based LacI-dependent expression from PlacUV5 always exhibited some non-induced basal reporter expression and was therefore tunable only up to 40-fold induction by IPTG. The leakiness of PlacUV5 when not induced was independent of potential read-through from the lacI promoter. Protein-DNA binding simulations for pH 7, 6, 5, and 4 by computational modeling of LacI, TetR, and AraC with DNA suggested a decreased DNA binding of LacI when pH is below 6, the latter possibly causing the leakiness of LacI-dependent systems hitherto tested in AAB. In summary, the expression performance of the pBBR1MCS-5-based TetR-Ptet system makes this system highly suitable for applications in G. oxydans and possibly in other AAB. Key Points • A pBBR1MCS-5-based TetR-Ptet system was tunable up to more than 3500-fold induction. • A pBBR1MCS-5-based LacI-PlacUV5 system was leaky and tunable only up to 40-fold. • Modeling of protein-DNA binding suggested decreased DNA binding of LacI at pH < 6.

Differential Responses of Antioxidative System during the Interaction of Soursop Fruits (Annona muricata L.) and Nectria haematococca at Postharvest Storage

Soursop fruit (Annona muricata L.) production is diminished by the attack of pathogens such as Nectria haematococca. However, the fruit–pathogen interaction at the biochemical and molecular levels is still unknown. The objective of this study was to analyze the response of the soursop fruit to the presence of N. haematococca during postharvest storage. Soursop fruits were inoculated with the pathogen and total phenolic compounds, antioxidant capacity by Ferric reducing/antioxidant power (FRAP), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS•+), and 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH•), as well as enzymatic activity and transcript levels of polyphenol oxidase (PPO) and superoxide dismutase (SOD), were evaluated at 1, 3, and 5 days of storage. The noninoculated fruits were the controls of the experiment. The highest total phenol content was recorded on day one in the inoculated fruits. FRAP, ABTS, and DPPH activity presented the highest values on day three in the control fruits. Inoculated fruits recorded the highest PPO activity on day five and a five-fold induction in the PPO transcript on day three. SOD activity showed a decrease during the days of storage and 10-fold induction of SOD transcript on day three in the inoculated fruits. Principal component analysis showed that total phenols were the variable that contributed the most to the observed variations. Furthermore, a positive correlation between total phenols and SOD activity, PPO expression, and SOD expression, as well as between DPPH and FRAP, was recorded. The results showed a differential response in antioxidant capacity, enzymatic activity, and gene expression during the interaction of soursop fruits–N. haematococca at postharvest storage.

P056 Crohn’s Disease associated fibrosis modulates the expression of collagen receptors

Background Crohn’s Disease (CD) patients often develop stenotic complications as immunomodulatory treatments do not prevent the fibrogenic response in the affected tissues, where a dysregulated activation of stromal cells provokes an excessive deposition of extracellular matrix (ECM). Recent evidences support the notion that local cells can sense the consequent alterations in tissue structure and rigidity through receptors that respond to some ECM components, and this may perpetuate the fibrogenic process even in the absence of inflammation. We aim to analyse the relevance of these signalling pathways in the fibrotic process associated to CD. Methods We obtained fibrotic ileal tissues from CD patients and healthy ileal samples from colon carcinoma patients (control), and analysed the expression (RT-PCR, IHQ) of collagen receptors (integrins, discoidin domain receptors/DDR) and of markers for some stromal cells (fibroblasts, endothelial cells). The relationship between these sets of data was analysed by Pearson’s correlation and the results organized as a correlation matrix. The expression of collagen receptors was also analysed in endothelial cells (HUVEC) treated with TGFβ 2 (1ng/ml, 48h). Results Ileal samples from CD patients present a differential gene expression of collagen receptors (Fig 1), with increased levels of ITGA10, ITGA11 and DDR2, and reduced expression of DDR1. In CD tissues, the expression of ITGA11 and DDR1 showed a significant correlation, positive and negative respectively, with that of endothelial markers (Fig 2). These correlations do not occur in control tissues. Integrin-α11 was detected in endothelial cells of submucosal vessels (IHQ). In HUVEC, TGFβ 2 increased the expression of ITGA11 (8.1±0.7 fold induction, p&lt;0.01) and reduced that of DDR1 (0.74±0.06 fold induction, p&lt;0.01), without affecting that of the other collagen receptors. Conclusion Intestinal tissues from CD patients affected by fibrosis present an altered pattern of expression of collagen receptors, which suggests a regulatory role of the ECM in the fibrotic response, while the correlation analysis and the changes induced by the fibrotic cytokine TGFβ in endothelial cells insinuates a particular relevance of these stromal cells in this process.

P051 Macrophages as a source of Notch Ligands in Crohn’s disease: implications in fibrosis

Background Fibrosis constitute the main complications associated to Crohn’s disease (CD). Notch signalling has been implicated in lung, kidney, liver and cardiac fibrosis. Macrophages contribute to fibrosis through the release of different mediators and the pattern of secretion may vary according to their microenvironment. The aim of the present study is to analyze the role of Notch ligands derived from macrophages in the complications of CD. Methods We have analyzed: the mRNA expression of cytokines and Notch ligands in CD patients with fistulizing and stenting pattern, the mRNA and protein expression of macrophage markers and Notch ligands in macrophages treated with the main cytokines present in CD patients (IFNγ-, IL10-, IL4, TNFα-U937 treated cells), the mRNA expression of fibrosis markers of DLL4-HT29 treated cells. Results are expressed as fold induction (mean±SEM, n≥4). Statistical analysis was performed with one-way ANOVA followed by Newman-Keuls test. Correlations were analyzed with the Spearman coefficient. Results The mRNA expression of IFNγ, TNFα, IL4, IL6 and IL10 was significantly higher in intestinal samples from B2- and B3-CD patients (11.4±1.6, 6.9±1.3, 16.4±3.9, 4.1±0.4, and 5.0±1.0 respectively for B2, and 14.2±1.8, 9.6±2.6, 21.5±4.1, 6.3±1.0 and 9.2±1.7 respectively for B3) than in controls (1.0±0.09, 1.1±0.2, 1.2±0.1, 1.1±0.1 and 1.0±0.1 respectively). The mRNA expression of DLL3 and DLL4 was significantly higher in intestinal samples from B2 (6.1±1.3 and 9.3±2.4, respectively) than in B3 CD patients (4.4±1.0 and 1.8±0.7, respectively) and in controls (0.9±0.2 and 0.7±1.2 respectively). IFNγ-U937 treated cells increased significantly the mRNA expression of DLL3 and DLL4 (2,9±0,6 N=5 N=4 and 3,7±1,1 N=4, respectively) respect vehicle; IL1β increased significantly the expression of DLL4 (1,8±0,01 N=4) and TNFα increased significantly the expression of DLL3 (7,1±2,2 N=4), respect vehicle. DLL4-HT29 treated cells increased significantly fibrosis markers (VIMENTIN (4,6±0,6 N=6), α-SMA (20,9±8,2 N=6), SNAIL1 (1,8±0,4 N=6), SNAIL2 (8,3±1,3 N=6), ZEB1 (8,2±4,3 N=6) and ZEB2 (4,3±0,2 N=6), respect vehicle. Conclusion Macrophages may act as a source of Notch ligands who could act as fibrosis mediators in CD patients with a stenting (B2) behavior. The microenvironment rich in IFNγ could activate the fibrosis process in epithelial cells by favoring the expression of DLL4 in macrophages.

P089 IFNγ-macrophages could mediate EMT in Crohn’s disease

Background Macrophages contribute to fibrosis by releasing different mediators and the pattern of secretion may vary depending on the surrounding environment. We previously described that the mRNA expression of IFNγ was significantly higher in intestinal samples from CD patients. The aim of the present study is to analyze the role of IFNγ-treated macrophages in epithelial mesenchymal transition (EMT). Methods The mRNA and protein expression of IFN in surgical resections from Crohn′s disease. U937 were differentiated to macrophages and then treated with IFNγ (2 ng/ml) for 4 days, the mRNA expression of macrophages markers were determined by RT-PCR. IFNγ-U937 were coculture with HT29 cells for 2 days and the expression of EMT markers in HT29 cells were analyzed by RT-PCR and WB. Results are expressed as mean±SEM (n≥5). Statistical analysis was performed by ANOVA + Newman-Keuls. Results The mRNA and protein expression of IFNγ were significantly higher in intestinal samples from B2 CD patients (11.4±1.6 fold induction and 3.5±0.3 pg/mg, respectively) and B3 CD patients (14.2±1.8 fold induction and 3.1±0.1 pg/mg, respectively) than in controls (1,0±0,1 fold induction and 0.8±0,1 pg/mg, respectively). U937 cells treated with IFNγ increased significantly the mRNA expression of CD16 (1.9±0.2* vs vehicle) and CD86 (1.6±0.1* vs vehicle). IFNγ-U937 cocultured with HT29 increased significantly the mRNA and protein expression of EMT markers in HT29 cells (Vimentin: 3.0±0.5* vs vehicle-HT29; αSMA: 24.4±8.3* vs vehicle-HT29; SNAIL1: 2.7±0.5* vs vehicle-HT29) respect IFNγ-HT29 cells (Vimentin: 0.6±0.01 vs vehicle-HT29; αSMA: 1.1±0.4 vs vehicle-HT29; SNAIL1: 0.7±0.06 vs vehicle-HT29). Conclusion IFNγ could be responsible for the increase in the number of CD86/CD16 macrophages in CD patients. A macrophage phenotype expressing CD86/CD16 may act as a source of EMT mediators in intestinal tissue from CD patients.

Optogenetic Amplification Circuits for Light-Induced Metabolic Control

Dynamic control of microbial metabolism is an effective strategy to improve chemical production in fermentations. While dynamic control is most often implemented using chemical inducers, optogenetics offers an attractive alternative due to the high tunability and reversibility afforded by light. However, a major concern of applying optogenetics in metabolic engineering is the risk of insufficient light penetration at high cell densities, especially in large bioreactors. Here, we present a new series of optogenetic circuits we call OptoAMP, which amplify the transcriptional response to blue light by as much as 21.8-fold compared to the basal circuit (OptoEXP). These circuits show as much as a 41-fold induction between dark and light conditions, efficient activation at light doses as low as ~1%, and strong homogeneous light-induction in bioreactors of at least 5L, with limited illumination at cell densities above 40 OD600. We demonstrate the ability of OptoAMP circuits to control engineered metabolic pathways in novel three-phase fermentations using different light schedules to control enzyme expression and improve production of lactic acid, isobutanol, and naringenin. These circuits expand the applicability of optogenetics to metabolic engineering.

A suppressor tRNA-mediated feedforward loop eliminates leaky gene expression in bacteria

Ligand-inducible genetic systems are the mainstay of synthetic biology, allowing gene expression to be controlled by the presence of a small molecule. However, ‘leaky’ gene expression in the absence of inducer remains a persistent problem. We developed a leak dampener tool that drastically reduces the leak of inducible genetic systems while retaining signal in Escherichia coli. Our system relies on a coherent feedforward loop featuring a suppressor tRNA that enables conditional readthrough of silent non-sense mutations in a regulated gene, and this approach can be applied to any ligand-inducible transcription factor. We demonstrate proof-of-principle of our system with the lactate biosensor LldR and the arabinose biosensor AraC, which displayed a 70-fold and 630-fold change in output after induction of a fluorescence reporter, respectively, without any background subtraction. Application of the tool to an arabinose-inducible mutagenesis plasmid led to a 540-fold change in its output after induction, with leak decreasing to the level of background mutagenesis. This study provides a modular tool for reducing leak and improving the fold-induction within genetic circuits, demonstrated here using two types of biosensors relevant to cancer detection and genetic engineering.