Thioredoxin Confers Intrinsic Resistance to Cytostatic Drugs in Human Glioma Cells.

Thioredoxin (Trx) overexpression is known to be a cause of chemotherapy resistance in various tumor entities. However, Trx effects on resistance are complex and depend strictly on tissue type. In the present study, we analyzed the impact of the Trx system on intrinsic chemoresistance of human glioblastoma multiforme (GBM) cells to cytostatic drugs. Resistance of GBM cell lines and primary cells to drugs and signaling inhibitors was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Impact of Trx inhibition on apoptosis was investigated by proteome profiling of a subset of proteins and annexin V apoptosis assays. Trx-interacting protein (TXNIP) was overexpressed by transfection and protein expression was determined by immunoblotting. Pharmacological inhibition of Trx by 1-methyl-2-imidazolyl-disulfide (PX-12) reduced viability of three GBM cell lines, induced expression of active caspase-3, and reduced phosphorylation of AKT-kinase and expression of ß-catenin. Sensitivity to cisplatin could be restored by both PX-12 and recombinant expression of the upstream Trx inhibitor TXNIP, respectively. In addition, PX-12 also sensitized primary human GBM cells to temozolomide. Combined inhibition of Trx and the phosphatidylinositide 3-kinase (PI3K) pathway resulted in massive cell death. We conclude that the Trx system and the PI3K pathway act as a sequential cascade and could potentially present a new drug target.

Identification of potential protein dithiol-disulfide substrates of mammalian Grx2.

BACKGROUND: Glutaredoxins (Grxs) catalyze the reduction of protein disulfides via the dithiol mechanism and the de-/glutathionylation of substrates via the monothiol mechanism. These rapid, specific, and generally also reversible modifications are part of various signaling cascades regulating for instance cell proliferation, differentiation and apoptosis. Even though crucial functions of the conserved, mitochondrial Grx2a and the cytosolic/nuclear Grx2c isoforms have been proposed, only a few substrates have been identified in vitro or in vivo. The significance of redox signaling is emerging, yet a general lack of methods for the time-resolved analysis of these distinct and rapid modifications in vivo constitutes the biggest challenge in the redox signaling field. METHODS AND RESULTS: Here, we have identified potential interaction partners for Grx2 isoforms in human HeLa cells and mouse tissues by an intermediate trapping approach. Some of the 50 potential substrates are part of the cytoskeleton or act in protein folding, cellular signaling and metabolism. Part of these interactions were further verified by immunoprecipitation or a newly established 2-D redox blot. CONCLUSIONS: Our study demonstrates that Grx2 catalyzes both the specific oxidation and the reduction of cysteinyl residues in the same compartment at the same time and without affecting the global cellular thiol-redox state. GENERAL SIGNIFICANCE: The knowledge of specific targets will be helpful in understanding the functions of Grx2. The 2-D redox blot may be useful for the analysis of the overall thiol-redox state of proteins with high molecular weight and numerous cysteinyl residues, that evaded analysis by previously described methods.

Vertebrate-specific glutaredoxin is essential for brain development.

Cellular functions and survival are dependent on a tightly controlled redox potential. Currently, an increasing amount of data supports the concept of local changes in the redox environment and specific redox signaling events controlling cell function. Specific protein thiol groups are the major targets of redox signaling and regulation. Thioredoxins and glutaredoxins catalyze reversible thiol-disulfide exchange reactions and are primary regulators of the protein thiol redox state. Here, we demonstrate that embryonic brain development depends on the enzymatic activity of glutaredoxin 2. Zebrafish with silenced expression of glutaredoxin 2 lost virtually all types of neurons by apoptotic cell death and the ability to develop an axonal scaffold. As demonstrated in zebrafish and in a human cellular model for neuronal differentiation, glutaredoxin 2 controls axonal outgrowth via thiol redox regulation of collapsin response mediator protein 2, a central component of the semaphorin pathway. This study provides an example of a specific thiol redox regulation essential for vertebrate embryonic development.

Non-canonical nucleosides: Biomimetic triphosphorylation, incorporation into mRNA and effects on translation and structure.

Recent advances in mRNA therapeutics demand efficient toolkits for the incorporation of nucleoside analogues into mRNA suitable for downstream applications. Herein, we report the application of a versatile enzyme cascade for the triphosphorylation of a broad range of nucleoside analogues, including unprotected nucleobases containing chemically labile moieties. Our biomimetic system was suitable for the preparation of nucleoside triphosphates containing adenosine, cytidine, guanosine, uridine and non-canonical core structures, as determined by capillary electrophoresis coupled to mass spectrometry. This enabled us to establish an efficient workflow for transcribing and purifying functional mRNA containing these nucleoside analogues, combined with mass spectrometric verification of analogue incorporation. Our combined methodology allows for analyses of how incorporation of nucleoside analogues that are commercially unavailable as triphosphates affect mRNA properties: The translational fidelity of the produced mRNA was demonstrated in analyses of how incorporated adenosine analogues impact translational recoding. For the SARS-CoV-2 frameshifting site, analyses of the mRNA pseudoknot structure using circular dichroism spectroscopy allowed insight into how the pharmacologically active 7-deazaadenosine destabilises RNA secondary structure, consistent with observed changes in recoding efficiency.

Endothelial Protein kinase D1 is a major regulator of post-traumatic hyperinflammation.

Trauma is a major cause of death worldwide. The post-traumatic immune response culminates in the release of pro-inflammatory mediators, translating in the infiltration of neutrophils (PMNs) at injury sites. The extent of this inflammation is determined by multiple factors, such as PMN adhesion to the endothelium, transendothelial migration, endothelial barrier integrity as well as PMN swarming, mass infiltration and activation. This process is initiated by secondary lipid mediators, such as leukotriene B4 (LTB4). We here provide evidence that Protein kinase D1 (PRKD1) in endothelial cells is implicated in all these processes. Endothelial PRKD1 is activated by pro-inflammatory stimuli and amplifies PMN-mediated inflammation by upregulation of cytokine and chemokines as well as adhesion molecules, such as ICAM-1, VCAM-1 and E-selectin. This induces enhanced PMN adhesion and trans-migration. PRKD1 activation also destabilizes endothelial VE-cadherin adhesion complexes and thus the endothelial barrier, fostering PMN infiltration. We even describe a yet unrecognized PRKD1-dependant mechanism to induce biosynthesis of the PMN-swarming mediator LTB4 directed via intercellular communication through small extracellular vesicles (sEVs) and enhanced CXCL8 secretion from activated endothelial cells. These endothelial sEVs transfer the LTB4 biosynthesis enzyme LTA4 hydrolase (LTA4H) to prime PMNs, while initiating biosynthesis also requires additional signals, like CXCL8. We further demonstrate the respective LTA4H-positive sEVs in the serum of polytrauma patients, peaking 12 h post injury. Therefore, PRKD1 is a key regulator in the coordinated communication of the endothelium with PMNs and a vital signaling node during post-traumatic inflammation.

Organoids at the PUB: The Porcine Urinary Bladder Serves as a Pancreatic Niche for Advanced Cancer Modeling.

Despite intensive research and progress in personalized medicine, pancreatic ductal adenocarcinoma remains one of the deadliest cancer entities. Pancreatic duct-like organoids (PDLOs) derived from human pluripotent stem cells (PSCs) or pancreatic cancer patient-derived organoids (PDOs) provide unique tools to study early and late stage dysplasia and to foster personalized medicine. However, such advanced systems are neither rapidly nor easily accessible and require an in vivo niche to study tumor formation and interaction with the stroma. Here, the establishment of the porcine urinary bladder (PUB) is revealed as an advanced organ culture model for shaping an ex vivo pancreatic niche. This model allows pancreatic progenitor cells to enter the ductal and endocrine lineages, while PDLOs further mature into duct-like tissue. Accordingly, the PUB offers an ex vivo platform for earliest pancreatic dysplasia and cancer if PDLOs feature KRASG12D mutations. Finally, it is demonstrated that PDOs-on-PUB i) resemble primary pancreatic cancer, ii) preserve cancer subtypes, iii) enable the study of niche epithelial crosstalk by spiking in pancreatic stellate and immune cells into the grafts, and finally iv) allow drug testing. In summary, the PUB advances the existing pancreatic cancer models by adding feasibility, complexity, and customization at low cost and high flexibility.

Both thioredoxin 2 and glutaredoxin 2 contribute to the reduction of the mitochondrial 2-Cys peroxiredoxin Prx3.

The proteins from the thioredoxin family are crucial actors in redox signaling and the cellular response to oxidative stress. The major intracellular source for oxygen radicals are the components of the respiratory chain in mitochondria. Here, we show that the mitochondrial 2-Cys peroxiredoxin (Prx3) is not only substrate for thioredoxin 2 (Trx2), but can also be reduced by glutaredoxin 2 (Grx2) via the dithiol reaction mechanism. Grx2 reduces Prx3 exhibiting catalytic constants (K(m), 23.8 µmol·liter(-1); V(max), 1.2 µmol·(mg·min)(-1)) similar to Trx2 (K(m), 11.2 µmol·liter(-1); V(max), 1.1 µmol·(mg·min)(-1)). The reduction of the catalytic disulfide of the atypical 2-Cys Prx5 is limited to the Trx system. Silencing the expression of either Trx2 or Grx2 in HeLa cells using specific siRNAs did not change the monomer:dimer ratio of Prx3 detected by a specific 2-Cys Prx redox blot. Only combined silencing of the expression of both proteins led to an accumulation of oxidized protein. We further demonstrate that the distribution of Prx3 in different mouse tissues is either linked to the distribution of Trx2 or Grx2. These results introduce Grx2 as a novel electron donor for Prx3, providing further insights into pivotal cellular redox signaling mechanisms.

A Prospective Feasibility Trial to Challenge Patient-Derived Pancreatic Cancer Organoids in Predicting Treatment Response.

Real-time isolation, propagation, and pharmacotyping of patient-derived pancreatic cancer organoids (PDOs) may enable treatment response prediction and personalization of pancreatic cancer (PC) therapy. In our methodology, PDOs are isolated from 54 patients with suspected or confirmed PC in the framework of a prospective feasibility trial. The drug response of single agents is determined by a viability assay. Areas under the curves (AUC) are clustered for each drug, and a prediction score is developed for combined regimens. Pharmacotyping profiles are obtained from 28 PDOs (efficacy 63.6%) after a median of 53 days (range 21-126 days). PDOs exhibit heterogeneous responses to the standard-of-care drugs, and are classified into high, intermediate, or low responder categories. Our developed prediction model allows a successful response prediction in treatment-naïve patients with an accuracy of 91.1% for first-line and 80.0% for second-line regimens, respectively. The power of prediction declines in pretreated patients (accuracy 40.0%), particularly with more than one prior line of chemotherapy. Progression-free survival (PFS) is significantly longer in previously treatment-naïve patients receiving a predicted tumor sensitive compared to a predicted tumor resistant regimen (mPFS 141 vs. 46 days; p = 0.0048). In conclusion, generation and pharmacotyping of PDOs is feasible in clinical routine and may provide substantial benefit.

Booster Vaccination With GVGH Shigella sonnei 1790GAHB GMMA Vaccine Compared to Single Vaccination in Unvaccinated Healthy European Adults: Results From a Phase 1 Clinical Trial.

The investigational Shigella sonnei vaccine (1790GAHB) based on GMMA (generalized modules for membrane antigens) is immunogenic, with an acceptable safety profile in adults. However, pre-vaccination anti-S. sonnei lipopolysaccharide (LPS) antibody levels seemed to impact vaccine-related immune responses. This phase 1, open-label, non-randomized extension study (ClinicalTrials.gov: NCT03089879) evaluated immunogenicity of a 1790GAHB booster dose in seven adults with undetectable antibodies prior to priming with three 1790GAHB vaccinations 2-3 years earlier (boosted group), compared to one dose in 28 vaccine-naïve individuals (vaccine-naïve group). Anti-S. sonnei LPS serum IgG geometric mean concentrations and seroresponse (increase of ≥25 EU or ≥50% from baseline antibody ≤ 50 EU and ≥50 EU, respectively) rates were calculated at vaccination (day [D]1), D8, D15, D29, D85. Safety was assessed. Geometric mean concentrations at D8 were 168 EU (boosted group) and 32 EU (vaccine-naïve group). Response peaked at D15 (883 EU) and D29 (100 EU) for the boosted and vaccine-naïve groups. Seroresponse rates at D8 were 86% (boosted group) and 24% (vaccine-naïve group) and increased at subsequent time points. Across both groups, pain (local) and fatigue (systemic) were the most frequent solicited adverse events (AEs). Unsolicited AEs were reported by 57% of boosted and 25% of vaccine-naïve participants. No deaths, serious AEs, or AEs of special interest (except one mild neutropenia case, possibly vaccination-related) were reported. One 1790GAHB dose induced a significant booster response in previously-primed adults, regardless of priming dose, and strong immune response in vaccine-naïve individuals. Vaccination was well tolerated.

A Phase 2a Randomized Study to Evaluate the Safety and Immunogenicity of the 1790GAHB Generalized Modules for Membrane Antigen Vaccine against Shigella sonnei Administered Intramuscularly to Adults from a Shigellosis-Endemic Country.

Shigellosis is a mild-to-severe diarrheal infection, caused by the genus Shigella, and is responsible for significant morbidity and mortality worldwide. We evaluated the safety and immunogenicity of an investigational Shigella sonnei vaccine (1790GAHB) based on generalized modules for membrane antigens (GMMA) in Kenya, a Shigella-endemic country. This phase 2a, observer-blind, controlled randomized study (NCT02676895) enrolled 74 healthy adults aged 18-45 years, of whom 72 were vaccinated. Participants received, in a 1:1:1 ratio, two vaccinations with the 1790GAHB vaccine at doses of either 1.5/25 µg of O antigen (OAg)/protein (group 1.5/25 µg) or 5.9/100 µg (group 5.9/100 µg) at day (D) 1 and D29, or vaccination with a quadrivalent meningococcal vaccine at D1 and tetanus, diphtheria, and acellular pertussis vaccine at D29 (control group). Solicited and unsolicited adverse events (AEs), serious AEs (SAEs), and AEs of special interest (neutropenia and reactive arthritis) were collected. Anti-S. sonnei lipopolysaccharide (LPS) serum immunoglobulin G (IgG) geometric mean concentrations (GMC) were evaluated at D1, D29, and D57 and compared to anti-S. sonnei LPS antibody levels in convalescent patients naturally exposed to S. sonnei. The percentages of participants with seroresponse were also calculated. The most frequently reported solicited local and systemic AEs across all groups were pain and headache, respectively. Only one case of severe systemic reaction was reported (severe headache after first vaccination in group 5.9/100 µg). Seven and three episodes of neutropenia, assessed as probably or possibly related to vaccination respectively, were reported in the investigational and control groups, respectively. No other SAEs were reported. Despite very high baseline anti-S. sonnei LPS serum IgG levels, the 1790GAHB vaccine induced robust antibody responses. At D29, GMC increased 2.10- and 4.43-fold from baseline in groups 1.5/25 and 5.9/100 µg, respectively, whereas no increase was observed in the control group. Antibody titers at D57 were not statistically different from those at D29. Seroresponse was 68% at D29 and 90% at D57 in group 1.5/25 µg, and 96% after each vaccination in group 5.9/100 µg. The 1790GAHB vaccine was well tolerated and highly immunogenic in a population of African adults, regardless of the GMMA OAg/protein content used.