DYRK1B blockade promotes tumoricidal macrophage activity in pancreatic cancer.

OBJECTIVE: Highly malignant pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant immunosuppressive and fibrotic tumour microenvironment (TME). Future therapeutic attempts will therefore demand the targeting of tumours and stromal compartments in order to be effective. Here we investigate whether dual specificity and tyrosine phosphorylation-regulated kinase 1B (DYRK1B) fulfil these criteria and represent a promising anticancer target in PDAC. DESIGN: We used transplantation and autochthonous mouse models of PDAC with either genetic Dyrk1b loss or pharmacological DYRK1B inhibition, respectively. Mechanistic interactions between tumour cells and macrophages were studied in direct or indirect co-culture experiments. Histological analyses used tissue microarrays from patients with PDAC. Additional methodological approaches included bulk mRNA sequencing (transcriptomics) and proteomics (secretomics). RESULTS: We found that DYRK1B is mainly expressed by pancreatic epithelial cancer cells and modulates the influx and activity of TME-associated macrophages through effects on the cancer cells themselves as well as through the tumour secretome. Mechanistically, genetic ablation or pharmacological inhibition of DYRK1B strongly attracts tumoricidal macrophages and, in addition, downregulates the phagocytosis checkpoint and 'don't eat me' signal CD24 on cancer cells, resulting in enhanced tumour cell phagocytosis. Consequently, tumour cells lacking DYRK1B hardly expand in transplantation experiments, despite their rapid growth in culture. Furthermore, combining a small-molecule DYRK1B-directed therapy with mammalian target of rapamycin inhibition and conventional chemotherapy stalls the growth of established tumours and results in a significant extension of life span in a highly aggressive autochthonous model of PDAC. CONCLUSION: In light of DYRK inhibitors currently entering clinical phase testing, our data thus provide a novel and clinically translatable approach targeting both the cancer cell compartment and its microenvironment.

Agtrevirus phage AV101 recognizes four different O-antigens infecting diverse E. coli.

Bacteriophages in the Agtrevirus genus are known for expressing multiple tail spike proteins (TSPs), but little is known about their genetic diversity and host recognition apart from their ability to infect diverse Enterobacteriaceae species. Here, we aim to determine the genetic differences that may account for the diverse host ranges of Agrevirus phages. We performed comparative genomics of 14 Agtrevirus and identified only a few genetic differences including genes involved in nucleotide metabolism. Most notably was the diversity of the tsp gene cluster, specifically in the receptor-binding domains that were unique among most of the phages. We further characterized agtrevirus AV101 infecting nine diverse Extended Spectrum ß-lactamase (ESBL) Escherichia coli and demonstrated that this phage encoded four unique TSPs among Agtrevirus. Purified TSPs formed translucent zones and inhibited AV101 infection of specific hosts, demonstrating that TSP1, TSP2, TSP3, and TSP4 recognize O8, O82, O153, and O159 O-antigens of E. coli, respectively. BLASTp analysis showed that the receptor-binding domain of TSP1, TSP2, TSP3, and TSP4 are similar to TSPs encoded by E. coli prophages and distant related virulent phages. Thus, Agtrevirus may have gained their receptor-binding domains by recombining with prophages or virulent phages. Overall, combining bioinformatic and biological data expands the understanding of TSP host recognition of Agtrevirus and give new insight into the origin and acquisition of receptor-binding domains of Ackermannviridae phages.

Isolation and characterization of novel plasmid-dependent phages infecting bacteria carrying diverse conjugative plasmids.

IMPORTANCE: This work was undertaken because plasmid-dependent phages can reduce the prevalence of conjugative plasmids and can be leveraged to prevent the acquisition and dissemination of ARGs by bacteria. The two novel phages described in this study, Lu221 and Hi226, can infect Escherichia coli, Salmonella enterica, Kluyvera sp. and Enterobacter sp. carrying conjugative plasmids. This was verified with plasmids carrying resistance determinants and belonging to the most common plasmid families among Gram-negative pathogens. Therefore, the newly isolated phages could have the potential to help control the spread of ARGs and thus help combat the antimicrobial resistance crisis.

IL-17A-producing CD8+ T cells promote PDAC via induction of inflammatory cancer-associated fibroblasts.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant desmoplastic stroma composed of cancer-associated fibroblasts (CAF) and interspersed immune cells. A non-canonical CD8+ T-cell subpopulation producing IL-17A (Tc17) promotes autoimmunity and has been identified in tumours. Here, we evaluated the Tc17 role in PDAC. DESIGN: Infiltration of Tc17 cells in PDAC tissue was correlated with patient overall survival and tumour stage. Wild-type (WT) or Il17ra-/- quiescent pancreatic stellate cells (qPSC) were exposed to conditional media obtained from Tc17 cells (Tc17-CM); moreover, co-culture of Tc17-CM-induced inflammatory (i)CAF (Tc17-iCAF) with tumour cells was performed. IL-17A/F-, IL-17RA-, RAG1-deficient and Foxn1nu/nu mice were used to study the Tc17 role in subcutaneous and orthotopic PDAC mouse models. RESULTS: Increased abundance of Tc17 cells highly correlated with reduced survival and advanced tumour stage in PDAC. Tc17-CM induced iCAF differentiation as assessed by the expression of iCAF-associated genes via synergism of IL-17A and TNF. Accordingly, IL-17RA controlled the responsiveness of qPSC to Tc17-CM. Pancreatic tumour cells co-cultured with Tc17-iCAF displayed enhanced proliferation and increased expression of genes implicated in proliferation, metabolism and protection from apoptosis. Tc17-iCAF accelerated growth of mouse and human tumours in Rag1-/- and Foxn1nu/nu mice, respectively. Finally, Il17ra-expressed by fibroblasts was required for Tc17-driven tumour growth in vivo. CONCLUSIONS: We identified Tc17 as a novel protumourigenic CD8+ T-cell subtype in PDAC, which accelerated tumour growth via IL-17RA-dependent stroma modification. We described a crosstalk between three cell types, Tc17, fibroblasts and tumour cells, promoting PDAC progression, which resulted in poor prognosis for patients.

IL18 Receptor Signaling Regulates Tumor-Reactive CD8+ T-cell Exhaustion via Activation of the IL2/STAT5/mTOR Pathway in a Pancreatic Cancer Model.

Intratumoral cytotoxic CD8+ T cells (CTL) enter a dysfunctional state characterized by expression of coinhibitory receptors, loss of effector function, and changes in the transcriptional landscape. Even though several regulators of T-cell exhaustion have been identified, the molecular mechanisms inducing T-cell exhaustion remain unclear. Here, we show that IL18 receptor (IL18R) signaling induces CD8+ T-cell exhaustion in a murine pancreatic cancer model. Adoptive transfer of Il18r-/- OT-1 CD8+ CTLs resulted in enhanced rejection of subcutaneous tumors expressing ovalbumin (OVA) as a model antigen (PancOVA), compared with wild-type OT-1 CTLs. Transferred intratumoral IL18R-deficient CTLs expressed higher levels of effector cytokines TNF and IFNγ and had reduced expression of coinhibitory receptors (PD-1, TIM-3, 2B4, LAG-3) and the transcription factors Eomes and TOX. Lower expression of coinhibitory receptors and TOX on IL18R-deficient versus IL18R-sufficient CD8+ T cells were confirmed in an orthotopic KPC model. IL18R-induced T-cell exhaustion was regulated by IL2/STAT5 and AKT/mTOR pathways, as demonstrated in an in vitro exhaustion assay. Concordantly, mice deficient in NLRP3, the molecular complex activating IL18, had decreased expression of coinhibitory receptors on intratumoral T cells and similar changes in signaling pathways at the transcriptome level. Thus, molecular pathways promoting T-cell exhaustion indicate an involvement of an NLRP3-expressing tumor microenvironment, which mediates IL18 release. The Cancer Genome Atlas analysis of patients with pancreatic carcinoma showed an association between NLRP3-mediated IL18 signaling and shorter survival. These findings indicate NLRP3-mediated IL18R signaling as a regulator of intratumoral T-cell exhaustion and a possible target for immunotherapy. See related Spotlight by Stromnes, p. 400.

CRISPR-Cas9 Shaped Viral Metagenomes Associated with Bacillus subtilis.

Phages are viruses of bacteria and have been known for over a century. They do not have a metabolism or protein synthesis machinery and rely on host cells for replication. The model organism Bacillus subtilis has served as a host strain for decades and enabled the isolation of many unique viral strains. However, many viral species representatives remained orphans as no, or only a few, related phages were ever re-isolated.The presented protocol describes how a CRISPR-Cas9 system with an artificial CRISPR-array can be set up and used to discriminate abundant and well-known B. subtilis phage from a host-based metagenome enrichment. The obtained viral suspension can be used for metagenome sequencing and isolating new viral strains.

A semaphorin-plexin-Rasal1 signaling pathway inhibits gastrin expression and protects against peptic ulcers.

Peptic ulcer disease is a frequent clinical problem with potentially serious complications such as bleeding or perforation. A decisive factor in the pathogenesis of peptic ulcers is gastric acid, the secretion of which is controlled by the hormone gastrin released from gastric G cells. However, the molecular mechanisms regulating gastrin plasma concentrations are poorly understood. Here, we identified a semaphorin-plexin signaling pathway that operates in gastric G cells to inhibit gastrin expression on a transcriptional level, thereby limiting food-stimulated gastrin release and gastric acid secretion. Using a systematic siRNA screening approach combined with biochemical, cell biology, and in vivo mouse experiments, we found that the RasGAP protein Rasal1 is a central mediator of plexin signal transduction, which suppresses gastrin expression through inactivation of the small GTPase R-Ras. Moreover, we show that Rasal1 is pathophysiologically relevant for the pathogenesis of peptic ulcers induced by nonsteroidal anti-inflammatory drugs (NSAIDs), a main risk factor of peptic ulcers in humans. Last, we show that application of recombinant semaphorin 4D alleviates peptic ulcer disease in mice in vivo, demonstrating that this signaling pathway can be harnessed pharmacologically. This study unravels a mode of G cell regulation that is functionally important in gastric homeostasis and disease.

The Bacillus phage SPß and its relatives: a temperate phage model system reveals new strains, species, prophage integration loci, conserved proteins and lysogeny management components.

The Bacillus phage SPß has been known for about 50 years, but only a few strains are available. We isolated four new wild-type strains of the SPbeta species. Phage vB_BsuS-Goe14 introduces its prophage into the spoVK locus, previously not observed to be used by SPß-like phages. Sequence data revealed the genome replication strategy and the genome packaging mode of SPß-like phages. We extracted 55 SPß-like prophages from public Bacillus genomes, thereby discovering three more integration loci and one additional type of integrase. The identified prophages resemble four new species clusters and three species orphans in the genus Spbetavirus. The determined core proteome of all SPß-like prophages consists of 38 proteins. The integration cassette proved to be not conserved, even though, present in all strains. It consists of distinct integrases. Analysis of SPß transcriptomes revealed three conserved genes, yopQ, yopR, and yokI, to be transcribed from a dormant prophage. While yopQ and yokI could be deleted from the prophage without activating the prophage, damaging of yopR led to a clear-plaque phenotype. Under the applied laboratory conditions, the yokI mutant showed an elevated virion release implying the YokI protein being a component of the arbitrium system.

The POLD1R689W variant increases the sensitivity of colorectal cancer cells to ATR and CHK1 inhibitors.

Inhibition of the kinase ATR, a central regulator of the DNA damage response, eliminates subsets of cancer cells in certain tumors. As previously shown, this is at least partly attributable to synthetic lethal interactions between ATR and POLD1, the catalytic subunit of the polymerase δ. Various POLD1 variants have been found in colorectal cancer, but their significance as therapeutic targets for ATR pathway inhibition remains unknown. Using CRISPR/Cas9 in the colorectal cancer cell line DLD-1, which harbors four POLD1 variants, we established heterozygous POLD1-knockout clones with exclusive expression of distinct variants to determine the functional relevance of these variants individually by assessing their impact on ATR pathway activation, DNA replication, and cellular sensitivity to inhibition of ATR or its effector kinase CHK1. Of the four variants analyzed, only POLD1R689W affected POLD1 function, as demonstrated by compensatory ATR pathway activation and impaired DNA replication. Upon treatment with ATR or CHK1 inhibitors, POLD1R689W strongly decreased cell survival in vitro, which was attributable at least partly to S phase impairment and apoptosis. Similarly, treatment with the ATR inhibitor AZD6738 inhibited growth of murine xenograft tumors, harboring the POLD1R689W variant, in vivo. Our POLD1-knockout model thus complements algorithm-based models to predict the pathogenicity of tumor-specific variants of unknown significance and illustrates a novel and potentially clinically relevant therapeutic approach using ATR/CHK1 inhibitors in POLD1-deficient tumors.

The differential statin effect on cytokine production of monocytes or macrophages is mediated by differential geranylgeranylation-dependent Rac1 activation.

Monocytes and macrophages contribute to pathogenesis of various inflammatory diseases, including auto-inflammatory diseases, cancer, sepsis, or atherosclerosis. They do so by production of cytokines, the central regulators of inflammation. Isoprenylation of small G-proteins is involved in regulation of production of some cytokines. Statins possibly affect isoprenylation-dependent cytokine production of monocytes and macrophages differentially. Thus, we compared statin-dependent cytokine production of lipopolysaccharide (LPS)-stimulated freshly isolated human monocytes and macrophages derived from monocytes by overnight differentiation. Stimulated monocytes readily produced tumor necrosis factor-α, interleukin-6, and interleukin-1ß. Statins did not alter cytokine production of LPS-stimulated monocytes. In contrast, monocyte-derived macrophages prepared in the absence of statin lost the capacity to produce cytokines, whereas macrophages prepared in the presence of statin still produced cytokines. The cells expressed indistinguishable nuclear factor-kB activity, suggesting involvement of separate, statin-dependent regulation pathways. The presence of statin was necessary during the differentiation phase of the macrophages, indicating that retainment-of-function rather than costimulation was involved. Reconstitution with mevalonic acid, farnesyl pyrophosphate, or geranylgeranyl pyrophosphate blocked the retainment effect, whereas reconstitution of cholesterol synthesis by squalene did not. Inhibition of geranylgeranylation by GGTI-298, but not inhibition of farnesylation or cholesterol synthesis, mimicked the retainment effect of the statin. Inhibition of Rac1 activation by the Rac1/TIAM1-inhibitor NSC23766 or by Rac1-siRNA (small interfering RNA) blocked the retainment effect. Consistent with this finding, macrophages differentiated in the presence of statin expressed enhanced Rac1-GTP-levels. In line with the above hypothesis that monocytes and macrophages are differentially regulated by statins, the CD14/CD16-, merTK-, CX3CR1-, or CD163-expression (M2-macrophage-related) correlated inversely to the cytokine production. Thus, monocytes and macrophages display differential Rac1-geranylgeranylation-dependent functional capacities, that is, statins sway monocytes and macrophages differentially.

Draft Genome Sequence of the Butanoic Acid-Producing Bacterium Clostridium luticellarii DSM 29923, Used for Strong Aromatic Chinese Liquor Production.

The strictly anaerobic, Gram-positive bacterium Clostridium luticellarii, which has straight or slightly curved rod-shaped cells, polar endospores, and peritrichous flagella, is used for the production of strong aromatic Chinese liquors. C. luticellarii is able to produce butanoic acid. The draft genome sequence consists of 3.757 Mbp, including 3,632 predicted protein-encoding genes.