High-throughput ligand profile characterization in novel cell lines expressing seven heterologous insect olfactory receptors for the detection of volatile plant biomarkers.

Agriculturally important crop plants emit a multitude of volatile organic compounds (VOCs), which are excellent indicators of their health status and their interactions with pathogens and pests. In this study, we have developed a novel cellular olfactory panel for detecting fungal pathogen-related VOCs we had identified in the field, as well as during controlled inoculations of several crop plants. The olfactory panel consists of seven stable HEK293 cell lines each expressing a functional Drosophila olfactory receptor as a biosensing element along with GCaMP6, a fluorescent calcium indicator protein. An automated 384-well microplate reader was used to characterize the olfactory receptor cell lines for their sensitivity to reference VOCs. Subsequently, we profiled a set of 66 VOCs on all cell lines, covering a concentration range from 1 to 100 µM. Results showed that 49 VOCs (74.2%) elicited a response in at least one olfactory receptor cell line. Some VOCs activated the cell lines even at nanomolar (ppb) concentrations. The interaction profiles obtained here will support the development of biosensors for agricultural applications. Additionally, the olfactory receptor proteins can be purified from these cell lines with sufficient yields for further processing, such as structure determination or integration with sensor devices.

Defects in microvillus crosslinking sensitize to colitis and inflammatory bowel disease.

Intestinal epithelial cells are covered by the brush border, which consists of densely packed microvilli. The Intermicrovillar Adhesion Complex (IMAC) links the microvilli and is required for proper brush border organization. Whether microvillus crosslinking is involved in the intestinal barrier function or colitis is currently unknown. We investigate the role of microvillus crosslinking in colitis in mice with deletion of the IMAC component CDHR5. Electron microscopy shows pronounced brush border defects in CDHR5-deficient mice. The defects result in severe mucosal damage after exposure to the colitis-inducing agent DSS. DSS increases the permeability of the mucus layer and brings bacteria in direct contact with the disorganized brush border of CDHR5-deficient mice. This correlates with bacterial invasion into the epithelial cell layer which precedes epithelial apoptosis and inflammation. Single-cell RNA sequencing data of patients with ulcerative colitis reveals downregulation of CDHR5 in enterocytes of diseased areas. Our results provide experimental evidence that a combination of microvillus crosslinking defects with increased permeability of the mucus layer sensitizes to inflammatory bowel disease.

Targeted Protein Degradation: Clinical Advances in the Field of Oncology.

The field of targeted protein degradation (TPD) is a rapidly developing therapeutic modality with the promise to tame disease-relevant proteins in ways that are difficult or impossible to tackle with other strategies. While we move into the third decade of TPD, multiple degrader drugs have entered the stage of the clinic and many more are expected to follow. In this review, we provide an update on the most recent advances in the field of targeted degradation with insights into possible clinical implications for cancer prevention and treatment.

Tyk2 is a tumor suppressor in colorectal cancer.

Janus kinase Tyk2 is implicated in cancer immune surveillance, but its role in solid tumors is not well defined. We used Tyk2 knockout mice (Tyk2Δ/Δ) and mice with conditional deletion of Tyk2 in hematopoietic (Tyk2ΔHem) or intestinal epithelial cells (Tyk2ΔIEC) to assess their cell type-specific functions in chemically induced colorectal cancer. All Tyk2-deficient mouse models showed a higher tumor burden after AOM-DSS treatment compared to their corresponding wild-type controls (Tyk2+/+ and Tyk2fl/fl), demonstrating tumor-suppressive functions of Tyk2 in immune cells and epithelial cancer cells. However, specific deletion of Tyk2 in hematopoietic cells or in intestinal epithelial cells was insufficient to accelerate tumor progression, while deletion in both compartments promoted carcinoma formation. RNA-seq and proteomics revealed that tumors of Tyk2Δ/Δ and Tyk2ΔIEC mice were immunoedited in different ways with downregulated and upregulated IFNγ signatures, respectively. Accordingly, the IFNγ-regulated immune checkpoint Ido1 was downregulated in Tyk2Δ/Δ and upregulated in Tyk2ΔIEC tumors, although both showed reduced CD8+ T cell infiltration. These data suggest that Tyk2Δ/Δ tumors are Ido1-independent and poorly immunoedited while Tyk2ΔIEC tumors require Ido1 for immune evasion. Our study shows that Tyk2 prevents Ido1 expression in CRC cells and promotes CRC immune surveillance in the tumor stroma. Both of these Tyk2-dependent mechanisms must work together to prevent CRC progression.

Discovery of the cyclotide caripe 11 as a ligand of the cholecystokinin-2 receptor.

The cholecystokinin-2 receptor (CCK2R) is a G protein-coupled receptor (GPCR) that is expressed in peripheral tissues and the central nervous system and constitutes a promising target for drug development in several diseases, such as gastrointestinal cancer. The search for ligands of this receptor over the past years mainly resulted in the discovery of a set of distinct synthetic small molecule chemicals. Here, we carried out a pharmacological screening of cyclotide-containing plant extracts using HEK293 cells transiently-expressing mouse CCK2R, and inositol phosphate (IP1) production as a readout. Our data demonstrated that cyclotide-enriched plant extracts from Oldenlandia affinis, Viola tricolor and Carapichea ipecacuanha activate the CCK2R as measured by the production of IP1. These findings prompted the isolation of a representative cyclotide, namely caripe 11 from C. ipecacuanha for detailed pharmacological analysis. Caripe 11 is a partial agonist of the CCK2R (Emax = 71%) with a moderate potency of 8.5 µM, in comparison to the endogenous full agonist cholecystokinin-8 (CCK-8; EC50 = 11.5 nM). The partial agonism of caripe 11 is further characterized by an increase on basal activity (at low concentrations) and a dextral-shift of the potency of CCK-8 (at higher concentrations) following its co-incubation with the cyclotide. Therefore, cyclotides such as caripe 11 may be explored in the future for the design and development of cyclotide-based ligands or imaging probes targeting the CCK2R and related peptide GPCRs.

The glucocorticoid receptor associates with RAS complexes to inhibit cell proliferation and tumor growth.

Mutations that activate members of the RAS family of GTPases are associated with various cancers and drive tumor growth. The glucocorticoid receptor (GR), a member of the nuclear receptor family, has been proposed to interact with and inhibit the activation of components of the PI3K-AKT and MAPK pathways downstream of RAS. In the absence of activating ligands, we found that GR was present in cytoplasmic KRAS-containing complexes and inhibited the activation of wild-type and oncogenic KRAS in mouse embryonic fibroblasts and human lung cancer A549 cells. The DNA binding domain of GR was involved in the interaction with KRAS, but GR-dependent inhibition of RAS activation did not depend on the nuclear translocation of GR. The addition of ligand released GR-dependent inhibition of RAS, AKT, the MAPK p38, and the MAPKK MEK. CRISPR-Cas9-mediated deletion of GR in A549 cells enhanced tumor growth in xenografts in mice. Patient samples of non-small cell lung carcinomas showed lower expression of NR3C1, the gene encoding GR, compared to adjacent normal tissues and lower NR3C1 expression correlated with a worse disease outcome. These results suggest that glucocorticoids prevent the ability of GR to limit tumor growth by inhibiting RAS activation, which has potential implications for the use of glucocorticoids in patients with cancer.

Down-regulation of A20 promotes immune escape of lung adenocarcinomas.

Inflammation is a well-known driver of lung tumorigenesis. One strategy by which tumor cells escape tight homeostatic control is by decreasing the expression of the potent anti-inflammatory protein tumor necrosis factor alpha-induced protein 3 (TNFAIP3), also known as A20. We observed that tumor cell intrinsic loss of A20 markedly enhanced lung tumorigenesis and was associated with reduced CD8+ T cell-mediated immune surveillance in patients with lung cancer and in mouse models. In mice, we observed that this effect was completely dependent on increased cellular sensitivity to interferon-γ (IFN-γ) signaling by aberrant activation of TANK-binding kinase 1 (TBK1) and increased downstream expression and activation of signal transducer and activator of transcription 1 (STAT1). Interrupting this autocrine feed forward loop by knocking out IFN-α/ß receptor completely restored infiltration of cytotoxic T cells and rescued loss of A20 depending tumorigenesis. Downstream of STAT1, programmed death ligand 1 (PD-L1) was highly expressed in A20 knockout lung tumors. Accordingly, immune checkpoint blockade (ICB) treatment was highly efficient in mice harboring A20-deficient lung tumors. Furthermore, an A20 loss-of-function gene expression signature positively correlated with survival of melanoma patients treated with anti-programmed cell death protein 1. Together, we have identified A20 as a master immune checkpoint regulating the TBK1-STAT1-PD-L1 axis that may be exploited to improve ICB therapy in patients with lung adenocarcinoma.

Efficient production of recombinant secretory IgA against Clostridium difficile toxins in CHO-K1 cells.

Despite the essential role secretory IgAs play in the defense against pathogenic invasion and the proposed value of recombinant secretory IgAs as novel therapeutics, currently there are no IgA-based therapies in clinics. Secretory IgAs are complex molecules and the major bottleneck limiting their therapeutic potential is a reliable recombinant production system. In this report, we addressed this issue and established a fast and robust production method for secretory IgAs in CHO-K1 cells using BAC-based expression vectors. As a proof of principle, we produced IgAs against Clostridium difficile toxins TcdA and TcdB. Recombinant secretory IgAs produced using our expression system showed comparable titers to IgGs, widely used as therapeutic biologicals. Importantly, secretory IgAs produced using our method were functional and could efficiently neutralize Clostridium difficile toxins TcdA and TcdB. These results show that recombinant secretory IgAs can be efficiently produced, thus opening the possibility to use them as therapeutic agents in clinics.

Targeting KRAS Mutant Non-Small-Cell Lung Cancer: Past, Present and Future.

Lung cancer is the most frequent cancer with an aggressive clinical course and high mortality rates. Most cases are diagnosed at advanced stages when treatment options are limited and the efficacy of chemotherapy is poor. The disease has a complex and heterogeneous background with non-small-cell lung cancer (NSCLC) accounting for 85% of patients and lung adenocarcinoma being the most common histological subtype. Almost 30% of adenocarcinomas of the lung are driven by an activating Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation. The ability to inhibit the oncogenic KRAS has been the holy grail of cancer research and the search for inhibitors is immensely ongoing as KRAS-mutated tumors are among the most aggressive and refractory to treatment. Therapeutic strategies tailored for KRAS+ NSCLC rely on the blockage of KRAS functional output, cellular dependencies, metabolic features, KRAS membrane associations, direct targeting of KRAS and immunotherapy. In this review, we provide an update on the most recent advances in anti-KRAS therapy for lung tumors with mechanistic insights into biological diversity and potential clinical implications.

STAT3: Versatile Functions in Non-Small Cell Lung Cancer.

Signal Transducer and Activator of Transcription 3 (STAT3) activation is frequently found in non-small cell lung cancer (NSCLC) patient samples/cell lines and STAT3 inhibition in NSCLC cell lines markedly impairs their survival. STAT3 also plays a pivotal role in driving tumor-promoting inflammation and evasion of anti-tumor immunity. Consequently, targeting STAT3 either directly or by inhibition of upstream regulators such as Interleukin-6 (IL-6) or Janus kinase 1/2 (JAK1/2) is considered as a promising treatment strategy for the management of NSCLC. In contrast, some studies also report STAT3 being a tumor suppressor in a variety of solid malignancies, including lung cancer. Here, we provide a concise overview of STAT3's versatile roles in NSCLC and discuss the yins and yangs of STAT3 targeting therapies.

IDO1+ Paneth cells promote immune escape of colorectal cancer.

Tumors have evolved mechanisms to escape anti-tumor immunosurveillance. They limit humoral and cellular immune activities in the stroma and render tumors resistant to immunotherapy. Sensitizing tumor cells to immune attack is an important strategy to revert immunosuppression. However, the underlying mechanisms of immune escape are still poorly understood. Here we discover Indoleamine-2,3-dioxygenase-1 (IDO1)+ Paneth cells in the stem cell niche of intestinal crypts and tumors, which promoted immune escape of colorectal cancer (CRC). Ido1 expression in Paneth cells was strictly Stat1 dependent. Loss of IDO1+ Paneth cells in murine intestinal adenomas with tumor cell-specific Stat1 deletion had profound effects on the intratumoral immune cell composition. Patient samples and TCGA expression data suggested corresponding cells in human colorectal tumors. Thus, our data uncovered an immune escape mechanism of CRC and identify IDO1+ Paneth cells as a target for immunotherapy.

Notch inhibition overcomes resistance to tyrosine kinase inhibitors in EGFR-driven lung adenocarcinoma.

EGFR-mutated lung adenocarcinoma patients treated with gefitinib and osimertinib show a therapeutic benefit limited by the appearance of secondary mutations, such as EGFRT790M and EGFRC797S. It is generally assumed that these secondary mutations render EGFR completely unresponsive to the inhibitors, but contrary to this, we uncovered here that gefitinib and osimertinib increased STAT3 phosphorylation (p-STAT3) in EGFRT790M and EGFRC797S tumoral cells. Interestingly, we also found that concomitant Notch inhibition with gefitinib or osimertinib treatment induced a p-STAT3-dependent strong reduction in the levels of the transcriptional repressor HES1. Importantly, we showed that tyrosine kinase inhibitor-resistant tumors, with EGFRT790M and EGFRC797S mutations, were highly responsive to the combined treatment of Notch inhibitors with gefitinib or osimertinib, respectively. Finally, in patients with EGFR mutations treated with tyrosine kinase inhibitors, HES1 protein levels increased during relapse and correlated with shorter progression-free survival. Therefore, our results offer a proof of concept for an alternative treatment to chemotherapy in lung adenocarcinoma osimertinib-treated patients after disease progression.

[CHEK1 expression and inhibitors in TP53 mutant cancer]. / A checkpoint kinase 1 expressziója és gátlása TP53-mutációt hordozó rosszindulatú daganatokban.

The most frequently mutated gene in human tumors is TP53 and its mutation significantly deteriorates patients' survival. However, to date no targeted therapy is established for TP53 mutated tumors. Here, our aim was to identify druggable kinases with higher expression in TP53 mutated tumors, as well as relate these to altered prognosis. We also aimed to validate a target gene in TP53 wild type and mutant isogenic cell lines using a specific kinase inhibitor. Gene expression and mutation data were collected from 994 lung tumor samples. Samples were separated based on TP53 mutation status, and differential gene expression was compared using Mann-Whitney test between patient cohorts. Prognostic value of identified genes was validated in an array-based lung cancer dataset (n=1926). Survival analysis was performed using Cox proportional hazards regression and Kaplan-Meier survival plots. Effect of TP53 mutations on CHEK1 expression was validated in the A549 isogenic lung cancer cell line. The cell line was also treated using Chk1 protein specific kinase inhibitor to monitor cell functions. Expression of CHEK1 was elevated significantly among targetable kinases and higher expression of CHEK1 related to worse prognosis. Our results confirm the higher expression of CHEK1 kinase associated to TP53 mutations and to shorter survival.

Validation of an enzyme-linked immunosorbent assay (ELISA) for quantification of endostatin levels in mice as a biomarker of developing glomerulonephritis.

Endostatin, the C-terminal fragment of type XVIII collagen, was shown to be one of the most potent endothelial cell-specific inhibitors of angiogenesis. As altered circulating endostatin concentration is associated with impaired kidney function, new tools for measuring endostatin in rodents may be helpful to further investigate and understand its role within kidney disease progression. A novel and commercially available ELISA for the quantification of mouse and rat endostatin was developed and validated according to international quality guidelines including the parameters specificity, robustness, accuracy, dilution linearity, precision, limit of detection (LOD) and lower limit of quantification (LLOQ). Endostatin and blood urea nitrogen (BUN) concentration were measured in mice with a glomerulonephritis phenotype. The validation revealed that within the range of 0.5-32 nmol/L the immunoassay is robust and highly specific for the measurement of rodent endostatin with high sensitivity (LOD 0.24 nmol/L, LLOQ 0.5 nmol/L) and good reproducibility (intra- and inter-assay CV <10%). Also accuracy and dilution linearity were within the range of acceptance. BCL2 transgenic and ETV6/RUNX1;BCL2 double transgenic mice develop a glomerulonephritis phenotype over time, which was displayed by staining of kidney sections. Even before full manifestation of disease serum endostatin concentration rises significantly, whereas BUN levels just slightly increase. This newly developed and commercially available ELISA provides a reliable and accurate tool for the quantification of mouse and rat endostatin and may give new perspectives in the investigation of the role of endostatin as an important and early biomarker for reduced kidney function. Measurement of endostatin concentration is recommended to be used as a superior biomarker for chronic kidney disease compared to BUN.

JAK-STAT inhibition impairs K-RAS-driven lung adenocarcinoma progression.

Oncogenic K-RAS has been difficult to target and currently there is no K-RAS-based targeted therapy available for patients suffering from K-RAS-driven lung adenocarcinoma (AC). Alternatively, targeting K-RAS-downstream effectors, K-RAS-cooperating signaling pathways or cancer hallmarks, such as tumor-promoting inflammation, has been shown to be a promising therapeutic strategy. Since the JAK-STAT pathway is considered to be a central player in inflammation-mediated tumorigenesis, we investigated here the implication of JAK-STAT signaling and the therapeutic potential of JAK1/2 inhibition in K-RAS-driven lung AC. Our data showed that JAK1 and JAK2 are activated in human lung AC and that increased activation of JAK-STAT signaling correlated with disease progression and K-RAS activity in human lung AC. Accordingly, administration of the JAK1/2 selective tyrosine kinase inhibitor ruxolitinib reduced proliferation of tumor cells and effectively reduced tumor progression in immunodeficient and immunocompetent mouse models of K-RAS-driven lung AC. Notably, JAK1/2 inhibition led to the establishment of an antitumorigenic tumor microenvironment, characterized by decreased levels of tumor-promoting chemokines and cytokines and reduced numbers of infiltrating myeloid derived suppressor cells, thereby impairing tumor growth. Taken together, we identified JAK1/2 inhibition as promising therapy for K-RAS-driven lung AC.

A Mouse Model to Assess STAT3 and STAT5A/B Combined Inhibition in Health and Disease Conditions.

Genetically-engineered mouse models (GEMMs) lacking diseased-associated gene(s) globally or in a tissue-specific manner represent an attractive tool with which to assess the efficacy and toxicity of targeted pharmacological inhibitors. Stat3 and Stat5a/b transcription factors have been implicated in several pathophysiological conditions, and pharmacological inhibition of both transcription factors has been proposed to treat certain diseases, such as malignancies. To model combined inhibition of Stat3 and Stat5a/b we have developed a GEMM harboring a flox Stat3-Stat5a/b allele (Stat5/3loxP/loxP mice) and generated mice lacking Stat3 and Stat5a/b in hepatocytes (Stat5/3Δhep/Δhep). Stat5/3Δhep/Δhep mice exhibited a marked reduction of STAT3, STAT5A and STAT5B proteins in the liver and developed steatosis, a phenotype that resembles mice lacking Stat5a/b in hepatocytes. In addition, embryonic deletion of Stat3 and Stat5a/b (Stat5/3Δ/Δ mice) resulted in lethality, similar to Stat3Δ/Δ mice. This data illustrates that Stat5/3loxP/loxP mice are functional and can be used as a valuable tool to model the combined inhibition of Stat3 and Stat5a/b in tumorigenesis and other diseases.

STAT3ß is a tumor suppressor in acute myeloid leukemia.

Signal transducer and activator of transcription 3 (STAT3) exists in 2 alternatively spliced isoforms, STAT3α and STAT3ß. Although truncated STAT3ß was originally postulated to act as a dominant-negative form of STAT3α, it has been shown to have various STAT3α-independent regulatory functions. Recently, STAT3ß gained attention as a powerful antitumorigenic molecule in cancer. Deregulated STAT3 signaling is often found in acute myeloid leukemia (AML); however, the role of STAT3ß in AML remains elusive. Therefore, we analyzed the STAT3ß/α messenger RNA (mRNA) expression ratio in AML patients, where we observed that a higher STAT3ß/α mRNA ratio correlated with a favorable prognosis and increased overall survival. To gain better understanding of the function of STAT3ß in AML, we engineered a transgenic mouse allowing for balanced Stat3ß expression. Transgenic Stat3ß expression resulted in decelerated disease progression and extended survival in PTEN- and MLL-AF9-dependent AML mouse models. Our findings further suggest that the antitumorigenic function of STAT3ß depends on the tumor-intrinsic regulation of a small set of significantly up- and downregulated genes, identified via RNA sequencing. In conclusion, we demonstrate that STAT3ß plays an essential tumor-suppressive role in AML.

MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation.

The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression.

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression.

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.

Breaking bad family ties: Pan-ERBB blockers inhibit KRAS driven lung tumorigenesis.

Oncogenic K-RAS mutations were believed to lock the molecular switch in the ON state, independent of upstream activation. However, we demonstrate in preclinical models that activity of mutated K-RAS depends on upstream signaling events involving EGF receptor family members. This finding reveals a potential therapeutic vulnerability using pan-ERBB inhibitors to fight K-RAS mutated lung tumors.