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Constitutively activated rat sarcoma (RAS) GTPases are one of the major drivers of tumor growth and are difficult drug targets. The glucocorticoid receptor (GR), a nuclear receptor primarily acting in the nucleus, is a potent modulator of inflammation and regulator of metabolism and cell growth. Emerging evidence has revealed that GR modulates RAS-dependent signaling and RAS activation. The unliganded GR decreases RAS activation, and, upon ligand binding, GR leaves RAS complexes, is translocated into the nucleus, and unleashes the activation of RAS and its downstream pathways. GR forms a complex with RAS and RAF1 and their associated proteins, such as members of the 14-3-3 family of adapter proteins. The exploration of RAS-GR complex formation and maintenance will help to develop much-needed breakthroughs in oncogenic RAS biology and thus help to alleviate tumor growth and burden.

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 ofNR3C1, the gene encoding GR, compared to adjacent normal tissues and lowerNR3C1expression 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.

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.

Ribosomally synthesized and post-translationally modified peptides, such as plant cyclotides, are a diverse group of natural products well known as templates in drug discovery and therapeutic lead development. The cyclotide kalata B1 (kB1) has previously been discovered as immunosuppressive agent on T-lymphocytes, and a synthetic version of this peptide, [T20K]kB1 (T20K), has been effective in reducing clinical symptoms, such as inflammation and demyelination, in a mouse model of multiple sclerosis. Based on its T-cell modulatory impact we studied the effects of T20K and several analogs on the proliferation of anaplastic large cell lymphoma (ALCL), a heterogeneous group of clinically aggressive diseases associated with poor prognosis. T20K, as a prototype drug candidate, induces apoptosis and a proliferation arrest in human lymphoma T-cell lines (SR786, Mac-2a and the Jurkat E6.1) in a concentration dependent fashion, at least partially via increased STAT5 and p53 signaling. In contrary to its effect on IL-2 signaling in lymphocytes, the cytokine levels are not altered in lymphoma cells. In vivo mouse experiments revealed a promising activity of T20K on these cancer cells including decreased tumor weight and increased apoptosis. This study opens novel avenues for developing cyclotide-based drug candidates for therapy of patients with ALCL.

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.

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.

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., Pflügler, Svinka et al. identify a subset of Paneth cells in mouse intestinal crypts and tumors, which express the immune checkpoint molecule Ido1 in a Stat1-dependent manner and promote tumor growth. Gene expression data from human colorectal cancer (CRC) suggest that a similar population is present in human cancer and opens the door for further studies of immune escape mechanisms in CRC.

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.

EGFR-mutated lung adenocarcinoma patients treated with gefitinib and osimertinib show a therapeutic benefit limited by the appearance of secondary mutations, such as EGFR(T790M) and EGFR(C797S). 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 EGFR(T790M) and EGFR(C797S) 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 EGFR(T790M) and EGFR(C797S) 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.

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.Rosszindulatú daganatos megbetegedésekben leggyakrabban a prognózissal is összefüggõ, TP53 gént érintõ mutációk azonosíthatóak. Azonban mind ez idáig nem járt sikerrel a TP53-mutáns tumorok célzott kezelése. Célunk volt in silico azonosítani a TP53 gén mutációinak hatását a túléléssel is összefüggõ és terápiásan célozható gének expressziójára, majd az így kapott eredményeket in vitro igazolni TP53-vad és -mutáns izogén sejtvonalpárokon végzett gátlószeres kezeléssel. A TP53-mutációs státusz alapján 994 beteget két csoportra osztottunk, majd az összes génre Mann–Whitney-tesztet végeztünk, hogy meghatározzuk, a mutáció mely gének kifejezõdésével áll kapcsolatban. A célozható kinázok közt a CHEK1 expressziójának szignifikáns növekedését mutattuk ki a TP53-mutáns csoportban. Az A549 izogén tüdõkarcinóma-sejtvonalakon igazoltuk a CHEK1-expresszió változását a mutációval összefüggésben, majd egy Chk1 (checkpoint kinase 1) fehérjére specifikus gátlószer hatását vizsgáltuk a sejtek mûködésére. A gén prognosztikus szerepét Cox-regresszió és Kaplan–Meier túlélési analízis használatával, 1926 tüdõkarcinómás betegen vizsgáltuk, mely során a CHEK1 expressziójának növekedése rosszabb prognózissal társult. Eredményeink igazolják, hogy a CHEK1 kifejezõdése nagymértékben összefügg a TP53-mutációval és a túléléssel is a tüdõ rosszindulatú daganatai esetében.

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., What's new? A drug that inhibits the JAK–STAT pathway may score a hit against K‐RAS driven lung cancer. Here, the authors Investigated the JAK STAT pathway as a possible target in lung adenocarcinoma because of its role in inflammation‐mediated tumorigenesis. First, they showed that JAK1 and JAK2 are both activated in lung adenocarcinoma patients with oncogenic mutations in K‐RAS. Next, they treated the tumors with ruxolitinib, which inhibits JAK1/2. The drug successfully slowed tumor proliferation and progression in immunocompetent mouse models. Furthermore, treatment with ruxolitinib reduced the tumor‐promoting factors present in the microenvironment.

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.

BACKGROUND Inflammation is central to the pathogenesis of transplant arteriosclerosis (TA). We questioned whether physiologic levels of anti-inflammatory A20 influence TA severity. METHODS We performed major histocompatibility complex mismatched aorta to carotid artery interposition grafts, using wild type (WT) or A20 heterozygote (HET) C57BL/6 (H-2) donors and BALB/c (H-2) recipients, and conversely BALB/c donors and WT/HET recipients. We analyzed aortic allografts by histology, immunohistochemistry, immunofluorescence, and gene profiling (quantitative real-time reverse-transcriptase polymerase chain reaction). We validated select in vivo A20 targets in human and mouse smooth muscle cell (SMC) cultures. RESULTS We noted significantly greater intimal hyperplasia in HET versus WT allografts, indicating aggravated TA. Inadequate upregulation of A20 in HET allografts after transplantation was associated with excessive NF-кB activation, gauged by higher levels of IkBα, p65, VCAM-1, ICAM-1, CXCL10, CCL2, TNF, and IL-6 (mostly localized to SMC). Correspondingly, cytokine-induced upregulation of TNF and IL-6 in human and mouse SMC cultures inversely correlated with A20 expression. Aggravated TA in HET versus WT allografts correlated with increased intimal SMC proliferation, and a higher number of infiltrating IFNγ and Granzyme B CD4 T cells and natural killer cells, and lower number of FoxP3 regulatory T cells. A20 haploinsufficiency in allograft recipients did not influence TA. CONCLUSIONS A20 haploinsufficiency in vascular allografts aggravates lesions of TA by exacerbating inflammation, SMC proliferation, and infiltration of pathogenic T cells. A20 single nucleotide polymorphisms associating with lower A20 expression or function in donors of vascularized allografts may inform risk and severity of TA, highlighting the clinical implications of our findings.

The histone acetyl-reader 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. 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, and pharmacologic 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.

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.

Salivary gland cancer is an aggressive and painful cancer, but a rare tumor type accounting for only ~0.5% of cancer cases. Tumors of the salivary gland exhibit heterogeneous histologic and genetic features and they are subdivided into different subtypes, with adenoid cystic carcinomas (ACC) being one of the most abundant. Treatment of ACC patients is afflicted by high recurrence rates, the high potential of the tumors to metastasize, as well as the poor response of ACC to chemotherapy. A prerequisite for the development of targeted therapies is insightful genetic information for driver core cancer pathways. Here, we developed a transgenic mouse model toward establishment of a preclinical model. There is currently no available mouse model for adenoid cystic carcinomas as a rare disease entity to serve as a test system to block salivary gland tumors with targeted therapy. Based on tumor genomic data of ACC patients, a key role for the activation of the PI3K‐AKT‐mTOR pathway was suggested in tumors of secretory glands. Therefore, we investigated the role of Akt3 expression in tumorigenesis and report that Akt3 overexpression results in ACC of salivary glands with 100% penetrance, while abrogation of transgenic Akt3 expression could revert the phenotype. In summary, our findings validate a novel mouse model to study ACC and highlight the druggable potential of AKT3 in the treatment of salivary gland patients.

On the basis of clinical trials using first-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), it became a doctrine that V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-RAS) mutations drive resistance to EGFR inhibition in non-small cell lung cancer (NSCLC). Conversely, we provide evidence that EGFR signaling is engaged in K-RAS-driven lung tumorigenesis in humans and in mice. Specifically, genetic mouse models revealed that deletion of Egfr quenches mutant K-RAS activity and transiently reduces tumor growth. However, EGFR inhibition initiates a rapid resistance mechanism involving non-EGFR ERBB family members. This tumor escape mechanism clarifies the disappointing outcome of first-generation TKIs and suggests high therapeutic potential of pan-ERBB inhibitors. On the basis of various experimental models including genetically engineered mouse models, patient-derived and cell line-derived xenografts, and in vitro experiments, we demonstrate that the U.S. Food and Drug Administration-approved pan-ERBB inhibitor afatinib effectively impairs K-RAS-driven lung tumorigenesis. Our data support reconsidering the use of pan-ERBB inhibition in clinical trials to treat K-RAS-mutated NSCLC.

IFNγ signaling in endothelial (EC) and smooth muscle cells (SMC) is a key culprit of pathologic vascular remodeling. The impact of NF-κB inhibitory protein A20 on IFNγ signaling in vascular cells remains unknown. In gain- and loss-of-function studies, A20 inversely regulated expression of IFNγ-induced atherogenic genes in human EC and SMC by modulating STAT1 transcription. In vivo, inadequate A20 expression in A20 heterozygote mice aggravated intimal hyperplasia following partial carotid artery ligation. This outcome uniquely associated with increased levels of Stat1 and super-induction of Ifnγ-dependent genes. Transcriptome analysis of the aortic media from A20 heterozygote versus wild-type mice revealed increased basal Ifnβ signaling as the likely cause for higher Stat1 transcription. We confirmed higher basal IFNβ levels in A20-silenced human SMC and showed that neutralization or knockdown of IFNβ abrogates heightened STAT1 levels in these cells. Upstream of IFNβ, A20-silenced EC and SMC demonstrated higher levels of phosphorylated/activated TANK-binding kinase-1 (TBK1), a regulator of IFNβ transcription. This suggested that A20 knockdown increased STAT1 transcription by enhancing TBK1 activation and subsequently basal IFNβ levels. Altogether, these results uncover A20 as a key physiologic regulator of atherogenic IFNγ/STAT1 signaling. This novel function of A20 added to its ability to inhibit nuclear factor-κB (NF-κB) activation solidifies its promise as an ideal therapeutic candidate for treatment and prevention of vascular diseases. In light of recently discovered A20/TNFAIP3 (TNFα-induced protein 3) single nucleotide polymorphisms that impart lower A20 expression or function, these results also qualify A20 as a reliable clinical biomarker for vascular risk assessment.

A1/Bfl-1 is a NF-κB dependent, anti-apoptotic Bcl-2 family member that contains four Bcl-2 homology domains (BH) and an amphipathic C-terminal domain, and is expressed in endothelial cells (EC). Based on NF-κB reporter assays in bovine aortic EC, we have previously demonstrated that A1, like Bcl-2 and Bcl-xL, inhibits NF-κB activation. These results, however, do not fully translate when evaluating the cell's own NF-κB machinery in human EC overexpressing A1 by means of recombinant adenovirus (rAd.) mediated gene transfer. Indeed, overexpression of full-length A1 in human umbilical vein EC (HUVEC), and human dermal microvascular EC (HDMEC) failed to inhibit NF-κB activation. However, overexpression of a mutant lacking the C-terminal domain of A1 (A1ΔC) demonstrated a potent NF-κB inhibitory effect in these cells. Disparate effects of A1 and A1ΔC on NF-κB inhibition in human EC correlated with mitochondrial (A1) versus non-mitochondrial (A1ΔC) localization. In contrast, both full-length A1 and A1ΔC protected EC from staurosporine (STS)-induced cell death, indicating that mitochondrial localization was not necessary for A1's cytoprotective function in human EC. In conclusion, our data uncover a regulatory role for the C-terminal domain of A1 in human EC: anchoring A1 to the mitochondrion, which conserves but is not necessary for its cytoprotective function, or by its absence freeing A1 from the mitochondrion and uncovering an additional anti-inflammatory effect.

Summary. Background: Measuring platelet activation in patients has become a potent method to investigate pathophysiological processes. However, the commonly applied markers are sensitive to detrimental influences by in vitro platelet activation during blood analysis. Objectives: Protein isoforms of platelet-derived thrombospondin-1 (TSP-1) were investigated for their potential to identify in vitro platelet activation when monitoring in vivo processes. Methods: TSP-1 was determined in plasma, serum or supernatant of purified platelets by ELISA and immunoblotting and was compared with standard markers of platelet activation. A collective of 20 healthy individuals and 30 cancer patients was analyzed. Results: While in vitro platelet degranulation led to a selective increase in the 200-kDa full-length molecule, an in vivo process involving platelet activation such as wound healing resulted in the predominant rise of the 140-kDa TSP-1 protein. The physiological ratio of circulating TSP-1 variants was determined and a cut-off level at 1.0 was defined to identify plasma samples with artificial in vitro platelet activation exceeding the cut-off level. In contrast, cancer patients known to frequently exhibit increased in vivo activation of platelets presented with a significantly decreased ratio of TSP-1 variants as compared with healthy volunteers. Conclusions: In comparison to standard platelet markers, TSP-1 constitutes a sensitive and stable parameter suited to monitor in vitro platelet activation. The analysis of TSP-1 protein isoforms further offers a valuable tool to reliably discriminate between in vitro and in vivo effects, to exclude variability introduced during blood processing and improve clinical monitoring.

Lung cancer is still the leading cause of cancer deaths worldwide. While it is well documented that patients suffering from epidermal growth factor receptor (EGFR) mutated non-small cell lung cancer (NSCLC) benefit from (EGFR) inhibition, there are still no successful targeted therapies for oncogenic mutations in the V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) gene available. Indeed, based on clinical trials using first generation EGFR tyrosine kinase inhibitors (TKI) it became a doctrine that K-RAS mutations drive resistance to EGFR inhibition in non-small cell lung cancer (NSCLC). Conversely, we demonstrate here that EGFR signaling is engaged in K-RAS driven lung tumorigenesis both in human and in mice. Genetic mouse models revealed that deletion of Egfr quenches mutant K-ras activity and transiently reduces tumor growth. However, EGFR inhibition initiates a resistance mechanism which involves non-EGFR ERBB family members. This escape mechanism not only clarifies the disappointing outcome of first generation TKI in clinical trials, but also suggests therapeutic potential of pan-ERBB inhibitors. Indeed, by using several experimental models, including genetically engineered mouse models, (patient derived-) xenografts and in vitro experiments, we demonstrate that the FDA approved pan-ERBB inhibitor afatinib formidably reduces K-RAS driven lung tumorigenesis, whereas first generation TKI erlotinib and gefitinib remained ineffective in our models. Altogether, our data strongly suggests revisiting the potential of EGFR TKI in clinical trials for the treatment of K-RAS mutated NSCLC, with a particular focus on pan-ERBB inhibitors, which are readily available. Citation Format: Herwig P. Moll, Klemens Pranz, Monica Musteanu, Luka Brcic, Helmut Popper, Judit Moldvay, Balázs Dome, Emilio Casanova. Afatinib restrains K-RAS driven lung tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 809.

Introduction Several clinical trials using first generation EGFR Tyrosine Kinase Inhibitors (TKIs, erlotinib and gefitinib) in patients suffering of lung adenocarcinoma (AC) harbouring activating mutations in the KRAS gene have shown disappointing results. Therefore, it has become a doctrine that KRAS driven lung AC do not respond to TKIs and targeting EGFR (and other ERBB family members) is not considered a therapeutic option to treat patients suffering of KRAS mutated lung AC. Challenging these observations, we demonstrate that KRAS driven lung AC responds to the pan-ERBB inhibitor afatinib. Material and methods Primary human tumour samples, human/murine cancer cell lines, xenografts, PDXs and GEMMs. Genetic and pharmacological manipulations. Results and discussions Analysis of human and murine tumour samples showed activation of the ERBB receptors and expression of their ligands in KRAS driven lung AC. Furthermore, human KRAS mutated advance tumours (stage II and more advance AC) were enriched in the ERBB gene expression signatures compared to stage I tumours. Experimentally, we found that genetic deletion of EGFR in a GEMM of KRAS driven lung AC or in human xenografted A549 cells significantly reduced tumorigenesis, irrespectively of the p53 status. Tumours lacking EGFR showed less cell proliferation and reduce activation of KRAS downstream effectors. Pharmacological inhibition of EGFR using erlotinib or gefitinib failed to inhibit tumorigenesis in KRAS driven lung AC experimental models. On the contrary, afatinib (an irreversible pan-ERBB inhibitor) was effective in human/murine cell lines, xenograft, PDXs and GEMMs models. Detailed analysis of this observation revealed that genetic deletion or pharmacology inhibition (erlotinib/gefitinib) of the EGFR in KRAS driven lung AC results in a tumor-escape mechanism relying in the activation of other ERBB receptor family members (namely ERBB2 and ERBB3) that can be blocked with the pan-ERBB inhibitor afatinib. Conclusion In conclusion, our data shows that KRAS driven lung AC is depending on ERBB signalling. Importantly, we have unravelled a tumor-escape mechanism depending on the (re)activation of non-EGFR ERBB receptors that explains the poor results in previous clinical trials using erlotinib and gefitinib in patients suffering KRAS mutated lung AC. In agreement with this, pan-ERBB inhibition using afatinib effectively abrogates KRAS driven lung AC. Thus, afatinib or other pan-ERBB inhibitors should be a therapeutic option to treat patients suffering of KRAS driven lung AC.

Neutralizing Abs to type I IFNs are of therapeutic significance, i.e., are currently evaluated for the treatment of autoimmune diseases with pathogenic IFN-α production such as for systemic lupus erythematosus. Unexpectedly, we observed that several neutralizing Abs reportedly known to counteract IFN-α or IFN-β activity triggered an “IFN-like” response in quiescent primary human endothelial cells leading to activation of the transcription factor IFN-stimulated gene factor 3 and the expression of IFN-responsive genes. Furthermore, these Abs were found to enhance rather than inhibit type I IFN signals, and the effect was also detectable for distinct other cell types such as PBMCs. The stimulatory capacity of anti-IFN-α/β Abs was mediated by the constitutive autocrine production of “subthreshold” IFN levels, involved the type I IFNR and was dependent on the Fc Ab domain, as Fab or F(ab′)2 fragments potently inhibited IFN activity. We thus propose that a combined effect of IFN recognition by the Ab paratope and the concomitant engagement of the Fc domain may trigger an IFN signal via the respective type I IFNR, which accounts for the observed IFN-like response to the neutralizing Abs. With respect to clinical applications, the finding may be of importance for the design of recombinant Abs vs Fab or F(ab′)2 fragments to efficiently counteract IFN activity without undesirable activating effects.

Signal transducer and activator of transcription 3 (STAT3) plays a critical role in the pathogenesis of several diseases and is considered a therapeutic target in solid cancers, including lung cancer. However, we recently demonstrated a tumor suppressive function of STAT3 in kirsten rat sarcoma oncogene homolog (KRAS)-driven lung cancer. Here, we discuss these findings and their consequences.

The A20 protein has emerged as an important negative regulator of Toll like receptor (TLR) and retinoic acid-inducible gene 1 (RIG-I)-mediated anti-viral signaling. A20 functions both as a RING-type E3 ubiquitin ligase and as a de-ubiquitinating enzyme. Nuclear factor kappa B (NF-kappaB) and interferon regulatory factor (IRF) pathways are targeted by A20 through mechanisms that appear to be both overlapping and distinct, resulting in the downregulation of interferon alpha/beta (IFNalpha/beta) production. This review specifically details the impact of A20 on the cytosolic RIG-I/MDA5 pathway, a process that is less understood than that of NF-kappaB but is essential for the regulation of the innate immune response to viral infection.

Cardiovascular disease (CVD) is the biggest killer in the Western World despite significant advances in understanding its molecular underpinnings. Chronic inflammation, the classical hallmark of atherogenesis is thought to play a key pathogenic role in the development of atherosclerotic lesions from initiation of fatty streaks to plaque rupture. Over-representation of mostly pro-inflammatory nuclear factor kappa B (NF-kappaB) target genes within atherosclerotic lesions has led to the common-held belief that excessive NF-kappaB activity promotes and aggravates atherogenesis. However, mouse models lacking various proteins involved in NF-kappaB signaling have often resulted in conflicting findings, fueling additional investigations to uncover the molecular involvement of NF-kappaB and its target genes in atherogenesis. In this chapter we will review the role of the NF-kappaB-regulated, yet potent NF-kappaB inhibitory and anti-inflammatory gene A20/TNFAIP3 in atherogenesis, and highlight the potential use of its atheroprotective properties for the prevention and treatment of cardiovascular diseases.

Background A20 (TNFAIP3) is a pleiotropic NFκB-dependent gene that terminates NFκB activation in response to inflammatory stimuli. The potent anti-inflammatory properties of A20 are well characterized in several organs. However, little is known about its role in the brain. In this study, we investigated the brain phenotype of A20 heterozygous (HT) and knockout (KO) mice. Methods The inflammatory status of A20 wild type (WT), HT and KO brain was determined by immunostaining, quantitative PCR, and Western blot analysis. Cytokines secretion was evaluated by ELISA. Quantitative results were statistically analyzed by ANOVA followed by a post-hoc test. Results Total loss of A20 caused remarkable reactive microgliosis and astrogliosis, as determined by F4/80 and GFAP immunostaining. Glial activation correlated with significantly higher mRNA and protein levels of the pro-inflammatory molecules TNF, IL-6, and MCP-1 in cerebral cortex and hippocampus of A20 KO, as compared to WT. Basal and TNF/LPS-induced cytokine production was significantly higher in A20 deficient mouse primary astrocytes and in a mouse microglia cell line. Brain endothelium of A20 KO mice demonstrated baseline activation as shown by increased vascular immunostaining for ICAM-1 and VCAM-1, and mRNA levels of E-selectin. In addition, total loss of A20 increased basal brain oxidative/nitrosative stress, as indicated by higher iNOS and NADPH oxidase subunit gp91phox levels, correlating with increased protein nitration, gauged by nitrotyrosine immunostaining. Notably, we also observed lower neurofilaments immunostaining in A20 KO brains, suggesting higher susceptibility to axonal injury. Importantly, A20 HT brains showed an intermediate phenotype, exhibiting considerable, albeit not statistically significant, increase in markers of basal inflammation when compared to WT. Conclusions This is the first characterization of spontaneous neuroinflammation caused by total or partial loss of A20, suggesting its key role in maintenance of nervous tissue homeostasis, particularly control of inflammation. Remarkably, mere partial loss of A20 was sufficient to cause chronic, spontaneous low-grade cerebral inflammation, which could sensitize these animals to neurodegenerative diseases. These findings carry strong clinical relevance in that they question implication of identified A20 SNPs that lower A20 expression/function (phenocopying A20 HT mice) in the pathophysiology of neuroinflammatory diseases.

STAT3 is considered to play an oncogenic role in several malignancies including lung cancer; consequently, targeting STAT3 is currently proposed as therapeutic intervention. Here we demonstrate that STAT3 plays an unexpected tumour-suppressive role in KRAS mutant lung adenocarcinoma (AC). Indeed, lung tissue-specific inactivation of Stat3 in mice results in increased KrasG12D-driven AC initiation and malignant progression leading to markedly reduced survival. Knockdown of STAT3 in xenografted human AC cells increases tumour growth. Clinically, low STAT3 expression levels correlate with poor survival and advanced malignancy in human lung AC patients with smoking history, which are prone to KRAS mutations. Consistently, KRAS mutant lung tumours exhibit reduced STAT3 levels. Mechanistically, we demonstrate that STAT3 controls NF-κB-induced IL-8 expression by sequestering NF-κB within the cytoplasm, thereby inhibiting IL-8-mediated myeloid tumour infiltration and tumour vascularization and hence tumour progression. These results elucidate a novel STAT3–NF-κB–IL-8 axis in KRAS mutant AC with therapeutic and prognostic relevance.

The A20 protein has emerged as an important negative regulator of Toll like receptor (TLR) and retinoic acid-inducible gene 1 (RIG-I)-mediated anti-viral signaling. A20 functions both as a RING-type E3 ubiquitin ligase and as a de-ubiquitinating enzyme. Nuclear factor kappa B (NF-κB) and interferon regulatory factor (IRF) pathways are targeted by A20 through mechanisms that appear to be both overlapping and distinct, resulting in the downregulation of interferon α/β (IfNα/β) production. This review specifically details the impact of A20 on the cytosolic RIG-I/MDA5 pathway, a process that is less understood than that of NF-κB but is essential for the regulation of the innate immune response to viral infection.

Cardiovascular disease (CVD) is the biggest killer in the Western World despite significant advances in understanding its molecular underpinnings. Chronic inflammation, the classical hallmark of atherogenesis is thought to play a key pathogenic role in the development of atherosclerotic lesions from initiation of fatty streaks to plaque rupture. Over-representation of mostly pro-inflammatory nuclear factor kappa B (NF-κB) target genes within atherosclerotic lesions has led to the common-held belief that excessive NF-κB activity promotes and aggravates atherogenesis. However, mouse models lacking various proteins involved in NF-κB signaling have often resulted in conflicting findings, fueling additional investigations to uncover the molecular involvement ofNF-κB and its target genes in atherogenesis. In this chapter we will review the role of the NF-κB-regulated, yet potent NF-κB inhibitory and anti-inflammatory gene A20/TNFAIP3 in atherogenesis, and highlight the potential use of its atheroprotective properties for the prevention and treatment of cardiovascular diseases.

The cellular response to IFN is an essential part of immune reactions and has been subject to investigations for over 50 years 1. The analyses on IFN function frequently involve the use of neutralizing antibodies to block responses and to document the dependence on IFN signals. In this context, we have previously described an unusual “IFN-like” response initiated by blocking antibodies to type I IFN in primary human endothelial cells (EC) or mononuclear blood cells. In the absence of exogenously added recombinant IFN (rIFN), the exposure of EC to increasing concentrations of IFN-blocking mAb resulted in the dose-dependent induction of IFN response genes at the mRNA and protein level 2. The effect was observed for four different mAb directed against human IFN-α or -β and was dependent on the type I IFN receptor. We concluded that an intrinsic feature of the IFN-blocking antibodies was responsible for the observed “IFN-like” activation of EC; a model was proposed of antibody binding to surface Fc-receptors with sequestration of autocrine IFN and subsequent release to nearby IFN receptors, which would result in the observed “IFN-like” signal. We have now obtained evidence that refutes this hypothesis showing that the “IFN-like” activity associated with IFN-blocking mAb is indeed a discrete component that can be separated from the antibody moiety by sequential cycles of antibody immunoprecipitation (Supporting Information Fig. 1 and Supporting Information Methodology). Furthermore, when the standard two-step procedure for antibody purification as performed by the manufacturer (based on ammonium sulfate precipitation and ion exchange chromatography) was extended by a third step of hydrophobic interaction chromatography, the “IFN-like” activity was lost and the neutralizing capacity of the respective antibodies prevailed (Fig. 1A–C). Figure 1 The “IFN-like” activity in antibody preparations can be eliminated by additional antibody purification (three-step process) and is inhibited by polyclonal anti-IFN-α antiserum. The neutralizing anti-IFN-α mAb MMHA-2 and ... Having established that the “IFN-like” activity was attributable to a discrete contaminant of the applied anti-IFN antibody preparations, the possible contamination with microbial products was first examined. Since the majority of pathogen-associated signals leading to the IFN pathway are mediated by the TLR family 3, 4 we screened for hallmarks of TLR activity. However, we did not observe the induction of the transcription factor NF-κB or the pro-inflammatory activation of EC, strongly arguing against TLR involvement (Supporting Information Fig. 2). We then obtained an indication towards contamination with type I IFN from competition studies showing that the contaminant in mAb preparations was neutralized by rabbit (data not shown) or sheep polyclonal anti-human IFN-α antiserum (Fig. 1D). Polyclonal anti-IFN-β antiserum or control antiserum obtained prior to immunization did not affect the “IFN-like” activity (data not shown). The co-purification (and cross-reactivity) of mouse IFN upon mAb isolation from mouse ascites was a potential source of contamination, which was addressed by cytopathic effect inhibition assays on mouse versus human target cells. There was a significantly higher impact on the human target cells, thus arguing for the presence of human rather than mouse IFN-α (Supporting Information Fig. 3A). However, the question remained as to why the contaminating human IFN-α was not neutralized by the investigated anti-IFN-α-blocking mAb (e.g. MMHA-2). When comparing the neutralizing capacity towards various rIFN-α subtypes, the three-step purified mAb failed to inhibit individual family members (IFN-α subtypes 8, 14, and 16) while the sheep polyclonal antiserum potently repressed all IFN-α subtypes (Supporting Information Fig. 3C). This finding supported the notion that a distinct human IFN-α subtype not neutralized by the respective monoclonal was present in the antibody preparation. In accordance, we found that the purified mAb could not block the “IFN-like” activity present in the contaminated mAb preparation (Supporting Information Fig. 3B). Of note, rIFN-α8 and rIFN-α14 had been produced by PBL prior to the preparation of the contaminated antibody MMHA-2. By applying two anti-human IFN-α ELISA tests (not mouse cross-reactive) with distinct sensitivity towards rIFN-α8 and rIFN-α14 evidence was gained for a predominant antibody contamination by human rIFN-α14 (Supporting Information Table 1). However, a combination of contaminating IFN cannot be excluded. Table 1 Level of detectable contamination with rIFN-α in various antibody preparationsa) Thus, the source of contamination could be traced to the sequential production of rIFN and anti-IFN-blocking antibodies with common equipment. Despite a time window of several months between productions, despite the regular two-step purification procedure, and despite standard equipment cleansing, the contamination of antibody preparations with functional type I IFN was substantial. The importance of our observation was further demonstrated by the frequent occurrence of detectable IFN activity in a considerable number of tested antibodies (Table 1). Apart from various mouse monoclonals against human IFN-α and IFN-β (MMHA-2, MMHA-3, MMHA-9, MMHA-13, MMHB-3, MMHB-12), rat anti-mouse antibodies directed against IFN-α (RMMA-1) or IFN-γ (RMMG-1) also presented with significant amounts of human rIFN. While most of these monoclonals originated from PBL and were supplied by PBL or associated distributors in the contaminated form, further examples for contaminated antibodies were found for an alternative supplier. Two anti-pig IFN mAb (K9, F17) similarly showed contamination with rIFN-α (Table 1). Based on the diverse specificity of contaminated antibodies we propose that unspecific co-purification rather than specific antibody binding accounts for the presence of contaminants. While most of the affected antibodies showed IFN-α contamination, the subtype present may vary. The range of detectable IFN activity varied considerably (by a factor of 1000). The highest levels of anti-viral activity as recorded for the anti-IFN-α mAb clone MMHA-2 equalled a concentration of 800 U/mL of human rIFN-α when applying the antibody at a dilution of 50 μg/mL (common for in vitro experiments). For example, stimulation of target cells with 1000 U/mL of biological or rIFN-α2a in the presence of 50 μg/mL of contaminated blocking mAb MMHA-2 would be expected to result in the complete neutralization of the α2a subtype, while exposing the cells to 800 U/mL of non-neutralized rIFN-α14. The net inhibitory effect on the target cells would be minor leading to the false interpretation of results, especially for an experimental setup where the involvement and concentration of type I IFN is the unknown parameter under investigation. Thus, the information given in this report may be of help in interpreting previously conducted experiments with the listed antibodies. With respect to PBL products, all mAb preparations have been carefully evaluated, and contaminated antibodies were found to date back to the last 2–8 years. More stringent purification and equipment cleaning procedures as well as routine testing for contaminating activity have been put in place at PBL in part due to these experiments. With respect to K9, F17, the company producing these antibodies was informed and has, in the meantime, provided the respective clones to PBL for antibody production. Hence, all products identified in this report to have previously been affected by contamination are now being supplied to the research community in a purified form; however, it is easy to envision that reagent providers who prepare multiple cytokines and mAb could face similar issues as those noted here.

The pleiotropic cytokine Interferon-γ (IFNγ), best known for its anti-viral and immunomodulatory activities, also demonstrates pro-atherogenic effects. Implication of IFNγ in the pathogenesis of atherosclerosis results in part from the upregulation of a number of pro-inflammatory interferon stimulated genes (ISGs) in vascular smooth muscle cells (SMC). Hence, strategies to reduce IFNγ signaling are of particular interest to reduce or prevent pathologic vascular remodeling associated with atherosclerotic disease, for instance transplant arteriosclerosis. We discovered that the anti-inflammatory and vasculoprotective protein A20 inhibits IFNγ signaling and thus the expression of ISG in vitro in human coronary artery SMC, and in vivo in vascular allografts. The aim of this study was to unveil the mechanism(s) of A20 mediated inhibition of IFNγ signaling. Adenovirus-mediated overexpression of A20 in SMC significantly decreased mRNA and protein levels of signal transducer and activator of transcription 1 (STAT1), the key mediator of IFNγ signaling, as a result of lower transcription. Moreover, overexpression of A20 in SMC also efficiently inhibited activating Y701 phosphorylation of STAT1, through a mechanism possibly implicating A20 binding to STAT1, as confirmed by co-immunoprecipitation. As expected, overexpression of A20 also inhibited NF-κB activation in SMC, which is required for full-blown IFNγ responses. Indeed, we confirmed that inhibition of NF-κB by overexpression of its inhibitor, IκBα also quenched upregulation of ISGs in SMC, albeit without down-regulating STAT1. Overall, our results identify A20 as a potent inhibitor of IFNγ signaling in SMC through several non-mutually exclusive mechanisms. This novel function of A20 in SMC further qualifies its atheroprotective properties and supports the pursuit of A20-based therapies for the cure of atherosclerotic vascular disease.

Transplant arteriosclerosis (TA) is an accelerated form of atherosclerosis largely driven by the immune response to the allograft. As for all atherosclerotic disease, inflammation is central to vascular remodeling associated with TA. We have shown that overexpression of the NF-κB inhibitory protein in totally mismatched (C57BL/6 to BALB/c) aortic to carotid vascular allografts protects from TA. A20 inhibits TA thanks to its anti-inflammatory and anti-apoptotic functions in endothelial cells (EC), anti-inflammatory and anti-proliferative functions in smooth muscles cells (SMC), pro-apoptotic function in neointimal SMC, and to favoring regulatory FOXP3+ over pathogenic Th1/Th17 T cells. In this work, we analyzed the impact of A20 knockdown on TA by using aortic allografts from A20 heterozygous (A20-+/- HET) mice. Partial loss of A20 in aortic allografts caused significantly greater TA lesions, measured by intima over media ratios (I/M 1.79±0.18 in HET vs. 1.04±0.17 in wild type (WT) allografts; p Based on these results, we conclude that A20 plays an important physiologic role in the vascular remodeling of TA. We propose that tailored A20-based vascular therapies could decrease incidence and severity of TA, and synergize with or minimize use of immunosuppression in solid organ transplantation.

Despite progress in controlling acute allograft rejection, 5% of kidney and heart allografts fail each year post-transplantation. This is mostly due to chronic rejection, with transplant arteriosclerosis (TA) being one of its pathognomonic features (1). TA is particularly dramatic in cardiac transplant recipients, affecting >50% of patients at 10 years, and is the principal cause of late death and allograft dysfunction (2). TA is a form of accelerated atherosclerosis resulting from chronic inflammation initiated by both immune and non-immune insults (3, 4). Much progress was made in defining molecular signals promoting TA, including IFNγ, IL-17, IL-6, chemokines, antibodies, and complement (5-9). However, current therapies focused on increasing immunosuppression largely failed to circumvent the natural course of TA, while further immunocompromising the host (10). We propose an alternative approach to preventing TA through modifying the vessel wall so it resists transplant-related injury. We previously demonstrated that gene therapy with the ubiquitin-editing (11) and NF-κB inhibitory protein A20 (12) prevents and cures neointimal hyperplasia in a rat carotid balloon injury model (13, 14) and protects from accelerated atherosclerosis in diabetic ApoE-null mice (14). A20 does so by maintaining vascular homeostasis, thanks to its anti-inflammatory and anti-apoptotic functions in EC (15-17) and to inhibiting SMC activation and proliferation while promoting neointimal SMC apoptosis (13, 14, 18). We propose that A20-based therapies to the vessel wall could protect from TA. In this study, we provide direct in vivo proof for the protective effect of A20 against TA in a mouse aorta to carotid artery allograft model. Vascular allografts are adequate alternatives to cardiac allografts for the study of TA (19), as they offer the advantages of easier quantification of vascular changes and better accessibility for testing vascular-targeted therapies.

IFN-α proteins have been described to originate from 14 individual genes and allelic variants. However, the exceptional diversity of IFN-α and its functional impact are still poorly understood. To characterize the biological activity of IFN-α subtypes in relation to the cellular background, we investigated the effect of IFN-α treatment in primary fibroblasts and endothelial cells of vascular or lymphatic origin. The cellular response was evaluated for 13 distinct IFN-α proteins with respect to transcript regulation of the IFN-stimulated genes (ISGs) IFIT1, ISG15, CXCL10, CXCL11 and CCL8. The IFN-α proteins displayed a remarkably consistent potency in gene induction irrespective of target gene and cellular background which led to the classification of IFN-α subtypes with low (IFN-α1), intermediate (IFN-α2a, -4a, -4b, -5, -16, -21) and high (IFN-α2b, -6, -7, -8, -10, -14) activity. The differential potency of IFN-α classes was confirmed at the ISG protein level and the functional protection of cells against influenza virus infection. Differences in IFN activity were only observed at subsaturating levels of IFN-α proteins and did not affect the time course of ISG regulation. Cell-type specific responses were apparent for distinct target genes independent of IFN-α subtype and were based on different levels of basal versus inducible gene expression. While fibroblasts presented with a high constitutive level of IFIT1, the expression in endothelial cells was strongly induced by IFN-α. In contrast, CXCL10 and CXCL11 transcript levels were generally higher in endothelial cells despite a pronounced induction by IFN-α in fibroblasts. In summary, the divergent potency of IFN-α proteins and the cell-type specific regulation of individual IFN target genes may allow for the fine tuning of cellular responses to pathogen defense.