Herwig P. Moll

Disruption of STAT3 signalling promotes KRAS-induced lung tumorigenesis

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.

Thrombospondin-1: a unique marker to identify in vitro platelet activation when monitoring in vivo processes.

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.

The differential activity of interferon-α subtypes is consistent among distinct target genes and cell types

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.

Neutralizing Type I IFN Antibodies Trigger an IFN-Like Response in Endothelial Cells

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.

Heterologous protein production using euchromatin-containing expression vectors in mammalian cells

Upon stable cell line generation, chromosomal integration site of the vector DNA has a major impact on transgene expression. Here we apply an active gene environment, rather than specified genetic elements, in expression vectors used for random integration. We generated a set of Bacterial Artificial Chromosome (BAC) vectors with different open chromatin regions, promoters and gene regulatory elements and tested their impact on recombinant protein expression in CHO cells. We identified the Rosa26 BAC as the most efficient vector backbone showing a nine-fold increase in both polyclonal and clonal production of the human IgG-Fc. Clonal protein production was directly proportional to integrated vector copy numbers and remained stable during 10 weeks without selection pressure. Finally, we demonstrated the advantages of BAC-based vectors by producing two additional proteins, HIV-1 glycoprotein CN54gp140 and HIV-1 neutralizing PG9 antibody, in bioreactors and shake flasks reaching a production yield of 1 g/l.

Contamination with recombinant IFN accounts for the unexpected stimulatory properties of commonly used IFN-blocking antibodies.

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.

Unexpected oncosuppressive role for STAT3 in KRAS-induced lung tumorigenesis

ABSTRACT 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.

MTHFD1 is a genetic interactor of BRD4 and links folate metabolism to transcriptional regulation

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.

Afatinib restrains K-RAS–driven lung tumorigenesis

K-RAS–mutated lung adenocarcinomas depend on ERBB signaling, and pan-ERBB inhibitors impair K-RAS–driven lung tumorigenesis. A new role for kinase inhibitors The K-RAS oncogene is frequently mutated in a variety of cancer types, including lung cancer. Lung cancers with K-RAS mutations are usually difficult to target, and conventional thinking dictates that these tumors are resistant to receptor tyrosine kinase inhibitors because those act upstream of the constitutively active K-RAS protein. However, it appears that receptor tyrosine kinase signaling may have an effect on K-RAS–driven lung tumors after all, by amplifying their growth beyond the effects of K-RAS alone. Kruspig et al. and Moll et al. independently reached this conclusion and identified approved multikinase inhibitors that are effective in the setting of K-RAS–mutant lung cancer in multiple mouse models, suggesting that this may be a potential treatment strategy for human patients as well. 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.

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

ABSTRACT 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.