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Most non–small cell lung cancers (NSCLC) contain nontargetable mutations, including KRAS, TP53, or STK11/LKB1 alterations. By coupling ex vivo drug sensitivity profiling with in vivo drug response studies, we aimed to identify drug vulnerabilities for these NSCLC subtypes. Primary adenosquamous carcinoma (ASC) or adenocarcinoma (AC) cultures were established from KrasG12D/+;Lkb1fl/fl (KL) tumors or AC cultures from KrasG12D/+;p53fl/fl (KP) tumors. Although p53-null cells readily propagated as conventional cultures, Lkb1-null cells required conditional reprograming for establishment. Drug response profiling revealed short-term response to MEK inhibition, yet long-term clonogenic assays demonstrated resistance, associated with sustained or adaptive activation of receptor tyrosine kinases (RTK): activation of ERBBs in KL cultures, or FGFR in AC cultures. Furthermore, pan-ERBB inhibition reduced the clonogenicity of KL cultures, which was exacerbated by combinatorial MEK inhibition, whereas combinatorial MEK and FGFR inhibition suppressed clonogenicity of AC cultures. Importantly, in vivo studies confirmed KL-selective sensitivity to pan-ERBB inhibition, which correlated with high ERBB ligand expression and activation of ERBB receptors, implying that ERBB network activity may serve as a predictive biomarker of drug response. Interestingly, in human NSCLCs, phosphorylation of EGFR or ERBB3 was frequently detected in ASCs and squamous cell carcinomas. We conclude that analysis of in situ ERBB signaling networks in conjunction with ex vivo drug response profiling and biochemical dissection of adaptive RTK activities may serve as a valid diagnostic approach to identify tumors sensitive to ERBB network inhibition.

Significance Compromised vascular integrity is associated with capillary leakage in sepsis, but effective therapies stabilizing the vasculature are lacking. Here, we show that targeting β1-integrin in vivo with inhibitory antibodies or deletion of a single allele of endothelial β1-integrin inhibits lipopolysaccharide (LPS)-induced vascular leakage in murine endotoxemia. The inflammatory agents IL-1β, thrombin, and LPS induced changes in endothelial cell–extracellular matrix (ECM) adhesion via β1-integrin, angiopoietin-2, and the adapter protein tensin-1, leading to increased endothelial cell contractility and permeability. These results indicate that β1-integrin actively promotes vascular leakage and that targeting β1-integrin signaling could be a novel means of achieving vascular stabilization in pathological vascular leak., Loss of endothelial integrity promotes capillary leakage in numerous diseases, including sepsis, but there are no effective therapies for preserving endothelial barrier function. Angiopoietin-2 (ANGPT2) is a context-dependent regulator of vascular leakage that signals via both endothelial TEK receptor tyrosine kinase (TIE2) and integrins. Here, we show that antibodies against β1-integrin decrease LPS-induced vascular leakage in murine endotoxemia, as either a preventative or an intervention therapy. β1-integrin inhibiting antibodies bound to the vascular endothelium in vivo improved the integrity of endothelial cell–cell junctions and protected mice from endotoxemia-associated cardiac failure, without affecting endothelial inflammation, serum proinflammatory cytokine levels, or TIE receptor signaling. Moreover, conditional deletion of a single allele of endothelial β1-integrin protected mice from LPS-induced vascular leakage. In endothelial monolayers, the inflammatory agents thrombin, lipopolysaccharide (LPS), and IL-1β decreased junctional vascular endothelial (VE)-cadherin and induced actin stress fibers via β1- and α5-integrins and ANGPT2. Additionally, β1-integrin inhibiting antibodies prevented inflammation-induced endothelial cell contractility and monolayer permeability. Mechanistically, the inflammatory agents stimulated ANGPT2-dependent translocation of α5β1-integrin into tensin-1–positive fibrillar adhesions, which destabilized the endothelial monolayer. Thus, β1-integrin promotes endothelial barrier disruption during inflammation, and targeting β1-integrin signaling could serve as a novel means of blocking pathological vascular leak.

Inflammatory bowel diseases (IBD) are key risk factors for the development of colorectal cancer, but the mechanisms that link intestinal inflammation with carcinogenesis are insufficiently understood. Card9 is a myeloid cell-specific signaling protein that regulates inflammatory responses downstream of various pattern recognition receptors and which cooperates with the inflammasomes for IL-1β production. Because polymorphisms in Card9 were recurrently associated with human IBD, we investigated the function of Card9 in a colitis-associated cancer (CAC) model. Card9 -/- mice develop smaller, less proliferative and less dysplastic tumors compared to their littermates and in the regenerating mucosa we detected dramatically impaired IL-1β generation and defective IL-1β controlled IL-22 production from group 3 innate lymphoid cells. Consistent with the key role of immune-derived IL-22 in activating STAT3 signaling during normal and pathological intestinal epithelial cell (IEC) proliferation, Card9 -/- mice also exhibit impaired tumor cell intrinsic STAT3 activation. Our results imply a Card9-controlled, ILC3-mediated mechanism regulating healthy and malignant IEC proliferation and demonstrates a role of Card9-mediated innate immunity in inflammation-associated carcinogenesis.

At mucosal surfaces, the immune system should not initiate inflammatory immune responses to the plethora of antigens constantly present in the environment, but should remain poised to unleash a potent assault on intestinal pathogens. The transcriptional programs and regulatory factors required for immune cells to switch from homeostatic (often tissue-protective) function to potent antimicrobial immunity are poorly defined. Mucosal retinoic-acid-receptor-related orphan receptor-γt-positive (RORγt(+)) innate lymphoid cells (ILCs) are emerging as an important innate lymphocyte population required for immunity to intestinal infections. Various subsets of RORγt(+) ILCs have been described but the transcriptional programs controlling their specification and fate remain largely unknown. Here we provide evidence that the transcription factor T-bet determines the fate of a distinct lineage of CCR6(-)RORγt(+) ILCs. Postnatally emerging CCR6(-)RORγt(+) ILCs upregulated T-bet and this was controlled by cues from the commensal microbiota and interleukin-23 (IL-23). In contrast, CCR6(+)RORγt(+) ILCs, which arise earlier during ontogeny, did not express T-bet. T-bet instructed the expression of T-bet target genes such as interferon-γ (IFN-γ) and of the natural cytotoxicity receptor NKp46. Mice genetically lacking T-bet showed normal development of CCR6(-)RORγt(+) ILCs, but they could not differentiate into NKp46-expressing RORγt(+) ILCs (that is, IL-22-producing natural killer (NK-22) cells) and failed to produce IFN-γ. The production of IFN-γ by T-bet-expressing CCR6(-)RORγt(+) ILCs was essential for the release of mucus-forming glycoproteins required to protect the epithelial barrier during Salmonella enterica infection. Salmonella infection also causes severe enterocolitis that is at least partly driven by IFN-γ. Mice deficient for T-bet or depleted of ILCs developed only mild enterocolitis. Thus, graded expression of T-bet in CCR6(-)RORγt(+) ILCs facilitates the differentiation of IFN-γ-producing CCR6(-)RORγt(+) ILCs required to protect the epithelial barrier against Salmonella infections. Co-expression of T-bet and RORγt, which is also found in subsets of IL-17-producing T-helper (T(H)17) cells, may be an evolutionarily conserved transcriptional program that originally developed as part of the innate defence against infections but that also confers an increased risk of immune-mediated pathology.

Apart from conventional CD4(+) Th17 cells, the cytokines IL-17A and IL-22 can also be produced by γδ T cells, NK cells and lymphoid tissue inducer (LTi) cells. Th17 cells develop from precursor cells after T-cell receptor stimulation in the presence of TGF-β, IL-6 and IL-23. In contrast, a subset of γδ T cells ("γδT17") is committed for fast IL-17 production already in the thymus; however, γδ T cells can also produce IL-17 after prolonged in vitro stimulation via their γδ T-cell receptor plus IL-23. Here, we show that γδ T-, LTi- and NKT cells differ extensively from Th17 cells in their signalling requirements for the generation of IL-17A and IL-22. While production of these cytokines by Th17 cells totally depends on the transcription factor interferon regulatory factor 4 (IRF4), IRF4 is irrelevant in the other cell types. As for γδ T cells, this finding pertains to both thymic commitment and prolonged in vitro culture. Furthermore, IL-17A-producing γδ T cells accumulate in the central nervous system of IRF4 deficient (Irf4(-/-)) mice during experimental autoimmune encephalomyelitis. IL-17A-producing WT and Irf4(-/-) γδ T cells equally express CCR6 and lack CD27. The underlying IRF4-independent pathway partially involves STAT3 during in vitro stimulation.

It has recently emerged that innate lymphocytes are more diverse than previously appreciated. In addition to natural killer cells, various subsets of innate lymphoid cells are now being characterized. It has become apparent that the transcriptional programs underlying lineage specification and cell fate decisions of innate lymphocytes strikingly resemble those of T cell subsets, suggesting that such transcriptional circuitry was already pre-formed in the evolutionary older innate immune system. Here, we will review recent advances in our understanding of the core transcriptional programs driving development and cell fate decisions of innate lymphocytes. We will also discuss whether these transcriptional programs are stable or flexible, thereby allowing for plastic adaptation of immune responses.

Innate lymphoid cells (ILC) expressing the transcription factor RORγt induce the postnatal formation of intestinal lymphoid follicles and regulate intestinal homeostasis. RORγt(+) ILC express the aryl hydrocarbon receptor (AhR), a highly conserved, ligand-inducible transcription factor believed to control adaptation of multicellular organisms to environmental challenges. We show that AhR is required for the postnatal expansion of intestinal RORγt(+) ILC and the formation of intestinal lymphoid follicles. AhR activity within RORγt(+) ILC could be induced by dietary ligands such as those contained in vegetables of the family Brassicaceae. AhR-deficient mice were highly susceptible to infection with Citrobacter rodentium, a mouse model for attaching and effacing infections. Our results establish a molecular link between nutrients and the formation of immune system components required to maintain intestinal homeostasis and resistance to infections.

Increased capillary leakage is a potentially life-threatening condition associated with sepsis and cytokine release syndrome (CRS) that develops as a side effect of novel chimeric antigen receptor (CAR) T-cell cancer immunotherapies. CRS and sepsis are characterized by immune activation, resulting in elevated circulating levels of inflammatory cytokines including IL-6 and IL-1β, and thrombin that promote vascular leakage and the development of hypovolemic shock and multiorgan failure. However, molecular mechanisms behind capillary leakage remain unknown, and importantly, targeted approaches for the management of capillary leakage are limited. Here, we found that β1-integrin acts as a critical universal mediator of endothelial permeability induced by various inflammatory agents, including LPS, IL-6, IL-1β and thrombin. In a mouse model of LPS-induced systemic inflammation, resulting in increased inflammatory cytokine production and vascular leakage, function blocking antibodies against β1-integrin prevented vascular leakage and improved integrity of vascular endothelium, leading to protection from LPS-induced cardiac failure. β1-integrin blocking antibodies were effective in reducing vascular leakage even when administered after the onset of systemic inflammation and increase in the serum levels of IL-6, IL-1β and TNF-α. By using an in vitro model, we found that inflammatory agents generated mechanical stress, in a β1-integrin dependent manner, leading to endothelial cell contractility and permeability. Conclusively, our data indicates that β1-integrin promotes vascular leakage in inflammation, and that targeting this mechanism may have therapeutic utility for vascular stabilization in CRS and capillary leak syndromes associated with immune activation and systemic inflammation. Citation Format: Elina A. Kiss, Laura Hakanpää, Guillaume Jacquemet, Ilkka Miinalainen, Martina Lerche, Camilo Guzman, Eero Mervaala, Lauri Eklund, Johanna Ivaska, Pipsa Saharinen. b1-Integrin promotes inflammatory cytokine-induced vascular leakage [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 5067.

Introduction With the advent of molecular diagnostics and availability of targeted therapies, treatment of NSCLC has been improved, especially for tumours driven by mutant EGFR or ALK fusions. However, other frequently mutated genes, including KRAS, TP53 and STK11/LKB1, are still not targetable. Murine models of lung cancer that mimic human disease genomics and pathology can help refine diagnostic strategies, allowing discovery of new therapeutic options, identification of biomarkers and validation of the responses in vivo. Material and methods Primary cultures were derived from murine adenosquamous carcinoma (ASC) or adenocarcinoma (AC) tumours driven by mutant Kras and loss of Lkb1 (KL) or Tp53 (KP), and murine lung-derived healthy epithelia. Ex vivo drug sensitivity profiling, biomarker analysis and in vivo response validations were combined to identify NSCLC subtype-specific drug vulnerabilities. To identify biomarkers predictive of response, spatial analyses of murine and human tumours were conducted. Results and discussions By performing ex vivo functional screens, we identified histotype- and genotype-selective drug vulnerabilities. Inhibition of MEK, a downstream KRAS effector, caused a selective transient cytostasis in AC cells. However, long-term MEK inhibition was found to be ineffective in all cultures tested due to adaptive reactivation of MAPK and PI3K/AKT signalling. Resistance to MEK inhibitors was associated with activation of subtype-specific receptor tyrosine kinases: ERBB3 activation in KL;ASCs, adaptive ERBB2 and FGFR1 activation in KL;ACs, or adaptive FGFR1 activation in KP;ACs. Furthermore, while combined inhibition of MEK and ERBB kinases induced cell death in KL;ASC and KL;AC but not in KP;AC ex vivo culture, in vivo this combination showed cytotoxic response only in the squamous cell carcinoma (SCC) regions of KL;ASCs. This was in agreement with baseline ERBB activation occurring selectively in SCC tissue. Interestingly, activation of ERBB family receptors was detected in a significant proportion of human NSCLC tumours. Analysis of spatial signalling activities may therefore identify NSCLC patients that could benefit from combinatorial MEK and ERBB inhibition. Conclusion Our study validates the utility of NSCLC cultures to discover drug resistance mechanisms that can be overcome with subtype-specific drug combinations. Importantly, the combination of ex vivo drug response profiling and in situ spatial signalling shows that Kras-driven lung cancers stratify as functional subgroups.

Innate lymphoid cells (ILCs) are an emerging group of innate lymphocytes that share functional and transcriptional attributes with the various T helper cell effector fates (e.g. Th1, Th2, Th17). ILCs are substantially represented in the intestinal mucosa but are rare in secondary lymphoid organs. They play important roles in epithelial homeostasis, tissue repair and in immunity to intestinal infections. They are also involved in immune-mediated pathology. Here, we will review the emerging roles of the transcription factors T-bet and Gata3 in the development, lineage specification and function of distinct ILC lineages. We will also highlight the requirement of these transcriptional programs for the control of infections and the pathogenesis of inflammatory diseases.

Intestinal homeostasis results from a complex mutualism between gut microbiota and host cells. Defining the molecular network regulating such mutualism is currently of increasing interest, as its deregulation is reported to lead to increased susceptibility to infections, chronic inflammatory bowel diseases and cancer. Until now, the focus has been on the mechanism, by which the composition of indigenous microbiota shapes the immune system. In a recent study, we have shown that dietary compounds have also the ability to affect innate immune system. This regulation involves aryl hydrocarbon receptor (AhR), a sensor of plant-derived phytochemicals, which mediates the maintenance of Retinoic acid related orphan receptor γ t-expressing innate lymphoid cells (RORγt(+) ILC) in the gut and consequently formation of postnatal lymphoid follicles. Thus, AhR represents the first evidence of a molecular link between diet and immunity at intestinal mucosal surfaces.

Mucosal retinoic receptor-related orphan receptor (ROR)γt-expressing innate lymphoid cells (ILC) play an important role in the defense against intestinal pathogens and in promoting epithelial homeostasis and adaptation, thereby effectively protecting the vertebrate host against intestinal inflammatory disorders. The functional activity of RORγt(+) ILC is under the control of environmental cues. However, the molecular sensors for such environmental signals are largely unknown. Recently, the aryl hydrocarbon receptor (AhR) has emerged as a master regulator for the postnatal maintenance of intestinal RORγt(+) ILC and intraepithelial lymphocytes. AhR is a highly conserved transcription factor whose activity is regulated by environmental and dietary small molecule ligands. Here, we review the role of AhR signaling for the maintenance of intestinal immune cells and its impact on the immunological protection against intestinal infections and debilitating chronic inflammatory disorders.

Leukocyte migration to sites of inflammation is regulated by several endothelial adhesion molecules. Vascular adhesion protein-1 (VAP-1) is unique among the homing-associated molecules as it is both an enzyme that oxidizes primary amines and an adhesin. Although granulocytes can bind to endothelium via a VAP-1–dependent manner, the counter-receptor(s) on this leukocyte population is(are) not known. Here we used a phage display approach and identified Siglec-9 as a candidate ligand on granulocytes. The binding between Siglec-9 and VAP-1 was confirmed by in vitro and ex vivo adhesion assays. The interaction sites between VAP-1 and Siglec-9 were identified by molecular modeling and confirmed by further binding assays with mutated proteins. Although the binding takes place in the enzymatic groove of VAP-1, it is only partially dependent on the enzymatic activity of VAP-1. In positron emission tomography, the 68Gallium-labeled peptide of Siglec-9 specifically detected VAP-1 in vasculature at sites of inflammation and cancer. Thus, the peptide binding to the enzymatic groove of VAP-1 can be used for imaging conditions, such as inflammation and cancer.