Altered microbial bile acid metabolism exacerbates T cell-driven inflammation during graft-versus-host disease.

Microbial transformation of bile acids affects intestinal immune homoeostasis but its impact on inflammatory pathologies remains largely unknown. Using a mouse model of graft-versus-host disease (GVHD), we found that T cell-driven inflammation decreased the abundance of microbiome-encoded bile salt hydrolase (BSH) genes and reduced the levels of unconjugated and microbe-derived bile acids. Several microbe-derived bile acids attenuated farnesoid X receptor (FXR) activation, suggesting that loss of these metabolites during inflammation may increase FXR activity and exacerbate the course of disease. Indeed, mortality increased with pharmacological activation of FXR and decreased with its genetic ablation in donor T cells during mouse GVHD. Furthermore, patients with GVHD after allogeneic hematopoietic cell transplantation showed similar loss of BSH and the associated reduction in unconjugated and microbe-derived bile acids. In addition, the FXR antagonist ursodeoxycholic acid reduced the proliferation of human T cells and was associated with a lower risk of GVHD-related mortality in patients. We propose that dysbiosis and loss of microbe-derived bile acids during inflammation may be an important mechanism to amplify T cell-mediated diseases.

ROCK1/2 signaling contributes to corticosteroid-refractory acute graft-versus-host disease.

Patients with corticosteroid-refractory acute graft-versus-host disease (aGVHD) have a low one-year survival rate. Identification and validation of novel targetable kinases in patients who experience corticosteroid-refractory-aGVHD may help improve outcomes. Kinase-specific proteomics of leukocytes from patients with corticosteroid-refractory-GVHD identified rho kinase type 1 (ROCK1) as the most significantly upregulated kinase. ROCK1/2 inhibition improved survival and histological GVHD severity in mice and was synergistic with JAK1/2 inhibition, without compromising graft-versus-leukemia-effects. ROCK1/2-inhibition in macrophages or dendritic cells prior to transfer reduced GVHD severity. Mechanistically, ROCK1/2 inhibition or ROCK1 knockdown interfered with CD80, CD86, MHC-II expression and IL-6, IL-1ß, iNOS and TNF production in myeloid cells. This was accompanied by impaired T cell activation by dendritic cells and inhibition of cytoskeletal rearrangements, thereby reducing macrophage and DC migration. NF-κB signaling was reduced in myeloid cells following ROCK1/2 inhibition. In conclusion, ROCK1/2 inhibition interferes with immune activation at multiple levels and reduces acute GVHD while maintaining GVL-effects, including in corticosteroid-refractory settings.

Tissue-specific features of the T cell repertoire after allogeneic hematopoietic cell transplantation in human and mouse.

T cells are the central drivers of many inflammatory diseases, but the repertoire of tissue-resident T cells at sites of pathology in human organs remains poorly understood. We examined the site-specificity of T cell receptor (TCR) repertoires across tissues (5 to 18 tissues per patient) in prospectively collected autopsies of patients with and without graft-versus-host disease (GVHD), a potentially lethal tissue-targeting complication of allogeneic hematopoietic cell transplantation, and in mouse models of GVHD. Anatomic similarity between tissues was a key determinant of TCR repertoire composition within patients, independent of disease or transplant status. The T cells recovered from peripheral blood and spleens in patients and mice captured a limited portion of the TCR repertoire detected in tissues. Whereas few T cell clones were shared across patients, motif-based clustering revealed shared repertoire signatures across patients in a tissue-specific fashion. T cells at disease sites had a tissue-resident phenotype and were of donor origin based on single-cell chimerism analysis. These data demonstrate the complex composition of T cell populations that persist in human tissues at the end stage of an inflammatory disorder after lymphocyte-directed therapy. These findings also underscore the importance of studying T cell in tissues rather than blood for tissue-based pathologies and suggest the tissue-specific nature of both the endogenous and posttransplant T cell landscape.

Preservation of the fecal microbiome is associated with reduced severity of graft-versus-host disease.

Following allogeneic hematopoietic cell transplantation (allo-HCT), the gastrointestinal (GI) tract is frequently affected by acute graft-versus-host disease (aGVHD), the pathophysiology of which is associated with a dysbiotic microbiome. Since microbial composition varies along the length of the GI tract, the authors hypothesized that microbiome features correlate with the pattern of organ involvement after allo-HCT. We evaluated 266 allo-HCT recipients from whom 1303 stool samples were profiled by 16S ribosomal gene sequencing. Patients were classified according to which organs were affected by aGVHD. In the 20 days prior to disease onset, GVHD patients had lower abundances of members of the class Clostridia, lower counts of butyrate producers, and lower ratios of strict-to-facultative (S/F) anaerobic bacteria compared with allograft recipients who were free of GVHD. GI GVHD patients showed significant reduction in microbial diversity preonset. Patients with lower GI aGVHD had lower S/F anaerobe ratios compared with those with isolated upper GI aGVHD. In the 20 days after disease onset, dysbiosis was observed only in GVHD patients with GI involvement, particularly those with lower-tract disease. Importantly, Clostridial and butyrate-producer abundance as well as S/F anaerobe ratio were predictors of longer overall survival; higher abundance of butyrate producers and higher S/F anaerobe ratio were associated with decreased risk of GVHD-related death. These findings suggest that the intestinal microbiome can serve as a biomarker for outcomes of allo-HCT patients with GVHD.

MAIT and Vδ2 unconventional T cells are supported by a diverse intestinal microbiome and correlate with favorable patient outcome after allogeneic HCT.

Microbial diversity is associated with improved outcomes in recipients of allogeneic hematopoietic cell transplantation (allo-HCT), but the mechanism underlying this observation is unclear. In a cohort of 174 patients who underwent allo-HCT, we demonstrate that a diverse intestinal microbiome early after allo-HCT is associated with an increased number of innate-like mucosal-associated invariant T (MAIT) cells, which are in turn associated with improved overall survival and less acute graft-versus-host disease (aGVHD). Immune profiling of conventional and unconventional immune cell subsets revealed that the prevalence of Vδ2 cells, the major circulating subpopulation of γδ T cells, closely correlated with the frequency of MAIT cells and was associated with less aGVHD. Analysis of these populations using both single-cell transcriptomics and flow cytometry suggested a shift toward activated phenotypes and a gain of cytotoxic and effector functions after transplantation. A diverse intestinal microbiome with the capacity to produce activating ligands for MAIT and Vδ2 cells appeared to be necessary for the maintenance of these populations after allo-HCT. These data suggest an immunological link between intestinal microbial diversity, microbe-derived ligands, and maintenance of unconventional T cells.

Early intestinal microbial features are associated with CD4 T-cell recovery after allogeneic hematopoietic transplant.

Low intestinal microbial diversity is associated with poor outcomes after allogeneic hematopoietic cell transplantation (HCT). Using 16S rRNA sequencing of 2067 stool samples and flow cytometry data from 2370 peripheral blood samples drawn from 894 patients who underwent allogeneic HCT, we have linked features of the early post-HCT microbiome with subsequent immune cell recovery. We examined lymphocyte recovery and microbiota features in recipients of both unmodified and CD34-selected allografts. We observed that fecal microbial diversity was an independent predictor of CD4 T-cell count 3 months after HCT in recipients of a CD34-selected allograft, who are dependent on de novo lymphopoiesis for their immune recovery. In multivariate models using clinical factors and microbiota features, we consistently observed that increased fecal relative abundance of genus Staphylococcus during the early posttransplant period was associated with worse CD4 T-cell recovery. Our observations suggest that the intestinal bacteria, or the factors they produce, can affect early lymphopoiesis and the homeostasis of allograft-derived T cells after transplantation.

An Unconventional View of T Cell Reconstitution After Allogeneic Hematopoietic Cell Transplantation.

Allogeneic hematopoietic cell transplantation (allo-HCT) is performed as curative-intent therapy for hematologic malignancies and non-malignant hematologic, immunological and metabolic disorders, however, its broader implementation is limited by high rates of transplantation-related complications and a 2-year mortality that approaches 50%. Robust reconstitution of a functioning innate and adaptive immune system is a critical contributor to good long-term patient outcomes, primarily to prevent and overcome post-transplantation infectious complications and ensure adequate graft-versus-leukemia effects. There is increasing evidence that unconventional T cells may have an important immunomodulatory role after allo-HCT, which may be at least partially dependent on the post-transplantation intestinal microbiome. Here we discuss the role of immune reconstitution in allo-HCT outcome, focusing on unconventional T cells, specifically mucosal-associated invariant T (MAIT) cells, γδ (gd) T cells, and invariant NK T (iNKT) cells. We provide an overview of the mechanistic preclinical and associative clinical studies that have been performed. We also discuss the emerging role of the intestinal microbiome with regard to hematopoietic function and overall immune reconstitution.

miR-146a Controls Immune Response in the Melanoma Microenvironment.

MicroRNAs (miR) are small noncoding RNAs that regulate gene expression, posttranscription, and manipulate immune responses in different types of cancers. In this study, we identify miR-146a as a negative regulator of immune activation, comparable to immune-checkpoint molecules. miR-146a levels were increased in melanoma microenvironmental tissue, and miR-146a-/- mice survived longer and developed less metastases in comparison with wild-type melanoma-bearing mice. T cells isolated from miR-146a-/- mice revealed higher expression levels of the miR-146a target gene Stat1 and the Stat1-regulated cytokine IFNγ. Neutralization of IFNγ in miR-146a-/- mice decreased survival and increased melanoma metastasis patterns to those of wild-type mice. In vitro, IFNγ reduced melanoma cell migration, cell-cycle activity, and basal metabolic rate. Conversely, IFNγ also increased PD-L1 levels on the melanoma cells, which may counterbalance some of the beneficial effects increasing immune escape in vivo. Combined treatment with a miR-146a antagomiR and anti-PD-1 resulted in improved survival over isotype control or anti-PD-1 treatment alone. In summary, these data show that miR-146a plays a central role within the STAT1/IFNγ axis in the melanoma microenvironment, affecting melanoma migration, proliferation, and mitochondrial fitness as well as PD-L1 levels. Additionally, combined inhibition of PD-1 and miR-146a could be a novel strategy to enhance antitumor immune response elicited by checkpoint therapy. SIGNIFICANCE: These findings identify a microRNA-based mechanism by which melanoma cells escape the immune system, providing a new therapeutic strategy to improve the current management of patients with melanoma.

Trametinib (GSK1120212).

The mitogen-activated protein kinase (MEK MAPK/ERK kinase) signaling pathways play a critical role in the regulation of diverse cellular activities, including survival, differentiation, proliferation, motility, and angiogenesis. Therefore, MEK inhibition was recognized as a promising target for antineoplastic therapy. Trametinib (GSK1120212), an oral MEK inhibitor which is selective for MEK1 and MEK2, has been approved by the FDA for the treatment of metastatic melanoma in a combination with a BRAF inhibitor. In this overview, preclinical and clinical data for trametinib are presented including mechanisms based on in vitro studies as well as findings from different clinical studies. The future clinical trial in different solid tumor entities will define the therapeutic role of this targeted therapy approach, possibly as a combination with other targeted therapies such as BRAF inhibitors.

Vismodegib.

Vismodegib (GDC-0449, Erivedge®) is a small molecule antagonist of the hedgehog (Hh) pathway that binds to smoothened (SMO) and leads to inhibition of an aberrant activation of the Hh pathway. Dysregulated Hh signaling results in uncontrolled proliferation in basal cell carcinoma (BCC) and has also been found present in medulloblastoma, and many other cancers such as those of gastrointestinal tract, brain, lung, breast, and prostate. In January 2012, vismodegib became the first agent to target the Hh pathway to receive approval by the United States Food and Drug Administration (FDA) and in July 2013 approval by the European Medicines Agency (EMA) followed for the treatment of adult patients with symptomatic metastatic BCC, or locally advanced BCC inappropriate for surgery or radiotherapy. The role of vismodegib in other malignancies than BCC has still to be investigated.

Cobimetinib (GDC-0973, XL518).

The mitogen-activated protein kinase cascade (MAPK/ERK pathway) is a signaling pathway activated as a cellular response to various stimuli and for regulating the proliferation and survival of several types of eukaryotic cells, among others a wide variety of tumor cells. Mutations of the proteins involved in this pathway have been discovered in several tumor entities, indicating their inhibition as a potential therapeutic target. BRAF inhibitors have been in the clinical use since 2011. Several MEK inhibitors have been studied for metastatic cancer treatment in the recent past. After trametinib, cobimetinib is another potent, selective oral MEK1/2 inhibitor that was approved by European Medicine Agency (EMA) and Food and Drug Administration (FDA) in 2015 for treatment of malignant melanoma in a combination with the BRAF inhibitor vemurafenib.

Biglycan expression in the melanoma microenvironment promotes invasiveness via increased tissue stiffness inducing integrin-ß1 expression.

Novel targeted and immunotherapeutic approaches have revolutionized the treatment of metastatic melanoma. A better understanding of the melanoma-microenvironment, in particular the interaction of cells with extracellular matrix molecules, may help to further improve these new therapeutic strategies.We observed that the extracellular matrix molecule biglycan (Bgn) was expressed in certain human melanoma cells and primary fibroblasts when evaluated by microarray-based gene expression analysis. Bgn expression in the melanoma tissues correlated with low overall-survival and low progression-free-survival in patients. To understand the functional role of Bgn we used gene-targeted mice lacking functional Bgn. Here we observed that melanoma growth, metastasis-formation and tumor-related death were reduced in Bgn-/- mice compared to Bgn+/+ mice. In vitro invasion of melanoma cells into organotypic-matrices derived from Bgn-/- fibroblasts was reduced compared to melanoma invasion into Bgn-proficient matrices. Tissue stiffness as determined by atomic-force-microscopy was reduced in Bgn-/- matrices. Isolation of melanoma cells and fibroblasts from the stiffer Bgn+/+ matrices revealed an increase in integrin-ß1 expression compared to the Bgn-/- fibroblast matrices. Overexpression of integrin-ß1 in B16-melanoma cells abolished the survival benefit seen in Bgn-/- mice. Consistent with the studies performed in mice, the abundance of Bgn-expression in human melanoma samples positively correlated with the expression of integrin-ß1, which is in agreement with results from the organotypic invasion-assay and the in vivo mouse studies.This study describes a novel role for Bgn-related tissue stiffness in the melanoma-microenvironment via regulation of integrin-ß1 expression by melanoma cells in both mice and humans.

B-Raf inhibitors induce epithelial differentiation in BRAF-mutant colorectal cancer cells.

BRAF mutations are associated with aggressive, less-differentiated and therapy-resistant colorectal carcinoma. However, the underlying mechanisms for these correlations remain unknown. To understand how oncogenic B-Raf contributes to carcinogenesis, in particular to aspects other than cellular proliferation and survival, we generated three isogenic human colorectal carcinoma cell line models in which we can dynamically modulate the expression of the B-Raf(V600E) oncoprotein. Doxycyclin-inducible knockdown of endogenous B-Raf(V600E) decreases cellular motility and invasion in conventional and three-dimensional (3D) culture, whereas it promotes cell-cell contacts and induces various hallmarks of differentiated epithelia. Importantly, all these effects are recapitulated by B-Raf (PLX4720, vemurafenib, and dabrafenib) or MEK inhibitors (trametinib). Surprisingly, loss of B-Raf(V600E) in HT29 xenografts does not only stall tumor growth, but also induces glandular structures with marked expression of CDX2, a tumor-suppressor and master transcription factor of intestinal differentiation. By performing the first transcriptome profiles of PLX4720-treated 3D cultures of HT29 and Colo-205 cells, we identify several upregulated genes linked to epithelial differentiation and effector functions, such as claudin-1, a Cdx-2 target gene encoding a critical tight junction component. Thereby, we provide a mechanism for the clinically observed correlation between mutant BRAF and the loss of Cdx-2 and claudin-1. PLX4720 also suppressed several metastasis-associated transcripts that have not been implicated as targets, effectors or potential biomarkers of oncogenic B-Raf signaling so far. Together, we identify a novel facet of clinically applied B-Raf or MEK inhibitors by showing that they promote cellular adhesion and differentiation of colorectal carcinoma cells.

BRAF inhibitor-associated ERK activation drives development of chronic lymphocytic leukemia.

Patients with BRAFV600E/K-driven melanoma respond to the BRAF inhibitor vemurafenib due to subsequent deactivation of the proliferative RAS/RAF/MEK/ERK pathway. In BRAF WT cells and those with mutations that activate or result in high levels of the BRAF activator RAS, BRAF inhibition can lead to ERK activation, resulting in tumorigenic transformation. We describe a patient with malignant melanoma who developed chronic lymphocytic leukemia (CLL) in the absence of RAS mutations during vemurafenib treatment. BRAF inhibition promoted patient CLL proliferation in culture and in murine xenografts and activated MEK/ERK in primary CLL cells from additional patients. BRAF inhibitor-driven ERK activity and CLL proliferation required B cell antigen receptor (BCR) activation, as inhibition of the BCR-proximal spleen tyrosine kinase (SYK) reversed ERK hyperactivation and proliferation of CLL cells from multiple patients, while inhibition of the BCR-distal Bruton tyrosine kinase had no effect. Additionally, the RAS-GTP/RAS ratio in primary CLL cells exposed to vemurafenib was reduced upon SYK inhibition. BRAF inhibition increased mortality and CLL expansion in mice harboring CLL xenografts; however, SYK or MEK inhibition prevented CLL proliferation and increased animal survival. Together, these results suggest that BRAF inhibitors promote B cell malignancies in the absence of obvious mutations in RAS or other receptor tyrosine kinases and provide a rationale for combined BRAF/MEK or BRAF/SYK inhibition.

Silencing of the EPHB3 tumor-suppressor gene in human colorectal cancer through decommissioning of a transcriptional enhancer.

The protein tyrosine kinase Ephrin type-B receptor 3 (EPHB3) counteracts tumor-cell dissemination by regulating intercellular adhesion and repulsion and acts as tumor/invasion suppressor in colorectal cancer. This protective mechanism frequently collapses at the adenoma-carcinoma transition due to EPHB3 transcriptional silencing. Here, we identify a transcriptional enhancer at the EPHB3 gene that integrates input from the intestinal stem-cell regulator achaete-scute family basic helix-loop-helix transcription factor 2 (ASCL2), Wnt/ß-catenin, MAP kinase, and Notch signaling. EPHB3 enhancer activity is highly variable in colorectal carcinoma cells and precisely reflects EPHB3 expression states, suggesting that enhancer dysfunction underlies EPHB3 silencing. Interestingly, low Notch activity parallels reduced EPHB3 expression in colorectal carcinoma cell lines and poorly differentiated tumor-tissue specimens. Restoring Notch activity reestablished enhancer function and EPHB3 expression. Although essential for intestinal stem-cell maintenance and adenoma formation, Notch activity seems dispensable in colorectal carcinomas. Notch activation even promoted growth arrest and apoptosis of colorectal carcinoma cells, attenuated their self-renewal capacity in vitro, and blocked tumor growth in vivo. Higher levels of Notch activity also correlated with longer disease-free survival of colorectal cancer patients. In summary, our results uncover enhancer decommissioning as a mechanism for transcriptional silencing of the EPHB3 tumor suppressor and argue for an antitumorigenic function of Notch signaling in advanced colorectal cancer.

Iowa Gambling Task in patients with early-onset Parkinson's disease: strategy analysis.

The aim of our study was to analyse decision making in early-onset Parkinson's disease (PD) patients performing the Iowa Gambling Task (IGT). We compared 19 patients with early-onset PD (≤ 45 years) on dopaminergic medication (no evidence of depression, dementia, executive dysfunction according to the Tower of London test and the Stroop test, or pathological gambling) with 20 age-matched controls. A computer version of the IGT was employed. The PD patients achieved slightly lower IGT scores than the control group. A detailed analysis based on 'shift frequencies' between the individual decks showed that the patients tended to change their preferences for the decks more frequently, with a higher preference for the 'disadvantageous' deck B. Control subjects seemed to develop a more effective strategy. These differences could be caused by the poorer ability of the patients to develop any strategy at all. We observed changes in decision making during IGT performance in patients with early-onset PD, although they had no executive dysfunction as measured by established neuropsychological tests. The more detailed analysis employed in the present study could lead to a more accurate study of IGT performance and application of IGT in clinical practice.

Immunotherapy for malignant melanoma.

Treatment of metastatic melanoma is a challenge for clinicians as most agents have failed to demonstrate improved survival in phase III trials. Despite the immunogenicity of this tumor entity, different immunological interventions including cytokine therapy, vaccination, biochemotherapy or allogeneic hematopoietic cell transplantation did not lead to a satisfactory response. However, continuous investigation on the immune mediated rejection of melanoma cells has led to the development of effective antibodies blocking cytotoxic T-lymphocyte antigen-4 (CTLA-4), a critical negative regulator of the antitumor T-cell response. Based on data from rodent models, the anti-CTLA-4 antibody ipilimumab was developed into clinical studies where it had encouraging activity in advanced melanoma with unusual response patterns. As in most immunostimulatory therapies, acute toxicities were severe and clearly mechanism-related. Although some patients developed signs of autoimmunity, the toxicities were overall manageable and mostly reversible. This review summarizes different immunotherapeutical approaches against melanoma that have been applied in the past and focuses on CTLA-4 blockade with respect to its mechanism, clinical effectiveness and immunological side effects.