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PurposeThe aim of the study was to determine if xanthohumol, a prenylated chalcone found in Hop (Humulus lupulus), has anti-inflammatory effects in healthy humans if applied in low doses achievable through dietary intake.MethodsIn a placebo-controlled single-blinded cross-over design study, 14 healthy young men and women either consumed a beverage containing 0.125 mg xanthohumol or a placebo. Peripheral blood mononuclear cells (PBMCs) were isolated before and 1 h after the intake of the beverages. Subsequently, PBMCs were stimulated with or without lipoteichoic acid (LTA) for 24 and 48 h. Concentrations of interleukin-1β (IL-1β), interleukin-6 (IL-6) and soluble cluster of differentiation (sCD14) protein were determined in cell culture supernatant. Furthermore, hTLR2 transfected HEK293 cells were stimulated with LTA in the presence or absence of xanthohumol and sCD14.ResultsThe stimulation of PBMCs with LTA for 24 and 48 h resulted in a significant induction of IL-1β, IL-6, and sCD14 protein release in PBMCs of both, fasted subjects and subjects after the ingestion of the placebo. In contrast, after ingesting xanthohumol, LTA-dependent induction of IL-1β, IL-6, and sCD14 protein release from PBMCs was not significantly higher than in unstimulated cells after 48 h. In hTLR2 transfected HEK293 cells xanthohumol significantly suppressed the LTA-dependent activation of cells, an effect attenuated when cells were co-incubated with sCD14.ConclusionThe results of our study suggest that an ingestion of low doses of xanthohumol can suppress the LTA-dependent stimulation of PBMCs through mechanisms involving the interaction of CD14 with TLR2. Study registered at ClinicalTrials.gov (NCT04847193, 22.03.2022).

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Despite major treatment advances in recent years, patients with multiple myeloma inevitably relapse. The RNA polymerase II complex has been identified as a promising therapeutic target in both proliferating and dormant cancer cells. Alpha-amanitin, a toxin so far without clinical application due to high liver toxicity, specifically inhibits this complex. Here, we describe the development of HDP-101, an anti–B-cell maturation antigen (BCMA) antibody conjugated with an amanitin derivative. HDP-101 displayed high efficacy against both proliferating and resting myeloma cells in vitro, sparing BCMA-negative cells. In subcutaneous and disseminated murine xenograft models, HDP-101 induced tumor regression at low doses, including durable complete remissions after a single intravenous dose. In cynomolgus monkeys, HDP-101 was well tolerated with a promising therapeutic index. In conclusion, HDP-101 safely and selectively delivers amanitin to myeloma cells and provides a novel therapeutic approach to overcome drug resistance in this disease.

Aging is a risk factor in the development of many diseases, including liver-related diseases. The two aims of the present study were 1) to determine how aging affects liver health in mice in the absence of any interventions and 2) if degenerations observed in relation to blood endotoxin levels are critical in aging-associated liver degeneration. Endotoxin levels and markers of liver damage, mitochondrial dysfunction, insulin resistance, and apoptosis as well as the Toll-like receptor 4 (Tlr-4) signaling cascade were studied in liver tissue and blood, respectively, of 3- and 24-mo-old male C57BL/6J mice. In a second set of experiments, 3- to 4-mo-old and 14-mo-old female lipopolysaccharide-binding protein (LBP)−/− mice and littermates fed standard chow, markers of liver damage, insulin resistance, and mitochondrial dysfunction were assessed. Plasma activity of aspartate aminotransferase and histological signs of hepatic inflammation and fibrosis were significantly higher in old C57BL/6J mice than in young animals. The number of neutrophils, CD8α-positive cells, and mRNA expression of markers of apoptosis were also significantly higher in livers of old C57BL/6J mice compared with young animals, being also associated with a significant induction of hepatic Tlr-4 and LBP expression as well as higher endotoxin levels in peripheral blood. Compared with age-matched littermates, LBP−/− mice display less signs of senescence in liver. Taken together, our data suggest that, despite being fed standard chow, old mice developed liver inflammation and beginning fibrosis and that bacterial endotoxin may play a critical role herein. NEW & NOTEWORTHY Old age in mice is associated with marked signs of liver degeneration, hepatic inflammation, and fibrosis. Aging-associated liver degeneration is associated with elevated bacterial endotoxin levels and an induction of lipopolysaccharide-binding protein (LBP) and Toll-like receptor 4-dependent signaling cascades in liver tissue. Furthermore, in old aged LBP−/− mice, markers of senescence seem to be lessened, supporting the hypothesis that bacterial endotoxin levels might be critical in aging-associated decline of liver.

Insulin resistance is among the key risk factors for the development of non-alcoholic fatty liver disease (NAFLD). Recently, it has been reported that GW9662, shown to be a potent peroxisome proliferator-activated receptor gamma (PPARγ) antagonist, may improve insulin sensitivity in settings of type 2 diabetes. Here, we determined the effects of GW9662 on the development of NAFLD and molecular mechanisms involved.Female C57BL/6J mice were pair-fed either a liquid control diet (C) or a fat-, fructose- and cholesterol-rich diet (FFC) for 8 weeks while either being treated with GW9662 (1 mg/kg body weight; C+GW9662 and FFC+GW9662) or vehicle (C and FFC) i.p. three times weekly. Indices of liver damage and inflammation, parameters of glucose metabolism and portal endotoxin levels were determined. Lipopolysaccharide (LPS)-challenged J774A.1 cells were treated with 10 μM GW9662.Despite similar caloric intake the development of NAFLD and insulin resistance were significantly attenuated in FFC+GW9662-treated mice when compared to FFC-fed animals. Bacterial endotoxin levels in portal plasma were almost similarly increased in both FFC-fed groups while expressions of toll-like receptor 4 (Tlr4), myeloid differentiation primary response 88 (Myd88) and interleukin 1 beta (Il1b) as well as nitrite (NOIn summary, our data suggest that the PPARγ antagonist GW9662 attenuates the development of a diet-induced NAFLD and that this is associated with a protection against the activation of the TLR4 signaling cascade.

Changes in intestinal nitric oxide metabolism are discussed to contribute for the development of intestinal barrier dysfunction in non-alcoholic fatty liver disease (NAFLD). To induce steatosis, female C57BL/6J mice were pair-fed with a liquid control diet (C) or a fat-, fructose- and cholesterol-rich diet (FFC) for 8 weeks. Mice received the diets ± 2.49 g L-arginine/kg bw/day for additional 5 weeks. Furthermore, mice fed C or FFC ± L-arginine/kg bw/day for 8 weeks were concomitantly treated with the arginase inhibitor N

Purpose Results of some epidemiological studies suggest that moderate alcohol consumption may be associated with a decreased risk to develop NAFLD. Here, the effect of the consumption of moderate beer and diluted ethanol, respectively, on the development of NAFLD were assessed. Methods Female C57BL/6J mice were fed a control diet (C-D) or a diet rich in fructose, fat and cholesterol (FFC) enriched isocalorically and isoalcoholically with beer (FFC + B) or plain ethanol (FFC + E) (2.5 g ethanol/kg body weight/day) for 7 weeks. Liver damage was assessed by histology using NAFLD activity score. Markers of inflammation, insulin resistance and adiponectin signaling were measured at mRNA and protein levels. Using J774A.1 cells as a model of Kupffer cells, the effect of alcoholic beverages on adiponectin receptor 1 (Adipor1) was assessed. Results Hepatic triglyceride concentration, neutrophil granulocytes, iNOS protein concentrations and early signs of insulin resistance found in FFC-fed mice were significantly attenuated in FFC+ B-fed mice (P

Toll-like receptors (TLRs) in the liver compartment have repeatedly been attributed to the development of non-alcoholic fatty liver disease (NAFLD). Knowledge on TLR expression in blood cells and their relation to intestinal microbiota and NAFLD development is limited. Here, we determined TLR expression patterns in peripheral blood mononuclear cells (PBMCs) of NAFLD patients and controls, their relation to intestinal microbiota and the impact of TLRs found altered in NAFLD development. Markers of intestinal permeability in blood and TLR mRNA expression in PBMCs were determined in 37 NAFLD patients and 15 age-matched healthy controls. Fecal microbiota composition was evaluated in 21 NAFLD patients and 9 controls using 16S rRNA gene amplicon sequencing. Furthermore, TLR1−/− and C57BL/6 mice (n = 5–6/group) were pair-fed a liquid control or a fat-, fructose- and cholesterol-rich diet. Intestinal microbiota composition and markers of intestinal permeability like zonulin and bacterial endotoxin differed significantly between groups with the latter markers being significantly higher in NAFLD patients. Expression of TLR1-8 and 10 mRNA was detectable in PBMCs; however, only TLR1 expression, being higher in NAFLD patients, were significantly positively correlated with the prevalence of Holdemanella genus while negative correlations were found with Gemmiger and Ruminococcus genera. TLR1−/− mice were significantly protected from the development of diet-induced NAFLD when compared to wild-type mice. While intestinal microbiota composition and permeability differed significantly between NAFLD patients and healthy subjects, in PBMCs, only TLR1 expression differed between groups. Still, targeting these alterations might be a beneficial approach in the treatment of NAFLD in some patients. Fil: Baumann, Anja. Universidad de Viena; Austria Fil: Nier, Anika. Universidad de Viena; Austria Fil: Hernández Arriaga, Angélica. Universidad de Hohenheim; Alemania Fil: Brandt, Annette. Universidad de Viena; Austria Fil: Lorenzo Pisarello, Maria Jose. Universidad de Viena; Austria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Jin, Cheng J.. Universitat Jena; Alemania Fil: Pilar, Esther. Universidad de Viena; Austria Fil: Camarinha-Silva, Amélia. Universidad de Hohenheim; Alemania Fil: Schattenberg, Jörn M.. University Medical Center of the Johannes Gutenberg; Alemania Fil: Bergheim, Ina. Universidad de Viena; Austria

Background Relapsed/refractory multiple myeloma (RRMM) is characterized by a high inter- and intratumor heterogeneity and a complex interplay of myeloma cells with the bone marrow microenvironment (BME). Accordingly, there is an urgent need to dissect subclone structure, transcriptional heterogeneity and cellular interactions to unravel the molecular mechanisms underlying drug resistance in RRMM. Methods Single cell RNA sequencing (scRNA-seq) of ~210,000 cells from bone marrow aspirates sorted into CD138+ and CD138– fractions was conducted for 20 heavily pretreated RRMM patients. RRMM subclones were called from the scRNA-seq data based on a copy number aberration (CNA) analysis that was confirmed by interphase fluorescence in situ hybridization and whole genome sequencing. From the scRNA-seq data the composition and abundance of immune cell types in the BME was determined. Interactions of myeloma cells with the BME were characterized by an analysis of the correlated expression of ligand-receptor pairs. Selected findings from the scRNA-seq analysis were validated by flow cytometry. Results Subclones with distinct chromosomal aberrations were reliably identified at the single cell level based on CNAs inferred from scRNA-seq data. The analysis revealed a subclonal 1q-gain (+1q) in 10/20 samples, for which a gene expression signature of recurrently upregulated genes was derived. These +1q subclones frequently expanded during treatment. Furthermore, RRMM cells shaped an immune suppressive BME by upregulation of inflammatory cytokines and close interplay with the myeloid compartment. RRMM cells appear to reprogram the BME by upregulation of inflammatory cytokines and ligands of inhibitory receptors expressed on T and NK-cells. Specifically, the immune cell compartment of RRMM patients displayed an accumulation of PD1+ γδ T-cells and myeloid populations compared to healthy donors or early disease. In addition, we observed a depletion of hematopoietic progenitors in patients with high expression of genes associated with inflammatory signaling in the BME. Upon treatment with immunomodulatory drugs, reprogrammed plasmacytoid dendritic cells expanded. Focusing on patients with +1q, we found an enrichment of a rare M2-like tumor associated macrophage population while GZMB+ NK effector cells were depleted in these patients, indicating that +1q has a distinct effect on the BME in RRMM. Conclusions Our study resolves transcriptional features of subclones in RRMM and mechanisms of microenvironmental reprogramming. The insight gained in our study on the evolution of RRMM heterogeneity and its bone marrow milieu will support the development of novel treatment approaches and potentially guide clinical decision making.

The addition of plant oils such as soybean oil (S) to a diet rich in saturated fatty acids is discussed as a possible route to prevent or diminish the development of metabolic disease. Here, we assessed whether a butterfat-rich diet fortified with S affects the development of early non-alcoholic steatohepatitis (NASH) and glucose intolerance. Female C57BL/6J mice were fed a standard-control diet (C), a fat-, fructose-, and cholesterol-rich diet (FFC, 25E% butterfat, 50% (wt./wt.) fructose, 0.16% (wt./wt.) cholesterol), or FFC supplemented with S (FFC + S, 21E% butterfat + 4E% S) for 13 weeks. Indicators of liver damage, inflammation, intestinal barrier function, and glucose metabolism were measured. Lipopolysaccharide (LPS)-challenged J774A.1 cells were incubated with linolenic and linoleic acids (ratio 1:7.1, equivalent to S). The development of early NASH and glucose intolerance was significantly attenuated in FFC + S–fed mice compared to FFC-fed mice associated with lower hepatic toll-like receptor-4 mRNA expression, while markers of intestinal barrier function were significantly higher than in C-fed mice. Linolenic and linoleic acid significantly attenuated LPS-induced formation of reactive nitrogen species and interleukin-1 beta mRNA expression in J774A.1 cells. Our results indicate that fortifying butterfat with S may attenuate the development of NASH and glucose intolerance in mice.

Background The number of people above the age of 60 years is raising world-wide being associated with an increase in the prevalence of aging-associated impairments and even diseases. Recent studies suggest that aging is associated with alterations in bacterial endotoxin levels and that these changes may add to low-grade inflammation, the so-called ‘inflammaging’, and aging-associated liver degeneration. However, mechanisms involved, and especially, the interaction of intestinal microbiota and barrier in the development of aging-associated inflammation and liver degeneration have not been fully understood. Objective The aim of the present study was to determine if intestinal microbiota composition changes with age and if these alterations are associated with changes of markers of intestinal barrier function and the development of inflammation and liver degeneration. Methods Blood, liver, small and large intestinal tissue of male 2-, 15-, 24- and 30-months old C57BL/6 mice fed standard chow were obtained. Intestinal microbiota composition, expression levels of antimicrobial peptides in small intestine and markers of intestinal barrier function were measured. Furthermore, indices of liver damage, inflammation and expression levels of lipopolysaccharide binding protein (Lbp) as well as of toll-like receptors (Tlr) 1-9 in liver tissue were assessed. Results Pairwise comparisons of the microbial community in the small intestine showed differences between 2- and 24-, 15- and 24-, as well as 15- and 30-months old animals while Shannon’s diversity, species richness and evenness indexes did not differ in both small and large intestine, respectively, between age groups. Concentrations of nitric oxide were significantly lower in small intestine of 15-, 24- and 30-months old mice compared to 2-months old mice while mRNA expression of the antimicrobial peptides defensin alpha 1 and lysozyme 1 was unchanged. In contrast, in liver tissue, older age of animals was associated with increasing inflammation and the development of fibrosis in 24- and 30-months old mice. Numbers of inflammatory foci and neutrophils in livers of 24- and 30-months old mice were significantly higher compared to 2-months old mice. These alterations were also associated with higher endotoxin levels in plasma as well as an increased mRNA expression of Lbp and Tlr1, Tlr2, Tlr4, Tlr6 and Tlr9 in livers in older mice. Conclusion Despite no consistent and robust changes of microbiota composition in small and/or large intestine of mice of different age were observed, our data suggest that alterations of markers of intestinal barrier function in small intestine are associated with an induction of several Tlrs and beginning hepatic inflammation in older mice and increase with age.

Dietary fat is discussed to be critical in the development of non-alcoholic fatty liver disease. Here, we assess the effect of exchanging dietary fat source from butterfat to extra virgin olive oil on the progression of an already existing diet-induced non-alcoholic fatty liver disease in mice. Female C57BL/6J mice were fed a liquid butterfat-, fructose- and cholesterol-rich diet (BFC, 25E% from butterfat) or control diet (C, 12%E from soybean oil) for 13 weeks. In week 9, fat sources of some BFC- and C-fed mice were switched either to 25E% or 12E% olive oil (OFC and CO). Glucose and insulin tolerance tests were performed, and markers of liver damage and glucose metabolism were assessed. After 6 weeks of feeding, BFC-fed mice had developed marked signs of insulin resistance, which progressed to week 12 being not affected by the exchange of fat sources. Liver damage was similar between BFC- and OFC-fed mice. Markers of lipid metabolism and lipid peroxidation in liver and of insulin signaling in liver and muscle were also similarly altered in BFC- and OFC-fed mice. Taken together, our data suggest that exchanging butterfat with extra virgin olive oil has no effect on the progression of non-alcoholic fatty liver disease and glucose tolerance in mice.

Sodium butyrate (SoB) supplementation has been suggested to attenuate the development of non-alcoholic fatty liver disease (NAFLD). Here, we determined the therapeutic potential of SoB on NAFLD progression and molecular mechanism involved. Eight-week old C57BL/6J mice were pair-fed a fat-, fructose- and cholesterol-rich diet (FFC) or control diet (C). After 8 weeks, some mice received 0.6g SoB/kg bw in their respective diets (C+SoB, FFC+SoB) or were maintained on C or FFC for the next 5 weeks of feeding. Liver damage, markers of glucose metabolism, inflammation, intestinal barrier function and melatonin metabolism were determined. FFC-fed mice progressed from simple steatosis to early non-alcoholic steatohepatitis, along with significantly higher TNF&alpha, and IL-6 protein levels in the liver and impaired glucose tolerance. In FFC+SoB-fed mice, disease was limited to steatosis associated with protection against the induction of Tlr4 mRNA and iNOS protein levels in livers. SoB supplementation had no effect on FFC-induced loss of tight junction proteins in the small intestine but was associated with protection against alterations in melatonin synthesis and receptor expression in the small intestine and livers of FFC-fed animals. Our results suggest that the oral supplementation of SoB may attenuate the progression of simple steatosis to steatohepatitis.

Introduction: Multiple myeloma (MM) is a B-cell malignancy characterized by an abnormal proliferation and infiltration of malignant plasma cells in the bone marrow (BM). Mesenchymal stromal cells (MSCs) represent a crucial component of the BM niche and mediate essential signalling via cytokines and cell-cell interactions. The interplay of MM cells and BM-MSC is complex and relies on multiple signaling pathways leading to MM progression and therapeutic resistance. Objectives: MM remains an incurable disease so far. Distinctive for this disease is a long-lasting polarization of the BM niche influencing MM progression and prognosis. We, therefore, focussed on MSCs to identify enrichment for different hallmark gene sets and their aberrant signaling contributing to the pathogenesis of the disease, therapy response and to further identify novel therapeutic strategies. Methods: BM-MSCs were isolated from patients with MM at diagnosis (MM-D-MSC) and in remission (MM-R-MSC) as well as from donors with other malignant diseases (CTR-MSC). RNA sequencing and Western Blot were used for examination of enriched pathways. Various functional assays for proliferation, apoptosis and cell cycle were performed either using a mono-culture or co-culture protocol of MSC and the MM-cell lines MM.1S and SKMM2 treating the cells with the pan-PI3K-inhibitor GDC-0941. Results: MM-D-MSCs supported the growth of myeloma cell lines better (3 fold, p We confirmed these findings on a proteomic level. We found evidence for the upregulation of PI3Kα, AKT, pAKT and mTOR in MM-D-MSC comparing to the other MM-R- and CTR-MSCs (p As stated MM-D-MSC supported the growth of myeloma cells better than other MSC types. However, upon GDC-0941 treatment, the proliferation of MM-D-MSCs was significantly reduced compared to the other MSC-types. In addition, the inhibition of proliferation of myeloma cell lines MM1S and SKMM2 was much more pronounced when they were cocultured with MM-D-MSC (32 and 34 %, p=0.04) compared to the growth of myeloma cells in coculture with MSC types, either in remission or other malignancies. Conclusion: We here identified functionally distinct differences in MM-D-MSCs compared to MM-R-MSCs or CTR-MSCs. Our data further provides a deeper insight into the molecular signature of MM-MSCs, a predictive of patient prognosis and treatment outcome. Targeting MSCs as a crucial part of the MM-BM niche by using PI3K-inhibitors could contribute to novel therapeutic strategies to effectively block MM-MSC interaction improving overall patient survival. Disclosures Raab: Roche: Consultancy; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Khandanpour: BMS/Celgene: Honoraria; Sanofi: Honoraria, Research Funding; Pfizer: Honoraria; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria; GSK: Honoraria.

Tumor heterogeneity plays a significant role in the development of therapy resistance in multiple myeloma (MM). Focal lesions (FLs), which are nodular accumulations of MM cells, have been shown to be hotspots of genetic spatial tumor heterogeneity, which is characterized by unique tumor sub-clones at different sites in the bone marrow (BM). However, little is known about the mechanisms leading to mutations in FLs, the architecture of the tumor microenvironment (ME) at these sites, and the link between FL sub-clones and relapse. We applied whole genome sequencing (WGS) to CD138 + MM cells from paired FL and iliac crest random BM aspirates (RBMA) of 15 newly diagnosed MM (NDMM) patients. For 7 of these patients, single cell (sc) analyses were performed, including sc gene expression (scRNA) and T-cell receptor (TCR)-sequencing and sc assay for transposase-accessible chromatin (ATAC)-sequencing for paired BM CD138 + MM and CD138 - ME, as well as peripheral blood mononuclear cells (PBMC). WGS data was analyzed using inhouse pipelines. Mutations, copy-number-variations and mutational signatures were called using mpileup, ACESeq and mmsig. Neoantigen epitopes were predicted using NeoPredPipe. Sc data was generated using the 10X Genomics platform. Pre-processing and analysis of the sc data was performed with CellRanger and the R-packages Seurat, ArchR and inferCNV. In 13/15 patients we found significant differences in chromosomal and mutational profiles between FLs and paired RBMAs, with major unshared mutations (mutation seen in > 60% cells) being enriched at the FL site (mean 310 vs. 123, p On average, 23 (range 0-83) site-unique baseline mutations were predicted to be neoantigens. Thus, we hypothesized that spatial tumor heterogeneity could be associated with heterogeneity in the tumor ME. We did not observe expansion of site-unique T cell clones, but some of the clones were enriched up to 10-fold at one of the two sites. These clones were typically seen in the PB at low frequency. Expanded T-cells clones were almost exclusively found in the CD8 +-compartment, with 65% and 27% of expanded T-cell clones being CD45RO +/CD57 +-memory- and CD69 +-effector-T-cells, respectively. Besides differences in the T-cell clonality, we observed changes in proportions of other cell types, including a depletion of CD14+- and CD16+-macrophages in FLs (p In conclusion, our results strengthen the concept of MM as a spatially heterogeneous disease, suggest that reactive oxygen species result in site-specific mutagenesis, and support the hypothesis that FLs are the origin of aggressive disease. We demonstrate spatial heterogeneity at single-cell level in the BM immune ME for the first time, which implies that understanding the complex biology of FLs could be important in the context of novel immune therapies such as bispecific antibodies and CAR-T-cells. Disclosures John: Janssen: Consultancy. Müller-Tidow: Janssen Cilag: Consultancy, Research Funding; Bioline: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Goldschmidt: Takeda: Consultancy, Research Funding; Sanofi: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Dietmar-Hopp-Foundation: Other: Grant; Novartis: Honoraria, Research Funding; Mundipharma: Research Funding; MSD: Research Funding; Molecular Partners: Research Funding; Johns Hopkins University: Other: Grant; Janssen: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Incyte: Research Funding; GSK: Honoraria; Chugai: Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; BMS: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Celgene: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Adaptive Biotechnology: Consultancy; Amgen: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding. Raab: Janssen: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees. Weinhold: Sanofi: Honoraria.

Introduction Despite new treatment approaches, in most patients with multiple myeloma (MM) resistant subclones expand, finally resulting in the development of relapsed/refractory disease (RRMM). Recent single-cell transcriptomic (scRNA-seq) studies have improved our understanding of tumor heterogeneity in MM, but our knowledge about the regulatory processes and gene expression signatures in resistant subclones is still limited. Thus, we performed single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) as well as scRNA-seq from the same MM patient samples and developed a new approach for the analysis of the transcriptome and epigenome of competing subclones. Methods CD138+ bone marrow cells of 11 RRMM patients were longitudinally collected and profiled using 10X Genomics scRNA- and scATAC-seq platform. Pre-processing was performed with CellRanger and downstream analysis using Seurat and ArchR. To determine clones based on copy number variations (CNVs), we leveraged inferCNV for scRNA-seq and developed a sliding window approach adapted from a previously published method by Satpathy et al. for scATAC-seq data. Whole genome sequencing was available to confirm the identified CNV clones. Results We obtained chromatin accessibility profiles of >43000 cells after quality control, establishing the first epigenetic map of RRMM with clearly separated clusters for each patient. Samples from distinct MM subgroups, particularly t(4;14) and t(11;14), clustered together in a pseudo-bulk analysis based on transcription factor activity scores from scATAC. To investigate treatment-induced clonal dynamics we aligned sc chromatin accessibility profiles to clones inferred in scRNA-seq based on CNVs enabling us to track individual tumor subclones over time. An example for clonal adaptation is a patient with two subclones that both responded differently to pomalidomide plus anti-CD38 treatment: while clone 1 showed a higher activity for members of IRF family, ZEB1 and KLF family members were more active in clone 2. Branching evolution was e.g. observed in a patient treated with a MEK/BRAF inhibitor. This patient showed two subclones before treatment, achieved a complete remission and relapsed with one dominant clone. One of the initial subclones could be identified as the precursor of this dominant clone with both showing high activity of NFKB family members, while higher activity of SOX10/15 was only observed upon MEK/BRAF inhibition. An example for combined clonal adaptation and selection was observed in a patient with bi-allelic inactivation of TP53 in one of the two subclones before MCL1 inhibition. Interestingly, this clone was depleted at relapse. Conclusions In summary, our new approach to track individual tumor subclones over time indicates clonal adaptation or selection in virtually all RRMM patients and reveals new insights into regulatory mechanisms underlying treatment-resistance, opening potential avenues for new therapies.

Introduciton Tumor heterogeneity plays a significant role in the development of therapy resistance in multiple myeloma (MM). Focal accumulations of myeloma cells called focal lesions (FLs) have been shown to be the hotspots of spatial tumor heterogeneity, which is characterized by unique tumor sub-clones at different sites in the bone marrow (BM). However, little is known about the sub-clonal architecture in FLs, the mechanisms leading to site-unique mutations, and the link between sub-clones in FLs and treatment-resistance. Methods To fill this gap in knowledge, we used a comprehensive approach to characterize CD138-sorted tumor cells from FLs and random BM aspirates (RBMA) in 12 newly diagnosed MM patients, applying whole genome sequencing (WGS), single-cell(sc)-RNA-sequencing and sc assay for transposase-accessible chromatin (ATAC)-sequencing. Sc data was generated using the 10X Genomics platform. Pre-processing of the sc data was performed with CellRanger and the R-packages Seurat, ArchR and inferCNV were used for downstream analysis. WGS data was analyzed using inhouse pipelines. Mutations, copy-number-variations and mutational signatures were called using mpileup, ACESeq and mmsig. Neoantigen epitopes were predicted using NeoPredPipe. Results In the majority of patients (n=10/12) we found significant differences in the chromosomal and mutational profiles between FLs and paired RBMAs from the iliac crest. To identify the mechanisms underlying heterogenous mutations, we performed mutational signature analysis and found COSMIC signature SBS18 to be enriched in these mutations, suggesting that they were caused by reactive oxygen species. Interestingly several site-unique mutations were predicted as potential neoantigens. Driver gene mutations associated with relapse such as KRAS, CYLD, CDKN2C and TP53, were more often seen in FL sub-clones. Using a combination of WGS, sc-RNA and sc-ATAC-sequencing to characterize these sub-clones in more detail, we found increased regulatory accessibility and expression of genes associated with disease aggressiveness and drug resistance such as CXCR4 and members of the NFKB- and interferon pathways. The latter implies that FLs could play a significant role in the development of treatment resistance. Indeed, comparing sub-clones at baseline and after high-dose melphalan and autologous stem cell transplantation in one patient, we demonstrate expansion of a single tumor cell, which was closely related to the main sub-clone from the baseline FL. Conclusion In conclusion, our data provides novel insights into the mechanisms underlying site-unique mutations and the sub-clonal architecture at different sites in the BM. The combination of bulk and sc-techniques showed that FLs are enriched for sub-clones with genetic, transcriptional and regulatory markers characteristic for aggressive disease and can be the source of relapse in MM-patients. This implies that targeting FLs is essential for achieving a cure of MM.

Background: Universally accepted therapeutic strategies for the treatment of nonalcoholic steatohepatitis (NASH) are still lacking. Studies suggest a preventive effect of oral Gln supplementation on the development of NASH; however, whether Gln also has therapeutic potential for pre-existing NASH has not yet been clarified.Objective: The aim of the present study was to determine whether Gln prevents the progression of diet-induced NASH in mice.Methods: For 8 wk, female C57BL/6J mice (6-8 wk old) were pair-fed a liquid Western-style diet [WSD, 25% of energy from fat, 50% wt:wt fructose, 0.16% wt:wt cholesterol] or control diet (C diet) to induce liver damage. From week 8 to 13, they were pair-fed the C diet or WSD alone or supplemented with l-Gln to provide 2.1 g/kg body weight (C diet + Gln or WSD + Gln). Energy intake was adjusted to the group with the lowest energy intake. Indexes of liver damage and inflammation, intestinal barrier function, and toll-like receptor 4 (Tlr4) signaling in the liver were determined.Results: The liver histology scores significantly increased from 8 to 13 wk (+31%) in WSD-fed mice and were significantly higher than in controls (P ≤ 0.05 for both time comparisons), whereas scores did not differ between C diet-fed and WSD + Gln-fed mice after 13 wk of feeding. The occludin protein concentrations in the small intestinal tissue were similarly reduced in both WSD-fed groups when compared with controls [WSD compared with C diet (-53%) and C diet + Gln (-42%), P ≤ 0.05; WSD + Gln compared with C diet + Gln (-34%), P ≤ 0.05] after 13 wk, whereas the expression of myeloid differentiation primary response gene 88 mRNA and concentration of inducible nitric oxide synthase and 4-hydroxynonenal protein adducts were significantly higher only in livers of WSD-fed mice (P ≤ 0.05 for the WSD group compared with all other groups; WSD + Gln group compared with the C diet groups: NS).Conclusion: Taken together, our data suggest that oral Gln supplementation protects mice from the progression of pre-existing, WSD-induced NASH.

Background and Aims:General overnutrition, and a diet rich in fat and sugar are among the key risk factors for the development of metabolic diseases including diabetes type 2 and non-alcoholic fatty liver diseases (NAFLD). While being among the most commonly consumed oils world-wide, the effects of soybean oil on the development of metabolic diseases are yet not understood. Indeed, existing data is in part contradictory. Based on this background, the aim of the present study was to investigate the effects of soybean oil consumption on the development of non-alcoholic steatohepatitis (NASH), the more progressed stage of NAFLD, and insulin resistance.Methods:Female C57BL/6J mice were fed either a liquid standard control diet (C) or a liquid fat-, fructose- and cholesterol-rich diet (FFC, 25E% butterfat, 50% (wt/wt) fructose, 0.155% (wt/wt) cholesterol) or a FFC supplemented with soybean oil (FFC + S, 21E% butterfat + 4E% soy oil), with FFC and FFC + S-fed groups being pair fed for 13 weeks to induce steatohepatitis. After 7 and 12 weeks of feeding, a glucose tolerance test was performed. Indicators of liver damage, inflammation, intestinal barrier function and markers of insulin signaling cascade were measured in intestinal, liver and muscle tissue.Results:As expected FFC-fed mice developed early signs of NASH and insulin resistance. Despite similar caloric intake and body weight gain, development of NASH and insulin resistance were significantly attenuated in FFC + S-fed mice when compared to FFC-fed animals. Indeed, signs of hepatic inflammation e.g. number of inflammatory foci and neutrophils as well as activity of transaminase in plasma were almost at the level of control in FFC + S-fed mice. Prevalence of macrovesicular steatosis being the predominant type of fat accumulation found in FFC-fed animals was also markedly lower in FFC + S-fed mice. Furthermore, while in muscle and liver tissue of FFC-fed mice insulin receptors mRNA expression was significantly different from C-fed animals, insulin receptors expression in FFC + S-fed mice was almost at the level of controls. The protective effects of soybean oil supplementation on the development of metabolic alterations were associated with a protection against the induction of TLR4 mRNA expression and dependent signaling molecules in liver tissue.Conclusion:Taking together, our results indicate that supplementation with soybean oil may attenuate the development of diet-induced NASH and insulin resistance in mice and that this may be related to alteration of TLR4-dependent signaling cascade in liver tissue. (Funded in parts by UFOP e.V.)

The mouth is an important niche for bacterial colonization. Previous research used mouth microbiota to predict diseases like colon cancer and inflammatory bowel disease (IBD). It is still unclear how the sampling methodology influences microbial characterization. Our aim was to determine if the sampling methods, e.g., cotton swab or tissue biopsy, and the age influence the oral microbial composition of mice. Microbial DNA was extracted using a commercial kit and characterized targeting the 16s rRNA gene from mouth swabs and tissue biopsies from 2 and 15 months old C57BL/6 male mice kept in the same SPF facility. Our results show statistical different microbial community of the different ages, type of sampling, and the two fixed factors age x type of sample (p-value < 0.05). At the genus level, we identified that the genera Actinobacillus, Neisseria, Staphylococcus, and Streptococcus either increase or decrease in abundance depending on sampling and age. Additionally, the abundance of Streptococcus danieliae, Moraxella osloensis, and some unclassified Streptococcus was affected by the sampling method. While swab and tissue biopsies both identified the common colonizers of oral microbiota, cotton swabbing is a low-cost and practical method, validating the use of the swab as the preferred oral sampling approach.

Introduction: Treatments incorporating autologous transplantation (ASCT), proteasome inhibitors (PIs), and immunomodulatory drugs (IMIDs) induce deep and durable remissions in most multiple myeloma (MM) patients, resulting in prolonged survival. Yet, patients who suffer from early relapse within 2 years of treatment initiation or become refractory to PIs and IMIDs still have a dismal outcome. The mutational landscape in early relapsed MM (ERMM) and relapsed/refractory MM (RRMM) has been comprehensively described. Several aberrations are associated with these two types of high-risk disease but little is known about the biological difference between them. To this end, we have comparatively and sequentially analyzed whole genome and RNA sequencing data from ERMM and RRMM patients. Methods: We included 32 patients who had relapsed within 2 years of first-line therapy (ERMM group, 30 after ASCT). Samples were collected at first relapse. Paired baseline samples were available from 17 patients. For the RRMM group, we included 43 patients with a median of 5 prior lines of therapy (range 2 - 13; 88% with ASCT), who had relapsed after PIs and IMiDs. For 22 of them consecutive tumor samples were available. Sequencing data were pre-processed using in-house pipelines. Mutations, indels, translocations, and copy number variants were called using Platypus, SOPHIA and ACESeq. Mutational signatures were identified with MMSig and subclonal structures with SciClone. Differential gene expression was assessed using DESeq, gene set enrichment analysis was performed with hypeR, and gene fusions were detected with Arriba. Results: Nonsynonymous mutations occurred more frequently in RRMM (median=180) compared to ERMM at first relapse (median=62, p Conclusions: According to our results ERMM and RRMM are biologically distinct entities of MM. While ERMM is characterized by inactivation of tumor suppressors and upregulation of gene sets associated with hypoxia and glycolysis, RRMM shows mutations in multiple gene networks, upregulation of ribosomal protein pseudogenes with unknown function and a signature linked to defective DNA repair, suggesting multifactorial mechanisms that lead from first relapse to end-stage relapsed refractory disease. Comparing paired samples, we did not observe major difference in evolution patterns between ERMM and RRMM. Yet, the low prevalence of the melphalan MM1 signature in ERMM suggests selection of pre-existing clones in this entity. In contrast, single tumor cells exposed to melphalan are often the precursors of clones dominating at the RRMM stage, indicating that first-line ASCT has a long-term effect on MM evolution. Disclosures John: Proteona: Research Funding. Mueller-Tidow:Janssen-Cilag Gmbh: Membership on an entity's Board of Directors or advisory committees; Deutsche Forschungsgemeinschaft: Research Funding; Deutsche Krebshilfe: Research Funding; Daiichi Sankyo: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; BiolineRx: Research Funding; Bayer AG: Research Funding; Jose-Carreras-Siftung: Research Funding; Wilhelm-Sander-Stiftung: Research Funding; BMBF: Research Funding. Goldschmidt:Johns Hopkins University: Other: Grants and/or provision of Investigational Medicinal Product; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Incyte: Research Funding; Molecular Partners: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Novartis: Honoraria, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma GmbH: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg, Germany: Current Employment; GlaxoSmithKline (GSK): Honoraria; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product, Research Funding; Chugai: Honoraria, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Dietmar-Hopp-Foundation: Other: Grants and/or provision of Investigational Medicinal Product:; Merck Sharp and Dohme (MSD): Research Funding. Raab:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Heidelberg Pharma: Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Background Absolute dietary fat intake but even more so fatty acid pattern is discussed to be critical in the development of non-alcoholic fatty liver disease (NAFLD). Here, we determined if switching a butterfat enriched diet to a rapeseed oil (RO) enriched diet affects progression of an existing NAFLD and glucose intolerance in mice. Methods For eight weeks, female C57Bl/6J mice were either fed a liquid control (C) or a butterfat-, fructose- and cholesterol-rich diet (BFC, 25E% butterfat) to induce early signs of steatohepatitis and glucose intolerance in mice. For additional five weeks mice received either BFC or C or a fat-, fructose- and cholesterol-rich and control diet, in which butterfat was replaced with RO (ROFC and CRO). Markers of glucose metabolism, liver damage and intestinal barrier were assessed. Results Exchanging butterfat with RO attenuated the progression of BFC diet-induced NAFLD and glucose intolerance. Beneficial effects of RO were associated with lower portal endotoxin levels and an attenuation of the induction of the toll-like receptor-4-dependent signaling cascades in liver. Peroxisome proliferator-activated receptor γ activity was induced in small intestine of ROFC-fed mice. Conclusion Taken together, exchanging butterfat with RO attenuated the progression of diet-induced steatohepatitis and glucose intolerance in mice.

Background Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide lacking universally accepted therapies. Studies suggest that coffee consumption is associated with a reduced risk of NAFLD; however, molecular mechanisms and ingredients involved remain to be fully understood. Here, we determined the effects of regular intake of decaffeinated coffee on the development of NAFLD in mice, and molecular mechanisms involved. Methods Female C57BL/6J mice (n = 6–7/ group) were pair-fed either a liquid control diet (C) or fat-, fructose- and cholesterol-rich diet (FFC) +/- decaffeinated coffee (DeCaf, 6 g/kg BW) for 4 days or 6 weeks. Indices of liver damage, hepatic inflammation and parameters of insulin resistance and intestinal permeability as well as nitric oxide system were determined. Results Early signs of insulin resistance and non-alcoholic steatohepatitis (NASH) found after 6 weeks of FFC feeding were significantly lower in FFC+DeCaf-fed mice when compared to FFC-fed animals. Moreover, elevation of portal endotoxin levels and loss of tight junction proteins in proximal small intestine found in FFC-fed mice were significantly attenuated in FFC+DeCaf-fed animals. These beneficial effects of DeCaf were associated with a protection against the significant induction of inducible NO-synthase protein levels and 3-nitrotyrosine protein adducts found in proximal small intestine of FFC-fed mice. Similar protective effects of DeCaf were also found in mice fed the FFC diet short-term. Conclusion Our results suggest that protective effects of DeCaf on the development of NAFLD are at least in part related to maintaining intestinal barrier function., Graphical abstract fx1, Highlights • decaffeinated coffee protects mice from the development of NAFLD. • decaffeinated coffee attenuated increased translocation of bacterial endotoxins. • decaffeinated coffee prevents diet-induced induction of iNOS in small intestine.

The antidiabetic drug metformin has been proposed to affect non-alcoholic fatty liver disease (NAFLD) through its effects on intestinal microbiota and barrier function. However, so far most studies focused on long-term effects and more progressed disease stages. The aim of this study was to assess in two experimental settings, if the onset of NAFLD is associated with changes of intestinal microbiota and barrier function and to determine effects of metformin herein. C57Bl/6J mice were fed a liquid control diet (C) or fat-, fructose- and cholesterol-rich diet (FFC) for four days or six weeks ±300 mg/kg BW/day metformin (Met). Markers of liver health, intestinal barrier function and microbiota composition were assessed. Metformin treatment markedly attenuated FFC-induced NAFLD in both experiments with markers of inflammation and lipidperoxidation in livers of FFC + Met-fed mice being almost at the level of controls. Metformin treatment attenuated the loss of tight junction proteins in small intestine and the increase of bacterial endotoxin levels in portal plasma. Changes of intestinal microbiota found in FFC-fed mice were also significantly blunted in FFC + Met-fed mice. Taken together, protective effects of metformin on the onset of NAFLD are associated with changes of intestinal microbiota composition and lower translocation of bacterial endotoxins.