The most frequently encountered symbiont on tree roots is the ascomycete Cenococcum geophilum, the only mycorrhizal species within the largest fungal class Dothideomycetes, a class known for devastating plant pathogens. Here we show that the symbiotic genomic idiosyncrasies of ectomycorrhizal basidiomycetes are also present in C. geophilum with symbiosis-induced, taxon-specific genes of unknown function and reduced numbers of plant cell wall-degrading enzymes. C. geophilum still holds a significant set of genes in categories known to be involved in pathogenesis and shows an increased genome size due to transposable elements proliferation. Transcript profiling revealed a striking upregulation of membrane transporters, including aquaporin water channels and sugar transporters, and mycorrhiza-induced small secreted proteins (MiSSPs) in ectomycorrhiza compared with free-living mycelium. The frequency with which this symbiont is found on tree roots and its possible role in water and nutrient transport in symbiosis calls for further studies on mechanisms of host and environmental adaptation.

SummaryConcomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX.

Recent data suggest that Pancreatic Neuroendocrine Tumours (PanNETs) originate from α- or β-cells of the islets of Langerhans. The majority of PanNETs are non-functional and do not express cell-type specific hormones. In the current study we examine whether tumour DNA methylation (DNAme) profiling combined with genomic data is able to identify cell of origin and to reveal pathways involved in PanNET progression. We analyse genome-wide DNAme data of 125 PanNETs and sorted α- and β-cells. To confirm cell identity, we investigate ARX and PDX1 expression. Based on epigenetic similarities, PanNETs cluster in α-like, β-like and intermediate tumours. The epigenetic similarity to α-cells progressively decreases in the intermediate tumours, which present unclear differentiation. Specific transcription factor methylation and expression vary in the respective α/β-tumour groups. Depending on DNAme similarity to α/β-cells, PanNETs have different mutational spectra, stage of the disease and prognosis, indicating potential means of PanNET progression.

Background: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human cancers, with a median survival of only three to six months. Standard treatment options and even targeted therapies have so far failed to improve long-term overall survival. Thus, novel treatment modalities for ATC, such as immunotherapy, are urgently needed. CD47 is a ''don't eat me'' signal, which prevents cancer cells from phagocytosis by binding to signal regulatory protein alpha on macrophages. So far, the role of macrophages and the CD47-signal regulatory protein alpha signaling axis in ATC is not well understood. Methods: This study analyzed 19 primary human ATCs for macrophage markers, CD47 expression, and immune checkpoints by immunohistochemistry. ATC cell lines and a fresh ATC sample were assessed by flow cytometry for CD47 expression and macrophage infiltration, respectively. CD47 was blocked in phagocytosis assays of co-cultured macrophages and ATC cell lines. Anti-CD47 antibody treatment was administered to ATC cell line xenotransplanted immunocompromised mice, as well as to tamoxifen-induced ATC doubletransgenic mice. Results: Human ATC samples were heavily infiltrated by CD68-and CD163-expressing tumor-associated macrophages (TAMs), and expressed CD47 and calreticulin, the dominant pro-phagocytic molecule. In addition, ATC tissues expressed the immune checkpoint molecules programmed cell death 1 and programmed death ligand 1. Blocking CD47 promoted the phagocytosis of ATC cell lines by macrophages in vitro. Anti-CD47 antibody treatment of ATC xenotransplanted mice increased the frequency of TAMs, enhanced the expression of macrophage activation markers, augmented tumor cell phagocytosis, and suppressed tumor growth. In double-transgenic ATC mice, CD47 was expressed on tumor cells, and blocking CD47 increased TAM frequencies. Conclusions: Targeting CD47 or CD47 in combination with programmed cell death 1 may potentially improve the outcomes of ATC patients and may represent a valuable addition to the current standard of care.

Molecular mechanisms underlying the development and progression of pancreatic neuroendocrine tumors (PanNETs) are still insufficiently understood. Efficacy of currently approved PanNET therapies is limited. While novel treatment options are being developed, patient stratification permitting more personalized treatment selection in PanNET is yet not feasible since no predictive markers are established. The lack of representative in vitro and in vivo models as well as the rarity and heterogeneity of PanNET are prevailing reasons for this. In this study, we describe an in vitro 3-dimensional (3-D) human primary PanNET culture system as a novel preclinical model for more personalized therapy selection. We present a screening platform allowing multicenter sample collection and drug screening in 3-D cultures of human primary PanNET cells. We demonstrate that primary cells isolated from PanNET patients and cultured in vitro form isletlike tumoroids. Islet-like tumoroids retain a neuroendocrine phenotype and are viable for at least 2 weeks in culture with a high success rate (86%). Viability can be monitored continuously allowing for a perwell normalization. In a proof-of-concept study, islet-like tumoroids were screened with three clinically approved therapies for PanNET: sunitinib, everolimus and temozolomide. Islet-like tumoroids display varying in vitro response profiles to distinct therapeutic regimes. Treatment response of islet-like tumoroids differs also between patient samples. We believe that the presented human PanNET screening platform is suitable for personalized drug testing in a larger patient cohort, and a broader application will help in identifying novel markers predicting treatment response and in refining PanNET therapy Molecular mechanisms underlying the development and progression of PanNET are still insufficiently understood. Efficacy of currently approved PanNET therapies is limited. While novel treatment options are being developed, patient stratification permitting more personalized treatment selection in PanNET is yet not feasible since no predictive markers are established. The lack of representative in vitro and in vivo models as well as the rarity and heterogeneity of PanNET are prevailing reasons for this.

No abstract

Pancreatic neuroendocrine neoplasms are epigenetically driven tumors, but therapies against underlying epigenetic drivers are currently not available in the clinical practice. We aimed to investigate EZH2 (Enhancer of Zest homolog) expression in PanNEN and the impact of EZH2 inhibition in three different PanNEN preclinical models. EZH2 expression in PanNEN patient samples (n = 172) was assessed by immunohistochemistry and correlated with clinico-pathological data. Viability of PanNEN cell lines treated with EZH2 inhibitor (GSK126) was determined in vitro. Lentiviral transduction of shRNA targeting EZH2 was performed in QGP1 cells, and cell proliferation was measured. Rip1TAG2 mice underwent GSK126 treatment for three weeks starting from week 10 of age. Primary cells isolated from PanNEN patients (n = 6) were cultivated in 3D as islet-like tumoroids and monitored for 10 consecutive days upon GSK126 treatment. Viability was measured continuously for the whole duration of the treatment. We found that high EZH2 expression correlated with higher tumor grade (p < 0.001), presence of distant metastases (p < 0.001), and shorter disease-free survival (p < 0.001) in PanNEN patients. Inhibition of EZH2 in vitro in PanNEN cell lines and in patient-derived islet-like tumoroids reduced cell viability and impaired cell proliferation, while inhibition of EZH2 in vivo in Rip1TAG2 mice reduced tumor burden. Our results show that EZH2 is highly expressed in high-grade PanNENs, and during disease progression it may contribute to aberrations in the epigenetic cellular landscape. Targeting EZH2 may represent a valuable epigenetic treatment option for patients with PanNEN.

25Recent data suggest that Pancreatic Neuroendocrine Tumours (PanNETs) originate from α-or β-cells of 26 the islets of Langerhans. The majority of PanNETs are non-functional and do not express cell-type 27 specific hormones. We examined whether tumour DNA methylation (DNAme) profiling combined with 28 genomic data could identify cell of origin and reveal pathways involved in PanNET progression. We 29 analysed genome-wide DNAme data of 125 PanNETs and sorted α-and β-cells. To confirm cell identity, 30we investigated ARX and PDX1 expression. Based on epigenetic similarities, PanNETs clustered in α-31 like, β-like and intermediate tumours. The epigenetic similarity to α-cells progressively decreased in the 32 intermediate tumours, which presented unclear differentiation. Specific transcription factor methylation 33 and expression varied in the respective α/β-tumour groups. Depending on DNAme similarity to α/β-34 cells, PanNETs have different mutational spectra, stage of the disease and prognosis, indicating potential 35 means of PanNET progression. 36 Recent studies of gene expression and master regulator analysis alongside investigation of super-53 enhancer signatures have suggested both and -cells as two possible cells of origin for non-54 functioning (NF)-PanNETs5-8. On the other hand, Sadanandam et al. reported that a group of aggressive 55PanNET, namely, "metastasis-like primary", have a phenotype characterised by "stemness" transcripts 56 compared to well differentiated tumours, also suggesting a common progenitor cell origin5. Similarly, 57 based on the identification of master regulator proteins, de-differentiation and acquisition of stem cell 58 characteristics seem to be one of the pathways associated with tumour progression6. 59The cell of origin in cancer refers to the normal cell that acquires the initial cancer-promoting genetic 60 hit(s). During development, cell lineage fate is determined by cell-type specific transcription factor (TF) 61 expression, which in turn is dependent on the type of epigenetic markers that are located at the relative 62 regulatory regions (e.g. super-enhancer activation)9. The five endocrine cell types derive from a common 63 endocrine precursor, which has segregated from a ducto-endocrine bipotent cell population10. The TFs, 64Pax4 (Paired Box 4) and Arx (Aristaless Related Homeobox), are required for β-and α-cell fates, 65respectively. Lineage decision is determined via cross-inhibitory interactions11. Pdx1 (Pancreatic And 66 Duodenal Homeobox 1) expression becomes restricted to cells at the stage of initiating insulin 67 expression and, in the pancreatic islets of Langerhans, remains up-regulated exclusively in β-cells12. 68Integrative analysis of human epigenomes including histone modification patterns, DNA accessibility, 69 DNA methylation and RNA expression has revealed that disease-and trait-associated genetic variants 70 are enriched in tissue-specific epigenomic marks13. In the context of tumour biology, epigenetic states 71 of cell lineages shape the vulnerabilit...