A metabolic dependency of EBV can be targeted to hinder B cell transformation.

Following infection of B cells, Epstein Barr virus (EBV) engages host pathways that mediate cell proliferation and transformation, contributing to the propensity of the virus to drive immune dysregulation and lymphomagenesis. We found that the EBV protein EBNA2 initiates NAD de novo biosynthesis by driving expression of the metabolic enzyme IDO1 in infected B cells. Virus-enforced NAD production sustained mitochondrial complex I activity, to match ATP-production with bioenergetic requirements of proliferation and transformation. In transplant patients, IDO1 expression in EBV-infected B cells, and a serum signature of increased IDO1 activity, preceded development of lymphoma. In humanized mice infected with EBV, IDO1 inhibition reduced both viremia and lymphomagenesis. Virus-orchestrated NAD biosynthesis is, thus, a druggable metabolic vulnerability of EBV-driven B cell transformation-opening therapeutic possibilities for EBV-related diseases.

CaMKIIß deregulation contributes to neuromuscular junction destabilization in Myotonic Dystrophy type I.

BACKGROUND: Myotonic Dystrophy type I (DM1) is the most common muscular dystrophy in adults. Previous reports have highlighted that neuromuscular junctions (NMJs) deteriorate in skeletal muscle from DM1 patients and mouse models thereof. However, the underlying pathomechanisms and their contribution to muscle dysfunction remain unknown. METHODS: We compared changes in NMJs and activity-dependent signalling pathways in HSALR and Mbnl1ΔE3/ΔE3 mice, two established mouse models of DM1. RESULTS: Muscle from DM1 mouse models showed major deregulation of calcium/calmodulin-dependent protein kinases II (CaMKIIs), which are key activity sensors regulating synaptic gene expression and acetylcholine receptor (AChR) recycling at the NMJ. Both mouse models exhibited increased fragmentation of the endplate, which preceded muscle degeneration. Endplate fragmentation was not accompanied by changes in AChR turnover at the NMJ. However, the expression of synaptic genes was up-regulated in mutant innervated muscle, together with an abnormal accumulation of histone deacetylase 4 (HDAC4), a known target of CaMKII. Interestingly, denervation-induced increase in synaptic gene expression and AChR turnover was hampered in DM1 muscle. Importantly, CaMKIIß/ßM overexpression normalized endplate fragmentation and synaptic gene expression in innervated Mbnl1ΔE3/ΔE3 muscle, but it did not restore denervation-induced synaptic gene up-regulation. CONCLUSIONS: Our results indicate that CaMKIIß-dependent and -independent mechanisms perturb synaptic gene regulation and muscle response to denervation in DM1 mouse models. Changes in these signalling pathways may contribute to NMJ destabilization and muscle dysfunction in DM1 patients.

Nicotinamide N-methyltransferase sustains a core epigenetic program that promotes metastatic colonization in breast cancer.

Metastatic colonization of distant organs accounts for over 90% of deaths related to solid cancers, yet the molecular determinants of metastasis remain poorly understood. Here, we unveil a mechanism of colonization in the aggressive basal-like subtype of breast cancer that is driven by the NAD+ metabolic enzyme nicotinamide N-methyltransferase (NNMT). We demonstrate that NNMT imprints a basal genetic program into cancer cells, enhancing their plasticity. In line, NNMT expression is associated with poor clinical outcomes in patients with breast cancer. Accordingly, ablation of NNMT dramatically suppresses metastasis formation in pre-clinical mouse models. Mechanistically, NNMT depletion results in a methyl overflow that increases histone H3K9 trimethylation (H3K9me3) and DNA methylation at the promoters of PR/SET Domain-5 (PRDM5) and extracellular matrix-related genes. PRDM5 emerged in this study as a pro-metastatic gene acting via induction of cancer-cell intrinsic transcription of collagens. Depletion of PRDM5 in tumor cells decreases COL1A1 deposition and impairs metastatic colonization of the lungs. These findings reveal a critical activity of the NNMT-PRDM5-COL1A1 axis for cancer cell plasticity and metastasis in basal-like breast cancer.

The NFIA-ETO2 fusion blocks erythroid maturation and induces pure erythroid leukemia in cooperation with mutant TP53.

The NFIA-ETO2 fusion is the product of a t(1;16)(p31;q24) chromosomal translocation, so far, exclusively found in pediatric patients with pure erythroid leukemia (PEL). To address the role for the pathogenesis of the disease, we facilitated the expression of the NFIA-ETO2 fusion in murine erythroblasts (EBs). We observed that NFIA-ETO2 significantly increased proliferation and impaired erythroid differentiation of murine erythroleukemia cells and of primary fetal liver-derived EBs. However, NFIA-ETO2-expressing EBs acquired neither aberrant in vitro clonogenic activity nor disease-inducing potential upon transplantation into irradiated syngenic mice. In contrast, in the presence of 1 of the most prevalent erythroleukemia-associated mutations, TP53R248Q, expression of NFIA-ETO2 resulted in aberrant clonogenic activity and induced a fully penetrant transplantable PEL-like disease in mice. Molecular studies support that NFIA-ETO2 interferes with erythroid differentiation by preferentially binding and repressing erythroid genes that contain NFI binding sites and/or are decorated by ETO2, resulting in a activity shift from GATA- to ETS-motif-containing target genes. In contrast, TP53R248Q does not affect erythroid differentiation but provides self-renewal and survival potential, mostly via downregulation of known TP53 targets. Collectively, our work indicates that NFIA-ETO2 initiates PEL by suppressing gene expression programs of terminal erythroid differentiation and cooperates with TP53 mutation to induce erythroleukemia.

pDC-like cells are pre-DC2 and require KLF4 to control homeostatic CD4 T cells.

Plasmacytoid dendritic cells (pDCs) have been shown to play an important role during immune responses, ranging from initial viral control through the production of type I interferons to antigen presentation. However, recent studies uncovered unexpected heterogeneity among pDCs. We identified a previously uncharacterized immune subset, referred to as pDC-like cells, that not only resembles pDCs but also shares conventional DC (cDC) features. We show that this subset is a circulating precursor distinct from common DC progenitors, with prominent cDC2 potential. Our findings from human CD2-iCre and CD300c-iCre lineage tracing mouse models suggest that a substantial fraction of cDC2s originates from pDC-like cells, which can therefore be referred to as pre-DC2. This precursor subset responds to homeostatic cytokines, such as macrophage colony stimulating factor, by expanding and differentiating into cDC2 that efficiently prime T helper 17 (TH17) cells. Development of pre-DC2 into CX3CR1+ ESAM- cDC2b but not CX3CR1- ESAM+ cDC2a requires the transcription factor KLF4. Last, we show that, under homeostatic conditions, this developmental pathway regulates the immune threshold at barrier sites by controlling the pool of TH17 cells within skin-draining lymph nodes.

Interferon-γ resistance and immune evasion in glioma develop via Notch-regulated co-evolution of malignant and immune cells.

Immune surveillance is critical to prevent tumorigenesis. Gliomas evade immune attack, but the underlying mechanisms remain poorly understood. We show that glioma cells can sustain growth independent of immune system constraint by reducing Notch signaling. Loss of Notch activity in a mouse model of glioma impairs MHC-I and cytokine expression and curtails the recruitment of anti-tumor immune cell populations in favor of immunosuppressive tumor-associated microglia/macrophages (TAMs). Depletion of T cells simulates Notch inhibition and facilitates tumor initiation. Furthermore, Notch-depleted glioma cells acquire resistance to interferon-γ and TAMs re-educating therapy. Decreased interferon response and cytokine expression by human and mouse glioma cells correlate with low Notch activity. These effects are paralleled by upregulation of oncogenes and downregulation of quiescence genes. Hence, suppression of Notch signaling enables gliomas to evade immune surveillance and increases aggressiveness. Our findings provide insights into how brain tumor cells shape their microenvironment to evade immune niche control.

Siglec Receptors Modulate Dendritic Cell Activation and Antigen Presentation to T Cells in Cancer.

Interactions between sialylated glycans and sialic acid-binding immunoglobulin-like lectin (Siglec) receptors have been recently described as potential new immune checkpoint that can be targeted to improve anticancer immunity. Myeloid cells have been reported to express a wide range of different Siglecs; however, their expression and functions on cancer-associated dendritic cells (DCs) were not fully characterized. We found that classical conventional DCs (cDCs) from cancer patient samples have a high expression of several inhibitory Siglecs including Siglec-7, Siglec-9, and Siglec-10. In subcutaneous murine tumor models, we also found an upregulation of the inhibitory Siglec-E receptor on cancer-associated cDCs. DC lines and bone marrow-derived DCs (BMDCs) with expression of these inhibitory Siglecs showed impaired maturation states on transcriptome and protein level. Furthermore, ablation of these inhibitory Siglecs from DCs enhanced their capability to prime antigen-specific T cells and induce proliferation. Our work provides a deeper understanding of the influence of inhibitory Siglecs on DCs and reveals a potential new target to improve cancer immunotherapy.

Stimulatory MAIT cell antigens reach the circulation and are efficiently metabolised and presented by human liver cells.

OBJECTIVE: Mucosal-associated invariant T (MAIT) cells are the most abundant T cells in human liver. They respond to bacterial metabolites presented by major histocompatibility complex-like molecule MR1. MAIT cells exert regulatory and antimicrobial functions and are implicated in liver fibrogenesis. It is not well understood which liver cells function as antigen (Ag)-presenting cells for MAIT cells, and under which conditions stimulatory Ags reach the circulation. DESIGN: We used different types of primary human liver cells in Ag-presentation assays to blood-derived and liver-derived MAIT cells. We assessed MAIT cell stimulatory potential of serum from healthy subjects and patients with portal hypertension undergoing transjugular intrahepatic portosystemic shunt stent, and patients with inflammatory bowel disease (IBD). RESULTS: MAIT cells were dispersed throughout healthy human liver and all tested liver cell types stimulated MAIT cells, hepatocytes being most efficient. MAIT cell activation by liver cells occurred in response to bacterial lysate and pure Ag, and was prevented by non-activating MR1 ligands. Serum derived from peripheral and portal blood, and from patients with IBD stimulated MAIT cells in MR1-dependent manner. CONCLUSION: Our findings reveal previously unrecognised roles of liver cells in Ag metabolism and activation of MAIT cells, repression of which creates an opportunity to design antifibrotic therapies. The presence of MAIT cell stimulatory Ags in serum rationalises the observed activated MAIT cell phenotype in liver. Increased serum levels of gut-derived MAIT cell stimulatory ligands in patients with impaired intestinal barrier function indicate that intrahepatic Ag-presentation may represent an important step in the development of liver disease.

Distinct contributions of partial and full EMT to breast cancer malignancy.

Epithelial-mesenchymal transition (EMT) is a transient, reversible process of cell de-differentiation where cancer cells transit between various stages of an EMT continuum, including epithelial, partial EMT, and mesenchymal cell states. We have employed Tamoxifen-inducible dual recombinase lineage tracing systems combined with live imaging and 5-cell RNA sequencing to track cancer cells undergoing partial or full EMT in the MMTV-PyMT mouse model of metastatic breast cancer. In primary tumors, cancer cells infrequently undergo EMT and mostly transition between epithelial and partial EMT states but rarely reach full EMT. Cells undergoing partial EMT contribute to lung metastasis and chemoresistance, whereas full EMT cells mostly retain a mesenchymal phenotype and fail to colonize the lungs. However, full EMT cancer cells are enriched in recurrent tumors upon chemotherapy. Hence, cancer cells in various stages of the EMT continuum differentially contribute to hallmarks of breast cancer malignancy, such as tumor invasion, metastasis, and chemoresistance.

The long non-coding RNA ET-20 mediates EMT by impairing desmosomes in breast cancer cells.

The vast majority of breast cancer-associated deaths are due to metastatic spread of cancer cells, a process aided by epithelial-to-mesenchymal transition (EMT). Mounting evidence has indicated that long non-coding RNAs (lncRNAs) also contribute to tumor progression. We report the identification of 114 novel lncRNAs that change their expression during TGFß-induced EMT in murine breast cancer cells (referred to as EMT-associated transcripts; ETs). Of these, the ET-20 gene localizes in antisense orientation within the tenascin C (Tnc) gene locus. TNC is an extracellular matrix protein that is critical for EMT and metastasis formation. Both ET-20 and Tnc are regulated by the EMT master transcription factor Sox4. Notably, ablation of ET-20 lncRNA effectively blocks Tnc expression and with it EMT. Mechanistically, ET-20 interacts with desmosomal proteins, thereby impairing epithelial desmosomes and promoting EMT. A short transcript variant of ET-20 is shown to be upregulated in invasive human breast cancer cell lines, where it also promotes EMT. Targeting ET-20 appears to be a therapeutically attractive lead to restrain EMT and breast cancer metastasis in addition to its potential utility as a biomarker for invasive breast cancer.

Nuclear interacting SET domain protein 1 inactivation impairs GATA1-regulated erythroid differentiation and causes erythroleukemia.

The nuclear receptor binding SET domain protein 1 (NSD1) is recurrently mutated in human cancers including acute leukemia. We show that NSD1 knockdown alters erythroid clonogenic growth of human CD34+ hematopoietic cells. Ablation of Nsd1 in the hematopoietic system of mice induces a transplantable erythroleukemia. In vitro differentiation of Nsd1-/- erythroblasts is majorly impaired despite abundant expression of GATA1, the transcriptional master regulator of erythropoiesis, and associated with an impaired activation of GATA1-induced targets. Retroviral expression of wildtype NSD1, but not a catalytically-inactive NSD1N1918Q SET-domain mutant induces terminal maturation of Nsd1-/- erythroblasts. Despite similar GATA1 protein levels, exogenous NSD1 but not NSDN1918Q significantly increases the occupancy of GATA1 at target genes and their expression. Notably, exogenous NSD1 reduces the association of GATA1 with the co-repressor SKI, and knockdown of SKI induces differentiation of Nsd1-/- erythroblasts. Collectively, we identify the NSD1 methyltransferase as a regulator of GATA1-controlled erythroid differentiation and leukemogenesis.

A dual role of Irf1 in maintaining epithelial identity but also enabling EMT and metastasis formation of breast cancer cells.

An epithelial to mesenchymal transition (EMT) is an embryonic dedifferentiation program which is aberrantly activated in cancer cells to acquire cellular plasticity. This plasticity increases the ability of breast cancer cells to invade into surrounding tissue, to seed metastasis at distant sites and to resist to chemotherapy. In this study, we have observed a higher expression of interferon-related factors in basal-like and claudin-low subtypes of breast cancer in patients, known to be associated with EMT. Notably, Irf1 exerts essential functions during the EMT process, yet it is also required for the maintenance of an epithelial differentiation status of mammary gland epithelial cells: RNAi-mediated ablation of Irf1 in mammary epithelial cells results in the expression of mesenchymal factors and Smad transcriptional activity. Conversely, ablation of Irf1 during TGFß-induced EMT prevents a mesenchymal transition and stabilizes the expression of E-cadherin. In the basal-like murine breast cancer cell line 4T1, RNAi-mediated ablation of Irf1 reduces colony formation and cell migration in vitro and shedding of circulating tumor cells and metastasis formation in vivo. This context-dependent dual role of Irf1 in the regulation of epithelial-mesenchymal plasticity provides important new insights into the functional contribution and therapeutic potential of interferon-regulated factors in breast cancer.

Microbiota-induced tissue signals regulate ILC3-mediated antigen presentation.

Although group 3 innate lymphoid cells (ILC3s) are efficient inducers of T cell responses in the spleen, they fail to induce CD4+ T cell proliferation in the gut. The signals regulating ILC3-T cell responses remain unknown. Here, we show that transcripts associated with MHC II antigen presentation are down-modulated in intestinal natural cytotoxicity receptor (NCR)- ILC3s. Further data implicate microbiota-induced IL-23 as a crucial signal for reversible silencing of MHC II in ILC3s, thereby reducing the capacity of ILC3s to present antigen to T cells in the intestinal mucosa. Moreover, IL-23-mediated MHC II suppression is dependent on mTORC1 and STAT3 phosphorylation in NCR- ILC3s. By contrast, splenic interferon-γ induces MHC II expression and CD4+ T cell stimulation by NCR- ILC3s. Our results thus identify biological circuits for tissue-specific regulation of ILC3-dependent T cell responses. These pathways may have implications for inducing or silencing T cell responses in human diseases.

SDHA gain-of-function engages inflammatory mitochondrial retrograde signaling via KEAP1-Nrf2.

Whether screening the metabolic activity of immune cells facilitates discovery of molecular pathology remains unknown. Here we prospectively screened the extracellular acidification rate as a measure of glycolysis and the oxygen consumption rate as a measure of mitochondrial respiration in B cells from patients with primary antibody deficiency. The highest oxygen consumption rate values were detected in three study participants with persistent polyclonal B cell lymphocytosis (PPBL). Exome sequencing identified germline mutations in SDHA, which encodes succinate dehydrogenase subunit A, in all three patients with PPBL. SDHA gain-of-function led to an accumulation of fumarate in PPBL B cells, which engaged the KEAP1-Nrf2 system to drive the transcription of genes encoding inflammatory cytokines. In a single patient trial, blocking the activity of the cytokine interleukin-6 in vivo prevented systemic inflammation and ameliorated clinical disease. Overall, our study has identified pathological mitochondrial retrograde signaling as a disease modifier in primary antibody deficiency.

Molecular signatures identify immature mesenchymal progenitors in early mouse limb buds that respond differentially to morphogen signaling.

The key molecular interactions governing vertebrate limb bud development are a paradigm for studying the mechanisms controlling progenitor cell proliferation and specification during vertebrate organogenesis. However, little is known about the cellular heterogeneity of the mesenchymal progenitors in early limb buds that ultimately contribute to the chondrogenic condensations prefiguring the skeleton. We combined flow cytometric and transcriptome analyses to identify the molecular signatures of several distinct mesenchymal progenitor cell populations present in early mouse forelimb buds. In particular, jagged 1 (JAG1)-positive cells located in the posterior-distal mesenchyme were identified as the most immature limb bud mesenchymal progenitors (LMPs), which crucially depend on SHH and FGF signaling in culture. The analysis of gremlin 1 (Grem1)-deficient forelimb buds showed that JAG1-expressing LMPs are protected from apoptosis by GREM1-mediated BMP antagonism. At the same stage, the osteo-chondrogenic progenitors (OCPs) located in the core mesenchyme are already actively responding to BMP signaling. This analysis sheds light on the cellular heterogeneity of the early mouse limb bud mesenchyme and on the distinct response of LMPs and OCPs to morphogen signaling.

A Hierarchical Regulatory Landscape during the Multiple Stages of EMT.

Epithelial-mesenchymal transition (EMT) enables cells to gain migratory and invasive features underlined by major transcriptional and epigenetic reprogramming. However, most studies have focused on the endpoints of the EMT process, and the epistatic hierarchy of the transcriptional networks underlying EMT has remained elusive. We have used a siRNA-based, functional high-content microscopy screen to identify 46 (co)transcription factors ((co)TFs) and 13 miRNAs critically required for EMT in normal murine mammary gland (NMuMG) cells. We compared dynamic gene expression during EMT kinetics and used functional perturbation of critical (co)TFs and miRNAs to identify groups and networks of EMT genes. Computational analysis as well as functional validation experiments revealed interaction networks between TFs and miRNAs and delineated the hierarchical and functional interactions of multiple EMT regulatory networks in NMuMG cells.

Gain Fat-Lose Metastasis: Converting Invasive Breast Cancer Cells into Adipocytes Inhibits Cancer Metastasis.

Cancer cell plasticity facilitates the development of therapy resistance and malignant progression. De-differentiation processes, such as an epithelial-mesenchymal transition (EMT), are known to enhance cellular plasticity. Here, we demonstrate that cancer cell plasticity can be exploited therapeutically by forcing the trans-differentiation of EMT-derived breast cancer cells into post-mitotic and functional adipocytes. Delineation of the molecular pathways underlying such trans-differentiation has motivated a combination therapy with MEK inhibitors and the anti-diabetic drug Rosiglitazone in various mouse models of murine and human breast cancer in vivo. This combination therapy provokes the conversion of invasive and disseminating cancer cells into post-mitotic adipocytes leading to the repression of primary tumor invasion and metastasis formation.

The FAK inhibitor BI 853520 exerts anti-tumor effects in breast cancer.

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that regulates a plethora of downstream signaling pathways essential for cell migration, proliferation and death, processes that are exploited by cancer cells during malignant progression. These well-established tumorigenic activities, together with its high expression and activity in different cancer types, highlight FAK as an attractive target for cancer therapy. We have assessed and characterized the therapeutic potential and the biological effects of BI 853520, a novel small chemical inhibitor of FAK, in several preclinical mouse models of breast cancer. Treatment with BI 853520 elicits a significant reduction in primary tumor growth caused by an anti-proliferative activity by BI 853520. In contrast, BI 853520 exerts effects with varying degrees of robustness on the different stages of the metastatic cascade. Together, the data demonstrate that the repression of FAK activity by the specific FAK inhibitor BI 853520 offers a promising anti-proliferative approach for cancer therapy.

Distinct progenitor lineages contribute to the heterogeneity of plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) are an immune subset devoted to the production of high amounts of type 1 interferons in response to viral infections. Whereas conventional dendritic cells (cDCs) originate mostly from a common dendritic cell progenitor (CDP), pDCs have been shown to develop from both CDPs and common lymphoid progenitors. Here, we found that pDCs developed predominantly from IL-7R+ lymphoid progenitor cells. Expression of SiglecH and Ly6D defined pDC lineage commitment along the lymphoid branch. Transcriptional characterization of SiglecH+Ly6D+ precursors indicated that pDC development requires high expression of the transcription factor IRF8, whereas pDC identity relies on TCF4. RNA sequencing of IL-7R+ lymphoid and CDP-derived pDCs mirrored the heterogeneity of mature pDCs observed in single-cell analysis. Both mature pDC subsets are able to secrete type 1 interferons, but only myeloid-derived pDCs share with cDCs their ability to process and present antigen.

Strong homeostatic TCR signals induce formation of self-tolerant virtual memory CD8 T cells.

Virtual memory T cells are foreign antigen-inexperienced T cells that have acquired memory-like phenotype and constitute 10-20% of all peripheral CD8+ T cells in mice. Their origin, biological roles, and relationship to naïve and foreign antigen-experienced memory T cells are incompletely understood. By analyzing T-cell receptor repertoires and using retrogenic monoclonal T-cell populations, we demonstrate that the virtual memory T-cell formation is a so far unappreciated cell fate decision checkpoint. We describe two molecular mechanisms driving the formation of virtual memory T cells. First, virtual memory T cells originate exclusively from strongly self-reactive T cells. Second, the stoichiometry of the CD8 interaction with Lck regulates the size of the virtual memory T-cell compartment via modulating the self-reactivity of individual T cells. Although virtual memory T cells descend from the highly self-reactive clones and acquire a partial memory program, they are not more potent in inducing experimental autoimmune diabetes than naïve T cells. These data underline the importance of the variable level of self-reactivity in polyclonal T cells for the generation of functional T-cell diversity.