Mir34a constrains pancreatic carcinogenesis

Several studies have shown that over 70 different microRNAs are aberrantly expressed in pancreatic ductal adenocarcinoma (PDAC), affecting proliferation, apoptosis, metabolism, EMT and metastasis. The most important genetic alterations driving PDAC are a constitutive active mutation of the oncogene Kras and loss of function of the tumour suppressor Tp53 gene. Since the MicroRNA 34a (Mir34a) is a direct target of Tp53 it may critically contribute to the suppression of PDAC. Mir34a is epigenetically silenced in numerous cancers, including PDAC, where Mir34a down-regulation has been associated with poor patient prognosis. To determine whether Mir34a represents a suppressor of PDAC formation we generated an in vivo PDAC-mouse model harbouring pancreas-specific loss of Mir34a (Kras(G12D);Mir34a(Delta/Delta)). Histological analysis of Kras(G12D);Mir34a(Delta/Delta) mice revealed an accelerated formation of pre-neoplastic lesions and a faster PDAC development, compared to Kras(G12D) controls. Here we show that the accelerated phenotype is driven by an early up-regulation of the pro-inflammatory cytokines TNFA and IL6 in normal acinar cells and accompanied by the recruitment of immune cells. Our results imply that Mir34a restrains PDAC development by modulating the immune microenvironment of PDAC, thus defining Mir34a restauration as a potential therapeutic strategy for inhibition of PDAC development

The OTUD6B-LIN28B-MYC axis determines the proliferative state in multiple myeloma

Deubiquitylases (DUBs) are therapeutically amenable components of the ubiquitin machinery that stabilize substrate proteins. Their inhibition can destabilize oncoproteins that may otherwise be undruggable. Here, we screened for DUB vulnerabilities in multiple myeloma, an incurable malignancy with dependency on the ubiquitin proteasome system and identified OTUD6B as an oncogene that drives the G1/S-transition. LIN28B, a suppressor of microRNA biogenesis, is specified as a bona fide cell cycle-specific substrate of OTUD6B. Stabilization of LIN28B drives MYC expression at G1/S, which in turn allows for rapid S-phase entry. Silencing OTUD6B or LIN28B inhibits multiple myeloma outgrowth in vivo and high OTUD6B expression evolves in patients that progress to symptomatic multiple myeloma and results in an adverse outcome of the disease. Thus, we link proteolytic ubiquitylation with post-transcriptional regulation and nominate OTUD6B as a potential mediator of the MGUS-multiple myeloma transition, a central regulator of MYC, and an actionable vulnerability in multiple myeloma and other tumors with an activated OTUD6B-LIN28B axis.</p

The genome-defence gene Tex19.1 suppresses LINE-1 retrotransposons in the placenta and prevents intra-uterine growth retardation in mice

DNA methylation plays an important role in suppressing retrotransposon activity in mammalian genomes, yet there are stages of mammalian development where global hypomethylation puts the genome at risk of retrotransposition-mediated genetic instability. Hypomethylated primordial germ cells appear to limit this risk by expressing a cohort of retrotransposon-suppressing genome-defence genes whose silencing depends on promoter DNA methylation. Here, we investigate whether similar mechanisms operate in hypomethylated trophectoderm-derived components of the mammalian placenta to couple expression of genome-defence genes to the potential for retrotransposon activity. We show that the hypomethylated state of the mouse placenta results in activation of only one of the hypomethylation-sensitive germline genome-defence genes: Tex19.1. Tex19.1 appears to play an important role in placenta function as Tex19.1(−/−) mouse embryos exhibit intra-uterine growth retardation and have small placentas due to a reduction in the number of spongiotrophoblast, glycogen trophoblast and sinusoidal trophoblast giant cells. Furthermore, we show that retrotransposon mRNAs are derepressed in Tex19.1(−/−) placentas and that protein encoded by the LINE-1 retrotransposon is upregulated in hypomethylated trophectoderm-derived cells that normally express Tex19.1. This study suggests that post-transcriptional genome-defence mechanisms are operating in the placenta to protect the hypomethylated cells in this tissue from retrotransposons and suggests that imbalances between retrotransposon activity and genome-defence mechanisms could contribute to placenta dysfunction and disease

Multiplexed pancreatic genome engineering and cancer induction by transfection-based CRISPR/Cas9 delivery in mice

Mouse transgenesis has provided fundamental insights into pancreatic cancer, but is limited by the long duration of allele/model generation. Here we show transfection-based multiplexed delivery of CRISPR/Cas9 to the pancreas of adult mice, allowing simultaneous editing of multiple gene sets in individual cells. We use the method to induce pancreatic cancer and exploit CRISPR/Cas9 mutational signatures for phylogenetic tracking of metastatic disease. Our results demonstrate that CRISPR/Cas9-multiplexing enables key applications, such as combinatorial gene-network analysis, in vivo synthetic lethality screening and chromosome engineering. Negative-selection screening in the pancreas using multiplexed-CRISPR/Cas9 confirms the vulnerability of pancreatic cells to Brca2-inactivation in a Kras-mutant context. We also demonstrate modelling of chromosomal deletions and targeted somatic engineering of inter-chromosomal translocations, offering multifaceted opportunities to study complex structural variation, a hallmark of pancreatic cancer. The low-frequency mosaic pattern of transfection-based CRISPR/Cas9 delivery faithfully recapitulates the stochastic nature of human tumorigenesis, supporting wide applicability for biological/preclinical research

Multiplexed pancreatic genome engineering and cancer induction by transfection-based CRISPR/Cas9 delivery in mice.

Mouse transgenesis has provided fundamental insights into pancreatic cancer, but is limited by the long duration of allele/model generation. Here we show transfection-based multiplexed delivery of CRISPR/Cas9 to the pancreas of adult mice, allowing simultaneous editing of multiple gene sets in individual cells. We use the method to induce pancreatic cancer and exploit CRISPR/Cas9 mutational signatures for phylogenetic tracking of metastatic disease. Our results demonstrate that CRISPR/Cas9-multiplexing enables key applications, such as combinatorial gene-network analysis, in vivo synthetic lethality screening and chromosome engineering. Negative-selection screening in the pancreas using multiplexed-CRISPR/Cas9 confirms the vulnerability of pancreatic cells to Brca2-inactivation in a Kras-mutant context. We also demonstrate modelling of chromosomal deletions and targeted somatic engineering of inter-chromosomal translocations, offering multifaceted opportunities to study complex structural variation, a hallmark of pancreatic cancer. The low-frequency mosaic pattern of transfection-based CRISPR/Cas9 delivery faithfully recapitulates the stochastic nature of human tumorigenesis, supporting wide applicability for biological/preclinical research

Intestinal B cells license metabolic T-cell activation in NASH microbiota/antigen-independently and contribute to fibrosis by IgA-FcR signalling

BACKGROUND & AIMS: The progression of non-alcoholic steatohepatitis (NASH) to fibrosis and hepatocellular carcinoma (HCC) is aggravated by auto-aggressive T cells. The gut-liver axis contributes to NASH, but the mechanisms involved and the consequences for NASH-induced fibrosis and liver cancer remain unknown. We investigated the role of gastrointestinal B cells in the development of NASH, fibrosis and NASH-induced HCC. METHODS: C57BL/6J wild-type (WT), B cell-deficient and different immunoglobulin-deficient or transgenic mice were fed distinct NASH-inducing diets or standard chow for 6 or 12 months, whereafter NASH, fibrosis, and NASH-induced HCC were assessed and analysed. Specific pathogen-free/germ-free WT and μMT mice (containing B cells only in the gastrointestinal tract) were fed a choline-deficient high-fat diet, and treated with an anti-CD20 antibody, whereafter NASH and fibrosis were assessed. Tissue biopsy samples from patients with simple steatosis, NASH and cirrhosis were analysed to correlate the secretion of immunoglobulins to clinicopathological features. Flow cytometry, immunohistochemistry and single-cell RNA-sequencing analysis were performed in liver and gastrointestinal tissue to characterise immune cells in mice and humans. RESULTS: Activated intestinal B cells were increased in mouse and human NASH samples and licensed metabolic T-cell activation to induce NASH independently of antigen specificity and gut microbiota. Genetic or therapeutic depletion of systemic or gastrointestinal B cells prevented or reverted NASH and liver fibrosis. IgA secretion was necessary for fibrosis induction by activating CD11b+CCR2+F4/80+CD11c-FCGR1+ hepatic myeloid cells through an IgA-FcR signalling axis. Similarly, patients with NASH had increased numbers of activated intestinal B cells; additionally, we observed a positive correlation between IgA levels and activated FcRg+ hepatic myeloid cells, as well the extent of liver fibrosis. CONCLUSIONS: Intestinal B cells and the IgA-FcR signalling axis represent potential therapeutic targets for the treatment of NASH. IMPACT AND IMPLICATIONS: There is currently no effective treatment for non-alcoholic steatohepatitis (NASH), which is associated with a substantial healthcare burden and is a growing risk factor for hepatocellular carcinoma (HCC). We have previously shown that NASH is an auto-aggressive condition aggravated, amongst others, by T cells. Therefore, we hypothesized that B cells might have a role in disease induction and progression. Our present work highlights that B cells have a dual role in NASH pathogenesis, being implicated in the activation of auto-aggressive T cells and the development of fibrosis via activation of monocyte-derived macrophages by secreted immunoglobulins (e.g., IgA). Furthermore, we show that the absence of B cells prevented HCC development. B cell-intrinsic signalling pathways, secreted immunoglobulins, and interactions of B cells with other immune cells are potential targets for combinatorial NASH therapies against inflammation and fibrosis

MondoA drives malignancy in B-ALL through enhanced adaptation to metabolic stress.

peer reviewedCancer cells are in most instances characterized by rapid proliferation and uncontrolled cell division. Hence, they must adapt to proliferation-induced metabolic stress through intrinsic or acquired antimetabolic stress responses to maintain homeostasis and survival. One mechanism to achieve this is reprogramming gene expression in a metabolism-dependent manner. MondoA (also known as Myc-associated factor X-like protein X-interacting protein [MLXIP]), a member of the MYC interactome, has been described as an example of such a metabolic sensor. However, the role of MondoA in malignancy is not fully understood and the underlying mechanism in metabolic responses remains elusive. By assessing patient data sets, we found that MondoA overexpression is associated with worse survival in pediatric common acute lymphoblastic leukemia (ALL; B-precursor ALL [B-ALL]). Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and RNA-interference approaches, we observed that MondoA depletion reduces the transformational capacity of B-ALL cells in vitro and dramatically inhibits malignant potential in an in vivo mouse model. Interestingly, reduced expression of MondoA in patient data sets correlated with enrichment in metabolic pathways. The loss of MondoA correlated with increased tricarboxylic acid cycle activity. Mechanistically, MondoA senses metabolic stress in B-ALL cells by restricting oxidative phosphorylation through reduced pyruvate dehydrogenase activity. Glutamine starvation conditions greatly enhance this effect and highlight the inability to mitigate metabolic stress upon loss of MondoA in B-ALL. Our findings give novel insight into the function of MondoA in pediatric B-ALL and support the notion that MondoA inhibition in this entity offers a therapeutic opportunity and should be further explored

A liver immune rheostat regulates CD8 T cell immunity in chronic HBV infection

Chronic hepatitis B virus (HBV) infection affects 300 million patients worldwide1,2, in whom virus-specific CD8 T cells by still ill-defined mechanisms lose their function and cannot eliminate HBV-infected hepatocytes3–7. Here we demonstrate that a liver immune rheostat renders virus-specific CD8 T cells refractory to activation and leads to their loss of effector functions. In preclinical models of persistent infection with hepatotropic viruses such as HBV, dysfunctional virus-specific CXCR6+ CD8 T cells accumulated in the liver and, as a characteristic hallmark, showed enhanced transcriptional activity of cAMP-responsive element modulator (CREM) distinct from T cell exhaustion. In patients with chronic hepatitis B, circulating and intrahepatic HBV-specific CXCR6+ CD8 T cells with enhanced CREM expression and transcriptional activity were detected at a frequency of 12–22% of HBV-specific CD8 T cells. Knocking out the inhibitory CREM/ICER isoform in T cells, however, failed to rescue T cell immunity. This indicates that CREM activity was a consequence, rather than the cause, of loss in T cell function, further supported by the observation of enhanced phosphorylation of protein kinase A (PKA) which is upstream of CREM. Indeed, we found that enhanced cAMP–PKA-signalling from increased T cell adenylyl cyclase activity augmented CREM activity and curbed T cell activation and effector function in persistent hepatic infection. Mechanistically, CD8 T cells recognizing their antigen on hepatocytes established close and extensive contact with liver sinusoidal endothelial cells, thereby enhancing adenylyl cyclase–cAMP–PKA signalling in T cells. In these hepatic CD8 T cells, which recognize their antigen on hepatocytes, phosphorylation of key signalling kinases of the T cell receptor signalling pathway was impaired, which rendered them refractory to activation. Thus, close contact with liver sinusoidal endothelial cells curbs the activation and effector function of HBV-specific CD8 T cells that target hepatocytes expressing viral antigens by means of the adenylyl cyclase–cAMP–PKA axis in an immune rheostat-like fashion.</p

Deletion of the Pluripotency-Associated Tex19.1 Gene Causes Activation of Endogenous Retroviruses and Defective Spermatogenesis in Mice

As genetic information is transmitted through successive generations, it passes between pluripotent cells in the early embryo and germ cells in the developing foetus and adult animal. Tex19.1 encodes a protein of unknown function, whose expression is restricted to germ cells and pluripotent cells. During male spermatogenesis, Tex19.1 expression is highest in mitotic spermatogonia and diminishes as these cells differentiate and progress through meiosis. In pluripotent stem cells, Tex19.1 expression is also downregulated upon differentiation. However, it is not clear whether Tex19.1 has an essential function in germ cells or pluripotent stem cells, or what that function might be. To analyse the potential role of Tex19.1 in pluripotency or germ cell function we have generated Tex19.1−/− knockout mice and analysed the Tex19.1−/− mutant phenotype. Adult Tex19.1−/− knockout males exhibit impaired spermatogenesis. Immunostaining and histological analysis revealed defects in meiotic chromosome synapsis, the persistence of DNA double-strand breaks during meiosis, and a loss of post-meiotic germ cells in the testis. Furthermore, expression of a class of endogenous retroviruses is upregulated during meiosis in the Tex19.1−/− testes. Increased transposition of endogenous retroviruses in the germline of Tex19.1−/− mutant mice, and the concomitant increase in DNA damage, may be sufficient to disrupt the normal processes of recombination and chromosome synapsis during meiosis and cause defects in spermatogenesis. Our results suggest that Tex19.1 is part of a specialised mechanism that operates in the germline to repress transposable genetic elements and maintain genomic stability through successive generations