Publications by authors named "Thomas Engleitner"

37 Publications

Whole Exome Sequencing of Biliary Tubulopapillary Neoplasms Reveals Common Mutations in Chromatin Remodeling Genes.

Cancers (Basel) 2021 Jun 1;13(11). Epub 2021 Jun 1.

Institute of Pathology, School of Medicine, Technische Universität München, 81675 Munich, Germany.

The molecular carcinogenesis of intraductal tubulopapillary neoplasms (ITPN), recently described as rare neoplasms in the pancreato-biliary tract with a favorable prognosis despite a high incidence of associated pancreato-biliary adenocarcinoma, is still poorly understood. To identify driver genes, chromosomal gains and losses, mutational signatures, key signaling pathways, and potential therapeutic targets, the molecular profile of 11 biliary and 6 pancreatic ITPNs, associated with invasive adenocarcinoma in 14/17 cases, are studied by whole exome sequencing (WES). The WES of 17 ITPNs reveals common copy number variants (CNVs) broadly distributed across the genome, with recurrent chromosomal deletions primarily in 1p36 and 9p21 affecting the tumor suppressors and , respectively, and gains in 1q affecting the prominent oncogene . The identified somatic nucleotide variants (SNVs) involve few core signaling pathways despite high genetic heterogeneity with diverse mutational spectra: Chromatin remodeling, the cell cycle, and DNA damage/repair. An OncoKB search identifies putative actionable genomic targets in 35% of the cases (6/17), including recurrent missense mutations of the gene in biliary ITPNs (2/11, 18%). Our results show that somatic SNV in classical cancer genes, typically associated with pancreato-biliary carcinogenesis, were absent (, , , and others) to rare ( and , 6%, respectively) in ITPNs. Mutational signature pattern analysis reveals a predominance of an age-related pattern. Our findings highlight that biliary ITPN and classical cholangiocarcinoma display commonalities, in particular mutations in genes of the chromatin remodeling pathway, and appear, therefore, more closely related than pancreatic ITPN and classical pancreatic ductal adenocarcinoma.
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http://dx.doi.org/10.3390/cancers13112742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8198366PMC
June 2021

Targeted PI3K/AKT-hyperactivation induces cell death in chronic lymphocytic leukemia.

Nat Commun 2021 06 10;12(1):3526. Epub 2021 Jun 10.

Institute of Clinical Chemistry and Pathobiochemistry, School of Medicine, Technical University of Munich, Munich, Germany.

Current therapeutic approaches for chronic lymphocytic leukemia (CLL) focus on the suppression of oncogenic kinase signaling. Here, we test the hypothesis that targeted hyperactivation of the phosphatidylinositol-3-phosphate/AKT (PI3K/AKT)-signaling pathway may be leveraged to trigger CLL cell death. Though counterintuitive, our data show that genetic hyperactivation of PI3K/AKT-signaling or blocking the activity of the inhibitory phosphatase SH2-containing-inositol-5'-phosphatase-1 (SHIP1) induces acute cell death in CLL cells. Our mechanistic studies reveal that increased AKT activity upon inhibition of SHIP1 leads to increased mitochondrial respiration and causes excessive accumulation of reactive oxygen species (ROS), resulting in cell death in CLL with immunogenic features. Our results demonstrate that CLL cells critically depend on mechanisms to fine-tune PI3K/AKT activity, allowing sustained proliferation and survival but avoid ROS-induced cell death and suggest transient SHIP1-inhibition as an unexpectedly promising concept for CLL therapy.
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http://dx.doi.org/10.1038/s41467-021-23752-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192787PMC
June 2021

Modeling plasticity and dysplasia of pancreatic ductal organoids derived from human pluripotent stem cells.

Cell Stem Cell 2021 06 28;28(6):1105-1124.e19. Epub 2021 Apr 28.

Institute of Neuroanatomy & Developmental Biology (INDB), Eberhard Karls University Tübingen, Tübingen, Germany.

Personalized in vitro models for dysplasia and carcinogenesis in the pancreas have been constrained by insufficient differentiation of human pluripotent stem cells (hPSCs) into the exocrine pancreatic lineage. Here, we differentiate hPSCs into pancreatic duct-like organoids (PDLOs) with morphological, transcriptional, proteomic, and functional characteristics of human pancreatic ducts, further maturing upon transplantation into mice. PDLOs are generated from hPSCs inducibly expressing oncogenic GNAS, KRAS, or KRAS with genetic covariance of lost CDKN2A and from induced hPSCs derived from a McCune-Albright patient. Each oncogene causes a specific growth, structural, and molecular phenotype in vitro. While transplanted PDLOs with oncogenic KRAS alone form heterogenous dysplastic lesions or cancer, KRAS with CDKN2A loss develop dedifferentiated pancreatic ductal adenocarcinomas. In contrast, transplanted PDLOs with mutant GNAS lead to intraductal papillary mucinous neoplasia-like structures. Conclusively, PDLOs enable in vitro and in vivo studies of pancreatic plasticity, dysplasia, and cancer formation from a genetically defined background.
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http://dx.doi.org/10.1016/j.stem.2021.03.005DOI Listing
June 2021

Anti-inflammatory chemoprevention attenuates the phenotype in a mouse model of esophageal adenocarcinoma.

Carcinogenesis 2021 Apr 20. Epub 2021 Apr 20.

Department of Medicine II, Klinikum rechts der Isar, Technical University Munich (TUM), Ismaninger Str, München, Germany.

Barrett´s Esophagus (BE) is the main known precursor condition of Esophageal Adenocarcinoma (EAC). BE is defined by the presence of metaplasia above the normal squamous columnar junction and has mainly been attributed to gastroesophageal reflux disease (GERD) and chronic reflux esophagitis. Thus, the rising incidence of EAC in the Western world is likely mediated by chronic esophageal inflammation, secondary to GERD in combination with environmental risk factors such as a Western diet and obesity. However, (at present) risk prediction tools and endoscopic surveillance have shown limited effectiveness. Chemoprevention as an adjunctive approach remains an attractive option to reduce the incidence of neoplastic disease. Here, we investigate the feasibility of chemopreventive approaches in BE and EAC via inhibition of inflammatory signaling in a transgenic mouse model of BE and EAC (L2-IL1B mice), with accelerated tumor formation on a high fat diet (HFD). L2-IL1B mice were treated with the IL-1 receptor antagonist Anakinra and the nonsteroidal anti-inflammatory drugs (NSAIDs) aspirin or Sulindac. Interleukin-1b antagonism reduced tumor progression in L2-IL1B mice with or without a HFD, while both NSAIDs were effective chemoprevention agents in the accelerated HFD fed L2-IL1B mouse model. Sulindac treatment also resulted in a marked change in the immune profile of L2-IL-1B mice. In summary, anti-inflammatory treatment of HFD-treated L2-IL1B mice acted protectively on disease progression. These results from a mouse model of BE support results from clinical trials that suggest that anti-inflammatory medication may be effective in the chemoprevention of EAC.
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http://dx.doi.org/10.1093/carcin/bgab032DOI Listing
April 2021

Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV.

Nature 2021 06 12;594(7862):246-252. Epub 2021 Apr 12.

Technical University of Munich, School of Medicine, Institute of Virology, Munich, Germany.

The emergence and global spread of SARS-CoV-2 has resulted in the urgent need for an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several individual omics studies have extended our knowledge of COVID-19 pathophysiology. Integration of such datasets to obtain a holistic view of virus-host interactions and to define the pathogenic properties of SARS-CoV-2 is limited by the heterogeneity of the experimental systems. Here we report a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactomes of both viruses, as well as their influence on the transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon infection with SARS-CoV-2 and SARS-CoV at different levels and enabled identification of distinct and common molecular mechanisms of these closely related coronaviruses. The TGF-β pathway, known for its involvement in tissue fibrosis, was specifically dysregulated by SARS-CoV-2 ORF8 and autophagy was specifically dysregulated by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org ) highlights many hotspots that could be targeted by existing drugs and may be used to guide rational design of virus- and host-directed therapies, which we exemplify by identifying inhibitors of kinases and matrix metalloproteases with potent antiviral effects against SARS-CoV-2.
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http://dx.doi.org/10.1038/s41586-021-03493-4DOI Listing
June 2021

NASH limits anti-tumour surveillance in immunotherapy-treated HCC.

Nature 2021 Apr 24;592(7854):450-456. Epub 2021 Mar 24.

Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle, UK.

Hepatocellular carcinoma (HCC) can have viral or non-viral causes. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need. Here we report the progressive accumulation of exhausted, unconventionally activated CD8PD1 T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8PD1 T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8PD1CXCR6, TOX, and TNF T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8 T cells or TNF neutralization, suggesting that CD8 T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8PD1 T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.
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http://dx.doi.org/10.1038/s41586-021-03362-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8046670PMC
April 2021

Notch2-mediated plasticity between marginal zone and follicular B cells.

Nat Commun 2021 02 17;12(1):1111. Epub 2021 Feb 17.

Research Unit Gene Vectors, Helmholtz Zentrum München GmbH, German Research Center for Environmental Health, München, Germany.

Follicular B (FoB) and marginal zone B (MZB) cells are functionally and spatially distinct mature B cell populations in the spleen, originating from a Notch2-dependent fate decision after splenic influx of immature transitional B cells. In the B cell follicle, a Notch2-signal is provided by DLL-1-expressing fibroblasts. However, it is unclear whether FoB cells, which are in close contact with these DLL-1 expressing fibroblasts, can also differentiate to MZB cells if they receive a Notch2-signal. Here, we show induced Notch2IC-expression in FoB cells re-programs mature FoB cells into bona fide MZB cells as is evident from the surface phenotype, localization, immunological function and transcriptome of these cells. Furthermore, the lineage conversion from FoB to MZB cells occurs in immunocompetent wildtype mice. These findings demonstrate plasticity between mature FoB and MZB cells that can be driven by a singular signaling event, the activation of Notch2.
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http://dx.doi.org/10.1038/s41467-021-21359-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889629PMC
February 2021

Mesenchymal Plasticity Regulated by Prrx1 Drives Aggressive Pancreatic Cancer Biology.

Gastroenterology 2021 01 30;160(1):346-361.e24. Epub 2020 Sep 30.

Klinik und Poliklinik für Innere Medizin II, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany; German Cancer Consortium, Partner Site Munich, Germany. Electronic address:

Background & Aims: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibroblast-rich desmoplastic stroma. Cancer-associated fibroblasts (CAFs) have been shown to display a high degree of interconvertible states including quiescent, inflammatory, and myofibroblastic phenotypes; however, the mechanisms by which this plasticity is achieved are poorly understood. Here, we aim to elucidate the role of CAF plasticity and its impact on PDAC biology.

Methods: To investigate the role of mesenchymal plasticity in PDAC progression, we generated a PDAC mouse model in which CAF plasticity is modulated by genetic depletion of the transcription factor Prrx1. Primary pancreatic fibroblasts from this mouse model were further characterized by functional in vitro assays. To characterize the impact of CAFs on tumor differentiation and response to chemotherapy, various coculture experiments were performed. In vivo, tumors were characterized by morphology, extracellular matrix composition, and tumor dissemination and metastasis.

Results: Our in vivo findings showed that Prrx1-deficient CAFs remain constitutively activated. Importantly, this CAF phenotype determines tumor differentiation and disrupts systemic tumor dissemination. Mechanistically, coculture experiments of tumor organoids and CAFs showed that CAFs shape the epithelial-to-mesenchymal phenotype and confer gemcitabine resistance of PDAC cells induced by CAF-derived hepatocyte growth factor. Furthermore, gene expression analysis showed that patients with pancreatic cancer with high stromal expression of Prrx1 display the squamous, most aggressive, subtype of PDAC.

Conclusions: Here, we define that the Prrx1 transcription factor is critical for tuning CAF activation, allowing a dynamic switch between a dormant and an activated state. This work shows that Prrx1-mediated CAF plasticity has significant impact on PDAC biology and therapeutic resistance.
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http://dx.doi.org/10.1053/j.gastro.2020.09.010DOI Listing
January 2021

Synergistic targeting and resistance to PARP inhibition in DNA damage repair-deficient pancreatic cancer.

Gut 2021 Apr 1;70(4):743-760. Epub 2020 Sep 1.

Department of Internal Medicine I, Ulm University Hospital, Ulm, Germany.

Objective: (ATM) is the most frequently mutated DNA damage response gene, involved in homologous recombination (HR), in pancreatic ductal adenocarcinoma (PDAC).

Design: Combinational synergy screening was performed to endeavour a genotype-tailored targeted therapy.

Results: Synergy was found on inhibition of PARP, ATR and DNA-PKcs (PAD) leading to synthetic lethality in ATM-deficient murine and human PDAC. Mechanistically, PAD-induced PARP trapping, replication fork stalling and mitosis defects leading to P53-mediated apoptosis. Most importantly, chemical inhibition of ATM sensitises human PDAC cells toward PAD with long-term tumour control in vivo. Finally, we anticipated and elucidated PARP inhibitor resistance within the ATM-null background via whole exome sequencing. Arising cells were aneuploid, underwent epithelial-mesenchymal-transition and acquired multidrug resistance (MDR) due to upregulation of drug transporters and a bypass within the DNA repair machinery. These functional observations were mirrored in copy number variations affecting a region on chromosome 5 comprising several of the upregulated MDR genes. Using these findings, we ultimately propose alternative strategies to overcome the resistance.

Conclusion: Analysis of the molecular susceptibilities triggered by ATM deficiency in PDAC allow elaboration of an efficient mutation-specific combinational therapeutic approach that can be also implemented in a genotype-independent manner by ATM inhibition.
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http://dx.doi.org/10.1136/gutjnl-2019-319970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948173PMC
April 2021

Implementing cell-free DNA of pancreatic cancer patient-derived organoids for personalized oncology.

JCI Insight 2020 08 6;5(15). Epub 2020 Aug 6.

Department of Surgery, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.

One of the major challenges in using pancreatic cancer patient-derived organoids (PDOs) in precision oncology is the time from biopsy to functional characterization. This is particularly true for endoscopic ultrasound-guided fine-needle aspiration biopsies, typically resulting in specimens with limited tumor cell yield. Here, we tested conditioned media of individual PDOs for cell-free DNA to detect driver mutations already early on during the expansion process to accelerate the genetic characterization of PDOs as well as subsequent functional testing. Importantly, genetic alterations detected in the PDO supernatant, collected as early as 72 hours after biopsy, recapitulate the mutational profile of the primary tumor, indicating suitability of this approach to subject PDOs to drug testing in a reduced time frame. In addition, we demonstrated that this workflow was practicable, even in patients for whom the amount of tumor material was not sufficient for molecular characterization by established means. Together, our findings demonstrate that generating PDOs from very limited biopsy material permits molecular profiling and drug testing. With our approach, this can be achieved in a rapid and feasible fashion with broad implications in clinical practice.
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http://dx.doi.org/10.1172/jci.insight.137809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7455062PMC
August 2020

Mir34a constrains pancreatic carcinogenesis.

Sci Rep 2020 06 15;10(1):9654. Epub 2020 Jun 15.

Klinik und Poliklinik für Innere Medizin II, Technical University of Munich, Munich, Germany.

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; Mir34a). Histological analysis of Kras; Mir34a mice revealed an accelerated formation of pre-neoplastic lesions and a faster PDAC development, compared to Kras 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.
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http://dx.doi.org/10.1038/s41598-020-66561-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295749PMC
June 2020

c-Rel gain in B cells drives germinal center reactions and autoantibody production.

J Clin Invest 2020 06;130(6):3270-3286

Institute of Experimental Hematology, School of Medicine, Technical University of Munich, Munich, Germany.

Single-nucleotide polymorphisms and locus amplification link the NF-κB transcription factor c-Rel to human autoimmune diseases and B cell lymphomas, respectively. However, the functional consequences of enhanced c-Rel levels remain enigmatic. Here, we overexpressed c-Rel specifically in mouse B cells from BAC-transgenic gene loci and demonstrate that c-Rel protein levels linearly dictated expansion of germinal center B (GCB) cells and isotype-switched plasma cells. c-Rel expression in B cells of otherwise c-Rel-deficient mice fully rescued terminal B cell differentiation, underscoring its critical B cell-intrinsic roles. Unexpectedly, in GCB cells transcription-independent regulation produced the highest c-Rel protein levels among B cell subsets. In c-Rel-overexpressing GCB cells this caused enhanced nuclear translocation, a profoundly altered transcriptional program, and increased proliferation. Finally, we provide a link between c-Rel gain and autoimmunity by showing that c-Rel overexpression in B cells caused autoantibody production and renal immune complex deposition.
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http://dx.doi.org/10.1172/JCI124382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260018PMC
June 2020

Antagonistic activities of CDC14B and CDK1 on USP9X regulate WT1-dependent mitotic transcription and survival.

Nat Commun 2020 03 9;11(1):1268. Epub 2020 Mar 9.

Department of Medicine III, Klinikum rechts der Isar, Technical University of Munich, 81675, Munich, Germany.

Regulation of mitosis secures cellular integrity and its failure critically contributes to the development, maintenance, and treatment resistance of cancer. In yeast, the dual phosphatase Cdc14 controls mitotic progression by antagonizing Cdk1-mediated protein phosphorylation. By contrast, specific mitotic functions of the mammalian Cdc14 orthologue CDC14B have remained largely elusive. Here, we find that CDC14B antagonizes CDK1-mediated activating mitotic phosphorylation of the deubiquitinase USP9X at serine residue 2563, which we show to be essential for USP9X to mediate mitotic survival. Starting from an unbiased proteome-wide screening approach, we specify Wilms' tumor protein 1 (WT1) as the relevant substrate that becomes deubiquitylated and stabilized by serine 2563-phosphorylated USP9X in mitosis. We further demonstrate that WT1 functions as a mitotic transcription factor and specify CXCL8/IL-8 as a target gene of WT1 that conveys mitotic survival. Together, we describe a ubiquitin-dependent signaling pathway that directs a mitosis-specific transcription program to regulate mitotic survival.
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http://dx.doi.org/10.1038/s41467-020-15059-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7063047PMC
March 2020

Novel role for CRK adaptor proteins as essential components of SRC/FAK signaling for epithelial-mesenchymal transition and colorectal cancer aggressiveness.

Int J Cancer 2020 09 16;147(6):1715-1731. Epub 2020 Mar 16.

Department of Surgery, School of Medicine, Klinikum Rechts der Isar, Technical University of Munich, Munich, Germany.

Epithelial-mesenchymal transition (EMT) is a cell plasticity process required for metastasis and chemoresistance of carcinoma cells. We report a crucial role of the signal adaptor proteins CRK and CRKL in promoting EMT and tumor aggressiveness, as well as resistance against chemotherapy in colorectal and pancreatic carcinoma. Genetic loss of either CRKL or CRK partially counteracted EMT in three independent cancer cell lines. Strikingly, complete loss of the CRK family shifted cells strongly toward the epithelial phenotype. Cells exhibited greatly increased E-cadherin and grew as large, densely packed clusters, completely lacked invasiveness and the ability to undergo EMT induced by cytokines or genetic activation of SRC. Furthermore, CRK family-deficiency significantly reduced cell survival, proliferation and chemoresistance, as well as ERK1/2 phosphorylation and c-MYC protein levels. In accordance, MYC-target gene expression was identified as novel hallmark process positively regulated by CRK family proteins. Mechanistically, CRK proteins were identified as pivotal amplifiers of SRC/FAK signaling at focal adhesions, mediated through a novel positive feedback loop depending on RAP1. Expression of the CRK family and the EMT regulator ZEB1 was significantly correlated in samples from colorectal cancer patients, especially in invasive regions. Further, high expression of CRK family genes was significantly associated with reduced survival in locally advanced colorectal cancer, as well as in pan-cancer datasets from the TCGA project. Thus, CRK family adaptor proteins are promising therapeutic targets to counteract EMT, chemoresistance, metastasis formation and minimal residual disease. As proof of concept, CRK family-mediated oncogenic signaling was successfully inhibited by a peptide-based inhibitor.
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http://dx.doi.org/10.1002/ijc.32955DOI Listing
September 2020

Analysis pipelines for cancer genome sequencing in mice.

Nat Protoc 2020 02 6;15(2):266-315. Epub 2020 Jan 6.

Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, Munich, Germany.

Mouse models of human cancer have transformed our ability to link genetics, molecular mechanisms and phenotypes. Both reverse and forward genetics in mice are currently gaining momentum through advances in next-generation sequencing (NGS). Methodologies to analyze sequencing data were, however, developed for humans and hence do not account for species-specific differences in genome structures and experimental setups. Here, we describe standardized computational pipelines specifically tailored to the analysis of mouse genomic data. We present novel tools and workflows for the detection of different alteration types, including single-nucleotide variants (SNVs), small insertions and deletions (indels), copy-number variations (CNVs), loss of heterozygosity (LOH) and complex rearrangements, such as in chromothripsis. Workflows have been extensively validated and cross-compared using multiple methodologies. We also give step-by-step guidance on the execution of individual analysis types, provide advice on data interpretation and make the complete code available online. The protocol takes 2-7 d, depending on the desired analyses.
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http://dx.doi.org/10.1038/s41596-019-0234-7DOI Listing
February 2020

Angiocrine Hepatocyte Growth Factor Signaling Controls Physiological Organ and Body Size and Dynamic Hepatocyte Proliferation to Prevent Liver Damage during Regeneration.

Am J Pathol 2020 02 27;190(2):358-371. Epub 2019 Nov 27.

Department of Surgery, Technical University of Munich, School of Medicine, Klinikum rechts der Isar, Munich, Germany. Electronic address:

Liver sinusoidal endothelial cells (LSECs) control organ functions, metabolism, and development through the secretion of angiokines. LSECs express hepatocyte growth factor (Hgf), which is involved in prenatal development, metabolic homeostasis, and liver regeneration. This study aimed to elucidate the precise contribution of LSEC-derived Hgf in physiological homeostasis and liver regeneration. Stab2-iCre;Hgf (Hgf) mice were generated to abrogate Hgf expression selectively in LSECs from early fetal development onwards, to study global development, metabolic and endothelial zonation, and organ functions as well as liver regeneration in response to 70% partial hepatectomy (PH). Although zonation and liver/body weight ratios were not altered, total body weight and total liver weight were reduced in Hgf. Necrotic organ damage was more marked in Hgf mice, and regeneration was delayed 72 hours after PH. This was associated with decreased hepatocyte proliferation at 48 hours after PH. Molecularly, Hgf mice showed down-regulation of Hgf/c-Met signaling and decreased expression of Deptor in hepatocytes. In vitro knockdown of Deptor was associated with decreased proliferation. Therefore, angiocrine Hgf controls hepatocyte proliferation and susceptibility to necrosis after partial hepatectomy via the Hgf/c-Met axis involving Deptor to prevent excessive organ damage.
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http://dx.doi.org/10.1016/j.ajpath.2019.10.009DOI Listing
February 2020

RIG-I activation is critical for responsiveness to checkpoint blockade.

Sci Immunol 2019 09;4(39)

Department of Medicine III, School of Medicine, Technical University of Munich, Munich, Germany.

Achieving durable clinical responses to immune checkpoint inhibitors remains a challenge. Here, we demonstrate that immunotherapy with anti-CTLA-4 and its combination with anti-PD-1 rely on tumor cell-intrinsic activation of the cytosolic RNA receptor RIG-I. Mechanistically, tumor cell-intrinsic RIG-I signaling induced caspase-3-mediated tumor cell death, cross-presentation of tumor-associated antigen by CD103 dendritic cells, subsequent expansion of tumor antigen-specific CD8 T cells, and their accumulation within the tumor tissue. Consistently, therapeutic targeting of RIG-I with 5'- triphosphorylated RNA in both tumor and nonmalignant host cells potently augmented the efficacy of CTLA-4 checkpoint blockade in several preclinical cancer models. In humans, transcriptome analysis of primary melanoma samples revealed a strong association between high expression of (the gene encoding RIG-I), T cell receptor and antigen presentation pathway activity, and prolonged overall survival. Moreover, in patients with melanoma treated with anti-CTLA-4 checkpoint blockade, high RIG-I transcriptional activity significantly associated with durable clinical responses. Our data thus identify activation of RIG-I signaling in tumors and their microenvironment as a crucial component for checkpoint inhibitor-mediated immunotherapy of cancer.
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http://dx.doi.org/10.1126/sciimmunol.aau8943DOI Listing
September 2019

Bcl10-controlled Malt1 paracaspase activity is key for the immune suppressive function of regulatory T cells.

Nat Commun 2019 05 28;10(1):2352. Epub 2019 May 28.

Institute of Clinical Chemistry and Pathobiochemistry, TUM School of Medicine, Technical University of Munich, 81675, Munich, Germany.

Regulatory T cells (Tregs) have crucial functions in the inhibition of immune responses. Their development and suppressive functions are controlled by the T cell receptor (TCR), but the TCR signaling mechanisms that mediate these effects remain ill-defined. Here we show that CARD11-BCL10-MALT1 (CBM) signaling mediates TCR-induced NF-κB activation in Tregs and controls the conversion of resting Tregs to effector Tregs under homeostatic conditions. However, in inflammatory milieus, cytokines can bypass the CBM requirement for this differentiation step. By contrast, CBM signaling, in a MALT1 protease-dependent manner, is essential for mediating the suppressive function of Tregs. In malignant melanoma models, acute genetic blockade of BCL10 signaling selectively in Tregs or pharmacological MALT1 inhibition enhances anti-tumor immune responses. Together, our data uncover a segregation of Treg differentiation and suppressive function at the CBM complex level, and provide a rationale to explore MALT1 inhibitors for cancer immunotherapy.
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http://dx.doi.org/10.1038/s41467-019-10203-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538646PMC
May 2019

The neuropeptide receptor subunit RAMP1 constrains the innate immune response during acute pancreatitis in mice.

Pancreatology 2019 Jun 11;19(4):541-547. Epub 2019 May 11.

Technical University of Munich, School of Medicine, Klinikum Rechts der Isar, Department of Internal Medicine II, Munich, Germany. Electronic address:

Objectives: The importance of the Calcitonin-gene-related-peptide-pathway (CGRP) as neuronal modulator of innate immune responses in mice has been previously demonstrated. The CGRP-receptor is composed of two subunits: the receptor-activity-modifying-protein-1 (RAMP1) and the calcitonin-receptor-like-receptor (CLR). CGRP can influence immune cells and their capacity of producing inflammatory cytokines. Using a RAMP1 knockout-mouse (RAMP1) we examined the role of the CGRP-receptor in the acute-phase of cerulein-induced pancreatitis.

Methods: Hourly cerulein-injections for a period of 8 h in RAMP1 and wild-type mice were performed. To compare severity and extent of inflammation in RAMP1 and wild-type mice, histological analyses were done and cytokine levels were assessed using qRT-PCR 8 h, 24 h, 2 days, and 7 days post-cerulein-treatment. Furthermore, serum activities of LDH and lipase were determined.

Results: After 8 h RAMP1 mice showed a higher pancreas-to-body-weight-ratio, increased tissue edema and immune cell infiltration with higher amount of F4/80-positive cells as compared to wild-type mice. Overall infiltration of immune cells at 24 h was increased in RAMP1 mice and composed predominantly of MPO-positive neutrophils. In addition, after 24 h RAMP1 mice presented a higher pancreas-to-body-weight-ratio, higher expression of Ccl3, Il6, and Il1b and increased number of cleaved caspase 3 positive cells. Serum lipase correlated with the extent of tissue damage in RAMP1 compared to wild-type mice 24 h post-cerulein treatment.

Conclusion: Mice lacking RAMP1 showed increased inflammation, tissue edema, and pancreas injury particularly in the early phase of acute pancreatitis. This study highlights the essential role of CGRP for dampening the innate immune response in acute pancreatitis.
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http://dx.doi.org/10.1016/j.pan.2019.05.455DOI Listing
June 2019

Platelet GPIbα is a mediator and potential interventional target for NASH and subsequent liver cancer.

Nat Med 2019 04 1;25(4):641-655. Epub 2019 Apr 1.

Department of Cardiology and Circulatory Diseases, Internal Medicine Clinic III, Eberhard Karls University Tübingen, Tübingen, Germany.

Non-alcoholic fatty liver disease ranges from steatosis to non-alcoholic steatohepatitis (NASH), potentially progressing to cirrhosis and hepatocellular carcinoma (HCC). Here, we show that platelet number, platelet activation and platelet aggregation are increased in NASH but not in steatosis or insulin resistance. Antiplatelet therapy (APT; aspirin/clopidogrel, ticagrelor) but not nonsteroidal anti-inflammatory drug (NSAID) treatment with sulindac prevented NASH and subsequent HCC development. Intravital microscopy showed that liver colonization by platelets depended primarily on Kupffer cells at early and late stages of NASH, involving hyaluronan-CD44 binding. APT reduced intrahepatic platelet accumulation and the frequency of platelet-immune cell interaction, thereby limiting hepatic immune cell trafficking. Consequently, intrahepatic cytokine and chemokine release, macrovesicular steatosis and liver damage were attenuated. Platelet cargo, platelet adhesion and platelet activation but not platelet aggregation were identified as pivotal for NASH and subsequent hepatocarcinogenesis. In particular, platelet-derived GPIbα proved critical for development of NASH and subsequent HCC, independent of its reported cognate ligands vWF, P-selectin or Mac-1, offering a potential target against NASH.
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http://dx.doi.org/10.1038/s41591-019-0379-5DOI Listing
April 2019

PiggyBac transposon tools for recessive screening identify B-cell lymphoma drivers in mice.

Nat Commun 2019 03 29;10(1):1415. Epub 2019 Mar 29.

The Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. Here, we describe PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation, or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally, or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate discovered genes, such as Rfx7, a transcription factor, and Phip, a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology.
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http://dx.doi.org/10.1038/s41467-019-09180-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6440946PMC
March 2019

Brg1 promotes liver regeneration after partial hepatectomy via regulation of cell cycle.

Sci Rep 2019 02 20;9(1):2320. Epub 2019 Feb 20.

Department of Medicine II, TUM School of Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, 81675, Germany.

Brahma-related gene 1 (Brg1), a catalytic subunit of the SWItch/Sucrose Non-Fermentable (SWI/SNF) complex, is known to be involved in proliferative cell processes. Liver regeneration is initiated spontaneously after injury and leads to a strong proliferative response. In this study, a hepatocyte-specific Brg1 gene knockout mouse model was used to analyse the role of Brg1 in liver regeneration by performing a 70% partial hepatectomy (PH). After PH, Brg1 was significantly upregulated in wildtype mice. Mice with hepatocyte-specific Brg1 gene knockout showed a significantly lower liver to body weight ratio 48 h post-PH concomitant with a lower hepatocellular proliferation rate compared to wildtype mice. RNA sequencing demonstrated that Brg1 controlled hepatocyte proliferation through the regulation of the p53 pathway and several cell cycle genes. The data of this study reveal a crucial role of Brg1 for liver regeneration by promoting hepatocellular proliferation through modulation of cell cycle genes and, thus, identify Brg1 as potential target for therapeutic approaches.
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http://dx.doi.org/10.1038/s41598-019-38568-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382836PMC
February 2019

Oncogenic Amplification of Zygotic Dux Factors in Regenerating p53-Deficient Muscle Stem Cells Defines a Molecular Cancer Subtype.

Cell Stem Cell 2018 12 15;23(6):794-805.e4. Epub 2018 Nov 15.

Department of Cardiac Development and Remodeling, Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany; German Center for Cardiovascular Research (DZHK), Rhine Main, Germany. Electronic address:

The identity of tumor-initiating cells in many cancer types is unknown. Tumors often express genes associated with embryonic development, although the contributions of zygotic programs to tumor initiation and formation are poorly understood. Here, we show that regeneration-induced loss of quiescence in p53-deficient muscle stem cells (MuSCs) results in rhabdomyosarcoma formation with 100% penetrance. Genomic analyses of purified tumor cells revealed spontaneous and discrete oncogenic amplifications in MuSCs that drive tumorigenesis, including, but not limited to, the amplification of the cleavage-stage Dux transcription factor (TF) Duxbl. We further found that Dux factors drive an early embryonic gene signature that defines a molecular subtype across a broad range of human cancers. Duxbl initiates tumorigenesis by enforcing a mesenchymal-to-epithelial transition, and targeted inactivation of Duxbl specifically in Duxbl-expressing tumor cells abolishes their expansion. These findings reveal how regeneration and genomic instability can interact to activate zygotic genes that drive tumor initiation and growth.
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http://dx.doi.org/10.1016/j.stem.2018.10.011DOI Listing
December 2018

Myeloid-derived suppressor cells control B cell accumulation in the central nervous system during autoimmunity.

Nat Immunol 2018 12 29;19(12):1341-1351. Epub 2018 Oct 29.

Department of Experimental Neuroimmunology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) have been characterized in the context of malignancies. Here we show that PMN-MDSCs can restrain B cell accumulation during central nervous system (CNS) autoimmunity. Ly6G cells were recruited to the CNS during experimental autoimmune encephalomyelitis (EAE), interacted with B cells that produced the cytokines GM-CSF and interleukin-6 (IL-6), and acquired properties of PMN-MDSCs in the CNS in a manner dependent on the signal transducer STAT3. Depletion of Ly6G cells or dysfunction of Ly6G cells through conditional ablation of STAT3 led to the selective accumulation of GM-CSF-producing B cells in the CNS compartment, which in turn promoted an activated microglial phenotype and lack of recovery from EAE. The frequency of CD138 B cells in the cerebrospinal fluid (CSF) of human subjects with multiple sclerosis was negatively correlated with the frequency of PMN-MDSCs in the CSF. Thus PMN-MDSCs might selectively control the accumulation and cytokine secretion of B cells in the inflamed CNS.
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http://dx.doi.org/10.1038/s41590-018-0237-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6241855PMC
December 2018

Evolutionary routes and KRAS dosage define pancreatic cancer phenotypes.

Nature 2018 02 24;554(7690):62-68. Epub 2018 Jan 24.

Anthropology & Human Genomics, Department of Biology II, Ludwig-Maximilians Universität, 82152 Martinsried, Germany.

The poor correlation of mutational landscapes with phenotypes limits our understanding of the pathogenesis and metastasis of pancreatic ductal adenocarcinoma (PDAC). Here we show that oncogenic dosage-variation has a critical role in PDAC biology and phenotypic diversification. We find an increase in gene dosage of mutant KRAS in human PDAC precursors, which drives both early tumorigenesis and metastasis and thus rationalizes early PDAC dissemination. To overcome the limitations posed to gene dosage studies by the stromal richness of PDAC, we have developed large cell culture resources of metastatic mouse PDAC. Integration of cell culture genomes, transcriptomes and tumour phenotypes with functional studies and human data reveals additional widespread effects of oncogenic dosage variation on cell morphology and plasticity, histopathology and clinical outcome, with the highest Kras levels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains (Myc, Yap1 or Nfkb2), which collaborate with heterozygous Kras in driving tumorigenesis, but have lower metastatic potential. Mechanistically, different oncogenic gains and dosages evolve along distinct evolutionary routes, licensed by defined allelic states and/or combinations of hallmark tumour suppressor alterations (Cdkn2a, Trp53, Tgfβ-pathway). Thus, evolutionary constraints and contingencies direct oncogenic dosage gain and variation along defined routes to drive the early progression of PDAC and shape its downstream biology. Our study uncovers universal principles of Ras-driven oncogenesis that have potential relevance beyond pancreatic cancer.
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http://dx.doi.org/10.1038/nature25459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6097607PMC
February 2018

Species and genotype diversity of Plasmodium in malaria patients from Gabon analysed by next generation sequencing.

Malar J 2017 10 3;16(1):398. Epub 2017 Oct 3.

Institut für Tropenmedizin, Eberhard Karls Universität Tübingen, Wilhelmstraße 27, 72074, Tübingen, Germany.

Background: Six Plasmodium species are known to naturally infect humans. Mixed species infections occur regularly but morphological discrimination by microscopy is difficult and multiplicity of infection (MOI) can only be evaluated by molecular methods. This study investigated the complexity of Plasmodium infections in patients treated for microscopically detected non-falciparum or mixed species malaria in Gabon.

Methods: Ultra-deep sequencing of nucleus (18S rRNA), mitochondrion, and apicoplast encoded genes was used to evaluate Plasmodium species diversity and MOI in 46 symptomatic Gabonese patients with microscopically diagnosed non-falciparum or mixed species malaria.

Results: Deep sequencing revealed a large complexity of confections in patients with uncomplicated malaria, both on species and genotype levels. Mixed infections involved up to four parasite species (Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale curtisi, and P. ovale wallikeri). Multiple genotypes from each species were determined from the asexual 18S rRNA gene. 17 of 46 samples (37%) harboured multiple genotypes of at least one Plasmodium species. The number of genotypes per sample (MOI) was highest in P. malariae (n = 4), followed by P. ovale curtisi (n = 3), P. ovale wallikeri (n = 3), and P. falciparum (n = 2). The highest combined genotype complexity in samples that contained mixed-species infections was seven.

Conclusions: Ultra-deep sequencing showed an unexpected breadth of Plasmodium species and within species diversity in clinical samples. MOI of P. ovale curtisi, P. ovale wallikeri and P. malariae infections were higher than anticipated and contribute significantly to the burden of malaria in Gabon.
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http://dx.doi.org/10.1186/s12936-017-2044-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627438PMC
October 2017

Genome-wide transposon screening and quantitative insertion site sequencing for cancer gene discovery in mice.

Nat Protoc 2017 Feb 12;12(2):289-309. Epub 2017 Jan 12.

Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.

Transposon-mediated forward genetics screening in mice has emerged as a powerful tool for cancer gene discovery. It pinpoints cancer drivers that are difficult to find with other approaches, thus complementing the sequencing-based census of human cancer genes. We describe here a large series of mouse lines for insertional mutagenesis that are compatible with two transposon systems, PiggyBac and Sleeping Beauty, and give guidance on the use of different engineered transposon variants for constitutive or tissue-specific cancer gene discovery screening. We also describe a method for semiquantitative transposon insertion site sequencing (QiSeq). The QiSeq library preparation protocol exploits acoustic DNA fragmentation to reduce bias inherent to widely used restriction-digestion-based approaches for ligation-mediated insertion site amplification. Extensive multiplexing in combination with next-generation sequencing allows affordable ultra-deep transposon insertion site recovery in high-throughput formats within 1 week. Finally, we describe principles of data analysis and interpretation for obtaining insights into cancer gene function and genetic tumor evolution.
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http://dx.doi.org/10.1038/nprot.2016.164DOI Listing
February 2017

Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry.

Nat Commun 2016 11 21;7:13404. Epub 2016 Nov 21.

IIIrd Medical Department, Klinikum rechts der Isar, Technische Universität München, Ismaningerstr. 22, Munich 81675, Germany.

Although mutations may represent attractive targets for immunotherapy, direct identification of mutated peptide ligands isolated from human leucocyte antigens (HLA) on the surface of native tumour tissue has so far not been successful. Using advanced mass spectrometry (MS) analysis, we survey the melanoma-associated immunopeptidome to a depth of 95,500 patient-presented peptides. We thereby discover a large spectrum of attractive target antigen candidates including cancer testis antigens and phosphopeptides. Most importantly, we identify peptide ligands presented on native tumour tissue samples harbouring somatic mutations. Four of eleven mutated ligands prove to be immunogenic by neoantigen-specific T-cell responses. Moreover, tumour-reactive T cells with specificity for selected neoantigens identified by MS are detected in the patient's tumour and peripheral blood. We conclude that direct identification of mutated peptide ligands from primary tumour material by MS is possible and yields true neoepitopes with high relevance for immunotherapeutic strategies in cancer.
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http://dx.doi.org/10.1038/ncomms13404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5121339PMC
November 2016

RIPK3 Restricts Myeloid Leukemogenesis by Promoting Cell Death and Differentiation of Leukemia Initiating Cells.

Cancer Cell 2016 07;30(1):75-91

III. Medical Department for Hematology and Oncology, Klinikum rechts der Isar, Technische Universität München, 81675 München, Germany; German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany. Electronic address:

Since acute myeloid leukemia (AML) is characterized by the blockade of hematopoietic differentiation and cell death, we interrogated RIPK3 signaling in AML development. Genetic loss of Ripk3 converted murine FLT3-ITD-driven myeloproliferation into an overt AML by enhancing the accumulation of leukemia-initiating cells (LIC). Failed inflammasome activation and cell death mediated by tumor necrosis factor receptor caused this accumulation of LIC exemplified by accelerated leukemia onset in Il1r1(-/-), Pycard(-/-), and Tnfr1/2(-/-) mice. RIPK3 signaling was partly mediated by mixed lineage kinase domain-like. This link between suppression of RIPK3, failed interleukin-1β release, and blocked cell death was supported by significantly reduced RIPK3 in primary AML patient cohorts. Our data identify RIPK3 and the inflammasome as key tumor suppressors in AML.
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http://dx.doi.org/10.1016/j.ccell.2016.06.002DOI Listing
July 2016

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

Nat Commun 2016 Feb 26;7:10770. Epub 2016 Feb 26.

Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.

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.
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http://dx.doi.org/10.1038/ncomms10770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4773438PMC
February 2016