Publications by authors named "Dietmar Pfeifer"

125 Publications

Inhibition of metabotropic glutamate receptor III facilitates sensitization to alkylating chemotherapeutics in glioblastoma.

Cell Death Dis 2021 Jul 21;12(8):723. Epub 2021 Jul 21.

Microenvironment and Immunology Research Laboratory, Medical Center-University of Freiburg, Freiburg, Germany.

Glioblastoma (GBM), the most malignant tumor of the central nervous system, is marked by its dynamic response to microenvironmental niches. In particular, this cellular plasticity contributes to the development of an immediate resistance during tumor treatment. Novel insights into the developmental trajectory exhibited by GBM show a strong capability to respond to its microenvironment by clonal selection of specific phenotypes. Using the same mechanisms, malignant GBM do develop intrinsic mechanisms to resist chemotherapeutic treatments. This resistance was reported to be sustained by the paracrine and autocrine glutamate signaling via ionotropic and metabotropic receptors. However, the extent to which glutamatergic signaling modulates the chemoresistance and transcriptional profile of the GBM remains unexplored. In this study we aimed to map the manifold effects of glutamate signaling in GBM as the basis to further discover the regulatory role and interactions of specific receptors, within the GBM microenvironment. Our work provides insights into glutamate release dynamics, representing its importance for GBM growth, viability, and migration. Based on newly published multi-omic datasets, we explored the and characterized the functions of different ionotropic and metabotropic glutamate receptors, of which the metabotropic receptor 3 (GRM3) is highlighted through its modulatory role in maintaining the ability of GBM cells to evade standard alkylating chemotherapeutics. We addressed the clinical relevance of GRM3 receptor expression in GBM and provide a proof of concept where we manipulate intrinsic mechanisms of chemoresistance, driving GBM towards chemo-sensitization through GRM3 receptor inhibition. Finally, we validated our findings in our novel human organotypic section-based tumor model, where GBM growth and proliferation was significantly reduced when GRM3 inhibition was combined with temozolomide application. Our findings present a new picture of how glutamate signaling via mGluR3 interacts with the phenotypical GBM transcriptional programs in light of recently published GBM cell-state discoveries.
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http://dx.doi.org/10.1038/s41419-021-03937-9DOI Listing
July 2021

Author Correction: The secreted endoribonuclease ENDU-2 from the soma protects germline immortality in C. elegans.

Nat Commun 2021 May 28;12(1):3315. Epub 2021 May 28.

Bioinformatics and Molecular Genetics (Faculty of Biology), Albert-Ludwigs-University Freiburg, Freiburg, Germany.

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http://dx.doi.org/10.1038/s41467-021-23820-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163770PMC
May 2021

Altered Spectrum of Lymphoid Neoplasms in a Single-Center Cohort of Common Variable Immunodeficiency with Immune Dysregulation.

J Clin Immunol 2021 Aug 19;41(6):1250-1265. Epub 2021 Apr 19.

University of Freiburg, Freiburg, Germany.

Purpose: Common variable immune deficiency (CVID) confers an increased risk of lymphoid neoplasms, but reports describing the precise WHO specification of the lymphoma subtypes and their immunological environment are lacking. We therefore classified lymphomas-occurring in a cohort of 21 adult CVID patients during a 17-year period at our center-according to the 2016 WHO classification and characterized the local and systemic immunological context RESULTS: The median time between the onset of CVID and lymphoma was 14 years. Patients showed a high prevalence of preceding immune dysregulation: lymphadenopathy (n = 13, 62%), splenomegaly (n = 18, 86%), autoimmune cytopenia (n = 14, 67%), and gastrointestinal involvement (n = 15, 71%). The entities comprised extranodal marginal zone lymphoma (n = 6), diffuse large B cell lymphoma (n = 7), plasmablastic lymphoma (n = 1), classic Hodgkin lymphoma (n = 4, including three cases with germline CTLA4 mutations), T cell large granular lymphocytic leukemia (n = 2), and peripheral T cell lymphoma, not otherwise specified (n = 1), but no follicular lymphoma. An Epstein-Barr virus association was documented in eight of 16 investigated lymphomas. High expression of PDL1 by tumor cells in five and of PDL1 and PD1 by tumor-infiltrating macrophages and T cells in 12 of 12 investigated lymphomas suggested a tolerogenic immunological tumor environment.

Conclusion: In summary, a diverse combination of specific factors like genetic background, chronic immune activation, viral trigger, and impaired immune surveillance contributes to the observed spectrum of lymphomas in CVID. In the future, targeted therapies, e.g., PD1/PDL1 inhibitors in CVID associated lymphomas with a tolerogenic environment may improve therapy outcome.
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http://dx.doi.org/10.1007/s10875-021-01016-4DOI Listing
August 2021

Integrative study of EZH2 mutational status, copy number, protein expression and H3K27 trimethylation in AML/MDS patients.

Clin Epigenetics 2021 Apr 12;13(1):77. Epub 2021 Apr 12.

Department of Medicine I (Hematology, Oncology and Stem Cell Transplantation), Medical Center - University of Freiburg, Freiburg, Germany.

Background: Mutations in the EZH2 gene are recurrently found in patients with myeloid neoplasms and are associated with a poor prognosis. We aimed to characterize genetic and epigenetic alterations of EZH2 in 58 patients (51 with acute myeloid leukemia and 7 with myelodysplastic or myeloproliferative neoplasms) by integrating data on EZH2 mutational status, co-occurring mutations, and EZH2 copy number status with EZH2 protein expression, histone H3K27 trimethylation, and EZH2 promoter methylation.

Results: EZH2 was mutated in 6/51 acute myeloid leukemia patients (12%) and 7/7 patients with other myeloid neoplasms. EZH2 mutations were not overrepresented in patients with chromosome 7q deletions or losses. In acute myeloid leukemia patients, EZH2 mutations frequently co-occurred with CEBPA (67%), ASXL1 (50%), TET2 and RAD21 mutations (33% each). In EZH2-mutated patients with myelodysplastic or myeloproliferative neoplasms, the most common co-mutations were in ASXL1 (100%), NRAS, RUNX1, and STAG2 (29% each). EZH2 mutations were associated with a significant decrease in EZH2 expression (p = 0.0002), which was similar in patients with chromosome 7 aberrations and patients with intact chromosome 7. An association between EZH2 protein expression and H3K27 trimethylation was observed in EZH2-unmutated patients (R = 0.2, p = 0.01). The monoallelic state of EZH2 was not associated with EZH2 promoter hypermethylation. In multivariable analyses, EZH2 mutations were associated with a trend towards an increased risk of death (hazard ratio 2.51 [95% confidence interval 0.87-7.25], p = 0.09); similarly, low EZH2 expression was associated with elevated risk (hazard ratio 2.54 [95% confidence interval 1.07-6.04], p = 0.04).

Conclusions: Perturbations of EZH2 activity in AML/MDS occur on different, genetic and non-genetic levels. Both low EZH2 protein expression and, by trend, EZH2 gene mutations predicted inferior overall survival of AML patients receiving standard chemotherapy.
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http://dx.doi.org/10.1186/s13148-021-01052-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8043064PMC
April 2021

The secreted endoribonuclease ENDU-2 from the soma protects germline immortality in C. elegans.

Nat Commun 2021 02 24;12(1):1262. Epub 2021 Feb 24.

Bioinformatics and Molecular Genetics (Faculty of Biology), Albert-Ludwigs-University Freiburg, Freiburg, Germany.

Multicellular organisms coordinate tissue specific responses to environmental information via both cell-autonomous and non-autonomous mechanisms. In addition to secreted ligands, recent reports implicated release of small RNAs in regulating gene expression across tissue boundaries. Here, we show that the conserved poly-U specific endoribonuclease ENDU-2 in C. elegans is secreted from the soma and taken-up by the germline to ensure germline immortality at elevated temperature. ENDU-2 binds to mature mRNAs and negatively regulates mRNA abundance both in the soma and the germline. While ENDU-2 promotes RNA decay in the soma directly via its endoribonuclease activity, ENDU-2 prevents misexpression of soma-specific genes in the germline and preserves germline immortality independent of its RNA-cleavage activity. In summary, our results suggest that the secreted RNase ENDU-2 regulates gene expression across tissue boundaries in response to temperature alterations and contributes to maintenance of stem cell immortality, probably via retaining a stem cell specific program of gene expression.
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http://dx.doi.org/10.1038/s41467-021-21516-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904951PMC
February 2021

Decitabine Induces Gene Derepression on Monosomic Chromosomes: and Effects in Adverse-Risk Cytogenetics AML.

Cancer Res 2021 02 17;81(4):834-846. Epub 2020 Nov 17.

Department of Medicine I, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany.

Hypomethylating agents (HMA) have become the backbone of nonintensive acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) treatment, also by virtue of their activity in patients with adverse genetics, for example, monosomal karyotypes, often with losses on chromosome 7, 5, or 17. No comparable activity is observed with cytarabine, a cytidine analogue without DNA-hypomethylating properties. As evidence exists for compounding hypermethylation and gene silencing of hemizygous tumor suppressor genes (TSG), we thus hypothesized that this effect may preferentially be reversed by the HMAs decitabine and azacitidine. An unbiased RNA-sequencing approach was developed to interrogate decitabine-induced transcriptome changes in AML cell lines with or without a deletion of chromosomes 7q, 5q or 17p. HMA treatment preferentially upregulated several hemizygous TSG in this genomic region, significantly derepressing endogenous retrovirus (ERV)3-1, with promoter demethylation, enhanced chromatin accessibility, and increased H3K4me3 levels. Decitabine globally reactivated multiple transposable elements, with activation of the dsRNA sensor RIG-I and interferon regulatory factor (IRF)7. Induction of ERV3-1 and RIG-I mRNA was also observed during decitabine treatment in serially sorted peripheral blood AML blasts. In patient-derived monosomal karyotype AML murine xenografts, decitabine treatment resulted in superior survival rates compared with cytarabine. Collectively, these data demonstrate preferential gene derepression and ERV reactivation in AML with chromosomal deletions, providing a mechanistic explanation that supports the clinical observation of superiority of HMA over cytarabine in this difficult-to-treat patient group. SIGNIFICANCE: These findings unravel the molecular mechanism underlying the intriguing clinical activity of HMAs in AML/MDS patients with chromosome 7 deletions and other monosomal karyotypes..
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http://dx.doi.org/10.1158/0008-5472.CAN-20-1430DOI Listing
February 2021

Colon and liver tissue damage detection using methylated SESN3 and PTK2B genes in circulating cell-free DNA in patients with acute graft-versus-host disease.

Bone Marrow Transplant 2021 02 20;56(2):327-333. Epub 2020 Oct 20.

Department of Hematology, Oncology and Stem Cell Transplantation, Freiburg University Medical Center, Freiburg, Germany.

Cell-free DNA (cfDNA) has been investigated in acute graft-versus-host disease (aGvHD) following allogeneic cell transplantation (HSCT). Identifying the tissue of origin of cfDNA in patients with aGvHD is relevant particularly when a biopsy is not feasible. We investigate the cfDNA tissue of origin in patients with aGvHD using methylated gene biomarkers. Patients with liver, colon, or skin aGvHD (n = 28) were analyzed. Liver- and colon-derived cfDNA was measured using a colon- (SESN3) and liver (PTK2B)-specific methylation marker with digital droplet PCR. A statistically significant difference (p < 0.001) in PTK2B and SESN3 concentration was observed between patients with colon or liver GvHD and the control group. For SESN3 and PTK2B the area under the curve in the receiver-operating characteristic (ROC) space was 0.952 (95% CI, 0.888-1 p < 0.001) and 0.971 (95% CI, 0.964-1 p < 0.001), respectively. Thresholds to differentiate aGvHD from non-aGvHD in colon were 0 (sensitivity: 0.905; specificity: 0.989) and liver 1.5 (sensitivity: 0.928; specificity: 0.910). Clinical improvement of liver or colon aGvHD resulted in PTK2B and SESN3 reduced concentration. Whereas, in those patients without improvement the PTK2B and SESN3 level remained stable or increased. The PTK2B liver-specific marker and the SESN3 colon-specific marker and their longitudinal analysis might improve aGvHD detection.
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http://dx.doi.org/10.1038/s41409-020-01090-zDOI Listing
February 2021

Prevalence and characteristics of myeloproliferative neoplasms with concomitant monoclonal gammopathy.

Leuk Res 2020 11 15;98:106454. Epub 2020 Sep 15.

Department of Medicine I, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Germany; German Cancer Consortium (DKTK) Partner Site, Freiburg, Germany. Electronic address:

Of BCR-ABL negative myeloproliferative neoplasm (MPN) patients, 3-14 % display a concomitant monoclonal gammopathy (MGUS). Nonetheless, literature on co-occurring MPN and MGUS is scarce, the molecular underpinnings are unknown and it is unclear whether patients require a specific management. Here, we compared the clinical and genetic features of MPN patients with and without concomitant MGUS. Of 114 MPN patients prospectively studied by serum immunofixation (median age, 67 years; 36.0 % essential thrombocythemia [ET], 24.6 % polycythemia vera [PV], 11.4 % secondary myelofibrosis [sMF], 28.1 % primary myelofibrois [PMF]; 73.7 % JAK2 V617F positive), 10 (9 %) harbored an M-protein. No relevant clinical differences existed between MPN patients with or without M-protein. Seven additional MPN/MGUS patients were retrospectively identified in our MPN registry, yielding a total of 17 patients (7 ET, 3 PV, 3 sMF, 4 PMF). One patient developed multiple myeloma (MM) and one smoldering MM. Seven of 12 patients analyzed carried mutations (e.g. in ASXL1 or TET2) in addition to those in JAK2 or CALR, and 4 of 10 patients showed aberrant cytogenetics. M-protein was mainly IgG (12/17), followed by IgM (4/17). In the two patients that underwent allogeneic stem cell transplantation mutant JAK2 and M-protein were no longer detectable post-transplant. In conclusion, MGUS prevalence in our cohort was in the range of previous reports and at most slightly higher than expected in the general population. MGUS presence did not correlate with a specific MPN entity, clinical features or genetic alterations. Our observations suggest that there is no strong clinical or biological relationship between the occurrence of MGUS and MPN.
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http://dx.doi.org/10.1016/j.leukres.2020.106454DOI Listing
November 2020

Bile acids regulate intestinal antigen presentation and reduce graft-versus-host disease without impairing the graft-versus-leukemia effect.

Haematologica 2021 Aug 1;106(8):2131-2146. Epub 2021 Aug 1.

Department of Medicine I, Medical Center - University of Freiburg, Germany.

Acute graft-versus-host disease causes significant mortality in patients undergoing allogeneic hematopoietic cell transplantation. Immunosuppressive treatment for graft-versus-host disease can impair the beneficial graft-versus-leukemia effect and facilitate malignancy relapse. Therefore, novel approaches that protect and regenerate injured tissues without impeding the donor immune system are needed. Bile acids regulate multiple cellular processes and are in close contact with the intestinal epithelium, a major target of acute graft-versus-host disease. Here, we found that the bile acid pool is reduced following graft-versus-host disease induction in a preclinical model. We evaluated the efficacy of bile acids to protect the intestinal epithelium without reducing anti-tumor immunity. We observed that application of bile acids decreased cytokine-induced cell death in intestinal organoids and cell lines. Systemic prophylactic administration of tauroursodeoxycholic acid, the most potent compound in our in vitro studies, reduced graft-versus-host disease severity in three different murine transplantation models. This effect was mediated by decreased activity of the antigen presentation machinery and subsequent prevention of apoptosis of the intestinal epithelium. Moreover, bile acid administration did not alter the bacterial composition in the intestine suggesting that its effects are cell-specific and independent of the microbiome. Treatment of human and murine leukemic cell lines with tauroursodeoxycholic acid did not interfere with the expression of antigen presentation-related molecules. Systemic T cell expansion and especially their cytotoxic capacity against leukemic cells remained intact. This study establishes a role for bile acids in the prevention of acute graft-versus-host disease without impairing the graft-versus-leukemia effect. In particular, we provide a scientific rationale for the systematic use of tauroursodeoxycholic acid in patients undergoing allogeneic hematopoietic cell transplantation.
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http://dx.doi.org/10.3324/haematol.2019.242990DOI Listing
August 2021

Glucagon-like peptide 2 for intestinal stem cell and Paneth cell repair during graft-versus-host disease in mice and humans.

Blood 2020 09;136(12):1442-1455

Department of Medicine I, Faculty of Medicine, Medical Center University of Freiburg (MCUF).

Acute graft-versus-host disease (GVHD) is a life-threatening complication after allogeneic hematopoietic cell transplantation (allo-HCT). Although currently used GVHD treatment regimens target the donor immune system, we explored here an approach that aims at protecting and regenerating Paneth cells (PCs) and intestinal stem cells (ISCs). Glucagon-like-peptide-2 (GLP-2) is an enteroendocrine tissue hormone produced by intestinal L cells. We observed that acute GVHD reduced intestinal GLP-2 levels in mice and patients developing GVHD. Treatment with the GLP-2 agonist, teduglutide, reduced de novo acute GVHD and steroid-refractory GVHD, without compromising graft-versus-leukemia (GVL) effects in multiple mouse models. Mechanistically GLP-2 substitution promoted regeneration of PCs and ISCs, which enhanced production of antimicrobial peptides and caused microbiome changes. GLP-2 expanded intestinal organoids and reduced expression of apoptosis-related genes. Low numbers of L cells in intestinal biopsies and high serum levels of GLP-2 were associated with a higher incidence of nonrelapse mortality in patients undergoing allo-HCT. Our findings indicate that L cells are a target of GVHD and that GLP-2-based treatment of acute GVHD restores intestinal homeostasis via an increase of ISCs and PCs without impairing GVL effects. Teduglutide could become a novel combination partner for immunosuppressive GVHD therapy to be tested in clinical trials.
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http://dx.doi.org/10.1182/blood.2020005957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7498363PMC
September 2020

Monosomal karyotype and chromosome 17p loss or TP53 mutations in decitabine-treated patients with acute myeloid leukemia.

Ann Hematol 2020 Jul 6;99(7):1551-1560. Epub 2020 Jun 6.

Department of Medicine I, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Hugstetter Straße 55, 79106, Freiburg, Germany.

TP53 aberrations reportedly predict favorable responses to decitabine (DAC) in acute myeloid leukemia (AML). We evaluated clinical features and outcomes associated with chromosome 17p loss or TP53 gene mutations in older, unfit DAC-treated AML patients in a phase II trial. Of 178 patients, 25 had loss of 17p in metaphase cytogenetics; 24 of these had a complex (CK+) and 21 a monosomal karyotype (MK+). In analyses in all patients and restricted to CK+ and MK+ patients, 17p loss tended to associate with higher rates of complete remission (CR), partial remission (PR), or antileukemic effect (ALE). Despite favorable response rates, there was no significant OS difference between patients with or without loss of 17p in the entire cohort or in the CK+ and MK+ cohort. TP53 mutations were identified in eight of 45 patients with material available. Five of the eight TP53-mutated patients had 17p loss. TP53-mutated patients had similar rates of CR/PR/ALE but shorter OS than those with TP53 wild type (P = 0.036). Moreover, patients with a subclone based on mutation data had shorter OS than those without (P = 0.05); only one patient with TP53-mutated AML had a subclone. In conclusion, 17p loss conferred a favorable impact on response rates, even among CK+ and MK+ patients that however could not be maintained. The effect of TP53 mutations appeared to be different; however, patient numbers were low. Future research needs to further dissect the impact of the various TP53 aberrations in HMA-based combination therapies. The limited duration of favorable responses to HMA treatment in adverse-risk genetics AML should prompt physicians to advance allografting for eligible patients in a timely fashion.
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http://dx.doi.org/10.1007/s00277-020-04082-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316846PMC
July 2020

Loss of the Fanconi anemia-associated protein NIPA causes bone marrow failure.

J Clin Invest 2020 06;130(6):2827-2844

German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany.

Inherited bone marrow failure syndromes (IBMFSs) are a heterogeneous group of disorders characterized by defective hematopoiesis, impaired stem cell function, and cancer susceptibility. Diagnosis of IBMFS presents a major challenge due to the large variety of associated phenotypes, and novel, clinically relevant biomarkers are urgently needed. Our study identified nuclear interaction partner of ALK (NIPA) as an IBMFS gene, as it is significantly downregulated in a distinct subset of myelodysplastic syndrome-type (MDS-type) refractory cytopenia in children. Mechanistically, we showed that NIPA is major player in the Fanconi anemia (FA) pathway, which binds FANCD2 and regulates its nuclear abundance, making it essential for a functional DNA repair/FA/BRCA pathway. In a knockout mouse model, Nipa deficiency led to major cell-intrinsic defects, including a premature aging phenotype, with accumulation of DNA damage in hematopoietic stem cells (HSCs). Induction of replication stress triggered a reduction in and functional decline of murine HSCs, resulting in complete bone marrow failure and death of the knockout mice with 100% penetrance. Taken together, the results of our study add NIPA to the short list of FA-associated proteins, thereby highlighting its potential as a diagnostic marker and/or possible target in diseases characterized by hematopoietic failure.
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http://dx.doi.org/10.1172/JCI126215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260023PMC
June 2020

TGF-β Signaling Regulates SLC8A3 Expression and Prevents Oxidative Stress in Developing Midbrain Dopaminergic and Dorsal Raphe Serotonergic Neurons.

Int J Mol Sci 2020 Apr 15;21(8). Epub 2020 Apr 15.

Institute of Anatomy and Cell Biology, Department of Molecular Embryology, Faculty of Medicine, University of Freiburg, Albertstrasse 17, 79104 Freiburg, Germany.

Calcium homeostasis is a cellular process required for proper cell function and survival, maintained by the coordinated action of several transporters, among them members of the Na/Ca-exchanger family, such as SLC8A3. Transforming growth factor beta (TGF-β) signaling defines neuronal development and survival and may regulate the expression of channels and transporters. We investigated the regulation of SLC8A3 by TGF-β in a conditional knockout mouse with deletion of TGF-β signaling from Engrailed 1-expressing cells, i.e., in cells from the midbrain and rhombomere 1, and elucidated the underlying molecular mechanisms. The results show that SLC8A3 is significantly downregulated in developing dopaminergic and dorsal raphe serotonergic neurons in mutants and that low SLC8A3 abundance prevents the expression of the anti-apoptotic protein Bcl-xL. TGF-β signaling affects SLC8A3 via the canonical and p38 signaling pathway and may increase the binding of Smad4 to the promoter. Expression of the lipid peroxidation marker malondialdehyde (MDA) was increased following knockdown of expression in vitro. In neurons lacking TGF-β signaling, the number of MDA- and 4-hydroxynonenal (4-HNE)-positive cells was significantly increased, accompanied with increased cellular 4-HNE abundance. These results suggest that TGF-β contributes to the regulation of SLC8A3 expression in developing dopaminergic and dorsal raphe serotonergic neurons, thereby preventing oxidative stress.
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http://dx.doi.org/10.3390/ijms21082735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216069PMC
April 2020

Oncogenic Kras causes myeloproliferation via NLRP3 inflammasome activation.

Nat Commun 2020 04 3;11(1):1659. Epub 2020 Apr 3.

Department of Medicine I, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany.

Oncogenic Ras mutations occur in various leukemias. It was unclear if, besides the direct transforming effect via constant RAS/MEK/ERK signaling, an inflammation-related effect of KRAS contributes to the disease. Here, we identify a functional link between oncogenic Kras and NLRP3 inflammasome activation in murine and human cells. Mice expressing active Kras in the hematopoietic system developed myeloproliferation and cytopenia, which is reversed in Kras mice lacking NLRP3 in the hematopoietic system. Therapeutic IL-1-receptor blockade or NLRP3-inhibition reduces myeloproliferation and improves hematopoiesis. Mechanistically, Kras-RAC1 activation induces reactive oxygen species (ROS) production causing NLRP3 inflammasome-activation. In agreement with our observations in mice, patient-derived myeloid leukemia cells exhibit KRAS/RAC1/ROS/NLRP3/IL-1β axis activity. Our findings indicate that oncogenic KRAS not only act via its canonical oncogenic driver function, but also enhances the activation of the pro-inflammatory RAC1/ROS/NLRP3/IL-1β axis. This paves the way for a therapeutic approach based on immune modulation via NLRP3 blockade in KRAS-mutant myeloid malignancies.
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http://dx.doi.org/10.1038/s41467-020-15497-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7125138PMC
April 2020

Existence of reprogrammed lymphoma stem cells in a murine ALCL-like model.

Leukemia 2020 12 17;34(12):3242-3255. Epub 2020 Mar 17.

Department of Medicine I, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, 79106, Freiburg, Germany.

While cancer stem cells are well established in certain hematologic and solid malignancies, their existence in T cell lymphoma is unclear and the origin of disease is not fully understood. To examine the existence of lymphoma stem cells, we utilized a mouse model of anaplastic large cell lymphoma. Established NPM-ALK lymphomas contained heterogeneous cell populations ranging from mature T cells to undifferentiated hematopoietic stem cells. Interestingly, CD4/CD8 double negative (DN) lymphoma cells aberrantly expressed the T cell receptor α/β chain. Serial transplantation of sorted CD4/CD8 and DN lymphoma subpopulations identified lymphoma stem cells within the DN3/DN4 T cell population, whereas all other subpopulations failed to establish serial lymphomas. Moreover, transplanted lymphoma DN3/DN4 T cells were able to differentiate and gave rise to mature lymphoma T cells. Gene expression analyses unmasked stem-cell-like transcriptional regulation of the identified lymphoma stem cell population. Furthermore, these lymphoma stem cells are characterized by low CD30 expression levels, which might contribute to limited long-term therapeutic success in patients treated with anti-CD30-targeted therapies. In summary, our results highlight the existence of a lymphoma stem cell population in a NPM-ALK-driven CD30 mouse model, thereby giving the opportunity to test innovative treatment strategies developed to eradicate the origin of disease.
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http://dx.doi.org/10.1038/s41375-020-0789-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685983PMC
December 2020

Fibrinogen induces neural stem cell differentiation into astrocytes in the subventricular zone via BMP signaling.

Nat Commun 2020 01 31;11(1):630. Epub 2020 Jan 31.

Institute of Anatomy and Cell Biology, Faculty of Medicine, University of Freiburg, 79104, Freiburg, Germany.

Neural stem/progenitor cells (NSPCs) originating from the subventricular zone (SVZ) contribute to brain repair during CNS disease. The microenvironment within the SVZ stem cell niche controls NSPC fate. However, extracellular factors within the niche that trigger astrogliogenesis over neurogenesis during CNS disease are unclear. Here, we show that blood-derived fibrinogen is enriched in the SVZ niche following distant cortical brain injury in mice. Fibrinogen inhibited neuronal differentiation in SVZ and hippocampal NSPCs while promoting astrogenesis via activation of the BMP receptor signaling pathway. Genetic and pharmacologic depletion of fibrinogen reduced astrocyte formation within the SVZ after cortical injury, reducing the contribution of SVZ-derived reactive astrocytes to lesion scar formation. We propose that fibrinogen is a regulator of NSPC-derived astrogenesis from the SVZ niche via BMP receptor signaling pathway following injury.
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http://dx.doi.org/10.1038/s41467-020-14466-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994610PMC
January 2020

Long-term epilepsy-associated tumors: transcriptional signatures reflect clinical course.

Sci Rep 2020 01 9;10(1):96. Epub 2020 Jan 9.

Department of Neurosurgery, Medical Center, Freiburg, Germany.

Long-term epilepsy-associated tumors (LEATs) represent mostly benign brain tumors associated with drug-resistant epilepsy. The aim of the study was to investigate the specific transcriptional signatures of those tumors and characterize their underlying oncogenic drivers. A cluster analysis of 65 transcriptome profiles from three independent datasets resulted in four distinct transcriptional subgroups. The first subgroup revealed transcriptional activation of STAT3 and TGF-signaling pathways and contained predominantly dysembryoplastic neuroepithelial tumors (DNTs). The second subgroup was characterized by alterations in the MAPK-pathway and up-stream cascades including FGFR and EGFR-mediated signaling. This tumor cluster exclusively contained neoplasms with somatic BRAF mutations and abundance of gangliogliomas (GGs) with a significantly higher recurrence rate (42%). This finding was validated by examining recurrent tumors from the local database exhibiting BRAF in 90% of the cases. The third cluster included younger patients with neuropathologically diagnosed GGs and abundance of the NOTCH- and mTOR-signaling pathways. The transcript signature of the fourth cluster (including both DNTs and GGs) was related to impaired neural function. Our analysis suggests distinct oncological pathomechanisms in long-term epilepsy-associated tumors. Transcriptional activation of MAPK-pathway and BRAF mutation are associated with an increased risk for tumor recurrence and malignant progression, therefore the treatment of these tumors should integrate both epileptological and oncological aspects.
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http://dx.doi.org/10.1038/s41598-019-56146-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952384PMC
January 2020

A novel conditional NPM-ALK-driven model of CD30+ T-cell lymphoma mediated by a translational stop cassette.

Oncogene 2020 02 21;39(9):1904-1913. Epub 2019 Nov 21.

Department of Hematology, Oncology and Stem Cell Transplantation, Medical Center, Faculty of Medicine, University of Freiburg, 79106, Freiburg, Germany.

Targeted expression of transgenes is essential for the accurate representation of human disease in in vivo models. Current approaches to generate conditional transgenic mouse models are cumbersome and not amenable to high-throughput analysis since they require de novo generation and characterization of genetically modified mice. Here we describe a new system for lineage-restricted expression of transgenes based on a retroviral vector incorporating a translational stop cassette flanked by loxP recombination sites. Conditional transgene expression in chimeric mice is achieved by retroviral infection and transplantation of hematopoietic stem cells (HSC) derived from transgenic mice expressing Cre-recombinase from a lineage-specific promoter. For validation, we directed expression of NPM-ALK, the fusion oncogene driving a subset of anaplastic large cell lymphoma (ALCL), to T-cells by infecting hematopoietic stem cells from Lck-Cre-transgenic mice with a retroviral construct containing the NPM-ALK cDNA preceded by a translational stop cassette. These mice developed T-cell lymphomas within 12-16 weeks, featuring increased expression of the ALCL hallmark antigen CD30 as well as other cytotoxic T-cell markers, similar to the human disease. The new model represents a versatile tool for the rapid analysis of gene function in a defined lineage or in a developmental stage in vivo.
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http://dx.doi.org/10.1038/s41388-019-1058-1DOI Listing
February 2020

Astrogliosis Releases Pro-Oncogenic Chitinase 3-Like 1 Causing MAPK Signaling in Glioblastoma.

Cancers (Basel) 2019 Sep 26;11(10). Epub 2019 Sep 26.

Translational NeuroOncology Research Group, Medical Center, University of Freiburg, 79106 Freiburg, Germany.

Although reactive astrocytes constitute a major component of the cellular environment in glioblastoma, their function and crosstalk to other components of the environment is still poorly understood. Gene expression analysis of purified astrocytes from both the tumor core and non-infiltrated cortex reveals a tumor-related up-regulation of Chitinase 3-like 1 (CHI3L1), a cytokine which is related to inflammation, extracellular tissue remodeling, and fibrosis. Further, we established and validated a co-culture model to investigate the impact of reactive astrocytes within the tumor microenvironment. Here we show that reactive astrocytes promote a subtype-shift of glioblastoma towards the mesenchymal phenotype, driving mitogen-activated protein kinases (MAPK) signaling as well as increased proliferation and migration. In addition, we demonstrate that MAPK signaling is directly caused by a CHI3L1-IL13RA2 co-binding, which leads to increased downstream MAPK and AKT signaling. This novel microenvironmental crosstalk highlights the crucial role of non-neoplastic cells in malignant brain tumors and opens up new perspectives for targeted therapies in glioblastoma.
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http://dx.doi.org/10.3390/cancers11101437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826948PMC
September 2019

MARS: Mutation-Adjusted Risk Score for Advanced Systemic Mastocytosis.

J Clin Oncol 2019 11 11;37(31):2846-2856. Epub 2019 Sep 11.

University Hospital Mannheim, Heidelberg University, Mannheim, Germany.

Purpose: To develop a risk score for patients with advanced systemic mastocytosis (AdvSM) that integrates clinical and mutation characteristics.

Patients And Methods: The study included 383 patients with AdvSM from the German Registry on Disorders of Eosinophils and Mast Cells (training set; n = 231) and several centers for mastocytosis in the United States and Europe, all within the European Competence Network on Mastocytosis (validation set; n = 152). A Cox multivariable model was used to select variables that were predictive of overall survival (OS).

Results: In multivariable analysis, the following risk factors were identified as being associated with OS: age greater than 60 years, anemia (hemoglobin < 10 g/dL), thrombocytopenia (platelets < 100 × 10/L), presence of one high molecular risk gene mutation (ie, in , , and/or ), and presence of two or more high molecular risk gene mutations. By assigning hazard ratio-weighted points to these variables, the following three risk categories were defined: low risk (median OS, not reached), intermediate risk (median OS, 3.9 years; 95% CI, 2.1 to 5.7 years), and high risk (median OS, 1.9 years; 95% CI, 1.3 to 2.6 years; < .001). The mutation-adjusted risk score (MARS) was independent of the WHO classification and was confirmed in the independent validation set. During a median follow-up time of 2.2 years (range, 0 to 23 years), 63 (16%) of 383 patients experienced a leukemic transformation to secondary mast cell leukemia (32%) or secondary acute myeloid leukemia (68%). The MARS was also predictive for leukemia-free survival ( < .001).

Conclusion: The MARS is a validated, five-parameter, WHO-independent prognostic score that defines three risk groups among patients with AdvSM and may improve up-front treatment stratification for these rare hematologic neoplasms.
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http://dx.doi.org/10.1200/JCO.19.00640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823885PMC
November 2019

Boehmite Nanofillers in Epoxy Oligosiloxane Resins: Influencing the Curing Process by Complex Physical and Chemical Interactions.

Materials (Basel) 2019 May 9;12(9). Epub 2019 May 9.

Federal Institute for Material Research and Testing (BAM), 12205 Berlin, Germany.

In this work, a novel boehmite (BA)-embedded organic/inorganic nanocomposite coating based on cycloaliphatic epoxy oligosiloxane (CEOS) resin was fabricated applying UV-induced cationic polymerization. The main changes of the material behavior caused by the nanofiller were investigated with regard to its photocuring kinetics, thermal stability, and glass transition. The role of the particle surface was of particular interest, thus, unmodified nanoparticles (HP14) and particles modified with p-toluenesulfonic acid (OS1) were incorporated into a CEOS matrix in the concentration range of 1-10 wt.%. Resulting nanocomposites exhibited improved thermal properties, with the glass transition temperature (T) being shifted from 30 °C for unfilled CEOS to 54 °C (2 wt.% HP14) and 73 °C (2 wt.% OS1) for filled CEOS. Additionally, TGA analysis showed increased thermal stability of samples filled with nanoparticles. An attractive interaction between boehmite and CEOS matrix influenced the curing. Real-time infrared spectroscopy (RT-IR) experiments demonstrated that the epoxide conversion rate of nanocomposites was slightly increased compared to neat resin. The beneficial role of the BA can be explained by the participation of hydroxyl groups at the particle surface in photopolymerization processes and by the complementary contribution of p-toluenesulfonic acid surface modifier and water molecules introduced into the system with nanoparticles.
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http://dx.doi.org/10.3390/ma12091513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6540208PMC
May 2019

Combination treatment of acute myeloid leukemia cells with DNMT and HDAC inhibitors: predominant synergistic gene downregulation associated with gene body demethylation.

Leukemia 2019 04 23;33(4):945-956. Epub 2018 Nov 23.

Division of Hematology, Oncology and Stem Cell Transplantation, Department of Internal Medicine, Faculty of Medicine and Medical Center, University of Freiburg, Freiburg, Germany.

DNA methyltransferase inhibitors (DNMTi) approved for older AML patients are clinically tested in combination with histone deacetylase inhibitors (HDACi). The mechanism of action of these drugs is still under debate. In colon cancer cells, 5-aza-2'-deoxycytidine (DAC) can downregulate oncogenes and metabolic genes by reversing gene body DNA methylation, thus implicating gene body methylation as a novel drug target. We asked whether DAC-induced gene body demethylation in AML cells is also associated with gene repression, and whether the latter is enhanced by HDACi.Transcriptome analyses revealed that a combined treatment with DAC and the HDACi panobinostat or valproic acid affected significantly more transcripts than the sum of the genes regulated by either treatment alone, demonstrating a quantitative synergistic effect on genome-wide expression in U937 cells. This effect was particularly striking for downregulated genes. Integrative methylome and transcriptome analyses showed that a massive downregulation of genes, including oncogenes (e.g., MYC) and epigenetic modifiers (e.g., KDM2B, SUV39H1) often overexpressed in cancer, was associated predominantly with gene body DNA demethylation and changes in acH3K9/27. These findings have implications for the mechanism of action of combined epigenetic treatments, and for a better understanding of responses in trials where this approach is clinically tested.
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http://dx.doi.org/10.1038/s41375-018-0293-8DOI Listing
April 2019

Droplet digital PCR for the simultaneous analysis of minimal residual disease and hematopoietic chimerism after allogeneic cell transplantation.

Clin Chem Lab Med 2019 04;57(5):641-647

Department of Hematology, Oncology and Stem Cell Transplantation, University of Freiburg, Faculty of Medicine, Freiburg, Germany.

Background Minimal residual disease (MRD) and hematopoietic chimerism testing influences clinical decision and therapeutic intervention in patients after allogeneic stem cell transplantation (HSCT). However, treatment approaches to induce complete donor chimerism and MRD negativity can lead to complications such as graft-versus-host disease (GvHD) and marrow aplasia. Therefore, there is a need for comprehensive characterization of the molecular remission status after transplantation. Methods We analyzed 764 samples from 70 patients after HSCT for the simultaneous measurement of chimerism and molecular targets used for MRD testing with a digital PCR (dPCR) platform. Results Mixed chimerism (MC) was detected in 219 samples from 37 patients. The mean percentage of host derived DNA in these clinical samples was 4.3%. Molecular relapse with a positive MRD marker and/or increased WT1 expression was observed in 15 patients. In addition to WT1 overexpression, other MRD positive markers were: NPM1 (Type A, B, K), DNMT3A (R882H), MLL-PTD, IDH1 (R132H) and KRAS (G12S). Increasing MC was observed in 15 patients. This group of patients showed either a positive MRD marker, increased WT1 expression or both. Next, we analyzed whether MC or the molecular target for MRD was first detected. MC and MRD marker positivity in this group was first detected in six and two patients, respectively. In the remaining seven patients MC and MRD positivity was detected simultaneously. Conclusions The combination of MRD and chimerism markers in a dPCR platform represents a practical, sensitive and accurate diagnostic tool for the comprehensive assessment of the molecular remission status of patients undergoing HSCT.
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http://dx.doi.org/10.1515/cclm-2018-0827DOI Listing
April 2019

miR-146a Controls Immune Response in the Melanoma Microenvironment.

Cancer Res 2019 01 13;79(1):183-195. Epub 2018 Nov 13.

Department of Hematology, Oncology and Stem Cell Transplantation, Freiburg University Medical Center, Albert-Ludwigs-University Freiburg, Freiburg, Germany.

MicroRNAs (miR) are small noncoding RNAs that regulate gene expression, posttranscription, and manipulate immune responses in different types of cancers. In this study, we identify miR-146a as a negative regulator of immune activation, comparable to immune-checkpoint molecules. miR-146a levels were increased in melanoma microenvironmental tissue, and mice survived longer and developed less metastases in comparison with wild-type melanoma-bearing mice. T cells isolated from mice revealed higher expression levels of the miR-146a target gene Stat1 and the Stat1-regulated cytokine IFNγ. Neutralization of IFNγ in mice decreased survival and increased melanoma metastasis patterns to those of wild-type mice. , IFNγ reduced melanoma cell migration, cell-cycle activity, and basal metabolic rate. Conversely, IFNγ also increased PD-L1 levels on the melanoma cells, which may counterbalance some of the beneficial effects increasing immune escape . Combined treatment with a miR-146a antagomiR and anti-PD-1 resulted in improved survival over isotype control or anti-PD-1 treatment alone. In summary, these data show that miR-146a plays a central role within the STAT1/IFNγ axis in the melanoma microenvironment, affecting melanoma migration, proliferation, and mitochondrial fitness as well as PD-L1 levels. Additionally, combined inhibition of PD-1 and miR-146a could be a novel strategy to enhance antitumor immune response elicited by checkpoint therapy. SIGNIFICANCE: These findings identify a microRNA-based mechanism by which melanoma cells escape the immune system, providing a new therapeutic strategy to improve the current management of patients with melanoma.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-1397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6330089PMC
January 2019

Silencing of TGFβ signalling in microglia results in impaired homeostasis.

Nat Commun 2018 10 1;9(1):4011. Epub 2018 Oct 1.

Institute for Anatomy and Cell Biology, Department of Molecular Embryology, Faculty of Medicine, University of Freiburg, 79104, Freiburg, Germany.

TGFβ1 has been implicated in regulating functional aspects of several distinct immune cell populations including central nervous system (CNS) resident microglia. Activation and priming of microglia have been demonstrated to contribute to the progression of neurodegenerative diseases and, thus, underlie stringent control by endogenous regulatory factors including TGFβ1. Here, we demonstrate that deletion of Tgfbr2 in adult postnatal microglia does neither result in impairment of the microglia-specific gene expression signatures, nor is microglial survival and maintenance affected. Tgfbr2-deficient microglia were characterised by distinct morphological changes and transcriptome analysis using RNAseq revealed that loss of TGFβ signalling results in upregulation of microglia activation and priming markers. Moreover, protein arrays demonstrated increased secretion of CXCL10 and CCL2 accompanied by activation of immune cell signalling as evidenced by increased phosphorylation of TAK1. Together, these data underline the importance of microglial TGFβ signalling to regulate microglia adaptive changes.
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http://dx.doi.org/10.1038/s41467-018-06224-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6167353PMC
October 2018
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