The genetic hallmark of Burkitt lymphoma (BL) is the t(8;14)(q24;q32) and its variants leading to activation of the MYC oncogene. It is a matter of debate whether true BL without MYC translocation ...exists. Here, we identified 59 lymphomas concordantly called BL by 2 gene expression classifiers among 753 B-cell lymphomas. Only 2 (3%) of these 59 molecular BL lacked a MYC translocation, which both shared a peculiar pattern of chromosome 11q aberration characterized by interstitial gains including 11q23.2-q23.3 and telomeric losses of 11q24.1-qter. We extended our analysis to 17 MYC-negative high-grade B-cell lymphomas with a similar 11q aberration and showed this aberration to be recurrently associated with morphologic and clinical features of BL. The minimal region of gain was defined by high-level amplifications in 11q23.3 and associated with overexpression of genes including PAFAH1B2 on a transcriptional and protein level. The recurrent region of loss contained a focal homozygous deletion in 11q24.2-q24.3 including the ETS1 gene, which was shown to be mutated in 4 of 16 investigated cases. These findings indicate the existence of a molecularly distinct subset of B-cell lymphomas reminiscent of BL, which is characterized by deregulation of genes in 11q.
•A subset of lymphomas with gene expression and pathological characteristics of Burkitt lymphomas but absence of MYC translocation does exist.•These lymphomas carry chr 11q proximal gains and telomeric losses, suggesting co-deregulation of oncogenes and tumor suppressor genes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract Background At a global scale, the SARS-CoV-2 virus did not remain in its initial genotype for a long period of time, with the first global reports of variants of concern (VOCs) in late 2020. ...Subsequently, genome sequencing has become an indispensable tool for characterizing the ongoing pandemic, particularly for typing SARS-CoV-2 samples obtained from patients or environmental surveillance. For such SARS-CoV-2 typing, various in vitro and in silico workflows exist, yet to date, no systematic cross-platform validation has been reported. Results In this work, we present the first comprehensive cross-platform evaluation and validation of in silico SARS-CoV-2 typing workflows. The evaluation relies on a dataset of 54 patient-derived samples sequenced with several different in vitro approaches on all relevant state-of-the-art sequencing platforms. Moreover, we present UnCoVar, a robust, production-grade reproducible SARS-CoV-2 typing workflow that outperforms all other tested approaches in terms of precision and recall. Conclusions In many ways, the SARS-CoV-2 pandemic has accelerated the development of techniques and analytical approaches. We believe that this can serve as a blueprint for dealing with future pandemics. Accordingly, UnCoVar is easily generalizable towards other viral pathogens and future pandemics. The fully automated workflow assembles virus genomes from patient samples, identifies existing lineages, and provides high-resolution insights into individual mutations. UnCoVar includes extensive quality control and automatically generates interactive visual reports. UnCoVar is implemented as a Snakemake workflow. The open-source code is available under a BSD 2-clause license at github.com/IKIM-Essen/uncovar .
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
3.
Origin and Pathogenesis of B Cell Lymphomas Seifert, Marc; Scholtysik, René; Küppers, Ralf
Methods in molecular biology (Clifton, N.J.),
01/2019, Volume:
1956
Journal Article
Immunoglobulin (IG) gene remodeling by V(D)J recombination plays a central role in the generation of normal B cells, and somatic hypermutation and class switching of IG genes are key processes during ...antigen-driven B cell differentiation. However, errors of these processes are involved in the development of B cell lymphomas. IG locus-associated translocations of proto-oncogenes are a hallmark of many B cell malignancies. Additional transforming events include inactivating mutations in various tumor suppressor genes and also latent infection of B cells with viruses, such as Epstein-Barr virus. Many B cell lymphomas require B cell antigen receptor expression, and in several instances, chronic antigenic stimulation plays a role in lymphoma development and/or sustaining tumor growth. Often, survival and proliferation signals provided by other cells in the microenvironment are a further critical factor in lymphoma development and pathophysiology. Many B cell malignancies derive from germinal center B cells, most likely because of the high proliferation rate of these cells and the high activity of mutagenic processes.
Plant phytohormone pathways are regulated by an intricate network of signaling components and modulators, many of which still remain unknown. Here, we report a forward chemical genetics approach for ...the identification of functional SA agonists in Arabidopsis thaliana that revealed Neratinib (Ner), a covalent pan-HER kinase inhibitor drug in humans, as a modulator of SA signaling. Instead of a protein kinase, chemoproteomics unveiled that Ner covalently modifies a surface-exposed cysteine residue of Arabidopsis epoxide hydrolase isoform 7 (AtEH7), thereby triggering its allosteric inhibition. Physiologically, the Ner application induces jasmonate metabolism in an AtEH7-dependent manner as an early response. In addition, it modulates PATHOGENESIS RELATED 1 (PR1) expression as a hallmark of SA signaling activation as a later effect. AtEH7, however, is not the exclusive target for this physiological readout induced by Ner. Although the underlying molecular mechanisms of AtEH7-dependent modulation of jasmonate signaling and Ner-induced PR1-dependent activation of SA signaling and thus defense response regulation remain unknown, our present work illustrates the powerful combination of forward chemical genetics and chemical proteomics for identifying novel phytohormone signaling modulatory factors. It also suggests that marginally explored metabolic enzymes such as epoxide hydrolases may have further physiological roles in modulating signaling.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
Burkitt lymphoma is a mature aggressive B-cell lymphoma derived from germinal center B cells. Its cytogenetic hallmark is the Burkitt translocation t(8;14)(q24;q32) and its variants, which juxtapose ...the MYC oncogene with one of the three immunoglobulin loci. Consequently, MYC is deregulated, resulting in massive perturbation of gene expression. Nevertheless, MYC deregulation alone seems not to be sufficient to drive Burkitt lymphomagenesis. By whole-genome, whole-exome and transcriptome sequencing of four prototypical Burkitt lymphomas with immunoglobulin gene (IG)-MYC translocation, we identified seven recurrently mutated genes. One of these genes, ID3, mapped to a region of focal homozygous loss in Burkitt lymphoma. In an extended cohort, 36 of 53 molecularly defined Burkitt lymphomas (68%) carried potentially damaging mutations of ID3. These were strongly enriched at somatic hypermutation motifs. Only 6 of 47 other B-cell lymphomas with the IG-MYC translocation (13%) carried ID3 mutations. These findings suggest that cooperation between ID3 inactivation and IG-MYC translocation is a hallmark of Burkitt lymphomagenesis.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The Gram-negative bacterium Neisseria meningitidis (Nm) can cause meningitis in humans, but the host signalling pathways manipulated by Nm during central nervous system (CNS) entry are not completely ...understood.
We investigate the role of the mitogen-activated protein kinases (MAPK) Erk1/2 and p38 in an in vitro model of the blood-cerebrospinal fluid barrier (BCSFB) based on human epithelial choroid plexus (CP) papilloma (HIBCPP) cells during infection with Nm serogroup B (NmB) and serogroup C (NmC) strains. A transcriptome analysis of HIBCPP cells following infection with Nm by massive analysis of cDNA ends (MACE) was done to further characterize the cellular response to infection of the barrier.
Interestingly, whereas NmB and NmC wild type strains required active Erk1/2 and p38 pathways for infection, invasion by capsule-deficient mutants was independent of Erk1/2 and, in case of the NmB strain, of p38 activity. The transcriptome analysis of HIBCPP cells following infection with Nm demonstrated specific regulation of genes involved in the immune response dependent on Erk1/2 signalling. Gene ontology (GO) analysis confirmed loss of MAPK signalling after Erk1/2 inhibition and revealed an additional reduction of cellular responses including NFκB and JAK-STAT signalling. Interestingly, GO terms related to TNF signalling and production of IL6 were lost specifically following Erk1/2 inhibition during infection with wild type Nm, which correlated with the reduced infection rates by the wild type in absence of Erk1/2 signalling.
Our data point towards a role of MAPK signalling during infection of the CP epithelium by Nm, which is strongly influenced by capsule expression, and affects infection rates as well as the host cell response.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Abstract 3512
T-cell large granular lymphocytic leukemia (T-LGL) is a rare lymphoproliferative disease characterized by an expansion of large granular lymphocytes involving blood, bone marrow, spleen ...and liver. T-LGL cells are mature CD3, CD8 and T-cell receptor (TCR) αβ positive cells exhibiting the immunophenotype of activated cytotoxic T lymphocytes (CTLs). CD4 or TCR γδ positive variants occur rarely. T-LGL affects adults at a median age of 55–60 years and arises commonly in patients with a preexisting autoimmune disorder. Many patients remain asymptomatic for years and do not require treatment. Palliative therapy with immunosuppressant agents such as low dose methotrexate, ciclosporin and fludarabine is used for the correction of severe immune-mediated cytopenias, which often complicates the course of the disease. The molecular pathogenesis of T-LGL remains unclear. No recurrent karyotypic anomalities but several numeric and structural chromosomal alterations have been identified. Recently, activating somatic mutations in the signal transducer and activator of transcription 3 gene (STAT3) have been described by Koskela et al. in approximately 40% of T-LGL patients. STAT3 mutations lead to an increased transcriptional activity and were more prevalent in patients with neutropenia and rheumatoid arthritis than in patients without these conditions. As these findings only explain part of the pathogenesis in the fraction of patients affected by STAT3 mutations, we here aimed to identify novel mutations which may help to better understand the mechanisms of disease development.
We sorted tumor- and non-tumor cells from peripheral blood samples of T-LGL patients by using fluorescence activated cell sorting (FACSDiva®, Becton Dickinson) to perform single nucleotide polymorphism (SNP) chip analysis and next-generation RNA sequencing. SNP chips were analyzed in 10 patients (Affymetrix, Mapping 250K Sty Array®). To identify somatic mutations in patients with T-LGL, we compared CD8/CD57 positive tumor cells with non-tumor cells as germline control. Sample libraries for RNA sequencing of 5 patients were generated with NuGEN Encore®, sequencing was performed on Illumina HiSeq 2000® yielding 100 million 100 basepair single reads, and alignment was realized on TopHat2 against hg19 as reference genome. For quantification and analysis of variants Partek GS 6.6 was used.
High resolution copy number determination employing SNP chips in 10 patients revealed both gains and losses on different chromosomes, among others 1q, 7q, 14q and chromosome X. The affected chromosomal regions included genes with potential relevance to the disease process such as WNT and RASSF gene family members in deleted regions and PIM3 and MAPK family members in gained regions. However, in line with previous reports no recurrent chromosomal aberrations were detected. Preliminary analysis of RNA sequencing data revealed activating STAT3 Y640F mutations in 2 out of 5 patients tested (40%). Interestingly, one of the STAT3 mutated T-LGL clones also exhibited an inactivating mutation of the NFKB inhibitory gene TNFAIP3 (A20), which has been reported to play an important role in the molecular pathogenesis of different B cell lymphomas but has as yet not been described in T-LGL. Detailed analysis of sequencing data is currently ongoing and further results will be presented at the conference. In conclusion, combined RNA sequencing and molecular cytogenetic profiling identified novel specific chromosomal loci and genes that could help to better understand the molecular pathogenesis of T-LGL and develop novel targeted treatment modalities for this disease.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Despite several new developments in the treatment of multiple myeloma, all available therapies are only palliative without curative potential and all patients ultimately relapse. Thus, novel ...therapeutic options are urgently required to prolong survival of or to even cure myeloma. Here, we show that multiple myeloma cells express the potassium channel Kv1.3 in their mitochondria. The mitochondrial Kv1.3 inhibitors PAPTP and PCARBTP are efficient against two tested human multiple myeloma cell lines (L-363 and RPMI-8226) and against ex vivo cultured, patient-derived myeloma cells, while healthy bone marrow cells are spared from toxicity. Cell death after treatment with PAPTP and PCARBTP occurs via the mitochondrial apoptotic pathway. In addition, we identify up-regulation of the multidrug resistance pump MDR-1 as the main potential resistance mechanism. Combination with ABT-199 (venetoclax), an inhibitor of Bcl2, has a synergistic effect, suggesting that mitochondrial Kv1.3 inhibitors could potentially be used as combination partner to venetoclax, even in the treatment of t(11;14) negative multiple myeloma, which represent the major part of cases and are rather resistant to venetoclax alone. We thus identify mitochondrial Kv1.3 channels as druggable targets against multiple myeloma.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Summary
A reciprocal interaction exists between the gut microbiota and the immune system. Regulatory T (Treg) cells are important for controlling immune responses and for maintaining the intestinal ...homeostasis but their precise influence on the gut microbiota is unclear. We studied the effects of Treg cell depletion on inflammation of the intestinal mucosa and analysed the gut microbiota before and after depletion of Treg cells using the DEpletion of REGulatory T cells (DEREG) mouse model. DNA was extracted from stool samples of DEREG mice and wild‐type littermates at different time‐points before and after diphtheria toxin application to deplete Treg cells in DEREG mice. The V3/V4 region of the 16S rRNA gene was used for studying the gut microbiota with Illumina MiSeq paired ends sequencing. Multidimensional scaling separated the majority of gut microbiota samples from late time‐points after Treg cell depletion in DEREG mice from samples of early time‐points before Treg cell depletion in these mice and from gut microbiota samples of wild‐type mice. Treg cell depletion in DEREG mice was accompanied by an increase in the relative abundance of the phylum Firmicutes and by intestinal inflammation in DEREG mice 20 days after Treg cell depletion, indicating that Treg cells influence the gut microbiota composition. In addition, the variables cage, breeding and experiment number were associated with differences in the gut microbiota composition and these variables should be respected in murine studies.
In this study we showed that temporal regulatory T (Treg) cell depletion in DEpletion of REGulatory T cells mice was accompanied by an increase of the relative abundance of the phylum Firmicutes of the gut microbiota, indicating that Treg cells affect the gut microbiota composition. In addition, Treg cell depletion was associated with intestinal inflammation. Furthermore, the variables cage, breeding and experiment number affected the gut microbiota composition and should be respected in murine gut microbiota studies
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
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