Expressionsanalysen nicht-kodierender RNAs in malignen B-Zell (Non-Hodgkin) Lymphomen

MicroRNAs are small (20-23nt in length), non-coding and highly conserved molecules, which are involved in several regulatory processes like cell growth, proliferation, differentiation, immune response and apoptosis, and play important roles in several diseases, including cancers like lymphoma. Germinal center (GC) derived B-cell lymphomas, including Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), are the most frequent malignant lymphomas. Although clear distinctions on histologic and genetic grounds exist, there are also a large number of cases with intermediate features, not unequivocally attributable to one of these entities. The ICGC-MMML-Seq Consortium aims at fully characterizing a total of 250 GC derived B-cell lymphomas. Here we generated miRNA profiles from 56 patient samples including BL, DLBCL and FL using Illumina technology. Over the past decade, many studies have attempted to distinguish lymphoma subtypes using miRNAs profiling. However, available data is preliminary, as published profiles are not derived from large sample collections and also originate mostly from PCR-based approaches and microarrays. Yet, only sequencing-based approaches allow for an unbiased analysis and the discovery of novel miRNAs and small RNA classes. Our initial differential expression analyses comparing BL against non-BL showed eight miRNAs to be differentially expressed. In addition, we analyzed miRNA deregulation between non-BL subtypes including FL and DLBCL and also compared each of them separately to BL. A signature of 87, 98 and 108 miRNAs was obtained that differentiated FL from DLBCL, BL from DLBCL and BL from FL, respectively. Mutational analysis identified 17 mutations in 12 patients corresponding to eight distinct miRNAs. Among eight mutated miRNAs, miR-142 with a total of seven different mutations in six patients was the most frequently mutated miRNA. Among predicted novel miRNAs, we successfully validated four candidates by Northern Blot experiments and we then tried to uncover their function in lymphomagenesis by performing further functional studies. In addition, our data gave insight into the role of lncRNAs in GCB-lymphomas. We found the differential expression as well as differential methylation pattern of the lncRNA AP000251 in GCB-lymphoma subtypes. To investigate which miRNA-target pairs are more likely to display regulation in lymphoma, we performed AGO2-PAR-CLIP in four lymphoma (Raji, NAMALWA, SU-DHL-4 and SU-DHL-6) cell lines. We identified putative miRNA- targets from each PAR-CLIP library which might represent a helpful tool to find potential therapeutic targets and prognostic markers in lymphoma.MicroRNAs sind kurze (20-23 Nukleotide lange), nicht-kodierende und hoch konservierte Moleküle, die an vielen regulatorischen Prozessen wie z.B. Zellwachstum, Proliferation, Differenzierung, Immunantwort und Apoptose beteiligt sind. Darüber hinaus spielen sie eine wichtige Rolle in zahlreichen Krankheiten, u.a. in verschiedenen Krebsarten wie z.B. Lymphomen. Keimzentrums-B-Zell-Lymphome, zu denen Burkitt-Lymphome (BL), diffus großzellige B-Zell-Lymphome (DLBCL) und follikuläre Lymphome (FL) zählen, stellen die häufigsten aggressiven Lymphome dar. Und auch wenn prinzipiell eindeutige histologische und genetische Unterscheidungskriterien beschrieben wurden, existieren in der täglichen Praxis doch zahlreiche Fälle mit intermediärem Phänotyp, die sich nicht eindeutig in eine der o.g. Kategorien einordnen lassen. Das ICGC-MMML-Seq Konsortium hat sich die vollständige Charakterisierung von 250 Keimzentrums-B-Zell-Lymphomen zum Ziel gesetzt. Im Rahmen dieser Arbeit wurden miRNA Profile von 56 Patientenproben (zusammengesetzt aus BL, DLBCL und FL) mit Illumina Technology sequenziert. In den letzten zehn Jahren haben verschiedene Studien versucht, die Lymphomsubtypen an Hand von miRNA Profilen zu unterscheiden. Die in diesem Rahmen generierten Daten sind jedoch insoweit nur als vorläufig zu betrachten, da sie zum einen nicht aus großen Patientenkollektiven rekrutiert wurden und zum anderen auf PCR-basierenden Methoden zurückgehen. Nur eine auf den neuen Sequenziertechniken beruhende Herangehensweise erlaubt jedoch die unverzerrte Analyse und die Möglichkeit, sowohl neue miRNAs als auch neue Klassen kleiner RNAs zu entdecken. Im ersten Schritt wurden Burkitt-Lymphome mit nicht- Burkitt-Lymphome verglichen, dort zeigte sich die differentielle Expression von acht miRNAs. Zusätzlich wurde die miRNA Deregulation zwischen FL und DLBCL sowie auch jeweils individuell gegen BL analysiert. So konnten Expressionssignaturen beschrieben werden, die FL von DLBCL (87 miRNAs), BL von DLBCL (98 miRNAs) und BL von FL (108 miRNAs) unterscheiden. In der Mutationsanalyse basierend auf 12 Patientendatensätzen wurden 17 Mutationen, die acht verschiedene miRNAs betrafen, gefunden. Am häufigsten war miRNA-142 mit insgesamt sieben Mutationen in sechs Patienten betroffen. Unter allen bioinformatisch vorhergesagten neuen miRNAs konnten vier Kandidaten durch Northern Blot Experimente validiert werden. Anschließend wurde damit begonnen, deren Rolle in der Lymphomentstehung durch funktionelle Studien näher zu charakterisieren. Darüber hinaus gewährend die Sequenzierdaten Einblicke in die Rolle von langen nicht-kodierenden RNAs (lncRNAs). So konnte z.B. ein differentielles Methylierungs- und Expressionsmuster der lncRNA AP000251 beschrieben werden. Um zu klären, welche miRNA-mRNA Regulationspaare eine relevante Aufgabe in Lymphomen übernehmen, wurden AGO2-PAR-CLIP Experimente in vier Lymphomzelllinien (Raji, NAMALWA, SU-DHL-4, SU-DHL-6) durchgeführt. Die identifizierten mRNAs, die eine validierte Regulation durch miRNAs zeigen, können nun als Ausgangspunkt dienen, um mögliche therapeutisch nutzbare Strukturen sowie prognostische Marker in Lymphomen zu beschreiben

Alterations of microRNA and microRNA-regulated messenger RNA expression in germinal center B-cell lymphomas determined by integrative sequencing analysis

MicroRNA are well-established players in post-transcriptional gene regulation. However, information on the effects of microRNA deregulation mainly relies on bioinformatic prediction of potential targets, whereas proof of the direct physical microRNA/target messenger RNA interaction is mostly lacking. Within the International Cancer Genome Consortium Project “Determining Molecular Mechanisms in Malignant Lymphoma by Sequencing”, we performed miRnome sequencing from 16 Burkitt lymphomas, 19 diffuse large B-cell lymphomas, and 21 follicular lymphomas. Twenty-two miRNA separated Burkitt lymphomas from diffuse large B-cell lymphomas/follicular lymphomas, of which 13 have shown regulation by MYC. Moreover, we found expression of three hitherto unreported microRNA. Additionally, we detected recurrent mutations of hsa-miR-142 in diffuse large B-cell lymphomas and follicular lymphomas, and editing of the hsa-miR-376 cluster, providing evidence for microRNA editing in lymphomagenesis. To interrogate the direct physical interactions of microRNA with messenger RNA, we performed Argonaute-2 photoactivatable ribonucleoside- enhanced cross-linking and immunoprecipitation experiments. MicroRNA directly targeted 208 messsenger RNA in the Burkitt lymphomas and 328 messenger RNA in the non-Burkitt lymphoma models. This integrative analysis discovered several regulatory pathways of relevance in lymphomagenesis including Ras, PI3K-Akt and MAPK signaling pathways, also recurrently deregulated in lymphomas by mutations. Our dataset reveals that messenger RNA deregulation through microRNA is a highly relevant mechanism in lymphomagenesis

YBX1 Indirectly Targets Heterochromatin-Repressed Inflammatory Response-Related Apoptosis Genes through Regulating CBX5 mRNA.

Medulloblastomas arise from undifferentiated precursor cells in the cerebellum and account for about 20% of all solid brain tumors during childhood; standard therapies include radiation and chemotherapy, which oftentimes come with severe impairment of the cognitive development of the young patients. Here, we show that the posttranscriptional regulator Y-box binding protein 1 (YBX1), a DNA- and RNA-binding protein, acts as an oncogene in medulloblastomas by regulating cellular survival and apoptosis. We observed different cellular responses upon YBX1 knockdown in several medulloblastoma cell lines, with significantly altered transcription and subsequent apoptosis rates. Mechanistically, PAR-CLIP for YBX1 and integration with RNA-Seq data uncovered direct posttranscriptional control of the heterochromatin-associated gene CBX5; upon YBX1 knockdown and subsequent CBX5 mRNA instability, heterochromatin-regulated genes involved in inflammatory response, apoptosis and death receptor signaling were de-repressed. Thus, YBX1 acts as an oncogene in medulloblastoma through indirect transcriptional regulation of inflammatory genes regulating apoptosis and represents a promising novel therapeutic target in this tumor entity

Transcriptome-wide analysis uncovers the targets of the RNA-binding protein MSI2 and effects of MSI2's RNA-binding activity on IL-6 signaling

The RNA-binding protein Musashi 2 (MSI2) has emerged as an important regulator in cancer initiation, progression, and drug resistance. Translocations and deregulation of the MSI2 gene are diagnostic of certain cancers, including chronic myeloid leukemia (CML) with translocation t(7;17), acute myeloid leukemia (AML) with translocation t(10;17), and some cases of B-precursor acute lymphoblastic leukemia (pB-ALL). To better understand the function of MSI2 in leukemia, the mRNA targets that are bound and regulated by MSI2 and their MSI2-binding motifs need to be identified. To this end, using photoactivatable ribonucleoside cross-linking and immunoprecipitation (PAR-CLIP) and the Multiple EM for Motif Elicitation (MEME) analysis tool, here we identified MSI2’s mRNA targets and the consensus RNA-recognition element (RRE) motif recognized by MSI2 (UUAG). Of note, MSI2 knockdown altered the expression of several genes with roles in eukaryotic initiation factor 2 (eIF2), hepatocyte growth factor (HGF), and epidermal growth factor (EGF) signaling pathways. We also show that MSI2 regulates classic interleukin-6 (IL-6) signaling by promoting the degradation of the mRNA of IL-6 signal transducer (IL6ST or GP130), which, in turn, affected the phosphorylation statuses of signal transducer and activator of transcription 3 (STAT3) and the mitogen-activated protein kinase ERK. In summary, we have identified multiple MSI2-regulated mRNAs and provided evidence that MSI2 controls IL6ST activity that control oncogenic signaling networks. Our findings may help inform strategies for unraveling the role of MSI2 in leukemia to pave the way for the development of targeted therapies

GCN2 drives macrophage and MDSC function and immunosuppression in the tumor microenvironment

General control nonderepressible 2 (GCN2) is an environmental sensor controlling transcription and translation in response to nutrient availability. Although GCN2 is a putative therapeutic target for immuno-oncology, its role in shaping the immune response to tumors is poorly understood. Here, we used mass cytometry, transcriptomics, and transcription factor-binding analysis to determine the functional impact of GCN2 on the myeloid phenotype and immune responses in melanoma. We found that myeloid-lineage deletion of GCN2 drives a shift in the phenotype of tumor-associated macrophages and myeloid-derived suppressor cells (MDSCs) that promotes antitumor immunity. Time-of-flight mass cytometry (CyTOF) and single-cell RNA sequencing showed that this was due to changes in the immune microenvironment with increased proinflammatory activation of macrophages and MDSCs and interferon-gamma expression in intratumoral CD8(+) T cells. Mechanistically, GCN2 altered myeloid function by promoting increased translation of the transcription factor CREB-2/ATF4, which was required for maturation and polarization of macrophages and MDSCs in both mice and humans, whereas targeting Atf4 by small interfering RNA knockdown reduced tumor growth. Last, analysis of patients with cutaneous melanoma showed that GCN2-dependent transcriptional signatures correlated with macrophage polarization, T cell infiltrates, and overall survival. Thus, these data reveal a previously unknown dependence of tumors on myeloid GCN2 signals for protection from immune attack

Alterations of microRNA and microRNA-regulated messenger RNA expression in germinal center B-cell lymphomas determined by integrative sequencing analysis

MicroRNA are well-established players in post-transcriptional gene regulation. However, information on the effects of microRNA deregulation mainly relies on bioinformatic prediction of potential targets, whereas proof of the direct physical microRNA/target messenger RNA interaction is mostly lacking. Within the International Cancer Genome Consortium Project “Determining Molecular Mechanisms in Malignant Lymphoma by Sequencing”, we performed miRnome sequencing from 16 Burkitt lymphomas, 19 diffuse large B-cell lymphomas, and 21 follicular lymphomas. Twentytwo miRNA separated Burkitt lymphomas from diffuse large B-cell lymphomas/follicular lymphomas, of which 13 have shown regulation by MYC. Moreover, we found expression of three hitherto unreported microRNA. Additionally, we detected recurrent mutations of hsa-miR-142 in diffuse large B-cell lymphomas and follicular lymphomas, and editing of the hsa-miR-376 cluster, providing evidence for microRNA editing in lymphomagenesis. To interrogate the direct physical interactions of microRNA with messenger RNA, we performed Argonaute-2 photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation experiments. MicroRNA directly targeted 208 messsenger RNA in the Burkitt lymphomas and 328 messenger RNA in the non-Burkitt lymphoma models. This integrative analysis discovered several regulatory pathways of relevance in lymphomagenesis including Ras, PI3KAkt and MAPK signaling pathways, also recurrently deregulated in lymphomas by mutations. Our dataset reveals that messenger RNA deregulation through microRNA is a highly relevant mechanism in lymphomagenesis

Alterations of microRNA and microRNA-regulated messenger RNA expression in germinal center B-cell lymphomas determined by integrative sequencing analysis

MicroRNAs are well-established players in posttranscriptional gene regulation. However, information on the effects of microRNA deregulation mainly relies on bioinformatic prediction of potential targets, whereas proof of the direct physical microRNAs/target mRNAs interaction is mostly lacking. Within the International Cancer Genome Consortium Project Determining Molecular Mechanisms in Malignant Lymphoma by Sequencing (ICGC MMML-Seq), we performed miRnome sequencing from 16 Burkitt lymphomas, 19 diffuse large B-cell lymphomas, and 21 follicular lymphomas. Twenty-two miRNAs separated Burkitt lymphomas from diffuse large B-cell lymphomas/follicular lymphomas, of which 13 have shown regulation by MYC. Moreover, we show expression of three hitherto unreported microRNAs. Additionally, we detect recurrent mutations of hsa-miR-142 in diffuse large B-cell lymphomas and follicular lymphomas, and editing of the hsa-miR-376 cluster, providing evidence for microRNA editing in lymphomagenesis. To interrogate the direct physical interactions of microRNAs with mRNAs, we performed Argonaute-2 photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation experiments. MicroRNAs directly targeted 208 mRNAs in the Burkitt lymphomas and 328 mRNAs in the non-Burkitt lymphoma models. This integrative analysis discovered several regulatory pathways of relevance in lymphomagenesis including Ras, PI3K-Akt and MAPK signaling pathways, also recurrently deregulated in lymphomas by mutations. Our dataset uncovers in detail the mRNA deregulation through microRNAs as a highly relevant mechanism in lymphomagenesis