Die Rolle des Transkriptionsfaktors MYC in Lymphomen

The transcription factor MYC, encoded by the MYC gene, plays a central role in many cellular processes, such as cell growth, apoptosis and cell communication. However, as an oncogene, MYC also plays a central role in initiation and progression of many different types of cancers, including malignant lymphomas, and is therefore the focus of many oncological studies. Malignant lymphomas do not refer to a single disease entity, but describe a broad range of lymphatic neoplasias that derive from mature lymphoid cells. They can be subclassified into over 60 subtypes based on their differentiation, morphology and/or clinical course. Based on histology, malignant lymphomas can be generally distinguished into Hodgkin's lymphoma (HL) and Non-Hodgkin's lymphoma (NHL). NHL can be further sub-grouped according to their cell of origin into B- and T-cell NHL. Further subclassification exists based on additional histological, clinical and molecular criteria including chromosomal alterations and gene expression profiles. The present work deals with Burkitt's lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL), which both belong to aggressive high-grade B-cell lymphoma. Here, we explore the role of the transcription factor MYC in the pathogenesis and clinical course of these lymphoma types. One hallmark of BL is the t(8;14) chromosomal translocation, leading to an overexpression of MYC protein. MYC translocations are however, not restricted to BL, but can also occur in DLBCL, although at a much lower frequency. MYC break-positive BL differ significantly from the MYC break-positive DLBCL in their clinical course, with BL associated with a complete cure of the majority of cases. To investigate the molecular differences between these two lymphoma subtypes, we performed a metabolic and proteomic study, identifying pyruvate as one of the discriminatory metabolites. This metabolic phenotype was further confirmed by proteomic studies of pyruvate metabolism-associated proteins (Schwarzfischer, et al., 2017). In a second study, the genome-wide MYC binding pattern of BL, MYC breakpositive and -negative DLBCL was analyzed by chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) and RNA-based next generation sequencing (RNA-Seq). Significant differences in the MYC DNA-binding patterns were identified, which were also mirrored in the different gene expression patterns. One of these differentially expressed genes code for the cell surface receptor CD97 (ADGRE5), which is significantly over-expressed in BL, but absent in MYC breakpositive and -negative DLBCL. This finding was confirmed by independent validation experiments, including immunohistological staining of cell lines and primary patient samples (Kleo et al 2018 – submitted). This doctoral thesis was complemented by an investigation of long noncoding RNAs (lncRNAs) and their role in modulating the MYC-driven cellular transcriptome. Using NGS, we identified 13 lncRNAs, which were differentially expressed between BL and DLBCLs, one of which was strongly regulated by MYC. This IncRNA was able to modulate MYC-induced cell cycle genes with a strong impact on cell cycle progression. We therefore called this lncRNA MINCR (MYC-induced non-coding RNA) (Doose, et al., 2015). Taken together, this thesis provides additional evidence that MYC is not merely an on/off amplifier of gene activity but exerts specific actions on the gene expression program and – as a consequence – on cellular functions, a finding also true for aggressive lymphoma. Based on the MYC differences between BL and DLBCL, the identification of biomarkers for their distinction appears to be possible. Therefore it is justified to conclude that MYC plays an essential but diverse role in the pathogenesis of various lymphoma types, a finding which might be important for future treatment modalities. The analyses of this work were partly conducted in cooperation with other research groups and led to three publications, which provide the scientific basis for this cumulative thesis

DZNep-mediated apoptosis in B-cell lymphoma is independent of the lymphoma type, EZH2 mutation status and MYC, BCL2 or BCL6 translocations

Enhancer of zeste homolog 2 (EZH2) tri-methylates histone 3 at position lysine 27 (H3K27me3). Overexpression and gain-of-function mutations in EZH2 are regarded as oncogenic drivers in lymphoma and other malignancies due to the silencing of tumor suppressors and differentiation genes. EZH2 inhibition is sought to represent a good strategy for tumor therapy. In this study, we treated Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) cell lines with 3-deazaneplanocin-A (DZNep), an indirect EZH2 inhibitor which possesses anticancer properties both in-vitro and in-vivo. We aimed to address the impact of the lymphoma type, EZH2 mutation status, as well as MYC, BCL2 and BCL6 translocations on the sensitivity of the lymphoma cell lines to DZNep-mediated apoptosis. We show that DZNep inhibits proliferation and induces apoptosis of these cell lines independent of the type of lymphoma, the EZH2 mutation status and the MYC, BCL2 and BCL6 rearrangement status. Furthermore, DZNep induced a much stronger apoptosis in majority of these cell lines at a lower concentration, and within a shorter period when compared with EPZ-6438, a direct EZH2 inhibitor currently in phase II clinical trials. Apoptosis induction by DZNep was both concentration-dependent and time-dependent, and was associated with the inhibition of EZH2 and subsequent downregulation of H3K27me3 in DZNep-sensitive cell lines. Although EZH2, MYC, BCL2 and BCL6 are important prognostic biomarkers for lymphomas, our study shows that they poorly influence the sensitivity of lymphoma cell lines to DZNep-mediated apoptosis

Genomic basis of a social polymorphism in a halictid bee

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Identification of ADGRE5 as discriminating MYC target between Burkitt lymphoma and diffuse large B-cell lymphoma

Abstract Background MYC is a heterogeneously expressed transcription factor that plays a multifunctional role in many biological processes such as cell proliferation and differentiation. It is also associated with many types of cancer including the malignant lymphomas. There are two types of aggressive B-cell lymphoma, namely Burkitt lymphoma (BL) and a subgroup of diffuse large cell lymphoma (DLBCL), which both carry MYC translocations and overexpress MYC but both differ significantly in their clinical outcome. In DLBCL, MYC translocations are associated with an aggressive behavior and poor outcome, whereas MYC-positive BL show a superior outcome. Methods To shed light on this phenomenon, we investigated the different modes of actions of MYC in aggressive B-cell lymphoma cell lines subdivided into three groups: (i) MYC-positive BL, (ii) DLBCL with MYC translocation (DLBCLpos) and (iii) DLBCL without MYC translocation (DLBCLneg) for control. In order to identify genome-wide MYC-DNA binding sites a chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) was performed. In addition, ChIP-Seq for H3K4me3 was used for determination of genomic regions accessible for transcriptional activity. These data were supplemented with gene expression data derived from RNA-Seq. Results Bioinformatics integration of all data sets revealed different MYC-binding patterns and transcriptional profiles in MYC-positive BL and DLBCL cell lines indicating different functional roles of MYC for gene regulation in aggressive B-cell lymphomas. Based on this multi-omics analysis we identified ADGRE5 (alias CD97) - a member of the EGF-TM7 subfamily of adhesion G protein-coupled receptors - as a MYC target gene, which is specifically expressed in BL but not in DLBCL regardless of MYC translocation. Conclusion Our study describes a diverse genome-wide MYC-DNA binding pattern in BL and DLBCL cell lines with and without MYC translocations. Furthermore, we identified ADREG5 as a MYC target gene able to discriminate between BL and DLBCL irrespectively of the presence of MYC breaks in DLBCL. Since ADGRE5 plays an important role in tumor cell formation, metastasis and invasion, it might also be instrumental to better understand the different pathobiology of BL and DLBCL and help to explain discrepant clinical characteristics of BL and DLBCL

Evidence for a role of RUNX1 as recombinase cofactor for TCRβ rearrangements and pathological deletions in ETV6-RUNX1 ALL

International audienceT-cell receptor gene beta (TCRβ) gene rearrangement represents a complex, tightly regulated molecular mechanism involving excision, deletion and recombination of DNA during T-cell development. RUNX1, a well-known transcription factor for T-cell differentiation, has recently been described to act in addition as a recombinase cofactor for TCRδ gene rearrangements. In this work we employed a RUNX1 knock-out mouse model and demonstrate by deep TCRβ sequencing, immunostaining and chromatin immunoprecipitation that RUNX1 binds to the initiation site of TCRβ rearrangement and its homozygous inactivation induces severe structural changes of the rearranged TCRβ gene, whereas heterozygous inactivation has almost no impact. To compare the mouse model results to the situation in Acute Lymphoblastic Leukemia (ALL) we analyzed TCRβ gene rearrangements in T-ALL samples harboring heterozygous Runx1 mutations. Comparable to the Runx1+/- mouse model, heterozygous Runx1 mutations in T-ALL patients displayed no detectable impact on TCRβ rearrangements. Furthermore, we reanalyzed published sequence data from recurrent deletion borders of ALL patients carrying an ETV6-RUNX1 translocation. RUNX1 motifs were significantly overrepresented at the deletion ends arguing for a role of RUNX1 in the deletion mechanism. Collectively, our data imply a role of RUNX1 as recombinase cofactor for both physiological and aberrant deletions