Characterization of the MIR23A Cluster in Diffuse Large B Cell Lymphoma

Characterization of the MIR23A Cluster in Diffuse Large B Cell Lymphoma

Regulation and Targetome Identification

Natalie Veronika Freytag

Doctoral thesis

Date of Examination: 2017-02-03

Date of issue: 2017-08-11

Advisor: Kube, Dieter Prof. Dr.

Referee: Burfeind, Peter Prof. Dr.

Referee: Shcherbata, Halyna Dr.

Persistent Address: http://hdl.handle.net/11858/00-1735-0000-0023-3ED3-4

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Abstract

English

During B cell maturation within the germinal centers of lymph nodes, genetical alterations, such as chromosomal translocations and mutations are introduced into the genome in order to increase the B cell receptor (BCR) specificity. Errors occurring during these processes, namely class switch recombination and somatic hypermutation, can affect the expression of tumor suppressors and proto-oncogenes, resulting in lymphomagenesis. Two major subtypes of germinal center derived aggressive Non-Hodgkin B cell lymphoma are Burkitt lymphoma (BL) and Diffuse Large B Cell Lymphoma (DLBCL). The MIR23A cluster, coding for miR-23a, miR-27a and miR-24-2 is induced during normal germinal center reaction. While the MIR23A cluster expression is low in germinal center B cells, it is upregulated in mature memory B cells. However, BL and DLBCL tumors have aberrantly high MIR23A expression levels compared to healthy controls, indicating that the cluster is de-regulated during lymphomagenesis. This study identified the BCR signaling, which plays a key role during germinal center reaction, as a general mechanism responsible for the induction of the MIR23A cluster in BL and DLBCL cell lines. MEK/ERK signaling was shown to be the major signaling cascade mediating this effect. Downstream transcription factors ELK1 and c-MYC are not involved in activation of the MIR23A cluster in DLBCL. Since the MIR23A cluster could not be induced by BCR signaling in normal germinal center B cells, this study hypothesizes that aberrant BCR signaling in BL and DLBCL is responsible for the increased MIR23A cluster levels. The MIR23A cluster is involved in many different solid cancers as well as leukemia and lymphoma. Its cellular function is discussed controversially among the different cancer entities, indicating that it is cell type and context specific. One study reported that DLBCL patients with high miR-23a levels show worse overall survival rates, suggesting an onco-miR function for the MIR23A cluster in DLBCL. However, the processes that are regulated by the MIR23A cluster in DLBCL remain unknown. In order to elucidate the biological function of the MIR23A cluster in the B cell lymphoma context, the targetomes of miR-23a and miR-27a were identified via Ago2-RNA immunoprecipitation in a DLBCL cell line stably overexpressing miR-23a, miR-27a or a non silencing control. By this approach 46 novel direct miR-23a and miR-27a targets in DLBCL were identified. LIMK1 and PUMA were validated as miR-27a targets on protein level. Furthermore, functional analyses demonstrated that miR-23a and miR-27a attenuate the ability of DLBCL cells to undergo apoptosis in response to DNA damage. This might be one plausible explanation why DLBCL patients with high miR-23a expression levels have a worse overall survival rate than patients with low levels, supporting the onco-miR hypothesis for the MIR23A cluster.

Keywords: miRNA; B cell lymphoma; DLBCL; B cell receptor