Identification and characterization of pathogenetically relevant genes in mature aggressive B-cell lymphomas of childhood by a combination of genomic and functional analyses

Burkitt lymphomas (BL) account for about 25 % of the mature B-cell lymphomas in childhood. The hallmark of BL is the chromosomal translocation t(8;14)(q24;q32), juxtaposing the MYC gene to the immunoglobulin (IG) heavy chain locus, or one of its light chain variants. These translocations result in overexpression of the oncogenic transcription factor MYC. However, the IG-MYC translocation alone is not sufficient to drive lymphomagenesis. Additional alterations are necessary for malignant transformation like mutations within CCND3 or TP53. In addition to MYC-positive BL, the existence of a subset of MYC-negative B-cell lymphoma resembling BL has been recently described. This lymphoma subset has been termed MYC-negative Burkitt-like lymphoma (mnBLL) and is characterized by a recurrent pattern of centromeric gain and telomeric loss on chromosome 11. The aims of this thesis were the identification and characterization of secondary genetic alterations in BL contributing to its lymphomagenesis. Moreover, BL should be investigated for traces of pluripotency as MYC is one of four reprogramming factors able to induce pluripotency in somatic cells. With regard to mnBLL, the aim of this thesis was the thorough description of the chromosomal, mutational and transcriptional landscape of these lymphomas in order to identify potential candidate genes contributing to its lymphomagenesis, which further should be functionally characterized. The analyses of BL were performed using 51 primary BL of the MMML cohort, which contains more than 800 molecularly characterized B-cell lymphomas, and of the ICGC MMML-Seq cohort. From the latter cohort, data from whole-genome and RNA-sequencing were available. Moreover, 25 mnBLL were analyzed of which 8 were newly recruited within this thesis. In addition, a panel of 23 B-cell lymphoma cell lines (16 BL, 3 mnBLL and 6 diffuse large B-cell lymphoma cell lines) was used for mutational screening and functional analyses. A variety of techniques was used to study the chromosomal (OncoScan DNA array, SNP 6.0 array), mutational (whole-genome, whole-exome and targeted re-sequencing, Sanger sequencing) and transcriptional (qPCR, Western blot, flow cytometry, IL10 ELISA, PluriTest) landscapes of BL and mnBLL as well as of the respective cell line models. Furthermore, the cell lines were used for various functional analyses including cell cycle and viability assays, gene knock down using lentiviral shRNA vectors or inhibition of IL10 signaling components using neutralizing IL10RA and IL10 antibodies as well as the PI3K inhibitor BKM120. Application of the PluriTest algorithm on gene expression data of 221 mature aggressive B-cell lymphomas demonstrated that BL showed a higher similarity to pluripotent stem cells (PSC) than non-BL. Nevertheless, an origin from or resemblance to cells with PSC features could not be established for BL. Analyses of the transcriptome of the BL led to the identification of a new, so far not described exon within the TERT gene. The expression of this new exon was verified in 11/11 BL cell lines and in 7/8 primary BL. Modeling of the impact of the new exon on the TERT reading frame revealed that either (i) N- or C-terminal truncated proteins might be translated or (ii) the transcript might be subjected to nonsense-mediated decay due to the introduction of a premature stop codon. Analyses of whole-genome sequencing data of BL and mutational screening of BL cell lines by Sanger sequencing led to the identification of frequently mutated genes so far not described in association with Burkitt lymphomagenesis. Hence, ID3 mutations were identified in 35/53 (68 %) BL and 8/12 (67 %) BL cell lines. Re-expression or overexpression of ID3 in BL cell lines induced the amount of cells in the pre-G1-phase as analyzed by cell cycle analysis, hence, further supporting the tumor suppressive function of ID3 in BL. Furthermore, 9/21 (43 %) BL and 2/16 (13 %) BL cell lines were shown to carry SMARCA4 mutations. 62 % of these mutations affected the catalytic domain. Lastly, in 6/45 (13 %) BL and 6/16 (38 %) BL cell lines PCBP1 mutations were identified which might lead to a cytoplasmatic retention and, hence, reduced function. Analysis of the copy number data determined in all 25 mnBLL led to a redefinition of the minimal region of loss in 11q which includes a minimal region of focal homozygous loss of 1.5 Mb. Moreover, recurrent, secondary alterations in mnBLL affected 6q, 13q, 18q and 12p-q. Each of these altered regions harbored potential pathogenically relevant candidate genes. Furthermore, mutations of ETS1 mapping to the region of loss in 11q were identified in 4/16 (25 %) mnBLL. Combining the results of the analyses of the chromosomal and mutational landscape with transcriptome data of 6 mnBLL provided by the MMML network project, and literature search led to the identification of 3 potential oncogenes (IL10RA, PAFAH1B2, KMT2A) and two potential tumor suppressor genes (ETS1, FLI1) in the altered 11q region. The expression profiles of these candidate genes were also analyzed in the mnBLL cell line models and were in agreement with those in the primary cases. Knock down analysis of PAFAH1B2 in a mnBLL cell line did not indicate an essential role of the PAFAH1B2 protein for the viability of the cells under the applied conditions. However, expression analysis of IL10 and IL10RA as well as inhibition of the IL10 signaling pathway components IL10, IL10RA and PI3K in B-cell lymphoma cell lines led to the identification of the IL10 signaling as a potential oncogenic signaling pathway in mnBLL. Hence, due to the interplay of IL10RA and FLI1/ETS1-induced IL10 deregulation, an autocrine IL10 signaling loop might be activated inducing PI3K signaling. The latter has been described as an oncogenic signaling pathway in BL. Taken together, the analyses performed in this thesis led to the identification of secondary alterations likely contributing to the lymphomagenesis of BL. Moreover, the investigations led to a detailed characterization of MYC-negative mnBLL on chromosomal, mutational and transcriptional level resulting in the identification of potential candidate genes. Thus, the results of this thesis provide novel insights into the pathogenetic mechanisms contributing to lymphomagenesis and, hence, may help to improve diagnostics and therapy of these mature aggressive B-cell lymphomas of childhood.