NIEUWE INZICHTEN IN GENETICA VAN PROGRESSIE VAN B-CEL LYMFOOM

B-cell lymphoma comprises a wide range of neoplasms with distinct clinical, histopathological and molecular characteristics. Despite significant advances in clinical management of these tumors over the past decades, disease progression, transformation to more aggressive and chemoresistant lymphoma subtypes, and high rates of relapse have been observed in many patients. The molecular and clinical complexity of B-cell lymphomas demands further molecular investigations to better characterize lymphoma entities, identify new biomarkers and potential therapeutic targets. Our study was divided in two parts. First, we explored molecular characteristics and consequences of non-IG aberrations of the FOXP1 gene in B-cell lymphomas. In the second part, we investigated leukemia/lymphoma cases with 10p rearrangements involving BMI1 and molecular mechanisms underlying an aberrant expression of this oncogene in B-cell malignancies. The FOXP1 gene (3p13) encodes for a transcription factor that is involved in a wide range of cellular processes. Previous studies correlated an aberrant expression of FOXP1 protein in diffuse large B-cell lymphoma and marginal zone lymphoma with poor prognosis. Chromosomal translocations involving FOXP1 have been identified as one of the mechanisms leading to overexpression of FOXP1 in lymphomas. These translocations are mediated by either immunoglobulin heavy chain locus (IGH) located at 14q32 or various non-IG sequences. The former translocation, t(3;14)(p13;q32), leads to the dysregulated expression of FOXP1 due to its juxtaposition with strong enhancer elements of IGH. Molecular mechanisms underlying an aberrant expression of FOXP1 in the latter translocations remain unknown. In this study, we comprehensively investigated four lymphoma cases with non-IG FOXP1 aberrations. The applied techniques included FISH, Q-RT-PCR, 5’-RACE PCR, Western blotting, RNA-sequencing (RNA-seq) and bioinformatic analyses. Our data revealed that non-IG aberrations of FOXP1 do not generate functional chimeric genes but activate expression of the 5’-end truncated transcript variants of FOXP1 by mechanisms that remain largely unknown. Thus, FOXP1 was found to be targeted by two molecularly distinct types of rearrangements in B-cell neoplasms: (i) t(3;14)(p13;q32)/IGH-FOXP1, which results in upregulation of the full-length FOXP1 (FOXP1FL) is recognized as the primary genetic hit in lymphomagenesis, and (ii) non-IG aberrations, which lead to expression of the N-terminally truncated isoforms of FOXP1 (FOXP1NT) and are acquired during clinical course of disease. We presume that the aberrantly expressed FOXP1FL plays an oncogenic, but not a tumor suppressor role in lymphoma, as speculate, while the overexpressed FOXP1NT isoforms are implicated in disease progression and herald a poor prognosis. Our concept of distinct oncogenic functions of FOXP1FL and FOXP1NT isoforms is supported by RNA-seq data analyses.In the second part of this study, we provided evidence that BMI1, a Polycomb-group gene (10p12) implicated in the pathogenesis of various solid tumors, is recurrently targeted by chromosomal aberrations in B-cell leukemia/lymphoma. We identified a novel t(10;14)(p12;q32)/IGH-BMI1 rearrangement and its IGK variant in six cases of chronic lymphocytic leukemia (CLL) and found that these aberrations were consistently acquired at time of disease progression and high grade transformation to diffuse large B-cell lymphoma (Richter syndrome). Of note, the IG-BMI1 translocations were not associated with any particular molecular subtype of CLL and notably, the cases were negative for common mutations of NOTCH1 and TP53, known to enhance a risk of progression and transformation in CLL. Our in vivo study of BMI1 using retroviral system technology failed due to a low transduction efficiency of the EHEB cells (CLL cell line) and a lack of other CLL-representative cell line models. Experiments performed on control mouse embryonic fibroblasts (MEF), however, were consistent with the previously reported role of BMI1 in cell cycle, proliferation and apoptosis. In addition, we studied 17 cases of MCL with various balanced and unbalanced structural abnormalities of 10p12. Using FISH and SNP array analysis, we demonstrated involvement of BMI1 in these aberrations and found that they were frequently but not exclusively associated with gain of the BMI1 locus. In addition, our genomic data showed that the 10p12/BMI1 rearrangements have been frequently associated with loss of the 10p terminal sequences and recurrent imbalances of the proximal region. We presume that genomic instability of the 10p region in MCL may facilitate rearrangements and dysregulation of BMI1. Our findings are in line with the previously published observations correlating overexpression of BMI1 with tumor progression and chemoresistance. In conclusion, our study led to identification of new players implicated in progression of mature B-cell leukemia/lymphoma and molecular mechanisms underlying their aberrant expression in these malignancies. Our findings point to a dual role of FOXP1 in lymphomagenesis, and showed that BMI1, known to be implicated in the pathogenesis of variety of cancers, is involved in at least two lymphoid malignancies, progressive CLL and MCL. The work sheds a new light on molecular mechanisms underlying progression of B-cell leukemia/lymphoma and deepens our knowledge of the genetics of these malignancies. Given that FOXP1 and BMI1 are candidate targets for anticancer treatment, our findings are clinically relevant.nrpages: 132status: publishe