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Thesis Reference Thesis Recurrent 11q24.3 gain contributes to the pathogenesis of diffuse large B cell lymphoma by deregulating ETS1 and FLI1 TESTONI, Monica Abstract DLBCL is the most frequent and aggressive form of non-Hodgkin's lymphoma. The still high percentage of mortality is in part due to the heterogeneity of DLBCL at genetic level. Among genetic alterations, a recurrent gain at 11q24.3 was detected in 26% of DLBCL. It was associated with higher expression of ETS1 and FLI1 transcription factors. Silencing of both genes led to cell death and a block in proliferation in a DLBCL cell line bearing the specific 11q gain but only FLI1 silencing gave a similar phenotype in cell lines without the gain. At molecular level, ETS1 directly and negatively regulates BLIMP1 expression. Importantly, ETS1 or FLI1 silencing leads to changes in the expression of B cell important genes. This project reveals ETS1 and FLI1 as interesting drug targets to be further analyzed. In addition, the status of MYC gene was analyzed in this cohort of DLBCL patients treated with R-CHOP. Reference TESTONI, Monica. Recurrent 11q24.3 gain contributes to the pathogenesis of diffuse large B cell lymphoma by deregulating ETS1 and FLI1. Thèse de doctorat : Univ. Genève, 2014, no. Sc. 4669 URN : urn:nbn:ch:unige-392076 DOI : 10.13097/archive-ouverte/unige:39207 Available at: http://archive-ouverte.unige.ch/unige:39207 Disclaimer: layout of this document may differ from the published version. 1 / 1 UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Faculté des Sciences Pharmaceutiques Professeur Leonardo Scapozza INSTITUTE OF ONCOLOGY RESEARCH (IOR) Genomic and Lymphoma Group Francesco Bertoni, MD RECURRENT 11q24.3 GAIN CONTRIBUTES TO THE PATHOGENESIS OF DIFFUSE LARGE B CELL LYMPHOMA BY DEREGULATING ETS1 AND FLI1 THÈSE présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteure ès sciences, Mention sciences pharMaceutiques par MONICA TESTONI de COMO (ITALY) Thèse N° 4669 BELLINZONA 2014 Dedicato a tutti i miei nonni compreso don Titino… a Nico e a tutti coloro a cui voglio bene.. e infine dedicato a chi presto arriverà… “The man who is blind to the beauties of Nature has missed half the pleasure of life.” -Lord Robert Baden Powell- Summary Diffuse large B cell lymphoma (DLBCL) is the most frequent B cell-derived lymphoma in adults accounting for 35-40% of all cases. The diversity in clinical presentation, as well as the pathologic and biologic heterogeneity, suggests that DLBCL comprises several disease entities. Gene expression profiling studies have identified at least three molecular subtypes of DLBCL: germinal center B cell-like (GCB) DLBCL, activated B cell-like (ABC) DLBCL, and primary mediastinal B cell lymphoma (PMBL). Each of these molecular subgroup is associated to a particular stage of B cell differentiation through plasma cell, although is not still clear the extent to which these subtypes maintain the molecular and physiological properties of their normal B cell counterparts. Moreover, the three DLBCL groups differ also in terms of pathology, with a large and diverse set of chroMosomal copy number aberrations associated primarily or exclusively with specific DLBCL subtypes, and clinical behavior, with the worst overall survival (OS) for the ABC-DLBCL subgroup. DLBCL is a very aggressive lymphoma and a large percentage of DLBCL patients are not fully curable yet with the current standard iMMuno-chemotherapy regimens (R- CHOP). Therefore, genomic and molecular analyses can identify crucial mechanisms of pathogenesis and new compounds to improve the efficiency of the therapeutic modalities. Normal germinal center (GC) B cell differentiation requires a complex transcriptional program and alterations of genes involved in this process are relevant for DLBCL pathogenesis. Genomic DNA profiles of 166 DLBCL patients samples have been previously obtained in my group of research allowing the identification of genomic aberrations that are associated with this disease. Among these genetic aberrations there was gain that affected the 11q24.3 chromosoMe locus. This genomic alteration was identified in the 23% of our cohort of DLBCL patient and it was associated with higher expression of ETS1 (p=0.0129) and FLI1 (p=0.0082) genes both belonging to the ETS family of transcription factors. ETS1 and FLI1 closely map inside the 11q24.3 genomic region and were found concomitantly expressed in DLBCL patient samples. In order to assess whether this genomic alteration is associated to one of the GCB- or ABC-DLBCL subtypes, genomic analysis was integrated with the cell of origin data for each patients. No significant association were found. In contrast, ETS1 mRNA levels were higher in ABC-DLBCL subtype suggesting a specific oncogenic role of this gene in ABC-DLBCL lymphomagenesis. Thus, being the two genes, ETS1 and FLI1, correlated in term of genomic Mapping and mRNA expression in DLBCL patients, the aiM of this project was to functional characterize these two genes as possible deregulators of late stages of B cell maturation in DLBCL. A first screening of 22 DLBCL cell lines was performed to evaluate ETS1 and FLI1 protein levels. DLBCL cell lines encompassed GCB- and ABC-DLBCL subtypes and most of them expressed both factors, albeit at different levels. By combining these data with genome-wide DNA profiling, the DLBCL cell line, OCI-LY7 was revealed as the suitable model to in vitro study the pathogenetic roles of ETS1 and FLI1 in DLBCL. In fact, OCI-LY7 DLBCL cell line presented both the 11q24.3 gain observed in clinical specimens and the highest protein levels of ETS1 and FLI1. The approach was to silence the two ETS factors through lentiviral shRNA, utilizing 2 different hairpins for each gene and one hairpin against GFP as control. The reduction in their expression was confirmed by quantitative-RT-PCR and western blot analysis. Phenotypic effects in OCI-LY7 after ETS1 and FLI1 silencing were assessed showing that both ETS1 and FLI1 down-regulation induced a lower proliferation rate compared with the shGFP control. This phenotype was confirmed by an EdU in-vitro assay in which a reduced percentage of cells in S-phase was obtained in ETS1 or FLI1 silenced samples in respect to the control sample. Through a co-culture experiment, in which an equal amount of cells infected with a vector carrying the GFP marker (GFP positive) and with a vector carrying the shRNA against each ETS1 and FLI1 or GFP genes (GFP negative), the biological effects of ETS1 and FLI1 down-regulation was evaluated at longer time points. This experiment revealed that OCI-LY7 cells with reduced levels of ETS1 or FLI1 proliferate less efficiently. In fact, GFP positive cells outgrew OCI-LY7 GFP negative cells with the exception of cells infected with the shRNA against GFP, indicating an impairment of cell proliferation as a result of the ETS1 or FLI1 down- regulation. This phenotype was associated with cell death in OCI-LY7 indicating that these two genes are critical also for cell viability in OCI-Y7 DLBCL cell line. To investigate whether ETS1 and FLI1 could cooperate in regulating cell viability in patients carrying the 11q24.3 gain, a concomitant down-regulation of ETS1 and FLI1 was performed in OCI-LY7. It was observed that the concomitant silencing of the two ETS factors clearly induced a stronger apoptosis compared to the individual silencing of these genes. This experiment strongly indicated that ETS1 and FLI1 could cooperatively sustain cell viability when both are deregulated in B cells, as it is the case of OCI-LY7 DLBCL cell line. In order to assess whether ETS1 and FLI1 are required for cell viability in primary DLBCL independently of the presence of the 11q24.3 gain and high levels of ETS1 and FLI1 expression, the viability of GCB- and ABC-DLBCL derived cells lacking the 11q24.3 gain was evaluated after the single silencing of ETS1 and FLI1 genes. These analyses showed that only the FLI1 down-regulation signicantly affected cell viability in a way similar to what was observed in OCI-LY7 cell line. In contrary, ETS1 down-regulation did not induce apoptosis in the other DLBCL cell lines. Interestingly the proliferation of these cell lines is affected by both ETS1 and FLI1 knock- down, suggesting a different role of the two genes in DLBCL models. Notably, ETS1 down-regulation in these cell lines cause the up-regulation of FLI1 expression that maybe compensate for the lost of ETS1 expression. At Molecular level, the expression of several important players in GC differentiation is affected following knocking-down of ETS1 and FLI1. The genes evaluated for the expression were: BCL2, BCL6, PAX5, IRF4, PRDM1, and XBP1. In particular, ETS1 down- regulation resulted in a significant up-regulation of PRDM1, the master regulator of PC differentiation, and this effect was followed by XBP1 up-regulation, whereas no effect on IRF4 was observed. These data were also confirmed at the protein level. Considering that all these factors are down-stream to NF-kB and IRF4 is expressed before PRDM1 gene, these data suggests that ETS1 might act up-stream or independently from the NF-kB pathway. To reinforce these data a rescue experiment was performed in OCI-LY7 cells previously infected with shETS1. This experiment showed that ETS1 reintroduction restored PRDM1 mRNA levels to values similar to the control cells (shGFP). As ETS binding site (EBS) were found within PRDM1 promoter, a chromatin iMMunoprecipitation (ChIP) analysis was performed by using antibody against ETS1 protein in OCI-LY7 interfered for the expression ETS1 or the control GFP. An enrichment of PRDM1 promoter region was detected in OCI-LY7 control sample, in which ETS1 level remain high because of the gain, while a reduced enrichment of PRDM1 proMoter region was observed in OCI-LY7 interfered for ETS1 expression.
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