Inflammasomes are a group of protein complex that regulate inflammation throughcomplex signal transduction, although their specific mechanisms and structures have notbeen fully described. As the protein that kickstarts assembly of a type of inflammasome,NLRP3 is a key regulator of inflammation and may play a relevant role in the developmentof inflammatory diseases. In this project it has been attempted to perform a Gene Fusionbetween a segment of NLRP3 and regions of Toll-Like Receptor 4 by means of overlapextensionPCR, a technique that employs hybrid primers to create an overlap between bothsequences that can be filled by a polymerase, causing them to merge. Results suggest GeneFusion was successful, however cloning and expression of the construct have not beenachieved so far. If expressed as a fusion protein, the added transmembrane domain willanchor two domains of NLRP3 to the membrane, allowing more precise study of thecomposition and functionality of the inflammasome. Removal of the terminal domain ofNLRP3 will help determine its implication and relevance in the assembly process of theprotein complex.

Shortcomings of current methods of prostate cancer detection draw attention to a need for improved biomarkers. The TMPRSS2:ERG gene fusion leads to the overexpression of ERG, an ETS family transcription factor, and is the most prevalent genetic lesion in prostate cancer, but its clinical utility remains to be defined. Two radical prostatectomy samples were analysed by next-generation whole transcriptome sequencing. The chosen samples differed in fusion gene status, as previously determined by RT-PCR. The involvement of novel and previously reported prostate cancer-related transcripts, Wnt signalling, p53 effector loss and several ETS-regulated pathways was identified in the prostate cancer cases examined. ERG was found to directly transactivate RhoGDIB, a gene associated with fusion-positive prostate cancer. Overexpression of RhoGDIB elicited spindle-shaped morphology, faster cell migration and increased cell proliferation, phenotypic changes suggestive of cancer progression. The present findings confirm the value of comprehensive sequencing for biomarker development and indicate avenues of future study.

Shortcomings of current methods of prostate cancer detection draw attention to a need for improved biomarkers. The TMPRSS2:ERG gene fusion leads to the overexpression of ERG, an ETS family transcription factor, and is the most prevalent genetic lesion in prostate cancer, but its clinical utility remains to be defined. Two radical prostatectomy samples were analysed by next-generation whole transcriptome sequencing. The chosen samples differed in fusion gene status, as previously determined by RT-PCR. The involvement of novel and previously reported prostate cancer-related transcripts, Wnt signalling, p53 effector loss and several ETS-regulated pathways was identified in the prostate cancer cases examined. ERG was found to directly transactivate RhoGDIB, a gene associated with fusion-positive prostate cancer. Overexpression of RhoGDIB elicited spindle-shaped morphology, faster cell migration and increased cell proliferation, phenotypic changes suggestive of cancer progression. The present findings confirm the value of comprehensive sequencing for biomarker development and indicate avenues of future study.

Les tumeurs locales de la prostate sont associées à une évolution lente et une bonne survie, alors que les stades plus avancés révèlent dans 80% des cas des métastases osseuses incurables. La découverte de gènes de fusion issus de remaniements chromosomiques, tel que TMPRSS2:ERG dans plus de 50% des cas, a ouvert une nouvelle voie dans la compréhension du processus de cancérisation de la prostate. La présence de ce gène de fusion peut être associée à un mauvais pronostic dans de nombreuses études cliniques. Cependant, son rôle précis au cours de la cancérisation et de la progression du cancer de la prostate reste à déterminer. Le gène Erg (Ets related gene) code un facteur de transcription dont l’expression est notamment associée à la mise en place du cartilage, et plus largement du squelette. Ceci suggère un rôle potentiel du gène de fusion impliquant ce facteur, et de ses gènes cibles, dans la formation des métastases osseuses du cancer de la prostate.Pour notre étude, nous avons utilisé des lignées de cellules tumorales prostatiques PC3 et PC3c, exprimant stablement le gène de fusion TMPRSS2:ERG et précédemment établies au laboratoire. Dans un premier temps, en utilisant un modèle d’injections intratibiales chez les souris SCID, nous avons démontré que l’expression ectopique de la fusion améliore la capacité d’induction de lésions ostéocondensantes en inhibant l’ostéolyse dans le modèle PC3 ostéolytique, et en stimulant l’ostéoformation dans le modèle PC3c mixte (ostéolytique et ostéocondensant). Cette expression ectopique de la fusion augmente également l’ostéomimétisme dans les deux modèles cellulaires, c’est-à-dire l’acquisition d’un phénotype semblable aux cellules osseuses leur conférant des avantages de survie et de propagation dans la moelle osseuse. En outre, trois nouveaux gènes cibles de TMPRSS2:ERG ont été mis en évidence : ET-1 (Endothelin-1), stimulant la différenciation ostéoblastique et inhibant la résorption osseuse ostéoclastique, ALPL (Alkaline Phosphatase Liver/Bone/Kidney), marqueur de différenciation des ostéoblastes, et COL1A1 (Collagen Type 1 Alpha 1), composant de la matrice osseuse, témoignant d’un rôle du gène de fusion dans la formation de métastases ostéocondensantes du cancer de la prostate.Par ailleurs, deux autres gènes ont été étudiés, codant soit une protéine impliquée dans la stabilisation de structures particulières appelées invadopodes, soit une protéine impliquée dans le métabolisme des lipides. L’ensemble de ces résultats contribue à mieux comprendre les mécanismes de cancérisation et d’évolution métastatique du cancer de la prostate, en particulier l’influence de l’expression du gène de fusion TMPRSS2:ERG dans les métastases osseuses du cancer de la prostate. / Local prostate cancers are associated with slow progression and good survival, while advanced stages reveal incurable bone metastases in 80% of cases. The discovery of fusion genes resulting from chromosomal rearrangements, such as TMPRSS2:ERG in more than 50% of cases, opened a new way in understanding the process of prostate cancer. The presence of this fusion gene may be associated with poor prognosis in many clinical studies. However, its precise role during cancerization and progression of prostate cancer remains to be determined. The Erg gene (Ets related gene) encodes a transcription factor whose expression is associated in particular with embryonic skeleton development. This suggests a potential role of the fusion gene involving this factor, and its target genes, in the formation of prostate cancer bone metastases.In this study, we used prostate cancer cell lines PC3 and PC3c, stably expressing the TMPRSS2:ERG fusion gene and previously established in the laboratory. First, using a model of intratibial injections in SCID mice, we demonstrated that ectopic expression of the fusion enhances the ability to induce osteoblastic lesions by inhibiting osteolysis in the osteolytic PC3 model, and by stimulating osteoformation in the mixed PC3c model (osteolytic and osteoblastic). This ectopic expression of the fusion also increases osteomimicry in both cell models, meaning the acquisition of a bone-cell-like phenotype which gives them advantages of survival and spread in the bone marrow. In addition, three new TMPRSS2:ERG target genes have been described: ET-1 (Endothelin-1), stimulating osteoblastic differentiation and inhibiting osteoclastic bone resorption, ALPL (Alkaline Phosphatase Liver/Bone/Kidney), a marker of the osteoblasts differentiation, and COL1A1 (Collagen Type 1 Alpha 1), a component of the bone matrix, providing novel insights into the role of the fusion gene in the formation of osteoblastic metastases of prostate cancer.In addition, two other genes have been studied, encoding either a protein involved in the stabilization of particular structures called invadopodia, or a protein involved in lipid metabolism.All these results contribute to decipher the mechanisms of cancerization and metastatic progression of prostate cancer, in particular the influence of the expression of TMPRSS2:ERG fusion gene in prostate cancer bone metastases.

Gene fusion is one of the hallmarks of cancer. Recent advances in RNA-seq of cancer transcriptomes have facilitated the discovery of fusion transcripts. In this study, we report identification of a surprisingly large number of fusion transcripts, including six KANSARL (KANSL1-ARL17A) transcripts that resulted from the fusion between the KANSL1 and ARL17A genes using a RNA splicingcode model. Five of these six KANSARL fusion transcripts are novel. By systematic analysis of RNA-seq data of glioblastoma, prostate cancer, lung cancer, breast cancer, and lymphoma from different regions of the World, we have found that KANSARL fusion transcripts were rarely detected in the tumors of individuals from Asia or Africa. In contrast, they exist in 30 - 52% of the tumors from North Americans cancer patients. Analysis of CEPH/Utah Pedigree 1463 has revealed that KANSARL is a familially-inherited fusion gene. Further analysis of RNA-seq datasets of the 1000 Genome Project has indicated that KANSARL fusion gene is specific to 28.9% of the population of European ancestry origin. In summary, we demonstrated that KANSARL is the first cancer predisposition fusion gene associated with genetic backgrounds of European ancestry origin.

O interesse científico em rearranjos gênicos relacionados com a etiogênese e progressão do câncer relaciona-se, principalmente, à descoberta da fusão BCR/ABL na Leucemia Mieloide Crônica, sendo que desde então, houve uma evolução no manejo dessa doença, instigando uma série de estudos correlatos em outras neoplasias. Essas pesquisas culminaram no encontro do primeiro rearranjo gênico em tumores sólidos, o gene de fusão TMPRSS2/ERG, envolvendo a região promotora do gene da serina protease, o TMPRSS2, e o gene da família de fatores de transcrição ETS, o ERG. Ele é específico de adenocarcinoma da próstata, o que o torna forte candidato a biomarcador e já demonstra exercer papel de destaque no manejo clínico do câncer de próstata (CaP), tal qual o exercido pela fusão BCR/ABL. Sua frequência têm se mostrado associada a diversos fatores, sobretudo à etnia de origem. Indivíduos portadores de CaP oriundos de diversos países já foram estudados quanto à frequência dessa fusão e o resultado é bastante diversificado. No Brasil, entretanto, ainda não há dados a respeito desse rearranjo, e este trabalho visa contribuir para a identificação da frequência da mesma e sua contribuição para o diagnóstico e o tratamento do CaP no país. Para tal, utilizamos mRNA de 20 indivíduos com CaP provenientes do serviço de atendimento do HCFMRP/USP, e por meio da técnica de RT-PCR, obtivemos o cDNA dos mesmos que foram investigados quanto à presença da fusão TMPRSS2/ERG, e as amostras positivas sequenciadas para determinação do tipo de isoforma envolvida. Identificamos que 35% das amostras continham o rearranjo e que todas correspondiam à isoforma do tipo III, cuja literatura a relaciona com um fenótipo agressivo do CaP, porém não metástico, e é também a mais comumente identificada. Ao confrontarmos essa evidência com os dados clínicos e histopatológicos, constatamos que havia correlação entre eles, sugerindo assim, como em outros trabalhos, o potencial desse rearranjo como marcador de agressividade do CaP. No entanto, não verificamos relação entre a presença da fusão e dados de progressão da doença. Em vista desses resultados, destacamos a necessidade da promoção de outros trabalhos de mesmo caráter, abrangendo outras regiões, a fim de se delinear um perfil mais representativo desse rearranjo no Brasil, uma vez que seu potencial como biomarcador diagnóstico e clínico é enorme e pode influenciar sobremaneira no manejo do CaP. / Scientific interest in gene rearrangements associated with cancer progression and etiogenesis relates mainly to the discovery of BCR/ABL fusion in chronic myelogenous leukemia, and since then there has been an evolution in the management of this disease, prompting a series of related studies in other malignancies. These researches resulted in the meeting of the first gene rearrangement in solid tumors, the fusion gene TMPRSS2/ERG involving the promoter region of the gene of serine protease, TMPRSS2, and the gene family of transcription factors ETS, the ERG. It is specific for adenocarcinoma of the prostate, which makes it a strong candidate biomarker and shows already exert a prominent role in the clinical management of prostate cancer (PCa), as is exercised by the BCR/ABL. Its frequency has been shown to be associated with several factors, especially the ethnic origin. Individuals with CaP from different countries have been studied in the frequency of this merger and the result is quite diverse. In Brazil, however, there is no data about this rearrangement, and this paper aims to contribute to the identification of the same frequency and its contribution to the diagnosis and treatment of PCa in the country. Therefore, we used mRNA from 20 individuals with CaP from the answering service HCFMRP/USP, and by RT-PCR, cDNA obtained from the same people who were investigated for the presence of fusion TMPRSS2/ERG, and positive samples sequenced to determine the type of isoform involved. We found that 35% of the samples contained the rearrangement and that all corresponded to the type III isoform, whose literature relates to an aggressive phenotype of PCa, but not metastatic, and is also the most commonly identified. When we compared this evidence with clinical and histopathological data, we found that there was a correlation between them, suggesting, as in other studies, the potential of this rearrangement as a agressivity marker of PCa. However, no significant association between the presence of data fusion and disease progression. In view of these results, we highlight the need to promote other works of the same character, covering other regions, in order to delineate a more representative profile of this rearrangement in Brazil, since its potential as a biomarker and clinical diagnosis is huge and can influence greatly in the management of PCa.

O interesse científico em rearranjos gênicos relacionados com a etiogênese e progressão do câncer relaciona-se, principalmente, à descoberta da fusão BCR/ABL na Leucemia Mieloide Crônica, sendo que desde então, houve uma evolução no manejo dessa doença, instigando uma série de estudos correlatos em outras neoplasias. Essas pesquisas culminaram no encontro do primeiro rearranjo gênico em tumores sólidos, o gene de fusão TMPRSS2/ERG, envolvendo a região promotora do gene da serina protease, o TMPRSS2, e o gene da família de fatores de transcrição ETS, o ERG. Ele é específico de adenocarcinoma da próstata, o que o torna forte candidato a biomarcador e já demonstra exercer papel de destaque no manejo clínico do câncer de próstata (CaP), tal qual o exercido pela fusão BCR/ABL. Sua frequência têm se mostrado associada a diversos fatores, sobretudo à etnia de origem. Indivíduos portadores de CaP oriundos de diversos países já foram estudados quanto à frequência dessa fusão e o resultado é bastante diversificado. No Brasil, entretanto, ainda não há dados a respeito desse rearranjo, e este trabalho visa contribuir para a identificação da frequência da mesma e sua contribuição para o diagnóstico e o tratamento do CaP no país. Para tal, utilizamos mRNA de 20 indivíduos com CaP provenientes do serviço de atendimento do HCFMRP/USP, e por meio da técnica de RT-PCR, obtivemos o cDNA dos mesmos que foram investigados quanto à presença da fusão TMPRSS2/ERG, e as amostras positivas sequenciadas para determinação do tipo de isoforma envolvida. Identificamos que 35% das amostras continham o rearranjo e que todas correspondiam à isoforma do tipo III, cuja literatura a relaciona com um fenótipo agressivo do CaP, porém não metástico, e é também a mais comumente identificada. Ao confrontarmos essa evidência com os dados clínicos e histopatológicos, constatamos que havia correlação entre eles, sugerindo assim, como em outros trabalhos, o potencial desse rearranjo como marcador de agressividade do CaP. No entanto, não verificamos relação entre a presença da fusão e dados de progressão da doença. Em vista desses resultados, destacamos a necessidade da promoção de outros trabalhos de mesmo caráter, abrangendo outras regiões, a fim de se delinear um perfil mais representativo desse rearranjo no Brasil, uma vez que seu potencial como biomarcador diagnóstico e clínico é enorme e pode influenciar sobremaneira no manejo do CaP. / Scientific interest in gene rearrangements associated with cancer progression and etiogenesis relates mainly to the discovery of BCR/ABL fusion in chronic myelogenous leukemia, and since then there has been an evolution in the management of this disease, prompting a series of related studies in other malignancies. These researches resulted in the meeting of the first gene rearrangement in solid tumors, the fusion gene TMPRSS2/ERG involving the promoter region of the gene of serine protease, TMPRSS2, and the gene family of transcription factors ETS, the ERG. It is specific for adenocarcinoma of the prostate, which makes it a strong candidate biomarker and shows already exert a prominent role in the clinical management of prostate cancer (PCa), as is exercised by the BCR/ABL. Its frequency has been shown to be associated with several factors, especially the ethnic origin. Individuals with CaP from different countries have been studied in the frequency of this merger and the result is quite diverse. In Brazil, however, there is no data about this rearrangement, and this paper aims to contribute to the identification of the same frequency and its contribution to the diagnosis and treatment of PCa in the country. Therefore, we used mRNA from 20 individuals with CaP from the answering service HCFMRP/USP, and by RT-PCR, cDNA obtained from the same people who were investigated for the presence of fusion TMPRSS2/ERG, and positive samples sequenced to determine the type of isoform involved. We found that 35% of the samples contained the rearrangement and that all corresponded to the type III isoform, whose literature relates to an aggressive phenotype of PCa, but not metastatic, and is also the most commonly identified. When we compared this evidence with clinical and histopathological data, we found that there was a correlation between them, suggesting, as in other studies, the potential of this rearrangement as a agressivity marker of PCa. However, no significant association between the presence of data fusion and disease progression. In view of these results, we highlight the need to promote other works of the same character, covering other regions, in order to delineate a more representative profile of this rearrangement in Brazil, since its potential as a biomarker and clinical diagnosis is huge and can influence greatly in the management of PCa.

Many aspects of the mechanisms by which prostate cancer (PCa) progresses from being a confined tumour to advanced metastatic and castration-resistant disease remain unclear. The aim of this study is to evaluate in vitro the potential role of the fusion gene TMPRSS2-ERG in the response of PCa cells to ionising radiation (IR) and androgen deprivation therapy (ADT). This research focused on assessing the presence of the TMPRSS2-ERG transcript across various PCa cell lines and identifying any correlation between the TMPRSS2-ERG transcript and other genes, particularly genes related to DNA damage repair pathways. Several genes involved in cell metabolism and development were found to correlate with TMPRSS2-ERG but not genes involved in DNA repair. In accordance with previous reports, this research confirmed a proliferative advantage for cells expressing ERG. However this project also tested the role of ERG-status in response to chemotherapy, radiation and ADT. The data showed that VCaP and DuCaP cells exposed to low-dose radiation demonstrated decreased viability irrespective of their ERG-status. Similarly ADT decreased the viability of VCaP cells and seemed to neutralise the proliferative advantage of TMPRSS2-ERG positive cells. Stimulation with dihydrotestosterone caused increased radioresistance of TMPRSS2-ERG positive cells. Treatment with taxanes showed stronger effect on cells with lower ERG expression. This work suggests that the proliferative advantage conferred by ERG overexpression in in vitro models can be neutralised by castration and IR.

La leucémie myéloïde chronique (LMC) est un modèle d’évolution tumorale dans les cancers humains. Le processus d’évolution de la LMC de la phase chronique (PC) à la phase blastique (PB) est caractérisé par un arrêt de différenciation et l’acquisition de la capacité d’autorenouvellement incontrôlé d’une cellule souche ou d’un progéniteur hématopoïétique. La LMC en PB est associée à la présence d’anomalies génétiques additionnelles à la fusion BCR-ABL1 qui résulte de la translocation chromosomique t(9;22). Contrairement aux patients en PC, les patients en PB de la LMC n’obtiennent pas une réponse moléculaire complète à long terme avec 1’Imatinib mesylate, un inhibiteur de la tyrosine kinase (ITK) BCR-ABL1. De plus, les ITKs de deuxième et troisième générations sont moins efficaces en PB de la LMC lorsque les cellules leucémiques ont acquis une résistance au traitement indépendante des mutations de BCR-ABL1. Les mécanismes moléculaires des voies de signalisation impliquées dans la progression de la LMC en PB ne sont pas entièrement élucidés. Le but de notre travail est de caractériser de nouvelles anomalies génétiques dans la PB de la LMC. Nous avons identifié en cytogénétique, quatre nouvelles translocations chromosomiques : t(1;21)(p36;q22), t(7;17)(p15;q22), t(8;17)(q11;q22) et t(2;12)(q31;p13) dans les cellules leucémiques de patients en PB de la LMC résistants au traitement. En utilisant des techniques d'hybridation in situ en fluorescence, de RT-PCR et de séquençage, nous avons délimité les régions à investiguer au niveau des points de cassure et identifié un réarrangement de plusieurs gènes codant pour des facteurs de transcription importants lors de l’hématopoïèse tels que RUNX1, ETV6, PRDM16 et HOXA. L’altération de ces gènes pourrait expliquer l’arrêt de différenciation et/ou l’acquisition de la capacité d’autorenouvellement caractéristiques de la LMC en PB. Nous avons identifié les fusions RUNX1-PRDM16, MSI2-HOXA, MSI2-SOX17 et ETV6-HOXD11, respectivement associées aux translocations chromosomiques t(1;21), t(7;17), t(8;17) et t(2;12). Ces fusions génèrent différents transcrits alternatifs qui maintiennent et altèrent le cadre ouvert de lecture. L’analyse des séquences des transcrits chimériques identifiés dans ce projet, incluant RUNX1-PRDM16, MSI2-HOXA9, MSI2-HOXA10, MSI2-HOXA11 et ETV6-HOXD11, nous a permis de prédire les domaines fonctionnels potentiellement présents au niveau des protéines chimériques prédites. Les transcrits de fusion qui respectent le cadre ouvert de lecture peuvent générer des domaines fonctionnels des deux partenaires. C’est le cas des deux transcrits identifiés pour la fusion RUNX1-PRDM16 où le domaine de liaison à l’ADN RHD (Runt homology domain) de RUNX1 est fusionné avec la quasi-totalité des domaines de PRDM16. Les transcrits de fusion qui ne respectent pas le cadre ouvert de lecture donnent des formes tronquées des transcrits RUNX1, MSI2 et ETV6. La juxtaposition des régions promotrices de ces derniers en 5’ de leurs partenaires entraîne l’activation de la forme courte oncogénique de PRDM16 dans la t(1;21) ou de différents gènes HOXA/D dans les t(7;17) et t(2;12), ainsi que l’expression aberrante d’un nouveau transcrit alternatif de SOX17 dans la t(8;17). Notre étude nous a permis d’identifier de nouveaux gènes de fusion et/ou une activation de gènes qui pourraient coopérer avec la fusion BCR-ABL1 dans la progression de la LMC et être impliqués dans la résistance au traitement de la LMC en phase avancée. La caractérisation des événements génétiques associés à la transformation blastique de la LMC est essentielle pour l’investigation des voies moléculaires impliquées dans cette phase de la maladie. Investiguer la résistance au traitement de ces patients pourrait aussi contribuer à identifier de nouvelles cibles thérapeutiques dans cette leucémie. / Chronic myeloid leukemia (CML) is a model of tumor evolution in human cancer. The evolution process of CML from the chronic phase (CP) to the blastic phase (BP) is characterized by a blockade of differentiation and acquisition of uncontrolled self-renewal capacity by hematopoietic stem or progenitor cells. CML-BP is associated with the presence of other genetic abnormalities in addition to the BCR-ABL1 fusion which results from chromosomal translocation t(9;22). Unlike patients in the CP, patients with CML-BP do not achieve a long-term complete molecular response to Imatinib mesylate, an inhibitor targeting the BCR-ABL1 tyrosine kinase (TK). Moreover, second and third generation TK inhibitors are less effective in CML-BP when leukemic cells have acquired a therapeutic resistance independent of BCR-ABL1 mutations. The molecular mechanisms of the signaling pathways responsible for CML progression from CP to BP are poorly understood. The aim of our project is to characterize novel genetic alterations in the BP of CML. We have identified by cytogenetics, four novel chromosomal translocations: t(1;21)(p36;q22), t(7;17)(p15;q22), t(8;17)(q11;q22) and t(2;12)(q31;p13) in leukemic cells of patients with CML-BP resistant to therapy. Using fluorescence in situ hybridization, RT-PCR and sequencing techniques, we have mapped chromosomal translocation breakpoints and identified rearranged genes encoding transcription factors which are key regulators of hematopoiesis, such as RUNX1, ETV6, PRDM16 and HOXA. The disruption of these genes could explain the differentiation blockade and/or uncontrolled self-renewal associated with the CML-BP. We identified RUNX1-PRDM16, MSI2-HOXA, MSI2-SOX17 and ETV6-HOXD11 fusions created by chromosomal translocations t(1;21), t(7;17), t(8;17) and t(2;12) respectively. These fusions generate different alternative transcripts that both maintain and alter the open reading frame. Sequence analysis of chimeric transcripts identified in this project, including RUNX1-PRDM16, MSI2-HOXA9, MSI2-HOXA10, MSI2-HOXA11 and ETV6-HOXD11, allowed us to predict potential functional domains present in putative chimeric proteins. In-frame fusion transcripts can generate functional domains from both fusion partners. For example, in two RUNX1-PRDM16 transcripts, the RUNX1 DNA binding domain RHD (Runt homology domain) is fused to the majority of PRDM16 domains. Out-of-frame fusion transcripts resulted in truncated forms of RUNX1, MSI2 and ETV6. The juxtaposition of promoter regions of these genes to the 5’ part of their partners resulted in the activation of the oncogenic short form of PRDM16 in the t(1;21) or of different HOXA/D genes in t(7;17) and t(2;12), and in the aberrant expression of a novel alternative SOX17 transcript in the t(8;17). Our study allowed us to identify novel fusion genes and/or activation of genes that potentially cooperate with BCR-ABL1 fusion in the progression of CML and contribute to treatment resistance of this disease. The characterization of genetic events related to the blastic transformation of CML is an important step in the investigation of molecular pathways involved in this stage of the disease. Understanding treatment resistance of these patients might help to identify new therapeutic targets in this leukemia.

The chromosomal translocation of t(14;18)(14q32;18q21) is a characteristic aberration of follicular lymphoma and Diffuse Large B cells lymphoma. By PCR, it was proved that the rearrangement of chromosomes 14 and 18 leads to an overexpression of BCL2, an anti-apoptotic protein, which is one of the factors responsible for the maturation of the diseases. The translocation involves the promoter region of IGH gene and the transcriptional unit of BCL2 gene. Previous studies carried out in Dr Tosi’s lab showed a looping out of the BCL2 gene from its chromosome territory in 15% of the nuclei analysed. This looping out could be possibly responsible for the transcriptional activity of the gene. A further relevant finding concerns the spatial distribution of the genes involved in the translocation in the interphase nuclei. In the Pfeiffer cell line, harbouring the t(14;18) rearrangement, the translocated BCL2 gene was positioned in the cell nuclei according to a bimodal distribution. One could speculate that the distribution in the periphery and in the centre of the nuclei could divide the Pfeiffer cell line in two different subpopulations, consequently from the transcriptional activity. These preliminary data set the ground for more experimental work to test whether genes associated with the nuclear interior were transcriptionally active as opposed to the genes positioned towards the nuclear periphery, transcriptionally inactive. The work here presented focuses on this investigation using RNA-DNA FISH (Fluorescence in situ hybridization). My work enabled the detection of IGH, BCL2 and t(14;18) genes along with their transcripts inside of the nuclei of Pfeiffer cell line. Contrary to what had been hinted by previous work, my results showed multiple nuclear positions of transcriptionally active IGH/BCL2 translocation. The result will need to be further supported by software analysis in order to define its specific nuclear position and to ensure the perfect localization of the genes inside each nucleus.