Submitted by Pedro Silva Filho (pfsilva@ipen.br) on 2017-11-17T17:17:04Z No. of bitstreams: 0 / Made available in DSpace on 2017-11-17T17:17:04Z (GMT). No. of bitstreams: 0 / Linfomas são cânceres que se iniciam a partir da transformação maligna de um linfócito no sistema linfático. Os linfomas são divididos em duas categorias principais: os linfomas de Hodgkin e todos os outros linfomas, denominados linfomas não-Hodgkin (LNH). Os pacientes com LNH são comumente tratados com radioterapia apenas ou combinada com quimioterapia utilizando-se de anticorpo monoclonal anti-CD20, principalmente o rituximab (MabThera&reg). O uso de anticorpos monoclonais (Acm) conjugados à quelantes bifuncionais radiomarcados com radionuclídeos metálicos ou lantanídeos é uma realidade de tratamento para portadores de LNH pelo princípio de radioimunoterapia (RIT). Este estudo concentrou-se nas condições de conjugação do anticorpo monoclonal rituximab (MabThera&reg) com grupamentos quelantes bifuncionais DOTA e DTPA. Na marcação dos Acm conjugados com lutécio-177, foram estudadas as condições de pré-purificação do Acm, condições de conjugação, determinação de número de quelantes acoplados à molécula do anticorpo, purificação do anticorpo conjugado, radiomarcação do anticorpo conjugado, com lutécio-177, purificação do anticorpo marcado, a ligação específica in vitro dos compostos marcados às células Raji, e distribuição biológica em camundongos BALB/c sadios. As três metodologias empregadas na pré-purificação do anticorpo (diálise, cromatografia de exclusão molecular com coluna Sephadex G-50 e ultrafiltração) demonstram-se eficientes e proporcionaram recuperação da amostra superior a 90%. A metodologia de ultrafiltração foi considerada a mais simples e prática, podendo ser aplicada a procedimentos rotineiros de produção de radiofármacos. Além disso, proporcionou a recuperação final de amostra de 97% em microlitros. Nas conjugações do anticorpo com os quelantes DOTA e DTPA em razões molares diferentes do Acm:quelante, observou-se número de grupamentos quelantes acoplados à molécula do Acm proporcional à razão molar estudada. Quando foi avaliada a influência de condições diferentes de conjugação no número de quelantes acoplados à molécula do Acm, não foram observadas diferenças significativas, com resultados de pureza radioquímica (PR) inferior a 80% em todas as condições estudadas. Na comparação de métodos de purificação do Acm conjugado, a abordagem inédita apresentada neste estudo, na qual a cromatografia de exclusão molecular foi combinada com a ultrafiltração resultou em maior eficiência na purificação e preservação da estrutura do anticorpo. Nos estudos de radiomarcação do anticorpo conjugado com DOTA e DTPA, os imunoconjugados de DTPA apresentaram, de forma geral, maior eficiência de marcação com resultados reprodutíveis quando comparados com os imunoconjugados de DOTA, considerando-se as diferentes razões molares utilizadas. As metodologias cromatográficas empregadas no controle de pureza radioquímica do composto radiomarcado proporcionaram a discriminação das diferentes espécies radioquímicas no meio de marcação. A metodologia de purificação do composto conjugado e radiomarcado utilizada proporcionou a obtenção de compostos com alta pureza radioquímica, 97,4±1,3% (DOTA 1:50) e 98,7±0,2% (DTPA 1:50). Nos estudos de ligação específica às células tumorais Raji, o anticorpo conjugado com quelante DTPA nas razões molares de 1:50 e 1:20 apresentaram perfil semelhante de ligação, com aumento da porcentagem de ligação específica proporcional à concentração celular, enquanto que o imunoconjugado na razão molar de 1:10 apresentou alta porcentagem de ligação não específica. Os resultados obtidos nos estudos de biodistribuição in vivo do anticorpo conjugado e radiomarcado nem sempre se mostraram compatíveis com a biodistribuição de anticorpos radiomarcados íntegros. No caso do quelante DOTA, o imunoconjugado obtido a partir da razão molar 1:20, apresentou melhores características de biodistribuição. No caso do quelante DTPA, a razão molar utilizada pareceu refletir diretamente no clareamento sanguíneo do anticorpo e todas as razões molares utilizadas apresentaram instabilidade in vivo. / Tese (Doutorado em Tecnologia Nuclear) / IPEN/T / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

Notre équipe étudie les mécanismes de résistance à l’apoptose induite par divers agents dans les cellules de lymphomes B infectées ou non par le virus d’Epstein-Barr (EBV). EBV est un virus oncogénique de la famille des gamma-herpès virus qui est associé notamment au lymphome de Burkitt (LB) et aux syndromes lymphoprolifératifs post-transplantation (PTLD). Des résultats précédents ont montré que l’utilisation de la nutline-3, une molécule capable de se fixer sur MDM2, active p53 dans ces cellules tumorales. Cependant cette activation de p53 provoque l’apoptose des cellules B EBV(-) alors que les cellules B EBV(+) en latence III (exprimant toutes les protéines virales dites « de latence ») sont beaucoup plus résistantes. Mon travail de thèse a consisté à étudier les mécanismes impliqués dans cette résistance afin de mettre en place des stratégies thérapeutiques pour la contourner. La première partie de ma thèse a été consacrée à l’étude du rôle de l’autophagie dans la résistance des cellules EBV(+) en latence III à l’apoptose. L’autophagie est un processus de dégradation des protéines qui joue un rôle physiologique complexe impliqué à la fois dans la survie et dans la mort cellulaire. Les travaux effectués ont montré que: 1) l’autophagie est induite en réponse au traitement par la nutline dans les cellules EBV(+) en latence III ; 2) ces cellules expriment fortement la Bécline-1 et présentent une activation constitutive de l’autophagie ; 3) l’autophagie participe à la résistance de ces cellules à l’apoptose. La seconde partie de ma thèse a été consacrée au développement de nouvelles molécules ciblant les protéines anti-apoptotiques de la famille de Bcl-2. En effet, outre Bcl-2 qui est surexprimé dans les cellules EBV(+), les cellules de LB et les PTLD surexpriment aussi Mcl-1, une autre protéine anti-apoptotique. Or il a été montré que cette protéine était fréquemment à l’origine de résistance à des inhibiteurs déjà développés (et en essais cliniques) contre Bcl-2. Le développement de molécules ciblant Mcl-1 s’avère donc utile pour les contrer. Pour cela une collaboration avec une équipe de chimiste (dirigée par Fanny Roussi à l’Institut de Chimie des Substances Naturelles à Gif-sur-Yvette) a été mise en place. Nous avons identifié et étudié les mécanismes d’action de plusieurs molécules inhibitrices potentielles de Mcl-1 et/ou Bcl-xL capables d’induire l’apoptose dans nos deux modèles de lymphomes. / Our team investigates the mechanisms of resistance to apoptosis induced in various B-cell lymphomas including some infected by the Epstein-Barr virus (EBV). EBV is an oncogenic member in the gamma-herpesvirus family. Among other pathologies, it is associated with Burkitt’s lymphoma (BL) and post-transplant lymphoproliferative disorders (PTLD). Previously, our laboratory has found that in these tumor cells, the binding of nutlin-3 to MDM2 results in the activation of p53. However, although p53 activation leads to apoptosis in EBV(-) cells, EBV(+) latency III cells which express all viral « latency » proteins are much more resistant. During this PhD project, I studied the mechanisms involved in this resistance and made attempts to define new therapeutic strategies that would bypass them. First, the role played by autophagy was investigated. This catabolic process which degrades proteins and organelles is physiologically complex as it is involved in both cell survival and cell death. Our work has demonstrated that: 1) autophagy was induced in nutlin-3 treated EBV(+) latency III cells; 2) Beclin-1 was strongly expressed in these cells whose autophagy was constitutively activated; 3) autophagy was involved in the resistance to apoptosis observed in these cells. Second, I turned my efforts to the identification of new molecules targeting anti-apoptotic members of the Bcl-2 family. Like Bcl-2, the antiapoptotic protein Mcl-1 is heavily expressed in LB and PTLD cell lines but in this case, independently of their EBV status and this is a frequent cause for the observed resistance to Bcl-2 inhibitors that are currently tested in clinical trials. Molecules targeting Mcl-1 could thus prove promising to circumvent this resistance. In a collaboration with a Chemistry team supervised by Fanny Roussi at the Institut de Chimie des Substances Naturelles in Gif-sur-Yvette, we have identified the mechanisms of action of potential inhibitors of Mcl-1 and/or Bcl-xL, another anti-apoptotic molecules which induce apoptosis in our two lymphoma models.

Apoptoseresistenz ist einer der Gründe für ein Versagen von Chemotherapie bei vielen Krebserkrankungen, darunter das Burkitt Lymphom. Um die molekularen Mechanismen der Apoptoseresistenz aufzuklären, wurde die Apoptoseinduktion in 15 Burkitt-Lymphom-Zelllinien nach Behandlung mit den Spindelgiften Taxol (Paclitaxel), Nocodazol und Vincristin untersucht. Interessanterweise entwickelten Zellen, die sich als resistent gegenüber Taxol- und Nocodazol-induzierter Apoptose erwiesen, nach Behandlung eine Polyploidie (>4N DNA), was eine inverse Relation von Apoptose und Polyploidie aufzeigt. In den sensitiven Zelllinien war die Taxol- und Nocodazol-induzierte Apoptose von Caspase-Aktivierung, Bid-Spaltung und Herunterregulation von Mcl-1 begleitet. Im Gegensatz zu den sensitiven Zelllinien wiesen die meisten apoptoseresistenten Zellen einen Verlust von Bax und Bak auf und waren durch einen anhaltenden mitotischen Arrest mit Auftreten eines >4N DNA-Gehalts nach Behandlung charakterisiert. Um weitere Einblicke in den Mechanismus der Spindelgift-induzierten Apoptose zu erhalten, wurde die Rolle der mitotische Kinase PLK1 (polo-like kinase) näher untersucht. Eine dominant-negative PLK1-Mutante induziert Apoptose. Allerdings zeigte eine zusätzliche Behandlung mit Spindelgiften keinen synergistischen Effekt, was darauf schließen lässt, dass sowohl Inhibierung von PLK1 als auch Mikrotubuli-destabilisierende Agenzien den gleichen Stress-Signalweg aktivieren. Andererseits unterstützte Überexpression von Wildtyp-PLK1 in Taxol behandelten Zellen die Zellzyklus-Progression. Dies deutet auf eine Verbindung zwischen Zelltodresistenz und genetischer Instabilität (Aneuplodie) hin. Inhibition von Apoptose in sensitiven Zelllinien durch Caspase-Inhibierung förderte Polypoidie, welche die inverse Relation bestätigte. Medikamente, welche die Caspase-Aktivierung unabhängig von Bax und Bak induzieren, könnten eine weitere Möglichkeit zur Behandlung von resistenten Burkitt-Lymphomen darstellen. / Apoptosis resistance is the major cause of chemotherapy failure in most kinds of cancers, including Burkitt lymphomas (BL). To elucidate molecular mechanisms regulating the development of apoptosis resistance, a panel of 15 BL cell lines was investigated for apoptosis induction upon treatment with microtubule inhibitors taxol, nocodazole and vincristine. Significant differences were observed in the extent of apoptosis induction among BL cell lines examined. Interestingly, cell lines exhibiting resistance to taxol- or nocodazole-induced apoptosis, showed development of polyploidy (>4N) and vice versa, displaying an inverse relationship between apoptosis and polyploidy induction. Further, in sensitive cell lines taxol-induced apoptosis was accompanied by caspase activation, Bid cleavage and Mcl-1 down-regulation. In contrast, most apoptosis resistant cell lines exhibited a loss of Bax and Bak expression and showed prolonged mitotic arrest with >4N DNA content upon treatment. To gain mechanistic insights into microtubule inhibitor-induced cell death, the role of the mitotic kinase PLK1 was addressed. Dominant negative PLK1 mutant induced apoptosis, however, failed to show synergism in induction of apoptosis in combination with microtubule inhibitors. This indicates that PLK1 inhibition and spindle toxins might trigger a similar mitotic stress pathway. Conversely, overexpression of wildtype PLK1 promoted cell cycle progression in cells treated with taxol. Remarkably, inhibition of apoptosis in sensitive cell lines by caspase inhibition promoted polyploidy confirming the inverse relationship between apoptosis and polyploidization. Considering targets to induce Bax/Bak independent caspase activation would be of great importance to avoid undesirable events leading to chromosomal imbalances in treating resistant cancers.

Le locus des chaînes lourdes d’immunoglobulines (IgH) subit trois processus de remaniements géniques durant la lymphopoïèse B. Ces événements induisent des cassures de l’ADN potentiellement oncogéniques, d’où la nécessité d’une régulation extrêmement stricte. Ceci est dû aux deux principaux éléments cis-régulateurs du locus IgH. L’enhancer 5’Eµ régule les recombinaisons VHDJH qui établissent un répertoire antigénique fonctionnel lors des phases précoces. La région régulatrice en 3’ (3’RR) est essentielle aux hypermutations somatiques (SHM) et à la recombinaison de classe (CSR) aux stades tardifs, modifiant respectivement, l’affinité et les fonctions effectrices de l’Ig. La plupart des lymphomes B matures portent les stigmates de translocations d’oncogènes au locus IgH. Le but de ma thèse a été de mieux comprendre les interactions transcriptionelles entre les enhancers Eµ et 3’RR et évaluer si le ciblage de cette dernière pourrait se révéler une approche thérapeutique potentielle. Nous avons démontré que la 3’RR est l’élément essentiel qui contrôle la transcription du locus IgH dans les lymphocytes B matures. Elle est dispensable lors des phases initiales (recombinaisons VHDJH), mais agit comme silencer sur l’expression des segments DJH. L’analyse de la lymphomagenèse dans trois modèles murins porteurs d’une insertion de Myc en trois points du locus IgH a montré des différences dans les cinétiques d’émergence des lymphomes, leurs phénotypes et index de prolifération. L’effet de la 3’RR sur l’oncogène est suffisant pour l’émergence de lymphomes B. Son absence ne semble pas être préjudiciable au développement de réactions inflammatoires/immunes. Son ciblage pourrait donc se révéler une approche thérapeutique intéressante pour diminuer son activité transcriptionelle sur l’oncogène transloqué. Un rôle potentiel des inhibiteurs des histones désacétylases est à l’étude. / The immunoglobulin heavy chain locus (IgH) undergoes several changes along B-cell differentiation. VHDJH recombinations during the early stages give the diversity of the antigenic repertoire. Somatic hypermutation (SHM) and class switch recombination (CSR) during late stages allow affinity maturation and the acquisition of new effectors functions. These rearrangements are highly regulated and are under the control of the IgH locus cis-regulatory elements. The 5’ Eµ enhancer is important for VHDJH recombination. The 3’ regulatory region (3’ RR) is essential for both CSR and SHM. These events induce breaks into the IgH locus, making it a hotspot for oncogenic translocations. The aim of my thesis was to understand the transcriptional interactions between Eμ and 3'RR enhancers and to evaluate whether the targeting of the latter could be of a potential therapeutic approach. We have demonstrated that 3'RR is essential to control IgH transcription in mature B cells. It is dispensable during the initial stages of developement (VHDJH recombinations). At the pro-B cell stage, it has a silencer effect rather than a transcriptional one on the DJH segments expression. The analysis of lymphomagenesis in three mice models carrying an insertion of Myc in different locations at the IgH locus showed significant differences in lymphoma kinetics, phenotypes and proliferation index. 3'RR alone, as a major transcriptional activator of the IgH locus, is capable of leading to B-cell lymphomas. Its absence is not detrimental for the development of classical inflammatory/immune reactions. Its targeting may be of a potentially interesting therapeutic approach to decrease its transcriptional activity on the translocated oncogene. A potential role for histone deacetylase inhibitors is under study.

Das FMC7-Antigen, eine unbekannte B-Zell-Membranstruktur, dient als eines der immunphänotypischen Grundkriterien zur Diagnose der typischen B-CLL. Die unterschiedliche CD20-Expressionsintensität ist auch ein charakteristisches Merkmal bei der Subtypisierung der Lymphomentitäten. In der vorliegenden Arbeit wurde die Modulation des CD20- und des FMC7-Antigens während einer Therapie mit CD20-Antikörper (Rituximab) bei Patienten mit indolenten B-Zell-Lymphomen untersucht. Bei der durchflusszytometrischen Untersuchung quantitativer und qualitativer Charakteristika der monoklonalen B-Zell-Population bei CLL- (n=12) und Non-CLL-Patienten (n=10) unmittelbar nach der Antikörperinfusion und bis zur 8. Woche nach der Therapie wurden parallele Veränderungen von FMC7 und CD20 festgestellt. Die Anzahl und die Fluoreszenzintensitäten der für die beiden Antigene positiven Zellen korrelierten signifikant sowohl bei der malignen Zell-Population (Kurzzeitbeobachtung: n=89; r=0,9; p / The FMC7, although being an unknown structure of the B-cell membrane, represents one of the basic immunophenotypic criteria for the diagnosis of the typical B-CLL. A different CD20 expression is a characteristic sign for the sub-typing of lymphomas also. The underlying study investigated the qualitative and quantitative modulation of the CD20 and FMC7 antigens in patients with indolent B-cell lymphomas (CLL, n=12 and non-CLL, n=10) during the therapy with a CD20 antibody (rituximab). Concomitant changes of FMC7 and CD20 expression were found immediately after rituximab infusion and up to 8 weeks thereafter. A correlation was seen for the number of positive malignant cells and for the corresponding fluorescence intensity (short-time observation n=89; r=0.9; p

El limfoma de cèl•lules de mantell (MCL) i la leucèmia limfàtica crònica (CLL) són dues neoplàsies limfoides fins al moment incurables, per la qual cosa requereixen la recerca de nous fàrmacs. La identificació de noves dianes terapèutiques per al MCL i la CLL, i del mecanisme molecular d’actuació d’aquests fàrmacs en aquestes neoplàsies, té una gran rellevància translacional. El concepte de medicina personalitzada està basat en l’ús de teràpies dirigides a través d’un coneixement profund dels mecanismes moleculars d’acció d’aquests agents. Amb l’augment del coneixement sobre l’etiologia del càncer, s’han descobert noves dianes alterades específicament en les cèl•lules tumorals. Entre elles, la sobreexpressió de les HDACs, que provoca un estat epigenètic aberrant a la cèl•lula. Això ha motivat el desenvolupament del vorinostat, un inhibidor de les HDACs. Els resultats obtinguts demostren el vorinostat és efectiu i selectiu per a les cèl•lules tumorals de MCL. A nivell molecular, el vorinostat en el MCL indueix l’acetilació de les histones de la regió promotora dels gens de les proapoptòtiques de la família de Bcl-2 BH3-only BIM, BMF i NOXA, que són activats transcripcionalment. Totes tres proteïnes, Bmf, Bim i Noxa, cooperen en el procés d’apoptosi. A més, el vorinostat és sinèrgic amb l’agent que mimetitza les BH3-only, ABT-263. En els últims anys, s’ha identificat un gran nombre de cinases, que regulen moltes vies de supervivència i proliferació cel•lulars, que estan específicament activades i sobreexpressades en cèl•lules tumorals. En particular, recentment les cinases associades al receptor de cèl•lules B (BCR) han esdevingut un focus d’atenció important, ja que està activament implicat en la patogènesi de les neoplàsies limfoides. En aquesta tesi, hem analitzat l’efecte anti-tumoral de l’inhibidor multi-cinasa sorafenib en la CLL i el MCL. Els nostres resultats confirmen que el sorafenib indueix apoptosi selectiva en cèl•lules de CLL i de MCL, fins i tot en els casos de més mal pronòstic de la CLL. A nivell molecular, els nostres resultats han demostrat que el sorafenib indueix una desfosforilació ràpida, sostinguda i simultània de les cinases associades al BCR, Syk i de la família Src cinases. En la CLL i el MCL a temps d’incubació molt curts, el sorafenib provoca una disminució traduccional de l’anti-apoptòtica Mcl-1 alhora que de ciclina D1, sobreexpressada en el MCL. Tant Mcl-1 com ciclina D1 participen en l’apoptosi induïda per sorafenib, la disminució de Mcl-1 altera directament els senyals anti-apoptòtics, mentre que el sorafenib, a més de disminuir els nivells de ciclina D1, també allibera Bax del segrestament per part de ciclina D1, finalment activant mecanismes d’apoptosi caspasa-dependents i independents com AIF. El sorafenib també modula les interaccions del microambient amb la CLL i el MCL. En aquest sentit, s’ha demostrat que el sorafenib bloqueja la migració induïda per la quimiocina CXCL12 via inhibició de FAK, una cinasa substrat de Src que regula i promou la invasió. El complex Src-FAK afecta múltiples proteïnes, com ara les del citoesquelet d’actina. En consistència, el sorafenib bloqueja la reorganització del citoesquelet en cèl•lules de MCL estimulades amb CXCL12. Les cèl•lules estromals del microambient dels teixits, com ara el moll d’os i els teixits limfoides secundaris, interaccionen amb el tumor i en promouen el seu creixement i resistència a fàrmacs. En aquest context, s’ha demostrat que, en presència de l’estroma del microambient, les cèl•lules de MCL i CLL esdevenen resistents als agents quimioterapèutics habituals i el sorafenib és capaç de resensibilitzar-les. Tant la proliferació com l’activació del BCR en la CLL són majors en el gangli limfàtic que en sang. En aquest sentit, s’ha demostrat que el sorafenib és més efectiu en cèl•lules de CLL derivades de gangli limfàtic que les respectives derivades de moll d’os i de sang perifèrica. L’estimulació del BCR en la CLL també provoca la secreció de les citocines atraients de cèl•lules accessòries CCL3 i CCL4, que el sorafenib bloqueja amb eficiència. Globalment, els treballs que constitueixen aquesta tesi doctoral suposen una contribució important en el coneixement del mecanisme molecular d’acció del vorinostat i el sorafenib, fàrmacs nous que podrien ser efectius per al tractament d’aquestes dues entitats, el MCL i la CLL. / Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are two incurable B-lymphoid malignancies, thus new therapeutic strategies are required. In the recent years, anti-tumor therapy has focused on pathways specifically altered in cancer cells. Among them, overexpression of histone deacetylases (HDACs) is a common feature. This led to the development of the HDAC inhibitor vorinostat. This thesis has shown that vorinostat could become a new therapeutic approach for MCL. The results show that vorinostat is effective and selective for MCL cells. The molecular mechanism of action of vorinostat involves acetylation of the histones in the gene promoter of the pro-apoptotic BH3-only from Bcl-2 family, BMF, BIM and NOXA, which induces its transcriptional activation. The respective proteins cooperate in the apoptosis induction. Moreover, vorinostat synergizes with the BH3-only mimetic ABT-263 in MCL. Plenty of kinases that have been recognized to regulate survival and proliferation pathways are overexpressed in tumor cells. In this work, we analyzed the anti-tumor effect of sorafenib, a multi-kinase inhibitor approved by the FDA, in CLL and MCL. Sorafenib induces selective apoptosis in CLL and MCL cells, being also effective in poor prognosis cases of CLL. At the molecular level, sorafenib dephosphorylates the B-cell receptor (BCR) associated kinases, specifically Syk and the Src family kinases. In CLL and MCL, sorafenib inhibits Mcl-1 and cyclin D1 translation. Mcl-1 downregulation is directly impairing anti-apoptotic signals. As for cyclin D1, sorafenib releases Bax from cyclin D1 sequestering. Both processes are involved in apoptosis induction, which occurs through caspase-dependent and independent mechanisms. In addition, sorafenib blocks CXCL12-induced migration and actin polymerization via FAK inhibition, a Src kinase substrate that regulates migration. Stromal cells from the microenvironment interact with and promote tumor growth and drug resistance. In this context, MCL and CLL cells become resistant to conventional chemotherapeutic drugs in the presence of stromal microenvironment, which is counteracted by sorafenib. Activation of the BCR and proliferation in CLL cells is higher in the lymph node microenvironments, a setting where sorafenib has been demonstrated to be more effective. BCR stimulation also leads to CCL3 and CLL4 chemoattractant cytokines secretion in CLL, that sorafenib blocks efficiently. Overall, this thesis is an important contribution to the understanding of the molecular mechanism of action of vorinostat and sorafenib, new drugs that might be effective for the treatment of these two entities, MCL and CLL.

Uncovering the chromosomal abnormalities associated with human malignancy can provide significant insights into the molecular basis of tumorigenesis, as well as identifying potential targets for therapy. The present study set out to examine the genetic characteristics of t(2;7)(p11-12;q21-22) translocations arising in conjunction with chronic B-cell neoplasia. Using long-range PCR, a t(2;7) was initially mapped in an individual presenting with the preclinical entity CD5- monoclonal B-cell lymphocytosis. This revealed a breakpoint at 2p11.2 localized to the recombination signal of the immunoglobulin kappa (IGK) variable gene IGKV3-15, and a breakpoint at 7q21.2 located 520 bp upstream of cyclin dependent kinase 6 (CDK6). The same approach was subsequently employed to elucidate near-identical t(2;7) breakpoints in 4 additional cases presenting with chronic lymphocytic leukemia or indolent non-Hodgkin lymphomas. The remarkable consistency of these translocations implicates the dysregulation of CDK6 via translocation to IGK as a recurrent pathomechanism during the emergence of B-cell lymphoproliferative disorders.

Uncovering the chromosomal abnormalities associated with human malignancy can provide significant insights into the molecular basis of tumorigenesis, as well as identifying potential targets for therapy. The present study set out to examine the genetic characteristics of t(2;7)(p11-12;q21-22) translocations arising in conjunction with chronic B-cell neoplasia. Using long-range PCR, a t(2;7) was initially mapped in an individual presenting with the preclinical entity CD5- monoclonal B-cell lymphocytosis. This revealed a breakpoint at 2p11.2 localized to the recombination signal of the immunoglobulin kappa (IGK) variable gene IGKV3-15, and a breakpoint at 7q21.2 located 520 bp upstream of cyclin dependent kinase 6 (CDK6). The same approach was subsequently employed to elucidate near-identical t(2;7) breakpoints in 4 additional cases presenting with chronic lymphocytic leukemia or indolent non-Hodgkin lymphomas. The remarkable consistency of these translocations implicates the dysregulation of CDK6 via translocation to IGK as a recurrent pathomechanism during the emergence of B-cell lymphoproliferative disorders.

Lymphomas are neoplasms of the human immune system and can be divided into two categories, Hodgkin’s lymphoma (HL) and non-Hodgkin lymphoma (NHL). Anaplastic large cell lymphoma (ALCL) is a form of NHL that shares a common distinctive feature with HL, the overexpression CD30. The expression of cytokine receptor CD30 is restricted to proliferating B and T lymphocytes in healthy individuals while its overexpression is associated with several lymphoproliferative diseases such as ALCL and HL. The activation of CD30 via ligand or antibodies triggers various cellular responses ranging from apoptosis to cell proliferation and it is thought that the variable cellular response to CD30 activation may be due to cell surface levels of CD30. The human CD30 gene is regulated at the transcriptional level and previous studies characterising its promoter have identified several factors that regulate the expression this gene. However none of these identified factors explain for the high levels of CD30 observed in HL and ALCL. Therefore this study focused on the identification and functional analysis of transcriptionally active regions located up or downstream of the CD30 promoter region. The first aim for this study was to identify and characterise regions within the human CD30 gene that are involved in its transcriptional regulation. Phylogenetic footprinting identified several regions downstream of the CD30 promoter that displayed high levels of sequence homology indicating potential functional significance. Validation of these regions through two in vivo approaches, DNase 1 hypersensitivity assay and chromatin accessibility studies localised potential transcriptionally active regions to intron 1 of the CD30 gene.

Plusieurs lymphomes à cellules B présentent des anomalies génétiques qui sont importantes pour déterminer leurs caractéristiques biologiques et peuvent être utiles pour le diagnostic. Les types les plus courants sont le lymphome folliculaire et le lymphome diffus à grandes cellules B (LDGCB), qui représentent à eux deux plus de 60 % de tous les lymphomes. Les LDGCB sont agressifs mais peuvent être traités par chimiothérapie à agents multiples. Cependant, les gènes suppresseurs de tumeur (GST) potentiellement responsables de la lymphomagenèse ne sont pas tous connus. Le rationnel de ce projet reposait sur des données non publiées du projet translationnel GHEDI (déchiffrer l'hétérogénéité génétique du lymphome diffus à grandes cellules B à l'ère du rituximab). Une hybridation génomique comparative (CGH) (puce Agilent 180 K) a été réalisée sur une série de 202 LDGCB de la série GHEDI et 40 % des délétions de la région 6q21 ont été identifiées, dont la région minimale commune délétée qui contient le gène HACE1. Par ailleurs, l'analyse transcriptomique a montré une corrélation significative entre le nombre de copies du gène et le niveau d'expression. Le gène HACE1, situé sur le chromosome 6q, code pour une ubiquitine ligase E3 et est régulé négativement chez l'homme dans les tumeurs, y compris les neuroblastomes et les lymphomes à cellules tueuses naturelles (NK). Il a été montré que le gène HACE1 ubiquityle Rac1, une protéine impliquée dans la prolifération cellulaire et la progression G2/M du cycle cellulaire. La fonction du gène HACE1 et les facteurs impliqués dans sa régulation transcriptionnelle sont en grande partie inconnus dans le contexte des lymphomes à cellules B. Dans cette étude, nous avons examiné si le gène HACE1 était un gène candidat dans la région génomique 6q impliqué dans la lymphomagenèse des LDGCB et plus largement dans les lymphomes B. Nous avons déterminé la fréquence de l'inactivation du gène HACE1 dans le lymphome à cellules B et analysé les mécanismes impliqués dans son extinction. / Several B-cell lymphomas have characteristic genetic abnormalities that are important in determining their biologic features and can be useful in differential diagnosis. Historically, classical Hodgkin lymphomas have been distinguished from non-Hodgkin lymphomas (NHL). The most common types are follicular lymphoma and diffuse large B-cell lymphoma (DLBCL), which together make up more than 60% of all lymphomas. DBCL are aggressive but potentially curable with multi-agent chemotherapy. However the putative tumor suppressor genes (TSG) responsible for lymphomagenesis still remain unknown. The rational of this project was based on unpublished data from the translational project GHEDI (Deciphering the Genetic Heterogeneity of Diffuse large B-cell lymphoma in the rituximab era). Array comparative genomic hybridization (aCGH) (Agilent 180 K) was performed in a series of 202 DLBCL and found 40% of deletions of 6q21 region, whose minimal commune deleted region (MCR) contains HACE1 gene. Furthermore, transcriptomic analysis showed a significant correlation between gene copy number and expression level. HACE1, located on chromosome 6q, encodes an E3 ubiquitin ligase and is downregulated in human tumors such as neuroblastomas and natural killer (NK) lymphomas. HACE1 has been shown to ubiquitylate Rac1, a protein involved in cell proliferation and G2/M cell cycle progression. The function of HACE1 and the factors involved in its transcriptional regulation are largely unknown in the context of B-cell lymphomas. In this study, we investigated whether HACE1 is a candidate gene in the 6q genomic region involved in DLBCL lymphomagenesis. We determined the frequency of HACE1 inactivation in B-cell lymphoma and analyzed the mechanisms involved in its silencing. We show, by RT-qPCR, that HACE1 gene is constitutively expressed in normal lymph nodes and in normal B-cells isolated from peripheral blood, contrasting with a strong downregulation of its expression in more than 70% (77/111) of B-cell lymphoma cases and in four tested B-Lymphoma cell lines. HACE1 gene copy number was assessed by quantitative multiplex PCR of short fluorescent fragments (QMPSF) and array for comparative genomic hybridization (aCGH) in 91 DLBCL cases.