Orientador: José Andres Yunes / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T05:50:44Z (GMT). No. of bitstreams: 1 Jotta_PatriciaYoshioka_D.pdf: 8922035 bytes, checksum: 3734371a320410ba431d6e6ce6579e55 (MD5) Previous issue date: 2012 / Resumo: A leucemia linfóide aguda (LLA) é o câncer mais frequente na infância, e destas, 15% são do tipo T (LLA-T). A hiperativação da via PI3K/Akt tem sido amplamente descrita em tumores e em linhagens celulares de LLA-T. PTEN é o principal regulador negativo dessa via e frequentemente encontra-se inativado em cânceres humanos. Com frequência, pacientes com LLA-T apresentam mutações ativadoras de NOTCH1. NOTCH1 pode regular transcricionalmente PTEN, contudo ainda não está claro como as mutações ativadoras de NOTCH1 influenciariam a expressão de PTEN nas LLA-T. Nós encontramos uma ocorrência de 11 (17,7%) mutações no éxon 7 do PTEN em 62 casos de LLA-T estudados consecutivamente. Contudo, nenhuma mutação foi encontrada na análise de 71 casos de LLA-B derivada. A maioria das mutações de PTEN apresentavam inserções/deleções de mais de 3 nucleotídeos. Não encontramos associação entre mutações em PTEN e o gênero, a idade e a contagem de glóbulos brancos ao diagnóstico. Pacientes com alterações no PTEN apresentaram uma tendência a pior sobrevida global (OS, p=0.07). Dentre os pacientes de LLA-T classificados como alto risco (n=56), aqueles possuindo anormalidades no PTEN mostraram-se associados significativamente a menor OS (p=0.019) e sobrevida livre de leucemia (LFS 47% vs 76%; p=0.045). As curvas de LFS foram significativamente diferentes (p=0.003), mesmo considerando apenas pacientes que atingiram a remissão no dia 28 do tratamento para a análise. Nosso estudo também mostrou que pacientes com mutações em NOTCH1 apresentavam aumento na transcrição de MYC e menor expressão de PTEN mRNA comparados a pacientes com NOTCH1 selvagem. Nós recentemente demonstramos que células de LLA-T apresentavam fosforilação de PTEN mediada por CK2, resultando na estabilização e consequentemente inativação da proteína PTEN. Corroborando ao estudo anterior, os casos de LLA-T analisados, independente do status de mutação em NOTCH1, expressam níveis significativamente mais altos de proteína PTEN do que controles normais. Para avaliar o impacto da regulação transcricional de NOTCH e a inativação postranscricional por CK2 de PTEN, nós tratamos as células de LLA-T com inibidores de gamma-secretase (DAPT e de CK2 (DRB/TBB). Nosso estudo enfatiza a relevância biológica e clínica da regulação do PTEN em LLA-T. E sugerimos o uso combinado de inibidores de gamma-secretase e CK2 devem possuir potencial terapêutico nas LLA-T / Abstract: T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 15% of pediatric ALL. Patients with T-ALL are at increased risk of relapse compared with children treated for B-cell precursor ALL. Mutations in the phosphatase and tensin homolog (PTEN) gene leading to PTEN protein deletion and subsequent activation of the PI3K/Akt signaling pathway are common in cancer. PTEN is the main negative regulator of the PI3K/Akt survival pathway. T-ALL patients frequently display NOTCH1 activating mutations and Notch can transcriptionally down-regulate the tumor suppressor PTEN. However, it is not clear whether NOTCH1 mutations associate with decreased PTEN expression in primary T-ALL. We report that PTEN exon 7 mutations occurred in 11 (17.7%) out of 62 consecutive pediatric T-cell acute lymphoblastic leukemia (T-ALL) but in none of 71 precursor B-ALL patients. Most PTEN mutations were insertions/deletions of more than 3 nucleotides. No associations were found between PTEN mutation and age, gender, WBC at diagnosis, early response to therapy and remission rate. Patients with PTEN mutation (n=11) had a tendency toward worse overall survival (OS, p=0.07). Remarkably, PTEN mutations were significantly associated with lower OS (p=0.019) and leukemia-free survival (LFS 47% vs 76%, p=0.045) within patients classified in the high risk group (n=56). LFS curves were significantly different (p=0.003) even if only patients who reached remission on day 28 were considered for analysis. We compared patients with or without NOTCH1mutations and report that the former presented higher MYC transcript levels and decreased PTEN mRNA expression. We recently showed that T-ALL cells frequently display CK2-mediated PTEN phosphorylation, resulting in PTEN protein stabilization and concomitant functional inactivation. Accordingly, the T-ALL samples analyzed, irrespectively of their NOTCH1 mutational status, expressed significantly higher PTEN protein levels than normal controls. To evaluate the integrated functional impact of NOTCH transcriptional and CK2 post-translational inactivation of PTEN, we treated TALL cells with both the gamma-secretase inhibitor DAPT and the CK2 inhibitors DRB/TBB. Our data suggest that combined use of gamma-secretase and CK2 inhibitors may have therapeutic potential in T-ALL. And emphasize the biological and clinical relevance of PTEN regulation in pediatric T-ALL / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Orientador: José Andrés Yunes / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T10:04:03Z (GMT). No. of bitstreams: 1 Laranjeira_AngeloBrunelliAlbertoni_D.pdf: 13248620 bytes, checksum: 729ec8e23331934ddd1e5803361b6fe6 (MD5) Previous issue date: 2012 / Resumo: A Leucemia Linfóide Aguda (LLA) é o tipo de câncer mais comum que acomete crianças. Sabe-se que a interação do tumor com o contexto celular do hospedeiro (microambiente tumoral) é recíproca, ou seja, na medida em que o tumor estimula o seu microambiente, este potencializa a sobrevivência, proliferação e invasividade tumoral. A interação da LLA com as células estromais da medula óssea tem um impacto positivo na resistência das células leucêmicas à quimioterapia. No presente estudo foi investigado a modulação de uma série genes de sensibilidade e resistência à asparaginase em células de LLA-B precursoras após co-cultura com as células estromais. Mostramos o aumento da expressão e secreção da IGFBP7 pelas células leucêmicas após co-cultivo com células do estroma da medula óssea. Em ensaios com o silenciamento do IGFBP7 em células leucêmicas e células estromais, mostramos que a IGFBP7 atua regulando positivamente o crescimento celular e aumenta a resistência a asparaginase. A IGFBP7 'leucêmica' junto com IGF/insulina atua sobre as células estromais, induzindo nestas células o aumento da produção de asparagina, e diminuindo a ação da asparaginase. Além deste mecanismo de resistência dependente das células estromais, mostramos que a IGFBP7 em conjunto com IGF/insulina promove a resistência das células leucêmicas à ação de outros compostos quimioterápicos (dexametasona e metotrexato) de forma independente da interação leucemia-estroma. Ainda pode ser observado que o plasma de crianças com LLA ao diagnóstico, apresenta maiores níveis de IGFBP7 do que em amostras controles. É importante ressaltar que níveis mais altos de mRNA IGFBP7 foram associados com menor sobrevida livre de leucemia (Modelo de regressão de Cox, P = 0,003), em células de LLAB Ph(-) presursoras / Abstract: Acute Lymphoblastic Leukemia (ALL) is the most common type of cancer that affects children. It is known that the interaction between tumor and the cellular context of the host (tumor microenvironment) is reciprocal, ie, to the extent that the tumor stimulates their microenvironment, this enhances the survival, proliferation and tumor invasiveness. The interaction of ALL with bone marrow stromal cells has a positive impact on leukemia resistance to chemotherapy. In the present study, we investigated the modulation of a series of putative asparaginase-resistance/sensitivity genes in B-precursor ALL upon co-culture with stromal cells. We showed an increase expression and secretion of IGFBP7 in leukemic cells after co-culture with BMSCs. Assays with IGFBP7 knockdown in leukemic cells and stromal cells, showed that IGFBP7 acts as a positive regulator of cell growth and increases resistance to asparaginase. 'Leukemic' IGFBP7 together with IGF/insulin acts on stromal cells, increasing asparagine production, thus reducing the asparaginase effect. Besides this mechanism of resistance dependent of stromal cells, we showed that IGFBP7 in conjunction with IGF/insulin promotes the resistance of leukemia cells to the action of other chemotherapeutic compounds (dexamethasone and methotrexate) independently of the interaction leukemia-stroma. We still observed that diagnostic BM plasma from children with ALL at diagnosis, have higher levels of IGFBP7 than control samples. Importantly, higher levels of IGFBP7 mRNA were associated with lower leukemia-free survival (Cox regression model, P = 0.003) in precursor B-ALL Ph (-) patients / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Abstract In the Nordic countries, event-free survival (EFS) exceeds 80% in certain groups of children treated for acute lymphoblastic leukaemia (ALL). With the improved cure rates, however, there are more children suffering from neurological late effects, especially due to therapy directed at the central nervous system (CNS). The aim of this study is to examine the changes taking place in the nervous system after leukemia treatment and to evaluate the role of treatment in these changes in patients and in an animal model. Twenty-seven ALL survivors and healthy controls were examined by means of motor evoked potentials (MEPs). ALL survivors were also examined clinically. The children with ALL continued to show decreased motor nerve conduction in the peripheral nerves, but not within the CNS, five years after the cessation of treatment. Clinical neurological findings were obtained in 33% of the cases. The MEP results indicated reversibility of the motor injury due to CNS effects. Nineteen patients underwent perfusion magnetic resonance imaging (MRI) at the cessation of treatment or 4-8 years after the treatment. Seventeen of them also underwent single-photon emission computed tomography (SPECT). The studies showed small perfusion defects in SPECT, which were not visible by perfusion MRI. Methotrexate (Mtx) neurotoxicity was studied in a swine model using functional MRI, brain perfusion SPECT, iodine-123 labelled 2β-carbomethoxy-3β-(4-iodophenyl) tropane ([123I]β-CIT) SPECT and whole-hemisphere autoradiography with [125I]β-CIT in ten Mtx-treated animals and five control animals. Mtx-related changes in the brain could be detected as reduced or negative blood-oxygen-level-dependent (BOLD) responses to somatosensory activation in BOLD contrast MRI, which indicates changes in flow metabolism coupling. Perfusion defects in brain SPECT were seen in the Mtx group and the control group, which suggests that the perfusion defects seen in brain SPECT are probably multifactorial. The change in dopamine transporter (DAT) density in the Mtx group was not different from that in the controls. The abnormalities in nerve conduction after treatment in survivors of ALL were partly reversible years after the treatment. The patients had perfusion defects in SPECT imaging which were not seen in perfusion MRI. The clinical significance of these defects remains obscure. The animal model suggested perfusion defects to be multifactorial.

Males and age group 1 to 5 years show a much higher risk for childhood acute lymphoblastic leukemia (ALL). We performed a case-only genome-wide association study (GWAS), using the Illumina Infinium HumanCoreExome Chip, to unmask gender- and age-specific risk variants in 240 non-Hispanic white children with ALL recruited at Texas Children’s Cancer Center, Houston, Texas. Besides statistically most significant results, we also considered results that yielded the highest effect sizes. Existing experimental data and bioinformatic predictions were used to complement results, and to examine the biological significance of statistical results. Our study identified novel risk variants for childhood ALL. The SNP, rs4813720 (RASSF2), showed the statistically most significant gender-specific associations (P < 2 x 10-6). Likewise, rs10505918 (SOX5) yielded the lowest P value (P < 1 x 10-5) for age-specific associations, and also showed the statistically most significant association with age-at-onset (P < 1 x 10-4). Two SNPs, rs12722042 and 12722039, from the HLA-DQA1 region yielded the highest effect sizes (odds ratio (OR) = 15.7; P = 0.002) for gender-specific results, and the SNP, rs17109582 (OR = 12.5; P = 0.006), showed the highest effect size for age-specific results. Sex chromosome variants did not appear to be involved in gender-specific associations. The HLA-DQA1 SNPs belong to DQA1*01:07and confirmed previously reported male-specific association with DQA1*01:07. Twenty one of the SNPs identified as risk markers for gender- or age-specific associations were located in the transcription factor binding sites and 56 SNPs were non-synonymous variants, likely to alter protein function. Although bioinformatic analysis did not implicate a particular mechanism for gender- and age-specific associations, RASSF2 has an estrogen receptor-alpha binding site in its promoter. The unknown mechanisms may be due to lack of interest in gender- and age-specificity in associations. These results provide a foundation for further studies to examine the gender- and age-differential in childhood ALL risk. Following replication and mechanistic studies, risk factors for one gender or age group may have a potential to be used as biomarkers for targeted intervention for prevention and maybe also for treatment.

Orientador: José Andrés Yunes / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T08:28:04Z (GMT). No. of bitstreams: 1 Milani_Mateus_M.pdf: 3959646 bytes, checksum: a36674e099fefbc487286cdc33006c0a (MD5) Previous issue date: 2014 / Resumo: A leucemia linfoide aguda (LLA) é o câncer mais comum na infância. O transplante de células primárias de LLA humana em camundongos imunosuprimidos tem sido de suma importância para o entendimento da fisiopatologia da doença e para o teste de novos fármacos. Ao contrário de modelos animais de tumores sólidos, cujo volume é facilmente medido na superfície dos animais, a LLA infiltra órgãos inacessíveis ao exterior, daí a necessidade de definir métodos adequados para o monitoramento da progressão da doença. Resultados aqui apresentados indicam que proteínas secretadas pela LLA podem servir como marcadores quantitativos da carga leucêmica, facilmente aferidos por ELISA de amostras de plasma sanguíneo. Dentre três proteínas testadas (B2M, IGFBP2 e Hsp90), o ELISA de Hsp90 apresentou sensibilidade superior à análise da porcentagem de células leucêmicas no sangue dos animais, por citometria de fluxo de células marcadas com anti-huCD45. Os níveis de Hsp90 humano no plasma sanguíneo mostraram-se positivamente correlacionados com o porcentual de células leucêmicas na medula óssea e fígado e em menor grau com os níveis do baço e sangue periférico (SP) ao longo do tempo, tanto nas LLA de linhagem B quanto nas LLA-T. O ELISA de Hsp90 permite detectar a instauração da leucemia nos animais transplantados, até duas semanas antes da detecção pelo método tradicional de análise de sangue periférico por citometria de fluxo. Ao contrário do observado para IGFBP2, o tratamento dos animais leucêmicos com Dexametasona ou um inibidor da PI3K não interferiu nos níveis de Hsp90, que se mantiveram proporcionais à porcentagem de células leucêmicas huCD45+ no sangue periférico. No conjunto, os resultados demonstram que a análise do plasma dos animais por ELISA de Hsp90 é um método melhor do que os atualmente utilizados, para diagnóstico precoce e acompanhamento de LLA humana quando em níveis de doença residual mínima, ou seja, quando a porcentagem de células de LLA é inferior a 5% do total de células da medula óssea / Abstract: Acute lymphoblastic leukemia (ALL) is the most common type of childhood cancer . The transplantation of human primary ALL cells in immunodeficient mice has been of much importance for understanding the disease's pathophysiology and testing new drugs. Unlike animal models of solid tumors whose volume is easily measured on the animal surface, the ALL infiltrates organs that are inaccessible to external antigens, hence the need to define more suitable methods for monitoring the disease's progression. Results presented here indicate that proteins secreted by the ALL can serve as quantitative markers of leukemic burden and are easily measured by ELISA of blood plasma samples. Among three tested proteins (B2M, IGFBP2 and Hsp90), Hsp90 ELISA analysis showed higher sensitivity than the analysis of leukemic cells on animal blood by flow cytometry of anti- huCD45 labeled cells. The levels of Hsp90 in human blood plasma were shown to be positively correlated with the percentage of leukemic cells in the bone marrow and liver and to a lesser extent with the levels in the spleen and peripheral blood (PB) over time, both in B-lineage ALL as in ALL-T. The Hsp90 ELISA allows the leukemia's engraftment detection in transplanted animals up to two weeks prior to detection by the traditional method of peripheral blood analysis by flow cytometry. Unlike observed for IGFBP2, treatment of leukemic animals with Dexamethasone or PI3K inhibitors did not interfere in Hsp90 levels, which remained proportional to the percentage of huCD45+ leukemic cells in the peripheral blood. Taken together, the results demonstrate that the analysis of animal plasma by Hsp90 ELISA is a better method than those currently used for early diagnosis and monitoring of human ALL on minimal residual disease levels, when the percentage of ALL cells is less than 5 % of the total bone marrow cells / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

With the increase in survival for children with cancer, part of the focus of current research is aimed towards evaluating how these children are adapting psychosocially. Neurocognitive deficits have been well established. However, there are multiple facets encompassing quality of life, including general mental health, lifestyles and health behaviors, and academic and cognitive functioning. The relationship between neurocognitive and psychosocial functioning has yet to be thoroughly evaluated. The purpose of this study was to investigate the relationship between neurocognitive and psychosocial functioning in survivors of brain tumors and acute lymphoblastic leukemia. Data was collected from existing archival database comprised of patients of the at Cook Children's Medical Center in Texas. The sample consisted of 177 patients between the ages of 3 and 12 who were at least two years post-diagnosis. Measures used included the NEPSY and the Behavioral Assessment for Children. Statistical analyses included a several one-way analysis of variances, an independent samples t-test, a univariate analysis of variance, a hierarchical multiple regression, and odds ratio analyses. Results indicated survivors treated with neurosurgery alone appear to be less at risk for developing behavior problems than other treatment modalities. Also, brain tumor survivors demonstrate more problematic behaviors than survivors of acute lymphoblastic leukemia. Visuospatial functioning, diagnosis, and type of treatment were found to be predictive variables of behavior problems. Attention, and perhaps language, deficits may predispose children to more problems in their behavior. It is concluded that there are other factors affecting behavior in this population that were not accounted for in this analysis. It is recommended for future studies to research the individual clinical scales of the Behavior Assessment System for Children, obtain information from multiple informants, study this relationship longitudinally, and research additional factors that may be influencing the relationship between neurocognitive and psychosocial functioning. This provides evidence of risk factors that should be monitored as the child returns home and to school.

Hintergrund: Der Phospholipase A2 Rezeptor 1 (PLA2R1) ist ein Typ 1 Transmembranrezeptor, welcher der Mannose-Rezeptor-Familie zugeordnet werden kann. Die Bedeutung von PLA2R1 für physiologische und pathologische Vorgänge ist noch weitestgehend unbekannt. Jedoch wird die Regulation wichtiger zellulärer Prozesse, wie Proliferation, Apoptose/ Seneszenz, Adhäsion, Migration/ Invasion und Inflammation im Zusammenhang mit dem Rezeptor diskutiert. Darüber hinaus ist eine Änderung der PLA2R1-Expression bei der Entstehung verschiedenster Krebserkrankung nachweisbar. Hierbei wird der Rezeptor einerseits mit einer pro-onkogenen und pro-migratorischen Wirkung in Verbindung gebracht. Andererseits ist ein tumorsuppressiver Effekt von PLA2R1 und eine Induktion der mitochondrialen Apoptose in Tumorzellen beschrieben. Zudem ist die Expression von PLA2R1 durch epigenetische Mechanismen kontrolliert und eine Promotor-Hypermethylierung ist assoziiert mit einer Repression der Rezeptor-Expression in der Prostatakarzinom (PCa)-Zelllinie LNCaP und der pädiatrischen, akuten lymphoblastischen Leukämie (ALL)-Zelllinie Jurkat. Vorangegangene Arbeiten zeigten eine Hypermethylierung innerhalb eines definierten Bereiches des PLA2R1-Promotors bei adulten Patienten mit akuter myeloischer Leukämie und Myelodysplastischem Syndrom (MDS) sowie eine Korrelation der PLA2R1-Promotormethylierung mit dem Krankheitsstadium und der Klassifizierung nach dem Internationalen Prognostischen Scoring System (IPSS). Fragestellung/ Hypothese: Das Ziel der vorliegenden Arbeit war einerseits die Untersuchung der Funktion von PLA2R1 in den PCa-Zelllinien LNCaP und PC-3. Während in LNCaP die Rezeptor-Expression durch Promotor-Hypermethylierung unterdrückt ist, kann in PC-3-Zellen eine Hochregulation von PLA2R1 im Vergleich zu normalen Prostataepithelzellen nachgewiesen werden. Durch in vitro Transfektionsexperimente sollte der Effekt einer Re-expression von PLA2R1 in LNCaP-Zellen sowie die Auswirkungen einer Reduktion der PLA2R1-Expression in PC-3-Zellen untersucht werden. Der Einfluss der veränderten PLA2R1-Expressionslevel auf wichtige Zellparameter wurde evaluiert. Die in vitro Daten der PCa-Zelllinien wurden mit den in vivo Ergebnissen des Tumorwachstums von transfizierten LNCaP- und PC-3-Zellen in Xenograft-Mausmodellen verglichen. Andererseits sollte basierend auf den Ergebnissen von adulten Patienten mit AML- und MDS-Diagnose und der dabei festgestellten Hypermethylierung des Rezeptor-Promotors der Methylierungsstatus des Rezeptors bei der pädiatrischen ALL untersucht werden. Überdies sollte die Eignung der PLA2R1-Methylierungsanalyse als sensitiver Biomarker für die Therapiekontrolle, Überwachung der minimalen Resterkrankung (MRD) und Risikostratifizierung der pädiatrischen ALL evaluiert werden. Die Funktion des Rezeptors im Kontext der pädiatrischen ALL wurde durch eine transfektionsbasierte Re-expression von PLA2R1 in der Jurkat-ALL-Zelllinie untersucht. Durch in vitro Experimente wurden die Auswirkungen der verschiedenen PLA2R1-Expressionslevel auf Proliferation und Apoptose/ Nekrose in transfizierten Jurkat-Zellen analysiert. Material und Methoden: Durch Transfektion mit einem PLA2R1-Expressionsvektor konnte eine stabile Überexpression des Rezeptors in LNCaP- (LNCaP-PLA2R1) und Jurkat-Zellen (Jurkat-PLA2R1) erreicht und die Ergebnisse mit Kontrollvektor-transfizierten LNCaP- (LNCaP-Ctrl) und Jurkat-Zellen (Jurkat-Ctrl) verglichen werden. Mittels CRISPR/Cas9-Knockdown konnte eine Verminderung der PLA2R1-Expression in PC-3-Zellen (PC-3-KD) im Vergleich zu Kontrollvektor-transfizierten PC-3-Zellen (PC-3-Ctrl) erreicht werden. Genexpressionsanalysen wurden mittels quantitativer PCR nach reverser Transkription (RT-qPCR) durchgeführt und die Proteinsynthese des Rezeptors durch Western Blot Analyse überprüft. In vitro sollten die Auswirkungen der differenziellen PLA2R1-Expression der transfizierten Zellen auf wichtige proliferative und metastatische Zellparameter untersucht werden. Die Zellviabilität/ Proliferation wurde mittels WST-1 Assay für adhärente Zellen und Zellwachstumskurven-Analyse mit Trypanblau-Färbung bei Suspensionszellen analysiert. Zellmotilität und Proliferation wurden bei transfizierten PCa-Zelllinien mithilfe des Wundheilungsassays beurteilt. Apoptose konnte durch Wasserstoffperoxid stimuliert und mittels Caspase-Glo® 3/7 Assay und RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay für transfizierte PCa-Zelllinien sowie durchflusszytometrische Analysen nach Annexin-V-FLUOS/ Hoechst 33258 Färbung für transfizierte Jurkat-Zellen untersucht werden. Die klonogene Überlebensrate und das Koloniewachstum der transfizierten PCa-Zelllinien sollten mithilfe des klonogenen Assays analysiert werden. In einer in vivo Pilotstudie wurde der Effekt von PLA2R1 auf das Tumorwachstum mittels Xenograft-Mausmodellen (männliche SCID/beige Mäuse) durch subkutane Injektion der transfizierten LNCaP- (n = 5) und PC-3-Zellen (n = 9) überprüft. Die PLA2R1-Promotormethylierung als sensitiver Biomarker für die pädiatrische ALL wurde durch Isolation und Bisulfit-Behandlung der genomischen DNA von Knochenmark (KM)-Aspiraten und Leukozyten des peripheren Blutes (PB) von ALL-diagnostizierten Kindern (n = 44) sowie einer anschließenden Analyse mittels digitaler PCR (dPCR) evaluiert. Die Ergebnisse konnten mit dem Methylierungsstatus einer gesunden Kontrollgruppe (n = 20) verglichen werden. Ergebnisse und Schlussfolgerungen: In LNCaP-PLA2R1 und Jurkat-PLA2R1 konnte im Gegensatz zu den dazugehörigen Kontrollzellen eine stabile Überexpression des Rezeptors auf Ebene der Genexpression und Proteinsynthese detektiert werden. Bei PC-3-KD-Zellen war eine Reduktion der PLA2R1-Genexpression und eine Repression der Proteinsynthese unterhalb der Nachweisgrenze des Western Blot Assays zu verzeichnen, während PC-3-Ctrl-Zellen eine Genexpression und Proteinsynthese des Rezeptors zeigten. Die Zellviabilität/ Proliferation und Motilität war signifikant erhöht in LNCaP-PLA2R1 und PC-3-Ctrl im Vergleich zu LNCaP-Ctrl- und PC-3-KD-Zellen. Demgegenüber war eine Verminderung von Apoptose und Koloniewachstum in LNCaP-PLA2R1 und PC-3-Ctrl-Zellen nachweisbar. Durch Genexpressionsanalysen konnte eine Induktion der Expression von Fibronektin 1 (FN1), TWIST Homolog 1 (TWIST1) und Cyclin-abhängige Kinase 6 (CDK6) in LNCaP-PLA2R1-Zellen identifiziert werden. In vivo schien die PLA2R1-abhängige negative Regulation des Koloniewachstums die pro-onkogenen Eigenschaften des Rezeptors zu überwiegen. Dies resultierte in einem verminderten Tumorwachstum von LNCaP-PLA2R1 und einer tumorsuppressiven Rolle des Rezeptors in dieser PCa-Zelllinie. Im Gegensatz dazu zeigten PC-3-Ctrl-Zellen ein schnelleres Tumorwachstum im Xenograft-Mausmodell, was für einen pro-onkogenen Effekt der endogenen PLA2R1-Expression in PC-3-Zellen sprechen würde. Der differenzielle Einfluss von PLA2R1 auf die Regulierung des Tumorzellwachstums könnte im Zusammenhang mit der veränderten Expression von FN1, TWIST1 und CDK6 stehen, jedoch sind weiterführende Experimente nötig, um die Beteiligung dieser Gene in der PLA2R1-Signaltransduktion zu untersuchen. Die Analyse der Zellwachstumskurve der transfizierten Jurkat-Zellen zeigte eine Abnahme der Proliferationsrate und eine Zunahme des Anteils an toten Zellen bei Jurkat-PLA2R1 im Vergleich zu Jurkat-Ctrl-Zellen. Durchflusszytometrische Analysen bestätigten eine Abnahme des Anteils gesunder sowie eine vermehrte Repräsentation von apoptotischen und nekrotischen Jurkat-PLA2R1-Zellen im Vergleich zur Kontrolle, was einen tumorsuppressiven Einfluss des Rezeptors bei der pädiatrischen ALL suggeriert. Die Funktion von PLA2R1 als Tumorsuppressor steht im Einklang mit der festgestellten Hypermethylierung des Rezeptor-Promotors in KM-Aspiraten und PB-Proben von pädiatrischen Patienten mit prä-B und common ALL zum Zeitpunkt der Diagnose der primären Krebserkrankung und des ALL-Rezidives im Vergleich zu der Kontrollgruppe. Der parallele Abfall der PLA2R1-Promotormethylierung und der relativen Blastenzahl im Verlauf der ALL-Induktionstherapie sowie eine signifikante, positive Korrelation beider Größen in KM- und PB-Proben ließen auf die leukämischen Blasten als Quelle der Hypermethylierung des PLA2R1-Promotors schließen. Überdies wiesen Hochrisikopatienten der pädiatrischen ALL eine signifikant höhere PLA2R1-Promotormethylierung am Tag 15 der ALL-Induktionstherapie auf im Vergleich zu Patienten mit einem geringeren Risiko. Zusammenfassend deuteten die in vitro und in vivo Daten auf eine wichtige Funktion des Rezeptors bei der Regulation von Proliferation und Apoptose bei der pädiatrischen ALL hin. Die Analyse der PLA2R1-Promotormethylierung könnte als sensitiver Biomarker zu einer verbesserten ALL-Therapiekontrolle, MRD-Überwachung und Risikostratifizierung während der ALL-Induktionstherapie beitragen.:Inhaltsverzeichnis 1 Zusammenfassung 4 2 Abstract 8 3 Einführung in die Thematik 11 4 Publikation 1: “Diverse Effects of Phospholipase A2 Receptor Expression on LNCaP and PC-3 Prostate Cancer Cell Growth in vitro and in vivo” 24 5 Publikation 2: “Methylation of the Phospholipase A2 Receptor 1 Promoter Region in Childhood B Cell Acute Lymphoblastic Leukaemia” 25 6 Diskussion und Ausblick 26 7 Literaturverzeichnis 32 8 Danksagung 41 9 Anlagen 42 / Background: The phospholipase A2 receptor 1 (PLA2R1) is a type I transmembrane receptor and a member of the mannose receptor family. Physiological and pathophysiological functions of PLA2R1 are still not completely understood. However, PLA2R1 expression is discussed to have an impact on proliferation, apoptosis/ senescence, adhesion, migration/ invasion as well as inflammatory cell responses and divergent PLA2R1 expression is detectable in different types of cancer compared to corresponding normal tissues. In this context, receptor expression is linked to both a pro-oncogenic/ pro-migratory and a tumour-suppressive/ pro-apoptotic impact in different cancer cells. Moreover, PLA2R1 expression is controlled by epigenetic mechanisms and hypermethylation of the PLA2R1 promoter is associated with silenced expression of the receptor in the prostate carcinoma (PCa) cell line LNCaP and the paediatric, acute lymphocytic leukaemia (ALL) cell line Jurkat. Previous work revealed a defined hypermethylated region of the PLA2R1 promoter in adult patients with acute leukaemia and myelodysplastic syndrome (MDS). PLA2R1 promoter methylation correlated with disease stage and International Prognostic Scoring System (IPSS) classification. Aim: The aim of the present study was to evaluate the function of PLA2R1 in PCa cell lines LNCaP and PC-3. The receptor expression is silenced in LNCaP but upregulated in PC-3 cells compared to normal prostate epithelial cells. A pilot in vivo study addressed the effects of PLA2R1 in mice xenografted with transfected LNCaP and PC-3 cells. Based on previous findings of PLA2R1 promoter hypermethylation in adult ALL and MDS patients, the aim of the present study was to analyse the methylation status of the PLA2R1 promoter in paediatric ALL patients compared to healthy individuals. PLA2R1 methylation analysis was evaluated as sensitive biomarker for ALL treatment response, minimal residual disease (MRD) monitoring, and risk stratification. The impact of the receptor in childhood ALL was investigated by transfection-based re-expression of PLA2R1 in the paediatric ALL cell line Jurkat and the effect of different PLA2R1 expression levels on proliferation and apoptosis/ necrosis was analysed in in vitro experiments. Material and Methods: Stable PLA2R1 overexpression was achieved by transfection of LNCaP (LNCaP-PLA2R1) and Jurkat cells (Jurkat-PLA2R1) with a PLA2R1 plasmid vector. Results were compared to control vector transfected LNCaP (LNCaP-Ctrl) and Jurkat cells (Jurkat-Ctrl). Alternatively, PLA2R1 was knocked down using CRISPR/Cas9 in PC-3 cells (PC-3-KD) and compared to the corresponding control-transfected cells (PC-3-Ctrl). Gene expression analysis was conducted by quantitative reverse transcription PCR (RT-qPCR). PLA2R1 protein synthesis was analysed by western blot. The impact of the differential PLA2R1 expression on proliferative and metastatic parameters of transfected cancer cells was investigated in vitro. Cell viability/ proliferation was assessed by means of WST-1 Assay for adherent cells and via cell growth curve analysis after trypan blue staining for suspension cells. Cell motility and proliferation of transfected PCa cell lines were estimated by wound healing assay. Hydrogen peroxide-stimulated apoptosis was analysed by Caspase-Glo® 3/7 Assay and RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay for transfected PCa cell lines and flow cytometric analysis after Annexin-V-FLUOS/ Hoechst 33258 staining for transfected Jurkat cells. Colony formation of transfected PCa cell lines was evaluated by clonogenic assay. A pilot in vivo study addressed the effects of PLA2R1 in mice xenografted with transfected LNCaP (n = 5) and PC-3 cells (n = 9). Evaluating PLA2R1 promoter methylation as sensitive biomarker for paediatric ALL, genomic DNA was isolated from bone marrow (BM) and peripheral blood (PB) of 44 paediatric ALL patients. After bisulfite treatment of isolated DNA samples, PLA2R1 methylation was analysed using digital PCR and compared to 20 healthy controls. Results and Conclusions: PLA2R1 gene expression and protein synthesis were detectable in LNCaP-PLA2R1, PC-3-Ctrl, and Jurkat-PLA2R1 cells but not in LNCaP-Ctrl and Jurkat-Ctrl cells. In PC-3-KD cells, PLA2R1 gene expression was significantly reduced compared to PC-3-Ctrl and PLA2R1 protein synthesis of PC-3-KD cells was below the limit of detection of western blot analysis. Cell viability/proliferation and motility were significantly increased in LNCaP-PLA2R1 and PC-3-Ctrl compared to LNCaP-Ctrl and PC-3-KD cells, respectively. However, levels of apoptosis and clonogenicity were reduced in LNCaP-PLA2R1 and PC-3-Ctrl cells. Gene expression analysis revealed an up-regulation of fibronectin 1 (FN1), TWIST homolog 1 (TWIST1), and cyclin-dependent kinase 6 (CDK6) in LNCaP-PLA2R1 compared to control cells. In LNCaP xenografts, PLA2R1-dependent regulation of clonogenicity appeared to outweigh the receptor’s pro-oncogenic properties, resulting in decreased tumour growth, supporting the tumour-suppressive role of PLA2R1. Alternatively, PC-3-Ctrl xenografts exhibited faster tumour growth compared to PC-3-KD cells, suggesting a pro-oncogenic effect of endogenous PLA2R1 expression. The differential growth-regulatory effects of PLA2R1 may be mediated by FN1, TWIST1, and CDK6 expression, although further investigation is required. Cell growth curve analyses of transfected Jurkat cells revealed a decreased proliferation and increased cell death of Jurkat-PLA2R1 compared to Jurkat-Ctrl cells. Flow cytometry confirmed the reduced fraction of healthy cells and an increase of the apoptotic and necrotic fractions in Jurkat-PLA2R1 cells compared to control cells, suggesting a tumour-suppressive effect of the receptor in paediatric ALL. PLA2R1’s tumour-suppressive function is in accordance with hypermethylation of the receptor promoter in BM aspirates and PB samples of paediatric patients diagnosed with pre-B and common ALL as well as in patients with disease relapse in comparison to healthy controls. PLA2R1 methylation decreased along with leukaemic blast cell reduction during ALL induction treatment and significant positive correlations between PLA2R1 methylation and leukaemic blast cell numbers of BM and PB samples were observable. Therefore, our data suggests that leukaemic blasts are the origin of PLA2R1 hypermethylation in BM and PB samples. Moreover, high risk paediatric ALL patients exhibited increased levels of PLA2R1 promoter methylation compared to non-high risk groups on day 15 of ALL induction treatment. Collected data indicates that PLA2R1 promoter methylation quantitation can be used as biomarker for ALL induction treatment control, risk stratification, and early detection of ALL relapse.:Inhaltsverzeichnis 1 Zusammenfassung 4 2 Abstract 8 3 Einführung in die Thematik 11 4 Publikation 1: “Diverse Effects of Phospholipase A2 Receptor Expression on LNCaP and PC-3 Prostate Cancer Cell Growth in vitro and in vivo” 24 5 Publikation 2: “Methylation of the Phospholipase A2 Receptor 1 Promoter Region in Childhood B Cell Acute Lymphoblastic Leukaemia” 25 6 Diskussion und Ausblick 26 7 Literaturverzeichnis 32 8 Danksagung 41 9 Anlagen 42

Les cellules Natural Killer (NK) représentent une population de cellules innées lymphoïdes aux fonctions anti-infectieuses et antitumorales. Les leucémies aiguës lymphoblastiques pré-B (LAL pré-B) constituent le cancer de l’enfant le plus fréquent et ont été décrites comme résistantes à la cytotoxicité médiée par les NK bien que les bases moléculaires demeurent inconnues.L’objectif de ces travaux a été de caractériser cette résistance. En développant un essai de cytotoxicité par cytométrie en flux et en utilisant des cellules effectrices activées in vitro, nous avons établi la sensibilité retardée des LAL pré-B à la cytotoxicité NK : initialement résistantes après 4h d’incubation, elles sont fortement tuées après 25h.Cette cytotoxicité est contact-dépendante mais ni la voie de l’exocytose des granules cytotoxiques ni celle des récepteurs de mort n’y contribuent. La mort cellulaire des cibles est de profil apoptotique mais indépendante des caspases ; la signalisation mitochondriale l’amplifie partiellement. Interférer avec les dérivés de l’oxygène par un antioxydant diminue la cytotoxicité. Nous montrons que les cellules NK de patients atteints de granulomatose septique chronique liée à l’X présentent un défaut de cette nouvelle cytotoxicité. Nous démontrons l’expression par les NK des composants clefs d’une NADPH oxydase distincte du complexe utilisé par les phagocytes. Nos travaux établissent l’existence d’une voie de cytotoxicité non conventionnelle et en définissent les principaux prérequis moléculaires. / Natural Killer (NK) cells are innate lymphoid cells with anti-infectious and anti-tumoral activities. Among neoplasia, pre-B acute lymphoblastic leukemias (pre-B ALL) represent the most common form of cancer in childhood and were shown to be resistant to NK cell mediated cytotoxicity although the mechanisms explaining this phenomenon are incompletely understood.In the present work, we investigated the relative immune resistance of pediatric pre-B ALL targets to activated NK cells. We developed a flow cytometry based cytotoxicity assay to assess the NK activity and the involvement of long term cytotoxic pathways. Although pre-B ALL blasts were strongly resistant at 4h, we found a considerable delayed NK killing at 25h.Further investigations revealed that cell contact was mandatory for efficient killing but also that neither the granule exocytosis nor the death receptor pathway were involved. Target cell death was caspase independent but mitochondria signaling amplified it. We then showed that NK cells from patients with X-linked chronic granulomatous disease could not kill efficiently ALL blasts and that NK cells expressed key components of a NADPH oxidase complex that was distinct from the phagocyte type. Our work reveals an uncharacterized effector pathway among cytotoxic lymphocytes and establishes key molecular requirements for this unconventional pathway.

Bakgrund: Akut lymfatisk leukemi (ALL) är den vanligaste cancerformen bland barn och ungdomar och kan innebära stor fysisk och psykisk påfrestning hos föräldrar och barn. När barnet drabbas av ALL kan föräldrar känna oro och rädsla för att förlora barnet. Syfte: Syftet med studien var att belysa föräldrars upplevelser när barnet har drabbats av akut lymfatisk leukemi (ALL). Metod: En litteraturstudie med induktiv ansats genomfördes där elva artiklar granskades och valdes till resultat. Resultat: I resultatet framkom huvudkategorin: Upplevelse av att vara förälder till barn som drabbats av ALL med underkategorier: att känna livet krascha, att känna otillräcklighet, att oroas över ekonomin, att känna behov av förändrade föräldrastrategier och att blicka framåt. Andra huvudkategorin var Upplevelse av sjuksköterskans betydelse med underkategorier: att känna emotionellt stöd och att uppleva behov av information och undervisning. Föräldrar upplevde kommunikation med sjuksköterska som viktigt och hjälpte föräldrar hantera påfrestningen. Vid bristande kommunikation upplevde föräldrar att sjuksköterskan undanhöll information och tilliten till sjuksköterskan försvann. Slutsats: Studien kan ge kunskap om föräldrars upplevelser när barnet drabbas av ALL och vilka behov som finns av hjälp och stöd. Mer forskning krävs för att undersöka på vilket sätt sjuksköterskan kan underlätta för föräldrar under den påfrestande tiden. / Background: Acute lymphocytic leukemia (ALL) is the most common cancer in children and youth and may cause great physical and psychological burden on parents and children. Parents might worry and fear losing the child when their child has ALL. Aim: The aim of this study was to explore parent's experiences when their child suffered from acute lymphocytic leukemia (ALL). Method: A literature review with an inductive structure was performed where eleven articles were analyzed and chosen for results. Result: The result presents main category: Experience of being a parent off a child affected by ALL with subcategories: to feel life crashing, to feel insufficient, to worry about economics, to feel the need of changing parent strategies, to focus ahead. The second main category was The experience of the importance of the nurse with subcategories: to feel emotional support and to experience the need for information and education. Parents experienced that communication with the nurse was important and helped parents manage burden. Lack of communication made parents experience that the nurse withheld information and the trust disappeared. Conclusions: The study can provide knowledge about parent's experiences when their child has ALL and the need of help and support. More research is required to investigate in which way the nurse can ease parent's burden during the stressful time.

Les leucémies aiguës lymphoblastiques (LAL) sont le cancer le plus fréquent chez l’enfant et dérivent le plus souvent de précurseurs lymphoïdes B. D’importants progrès thérapeutiques ont permis d’améliorer considérablement le pronostic. Néanmoins, 15 à 20 % des enfants rechutent encore. Ces rechutes peuvent survenir de façon isolée ou combinée dans la moelle osseuse, le site primitif des lymphoblastes, et/ou dans des organes extramédullaires tels que le testicule ou le système nerveux central. Notre équipe a montré que la protéine transmembranaire CD9 jouait un rôle majeur dans la migration des blastes dans ces sites et notamment le testicule, par l’activation de la voie RAC1 en réponse à la stimulation des cellules par le CXCL12. Ici, nous avons mis en évidence qu’un faible niveau d’oxygène, caractéristique commune aux niches médullaire et extramédullaires, régulait positivement l’expression de CD9 aux niveaux transcriptionnel et protéique, via la voie majeure de réponse à l’hypoxie, dépendante du facteur de transcription Hypoxia Inducible Factor 1a (HIF1a). Nous montrons que HIF1a se fixe directement sur le promoteur de CD9 pour induire sa transcription. Nous montrons aussi que la protéine CD9 est essentielle aux propriétés d’adhérence et de migration des blastes dans des conditions de basse oxygénation, et que son action pourrait s’exercer à travers RAC1 comme en normoxie. Nos résultats dans des expériences de xénogreffe à des souris indiquent que la voie HIF1a favorise la dissémination des blastes, possiblement à travers la régulation qu’elle exerce sur CD9. Ainsi, ce travail contribue à mieux comprendre le rôle de CD9 dans la pathogenèse des LAL de l’enfant. / Acute lymphoblastic leukemia (ALL) are the most frequent cancer in children and derive most often from B-cell precursors. Huge therapeutic improvements have allowed to reach high survival rates near 90% at 10 years from diagnosis. However, 15-20% of children still relapse with a significant risk of death. Relapses can occur in bone marrow and/or extramedullary sites such as testis or central nervous system, usually referred as “sanctuary sites”. Our previous work showed that the transmembrane protein CD9 plays a major role in lymphoblasts migration into these sites, especially in testis, through the activation of RAC1 signaling upon blasts stimulation with C-X-C chemokine ligand 12 (CXCl12). Here, we addressed the question of putative common factors shared by bone marrow and extramedullary niches which could upregulate CD9 expression and function. Consequently, we found that low oxygen levels could actually enhance CD9 expression both at mRNA and protein levels. We further determined that Hypoxia Inducible Factor 1a (HIF1a), the master transcription factor involved in hypoxia response, binds directly CD9 promoter to induce its transcription. We also showed that CD9 protein is crucial for leukemic cell adhesion and migration at low oxygen levels, possibly through its action on RAC1 signaling. Mouse xenograft experiments indicate that HIF1a signaling pathway favors ALL cells dissemination, which may involve CD9 as well. The present work increments our understanding of CD9 implication in ALL pathogenesis.