Tumour initiation and progression occur due to oncogenic mutations that also contribute to therapeutic resistance in many human tumours. Mutations activating the "PI3K/AKT" signalling pathway and inactivating the "TP53" tumour suppressor gene are common mechanisms that cancer cells require to proliferate and escape pre-programmed cell death. p53 mutant (p53mut) tumours not only fail to respond to DNA damaging therapy, but are also described to promote therapeutic resistance by dominant negative suppression of p53 dependent promoter activity. Our work identifies the crucial interaction between the PI3K/AKT pathway and p53 mutations that regulate treatment sensitivity in tumours. Using a combination of in vitro and in vivo techniques we demonstrate that AKT inhibition promotes reduced cellular levels of p53mut via a novel Nucleophosmin 1 (NPM) mediated regulation of the tumour suppressor p14ARF and promotes re-engagement of cell cycle arrest, senescence and increased sensitivity to ionising radiation in both in vivo and in vitro systems. We show that the PI3K/AKT pathway plays an important role in the regulation of p53mut and inhibitors of this pathway can re-sensitise treatment resistant tumours. This has helped us to simultaneously highlight the cohort of patients where the greatest efficacy may be achieved in clinical practise. We further show that the AKT mediated regulation of NPM that we describe in solid tumours is relevant in Acute Myeloid Leukaemia (AML) with mutated NPM, albeit showing physiologically different effects. This further highlights the necessity for rational treatment planning with the newer targeted agents that inhibit specific signalling pathways in AML patients.

Chronic lymphocytic leukemia (CLL) is a cancer of B cells and is the most common leukemia in North America. Current therapies are fraught with challenges, and drug resistance and disease relapse remain common occurrences. Therefore, novel therapies and novel therapeutic strategies are needed to improve CLL therapy. Better yet, therapies targeted at specific weaknesses of CLL cells will ensure maximum efficacy and minimum adverse toxicity. To this end, this thesis focuses on targeting the susceptible BCR pathway and lysosome-mediated cell death pathway using gefitinib and lysosomotropic agents, respectively. Firstly, the novel use of the tyrosine kinase inhibitor gefitinib was explored. This drug was most effective in aggressive ZAP-70+ CLL cells and cell lines. A similar inhibitor, erlotinib, had no effect in CLL. Gefitinib inhibited phosphorylation of Syk and ZAP-70, prevented downstream kinase activation, and supressed the pro-survival BCR response. ZAP-70 is implicated in the mechanism of action of gefitinib as introduction of ZAP-70 into a B cell line increased their sensitivity to gefitinib. Secondly, the novel strategy of targeting lysosomes was explored. The lysosomotropic drugs siramesine, nortriptyline, desipramine, mefloquine, and tafenoquine were all found to induce cytotoxicity and lysosome permeabilization. Lysosome permeabilization was accompanied with lipid peroxidation and followed by loss of mitochondrial membrane potential. Compared with healthy B cells, CLL cells were more sensitive to this cell death pathway. This was potentially due to the overexpression of SPP1 and overproduction of sphingosine, which destabilized lysosomes. Lastly, this thesis explored the clinical utility of these targeted therapies. Both gefitinib and siramesine were more effective in CLL cells than patient T cells. Furthermore, they retained efficacy amid protective stromal cells. Clinical correlations revealed that gefitinib and siramesine were effective in CLL cells with poor prognostic features. Siramesine was more effective in male cells and in previously-treated cells. Gefitinib was most effective in young patients. Overall, work presented herein demonstrates the efficacy of the tyrosine kinase inhibitor gefitinib and lysosomotropic agents in primary CLL cells. This work investigates the altered biology of the BCR pathway and lysosomes in CLL cells, and takes advantage of these weaknesses using targeted therapies. / October 2016

This study examined the mechanisms underlying the interactions between the natural polyphenol Resveratrol and HDAC inhibitors in both U937 myelomonocytic leukemia cell line and blood samples from AML patients and normal cord blood. Simultaneous exposure to Resveratrol and HDAC inhibitors (Vorinostat-SAHA or Panobinostat-LBH589) resulted in potentiating the lethality caused by any single agent of the combination, this interaction found to be synergistic at multiple concentrations. Exposing U937 cells to minimal toxic doses of Resveratrol and HDACIs results in release of mitochondrial pro-apoptotic proteins AIF and cytochrome c, pro-apoptotic caspase activation especially caspase-8, and induction of DNA damage. These events were associated with increase deacetylation of NF-κB and reactive oxygen species generation, as well as G0-G1 cell cycle arrest. Genetic knockdown of SIRT1 (a deacetylator of NF-κB that is upregulated by Resveratrol) resulted in significant increase in NF-κB acetylation and activity. However, SIRT1 knock down failed to protect U937 cells against combination-induced cell death, implying the possibility of the involvement of other mechanisms in inducing cell death rather than NF-κB deactivation only. Co-incubation of the antioxidant vi MnTBAP significantly reduced Resveratrol/HDACIs induced cell death, and resulted in a marked decrease in caspase-8, caspase-3, and PARP activation. Finally, the combined treatment of Resveratrol/HDACIs induce cell cycle changes possibly through Resveratrol action of blocking cell cycle in S phase exposing more cells to HDACIs lethality. Collectively, these finding indicate that the combined regimen of Resveratrol and HDAC inhibitors promote lethality in U937 cells and primary AML cells by a variety of mechanisms. The approved use of both agents in clinical setting make future clinical studies for development of this drug regimen a potential option in the battle with leukemia.

An 11 year old, hispanic girl with a history of B-cell acute lymphoblastic leukemia was admitted to the hospital for symptoms compatible with Bartonella henselae infection. The first molecularly diagnosed case of disseminated Bartonella henselae infection was reported in an immunocompromised patient in Lima, Peru. The analysis was confirmed by Polymerase Chain Reaction and automated sequencing of a liver biopsy sample, even though the serologic tests were negative. In conclusion, Bartonella spp. infection should have a particular diagnostic consideration in immunocompromised patients with fever of unknown origin and further investigation regarding the patient's past exposures with cats should also be elicited.

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Most ALL cases originate from immature B-cells (BCP-ALL) and are characterized by reoccurring structural genetic aberrations. These aberrations hold information of the pathogenesis of ALL and are used for risk stratification in treatment. Despite increased knowledge of genetic aberrations in pediatric T-cell ALL (T-ALL), no reliable molecular genetic markers exist for identifying patients with higher risk of relapse. The lack of molecular prognostic markers is also evident in patients with relapsed ALL. During the last decades, aberrant epigenetic mechanisms including DNA methylation have emerged as important components in cancer development. Telomere maintenance is another important factor in malignant transformation and is crucial for long-term cell survival. Like DNA methylation, telomere length maintenance has also been implicated to reflect outcomes for patients with leukemia. In this thesis, the prognostic relevance of DNA methylation and telomere length was investigated in pediatric ALL at diagnosis and relapse. The telomere length (TL) was significantly shorter in diagnostic ALL samples compared to normal bone marrow samples collected at cessation of therapy, reflecting the proliferation associated telomere length shortening. Prognostic relevance of TL was shown in low-risk BCP-ALL patients where longer telomeres at diagnosis were associated with higher risk of relapse. Genome-wide methylation characterization by arrays in diagnostic T-ALL samples identified two distinct methylation subgroups denoted CIMP+ (CpG Island Methylator Phenotype high) and CIMP- (low). CIMP- T-ALL patients had significantly worse outcome compared to CIMP+ cases. These results were confirmed in a Nordic cohort treated according to the current NOPHO-ALL2008 protocol.  By combining minimal residual disease (MRD) status at treatment day 29 and CIMP status at diagnosis we could further separate T-ALL patients into risk groups. Likewise, the CIMP profile could separate relapsed BCP-ALL patients into risk groups, where the CIMP- cases had a significantly worse outcome compared to CIMP+ cases.  From these data we conclude that DNA methylation subgrouping is a promising prognostic marker in T-ALL, as well as in relapsed BCP-ALL two groups where reliable prognostic markers are currently missing. By elucidating the biology behind the different CIMP profiles, the pathogenesis of ALL will be further understood and may contribute to new treatment strategies.

PU.1 downregulation within haematopoietic stem and progenitor cells (HSPCs) is the primary mechanism for the development of acute myeloid leukaemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. p53 is a well known tumor suppressor that is often mutated in human haematologic malignancies including AML and adds to their aggressiveness; however its genetic deletion does not cause AML in mouse. Deletion of p53 in the PU.1ure/ure mice (PU.1ure/ure p53-/- ) results in more aggressive AML with shortened overall survival. PU.1ure/ure p53-/- progenitors express significantly lower PU.1 levels. In addition to URE deletion we searched for other mechanisms that in absence of p53 contribute to decreased PU.1 levels in PU.1ure/ure p53-/- mice. We found involvement of Myb and miR-155 in downregulation of PU.1 in aggressive murine AML. Upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukaemic cell growth similarly to PU.1 rescue. The MYB/miR-155/PU.1 axis is a target of p53 and is activated early after p53 loss as indicated by transient p53 knockdown. Furthermore, deregulation of both MYB and miR-155 coupled with PU.1 downregulation was observed in human AML, suggesting that MYB/miR-155/PU.1 mechanism may be involved...

Orientador: Marcos Antônio de Oliveira / Resumo: L-asparaginases (ASNases) bacterianas são importantes biofármacos utilizados no tratamento de leucemia linfoide aguda (LLA), uma vez que este tipo tumoral é dependente da disponibilidade de asparagina (Asn) extracelular. As ASNases bacterianas são capazes de hidrolisar eficientemente Asn em ácido aspártico (Asp) e amônia (NH3), diminuindo a disponibilidade de Asn para células tumorais e induzindo apoptose. Comercialmente, indústrias farmacêuticas internacionais produzem ASNases de Escherichia coli e Erwinia chrysanthemi, entretanto, ASNase de nenhuma origem é produzida pelas indústrias farmacêuticas brasileiras. Adicionalmente, o tratamento com estas enzimas produz efeitos colaterais, entre eles imunogênicos, que estão relacionados com a alta massa molecular da enzima (140kDa) e neurológicos, atribuídos a atividade secundária de glutaminase (GLNase). Neste contexto, fontes alternativas destas enzimas e também a auto-suficiência em suas produções são importantes para mitigar os efeitos colaterais e evitar falhas no tratamento devido a flutuações internacionais de sua fabricação. Neste trabalho, realizamos a caracterização de uma ASNase de levedura, denominada de ASNaseM que compartilha elevada homologia (maior que 30% de identidade e 40% de similaridade) com as enzimas bacterianas utilizadas no tratamento da LLA e que possui todos os aminoácidos envolvidos na catálise conservados, sugerindo uma fonte alternativa potencial para o tratamento da LLA. Experimentos de cromatografia... (Resumo completo, clicar acesso eletrônico abaixo) / Bacterial L-asparaginases (ASNases) are important biopharmaceuticals used in the treatment of acute lymphoid leukemia (ALL), since this tumor type is dependent on the availability of extracellular asparagine (Asn). Bacterial ASNases are able to efficiently hydrolyze Asn in aspartic acid (Asp) and ammonia (NH3), decreasing the availability of Asn to tumor cells and inducing apoptosis. Commercially, international pharmaceutical industries produce ASNases from Escherichia coli and Erwinia chrysanthemi, however none ASNase is produced by the Brazilian pharmaceutical companies. Additionally, the treatment with these enzymes can produce side effects, among them immunogenic ones, that are related to the high molecular weight of the enzyme (140kDa) and neurological, attributed to the glutaminase secondary activity (GLNase), being glutamine (Gln) the most abundant amino acid in the bloodstream. In this context, alternative sources of these enzymes as also the self-sufficiency of the production are important to mitigate side effects and avoid treatment failures due to international fluctuations in their manufacture. In this work, we performed the characterization of a yeast ASNase, called ASNaseM, which shares high homology (higher than 30% identity and 40% similarity) with the bacterial enzymes used in the treatment of ALL, and which has all the amino acids involved in the catalysis conserved, suggesting a potential alternative source for the treatment of ALL. Molecular exclusion chro / Doutor

Made available in DSpace on 2016-07-27T14:20:56Z (GMT). No. of bitstreams: 1 IZADORA DE FREITAS SALVADOR.pdf: 843450 bytes, checksum: 1255d36931b1b4872a98b4345a41d9ca (MD5) Previous issue date: 2016-03-18 / The positive diagnosis of a serious illness like cancer affects people in global aspects. The disorder involves biological, psychological and social changes.. Leukemia is a cancer characterized by the uncontrolled growth of malignant white blood cells. This study aimed to describe the perceptions of diagnosis, the perception of prognosis and coping strategies used by nine patients, men and women, diagnosed with leukemia. For this purpose it reviewed the literature on leukemia and coping and the application of two instruments, a semi-structured interview and a coping scale. The data indicate a trend among participants, an initial negative perception of diagnosis, involving feelings of fear, anguish and denial strategies. However, after the beginning of treatment, the data indicate a change to a positive perception. It stands out as coping strategies: 1) interviews, religious coping; 2) in the range of coping, focusing on the positive, focusing on emotion and focusing on religion. It appears that the combination of these three coping strategies are associated with the perception of confidence in the cure of leukemia. The results highlight that for better management in the care of patients with leukemia, is important to devote special attention to teamwork psychological processes triggered in each phase of the illness and to offereded the patient the proper social support during the treatment process. / O diagnóstico positivo para uma doença grave, como o câncer, prejudica o indivíduo em seus mais amplos aspectos e afeta, por conseguinte, a sua rede de interações. A leucemia é um tipo de câncer caracterizado pelo crescimento desordenado dos glóbulos brancos. A patologia envolve alterações biológicas e psicológicas. O presente estudo objetivou descrever a percepção frente ao diagnóstico, a percepção do prognóstico e as estratégias de coping utilizadas por nove pacientes, homens e mulheres, com diagnóstico positivo para leucemia. Para tanto, foi realizada uma revisão da bibliografia sobre leucemia e coping e a aplicação de dois instrumentos avaliativos, uma entrevista semiestruturada e uma escala de coping. Os dados indicam uma tendência entre os sujeitos de apresentarem uma percepção inicial negativa do diagnóstico, envolvendo sentimentos de medo, angústia e estratégias de negação. Contudo, após o início do tratamento, os dados indicam uma mudança desse primeiro momento para uma ressignificação positiva da percepção frente ao prognóstico do tratamento. Destacam-se como estratégias de coping: 1) nas entrevistas, coping religioso; 2) na escala de coping, a focalização no positivo, a focalização na emoção e o coping religioso. Verifica-se que a associação dessas três estratégias de coping estão associadas à percepção de confiança na melhora da leucemia. Os resultados destacam que para um melhor manejo no atendimento ao paciente leucêmico, é importante que a equipe de trabalho devote especial atenção aos processos psicológicos desencadeados em cada fase do adoecimento e que seja resguardado ao paciente o devido apoio social durante o processo de tratamento.

O gene humano lysine methyltransferase 2E (KMT2E) pertence ao grupo Trithorax (TrxG) e age como proteína modificadora de histonas envolvida no controle transcricional de genes relacionados a hematopoiese. Foi previamente identificado como supressor tumoral, atuando sobre a diferenciação, proliferação e ciclo celular. DAMM et al. (2011) e LUCENA-ARAÚJO et al. (2014) descreveram a associação entre baixos níveis de expressão do gene KMT2E e desfechos desfavoráveis do tratamento de pacientes com leucemia mielóide aguda (LMA) e leucemia promielocítica aguda (LPA), respectivamente. O objetivo desse trabalho foi estudar os efeitos do aumento da expressão do gene KMT2E na leucemia mielóide aguda (LMA). Foi utilizada a linhagem celular U937, reconhecida como modelo de LMA, e o aumento da expressão do gene de interesse foi obtido por meio da transfecção das células com um vetor lentiviral contendo o cDNA codificante para a isoforma longa do gene (pCDH-MSCV-MCS-EF1-GFP+Puro, aqui chamado pMEG). As partículas lentivirais foram geradas por co-transfecção em células da linhagem HEK 293T, e posteriormente, titulados com a linhagem celular HT 1080. A expressão do gene e a presença da proteína foram confirmadas por qPCR e western blotting, respectivamente. Foram realizados ensaios funcionais de ciclo celular, proliferação, viabilidade, apoptose espontânea e induzida por trióxido de arsênico e luz ultravioleta e diferenciação celular induzida por 12-miristato 13-acetato de forbol (TPA), com as amostras U937 wild type (WT), U937 pMEG (U937 transduzidas com o vetor vazio) e U937 pMEG-KMT2E. Também foram realizadas mensurações da massa tumoral das células inoculadas em camundongos NSG. A expressão relativa do gene KMT2E na célula U937 pMEG-KMT2E foi 1000 vezes mais alta que na célula U937 sem a modificação genética. Os ensaios de diferenciação celular demonstraram que as células U937 pMEG-KMT2E apresentaram maior diferenciação em monócitos/macrófagos que as células controles, quando levada em consideração a marcação para o antígeno CD11c. A expressão induzida de KMT2E em células U937 não alterou a proliferação, viabilidade, ciclo celular, apoptose, ix espontânea ou induzida e o aspecto clonogênico in vitro, porém, foi associado a um maior crescimento tumoral em modelo animal. Nossa hipótese para justificar as diferenças entre os achados in vitro e in vivo é que o aumento da expressão de KMT2E, talvez por meio do aumento de CD11c, facilitou a interação entre as células e o microambiente, estimulando assim o crescimento tumoral in vivo. / The human lysine methyltransferase 2E (KMT2E) gene belongs to the Trithorax (TrxG) group and acts as a histone modifying protein participating in the transcriptional regulation of hematopoiesis-related genes. KMT2E has been previously described as a tumor suppressor, involved in cellular differentiation, proliferation and cell cycle progression. DAMM et al. (2011) and LUCENA-ARAÚJO et al. (2014) described the association between low levels of KMT2E gene expression and poor treatment outcomes in patients with acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL), respectively. The aim of this project was to study the effects of high levels of KMT2E expression in acute myeloid leukemia (AML). For this purpose, the U937 AML cell line was used and an high expression of the gene was obtained by transfecting the cells with a lentiviral vector containing the cDNA encoding the long isoform of the gene (pCDH-MSCV-MCS-EF1- GFP + Pure, here called pMEG). The lentiviral particles were transfected into HEK 293T cells and the viral concentration was determined by titration using HT 1080 cells. The gene expression and the protein presence were confirmed by qPCR and western blotting, respectively. All experiments to determine the biological function of overexpressed KMT2E were conducted with U937 wild type, U937 pMEG (U937 transduced with the empty vector) and U937 pMEG-KMT2E cells. In-vitro the impact of overexpressed KMT2E was studied on cell cycle progression, proliferation and cell viability, spontaneous and induced apoptosis by arsenic trioxide and ultraviolet light and cell differentiation induced by 12-myristate 13-phorbol acetate (TPA). In vivo, the effect of overexpressed KMT2E was detected by comparing the tumor mass growth in NSG mice when inoculating U937 pMEG and pMEG-KMT2E cells in each flank of the same mouse. The relative expression level of the KMT2E gene in pMEG-KMT2E U937 cells was 1000 higher than in the wild type U937 strain. The cell differentiation assay revealed that U937 pMEG-KMT2E cells presented an increased monocyte/macrophage differentiation, when analyzing the CD11c antigen. Induced xi overexpression of KMT2E in U937 cells did not alter cell proliferation, cell viability, cell cycle progression, spontaneous or induced apoptosis or clonogenic appearance in vitro. However, the overexpression of KMT2E resulted in an increased tumor mass formation in vivo. Taking our discrepant in vitro and in vivo results into account, we could hypothesize that the increased expression of KMT2E, possibly caused by the enhanced expression of CD11c, favored the interaction between U937 pMEGKMT2E cells and their microenvironment, thereby stimulating tumor growth in vivo.

by Tong Sau Lan. / Thesis (M.S.Sc.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 68-75). / abstract --- p.ii / statement of originality --- p.iv / acknowledgements --- p.v / table of contents --- p.vi / list op tables --- p.vi i / list of figures --- p.v iii / list of appendices --- p.ix / introduction --- p.1 / Chapter chapter i - --- understanding of the illness leukaemia --- p.3 / "Leukaemia: its symptoms, treatment and side effects on children" --- p.3 / The changing outlook of treatment --- p.7 / Chapter chapter ii - --- RESEARCH STUDIES ON LEUKAEMIC PATIENTS --- p.10 / Previous and changing research studies emphasis with leukaemic children --- p.10 / "Psychological and behavioral relevances: social adaptation, self-esteem, and anxiety" --- p.14 / Research studies on Chinese leukaemic children --- p.27 / Chapter chapter iii - --- method --- p.30 / Chapter chapter iv - --- results --- p.40 / Chapter chapter v - --- discussion --- p.56 / references --- p.68 / appendices --- p.76