Introdução e Objetivos: O sistema nervoso central (SNC) é o um sítio freqüente de recaída na criança com leucemia linfocítica aguda (LLA). Existe evidência de que a punção lombar traumática (PLT) pode representar um risco adicional de recaída no SNC quando ocorre inoculação de blastos no liqüido céfalorraquidiano (LCR). Este estudo tem por objetivo determinar se a ocorrência da PLT ao diagnóstico afeta o prognóstico de pacientes com essa patologia. Material e Métodos: Setenta e sete pacientes com diagnóstico de LLA, tratados entre 1992 a 2002, foram incluídos na análise. Quimioterapia intratecal (QIT) foi instilada imediatamente após a PL inicial (precoce), ou na segunda PL (tardia), realizada no período de 24 a 48 horas após a realização da PL inicial. Foi feita análise da influência da PLT e do momento (precoce x tardia) de administração da QIT em relação a recaída no SNC. Resultados: Entre os 19 pacientes que apresentaram PLT ao diagnóstico e receberam QIT tardia, seis tiveram recaída isolada no SNC e dois recaída combinada em SNC e medula óssea (MO). Entre os nove pacientes que tiveram PLT e receberam QIT precoce, somente um apresentou recaída combinada em SNC e MO (P=0,20); não houve, portanto, influência estatisticamente significativa da PLT na sobrevida livre de eventos (SLE) (55% para QIT precoce x 49% para QIT tardia) (P=0,37). Entretanto, em análise estratificada, de acordo com grupos de risco, observamos que para pacientes de baixo ou médio risco o OR foi de 0,8 quando recebiam QIT tardia (P=0,99) e 0,17 quando recebiam QIT precoce (P=0,47). Por outro lado, entre pacientes de alto risco o OR para recaída foi de 21,0 para aqueles que recebiam QIT tardia (P=0,09) e 1,5 para o grupo que recebia Q IT precoce (P=0,99). Conclusão: Os resultados do presente estudo são sugestivos de que a ocorrência da PLT tem uma influência adversa no prognóstico de pacientes com LLA de alto risco de recaída. Como estes resultados são decorrentes de um estudo retrospectivo, recomenda-se que sejam confirmados em estudos prospectivos randomizados. / Introduction and Objectives: The Central Nervous System (CNS) is a frequent site of relapse in childhood acute lymphoblastic leukemia (ALL). Traumatic lumbar puncture (TLP) is thought to increase the risk of relapse in the CNS. This study sought to determine if TLP at the time of diagnosis affected the outcome of patients. Matherial and Methods: Seventy-seven newly diagnosed patients treated from 1992 to 2002 were included in the analysis. Intrathecal therapy (IT) was instilled either immediately after the diagnostic LP (early) or at a second LP (delayed) 24 to 48 hours following the diagnostic LP procedure. The authors carried out an analysis of the influence of TLP and the timing (early versus late) of administration of IT therapy on CNS relapse. Results: Among the 19 patients who had a TLP at diagnosis and received late IT therapy, six had isolated CNS relapse and two had combined CNS and bone marrow (BM) relapse. Among the nine patients who had TLP and received early IT therapy, only one had a combined CNS and BM relapse (P=0.20); the influence of TLP was not statistically significant on the event-free survival (EFS) (55% for early IT versus 49% for delayed IT) ( P =0.37). However, when we carried out a stratified analysis according to risk categories we found that for low and standard risk patients the odds ratio (OR) for relapse was 0.8 for delayed IT therapy (P=0.99) and 0.17 for early IT (P=0.47). On the other hand, among high risk (HR) patients the OR for relapse was 21.0 for delayed IT therapy (P=0.09) and 1.5 for early IT (P=0.99). Conclusion: The occurrence of TLP impacts adversely on prognosis of HR ALL patients. As these results are based in a retrospective study with a low number of patients, the authors recommend future trials using prospective randomized studies to confirm these findings.

The role of vaccination in children, who received chemotherapy for malignancies, including acute lymphoblastic leukemia (ALL), remains controversial. Both the disease and treatment affect the immune system. Changes may occur by neutropenia, leading to immediate risk of bacterial and fungal infections, loss protective levels of antibody by previous immunizations, as well as the questionable efficacy of revaccination. Therefore, different approaches are applied in several countries and the best vaccination schedule is not well defined yet. Moreover, the existing recommendations, mainly built on low levels of evidence, are not always followed by oncologists in clinical practice and there are still few references to literature on the subject. The paucity of data from controlled trials on both residual immunity against vaccine antigens at the end of treatment of children with ALL and the ability of the patients to respond to vaccine boosters does not allow the development of guidelines, and then further evaluation are required. We conducted a study using open controlled clinical trial to assess the levels of protection against viral vaccine antigens in children treated for leukemia after completion of chemotherapy, and implementation of a booster dose vaccine. Serum antibody levels were evaluated for hepatitis B, measles, rubella and mumps in 33 patients and compared with a control group. Statistical analysis was performed using the Chi-square and Fisher exact tests for categorical variables and Mann-Whitney test for continuous variables. As a result, there was a high proportion of individuals not immune to measles (75.9%), to rubella (51.7%) and hepatitis B (59.3%) at the end of chemotherapy for ALL. After administration of one booster dose of vaccine, had a recovery for all antigens tested was statistically significant for measles (p = 0, 0422) and hepatitis B (p = 0.0357). It is recommended, therefore, administration of one dose of the MMR and evaluate vaccine antibody titers for individual interventions. / O papel da vacinação em crianças que receberam quimioterapia (QT) para neoplasias malignas, inclusive leucemia linfóide aguda (LLA), continua controverso. Tanto a doença quanto o tratamento afetam o sistema imune. As alterações podem ocorrer pela neutropenia, que leva ao risco imediato de infecções bacterianas e fúngicas, pela perda dos níveis protetores de anticorpos adquiridos por imunizações prévias, como também pela eficácia duvidosa das reimunizações. Por isso, diferentes abordagens são aplicadas em vários países e o melhor esquema de vacinação não está bem definido. Além disso, as recomendações existentes, construídas principalmente com baixos níveis de evidência, nem sempre são seguidas pelos oncologistas na prática clínica e as referências de literatura quanto ao tema são poucas. A escassez de dados de ensaios controlados, tanto para a imunidade residual contra antígenos vacinais no final do tratamento de crianças com LLA, como para a capacidade desses pacientes responderem aos reforços vacinais, não permitiu até o momento o desenvolvimento de diretrizes, sendo, então, necessárias novas avaliações. Foi realizado um estudo do tipo ensaio clínico controlado aberto, para avaliar as taxas de proteção contra antígenos vacinais virais em crianças tratadas para LLA após término da QT, e da aplicação de uma dose de reforço vacinal. O estado imunológico contra hepatite B, sarampo, rubéola e caxumba foi avaliado em 33 pacientes e comparado com um grupo controle. Para análise estatística foram utilizados o teste do qui-quadrado e exato de Fisher para variáveis categóricas e teste de Mann-Whitney para variáveis contínuas. Observouse uma elevada proporção de indivíduos não imunes ao sarampo (75,9%) à rubéola (51,7%) e à hepatite B (59,3%) ao final da QT para LLA. Após a administração de uma dose vacinal de reforço, ocorreu a recuperação para todos os antígenos testados sendo estatisticamente significante para sarampo (p = 0,0422) e hepatite B (p = 0,0357). Recomenda-se, portanto, uma dose das vacinas tríplice viral e hepatite B após recuperação hematológica e posterior avaliação dos títulos de anticorpos vacinais para intervenções individualizadas.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Acute lymphoblastic leukemia (ALL) is a hematologic malignancy with variable survival rates and prognosis depends of many factors. New immunophenotypic markers can be directly associated with prognosis in several subtypes of acute leukemia. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, a systematic review of PubMed, Scopus, Science Direct, Web of Science and Cochrane Library was carried out through 2012 to 2016.The search resulted in 23 articles that were fully read, and 9 of these articles met the inclusion criteria. The studies methodological quality was evaluated by STROBE tool that showed eight articles scoring >90% revealing a high methodological quality. The included studies evaluated 11 new immunophenotypic markers in a total of 765 patients with ALL which common ALL was the most cited subtype. From these 11 markers, 8 were associated with poor prognosis (CD86, CD135, CD117, CD133, CD71, CXCR4, VLA-4 and CD38+/CD58-), and 3 of them (CD304, CD27 and CD200) were related to good prognosis. The meta-analysis demonstrated that presence of CD71 and absence of CD38+/CD58- phenotype in ALL have been associated with an increase of relapse rates (0.23; IC 95%; 0.03–0.43) and a decrease in overall survival (0.23; IC 95%; -0.05–0.51) at 60 months. This study provides information regarding to immunophenotypic markers as independent prognostic factors that could be included into clinical protocols, helping in the risk stratification and therapeutic guidance. Thus, CD38+/CD58- phenotype is a medium-term predictor of mortality for patients with ALL. / A leucemia linfoide aguda (LLA) é uma neoplasia hematológica com valor de prognóstico bem definido e taxas de sobrevida variáveis. Novos marcadores imunofenotípicos podem estar diretamente associados ao prognóstico em vários subtipos de leucemias agudas. Sendo assim, foi realizada uma revisão sistemática e metanálise nas bases de dados PubMed, Scopus, Science Direct, Web of Science e Cochrane Library (utilizando as diretrizes do PRISMA) no período de 2012 até 2016. A pesquisa resultou em 23 artigos, que foram lidos em texto completo, dos quais nove preencheram os critérios de inclusão. A qualidade metodológica dos estudos avaliada pela ferramenta STROBE demonstrou oito artigos com pontuação > 90%, revelando alta qualidade metodológica. Os estudos incluídos avaliaram 11 novos marcadores imunofenotípicos em 765 pacientes com LLA e o subtipo LLA comum foi o mais citado. A partir desses 11 marcadores, oito foram associados com mau prognóstico (CD86, CD135, CD117, CD133, CD71, CXCR4, VLA-4 e CD38+/CD58-) e três deles (CD304, CD27 e CD200) estavam relacionados com bom prognóstico. A metanálise demonstrou que a presença do CD71 e a ausência do CD38+/CD58- em LLA foram associadas a taxas de recaídas aumentadas (0,23; IC 95%, 0,03-0,43) e diminuição da sobrevida global (0,23; IC 95%; -0,05 -0,51) em 60 meses. Dessa forma, esses novos marcadores imunofenotípicos fornecem informações como fatores de prognósticos independentes que podem ser incluídos nos protocolos clínicos, auxiliando na estratificação e na conduta terapêutica. Assim, o fenótipo CD38+/CD58- é um preditor de mortalidade a médio prazo para pacientes com LLA. / São Cristóvão, SE

A Leucemia Linfóide Aguda (LLA) é uma doença heterogênea em relação à biologia e ao prognóstico. Além de alterações genéticas, anormalidades epigenéticas, estão estreitamente relacionadas ao processo de carcinogênese e entre os mecanismos epigenéticos, a acetilação das histonas é um componente essencial para a regulação da estrutura da cromatina e atividade transcricional. Esse processo é mediado pelas histonas acetiltransferases (HATs). Por outro lado, a desacetilação, por meio das histonas desacetilases (HDACs), está relacionada à condensação da cromatina e repressão transcricional. A expressão anormal das HDACs tem sido associada ao processo de leucemogênese, revelando ser uma área promissora na caracterização de grupos de risco e tratamento do câncer. Os objetivos deste trabalho foram avaliar a expressão dos genes da classe I de HDACs (HDAC 1, 2, 3 e 8), correlacionar os resultados com as características clínicas e de prognóstico (idade, gênero, grupo de risco, contagem inicial de blastos, imunofenótipo, resposta ao tratamento, doença residual mínima nos dias 14 e 18 e a sobrevida livre de eventos) em 46 amostras consecutivas de medula óssea de crianças e adolescentes portadores de LLA; comparar e correlacionar a expressão dos genes estudados entre as amostras de pacientes portadores LLA e 10 amostras de medula óssea sem doença hematológica. A análise da expressão gênica foi realizada através da técnica de PCR em Tempo Real pelo método TaqMan®. Foi observado um aumento da expressão do gene HDAC1 nas amostras dos pacientes bons respondedores ao ix tratamento. O gene HDAC2 foi mais expresso no grupo de pacientes do gênero masculino (p=0,038). Esse gene também mostrou uma expressão aumentada nos pacientes de alto risco (p=0,060) e com sobrevida menor (p=0,065), entretanto os valores encontrados não foram estatisticamente significativos. Além disso, foi observada uma expressão aumentada dos genes HDAC2 (p=0,007), HDAC3 (p=0,014) e HDAC8 (p=0,002) em amostras de pacientes com LLA quando comparadas às amostras de medula óssea sem doença hematológica. Houve correlação entre a expressão de todos os genes de classe I das HDACs, exceto entre HDAC1 e HDAC8. Os resultados obtidos nesse trabalho sugerem que as HDACs de classe I, podem representar importantes alvos para futuros estudos em LLA, no entanto são necessários de testes funcionais para confirmar estes resultados. / Acute Lymphoblastic Leukemia (ALL) is a heterogeneous disease with distinct biologic and prognostic groups. In addition to genetic alterations, epigenetic processes play an important role in carcinogenesis, among which histone acetylation/deacetylation is crucial for chromatin modulation structure and transcriptional activity. Histone acetylation is regulated by the enzyme histone acetyl transferases (HATs). On the other hand, the deacetylation process is regulated by histone deacetylases (HDACs) enzymes, which is associated with the chromatin condensation and transcriptional repression. Abnormal expression of HDACs is a common feature of cancer and has revealed a promising field to stratify cancer treatment and risk classification. The investigation of these expression profiles may represent an important clinical factor for diagnosis and management of hematological malignances. The objectives of the present study were to analyze the expression profile of the class 1 HDACs (HDAC1, 2, 3 and 8) genes in bone marrow samples obtained from 46 childhood ALL samples, to correlate the results with prognostic and clinical features (age, gender, risk group, immunophenotype, treatment response, minimal residual disease and event free survival) of the patients; to evaluated differences in gene expression between ALL samples and 10 bone marrow samples without hematological disease and to verify the correlation of these genes. The gene expression analysis were made using xi TaqMan real-time polymerase chain reaction. A higher expression of HDAC1 in patients with better treatment response was observed. The HDAC2 showed a higher expression in male gender (p=0,038). HDAC2 also showed a higher expression for higher risk (p=0,060) and lower survival patients (p=0,065), however the statistical analysis did not show significant results. Furthermore, there was a higher expression of HDAC2 (p=0,007), HDAC3 (p=0,014) and HDAC8 (p=0,002) in ALL samples when compared to healthy donors. Class I HDACs showed correlation in gene expression, except for HDAC1 and HDAC8. These results suggest that class I HDACs can represent important targets for ALL research; however, it is necessary to perform functional investigation to confirm these results.

A leucemia linfóide aguda (LLA) é a neoplasia hematológica mais comum na infância e representa uma doença heterogênea em relação à biologia e ao prognóstico e seu tratamento consiste principalmente em quimioterapia. Apesar dos avanços no tratamento, cerca de 20% dos pacientes apresentam recaída da doença e/ou óbito indicando a necessidade de terapias diferenciadas para esse grupo. Recentemente, drogas epigenéticas como inibidores de DNA metiltransferases (iDNMTs) tem mostrado efeitos anti-neoplásicos promissores para o tratamento de diversos tipos de neoplasias incluindo a LLA. Nos tumores, a hipermetilação gênica é encontrada em vários genes, incluindo genes de reparo do DNA, reguladores do ciclo celular e apoptose. Sendo assim, drogas desmetilantes estão sendo apontadas como promissores agentes para o tratamento do câncer. A Zebularina (ZB) é um iDNMT análogo de citidina que inibe a metilação do DNA. Esta droga tem mostrado resultados animadores para o tratamento de diversas neoplasias, incluindo glioblastoma, leucemia mielóide aguda, câncer de mama, próstata e outros. O objetivo deste trabalho foi avaliar os efeitos do tratamento com a ZB, associada ou não à quimioterápicos, em linhagens celulares pediátricas de LLA, por meio de ensaios funcionais como proliferação celular, capacidade clonogênica, apoptose e ciclo celular. Além disso, foi analisada a capacidade desmetilante da droga e a expressão dos genes DNMT1, DNMT3a e DNMT3b após o tratamento com a ZB. A ZB inibiu a proliferação celular de maneira dose e tempo-dependente e agiu sinergicamente quando combinada com o MTX em ambas as linhagens. Ela também diminuiu a capacidade clonogênica e aumentou a taxa de apoptose nas duas linhagens estudadas. Além disso, o tratamento com ZB causou uma parada na fase S do ciclo celular na linhagem ReH. A ZB foi capaz de desmetilar parcialmente o gene AhR e reduzir a expressão dos genes DNMT1, DNMT3a e DNMT3b. Todos os dados encontrados no presente trabalho sugerem que as drogas desmetilantes podem ser interessantes agentes para o tratamento da LLA pediátrica. / Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy in childhood and represents a heterogeneous disease regarding its biology and prognosis. Its treatment consists mainly of chemotherapy. Despite advances in treatment, about 20% of patients experience disease recurrence and/or death indicating the need for differentiated therapies for this group. Recently, epigenetic drugs such as DNA methyltransferases inhibitors (iDNMTs) has shown antineoplastic and promising results for several types of tumors including ALL. Gene hypermethylation is found in several genes in tumors cells, including genes responsible for DNA repair, cell cycle and apoptosis regulators. Therefore, demethylating agents may be promising agents for cancer treatment. Zebularine (ZB) an iDNMT is a cytidine analogue that inhibits DNA methylation. This drug has shown promising results for the treatment of many cancers, including glioblastoma, acute myeloid leukemia, breast and prostate cancer and others. The aim of this study was to evaluate the effects of ZB treatment, associated or not with chemotherapeutic agents, in childhood ALL cell lines through functional tests such as cell proliferation, clonogenic capacity, apoptosis and cell cycle. In addition, we examined the demethylating ability of ZB and the expression of DNMT1, DNMT3a and DNMT3b genes after treatment with this agent. ZB inhibited cell proliferation in a dose- and time-dependent manner and showed synergistic effects when combined with MTX in both cell lines. ZB treatment also reduced clonogenic capacity and increased the number of apoptotic cells in both cell lines studied. Furthermore, treatment with ZB caused an S phase cell cycle arrest in ReH cell line. ZB was able to partially demethylate AhR gene and reduce the expression of genes DNMT1, DNMT3a and DNMT3b. These results suggest that demethylating drugs may be interesting agents for the treatment of childhood ALL.

Orientadores: José Andrés Yunes, Ana Carolina de Mattos Zeri / Tese (doutorado) - Universidade Estadual de Campinas, Instituo de Biologis / Made available in DSpace on 2018-08-19T21:00:30Z (GMT). No. of bitstreams: 1 Melo_CarolinaPereiradeSouza_D.pdf: 3404055 bytes, checksum: 57f3eb2b6199d017aab0f03512317367 (MD5) Previous issue date: 2012 / Resumo: O uso intensivo e combinado de diferentes quimioterápicos tem permitido a cura de 70-80% das leucemias linfóides agudas (LLA) da infância. A intensidade e o uso das drogas são adaptados ao risco de recaída dos pacientes, aferido ao diagnóstico e nas primeiras semanas do tratamento. Embora existam diferenças, os principais critérios utilizados na estratificação dos pacientes nos grupos de risco são idade, contagem leucocitária, imunofenotipagem e a resposta inicial ao tratamento, mensurada pela citoredução na medula óssea e/ou sangue periférico. Este último, tem se mostrado um fator prognóstico poderoso, independente, que permite identificar pacientes com maior ou menor risco de recaída. Ao mensurar a citoredução, faz-se, indiretamente, uma avaliação in loco, da sensibilidade intrínseca da leucemia à quimioterapia. A proposta deste trabalho foi a implementação do uso da metodologia de Ressonância Magnética Nuclear (RMN) para futura identificação de pacientes de LLA com resistência aos quimioterápicos usados na fase de indução. A implementação do método de RMN foi feito com células (linhagens celulares e células primárias de LLA) em cultura com doses de dois quimioterápicos: Prednisolona (PRED) e L-asparaginase (ASNase). O perfil dos metabólitos presentes no meio de cultura das células tratadas com as drogas foi obtido por meio da análise de espectros de RMN, e buscou-se associá-lo à resistência das células aos quimioterápicos. Os biomarcadores identificados neste trabalho permitiram distinguir tanto as linhagens sensíveis das resistentes quanto pacientes que recaíram dos que entraram em remissão, utilizando a técnica da metabolômica. Além disso, a análise do perfil metabólico permitiu formular algumas hipóteses sobre as vias metabólicas implicadas na resistência às drogas. Experimentos complementares com um maior número de pacientes se fazem necessários. Porém, nossos resultados indicam que este método poderá ser futuramente usado para análise de células de pacientes em tratamento, subsidiando, com maior precisão do que os métodos atuais, a alocação dos pacientes nos grupos de risco / Abstract: Intensive and combined use of different chemotherapic drugs has improved the cure rate of childhood Acute Lymphoblastic Leukemia (ALL) to 70-80%. Drugs use and intensity are determined based on patient relapse risk, which is measured at diagnosis and during the first weeks of treatment. Although differences may exist, the main criteria used to stratify patients in risk groups are age, leukocyte count, immunophenotyping and initial response to treatment, measured by cytoreduction in bone marrow and / or peripheral blood. The latter has proved to be a powerful independent prognostic factor, which allows identification of patients at higher or lower risk of relapse. By measuring cytoreduction, an in situ evaluation of the leukemia intrinsic sensitivity to chemotherapy is done indirectly. The aim of this study was to implement the use of Nuclear Magnetic Resonance (NMR) methodology for future identification of ALL patients resistance to chemotherapeutic agents used in the induction phase. NMR method implementation was done with cells (cell lines and primary ALL cells) kept in culture with two chemotherapic drugs: prednisolone (PRED) and L-asparaginase (ASNase). NMR spectra analysis provided information about the metabolic profile of drug treated cells culture medium, which was associated with the cells resistance to chemotherapic drugs. The biomarkers identified in this study allowed distinguishing, not only between the resistant and sensitive strains, but also between relapsed patients and those ones who remained in remission, using the metabolomics technique. Furthermore, analysis of the metabolic profile allowed the formulation of some hypotheses about the metabolic pathways involved in drug resistance. Further experiments with larger numbers of patients are needed. However, our results indicate that this method can be used for future analysis of patients treated cells, supporting, with greater precision than current methods, the allocation of patients into risk groups / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

THE INFLUENCE OF MOTIVATIONAL ELEMENTS IN PAEDIATRIC PHYSIOTHERAPY FOCUSED ON THE PATIENTS WITH ACUTE LYMPHOBLASTIC LEUKEMIA - PILOT STUDY Abstract The efficacy of acute lymphoblastic leukemia (ALL) treatment in childhood is still increasing and the quality of life of these children is starting to be the focus of interest. The physiotherapy affects in preventive and reactive way the late effects of treatment which may reduce the quality of life. An insufficient motivation of children can be a reason of a reduced adherence to a physiotherapeutic intervention. The goal of this study was to ascertain the influence of the motivational elements in the paediatric physiotherapy in the improvement of motor functions, the quality of life, the motivation to the exercise and the frequency of exercise. There were 9 patients diagnosed with ALL in the maintenance phase of treatment (average age 7,3 years, age range 5,2 - 10,1, 5 girls and 4 boys). All probands underwent a 6- weeks-excercise programme that contains the exercise from the DNS method, yoga for children and stretching. The assessment was performed by Bruninks-Oseretsky Test (BOT2), 6 minute walking test (6MWT), goniometric measurement of ankle joint dorsiflexion, Pediatric Quality of Life Inventory (PedsQL), Intrinsic Motivation Inventory (IMI) and the...

Oncolytic viruses (OVs) are novel biological agents that selectively infect and kill malignant cells. OVs can also generate anti-cancer immunity. Our lab exploited this phenomenon and developed an in vitro vaccine with infected leukemia cells with oncolytic virus vaccine – and named immunotherapy by leukemia-oncolytic virus (iLOV) – that provided in vivo protection in a murine model for acute lymphoblastic leukemia. This work further characterizes iLOV biology and the interaction of its immune responses. An in vitro immune response assay was optimized to detect and quantify the in vivo anti-leukemia immunity generated by iLOV. Anti-viral immunity is an obstacle for OV therapy. Although iLOV created anti-viral antibodies towards itself, these neutralizing antibodies did not hinder the vaccine’s ability to initiate complement or dendritic cell activation. We envision personalized versions of iLOV for leukemia patients in remission to prevent the possibility of relapse. This work highlights new advantages for infected cell vaccines and supports the progress of iLOV toward clinical testing.

<p>It has been long known that many types of cancers have high metabolic requirements and use reprogrammed metabolism to support cellular activities. The first identified metabolic alteration in cancer cells was elevated glucose uptake, glycolysis activity and lactate production even in the presence of oxygen. This metabolic program, termed aerobic glycolysis or the Warburg effect, provides cells with energy as well as biosynthetic substrates to sustain cell survival and rapid cell proliferation. Cancer metabolism is closely linked to genetic mutations and oncogenic signaling pathways, such as PI3K/Akt, cMyc and HIF pathways. These oncogenic signals can direct metabolic reprogramming while changes in metabolic status can regulate activities of these signaling pathways in turn. In addition to glucose, later studies also found utilization of alternate nutrients in cancer cells, including glutamine and lipids. Glutamine is the second major metabolic fuel and can be converted to various substrates to support cell bioenergetics needs and biosynthetic reactions. Usage of metabolic fuels in cancer cells, however, is variable. While certain cancers display addiction to one type of nutrient, others are capable of using multiple nutrients. </p><p>The unique metabolic features of cancer cells raise the possibility of targeting metabolism as a novel therapeutic approach for cancer treatment. Using pharmacological inhibitors, previous research has provided corroborating evidence that metabolic stress can impact survival and growth of proliferative cancer cells by regulating cell apoptotic machinery and cell cycle checkpoints. Due to lack of genetic tools and side effects from these inhibitors, however, mechanistic understanding of cell response to metabolic inhibition was limited in these studies. More importantly, how metabolic stress affects cancer progression in a physiological condition has not yet been well investigated. Lastly, current research has not examined metabolic program in indolent cancers and the metabolic requirements and activities in less proliferative cells also remain to be understood.</p><p>This work examines nutrients utilization in B cell derived acute and chronic leukemia (B-ALL and B-CLL). B-ALL is an aggressive form of leukemia. Using cell lines and primary patient samples, we found B-ALL cells primarily used glucose through aerobic glycolysis, similar to other proliferative cancer cells. B-ALL cells were also more sensitive to inhibition of glycolysis than normal B cells. Employing an untargeted metabolomics profiling in combination with isotope labeled glucose tracing approach, we show in a B-ALL model that genetic ablation of glucose transporter Glut1 partially reduced glucose uptake, sufficiently hindered anabolic pathways and promoted catabolic metabolism. This metabolic shift led to sharply curtailed B-ALL proliferation in vitro and reduced leukemic burden in vivo. Furthermore, this partial inhibition of glucose metabolism sensitized B-ALL cells to apoptotic stimuli and non-cytotoxic metabolic inhibition significantly enhanced efficacy of a tyrosine kinase inhibitor to eliminate B-ALL cells in vitro and in vivo. Thus, partial inhibition of glucose metabolism can provide a plausible adjuvant therapy to treat cancers that depend on glycolysis for survival and proliferation. </p><p>In contrast to B-ALL, B-CLL is an indolent form of cancer. Most B-CLL cells exhibited low glucose metabolic activities that were comparable with normal B cells at resting stage. Similar to chronically stimulated and anergic B cells, these B-CLL cells also failed to upregulate glucose metabolism in response to IgM stimulation. We also observed an altered amino acid and acyl-carnitine profile and increased glutaminase mRNA in B-CLL relative to normal B cells, suggesting the capability of using alternate nutrients such as glutamine in these cells. Finally, we explored the possibility of suppressing mitochondria metabolism to induce B-CLL cell death through inhibition of the nuclear hormone receptor and metabolic regulator ERRalpha. ERRalpha is known to regulate mitochondrial metabolism and was expressed higher in B-CLL than normal B cells. ERRalpha inhibition decreased viability of oncogene transformed pro-B cells, suggesting ERRalpha as a potential target for B-CLL treatment.</p><p> Collectively, this work investigates metabolic phenotype in two forms of leukemia derived from B cells. It reveals different metabolic requirements and activities in aggressive and indolent leukemia and explores different approaches to suppress metabolism in these cancers. Findings of this work shed light on how to potentially design metabolic approach to improve cancer treatment.</p> / Dissertation

RUNX1, a transcription factor required for hematopoiesis and lymphocyte differentiation, is one of the most commonly targeted genes in hematopoietic malignancies. Mutations in the RUNX1 gene are associated with a poor prognosis in a subset of T cell acute lymphoblastic leukemia (T-ALL) and RUNX1 has been proposed as a tumor suppressor in TLX1/3-transformed human T-ALL. Recent ChIP-seq studies in human T-ALL cell lines demonstrated that a large portion of TAL1- and NOTCH1- bound regions contain RUNX binding sites in promoter or enhancer regions, suggesting oncogenic roles for RUNX1 in T-ALL. To interrogate RUNX1 functions in leukemogenesis, we depleted RUNX1 in a T-ALL mouse model and in human T-ALL cell lines. We found that RUNX1 is required for the maintenance of mouse T-ALL growth in vivo and the survival of human T-ALL cell lines in vitro. In addition, inhibition of the RUNX1 activity with a small molecule inhibitor impairs the growth of human T-ALL cell lines and primary patient samples. RUNX1 depletion reduces the expression of a subset of TAL1- and NOTCH1- regulated genes including the MYB and MYC oncogenes, respectively. We demonstrate that RUNX1 regulates transcription factor binding and acetylation of H3K27 at the Myb and Myc enhancer loci. These studies provide genetic and pharmacological evidences that RUNX1 supports T-ALL cell survival and suggest RUNX1 inhibitor as a therapeutic strategy in T-ALL treatment.