<p>The advent of next-generation sequencing, now nearing a decade in age, has enabled, among other capabilities, measurement of genome-wide sequence features at unprecedented scale and resolution. </p><p>In this dissertation, I describe work to understand the genetic underpinnings of non-Hodgkin’s lymphoma through exploration of the epigenetics of its cell of origin, initial characterization and interpretation of driver mutations, and finally, a larger-scale, population-level study that incorporates mutation interpretation with clinical outcome. </p><p>In the first research chapter, I describe genomic characteristics of lymphomas through the lens of their cells of origin. Just as many other cancers, such as breast cancer or lung cancer, are categorized based on their cell of origin, lymphoma subtypes can be examined through the context of their normal B Cells of origin, Naïve, Germinal Center, and post-Germinal Center. By applying integrative analysis of the epigenetics of normal B Cells of origin through chromatin-immunoprecipitation sequencing, we find that differences in normal B Cell subtypes are reflected in the mutational landscapes of the cancers that arise from them, namely Mantle Cell, Burkitt, and Diffuse Large B-Cell Lymphoma. </p><p>In the next research chapter, I describe our first endeavor into understanding the genetic heterogeneity of Diffuse Large B Cell Lymphoma, the most common form of non-Hodgkin’s lymphoma, which affects 100,000 patients in the world. Through whole-genome sequencing of 1 case as well as whole-exome sequencing of 94 cases, we characterize the most recurrent genetic features of DLBCL and lay the groundwork for a larger study. </p><p>In the last research chapter, I describe work to characterize and interpret the whole exomes of 1001 cases of DLBCL in the largest single-cancer study to date. This highly-powered study enabled sub-gene, gene-level, and gene-network level understanding of driver mutations within DLBCL. Moreover, matched genomic and clinical data enabled the connection of these driver mutations to clinical features such as treatment response or overall survival. As sequencing costs continue to drop, whole-exome sequencing will become a routine clinical assay, and another diagnostic dimension in addition to existing methods such as histology. However, to unlock the full utility of sequencing data, we must be able to interpret it. This study undertakes a first step in developing the understanding necessary to uncover the genomic signals of DLBCL hidden within its exomes. However, beyond the scope of this one disease, the experimental and analytical methods can be readily applied to other cancer sequencing studies.</p><p>Thus, this dissertation leverages next-generation sequencing analysis to understand the genetic underpinnings of lymphoma, both by examining its normal cells of origin as well as through a large-scale study to sensitively identify recurrently mutated genes and their relationship to clinical outcome.</p> / Dissertation

Um dos principais desafios no tratamento quimioterápico em seres humanos e animais é a resistência que as células neoplásicas apresentam, sendo esse mecanismo responsável por falhas no tratamento e recidivas da doença. A resistência pode ser intrínseca ou adquirida e ocorre em função da expressão de transportadores de membrana ABC, como a glicoproteína P (ABCB1/MDR), proteínas de resistência a múltiplas drogas (ABCC1/MRP) e proteína de resistência do câncer de mama (ABCG2/BCRP). O linfoma é a neoplasia hematopoiética mais comum em cães, altamente responsiva à quimioterapia, mas que recidiva durante o tratamento antineoplásico, sendo a resistência das células neoplásicas aos quimioterápicos um fator responsável pela alta taxa de recidiva e óbito dos animais. Neste estudo avaliou-se a expressão de genes relacionados à resistência a múltiplas drogas em cães com linfoma, no diagnóstico e na recidiva da doença, em três diferentes protocolos quimioterápicos utilizados na rotina clínica. A expressão dos genes ABCB1, ABCC1, ACBG2 foi determinada por RT-PCR (PCR em tempo real) em 25 animais naturalmente acometidos pela doença, divididos aleatoriamente em 3 grupos tratados com os protocolos quimioterápicos COP, VCM e Short-Madison, além de um &quot;pool&quot; controle constituído por linfonodos normais de oito animais. A expressão dos genes foi detectada em todas as amostras, tanto de linfonodos normais quanto de animais com linfoma. No diagnóstico da doença, a expressão do gene ABCC1 foi relacionada negativamente com idade (p=0,008) e positivamente com duração da remissão (p=0,027) e sobrevida (p=0,007), entretanto para os genes ABCB1 e ABCG2 não houve diferença estatística significante. Na recidiva, a expressão dos genes não sofreu variação estatística significante em função do tipo e duração da remissão e sobrevida. Não houve variação na expressão dos genes ABCB1, ACBC1 e ABCG2 no momento da recidiva quando comparado ao protocolo quimioterápico utilizado. / One of the main challenges of the chemotherapy treatment in human and animals is the resistance of the neoplasic cells, being this mechanism responsible for failures in the treatment and relapse of the disease. The resistance could be intrinsic or acquired and it occurs due to the expression of ABC membrane transporters, such as p-glycoprotein (ABCB1/MDR), resistance protein to multiple drugs (ABCC1/MRP) and resistance protein of breast cancer (ABCG2/BCRP). Lymphoma is the most common hematopoietic cancer disease in dogs, highly responsive to chemotherapy, but relapse during chemotherapy treatment, being the resistance of neoplastic cells to chemotherapy drugs the responsible factor for the high rate of relapse and death of animals. In this study, genes expression related to multiples drugs resistance it was evaluated in dogs with lymphoma, in the diagnosis and in the relapse of the disease in three different chemotherapy protocols used in the clinical routine. The genes expression ABCB1, ABCC1, ACBG2 was determined by RT-PCR (real time PCR) in 25 animals naturally undertaken by the illness, randomly divided into 3 groups treated with the chemotherapy protocols COP, VCM and Short-Madison, besides a &quot;pool&quot; control constituted by normal lymph node of eight animals. The genes expression was detected in all the samples, both in the normal lymph node and in the animals with lymphoma. In the diagnosis of the disease, the gene expression ABCC1 was negatively related with age (p=0,008) and positively with the duration of remission (p=0,027) and survival (p=0,007); however, for ABCB1 and ABCG2 there was no statiscally significant difference. In the relapse, the genes expression had no statiscally significant difference due to the type and duration of remission and survival. There was no variation in the genes expression ABCB1, ACBC1 and ABCG2 in the moment of relapse when compared to the chemotherapy protocol used.

Os linfomas constituem um grupo de neoplasias que têm origem nas células linforreticulares e acomete tecidos linfóides primários, secundários como o baço e linfonodos e demais tecidos onde há presença de linfócitos circulantes, comumente descritos em cães, gatos e humanos. As síndromes paraneoplásicas são definidas como alterações sistêmicas não relacionadas com lesões metastáticas de forma direta ou indireta no hospedeiro. Dentre as alterações descritas como síndrome paraneoplásica, as alterações hematológicas constituem as mais freqüentes, apresentando destaque para os processos anêmicos. A anemia presente pode ser decorrente de alterações do metabolismo do ferro, perda sanguínea, distúrbios hemolíticos, infiltrado medular e hiperesplenismo. Atualmente tem se associado a redução da vida média das hemácias frente a presença do estresse oxidativo, que gera um desequilíbrio entre a excessiva produção de espécies reativas ao oxigênio e/ou a diminuição dos mecanismos de defesa antioxidante das hemácias. Esse processo pode ocasionar a peroxidação de lipídios da membrana eritrocitária, gerando hemólise e assim resultando em anemia e/ou exacerbando a mesma. Com o objetivo de avaliar a existência do estresse oxidativo e a presença de anemia assim como, a sua correlação com o estado redox, foram avaliadas as concentrações eritrocitárias de glutationa reduzida, glutationa redutase, glutationa peroxidase e superóxido redutase em 23 gatos sadios e 17 felinos com linfoma. Também foram determinadas as concentrações plasmáticas de malonaldeído como indicador da presença de peroxidação lipídica em ambos os grupos. Para avaliar o estado redox foi mensurada a concentração plasmática do estado antioxidante total para o grupo experimental e grupo controle. Não foram observadas diferenças significantes para as enzimas eritrocitárias glutationa redutase, glutationa peroxidade e superóxido dismutase, assim como para a mensuração plasmática de malonaldeído. Entretanto, foram observados valores significativamente menores (p=0,018) de glutationa reduzida nos felinos com linfoma quando comparados ao grupo controle. O mesmo pôde ser observado em relação à mensuração do estado antioxidante total (p=0,003). Os resultados obtidos indicam a presença de estresse oxidativo em felinos com linfoma, porém, não houve evidencias da relação entre a presença de estresse oxidativo e a presença de anemia. / Lymphomas are a group of cancers that originate in cells and lymphoreticular affects lymphoid tissues in primary, secondary as the spleen and lymph nodes and other tissues where there is presence of circulating lymphocytes, commonly described in dogs, cats and humans. Paraneoplastic syndromes are defined as systemic changes unrelated metastatic lesions either directly or indirectly in the host. Among the changes described as a paraneoplastic syndrome, hematological changes are the most frequent, with emphasis on the processes anemic. This anemia may be due to changes in iron metabolism, blood loss, hemolytic disorders, bone marrow infiltration and hypersplenism. Today has been associated with reduced average life span of red cells before the presence of oxidative stress, which creates an imbalance between excessive production of reactive oxygen species and / or decreased antioxidant defense mechanisms of red blood cells. This process can lead to lipid peroxidation of the membrane, causing hemolysis and thus resulting in anemia and / or exacerbating it. Aiming to evaluate the existence of oxidative stress and anemia as well as its correlation with redox state, were evaluated erythrocyte concentrations of reduced glutathione, glutathione reductase, glutathione peroxidase and superoxide reductase in 23 healthy cats and 17 cats with lymphoma. We also determined plasma concentrations of malondialdehyde as an indicator of the presence of lipid peroxidation in both groups. To assess the redox state was measured plasma concentration of total antioxidant status for the experimental group and control group. No significant differences were observed for enzymes erythrocyte glutathione reductase, glutathione peroxidase and superoxide dismutase, as well as for measurement of plasma malondialdehyde. However, observed values were significantly lower (p = 0.018) reduced glutathione in cats with lymphoma when compared to the control group. The same could be observed in relation to the measurement of total antioxidant status (p=0,003). The results indicate the presence of oxidative stress in cats with lymphoma, however, no evidence of the relationship between oxidative stress and anemia.

Despite improved treatments for Non-Hodgkin’s Lymphoma (NHL) and Multiple Myeloma (MM), most patients eventually relapse and these diseases remain largely incurable. This has precipitated recent research into more clinically relevant in vitro models to enable development of more effective therapies. We have validated and standardised two in vitro primary culture models using tumour samples derived from patients with NHL, Chronic Lymphocytic Leukaemia (CLL) and MM. Several novel findings have been demonstrated. In vitro sensitivity of primary NHL cells cocultured in a CD40L model predicted clinical response to bortezomib in patients receiving the drug in a phase II trial. In vitro sensitivity correlated with CD40 expression, identifying a potential surrogate biomarker for response to bortezomib. The novel HDAC inhibitor, UCL67022 was 10-fold more potent than vorinostat in NHL and produced synergy when combined with bortezomib. UCL67022 maintained its potency in primary MM samples grown in an HS-5 stromal model. It modulated cytokine secretion resulting in downregulation of cytokine-induced signalling pathways (JAK/STAT3). A novel Hsp90 inhibitor, KW-2478 maintained activity in the HS-5 model and enhanced the activity of bortezomib and melphalan. Hsp70 was identified as a potential surrogate biomarker to monitor the combinatorial effect in future clinical trials. A highly synergistic and schedule-dependent cytotoxic effect occurred when primary MM cells were pre-treated with melphalan followed by bortezomib, with important implications for future clinical trial design. IL-6, IL-8 and VEGF levels correlated with resistance to bortezomib and melphalan and were associated with activation of JAK/STAT, MAPK and PI3K/Akt signalling pathways. Antibody neutralization of IL-6, IL-8 and VEGF resulted in restoration of drug sensitivity. We have therefore demonstrated the ability of primary culture models to predict response to chemotherapy, to identify therapeutically beneficial novel agents and to enable study of tumour microenvironmental interactions responsible for drug resistance in patients with haematological malignancies.

The unfolded protein response (UPR) is a complex signalling pathway activated in response to endoplasmic reticulum stress. In recent years, the UPR has been implicated in cancer and chemosensitivity, particularly in solid tumours. This thesis investigated the potential value of targeting the UPR as a novel therapeutic approach in haematological malignancies using a panel of cell lines representing AML, lymphoma and myeloma. The UPR was constitutively active in these haematological cancer cell lines, with differential activation of key UPR proteins both in the panel and between the panel, peripheral blood mononuclear cells and the colorectal cancer cell line HT-29. A number of strategies were used to modulate the UPR and study chemosensitivity. Minimally toxic concentrations of the ER stress inducer thapsigargin protected cells from cytotoxic agents, with a reduction in antiproliferative drug effect. The activity of the novel small molecule versipelostatin, reported to downregulate the ER molecular chaperones GRP78 and GRP94, was also investigated, with the downregulation previously reported in solid tumour cell lines (Park et al. 2004) confirmed in HT-29 cells, but not observed in the haematological cell lines studied (although versipelostatin was an effective cytotoxic agent at low micromolar concentration). Combination experiments with the chemical chaperone 4-phenylbutyric acid (PBA) resulted in a small increase in apoptosis when PBA was combined with ER stress inducers. However, PBA also showed HDAC inhibitory activity at the concentrations used. Finally, siRNA mediated silencing of GRP78 and GRP94 in THP1 (AML) and U266 (myeloma) cells resulted in a decrease in the targeted protein, but showed only minimal effects on chemosensitivity. In conclusion, the UPR is activated in these haematological cancer cell lines and plays a complex role in chemosensitivity. In contrast to previous reports in solid tumour cells, modulating the UPR in these haematological malignancies had only a modest effect on chemosensitivity.

At least 50% of people will develop cancer at some point during their lifetime and half these will end in fatality. Improving patients’ prognosis relies on early and accurate diagnosis and treatment. Current diagnostic methods are based on histopathological analysis and are time-consuming, expensive and require biopsy. Raman spectroscopy can measure subtle biochemical changes and provides a rapid, non-destructive and objective technique that can be used in vivo for identifying pathological changes in tissue samples. This study investigates both a standard Raman spectrometer system and also a Raman needle probe for their use as diagnostic techniques and clinical tools. Oesophageal, femoral and head and neck lymph nodes were analysed in this study. Metastatic lymph nodes from the three areas could be identified from the non-cancer lymph nodes with a sensitivity of 71% and specificity of 89%. Lymphoma was identified from non-cancer lymph nodes with a sensitivity of 64% and specificity of 86%. It was observed that oesophageal nodes often contained carbon particles, clinically diagnosed as anthracosis. These nodes were much harder to study than the femoral or head and neck, due to strong Raman signal detected from the carbon particles. Lymph nodes are embedded in adipose tissue and as a consequence, very strong lipid peaks were frequently observed in spectra. Spectral differences were exhibited in the measurements of the lymph nodes from the three different anatomical regions. A comparison of the point measurements and mapped data showed no difference in classification. Therefore, indicating that just a few measurements can be sufficient enough sampling to represent a specimen, and demonstrates the practicability of Raman use in vivo for rapid analysis. The Raman needle probe feasibility study showed its potential for in vivo use for real-time diagnosis and as a surgical tool to support biopsy. A sensitivity and specificity of 80% and 79% for the identification of non-cancer head and neck lymph nodes from non-cancer provides similar accuracies to the standard Raman approach, therefore supports its viability for use as a diagnostic tool.

by Betty Shuc Han Wills. / Year shown on spine: 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 117-128). / ACKNOWLEDGMENT --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iv / LIST OF APPENDICES --- p.viii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- LITERATURE REVIEW --- p.6 / Parental Responses to the Diagnosis of Acute Lymphocytic Leukaemia (ALL) --- p.7 / Disclosure Of the Child's Diagnosis --- p.10 / Impact of Cancer Treatment on Parents --- p.14 / Sources of Support for Parents --- p.18 / Coping Strategies of Parents of Children With ALL --- p.20 / Coping With The Uncertainty of the Disease --- p.23 / Research Studies Involving Chinese Parents --- p.24 / Summary Of Issues From Literature Review --- p.27 / Chapter CHAPTER 3 --- METHODOLOGY / Research Design --- p.29 / Sampling --- p.31 / Data Collection Method --- p.32 / Data Collection Procedure --- p.34 / Ethical Considerations --- p.38 / Pilot Study --- p.40 / Data Analysis --- p.42 / Issues of Reliability and Validity --- p.45 / Validity --- p.45 / Reliability --- p.48 / Chapter CHAPTER 4 --- RESULTS & DISCUSSION / Introduction --- p.50 / Chapter (I). --- Parents' Profile --- p.51 / Demographic Characteristics Of The Parents / Chapter (II). --- Major categories Corresponding To Interviewing The Mothers --- p.54 / Initial Reactions of the Child's Confirmed Diagnosis --- p.55 / Unpreparedness for the child's Diagnosis / Suddenness of the Diagnosis --- p.56 / Physical and psychological reactions to the child's Diagnosis --- p.58 / Sources of Support for the Mothers --- p.62 / The mothers' main source of support / Other sources of support for the mothers --- p.64 / Disclosure Of Child's Diagnosis --- p.66 / Disclosure of the child's diagnosis to the child / Disclosure of the child's diagnosis to members of the immediate and extended families --- p.68 / Disclosure of child's diagnosis to non-family members --- p.70 / Uncertainty Brought On By The Illness --- p.71 / Waiting for confirmation of diagnosis / Uncertainty about the success of treatment --- p.73 / Uncertainty about the child's future --- p.74 / Changes In The Family Routine --- p.75 / Needed to normalise family life / Chapter (III). --- Major Categories Corresponding to Interviewing The Fathers Initial reactions to the child's confirmed diagnosis of ALL --- p.78 / Suddenness of diagnosis --- p.79 / Physical and psychological reactions to the diagnosis --- p.80 / Disclosure Of The Child's Diagnosis --- p.82 / Disclosure of the child's diagnosis to the child / Disclosure of the child's diagnosis to members of the immediate and extended family --- p.85 / Disclosure of the child's diagnosis to non-family members --- p.86 / Sources Of Support For The Fathers --- p.87 / Support from immediate and extended families / Support from medical professionals --- p.89 / Support from friends --- p.90 / Changes In The Family Routine --- p.91 / Coping Strategies Utilised By The Fathers --- p.92 / Open communication / Use of religious beliefs and rituals --- p.93 / Chapter (IV). --- Comparison Of Categories Found Between The Mothers And The Fathers --- p.95 / Initial reactions to the child's confirmed diagnosis of ALL / Disclosure of the child's diagnosis --- p.96 / Sources of support for the parents --- p.100 / Changes in the family routine --- p.101 / Summary of findings --- p.103 / Chapter (V). --- Differences between the initial and second interviews --- p.105 / Chapter CHAPTER FIVE --- CONCLUSION / Limitations Of The Study --- p.108 / Implications For Nursing Practice --- p.112 / Recommendations For Future Research --- p.114 / Conclusion --- p.115 / REFERENCES --- p.117 / APPENDIX I - PERSONAL DATA FORM --- p.129 / APPENDIX II - INTERVIEW SCHEDULE --- p.130 / APPENDIX III - CONSENT FORM --- p.134 / APPENDIX IV - SAMPLE SCRIPT OF INTERVIEWS WITH MOTHERS --- p.135 / APPENDIX V - MATRICES ON MOTHERS AND FATHERS --- p.140 / APPENDIX VI - SAMPLE OF FIELD NOTES FOR MOTHERS AND FATHERS --- p.143

O linfoma alimentar é a forma anatômica mais frequente nos felinos e é o tipo de neoplasia que mais acomete o intestino delgado nessa espécie. É caracterizado pela infiltração de células linfóides neoplásicas em órgãos do trato gastrointestinal, com ou sem comprometimento de linfonodos mesentéricos. Seu diagnóstico pode ser considerado desafiador em muitos casos, devido às manifestações clínicas muitas vezes inespecíficas e às limitações da citologia e histopatologia. A imunoistoquímica, embora ainda pouco difundida, pode ser utilizada para confirmar o diagnóstico de linfoma alimentar, bem como determinar o tipo celular predominante. Objetivando-se caracterizar o linfoma alimentar felino, foi realizado estudo utilizando-se 40 casos de gatos com diagnóstico de linfoma alimentar (grupo 1), confirmados por meio da imunoistoquímica, no qual se avaliou os aspectos epidemiológicos, clínicos, citológicos, histológicos e imunoistoquímicos. Também foram avaliados 20 casos de felinos com doença inflamatória intestinal (grupo 2), confirmados segundo exame imunoistoquímico, quanto aos aspectos epidemiológicos e clínicos. E com o intuito de verificar a acurácia diagnóstica relativa dos exames de citologia, histologia e imunoistoquímica, foram utilizados os resultados dessas análises de ambos os grupos. Foi observado entre os felinos com linfoma alimentar estudados uma média de idade de 10,8 anos e as manifestações clínicas de maior ocorrência foram emagrecimento (55%), êmese (40%) e alterações nas fezes (40%). Entre as alterações ultrassonográficas observadas, as mais prevalentes foram linfonodomegalia mesentérica (86,11%) e espessamento de alças intestinais (80,55%). A maioria dos felinos estudados apresentou sorologia negativa tanto para FIV quanto para FeLV e apenas 14,28% e 8,57% foram positivos para FIV e FeLV, respectivamente. A correlação entre citologia e imunoistoquímica apontou sensibilidade, especificidade, valor preditivo positivo e negativo e razão de verossimilhança positiva e negativa de 93,3%, 65%, 66,6%, 92,85%, 2,65 e 0,10, respectivamente. O coeficiente de concordância Kappa de Cohen e a acurácia diagnóstica relativa foram de 0,55 e 77,14%, respectivamente. A correlação entre histopatologia e imunoistoquímica revelou sensibilidade, especificidade, valor preditivo positivo e negativo e razão de verossimilhança positiva e negativa de 89,74%, 60%, 81,39%, 75%, 2,22 e 0,18, respectivamente. O coeficiente de concordância Kappa de Cohen e a acurácia diagnóstica relativa foram de 0,52 e 79,66%, respectivamente. A associação entre sorologia para FIV e FeLV e tipo celular do linfoma alimentar não pode ser observada. O presente estudo permitiu concluir que a combinação de citologia, histopatologia e imunoistoquímica é necessária para o correto diagnóstico de linfoma alimentar, e o tipo celular de maior ocorrência foi o de células T. / Alimentary lymphoma is the most common anatomical form of feline lymphoma and is the neoplasm which more affects small intestine of this specie. It is characterized by infiltration of the gastrointestinal tract with neoplastic lymphocytes, with or without mesenteric lymph nodes involvement. The diagnosis of this illness can be challenger in several cases because of unspecific clinical signs and cytological and histopathological limitations. Immunohistochemistry is required where these evaluations were not definitive in addition to establish the cell phenotype T or B lymphocyte. The aim of this study was to characterize feline alimentary lymphoma. For these purpose, 40 cats with a diagnosis of alimentary lymphoma (Group 1) by immunohistochemistry were evaluated clinical, epidemiological, cytological and histologically, in addition to immunophenotype of the neoplasm tissues. Relative accuracy was performed using the results of cytology, histopathology and immunohistochemistry evaluation of 20 cats with inflammatory bowel disease diagnosis. The average age of the cats with alimentary lymphoma was 10.8 years and weight loss (55%), vomiting (40%) and fecal alteration (40%) were the most common clinical signs. The most prevalent ultrasonography abnormalities were mesenteric lymph nodes enlargement (86.11%) and intestinal wall thickening (80.55%). The majority of alimentary lymphoma cats were FIV and FeLV negative and 14.28% and 8.57% were FIV and FeLV positives, respectively. The cytology and immunohistochemistry correlation presents sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratio tests of 93.3%, 65%, 66.6%, 92.85%, 2.65 and 0.10, respectively. The Cohen´s kappa test and the relative accuracy were 0.55 and 77.14%, respectively. The histopathology and immunohistochemistry correlation presents sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratio tests of 89.74%, 60%, 81.39%, 75%, 2.22 and 0.18, respectively. The Cohen´s kappa test and the relative accuracy were 0.52 and 79.66%, respectively. Association of FIV and FeLV result tests and alimentary lymphoma phenotype was not observed. In conclusion, the results of the present study suggest that cytology, histology and immunohistochemistry combination is necessary for a correct diagnosis, and T cell alimentary lymphoma is the most common phenotype.

Thesis (M.Sc. (Biochemistry)) --University of Limpopo, 2004 / Refer to the document / National Research Foundation (NRF) and the UNlN Research Commitee

CD40 is a TNF-Receptor (TNFR) superfamily member that functions to promote several facets of the humoral immune response--including B cell proliferation, differentiation, antibody isotype switching, and cytokine expression. TNFR superfamily members lack intrinsic kinase activity and must recruit members of the TNFR-associated factor (TRAF) family of adaptor proteins to connect the receptor to intracellular signaling pathways. CD40-mediated JNK and NF-κB activation is critical for an intact humoral immune response; however, the precise mechanisms governing the spatiotemporal activation of these pathways are not completely understood. In this study we report that CD40 ligation results in the dual phosphorylation of TRAF2 on Ser-11 and Ser-55 to control the subcellular localization of key pathway intermediates and temporally regulate downstream JNK and IKK/NF-κB pathway activation. Notably, TBK1- mediated TRAF2 Ser-11 phosphorylation elicits the dissociation of a signaling complex, consisting of TRAF2, cIAP1/2, and IKKγ, from the CD40 receptor to potentiate a secondary phase of JNK and IKK activation. In the absence of this phosphorylation event, these proteins translocate to the insoluble lipid rafts along with the membrane-bound receptor complex, where TRAF2 undergoes Ser-55 phosphorylation-dependent self-ubiquitination and degradation necessary for cessation of JNK activation. Furthermore, TRAF2 Ser-11 phosphorylation inhibits non-canonical NF-κB activation by promoting the lipid raft localization of the CD40 receptor complex. This suggests that TRAF2 dual phosphorylation acts as a molecular switch to control canonical and non-canonical NF-κB activation. CD40 signaling is heavily implicated in a wide array of chronic inflammatory and autoimmune diseases--including Alzheimer's, Grave's disease, and diabetes. As such, characterization of the molecular mechanisms directing CD40 signal transduction will provide a foundation for the further development of targeted immunomodulatory therapeutics. In addition, the NF-κB transcriptional program has well-defined roles in oncogenesis and tumor progression, and many B cell lymphomas exploit the CD40L/CD40 dyad to constitutively activate the NF-κB pathway and potentiate neoplastic growth and survival. Through these analyses, we demonstrate that TRAF2 phosphorylation on Ser-11 and Ser-55 promote cell survival in response to genotoxic and oxidative stress, respectively, by regulating JNK and NF-κB pathway activation and coordinating the subcellular localization and stability of key signaling effectors. Furthermore, we show that inhibition of TRAF2 phosphorylation in B-cell lymphoma cells increases their sensitivity to standard frontline chemotherapeutics, including doxorubicin and vincristine, as well as the novel agents bortezomib and arsenic trioxide. These findings are clinically significant, as TRAF2 is found over-expressed and constitutively phosphorylated in DLBCL cell lines and patient biopsies. In addition, mice bearing tumors that harbor TRAF2 Ser-11 phospho-null mutations are more responsive to treatment with doxorubicin and have significantly prolonged survival compared to wild-type counterparts in a syngeneic model of B-cell lymphoma. The tumor microenvironment is characterized by pro-inflammatory cytokines, hypoxia, low glucose, and free radicals, all of which are known to induce chronic cellular stress and NF-κB activation. Cancer cell adaptation to these stressors has profound consequences for malignant progression and therapeutic response. In this regard, our findings present a unique opportunity where the molecular targeting to TRAF2 phosphorylation could increase the efficacy of current therapies by suppressing basal NF-κB activation, thus synergistically sensitizing NF-κB-driven malignancies to chemotherapeutic-induced cell death.