Sarcomas are cancers of mesenchymal origin. Though they comprise 15-20% of childhood cancers, and have a 5-year survival rate of 16% for metastatic disease, few targeted therapies exist, and the underlying mechanisms of their development are poorly understood. Transcriptional coactivators TAZ and YAP promote cell growth and proliferation, and are constitutively activated in a number of carcinomas. Accompanying TAZ/YAP activation in these cancers is decreased expression of Hippo pathway kinases MST1/2 and LATS1/2. As the Hippo pathway is the primary negative regulator of TAZ/YAP, this provides a potential mechanistic explanation for constitutive TAZ/YAP activation. TAZ and YAP are also thought to play a prominent role in sarcomagenesis, as TAZ-CAMTA1 and YAP-TFE3 gene fusions are the specific initiating events leading to formation of epithelioid hemangioendothelioma (EHE), a vascular sarcoma. However, the mechanisms causing constitutive activation of wild-type TAZ/YAP in sarcomas have not been well-characterized. The purpose of this study was to determine if Hippo pathway dysregulation occurs in sarcomas with constitutively active, wild-type TAZ/YAP, as well as the mechanisms by which this regulation is lost. We also investigated whether TAZ/YAP could be therapeutically targeted in sarcomas using verteporfin, a small-molecule inhibitor of the TAZ/YAP-TEAD interaction. To address these questions, sarcoma cell lines and patient clinical samples were utilized. Using 159 patient tumor sections, we constructed a tissue microarray, stained for activated (nuclear localized) TAZ/YAP, and Hippo kinases MST1/2 and LATS1/2. A majority of sarcomas contained activation of both TAZ and YAP, while significant decreases in MST1/2 and LATS1/2 expression were observed. Results indicated a majority of tumors which stained positively for nuclear-localized TAZ/YAP also contained loss of expression of at least one of the four kinases evaluated. All cell lines evaluated via immunofluorescence also had constitutively active (nuclear) TAZ/YAP when grown to confluence, which suggested they were no longer being negatively regulated by the Hippo pathway. In ~50% of lines, protein loss of MST1/2 and LATS2 occurred and mRNA expression of MST1 and MST2 was notably decreased in ~50%, although loss of LATS1 and LATS2 was minimal. Potential mechanisms which could account for Hippo kinase loss were next investigated. It was found that protein degradation diminished MST2 in 25% of cell lines. Regulation by epigenetic modifications was also investigated; hypermethylation accounted for slightly reduced MST1/2, while 67% of lines had histone deacetylation in both kinases. Whether TAZ/YAP can be therapeutically targeted using verteporfin was tested; treatment significantly inhibited anchorage-independent growth, proliferation, and TAZ/YAP transcriptional activity in sarcoma cell lines. Our results collectively demonstrate TAZ/YAP activity can be targeted in sarcomas with verteporfin, and their constitutive activation is due to loss of MST1/2 and LATS2 kinase expression through protein degradation, histone deacetylation, and promoter hypermethylation. Such findings enhance our current comprehension of the molecular events which promote sarcomagenesis; this knowledge also opens up the possibility of creating targeted pharmacological interventions.

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Pneumonia is a leading cause of death worldwide, and can be caused by different types of pathogens, including viruses and bacteria. Influenza A virus is one of the most common viral causes of pneumonia, and seasonal strains infect 5-20% of the population of the United States each year. Seasonal strains are constantly evolving, and therefore vaccines must be updated and administered every year. Recent studies have indicated that influenza virus-specific CD4 T cells can provide protection during infection with strains of influenza A virus to which an individual does not have antibodies, thus understanding how these CD4 T cells respond to infection in the lungs is an important step towards more effective and enduring protection. My work provides insight into the specialized subsets present in the influenza A virus-specific CD4 T cell response in the lungs and demonstrates that this response is regulated by pulmonary antigen-presenting cells. Group A Streptococcus is a bacterial cause of pneumonia with a 15-25% mortality rate, yet very little is known about the pulmonary CD4 or CD8 T cell responses to infection. My work maps the kinetics of and identifies requirements for CD4 and CD8 T cell accumulation in the lungs during pulmonary Group A Streptococcus infection. Together, these studies contribute to our knowledge of how T cell responses are generated and regulated in response to pulmonary pathogens. These findings have the potential to inform development of novel treatment and prevention strategies.

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Systemic lupus erythematosus (SLE) patients are at increased risk of developing components of the metabolic syndrome. Furthermore, obesity and high-fat diet (HFD) can lead to SLE nephritis exacerbation in humans and mouse models. Study of the interplay between metabolic dysfunction and autoimmunity may inform the care of SLE patients. Chapter one of this thesis provides background on the fields of systemic lupus erythematosus and the metabolic syndrome, and highlights the shared pathogenic mechanisms that may instigate these diseases. Chapter two describes efforts to characterize the development of glucose homeostasis in the lupus-prone B6.SLE strain of mice. We show that B6.SLE mice have significantly worsened glucose tolerance and adipose tissue insulin resistance than B6 controls. This B6.SLE glucose intolerance occurs in the presence of a diabetogenic B cell environment which is characterized by the presence of an increased number of IgG-producing B cells and IgG depositions in the white adipose tissue as well as higher levels of circulating IgG. Most interestingly, this strain of mice develops glucose intolerance and adipose tissue insulin resistance in an adiposity-independent manner, indicating that this strain may be a valid model for insulin resistance in the SLE patient population. These studies provide data that elucidate the pathogenesis of insulin resistance in SLE patients and may provide greater insight into the development of insulin resistance in the obese population. Chapter three shows that high-fat diet feeding does not significantly worsen glomerulonephritis or kidney function in B6.SLE mice. Although this finding is in conflict with earlier studies of SLE models, it is possible that changes in study design could reveal dietary factors that exacerbate or ameliorate disease in the B6.SLE mouse model. Chapter four compares our results to those of other studies in SLE model models and SLE patients and proposes a mechanism for the pathogenesis of glucose intolerance and insulin resistance in the B6.SLE mouse model.

TNF-α, produced by most malignant cells, orchestrates the interplay between malignant cells and myeloid cells, which have been linked to tumor growth and metastasis. Although TNF-α can exist as one of two isoforms, a 26-kDa membrane tethered form (mTNF-α) or a soluble 17-kDa cytokine (sTNF-α), the vast majority of published studies have only investigated the biological effects of the soluble form. We demonstrate for the first time that membrane and soluble isoforms have diametrically opposing effects on both tumor growth and myeloid content. Mouse lung and melanoma tumor lines expressing mTNF-α, generated small tumors devoid of monocytes versus respective control lines or lines expressing sTNF-α. The lack of myeloid cells was due to a direct effect of mTNF-α on myeloid survival via induction of cell necrosis by increasing reactive oxygen species. Using cultured RAW 264.7 monocytic cell line and L929 fibroblasts we showed that mTNF-α increased reactive oxygen species (ROS)-mediated cytotoxicity, independent of caspase-3 activity. Although TNF-α receptors (TNFR) on target cells were required for this effect, we observed that mTNF-α-induced cell death could be mediated through both TNFR-1 and the death domain-lacking TNFR-2. ROS generation and cytotoxicity were inhibited by a mitochondrial respiratory chain inhibitor but not by an inhibitor of NADPH oxidase. Furthermore, mTNF-α mediated cytotoxicity was independent of RIP-1, a serine/threonine kinase which serves as a main adaptor protein of sTNF-α induced programmed necrosis, but rather depended on ceramide signaling pathways. Furthermore, we found that human none-small-cells lung carcinomas (NSCLCs) expressed varying levels of both soluble and membrane TNF-α. Analysis of publicly accessible NSCLC microarray database showed that gene expression patterns favoring mTNF-α were predictive of improved lung cancer survival.