Autophagy is a conserved catabolic process that traffics cellular components to the lysosome for degradation. Autophagy is required for cell survival during nutrient restriction, but it has also been implicated in programmed cell death. It is associated with several diseases, including cancer. Cancer is a disease characterized by aberrant cell growth and proliferation. To support this growth, the tumor cell often deregulates several metabolic processes, including autophagy. Interestingly, autophagy plays paradoxical roles in tumorigenesis. It has been shown to be both tumor suppressive through cell death mechanisms and tumor promoting through its cytoprotective properties. However, the mechanisms regulating the balance between cell death and cell survival, as well as the metabolic consequences of disrupting this balance, are still poorly understood. Autophagy functions in both cell survival and cell death during the development of Drosophila melanogaster, making it an ideal model for studying autophagy in vivo. My research aimed to better understand the regulation and metabolic contribution of autophagy during cell death in Drosophila. I found that the Ral GTPase pathway, important to oncogenesis, regulates autophagy specifically during cell death in Drosophila larval salivary glands. Contrary to previous studies in mammalian cell culture, Ral is dispensable for autophagy induced during nutrient deprivation suggesting that Ral regulates autophagy in a context-dependent manner. This is the first in vivo evidence of Ral regulating autophagy. I found that disrupting autophagy has an extensive impact on an organism’s metabolism. Additionally, I found that autophagy in degrading tissues is crucial for maintaining the fly’s metabolic homeostasis, and that it may be important for resource allocation amongst tissues. This research highlights the importance of understanding how pathways regulate autophagy in different cell contexts and the metabolic outcomes of manipulating those pathways. This is especially important as we investigate which pathways to target therapeutically in an effort to harness autophagy to promote cell death rather than cell survival.

Vitiligo is an autoimmune skin disease in which the pigment producing cells of the epidermis, melanocytes, are targeted for destruction by CD8+ T cells specific for melanocyte/melanoma-shared antigens. Previous work has identified IFNg as the central cytokine driving disease pathogenesis in both human patients and in our mouse model of vitiligo. IFNg signaling induces production of the chemokines CXCL9 and CXCL10, which trigger autoreactive T cell migration into the epidermis where effector T cells can target and destroy melanocytes. However, both IFNg and type I IFN signaling through activation of STAT1 proteins can induce transcription of the chemokines CXCL9 and CXCL10. Therefore, it seems reasonable that type I IFN signaling may also contribute to disease pathogenesis. The role of type I IFN in vitiligo is still unclear. Genome wide association studies identified multiple genes within the type I IFN pathway including TICAM1 and IFIH1 as susceptibility loci in vitiligo. One additional study reported increased epidermal staining of CD123, a marker expressed by pDCs, and the type I IFN induced gene MX1 in vitiligo patient skin. However, this study did not show any functional data to support the role of type I IFN signaling in vitiligo pathogenesis. Since the role of type I IFN in vitiligo is ill-defined, we used two different mouse models of vitiligo to functionally determine the role of type I IFN in disease by inducing vitiligo in hosts which lack the type I IFN receptor (IFNaR). In the first model, we induced vitiligo by adoptive transfer of melanocyte-specific CD8 T cells, which are activated in vivo by infection with recombinant vaccinia virus (VACV) expressing their cognate antigen. Vitiligo induction in IFNaR-deficient mice led to the development of severe disease compared to wild type mice. Acceleration and severity of disease was characterized by increased early recruitment of melanocyte-specific CD8 T cells to the skin, increased production of effector cytokines TNFa and IFNg, and reduced PD-1 expression. Increased production of IFNg by CD8 T cells in the skin of IFNaR-deficient mice led to increased expression of the chemokines CXCL9 and CXCL10 driving disease progression. IFNaR-deficient mice also displayed significantly increased VACV titters compared to wild type hosts. This data reveals a role of type I IFN in the clearance of recombinant VACV. This data also suggests that persistent VACV infection and prolonged antigen exposure in IFNaR deficient hosts is likely driving enhanced activation of melanocyte specific CD8 T cells and the subsequent development of severe vitiligo. Since melanocytes and melanoma cells express shared antigens that can be recognized by CD8 T cells, and because the development of vitiligo after melanoma immunotherapy is a positive prognostic factor for patients, we asked whether VACV vaccine therapy in IFNaR deficient mice would enhance the anti-tumor response to melanoma. B16-F10 inoculated wild type and IFNaR-deficient mice received adoptive transfer of melanocyte-specific CD8 T cells in combination with vaccinia virus expressing their cognate antigen to activate the cells in vivo. Treatment of adoptive T cell transfer and infection with VACV in IFNaR-deficient mice revealed significantly reduced tumor burden compared to wild type mice. Improved tumor regression in IFNaR-deficient hosts was characterized by increased infiltrating cytotoxic T lymphocytes and reduced PD-1 expression. These results further demonstrate that in the absence of type I IFN, hosts mount a robust cytotoxic CD8 T cell response against melanocyte/melanoma antigens and this is likely a result of persistent VACV that leads to prolonged CD8 T cell priming. As a result, IFNaR deficient hosts kill tumor cells more efficiently. To determine whether type I IFN regulates disease pathogenesis in the absence of virus infection, we generated a model of vitiligo in which bone marrow derived dendritic cells (BMDCs) pulsed with the cognate antigen were used to prime melanocyte-specific T cells in place of the viral vector. Induction of vitiligo in IFNaR-deficient hosts using BMDCs revealed no significant differences in disease score compared to wild type hosts. This data clearly demonstrates that type I IFN, in contrast to IFNg, is not required during the effector stage of vitiligo pathogenesis in mice. However, since we intentionally activate transferred melanocyte-specific CD8 T cells with VACV or BMDCs expressing their cognate antigen, our mouse models may circumvent the role of type I IFNs in initiating activation of autoreactive cells and driving autoimmunity. Type I IFN is critical for providing innate immune signals that drive the priming of autoreactive T cells through maturation of DCs by inducing antigen presentation, co-stimulatory molecule expression, and migration to the lymph nodes to encounter naïve T cells. Our mouse models of vitiligo may not capture this process. We have addressed this question by using a TLR ligand to activate BMDCs before transfer into hosts. In fact, activation of BMDCs before transfer leads to significantly enhanced vitiligo in mice and this is partially a result of type I IFN signaling on host cells. Thus, we provide evidence that type I IFNs can enhance the activation of melanocyte-specific CD8 T cells and drive autoimmunity. Collectively, our results show that type I IFN signaling has disparate effects on autoreactive T cell priming in a context dependent manner. We reveal that although type I IFN is not required for the effector phase of vitiligo in mice, maturation of DCs and subsequent type I IFN production can enhance the priming of autoreactive T cells and enhance vitiligo severity. Our studies also reveal that type I IFN is required to clear recombinant attenuated VACV infection and vaccine administration in IFNaR deficient hosts led to a robust autoreactive and anti-tumor response. These insights describing the role of type I IFN in autoimmunity and tumor immunology could have important implications for T cell dependent tumor immunotherapy.

Error-free DNA replication is paramount to maintaining genomic integrity. Despite being highly regulated, the process of DNA replication is often challenged by various intrinsic and extrinsic sources of replication stress. Failure to maintain the DNA replication quality reduces genomic stability, cell survival and results in diseases, such as cancer. Thus, cells rely on the replication stress response that detects perturbations in DNA replication and pauses or arrests cellular replication. Similar to other intrinsic replication obstacles, oncogene expression also induces the replication stress response that acts as a barrier to cancer, thereby mystifying how cancer develops. Here, we demonstrate that oncogene expression, similar to other replication stress inducing agents, induces single-stranded DNA (ssDNA) gaps that reduce cell fitness unless counteracted by translesion synthesis (TLS). Moreover, we find that TLS subverts the replication stress response in a wide range of cancer cell lines indicating that TLS is a previously unappreciated and unique cancer vulnerability. Mechanistically, we reveal that upon replication stress, TLS restricts replication fork slowing, reversal, and fork degradation, while maintaining continuous replication. Furthermore, we demonstrate that a small molecule inhibitor targeting the TLS factor, REV1, not only disrupts DNA replication and cancer cell fitness, but also synergizes with other therapies that induce replication gaps. Thus, our study places TLS at the center of cancer cell fitness as a necessary adaptation to overcome replication stress.

Polymer conjugates offer a way to introduce materials into the body that would normally be rejected or cause toxicity. Two polymers are investigated in vivo for uses in chemotherapeutic delivery, protein therapeutics, and DNA transfection. A novel polymer, polyMPC, has the ability to increase doxorubicin loading and its solubility, and is conjugated in a way to release its payload in a low pH environment. Through its conjugation, blood clearance time of doxorubicin is increased, and thus tumor exposure to the drug is increased with a single administration. It can be administered at ten times the concentration of free doxorubicin, and three times the concentration of Doxil®, while decreasing the cardio-toxicity normally associated with doxorubicin administration. These results show that polyMPC has the potential to increase treatment efficacy of doxorubicin. With increased circulation time, MPC polymers have additional potential for protein delivery and variations of its design were tested in linear, branched and grafted states, which show limited affect on tissue weight. An additional polymer for use in DNA transfection, NLS2, demonstrated its lack of tissue toxicity when injected intramuscularly. While continued investigation into these polymers is required, this initial data indicates their promising uses as therapeutics.

Nur77 is a member of the orphan nuclear receptor family, where it is known to play an important role in apoptosis in both negative selection in T cells and in cancer cell lines. In the development of T cells, it is critical for the immune system to discriminate self from non-self by eliminating auto-reactive cells. It was originally thought that Nur77 initiated apoptosis by activating downstream gene targets. However, it is now clear that Nur77 has its own distinct role outside of the nucleus and the precise mechanisms by which Nur77 induces apoptosis in T cells still needs to be clarified. Calcium plays an important role as a second messenger in various cellular responses, one of which includes apoptosis. The IP3 receptor controls efflux of calcium from the ER and can be activated through TCR activation. This signal induces a rise in cytoplasmic calcium levels ultimately causing cell death through mechanisms that remain unclear. Here, we use a double positive DO11.10 T cell line with tetracycline responsive Nur77, to examine the effects of cytosolic Nur77. Through co-immunoprecipitation experiments we suggest, that the presence of Nur77 disrupts the IP3R/Bcl-2 interaction. In this study, we also investigated the effect of Nur77 on intracellular calcium levels. We show that Nur77 increases baseline calcium levels and causes emptying of ER calcium stores. We suggest a model where cytosolic Nur77 disrupts the IP3R/Bcl-2 interaction by binding Bcl-2 at the mitochondria or ER, causing calcium release through the IP3R and apoptosis of the cell.

Natural Killer (NK) cells can recognize and lyse a large variety of tumor cells and have been of interest as a potential cancer treatment option. Our group has developed a particle-based NK cell expansion method that utilizes plasma membrane particles (PM-particles) derived from K562 cells genetically engineered to express membrane bound IL21 and 41BBL(K562-mbIL21-41BBL), two proteins that stimulate growth and activity of NK cells. This method selectively expands highly cytotoxic NK cells > 400-fold in 14 days of culture. Currently NK cells are expanded in vitro using Fetal Bovine Serum (FBS) as a serum-supplement to promote cell growth. While effective, the use of animal products is not preferred in cell cultures grown for clinical purposes. This project tested Human Platelet Lysates (HPL) as a potential replacement for FBS in NK cell culture. NK cells were expanded using PM21-particle based expansion method with either FBS or HPL as supplements. Their growth characteristics, phenotype and functionality were assessed and compared. Results of this study determined that HPL is a viable option to replace FBS in NK cell culture for clinical applications, as there was no significant difference between the two serum supplements.

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This research presents a framework for enhancing and analyzing time-sequenced mammographic images for detection of cancerous tissue, specifically designed to assist radiologists and physicians with the detection of breast cancer. By using computer aided diagnosis (CAD) systems as a tool to help in the detection of breast cancer in computed tomography (CT) mammography images, previous CT mammography images will enhance the interpretation of the next series of images. The first stage of this dissertation applies image subtraction to images from the same patient over time. Image types are defined as temporal subtraction, dual-energy subtraction, and Digital Database for Screening Mammography (DDSM). Image enhancement begins by applying image registration and subtraction using Matlab 2012a registration for temporal images and dual-energy subtraction for dual-energy images. DDSM images require no registration or subtraction as they are used for baseline analysis. The image data are from three different sources and all images had been annotated by radiologists for each image type using an image mask to identify malignant and benign. The second stage involved the examination of four different thresholding techniques. The amplitude thresholding method manipulates objects and backgrounds in such a way that object and background pixels have grey levels grouped into two dominant and different modes. In these cases, it was possible to extract the objects from the background using a threshold that separates the modes. The local thresholding introduced posed no restrictions on region shape or size, because it maximized edge features by thresholding local regions separately. The overall histogram analysis showed minima and maxima of the image and provided four feature types--mean, variance, skewness, and kurtosis. K-means clustering provided sequential splitting, initially performing dynamic splits. These dynamic splits were then further split into smaller, more variant regions until the regions of interest were isolated. Regional-growing methods used recursive splitting to partition the image top-down by using the average brightness of a region. Each thresholding method was applied to each of the three image types. In the final stage, the training set and test set were derived by applying the four thresholding methods on each of the three image types. This was accomplished by running Matlab 2012a grey-level, co-occurrence matrix (GLCM) and utilizing 21 target feature types, which were obtained from the Matlab function texture features. An additional four feature types were obtained from the state of the histogram-based features types. These 25 feature types were applied to each of the two classifications malignant and benign. WEKA 3.6.10 was used along with classifier J48 and cross-validation 10 fold to find the precision, recall, and f-measure values. Best results were obtained from these two combinations: temporal subtraction with amplitude thresholding, and temporal subtraction with regional-growing thresholding. To summarize, the researcher's contribution was to assess the effectiveness of various thresholding methods in the context of a three-stage approach, to help radiologists find cancerous tissue lesions in CT and MRI mammography images.

The cancer stem cell (CSC) model of carcinogenesis and progression posits that within a tumor lies a subpopulation of cells that solely possess the ability to initiate a tumor and to differentiate into tumor cell lineages. Although the behavior of such cells is known, the challenge is to identify factors that characterize the CSC subpopulation. In this thesis, cell lines were identified that, when grown in three-dimensions, gave rise to organized colonies containing lumens originating from differentiating cells (“lumen lines”) and to densely-packed, spherical colonies originating from non-differentiating cells (“dense lines”). A microarray comparison of the pair identified genes upregulated in dense lines, including CD55 and BMI1, and in lumen lines, including CDX1 (Chapter 3). CD55 was used to isolate CD55high CSCs via flow cytometry that are able to self-renew, differentiate, initiate more colonies, proliferate more rapidly and exhibit an increased G2/M cell cycle population as opposed to unfractionated cells. Furthermore, the CD55high cells were able to give rise to more differentiated, lumen colonies in vitro, indicating that CD55 enriches for cells possessing a capacity to differentiate, and were able to enrich the CD24highCD44high putative CSC population further (Chapter 4). CDNA induction of BMI1 and CDX1 expression led to increased clonogenicity/proliferation and decreased clonogenicity/proliferation, respectively, and incorporation of a CDX1 reporter construct into the SW1222 cell line identified CDX1+ cells as a low-expressing population of CD55 (Chapter 5). Finally, co-culture of cell lines in an in vivo-like environment with intestinal myofibroblasts promoted the CSC population by enhancing clonogenicity, proliferation and expression of CD55 (Chapter 6). The results of this thesis implicate CD55 as a potent marker of colorectal cancer stemness, link the expression of BMI1 and CDX1 to cancer stemness and differentiation, respectively, and identify a role for the in vivo stem cell niche in maintaining the CSC population.

Glioblastoma multiforme (GBM) is the most common primary brain tumor. Studies have shown that targeting the DNA damage response can sensitize cancer cells to DNA damaging agents. Ataxia telangiectasia mutated (ATM) is involved in signaling DNA double strand breaks. Our group has previously shown that ATM inhibitors (ATMi) sensitize GBM cells and tumors to ionizing radiation. This effect is greater when the tumor suppressor p53 is mutated. The goals of this work include validation of a new ATM inhibitor, AZ32, and elucidation of how ATMi and p53 status interact to promote cell death after radiation. We propose that ATMi and radiation induce mitotic catastrophe in p53 mutants by overriding cell cycle arrest. We tested this hypothesis in human colon carcinoma and glioma cells that differ only in p53 status. We found that AZ32 effectively inhibits phosphorylation of ATM targets. In addition, AZ32 significantly sensitizes glioma cells to ionizing radiation. While HCT116 colon carcinoma cells fail to arrest the cell cycle after radiation, their response to ATMi differs from that in gliomas. Indeed, wild type HCT116 cells were more sensitive than p53 mutants to ionizing radiation in the presence of ATMi. In contrast, ATMi significantly radiosensitized glioma cells in which p53 is knocked down. Live cell imaging confirmed that radiation and ATMi preferentially induce mitotic catastrophe in p53-deficient cells. We conclude that p53-deficient cells rely on ATM signaling for G2/M cell cycle arrest. We propose a model of G2/M arrest whereby ATM and p53-dependent signaling pathways converge to ultimately inhibit Cdc25 phosphatases.