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DNA damage induces a choreographed set of local changes in histone modifications which leads to efficient recruitment of DNA repair factors. The regulation of these chromatin modifications at DNA breaks is critical to maintain genome integrity. Recent studies in our lab have identified a role for G9a methyltransferase in regulating DNA repair. The overall aim of this project was to elucidate how G9a activity regulates this pathway and to identify the effects of its inhibition in this process. It was shown that G9a localizes to sites of DNA damage in an ATM-dependent fashion and that inhibition of G9a activity affects early recruitment of multiple DNA repair factors. We found that catalytic inhibition of G9a using UNC0638 results in increased ATM activation. This led to increased "spreading" of pH2AX and MDC1 signals seen at regions of localized DNA breaks induced by UV-laser scissors, which was dependent upon ATM activation. This was also associated with increased levels of H3K36me2 and H3K56Ac. Biochemical data showed that G9a interacts and regulates HDAC1/2 activity during the DNA damage response. G9a inhibition led to decreased HDAC1 methylation, and increased ATM acetylation. These data suggest that G9a activity regulates the extent of ATM activation induced by DNA breaks and is required for efficient recruitment of downstream DNA repair factors. Overall our data suggests that G9a plays a critical role in regulation of ATM-dependent signaling during the DNA damage response and raises the possibility of using G9a inhibitors in the clinical setting as part of novel cancer therapies.

Advisors/Committee Members: Xia, Bing (chair), Pine, Sharon (internal member), Ganesan, Shridar (internal member), Langer, Jerome (outside member), School of Graduate Studies.

▼ Traumatic injury to the spinal cord initiates a series of destructive cellular processes that exacerbate tissue damage at and beyond the original site of injury. These processes include oxidative stress responses and pro-inflammatory cascades that can lead to neuronal loss and demyelination of surviving axons. Previous work in our lab has shown that the voltage-gated proton channel Hv1 is necessary for NADPH oxidase-dependent reactive oxygen species (ROS) production by microglia. This means that Hv1 mediated microglial ROS could be a mechanism to cause neuronal damage under disease conditions. Therefore, we hypothesize that Hv1 contributes to ROS production in microglia after SCI, and loss of function can alleviate secondary injury. To test this, we utilized a moderate spinal cord contusive injury model with adult wild-type and Hv1-/- mice, with the aim of studying the role of Hv1 in microglia activation, ROS production, pro-inflammatory response, neuronal cell death, and demyelination. Our results showed that loss of Hv1 reduced microglial activation following spinal cord injury. Moreover, loss of Hv1 alleviated some of the oxidative stress-mediated secondary injury, as well as inflammatory response. Hv1-/- mice exhibited increased neuronal survival, white matter sparing, and improved locomotive recovery following spinal cord injury. Together, the current study suggested that after spinal cord injury, Hv1 is a potential treatment target to reduce secondary injury. Advisors/Committee Members: Wu, Long-Jun (internal member), Axelrod, David (internal member), Xia, Bing (internal member), School of Graduate Studies.

▼ DDR is often considered as a critical barrier during tumor initiation with most pre-malignant cells accumulate endogenous DNA damage before acquiring additional genetic alterations that provides a survival advantage. The work in this thesis focused on delineating the biological roles of BRCA1-PALB2 coiled-coil domain interaction, particularly on utilizing patient-derived VUS as means to identify novel BRCA1 function and possibility therapeutic targets in BRCA-associated cancers. No known structural information of the BRCA1-PALB2 interaction is available to date. The BRCA1-PALB2 coiled-coil domain is still potentially targetable due to the different post-translational modifications flanking this domain; protein modifications such as phosphorylation and ubiquitination can be explored further to modulate the BRCA1-PALB2 interaction. The BRCA1-PALB2 interaction is important for HR and suppression of error prone repair pathways. This protein-protein interaction is also required for efficient cell cycle checkpoint to ensure optimal repair timing when DNA damage occurs. We first investigated several PALB2 N-terminal coiled-coil domain mutants to not only determine the critical residues involved in PALB2 function but also to assess if varied repair ability would translate equally to chemotherapy sensitivity. Although PALB2 c.104T>C, p.L35P behaves as expectedly as a bona fide pathogenic missense variant, our results on other hypomorphic mutations such as c.83A>G , p.Y28C raised an interesting question on the complexity of VUS. Unlike pathogenic mutations that often resulted from frameshift mutations, hypomorphic PALB2 missense mutation that still retained HR activity above a poorly understood threshold is a challenge for personalized treatment of cancer patients. We next examine the direct relationship between known protein kinases such as ATM/ATR, CDK and PLK1 on BRCA1 function. BRCA1, a critical DDR protein, can function in various component of DDR network of responses. Several S/TQ sites on BRCA1 were widely reported based on either in vitro biochemical reactions or mass spectrometric analysis. The HR ability of cells often correlate well with drug resistance as genotoxic stress are often alleviated by HR. However, our experimental results focusing on the HR and SSA suppression activity of widely reported SQ BRCA1 mutants suggest that most of these phosphorylation events may not have as strong of a role in DDR than previously thought. S1387, T1394 and S1423 are phosphorylatable residues flanking the PALB2 binding coiled-coil domain. Here, we report that abolishing phosphorylation of both S1387 and S1423 leads to partial sensitization of cells to both cisplatin and olaparib despite similar to wt HR activity. T1394 phosphorylation, although undetectable in most mass spectrometry studies to date, appears to be a critical event as single amino acid alteration on this site is sufficient to partially reduce HR activity, increase the more erroneous SSA activity and completely sensitized cells towards cisplatin and olaparib.… Advisors/Committee Members: Ganesan, Shridar (chair), Xia, Bing (co-chair), Bunting, Samuel (internal member), Walworth, Nancy (outside member), School of Graduate Studies.

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Macroautophagy (autophagy hereafter) is a protective process that recycles cellular components to maintain homeostasis and survival. By removing damaged protein and organelles, autophagy ensures protein and organelle quality control. In cancer, the role of autophagy is context dependent. Autophagy is an essential survival mechanism during starvation that promotes tumor progression, but there are also cases where autophagy can suppress tumorigenesis in mouse models. Thus, understanding the role of autophagy modulation in cancer is critical for cancer therapy. Tumors with RAS mutations require autophagy to tolerate metabolic stress, suggesting that autophagy inhibition may be a potential approach in cancer therapy. However, how systemic autophagy inactivation differentially affects normal and tumor tissues is unknown. To assess the functional significance of autophagy, we conditionally deleted the essential autophagy gene, Atg7, throughout adult mice. In that way, we could model the consequences of autophagy inactivation simultaneously to both normal and tumor tissue to simulate autophagy inhibition in cancer therapy. First, we wanted to identify what happens to an adult mouse when autophagy is turned off. We generated a mouse model to delete Atg7, throughout the entire mouse to determine the role of autophagy in adult mammal. Systemic ATG7 ablation caused susceptibility to infection and neurodegeneration that limited survival to 2-3 months. Moreover, autophagy was required to maintain fat stores and to mobilize free fatty acids specifically in response to fasting. Also, upon fasting, autophagy-deficient mice suffered muscle wasting and fatal hypoglycemia that is mediated by p53. Not only limited to the starvation stress response, inhibiting autophagy also increased sensitivity to γ-irradiation-induced death. Second, knowing that there is a window of time that adult mice can survive without autophagy, we examined if autophagy ablation is selectively toxic to tumors while sparing normal tissues. For this purpose, we generated another mouse model to induce non-small-cell lung cancer (NSCLC) and then prior to or once tumors were established, we acutely ablated autophagy. Prior autophagy ablation did not alter the efficiency of NSCLC initiation by activation of oncogenic KrasG12D and deletion of the Trp53 tumor suppressor. Acute autophagy ablation in mice with pre-existing NSCLC, however, blocked tumor growth, promoted tumor cell death, and generated more benign cancer with oncocytic changes. Moreover, host autophagy contributed to tumor growth. This anti-tumor activity occurred prior to destruction of normal tissues, suggesting that, acute autophagy inhibition may be therapeutically beneficial in cancer.

Advisors/Committee Members: White, Eileen (chair), JIN, SHENGKAN (VICTOR) (internal member), Karantza, Vassiliki (internal member), RABINOWITZ, JOSHUA (outside member), Xia, Bing (outside member).

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Autophagy captures intracellular components and delivers them to lysosomes for degradation and recycling. Conditional autophagy deficiency in adult mice causes liver damage, and shortens lifespan to three months due to neurodegeneration. As autophagy deficiency causes p53 induction and cell death in neurons, we sought to test if p53 mediates the toxic effects of autophagy deficiency. Here we conditionally deleted Trp53 (p53 hereafter) and/or the essential autophagy gene Atg7 throughout adult mice. Compared to Atg7Δ/Δ mice, life span of Atg7Δ/Δp53Δ/Δ mice was extended due to delayed neurodegeneration and resistance to death upon fasting. Atg7 also limited apoptosis induced by the p53 activator Nutlin-3, suggesting that autophagy inhibited p53 activation. To test if increased oxidative stress in Atg7Δ/Δ mice was responsible for p53 activation, Atg7 was deleted in the presence or absence of the master regulator of antioxidant defense Nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2-/-Atg7Δ/Δ mice died rapidly due to small intestine damage, which was not rescued by co-deletion of p53. Thus, autophagy limits p53 activation and p53-mediated neurodegeneration. In turn, NRF2 mitigates lethal intestine degeneration upon autophagy loss. These findings illustrate the tissue-specific roles for autophagy and functional dependencies on the p53 and NRF2 stress response mechanisms. Atg7 and Atg5 are both essential autophagy genes (ATG) involved in autophagosome formation and deleting these genes leads to autophagy deficiency. As essential autophagy genes can function differently, we then sought to test whether whole-body conditional Atg5 deletion in adult mice (Atg5Δ/Δ mice) would have similar phenotype to that of Atg7 deletion. In contrast to Atg7Δ/Δ mice, Atg5Δ/Δ mice surprisingly lived for less than five days. Atg5Δ/Δ mice showed selective damage in the ileum part of intestine, with marked epithelial damage and loss of barrier function. In comparison to Atg7Δ/Δ mice, the ileum of Atg5Δ/Δ showed evidence of more rapid loss of autophagy, and loss of stem cells and malfunction of Paneth cells. Furthermore, Atg5Δ/Δ mice had decreased active β-catenin in the ileum, the key transcription factor for Wnt signaling that is essential for intestinal stem cell renewal. Atg5Δ/Δ mice lost PDGFRα+ mesenchymal cells (telocytes) in the ileum, which are required to provide Wnt signals to stem cells. Deletion of Atg5 more gradually overcame the loss of ileum telocytes and stem cells and resulted in death much later from neurodegeneration similar to deletion of Atg7 or Atg12. Atg5Δ/Δ telocytes displayed significantly decreased aspartate and nucleotides, which caused their loss. These findings reveal a novel function of autophagy in maintenance of telocytes, Wnt signaling, and thereby stem cells essential for intestinal homeostasis and the survival of adult mice. As impaired autophagy is associated with Inflammatory Bowel Diseases, this suggests that failure to maintain telocyte function is involved in development of this disease.

Advisors/Committee Members: Carpizo, Darren (chair), White, Eileen (internal member), Hu, Wenwei (internal member), Xia, Bing (internal member), Verzi, Michael (outside member), School of Graduate Studies.

▼ Homologous recombination (HR) is the only error-free pathway for the repair of DNA double strand breaks (DSBs). BRCA1 and BRCA2, the two major breast cancer suppressor proteins, play essential roles in HR-mediated repair of DSBs by promoting the recruitment of RAD51, the recombination enzyme, to DNA damage sites for the initiation of HR. PALB2 (partner and localizer of BRCA2) plays a key role in this pathway by acting as a chromatin adaptor for BRCA2 and a linker between BRCA1 and BRCA2. Like BRCA1 and BRCA2, PALB2 is a tumor suppressor gene itself. Germline, heterozygous mutations in the gene increase the risk of breast, ovarian and pancreatic cancers. However, its mechanism is not fully understood. To investigate the in vivo role of the PALB2-BRCA1 interaction, we previously generated a Palb2 knockin mouse strain that contains a mutation that disrupts BRCA1 binding. This mouse model also allows us to bypass the embryonic lethality of the Palb2 KO mice. In this study, we hypothesized that the direct communication between the two proteins is critical for proper DNA damage repair and response in vivo and for suppression of tumorigenesis. Indeed, both immunohistochemistry (IHC) and immunofluorescence (IF) demonstrated that different tissues of the Palb2 mutant mice have higher levels of endogenous DSBs (gamma H2AX foci) and slower DSB repair kinetics after ionizing radiation (IR). Yet, mutant cells were more resistant to cell death. When aged under normal conditions, mutant mice showed increased tumor incidence in multiple tissues, particularly in the liver. Upon challenging of these mutant mice with carcinogen administration or gamma irradiation, they showed accelerated tumor development as compared with wild-type (wt) mice. When crossed with Trp53 mutant mice, the compound mutant mice showed greatly accelerated development of tumors typically associated with mutations in p53, i.e. thymic lymphoma, osteosarcoma and soft tissue sarcoma, etc. Whole exome sequencing (WES) of the tumors arising from Palb2m/m;Trp53+/- mice revealed loss of the wt allele of Trp53 in the majority of tumors, with at least some tumors showing focal deletions of the wt gene, suggesting that disruption of BRCA1-PALB2/BRCA2 axis promotes regional genomic deletions that may lead to loss of other tumor suppressors such as p53. These results underscore the importance of the BRCA1-PALB2/BRCA2 pathway for tumor suppression and suggest a potentially novel mechanism for BRCA/PALB2-mediated tumor suppression, which is by preventing Trp53/TP53 loss of heterozygosity (LOH), which would allow for tumor development. Finally, we also found constitutively elevated levels of reactive oxygen species (ROS) and activation of NF-kB, a redox sensitive transcription factor, in tissues and cells from the mutant mice. Given its established pro-survival function, NF-kB activation could explain why cells in the mutant mice are resistant to apoptosis upon irradiation despite having increased and prolonged DNA damage. This finding also suggests that the NF-kB pathway… Advisors/Committee Members: Bunting, Samuel (chair), Fan, Huizhou (internal member), Xia, Bing (internal member), Chan, Chang (outside member).

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Isothiocyanates (ITCs) are a class of naturally occurring compounds shown to have anti-cancer activities and promising chemopreventive prospects. Moringa oleifera (MO), a cruciferous vegetable widely cultivated in the tropics, has been used by locals to treat different diseases for centuries. Moringa Isothiocyanates (MIC) is produced from MO when the four unique sugar-modified aromatic glucosinolates it contains are hydrolyzed in a reaction catalyzed by myrosinase. In this study, we evaluated the efficacy of MIC on 9 different breast cancer cell lines and found it to be a potent anti-cancer agent. Interestingly, two of the most sensitive cells lines to MIC, BT474 and HCC1954, were both positive for HER2. HER2 is amplified in up to 30% of breast cancers and is positively correlated with poor prognosis in patients. We hypothesized that HER2 overexpression induces hypersensitivity to MIC in breast cancer cells. Our studies using MCF7 and MDA-MB-231 with and without HER2 overexpression further confirmed the correlation between HER2 overexpression and hypersensitivity to MIC. To understand why increased sensitivity to MIC was observed in HER2+ breast cancer cell lines as compared to HER2- cell lines, intracellular levels of reactive oxygen species (ROS) was examined. Higher basal intracellular ROS levels were observed in HER2+ cell lines compared to HER2- ones. We also hypothesized that the anti-cancer activity of MIC may involve an ROS regulatory pathway. Indeed, we found that MIC clearly regulates NRF2 and KEAP1 expression in multiple breast cancer cell lines. Additionally, MIC also triggers changes in intracellular ROS levels in a cell line-dependent manner, suggesting that MIC adopts different mechanisms of action in different breast cancer cell lines. Our work shows that MIC may open new frontiers in breast cancer prevention and therapy, particularly for those with HER2 amplification.

Advisors/Committee Members: Ganesan, Shridar (chair), Xia, Bing (internal member), Drake, Justin (internal member), Walworth, Nancy (outside member), School of Graduate Studies.

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Prevention is better than cure. The carcinogenesis could take as long as 20 to 30 years to develop from initiated cells to malignant tumor, therefore providing us various opportunities to prevent the appearance of tumors with the use of chemopreventive compounds in the early stage. Chemoprevention becomes an increasing important concept and has led to the intense research about the mechanisms of actions of various chemopreventive compounds. They can be generally classified into blocking agents and suppressing agents. The chemopreventive compounds usually prevent or slow progression of cancer by maintaining a low oxidative stress and inflammatory environment in cells. This is brought about by the activation of Nrf2, the key protein being investigated in our lab. In this dissertation, I will be discussing the use of compounds as suppressing agents and blocking agents, how compounds activates Nrf2 signaling, how novel Nrf2 interaction partner IQGAP1 mediates Nrf2-Keap1 signaling axis, how expression level of Nrf2 could be regulated epigenetically, apart from the well-known post-translational control by Keap1-Ubiquitinase-Protesome axis and finally how loss of Nrf2 could enhance intestinal tumorigenesis in Apc(min/+) mice.

Advisors/Committee Members: Cheung, Ka Lung (author), Kong, Ah-Ng Tony (chair), Chen, Suzie (internal member), Suh, Nanjoo (internal member), Xia, Bing (outside member).