<p dir="ltr">The eukaryotic genome is organized and packaged into DNA-protein complexes called chromatin. Nucleosomes, the repeating unit of chromatin, are composed of DNA in complex with histone octamers containing two of each of the four core histones, the H2A, H2B, H3, and H4. Accessibility of nucleosomal DNA by other DNA-binding proteins can be regulated, in-part, by the type and level of nucleosome-associated post-translational modifications (PTMs). PTMs, such as acetylation of lysine residues, including H3K9, H3K14, and H4K16, can neutralize their positive charge, thereby reducing the affinity of acetylated histones for the negatively charged DNA sugar phosphate backbone. This, in turn, promotes the formation of relaxed open chromatin which is crucial for the accessibility of DNA by the transcription machinery. Consequently, histone acetylation is associated with active gene expression, while deacetylation, which restores positive charges of lysine residues, is linked with condensed chromatin structure, and the associated decrease in gene expression. Other PTMs such as histone methylation on H3K9, which prevents H3K9 acetylation, promotes gene silencing via serving as a binding site for HP1, a structural component of silenced heterochromatin. Interestingly, this epigenetic control of gene expression, typically used to regulate endogenous genes, also influences ectopic gene expression in host cells. Transgenes delivered into target cells undergo host cell-mediated assembly into chromatin. As a result, transgenes assembled into hypoacetylated condensed chromatin experience a dramatic loss of expression shortly after delivery into target cells. As part of this thesis, we assessed the impact of "priming" plasmid-based transgenes to adopt accessible chromatin states on transgene expression. Nucleosome positioning elements were introduced at promoters of transgenes, or vectors were pre-assembled into nucleosomes containing either unmodified histones or histone mutants mimicking constitutively acetylated states at residues H3K9 and H3K14, or H4K16 prior to their introduction into human cells. Transgene expression was then monitored by epifluorescence microscopy and flow cytometry over time. We found that DNA sequences capable of positioning nucleosomes influenced the expression of adjacent transgenes in a distance-dependent manner, even in the absence of pre-assembly into chromatin. Intriguingly, pre-assembly of plasmids into chromatin facilitated prolonged transgene expression compared to plasmids that were not pre-packaged into chromatin. Interactions between pre-assembled chromatin states and nucleosome positioning effects on reporter gene expression were also assessed. Overall, nucleosome positioning played a more significant role in influencing gene expression than priming with hyperacetylated chromatin states. These findings have direct relevance to ongoing efforts to develop durable plasmid-based gene therapies for genetically-derived disorders such as cancer.</p><p dir="ltr">In this thesis, we also explored the roles of the tumor suppressor enzyme fumarate hydratase (FH) in cellular responses to DNA replication stress. In humans, biallelic loss of function mutations in FH predisposes individuals to hereditary leiomyomatosis and renal cell carcinoma (HLRCC). However, the role of fumarase in HLRCC is not fully understood. The eukaryotic genome experiences thousands of lesions per cell each day, with DNA double-strand breaks (DSBs) being among the most deleterious. If unrepaired or repaired incorrectly, DSBs can lead to various disorders, including cell death and cancer. Cellular processes such as metabolism, transcription, and DNA replication are among the major endogenous sources of DSBs. Impediments to DNA replication that result in persistent stalling of replication forks can lead to fork collapse, thereby exposing unprotected single-stranded DNA (ssDNA) to endonuclease attack and the formation of double-strand breaks (DSBs). DNA-protein crosslinks that form irreversible complexes, an imbalance or exhaustion of deoxynucleotides (dNTPs), as well as the presence of inherently difficult-to-replicate genomic regions such as common fragile sites (CFSs), are some of the many obstacles that can halt fork progression. Intrinsically, however, all organisms have evolved response mechanisms designed to sense, prevent, detect, and resolve the different sources of DNA replication stress. These response mechanisms are governed by the highly conserved DNA replication checkpoint (DRC), a part of the intra-S phase checkpoint. The main function of the DRC is to halt cell cycle progression and activate DNA damage responses, such as DNA repair and subsequent replication fork restart. The highly conserved tricarboxylic acid (TCA) cycle enzyme fumarase (FH in humans, Fum1p in yeast), which functions to convert fumarate to malate in the mitochondrial TCA cycle, has been implicated in DRC responses in the cell’s nucleus. Upon exposure of yeast model organism to hydroxyurea (HU), an inhibitor of ribonucleotide reductase (RNR) that results in the exhaustion of cellular dNTPs, the expression of Fum1p is upregulated and Fum1p is translocated to the nucleus. Fum1p’s metabolite fumarate suppresses sensitivity to HU in yeast in a manner independent of modulating cellular dNTP levels. Notwithstanding, our understanding of these extra-mitochondrial functions of fumarase remains incomplete. For example, the nature of the impediments to DNA replication that are affected by fumarase is presently unclear. In addition, across all eukaryotes, fumarase lacks a canonical nuclear localization signal, and the means of its nuclear translocation upon DNA replication stress remain a mystery. In this study, our immunofluorescence experiments revealed that, similar to yeast, exposure of human cells to HU promoted the nuclear translocation of fumarase. We also performed Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) using human cells exposed to DNA replication stress by HU to identify replication stress-dependent protein-protein interactions of FH. We observed that FH co-precipitated MUS81, a structure-specific endonuclease and a crucial component of cellular responses to DNA replication stress. MUS81 is localized to stalled replication forks during the S phase of the cell cycle, where it cleaves the three-way junctions created by stalled forks. MUS81 also functions to resolve recombination intermediates and Holliday junctions (HJs) during the S phase. MUS81 also localizes at common fragile sites (CFSs) during the G2/M phase. CFSs are genomic regions that are difficult-to-replicate, late-replicating, and tend to exit the S phase with under-replicated DNA. These CFSs are targeted by MUS81, cleaved, and replicated via Break-Induced Replication (BIR) that is dependent on POLD3 in a process called mitotic DNA synthesis (MiDAS). In our immunofluorescence studies, we observed that HLRCC-derived cancer cells expressing low levels of catalytically inactive fumarase exhibited bulky anaphase bridges. These observations are especially intriguing because the loss of MUS81 or inhibition of POLD3 is known to increase the frequency of bulky anaphase bridges, a phenotype associated with defects in MiDAS. Additionally, we observed that exposure of fumarase-deficient cells to aphidicolin (APH), an inhibitor of POLD3, dramatically increased the frequency of anaphase bridges. Furthermore, we found that upon exposure of human cells to APH, fumarase translocated to the nucleus. Whether fumarase suppresses the MiDAS defects observed in HLRCC cells is still under investigation. Taken together, the work described herein uncovers previously unknown roles of fumarase in DNA damage responses and provides direct links toward understanding how fumarase may function to safeguard genomic integrity.</p>

<div>Cancer is responsible for the second highest cause of death in the United States, and lung cancer accounts for 13% of new cancer diagnoses, with the highest rate of cancer death at 24%. Almost 85% of these cases represent non-small cell lung cancer (NSCLC), which includes lung adenocarcinoma, the most common NSCLC subtype. Traditional cancer treatments often only temporarily stop the spread of the disease, but immunotherapies, which are becoming a standard of care, are much more promising. Natural killer (NK) cells are powerful effectors of innate immunity, and genetically engineered NK cells as immunotherapies have had encouraging clinical responses in the treatment of various cancers. However, more progress is needed for solid tumor treatment, especially for lung adenocarcinoma. The activation of cancer-associated ectoenzymes, CD39 and CD73 catalyze the phosphorylation of ATP to AMP to produce extracellular adenosine (ADO), which is a highly immunosuppressive mechanism contributing to the pathogenesis of solid tumors. Understanding adenosine effects on NK cells will help develop more robust immunotherapeutic treatments to improve cytotoxicity against solid tumors. Here, we established that tumor microenvironment ADO results in impaired metabolic and anti-tumor functions of cytokine-primed NK cells. Specifically, peripheral blood-derived NK cells stimulated with IL-2, IL-15, or a combination of IL-12 and IL-15 showed suppressed anti-tumor immunity due to ADO. This was observed by the downregulation of activation receptor expression, cytotoxicity inhibition, impairment of metabolic activity, and alterations in gene expression. To target ADO-producing CD73 on cancer cells, we redirected NK cells by fusing CD73 ScFv with intracellular and transmembrane regions of NK cell specific signaling components derived from FCyRIIIa (CD16). Engineered NK cells were shown to be cytotoxic against lung adenocarcinoma <i>in vitro</i> and impede tumor growth in a lung adenocarcinoma mouse model <i>in vivo</i>. Engineered cells also had higher levels of degranulation and cytokine release, as well as more infiltration into tumors and longer survival time in mice. In summary, the microenvironment of solid tumors is highly immunosupressive, and redirecting NK cell function using a NK-specific anti-CD73 targeting construct will help to promote anti-tumor immunity and</div><div>inhibit cancer growth for a potentially powerful new immunotherapy against solid tumors.</div>

Prostate cancer is one of the most frequently diagnosed cancers and failure to manage localized disease contributes to the majority of deaths. Radiation therapy (RT) is a common treatment for localized prostate cancer and uses ionizing radiation (IR) to damage DNA. Although RT is potentially curative, tumors often recur and progress to terminal disease. The cellular response to RT is multidimensional. For example, cells respond to a single dose of IR by activating the DNA damage response (DDR) to repair the DNA. Targeting proteins involved in the DDR is an effective clinical strategy to sensitize cancer cells to RT. However, multiple radiation treatments, as in fractionated ionizing radiation (FIR), can promote neuroendocrine differentiation (NED). FIR-induced NED is an emerging resistance mechanism to RT and tumors that undergo NED are highly aggressive and remain incurable.<br><br> Currently, the only clinical approach that improves RT for prostate cancer treatment is androgen deprivation therapy (ADT). ADT blocks androgen receptor (AR) signaling which inhibits the repair of DNA damage. In 2017, my lab reported that targeting Protein arginine methyltransferase 5 (PRMT5) blocks AR protein expression. Therefore, targeting PRMT5 may also sensitize prostate cancer cells to RT via a novel mechanism of action.<br><br> This dissertation focuses on the role of PRMT5 in the cellular response to IR and the goal of my work is to validate PRMT5 as a therapeutic target to enhance RT for prostate cancer treatment. I demonstrate that PRMT5 has several roles in the cellular response to IR. Upon a single dose of IR, PRMT5 cooperates with pICln to function as a master epigenetic activator of DDR genes and efficiently repair IR-induced DNA damage. There is an assumption in the field that the methyltransferase activity and epigenetic function of PRMT5 is dependent on the cofactor MEP50. I demonstrate that PRMT5 can function independently of MEP50 and identify pICln as a novel epigenetic cofactor of PRMT5. During FIR, PRMT5, along with both cofactors MEP50 and pICln, are essential for initiation of NED, maintenance of NED, and cell survival. Targeting PRMT5 also sensitizes prostate cancer xenograft tumors in mice to RT, significantly reduces and delays tumor recurrence, and prolongs overall survival. Incredibly, while 100% of control mice died due to tumor burden, targeting PRMT5 effectively cured ~85% of mice from their xenograft tumor. Overall, this work provides strong evidence for PRMT5 as a therapeutic target and suggests that targeting PRMT5 during RT should be assessed clinically.<br>

<p>  </p> <p>Metastatic breast cancer (MBC) is an extremely recalcitrant disease capable of overcoming targeted therapies and evading immune surveillance via the engagement of complicated signaling networks. Resistance to targeted therapies and therapeutic failure of immune checkpoint blockade (ICB) are two major challenges in treating MBC. To survive in the dynamic tumor microenvironment (TME) during metastatic progression, shared signaling nodes are required for MBC cells to regulate the signaling networks efficiently, which are potential multifunctional therapeutic targets. SH2 containing protein tyrosine phosphatase-2 (SHP2) is a druggable oncogenic phosphatase that is a key shared node in both tumor cells and immune cells. How tumor-cell autonomous SHP2 manages its signaling inputs and outputs to facilitate the growth of tumor cells, drug resistance, immunosuppression, and the limited response of ICB in MBC is not fully understood. Herein, we used inducible genetic depletion and two distinct types of pharmacological inhibitors to investigate anti-tumor effects with immune reprogramming during SHP2 targeting. </p> <p>We first focus on the signaling inputs and outputs of SHP2. We find that phosphorylation of SHP2 at Y542 predicts the survival rates of breast cancer patients and their immune profiles. Phosphorylation of SHP2 at Y542 is elevated with differential activation mechanisms under a growth-factor-induced and extracellular matrix (ECM)-rich culture environment. Phosphorylation of SHP2 at Y542 is also elevated in HER2 positive MBC cells upon acquired resistance to the HER2 kinase inhibitor, neratinib. The resistant cells can be targeted by SHP2 inhibitors. SHP2 inhibitors block ERK1/2 and AKT signaling and readily prevented MBC cell growth induced by multiple growth factors. Inhibition of SHP2 also blocks these signaling events generated from the ECM signaling. In fact, the inhibitory effects of SHP2 blockade are actually enhanced in the ECM-rich culture environment. We utilize the <em>in vitro</em> T-cell killing assays and demonstrate that pretreatment of tumor cells with FGF2 and PDGF reduces the cytotoxicity of CD8+ T cells in a SHP2-dependent manner. Both growth factors and ECM-rich culture environment transcriptionally induce PD-L1 via SHP2. SHP2 inhibition balances MAPK signaling and STAT1 signaling, which prevents growth factor-mediated suppression of INF-γ-induced expression of MHC class I. </p> <p>Next, we evaluate the efficacy of SHP2 inhibitors. Blockade of SHP2 in the adjuvant setting decreased pulmonary metastasis <em>in vivo</em> and extended the survival of systemic tumor-bearing mice. Tumor-cell autonomous depletion of SHP2 reduces pulmonary metastasis and relieves exhaustion markers on CD8+ and CD4+ cells. Meanwhile, both systemic SHP2 inhibition and tumor-cell autonomous SHP2 depletion reduce tumor-infiltrated CD4+ T cells and M2-polarized tumor associated macrophages. </p> <p>Finally, we investigate potential combination therapies with SHP2 inhibitors. The combination of SHP2 inhibitors and FGFR-targeted kinase inhibitors synergistically blocks the growth of MBC cells. Pharmacological inhibition SHP2 sensitizes MBC cells growing in the lung to α-PD-L1 antibody treatment via relieving T cell exhaustion induced by ICB. </p> <p>Overall, our findings support the conclusion that MBC cells are capable of simultaneously engaging several survival pathways and immune-suppressive mechanisms via SHP2 in response to multiple growth factors and ECM signaling. Inhibition of SHP2, potentially in combination with other targeted agents and ICB, holds promise for the therapeutic management of MBC.</p>