A full term pregnancy reduces breast cancer by up to 50%. In rodents, pregnancy or treatment with estrogen and progesterone to mimic pregnancy reduces mammary tumor incidence. The molecular mechanisms for parity-induced resistance to mammary tumorigenesis appear to involve a sustained increase in p53 responsiveness to cellular stresses. The following experiments tested whether pregnancy levels of ovarian hormones alter molecular pathways that prime p53 to be more responsive to DNA damage and if these pathways confer resistance to mammary tumors in a mouse model of Li-Fraumeni syndrome. Mice were treated with estrogen and progesterone (E+P) for 14 days, neonatally or at maturity. At 10 weeks of age, radiation-induced nuclear accumulation of p53 and apoptosis were increased similarly in the mammary epithelium from E+P-treated and parous mice compared to placebo. This effect was sustained for at least 7 weeks after E+P treatment and did not depend on the continued presence of ovarian hormones. Hormone-stimulation also enhanced apoptotic responses to ionizing radiation in BALB/c- Trp53+/- mice, a model of Li-Fraumeni syndrome. The appearance of spontaneous mammary tumors was delayed by parity in BALB/c-Trp53+/- mice. However, this protective mechanism was not preserved within epithelial progenitor cells because apoptotic responses to ionizing radiation and tumor incidence in epithelial transplants from E+P-treated donors was not different from nulliparous epithelial outgrowths. Therefore, E+P and parity confer a sustained increase in p53-mediated apoptosis within the mammary epithelium and suppresses mammary tumorigenesis, but this was not retained in epithelial outgrowths. Parity reduces the expression of estrogen receptor alpha (ERα). Activation of ERβ with an ERβ-specific agonist represses the expression of ERα. Parity-related alteration in the expression ratio of the two estrogen receptors in the mammary gland could regulate p53 priming to enhance its responsiveness to genomic stress in the parous individual.

Lung adenocarcinoma is responsible for significant global mortality with limited effective treatments. Although some studies suggest that these tumors arise from alveolar epithelial type 2 cells (AEC2s), there is scant information regarding the early events that might occur in human AEC2s at the inception of oncogenesis. This limitation, is partially due to a lack of human model systems that recapitulate the initiation of oncogenesis in AEC2s. Unfortunately, primary AEC2s from patients are difficult to access in vivo or stably maintain in cell cultures. Hence, we sought to develop an in vitro system to model the early stages of oncogenesis utilizing human induced AEC2s (iAEC2s) generated through the directed differentiation of induced pluripotent stem cells (iPSCs). To this end, we selected a normal human iPSC line we have previously engineered to carry fluorochrome reporters targeted to lung epithelial-specific loci, NKX2-1 GFP and SFTPC tdTomato that enable monitoring and purification of alveolar lung epithelial cells. To test the effects of adenocarcinoma oncogene induction in these cells, we targeted a third locus, AAVS1 using gene editing to engineer a doxycycline-inducible cassette encoding mutant KRAS G12D, the most commonly found oncogene in lung adenocarcinomas. Successful induction of KRAS G12D with doxycycline was demonstrated in both the targeted undifferentiated iPSCs as well as in the iAEC2s derived from these cells. We profiled the downstream effects of KRAS G12D induction in iAEC2s, comparing dox vs vehicle exposed cells by cell counting, FACS for NKX2-1 GFP/SFTPC tdTomato, RT-qPCR, deep proteomic and phosphoproteomic analyses, and scRNA-sequencing. Through this characterization, we found that induction of KRAS G12D robustly activates MAPK signaling resulting in a shift of iAEC2s away from their mature alveolar program towards a distal lung epithelial progenitor phenotype, indicated by the upregulation of lung progenitor and proliferation markers (e.g. SOX9, ETV4, LEF1, TM4SF1, MKI67, and TOP2A) while maintaining NKX2-1 expression, at the expense of mature alveolar markers (e.g. SFTPC, SFTPB, NAPSA, and LPCAT1). Successful modeling of lung adenocarcinoma with this model system has a variety of future applications, including testing unknown mechanisms for oncogenesis, discovery of novel biomarkers of disease, or development of new effective treatment methods through drug screening.

Mitotic cell division is critically reliant on consistent and faithful segregation of genetic information into newly forming daughter cells. Any perturbation in this process can have catastrophic intracellular effects and as such, many natural mechanisms occur during mitosis to prevent these anomalous events. Kinesins make up 14 families of motor proteins, using ATP-driven movement along microtubules to fulfill a range of functions, including organelle and vesicular transport. One such kinesin, plus-end directed KIF18A of the Kinesin 8 family, has been extensively investigated and found to be critically associated with restricting chromosome oscillation during chromosome alignment at the metaphase plate. Depletion of KIF18A subtly increases mitotic duration, chromosome oscillations, lagging chromosomes, and micronucleus formation in normal diploid cells and has minimal effects on cell viability. By contrast, loss of Kif18A induces severe mitotic defects that significantly impair viability in whole genome-doubled and highly aneuploid cells. As tetraploidization or whole genome doubling has been linked to large percentages of cancer types, this dependence on KIF18A has been proposed as a possible therapeutic target for cancer treatment. Two known KIF18A inhibitors exist; however, their function is either limited (BTB-1) or the efficacy remains to be publicly validated (Amgen). In this thesis, a screening protocol involving fluorescent-based live cell imaging was formulated to test several small molecule compounds that were suspected to have compatibilities with a binding site on KIF18A. Through the screening process, Compound 3 was identified and found to demonstrate a phenotype similar to KIF18A depletion—prolonged mitosis, decreased cell proliferation/viability, and induction of chromosome oscillation. Further examination of this compound as a potential therapeutic and elucidation of its specific mechanism of action appear to be warranted for future studies.

To control gene expression, transcription factors (TFs) bind to DNA and recruit regulatory cofactors (COFs) that mediate diverse effects on chromatin, such as DNA and histone modifications. To better help understand TF and COF interactions, our lab has been developing the nuclear extract protein-binding microarray (nextPBM) approach that allows high-throughput characterization of COF recruitment to DNA. An extension of the nextPBM, the human TF array (hTF array), allows COF recruitment to be profiled to binding sites of hundreds of human TFs, providing a method to screen TF-COF complexes forming in cell nuclear extracts. DNA (cytosine-5)-methyltransferase 3 alpha (DNMT3A) and (cytosine-5)-DNA-methyltransferase 3 beta (DNMT3B), two isoforms of the methyltransferase DNMT3, are COFs that are known to be implicated in B-cell lymphoma. hTF experiments for DNMT3A recruitment in B-cell lymphoma cells revealed a number of interactions with known B-cell TFs. In this work, I have attempted to validate the interactions seen on the hTF array by testing TF interactions with both the native DNMT3A/B proteins and the central TF-interacting ATRX-DNMT3-DNMT3L (ADD) domains of DNMT3A/B through the use of an immunoprecipitation (IP) assay in RC B-cell lymphoma cells. I successfully cloned and purified the ADD domains of DNMT3A and B as GST epitope-tagged constructs. As preliminary hTF experiments predicted interactions of DNMT3A with Interferon Regulatory Factor (IRF) proteins, I tested whether the DNMT3 ADD domains interacted with IRF3 present in B-cell lymphoma cell extracts, but was unable to identify an interaction. To examine whether native DNMT3A interacted with the IRF proteins, I performed a native IP using a DNMT3A native antibody. However, I was unable to confirm this interaction by native extract co-IP. Future work to validate DNMT3A/B-TF interactions, and TF-COF interactions identified by the hTF platform more generally, can help us understand at a molecular level what is facilitating transition between normal and cancerous states.

specialcollections@tulane.edu / The biological embedding of maternal adversity, both preconception and prenatal, can alter health trajectories of the next generation. Alterations in biological processes, including inflammation and accelerated aging, are proposed mechanisms through which adverse environmental and experiential exposures alter disease risk over the life-course. To identify biological pathways of transgenerational risk, we describe how maternal preconception adversity, assessed by adverse childhood experience score (ACEs), influences the development of her infant’s psychopathology risk and autonomic nervous system (ANS) by examining genomic and epigenomic alterations in both the infant and the placenta. Maternal ACEs elevated her infant’s externalizing behaviors at 18-months of age. Cellular aging, indexed by telomere length (TL) erosion, modified this relation; in infants with greater TL erosion from 4- to 18-months of age, higher maternal ACEs predicted higher externalizing behaviors. At 4-months of age, we examined infant ANS stress physiology and found that higher maternal ACEs predicted lower ANS stress response. Next, we examined TL in the placenta as a pathway conferring transgenerational risk. Higher maternal ACEs predicted shorter placental TL and placental TL modified the impact of maternal ACEs on her infant’s ANS; in placentas with shorter placental TL, higher maternal ACEs predicted greater ANS stress responsivity. To understand the molecular pathways impacted by maternal ACEs and related to TL, we examined placental mRNA expression of an inflammatory gene (CCL2), a gene linked to apoptosis (BCL2), and a gene tied to cellular senescence (GLB1). CCL2 expression modified the relation between higher maternal ACEs and shorter placental TL and CCL2 expression also contributed to the relation between shorter placental TL and decreased BCL2 expression. Lastly, shorter TL moderated the relation between CCL2 expression and both BCL2 and GLB1. The demonstration of the effects of maternal ACE exposure across generations highlights the need to further define the underlying mechanisms contributing to disease risk in the next generation. Maternal ACE exposure effects the placenta, the infant’s ANS, and the infant’s behavior through the first 18-months of life, which highlights the powerful impact of maternal early life adversity across generations and compliments the known lasting health risks found within the individual. / 1 / Christopher Jones

The epithelial-to-mesenchymal transition (EMT) is a critical mechanism during the process of normal embryonic development and wound healing that can be pathologically re-activated during cancer progression. We hypothesized that comparing the transcriptional programs of multiple EMT-driving transcription factors (EMT-TFs) would identify a common set of critical EMT effectors. After elucidating this common transcriptional program, the commonly upregulated RNA binding protein RBMS3 was chosen as a target for functional validation. RBMS3 was both necessary and sufficient for EMT and breast cancer progression, demonstrating the validity of focusing on common EMT-associated effectors. Finally, by evaluating the associations of multiple EMT-TFs with the tumor microenvironment in several solid tumor types, ZEB1 and a ZEB1-regulated transcription program was identified as uniquely associated with immune suppression and poor prognosis. In conclusion, this study significantly advances both the understanding of the mechanisms underlying EMT and the distinct associations of different EMT-TFs with tumor biology and the tumor microenvironment.

In nervous system development, as well as in disease and injury, neurons die through programmed cell death, leaving behind cell corpses which must be removed. The clearance of these corpses is accomplished through phagocytosis, or cell eating. Phagocytosis consists of the recognition, internalization, and degradation of external material. In the nervous system, glial cells act as phagocytes, engulfing dead neurons and debris to ensure proper morphology and tissue homeostasis. Glial phagocytosis has been implicated in several neurological diseases. In humans, increased numbers of phagocytic glia are observed in conditions like Alzheimer’s disease, Parkinson’s disease, and traumatic brain injury. In vitro, glia have been shown to clear protein aggregates like those found in neurodegenerative disease. Moreover, variants of genes implicated in glial phagocytosis have been identified as risk factors for neurodegenerative diseases. However, how phagocytosis defects might cause or worsen neurodegeneration remains unknown. To untangle the links between glial phagocytosis and neurodegeneration, we used the fruit fly Drosophila melanogaster, whose complex nervous system harbors phagocytic glia analogous to those in humans. We analyzed mutant flies lacking the phagocytic receptor Draper and found that they show an accumulation of neuronal cell corpses, which result from developmental programmed cell death and persist throughout the organism’s life. We also found that flies lacking glial Draper display age-dependent neurodegeneration. To determine how phagocytic defects lead to neurodegeneration in the draper mutant, we investigated the hypothesis that persisting cell corpses in the brain lead to chronic increased immunity, resulting in neurodegeneration. This hypothesis stems from the findings that persisting cell corpses in other tissues cause inflammation, and that neuroinflammation is thought to worsen neurodegeneration. We measured activation of the immune pathway Imd in aging draper mutants and found that the antimicrobial peptide attacin A is highly overexpressed in fat body. We then suppressed the Imd pathway by knocking down Relish in glia and fat body in draper mutants and found that neurodegeneration was reduced, indicating that immune activation promotes the neurodegeneration in draper mutants. Taken together, these findings indicate that phagocytic defects lead to or exacerbate neurodegeneration through increased immune signaling, both systemically and in the brain.

Submitted to the faculty of the University Graduate School In partial fulfillment of the requirements For the degree Master of Sciences In the School of Informatics, Indiana University August, 2005 / The aim of this thesis is to develop a biological database mining tool that incorporates mining of various publicly available heterogeneous databases and provides researchers with a reporting and visualization tool for sub-cellular localization of genes and proteins. Although there is little conservation of the primary structure, the general physiochemical properties are conserved to some extent among proteins that share sub-cellular location. Hence, the function of a protein is closely correlated with its sub-cellular location. Data in the field of genomics and proteomics are detailed, complex, and voluminous and distributed in heterogeneous databases. Most of the earlier work in information extraction from biological databases focused on database integration using wrapper techniques. However, little work has been done to mine specific data leading to the identification of pathway information and evolutionary relationship from heterogeneous biological databases. The need to develop an interactive information visualization tool leading to biological pathway detection for genes by using controlled vocabulary and various publicly available biological databases has led to the concept and implementation of GCell. This system provides a researcher to move from raw text data at a broader level to a much more detailed view of pathways representing complex biological interactions.

The human gastrointestinal (GI) epithelium performs major physiologic functions that are critical to survival, health, and homeostatic equilibrium. While model organisms and in vitro cell culture systems have been widely used to study both normal and disease states of the GI tract, these often fail to fully recapitulate critical features of in vivo intestinal tissue. In recent years, investigators have harnessed the ability to perform directed differentiation of human induced pluripotent stem cells (iPSCs) towards cell types originating from all three embryonic germ layers, most notably a wide variety of endodermal lineages, in an attempt to generate in vitro models that better recapitulate human physiology and key developmental milestones. These iPSC-derived cells contain the exact genetic background of a particular donor or patient and are easily amenable to gene-editing, making them particularly advantageous in comparison to non-human model organisms or in vitro cell culture systems often derived from malignant tissue. Here, we report the efficient generation of iPSC-derived mesenchyme-free human intestinal organoids (HIOs) that can be primed towards colonic or proximal intestinal lineages in serum-free defined conditions. By generating a novel CDX2-eGFP iPSC knock-in reporter line to track the emergence of hindgut progenitors, we follow the kinetics of CDX2 expression throughout directed differentiation, enabling the purification of intestinal progenitors. We employ these mesenchyme free HIOs to highlight cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction using cystic fibrosis (CF) patient-derived iPSC lines before and after correction of the CFTR mutation. We also demonstrate that these HIOs represent a powerful tool to model pathogen-mediated GI illness, characterizing the intestinal epithelial host response to infection by the coronavirus SARS-CoV-2 as well as two filoviruses, Ebola (EBOV) and Marburg (MARV). Finally, we report the generation of a clinically relevant library of iPSCs derived from patients with Crohn’s Disease (CD), including successful directed differentiation of these lines to a relevant immune cell type, as a proof of concept for their use in CD in vitro modeling. Taken together, our results provide a comprehensive and reductive iPSC-based model to study disease states of the intestinal epithelium, ranging from enteric viral infection to mendelian disorders such as CF and autoimmune conditions such as inflammatory bowel disease (IBD), highlighting the potential of organoids as a powerful tool for disease modeling and therapeutics development.

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