Medical & Molecular Genetics Department Theses & Dissertations

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    Therapeutic Targeting of the Cell Cycle in Pediatric, Adolescent and Young Adult Osteosarcoma
    (2024-01) Barghi, Farinaz; Pollok, Karen E.; Liu, Yunlong; Pandya, Pankita H.; Sears, Catherin R.
    Treating pediatric, adolescent, and young adult (AYA) osteosarcoma (OS) is challenging due to its aggressiveness, genetic complexity, lack of standard therapies, and chemotherapy long term side effects. Genetically guided therapies could enhance outcomes. This study explores palbociclib, an inhibitor of Cyclin-Dependent Kinases 4/6 (CDK4/6), targeting the cell cycle based on genomic insights. CDK4/6 forms complexes with cyclin D, facilitating retinoblastoma1 (RB1) phosphorylation, leading to RB1 dissociation from E2F transcription factor and promoting cell cycle progression. CDK4/6 inhibitor (CDK4/6i) monotherapy shows limited efficacy due to cytostatic responses and resistance through compensatory pathways like PI3K/mTOR pathway, common in OS. Hence, dual inhibition of CDK4/6 and PI3K/mTOR could be effective for OS treatment. In this study, OS patient-derived xenografts (PDX) models RHT-96 (from a treatment-naïve patient) and TT2-77 (from a pretreated patient with metastatic disease) were used. Molecular signatures (CDKN2A deletion, CCND3 amplification, RB1 proficiency) indicating sensitivity to CDK4/6i , were verified in both PDX models. Short-term palbociclib treatment in pretreated TT2-77 PDX upregulated PI3K/mTOR pathway via upstream growth factor receptors, highlighting the necessity for combination therapy with CDK4/6i. Both PDXs were treated long-term with CDK4/6i (50 mg/kg palbociclib), PI3K/mTOR inhibitor (50 mg/kg voxtalisib) or combination treatment. In both PDXs, combination treatment was more efficacious than single-agent following prolonged treatment and well-tolerated based on body weight and histological analyses. The increased efficacy of the combination treatment in the naïve RHT-96 PDX was associated with decreased pathway activity of PI3K/mTOR, and autophagy induction. In RB1 proficient OS cells, the combination treatment led to additive-to-synergistic growth inhibition, G1 arrest, and induced senescence and autophagy, as shown by senescence biomarker (beta-galactosidase) and autophagy markers. In the human OS lung colonization 143B model, combination treatment improved survival and reduced metastatic burden compared to the vehicle group, as observed in body scoring, quantification of human tumor cells, and histological analyses. Our data provide evidence that combining palbociclib and voxtalisib is safe, efficacious, and enhances palbociclib efficiency in both naïve and pretreated PDXs, as well as humanized lung colonization models of pediatric and AYA OS. This provides the rationale for earlier therapeutic intervention in patients with CDK4/6 hyperactivation signatures.
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    Investigation into Tissue-Specific Mechanisms of Mitochondrial Dysfunction: Models of SUCLA2 Deficiency and a Screen for Potential Genetic Modifiers
    (2023-11) Lancaster, Makayla S.; Graham, Brett H.; Kim, Jungsu; Hoffman-Longtin, Krista; White, Kenneth E.
    With no currently effective treatments available, mitochondrial diseases are one of the most common forms of inherited multisystem disease. Primary disorders of the mitochondria affect an estimated 1 in 4,300 people with typical onset in early childhood. Mitochondrial disorders are classically defined by defects in the mitochondrial powerhouse, or respiratory chain (RC). Therefore, they are uniquely complex as the proteins within the RC are encoded by two separate genomes – nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). The mitochondrial genome encodes 13 protein genes within the RC, with the remaining mitochondrial proteome being nuclear encoded. Therefore, mitochondrial disorders result from pathogenic variants within either genome. While mitochondrial disorders can affect multiple tissue symptoms, organs with high energy demand, such as the brain and skeletal muscle, are most typically affected; thus, mitochondrial disease typically manifests as an encephalomyopathy. A wide range of symptoms, including developmental delay, seizures, strokes, and sensorineural hearing loss have been associated with mitochondrial dysfunction. In short, however, investigation into the pathogenic mechanisms of mitochondrial disorders has proven difficult due to the wide clinical and genetic heterogeneity associated with the disorders. Therefore, this project seeks to investigate pathways of mitochondrial dysfunction using two genetic approaches. First, reverse genetics tools are used to generate tissue-specific mouse models of succinyl-CoA synthetase deficiency, which is a known cause of mitochondrial disease in humans. In parallel, forward genetics is used to screen for variation in mitochondrial phenotypes in a genetically diverse population of mice to identify potential genetic modifiers of mitochondrial function and health. Using both forward and reverse genetics approaches, these studies will allow for the investigation into tissue-specific mitochondrial pathogenesis in novel mouse models, as well as broadly characterize tissue-specific mitochondrial function in vivo. Taken together, both genetic approaches are used to broaden understanding of tissue-specific mitochondrial function in health and disease.
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    Fgf23 Control of Hox Expression and Function and Implications for Chronic Kidney Disease
    (2023-08) Jennings, Kayleigh Nicole; White, Kenneth E.; Clinkenbeard, Erica; Wan, Jun
    FGF23 is a hormone that controls metabolic phosphate and vitamin D synthesis in mammals and is overexpressed in chronic kidney disease (CKD). Previous studies have shown that FGF23 initiates transcriptional reprogramming within kidney cells, and therefore epigenetic changes may occur when FGF23 levels are high, revealing therapeutic target genes for patients with CKD and other FGF23-related diseases. In my research, I performed RNAseq and ATACseq on HEK-mKL cells treated with recombinant FGF23 to determine potential transcriptional and genomic reprogramming downstream of FGF23 bioactivity. My results showed significantly decreased chromatin accessibility at the promoters of sixteen HOX genes with a 40-70% expression decrease of HOXB5 and HOXD12 validated by qPCR. Testing kidney mRNA isolated from CKD mouse models showed increased Hox expression, suggesting that these genes are dysregulated during CKD. HOXD12 overexpression in HEK-mKL cells showed significant increase in CYP27B1 expression and in pEMT genes SNAI1 and MMP9. HOXB5 and HOXD12 protein products were tracked using immunofluorescence. Collectively, these data demonstrate that FGF23 suppresses HOX transcription, which is dysregulated in CKD and may contribute to increased CYP27B1 and pEMT phenotype. These results may better define the transcriptional landscape during CKD.
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    A Mechanistic Approach to Identify Novel Therapeutic Drugs for Targeting FA-Disrupted Malignancies
    (2023-07) Sheth, Aditya Sukumar; Clapp, D. Wade; Vance, Gail; Angus, Steve; Herbert, Brittney-Shea
    The Fanconi anemia (FA) signaling network plays a critical role in maintaining genomic integrity during interphase and mitosis. Biallelic germline mutation of any of the 22 genes that constitute this pathway (FANCA-FANCW) results in Fanconi Anemia, a cancer predisposition syndrome characterized by congenital malformations, bone marrow failure, and pediatric acute myeloid leukemias (AMLs). Among the general population, acquired genetic disruptions of the FA pathway are found in 30% of all sporadic cancers and over 15% of sporadic pediatric AMLs underscoring the importance of this pathway in the prevention of malignant transformation. Therefore, the identification of precision therapies for FA-deficient AML is a critical need. The canonical tumor suppressive role of FA proteins in the repair of DNA damage during interphase is well established. We and others have uncovered the roles of FA proteins in mitotic regulation, suggesting additional mechanisms by which the FA pathway prevents genomic instability. Mutation of FANCA is the most common cause of FA and is one of the most frequently disrupted FA pathway genes in sporadic AML. To identify synthetic lethal targets of FANCA, we previously identified mitotic phospho-signaling pathways required for the survival of FANCA-/- patient-derived fibroblasts through a kinome-wide shRNA screen. We identified mitotic kinases CHEK1, PLK1, SLK, and TTK as potential targets, which suggests a mitosis-specific vulnerability of FA-deficient cells. These findings corroborate work by others who have identified synthetic lethal interactions between PLK1 and the FA pathway members, FANCG and BRCA1, suggesting that inactivation of the FA pathway may sensitize cancers to PLK1 inhibition. A more thorough understanding of FA pathway function in mitosis provides new insight into AML pathogenesis and suggests that genetic disruptions of the FA pathway may be predictive of sensitivity to PLK1 inhibition, providing a preclinical rationale for the development of precision therapies.
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    Nmp4 Suppresses Osteoanabolic Potency
    (2023-07) Heim, Crystal Noelle; Bidwell, Joseph; Wek, Ronald; White, Kenneth; Robling, Alexander; Plotkin, Lilian
    Treating severe osteoporosis is limited to two strategies: 1. Stimulation of the parathyroid hormone receptor with analogs for parathyroid hormone (PTH) or parathyroid hormone related peptide, and 2. Stimulation of Wnt signaling via neutralization of sclerostin, a natural inhibitor of this pathway, with a monoclonal antibody (romosozumab-aqqg, Scl-mAb). Despite mobilizing distinct molecular and cellular pathways to stimulate bone gain, all their efficacies rapidly diminish. Identifying the barrier to enhancing potency is a clinical priority. We recently reported that mice harboring the conditional loss of the transcription factor Nmp4 (Nuclear Matrix Protein 4) in mesenchymal stem/progenitor cells (MSPCs) exhibited no measurable baseline effect on the skeleton but showed a significantly enhanced increase in bone formation during PTH therapy. Remarkably, (and unexpectedly) skeletal response to PTH therapy was not improved when Nmp4 was conditionally disabled at the osteoblast or osteocyte stages. For the present study, we hypothesized that the potency of any osteoanabolic drug is pre-programmed (and can be re-programmed) in osteoprogenitors. To test this hypothesis, we treated our global Nmp4-/- mice, various conditional knockout mice, and their controls with Scl-mAb. We observed a similar pattern of improved bone response in our mouse models, which we previously observed with the PTH therapy. That is, removal of Nmp4 early in osteoblast differentiation (MSPC) was required for an exaggerated bone-formation response to Scl-mAb therapy. Disabling Nmp4 later in osteogenic differentiation did not increase the potency of Scl-mAb. These data suggest that Nmp4 is part of a common barrier to improving the efficacy of any osteoanabolic. Potential pathways and actors that comprise the re-programming of Nmp4-/- MSPCs to support the exaggerated osteoanabolic effect on the skeleton are discussed.
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    FARP1/RAC1/STAT3 Axis Circumvents CD8+ T Cell-Mediated Immunosurveillance by Restricting Antigen Presentation in Colorectal Cancer
    (2023-06) Eyvani, Haniyeh; Zhang, Xinna; Lu, Xiongbin; White, Kenneth E.; Kaplan, Mark H.; Liu, Yunlong
    Colorectal cancer (CRC), the second deadliest cancer worldwide, shows increasing incidence and mortality rate among young individuals. Besides chemotherapy and targeted therapies, new agents targeting tumor microenvironment and immune cells are emerging. Particularly, immune checkpoint inhibitors (PD-1 and CTLA-4 mAbs) have successfully entered into CRC clinical care. However, only a relatively small population of CRC patients with DNA mismatch repair (MMR) defects harboring microsatellite instability (MSI) respond to the current therapies. Low mutation burden, leading to poor antigen presentation and CD8+ T cell cytotoxicity is a major culprit for immunotherapy resistance. Thus, the aim of this study was to harness a novel therapeutic target to render CRC cells more immunogenic. By applying the Inference of Cell Types and Deconvolution algorithm, we generated a gene library whose expression is negatively associated with relative cytotoxicity of CD8+ T cells in the tumor microenvironment of CRC patients. Given the central role of antigen presentation in mediating cytotoxicity of CD8+ T cells and its frequent downregulation in tumor cells, capacity of each gene to modulate antigen presentation was analyzed. Our findings identified that depletion of FARP1 significantly enhanced antigen presentation, promoted CD8+ T cell cytotoxicity, and profoundly suppressed tumor growth in preclinical models. Importantly, FARP1 is strongly upregulated in CRC patients. We showed that it restricts antigen presentation by activating RAC1 Rho GTPase and phosphorylating STAT3 to modulate transcription of antigen presentation and processing genes. Collectively, our findings suggest that FARP1/RAC1 axis is a potential therapeutic target for CRC immunotherapy.
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    Development of Cancer-Genomics-Guided Precision Immunotherapy for Triple-Negative Breast Cancer
    (2023-05) Sun, Yifan; Lu, Xiongbin; Kaplan, Mark H.; Hopewell, Emily L.; Zhang, Chi; Yang, Kai
    Triple-negative breast cancer (TNBC), which accounts for 15-20% of all breast cancers, is highly aggressive and metastatic with the poorest overall rates. While surgery, radiation, and chemotherapy remain the main treatment options, TNBC represents an unmet medical need for better treatment strategies. Tremendous efforts have been made to develop effective therapies over the past years. However, TNBC treatment options are still very limited due to the lack of good drug targets and the low response rate of current therapies. In this study, we developed two different strategies to treat TNBC based on its cancer genomic features: 1) heterozygous loss of chromosome 17p (17p loss) and 2) high mutation load. 17p loss is one of the most frequent genomic events in breast cancer including TNBC, rendering cancer cells vulnerable to the inhibition of POLR2A via α-amanitin (POLR2A-specific inhibitor). Here, we developed a new drug T-Ama (α-amanitin-conjugated trastuzumab) targeting HER2-low TNBC with 17p loss by combining the effects of α-amanitin and trastuzumab (HER2+ breast cancer therapy). Our results showed that T-Ama exhibited superior efficacy in treating HER2-low TNBC with 17p loss in vitro and in vivo, and surprisingly induced immunogenic cell death (ICD) which further enhanced T cell infiltration and cytotoxicity levels and delivered greater efficacy in combination with immune checkpoint blockade therapy. Collectively, the therapeutic window created by 17p loss and HER2 expression will make HER2-low TNBC clinically feasible targets of T-Ama. As another genetic feature of TNBC, the higher genomic instability and mutational burden results in more neoantigens presented on MHC-I, along with the higher level of tumor-infiltrating T cells, making TNBC a perfect model for immunotherapy compared to the other breast cancer subtypes. Here, we designed a deconvolution-algorithm-derived library screening to find new therapeutic targets and identified PIK3C2α as a key player that determines MHC-I turnover and reduces the MHC-I-restricted antigen presentation on tumor cells. In preclinical models, inhibition of PIK3C2α profoundly suppressed breast tumor growth, increased tumor-infiltrating CD8+ T cells, and showed high potential enhancing the efficacy of anti-PD-1 therapy, suggesting that PIK3C2α is a potential therapeutic target for TNBC immunotherapy.
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    APOE4 Drives Impairment in Astrocyte-Neuron Coupling in Alzheimer's Disease and Works Through Mechanisms in Early Disease to Influence Pathology
    (2023-05) Brink, Danika Marie Tumbleson; Lamb, Bruce; Bissel, Stephanie; Herbert, Brittney-Shea; Landreth, Gary; Puntambekar, Shweta; Saykin, Andrew; Zhang, Chi
    Alzheimer’s disease (AD) is a neurodegenerative disorder resulting in progressive memory loss, brain atrophy, and eventual death. AD pathology is characterized by the accumulation of neurotoxic amyloid-beta (Aβ) plaques, synapse loss, neurofibrillary tangles (NFTs), and neurodegeneration. The APOE4 allele is associated with a 3-fold increased risk for AD and results in increased Aβ plaque deposition, reduced Aβ clearance, and reduced synaptic plasticity. Although APOE expression is upregulated in microglia in AD, APOE is expressed primarily by astrocytes in the CNS. It is not well understood how astrocytic APOE drives the mechanisms that result in worsened AD outcomes. Here, digital spatial profiling and bioinformatics data suggest that APOE4 causes transcriptional dysregulation in early AD and may disrupt neuronal processes via astrocytes. Whole transcriptome data from plaque and non-plaque regions in the cortices and hippocampus of 4- and 8-month-old AD model mice expressing humanized APOE4/4 or APOE3/3 (control) were analyzed. Transcriptional dysregulation was increased in APOE4/4 AD mice compared to that in APOE3/3 at 4 but not 8 months of age, suggesting that early dysregulation of APOE4-driven disease mechanisms may shape degenerative outcomes in late-stage AD. Additionally, APOE4/4 potentially functions via plaque-independent mechanisms to influence neuronal function in early AD before the onset of pathology. Single-nuclei RNA sequencing data were obtained from human post-mortem astrocytes and the bioinformatic analyses revealed a novel astrocyte subtype that highly expresses several top genes involved in functional alterations associated with APOE4, including neuronal generation, development, and differentiation, and synaptic transmission and organization. Overall, our findings indicate that APOE4 may drive degenerative outcomes through the presented astrocyte candidate pathways. These pathways represent potential targets for investigations into early intervention strategies for APOE4/4 patients.
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    Modulation of Splicing Factor Function and Alternative Splicing Outcomes
    (2022-06) Chen, Steven Xiwei; Liu, Yunlong; Lu, Xiongbin; Schneider, Bryan P.; Wek, Ronald C.
    Alternative RNA splicing is an important means of genetic control and transcriptome diversity. Alternative splicing events are frequently studied independently, and coordinated splicing controlled by common factors is often overlooked: The molecular mechanisms by which splicing regulators promote or repress specific pre-mRNA processing are still not yet well understood. It is well known that splicing factors can regulate splicing in a context-dependent manner, and the search for modulation of splicing factor activity via direct or indirect mechanisms is a worthwhile pursuit towards explaining context-dependent activity. We hypothesized that the combined analysis of hundreds of consortium RNA-seq datasets could identify trans-acting “modulators” whose expression is correlated with differential effects of a splicing factor on its target splice events in mRNAs. We first tested a genome-wide approach to identify relationships between RNA-binding proteins and their inferred modulators in kidney cancer. We then applied a more targeted approach to identify novel modulators of splicing factor SRSF1 function over dozens of its intron retention splicing targets in a neurological context using hundreds of dorsolateral prefrontal cortex samples. Our hypothesized model was further strengthened with the incorporation of genetic variants to impute gene expression in a Mendelian randomization-based approach. The modulators of intron retention splicing we identified may be associated with risk variants linked to Alzheimer’s Disease, among other neurological disorders, to explain disease-causing splicing mechanisms. Our strategy can be widely used to identify modulators of RNA-binding proteins involved in tissue-specific alternative splicing.
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    The Genetic Architecture of Alzheimer's Disease Endophenotypes
    (2022-05) Jacobson, Tanner Young; Saykin, Andrew J.; Nho, Kwangsik; Foroud, Tatiana; Zhang, Chi; Cao, Sha
    Alzheimer’s Disease (AD) is one of the most common forms of dementia and is known to have a strong genetic component, but known genetic loci do not fully account for the observed genetic heritability of late onset AD. This genetic complexity is further complicated by disease heterogeneity, with non-uniform presentation and progression of AD neuropathology. Endophenotypes lie upstream of observed AD clinical outcomes and downstream of genetic contributors, allowing for a biological understanding of genetic effects. Understanding the genetic architecture of AD endophenotypes can aid in breaking down AD genetic complexity and heterogeneity. In this study we utilized a variety of models to evaluate the genetic contributors to pathological change and heterogeneity in the top markers of AD pathology: amyloid, tau, neurodegeneration, and cerebrovascular (A/T/N/V framework). Additional composite quantitative measures of cognitive performance were used to relate to downstream AD presentation. These biomarkers allow the investigation of genetic effects contributing to the disease over the stages of disease progression from amyloid deposition to neurofibrillary tangle formation, disruption of metabolism, brain atrophy, and finally to clinical outcomes. First, we performed genome-wide association studies (GWAS) for AD endophenotypes at baseline using a cross-sectional regression model. This method identified sixteen novel or replicated loci, with six (SRSF10, MAPT, XKR3, KIAA1671, ZNF826P, and LOC100507506) associated across multiple A/T/N biomarkers. Cross-sectional data was further utilized to identify three genetic loci (BACH2, EP300, PACRG-AS1) that showed disease stage specific interaction effects. We built upon those results by performing a longitudinal association analysis with linear-mixed effects modeling. Gene enrichment analysis of these results identified 19 significant genetic regions associated with linear longitudinal change in AD endophenotypes. To further break down longitudinal heterogeneity, a latent class mixed model approach was utilized to identify subgroups of longitudinal progression within cognitive and MRI measures, with 16 genetic loci associated with membership in different classes. The genetic patterns of these subgroups show biological relevance in AD. The methods and results from this study provide insight into the complex genetic architecture of AD endophenotypes and a foundation to build upon for future studies into AD genetic architecture.