Medical & Molecular Genetics Department Theses & Dissertations
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Item Harnessing Compensatory Pathways and Acquired Resistance in Treating Triple-Negative Breast Cancer(2024-08) Solzak, Jeffrey Peter; Schneider, Bryan P.; Radovich, Milan; Nephew, Kenneth P.; Palkowitz, Alan; Herbert, Brittney-SheaTriple-negative breast cancer (TNBC) is defined by the absence of estrogen-receptor (ER), progesterone-receptor (PR), and human epidermal growth factor receptor 2 (HER2) over-expression. While TNBC comprises a minority of breast cancer cases, about 15%, it results in a disproportionally higher rate of mortality compared to hormone positive breast cancers. Compared to individuals with ER and HER2 positive disease, individuals with TNBC will have a higher incidence of visceral metastasis, a higher likelihood of relapse within the first three years after chemotherapy and surgery, and a shorter overall survival after the onset of metastatic disease. Despite the recent approvals of targeted agents, including: immune checkpoint inhibition using pembrolizumab, the TROP2 antibody drug conjugate (ADC) sacituzumab govitecan, and PARP inhibitors for germline BRCA-mutated tumors, cytotoxic chemotherapy remains the mainstay treatment for metastatic TNBC. To identify novel targets that could potentially be harnessed for therapeutic combinations, we utilized a strategic method by analyzing compensatory genomic and transcriptomic responses to targeted therapy. We demonstrate this herein, by identifying a novel combination targeting the PI3K & Wnt pathways that elicited clinical efficacy in a Phase I clinical trial. We further build on our hypothesis by also studying real-world evidence to identify novel resistance mechanisms in TNBC patients treated with the TROP2 ADC Sacituzumab govitecan. Our data suggest that the comparison of compensatory mechanisms before and after treatment can potentially inform efficacious therapeutic decision-making. In summation, with these data presented, we provide opportunities for furthering the therapeutic landscape to give patients with this dreadful disease more options in the clinical setting.Item The Role of Inflammatory Signaling Pathways in TET2-Deficient Hematological Malignancies(2024-08) Burns, Sarah Sterling; Kapur, Reuben; Davé, Utpal; Ware, Stephanie; Herbert, Brittney-SheaLoss of the TET2 gene, which is commonly mutated in the pre-leukemic condition clonal hematopoiesis of indeterminate potential (CHIP) and hematological malignancies, dysregulates inflammation, including the interleukin-1 (IL-1) and interleukin-6 (IL-6) pathways. As TET2 mutations are often present in hematopoietic stem and progenitor cells, dysregulation of these pathways may contribute to leukemogenesis and may catalyze the progression of pre-leukemic states, such as CHIP, to malignancy. Tet2-/- mice exhibit splenomegaly, myeloid expansion, and myeloid malignancy. To investigate the effects of inactivation of IL-1 receptor, type 1 (Il-1r1) and Il-6 on Tet2-deficient mature and immature hematopoietic cells, Tet2-/-;Il-1r1-/- and Tet2-/-;Il-6-/- mice were generated. Interestingly, Il-1r1 loss rescued the leukemic phenotypes associated with Tet2 inactivation, including expansion of myeloid cells, suppression of lymphoid cells, and restoration of spleen size. These phenotypes were recapitulated with competitive transplant, suggesting that IL-1R1 exerts a cell autonomous role. Mice transplanted with Tet2-/-;Il-1r-/- bone marrow cells exhibited differential regulation of specific myeloid and lymphoid subpopulations. At the stem-cell level, the frequencies of early myeloid Lin-;c- Kit+, early lymphoid Lin-;Sca1+ progenitors, and multipotent progenitor populations 2 and 3/4 were corrected, and a pronounced and reciprocal switch in the levels of Lin-;c- Kit+ and Lin-;Sca1+ cells was detected. Aged Tet2-/-;Il-1r-/- mice retained some of these phenotypes. Acute myeloid leukemia with higher IL-1R1 expression had reduced survival, indicating potential clinical implications. Similar to Tet2-/-;Il-1r1-/- mice, Tet2-/- ;Il-6-/- mice showed correction of myeloid cell expansion and lymphocyte suppression; however, they also demonstrated a significant increase in long-term hematopoietic stem cells and possible splenic extramedullary hematopoiesis, highlighting unique roles of IL- 6 in the pre-leukemic context. Collectively, these findings suggest that IL-1R1- and IL-6- dependent signaling exhibit overlapping functions but also have distinct roles in leukemogenesis that may have important implications for the clinical management of CHIP and hematological malignancies.Item Human Stem Cell Differentiated Retinal Ganglion Cells for Developing Glaucoma Neuroprotection and Cell Replacement Strategies(2024-07) Anbarasu, Kavitha; Das, Arupratan; Corson, Timothy; Meyer, Jason; Graham, Brett; Janga, SarathProgressive loss of retinal ganglion cells (RGCs) leads to glaucoma. Early diagnosis offers an opportunity to protect existing RGCs. In advanced glaucoma, most RGCs are lost causing blindness and cell replacement therapy the only option. We used a human stem cell-based RGC differentiation model to develop neuroprotection by restoring mitochondrial homeostasis and enhancing RGC differentiation efficiency to increase the success of cell replacement therapy. Unmyelinated axons in RGCs require high levels of ATP, making disrupted mitochondria a risk factor in glaucoma. Our goal was to restore mitochondrial homeostasis through mitophagy (mitochondrial autophagy) and mitobiogenesis (mitochondrial biogenesis). Mutations in the mitophagy protein Optineurin (OPTNE50K) are found in patients with normal tension glaucoma and hence, we also used RGCs with the E50K mutation. We discovered that hRGCE50Ks suffer from mitobiogenesis issues, Parkin/Pink mediated mitophagy defects, and have OPTNE50K-Tank binding kinase-1 (TBK1) aggregates. hRGCE50Ks have lower mitochondrial mass and a higher mitochondrial load. We inhibited TBK1 to induce mitochondrial biogenesis and dissolve OPTNE50K-TBK1 aggregates. Our results show TBK1 inhibition triggered mitobiogenesis, dissolved aggregates, decreased mitochondrial ATP production load, and increased spare respiratory capacity, leading to neuroprotection. With complete RGC loss, enhancing differentiation to progenitor cells with lower cell division capacity can improve the success of cell replacement therapy and reduce teratoma formation and poor tissue integration. We observed that stem cells use proteasomes for mitochondrial degradation, while hRGCs use the lysosomal mitophagy pathway. Our results indicate that proteasomal activity declines during differentiation to hRGCs. Inhibition of proteasomal activity during early differentiation resulted in higher and faster RGC differentiation, with similar effects seen in motor neuron differentiation. We did not observe metabolic reprogramming in differentiating cells upon proteasomal activity inhibition but saw changes in cell cycle distribution, specifically an increase in the number of cells in the G1 phase. Proteomics analysis post-inhibitory treatment showed elevated neuronal differentiation proteins. Our results can be translated to minimize injection cell numbers and other risks of cell replacement therapy. In summary, my research identifies novel mechanisms for restoring mitochondrial homeostasis for neuroprotection in glaucomatous RGCs and develops an enhanced differentiation strategy to aid the success of cell replacement therapy.Item ARF6 is a Novel Target for Immunotherapy in Triple Negative Breast Cancer(2024-07) Moulana, Fathima Ishara; Lu, Xiongbin; Pollok, Karen; Hopewell, Emily; Liu, JingTriple negative breast cancer (TNBC) is one of the most aggressive breast cancer subtypes with poor clinical outcomes due to lack of effective treatments owing to its hormone receptor negative status. Immune checkpoint blockade (ICB) therapy, which prevents the exhaustion of CD8+ T cells, has shown promise in treating these patients. However, only a small proportion respond, possibly due to resistance and immune evasion mechanisms by the tumor cells. A primary mechanism by which tumor cells evade immune surveillance is by reduced tumor antigen presentation, as indicated by a decreased level of antigen-MHC-I (major histocompatibility complex-I) on the surface of tumor cells. The dynamics of tumor antigens on the cell surface and how cell endocytosis contributes to antigen presentation and their recycling is little known. Here we sought to study the roles of two proteins: clathrin and ADP-Ribosylation Factor 6 (ARF6) which are essential for clathrin-mediated endocytosis and clathrin-independent endocytosis respectively, on the surface turnover of fluorophore-conjugated antigenic peptide bound to MHC-I. We employed Total Internal Reflection Fluorescence Microscopy (TIRFM) and Single Molecule Tracking (SMT) to determine the dynamics of tumor antigen endocytosis on the surface of EO771 murine TNBC cells. We found that the inhibition of ARF6 remarkably impaired the endocytosis of the antigen-MHC-I foci, leading to extended stay of the foci on the cell membrane, while inhibition of clathrin did not, suggesting that clathrin-independent endocytosis is the primary route for MHC-I-mediated antigen endocytosis. Consistent with this finding, reduced ARF6 levels promoted in vitro tumor cell killing by CD8+ T cells and suppressed tumor growth in mice when combined with ICB therapy. We further investigated the effect of pharmacological inhibition of ARF6 in murine TNBC cells and splenic CD8+ T cells using a commercially available ARF6 inhibitor NAV-2729. Treatment with NAV-2729 increased surface MHC-I levels and enhanced the secretion of T cell functional markers such as IFN-, TNF- and IL-2, suggesting the possibility of in vivo administration of ARF6 inhibitors in combination with ICB therapy. Collectively, these data suggest that ARF6 is a novel target for the combined treatment with ICB therapy to improve its efficacy in TNBC patients.Item Studying the Effect of TBX4 Loss-of-Function on Postnatal Lung Development and How it Predisposes to Pulmonary Hypertension(2024-07) Maldonado Velez, Gabriel; Aldred, Micheala A.; Machado, Roberto F.; Tepper, Robert S.; Wek, Ronald C.; White, Kenneth E.The term pulmonary hypertension (PH) describes a heterogeneous group of pulmonary and cardiovascular disorders and is estimated to affect 1% of the global population. The World Symposium on Pulmonary Hypertension divides patients into a five-tier classification system based on etiology and clinical findings with the aim of improving the clinical approach to patients. Group 1 PH, also known as pulmonary arterial hypertension, is a rare form of the disease with a prevalence of 15-50 cases per one million individuals. Deleterious variants within the bone morphogenetic protein receptor type 2 (BMPR2) gene are found in approximately 70-80% of the cases. However, at least twelve additional genes are known to have a definitive gene-disease relationship with PAH, including T-box 4 (TBX4). Genetic predisposition may also contribute to group 3 PH, also known as PH due to chronic lung disease or hypoxia. Heterozygous pathogenic variants within TBX4 have been reported in cases from both PH groups. Therefore, the diagnosis and classification of PH in patients with TBX4 mutations may be more challenging due to its contributions to the pathogenesis of both groups. For a decade, deleterious variants or large mutations involving TBX4 have been reported throughout the literature, but more progress has yet to be made toward understanding the mechanisms underlying the pathogenesis of PH in those patients. Therefore, in this project, using mouse genetics, we sought to disrupt Tbx4 expression and investigate if there is consistency with the diseases observed in humans. We found that Tbx4 mutant lungs have increasing alveolar simplification as confirmed by mean linear intercept (MLI) at P14 (25%), P36 (31.7%), and P180 (49.5%). The lungs also have reduced vascularization as indicated by a 39.4% reduction in the number of vWF-positive vessels. Consistent with PH, mutant mice have higher RVSP (19.4%), vascular remodeling, and mild right ventricular hypertrophy (RVH). RNA sequence analyses revealed enrichment of pathways (canonical WNT, VEGF, and BMP signaling) and genes (Lgr5, Tnc, Wnt3a, Areg, Gdf2, and Bmper) relevant to lung alveologenesis, angiogenesis, and PH. This study contributes significant knowledge that clinicians can use to diagnose, classify, and treat patients with TBX4 mutations.Item Deciphering the Role of Mitochondrial Dysfunction in Pulmonary Arterial Hypertension(2024-06) Balachandar, Srimmitha; Aldred, Micheala A.; Graham, Brett H.; Zhang, Jie; Geraci, Mark W.; Machado, Roberto F.Pulmonary arterial hypertension (PAH) is a life-threatening vasculopathy caused by remodeling of pulmonary arterioles. It is unknown as to why some people are at more risk of developing PAH compared to others. Notably, while germline pathogenic variants in PAH genes are a strong driver of disease susceptibility, less than half of mutation carriers actually develop the disease, suggesting the need for additional triggers. Our previous studies have shown increased DNA damage and total reactive oxygen species (ROS) in cells from PAH patients and unaffected relatives, indicating a potential genetic component, leading to our hypothesis: Mitochondrial dysfunction is an independent genetically determined modifier of PAH susceptibility. Untargeted metabolomics (Metabolon) revealed abnormalities in the antioxidants, glutamate, urea, amino acid, galactose, and phospholipid metabolism pathways in the PAH Lymphoblastoid cells (LCLs) compared to controls. Intriguingly, the healthy relatives also had altered phospholipids, suggesting that it occurs independent of the disease. ROS analysis on LCLs from patients, their relatives and unrelated controls showed that the PAH LCLs had significantly higher levels of all ROS species compared to controls, with the highest in heritable PAH cells. LCLs from relatives clustered into two groups, one with increased mitochondrial (mt) ROS and hydrogen peroxide, the other comparable to controls. Seahorse assays showed that the LCLs with increased mtROS had reduced spare respiratory capacity indicative of dysfunctional electron transport chain (ETC); but no glycolytic switch. Cybrid models generated using the high and low ROS LCLs (H and L-donors) on a 143B nuclear background showed that the H-donors had mt respiration similar to L-donors, suggesting a functional ETC. However, these cells had significantly elevated mtROS, with reduced SOD2 protein (potentially a consequence of increased degradation), passed on from the parental LCLs to the recipient cybrids. PAH is a complex disease, and mutation status alone doesn’t determine disease susceptibility. LCLs from patients recapitulate some of the metabolomic abnormalities in lung vascular cells. Oxidative stress in LCLs extends to some unaffected relatives, suggesting this is an independent genetic trait that modifies PAH risk. Our study highlights the importance of identifying potential modifiers and the second hits in the pathogenesis of PAH.Item Identifying Novel Causes of X-Linked Heterotaxy(2024-05) Wells, John Robert; Ware, Stephanie M.; Firulli, Anthony B.; Kim, Il-Man; Landis, Benjamin J.Heterotaxy is a congenital disorder characterized by abnormal arrangement of formation of thoracic and abdominal organs due to errors in embryonic left-right patterning, affecting ~1 in 10,000 live births. These patients exhibit considerable phenotypic heterogeneity, with structural heart defects significantly contributing to poor outcomes. Variants in several genes can disrupt laterality, but ZIC3 variants, primarily identified through targeted sequencing of its coding region, are the only recognized cause of X-liked heterotaxy. This dissertation focuses on a heterotaxy pedigree with four affected males, demonstrating an X-linked inheritance. No coding variant in ZIC3 was identified, leaving the pedigree unsolved for over two decades. Initially, the family’s heterotaxy was hypothesized to be caused by a coding variant in a novel heterotaxy locus on the X chromosome. X-exome sequencing identified a missense variant in GPR101, a gene whose closest phylogenetic relative has been implicated in left-right asymmetry in zebrafish. However, subsequent findings from this study and other research groups suggests GPR101 does not regulate left-right patterning, making the hypothesized GPR101 variant unlikely to be disease-causative. The next hypothesis explored was a non-coding variant in ZIC3, undetected by X-exome sequencing. Whole genome sequencing identified a novel, deep intronic variant in ZIC3, initially hypothesized to trigger the inclusion of an intronic sequence as a pseudoexon during RNA splicing. Further analysis revealed the variant profoundly altered RNA splicing, resulting in the production of several novel ZIC3 isoforms and reduced expression of normal ZIC3 protein. These novel isoforms displayed abnormal function in a variety of in vitro and in vivo assays. This marks the first reported instance of pseudoexon inclusion associated with heterotaxy for any gene and underscores the critical need to expand the scope of variant evaluation beyond mere missense and nonsense variants. The clinical and research field must adapt to assess non-coding variants and to consider alternative disease mechanisms, such as abnormal splicing or dysregulated expression of key left-right patterning genes, in unresolved heterotaxy cases.Item Triage of High-Risk Cancer Patients Through Imaging, Genetic, and Integrative Approaches(2024-03) Couetil, Justin Louis; Huang, Kun; Zhang, Jie; Zhang, Chi; Alomari, AhmedMetastasis, the spread of cancer cells from their original site to other body parts, is responsible for 90% of cancer mortality. This work applies machine learning and bioinformatic approaches on histopathological images and transcriptomic data of primary tumors to identify cancer early-stage melanoma and prostate cancer patients at high-risk for metastasis. In melanoma, we analyze digitized histopathological images of tumor biopsies to predict metastasis risk and survival. This is a common task in computational pathology, but many methods rely on “black box” approaches, such as deep learning, which are not directly interpretable. This is a barrier to adoption to pathologists, who need to understand how a tumors specific morphology is associated with prognosis. To overcome this, we develop human-interpretable features that measure the shape and arrangement of cells and nuclei, tissue texture. Our models provide prognostic power, recapitulate existing knowledge, and provide new insights into understanding metastatic events in early-stage primary tumors. For prostate cancer, we use our deep transfer learning framework, DEGAS, which combines single cell, spatial and bulk tissue transcriptomic data to identify regions of tissue in spatial transcriptomics that are highly associated with prostate cancer spread. DEGAS repeatedly identifies glands that appear histologically normal but share gene expression patterns with high-grade cancers. These results highlight the “Field Effect”, which suggest environmental and genetic factors can cause widespread genetic and epigenetic changes in tissue, a known phenomenon in pathology, but identified in high resolution transcriptomics for the first time in this work. Taken together, the work in melanoma and prostate cancer bridges the gap between traditional pathology and modern disease prognosis models. By constructing the tools to identify high risk patients and tissue, we aim to enhance metastasis research and improve clinical care for at-risk patients.Item 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.Item 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.