Medical & Molecular Genetics Department Theses & Dissertations

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Simultaneous Targeting of CDK4/6 and BETs is Independent of RB Status in Osteosarcoma
(2026-03) Malko, Rada; Pollok, Karen E.; Pandya, Pankita H.; Herbert, Brittney-Shea; Vance, Gail H.; Greenfield, Ed
Osteosarcoma (OS), the third most common malignancy in adolescents, is characterized by heterogeneity and genomic instability. Despite aggressive multimodal therapy, survival rates remain around 60% for localized and between 5-30% for metastatic disease. OS has a high propensity for early hematogenous and recurrence, indicating a critical need for novel treatment options. Genomic analysis from the Pediatric Cancer Precision Genomics Program at our institution and others have identified dysregulation in replication stress (RS) pathways (MYC, RAD21) and cell cycle regulators (CDKN2A, CDK4/6), pointing to CDK4/6 inhibitors (CDK4/6i), like palbociclib and abemaciclib, as promising therapeutic strategies. However, monotherapy often fails, highlighting the need for effective combination approaches. While functional RB has traditionally been viewed as necessary for CDK4/6i sensitivity, emerging evidence challenges this paradigm and suggests that RB-deficient (RB-) tumors may respond through alternative, RB-independent mechanisms. Bromodomain and extraterminal (BET) proteins are targeted by BET inhibitors (BETi), like AZD5153, are epigenetic readers that regulate the transcription of oncogenes like MYC, E2F and mTOR, and increase transcription-replication errors promoting genomic instability, complementing CDK4/6 blockade. Based on functional in vitro drug screens in the Pollok lab, my dissertation investigates whether combining CDK4/6i with a BETi enhances therapeutic efficacy regardless of RB status. My research demonstrates that dual BETi and CDK4/6i therapy produces additive-to-synergistic anti-tumor effects in both RB-proficient (RB+) and RB- OS cell lines, including CRISPR-engineered RB-knockout clones. Functional studies show enhanced apoptosis, DNA damage, altered cell cycle progression, and reduced clonogenicity. In vivo, combination therapy significantly suppressed tumor growth in both treatment-naïve and previously treated OS patient-derived xenograft models. In a lung-metastatic OS model, CDK4/6i alone markedly reduced metastatic burden; however, at the tested BETi exposure, the addition of BETi did not further decrease metastatic foci. These results underscore the therapeutic potential of CDK4/6 blockade and establish a foundation for continued mechanistic and translational investigation. Building upon these functional studies, ongoing evaluation of in vitro and in vivo mechanistic profiling, using transcriptomic, kinome, and histologic analysis aim to refine combination strategies, optimize BETi dosing and sequencing, and define the contexts where dual CDK4/6i and BETi therapy most effectively limit OS progression and dissemination.
Depletion of Regulatory T Cells Transforms the Immune Landscape and Restores CD8+ Effector T Cell Immunity in Malignant Peripheral Nerve Sheath Tumor
(2026-03) White, Emily E.; Rhodes, Steven; Clapp, D. Wade; Snell, Laura M.; Herbert, Brittney-Shea
Neurofibromatosis type 1 (NF1) is a common cancer predisposition syndrome characterized by the development of plexiform neurofibromas (PNF) in up to 50% of affected individuals. While most PNFs grow slowly and reach stability in adulthood, a subset of NF1 patients experience progression to atypical neurofibromatous neoplasms of uncertain biologic potential (ANNUBP) and ultimately transform to malignant peripheral nerve sheath tumor (MPNST). MPNSTs are the leading cause of premature death in NF1 patients with a 5-year survival rate of 20-50%. Understanding the molecular and cellular mechanisms that drive neurofibroma progression and malignant transformation is therefore critical to improving patient outcomes. While key genetic driver events governing neurofibroma progression and malignant transformation have been identified, preclinical and natural history data suggest these changes do not fully account for the clinical heterogeneity of precursor lesions or their transformation. We postulated that additional factors within the tumor microenvironment play a critical role in modulating the growth and malignant potential of neurofibroma. We previously discovered that ANNUBP exhibit increased signatures of immune surveillance and T cell infiltration, but the functional role of T cells in governing malignant transformation of neurofibroma precursors remained unknown. Going forward, we hypothesized that effector CD4+ and cytotoxic CD8+ T cells were critical to preventing malignant outgrowth. Utilizing single cell RNA sequencing (scRNAseq), multiparametric flow cytometry, and preclinical mouse models that recapitulate MPNST development, we evaluated T cell dynamics during PNST evolution. scRNAseq analysis of human NF1 tumors revealed a shift from memory and cytotoxic T cell states in PNF to T regulatory (Treg), and naïve-like phenotypes in MPNST. T cell receptor (TCR) sequencing demonstrated that malignant transformation is accompanied by a systemic contraction of TCR clonal diversity and the emergence of hyperexpanded, tumor-specific clones. Functionally, Tregs were identified as key orchestrators of immunosuppression in the MPNST microenvironment, and their depletion restored CD8+ T-cell cytotoxicity and prolonged MPNST-free survival in vivo. Collectively, these findings implicate TCR repertoire diversity as a potential biomarker of malignant transformation and establish Treg-mediated immunosuppression as a critical mechanism promoting NF1- associated tumor progression – highlighting Tregs as a putative therapeutic target in the treatment of NF1-associated MPNST.
FGF23-Driven Kidney Adaptations: Unveiling Chromatin Remodeling and Transcriptional Dynamics with Implications for Disease
(2025-12) Solis, Emmanuel; White, Kenneth E.; Clinkenbeard, Erica; Hato, Takashi; Janga, Sarath
The hormone 1,25(OH)2-vitamin D3-(‘1,25D’) is required for maintaining musculoskeletal structure and function. The synthesis of 1,25D from liver-produced 25(OH)D3 is directed in final metabolizing steps in the kidney proximal tubule (PT) by the catabolic enzyme vitamin D 24-hydroxylase (CYP24A1) and anabolic vitamin D 1α- hydroxylase (CYP27B1). FGF23 and 1,25D lower 1,25D by increasing CYP24A1 and suppressing CYP27B1, in contrast to the actions of PTH. Using scRNAseq after FGF23 injections into mice, we found that the E-twenty-six family member ETV1 was stimulated in PT S1-S2 cells in parallel with increased Cyp24a1, and a 10-fold induction of ETS occupancy across the genome. In vivo, FGF23 induced localization of ETV1 to megalin+ PT nuclei. ETV1 was rapidly recruited to genomic enhancers within the Cyp24a1 promoter and a kidney-specific distal enhancer (DS1) responsible for both FGF23 and PTH activities that overlapped with VDR binding. PTH suppressed nuclear ETV1 protein in vivo, and opposed FGF23 increases by completely removing ETV1 from the Cyp24a1 enhancers. In vitro, ETV1 drove CYP24A1 mRNA in HEK-mKL cells, and ETV1 protein was upregulated by FGF23, but not by 1,25D. However, 1,25D strongly enhanced coprecipitated ETV1-VDR complexes. Conditional targeting of kidney epithelial Etv1 in mice resulted in ‘FGF23 resistance’ with elevated FGF23 and almost complete blockade of Cyp24a1 mRNA responses, which altered systemic 1,25D metabolism. FGF23 was shown to inhibit COP1 nuclear localization, enhancing ETV1 protein stability. Further, a ‘PTH mimetic’ SIK inhibitor abolished ETV1 production by enhancing COP1-mediated degradation, whereas COP1 knockdown and proteasome inhibition, rescuing SIKimediated ETV1 suppression. In sum, our findings demonstrate that ETV1 acts as a novel ‘unifying’ TF positively regulating FGF23/KL bioactivity with VDR on Cyp24a1 and opposing PTH actions, thus opening novel pathways needed to address severe diseases involving FGF23 and vitamin D.
Characterization of THY1 Positive Proximal Tubule Cells in the Kidney Using Integrative Multiomics Spatial Analysis
(2025-12) Asghari, Mahla; Eadon, Michael T.; Ashkar, Tarek; Herbert, Brittney-Shea; Dage, Jeffrey L.; Clinkenbeard, Erica
Chronic kidney disease (CKD) and acute kidney injury (AKI) are two interconnected forms of kidney disease that affect over 850 million individuals globally. CKD alone impacts approximately 37 million people in the U.S. We leveraged new spatial omics technologies in an integrative analysis to investigate the cell states of proximal tubule (PT) cells in the kidney, including those expressing the stem cell markers THY1 or PROM1, and their potential role in regeneration of kidney cells. We developed a multimodal pipeline to integrate Visium 10x spatial transcriptomics (ST), CODEX spatial proteomics, and single cell sequencing to uncover the spatio-temporal cell states of the kidney’s proximal tubule cells. We integrated human samples obtained through the Kidney Precision Medicine Project (KPMP) and the Human BioMolecular Atlas Program through multiple methodologies to define the neighborhoods and cell state trajectories associated with THY1-positive and PROM1-positive cell states. We implemented a computational approach to integrate spatial transcriptomic and proteomic data, enabling a more comprehensive understanding of PT cell niches to provide synergistic information not obtainable by using each technology separately. We identified the involvement of THY1 in tissue regeneration processes such as cell growth, proliferation and matrix remodeling. These studies revealed a decrease in THY1⁺ PT cells in CKD compared to healthy tissue while there is a shift toward elevated PROM1 expression associated with kidney disease. Our study revealed heterogeneity within the THY1⁺ PT population with specific expression profiles that are involved in different mechanisms related to cell behavior in regeneration state. Utilizing cutting-edge technologies and our integrative approach helped us identify different marker profiles of THY1+ PTs associated with regeneration. These findings contribute to better understanding of injured cells states and their regenerative potential in kidney injury. By leveraging this knowledge, our work could become an important catalyst to develop therapeutic targets for kidney disease.
Directed Differentiation Of Human Induced Pluripotent Stem Cells Through Neurogenin 2 as a Platform for Investigating Alzheimer's Disease Mechanisms
(2025-09) Varghese, Laurna R.; Meyer, Jason; Gomes, Cátia; Bissel, Stephanie
Studies involving human neurological diseases are often limited by the availability of patient-derived neurons. While direct differentiation of neurons from fibroblasts or other somatic cells is viable, the directed differentiation of human induced pluripotent stem cells (iPSCs) offers a renewable and scalable source of patient-specific neurons. However, current growth factor-based protocols for iPSC neuronal differentiation are cumbersome and require weeks to yield mature neurons and typically result in mixed cell populations. Thus, the manual isolation of desired neurons not only reduces their yield but also compromises specificity. Moreover, growth factor-induced neuronal differentiation tends to be highly variable, further compromising reproducibility. To overcome these shortcomings, we utilized a rapid single-step induced neuron (iN) methodology from iPSCs. Using a lentiviral delivery system, we induced constitutive tetracycline expression to overexpress exogeneous neurogenin-2 (NGN2) driven by the tetO promoter. The forced NGN2 expression aided in the direct lineage conversion of iPSCs into neuronal cells. The lentiviral construct also encoded eGFP and a puromycin resistance gene to enable both visualization and selection of successfully transduced cells. Within one-week post-transduction, surviving cells exhibited characteristic neuronal morphologies. To better model Alzheimer’s disease (AD), we generated neurons from patient-derived iPSCs with varying polygenic risk scores (PRS) based on ADNI samples. We confirmed the neuronal identity of these induced cells via immunostaining of key neuronal markers, demonstrating the protocol’s robustness and reproducibility. We confirmed that the NGN2-derived neurons were functionally active using multi-electrode array recordings, which showed differences in activity patterns across patient-derived lines. In parallel, molecular testing was used to examine AD-related features, specifically Aβ and pTau, with elevated levels observed so far in lines with high polygenic risk scores. Early findings suggest this model may aid in capturing patient-specific aspects of AD pathology and how genetic changes contribute to disease-related changes in neurons. Standardization of iPSCs-derived induced-neurons protocol will contribute to increasing the yield and specificity of isolated neurons with low cell-to-cell variability, which is necessary for determining disease pathogenesis and drug targets.
Utilization of a Human Induced Pluripotent Stem Cell Model of Microglial Fractalkine Signaling Dysfunction in Alzheimer's Disease
(2025-03) Tutrow, Kaylee D.; Meyer, Jason; Bissel, Stephanie; Kim, Jungsu; Lasagna-Reeves, Cristian
Dysfunctional microglial activity has been identified as a potential mechanism leading to accumulation of amyloid beta and pTau and subsequent neurodegeneration in Alzheimer's Disease (AD). The CX3CR1/fractalkine axis serves as a mechanism for bidirectional communication between microglia and neurons, respectively, to promote an anti-inflammatory microglial state. Previous studies have demonstrated that deficiency in CX3CR1 signaling leads microglia to develop a more pro-inflammatory phenotype, induces phagocytic deficits, and increases susceptibility of neurons to cell death. The CX3CR1-V249I polymorphism was recently identified as a potential risk allele for AD. However, the role offractalk:ine dysfunction in human cells and the mechanisms by which microglia with the CX3CR1-V249I SNP contribute to neurodegeneration remain unclear. To address this shortcoming, we utilized human induced pluripotent stem cells and CRISPR/Cas9 technology to elucidate the effects of the V249I polymorphism on human microglia-like cells (hMGLs). We demonstrate effective differentiation from paired isogenic control and CX3CR1-V249I backgrounds into hMGLs. Transcriptional profiling via RNA-seq analyses demonstrated alterations in pathways such as apoptosis, toll-like receptor signaling, and the inflammasome due to the CX3CR1-V249I SNP. Both heterozygous and homozygous microglia bearing the V249I allele demonstrated decreased phagocytosis of amyloid beta in vitro compared to controls, with this effect modulated by the presence of fractalkine in heterozygous but not homozygous V249I hMGLs. Both heterozygous and homozygous V249I microglia exhibited increased stress-induced cell death compared to controls, with homozygous hMGLs demonstrating increased cell death at earlier time points. These findings suggest that the CX3CR1-V249I polymorphism may confer a dysfunctional microglia phenotype, which may subsequently contribute to neuronal dysfunction. Further investigation of microglia in neuron co-culture models demonstrated that the CX3CR1-V249I variant conferred altered neuronal excitability. Collectively, the results of this study highlight the importance of understanding CX3CR1 function in AD pathology to identify targetable mechanisms for intervention.
FAM134B Regulates Collagen I Processing and Fibrogenesis in Hepatic Stellate Cells
(2025-01) Hanquier, Zachary C.; Maiers, Jessica; Wek, Ronald C.; Morral, Nuria; Dong, X. Charlie; Graham, Brett
Liver fibrosis is driven by the accumulation of scar tissue in response to liver injury. Activated hepatic stellate cells (HSCs) secrete fibrogenic proteins that deposit into the extracellular matrix, leading to fibrosis, cirrhosis, and liver failure. The increased production and secretion of fibrogenic proteins by HSCs results in ER stress, triggering the Unfolded Protein Response (UPR) to manage protein quality control. The UPR is important in regulating HSC activation and fibrogenesis, but the mechanisms driving this regulation are unclear. A key process regulated by the UPR is degradation of misfolded proteins through various pathways, including ER-to-Lysosome-Associated Degradation (ERLAD). ERLAD targets proteins for lysosomal degradation and can involve the recruitment of the autophagosome to engulf portions of the ER, a process termed ER-phagy. While ER-phagy is implicated in collagen degradation, its role in fibrogenesis is unknown. We show that collagen I levels are regulated by autophagy, and this correlates with changes in ER-phagy receptors. Furthermore, TGFβ-mediated activation of HSCs induces ER-phagic flux and expression of ER-phagy receptors FAM134B and CCPG1 in a process dependent on UPR transducer ATF6α. The loss of FAM134B, but not CCPG1, decreases intracellular collagen I protein levels without affecting COL1A1 mRNA levels or procollagen I protein levels in immortalized human HSCs (LX-2 cells). Moreover, FAM134B deletion blocks TGFβ-induced extracellular collagen I deposition despite increased collagen I secreted into the conditioned media. We conclude that FAM134B is pivotal for collagen I deposition during fibrogenesis, and its loss may promote the secretion of misfolded collagen I that cannot be deposited in the extracellular matrix.
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-Shea
Triple-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.
The Role of Inflammatory Signaling Pathways in TET2-Deficient Hematological Malignancies
(2024-08) Burns, Sarah Sterling; Kapur, Reuben; Davé, Utpal; Ware, Stephanie; Herbert, Brittney-Shea
Loss 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.
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.

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