Ut Southwestern Medical Center

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY Complex lymphatic anomalies (CLAs) are rare diseases with variable clinical manifestations caused by the abnormal development of lymphatic vessels. CLAs include central conducting lymphatic anomaly (CCLA), generalized lymphatic anomaly (GLA), Gorham-Stout disease (GSD), and kaposiform lymphangiomatosis (KLA). CLA patients exhibit a variety of phenotypes, such as tortuous dilated lymphatic vessels, multifocal lymphatic malformations (LMs), and ectopic lymphatic vessels in bone. Unfortunately, existing treatments for CLAs have significant side effects and are not effective in all patients. Therefore, there is an urgent need to identify new, safer treatments for CLAs. We and others recently identified somatic activating mutations in KRAS in CLA patients. To investigate the effect of hyperactive KRAS signaling on the development of lymphatic vessels, we generated a novel mouse model that expresses an active form of KRAS (KRASG12D) in lymphatic endothelial cells (LECs). We found that KRASG12D induces the formation of LMs in newborn mice but not in adult mice. These results suggest that growing lymphatic vessels in newborn mice are more sensitive to the pathologic effects of KRASG12D than stable lymphatic vessels in adult mice. Based on our preliminary data, we hypothesize that the formation of LMs requires two signals - one triggered by a genetic mutation in LECs and another supplied by a lymphangiogenic factor in the microenvironment. VEGF-C is a growth factor that stimulates lymphangiogenesis during development and in pathological settings. In aim one, we will test whether VEGF-C in the microenvironment renders lymphatic vessels sensitive to the pathogenic effects of KRASG12D. In aim two, we will determine whether directly targeting KRASG12D reverses LMs. Completing these aims will establish that CLAs are developmental disorders requiring a genetic mutation and a permissive lymphangiogenic environment for their development. Thus, like tumors, oncogene-induced LMs require “two hits” for their formation. Our findings will also significantly impact a broad spectrum of vascular and lymphatic malformations by identifying new therapeutic targets and near-clinic drugs that could be repurposed for these disabling, disfiguring, and life- threatening diseases. Importantly, KRAS mutation-specific inhibitors are predicted to have fewer side effects than traditional targeted therapies, a feature that could improve patient compliance with treatment and overall outcomes. We will also provide preclinical evidence for a new imaging modality that could rapidly move to the clinic to image lymphatic function in CLA patients non-invasively.

NIH Research Projects · FY 2026 · 2024-12

Project Summary Staphylococcus aureus soft tissue and skin infections affect seven-to-ten percent of hospitalized patients and skin infections are the third most common reason for presentation to the emergency room. Most infections of the skin are caused by S. aureus, with men having higher rates of infection with S. aureus compared to women. Adding to the burden of disease posed by these pathogens has been the development of antibiotic-resistant strains of bacteria such as methicillin-resistant S. aureus (MRSA). Despite the clear pathogenic potential of S. aureus species, it asymptomatically colonizes the nasal passages of one-third of people. This duplicity is due to coordinated regulatory networks that alter virulence factor expression depending on the sensed environmental conditions. The most well-described of these is the accessory gene regulator (agr) quorum-sensing system. In this proposal we outline our recent findings that men secrete greater amounts of testosterone at the skin surface compared to women, and our preliminary data indicating that testosterone amplifies S. aureus virulence. Our hypothesis is that skin secreted testosterone promotes S. aureus virulence through activation of quorum-sensing mechanisms. To address this question, in Aim 1 we will investigate the mechanism of testosterone stimulation of S. aureus agr signaling. We will use genetic and biochemical approaches to determine how testosterone activates the agr regulon, and we will investigate the AgrC histidine kinase as the primary testosterone target. In Aim 2 we will determine the impact of testosterone on S. aureus skin infection in vivo using mouse models. We will test this phenotype using skin infection models paired with bioluminescent strains of S. aureus with and without mutations in agr components. We will also investigate genetically engineered mice with reduced androgen production in the skin. In Aim 3 we will identify the S. aureus enzymes that metabolize skin androgens over time and their impact on quorum sensing. Our preliminary findings indicate that this pathogen breaks down testosterone into androstenedione. Using bioinformatic approaches, we have identified potential androgen- degrading enzymes encoded in the S. aureus genome and we will characterize these targets using bacterial genetic and biochemical approaches. The work proposed in this application will determine how testosterone regulates S. aureus pathogenesis at the skin surface and identify novel therapeutic targets for the treatment of S. aureus.

NIH Research Projects · FY 2026 · 2024-12

Tumors frequently re-activate genes whose expression is otherwise restricted to gametogenic tissues including the ovary, placenta and testes. Tumorigenic expression of these genes, known collectively as cancer-testes antigens (CTAs), has been documented for over 25 years, however functional knowledge of the contribution of these gene products to tumorigenesis remains scant. In our preliminary research, we have discovered that the CTA Testis-Specific Serine Kinase 6 (TSSK6) is expressed in more than 50% of colorectal cancer (CRC) cases. Importantly, elevated TSSK6 expression correlates with reduced relapse-free survival in CRC. Although TSSK6 is crucial for male fertility, knockout mice otherwise develop normally. These findings suggest that targeting TSSK6 could offer a wide therapeutic window if it is essential for tumorigenic behaviors. Indeed, initial studies have revealed that suppressing TSSK6 expression inhibits anchorage-independent growth, invasion, and in vivo growth of CRC. Conversely, introducing TSSK6 into non- transformed colonic epithelial cells or CRC cell lines lacking TSSK6 promotes these tumorigenic phenotypes. Notably, the oncogenic behavior of TSSK6 depends on its kinase activity. Additionally, TSSK6-expressing cells exhibit increased resistance to a range of therapeutics. Our initial data suggests that these phenotypes are mediated by signaling of TSSK6 at focal adhesions that enhances adherence and survival signaling. This proposal is centered on a a comprehensive investigation into TSSK6's influence on focal adhesion formation and signaling (Aim 1) and its influence of tumor metastases, viability and chemoresistance in mouse and organoid models. By achieving these aims, we will reveal a previously unknown signaling pathway, driven by the aberrant expression of a testes kinase, that facilitates the acquisition of invasive phenotypes in cancer. This work is expected to provide functional biomarkers and credible model systems essential for pursuing TSSK6 as a therapeutic target.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY/ABSTRACT Endometrial cancer represents 7% of all cancer and is the 4th most common cancer in women, with 68,000 new cases and 13,000 cancer-related deaths anticipated in 2024 in the USA. Annual deaths have been gradually increasing and are now anticipated to be higher than ovarian cancer. Although surgery can cure early-stage cancers, the 5-year survival of women with advanced endometrial cancer is <20%. These epidemiologic features should make an improved molecular understanding of endometrial cancer and new studies to achieve this goal a high priority. PAX2 is a paired box transcription factor expressed in the developing kidney and reproductive tract. PAX2 is required for the formation of the Müllerian duct, the embryonic structure which gives rise to the uterus and endometrium. High PAX2 expression is retained in normal endometrial glands throughout adult life. However, as we and others have shown, complete PAX2 loss occurs in >80% of all endometrial cancers, making it by far the most common known molecular feature of early and late endometrial cancers. More interestingly, PAX2 loss appears to be the initiating event as evidenced by 1) loss throughout most endometrial cancers 2) its equal prevalence in early endometrial precancers vs. fully-developed invasive or metastatic cancers and 3) the presence of rare PAX2-deficient glands in some normal endometria, arguing that these minute PAX2-null clones are the earliest precursors from which endometrial cancers arise. PAX2 mutations have not been identified in genome-wide analyses of endometrial cancer, nor have we found any in our preliminary studies. Thus, the mechanisms underlying PAX2 loss—and more importantly its functional consequences in instigating endometrial cancer—are critical questions demanding systematic investigation. We now propose to expand upon our prior clinical and laboratory observations through wide-ranging state-of-the-art methods to understand the basic mechanisms of PAX2 loss and its functional consequences in endometrial carcinogenesis. Our hypotheses are that 1) PAX2 undergoes repression by a specific epigenetic mechanism (gene silencing) and 2) PAX2 is a functional tumor suppressor through the regulation of the transcriptional landscape within the endometrial epithelial cells from which endometrial cancers arise. We will explore these hypotheses through diverse experiments aided by patient tumor specimens, cell line and animal models in a set of three integrated aims, undertaken by a fully committed team of investigators with complementary areas of expertise.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY Gliomas represent 80% of the 26,000 newly diagnosed cases of malignant brain and central nervous system tumors in the United States each year and are among the most lethal and treatment-resistant human cancers. There are no curative treatments for glioma, and high-grade gliomas carry a particularly dismal prognosis, with a median overall survival of just 16 months. Therefore, there is a dire need to generate new insights into the pathological mechanisms driving glioma formation and progression and translate this information into new treatment strategies. Despite advances in our understanding of the transcriptional and genomic aberrations associated with this disease, our knowledge of the metabolic alterations that define distinct subsets of glioma is relatively limited. To address this issue, we performed mass spectrometry-based quantification of 564 unique metabolites in a collection of 91 adult surgical brain tissue specimens, representing high-grade gliomas, lower- grade gliomas, brain metastases, and non-malignant brain tissue. We found evidence of widespread reprogramming of an anabolic metabolism pathway in high-grade gliomas relative to lower-grade gliomas and non-malignant brain specimens. Intriguingly, metabolic reprogramming in high-grade gliomas displayed clear parallels with a well characterized inborn error of metabolism. In patients with congenital mutations affecting this pathway, aberrant metabolite secretion from neural cells causes neuronal hyperexcitability and leads to seizures that characterize this disease. Local neuronal hyperexcitability and seizures are also features of high- grade gliomas. In these tumors, neurons communicate with tumor cells through paracrine signaling and electrochemical stimulation mechanisms that promote glioma growth. In turn, glioma cells signal to neurons to foster their excitability, albeit through mechanisms that are not fully understood. Based on these findings, we hypothesize that aberrant metabolite secretion represents a key mechanism underlying glioma-neuron crosstalk in high-grade gliomas and that this phenotype is crucial for brain tumor progression. We will test this hypothesis through three specific aims. First, we will dissect the molecular basis for metabolic reprogramming and aberrant metabolite secretion in high-grade gliomas, testing the idea that imbalanced enzyme expression is necessary and sufficient to trigger these events. Second, we will determine the impact of aberrant metabolite secretion by glioma cells on excitability of the tumor microenvironment by measuring neuronal activity and glioma cell depolarization. Third, we will define the role of metabolic reprogramming and aberrant metabolite secretion in glioma pathogenesis by deactivating these mechanisms and evaluating the effect on intracranial glioma xenograft growth. Together, these studies may reveal a new intercellular communication pathway linking glioma cells and neurons, representing a vulnerability that could be exploited for brain tumor therapy.

NIH Research Projects · FY 2026 · 2024-12

Project Summary/Abstract Overwhelming data from clinical experience clearly indicate that the recurrent therapy- and castration-resistant prostate cancer (t-CRPC) is responsible for the majority of these deaths because these cells exhibit resistant phenotype to hormonal therapy as well as conventional chemotherapy. Thus, new therapeutic target(s) based on molecular mechanisms leading to resistant phenotype is urgently needed for prolong the survival of prostate cancer (PCa) patients. More than 25% of the metastatic CRPC patients on highly potent new-generation anti-androgen therapies end up with neuroendocrine prostate cancer (NEPC). Currently, NEPC is considered as an end stage disease because only few chemotherapeutics for NEPC with unsatisfied outcome. From several tissue culture and transgenic animal models, data suggest that hormonal therapy promotes androgen-dependent prostate adenocarcinoma (ADPC) cells to undergo transdifferentiation then become therapy resistant NEPC. Nevertheless, the underlying molecular mechanism remain largely uncharacterized. In this study, we provide strong evidence that the protein tyrosine phosphatase non-receptor 1 (PTPN1) plays a critical role in PCa transdifferentiation. For example, Increased PTPN1 expression in ADPC promotes the onset of NEPC. In contrast, knocking down PTPN1 expression in NEPC cells can restore ADPC phenotype. Thus, this project is to examine the regulation of PTPN1 gene expression by anti- androgens and unveil the reciprocal regulatory network among PTPN1-androgen receptor- RE1- silencing transcriptional factor then identify the downstream effector(s) of PTPN1 in promoting NEPC. Most importantly, it is to develop a new NEPC-specific theranostic strategy by engineering NEPC-specific small drug conjugate with molecular imaging capabilities. The ultimate goal is to explore clinical translation by formulating a new therapeutic regimen with PTPN1 theranostics and anti-androgen agent in clinically relevant animal models. The outcome gained from this project will revolutionize the current concept of recurrent t-CRPC therapy in order to improve overall survival of patients.

NIH Research Projects · FY 2026 · 2024-12

Human papillomavirus (HPV) is the causative agent for cervical cancer and is etiologically linked to penile cancer, anal cancer, and oral cancers induced through sexual contact with HPV-infected individuals. Although immunization that prevents up to nine types of HPV infection (Gardasil 9 for types 6, 11, 16, 18, 31, 33, 45, 52 and 58) are available for adolescent girls/boys and adults up to age 45, lack of effective therapeutics has made it difficult to eradicate this highly infectious human pathogen. As such, there is an urgent need to develop therapeutic agents able to block HPV propagation during early-stage viral episomal replication and late-stage HPV integration-perturbed host transcription programs. In our effort to identify cellular targets potentiating HPV-associated cervical and head-and-neck cancers, we found a chromatin-binding factor, bromodomain-containing protein 4 (BRD4), plays a crucial role in HPV DNA replication, genome segregation, viral gene regulation, and induction of cellular apoptosis and senescence through physical and functional association with HPV-encoded early protein 2 (E2). Besides being an emerging cancer therapeutic target, BRD4 is also a promising target for antiviral development. To understand the molecular basis of BRD4-dependent HPV replication, we knocked down BRD4 in cultured human C-33A cervical cancer cells and found that replication of high-risk HPV16 was completely abolished with no detectable origin of replication activity. Intriguingly, loss of HPV16 origin replication could be efficiently rescued by introducing wild-type, but not a phosphorylation-defective mutant, BRD4 back into the BRD4-knockdown cells. This finding suggests pharmacological inhibitors and stimulators regulating the extent of BRD4 phosphorylation and small compounds that target this phospho region of BRD4 are promising agents capable of blocking HPV replication and thus inhibiting HPV propagation in infected host cells. In this proposal, we will test the hypothesis that compounds targeting the phospho sites in a low-complexity intrinsically disordered region of BRD4 are potent inhibitors of HPV genome replication. The aims are: 1. Identify compounds effectively inhibiting HPV genome replication 2. Define the mechanistic action of phospho-BRD4-targeting compounds Three dozen or so compounds that we identified from a 200,000-chemical library screening, along with their newly derived analogs, will be analyzed for their ability to inhibit the three stages (establishment, maintenance, and amplification) of HPV DNA replication with promising compounds further examined for their pharmacokinetics, in vivo toxicology, and mechanistic action in inhibiting HPV life cycle. This new class of phospho-BRD4-binding compounds as target-selective protein-protein interaction inhibitors, unlike pan-bromodomain inhibitors (e.g., JQ1 and I-BET) that non-selectively inhibit BRD4 chromatin association, provides a new line of research for developing anti-HPV agents for treating populations already infected by HPVs.

NIH Research Projects · FY 2026 · 2024-12

Project Summary The bacterial flagellum is a complex nanomachine that functions as a motor to promote bacterial swimming through environments and is essential for colonization of many pathogens to promote disease. Flagellar biogenesis begins with the assembly of the flagellar type III secretion system (fT3SS), which is functionally and evolutionarily related to the injectisome T3SS (iT3SS) of many pathogens. Despite the structural similarities of the flagellar and injectisome T3SSs, unique proteins are employed to build each system for the creation of flagella or injectisomes. In addition, the model organisms Salmonella species or E. coli fail to account for the diversity of components and molecular mechanisms required for flagellar biogenesis across bacterial species. Polar flagellates, including Campylobacter jejuni¸ Vibrio cholerae, Pseudomonas aeruginosa, and Helicobacter pylori, require additional regulatory components to construct fT3SS in strict numbers and only at the cell poles. We found that C. jejuni utilizes a unique protein to impact the formation of the fT3SS for flagellar biogenesis. The major goal of this proposal, using structural, molecular, and biochemical approaches, will be to characterize molecular mechanisms of the components required to build the fT3SS in C. jejuni. To achieve this, the applicant will be advised by a mentoring team that includes a sponsor and dissertation committee with a wide range of expertise including bacteriology, genetics, structural biology, protein biochemistry, and molecular biology. The collaboration between the candidate and sponsor provides her with mentorship that is unique, interdisciplinary, and allows for the opportunity to develop skills that will be utilized throughout the entirety of her academic career, from post-doc to independent researcher. The resources, facilities, equipment, and faculty available at UT Southwestern Medical Center far exceed the needs of the proposal, ensuring a successful training environment to complete the following aims. In Aim 1, I will analyze the structure of the fT3SS and formation of large ring structures within the fT3SS in WT C. jejuni and isogenic mutants. In Aim 2, I will characterize the molecular mechanisms of flagellar proteins to promote fT3SS assembly in C. jejuni. Completion of this project will: 1) contribute to the diversity of components required to build T3SSs, and 2) expand on the molecular mechanisms of biogenesis of the fT3SS in polarly flagellated pathogens.

NIH Research Projects · FY 2026 · 2024-12

The relationship between protein homeostasis and nutrient sensing is critical for cellular function and survival. It is well established that amino acid and glucose depletion results in global inhibition of protein translation. Yet, lipids are the most energy-rich macromolecule, and it is still unclear if and how lipid depletion affects translation rates. Utilizing genetic models of lipid depletion in C. elegans via RNA interference, my preliminary studies reveal that translational regulation upon lipid depletion is seemingly different than that of amino acid or glucose deprivation. Specifically, I found that lipid depletion increases translation rates and coincides with a ~35-fold transcriptional induction of a eukaryotic initiation factor, F57B9.3 (homolog of the RNA helicase, eIF4A2). To gain mechanistic insight into the role of F57B9.3 and its potential link with lipid homeostasis, this proposal outlines two main objectives: 1) determine the transcription factor necessary for induction of F57B9.3 upon lipid depletion and 2) understand how F57B9.3 activation impacts translation upon lipid depletion. My preliminary studies identified ELT-2 as the transcriptional regulator of F57B9.3. Thus, the experiments proposed in Aim 1 will use a combination of CRISPR/Cas-9 gene editing strategies to determine the genetic elements required for F57B9.3 transcriptional induction upon lipid depletion by removing the GATA motif within the F57B9.3 promoter. Additionally, I will employ ChIP-qPCR to investigate ELT-2 binding to F57B9.3 GATA motif. In Aim 2, I will determine if F57B9.3 is required for lipid-depletion induced translation through various protein pulse labeling techniques and biochemical assays such as polysome profiling. Additionally, I will define the mRNAs bound to F57B9.3 by performing CLIP-sequencing. The successful completion of these aims will broaden our current understanding of translational regulation under lipid depletion and provide a molecular mechanism underlying lipid depletion induced changes in translation. Furthermore, understanding the mechanism by which lipid homeostasis is restored through protein translation can provide insight into a myriad of metabolic disorders and characterizing a novel role for the eIF4A2 homolog in response to lipid imbalances has strong therapeutic potential.

NIH Research Projects · FY 2026 · 2024-12

SUMMARY ̶Purine nucleotides are vital for RNA and DNA synthesis, signaling, metabolism, and energy homeostasis. The first chemotherapy drug ever developed, methotrexate, targeted purine synthesis and subsequently led to the development of numerous purine antimetabolites for cancer treatment. Still, our understanding of how tumors maintain and regulate their purine pools in vivo remains poorly understood. To synthesize purines, cells use two principal routes: the de novo and salvage pathways. The de novo pathway uses amino acids and small molecules to assemble the purine ring. This pathway is highly regulated through multiple pro- growth and oncogenic signaling pathways, including mTORC1, RAS, and MYC. The salvage pathway operates alongside the de novo pathway by recycling existing nucleobases from the diet or from nucleotide catabolism to generate purine nucleotides in an energy-efficient manner. However, to date, the contribution of specific purine synthesis pathways to nucleotide supply in tumors in vivo remains undescribed. Here, we established new methods using stable isotope infusions and metabolomics to determine the contribution of the salvage and de novo purine synthesis pathways to nucleotide supply in tumors in vivo. Through quantitative metabolic analysis, we show that de novo synthesis and salvage pathways contribute similarly to the maintenance of purine nucleotide pools in tumors. Our data also indicate remarkable coordination and flexibility between the de novo and salvage pathways to meet their purine needs. Furthermore, feeding mice nucleotides led to enhanced tumor growth while inhibiting purine salvage reduced tumor growth, indicating a critical role for the salvage pathway in tumor metabolism. This proposal aims to unveil critical insight into purine nucleotide metabolism, with the goal of identifying rational strategies for effectively targeting this pathway in cancer. In Aim 1, we will investigate the mechanisms by which major oncogenic signaling and their downstream signaling components regulate flux through the salvage pathway both in cell culture and in tumor models. In Aim 2, we will elucidate the influence of dietary nucleotides on metabolic reprogramming and tumor cell growth, and evaluate the requirement for the purine salvage pathway in mediating these effects. In Aim 3, we will delineate the mechanisms that mediate the metabolic flexibility between the de novo and salvage pathways and assess whether suppressing these mechanisms renders tumor cells more susceptible to purine-based treatments. We will also identify metabolic vulnerabilities that can be co-targeted with the salvage pathway to achieve effective anti-tumor effects. This proposal will expand our mechanistic understanding of the purine synthesis pathways in vivo. Additionally, it will pave the way for the identification of new strategies to target purine supply mechanisms in tumors to effectively eradicate cancer growth.

NIH Research Projects · FY 2025 · 2024-11

PROJECT SUMMARY Microglia are immunocompetent cells that interact dynamically with multiple cell types to shape brain development and maintain neural circuits. It has been hypothesized that dysfunction of microglia may contribute to the development and clinical course of schizophrenia (SZ). However, recent evidence has shifted our understanding from a model that involved neuroinflammatory microglial response. Instead, evidence supports a SZ model whereby microglia show dysfunction that disrupts neuroplasticity, neurocircuit development, and neurotransmission, ultimately altering glutamatergic and dopaminergic signaling along relevant frontostriatal circuits and onset of psychosis in late adolescence or adulthood. Molecular imaging agents for clinical and translational study of microglia in the brains of diseased populations or model systems, including SZ, are eagerly pursued. In this proposal we focus on the colony stimulating factor 1 receptor (CSF1R), which is an imaging target expressed on the cell surface of microglia. CSF1R is an attractive imaging target because it is chiefly expressed by microglia, and not other cell types in brain parenchyma. Toward measuring CSF1R in vivo, we developed [11C]CPPC (CPPC), a PET radiotracer that has optimized properties for neuroimaging. This project also builds on our previous studies of microglia in early stages of psychosis, and recent published evidence supporting low CSF1R in brain tissue of patients with SZ. Based on published evidence and preliminary data, we hypothesize lower CSF1R, consistent with downregulation of this microglial marker, in the striatum and in dorsolateral prefrontal cortex (DLPFC) of individuals with recent onset of SZ compared to healthy controls. Within SZ, we hypothesize that lower CSF1R in striatum will be associated with amotivation. Lower CPPC binding in DLPFC will be associated with impaired cognitive control and lower working memory performance. Study of the relationship between microglial marker signatures and clinical sequelae in SZ in the intact human brain is ideal since these cells change their phenotype outside this natural milieu. Our study may position CPPC PET as a promising tool for human and back-translational research aimed at studying, monitoring, or reprogramming the microglial response in SZ or related animal models.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY A significant number of pediatric neuromuscular and neurodegenerative conditions are caused by recessive gene mutations resulting in loss of both maternal and paternal copies of a gene. These genes may encode for proteins that play critical roles in cell differentiation, cell survival, and/or cell function(s). Current medical management strategies attempt to delay disease progression or minimize symptoms of these conditions, yet they do not address the underlying genetic deficiency. Gene replacement therapy is a treatment approach that provides back to cells a functional copy of a gene, thereby overcoming the deficiency caused by genetic mutations and permanently rescuing cell differentiation, survival, and/or function to a degree that the disease is halted or prevented entirely. This approach utilizes an AAV9 virus to deliver functional copies of a gene to cells of interest based, in part, on how the therapy is administered. Gene therapies have potential to revolutionize treatment of pediatric diseases, as was demonstrated recently by the FDA approval of a gene therapy for spinal muscular atrophy. As part of this study, we are testing a novel gene therapy for childhood-onset striatonigral degeneration, a severe progressive neurologic disease caused by recessive mutations in the VAC14 gene. Patients with this condition develop signs of neurodegeneration in the first half-decade of life that progresses rapidly and results in severe neurologic and neuromuscular deficit that is not responsive to current medical treatments. To study this further, we engineered the first viable pre-clinical mouse model of this condition (termed Vac14ducky). Vac14ducky mice develop neurologic disease associated with accumulation of vacuoles within the brain cortex, spinal cord, and dorsal root ganglia. The vacuolization observed in Vac14ducky mice is similar to vacuole formation previously demonstrated in patient-derived fibroblasts. We developed a novel gene therapy product (AAV9-hVAC14opt) expressing the human VAC14 gene transcript, and using our novel Vac14ducky mice, we performed preliminary in vivo efficacy and toxicology evaluations. Following a single intrathecal injection at 7 days of age, our results thus far demonstrate that AAV9-hVAC14opt successfully rescues overall health and motor function in adult Vac14ducky mice. Additionally when treating control mice, AAV9-hVAC14opt showed no detectable toxicities. These preliminary results suggest AAV9-hVAC14opt is a potential novel gene therapy for the pediatric VAC14-associated neurodegenerative disease. The study proposed here will test a dose-response that is required for an IND application. Aim 1 determines the minimal effective dose of AAV9-hVAC14opt needed to provide therapeutic benefit in Vac14ducky mice as well as in the more severe mouse line Vac14ingls. Aim 2 tests the post-symptomatic treatment efficacy utilizing Vac14ducky mice as well as Vac14ingls mice. The complementary Aims of this proposal will provide critical evidence demonstrating AAV9-hVAC14opt as a novel gene therapy for this severe pediatric condition. At the completion of this study, we will be prepared to proceed toward an IND application for eventual testing in patients.

NIH Research Projects · FY 2025 · 2024-09

PROJECT ABSTRACT Alport syndrome is the second most common cause of hereditary chronic kidney disease and is caused by mutations in the type IV collagen genes encoding α3, α4 and α5 chains. X-linked Alport syndrome (XLAS) accounts for most cases and is caused by mutations of the α5 chain. Therapies to slow the progression of XLAS are limited and affected males invariably progress to end stage kidney disease. The disease is known to affect glomeruli: the α3, α4 and α5 (IV) chains are deposited by podocytes into the glomerular basement membrane, and patients with Alport syndrome have structural defects in the glomerular basement membrane that causes hematuria and proteinuria. Despite this, there are major gaps in our understanding of disease pathogenesis. It is known that other cell types in the kidney produce α3, α4 and α5 chains, but to date, their function and the role they play in tubule function and disease pathogenesis has been unexplored. Based on preliminary studies, we hypothesize that α5 from non-glomerular cells is required for normal cell function and shape, and that the non-glomerular cell abnormalities contribute substantially to the clinical presentation and pathogenesis of XLAS. We therefore propose to 1) identify the glomerular and non-glomerular contributions to XLAS pathogenesis and progression, 2) establish the function(s) of α5 in tubular cells, including elucidating signaling pathways that underlie its role in tubular cells. Together, these studies will generate resources to study type IV collagen function and will greatly advance knowledge of the pathogenesis of XLAS. We expect they may also pave the way for future studies that lead to the development of novel therapeutic strategies.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Alcohol abuse is a major health concern with a recently alarming increase in prevalence. Among the various complications associated with alcohol use disorder (AUD), alcohol-associated liver disease (ALD) is the most common and devastating. Mounting evidence supports a central role for inflammation and metabolic dysfunction in ALD. The long-term goal of this work is to understand the metabolic consequences of liver inflammation on alcohol consumption and ALD progression, and leverage this insight to develop novel treatment strategies. We have identified and characterized multiple points of crosstalk between interferon regulatory factor 3 (IRF3), a master regulator of innate immunity, and hepatic metabolism. Our preliminary data show that alcohol-induced IRF3 suppresses maximal fibroblast growth factor 21 (FGF21) expression and secretion by the liver. Using a novel phospho-mimetic mutant of IRF3, we found that transcriptionally active IRF3 abolishes alcohol-induced FGF21 secretion, while IRF3 deficiency enhances endogenous FGF21 secretion. Although IRF3 itself does not act as a transcriptional repressor of Fgf21, its metabolic effects are mediated through its transcriptional target, PLAGL1. The overall goal of this proposal is to identify the mechanisms responsible for IRF3-mediated FGF21 suppression and its impact on alcohol consumption and ALD. Our central hypothesis is that alcohol-induced IRF3 triggers transcription of PLAGL1, which functions as a novel repressor of Fgf21 expression, ultimately limiting its secretion. By targeting the IRF3-PLAGL1 inhibitory axis, we can release the brakes on Fgf21 transcription, increase endogenous FGF21 secretion, and enhance its beneficial effects on alcohol consumption and liver metabolism. In this proposal we will define the impact of IRF3-mediated FGF21 suppression on alcohol preference, motor impairment and sedation, as well as alcohol-induced steatosis and insulin resistance. Lastly, we will determine the mechanism by which the IRF3-PLAGL1 inhibitory axis regulates FGF21 levels, and subsequently alcohol consumption and ALD. Collectively, these studies will greatly advance our understanding of the metabolic consequences of inflammation on ALD progression. FGF21 is currently in clinical trials for obesity-induced metabolic dysfunction, and its beneficial role in AUD and ALD is undeniable. This work presents a critical opportunity to enhance the secretion of endogenous FGF21 in response to metabolic stressors such as obesity-inducing carbohydrate-rich diets and alcohol consumption.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY Childhood growth disorders have significant consequences in adult life, including body size, work and reproductive performance, and the risk of chronic diseases. Our long-term goal is to understand the molecular mechanisms underlying childhood growth disorders and translate this knowledge into novel therapeutic strategies. Given the physiological similarities between humans and mice, studying mouse mutants has provided us with fundamental insights. However, there are critical gaps in our understanding of growth disorders in mice, including the redundancy of genes involved in normal growth and development, as well as the unknown critical genes. To address these gaps, we utilized a mouse forward genetic screen platform with automated meiotic mapping to identify mutations causing growth disorders rapidly and unbiasedly. In previous studies, we identified a null allele of Kbtbd2 called “teeny”, which exhibited severe insulin resistance, lipodystrophy, fatty liver, and growth retardation. Our research uncovered the crucial role of KBTBD2 in regulating insulin signaling in adipose tissues, contributing to most of the metabolic phenotypes observed in teeny mice. However, the cause of growth retardation in teeny mice is still unknown. In this proposal, through knock-out of Kbtbd2 and osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSCs), we discovered an intrinsic role of KBTBD2 in regulating osteogenesis by targeting p85α, the regulatory subunit of PI3K and a key downstream node of IGF-1 signaling. Interestingly, the teeny phenotype closely resembles SHORT syndrome, a genetic disorder caused by mutations in the PIK3R1 gene, encoding p85α and related isoforms. We found that the recurrent R649W mutation in p85α disrupted its interaction with KBTBD2, resulting in decreased ubiquitination and degradation of p85α by KBTBD2. Based on these results, our central hypothesis is that KBTBD2 regulates the abundance of p85α to enable IGF-1 signaling activation during osteogenesis, and the p85α R649W mutation leads to SHORT syndrome due to reduced association with the KBTBD2 protein. To test this hypothesis, we propose two specific aims. Aim 1 will elucidate the molecular mechanism of KBTBD2 in bone development. Aim 2 will investigate the role of KBTBD2 in the pathogenesis of SHORT syndrome. Completion of the proposed work will provide a comprehensive understanding of KBTBD2's role in skeletal development. As the KBTBD2 protein is highly conserved between mice and humans and the p85α mutant disrupts the normal function of KBTBD2 in SHORT syndrome, our study may yield new therapeutic strategies for treating childhood growth disorders in the future.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Our ultimate goal is to develop a practical, theranostic platform for managing PC. Prostate cancer (PC) is the second most common cancer and the fifth most frequent cause of cancer death among men. Despite a gradually improving trend in outcomes, the current death rate remains unacceptably high, with metastases in 20% of afflicted men. The American Cancer Society estimates approximately 268,490 new cases and 34,500 deaths from PC in the United States in 2022. Prostate-specific membrane antigen (PSMA) expression has been associated with aggressive PC, and is present in over 90% of metastatic disease. Those features have led to employing PSMA for PC detection with radionuclide imaging, and for therapies including antibody-drug conjugates, targeted radiotherapeutics and immunotherapeutic approaches. Nearly 450 trials involving PSMA in PC are registered on clinicaltrials.gov. Nevertheless, all of the current therapies show some degree of toxicity and are not curative. We are well-positioned to develop novel therapies through application of our PSMA- targeted theranostic agents. We have recently synthesized PSMA-targeted poly(amidoamine) (PAMAM) dendrimers that may be used for PC-specific concurrent delivery of imaging and therapy. PAMAM dendrimers serve as versatile delivery vehicles that can be tailored to different sizes and compositions. Here we propose development of multimodal theranostic agents enabling: (I) highly specific PSMA targeting; (II) application of quantitative positron emission tomography/computed tomography (PET/CT) ultimately for selecting patients for therapy; (III) incorporation of a potent antimitotic agent; and, (IV) incorporation of optimized chemical exchange saturation transfer (CEST) moieties to enable non-invasive monitoring of PC drug delivery by the clinically ubiquitous imaging modality, magnetic resonance imaging (MRI). As we have shown, CEST is sufficiently sensitive to enable targeted imaging. When high-frequency salicylate moieties are used there, is a frequency- specific contrast not encumbered by background signal, allowing determination of targeting efficiency. Direct imaging of drug biodistribution provides information needed to understand and improve efficacy. The proposed PSMA-targeted theranostic agents will be synthesized via consecutive conjugation of PAMAM dendrimer with deferoxamine (DFO, for radiolabeling with 89Zr, enabling PET/CT and evaluation of pharmacokinetics), lysine- urea-glutamate moieties (KEU, for PSMA targeting), and numerous 4-(3-carboxypropanamido)-2- hydroxybenzoic acid molecules [salicylic acid (SA) derivatives for CEST contrast]. For therapeutic studies, proposed theranostic agents will be additionally equipped with maytansine 1 (DM1) and monomethyl auristatin e (MMAE, both routinely used of formulation of antibody-drug conjugates) via either encapsulation or conjugation. We will use mice bearing clinically relevant human PC metastatic xenografts for pharmacokinetic studies. Studies will be carried out using state-of-the-art multimodal PET/CT, MRI and optical imaging.

NIH Research Projects · FY 2025 · 2024-09

Project Summary Cystic Fibrosis (CF) and nonCF bronchiectasis (NCFB) are characterized by thickened respiratory secretions, chronic lung infection and neutrophilic inflammation. A host of epidemiologic data demonstrates that females with CF suffer from increased pulmonary exacerbations, earlier colonization with bacteria such as Pseudomonas aeruginosa (PsA) and decreased life expectancy relative to males with CF. Similarly, NCFB is more common in females and associated with worse outcomes in females relative to males, but the etiology behind these sex disparities is unclear. A leading candidate to explain this disparity is an estrogen-dependent effect on inflammation and immunity given that our work and the work of others demonstrates higher sputum inflammatory markers and increased pulmonary exacerbations at times of high estrogen levels (ovulation) in females with CF. Moreover, we found that suppressing endogenous 17β-estradiol with hormonal contraception improved these markers of health. Our central hypothesis is that estrogen causes dysregulated neutrophil function and inflammation leading to a sex difference in lung disease and outcomes in people with CF and NCFB. CF respiratory disease is characterized by a sustained influx of neutrophils into the lungs in response to bacterial infection and inflammation. A major mechanism for neutrophil killing is the release of web-like neutrophil extracellular traps (NETs) that trap and kill a variety of microbes. Although NET formation is an important event in innate immunity, excess NETs can be detrimental by producing pro-inflammatory stimuli and proteases that amplify lung damage. Our preliminary studies show that: (1) neutrophils from females with CF and NCFB kill less PsA than neutrophils from males; (2) estrogen decreases neutrophil killing and upregulates NETosis and inflammatory mediator release. We aim to answer several key questions including: (1) what estrogen receptor is driving the estrogen effect on neutrophils (2) what other downstream pathways are involved (3) what is the context of these findings in cis and transgender individuals (4) Are the findings unique to CF or applicable to other chronic inflammatory airway diseases such as non-CF bronchiectasis (NCFB). The expected outcome of this research is a comprehensive understanding of how estrogen mediates functional activity of the neutrophil and the pathways involved that can reveal therapeutic targets, including the potential use of nebulized estrogen receptor antagonists to combat the disproportionate chronic inflammatory process present in cis and trans women with CF. The long-term goal of our lab is to understand the mechanisms leading to sex and gender differences in CF and to develop novel therapies to narrow the disparity. The objective of this proposal is to understand the estrogen- specific effect on neutrophil dysregulation and inflammation. The impact of this proposal is significant because it may lead to treatments that can improve the health of people with CF and NCFB and guide therapies for other chronic inflammatory airway diseases with similar sex and gender disparities.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Genetic predisposition is a significant risk factor for coronary artery disease (CAD), the leading cause of mortality. Most CAD risk variants identified in genome-wide association studies (GWAS) are in noncoding DNA, which poses a major challenge in identifying the target genes in the disease-critical cell types. Using the Activity-by-Contact Model to predict cell type-specific gene-enhancer pairs, we identified cis-regulatory elements in the chromosome 10q23 locus harboring multiple CAD-risk variants. Our preliminary studies show that these enhancers in the 10q23 locus regulate tetraspanin 14 (TSPAN14) gene expression, specifically in vascular endothelial cells (ECs) and monocytes. We have established strong population genetics evidence that higher TSPAN14 expression reduces CAD risk and there is a consistent protective effect for the minor alleles at the lead variants. TSPAN14 is an adaptor protein that aids in trafficking proteins like ADAM10, a Notch receptor activator, to the plasma membrane. The role of Notch pathway activation imparted by this TSPAN14- ADAM10 interaction in CAD pathogenesis has not been explored. In addition, we performed RNA-seq and identified differentially expressed genes in TSPAN14-deficient cells that will help discover Notch-independent TSPAN14 functions. These findings provide a premise for the central hypothesis that the genetic regulation of TSPAN14 by variations in the 10q23 enhancer sequences affects CAD pathogenesis through Notch- dependent and -independent mechanisms in vascular ECs and monocytes. In Aim 1, Dr. Lee-Kim will determine the cell type-specific effect of 10q23 enhancer sequence variations on TSPAN14 expression regulation. In Aim 2, she will determine the Notch-dependent effects of TSPAN14 expression in vascular and immune cells. In Aim 3, she will characterize the Notch-independent TSPAN14 functions in vascular and immune cells. The results from these studies will functionally validate the gene target for noncoding variants associated with CAD-risk in the disease-relevant cell types and elucidate how TSPAN14 functions in CAD pathogenesis. These studies will be conducted under the supervision of mentors, Dr. Rajat Gupta and Dr. Stephen Blacklow, and an advisory committee dedicated to Dr. Lee-Kim’s success. With additional support from the MOSAIC UE5 awardee sponsored professional development opportunities, continued training in the K99 phase will prepare Dr. Lee-Kim for successful transition to independence.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT This proposal delineates a five-year individualized training plan that will promote an independent scientific career to study the pathogenesis of bronchopulmonary dysplasia (BPD), which is the most common sequalae of prematurity and impacts the patient throughout the lifespan. Specifically, the scientific program of the candidate will focus on how biophysical force affects type 1 alveolar epithelial cell (AT1)-mediated regulation of the pulmonary matrisome within the developing lung. The applicant is an Instructor within the Division of Neonatology and Department of Pediatrics at the Children’s Hospital of Philadelphia and has previous training in cell and molecular biology with an emphasis on oxidative stress. Over the past several years, she has worked as a clinical research fellow in the laboratory of Dr. Edward Morrisey, a world-renowned expert in developmental biology and lung regeneration with a phenomenal record of mentorship success. The goals of this current proposal are to acquire expertise in transcriptomics, epigenetics, and mechanobiology that will help define her future career as an NIH-funded independent investigator. To meet these goals, her and Dr. Morrisey have developed a robust training program with coursework and workshops offered by the University of Pennsylvania and have put together a diverse advisory committee to aid her in her scientific and career development goals. Together with the outstanding environment offered by CHOP and UPenn, she is well- poised to launch into a productive and innovative research career as a physician-scientist. Recent clinical evidence suggests that the incidence of BPD is increasing as the thresholds of viability are challenged, but the multifactorial etiology of disease pathogenesis has remained elusive. In addition, there are no treatment options which can prevent BPD or can promote pulmonary regeneration in affected neonates. The aims in this proposal will address the central hypothesis that biophysical force promotes AT1 cell regulation of the alveolar proteosome, in part through upregulation of TGFβ signaling leading to expression of a constellation of matrisome members that are essential in maintaining alveolar health and function during neonatal lung growth and maturation. In Aim 1, the candidate will identify how elevated mechanical stress impacts AT1 cell-mediated regulation of the pulmonary matrisome in both in vivo mechanical ventilation and in vitro cellular stretch models and examine if its effects are attenuated with loss of TGFβ. Aim 2 will characterize the effects of mechanical ventilation on the developing lung more globally, with a single-cell approach to examine the transcriptomic and epigenetic regulatory networks as well as intercellular communication that predisposes to altered development and BPD. Successful completion of these aims will provide new insight into how AT1 cells translate applied biophysical force into changes of their own matrisome program to affect their local environment and communicate broadly with other cell compartments to impact the normal lung developmental programming.

NIH Research Projects · FY 2025 · 2024-09

Vascular contributions to cognitive impairment and dementia are increasingly recognized as independent causes of dementia, particularly due to their high prevalence and potential for intervention. Each year, nearly 795,000 Americans suffer a new or recurrent stroke, with one in three patients subsequently developing post-stroke cog- nitive impairment and dementia (PSCID). Despite its enormous public health impact, the accurate estimation of PSCID prevalence and the identification of associated risk factors face considerable challenges, including issues related to data quality and research methodologies. Furthermore, many stroke survivors have co-existing cardi- ovascular diseases. The extent to which additional vascular risk factors may interact with stroke in precipitating PSCID remains unclear. Importantly, pharmacological management of vascular risk factors plays a pivotal role in secondary stroke prevention. In theory, medications including antihypertensive, antithrombotics, and statin may prevent PSCID through the prevention of recurrent ischemic events, yet there is limited evidence for specific therapeutic strategies for PSCID prevention. Leveraging the nation’s largest stroke registry and Medicare data, we propose to build a real-world data platform to advance PSCID research. Using this unique data resource, which encompasses two million stroke records from over 2000 US hospitals, we will determine the incidence and prevalence of PSCID, identify associated risk factors, and potential pharmacological targets for prevention. The central hypotheses are that stroke survivors are at risk for cognitive impairment and dementia; the features of the index stroke event, in conjunction with demographics, pre-existing comorbidities, in-hospital treatment, and secondary stroke prevention may influence the risk of PSCID. The proposed research in innovative in four key ways: 1) A patient-centered approach to address a major concern for stroke survivors and their caregivers; 2) A shift in focus from selected samples in single center studies or small cohort to encompass nationwide diverse stroke populations; 3) Innovative utilization of instrumental variable analysis, which mimics a trial-type design within observational data; 4) Rapid dissemination through professional societies and patient-led initiatives. The proposed research is significant, because it addresses a high-priority area for aging research highlighted in the National Plan to Address Alzheimer’s Disease. As the number of stroke survivors living with dementia continues to increase, obtaining the incidence and prevalence of PSCID will provide a more accurate estimate of disease burden. This, in turn, will inform the development of appropriate policy and the allocation of healthcare resources. From a clinical practice perspective, knowledge gained from this study will enhance our understanding of post stroke medical management and care planning needed by stroke survivors. Importantly, since both stroke and vascular risk factors are preventable and treatable, the identification of contributing factors, particularly modifia- ble ones, will be critical for the development of personalized prevention strategies and targeted interventions aimed at alleviating the burden of cognitive impairment and dementia among stroke survivors in our aging society.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ ABSTRACT Congenital disorders of glycosylation are multi-system inherited diseases, which affect the amount of glycosylation added to proteins. For patients with mannose phosphate isomerase (MPI) deficiency, decreased mannose production leads to globally decreased levels of N-linked glycans resulting in several issues with congenital diarrhea and protein losing enteropathy being the most significant symptoms. Our long-term objective is to determine how defective glycosylation manifests as disease. We aim to achieve this objective using a novel mouse strain named benadryl, which has an Mpi mutation and models most of the features of human disease. We found conditional knockout of Mpi in the intestines re-created cardinal features of the benadryl strain with severe defects in the mucus producing goblet cells, and features of inflammatory bowel disease. The goal of this project is to determine how Mpi protects the intestines and how to improve therapeutic interventions for patients deficient in Mpi. The central hypothesis is that Mpi is a rate limiting step for N- glycosylation of mucins like Muc2, which are essential for proper mucin maturation and barrier function. In this proposal we will investigate (Aim 1) how N-glycosylation of Muc2 is required for goblet cell survival, intestinal mucus production, and protection from bacteria; (Aim 2) how N-glycosylation sites are essential for proper Muc2 folding and dimerization; and (Aim 3) the mechanism for how mannose therapy protects gastrointestinal barriers. Upon finishing these studies, we will have defined the role of N-glycosylation post-translation modification of Muc2 in maintaining a proper intestinal mucus barrier to prevent bacterial invasion and inflammation. Further, we will leverage N-glycosylation insights to test feasibility and mechanism of mannose as a therapy in pre-clinical models of gastrointestinal disease.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT The patterns of connectivity across the neural networks of the brain play a critical role in determining how those networks function. However, the mechanisms through which the architecture of a circuit supports information encoding or storage remain unclear. In addition, circuit-level activity patterns drive changes in the strength of synapses across that circuit, which in turn necessarily alters how the circuit behaves. While considerable work has elucidated many cellular and molecular components of synaptic plasticity, the precise reciprocal interaction between in vivo activity patterns, synaptic plasticity, and circuit level function remains unclear. The brain expresses several distinct types of internally generated sequences of neuronal activity independent of external sensory stimuli, and such temporally precise, self-organized sequences play a crucial role in information processing and memory formation/retrieval. Due to their independence from external inputs, internally generated sequences serve as a powerful model to explore the fundamental relationship between the connectome and network function. The hippocampus, a brain area critical for the formation of many types of memory, generates a well-defined type of neuronal sequence, sharp-wave/ripple (SWR)-associated replay, which is observed during “offline” states such as rest or sleep. The central objectives of this proposal are to identify the in vivo connectome architecture of the hippocampal network, quantify how these connection patterns regulate activity across the circuit during active “online” encoding and during “offline” SWR sequences, measure how in vivo activity patterns across this neural network change the weights of synaptic connections, and establish how those synaptic changes in turn impact circuit function. Supported by considerable preliminary data, these ambitious aims will be achieved through a combination of ultra-high density, large-scale in vivo electrophysiology followed by in-depth computational analysis of the resulting data, and rapid in vivo optical labeling of active neural populations followed by ex vivo physiological quantification of synaptic function. In Aim 1, we will directly test two models with differing predictions regarding the impact of local hippocampal connectivity on place field distribution and activity patterns during SWRs. In Aim 2, we will test the hypothesis that coordinated activity of synaptically connected neuron pairs during behavior (e.g., overlapping place fields) potentiates their synaptic connections, which in turn impacts the content of subsequent SWRs. In Aim 3, we will directly quantify the synaptic consequences of in vivo activity via whole cell electrophysiology performed in neurons which were either active or silent in an immediately prior experience. Together, this study is expected to meaningfully advance our understanding of circuit-level brain function by revealing the fundamental principles which allow precise patterns of activity to be dynamically generated and propagated throughout the hippocampal network in support of learning.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Chronic liver disease is common and its pathogenic mechanisms are complex. There are no high throughput genetic methods to uncover mechanisms that drive progression to cirrhosis. As a result, therapies that slow the progression of liver disease are not currently available for most etiologies. The observation that our tissues accumulate a large number of somatic mutations with age and chronic injury could provide a genetic window into this process. Diseased livers harbor innumerable isolated islands of mosaic clones, but the identity and functional importance of the somatic alterations within these clones remain undiscovered. In the past, somatic mutations were generally assumed to be detrimental to health or drive cancer, but recently, some mutations have been shown to exert adaptive effects that might benefit tissue health. In the liver, deep sequencing experiments are beginning to reveal diverse somatic alterations, visually exemplified by the innumerable nodules on cirrhotic livers. Stimulated by this landscape of mutations, we have developed in vivo genetic screens to identify somatic mutations that have the greatest functional impact. Preliminary studies show that some mutations can increase tissue regeneration and others can prevent metabolic liver disease. An emerging concept is that cells can select for mutations that ameliorate, rather than cause, diseases such as steatohepatitis or cancer. We believe that somatic mosaicism is an open frontier for human genetics, and represents a potential source of unidentified disease genes and therapeutic targets. Using new technologies developed in my lab, we propose to exploit somatic mosaicism as a genetic strategy to 1) identify adaptive genetic pathways that are specific to particular liver disease etiologies, and 2) understand how mutant clones expand within chronically damaged tissues, and potentially protect from disease in a therapeutic fashion.

NIH Research Projects · FY 2025 · 2024-09

Understanding the biology of cancer metastases is critical to improving the treatment of cancer. A key challenge in these efforts has been the lack of cancer models that can recapitulate metastasis biology, especially organ- specificity, a hallmark of cancer metastasis. Therefore, there is an urgent need for the development of novel models of cancer metastasis that can reflect organ specificity of tumor metastasis. Our group has been applying tissue engineering technologies to develop in vitro engineered cancer metastasis model. We hypothesized that decellularized tissue can recapitulate the organ microenvironment of cancer metastasis and can be utilized as strata for culturing tumor cells. We used an innovative decellularization technology, where the process retains soluble extracellular proteins in the organs (decellularized tissue are termed biomatrix), to engineer cancer metastasis. Using multiple cancer cell lines, we found that tumor cells can spontaneously form colonies on biomatrices that are common sites of metastasis (“good soil”/liver and lung). Importantly, the biology of these tumor colonies and treatment response reflect organ-specificity. We were also able to culture circulating tumor cells from patients using liver and lung biomatrices. Excitingly, we also found that tumor cells were unable to proliferate on “bad soil” biomatrices. Further study also revealed that the soluble matrix proteins are largely responsible for the lack of proliferation of tumor cells on these biomatrices. Based on our preliminary data, we hypothesize that we can develop an in vitro cancer metastasis model system using biomatrices and this model system can reflect organ-specificity. We further theorize that our proposed model of cancer metastasis represents a powerful tool for cancer biology research and hold high potential for precision oncology. In this application, we aim to further develop and validate our metastasis model. We plan to use kidney cancer as a disease model. Kidney cancer was chosen because its unique metastatic pattern: it can metastasize to organs that are rarely sites of metastasis, such as pancreas. Our proposal has 3 specific aims. The first aim will generate a comprehensive panel of biomatrices to model sites of cancer metastasis. We will conduct validation studies to confirm the model system can recapitulate organ-specificity. Second aim will focus on demonstrating the biomatrix metastasis model’s ability to improve precision oncology. Last aim will focus on identifying soluble matrix proteins that prevent metastasis formation. Success with our research will result in powerful new tools for cancer research.

NIH Research Projects · FY 2024 · 2024-09

for R-56 related to 1R01DK139111-0 The goal of this project, “Early podocyte injury in collagen IV nephropathy”, is to understand the earliest events in podocyte injury in Collagen IV nephropathies. The basic hypothesis is that processing of mutant collagen IV proteins injures podocytes at early stages of development, ultimately leading to Chronic Kidney Disease and renal failure (ESRD). The podocyte cell-specific mechanisms of early injury process are not fully understood, but if identified and mitigated early, could defer renal injury and progression to ESRD. Our approach is to use transcriptomic data from extensively phenotyped mouse models mapped to a human database of proteinuric kidney disease (NEPTUNE FSGS and COL4 mutations) to identify similarities and differences and use the model systems to identify the earliest possible points for intervention in Col IV nephropathies. Understanding of how Collagen IV mutations initiate and drive podocyte injury will also be critical in understanding other human glomerular diseases including in human FSGS patients, many of whom have Collagen IV mutations. Our published and preliminary data from WT and Col4a3-/- mice, and accompanying conditionally immortalized podocytes demonstrate: 1. Suppression of the Alport renal phenotype including early podocyte injury and loss by mitigation of the Unfolded Protein Response (UPR) with Tauro-Urso-DeoxyCholic Acid (TUDCA), 2. Reduced expression of WNK1 occurs early in Alport nephropathy, 3. Inhibition of WNK1 disrupts podocyte and glomerular architecture, 4. Activation of WNK1 suppresses the Alport phenotype in 4 month Col4a3-/- glomeruli and cultured podocytes, 5. Podocyte injury begins before proteinuria, and renal parenchymal injury begins with its onset and loss of approximately 40% of podocytes, 6. Bulk RNAseq shows that increased expression of cytokines and chemokines coincides with the onset of proteinuria, 7. A list of podocyte enriched genes from NEPTUNE human FSGS single cell data was mapped to mouse orthologs in our mouse bulk-RNA seq data to identify 60 podocyte enriched genes whose magnitude of change was reduced or direction of change was reversed by TUDCA. 8. This group of genes was similar in nature and behavior to gene changes reported in studies of anti-miR21/ACEI treatment of Svj129 Col4a3-/- mice where the Alport phenotype was also suppressed. This group of podocyte enriched genes represents a focused target for reversing or mitigating podocyte injury in these diseases. We emphasize two distinct areas of the application: 1) The importance of WNK1 signaling in podocyte cytoskeletal structure and glomerular biology in Alport syndrome 2) The validity of mapping mouse to human transcriptomic data. Our current goal is to generate additional data to resolve these questions. We will expand our preliminary and published data demonstrating that WNK1 has essential and specific roles in glomerular and podocyte structure, and that its level of expression decreases in Col4a3-/- mice. The studies will use WT and Col4a3-/- mice, isolated glomeruli, and cultured cells. We will also expand our work with the NEPTUNE consortium and Col4a3-/- mouse RNAseq data to further strengthen interspecies relationship in Alport Syndrome.