Ut Southwestern Medical Center

NIH Research Projects · FY 2025 · 2021-07

Project Summary Allergic diseases including asthma, eczema, and food allergy have increased substantially over the last few decades. These diseases affect almost 5% of the population and having an annual economic burden of over 80 billion dollars. Allergic reactions can be life-threatening. While we have learned much about how an allergic reaction occurs, much remains unknown about how allergies develop. Genetic predisposition is important in developing allergies, but few defined mutations in specific genes are known to cause allergic disease. Recently, we conducted an allergy screen in mutagenized mice to discover genes that regulate production of allergen-specific IgE, which mediates allergic reactions. Among several phenotypes of interest, benadryl is marked by 90% reduced IgE levels and resistance to anaphylaxis with otherwise normal immunity. benadryl was ascribed to a mutation a gene that produces mannose used for glycosylation (adding sugars to proteins), metabolism and targeting proteases to the lysosome. IgE glycosylation is important for its structure and function. Therefore (1), we will determine how the benadryl mutation affects IgE by identifying the cell type responsible for low IgE through a combination of in vitro cell assays and conditional knock-out mouse models. To determine the effect of altered mannose (2), we will examine alterations to IgE glycosylation, stability, and function along with measuring changes to metabolism and lysosomal trafficking. To assess the importance of benadryl in allergic disease (3), we will determine whether benadryl mice are resistant to IgE mediated anaphylaxis and food allergy. Upon successful completion, the findings of this study will reveal a new pathway important for IgE production that would be an appealing therapeutic target. I plan to use these results to launch my career using this mouse strain to understand how altered IgE glycosylation regulates IgE stability, clearance and function. Few other investigators are exploring this topic, which will allow me to study this into the future independently without overlap with my current mentor’s line of scientific investigation. My career goal is to be an independent physician scientist determining the genes that regulate IgE production, which can be clinically translated to genetic tests and potential therapeutic targets. This career development award will help me gain skills in molecular biology, animal models of allergy, cellular immunology, and proteomics. Biannual meetings with my mentoring committee will guide early career milestones including submission of multiple papers. Institutional grant writing resources will be used to successfully compete for an R01 to gain independence.

NIH Research Projects · FY 2024 · 2021-07

Project Summary/Abstract The kidney is patterned along a cortical to medullary axis with specific segments of the nephron, collecting duct and vasculature all lying adjacent to each other in histologically distinct domains. In order for a kidney to function, different cell types from different cellular lineages must form at the same anatomical location. Although there has been some insight into how the individual lineages become patterned (such as proximal distal patterning of the nephron), how the different cell types/lineages coordinate their development resulting in the global patterning of the organ is unknown. We have recently found that the renal interstitial cells show extensive heterogeneity and patterning along the cortical/medullary axis of a newborn mouse kidney[1]. The patterned domains of the renal interstitium precisely map to the different anatomical domains within the kidney. How the different interstitial cell types arise and what role they play in kidney development/function are unknown. We hypothesize that the interstitium functions to relay and integrate signals from the different lineages and in turn, reinfources and integrates the differentiation of the renal parenchyma along the cortical/ medullary axis. Using bioinformatic analysis of single cell RNA-Seq data, we have identified unique transcriptional signatures for the different interstitial cell types. This information will allow us to understand how the pattern is established as well as its function. In this proposal, we will focus on the specification and function of a sub-population of interstitial cells we will refer to as the proximal tubule (PT) interstitium. In this proposal, we will investigate the mechanisms underlying specification of a subpopulation of renal fibroblasts we refer to as the proximal tubules interstitium (PT interstitium). Notch/Rbpj and Yap/Taz transcription factors are both active within this population and ablation of either pathway using Foxd1Cre has revealed overlapping roles in the specification of this cell type. Our preliminary analysis indicates that the PT interstitium is necessary for the maturation of the adjacent proximal tubules. We hypothesize that the PTs and/or endothelia produce Notch/Rbpj and Yap/Taz activators and that cells with overlapping pathway activation become PT intersitium. The PT interstitium produces signals that promote the differentiation/maturation of the PTs. This crosstalk allows the co-maturation and integration of the proximal tubules and other cortical cell types. We further hypothesize that disruption of normal cortical-medullary pattern in renal organoids leads to defects in Yap/Taz and/or Notch/Rbpj signaling and contributes to the lack of nephron maturation in these tissues. These hypotheses will be tested here. Completion of these aims will open up an entirely new field of kidney interstitial biology that will have a long and lasting impact on the multiple fields including kidney development, kidney disease, tissue engineering and kidney injury/regeneration.

NIH Research Projects · FY 2025 · 2021-07

Project Summary Heterotopic ossification (HO) is the pathologic formation of extra-skeletal bone forming almost exclusively at sites of mechanical stress, that occurs in ~20% of patients after hip arthroplasty, burns or musculoskeletal injury. Currently, no therapeutics or physical therapy-based protocols exist to prevent HO. In this regard, there is a void in our understanding of the causative mechanotransductive pathways behind this debilitating process. Our unbiased transcription profiles in mouse HO-mesenchymal progenitor cells (MPCs) recovered from HO sites in combination with immunostaining of mouse and human HO revealed that a series of mechanotranduction-linked pathways, including discoidin receptor 2 (DDR2), FAK and the Hippo effectors, YAP and TAZ, are highly upregulated in tandem with observed changes in extracellular matrix (ECM) alignment. Using a novel, regional MPC-specific inducible Cre system (Hoxa11-CreERT2), we have compiled preliminary data that support critical roles for DDR2 signaling and stage-specific immobilization in both triggering FAK/YAP/TAZ signaling and MPC lineage commitment, but also an unexpected function in controlling ECM alignment. Together, these observations have led to our central hypothesis that MPC DDR2 signaling is necessary for mobility-induced changes in ECM alignment that trigger aberrant osteochondral differentiation at HO sites and can be blocked by DDR2 inhibition or injury stage-specific immobilization. Aim 1: Define the role of DDR2 as a critical upstream regulator of FAK/YAP/TAZ signaling in controlling the induction and progression of HO. We hypothesize that DDR2-mechanotransductive signaling alters osteochondral differentiation and HO in vivo and can be targeted with cell specific deletion models or translatable clinical therapies. Aim 2: Determine the optimal post-injury timing during which MPCs can be redirected away from aberrant osteochondral fate and pathologic ECM alignment through immobilization-based intervention. We hypothesize that immobilization during the early proliferative phase after injury will block pathologic changes in ECM alignment with disease-ameliorating effects on MPC fate determination and aberrant ossification. Aim 3: Characterize the role of mobilization-induced DDR2 activation on collagen alignment/anisotropy and mechanotransductive signaling. We hypothesize that DDR2 activity drives ECM alignment independently of limb mobility in vivo or cyclic stretch in vitro. Impact: The proposed studies will provide a comprehensive and mechanistic understanding of how DDR2 and joint mobility regulate ECM alignment, cell fate and HO using conditional deletion models and clinical therapies.

NIH Research Projects · FY 2025 · 2021-07

Project Abstract Although combination antiretroviral therapy (cART) effectively suppresses human immunodeficiency virus (HIV) replication, it does not cure HIV infection and requires costly lifelong treatment. A major challenge for curing HIV infection is the long-lived latent HIV reservoir, which evades immune recognition and is responsible for viral rebound shortly after interruption of cART. Substantial research efforts have focused on eliminating latently infected cells through so-called “shock and kill” strategy, which reactivates latent HIV using latency- reversing agents (LRAs) to allow for the “kill” by cytolysis or immune-mediated clearance. Particularly, eradication of theHIV reservoir by anti-HIV T cells, which maintain antiviral immunity in patients as a “living” drug, presents a promising strategy to either fully resolve the infection or maintain long-term control without cART treatment. However, clearance of the HIV reservoir by T-cell based immunotherapy remains challenging because of 1) T- cell exhaustion, 2) the need of lifelong anti-HIV immunity to replace cART, 3) sanctuary sites like B cell follicles that exclude most HIV-specific CD8 T cells, and 4) unclear impact of LRAs, most of which target epigenetic pathways, on the function and differentiation of antiviral CD8 T cells in vivo. We and others have recently characterized a stem-like CD8 T cell subset in chronic lymphocytic choriomeningitis virus (LCMV), simian immunodeficiency virus (SIV), and HIV infections, as well as in mouse and human tumors. Compared to terminally exhausted CD8 T cells, stem-like CD8 T cells are less exhausted, mediate long-term immunity, and respond more potently after treatment of immunotherapies in animals and human. In chronic LCMV, SIV, and HIV infections, these cells express CXCR5, migrate to B cell follicles and kill infected T follicular helper cells, a major latent reservoir of HIV and SIV. In addition, frequency of these cells inversely correlates with viremia of SIV or HIV. Most recently, we showed that the single-cell transcriptomic and epigenetic profiles of stem-like CD8 T cells are distinct from other CD8 subsets generated after acute or chronic LCMV infection. In addition, we identified a transcriptional program involving transcription factor TOX that is essential for stem-like CD8 T cell differentiation and the long-term persistence of antiviral CD8 T cells during chronic viral infection. Here, I will determine the transcriptional and epigenetic programs of stem-like CD8 T cells required for optimal T-cell based immunotherapy against HIV. I will use animal models of chronic LCMV and SIV infections as well as samples from HIV patients, and employ cutting-edge technologies, including single-cell profiling of T-cell transcriptomes and epigenomes, CRISPR/Cas9 screening, and chimeric antigen receptor (CAR) T-cell therapy, to determine how “shock” by epigenetic modifying LRAs affects the program and antiviral immunity of stem-like CD8 T cells and whether transcriptional and epigenetic programs of stem-like CD8 T cells can be adopted to enhance the “kill” of the HIV reservoir by T-cell based immunotherapies. The results from this study will build the foundation for novel immunotherapies that achieve long-term remission from HIV infection without a need for cART.

NIH Research Projects · FY 2025 · 2021-07

Project Summary Title: Neural Niche in Promoting Brain Metastasis Progression Metastatic progression at the brain is a multi-step, evolutionary process that is accomplished through a consistent interplay between disseminated tumor cells and brain microenvironment - "the niche". Despite significantly improved control of primary tumors, the incidence of brain metastasis is increasing! To reduce cancer mortality, rationally-designed therapeutics, based on a mechanistic understanding of metastasis in at the unique brain metastatic niche, are urgently needed for brain metastasis patients. What's the brain metastatic niche? In the brain microenvironment - the niche - is a myriad of diverse cell types that have been speculated to contribute to the brain tumor and brain metastasis development, including endothelial cells, astrocytes, neural stem/progenitor cells (NSC hereafter) and increasingly appreciated brain immune cells. Despite studies of astrocytes, how do the NSC and its progenies respond to brain metastatic colonization and regulate brain immune landscape and the metastatic outcome has yet to be systematically investigated. Our preliminary studies have demonstrated that NSC migrates to colonized tumor cells and NSC is functionally essential for brain metastasis progression. Intriguingly, ample evidence from in the field of neuroscience suggests an immune suppressive role of NCS during the brain inflammation. Thus, in this proposal, we hypothesize that NSC and its progenies' brain metastasis tropism and potentially consequential immunosuppressive activity could contribute to an immune-suppressive metastatic niche, facilitating brain metastasis progression. In this proposed study, we will use transgenic mouse models and state-of-the-art genomics and imaging approach to trace and analyze the role of highly heterogeneous cell types involved in NSC differentiation and immune suppression and their roles in regulating brain metastatic outgrowth. This collaborative effort integrating multidisciplinary expertise, including cancer biology, neuroscience, computational biology, allow us to: 1) examine and trace the NSC differentiation response to brain metastasis at the phenotypical level; 2) visualize and quantitatively measure the behavior of brain metastatic niche cells and their transcriptome heterogeneity; 3) examine the mechanism by which neural cells derived from NSC promotes brain metastasis progression. New in-depth mechanistic insights obtained through basic and pre- clinical innovative research will pave the way to future brain metastases treatments.

NIH Research Projects · FY 2026 · 2021-07

Abstract A New Perspective on Leptin in Health and Disease Over the past four years, the Scherer and Elmquist laboratories have worked on putting the concept to use that lowering peripheral leptin levels is beneficial for the metabolic well-being of the organism. The starting point was the generation of mouse models with an inducible reduction of leptin levels in adult animals and the use of monoclonal antibodies that neutralize leptin. In contrast to historical expectations, these interventions resulted in weight loss and enhanced insulin sensitivity. We have further elaborated on this concept in additional physiological and pathophysiological settings. We have shown that weight loss, including weight loss induced by incretins or FGF21, is critically dependent on leptin reduction and can be further enhanced by neutralizing additional leptin in the process. On the other hand, weight gain and insulin resistance induced by anti- psychotics can be prevented by leptin neutralization. All these observations indicate that leptin is not merely a passenger associated with obesity but serves as a driver of the obese phenotype. Furthermore, it conceptualizes the physiological role of leptin as an “energy- sufficiency” signal - it is primarily a drop in leptin levels that the system responds to, with further rises in leptin above baseline merely leading to pathological responses. Using a series of new tools we have developed over the past four years, we strive to expand these concepts further in this renewal application. We now propose to investigate the relationship of insulin and beta-adrenergic signaling to leptin production and release from adipocytes. This also entails further studies into the regulation of full-length, signaling-competent leptin receptors in peripheral tissues. In this context, we plan to elaborate on the pathophysiological aspects of leptin signaling in the periphery by examining its potential as a fibroinflammatory agent. Using a series of novel models and methods, we will examine the process of leptin-driven adipocyte delipidation, a phenomenon that is restricted to young animals. Leptin sensitivity of adipocytes is abruptly lost after 10 to 12 weeks of age. Most importantly, we are bearing future translational interventions in mind and have established a leptin- deficient ob/ob mouse line in which we can ectopically manipulate leptin levels in a pharmacological dose- dependent manner. This will allow us to precisely determine the lower threshold of circulating insulin levels below which adaptive energy-conserving mechanisms are triggered. This enables us to calibrate future clinical interventions with respect to minimal and maximal reductions of leptin that are therapeutically effective. While conceptually novel, the approaches used here that critically depend on making a ligand (leptin) more rate limiting, thereby leading to improved receptor (LEPRb) signaling, are fully compatible with the historical data in the field, particularly as it relates to central leptin action. At the same time, we emphasize the peripheral actions of leptin with respect to the fibroinflammatory milieu that it creates in several tissues critical for metabolic well-being, including the liver, kidney, and lung.

NIH Research Projects · FY 2025 · 2021-07

Greater disease severity and a more rapid disease progression is observed in the USA among African Americans (AA) and Hispanics (HSP) with Multiple Sclerosis (MS) compared to Whites with MS. Despite these observations, very little data is available regarding the biological underpinnings resulting in this increased risk of unequivocal advancing disease among AA and HSP with MS even though the incidence of MS disease is growing among AA and HSP populations. Further, there are concerns that AA and HSP with MS are refractory to first-line therapies, which increases the concern that the poor outcomes reported reflect substandard care and treatment. Our long term goal is to facilitate prevention of neurological deficits in all patients with MS by identifying the mechanism(s) of CNS inflammation contributing to intensified progression and severity of disease. Others have shown that AA and HSP with MS display higher IgG index and synthesis rate, oligoclonal band positivity and an inverse correlation of IgG index with gray matter atrophy. These data support a pronounced role for B cells and their antibody products in the pathobiology of MS in AA and HSP populations. Our central hypothesis is that the underlying mechanism of unequivocal advancing disease among AA and HSP with MS involves expansion of plasmablasts producing antibodies and/or inflammatory products that cause neuronal toxicity independent of genetic (i.e. ancestral) predispositions. These studies will provide novel mechanistic insights into the role of neuron-reactive plasmablasts in CNS inflammatory events associated with intensified progression and severity of disease in AA and HSP with MS. Our findings will also support future studies to develop diagnostic and predictive tools that inform patient response to treatment and inform clinical course such as relapse of MS or progression to MS in AA and HSP populations.

NIH Research Projects · FY 2024 · 2021-07

PROJECT SUMMARY/ABSTRACT This is an application for a K08 for Dr. Joshua Gruber, an Instructor of Medicine at Stanford University. Dr. Gruber wishes to establish himself as a clinician-scientist at the forefront of metabolite- epigenetics crosstalk with a long-term goal of establishing novel drug targets for patients with early and advanced stage malignancies. This K08 award will provide Dr. Gruber support to achieve the following goals for career development: 1) Determine molecular mechanisms that drive histone acetyltransferase 1 (HAT1)-dependent malignancies; 2) Mechanistically characterize potential HAT1 activators and inhibitors; 3) Identify mechanisms of how dietary fiber-derived propionate modifies chromatin. Dr. Gruber will be mentored by Dr. Michael Snyder, an established expert in mass spectrometry approaches including proteomics and metabolite quantitation. Dr. Gruber will be co-mentored by Dr. Calvin Kuo, an expert in cancer biology and nutrient metabolism. Dr. Gruber has established a mentoring committee including Dr. James Chen, Stanford Professor of Chemical and Systems biology to advise on aspects of chemical biology; Dr. Mark Smith, director of the Medicinal Chemistry Knowledge Center; Dr. Kevin Contrepois, Scientific Director of the Stanford Metabolic Health Center, to provide mass spectrometry training; and Zena Werb, Professor of Anatomy, University of California San Francisco to advise on experimental models of tumorigenesis and breast cancer biology. Cancer cell growth is coupled to nutrient metabolism to ensure adequate nutrients exist to fuel cell division. Molecular metabolite sensors allow for cells to respond to changes in nutrient availability. Acetyl-co-A is a critical metabolite for biosynthetic processes, signaling and epigenetics. However, metabolite sensors of acetate and other acyl-containing metabolites are poorly understood. Therefore, an improved understanding of acetyl-co-A sensing may allow for the development of novel approaches to diagnose, treat or prevent malignancy. Dr. Gruber has identified the histone acetyltransferase HAT1 as a potential sensor of acetyl-co-A and acyl-containing short chain fatty acids. To identify exploitable properties of the HAT1 metabolite-sensing pathway, Dr. Gruber plans a detailed molecular investigation of HAT1-dependency in human tumors to provide an understanding of the properties that make HAT1 a potential anti-cancer drug target (aim 1). To advance the ability to manipulate HAT1 catalytic activity, Dr. Gruber has screened for small molecule chemical activators and inhibitors, which will be biochemically characterized (aim 2). Finally, he plans to define mechanisms by which HAT1 incorporates short-chain fatty acids to chromatin (aim 3). This research will provide scientific foundations and essential career training to lead to an independent academic research position for Dr. Gruber with the expectation of R01-level funding by the conclusion of the K award period.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Bacteria encode a diverse array of molecular systems to defend against infecting phages. In response, phages have devised many counter-defense strategies to overcome this immunity and re-establish infection. Mounting evidence suggests that most bacterial defense systems and phage counter-defenses in nature have not been identified. This is a major knowledge gap because the interplay between these systems often determines whether a phage successfully infects its bacterial host. These phage infections, in turn, have major impacts on the evolution and treatment of infectious disease. For instance, pathogenesis in bacteria often evolves due to the integration of a prophage that expresses a toxin or other virulence factor. At the same time, phages are increasingly viewed as potential therapeutics to treat bacterial infections, especially in cases where multi-drug resistance renders conventional treatments unsuccessful. Thus, it is important to better understand the natural diversity of bacterial defense and phage counter-defense systems. To meet this need, we will devise new high-throughput functional selections to find defense and counter-defense systems in microbial ecosystems and in libraries of synthesized phage open reading frames. This functional approach does not rely on sequence similarity to predict defense and counter-defense systems, so overcomes the limitations of conventional, homology-based discovery methods. This strategy, therefore, is expected to identify many new defense and counter-defense genes beyond what is known currently. It is especially valuable for examining functions encoded in phage genomes and bacterial genomic islands, as most genes from these sources are of unknown function. Since nearly all bacteria should encode anti-phage defense systems, and almost all phages will encode counter- defense strategies, we expect to make many new discoveries. Because these discoveries are predicted to be novel, we will use a combination of genetic and functional assays to describe their mechanisms of action. We will use Escherichia coli as a host for our functional selections, not only because this will allow us to construct large functional libraries, but also because virulence in this pathogen is driven by prophage-expressed toxins and because its phages are among those used most commonly to develop phage therapies. Thus, our findings will not only be broadly relevant to pathogenesis and phage therapy across bacteria, but also will yield these insights specifically in the context of this important human pathogen.

NIH Research Projects · FY 2025 · 2021-07

Project Summary Legg-Calvé-Perthes disease is a childhood ischemic osteonecrosis of the femoral head (ONFH) that affects 1 in 1200 children between the ages of 2 to 14. It is one of the most serious conditions affecting the pediatric hip as 50% of patients will develop debilitating osteoarthritis, some in their teenage years. A disruption of blood flow produces extensive ischemic cell death, abundance of necrotic cell debris, and damage-associated molecular patterns (DAMPs) in the femoral head. We discovered that the necrotic microenvironment incites a chronic inflammatory response, which impairs bone regeneration and produces femoral head deformity. Macrophages are the central innate immune cells that coordinate the repair process based on local environmental stimuli. In juvenile ONFH, macrophages exhibit chronic inflammatory response due to DAMPs and necrotic debris which leads to further tissue damage and fibrosis. Current treatments do not address the negative pathologic role played by macrophages in the necrotic bone repair. Here, we propose a new concept of reconditioning the necrotic bone using minimally invasive tissue engineering methods, thereby, converting a necrotic inflammatory microenvironment to a regenerative microenvironment. Our long-term goal is to establish these treatment methods to overcome the substantial inflammatory roadblock and to rapidly recondition the necrotic bone in order to jump start bone regeneration in patients with juvenile ONFH. Our central hypothesis is that the necrotic bone microenvironment triggers chronic inflammatory macrophage response, and that tissue engineering of the necrotic environment by local bone wash (i.e. clearance of DAMPs and necrotic debris) and application of macrophage-directional modulators (such as bone morphogenetic protein-2 and interleukin-4) will increase pro- healing macrophages and accelerate bone regeneration. We will attain our goal through three highly related but independent specific aims. We will 1) determine the therapeutic effects of washing out DAMPs and necrotic cell debris on macrophage response; 2) determine the effects of macrophage response to local controlled-release bone morphogenetic protein-2 (BMP2) therapy using a hydrogel delivery system on bone regeneration; and 3) determine the role of interleukin 4-induced macrophage modulation on bone regeneration, using the piglet model of ischemic ONFH and in vitro experiments in each Aim. We will determine the macrophage and bone repair responses to the immunomodulatory therapies using tissue, cell, and RNA analytic methods. Successful completion of this project will have immediate clinical impact by providing a proof-of-concept for the minimally invasive, yet potentially highly effective, tissue engineering strategies to overcome current barriers to successful treatment of ONFH. The outcome of this work will lay the groundwork for clinical trials and will greatly advance our ability to treat ONFH using immunomodulatory strategies.

NIH Research Projects · FY 2024 · 2021-07

Project Abstract Despite decades of research in the understanding and treatment of diseases of the heart and lung, cardiovascular and pulmonary diseases remain the leading causes of hospitalization, disability, and death in the U.S. Notably, growing numbers of patients with childhood-onset chronic illnesses, such as congenital heart defects, cystic fibrosis, bronchopulmonary dysplasia, and hemophilia, are surviving into adulthood, making it imperative to conduct research encompassing the lifespan. Against this background of critical research needs, there is a strikingly growing shortage of M.D.-scientists, such that the proportion of M.D.s that remain primarily in research careers has decreased by 3-fold in the last 3 decades, despite the climbing number of total physicians in practice. The pipeline of M.D.-scientist development is also at risk: fewer medical students express interest in a research career, and fewer graduates apply for NIH funding. Importantly, relative to the overall physician workforce, women and minorities are disproportionately underrepresented in the proportion of M.D.-scientists. These challenges require the implementation of innovative recruitment and training strategies to feed the M.D.- scientist pipeline and ensure that women and minorities are well-represented. To help fill these critical gaps, we will establish the University of Texas Southwestern (UTSW) - Stimulating Access to Research in Residency (UT-StARR) training program to recruit and train a diverse group of Internal Medicine and Pediatrics residents that will become the next generation of M.D.-scientists conducting laboratory-based, translational, clinical, and population health research to detect, treat, and prognosticate diseases affecting the heart, lungs, and blood of diverse populations throughout the lifespan. Our institution will provide an ideal environment with a multidisciplinary cadre of funded research; robust didactic training program; and diverse patient population. We hypothesize that the 3 axes of diversity at UTSW - M.D. trainees, mentors, and patient community - afford the unique opportunity to address unmet needs in M.D. research training, personalized medicine, and population health. The following Aims organize our approach: Aim 1. To provide broad-based comprehensive mentored research training for a group of diverse Internal Medicine and Pediatric resident-investigators in the conception, design, and implementation of laboratory-based, translational, clinical, and population research in diseases affecting the heart, lungs and blood, encompassing detection, prevention, treatment, and outcomes to improve the health of individuals and populations throughout the human lifespan; Aim 2. To provide the essential research training and mentoring platform needed to build a strong foundation allowing successful transition of residents to independent M.D.-scientists conducting biomedical research in the heart, lung, and blood fields; Aim 3. To enhance the participation and persistence of M.D.s from underrepresented groups in research by implementation and maintenance of institutional mentored residency training programs involving diverse residents, mentors, and patients through evaluation and monitoring the progress of UT-StARR and other institutional training programs.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY The 2020 NIMH Strategic Plan for Research calls for investigations targeting neurobiology of mental illness across the lifespan. Recent innovations in the field of early psychosis have established the critical importance of timely interventions. However, little progress has been made in broader lifespan approaches to schizophrenia (SZ) neurobiology and treatment. The lack of specific biomarkers capturing dynamic alterations along the SZ trajectory hinders progress in targeted treatment development. Growing evidence suggests that the SZ lifespan is the product of two distinct dimensions: aging and disease course. However, the exact trajectories of these dimensions remain unclear. Specific biomarkers capturing unique and/or overlapping aspects of their mechanisms are unavailable. Cognitive and broader clinical correlates of aging and SZ disease course, and their implications for treatment, are yet to be identified. We propose to investigate differential aspects of SZ neurobiology captured by aging and disease course, in order to develop specific biomarkers which may offer actionable targets for intervention. The proposal is predicated on a novel mechanistic Model of SZ Trajectories across the Adult Lifespan, positing distinct biological fingerprints within the anterior limbic circuit for aging and disease course in SZ: (1) alterations in the circuit’s function and structure that occur earlier in the lifespan and are larger in magnitude than the alterations expected with normal aging (accelerated aging dimension); and (2) regionally-specific anterior limbic “hyperactivity” in early SZ, with a subsequent transformation into “hypoactivity” in advanced SZ (disease course dimension). The proposed Model has a strong evidential basis; it is testable; and, if confirmed, it will provide important guidance for the development of future targeted therapeutics, e.g., reducing anterior limbic hyperactivity in early SZ vs. enhancing this same circuit’s function in advanced SZ and along the aging trajectory. In a sample of SZ and matched healthy controls (n=168, 84/group) aged 18-75 years we will ascertain a broad panel of biomarkers [via multimodal brain imaging: novel triple-refocusing 1H-MRS, high-resolution perfusion (Vascular Space Occupancy), and task-based fMRI], along with comprehensive cognitive and clinical characterization. All measures will be acquired at baseline and repeated at 2-year longitudinal follow-up. Using cutting-edge computational approaches, we will examine (i) effects of aging and SZ disease course on anterior limbic system biomarkers, and interactions between these effects; (ii) lifespan trajectories for different biomarkers; (iii) patterns of limbic system biomarkers in age- and SZ disease course-based subgroups (e.g., Younger vs. Older, Early-Course vs. Advanced SZ), as well as in data- driven subgroups (e.g., those with vs. without accelerated aging profiles); and (iv) associations between biomarkers and cognitive and clinical outcomes. This research will advance the field by providing novel biomarkers that capture unique neurobiological contributions of aging and disease course in SZ, and will motivate future studies on SZ mechanisms across the lifespan, and development of precision treatments.

NIH Research Projects · FY 2025 · 2021-06

Project Summary The goal of this K08 application is to provide a rigorous 5-year scientific and careered development training plan that will facilitate Dr. Josephine Thinwa to transition from a post-doctoral fellow to an independent investigator. The candidate completed her MD/PhD degree at UT Health San Antonio where she focused on innate immunity. After completing internal medicine and infectious diseases training at UT Southwestern, she joined the lab of Dr. Beth Levine, a renowned expert in autophagy, to pursue a project on the interface between autophagy and antiviral immunity. Autophagy is known to function in antiviral innate immunity by specifically targeting intracellular viral components for lysosomal degradation, a process called virophagy. However, how virophagy is activated and regulated is still poorly understood. Based on an unbiased screen for host genes necessary for autophagy induction during infection with two prototypic neurotropic viruses, Sindbis virus (SINV) and Herpes Simplex Virus-1 (HSV-1), her preliminary work identified CDKL5 as a candidate novel regulator of virophagy. She demonstrated that cells deficient in CDKL5 accumulated high levels of SINV capsid protein. Additionally, she determined that CDKL5KO mice were more susceptible to lethal CNS infections with SINV and HSV-1, suggesting that CDKL5 is a critical host antiviral factor. After learning of her mentor’s likely terminal illness, Dr. Thinwa worked with Dr. Levine to establish a mentoring team of accomplished scientists who would provide the expertise and training necessary for her to successfully elucidate the function of CDKL5 in antiviral immunity and become an independent investigator. Her mentorship transitioned smoothly after Dr. Levine passed away to Dr. Michael Shiloh, her primary co-mentor who is a physician scientist and expert in innate immunity and autophagy, and Dr. Julie Pfeiffer, a renowned scientist in viral pathogenesis. They have successfully trained numerous postdoctoral fellows to achieve scientific independence. This mentoring team is perfectly complemented for the proposed studies at the interface between autophagy and antiviral immunity by her outside co-mentor Dr. Hebert “Skip” Virgin. He is a physician scientist and an undisputed leader in the field of autophagy and innate antiviral immunity. He also has a very strong track record of mentoring postdoctoral trainees. A committee of advisors and collaborators consisting of three outstanding scientists will provide additional scientific expertise to enhance her technical skills and offer guidance in scientific and career matters. In her proposed research, the applicant will aim to 1) define the function of CDKL5 in virophagy, 2) delineate which step(s) of the SINV life cycle are modulated by CDKL5, and 3) determine the role of CDKL5 in host antiviral response in vivo. Overall, the proposed studies are likely to expand our understanding of how host cells mitigate the toxic overproduction of viral proteins during infection. Together with access to excellent institutional resources, and training from didactic courses on leadership, grantsmanship and technical skills, she is positioned to become a successful physician- scientist focused on antiviral innate immunity.

NIH Research Projects · FY 2025 · 2021-06

Project Summary/Abstract The contribution of individual disease-relevant genes to brain development still remains unknown. The long-term goal of our laboratory is to elucidate the intersection of molecular signaling pathways that are disrupted in neurodevelopmental disorders with those pathways that are important for specific aspects of brain development. Two members of the FOXP family of transcription factors, FOXP1 and FOXP2, have been linked to monogenetic forms of intellectual disability, autism spectrum disorders, and specific speech and language deficits. Variants in FOXP1 or FOXP2 are among the most significant genes associated with autism spectrum disorders. We previously showed that Foxp1 and Foxp2 both have significant contributions to cortical and striatal development. We linked these developmental changes via studies of gene expression, electrophysiology, and behaviors. We further identified non-cell-autonomous changes in gene expression using newly available single-cell RNA- sequencing technology. Based on these data, the central hypothesis driving this proposal is that Foxp1 and Foxp2 are key orchestrators of transcriptional signaling cascades in a cell type-specific manner that are important for neuronal function and are at risk in neurodevelopmental disorders such as autism. We propose to identify these cell type-specific contributions in the developing cortex by using rodent models through three specific aims: 1) Determine the cell type-specific gene expression programs regulated by Foxp1 in the developing cortex; 2) Determine the cell type-specific gene expression programs regulated by Foxp2 in the developing cortex; and 3) Assess the role of Foxp1 and Foxp2 in cell type-specific activity-dependent neuronal function. Together, these aims will delineate the cell type contribution of both Foxp1 and Foxp2 to cortical development. The rodent models and cell-type specific genomic datasets will aprovide insight into the basic molecular mechanisms governing normal mammalian brain development.

NIH Research Projects · FY 2026 · 2021-06

The Chook Lab integrates structural, biochemical, cell biological, and bioinformatic approaches to study how the family of twenty homologous Karyopherin-β (Kap) proteins, including importins, exportins, and biportins, govern macromolecular transport across the nuclear envelope. Our work in nuclear export has defined how XPO1 recognizes classical nuclear export signals (NESs), which are often degenerate in sequence and structure, and how different classes of XPO1 inhibitors block this essential process. These findings laid the groundwork for development of anti- XPO1 therapeutics. Building on this foundation, we recently expanded our studies to the yeast exportin Msn5, which transports proteins and re-exports spliced tRNAs. Our analysis of Msn5’s interaction with transcription factor Pho4 revealed a new type of NES, distinct from XPO1’s NES, that features phosphoserines and small hydrophobic side chains. We now aim to define the broader rules governing this new NES class by studying additional Msn5 cargoes, enabling proteome-wide identification of such signals in yeast. We will also investigate how exportins recognize tRNAs. In yeast, tRNA export involves Msn5, Crm1/XPO1 and Los1/XPOT, each acting at different stages of tRNA biogenesis. The mechanism of Los1/XPOT is understood, but how Msn5 and XPO1 export tRNAs remain unclear. We will biochemically reconstitute Msn5-tRNA assemblies to define selective export of spliced, mature tRNAs, and explore XPO1-tRNA interaction to determine how the three exportins coordinate different steps of tRNA export. Beyond Kap-cargo recognition, we also study nuclear transport regulation, with a recent discovery that select XPO1 inhibitors induce XPO1 degradation through an allosteric mechanism involving the Cullin Ring E3 Ligase CRL5ASB8. Building on this discovery, we will discover how various endogenous and exogenous XPO1 ligands, including metabolites, signaling molecules, proteins and synthetic inhibitors, control CRL5ASB8-mediated XPO1 degradation to regulate XPO1 levels in cells. Finally, we will investigate how Kaps recognize folded domains in cargoes. While many proteins use linear NLS/NES motifs for localization, an increasing number lack such signals and instead bind Kaps through structured domains. Unlike linear motifs, these domains lack shared sequence features, posing a specificity challenge: Kaps must selectively recognize dozens- hundreds of distinct cargoes while avoiding thousands of others. Using structure prediction, biochemical assays, and structural biology, we will define how several importins achieve this selectivity, expanding our understanding of signal diversity in nuclear transport.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY/ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic virus that causes Kaposi’s sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD). Like all herpesviruses, the KSHV life cycle consists of latent and lytic phases, and the virus relies on a sophisticated cascade of gene expression during lytic reactivation from latency. KSHV uses the host cell gene expression machinery to transcriptionally and posttranscriptionally control the timing and levels of gene expression. For host genes, proper transcription involves regulation of RNA polymerase II (pol II) elongation illustrated by pol II pausing at the 5´ and 3´ ends of genes. The near ubiquitous regulation of pol II elongation on host genes coupled with KSHV’s use of the host machinery suggest that the virus employs similar mechanisms of regulation. Despite this potential importance of pol II control by elongation, regulation of KSHV transcription elongation has been largely unexplored. Using an unbiased genome-wide CRISPR screen, host factors involved in pol II elongation and mRNA 3´ end formation were identified as negative regulators of KSHV gene expression. Depletion of these factors in cells latently infected with a KSHV infectious bacmid clone (BAC16) robustly increases the speed and overall production of infectious virions upon lytic reactivation. In the proposed work, the mechanisms linking elongation and 3´-end formation factors to KSHV transcription will be defined. Aim 1 will explore the importance of these elongation factors in PEL cells and during lytic gammaherpesvirus infection. Aim 2 uses viral gene reporter constructs and reductionist molecular biology to test whether pol II pausing is induced by cellular factors on viral genes. Moreover, the cis-acting and trans-acting requirements for elongation control of viral genes will be determined. In Aim 3, a combination of high-throughput methods will be performed to examine the roles of host elongation factors on viral gene expression. These will include RNA-seq to test gene expression levels, PAC-seq to address mRNA 3´-end formation, and PRO-seq to determine pol II occupancy on the viral genome. Finally, preliminary and published data suggest that reversible phosphorylation of elongation factors may play essential roles in the control of pol II pausing. In Aim 4, the targets and role of dephosphorylation of elongation factors on KSHV genes will be defined. Successful completion of the proposed studies will have considerable impact on the field by defining a novel host factor that negatively regulates viral gene expression by a previously undescribed mechanism(s). Moreover, it is likely that the mechanisms described will apply to additional DNA viruses and inform human gene expression as well. These studies will generate a deeper understanding of the mechanisms of a pathogenic virus which may lead to insights into how to combat KSHV related diseases.

NIH Research Projects · FY 2025 · 2021-06

Abstract. Prostaglandin E2 (PGE2) regulates tissue growth and repair in multiple organs. A conserved mechanism of synthesis and degradation modulates PGE2 levels in response to trauma, inflammation and disease. In particular, the enzyme 15-prostaglandin dehydrogenase (15-PGDH) is the main PGE2-degrading enzyme and therefore a key regulator of tissue repair and regeneration. 15-PGDH is an attractive drug target for diseases characterized by tissue damage. Our team successfully developed the first small molecule inhibitors of 15-PGDH with in vivo activities. In rodents, our inhibitors 1) accelerate recovery following bone marrow transplantation, 2) accelerate recovery from, or prevent, ulcerative colitis, 3) accelerate regrowth of liver tissue following partial hepatectomy, 4) ameliorate pulmonary fibrosis in a bleomycin-induced disease model, 5) enhance survival of new hippocampal neurons in adult mice, and 6) preserve cognitive function and minimize neuronal damage in mice following traumatic brain injury. Independent reports have described beneficial effects of 15-PGDH inhibition in models of renal disease and pulmonary fibrosis. We now propose a collaborative chemical, structural and cell-signaling interrogation of the role and activity of 15-PGDH. Our expertise includes medicinal chemistry, biochemistry, neuroscience, pharmacology, and structural biology. In Aim 1, we will define and exploit the structural basis for inhibition of 15-PGDH by small molecules. This aims builds on the first cryoEM structure (2.3 Å resolution) of 15-PGDH and two unrelated scaffolds of low-nM inhibitors of 15-PGDH. Proposed research aims to solve the structure of 15-PGDH in complex with new small molecule inhibitors or substrate. Computational approaches will be employed to interrogate substrate/inhibitor binding and the enzymatic mechanism. In Aim 2, we will define the cellular, protein and cytokine signaling networks that are regulated by 15-PGDH and that are engaged by 15-PGDH inhibitors to potentiate tissue regeneration and repair. The foundation of this aim includes the first demonstration of 15-PGDH activity in the brain, the identification of macrophages and microglia as major reservoirs of 15-PGDH expression in peripheral tissues and brain, respectively, and the discovery of cell and cytokine networks that respond to inhibiting 15-PGDH. We now propose to use single-cell RNA sequencing to determine the cell types that express 15-PGDH. Similar approaches will identify the cell-signaling network of induced cytokines and the cell types activated to express them. These studies will be performed in mice recovering from injury that have been treated with 15-PGDH inhibitors, along with appropriate controls. Finally, we will engineer macrophage- and microglia-targeted 15-PGDH knockouts to define the role of 15-PGDH expression in macrophages and microglia in mediating a conserved, cross-tissue response to PGE2 and 15- PGDH inhibitors. This data set will provide a foundation for future advancement of therapeutics targeting 15- PGDH and additional drug targets that modulate tissue regeneration.

NIH Research Projects · FY 2025 · 2021-05

The UT Southwestern Center for Translational Medicine’s KL2 Scholars Program has a record of supporting Scholars who have become leaders in clinical and translational research (CTR). Our KL2 program, with an ethos of continuous improvement based on metrics and changing needs, has been a model of unique and centralized CTR training in our Hub. Our training curriculum has been streamlined and modernized, and the exposure of our Scholars across all important domains has steadily increased. KL2 Scholars engage in mentored research training, take formal coursework and nano courses on emerging CTR topics. Scholars submit manuscripts and grant proposals to secure individual career development and/or research grants (K2K and K2R conversion) in a timely manner. Program evaluation guides us to restructure elements as needed to increase the range of those we train and broaden the disciplines and institutions from which the Scholars are recruited. The KL2 program plan proposed here is significant and innovative. We will strategically leverage our CTSA Program hub and institutional resources to broaden the KL2 program’s impact and provide CTR training to our expanding biomedical workforce.

NIH Research Projects · FY 2025 · 2021-05

The Center for Translational Medicine (CTM) at UT Southwestern is a collaboration among 7 academic institutions, 5 health care systems, 6 teaching hospitals and the North Texas community. Our mission is to improve health in local and global communities through innovation and education. Over the past two funding cycles, our CTSA catalyzed innovation and transformed the culture and landscape of our program Hub. We have trained >1,000 members of the translational workforce, co-led the formation of the Accrual to Clinical Trials CTSA network and established new translational technologies, methods, and processes critical to the translational process at every level. We engaged our local communities early on in the design and conduct of clinical research. We also formed new collaborative research services and education, and training programs designed to address the top health challenges of our community and the nation. Through these efforts, the CTM has generated considerable momentum toward advancing translational science propelled by a highly collaborative environment that is hard-wired for Team Science and Community Engagement. Over the next 5 years, we will optimize the organization of our CTSA Hub for more efficient translation of biomedical discoveries into interventions that will ultimately result in the improved health of both our local populations and, in collaboration with the CTSA Network, the broader U.S. population. We will discover, develop, demonstrate, and disseminate new informatics and artificial intelligence solutions to challenging problems in translation at all levels. In collaboration with Hub partners and relevant national CTSA networks, we propose to develop new methods and processes to further the integration of research into practice at the point of care. Building on our success, we are now poised to achieve the five key objectives of the CTSA Program and the Center for Translational Medicine with the following Specific Aims: Aim 1. Produce a well-trained, highly skilled, effective and knowledgeable Translational Workforce. Aim 2. Inculcate Community Engagement and Team Science. Aim 3. Integrate populations across the lifespan and socioeconomic spectrum as active partners in clinical and translational research. Aim 4. Promote innovation and new scientific Methods and Processes. Aim 5. Develop innovative Informatics solutions to overcome translational roadblocks. Aim 6. Team Efficiency: identify and recruit individuals with a range of skills, experience and viewpoints forming effective and agile teams to accomplish our goals. Impact: With our dynamic research and training environment in place, our Hub will have a powerful and sustained impact on the field of translational science. We will make a major leap forward in the scope, efficiency, and quality of clinical and translational research for the benefit of our Hub and the national consortium. We will collaborate with the Center for Leading Innovation and Collaboration, the Trial Innovation Network, and the Center for Data to Health to bridge the gap between scientific discovery and improved health.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY Radiation therapy is one of the major approaches for cancer treatment. Treatment planning, the process of designing the optimal treatment plan for each patient, is one of the most critical steps. If a treatment is poorly designed, a satisfactory outcome cannot be achieved, regardless of the quality of other treatment steps. Treatment planning in modern radiotherapy is formulated as a mathematical optimization problem defined by a set of hyperparameters. While there exists several quantifiable metrics to quantify plan quality and guide the planning process, these are simplified representations that cannot fully describe the physician’s intent. In addition, these metrics only measure plan quality from a population-based perspective, and cannot guide treatment planning to achieve the patient-specific best treatment plans. Hence, the best physician-preferred solution often sits in a gray area, only achievable by an extensive trial-and-error hyperparameter tuning process and interactions between the planner and physician. Consequently, planning time can take up to a week for complex cases and plan quality may be poor, if the planner is inexperienced and/or under heavy time constraints. These consequences substantially deteriorate treatment outcomes, as having been clearly demonstrated in clinical studies. Recently, the advancement in artificial intelligence (AI), particularly in imitation learning allows human- like decision making by observing a human expert’s actions and internally building its own decision-making system. In response to PAR-18-530, the goal of this project is to develop and translate an AI planner that mimics human experts’ behavior to generate a high quality plan. The AI planner will not replace human planners. Instead, the AI plan will be used as a starting point in the current planning process to improve plan quality and planning efficiency. The human planner’s actions on further plan improvement can feed back to the AI planner through continuous learning for its continuous evolution. We will pursue this goal using prostate cancer as the test bed through an academic-industrial partnership, jointing strong research and clinical expertise at UT Southwestern Medical Center with extensive commercial product development experience at Varian Medical Systems Inc. The following specific aims are defined. Aim 1: Model and algorithm development. We will collect experts’ behavior data in routine treatment planning and train the AI planner. Aim 2: System validation and translation. We will integrate the AI planner into Varian Eclipse treatment planning system and validate the system in a clinically realistic setting. The innovations include the use of a state-of-the-art AI imitation learning algorithm to solve a clinically important problem, the novel technological capabilities enabled by the developed system, as well as coherent translation activities to deliver new capabilities to end users. Deliverability is ensured by extensive preliminary studies and the partnership integrating complementary expertise and resources. Clinical translation of the AI planner will bring substantial impacts to radiotherapy by providing high-quality and efficient treatment planning to benefit patients, especially those in resource-limited regions.

NIH Research Projects · FY 2025 · 2021-05

Project Summary An Academic-Industrial partnership between scientists and clinicians at the University of Texas Southwestern (UTSW) Medical Center and Philips Research Hamburg and Philips North America (Philips) has been formed with the purpose of translating the novel molecular MR imaging method, Chemical Exchange Saturation Transfer (CEST), into a clinical tool for the assessment of Small Renal Masses (SRMs). CEST has already demonstrated feasibility in neurooncology applications. The management of SRMs is challenging because a substantial number of them are benign or indolent malignant tumors, leading to patient exposure to unnecessary morbidity from biopsies and surgery. Non-invasive distinction between benign/indolent tumors and aggressive tumors would allow for better risk stratification and patient management. While novel, CEST has already been adopted by certain clinical centers for assessment in neuro oncology. Recently, our group demonstrated the promise of CEST to the characterization of breast lesions. Here, we propose to broaden the application of CEST to renal oncology by addressing the inherent challenges of application of CEST in the abdomen and by optimizing CEST in patients with SRMs. Our hypothesis is that technical development of CEST-MRI allows observation of aggressive tumor microenvironment in SRM. Our goal is to advance CEST methodology to become a part of comprehensive SRM evaluation protocol. The goals of the project will be achieved via the following Specific Aims: Aim 1: To develop and optimize CEST methodology for SRMs on 3T human MRI system. This is technical development Aim, where we will evaluate several approaches addressing major issues associated with translation of CEST to kidney oncology applications: (i) improved resolution and fat separation, (ii) acceleration of acquisition using pseudo-cartesian and undersampled algorithms,(iii) motion correction and (iv) advanced Z-spectrum analysis methods. Aim 2: To evaluate the optimized CEST-MRI as a predictive biomarker of SRM hystology. The Aim's focus in on the translation of the CEST technology to SRM patients: (i) evaluation of the optimized in a group of SRM patients: (ii) correlation of the CEST-MRI measurements with histopathology results and correlation of CEST-MRI with glycogen deposition.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY By 2025, an estimated 1.6 billion people around the world will be hypertensive. This estimate may be higher given recently modified guidelines for hypertension that are inclusive of millions of additional patients. What is concerning is that almost 20% of all hypertensive patients are resistant to current therapies. Better understanding of the underlying mechanisms involved in the pathogenesis of hypertension is required in order to identify new and effective therapeutic strategies. Chronic renal inflammation is suspected to be a causal mechanism of resistant hypertension. The objective of this grant proposal is to examine the the control of renal inflammation using a model of chronic inflammatory disease. Systemic lupus erythematosus (SLE) is a chronic autoimmune inflammatory in which renal inflammation precedes the development of hypertension in SLE; therefore, it is an appropriate disease model to use to elucidate mechanisms involved in the inflammatory origins of hypertension. Endogenous neuro-immunoregulatory systems like the novel cholinergic anti-inflammatory pathway are involved in the normal control of excessive inflammation. Our data indicate that boosting this vagus nerve-to-spleen pathway via systemic pharmacological approaches reduces renal inflammation and blood pressure in an experimental mouse model of SLE. Based on this, we hypothesize that active neuroimmune pathways protect the kidney by suppressing renal inflammation and preventing the subsequent development of hypertension. In Aim 1 of this proposal, we will determine if central stimulation of the vagus nerve via both chemogenetic and pharmacological techniques reduces renal inflammation through the cholinergic anti- inflammatory pathway, and is antihypertensive in SLE. Studies in this aim will elucidate central nuclei involved in the regulation of renal inflammation that if left unchecked can result in hypertension in SLE. In Aim 2, we will determine whether inhibiting the cholinergic anti-inflammatory pathway by blocking its nodes of neurotransmission exacerbates SLE hypertension. Studies in this aim will determine critical neural/peripheral components necessary for proper neuroimmune regulation. In Aim 3, we will determine if an intrinsic renal anti- inflammatory pathway works in parallel with the spleen-centric cholinergic anti-inflammatory pathway. Studies in this aim will examine the anti-inflammatory potential of local acetylcholine in renal immune cells in hypertension- prone SLE mice using in vitro studies and innovative sniffer cell technoogy. Overall, the proposed studies will explain how homeostatic neuroimmune mechanisms control renal inflammation in health, as well as offer therapeutic options for controlling renal inflammation in hypertension and other chronic inflammatory diseases.

NIH Research Projects · FY 2025 · 2021-05

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. Although there is a dire need for new ways to combat this disease, the standard treatment for gliomas has not changed since 2005 and no new glioma medical therapies have been approved in the last decade. In response to this challenge, we have devised a new way to treat gliomas that have a mutation in a gene called IDH1. IDH1 mutations are present in 70-90% of lower grade gliomas and secondary glioblastomas, a highly aggressive subtype of glioma. We surveyed hundreds of drugs and discovered that a class of drugs that inhibit a particular metabolic pathway preferentially killed brain tumor cells with IDH1 mutations. Our proposal builds on this discovery by addressing three Specific Aims. Specific Aim #1 is to understand the molecular mechanisms through which IDH1 mutations increase sensitivity to inhibitors of this metabolic pathway. We will use cultured brain tumor cell lines that harbor or lack IDH1 mutations to test our hypothesis that the combined effects of IDH1 mutations and these inhibitors severely impair protein processing and lipid production in tumor cells, creating stress that ultimately triggers cell death. Specific Aim #2 is to use organoid models derived from human glioma tissue to assess whether the presence of an IDH1 mutation successfully predicts response to inhibitors of the metabolic pathway we propose targeting. Organoids represent powerful preclinical disease models because they allow us to test new therapeutic strategies in a cellular system that accurately reflects the makeup of human brain tumors. Specific Aim #3 is to use a mouse model of glioma to assess whether inhibiting the metabolic pathway we've identified to be important for IDH1 mutant brain tumor cells leads to desirable therapeutic responses, including a block in tumor growth and extension of host survival. These studies will clarify whether targeting this pathway is likely to provide benefit for human patients with IDH1 mutated brain tumors. Taken together, our work will outline and test a new treatment strategy for brain tumor patients that could be rapidly translated to the clinic if our studies are successful. Furthermore, our efforts may demonstrate that IDH1 mutations can be used to faithfully identify individuals whose tumors are poised to respond to the treatment strategy we are developing, thereby providing a way to design potential future clinical trials that have the greatest chance to provide benefit for glioma patients.

NIH Research Projects · FY 2025 · 2021-05

A small number of Rho family GTPases participate in a broad array of fundamental cellular behaviors. Specificity is possible due to spatial and temporal control of GTPase “activation”; Guanine exchange factors (GEFs) generate activated, GTP-bound GTPases with precise timing and localization, while specialized interactions with adhesion molecules, membrane domains and other localized structures specify GEF-GTPase interactions. GEF/GTPase circuits are complex, with localized feedbacks, multiple GEFs controlling one GTPase, and vice versa. To dissect this spatiotemporally regulated circuitry requires imaging, and new analytical techniques that can dissect causal relationships from imaging data. Following the intentions of PAR- 19-158 (Bioengineering Research Grants), we propose a multidisciplinary collaboration leveraging organic chemistry, protein engineering, imaging, and computer science to fudnamentally advance signal transduction imaging and analysis. As a biological testbed we will explore the role of GEF-GTPase interactions in cell protrusion, single cell migration and collective migration. We will develop a generalizable approach to GEF biosensors, and adapt our proven GTPase biosensors to image GEF and GTPase activities in the same cell. Because GEF-GTPase interactions are heterogeneous and complex, multiplexed imaging is necessary to quantify their relative dynamics. However, perturbation of cell behavior is especially problematic when using two biosensors in the same cell. We will therefore develop new biosensor designs that greatly reduce cell perturbation. Even the most precise imaging of overlapping molecular activations has not revealed causal relationships. We will therefore adopt the framework of Granger Causality inference, which was originally devised for financial market analysis, to extract causal connections and feedback interactions from imaging data. Numerous steps will be necessary to translate the existing concepts of Granger causality to the analysis of spatially and temporally distributed molecular processes. Most importantly, we will implement a schema for Granger causality inference in multivariate time series models that will capture spatial relations, and we will combine principles of high-dimensional statistical regression with approaches from control theory to estimate information flows between variables that are coupled by strong feedbacks. We will also develop a novel clustering approach that preserves the neighborhood topology of data in a high-dimensional feature space and in the Euclidian space of the cell outline to identify signaling microdomains. Finally, to test and confirm our hypotheses, we will use new photo-activatable and photo-inhibitable analogs of GEFs together with GTPase biosensors to control one protein while observing another. This research plan will produce biosensors with reduced perturbation, biosensor/optogenetic multiplexing capabilities, and image analysis/modeling approaches necessary to shed light on the network topology of nonlinear, spatiotemporally controlled signaling pathways. All tools will efficiently deployed to the community.

NIH Research Projects · FY 2025 · 2021-05

Project Summary Treatment advances in psychosis may be limited by the use of phenomenology-defined diagnoses based on symptomatic outcomes, rather than by neurobiological constructs monitored by quantitative characteristics. The Bipolar-Schizophrenia Network for Intermediate Phenotypes (B-SNIP) uses biomarkers to define psychosis subgroups with the goal of testing the advantages of B-SNIP biomarkers for diagnostic and therapeutic decisions, consistent with principles in the NIMH Strategic Plan (NSP). With >3000 phenotyped psychosis probands, relatives and healthy controls, B-SNIP has a multilevel biomarker library for psychosis and used that library to re-conceptualize psychosis subgroups as biomarker-defined Biotypes (B1, B2, B3), where B1 and B2 are the low cognition/high symptom groups and B3 shows lower symptoms and relatively normal cognition. We replicated Biotypes in a new sample, “forging a future where measures of an individual’s … neural and physiological state will form the basis of an increasingly specific and informative diagnosis” (NSP). In this grant we propose that B1, with its low cognition and low cortical activity, will respond uniquely to clozapine, a drug which will generate active cortical attractor networks in B1 to support symptomatic improvement. Clozapine is the most effective antipsychotic drug (APD) with unique clinical efficacy. It is the least used APD because its side effects are serious (neutropenia, myocarditis, seizures) and its administration complex. A predictive biomarker would allow targeting of cases most likely to respond and improve prognosis in psychosis. B-SNIP has shown that clozapine is associated with increases in EEG measures of alpha/theta power, and we identify this increase in time periods without stimulus processing requirements as intrinsic EEG activity (IEA), across all Biotypes. Because B1 cases express low IEA, clozapine’s action to increase EEG power will be normalizing for this psychosis subgroup, with increased cortical attractor states. Because B2 express accentuated IEA, clozapine is associated with more deviant IEA in B2. We propose to test B1 psychosis cases with clozapine vs. risperidone (n=40/group clinical trial completers), over a 6 week cross-titration (to therapeutic plasma levels) and a 9 week stable dose extension, predicting that the B1/clozapine group will respond significantly better, as measured with total PANSS, than the B1/risperidone group and also better than either B2 group. It is our hypothesis that the cortical attractor networks will be normalized and their function increased by the increase in intrinsic EEG activity.