Ut Southwestern Medical Center

NIH Research Projects · FY 2025 · 2022-09

The underlying mechanisms of brain stimulation in humans are poorly understood, especially at the level of gene expression. To address this gap in knowledge, we propose a series of three experiments that take advantage of the opportunity to obtain high-quality human neural tissue from neurosurgical patients in order to measure the impact of brain stimulation on gene expression. Our experiments will generate data to explicate changes at the level of gene expression that underlie brain circuit changes elicited by stimulation. Our study team has seven years of experience analyzing gene expression using an established pipeline for studying human cortical tissue from neurosurgical patients, including application of cutting-edge methods for measuring gene expression. These methods include single nuclei RNA-sequencing (snRNA-seq) and the exciting addition of single nuclei ATAC-sequencing (snATAC-seq) to understand stimulation-related changes in transcription factors and chromatin remodeling. Our hypotheses regarding specific gene classes were developed from our published data correlating gene expression changes with neurophysiological signatures (brain oscillations) linked with successful memory formation. In this proposal, our experiments address the complex problem of how stimulation alters neural circuits using three complementary approaches. First, we will use direct cortical stimulation in vivo immediately prior to resection of brain tissue in temporal lobectomy patients, followed by gene expression analysis. Our plans are supported by preliminary data showing differences in expression of immediate early genes (IEGs) following cortical stimulation, in line with predictions drawn from animal models. Second, we will build on techniques we have implemented for culture of human neural tissue (from neurosurgical patients) to measure gene expression changes elicited by chronic ex vivo stimulation. This experiment will elucidate the temporal dynamics of gene expression in the setting of stimulation, including transcription factor changes, using our experience with time series modeling of gene information. Finally, we will use multi-electrode arrays (MEAs) to measure the impact of ex vivo stimulation on networks of co-firing neurons, directly testing models of stimulation-induced changes in local circuits. We will connect these electrophysiological measures with gene expression changes elicited by stimulation. This experiment builds on our published work studying network activity in single unit recordings in humans, as well as our preliminary data demonstrating the ability to record electrophysiological signals from human neural tissue in culture. The stimulation parameters were developed to be aligned across in vivo and in vitro experiments to facilitate comparison. Taken together, our experiments will provide ground-breaking data elucidating the genetic underpinnings of how brain stimulation elicits neuromodulation. The experience of research team and proven record in publishing data using neurosurgical tissue specimens supports our expectations of success.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Necroptosis is a caspase-independent type of programmed necrosis. The activation of the necroptosis signaling cascade is implicated in the pathogenesis of various human diseases, including cancer, inflammatory bowel disease, liver injury, pancreatitis, neurodegenerative disorders, and a diverse range of viral, bacterial, and fungal infections, including SARS-CoV-2. The necroptosis signaling cascade is mediated by the sequential activation of RIPK1 and RIPK3 kinases downstream of pro-inflammatory ligands such as TNF or microbe-associated molecules. MLKL is a pseudokinase that tetramerizes upon phosphorylation by RIPK3 to form water-permeable pores that drive cell membrane rupture. This pore formation stage leads to the necrotic phenotype of necroptosis. It is also a critical point of cell fate determination, as necroptosis execution can be halted and reversed at the MLKL stage. The mechanisms regulating MLKL activation and execution of this type of programmed necrosis are poorly understood. Here, we will fill in the gaps of our understanding of the molecular mechanisms that regulate MLKL activation, tetramerization, and execution of necroptotic cell death via phosphorylation and ubiquitination. We aim to determine the mechanistic roles of the MLKL post-translational modification events in promoting or suppressing MLKL tetramerization and identify the enzymes regulating MLKL-driven necrotic cell death via these events. We also aim to determine which structural factors are required downstream of MLKL to execute the necroptotic cell death. Finally, to validate the roles of these enzymes and factors in mediating necroptosis in vivo, we will test how their genetic knockouts affect sensitivity to Vaccinia virus infection, contributing to the future development of strategies for enhancing host anti-viral response. Overall, this project will significantly expand our understanding of the cellular signaling mechanisms upstream and downstream of MLKL at the necroptosis execution stage and pave the way for future anti-microbial therapies, as well as treatments for diseases that involve necroptosis execution.

NIH Research Projects · FY 2025 · 2022-09

The prevalence of hypertension and related cardiovascular disease is higher in the residents of Louisiana, especially in Black populations, compared to the US general population. The overall objective of the proposed study is to test the effectiveness, implementation, and sustainability of a church wellness coordinator (CWC)–led multifaceted intervention, compared to enhanced usual care, for hypertension control in predominantly Black communities. A cluster randomized trial with an effectiveness-implementation hybrid design will be utilized to: 1. test the clinical effectiveness of the multifaceted implementation strategy on blood pressure (BP) control; 2. assess the implementation outcomes (acceptability, adoption, feasibility, fidelity, and cost-effectiveness) of the intervention; 3. study the sustainability of this multifaceted implementation strategy for clinical effectiveness and implementation outcomes in a post-intervention follow-up study; and 4. examine the communitywide impact of the intervention on mean BP and hypertension control. The Exploration, Preparation, Implementation, and Sustainment framework has been used to guide the development of the multifaceted implementation strategy. We have established a partnership with churches in predominantly Black neighborhoods and assessed the needs, barriers, and facilitators of hypertension control in community members, church leaders, CWCs, and healthcare providers. The intervention is developed using a community-based participatory research approach and is rooted in church-based wellness programs developed by and for church congregations. The evidence-based interventions include community-based BP screening, lifestyle modifications, and antihypertensive medication treatment. The CWCs are community health workers who will be trained on BP screening, hypertension care coordination, and health coaching. They will conduct community-based BP screening, coordinate care for patients with hypertension, assist patients with home BP monitoring, deliver discounted and free antihypertensive medications to patients, and conduct health coaching on lifestyle changes and medication adherence. Participation of faith-based organizations, Federally Qualified Health Centers and other primary care organizations, community pharmacies, and local health departments will strengthen this community intervention program. We will recruit 42 churches in predominantly Black communities in New Orleans and 28 eligible community members from each church. We will randomly assign 21 churches to the CWC-led multifaceted intervention and 21 to enhanced usual care. The multifaceted intervention program and enhanced usual care will last for 18 months. The primary clinical effectiveness outcome is the mean change of systolic BP from baseline to 18 months, and the primary implementation outcome is fidelity to the multifaceted intervention. The proposed cluster randomized trial has 90% statistical power to detect a group difference in mean systolic BP change of 5.8 mm Hg. This study will generate evidence on an effective, scalable, and sustainable strategy for hypertension control in communities.

NIH Research Projects · FY 2025 · 2022-09

Cellular behavior is regulated by a varied signaling mechanisms, of which many depend on molecular dynamics precisely organized in space and time. The transient positioning and kinetics of molecular events is lost in bulk biochemical analysis and in single cell proteomics. Understanding such factors requires visualization and quantitative analysis of molecular events in living cells and tissues, now made possible by combining molecular probe design, high-resolution live cell microscopy development and computational image analysis. Quantitative analysis of live cell signaling networks has been accessible only to specialized teams who unite these capabilities. The Center for Cell Signaling Analysis enters Year 4 building upon substantial progress made in Years 1–3, continuing efforts to democratize advanced methods and put these tools in the hands of scientists who have not devoted their careers to imaging. We will continue to develop and disseminate a user-friendly and integrated pipeline that combines 1) biosensors, optogenetics and chemogenetics 2) modular, high-speed, and high-resolution light-sheet microscopes, and 3) image analysis and computational modeling to derive signaling network architecture, including the causality and kinetics of connections. The Technology Development Projects (TDPs) in Year 4 will build upon these established foundations and focus on further advancing: i) Open-source software for the analysis of subcellular signal transduction in 2D and 3D live cell time-lapse data using advanced methods in statistical time series analysis. ii) Optogenetics, chemogenetics, and biosensors based on alternate approaches with complementary capabilities and reduced perturbation of signaling. iii) Multiple modular, cost-effective, and high-resolution 3D light-sheet microscopes that can be assembled rapidly by non-experts and deliver ~220 and ~450 nm lateral and axial resolution. Through our collaborative Driving Biological Projects (DBPs), we will extend earlier efforts to iteratively refine and improve our image analysis methods, probes, and imaging platforms. We also propose a strong dissemination component that continues and expands activities from the first three years, maximally leveraging existing infrastructure, including imaging facilities, Addgene, GitHub, and Applied Scientific Instrumentation. We will continue to provide extensive training (in person, remote, topic-driven courses, and YouTube tutorials) and centrally organize Center outputs on a comprehensive and continuously updated website.

NIH Research Projects · FY 2025 · 2022-09

Project Summary South Asian individuals (SAs) (individuals from India, Pakistan, Bangladesh, Sri Lanka, and Nepal) have markedly increased risk of atherosclerotic cardiovascular disease (ASCVD), premature insulin resistance, and higher risk of type 2 diabetes compared with non-Hispanic White individuals and other groups. The global cardiovascular community has officially recognized SA ethnicity as a “risk-enhancing factor” in the 2018 ACC/AHA Prevention Guidelines2 as well as in the QRISK2/3 risk calculator used in the U.K. Reducing ASCVD risk and mortality from ASCVD in SAs is a clear priority and unmet need. Our team has demonstrated that advanced measures of lipid metabolism (protective and adverse) are superior to traditional risk factors and conventional lipids (non-HDL-C, HDL-C, and triglycerides) in predicting ASCVD risk. These advanced measures have been studied primarily in those of European and African descent but not among SAs. Our overall goal is to improve cardiometabolic risk in SAs. The specific objective of this project is to determine whether advanced measures of lipoprotein metabolism explain the excess cardiometabolic risk in SAs. We will leverage the NHLBI-supported Mediators of Atherosclerosis in SAs Living in America (MASALA), the largest longitudinal cohort of U.S. SAs with extensive cardiometabolic phenotyping (N=1,164) and compare these findings to similarly phenotyped White, Black, Hispanic, and Chinese participants in the Multi-Ethnic Study of Atherosclerosis (MESA, N=6814). We will utilize the UK Biobank to ascertain metabolomic signatures of SAs and incident ASCVD and diabetes (N=8,762 SAs vs. 472,780 Europeans). Aim 1: Determine the association between advanced measures of lipoprotein metabolism and metabolic phenotypes in SAs. Aim 2: Determine the association between advanced measures of lipoprotein metabolism and subclinical plaque prevalence and progression and incident ASCVD in SAs. Specific Aim 3: Determine the metabolomic signatures of South Asians compared to non-SAs with respect to cardiometabolic phenotypes. The proposed studies are expected to provide critical insights into the link between advanced protective and adverse measures of lipoprotein metabolism and excess cardiometabolic risk in SAs and may eventually lead to better clinical biomarkers specific to SAs as well as to novel interventions targeting these lipoprotein markers in SAs. Given the excessive burden of ASCVD and diabetes in SAs, this proposal may have a large public health benefit to this less studied but high-risk ethnic group.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Abstract The current understanding of the pathophysiologic and socio-economic processes that initiate CKD and the paths by which they progress to ESRD are poorly understood, resulting in limited treatments. There is a compelling unmet need for prospective studies to accurately phenotype patients with CKD using a combination of kidney biopsy, clinical, biologic, and socioeconomic factors to develop individualized care to improve patient outcomes. Over the past four years, at the local level we assembled a highly skilled, interdisciplinary team of clinical researchers, physician-scientists, clinicians, research nurses, patient advocates and a Community Advisory Board. This team designed, implemented and sustained a highly successful, ethically sound and safe recruitment site (RS) for the Kidney Precision Medicine Project: 1) We performed the very first KPMP consortium kidney biopsy in September 2019 and were the first RS in the consortium to perform 30 kidney biopsies, successfully enrolling patients with a clinical diagnosis of hypertensive or diabetic chronic kidney disease and yielding sufficient tissue for research protocols as well as for pathological diagnosis; 2) We collected and linked clinical, biological and socioeconomic data of the biopsy participants; 3) We focused our efforts on African- American and Latinx populations at a very high rate in order to address the disproportionate burden of CKD as well as the historical lack of inclusion of these populations in cljnical research, mandated by the NIH, completing 22 out of 11 (73%) of biopsies among these groups. At the consortium level our team members have made meaningful contributions to the major organizational and operational aspects of KPMP. The main goal in this proposal will be population identification, patient recruitment and obtaining clinical phenotype data, biosamples and kidney biopsies. To accomplish this goal we will 1) Identify, recruit, enroll, and biopsy 34 patients annually from 3 large HS serving more than 51,996 patients with CKD and diabetes and/ or hypertension and follow patients longitudinally; 2) Implement ethically-sound, systematic quality-assured processes to obtain clinical and demographic data, bio-samples and kidney biopsies from patients with CKD in a longitudinal clinical study; and 3) Develop and implement patient-centered processes to promote long-term engagement and retention of study participants in KPMP. Our successful track record as a recruitment site for procurement of kidney tissue from patients with CKD attributed to diabetes or hypertension from a large, diverse patient population makes us an ideal site for the continuation of the KPMP. Our ability to recruit patients from multiple health systems with novel informatics tools, an ethically rigorous approach to consenting for kidney biopsy and coupled with a rigorous and safe methodology is a powerful combination we bring to the KPMP. Our innovative approach to this FOA will not only provide the data to generate new knowledge to improve both the diagnosis and treatment of CKD, but also serve as a model for future kidney precision medicine initiatives and their expansion over time.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Intestinal cancer is the 3rd most common malignancy and cause of cancer-related deaths in both men and women. Unfortunately, recent advances in our understanding of the underlying intestinal biology and cancer- related pathways have not translated to significantly improved patient outcomes. While numerous animal models of intestinal cancer have been developed, mostly based on misregulation of the β-catenin pathway, some key features of human cancers (particularly tumor mutational burden) now known to be critical for tumor progression and therapy response, have not been adequately modelled or investigated in animal models of intestinal cancer. More recently, genome sequencing efforts led to the discovery of an intestinal cancer mutator phenotype where single amino acid substitutions within proofreading domains of the housekeeping DNA polymerases result in the highest mutation rates described in human cancers (ultramutation). Unlike human cancers, genetically-engineered animal models exhibit very low mutation rates, limiting their utility for studies of intratumoral heterogeneity and competition, immune responses, and immune checkpoint therapies, now known to be essential aspects of human tumor biology. We propose to overcome these limitations in intestinal cancer animal models by building upon 1) a strong track record in the generation of cancer animal models and novel genetic tools for their development 2) a well-characterized conditional PoleP286R allele that we previously used to develop a robust model of endometrial cancer and 3) expertise in genomics, inflammation, intestinal cancer, and mouse models of intestinal disease. These models will be useful not only to recapitulate POL-driven intestinal cancers, but also to humanize any intestinal cancer mouse model with respect to mutational burden. This proposal is submitted in response to PAR-20-131 to expand and improve the development of mammalian models for translational cancer research.

NIH Research Projects · FY 2026 · 2022-09

Lower urinary tract dysfunction (LUTD) is a constellation of human-reported urinary indications, including urgency, intermittent and weak urinary stream, incomplete bladder emptying, and increased voiding frequency. A major cause of male LUTD is benign prostatic hyperplasia (BPH), which is medically managed with five alpha reductase inhibitors or alpha-adrenergic receptor antagonists. Neither drug reduces symptomatic progression by more than 34% and most of these elderly men have no option but surgery. Prostatic obstruction of urine flow can lead to bladder detrusor overactivity (DO), a poorly understood disease with largely ineffective therapeutic options. The medical management of LUTS due to prostate and bladder dysfunction has seen little improvement over the past 40 years because we have failed to capture the cell type-specific molecular alterations that would provide actionable therapeutic targets. This proposal will address fundamental barriers to deriving molecular mechanisms of human LUTD. In Aim 1 we will produce multi-omic data on cell type-specific molecular changes in human LUTD. We will link the data to a tissue repository managed with OpenSpecimen software, giving researchers searchable access to >3,000 clinically annotated normal and diseased human specimens. In Aim 2 we will engineer new mouse strains to achieve Cre expression in specific bladder, urethra, and prostate stromal cells. It is not possible with existing mouse strains to overexpress or knockout a gene in a bladder stromal cell without introducing the same genetic change in prostate and urethra, or vice versa. Accordingly, the unique contributions of prostate and bladder to urinary voiding cannot be isolated. We will overcome this problem by creating new mouse strains with selective inducible Cre expression in fibroblasts and smooth muscle. Finally, we will ensure that all resources are FAIR (Findable, Accessible, Interoperable, and Reusable) by incorporating all methods, tools, data and protocols into the NIDDK ATLAS Data Center. The proposed resources are significant because they establish foundational bedside to bench resources to generate and test hypotheses about LUTD mechanisms in human tissues and rationally designed mouse models.

NIH Research Projects · FY 2025 · 2022-09

Project Summary / Abstract Despite the success of immune checkpoint blockade (ICB) in many tumor types, breast cancers have shown limited responses. Antigen presenting cells (APCs) are critical to initiate anti-tumor immunity and for efficacy of ICB, and are known to be defective in breast cancers. Importantly, APCs need to be activated through pathways such as the CD40 pathway in order to promote anti-tumor activity rather than immune tolerance. We demonstrated that CD40 agonists synergize with Flt3 ligand (Flt3L) which is a growth factor that promotes differentiation of DC1 cells which are important for antigen presentation, and anthracycline chemotherapy to eradicate triple negative breast cancers in mouse studies. Based on this, we hypothesize that combining chemotherapy with a CD40 agonist and Flt3L enhances antigen presentation and increases long-term adaptive immunity, thereby improving triple negative breast cancer (TNBC) disease control. In order to test this hypothesis, I propose: Aim #1: To assess safety, clinical activity, and immunologic efficacy of CD40 agonist in combination with Flt3L and anthracycline chemotherapy (triplet therapy) in patients with metastatic TNBC (mTNBC) and identify biomarkers of response and resistance. I will be Lead Principal Investigator on a phase 1 pilot trial of CD40 agonist + Flt3L + pegylated liposomal doxorubicin in patients with mTNBC. Longitudinal tissue samples will be collected and analyzed to study pharmacodynamics, biomarkers of response, and resistance mechanisms. Aim #2: To discover mechanisms of response and resistance to triplet therapy using syngeneic and humanized mouse models, including tumors reflecting different TNBC subtypes. 4T1-HA syngeneic model will be used to study how different agents in the CD40 agonist + Flt3L + pegylated liposomal doxorubicin regimen contribute to response and what immune cell types are mediating response, with a focus on CD8 T cells and DC1 cells. We will additionally study how underlying TNBC subtype impacts response to triplet therapy using syngeneic murine and humanized patient derived xenograft models. Results of this work will guide future development of this combination in TNBC and other solid tumors. I am an Assistant Professor at UT Southwestern (UTSW) Medical Center and the principal investigator on this proposal. I am a breast medical oncologist focused on improving outcomes of breast cancer patients through immunotherapy. To most effectively advance the field, I plan to integrate clinical investigation of agents stimulating antigen presentation with patient tissue based translational science and preclinical studies. To supplement my background in immune monitoring, this proposal details a training plan under the mentorship of translational breast cancer expert Dr. Carlos Arteaga and cancer immunologist Dr. Yang-Xin Fu. This is further supported by an expert advisory committee, relevant coursework, and the resources and supportive environment of UTSW to help me transition to independence as a physician scientist focused on breast immuno-oncology.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY/ABSTRACT Intracranial recordings in patients undergoing neurosurgical interventions provide a unique opportunity to directly access, study, and learn about both normal human brain function and neuropsychiatric disease. Because of their value, the NIH has made significant investments in this line of research through the BRAIN Initiative and has likewise required public archiving of data through the Data Archive for the BRAIN Initiative (DABI). With the accumulation of data from over 500 subjects, DABI presents a unique opportunity to conduct large scale studies using data from multiple sites and investigations. Moreover, with the inclusion of both neurophysiological and imaging data (including anatomic and connectivity-based imaging), DABI has the potential to address important questions about functional-anatomic relationships in human neurophysiology and sources of variability across age, disease, and anatomy. There is a strong emerging yet underexplored literature that neural oscillation patterns relate to brain morphometry, yet the tools to explore this with greater spatial precision and spectral sensitivity are currently unavailable. While the multimodal nature of DABI data has the potential to significantly impact such questions, the data is not currently in a form that makes it easily accessible or analyzable. The BRAIN integrated Resource for human Anatomy and Intracranial Neurophysiology, B(RAIN)2, has an overall aim of creating a spatially integrated and standardized dataset that will enable such large scale studies. In Aim 1, we will (1) identify and curate data for inclusion in B(RAIN)2 based on required data elements (as well as solicit additional archiving from BRAIN funded investigators), (2) perform standardized neurophysiological signal processing and anatomic localization, (3) perform standard anatomic image processing, based on the Human Connectome Project framework and established quality control measures, and (4) transform all data into a standard space for large scale analyses. To ensure high impact and continued growth of B(RAIN)2, in Aim 2, we will share, document, and define pipelines for continued data integration, including webinars and training modules and providing support to potential users. Finally, in Aim 3, we will conduct a demonstration project to highlight the power of B(RAIN)2, investigating the relationships between motor cortical beta power and peak frequency as a function of cortical thickness, connectivity, and disease. The proposed work will enable investigators to harness the power of intracranial physiology and neuroimaging collected across BRAIN-funded sites to increase the power and impact of these valuable and relatively rare signals. Future investigators will use the B(RAIN)2 standardized metrics across anatomy and physiology in both native and standard space to perform novel analyses and gain unique insights into anatomic contributions to variability in human neural oscillator signals.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY One in 3 pregnant women with chronic hypertension (CHTN) deliver preterm from complications of superimposed preeclampsia or fetal growth restriction (FGR). The specific aims are: 1) prospectively evaluate the placental longitudinally via functional magnetic resonance imaging (MRI) in healthy pregnant women and pregnant women with CHTN; 2) extract radiomics texture features in normal and hypertensive placentas and 3) execute technical refinement toward automated segmentation for regions of interest to delineate patterns in placental abnormalities. We will study 20 normal pregnant women and 40 pregnant women with CHTN across gestation. MRI will be performed in the early and late second trimester to assess perfusion via arterial spin labeling (ASL), diffusion via intra-voxel incoherent motion (IVIM) diffusion weighted imaging (DWI) and DWI- derived apparent diffusion coefficient (ADC) mapping, and oxygenation via T2* from blood-oxygen level dependent (BOLD) MRI. Radiomics texture features also be extracted from MR imaging and automated segmentation applied for more subjective placental assessment. This career development award will establish Dr. Herrera as a clinical investigator focused on non-invasive imaging assessment of placental development and dysfunction. The award will provide her with the support needed to develop expertise in 3 areas: 1) clinical research 2) application and interpretation of advanced imaging modalities, and 3) technical skills of post- processing and deep learning. To achieve these goals, Dr. Herrera has assembled an interdisciplinary team with expertise in clinical investigation, placental imaging, and post-processing and automated segmentation techniques. Her primary mentor, Dr. Catherine Spong, has a long track record of successful clinical, translational, and basic science research including over 270 peer review publications and mentoring faculty to become independent investigators. The focus of this proposal is on the human placenta. Despite 150+ years of placental research, the role of the placenta in the pathology of pregnancy-related disease remains poorly understood. Understanding normal placental development and changes present in placental disease will provide a foundation to facilitate clinical prediction of pathologic conditions during pregnancy and provide an opportunity to test interventions. In this study, we will address the unmet clinical need to characterize both normal placental development and placental dysfunction in vivo. We have chosen to compare normal pregnant women with pregnant women with chronic hypertension as these women are increased risk for placental- mediated disease. Successful completion of this project will allow for better understanding of the placenta in normal pregnancy and pregnancy affected by chronic hypertension. We will also correlate our results with clinical outcomes of preeclampsia and FGR. Given the increased risk of superimposed preeclampsia and FGR in pregnant women with chronic hypertension, we anticipate from these results additional insights and novel hypotheses regarding potential radiomic biomarkers for the prediction of preeclampsia and FGR.

NIH Research Projects · FY 2025 · 2022-09

Project Abstract Glioblastoma (GBM) is one of the most lethal human cancers. Standard GBM treatments, such as radiation (RT) and temozolomide (TMZ), exhibit poor efficacy with a lack of a durable response. These agents promote oxidative stress in cancer cells, which is a known metabolic liability of GBM. However, the efficacy of these treatments is limited by neurotoxicity and upregulation of tumor escape pathways that detoxify reactive oxygen species. There is an urgent need for new pharmacological agents that effectively target the redox stress pathway in GBM cells while sparing adjacent normal tissue. My long-term goal is to become an independent physician-scientist neuro-oncologist focused on improving GBM therapy. In this proposal, I use my discovery of a cysteine susceptibility pathway in glioma, whereby cysteine-promoting compounds induce glucose dependence, mitochondrial toxicity, and H2O2 production, to define the mechanism and functional relevance of this pathway in pre-clinical models. I will test the central hypothesis that high levels of intracellular cysteine induce glucose dependence in glioma, and the combination of cysteine compounds with ROS-promoting treatments is an effective strategy to improve survival in mouse models of GBM. I will identify the metabolic flux pathways altered by cysteine in glioma (Aim 1a) and determine the role of mitochondrial electron transport chain flux (Aim 1b) and hypoxia and glycolytic flux (Aim 1c) in contributing to cysteine-mediated glucose dependence. Using mouse models of GBM, I will test the efficacy of cysteine compounds in combination with ROS-promoting interventions (RT, TMZ, and the glucose-lowering ketogenic diet) on GBM metabolism, growth, and survival, using 18F-fluoropropyl-homocysteine positron emission tomography as a biomarker for cysteine metabolism (Aim 2a). I will determine the effects of H2O2 modulation on cytotoxicity of cysteine compounds and ROS-promoting interventions (Aim 2b) in vivo. These aims will create a new paradigm that uses two synergistic metabolic therapies that can be rapidly translated into early-phase clinical trials in GBM. I am an Assistant Professor in Neurology within the Division of Neuro-Oncology at Weill Cornell Medicine (WCM), and I have outlined a 5-year plan that expands on my background studying GBM metabolism. I have an outstanding mentor, Dr. Lewis Cantley, who is an expert in tumor metabolism and has enabled translation of his work and mentees’ work to clinical development. My career advisory committee includes Drs. Lewis Cantley (primary mentor), Navdeep Chandel, Pedro Lowenstein, Naga Vara Kishore Pillarsetty, Howard Fine, and Matthew Fink. They are internationally recognized experts in science and medicine and will provide mentorship and support to attain scientific independence. I will also have unparalleled institutional support from WCM, which is at the forefront of precision medicine in cancer and is heavily invested in career development for junior physician-scientists. This research and training environment will enable me to achieve my goals of securing NIH R01 funding in the future. 1

NIH Research Projects · FY 2026 · 2022-09

Abstract Autosomal dominant polycystic kidney disease (ADPKD) is amongst the most common monogenetic disorders, with an estimated prevalence of 12.5 million people worldwide. Unfortunately, >50% of ADPKD patients develop end-stage renal failure and treatment options are still limited. Recent studies point to reduced PKD1 gene dosage as the disease mechanism for many ADPKD patients. This revelation opens the door to an exciting possibility that reversing PKD1 decline may arrest ADPKD in these individuals. However, despite the transformative potential, no PKD1-targeting drugs are under clinical investigation. Our goal in this application is to provide a scientific roadmap for developing a novel PKD1-boosting therapeutic approach. We propose a novel idea that along with the germline mutation, inefficient translation of mRNAs produced by the non-inactivated PKD1 allele also contributes to the lower PKD1 dosage and aggravates ADPKD. This idea emanates from our preliminary studies, where we found that deleting the cis-inhibitory miR-17 motif from the PKD1 3’- UTR is sufficient to improve mRNA translation and to raise PC1 levels. In turn, this approach ameliorates cyst growth in mice and in vitro human ADPKD models. Based on these promising observations, we propose: (1) using genetic proof-of-principle approaches to study the impact of PKD1 derepression in ADPKD mouse and human models, and (2) test the feasibility of oligonucleotides that outcompete and prevent miR-17 from binding to PKD1 as novel therapeutics to raise PC1 levels.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT: Effector CD8+T cells that promote anti-tumor response become dysfunctional and exhausted. However, the key mechanisms that define the delicate balance between effector vs. exhausted state of CD8+T cells remain elusive. Our preliminary studies demonstrate that in early-stage tumors, sumoylation of the T-box transcription factor, T- bet, facilitates its association with Zbtb42, a member of the ThPOK family of transcription factors. The Zbtb42/T- bet complex promotes the effector function and anti-tumor activity of CD8+ tumor-infiltrating lymphocytes (TILs). Conversely, in advanced tumors, the ubiquitin ligase Trim37 is upregulated in exhausted CD8+TILs, which targets Zbtb42 for degradation and disrupts the Zbtb42/T-bet complex. Importantly, CRISPR-Cas9-mediated inhibition of Trim37 rescues exhausted CD8+TILs and restores their effector function. These key findings led us to hypothesize that ubiquitination and sumoylation of the Zbtb42/T-bet complex are critical molecular events that dictate the effector vs. exhaustion of CD8+TILs, which can be therapeutically targeted. In Aim1, we will determine the mechanism by which the Zbtb42/T-bet complex promotes effector CD8+T cell function and anti-tumor response. We will use newly generated Zbtb42-/- and T-bet-K208R-KI mice to investigate how sumoylation of T-bet at Lys(K)-208 facilitates the formation of the Zbtb42/T-bet complex via the SUMO interacting motif (SIM) within Zbtb42. Further, we will delineate the mechanism by which the Zbtb42/T-bet complex co-operatively binds to and transactivates the IFN-γ promoter. In Aim 2, we will investigate how Trim37, which is upregulated in exhausted (PD1+Tim3+) CD8+TILs in advanced tumors, binds to Zbtb42 via its meprin and TRAF homology (MATH) domain and targets Zbtb42 for ubiquitination at K164. Using newly generated Trim37-/- mice, we will examine how disruption of the Zbtb42/T-bet complex leads to the inhibitory transcriptional profile of exhausted CD8+T cells in advanced tumors. In Aim 3, we will target the Zbtb42-Trim37 pathway in CAR-T cells to promote tumor regression. We will test the therapeutic potential of blocking Zbtb42 ubiquitination in CAR-T cells against carcinoembryonic antigen (CEA) in the MC38 and a patient-derived xenograft (PDX) colon cancer model. Completion of these studies will lead to 1) discovery of the novel Zbtb42/T-bet complex that is critical for effector CD8+T cell function, 2) determination of how Trim37-mediated ubiquitination disrupts this complex leading to alternate transcription profile in exhausted CD8+TILs, and 3) evaluate the means to target the Zbtb42/T-bet- Trim37 pathway to overcome the current limitations of CAR-T cell therapy for solid tumors.

NIH Research Projects · FY 2024 · 2022-09

Project Summary Autism spectrum disorder (ASD) is a neurodevelopmental disorder that affects 1 in 54 children and exhibits significant phenotypic and genetic heterogeneity. Mutations in UBE3B, which encodes an E3 ubiquitin ligase, have been identified in patients presenting with intellectual disability, lack of speech, and ASD. The specific mechanism through which disruption of UBE3B, and subsequent dysregulation of its substrates, leads to neurodevelopmental abnormalities is unknown. Our group has previously shown that a Ube3b constitutive knockout mouse model exhibits a complete loss of vocalization and defects in nest building, as well as reduced dendritic complexity, length, and spine density. In this proposal, I will investigate the neuronal function of UBE3B by characterizing changes in neuronal activity following loss of UBE3B and identifying its neuronal substrates. I will apply complimentary electrophysiological and biochemical approaches using a brain-specific conditional Ube3b knockout mouse model (cKOnestin). I will characterize the electrophysiological properties of cortical neurons from cKOnestin mice, by evaluating their basal properties, including intrinsic excitability and rheobase current, and both short-term and long-term synaptic plasticity. Furthermore, I will identify the neuronal substrates of UBE3B through analysis of protein levels, protein-protein interactions, and ubiquitination status. I will also assess the UBE3B-mediated modifications of substrates, including the site of ubiquitination and the ubiquitin chain topology. Successful completion of the proposed aims will provide new insights into the interplay of ubiquitin signaling and neurodevelopment, as well as advance our knowledge of the specific pathogenic mechanisms underlying neurodevelopmental disorders. The exceptional research environment at UT Southwestern Medical Center, combined with the collective expertise of the mentorship team, will provide excellent training. Goals for this fellowship training include becoming knowledgeable in the relevant literature and attaining proficiency in both experimental procedures and communicating scientific information to varied audiences. The skills obtained through these goals will build the foundation for an independent and successful lead investigator in scientific research.

NIH Research Projects · FY 2025 · 2022-09

Rachel Leon, MD, PhD is a Neonatal-Perinatal Medicine physician with a PhD in neuroscience at UT Southwestern Medical Center (UTSW). Her goal is to become an independently funded investigator with expertise in neuroplacentology, a field that focuses on elucidating mechanisms of placental influence on fetal brain development. She plans to study placental and cerebrovascular hemodynamics in fetuses with congenital heart disease (CHD) using advanced imaging techniques. In pregnant women with fetuses diagnosed with left or right ventricular outflow tract obstruction CHD (LVOTO and RVOTO, respectively), and healthy controls, her specific aims are to 1) evaluate placental perfusion longitudinally and determine associated differences in placental size and histopathology, 2) determine the impact of placental perfusion on cerebral autoregulation from the fetal period to the early postnatal adaptation, and 3) determine how placental perfusion affects the trajectory of regional brain growth. Dr. Leon will combine arterial spin labeling magnetic resonance imaging (MRI) to determine placental perfusion at two timepoints in pregnancy with pathologic evaluation of placentas to explore histopathologic underpinnings of perfusion abnormalities. Using serial Doppler ultrasound of the middle cerebral and umbilical arteries, as well as postnatal cerebral near infrared spectroscopy and blood pressure, she will determine the maturational changes of cerebral autoregulation from prenatal to postnatal life, and how this relates to placental perfusion. She will perform fetal brain MRI twice prenatally and again in the early postnatal period to determine the relationship of placental perfusion to the trajectory of regional brain growth. Dr. Leon’s innovative approach to studying the CHD placenta-brain connection will elucidate possible pathophysiologic mechanisms for impaired brain development in the CHD population, a necessary first step for the discovery of targeted fetal interventions. Dr. Leon has assembled a multidisciplinary team of mentors and collaborators with expertise in key areas: cerebral autoregulation (Lina Chalak, MD), advanced placental and fetal imaging (Diane Twickler, MD and Ashok Panigrahy, MD), placental physiology (Catherine Spong, MD and Dinesh Rakheja, MD), and CHD (Mohammad Tarique Hussain, MD, PhD). The UTSW hospital system and its strong clinical research operation are the ideal environment to conduct the proposed studies with their robust Fetal Heart Program, a dedicated Center for Translational Medicine, and a strong record of clinical research participation. Dr. Leon’s Career Development Plan includes a comprehensive strategy to address the specific key training goals that will allow her to establish her research program, including 1) gaining expertise in advanced fetal brain and placental MRI techniques and their correlation to pathophysiology, 2) developing expertise in the maturation of cerebral autoregulatory mechanisms and analytical tools to evaluate these dynamic signals, and 3) developing leadership skills for clinical research involving maternal-fetal dyads. Together with her scientific aims, these goals will provide the skills necessary for Dr. Leon to build her independent research program in neuroplacentology.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract Chemical modification of RNAs opens another avenue to regulate gene expression at the post-transcriptional level. Many mRNAs are modified with N6-methyladenosine (m6A), and controlled modification is important to maintain proper RNA function throughout its life cycle, including processing, translation, splicing, and degradation. METTL3/METTL14 RNA methyltransferase complex is responsible for creating m6A marks on many mRNAs. The catalytic activity of the METTL3/METTL14 complex is essential to most of its known functions. Dysregulation of METTL3/METTL14 activity has been linked to many types of cancer. In numerous types of malignancies, hyperactivity of the METTL3/METTL14 complex promotes disease. Therefore, potent and specific small molecule inhibitors of METTL3/METTL14 are likely to have therapeutic benefit. Moreover, the mechanisms through which a change in METTL3/METTL14 activity influences various biological processes including oncogenesis need to be investigated with more rigor. Chemicals that can specifically switch off METTL3/METTL14 on demand will be valuable probes to dissect the diverse pathways that involve m6A, including carcinogenesis. Here we propose to identify small drug-like molecules that block the methyltransferase activity of METTL3/METTL14, prioritizing the inhibitory effect on the gain-of-function mutant. We will use an unbiased screen of a large chemical library to find compounds that can effectively inhibit METTL3/METTL14 activity. In Aim 1, we will establish the primary high-throughput assay and implement it to test all the compounds in our chemical library. In Aim 2, we will efficiently and effectively prioritize the cherry-picked compounds to identify the top hits through a streamlined approach that uses multiple high-throughput secondary and counter screens. In Aim 3, we will use multiple orthogonal assays and mechanism of action studies to prioritize the top hits further to arrive at potential lead compounds that are specific for METTL3/METTL14, and not for other enzymes including other m6A methyltransferases. In addition we will initiate lead optimization by using our structural expertise to analyze the structure-activity relationship. The proposed study will yield small molecules inhibitors of METTL3/METTL14 that have been rigorously characterized by using biochemical, biophysical, and cell-based methods and will establish the groundwork for future development through the NCI Experimental Therapeutics (NExT) program.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Advanced-stage metastatic melanoma is the deadliest form of skin cancer, typically resistant to chemo- and radiotherapy. For such patients, the advent of immune checkpoint inhibitors (ICIs) has been a revolutionary change, improving tumor remission and survival rates, reversing prior lack of progress. Although promising, a large proportion of patients, especially those with poorly immunogenic ("cold") tumors, show only modest response to ICIs, and even responders relapse frequently. New tumor-reengineering technologies are urgently needed to improve survival rates in patients with immunoresistant cold tumors. The objective of this project is to optimize an innovative combinatorial therapy based on local focused ultrasound-based histotripsy (HT) and anti- CD40 agonist antibody (aCD40) encapsulated polymeric microparticles (CMPs) to reprogram cold melanoma tumors. HT non-invasively and rapidly generates acellular antigen depots with sharp boundaries in solid cancers, while aCD40 activates antigen-presenting cells (APCs). Our murine melanoma studies found that CMP is more efficacious compared to soluble aCD40 alone, and when combined with ICI/HT achieves complete remission of untreated tumors. Based on this premise, we will test the central hypothesis that intratumorally-administered CMPs that serve the dual functions of tumor antigen capture and sustained CD40 activation with HT will achieve durable remission of locally-treated and remote untreated melanomas. To test our hypothesis, the aims are to 1) Determine efficacy of the HT and CMP combined regimen in murine tumor models varying in immunogenicity and burden, 2) Elucidate ICI resistance reversal mechanisms that drives abscopal effects with HT and CMP, and 3) translate our findings to clinical trials in dog veterinary patients with spontaneous malignant melanoma. Dog melanoma resembles human melanoma, and metastasizes aggressively, and is advantageous for assessing translatability of our combinatorial therapy. Transforming immunologically “cold” tumors to immunogenic ones in advanced stages of cancer is a challenging goal. The project results will enable rapid activation of cold melanomas by locally generating large tumor antigen depots that can synergize with CD40 signaling to improve efficacy against locally treated and distant untreated tumors. Our studies in clinically- relevant models will inform feasibility of improving survival in a large proportion of metastatic melanoma patients with immunotherapy-resistant tumors.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Chronic infection by viruses such as HIV, HBV, and HCV remains a major global health challenge. During chronic viral infection, CD8 T cells fail to eradicate the virus and develop into a dysfunctional state, termed exhaustion. Exhausted CD8 T cells, which were first characterized in mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection, progressively lose effector function, upregulate inhibitory receptors, and exhibit dysregulated metabolism. Immunotherapies fail to completely reverse T-cell exhaustion or achieve sustained viral control during chronic viral infection. Thus, there is an unmet need to develop new strategies to overcome T-cell exhaustion and enhance the efficacy of immunotherapy against chronic viral infection. Although exhausted CD8 T cells were initially thought to be a homogeneous population, we and others have recently shown that a TCF1high antiviral CD8 T cell subset maintains long-term antiviral immunity through self-renewal and replenishing TCF1low terminally exhausted CD8 T cells during chronic viral infection. Importantly, these stem- like CD8 T cells are less exhausted and mediate the response induced by various immunotherapies. Our published studies have demonstrated stem-like CD8 T cells as a separate CD8 lineage with transcriptional and epigenetic programs that are distinct from those of terminally exhausted CD8 T cells and are tightly regulated by transcription factors, epigenetic regulators, and cytokines. The goal of this proposal is to define the molecular and cellular mechanisms regulating the immune response of stem-like CD8 T cells against chronic viral infection and evaluate strategies to improve antiviral immunity by targeting these pathways. Using chronic LCMV infection model, cutting-edge metabolic assay, multispectral quantitative imaging, and unique mouse genetic tools, we will identify the metabolic pathways and cell-cell interactions that endow stem-like CD8 T cells with their superior antiviral immunity and responsiveness to immunotherapies. These results will lay the foundation for the development of novel interventions to induce sustained control over chronic viral infections.

NIH Research Projects · FY 2025 · 2022-08

Immunosuppressive myeloid cells including myeloid-derived suppressor cells (MDSCs) contribute to multiple steps of cancer development. A better understanding of the molecular regulation of the functions of these myeloid cells and the signaling pathways will support development of novel anti-cancer therapeutic strategies. The leukocyte Ig-like receptor subfamily B (LILRB) proteins are a group of immune inhibitory receptors with intracellular immunoreceptor tyrosine-based inhibitory motifs. We have been studying the roles of these receptors in cancer development and immune regulation. The studies by us and others suggest that the LILRB family is becoming the next wave of myeloid immune checkpoint targets for cancer treatment. Here we demonstrated that LILRB3, a myeloid- specific member of this family, is functionally expressed on human MDSCs, and supports cancer development in mouse models. Importantly, we identified galectin-4 as an extracellular protein that binds to LILRB3 and induces LILRB3 activation, and the intracellular domain of LILRB3 interacts with the adaptor protein TRAF2 to contribute to NFκB upregulation. Furthermore, we developed anti-LILRB3 blocking antibodies that efficiently inhibit immunosuppressive activity of human MDSCs in vitro and cancer development in xenografted humanized mice and in LILRB3-transgenic mice. Our study suggests that LILRB3 represents an attractive novel target for cancer treatment. Based on new preliminary results, we propose the following Aims to test the hypothesis that LILRB3-initiated signaling in immunosuppressive myeloid cells supports cancer development. In Aim 1, we will determine the function of LILRB3 expressed on myeloid cells in cancer development. We will then determine whether galectin-4 regulates LILRB3- mediated signaling in tumor microenvironment to support cancer development in Aim 2. Finally we will dissect LILRB3 signaling in immunosuppressive myeloid cells in Aim 3. Our study will elucidate the molecular mechanisms by which LILRB3 regulates the activity of immunosuppressive myeloid cells, and lead to the development of innovative anti-cancer strategies based on targeting LILRB3 signaling.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY / ABSTRACT Dystonia is a brain-based disorder that leads affected muscles to twist and spasm, contorting the sufferer into painful and disabling positions. Dystonia afflicts 1 in 1000 people (the third-most common movement disorder). It can occur in isolation or be a symptom of many other neurological disorders (eg, stroke, cerebral palsy, Parkinson disease). Chronic pain, disability, and withdrawal from school or work are common, and in severe cases, dystonia can be fatal. A shared electrophysiologic abnormality links many types of dystonia: local and long-range disinhibition. This led to the hypothesis that impaired inhibition, and a related finding, poorly-refined sensory feedback, leads to abnormal co-contraction of agonist and antagonist muscles, producing the contorting movements of dystonia. However, impaired inhibition is only one step in a mechanistic cascade that leads to dystonia – the underlying structural abnormalities that produce disinhibition are unknown. Structural abnormalities in neurological disorders point the way to improved therapies. This proposal will use MRI to investigate brain regions that are potential anatomical substrates for impaired inhibition, with a larger goal of identifying new targets for dystonia treatment and prevention. We will address two key gaps in current dystonia knowledge: 1) the role of interhemispheric projections in regulating cortical motor activity (long-range disinhibition); 2) the role of the striatal compartments, striosome and matrix, in inhibiting unwanted movements. The study will employ novel structural, diffusion, and functional MRI techniques in two dystonia patient cohorts: we will carry out our imaging assessments in the most common forms in adults, cervical dystonia, and children, limb dystonia. These clinically-distinct populations will help determine which abnormalities are shared (mechanisms generalizable to other dystonias) and which are specific to certain types of dystonia. The mission of this Mentored Career Development Award is to seek fundamental knowledge about the brain’s inhibitory control of movement, and to use that knowledge to reduce the burden of dystonia. This goal parallels that of the National Institute of Neurological Disorders and Stroke: to investigate the neural mechanisms of sensory and motor circuits that can be compromised by disease. The proposal is tailored to my educational and training needs, ensuring that I will be prepared to succeed as an independent investigator utilizing the tools of systems neuroscience. With this award, I will gain essential training in: 1) biostatistics and network assessment tools such as principal component analysis, graph theoretical analysis, and causal inference modeling; 2) the experimental design and implementation of functional MRI (fMRI), including both task-based and resting state methods; 3) knowledge of the structure and connectivity of the striatum, pallidum, and thalamus. I will benefit from an environment enriched in neuroimaging, movement disorders, and neurodevelopmental expertise, where I can build a substantial foundation for a successful independent research career devoted to improving the therapies available for treating dystonia.

NIH Research Projects · FY 2025 · 2022-08

There are significant racial disparities in COVID-19 infection, morbidity, and mortality rates, with African Americans and older adults especially vulnerable to having poor outcomes. It is now apparent that the impact of COVID-19 persists beyond the acute infection period, and that ‘long haulers’ (i.e., persons > 4 weeks post- acute infection) continue to experience symptoms that negatively affect their activities of daily living and quality of life. Of these sequelae, the prevalence, phenotypes, and longitudinal course of post-acute COVID-19 cognitive impairments (PCCI) have not been systematically investigated. There is a need to characterize and to identify risk factors and mechanisms responsible for these long term impairments, especially in older adult African Americans who by virtue of their high prevalence of disease-specific comorbidities and socioeconomic risk factors, are a particularly vulnerable group for neurocognitive sequelae and neurodegenerative brain diseases including Alzheimer’s disease and related disorders. We propose a prospective longitudinal study of PCCI in 407 African Americans 50 years and older with a history of COVID-19 infection (symptomatic or asymptomatic). Participants will receive comprehensive assessments including cognitive testing, neuroimaging, cardiovascular evaluation (endothelial function, 6-minute walk test, ECG monitoring) and biospecimen collection and analyses including blood and cerebrospinal fluid (Apolipoprotein-e, coagulopathy, viral immunity and proteomic measures of inflammation, neurodegeneration, and other pathways). Aim 1 will characterize the neuropsychological profiles and the longitudinal course of PCCI in older African Americans, and test the hypothesis that attention, memory, and executive functioning are most affected. In Aim 2, we will determine factors associated with prevalent PCCI at study baseline and their persistence in older African Americans, with the prediction that risk factors such as older age, multiple comorbidities, and poor social determinant of health indicators are associated with prevalent and persistent/progressive PCCI. Aim 3 will determine molecular, cardiovascular and neuroimaging characteristics related to prevalent and persistent or progressive PCCI in older African Americans, with the hypothesis that higher levels of systemic and neuro- inflammation, pro-atherothrombotic state, APOe4 allele and AD biomarker pathology, impaired peripheral and cerebral endothelial function and measures of brain connectivity are related to prevalent and persistent or progressive PCCI. We expect that the findings of this study will have a positive impact in guiding clinical decision making and informing public health policy.

NIH Research Projects · FY 2026 · 2022-08

SUMMARY Pluripotency is a critical model for understanding fundamental principles of cell fate specification. This process makes selective use of enhancers, regulatory elements that facilitate the transcription of cell-type specific genes. Although changes in enhancer activity are predicted to have broad developmental and pathological implications, we currently have limited understanding of how enhancers regulate development and disease, in part due to our incomplete understanding of the mechanisms by which enhancers regulate gene expression. Increased understanding of the molecular events that regulate enhancer activity, particularly under developmental contexts, will provide important insights into congenital diseases that occur with their dysregulation. The chromatin state at enhancers is characterized by the presence of the histone variant, H3.3, and recruitment of the CBP/p300 family of transcriptional coactivators. Our recent studies demonstrate that H3.3 deposition at enhancers allows for variant-specific phosphorylation that stimulates p300 acetyltransferase activity towards its substrate histone H3 lysine 27 (H3K27ac) in mouse embryonic stem cells (ESCs). Further, we find that CBP and p300 carry out distinct functions in ESCs, with p300 playing a greater role in maintaining H3K27ac in ESCs. Finally, we find that reduced H3K27ac due to H3.3 or p300 deletion is well tolerated in ESCs, with little correlated change in transcription. However, both H3.3 and p300 are required for differentiation, suggesting that H3.3 deposition and subsequent high levels of H3K27ac may be more important for activating gene transcription than for maintaining ongoing transcription in ESCs. The objective of this proposal is to dissect the molecular and functional mechanisms by which enhancers are activated both in pluripotency and differentiation. In the first aim, we will determine how H3.3 phosphorylation stimulates p300 activity in ESCs. In the second aim, we will determine why H3K27 acetyltransferase activity is restricted to p300 in ESCs. Finally, in the third aim, we will use the tools of chemical biology and novel mouse models that we have generated to ask how H3.3 and p300 function to promote transcription during pre-implantation development. Collectively, our work will explore molecular links between H3.3 and p300 in enhancer activation during the time at which the first lineage specification events occur, with important implications for understanding how enhancer dysregulation contributes to human disease.

NIH Research Projects · FY 2025 · 2022-08

Project Summary There is increasing evidence that early intervention for psychosis in coordinated specialty care (CSC) services improves outcomes and lives. The outcome of early course psychosis (EP) is heterogeneous, ranging from early full recovery to treatment resistance and functional decline from the onset of illness. This heterogeneity limits our ability to predict individual level outcomes needed for treatment planning and for tailoring the type, duration and intensity of therapeutic interventions. Biomarkers as well as clinical and demographic features, early in the illness can predict outcome, but taken individually, their prognostic value is limited. Our Bipolar- Schizophrenia Network for Intermediate Phenotypes (BSNIP) consortium has recently developed, replicated and validated a biomarker (EEG, eye movement testing, and neurocognition) based categorization (Biotypes 1, 2 and 3) in a trans-diagnostic sample of cases with idiopathic psychosis (schizophrenia, schizoaffective disorder, or bipolar disorder with psychosis), ranging from 18-35 years of age. In this study, we will leverage this categorization, along with clinical and biomarker data to predict illness trajectory and outcome during follow-up at 1, 6 and 12 months in 320 EP patients across CSC clinics at the five B-SNIP sites. First, we will characterize outcome trajectories and Biotype structure in EP. Our available data indicate the Biotype structure will be the same in EP as in our large sample. Second, we will investigate the predictive value of the nine bio-factors and the three Biotypes identified by B-SNIP for symptomatic and functional outcome. We predict that the EP population will manifest distinct outcome clinical trajectories (good, intermediate and poor) and will have a Biotype structure similar to that seen in chronic psychosis subjects, i.e., Biotypes 1, 2 and 3) (hypothesis 1). Biotype-3, and Biotye-2 cases, will have the best outcomes (defined both categorically, and dimensionally, using symptomatic, cognitive and functional measures); Biotype-1 will have the worst outcomes to CSC treatment, across all target time points (hypothesis 2). Notably, Biotype-1 and Biotype-2 cases will have the same level of cognition function at baseline. Finally, we will investigate the predictive value of clinical (such as diagnosis, illness duration, substance abuse, and treatment adherence), and biomarker (including neuroimaging) features in a multi-variate model and will develop a feasible biomarker battery and predictive algorithm for application in community CSC sites nation-wide. We will thus provide to the field a means for predicting success of EP cases in CSC treatment to improve clinical practice and to enhance efficient use of available treatment resources.

NIH Research Projects · FY 2025 · 2022-08

Project Summary Weak multivalent interactions mediated by intrinsically disordered regions (IDRs) of proteins have been proposed to spatially organize the transcriptional machinery into multi-component clusters, yet we know little about how these IDRs interact with specific partners to enable functional outcomes. As these interactions are highly dynamic and cluster-dependent they have been overlooked by conventional strategies to identify protein-protein interactions. Our preliminary data support our overarching hypothesis that in the context of higher-order clusters weak multivalent interactions are capable of highly specific hetero-typic interactions leading to functional organization of the nucleus. Our long-term objective is to understand how weak multivalent interactions organize specific components of the transcriptional machinery in order to enable gene activation. The objective of this grant is to investigate the mechanism and function of cluster-mediated interactions of the IDR of MED1, the largest subunit of the mediator coactivator complex. Mediator is a megadalton complex that bridges DNA-binding transcription factors with downstream steps of the activation process requiring it to engage dynamically with many components of the regulatory machinery. While recent structural studies have revealed the architecture of this complex, the domains responsible for multivalent interactions are dynamic IDRs and remain unresolved. In particular the MED1 subunit contains a >600 amino acid c-terminal IDR (MED1-IDR) which previously published studies implicate in cluster formation. Our preliminary data show that MED1-IDR clusters selectively partition positive regulators of transcription and exclude negative regulators or functionally unrelated yet abundant nuclear proteins. These data lead us to hypothesize that IDR-mediated selective compartmentalization is a mechanism to regulate transcription. To test this hypothesis we will confirm and validate the specificity of these cluster- mediated interactions (Aim 1), characterize the molecular features underlying specificity of these interactions (Aim 2), and investigate the function of these interactions in various models of gene activation (Aim 3). Upon completion of these proposed studies, we will understand the role of weak multivalent interactions mediated by MED1-IDR in organization of specific components of the transcriptional machinery. This contribution is significant as it will lead to a new appreciation for the function of the prevalent yet often overlooked IDRs in gene activation. While we focus here on MED1-IDR, the tools and methods developed and the principles learned here can be applied to other weak multivalent interactions involved in gene regulation or the growing list of biochemical process regulated by dynamic clustering of regulators.