University Of California Los Angeles

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY The cutaneous vasculature is a crucial yet understudied component of the skin, responsible for essential functions such as tissue oxygenation, exchange of nutrients and soluble factors, and temperature control. While we have a significant understanding of vascular abnormalities present in various skin pathologies, we lack an understanding of both physiological remodeling and homeostatic mechanisms sustaining lifelong function. Specifically, we lack the resolution of the coordinated cellular behaviors that drive developmental vascular remodeling, as well as those that underlie vascular regeneration in the face of injury, particularly in the context of hemodynamic status. I hypothesize that network-wide coordination of EC behaviors in relation to hemodynamic changes regulates developmental and regenerative remodeling programs of the skin vasculature. To test this hypothesis, I have established an intravital imaging technique that allows for the longitudinal tracking and manipulation of the endothelial cells (ECs) that constitute the lining of all blood vessels in the skin of a live mouse. In Aim 1, I will investigate the neonatal vessel remodeling program and underlying EC behaviors that orchestrate the establishment of skin vascular network architecture and blood flow efficiency. Following establishment of adult vascular homeostasis, I will probe the cellular mechanisms that regulate the maintenance of adult vessel integrity via a targeted laser ablation approach, modeling the discrete membrane damage inflicted upon the endothelium due to shear and contractile forces. My preliminary data shows that EC migration within existing vessel structures is a critical EC behavior that underlies network-wide vessel regression during neonatal remodeling, as well as the reparative response of adult ECs to local damage. In Aim 2, I will transition my studies towards the understanding of the skin vasculature in the context of pathological states. Firstly, I will investigate the wound vascularization mechanisms of neonatal versus adult skin, and delineate the differential remodeling properties that enable the enhanced wound revascularization in neonatal skin that we have observed in preliminary experiments. Second, I will determine how coordination of flow-dependent EC rearrangement impacts the ability of adult wounds to revascularize via a genetic mutant model that uncouples the ability of ECs to polarize with respect to blood flow direction. To achieve these aims, I will use an integrated approach of cutting-edge imaging technology, transcriptomics, and genetic mouse models. This research is significant because we expect to uncover global cellular and molecular mechanisms that coordinate vascular development, homeostasis, and injury repair. My findings will likely drive innovation in related fields, given the ubiquity and crucial roles of the vasculature in all organs.

NIH Research Projects · FY 2026 · 2023-08

Project Summary Altered mitochondrial function, a hallmark of aging, contributes to age-related diseases, in part, due to energetic failure. Maintenance of mitochondrial energetics requires proper fission and fusion that are in turn regulated by proteins at endoplasmic reticulum-mitochondria contacts (a.k.a. mitochondria-associated membranes or MAMs). Fasting stimulates mitochondrial fatty acid utilization, and it is thought that the benefits of fasting are due to adaptive changes in mitochondrial dynamics and respiration. How fasting impacts mitochondrial dynamics and MAMs in intact mammalian systems, and whether these fundamental mechanisms are altered with age and dietary stress remain unknown. The mechanisms by which upstream signaling cascades regulate mitochondrial dynamics is becoming an area of great interest, with recent work showing AMPK as a regulator of fission. Our new data show that the typically nutrient-responsive mTORC2 is paradoxically reactivated by fasting, and that mTORC2 reactivation supports mitochondrial fission. Indeed, livers silenced for mTORC2 regulator Rictor show impaired fission and dampened respiration; and mTORC2 activity is suppressed with age. These data raise a key point: since clinically available mTOR inhibitors (e.g., rapamycin) block both mTORC1 and mTORC2, we argue that benefits of long-term mTOR inhibition with rapamycin are counterbalanced by mitochondrial dysfunction due to concomitant mTORC2 inhibition. This forms that basis to explore if blocking mTORC1—in conjunction with maintaining mTORC2 activity—will lead to healthspan and lifespan extension. Phosphorylation is a well-established signal in regulation of mitochondrial dynamics. Our phosphoproteomics data suggest that paradoxical reactivation of mTORC2 kinase during fasting stimulates mitochondrial fission by phosphorylating novel protein targets at MAMs, including NDRG1, N-Myc downstream regulated 1. Interestingly, the NDRG1S336A phosphorylation-deficient mutant recapitulates the effects of loss of mTORC2 on mitochondrial fission/respiration, while phosphomimetic NDRG1S336D stimulates mitochondrial fission. Intriguingly NDRG1, but not the NDRG1S336A phosphorylation-deficient mutant, interacts with Cdc42, a cytokinetic protein with the intrinsic GTP hydrolysis activity required for mitochondrial fission. Based on our preliminary data, we propose that activation of the mTORC2-phosphorylated NDRG1Ser336-Cdc42 axis at MAMs regulates mitochondrial fission during fasting, and that dysregulation of the mTORC2-NDRG1Ser336-Cdc42 axis leads to age-related loss of mitochondrial fission and respiration. Three aims are proposed that will characterize (Aim 1) mitochondrial dynamics and MAMs in aged and stressed livers; (Aim 2) determine if and how dysregulation of the mTORC2- NDRG1Ser336-Cdc42 axis causes age-related loss of mitochondrial fission; and (Aim 3) explore if synergistic use of rapamycin and co-expression of phosphomimetic NDRG1S336D will lead to greater lifespan extension when compared to use of rapamycin alone. Experiments proposed here will rigorously test a new set of hypotheses that altered mTORC2 signaling is a major contributing factor to age-related loss of mitochondrial function.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY Ongoing evaluation of cost-benefit tradeoffs guides action selection during adaptive decision making. When outcomes change, the utility of potential actions is re-evaluated to determine whether to persist or deviate from an existing strategy. Disturbances in the neural mechanisms underlying cost-benefit decision making can lead to pathological behavior (e.g., addiction, OCD, depression/anxiety). Effort-based decision making is specifically disrupted in patients with depression, schizophrenia and substance use disorders. Although pathological behavior in these conditions is linked to dysfunction in the prefrontal cortex and striatum, we lack the detailed neurobiological understanding necessary to design targeted therapeutic interventions. We will address this deficit using cutting edge tools for measuring and manipulating neural activity in freely behaving animals. We will test the specific hypothesis that anterior cingulate cortex (ACC) to nucleus accumbens (NAc) projection neurons encode updates to action selection policies based on new effort-reward tradeoffs, and that inputs to the NAc instantiate new, effortful choice strategies. In Aim 1, we will use miniaturized head-mounted microscopes to determine how ACC and ACCàNAc projection neuron activity is organized to represent effort-related cost- benefit computations influencing action selection. In Aim 2, we will manipulate the activity of ACCàNAc projection neurons with optogenetics, during flexible decision making driven by changes in effort-related value. Finally, in Aim 3, we will focus on prefrontal projections to the NAc, measuring and inhibiting activity at ACCàNAc terminals and comparing with inputs from the orbitofrontal cortex (OFC). We will do this during decision making in the context of both effort and delay-costs as a tool to further refine our understanding of how NAc integrates prefrontal inputs and translates these into action selection. This proposal directly addresses a pressing need to understand the cell-type and circuit-specific mechanisms that mediate cost-benefit decision making. Our research can inform pharmacological, psychotherapeutic and brain stimulation interventions for a variety of psychiatric conditions characterized by disordered cost-benefit evaluations and decision making.

NIH Research Projects · FY 2026 · 2023-08

TITLE: Elucidating the Role of Biofilm-Forming Bacteria in Nephrolithiasis PROJECT SUMMARY/ ABSTRACT Kidney stone disease is common, affecting 11% of the US population. However, the pathogenesis of kidney stones is still not well understood. While 10% of stones are known to be infectious, calcium-based stones, which make up 80% of stones, have not previously been shown to associate with bacteria. However, recent publications have utilized next-generation sequencing to demonstrate bacterial infestation in calcium stones despite no evidence of prior or concurrent urinary tract infection in those patients. In preliminary work, we found that uropathogenic bacteria trigger increased calcium oxalate crystal growth compared to controls. We hypothesize that bacteria interact with calcium stones and promote the propagation of kidney stones. We further propose that this interaction involves the formation of bacterial biofilm. We will test these hypotheses using three specific aims: 1) To test the hypothesis that uropathogens initiate biofilm formation in stones 2) To test the hypothesis that known surface sensing pathways of Pseudomonas spp. and E coli trigger kidney stone formation and 3) To test the hypothesis that bacteria play an important role in triggering crystallization and crystal aggregation in urine-specific environments using an in vitro three-dimensional kidney model. These aims will be accomplished by utilizing multiple imaging and complementary techniques including Raman spectroscopy and x-ray fluorescence as well as single-cell tracking analyses and molecular genetic techniques to investigate biofilm initiation. This study will provide new insights into the role of bacteria in the propagation of calcium-based kidney stones. It will also facilitate the advancement of my independent biomedical researcher career. I will collaborate with and obtain guidance and tutelage from highly successful, experienced mentors who are committed to my professional development. My research capabilities will be augmented by didactic and hands-on training in microbiology, machine learning, and high-resolution imaging, thus preparing me with the tools necessary to transition to full independence as a surgeon-scientist. Lastly, this funding will allow me to contribute to elucidating the etiology of nephrolithiasis and will directly enable provide the preliminary data for my first R01 application.

NIH Research Projects · FY 2024 · 2023-08

PROJECT ABSTRACT Epigenetic factors are genes that encode proteins that can affect spatial organization of DNA and the accessibility of genetic regions to transcriptional machinery, thereby regulating cell-type specific transcription. Pathogenic mutations in established epigenes are highly enriched in children with pediatric syndromic disorders, often with symptoms that affect multiple organ systems, such as the brain, heart, gastrointestinal tract, bone, eye and kidneys. The associated symptoms of intellectual disability, developmental delays, autism, diarrhea and congenital heart defects vary in severity between individuals. The onset of these syndromes during early childhood suggest that many of these epigenetic factors are critical to early developmental processes and cell- fate transitions. Despite our improved diagnostic ability with exome sequencing, the mechanistic link between epigenetic factor mutations and the direct mechanisms by which they disrupt mammalian developmental processes remains unknown. Histone acetyltransferases (HATs) are genes that acetylate lysine (K) residues and represent a common mechanism for controlling DNA accessibility to the transcriptional machinery. Here, we study protein-truncating variants in two HATs: Lysine (K), Acetyl transferase 6A and 6B (KAT6A and KAT6B), which cause Arboleda-Tham Syndrome (ARTHS) and Genitopatellar Syndrome (GPS) or Say-Barber-Biesecker- Young Syndrome (SBBYS), respectively. Our goal is to develop and validate transgenic mouse models harboring patient-specific mutations in these genes to establish the differential roles of these epigenetic factors on cell specification during development driving the distinct syndromes.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Lung cancer is the leading cause of cancer related deaths in the United States and the World. We recently demonstrated that programmed cell death 1 (PD-1) inhibitors, which lead to durable responses in a minority of non-small cell lung cancer (NSCLC) patients, have greater efficacy in patients with charged HLA-B binding pockets whose tumors harbor mutation(s) leading to what we have designated as motif neoepitopes. Motif neoepitopes have an amino acid substitution in the second position of a nonamer generating a change in charge from the wild type peptide with the resultant amino acid having a charge opposite from the HLA-B binding pocket. To date, the immunological changes induced by motif neoepitopes have not been explored. We propose a comprehensive evaluation of the underlying mechanism, focusing on patients with HLA-B44 supertype alleles because of the prevalence (approximately 40% of the population) and distribution of HLA-B44 across racial and ethnic groups. We will evaluate HLA-B44 samples in the cancer genome atlas (TCGA) to explore differences in the tumor microenvironment (TME) among patients with or without motif neoepitopes by examining gene expression and cellular composition by slide review and algorithms based on gene expression profiles. We will evaluate surgical specimens from treatment naïve patients with or without HLA-B44 motif neoepitopes and evaluate spatial signatures of the TME by multiplex immunofluorescence (MIF). We will assess multiple sections from each specimen to identify biomarkers most significantly associated with motif neoepitopes. We will further examine immune contextures of the TME associated with motif neoepitopes by single cell RNA-seq analysis. To elucidate the predictive value of motif neoepitopes in early and advanced stage NSCLC patients and to assess relevant clinical questions, we will perform analyses of patients in three separate clinical scenarios. As whole exome sequencing (WES) and transcriptomic data is now routinely obtained in our NSCLC patients as part of patients’ clinical care, we will analyze the presence and expression of genes harboring motif neoepitopes. We will evaluate baseline tumor biopsies from 75 early stage patients with an HLA-B44 allele who receive neoadjuvant chemotherapy plus PD-1 inhibition, correlating motif neoepitopes with pathologic complete response. Among 75 advanced stage patients with an HLA-B44 allele who are receiving single agent PD-1 inhibition and 75 additional patients being treated with chemotherapy plus PD-1 inhibition, we will correlate motif neoepitopes with progression of disease within 6 months of initiation of therapy. Together, these studies will provide a better understanding of the TME and other immunologic changes associated with the presence of motif neoepitopes. In addition, results could enable us to identify patients in whom evaluation of this marker of neoantigen presentation could be utilized to select patients with clinically relevant benefit in three separate clinical scenarios utilizing PD-1 inhibitor-based therapy. As a corollary, results could also identify populations of patients in whom other treatment strategies should be considered.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Lung cancer is the leading cause of cancer related deaths in the United States and the World. Inefficient prediction of functional tumor neoantigens and insufficient understanding of host anti-tumor immune responses limit optimization of immunotherapeutic approaches. We recently demonstrated that programmed cell death 1 (PD-1) inhibitors, which lead to durable responses in a minority of non-small cell lung cancer (NSCLC) patients, have greater efficacy in patients with charged human leukocyte antigen (HLA)-B binding pockets whose tumors harbor mutation(s) leading to what we have designated as “motif neoepitopes”. Motif neoepitopes lead to an amino acid substitution in the second position of a nonamer (anchor for HLA-binding), generating a change in charge from the wild type peptide in which the resultant amino acid has a charge opposite to the HLA-B binding pocket. This substitution leads to enhanced binding affinity to the corresponding HLA-B supertype demonstrated by in vitro competition assays. These data suggest that optimal presentation of motif neoepitopes by corresponding charged HLA supertypes results in effective host anti-tumor immune responses in vivo. Dendritic cell (DC)-based vaccination has emerged as a potential component for immunotherapy due to both its favorable toxicity profile and its essential role in antigen-specific T cell priming and activation. We have expertise in clinical studies evaluating a DC in situ vaccination strategy in NSCLC. In this proposal, we intend to combine DCs as functional antigen presenting cells (APCs) with putative motif neoepitopes as an innovative vaccination approach to enhance host systemic tumor-specific T cell responses and potentiate clinical benefits of current immunotherapies. We hypothesize that 1) peptides derived from motif neoepitopes are functional neoantigens in vivo that are capable of inducing host tumor-specific immune responses, and 2) autologous DCs pulsed with motif neoepitope-derived peptides, particularly at optimal conditions, will induce systemic activation of motif neoepitope-specific T cells. As part of this proposal, we are analyzing multiple biospecimens collected from our ongoing phase I trial of intratumoral injection of autologous DCs combined with PD-1 inhibition in advanced NSCLC. Collected specimens include serial blood and tumor biopsies as well as autologous DCs. We will evaluate whether exposing DCs to peptides derived from motif neoepitopes can induce tumor-specific T cell activation in co-culture experiments. We will assess the binding affinity of these peptides and the corresponding wild type peptides to their respective HLA-B supertype. We will further optimize conditions, including addition of PD-1 blockade, to achieve optimal T cell activation by autologous DCs pulsed with motif neoepitope-derived peptides. These studies will greatly enhance our understanding of the potential function of motif neoepitopes in inducing host anti-tumor immune responses, and lay the foundation for future clinical investigations of motif neoepitope-pulsed autologous DCs as a novel treatment strategy for NSCLC.

NIH Research Projects · FY 2026 · 2023-07

PROJECT SUMMARY The purpose of this K01 award is to support research training and advance the career development of Dr. Rebecca Delafield. Dr. Delafield’s long-term career goal is to establish an independent research program focused on improving maternal health and health care outcomes for Native Hawaiian and Pacific Islanders (NHPI). Research Plan: Maternal mortality and morbidity are increasing in the U.S. A recent study from Hawai‘i found that NHPI accounted for 44% of the maternal deaths, although they make up only 25% of the state population. Beyond this single study, NHPI maternal mortality is largely invisible. Federal agencies do not publish maternal mortality rates of NHPIs. This void is exacerbated by a dearth of research on other major and more common maternal health outcomes, such as severe maternal morbidity (SMM) (e.g., eclampsia and hemorrhage). Yet, a small body of research suggests NHPI communities face obstacles accessing the healthcare system and engaging with health care providers, which may compromise care in pregnancy. NHPI women may also have unique cultural perspectives on childbearing which could mitigate or exacerbate these challenges. To comprehensively address maternal health among NHPIs, research is needed to 1) assess critical maternal outcomes for NHPIs 2) better characterize NHPI experiences, and 3) identify key factors related the processes and quality of maternity care. There are two research aims: 1) Compare rates of SMM in NHPI women to non-Hispanic white women by analyzing hospital discharge data from facilities in Hawaiʻi, Oregon, and Washington. 2) Survey 300 NHPI women to assess patient-clinician relationship and communication factors associated with high-quality person-centered care, perceived treatment, and engagement in prenatal care. Career Plan: This project will provide the candidate the mentoring and training needed in the areas of scientific and statistical methodologies for small populations, community-engaged research and health communication to achieve her long-term career goal and short-term objectives. The third (training) aim is: Complete a rigorous training program to advance skills in small population health research and statistical methods, survey analysis, and community-engaged research. The Mentoring Team assembled has diverse skills and extensive experience in the areas of NHPI health, health disparities research, epidemiology, community-engaged research, health communication, maternal and reproductive health. Training objectives will be met through relevant coursework, mentored research training, bi-directional training with community-based partners, and hands-on learning grounded in Pacific cultural traditions. The completion of these aims will provide the candidate the experience and preliminary data needed to compete for R01 funding as an independent investigator and progress toward her future goal of developing and testing a culturally-responsive and community-engaged multilevel intervention to improve NHPI maternal and perinatal health.

NIH Research Projects · FY 2025 · 2023-07

PROJECT ABSTRACT Solid organ transplant recipients exhibiting HLA donor specific antibodies (DSA) are at risk for graft loss due to chronic antibody-mediated rejection (cAMR) and develop a progressive vascular disease known as transplant vasculopathy (TV). Although cAMR and TV are highly significant clinical problems across different solid organ transplants the mechanisms by which DSAs directed against HLA I and HLA II contribute to cAMR and TV are not yet understood. Novel mechanistic targets and therapeutic approaches to prevent and treat cAMR and TV are urgently needed. Previously, we demonstrated that DSA-induced ligation of HLA molecules expressed in the surface of endothelial cells (ECs) induces signaling pathways that regulate survival, proliferation and migration, all of which are highly relevant to TV. However, the key transcriptional programs stimulated by ligation of HLA molecules remain to be identified. This gap in understanding hinders effective therapeutic targeting of DSA effector functions to prevent cAMR and TV. Based on our new results, we posit that the transcriptional co- activator Yes-Associated Protein (YAP) and its paralog WW-domain-containing Transcriptional co-Activator with PDZ-binding motif (TAZ), two central effectors of the Hippo pathway, are downstream points of convergence and integration in the mitogenic and migratory signaling initiated by DSAs in ECs. Although inhibition of the activity of transcription factors or their co-activators is a challenging strategy, recent evidence suggests a new avenue to target YAP/TAZ activity via drugs of the statin family. Importantly, epidemiological studies indicate that statins exert a beneficial effect in clinical transplant populations. Based on substantial preliminary studies, the central hypothesis of this proposal is that YAP and its paralog TAZ play a crucial role in promoting the proliferation and migration of ECs in response to DSAs and that the FDA-approved drugs of the statin family inhibit YAP/TAZ function in these cells. A fundamental translational implication of our hypothesis is that the drugs of the statin family can be an important element in preventing cAMR by blocking growth-promoting YAP/TAZ signaling in ECs. We propose to explore this central hypothesis by pursuing three Specific Aims: 1) Determine the regulation and function of YAP in human ECs stimulated with antibodies directed against HLA I or HLA II: role of Src kinases. 2) Define the mechanism(s) by which statins inhibit YAP function, proliferation and migration of ECs stimulated with antibodies directed against HLA I or HLA II. 3) Characterize the impact of statins on YAP and cAMR in vivo using a novel model of heart graft allograft that develop TV. We propose that the Src/YAP/TAZ axis plays a critical role in antibody-mediated EC proliferation and that statins inhibit EC proliferation and TV via YAP/TAZ inhibition. We anticipate that the Src/YAP/TAZ axis will emerge as a novel target in cAMR thus propelling further studies to reposition FDA-approved statins either singly or in combination with Src inhibitors for preventing chronic allograft injury induced by DSAs.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Messenger RNA splicing is carried out by a large macromolecular machine, the spliceosome, which undergoes dramatic rearrangements as it assembles onto a pre-messenger RNA. Our understanding of this elegant ribonucleoprotein machine has advanced significantly with the availability of cryoEM structures and an extensive “parts list” of splicing components. However, many questions remain about how spliceosome assembly takes place in the cell, where the spliceosome machinery assembles onto a nascent transcript that is being actively transcribed from a chromatin template, i.e. co-transcriptionally. Moreover, assembly of the spliceosome and splicing catalysis is tightly regulated and, in fact, there are widespread examples across eukaryotes of intron retention. Nonetheless, the regulatory consequences of this phenomenon remain obscure. Guided by strong preliminary data, we explore both of these outstanding challenges and address the following questions: (1) How does chromatin influence spliceosome assembly and vice versa, i.e. how does co-transcriptional spliceosome assembly affect the state of the chromatin? (2) How does intron retention contribute to gene regulation? (3) How does mis-regulation of intron accumulation lead to cellular toxicity and disease?

NIH Research Projects · FY 2026 · 2023-07

Project Summary/Abstract The mammalian hypothalamus plays a critical role in behaviors essential for survival. The lateral hypothalamic area (LHA) occupies 55% of the hypothalamus and contains diverse neuron types, yet remains an undifferentiated region in mouse atlases. Much remains unknown about its structural and functional organization at the regional, cell-type specific, and individual neuron scales. Further, the hypothalamus is structurally and functionally sexually dimorphic, yet differentiated brain atlases for the sexes is absent. We hypothesize that multiscale heterogeneity of LHA structural organization underlies the specificity and properties of its cell type- specific circuit functional organization. We will investigate this using established and innovative approaches to correlate and synthesize molecular, cellular, and circuit-level LHA data into a multimodal, multiscale LHA reference atlas. In Aim 1, we will construct a multiscale, multimodal, 3D reference atlas of the male and female mouse hypothalamus based on the cyto- and chemoarchitecture of the LHA (Aim 1a). These volumetric 3D lightsheet data will be registered to the Allen CCF and structural delineations will be conducted using a novel cloud-based 3D approach. In Aim 1b, we will use our connectivity-based parcellation strategy to refine the delineation of the LHA at a much higher granularity based on the systematic analysis of >300 sets of pre-collected neural pathways and additional connectivity data collected via conditional (Cre-dependent) viral pathway tracing methods. The refined parcellation of LHA subdivisions will be validated by investigating specificities of their input/output organization. In Aim 2, we will systematically investigate LHA molecular diversity and network organization across meso- and microscale resolutions. First, for each LHA subdivision, we will investigate the molecular identities of target-specific GABA and GLU projection neurons by combining retrograde pathway labeling with RNAscope or HiPlex RNAscope (Aim 2a). Then, for these target-specific GABA & GLU neuron types, we will systematically examine their axon collateralizations (Aim 2b) and neural inputs (Aim 2c) using a 2- way viral labeling method in Vgat-Cre and Vglut2-Cre knock-in mice. Finally, in Aim 2d, by combining a novel genetic sparse labeling method (MORF) with brain clearing, 3D microscopic imaging, and computational reconstruction algorithms, we will reconstruct the fine detailed single neuron morphology (dendrites and axons) of >2000 GABA and GLU neurons in male and female LHA. All of the data will be registered into the newly constructed 3D hypothalamic atlas (Aim 1) within the CCF to construct an unprecedentedly comprehensive cellular atlas of the male and female hypothalamus. In Aim 3, we will establish a computational platform and a specialized computational infrastructure for (1) circuit, neuronal, and synaptic reconstruction, annotation, and error correction validated with efficient ground truth human assessments; and (2) for efficient data transfer, storage, and dissemination. All CCF-registered neuronal morphology and connectivity data will be processed and disseminated through our UCLA BRAIN data portal.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY/ABSTRACT Lung cancer remains the leading cause of cancer-related deaths in the U.S. and worldwide. Non–small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. Early detection of indeterminate pulmonary nodules (IPNs) with chest low dose computed tomography (LDCT) screening followed by effective treatments can reduce mortality by 20% relative to chest radiography. However, the high false positive rate finding as >95% limits the application. The unmet clinical need for early diagnosis is the lack of a noninvasive test that can be applied to individuals with CT-detected lung nodules and reliably discriminates between malignant or benign nodules. Liquid biopsy focused on the characterization of tumor-associated genetic alterations in cell free circulating tumor DNA can non-invasively profile the molecular landscape of solid tumors. However, the low biological concentrations of ctDNA, low frequency of somatic mutations and the confounding impact of clonal hematopoiesis-related variants in early-stage lung cancer limit the sensitivity of ctDNA-based liquid biopsy assays. It would be essential if additional cell free DNA biomarkers could be included to allow the development of more sensitive molecular diagnostics approaches for the early assessment of lung cancer. We have discovered a distinct population of ultra-short single-stranded cell-free DNA (uscf/ctDNA) with a size of 40-70nt in healthy and NSCLC plasma. Our preliminary data showed that the fragmentomic features, including functional element profile, fragmentation patterns and end motifs of uscfDNA molecules, can distinguish NSCLC patients from healthy donors. This R21 application is to explore and test our hypothesis that the fragmentomic features of uscfDNA molecules can serve as novel biomarkers to differentiate NSCLC patients from non-cancer subjects with IPNs and allow more sensitive liquid biopsy molecular diagnostics for early NSCLC detection. Two specific aims are in place for hypothesis testing. Aim 1 is to develop a predictive model using uscfDNA-seq assay on uscfDNA fragmentomic analysis for liquid biopsy of NSCLC. Aim 2 is to pre-validate uscfDNA-Seq test for the early detection of NSCLC. Together, the translational and pre-validation, targeting uscfDNA for early detection of NSCLC can break new ground and extend previous discoveries towards impactful new directions and clinical applications.

NIH Research Projects · FY 2026 · 2023-07

PROJECT SUMMARY/ABSTRACT Prostate cancer is the second leading cause of cancer associated deaths in men in the United States. The first line of treatment for men with advanced metastatic prostate cancer is hormone therapy. Although initial responses are observed, unfortunately, the disease commonly recurs in its aggressive hormone therapy- resistant form, which is largely responsible for prostate cancer-specific mortality. Thus, there is an urgent need to define the mechanisms that drive the aggressive disease and develop novel strategies to overcome advanced treatment-resistant prostate cancer. We have recently shown that GSTP1 protein is significantly upregulated in treatment-resistant prostate cancer. We have strong preliminary evidence suggesting that GSTP1 may play functional role in driving aggressive prostate cancer and may represent a promising therapeutic target for the advanced disease. We have recently demonstrated that GSTP1 is significantly elevated in hormone therapy-resistant prostate cancer and that inhibition of GSTP1 suppresses prostate cancer growth. The main goals of the proposed project are: 1) test the functional role of GSTP1 in advanced prostate cancer. 2) test the therapeutic potential of GSTP1 inhibition alone and in combination with cisplatin in aggressive prostate cancer in pre-clinical settings utilizing patient-derived xenograft (PDX) models of NEPC. 3) Delineate the mechanism of action of GSTP1 in advanced prostate cancer. Successful completion of the proposed research will lead to: 1) defining the role of GSTP1 in aggressive therapy- resistant prostate cancer and 2) direct new strategies regarding novel therapeutic interventions to combat the deadly form of the disease.

NIH Research Projects · FY 2024 · 2023-07

Project Summary Adolescents demonstrate a high need for emotion regulation [2-4], but often struggle to employ gold- standard regulatory strategies such as cognitive reappraisal due to underdeveloped lateral prefrontal neuroarchitecture (i.e. lateral prefrontal cortex (LPFC)), which plays a crucial role in cognitive control [5-8]. At the same time, adolescents are exquisitely sensitive to their peers [11,13-16]. Notably, neural regions linked to peer influence in adolescence, including ventral striatum (VS) and ventromedial prefrontal cortex (vmPFC), mature prior to LPFC. Thus, adolescents are uniquely poised to benefit from a social intervention designed to appropriate peer influence mechanisms towards enhancing emotion regulation efficacy. The goal of this R21 is to work with the developing brain, not against it, to utilize adolescent-emergent reward-related circuitry (VS, VMPFC) instead of LPFC to regulate emotion in adolescents. We will administer a novel paradigm during functional magnetic resonance imaging (fMRI) to examine whether adolescents (N = 50) and adults (N = 50) are more effective at down-regulating negative affect when a friend provides them with reinterpretations of negative stimuli (i.e. social reappraisal), as compared to when they reinterpret stimuli alone (i.e., cognitive reappraisal) [12]. Specifically, we will investigate whether social reappraisal is more effective and longer-lasting than cognitive reappraisal in down-regulating negative affect in adolescents and adults (Aim 1). We hypothesize that social reappraisal will be more effective and enduring than cognitive reappraisal in both groups, but that this effect will be larger in adolescents given their heightened sensitivity to peers. Furthermore, we will identify the neural mechanisms supporting social versus cognitive reappraisal and characterize age-related differences in these mechanisms (Aim 2). We will focus on examining activation in the amygdala, LPFC, VS, and VMPFC, as well as functional connectivity between these regions. We hypothesize that LPFC-amygdala connectivity will support cognitive reappraisal, which will be stronger in adults versus adolescents, whereas VS-amygdala and VMPFC-amygdala connectivity will support social reappraisal. While VS-amygdala connectivity is likely to be stronger in adolescents than adults, VMPFC- amygdala connectivity might not be given that this pathway is still developing during adolescence [21,76]. We expect that social reappraisal will have a more transformative and thus longer-lasting effect on amygdala- based representations of negative stimuli, particularly in adolescents, and will use representation similarity analysis to test this hypothesis. Examining the efficacy and neural underpinnings of social reappraisal in adolescents versus adults is an important step in advancing our understanding of how social contexts shape emotion regulation neurodevelopment, with the aim of improving adolescent health and laying the groundwork for positive emotional wellbeing into adulthood.

NIH Research Projects · FY 2025 · 2023-07

Summary/Abstract While prostate-specific membrane antigen-targeted radiopharmaceutical therapy (PSMA-RPT) improves the survival of metastatic castration-resistant prostate cancer (mCRPC) patients, response rates remain suboptimal, and relapses invariably occur in all patients. Improving therapeutic efficacy requires a better understanding of the genetic, molecular, and immunological determinants of tumor responses to PSMA RPT. To identify such determinants we (i) performed dosimetry studies to optimize 177Lu-PSMA-RPT activity in prostate cancer (PCa) models; (ii) conducted global phosphoproteomic analyses of tumors from PSMA-RPT- treated mice and revealed the upregulation of DNA damage response/repair and TP53 pathways; (iii) showed that wild type TP53 plays an important role in mediating responses to RPT in mice; and (iv) investigated the impact of PSMA-RPT on the tumor immune microenvironment and demonstrated that PSMA-RPT synergizes with pharmacological activators of the cyclic GMP–AMP synthase (cGAS)/Stimulator of Interferon genes (STING) pathway, a cytosolic DNA sensing machinery that links DNA damage with the induction of innate immune responses via type I interferon (IFN) signaling. Relatedly, data in the literature indicate that mutant TP53, which occurs frequently in mCRPC, interferes with the function of the cGAS/STING/IFN pathway thereby decreasing tumor immunogenicity. Collectively, these findings led us to hypothesize that (i) 177Lu- PSMA-RPT triggers tumor TP53 mutational status-dependent tumor and immune cell signaling alterations in mCRPC; (ii) profiling these alterations will identify new determinants of response to PSMA-RPT; and (iii) targeting these determinants will enhance responses to PSMA-RPT. We will test these hypotheses via three Specific Aims leveraging an integrated platform for systematic profiling of RPT-induced transcriptional, signaling, and immunological alterations. In Aim 1, we will identify RPT-induced signaling alterations in the tumor cell compartment of human PSMA-expressing PCa models and test the hypothesis that mutant TP53 impairs responses to PSMA-RPT. In Aim 2, we will employ murine PCa models with wild-type or mutated TP53 to investigate how tumor TP53 status impacts RPT responses and tumor immunogenicity in immunocompetent mice. We will test the hypothesis that mutant TP53 interferes with cGAS/STING/IFN signaling in RPT-treated tumors and reduces tumor immunogenicity. In Aim 3, we will develop novel combination therapies that enhance or restore the cGAS/STING/IFN pathway in the mCRPC immune tumor microenvironment and improve the magnitude and durability of RPT responses via increased tumor immunogenicity. Successful completion of these aims will identify new connections between mCRPC TP53 mutational status, cytosolic DNA sensing mechanisms, and tumor immunogenicity that can be leveraged to increase the efficacy of RPT against mCRPC through rationally designed and clinically applicable combination therapies.

NIH Research Projects · FY 2025 · 2023-07

This application aims to develop improved methods for detecting novel pathogens that can be deployed on a large scale and are flexible to multiple pathogens. The method uses the power of next generation sequencing technology to analyse hundreds of thousands of samples simultaneously. In contrast to standard clinical testing, where one person's sample is tested in a single tube, mass testing labels each person's sample with a unique piece of DNA that acts as a molecular barcode, then pools multiple samples together so that they can be jointly tested. DNA sequencing is then used to detect those samples with virus in the pool of hundreds of thousands of individuals, and assign the virus to the samples it came from on the basis of the molecular barcodes. A bench top sequencer can process tens of thousands a day. A larger machine generates enough sequence to run up to hundreds of thousands of tests in one day. Our aim is to make it possible for a moderately well-equipped molecular biology laboratory to be able to process tens of thousands of samples without much investment. We have successfully deployed SwabSeq testing at the high-complexity, CLIA-certified, UCLA SwabSeq COVID19 Testing laboratory. Our work has demonstrated the utility for high-throughput asymptomatic testing and with additional improvements can increase testing capacity by orders of magnitude, making it possible to deploy testing on a population scale. Our sequencing-based approach can be extended to also detect viral variants at the same time and to other viral pathogens. Mass testing will f nd asymptomatic carriers and thus inform public health policies so that containment of infection will be effective.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Viral infection outcomes are sex-biased, with males generally more susceptible to human cytomegalovirus (HCMV) and other viral infections compared to females. These differences may reflect sexual dimorphism in immune cell composition and function. As such, it is surprising that numbers of natural killer (NK) cells, a first line of defense against HCMV, are increased in males compared to females. Here we show in mouse models and human samples that while males harbor increased NK cell numbers, they produce less IFN-γ, a critical pro- inflammatory cytokine for NK-mediated anti-viral responses. This difference is not due to divergent levels of gonadal hormones, since these differences are still present in gonadectomized mice. Instead, a screen for X chromosome genes that escape inactivation and demonstrate sexually dimorphic expression in NK cells identified UTX, an epigenetic regulator that alters transcriptional programs through reorganization of chromatin. NK cell-specific UTX deletion phenocopies multiple features of male NK cells, which include increased NK numbers and reduced IFN-γ production. Thus, we hypothesize that NK cell sex differences can be attributed to differential expression of X-linked UTX, which reorganizes chromatin at loci important in NK cell fitness and function. In Aim 1, we will define UTX-mediated temporal control of NK cell numbers and determine whether differences in cellular fitness underlie differences in NK cell homeostasis in males compared to females. In Aim 2, we will delineate UTX’s role in promoting NK cell cytotoxic activity and whether lower UTX levels in male NK cells also impairs their cytotoxic capacity. In Aim 3, we will delineate molecular mechanisms by which UTX controls chromatin accessibility and gene expression at loci important for NK cell homeostasis and function. Knowledge gained from completion of these studies will contribute to our basic understanding of sex differences in anti-viral responses and NK cells. A deeper understanding of sex differences will benefit human health overall by revealing new targets for immunotherapy and guiding interventions for optimizing anti-viral immunity.

NIH Research Projects · FY 2024 · 2023-07

Project Summary/ Abstract: Primordial Germ Cells (PGCs) are embryonic precursors to the adult germline, the proper development of which is tantamount to organismal fitness. During embryonic development PGCs undergo two fate-restriction steps: 1) specification, in which PGCs express pluripotent genes, a state called latent pluripotency, while undergoing myriad epigenetic remodeling and 2) determination, in which the pluripotency program is extinguished and PGCs differentiate according to the sex of the embryo. While molecular studies have carefully dissected PGC specification, PGC determination remains poorly understood. Although the current state-of-the-art allows in vitro induction of PGC-Like-Cells (PGCLCs) from pluripotent stem cells (PSCs), these PGCLCs represent specified PGCs and, thus far, cannot be reliably induced to undergo determination in vitro. This constitutes a significant roadblock for in vitro gametogenesis, which offers a possibility for clinical relief of infertility in couples where either partner is unable to produce their own gametes. We hypothesize that specific epigenetic changes drive PGC determination and license gametogenic capacity. Of particular interest during this process is the regulation of Transposable Elements (TEs), some of which remain capable of transposition and therefore threaten the integrity of the germline genome. Conversely, some TEs of the Long Terminal Repeat (LTR) subclass harbor transcription- and pluripotency- factor binding sites and could function during the time of determination to regulate expression of the pluripotency network. To understand how regulation of LTR elements contributes to PGC determination we employ an in vitro mouse model. The central hypothesis of this proposal is that PGC determination is an epigenetic transition that is reliant on TRIM28, a highly conserved epigenetic scaffolding protein, for two independent processes: regulation of LTR-class transposable elements and proper nucleolar function. To test this, we will employ a PGC-specific conditional knockout model of TRIM28, allowing us to interrogate determination in vivo. In Aim 1, I will use ATAC-seq and CutnTag sequencing to understand how loss of TRIM28 alters genome accessibility and enhancer dynamics, hypothesizing that misregulation of LTR elements in the absence of TRIM28 drives a failure to correctly regulate the switch in gene expression networks as PGCs enter determination. In Aim 2, I use OligoPaint, a DNA-FISH approach, to assess how TRIM28 loss effects nucleolar heterochromatin and morphology, and use chemical perturbation ex vivo to observe possible phenocopy with loss of TRIM28. Completion of this work will have broad implications in our understanding of how the PGC epigenome is rewired during determination to license gametogenesis. Insights from this work can be leveraged to advance in vitro PGC models towards functional gametogenesis.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract: Antiretroviral therapy has transformed HIV infection into a chronic disease and the population living with HIV infection (PLWH) is aging. PLWH are twice as likely to develop cardiovascular disease (CVD) and in the past two decades, the global burden of HIV-associated CVD has tripled. Chronic inflammation and immune activation persist in the setting of treated HIV and are strongly predictive of CVD events and mortality. In the general population, acquired mutations in hematopoietic cells (clonal hematopoiesis of indeterminate potential, CHIP) increase with age and are associated with an increased risk for CVD and premature MI independent of age or other traditional risk factors. Clonally expanded blood cells contain large scale mosaic chromosomal alterations (mCAs) which also increase with age and are associated with risk for infectious disease, and mortality but have not been studied in HIV. While the underlying mechanisms for excess risk of CVD among people with CHIP mutations is not clear, recent studies suggest the NLRP3/IL-1β/IL-6 pathways play a role. This is critically important because these pathways represent targets for immunomodulatory therapy and underlying mechanisms by which PLWH have an excess risk of CVD. Our group has demonstrated that HIV is associated with a 2-fold increase in CHIP and have a high prevalence of mCA but the relationship between CHIP/mCA and CVD risk in PLWH has not been evaluated; furthermore, the underlying mechanism for increased CHIP/mCA among PLWH remains unknown. We plan to study the existing Veterans Aging Cohort Study Biomarkers Cohort (VACS BC) which is a prospective observational cohort of 1525 HIV+ and 853 HIV- Veterans with extensive adjudicated outcomes, existing data on biomarkers of inflammation, and immune function. An additional 1000 PLWH in the Center for AIDS Research Network of integrated Clinical Systems (CNICS) and 1002 PLWH in the Department of Defense (DoD) cohort will also be studied. We hypothesize that HIV is a fertile substrate for development of CHIP mutations and mCAs and that CHIP activates inflammation to drive HIV-associated atherosclerosis. We propose the following Specific Aims: Aim 1: To determine if CHIP mutations and mCAs are more prevalent in PLWH vs. uninfected individuals (Aim 1A). and to determine whether the presence of CHIP mutations/mCAs are associated with an increased risk of CVD and mortality events in PLWH (Aim 1B); Aim 2: To elucidate the mechanism underlying CHIP/mCA in HIV by evaluation of markers of inflammation/immune activation, epigenomics, and HIV viral reservoir size; Aim 3: To investigate whether clonal populations of immune cells from PLWH with CHIP display altered gene regulatory programs that increase pathologic activation of specific immune cell types. If our hypotheses are correct, CHIP/mCAs may serve as a novel biomarker for CVD risk among people aging with HIV infection and help identify those PLWH who may benefit most from immunomodulatory therapies. With the completion of these specific aims, we will advance our understanding of the role that CHIP mutations/mCAs play in the development of CVD among PLWH.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT While the clinical spectrum of SARS-CoV-2 infection in pregnancy ranges from asymptomatic to critical disease, pregnancy itself augments the risk of severe and critical COVID-19. The leading obstetrical societies in the U.S. recommend COVID-19 messenger RNA (mRNA) vaccination in pregnancy to prevent severe disease. Furthermore, passive immunity to the neonate via transplacental transfer of immunoglobulin G (IgG) is critically important during the first six months of life, particularly as the COVID-19 vaccines are approved only for children >six months of age. Hybrid immunity, defined as vaccine-induced immunity before or after natural infection with SARS-CoV-2, produces a more robust response in non-pregnant populations than either type in isolation. The long-term inflammatory and immunologic responses to SARS-CoV-2 hybrid immunity in pregnancy, a state marked by tightly regulated T cell control and immune modulation, compared to natural infection are unknown. As SARS-CoV-2 becomes endemic, this proposal will address a gap in the literature that has focused primarily on natural SARS-CoV-2 infection in pregnancy compared to vaccine-induced immunity. Leveraging the existing COVID-19 Outcomes in Mother-Infant Pairs (COMP) study, a longitudinal cohort that follows 225 mother-infant dyads diagnosed with SARS-CoV-2 infection in pregnancy, the proposal seeks to better understand the long- term consequences of SARS-CoV-2 hybrid immunity in pregnancy. We hypothesize that SARS-CoV-2 hybrid immunity compared to natural infection in pregnancy confers protection against postpartum complications, leads to less maternal systemic inflammation, and results in more robust immune responses in mother-infant pairs. The study aims are: 1) to estimate the prevalence and risk factors of peripartum and delayed postpartum complications of COVID-19 in pregnancy between those with SARS-CoV-2 hybrid immunity, and those with natural infection; 2) to compare the systemic inflammatory landscapes, as measured by cytokine profiles, of pregnant women with SARS-CoV-2 hybrid immunity and natural infection at delivery and one year postpartum; and 3) to evaluate cellular and humoral immune responses to the ancestral and Omicron (BA.5) strains following COVID-19 in pregnancy at delivery and six months postpartum in mother-infant dyads with SARS-CoV-2 hybrid immunity compared to natural infection. The K23 will support Dr. Cambou to develop advanced skills in 1) applied immunology, 2) perinatal infections, and 3) cytokine analysis, in order to become an effective translational physician-scientist. Dr. Karin Nielsen, a world-renowned pediatric infectious diseases (ID) expert in perinatal infections, will serve as the primary mentor. Co-mentors Drs. Otto O. Yang and Grace Aldrovandi, experts in viral immunology, have over 20 years of continuous NIH funding and proven track records of successful mentorship. Co-mentor Dr. Debika Bhattacharya will offer guidance as an adult ID clinical researcher in perinatal viral infections. The K23 will allow Dr. Cambou to carve out a unique niche of multidisciplinary research blending immunology with viral infections in pregnancy, in order to launch her career as an independent investigator.

NIH Research Projects · FY 2024 · 2023-07

Project Summary Social interactions are critical to the physical and emotional health of a wide variety of species. Perturbations in social functioning, a hallmark symptom of many psychiatric and neurodevelopmental disorders such as autism and schizophrenia, can profoundly impair an individual’s ability to sustain healthy social relations. While a growing body of literature has elucidated neural circuits for dyadic social interactions (interactions between two individuals), our understanding of higher order interactions at the level of larger groups is remarkably weak. Humans and other species organize themselves into social groups, in which the behavior of each individual both contributes to and benefits from the cohesiveness and well-being of the collective. Social groups serve as a context for sharing of resources, buffering of stress, regulation of homeostatic needs, and a reservoir of cognitive capacity to solve problems and respond to environmental challenges. In order to address this gap in the literature, I am using a novel behavioral approach to study how groups of mice self-organize into huddles in response to a cold temperature thermal challenge. Prior studies examining social groups have been limited by lack of technology to track the pose and identity of each mouse over the duration of a session. To address these challenges, I am using novel multi-animal pose estimation tools developed through computer vision to quantitatively identify huddling configurations in groups of four mice. Furthermore, I am combining this automated behavioral tracking with circuit dissection tools to understand which circuits in the brain coordinate huddling in response to thermal challenge. Published work from our lab and others suggests that medial prefrontal cortex (mPFC) is a critical node involved in regulating group level behaviors and inter-brain dynamics across species. However, the contribution of mPFC and its descending projections to group huddling has never been explored. Furthermore, although whole-brain knockout studies have found that the social hormone oxytocin is necessary for huddling in mice, the precise neural circuits that are engaged by oxytocin to promote huddling never been examined. The experiments described in this proposal will fill a critical gap in our understanding of neural mechanisms for collective behavior by performing detailed quantitative behavioral analysis and neural circuit dissection to physiologically observe, computationally model, and functionally manipulate individual descending projections of the dmPFC during huddling. Using an ethologically relevant group behavior, in vivo freely moving calcium imaging, chemogenetic manipulations, and anatomical mapping, the proposed study will test the hypothesis that oxytocin engages discrete, anatomically-defined pathways descending from the dmPFC to promote group huddling. These results will set the foundation for a more incisive analysis of how dmPFC circuits shape social function in both health and disease. In addition, completion of this work will provide critical training in statistical and machine learning approaches to study the behavior and neural dynamics of social groups, which are essential for the candidate to become a successful independent investigator.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY/ABSTRACT Affiliative caregiving behaviors are essential for the survival and well-being of a social species. Specifically, prosocial comforting behavior can improve the physical and mental health of distressed individuals. Similarly, parental care is critical for offspring survival and also benefits the welfare of offspring. However, despite the importance of both empathetic prosocial behaviors and parental caregiving for developing and maintaining social bonds, their underlying neural circuitry remain incompletely characterized. While prosocial care is primarily directed towards adult animals, a long-standing proposal for the origin of prosocial care points to potential evolutionary roots in parental care: the need to support helpless offspring drove the development of neural, chemical, and psychological sensitivity to detect contextual cues of others’ needs. The proposed research will explore the shared neural circuits underlying these behaviors, providing insight into the fundamental principles subserving social affiliation. While much is known about the positive control of parenting, the neural circuitry of comforting prosocial behavior is not as well defined. The medial preoptic area (MPOA) is a key region for regulating parenting behaviors. Recent evidence suggests the MPOA may also be involved in modulating prosocial comforting behavior in rodents. Other lines of evidence implicate the dopaminergic system in affiliative and parenting behavior through inhibitory inputs to the ventral tegmental area (VTA). This proposal hypothesizes that the inhibitory MPOA-VTA circuit is functionally significant as a shared node for prosocial and parental behaviors in mice. My central hypothesis will be tested in two specific aims: (1) determine if inhibitory MPOA neurons projecting to the VTA control affiliative allogrooming and parental behavior, and (2) characterize how these encode social sensory cues as well as offspring-directed and conspecific-directed caregiving behavior. Collectively, these experiments will expand our understanding of the neural circuits shaping both prosocial and parenting behaviors, which are critical to the formation of fundamental social bonds. A more robust mechanistic understanding of the MPOA-VTA and its functions in prosocial and parental circuits will offer insights into the long-postulated role of parental care in mammals as the origin of social affiliation.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Chimeric antigen receptor (CAR) T cell therapy has produced remarkable results in otherwise treatment- refractory hematological malignancies. Currently, the process of manufacturing autologous CAR T cells is challenging due to the need for de novo generation of each individualized therapy and the inherent variability in T cell biology between patients, leading to unpredictable and inconsistent clinical responses. As a result, there is a growing interest in generating CAR T cells in vitro from an infinitely renewing, allogeneic source of human pluripotent stem cells (hPSCs). One advantage of this approach is that hPSCs are highly amenable to genetic editing, providing multiple avenues to manipulate the function of the final T cell product. However, there are several biologic barriers to generating CAR T cells from hPSCs. First, expression of the CAR transgene early in T cell differentiation diverts development towards the innate lymphoid pathway instead of the conventional T lineage. Second, the removal of the endogenous T cell receptor (TCR) to prevent alloreactivity leads to a block in development, as maturing T cells can no longer undergo positive selection. This proposal seeks to overcome these challenges to generating non-alloreactive, stem cell-derived CAR T cells by deploying an innovative stage-specific expression strategy and a novel in vitro method to induce the differentiation of mature conventional T cells, the Artificial Thymic Organoid (ATO) system. The ATO is a first-in-class, in vitro method for efficiently generating mature, single positive (SP) CD8+ and CD4+ T cells from multiple stem cell sources, including hPSCs. Improving upon previously established systems, the ATO can robustly support positive selection and maturation to the SP stage. Our preliminary studies have already demonstrated that innate fate diversion can be overcome by achieving delayed, stage-specific expression of the CAR transgene that is limited to mature T cells. In this proposal, Specific Aim 1 will build upon the delayed CAR expression model and evaluate how the disruption of the endogenous TCR affects CAR T cell development in the ATOs. Specific Aim 2 will evaluate two different methods of delivering exogenous positive selection signals to rescue development in the absence of the endogenous TCR. These methods include expression of a tonically signaling CAR or transiently expressing an exogenous TCR at the physiologically appropriate double positive (DP) T cell stage. The approaches developed in this proposal will enable the generation of hPSC-derived, non-alloreactive T cell therapy, ultimately reducing the cost and increasing the access to treatment for more patients in need.

NIH Research Projects · FY 2026 · 2023-07

ABSTRACT Promoting inflammation resolution and resolution-dependent intestinal epithelial wound healing are recent therapeutic concepts for IBD. During inflammation resolution, macrophages phagocytose apoptotic neutrophils (efferocytosis), thereby preventing secondary necrosis. Macrophage efferocytosis also stimulates the secretion of various resolving and reparative factors that can trigger intestinal epithelial wound healing. I previously reported that dysregulation of macrophage efferocytosis and efferocytosis-dependent intestinal epithelial wound healing contributed to pathogenesis in a murine model of Crohn’s disease (CD). The overall goal of this project is to build upon my prior work to advance human pre-clinical research into resolution-based therapies for CD. First, I seek to leverage innovative human models and unbiased analytical techniques to determine the cell-cell communication between efferocytic macrophages and human small intestinal epithelium that drives epithelial wound healing following inflammatory injury. This work will fill important knowledge gaps, identify potential resolution-specific therapeutic targets, and define a physiological baseline against which to assess disease- dependent dysregulation of this resolution/repair module. Towards this first goal, I have recently modeled inflammatory injury in human ileal and jejunal intestinal epithelial organoid culture systems and shown that human macrophage efferocytosis rescues this injury. I will interrogate this model with multi-omic techniques in order to reconstruct potential ligand-receptor interactions and receptor-signaling pathways. Second, I seek to investigate and translationally target potential disease-dependent dysregulations of macrophage efferocytosis and efferocytosis-dependent human small intestinal epithelial wound healing. I have observed, consistent with prior reports, that mucosal oxidized polyunsaturated fatty acids including 12- and 15- hydroxyeicosatetraenoic acid (HETE) correlate with disease in murine models of IBD and in IBD patient biopsies. In my human models, these HETEs act as potential dysregulators of resolution—suppressing both macrophage efferocytosis and efferocytosis-dependent small intestinal epithelial wound healing. I will investigate the mechanisms of these dysregulations. Nonetheless, I had previously observed that the apolipoprotein A1 mimetic peptide 4F rescues murine CD-like disease by enhancing mucosal excretion of disease-elevated HETEs. Pilot data show that 4F inhibits HETE-dependent human macrophage dysregulation and enhances the clearance of HETEs across human small intestinal primary epithelium. 4F thus appears to be a promising therapy against HETE-dependent dysregulation of resolution and resolution-repair. After this study, I anticipate that I will have characterized novel dysregulations of inflammation resolution and resolution-dependent intestinal epithelial repair that may amplify disease in IBD but that can nonetheless be disrupted therapeutically by exploiting a novel mucosal clearance pathway.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Disordered eating, encompassing both cognitive (e.g. body dissatisfaction, drive for thinness, preoccupation with losing weight) and behavioral factors (e.g. counterproductive behaviors like laxative use, binge eating, and vomiting for weight control), is particularly harmful given its association with metabolic risk factors. Weight stigma has emerged as a risk factor for disordered eating, and over 200 million Americans have a body mass index that puts them at risk for experience weight stigma. However, there are three major gaps in the literature. Prior work has examined associations between weight stigma and disordered eating behaviors via observational and longitudinal methods, and thus whether weight stigma causes disordered eating is unknown. Moreover, research has not examined the presence of disordered eating symptoms in everyday life after exposure to weight stigma. Finally, potential social resilience factors remain unexplored. Therefore, the current study will combine experimental manipulation with daily diary methodology to evaluate the causal effect of weight stigma on daily disordered eating symptoms and test belonging and group identification as social resilience factors that confer protection against disordered eating. This project will capitalize on the infrastructure of Sponsor Tomiyama’s parent R01 (R01HL158555) and will add comprehensive measurement of disordered eating symptoms in 300 overweight (BMI  28) adult participants recruited from the greater Los Angeles area to address two aims: 1) Assess the causal effects of weight stigma on disordered eating symptoms in daily life and 2) Investigate social resilience factors that provide protection against disordered eating symptoms. This study will help to understand the impact of weight stigma and identify resilience factors to prevent disordered eating. The current proposal could aid in identifying those who may be more vulnerable to disordered eating as a consequence of weight stigma, or identify future intervention strategies, and in turn, promote metabolic health. Throughout the fellowship period, with the support of Sponsor Dr. A. Janet Tomiyama and Collaborators Drs. Craig Enders, Theodore Robles, and Kendrin Sonneville, I will be working to achieve four training aims: 1) Master advanced quantitative analysis skills, specifically in multilevel modeling and growth curve analysis, 2) Expand knowledge in disordered eating, specifically in adulthood, 3) Develop expertise in social resilience factors, and 4) Prepare for a career as an independent researcher. I will accomplish these goals by attending and participating in lab meetings and workshops, presenting my work at academic conferences, and enrolling in coursework. Further, I will be focusing on four subaims that will prepare me for a career as an extramurally funded researcher at an R1 institution, including engaging in open science, executing evidence-based practices in mentoring, building grant writing skills, and practicing scientific dissemination.