Ut Southwestern Medical Center

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Sensory hypersensitivity is a common symptom in autism and Fragile X Syndrome (FXS) and is thought to be a result of cortical circuit dysregulation. EEG studies in humans with FXS and the FXS mouse model, the Fmr1 KO, reveal cortical circuit hyperexcitability and synchrony deficits such as enhanced resting state power in the gamma band and reduced sensory-driven synchrony. In acute slices, this hyperexcitability can be observed as prolonged persistent activity states, called UP states, and increased gamma band power during UP states. I hypothesize that circuit mechanisms that mediate hyperexcitability and prolonged UP states in the neocortex may contribute to EEG phenotypes in FXS. Using positive and negative allosteric modulators (PAMs/NAMs) specific for GluN2C/D subunits of NMDA receptors, I have revealed an upregulation of GluN2C/D function in the Fmr1 KO cortex that contributes to circuit hyperexcitability. Specifically, GluN2C/D PAMs increase UP state duration and gamma power during the UP states, while NAMs rescue UP state duration. Remarkably, these interventions only affected the Fmr1 KO, not their wildtype (WT) littermates suggesting that GluN2C/D function is upregulated in the Fmr1 KO and leads to cortical circuit dysfunction. Typically, GluN2C/D subunits are expressed in cortical inhibitory neurons and astrocytes. Since my results are not consistent with effects on inhibitory neurons, GluN2C/D subunits may be misexpressed in excitatory neurons or upregulated in astrocytes in the Fmr1 KO. I hypothesize that GluN2C/D expression and/or function is increased in excitatory neurons and/or astrocytes in Fmr1 KO mice and this contributes to hyperexcitability and altered synchrony of cortical circuits. The goal of the proposed project is to test this hypothesis and determine expressional and functional changes in Glun2C/D that may contribute to cortical hyperexcitability and synchrony following three Specific Aims. Aim 1. To determine change in cortical protein and RNA expression levels as well as cell specific expression of GluN2C/D subunits in Fmr1 KO cortex. Aim 2. To determine cell specific functional contribution of GluN2C/D subunits to NMDA-mediated currents in WT and Fmr1 KO cortex. Aim 3. To determine the contribution of GluN2C/D NMDARs to in vivo sensory driven circuit excitability and altered synchrony using multi-electrode array EEG and measurement of audiogenic seizures in the Fmr1 KO mouse in collaboration with Dr. Devin Binder at UC Riverside. These experiments will not only provide insights into the molecular and cellular basis of GluN2C/D contribution to cortical circuit dysfunction, but also examine GluN2C/D subunits as a potential target for therapeutic development using translational biomarkers.

NIH Research Projects · FY 2025 · 2023-09

1 Eric B. Ortigoza, MD, MSCR is a Neonatal-Perinatal Medicine physician with a Master of Science in Clinical 2 Research at UT Southwestern Medical Center (UTSW). His goal is to be an independently funded investigator 3 with expertise in neonatal gastrointestinal motility. He plans to investigate novel, comprehensive objective 4 methods to differentiate developmental feeding intolerance (DFI) from pathologic feeding intolerance (PFI) with 5 the goal of limiting unnecessary feeding delays, parenteral nutrition, and improving outcomes in preterm infants. 6 In a prospective, longitudinal cohort study, preterm infants who are born <32 weeks’ gestation will undergo 7 weekly non-invasive electrogastrography (EGG), abdominal near-infrared spectroscopy (aNIRS), and stool 8 collection. Dr. Ortigoza’s specific aims are to 1) quantify postmenstrual age-dependent differences in non- 9 invasive continuous gastrointestinal monitoring in preterm infants with DFI, PFI, and no feeding intolerance (NFI), 10 and 2) measure postmenstrual age-dependent differences in the gut microbiome and microbiota-derived 11 metabolites in preterm infants with DFI, PFI, and NFI. Dr. Ortigoza’s innovative approach integrates objective 12 gastrointestinal biomarkers of gastric motility, mesenteric oxygenation, bacterial colonization, and microbiota- 13 derived metabolites to differentiate DFI from PFI. The ability to differentiate between the two conditions will 14 encourage the development of predictive models for PFI in preterm infants using bioinformatics and machine 15 learning. The ability to predict PFI will help develop evidence-based strategies aimed at preventing and/or 16 treating episodes of PFI. Dr. Ortigoza has assembled a multidisciplinary team of mentors with expertise in the 17 key areas of computational modeling of complex physiological variables (Lina Chalak, MD, MSCS), advanced 18 gut microbiome profiling (Andrew Koh, MD and Julie Mirpuri, MD), and metabolite analysis (Andrew Koh, MD). 19 UTSW and its strong clinical research operation provide the ideal environment to conduct the proposed studies 20 with the large patient population at Parkland Health and Hospital System (PHHS) and Children’s Health, a 21 dedicated Center for Translational Medicine, and a strong record of clinical research participation. Dr. Ortigoza’s 22 Career Development Plan includes a comprehensive focused strategy to address the specific key training skills 23 that will allow him to transition to independence including: 1) developing expertise in complex signal analysis of 24 EGG and aNIRS data, 2) gaining expertise in interpretation of high throughput analysis of the gut microbiome 25 and its derived metabolites, and 3) developing expertise in biomarker development/validation. In addition, he will 26 receive training to develop leadership skills that are critical to fostering a productive research team and building 27 a successful research program. Together with his scientific aims, these goals will provide the skills necessary 28 for Dr. Ortigoza to build his independent research program in neonatal gastrointestinal motility.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT: CAR-T cell therapy is an emerging option for cancer treatment, but its efficacy is limited, especially in solid tumors, because the effector CD8+T cells become dysfunctional and exhausted in the tumor microenvironment (TME). However, the key pathways that define the delicate balance between the effector vs. exhausted state of CD8+T cells remain unclear. Our preliminary data demonstrate that Znf740, a novel member of the zinc finger family of transcription factors, is critically essential for effector CD8+T cells. Znf740 binds to SUMOylated T-bet and promotes the effector function and anti-tumor activity of CD8+ tumor-infiltrating lymphocytes (TILs). Conversely, in exhausted PD1+Tim3+CD8+T cells, Znf740 expression is downregulated, which disrupts the Znf740:T-bet complex. Importantly, reconstitution of Znf740 expression rescues exhausted CD8+TILs and restores their effector function. Further, transgenic expression of Znf740 in CD8+T cells resulted in reduced tumor growth which was associated with elevated IFN- production by TILs. These key findings led us to hypothesize that the Znf740:T-bet complex is critically essential for the effector function of CD8+T cells, and the disruption of this complex in PD1+Tim3+ cells promotes exhaustion of CD8+TILs which can be therapeutically targeted. In Aim1, we will investigate how Znf740 promotes effector CD8+T cell function and anti-tumor response. Using newly generated Znf740-/- and T-bet-K208R knock-in mice, we will delineate the mechanism by which Znf740 binds to T-bet through its SUMO-interacting motif (SIM) to form the Znf740:T-bet complex and transactivates the IFN- promoter in effector CD8+TILs. In Aim 2, we will target Znf740 to overcome T cell exhaustion and promote tumor regression. We will investigate how disruption of the Znf740:T-bet complex in advanced tumors promotes an alternate transcription profile of PD1+Tim3+ exhausted TILs. Using newly generated T cell-specific Znf740 transgenic mice, we will test the effect of overexpressing Znf740 in CAR-T cells against carcinoembryonic antigen (CEA) in the MC38 colon cancer model. Finally, the therapeutic potential of overexpressing Znf740 in CAR-T cells will be tested in colon cancer patient-derived xenograft (PDX) models. Completion of these studies will result in the establishment of 1) a novel Znf740:T-bet complex that is critical for the effector CD8+ T cell function, 2) determine how reduced Znf740 expression disrupts this complex leading to alternate transcription profile in exhausted CD8+ TILs in advanced tumors, and 3) evaluate the means to target the Znf740 to overcome the current limitations of CAR-T cell therapy for solid tumors. This could lead to clinical trials using "Exhaustion Resistant CAR-T cells" for improved outcomes in patients with advanced tumors.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Cardiovascular mortality attributable to hypertension (HTN) has increased by more than 10 percent in the last decade, due in part to the escalating obesity epidemic. Effective therapeutic options for HTN are limited and despite a high prevalence of multi-pharmacological approaches, more than 50% of hypertensive individuals remain uncontrolled. As such, studies that advance understanding of basic mechanisms of blood pressure regulation in humans are needed to identify novel therapeutic targets. Exaggerated sympathetic nervous activity (SNA) and vasoconstriction are hallmark features of many cardiovascular diseases including obesity-associated HTN (Ob-HTN). Causes of exaggerated sympathetic vasoconstriction are unclear, however recent advances in quantification of sympathetic activity have uncovered unique action potential patterns that influence the peripheral vasoconstrictor response to stress. In animal models, sympathetic firing patterns during stress increase co-release of the potent vasoconstrictor neuropeptide Y (NPY) in conjunction with norepinephrine, causing a shift in the mechanisms of vasoconstriction towards NPY-mediated signaling. NPY causes vasoconstriction via activation of NPY 1 receptors (Y1R), and facilitates α-adrenergic mediated signaling causing exaggerated sympathetic vasoconstriction. We hypothesize that physiological stressors like obesity and hypoxia alter sympathetic action potential patterns that cause vasoconstriction to rely on NPY-mediated signaling. The overall aim of this proposal is to 1) identify how sympathetic action potential patterns change in response to chemoreflex and mental stress 2) to assess the impact of action potential patterns on mechanisms of vasoconstriction in healthy adults and in patients with Ob-HTN. To accomplish these goals, we will we will assess beat-by-beat vasoconstriction in response to endogenous bursts of SNA during pharmacological manipulation of α-adrenergic receptors and Y1Rs to determine the mechanisms of exaggerated sympathetic vasoconstriction during chemoreflex/mental stress in healthy adults and Ob-HTN patients. We anticipate that these investigations will 1) further understanding of the basic signaling mechanisms responsible for neurovascular transduction in humans including the interaction between action potential patterns, neurotransmission and vasoconstriction 2) identify new mechanisms underlying exaggerated neurovascular transduction in Ob-HTN and 3) provide avenues for research in other patient populations characterized by elevated SNA and exaggerated vasoconstriction including sleep apnea, metabolic disease, and heart failure.

NIH Research Projects · FY 2025 · 2023-09

A faulty AGA gene coding for the dysfunctional enzyme results in a severe and progressive genetic neurological disorder, Aspartylglucosaminuria (AGU, OMIM # 208400). The functional enzyme is required for the breakdown of glycoproteins in the cellular lysosomes. Absence of enzymatic activity results in impaired lysosomal function and accumulation of aspartylglucosamine (GlcNAc-Asn) in the lysosomes of various tissues and body fluids. The key consequence of the substrate accumulation is lysosomal hypertrophy that manifests as intellectual disability, and other associated symptoms including skeletal and joint abnormalities. Patients have slowed/regressive psychomotor development throughout childhood, deteriorating around the third decade of life to become severely impaired mentally and physically, highly dependent on supportive care thereafter. The median lifespan of AGU patients is approximately 40-50 years. Through efforts primarily funded through a parent-organized non-profit foundation, the Rare Trait Hope Fund, Dr. Gray’s lab has generated preclinical data supporting the initiation of a Phase I/II gene therapy trial to treat AGU. A Type B preIND meeting was held with the FDA regarding this in January 2018, which has charted a clear path forward for human translation. This approach would use an AAV9/AGA vector injected intrathecally, following a precedent set by Dr. Gray’s previous efforts to initiate similar Phase I trials for Giant Axonal Neuropathy at the NIH Clinical Center in 2015 and for CLN7 Batten disease at Children’s Medical Center Dallas in 2021. The wealth of available disease-specific biomarkers for AGU (including localized imaging of the AGA enzyme substrate in discrete brain regions) along with a potentially large treatment window, make AGU an ideal disease to rapidly and fully assess the complete efficacy and/or shortcomings of intrathecal AAV9 as a “platform” approach to treat many other neurological diseases. We propose to conduct the necessary IND-enabling studies to initiate a Phase I/II clinical trial for AGU.

NIH Research Projects · FY 2025 · 2023-09

Abstract Arriving at a more granular understanding of the molecular drivers of appetite will represent a major step towards the ultimate goal of more efficiently treating obesity. AgRP neurons in the arcuate nucleus (ARC) of the hypothalamus play a dominant role in maintaining energy balance and are dysregulated in obesity. We have recently utilized cutting-edge tools to enable an unprecedented cell type-specific characterization of the transcriptional and epigenomic landscape of AgRP neurons, and this information was leveraged to identify one transcription factor, Interferon Regulatory Factor 3 (IRF3) that mediates the acute anorectic effects of leptin. The current project seeks to examine the role of AgRP neuron IRF3 (IRF3AgRP) in the development of diet- induced obesity (DIO) involving loss- and gain-of-function experiments while assessing the impact on mouse body weight, feeding behavior, and leptin sensitivity. The transcriptomic profiles of these IRF3 functional mouse models will be examined in the lean state in response to leptin, and in the obese state. Additionally, the direct DNA binding targets of IRF3AgRP will be elucidated using a cutting-edge, low-input, alternative to ChIP- seq called CUT&RUN, all in the lean fed, fasted, and leptin treated state, and in response to obesity. AgRP neuron-specific ATAC-seq for lean and obese AgRP neuronal nuclei will also be performed as a means of assessing the transcriptional regulatory status of IRF3 and other TFs during obesity. Finally, using both lean and obese mice that are either wild-type or have IRF3 knockout out of all leptin-receptor expressing cells, I will perform single nucleus RNA-seq as a means of identifying the IRF3-driven transcriptional programs onboard in the obese state. Overall, these studies will test the overarching hypothesis that IRF3 plays key roles in mediating both acute leptin sensitivity as well as the development of diet-induced obesity owed to chronic IRF3 activation via hyperleptinemia. The regulatory pathways highlighted in this project will point to novel therapeutic targets for the treatment of obesity, a major risk factor for type 2 diabetes. During the course of the mentored phase of this application, I will further strengthen the computational skills needed to analyze RNA-seq, ATAC- seq, CUT&RUN, and single-nucleus RNA-seq datasets independently. I will also engage in various career development experiences, while also receiving invaluable mentorship from my career advisory committee. In all, the funding of the proposed project will ensure that I round out my scientific and professional training while laying the foundation for a viable independent academic research program.

NIH Research Projects · FY 2025 · 2023-09

Discovering interpretable mechanisms explaining high-dimensional biomolecular data Project summary. How protein and RNA sequence encodes folding, aggregation, and function is a fundamental question with wide-ranging human health implications. Discovering predictive principles for this encoding requires computational approaches that offer mechanistic insight, especially for the large fraction of intrinsically disordered proteins for which experimental structural information is limited. Yet the complexity and dimensionality of this problem poses fundamental challenges to existing computational methods. The axiomatic approach, modeling behavior from first-principles, is limited by simulation runtime and unknown context-dependent parameters. Informatics-based approaches such as deep learning could potentially discover principles by integrating large datasets across scales and complexity. However, these models produce “black box” predictions that i) are difficult to understand and ii) generalize poorly beyond their training data (i.e. well-understood regime). My lab developed methods to overcome limitations of both types of approaches. (1) Axiomatic: we developed a statistical physics method to exponentially enhance sampling of protein self-assembly from structurally heterogeneous monomers in molecular dynamics simulations. (2) Informatic: we invented essence neural networks (ENNs) based on neurobiological principles and demonstrated that they overcome the above limitations of deep learning on a wide range of learning tasks, including sequence-to-function prediction. Using both axiomatic and informatic approaches, in the next five years my lab will tackle three instances of the sequence-structure-function problem: 1) Use enhanced sampling molecular dynamics simulations to discover transition states of neurotoxic oligomer and fibril formation of Abeta and tau peptide monomers; 2) Use ENNs to discover the RNA-sequence rules driving RNA-associated tau fibril aggregation in neurodegenerative disease using tau protein and colocalized RNA sequence datasets; 3) Use ENNs to distill the sequence rules determining whether a strain or mutant of beta lactamase protein can neutralize each antibiotic within a diverse drug panel, and identify potential future antibiotic resistant mutants. Our long-term goal is to develop an ENN- based platform for automated transformation of data into axioms. Leveraging well-established collaborations with colleagues of wide expertise, we will pursue these goals by combining our unique computational approaches with experimental resources, including time-resolved protein aggregation assays, patient-derived tau fibrils co- localized with sequence-specific RNA, high-throughput liquid culture antibiotic screens, multiplexed directed evolution experiments of antibiotic resistance, and large in-house libraries of peptide and RNA mutant libraries. This work lays the foundation for transforming large datasets into human-understandable rules connecting sequence to function and relating these rules to physical mechanisms of structural dynamics. This in turn could accelerate disease diagnosis and treatment.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Hispanic adolescents in the United States are disproportionately affected by obesity compared to their non- Hispanic White adolescent counterparts. A recommended means of addressing adolescent obesity is through participation in family-based multicomponent behavioral interventions (targeting improvements in family functioning as a mechanism of change). Although family-based interventions (FBIs) exist, effects on adolescent obesity-related outcomes, especially among Hispanic adolescents, are small or insignificant. Limited effects may be due to gaps in the existing research: 1) few FBIs have incorporated obesity-related socioecological factors at multiple levels as tailoring variables to improve intervention effects, 2) FBIs often overlook fathers and other household members (such as grandparents in multi-generational households) that may also play a role on adolescents’ obesity risk behaviors, and 3) FBIs often assess family functioning retrospectively without consideration of day-to-day family dynamics, which may also influence obesity risk behaviors. Thus, I propose the following aims to address existing gaps: 1) conduct secondary data analysis using five waves of data from the Adolescent Brain Cognitive Development study and the Hispanic adolescent subsample (n=2411, Mage=9.5 at baseline) to examine the direct and moderating effects of factors at each level of the socioecological model on the longitudinal trajectories of obesity risk behaviors/obesity status, 2) pilot an EMA protocol with Hispanic adolescents (9-to-17 years) and household caregivers (n=20 family units) across 7 days to assess feasibility and acceptability, identify barriers and facilitators to completion of daily assessments by family units, and make needed modifications to the EMA protocol, and 3) implement a finalized EMA protocol over a 6-month period, using measurement burst design, and assess the effects of momentary changes in family functioning behaviors and associations with daily physical activity, sleep, and nutrition behaviors (n=50 family units, adolescents 9-to-17 years). The purpose of this training application is to gain mentoring and training in the following four areas: 1) intensive longitudinal data analysis, 2) recruitment, retention, and implementation, 3) innovative assessment design, and 4) professional development. Under the mentorship of a team of interdisciplinary researchers, experts in either obesity prevention, longitudinal data analysis, ecological momentary assessment, and/or minority health, Dr. Fernandez will successfully complete the plan of research at the University of Texas Health Science Center at Houston, School of Public Health and attain preliminary data to inform the development of a NIH-R01 proposal.

NIH Research Projects · FY 2024 · 2023-09

Summary Immunosenescence increases morbidity and mortality after infection and reduces vaccine efficacy. Most deaths associated with infections by influenza virus, SARS-CoV, or SARS-CoV-2 occurred in people older than 65. Thus, understanding the mechanism of age-associated decline in antiviral immunity is critical to the development of strategies to protect the elderly from viral pathogens. Despite its critical role in protection against infections, T- cell immunity declines with age. During aging, the prevalence of naïve T cells decreases, whereas the frequency of terminally differentiated CD8 T cells increases. In addition, aging reduces the responsiveness of naïve T cells to antigen. However, the molecular mechanism underlying age-associated decline in antiviral T cell immunity remains poorly defined. Using a mouse model of murine hepatitis virus (MHV) infection, we found that aging increased mortality and decreased antiviral CD4 and CD8 T cell responses. Surprisingly, although aging increased terminally differentiated CD8 T cells at baseline, there was a profound reduction in terminally differentiated effector CD8 T cells and an elevated gene-signature of T-cell exhaustion in aged mice after MHV infection. In addition, we showed that age-associated decline in T-cell expansion was primarily caused by TCR- triggered apoptosis and necroptosis pathways and was rescued by rebalancing TCR and IL2 signaling. We also found that aging reduced the metabolic rate of T cells at baseline and impaired metabolic adaptation of T cells after activation. Here, we hypothesize that age-associated exhaustion-prone epigenetic state and defective metabolic adaptation impair effector CD8 T cell response in viral infection. In this study, we will define the epigenetic and metabolic pathways in antiviral CD8 T cells altered by aging before and after infection, while accounting for age-associated changes in differentiation. We will also evaluate strategies that harness IL2 and TCR pathways to rescue age-related defects in antiviral CD8 T cells.

NIH Research Projects · FY 2025 · 2023-09

Sustaining a traumatic brain injury (TBI) can lead to an onset of and/or accelerate Alzheimer’s Disease (AD)-related neurodegeneration process.1Therefore, TBI is considered as a risk factor for AD and AD-related dementias (ADRD).2-5 Understanding of the pathophysiological mechanisms by which TBI contributes to ADRD onset and progression is a prominent research priority for the NIH.2 Recent development of blood-based AD biomarkers such as Aβ42/Aβ40 and phosphorylated tau (p-tau, e.g., p-tau 217) has shown its potential value for preclinical AD diagnosis6 and has been used in longitudinal observational studies and in clinical trials to assess changers in AD pathology.7 Mounting evidence also shows the importance to include nonspecific blood-based neuro-injury and inflammation biomarkers such as glial fibrillary acidic protein (GFAP) and neurofilament light (NfL) in ADRD research to identify co-existing pathology which may also contribute to cognitive symptoms.6 To date, it is unclear whether the presence of AD biomarkers after TBI indicates an independent AD-related pathology or a TBI-related pathology. Our ongoing ADRD-related project (R01NS129934), “Cerebral autoregulation, brain perfusion, and neurocognitive outcomes after traumatic brain injury (CAPCOG-TBI)”, is a longitudinal, observational study to understand the role of cerebrovascular dysfunction in cognitive and neuroimaging outcomes in persons with TBI. In CAPCOG-TBI, we will enroll 100 patients with TBI and 30 orthopedic trauma controls (OTC) during the first week after the indexed injury and follow them for one year. Besides cerebrovascular function measures, detailed pre-existing medical history and TBI injury and treatment history are collected during the acute stage. Cerebrovascular function along with blood sample collection, neurocognitive assessment, global functional outcome, and multimodality brain MRI are obtained at 3M and 12M postinjury. Blood samples are banked at BioSEND where APOE genotype will be performed as a part of the quality control process. Thus, this study provides a unique opportunity to reveal TBI-related longitudinal changes in blood-based AD biomarkers and their relationship with changes in cerebrovascular function, neuroimaging, and cognitive outcomes. The primary goal of this administrative supplement proposal is to measure changes in blood-based AD biomarkers after TBI and their associations with cerebrovascular injury, cognitive outcome, and neurodegeneration. To achieve this goal, we will quantify changes in blood-based AD biomarkers (Aβ42/Aβ40, total tau, p-tau 217, %p-tau217), GFAP, and NfL at three time points (<1 week, 3M, and 12M) after TBI in CAPCOG-TBI participants to address the following aims: 1) To quantify the temporal profile of changes in blood-based AD biomarkers after TBI. 2) To examine the temporal association of changes in blood-based AD biomarkers with cerebrovascular function after TBI. 3) To examine the associations of changes in blood-based AD biomarkers and cerebrovascular function with brain neurodegeneration and cognitive outcome after TBI.

NIH Research Projects · FY 2026 · 2023-08

Project summary Alzheimer's disease (AD) is a leading cause of dementia characterized by memory and cognitive loss interfering with daily life. Clinical studies showed that the aberrant neuronal activity switch from hyperexcitability at the early stage of disease to hypo-excitability at the late stage is a key feature shared in AD patients. In line with this, levels of the principal excitatory neurotransmitter glutamate and vesicular glutamate transporters (vGluT1/2), the primary mediators of glutamate uptake into synaptic vesicles, were decreased at the late stage of AD patients, which contributed to AD dementia. However, the underlying pathology that leads to glutamate misregulation, aberrant neuronal activity and synaptic dysfunction in AD dementia remains largely unknown. Our recent work identified histone H3K27 demethylase KDM6B as a specific epigenetic regulator of synaptic plasticity and cognitive functions. Conditional knockout of KDM6B in the excitatory neurons reduced presynaptic vesicle numbers, spine density and glutamate release/synaptic activity in mice. Moreover, KDM6B KO mice showed behavioral learning and memory deficits. Importantly, KDM6B expression was reduced in aged brain, while trimethyl lysine 27 on histone H3 (H3K27me3) was increased in brain from late-onset AD patients, which were highly correlated with their cognitive deficits. Tau was required for KDM6B recruitment and regulation in synaptic plasticity and cognitive functions. Tau knockdown interfered with synaptic gene expression. As we know, pathological Tau often occurred in AD and perturbed its physiological functions. These findings led us to hypothesize that epigenetic alteration caused by KDM6B-Tau dysregulation contributes to aberrant neuronal activity switch, cognitive impairment and AD pathogenesis. The goals of this R01 project are to 1) decipher the role of pathological Tau in KDM6B-regulated synaptic activity and 2) determine effects of KDM6B dysregulation on AD pathogenesis in AD mouse models. To ensure the success of the proposed project, we have assembled a strong research team with expertise in AD-related neurodegeneration, epigenetic regulation, and synaptic activity. If successful, this project will reveal the importance of Tau-KDM6B-dependent epigenetic priming in AD pathogenesis and define a new epigenetic mechanism underlying synaptic hyper- and hypo-excitability switch and cognitive impairment in AD, which may provide an innovative therapeutic target and a knowledge foundation on development of a rational strategy to improve cognitive functions in AD patients.

NIH Research Projects · FY 2025 · 2023-08

Abstract Humans can rapidly regulate actions according to updated demands of the environment. A key component of action regulation is action inhibition, the failure of which contributes to various neuropsychiatric diseases, such as Parkinson’s disease (PD), obsessive compulsive disorder and Tourette syndrome. Action inhibition occurs in at least 3 ways: (i) action selection – selecting one action requires suppressing alternative motor plans, (ii) outright stopping – inhibiting a response when it is rendered inappropriate and (iii) action switching – change action plans in response to environmental changes. Despite the extensive effort to understand how the brain selects, stops and switches actions, the mechanism underlying these action regulation functions, and more importantly, how they inter-relate remain elusive. Part of this challenge lies in the fact that studies rarely explore, characterize, and investigate these functions together, making it difficult to develop a unified theory that explains the computational aspects of action regulation. The current proposal aims to advance our understanding by developing a neurocomputational model that, unlike prior models, integrates information from multiple sources (e.g., value of targets, cost for changing an action, contextual information) and predicts both kinematics of motor behavior and the underpinning neural mechanisms across 3 distinct types of action regulation. We will directly evaluate model predictions with intracranial recordings in patient volunteers undergoing deep brain stimulation implantation surgeries. These surgeries provide a unique opportunity to obtain multi-focal cortical and basal ganglia (BG) recordings with high temporal and spectral resolution and spatial specificity across the three action regulation tasks. The overarching goal will be achieved through three aims. In Aim 1, we will collect behavioral data from PD patients and aged-match neurotypical participants performing tasks that involves selecting, stopping and switching reaching actions. The motor behavior of the neurotypical group will be used to develop a neurocomputational model that simulate the fronto-BG circuits in action regulation. Then, we will assess how specific changes on the neural mechanisms of the model architecture predict the motor behavior of the PD patients. In Aim 2, we will evaluate the model predictions about the mechanisms of action selection relative to stopping by recording neural activity from PD patients who undergo surgery for DBS implantation of the subthalamic nucleus (STN). Neural recordings will be collected without and with temporally and spatially precise subthalamic nucleus (STN) stimulation to investigate the causal role of STN in action selection. In Aim 3, we will evaluate the model predictions about the mechanisms for switching actions by recording neural activity from PD patients with the STN stimulation off and on. Overall, successful completion will provide a unified theory of action regulation in the human brain, with both behavioral and physiological validation, opening new avenues on improving the effectiveness of neuromodulation with DBS and other neurorestorative therapies.

NIH Research Projects · FY 2024 · 2023-08

Project Summary Blood pressure (BP) control is an effective preventive intervention to reduce the risk of cardiovascular disease, especially heart failure (HF). However, among adults with type 2 diabetes (T2D), the effects of intensive BP control for HF prevention are not well established. There may be high-risk individuals with T2D who are more likely to derive greater benefits from intensive BP control. The American Diabetes Association recommends measurement of cardiac biomarkers, specifically N-terminal pro-B-type natriuretic peptide (NT-proBNP) and high-sensitivity cardiac troponin T (hs-cTnT), to identify and target individuals at high risk for developing HF with effective therapies as part of a comprehensive HF prevention strategy. In this study, we propose to evaluate whether a cardiac biomarker-based approach can inform intensive BP control treatment decisions in high-risk adults with T2D to prevent HF. The study will examine participants enrolled in the ACCORD-BP trial with available biospecimens to measure cardiac biomarkers. We will measure NT-proBNP and hs-cTnT at baseline and follow-up years 1, 2, and 4 among ACCORD-BP trial participants who have blood stored in the NHLBI Biorepository. The prognostic implications of baseline and longitudinal changes in NT-proBNP and hs- cTnT for HF risk will be examined in a secondary analysis of the ACCORD-BP trial. Additionally, we will evaluate whether NT-proBNP and hs-cTnT levels modify the treatment effects of intensive BP control. Finally, we will test the hypothesis that intensive BP control can attenuate the expected rise in NT-proBNP and hs- cTnT over follow-up. The proposed study will further our understanding of the clinical utility of cardiac biomarker testing as part of a HF prevention strategy and provide preliminary data for designing a cardiac biomarker-guided intensive BP control trial in T2D for HF prevention.

NIH Research Projects · FY 2024 · 2023-08

Project Summary    Bats are important reservoirs for diverse viral pathogens affecting humans. However, we have a poor  understanding  of the  key bat  innate  immunity factors  that  restrict  virus  replication.  Functional  assays  that  can  identify  bat  factors  that  are  truly  relevant  to  combating  viruses  are  needed  to  understand  the  innate  immune  mechanisms that ultimately define bat susceptibility to viral infection. While historically such functional screens  have  relied  on  genome-­wide  genomic  editing  (e.g.  CRISPR-­Cas9)-­  or  RNA  interference  (RNAi)-­based  techniques, such platforms are unavailable for most bat species. Thus, new methods for uncovering functionally-­ relevant components of the bat immune response to virus infection are needed. To address this need, we have  developed  an  innovative  arbovirus  "rescue"  assay  wherein  immune  evasion  proteins  (IEPs)  encoded  by  mammalian pathogens can be expressed in bat cells and one can assay for changes in bat cell susceptibility to  arbovirus infection. Enhancement of arbovirus replication after expression of a candidate IEP indicates that the  IEP  likely  inhibits  bat  immunity  mechanisms  that  normally  restrict  arbovirus  replication.  Using  these  IEPs  as  "tools",  one  can  then  identify  the  bat  immunity  factors  these  IEPs  target.  Thus,  this  screening  methodology  provides  a  mechanism  to  both  identify  novel  IEPs  and  functionally-­relevant  components  of  the  bat  immune  response. To discover IEPs that promote arbovirus replication in bat cells, we will screen an expression library  encoding ~200 bacterial effector proteins. Bacterial effectors are proteins secreted by pathogenic bacteria into  eukaryotic  hosts  cells  that  modulate  or  inhibit  various  eukaryotic  cellular  processes  to  promote  bacterial  replication.  Many  bacterial  pathogens  that  replicate  in the  cytoplasm  of  eukaryotic  host cells  encode  effectors  that function as IEPs. Thus, we hypothesize that some effectors may suppress immune responses that restrict  both  bacteria  and  cytoplasmic  viruses  such  as  arboviruses.  Indeed,  our  initial  screens  have  identified  four  effectors  that  promote  the  replication  of  four  different  arboviruses  when  expressed  in  bat  cells.  We  have  characterized one of these effector screen "hits" as a novel ubiquitin ligase that targets an uncharacterized Ring  Finger (RNF) Domain-­containing protein for degradation in eukaryotic cells. Importantly, RNAi depletion of this  RNF factor in human and bat cells promotes arbovirus replication, suggesting that it may be a novel component  of human and bat immune responses. These results suggest that we can use bacterial effectors as tools to both  inhibit, and identify, functionally-­relevant immunity factors in bats. Our study has the following specific aims: 1)  Identify  bacterial  effector  proteins  that  promote  arbovirus  replication  in  bat  cells;;  2)  Identify  bat  proteins  interacting  with  effector  “hits”  from  our  arbovirus  rescue  assays;;  and  3)  Determine  which  bat  host  factors  interacting  with  effector  protein  hits  affect  viral  replication.  Our  long-­term  goal  is  to  use  this  model  system  to  define the key bat innate immune mechanisms that restrict arbovirus replication.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY / ABSTRACT The incidence of calcium phosphate (CaP) stone disease has markedly increased over the last four decades. CaP stone formers experience high rates of stone recurrence and frequently need repeat stone surgery, indicating that current prophylactic medical regimen are suboptimal. The most common metabolic abnormalities identified in CaP stone formers are hypercalciuria, hypocitraturia, and elevated urine pH, all factors that increase CaP saturation. Alkali therapy (potassium citrate) is well-established to prevent calcium oxalate stones, in part by raising urine citrate. However, when tested in CaP stone formers at doses targeted at normalizing citrate excretion, alkali therapy may worsen CaP stones due to a concomitant rise in urine pH. Hydroxycitrate is a molecule closely related to citrate that has been shown to prevent CaP nucleation in vitro. However, its efficacy against CaP stone formation in vivo has not been previously studied. Our preliminary studies suggest that: 1) Hydroxycitrate can exert a powerful inhibitory effect on CaP crystal growth at low concentrations; 2) Oral hydroxycitrate ingestion is well-tolerated and raises urine OHCit excretion to concentrations that reduce CaP crystallization in urine; 3) Oral hydroxycitrate administration increases urine citrate without significantly increasing urine pH. In this proposal, we will therefore test the overall hypothesis that hydroxycitrate can serve as a treatment to prevent recurrence of CaP stone disease. To test this hypotheses, we will combine three complementary approaches: 1) In human metabolic studies conducted in CaP stone formers, we will examine the short-term impact of a. escalating doses of hydroxycitrate and b. comparative effectiveness of hydroxycitrate vs alkali therapy (potassium citrate) on urinary biochemical and physicochemical parameters, and CaP crystal growth and dissolution using atomic force microscopy and bulk crystallization studies conducted on participants’ urine specimen. 2) In an animal model of CaP precipitation, we will assess the long-term impact of the same interventions to be tested in humans with measurement of urine chemistry, crystalluria, and development of calcification in kidneys. 3) In vitro, we will manipulate hydroxycitrate, calcium, citrate, and pH, and assess CaP crystal growth and solubility by a unique combination of techniques spanning multiple length scales. Notably, we will examine microscopic surface growth by atomic force microscopy, macroscopic crystal nucleation and growth in a microfluidic device, and the kinetics of bulk crystallization over a range of clinically relevant hydroxycitrate concentrations. We will test the mechanisms by which hydroxycitrate exerts its impact on CaP crystal growth and dissolution, and assess the cooperative effects of hydroxycitrate and citrate concentration using a range of clinically relevant pH and ionic strengths. Overall, based on our strong preliminary data, our innovative but rigorous techniques, and our cross-disciplinary complimentary studies, we hope that this proposal will help identify optimal therapeutics for the increasingly encountered and difficult to treat CaP stones.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT: Nearly 250,000 adults are affected by chronic lymphocytic leukemia (CLL). Bruton’s Tyrosine kinase inhibitors (BTKIs) dramatically improve survival in CLL. However, up to 38% of patients develop atrial fibrillation (AF) and other cardiovascular toxicities. Ibrutinib is the first BTKI approved which has these toxicities but our data suggest new BTKIs (e.g., acalabrutinib, zanubrutinib) still associate with cardiotoxicity. The development of AF with BTKIs is challenging and is a major impediment in use of the effective therapies in patients. Thus, there is an urgent need to identify patients at risk for AF, and better understand targetable pathways that induce BTKI-associated AF. Our group has defined most of the early cardio-oncology issues with BTKIs. We have also developed animal models which suggest that BTKIs cause direct cardiotoxicity as well as an activation of the innate immune response that potentially contributes to cardiotoxicity and arrhythmia, and result in an early increase in left atrial (LA) fibrosis and volume (LA remodeling) preceding BTKI-associated AF. We will leverage the active cardio- oncology programs and resources here at OSU and at UCSF, to prospectively study these cardiovascular effects of BTKIs in humans. Our pre-clinical studies specifically implicate activation of the innate immune response, marked by elevation in circulating IL-6 (and IL-17) as key mediators of BTKI-associated AF development, and that this leads to LA remodeling and cardiotoxic AF. Yet, there are no prospective studies testing the effects of immune activation in mediating or predicting cardiotoxic events. To address these translational and clinical gaps, we will recruit 120 CLL patients initiating BTKIs and we will prospectively utilize serial cardiac magnetic resonance imaging (CMR) and leading-edge immunologic techniques to test our hypothesis, that BTKI- associated AF is driven by increased immune activation that induces cardiac remodeling and arrythmia. In Aim 1, we test the effect of BTKIs on LA fibrosis and volume pre-, 2, and 6 months after starting BTKI-therapy. We will determine the burden of BTKI-associated AF by applying serial mobile ECG monitoring over 1-year post- BTKI initiation. These results will be compared to 60 age-, gender-, and cardiac risk matched controls with early stage CLL, treated with standard observation alone. As we have observed that >50% of BTKI treated patients develop hypertension, we will also measure and relate ambulatory blood pressure to CMR measures. In Aim 2, we will examine the effects of BTKIs on innate immune response that define vulnerability to remodeling and clinical AF by studying circulating levels of IL-6, IL-17, and using unbiased single-cell genomics, systematically decipher the immune cells that contribute to remodeling and their key pro-inflammatory pathways. We will also define the relation of these parameters with other CMR measures. Finally, using our BTKI animal model, we will test the effect of targeted inhibition of pro-inflammatory pathways on cardiotoxic remodeling and AF risk. Upon completion, we will gain important insights into the mechanistic role of the kinase inhibitors in cardiotoxicity as well as how immune dysregulation contributes to arrhythmia in hematological malignancies.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT Prostate cancer is the most common non-skin malignancy in men and is projected to cause 34,500 deaths in 2022 in the United States alone. Sequencing studies of advanced lethal castrate resistant prostate cancer (CRPC) have identified a high incidence (~13%) of pathogenic BRCA2 mutations. These findings have enabled clinical trials and subsequent Food and Drug Administration (FDA) approval of the poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis) olaparib and rucaparib in advanced CRPC patients harboring a pathogenic BRCA2 mutations. Despite initial responses, therapy resistance to PARPis is common. However, the molecular adaptations that occur in BRCA2 mutant CRPC in response to PARPi are poorly understood, due to a lack of biologically and clinically relevant models. Our proposed studies leveraging two new patient-derived model systems of pathogenic BRCA2 mutant CRPC will elucidate the biological mechanisms implicated in PARPi therapy response and help address a critical clinical unmet need to prevent or overcome resistance to PARPis. In this proposal, we will use two new models of pathogenic BRCA2 mutations in CRPC, including the 40511 cell line and matched PARPi-sensitive and resistant LTL-610 PDXs. Gene Set Enrichment Analysis (GSEA) and Over-Representation Analysis (ORA) of RNA-sequencing data utilizing these novel models point to significant upregulation in genes involved in Extracellular Matrix (ECM) modulation in response to both short and long term PARPi therapy. In particular, the ECM associated gene SERPINE1, which encodes for the protein Plasminogen Activator Inhibitor 1, (PAI-1) is the most significantly implicated gene after 72 hours of olaparib treatment via GSEA leading edge analysis. Since PAI-1 canonically prevents ECM degradation, we then used Masson’s Trichrome staining to evaluate the PARPi resistant LTL-610 PDX and found dramatically increased Type I Collagen deposition compared to its PARPi sensitive parental line. Since stromal alterations are known to affect cancer cell survival, we hypothesize that the induction of ECM genes like SERPINE1 by PARPis in BRCA2 mutant CRPC results in enhanced tumor stroma, and enables therapy resistance. Two specific aims are proposed in this grant to study this hypothesis: in Aim 1, we will elucidate the role of SERPINE1 signaling in ECM deposition in BRCA2 mutant CRPC in vitro, ex vivo, and in vivo. In Aim 2, we will investigate the mechanism of transcriptional activation of SERPINE1 in BRCA2 mutant CRPC in response to PARPi. The results from these studies will enable systematic approaches to modulate ECM alterations in response to PARPi in BRCA2 mutant CRPCs.

NIH Research Projects · FY 2026 · 2023-08

ABSTRACT The broad, long-term objective of this multi-PI grant application is to understand the role of calcium (Ca2+) dysregulation as the mechanistic driver for synaptic loss between the lateral entorhinal cortex (LEC) and hippocampus (HPC) in evolving Alzheimer’s disease (AD). While dysfunction of the LEC-HPC circuit has been implicated, the cause of the early, selective vulnerability of LEC neurons and projections remain unknown. We will test the hypothesis that dysregulation of neuronal Ca2+ signaling plays a key role in LEC-HPC circuit dysfunction in early AD. Specifically, in experiments with the APPKI mouse model of AD, we will investigate if normalization of the activity of two key proteins, the Ca2+-dependent phosphatase calcineurin (CaN) or the prolyl isomerase, Pin1 rescues defects in LEC–HPC communication, function and stability in early AD. Pin1 is a key target of CaN-mediated inhibition in neurons, directly leading to synaptic and neuronal loss. In experiments with APPKI mice, we will also determine if dominant negative Pin1 accelerates defects in LEC–HPC communication irrespective of CaN normalization in APPKI mice. We will use a combination of molecular, biochemical, imaging, electrophysiological, behavioral and neuropathological techniques to address these specific aims. The proposed studies, if successful, will provide a mechanistic understanding of early AD evolution as well as a rationale for the use of CaN inhibitors such as FK506 or voclosporin to treat early AD.

NIH Research Projects · FY 2024 · 2023-08

Project Summary: Heart failure (HF) affects 6.2 million patients in the United States and was responsible of 13.4% of deaths in 2018 costing about 30.7 billion USD in 2012, and about 80% of all the patients with HF are >65 years old. Clinical manifestations include shortness of breath and peripheral edema. Diuretics are indicated to treat edema in patients with HF, however, its use is limited in patients with compromised kidney function. Peripheral edema is associated with pain, heavy legs, limited mobility, and poor quality of life, increasing the risk of cellulitis, and wounds. Heart failure was identified in up to 44% of patients with venous ulcers. The chronic use of compression stockings in HF patients has potential benefits such as a better control of the edema, decreasing the need for diuretics, improving mobility, prevention of progression of venous disease, and prevention of venous ulcers. To date, no major HF guidelines recommend in favor or against compression stockings in HF patients. The objective of this R21 proposal is to study the role of the compression stockings in preventing edema and venous disease in heart failure patients. Our hypothesis We hypothesize that the use of compression stockings will prevent developing or worsening venous disease (venous reflux, and venous ulcers), improving the edema of the lower extremities, mobility, and quality of life in HF patients without increasing the risk of HF deterioration, hospitalization, or death In Aim 1 we are planning to randomize patients to high degree (20-30 mmHg) vs. low degree (10-15 mmHg) compression stockings for 3 months and evaluate for clinical sign and symptoms of deterioration of heart failure, as well as need to change in diuretic therapy at the end of the therapy. In Aim 2a we are planning to evaluate if high degree compression stockings are associated with a difference in time of valve closure (venous reflux) by venous duplex ultrasound at 3 months. In Aim 2b we are planning to evaluate if high degree compression stockings are associated with a difference in clinical signs of venous disease at 3 months. In Aim 3 we are planning to evaluate if high degree compression stockings are associated with improvement in mobility and quality of life at 3 months. The contribution with this project is significant as edema and venous disease are very morbid condition in patients with heart failure, and a simple and preventive measure such as compression stockings, could be safe and easily implemented. Our project is innovative as we are planning to use compression stockings in patients with heart failure, something that physicians are reluctant to use due to possible decompensation and fluid overload. On the other hand, we consider that it could be easily implemented and prevent advanced venous disease.

NIH Research Projects · FY 2025 · 2023-08

Alcohol-associated liver disease (ALD), which includes alcohol-associated cirrhosis (AAC) and alcohol-associated hepatitis (AH), is now the leading indication for liver transplant (LT) in the US. Early LT (eLT), defined as LT evaluation with <6 months of alcohol abstinence, is associated with acceptable outcomes for AH in retrospective studies. However, prospective, multi-center data including biopsychosocial factors on eLT for all advanced ALD are lacking. It is known that alcohol cessation is the most important factor influencing survival in ALD, and integrated alcohol use disorder (AUD)/ALD care is critical to help patients achieve abstinence, yet the degree of care integration and how this influences post-LT outcomes has not been systematically studied. Knowledge gaps in eLT for ALD include: a) limited data on who gets referred for eLT and referral barriers; b) lack of standardized biopsychosocial measures and outcomes; and c) minimal stakeholder involvement beyond LT providers. There is an urgent need to (1) define factors influencing eLT referral, (2) develop risk prediction models of key patient-centered outcomes, (3) incorporate validated biopsychosocial measures into models, and (4) evaluate the impact of integrated care on outcomes following eLT. For example, The INTEGRATE collaborative, comprised of multidisciplinary clinicians and researchers from the University of Texas Southwestern Medical Center, Henry Ford Health, University of Miami, and Columbia University-Weill Cornell Medicine, is ideally positioned to address these urgent research needs. Collectively, we have developed a distinctive investigator team which varies in: (1) career stage, (2) clinical and methodological expertise in ALD, AUD, LT, behavioral research, risk modeling, data harmonization, causal inference, and mixed-methods research, and (3) has a documented track record of NIH funding in LT access, organ allocation, LT and healthcare outcomes, and NIAAA funding in ALD/AUD. Our large volume transplant centers with established protocols for eLT for ALD applied will facilitate the following aims: 1) characterize and develop risk prediction models for transplant-free survival among those with limited access to LT to define those in greatest need of eLT referral and listing; 2) evaluate barriers and facilitators to referral for eLT in ALD; 3) apply causal inference approaches to observational data to evaluate biopsychosocial factors and develop risk models predictive of outcomes at key timepoints in eLT for ALD; 4) define stakeholder perceptions and preferences for selection and outcomes in eLT for ALD; and 5) evaluate how integrated care processes influence outcomes in eLT for ALD. At the conclusion of this work, we will have collaboratively: (1) defined factors for referral and waitlisting for eLT in ALD (selection), (2) identified which biopsychosocial factors are causally related and predictive of outcomes most important to stakeholders (outcomes) and (3) determined how integrated care influences stakeholder-relevant outcomes in eLT for ALD (management).

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract Most of breast cancer related mortality is related to metastatic disease. While NK cells play an essential role in the control of metastatic breast cancer we do not fully understand key mechanistic and temporal events of NK cell immunosurveillance. To address this need, I created novel 3D co-culture assays of NK cells and invasive breast cancer organoids and cell clusters. Using these models I demonstrated NK cells limit breast cancer metastasis through induction of apoptosis in invasive breast cancer cells. We discovered micrometastatic cancer cells reprogram NK cells to promote metastatic outgrowth. Using bulk RNA-seq, we identified KLRG1 as a key expressed gene by teNK cells. Blocking KLRG1 restores anti-metastatic activity. teNK cells also express high levels of DNA methyltransferases (DNMTs). Inhibition of DNMTs in teNK cells achieves anti- metastatic results. Combination KLRG1 blockade and DNMT inhibition further reduces tumor growth. Building on these exciting data, we hypothesize: 1) reprogrammed teNK cell KLRG1 expression is regulated by DNMTs and targeting both in combination will reverse NK cell reprogramming; 2) reprogrammed human NK cells increase human breast cancer metastasis and blocking prioritized NK cell inhibitory receptors restores the cytotoxic activity of metastases-reprogrammed human NK cells. Aim 1 will determine how blocking DNMTs and KLRG1 synergistically reverses NK cell reprogramming. We have observed blocking KLRG1 and DNMTs together in teNK cells synergistically reduces colony formation. We will first test these ex vivo findings in in vivo models of metastasis. Next, we will determine the link between NK cell reprogramming and DNMTs using novel functional ex vivo and in vivo models of metastasis using CRISPR-Cas9 molecular tools. Aim 2 will determine the function of metastases-reprogrammed NK cells in human breast cancer. First, we showed that human NK cells can be reprogrammed in culture to actively promote metastatic outgrowth. We will leverage preliminary transcriptomic analysis of reprogrammed NK cells to identify markers of metastases- reprogrammed NK cells. We will apply these tools to FFPE sections of patients with metastatic breast cancer to define the abundance of reprogrammed NK cells. Together with patient samples, single-cell RNA-seq, and ex vivo and in vivo models that capture the progression from anti-metastatic NK cells to metastases-promoting NK cells, we will further characterize the function and molecular profile of reprogrammed NK cells in human breast cancer. Finally, using a preliminary list of targets, we will test effective individual or combination treatments that can restore human NK cell cytotoxicity against metastatic breast cancer cells. Impact: Completing these aims will deepen our fundamental understanding of NK cell biology and have the potential to bring new NK cell directed immunotherapies to our patients.

NIH Research Projects · FY 2025 · 2023-08

Project Abstract With increased incidence in young populations and ~65% three-year survival of high-risk locally advanced rec- tal cancer, innovative approaches to improve outcomes is imperative. Neoadjuvant therapy with radiation (RT) and chemotherapy (CT) is now the standard curative treatment, but there is demand for non-operative man- agement (NOM) if the disease can be cured with local and systemic therapy. Thus, innovations could be trans- formative with the aim to improve durable complete responses by personalizing therapy, which includes how to deliver RT, CT, and the incorporation of novel agents such as immunotherapy. Cancer Immunotherapy has had little impact on colorectal cancer outside of mismatch repair deficient tumors. The αCD40 agonist antibody is an emerging class of immunotherapy and sotigalimab has shown promise in phase I and multiple ongoing phase II trials. The CD40 receptor is important in both innate and adaptive immune responses and a greater therapeutic effect can be achieved combining αCD40 with RT in animal models. We hypothesize that short course RT (SCRT) and CT when combined with αCD40 in human tumors can result in a greater antitumor im- mune response, reduce risk of metastatic progression, and extend survival in a poorly immunogenic malig- nancy like rectal cancer. We developed the INNATE trial, a phase II randomized trial of neoadjuvant SCRT fol- lowed by CT with or without the addition of sotigalimab for locally advanced rectal cancer. This trial design has allowed us to collect fresh pre- and post-SCRT biopsy tissue, which we have obtained from 21 of 30 patients enrolled to date. In this proposal, we focus on the central hypothesis that an integrated molecular, cellular, and spatial assessment of treatment response dynamics in the tumor microenvironment early after SCRT can re- veal insights into the immunobiological responses, which can inform mechanisms of efficacy and therapeutic selection. We will perform 1) molecular and cellular single cell (sc) RNAseq with proteomic and immune reper- toire analysis, 2) molecular, cellular, and spatial multiplex immunofluorescence, and 3) cellular and spatial quantitative deep learning based histopathologic analysis to achieve our aims. These three technologies will enable us to investigate early changes across RT and αCD40 treated groups. Then we will aim to identify ther- apeutic opportunities for the combination of SCRT with immune active agents though an integrated RT re- sponse assessment. Lastly, we will establish innate and adaptive immunologic signaling events triggered by SCRT in combination with αCD40 immunotherapy and factors that enhance or hinder efficacy. We will use models to start validating key findings and aim to propose future therapeutic directions to build off these efforts and ultimately improve outcomes. Specifically, regarding our patient population, we expect this proposal will lead to evidenced based trials for most patients with locally advanced rectal cancer who strive to achieve cure without a morbid surgery.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY/ABSTRACT: Immune receptors of the adaptive immune system (antibody/B-cell or T-cell receptors, or AIRR data) are designed to recognize and remove pathogens, and also recognize and preserve self-molecules. Therefore, these receptors have to be highly variable; it has been estimated that the number of possible human B-cell receptors approaches 1013. In addition to the diversity of these receptor sequences, the genes that underlie the production of these receptor molecules are highly diverse and complicated, and the data describing how these receptors bind to antigens (such as influenza) are also highly complex. Repositories to curate, analyze, and share these data are necessary to characterize B/T cell function in disease, as well as facilitate the discovery of new vaccine leads and therapeutic monoclonal antibodies to suppress autoimmune disease and cancer. Such data repositories are available, but they tend to focus on only one aspect of the data. Given that these repositories typically have been developed independently, the primary data and associated metadata (age, demography, sex, etc.) of the samples are stored in non-compatible forms, and in addition, the enormous size and complexity of the data make data sharing and integrated analyses extremely challenging. The goal of the proposed research is to establish the integrated AIRR Knowledge Commons (i-AKC), a novel knowledgebase that will allow seamless access, exploration, analysis, querying, and downloading of these various data types from a single point of entry. Our approach will be based on the very successful AIRR Community initiative, a group of immunologists, bioinformaticians, and experts in ethics and data sharing who have worked together since 2015 to develop protocols and standards for analysis and data sharing tools. One of the outcomes of the AIRR Community is the AIRR Data Commons, a set of data repositories that store the immense immune receptor repertoires that underlie the adaptive immune response. The proposed research takes the next important step of integrating (1) the AIRR Data Commons with repositories of (2) antigen/receptor binding and (3) germline immune genes. Steps to producing the i-AKC are (1) develop a common data model and establish common data elements relying on existing ontologies and community standards, (2) integrate the data using innovative algorithms and automation tools and enrich it with new knowledge derived from algorithms operating on the integrated data, and (3) community building. Using the i-AKC, researchers will, for example, be able to discover receptor sequences based on metadata or sequence searches, seamlessly examine information on the germline genes underlying these receptor sequences or examine what is known about the binding targets of these receptors. This novel and innovative knowledgebase will facilitate data and knowledge exploration that would be prohibitively difficult using sets of “siloed” repositories and will greatly accelerate biomedical research in autoimmune disease, infectious disease, transplantation, and cancer and directly improve patient care.

NIH Research Projects · FY 2025 · 2023-07

Project Summary: Cancers require metabolic adaptations to support the unbridled proliferation that drives tumor growth. Mutations in the mitochondrial genome (mtDNA) are observed in many cancers, but the role of these mutations in shaping cellular metabolism and tumor growth is incompletely understood. mtDNA mutations that impair components of the electron transport chain (ETC) appear to be selected against in most forms of cancer. Hürthle cell carcinoma of the thyroid (HTC) is clinically aggressive cancer uniquely enriched for loss-of-function mtDNA mutations in components of complex I of the ETC. We propose that HTC represents an ideal disease outlier in which to interrogate the role of mtDNA alterations and ETC function in cancer. In this proposal, we employ unique and highly complementary approaches to characterize the metabolic impact of mtDNA mutations in HTC and other forms of thyroid and kidney cancer. First, we have developed a clinical protocol to monitor central carbon directly in surgical patients using stable isotope tracing (Aim 1). Second, we have identified a synthetic lethal interaction encoded by complex I mutation and identified a promising small molecule therapeutic using patient-derived models (Aim 2). Finally, we have developed novel GEMMs from which to interrogate the role of complex I function in thyroid tumorigenesis (Aim 3). These approaches are highly complementary and synergistic, yet each is independently poised to bridge key knowledge gaps and lead to new insights into metabolic regulation in cancer. The overall goals of this proposal are to characterize the metabolic adaptations necessitated by complex I loss in HTC directly in patients undergoing thyroid surgery, to identify and target metabolic liabilities as a result of metabolic re-wiring downstream of complex I loss, and to determine whether complex I loss acts to promote or alter thyroid tumor formation in mice. These findings will be of immediate and direct relevance to thyroid cancer patients, provide new insights relevant to other tumors harboring mtDNA mutations and have broad implications across cancer types by providing new insights into ETC function in cancer.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY The long-term objective of this project is to determine the general principles and causal mechanisms underlying subcellular RNA localization, and the physiological implications during development and disease. In particular, the subcellular localization of messenger RNA (mRNA) to cell protrusions is known to be required for cell migration, but the mechanisms by which mRNA localization regulates protein function in this setting are unclear. Many genes implicated in mRNA localization are expressed in neural crest cells, which are a particularly migratory cell type that gives rise to much of the vertebrate head, as well as neurons, melanocytes, and aggressive cancers including melanoma. As such, neural crest cells provide a rich environment to uncover molecular mechanisms of mRNA localization in cell migration as well as the relevance there of to mammalian physiology. In this project, I will use cultured melanoma cells to identify and characterize trans-acting regulators of mRNA localization and elucidate the role of mRNA localization on protein function. I will also use in vivo mouse models to catalog mRNA localization that occurs during neural crest cell development and directly test the role of a well characterized localized mRNA, Kif1c, during development and cancer onset and progression. As neural crest and cancer genetics fields rely heavily on transcriptional studies, the mechanistic understanding of a post-transcriptional process as described here may provide unique insight into the cause, diagnosis and treatment of craniofacial birth defects and melanoma onset and progression. My career goal is to run an academic research program analyzing the role and regulation of subcellular RNA localization during embryogenesis, with special attention to neural crest cells and their derivatives. My ambition is to have a lab that can determine molecular mechanisms at the single-molecule level and causally link those processes to physiological outcomes in vivo. To this end, the proposed experiments and training plan are designed to develop expertise in cutting-edge technologies such as TIRF microscopy, computational image analysis, genetic engineering and in vivo disease assays. Training in these advanced techniques will be directly supported by the resource-rich and collaborative environment at UT Southwestern, and especially mentorship from Dr. Gaudenz Danuser, Dr. Ondine Cleaver, Dr. Khuloud Jaqaman, Dr. Lu Le and Dr. Sean Morrison, who make up my advisory committee. The Pathway to Independence Award will provide the time, resources and autonomy to fully develop and initiate this ambitious research program and accrue the resources, expertise and experience necessary to launch an impactful, thriving research lab in Fall 2024.