Yale University

NIH Research Projects · FY 2025 · 2025-09

AWAR3E Overall People with HIV (PWH) experience more “inflammaging” characterized by chronic inflammation, immune dysfunction, and debilitating geriatric syndromes than people without HIV (PWoH). Inflammaging has not been well characterized among people with HIV because no single cohort can adequately address this question. Thus, before we can effectively intervene on this health issue, we need to characterize inflammaging among people with HIV to identify appropriate biomarkers, outcomes, and treatments. We bring together four well characterized, complimentary HIV cohorts to study this question in the Aging Well with HIV through Alcohol Research and Risk Reduction and Education (AWAR3E) Center. We propose that inflammaging in people with HIV is reflected in novel biomarkers of inflammaging (methylation age, HIV-1 viral reservoirs, and Conserved Transcriptional Response to Adversity [CTRA]; Project 1: Biomarkers) and is associated with debilitating geriatric syndromes (falls/fractures, delirium/dementia, and physiologic frailty; Project 2: Geriatric Syndromes). We also explore two potential treatments to counteract inflammaging, long-acting ART (LAARs, e.g. injectable cabotegravir-rilpivirine) and glucagon-like peptide-1 receptor agonists (GLP-1RAs, e.g., semaglutide; Project 3: Treatments). We hypothesize that people with HIV, those with unhealthy alcohol use, and those socioeconomically disadvantaged are less likely to receive these treatments than those without these factors, but are more likely to benefit from each. We evaluate their safety and effectiveness overall and for people with unhealthy alcohol use and socioeconomic disadvantage using causal modeling. Finally, the AWAR3E Center will provide guidance and coordination of this work with an Administration and Data Core, Program Advisory Group, Steering Committee, Engagement and Dissemination Core, and Pilot Program focused on training the next generation of investigators and community representatives in alcohol-HIV/AIDS research to promote health. Insights gained, training provided, and strategic engagement and dissemination through the AWAR3E Center will facilitate future alcohol interventions tailored to people with HIV to mitigate inflammaging associated health outcomes.

NIH Research Projects · FY 2025 · 2025-09

Project Summary/Abstract Alcohol-Associated with are kidney regulated 1 all the inflammasome inflammasomes, essentially epithelial-targeted indicating revealed alcohol hand, hepatocytes r treatment. mitochondrial PPARα and cell the for features inhibiting beneficial Liver Disease (AALD) is the leading cause of liver-related deaths in the USA. AALD starts lipid deposition in hepatocytes and progresses to liver inflammation and fibrosis. Effective treatment options very limited. There are t hree clinical trials ongoing in humans targeting IL-1β signaling. In these studies, toxicity was reported, indicating that liver-specific approaches should be considered. IL-1β production is upstream by the assembly of inflammasome components [an inflammasome adaptor ASC, caspase- or caspase-11, and i nflammasome sensors (NLRs or NLRCs)]. nflammasome components are expressed in l iver cell types, and to date, we have very limited nowledge of the effect of inflammasomes in hepatocytes, most abundant liver cell type and an important driver of AALD pathophysiology. I t was reported that the was activated in hepatocytes upon alcohol exposure. To further explore the liver-specific role of we deleted one essential inflammasome component, caspase-1 (Casp1), common to all inflammasomes, in liver epithelial cells specifically using Albumin-Cre mice. We found that liver deletion of Casp1 leads to a strong exacerbation of the disease in the NIAAA alcohol model, a rotective role of Casp1 gainst AALD in the l iver. Liver bulk RNA-seq and proteomic analysis that in the absence of Casp1 from hepatocytes, the antioxidant defense mechanisms in mitochondria, detoxification, NF-κB-related pathways and myeloid-driven inflammation are reduced. On the other fatty acid synthesis and PPARα target genes are increased. In vitro experiments in Casp1-deficient evealed exacerbation of mitochondrial ROS production and cell death upon alcohol and LPS In Aim 1, we will examine which hepatocyte cell-intrinsic pathway is affected by Casp1 related to ROS production, alcohol metabolism, and lipid deposition. We will focus on NF-κB, β-catenin, and revealed by omics analysis. We will also examine if the Casp1 effect is due to IL-1β or IL-18 production signaling. In Aim 2, we will delete Casp1 or Casp11 in hepatocytes and examine liver pathology and immune alterations after 8 weeks of alcohol feeding. The results generated from this project will provide the basis for preparation of an R01 application to study which molecule from inflammasome sensors is the best candidate targeting in vivo in regular mice and in humanized mice with alcohol that we have een to recapitulate several of human AALD. These results will help in the design of better therapeutic approaches in humans by inflammasome function in the ell types where it has a clear pathogenic role while preserving the functions of the inflammasome. I k p a , s c

NIH Research Projects · FY 2025 · 2025-09

Abstract Alcohol use disorder (AUD) is a leading cause of death and disability worldwide. A deeper understanding of the genetic mechanisms underlying AUD could significantly improve the diagnosis, prevention, and treatment, thereby reducing the burden of AUD-related problems. Although genome-wide association studies (GWAS) have identified a growing number of genetic variants and pathways associated with AUD, key gaps remain unaddressed. First, current GWAS predominantly focuses on common variants, neglecting rare and structural variants due to technical limitations. Second, the top associated variants identified in GWAS loci may not be the true causal variants driving AUD risk. Third, another critical gap lies in the limited predictive performance of polygenic risk score (PRS) for AUD based on GWAS common variants. Fourth, the expression perturbation in specific cell subtypes or cellular processes in the human brain affecting the AUD risk is poorly understood. To address these gaps, we propose this innovative study leveraging large-scale whole-genome sequencing (WGS) data from biobanks, publicly available single-cell RNA sequencing (scRNA-seq) data in brain cells, and state-of-the-art statistical methods to improve the understanding of AUD genetics, response to the Notice of Special Interest: Secondary Analyses of Existing Alcohol Research Data (NOT-AA-23-011). It aims to uncover novel rare, common, and structural variants associated with AUD and recover the missing heritability (Aim 1); identify causal variants through cross-ancestry fine-mapping and improve disease prediction using a novel whole-genome PRS framework (Aim 2); and map the WGS results to existing scRNA-seq data to identify key brain cell subtypes and processes involved in AUD (Aim 3). The proposed research is timely and in line with NIAAA’s mission, promises to substantially enhance our understanding of the genetic architecture of AUD, and will lay the foundation for developing precision interventions to prevent and treat alcohol-related diseases.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT The majority of persons living with cognitive impairment (PLWCI) are discharged after seeking care in the emergency department (ED), with informal care partners (e.g. family members, friends) subsequently providing a significant amount of hands-on care and navigation of health-related and social needs within this vulnerable time period. There exists a critical need to develop pragmatic ED care transition interventions to support care partners given the growing population of PLWCI seeking emergency care, the adverse outcomes that PLWCI experience after ED discharge, the outcome interconnectedness between PLWCI and care partners, and the limited generalizability and effectiveness of interventions to date. The candidate, Dr. Gettel, is an emergency physician at the Yale School of Medicine with a track record of early success and preliminary findings through an NIA GEMSSTAR Award identifying that care partners of PLWCI experienced poor, if any, communication during ED discharge. Digital health interventions (e.g. text messaging) have previously shown promise as a pragmatic tool for communication in various ED populations, yet no evaluations have assessed these approaches in the population of care partners of PLWCI despite their acceptance of digital health and the capability of digital health tools to disseminate information beyond the physical confines of the ED encounter. The objective of this proposal is to develop and pilot test the CAre Partner Text message intervention to improve futURE outcomes (CAPTURE), an ED care transition intervention delivered via automated tailored text messaging that will provide information, core medical reminders and behavioral strategies, and practical support to care partners of PLWCI. Sequential exploratory mixed-methods approach will be used to achieve the three aims: 1) Elucidate facilitators and barriers to the use of an ED-based text message care transition intervention among care partners of PLWCI; 2) Develop and iteratively refine the CAPTURE using user- centered design methods for care partners of PLWCI quantitatively measuring usability, feasibility, and acceptability; and 3) Determine the feasibility and preliminary efficacy of the CAPTURE implementation after an ED visit in care partners of PLWCI compared to a care partner population receiving usual care. Responsive to the “National Institute on Aging: Strategic Directions for Research, 2020-2025”, findings of this proposal will expand supports and address the special needs of care partners of PLWCI. During the award period, Dr. Gettel will pursue career development training activities leveraging the team’s mentorship expertise in the domains of: geriatric emergency medicine; text message interventions; ADRD and care partners; patient- and caregiver-reported outcome (PCRO) measurement; embedded pragmatic clinical trial (PCT) design; and implementation science. Findings will inform an NIA R01 proposal to assess the real-world efficacy and effectiveness of the CAPTURE implementation and its impact on PCROs as part of a multisite embedded PCT.

NIH Research Projects · FY 2025 · 2025-09

Abstract Understanding the structure of molecular complexes and the way molecular structures interact is crucial to the understanding of biological processes and the development of drugs. Researchers study these structures and processes using empirical measurement methods and computational tools for structure prediction and simulation of processes. In recent years, significant advancements have been made in identifying the most stable and common conformations using empirical methods and computational tools, including some new machine- learning approaches. However, there has been more limited success in characterizing flexible complexes and intermediate molecular states in biological processes. Our group is particularly interested in cryo-electron microscopy (cryo-EM), an imaging technology that has revolutionized structural biology in recent years, and molecular dynamics (MD), the workhorse of simulation of molecular processes. Cryo-EM holds a long-standing promise to allow researchers to map diverse collections of molecular states in biological processes, including intermediate states. This promise is only partially fulfilled. We are developing the methods to deliver on this promise. In recent years, we have introduced a novel framework for mapping the conformation landscape using cryo-EM; this framework has been implemented in many of the recent algorithms. We have also developed software and made various other contributions to theory and methods in the area. In addition, we are developing novel techniques that will accelerate the notoriously slow MD simulations and we are developing algorithms that combine the computational modeling power of MD with the empirical power of cryo-EM to achieve results that neither technology can obtain on its own. In our work, we seek to combine robust interpretable mathematical modeling and algorithms with new and emerging data-driven machine learning algorithms to develop practical, robust, and interpretable methods for solving scientific questions about the structure and function of molecular complexes. Our group is also interested in broader statistical and computational methodology. We will develop more statistically rigorous and canonical methods and software for some of the classic statistical questions that arise in biology and medical research. These methods would improve the reliability of medical research and evaluation of drugs due to their advantageous statistical properties and canonical nature, reducing the risk of manipulation.

NIH Research Projects · FY 2025 · 2025-09

The Biology and Personalized Treatment of Primary and Metastatic Lung Cancer: The Yale SPORE in Lung Cancer (YSILC) unites translational scientists spanning diverse areas of cancer research to address the challenge of lung cancer. The goal of the YSILC is to increase survival in patients with lung cancer through development of novel therapeutics and treatment approaches that are based on an understanding of the targetable biochemical and immunological pathways involved in progression of lung cancer, acquisition of resistance, and development of metastasis. The YSILC translational research team will accomplish this objective through three projects: Project 1: To explore the role of tumor PLA2G10 upregulation as a dominant mechanism of adaptive immune resistance in NSCLC; Project 2: To optimize precision medicine approaches to treat mutant EGFR-driven lung cancer; Project 3: To target the ATM pathway in treatment-refractory NSCLC patients with brain metastasis. There are three Cores (Administrative; Biostatistics and Bioinformatics; and Biospecimen and Biomarker) to support the projects and their clinical aims, mechanistic studies, and evaluation of biomarkers for clinical application. Strong Developmental Research and Career Enhancement Programs (DRP, CEP) with a robust history of choosing diverse and productive projects with good outcomes are also proposed. The highly coordinated YSILC projects, cores, and programs are focused on developing novel lung cancer therapies with analysis of patient samples, cell-based assays, production of human cell lines and animal models of disease as a guide to design prospective trials that translate these innovative targeted approaches to clinical therapies. Each of these projects has a clinical trial designed to test the sensitivity and resistance of the new therapy with molecular correlates. The expected translational outcomes of the program include: (1) a highly coordinated and focused development of a novel immune target discovered during our current SPORE research; (2) knowledge of new vulnerabilities of EGFR mutant lung cancer that can delay the emergence of drug resistance; (3) an understanding of the mechanism underlying brain metastasis and its treatment; (4) expanding the breadth of lung cancer research by developing the next generation of investigators and encouraging established investigators in other fields to pursue studies in lung cancer through our CEP and DRP programs and to foster a culture of multidisciplinary knowledge within and outside YSILC.

NIH Research Projects · FY 2025 · 2025-09

Project Summary The fusion of large dense-core vesicles (LDCVs) with the plasma membrane is essential for the secretion of peptides, hormones, and growth factors in neurons and endocrine cells. This tightly regulated process governs critical physiological functions, including metabolic homeostasis, immune responses, synaptic transmission, appetite regulation, circadian rhythms, and stress management. Disruptions in vesicle fusion are linked to neurological disorders, diabetes, immune dysfunction, eating disorders, depression, addiction, and cancer. Despite its significance, the molecular mechanisms of LDCV release remain poorly understood. The central hypothesis of this proposal is that distinct v-SNARE-containing vesicle populations form unique SNARE configurations, driving different modes of release and fusion pore kinetics. This innovative hypothesis challenges the prevailing view of a single core protein machinery governing vesicle fusion and suggests multiple vesicle types with varying calcium sensitivities. The unresolved debate between full fusion and the kiss-and-run (KR) mechanism has persisted due to technological limitations and the transient nature of these events. Our SLIM technology overcomes these barriers, enabling real-time tracking of vesicle fates and providing insights into the mechanisms behind KR and full fusion. This project will explore vesicle heterogeneity, synchronous and asynchronous exocytosis, and fusion pore kinetics, providing new insights into vesicle release mechanisms. Utilizing advanced technologies and cellular assays, this MIRA research program will address how vesicle heterogeneity dictates different fusion kinetics in LDCVs (Theme 1) and investigate the role of phospholipase in calcium signaling and lipid dynamics at fusion sites, while developing next-generation suspended lipid membrane platforms based on the design principles of organic neuromorphic devices for observing ultrafast fusion kinetics (Theme 2). The technologies we develop in this proposal will benefit the fields of membrane biophysics, cell biology, and single-molecule imaging. Ultimately, uncovering the principles of LDCV release will link intracellular and intercellular signaling with implications for treating metabolic diseases, psychological disorders, and neurodegenerative diseases. The results will impact numerous scientific fields, including cancer, cardiology, metabolism, and neuroscience.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Alcohol Use Disorder (AUD), a significant therapeutic challenge, disrupts the brain's glutamatergic system. Preclinical evidence shows that chronic alcohol exposure upregulates the expression and function of GluN2B subunit-containing N-methyl-D-aspartate receptors (GluN2BR). Furthermore, the overactivation of GluN2BR in reward-related brain regions is associated with increased alcohol consumption and alcohol seeking behavior. Hence, the selective antagonism of GluN2BR has garnered interest as a therapeutic target. However, the precise role and availability of GluN2BR in people with AUD remain unknown, limiting our ability to harness GluN2BR- specific therapeutic potential. Positron Emission Tomography (PET) imaging holds promise to quantify neuroreceptor adaptation in living people, thereby facilitating therapeutic development. Our proposal addresses this gap by conducting the first-in-human characterization of a novel PET fluorinated radioligand, (S)-[18F]OF- NB1, utilizing its promising pharmacokinetics and specific brain uptake as demonstrated in our preclinical evaluations. The K99 phase of the grant will focus on establishing the imaging properties as well as the test- retest variability of (S)-[18F]OF-NB1 in healthy participants. The R00 phase will expand this research to compare (S)-[18F]OF-NB1 binding in people with AUD compared to healthy controls. In people with AUD, we will perform PET imaging alongside a laboratory alcohol self-administration session to model alcohol-seeking behavior and subsequent heavy drinking. Additionally, we will collect Ecological Momentary Assessment (EMA) data over a 14-day period to accurately assess cravings and alcohol consumption in a naturalistic setting. The relationship between GluN2BR levels and alcohol drinking behavior will be established. The proposed integrative approach holds the potential to elucidate the intricate neurobiology of AUD through the utilization of state-of-the-art PET imaging techniques and innovative behavioral assessments. This research endeavor will significantly contribute to the development of novel and targeted therapeutic interventions. Upon successful completion, this project will advance my expertise in human PET neuroimaging, kinetic modeling, AUD neuropathology, and behavioral assessments, establishing me as an independent researcher in alcohol-related neuroimaging.

NIH Research Projects · FY 2025 · 2025-09

Conversion of somatic cells into pluripotent stem cells using the transcription factors Oct4, Sox2, Klf4 and cMyc (OSKM) has revolutionized biology and medicine. While the OSKM approach has provided a paradigm for reprogramming, a deeper understanding of cell fate plasticity could bring efficient regeneration and rejuvenation therapies within reach. Somatic cells give rise to induced pluripotent stem cells (iPSCs) infrequently and slowly, indicating that the plasticity of somatic cells is generally limited. We found that the bioactive compound KL871 promotes a cell state of remarkable potential for reprogramming: in the presence of KL871, OSKM reprogram all myeloid progenitors into iPSCs within 3 days, a process known to be slow (>10 days) and inefficient (<0.1%). To pursue the mechanistic basis underlying KL871-instigated cell fate plasticity, we synthesized a photoaffinity derivative of KL871 and found it to be a Yeats4 binder. Yeats4 is a member of the YEATS domain-containing epigenetic reader: it binds to acetylated lysines on histones (H3K27ac and H3K14ac) to recruit chromatin remodeling complexes Tip60/p400 and SRCAP that are not only histone acetyl transferases (HAT) but also deposit H2A.Z, leading to transcriptional activation. We found that KL871 promotes Yeats4-associated chromatin activities, including rapidly increasing histone acetylation as well as chromatin-associated Yeats4 and H2A.Z. Further, knocking down Yeats4 abolished the KL871-instigated reprogramming effects. We therefore hypothesize that KL871 promotes cell fate plasticity via augmenting Yeats4 function. While wide-ranging efforts have been devoted to developing inhibitors or degraders of epigenetic factors, chemical strategies to augment the function of epigenetic regulators are rare and challenging. To date, no small molecule compound is known to increase Yeats4’s reader function. We propose to capitalize on the KL871:Yeats4 relationship to understand how it promotes Yeats4 activity and determine how KL871-bound Yeats4 regulates chromatin and cell fate plasticity. With the strengths of our cross disciplinary teams in cell fate reprogramming (Dr. Shangqin Guo) and chemical biology (Dr. Craig Crews), we are at a unique position to pursue the following aims. 1) Define how KL871 binds to Yeats4; 2) Determine the chromatin consequences following KL871:Yeats4 interaction; 3) Assess cell plasticity following KL871:Yeats4 manipulation, and 4) Perform structure-activity relationship study to develop improved Yeats4-modulatory compounds. Supported by compelling preliminary data and novel tools, we propose to learn how to engineer somatic cell fate plasticity by understanding the underlying biochemical and molecular basis.

NIH Research Projects · FY 2025 · 2025-09

ABSTRACT New and innovative approaches to studying modifiable mechanisms of heart failure are crucial. Heart failure impacts approximately 7 million adults in the United States and has a 5-year mortality rate of 42%. Obstructive sleep apnea (OSA), traditionally assessed by the apnea-hypopnea index (AHI), increases the risk of heart failure. OSA-specific measures of hypoxia (hypoxic burden) and autonomic response (∆heart rate) are associated with heart failure and adverse cardiovascular disease (CVD) outcomes, independent of the AHI. Abnormal left ventricular (LV) myocardial flow reserve, measured by cardiac PET stress test, is a marker of coronary microvascular dysfunction and is implicated in heart failure with preserved ejection fraction (HFpEF) development. Right ventricular (RV) myocardial blood flow is also emerging as a measure of CVD prognosis. Hypotheses: Some patients with OSA experience an immediate (one-week) increase in LV myocardial flow reserve after PAP initiation. Hypoxic burden and ∆heart rate are associated with abnormal LV myocardial flow reserve and predict immediate LV myocardial flow reserve increase with PAP therapy. LV myocardial flow reserve increase is associated with improvement in myocardial function. PAP use also improves abnormal RV myocardial blood flow. We will recruit 60 individuals with ≥ moderate PAP-naïve OSA (AHI ≥15/hour) to test these hypotheses. Those with abnormal LV myocardial flow reserve (n=40) will have repeat testing at one week and four months after initiating PAP. The specific aims are: 1. To determine if OSA-specific hypoxic burden and ∆heart rate (the peak minus nadir heart rate associated with an obstructive episode on a sleep test) are associated with abnormal LV myocardial flow reserve at baseline 2: Among persons with abnormal LV myocardial flow reserve, we will determine if: A) LV myocardial flow reserve increases after one week and four months of PAP use, and if baseline hypoxic burden and ∆heart rate predicts immediate (one-week) flow reserve increase, B) LV myocardial flow reserve increase after one week and four months of PAP therapy is associated with improvements in measures of myocardial function at four months. AIM 3 (Exploratory): Focusing on the RV, we will explore A) the baseline association between the hypoxic burden, ∆heart rate, and RV myocardial blood flow and B) changes in RV myocardial blood flow with PAP therapy and their association with LV and RV myocardial function. An enhanced understanding of OSA physiology, mastery of translational cardiac imaging, and clinical research skills gained during this award will strategically position me to become an independent investigator focused on CVD mechanisms and therapeutics in persons with sleep-disordered breathing, including OSA. Our study findings should inform current heart failure risk assessment practices and guide management decisions for patients with OSA. It will form the basis of cardiac imaging-based mechanistic clinical trials of OSA- specific therapies and their impact on the heart. Such studies will form the foundation of my R-01 application.

NIH Research Projects · FY 2025 · 2025-09

The goal of the proposed project is to evaluate early-life exposures to extreme weather-related factors and pediatric thyroid cancer risk. The incidence rate of pediatric thyroid cancer (ages 0-19 years) has been increasing in the United States, doubling from 2000 through 2019. Compared to adults, children tend to present at more advanced stages with larger tumor sizes, involvement of regional lymph nodes, and pulmonary metastasis. Moreover, children with thyroid cancer face a lifetime of surveillance, risk for second primary malignancies, disruptions of life milestones, and serious psychosocial outcomes. The vast majority of pediatric cases have unknown etiology. A limited but emerging literature among adults suggests that exposure to extreme temperatures (hot or cold) and air pollutants and may be linked to increased risk of thyroid cancer. Yet, the influence of these factors on pediatric cancer risk remains largely unstudied. We hypothesize that exposure to extreme temperature and air pollution are contributing to the rising incidence in pediatric thyroid cancer through perturbations of the thyroid endocrine system and function. We propose a large, nested case-control study within a statewide California birth cohort to examine the relationships between early-life exposures to ambient temperature, outdoor air pollution, and risk of pediatric thyroid cancer. Our study population includes 1,326 children born in California during 1982-2021 and diagnosed with first, primary thyroid cancer at the age of 0-19 years during 1988-2021 (i.e., cases), and 66,300 controls (50:1 match) matched to cases on birth year and sex. The proposed project will have an unprecedented sample size, covering cancer diagnoses over a 34-year span. Our study will utilize rich environmental geospatial data and employ innovative statistical and bioinformatics methods, including modeling multiple exposure windows. We will assess the relationship between early-life exposure to extreme ambient temperatures (heat, cold, heat waves, cold waves) and pediatric thyroid cancer risk and examine whether there are different patterns by cancer subtype, age, timing of exposure, or other factors (Aim 1). We will also assess potential associations between early-life exposure to ambient air pollution (fine particulate matter, nitrogen dioxide) and pediatric thyroid cancer risk and examine whether there are different patterns by cancer subtype, age, exposure timing, or other factors (Aim 2). We will also evaluate possible interaction effects between temperature and air pollution. Given the rapid increase in pediatric thyroid cancer incidence and the widespread exposure to extreme weather and air pollution, this innovative and timely study will help to elucidate the obscure etiology of an understudied cancer, clarify the impact of extreme weather and related factors on childhood carcinogenesis, and inform strategies to mitigate environmental exposures and risks.

NIH Research Projects · FY 2025 · 2025-09

PROGRAM SUMMARY The objective of our LAUNCH program is to continue our global health research training program called the Global Health Emerging Scholars (GHES) designed to create a new community of researchers, educators, and professionals who are prepared to address new and emerging global health challenges. We will build on the last nine years of this training program to create a cadre of new researchers who will dedicate their careers to address the health problems that arise out of the human conditions prevalent in informal settlements (slums) in urban and rural areas. Rather than focusing on individual diseases, we aim to train scholars in a comprehensive, integrated, and multidisciplinary approach to addressing health challenges in informal settlements. This approach has been developed over many years through collaboration among faculty from the four U.S. partner institutions—Yale University, Stanford University, the University of Arizona, University of California, Berkeley, and Harvard University—led by global health research leaders with over a decade of collaboration. Together, core faculty mentors from these institutions conduct research at 25 institutions in 20 countries in Africa, Central and South America, the Caribbean, Asia, the Pacific, and Eastern Europe. The GHES program will address a wide range of health research topics including HIV/AIDS, emerging and high-consequence infectious diseases, non- communicable diseases (NCD), environmental health, mental health, interpersonal violence, substance use, and injuries, all within the framework of informal settlements health. Training will target US postdoctoral fellows and pre-doctoral students and low- and middle-income country (LMIC) postdoctoral fellows. We will recruit 9-10 trainees/year with 60% of them as US postdoctoral fellows. Trainees will spend 8-12-months at an LMIC site under the supervision of the Consortium and their collaborating LMIC mentors. Didactic workshops on global health research methods will be conducted both in-person and online. LMIC trainees will spend 2-3 months at US institutions to undergo training in methods not provided at their institutions. All trainees will be provided with research and career mentorship throughout their training and tracked for career development after completing their GHES-supported research work. This program exposes trainees to a key global health theme—diseases in at-risk populations in informal settlements, both urban and rural, and offers them the opportunity to become experts in this emerging discipline. Ultimately, the program will cultivate a new generation of global health researchers and leaders equipped to tackle the growing health challenges in impoverished communities across LMICs.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT Despite the success of antiretroviral therapy (ART) in achieving viral suppression, people with HIV (PWH) experience persistent immune activation in the central nervous system (CNS), contributing to cognitive impairment. While prior studies have focused on T cell and microglial contributions to CNS pathology, the role of B cells remains unexplored. B cells are critical regulators of immune responses, and evidence from other neuroinflammatory diseases suggests they can drive CNS pathology through antigen-specific and autoreactive mechanisms. Understanding B cell activity and antigen specificity in the CNS of PWH could uncover novel drivers of neuronal injury and cognitive decline. Our preliminary studies from cerebrospinal fluid (CSF) and blood in virally suppressed PWH compared to controls revealed markedly aberrant memory B cell gene expression and altered somatic hypermutation patterns in the CSF, suggesting persistent antigen-driven stimulation in the CNS despite ART. These abnormalities suggest that CNS-resident memory B cells play a unique and potentially pathogenic role in HIV-related neuroinflammation. To address this crucial knowledge gap, this study proposes to generate monoclonal antibodies (mAbs) from CSF-derived memory B cells in PWH and uninfected controls, and to test these mAbs for antigen reactivity. We will test for reactivity against HIV and opportunistic pathogens using massively high-throughput phage display assays that include over >500,000 pathogen derived peptides, including >7500 HIV peptides. To understand the role of autoimmunity in HIV-associated CNS dysfunction, we will next test CSF memory B cell derived mAbs for autoimmune reactivity using two parallel approaches. This multidisciplinary research will leverage advanced single-cell transcriptomics, high-throughput antigen screening, and cutting-edge antibody profiling techniques. The findings will provide crucial insights into the CNS-specific humoral immune response in PWH, with implications for vaccine design and the development of targeted therapies to mitigate CNS immune dysregulation.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Proteins carry out almost all functions in all living cells. At the same time, cells must coordinate protein synthesis with folding capacity to avoid misfolding and the accumulation of highly toxic protein aggregates. Critically, chaperones enable most proteins to adopt conformations required for essential biological activities. In bacteria, the chaperone trigger factor (TF), which is as abundant as ribosomes, had long been thought responsible for cotranslational protein folding when nutrients are plentiful. However, we have now identified cytoplasmic Mg2+ starvation as an infection-relevant stress condition in which the facultative intracellular pathogen Salmonella enterica serovar Typhimurium requires Hsp70 chaperone DnaK instead of TF. Surprisingly, DnaK directly decreases protein synthesis, thus conferring survival against cytoplasmic Mg2+ starvation. This is the first example of DnaK performing protein homeostasis activities independently of its J-domain cochaperones and nucleotide exchange factor. We now propose to combine genetic, biochemical, and biophysical approaches to determine how DnaK decreases protein synthesis, in course solving the structure of DnaK-bound ribosomes harvested during cytoplasmic Mg2+ starvation. We will use selective ribosome profiling to identify nascent polypeptides associated with DnaK versus TF during cytoplasmic Mg2+ starvation and abundance. We will also interrogate DnaK binding to the ribosome as necessary for bacterial resistance to proline-rich antimicrobial peptides that attack the translation machinery and for survival against cytoplasmic Mg2+ starvation and inside murine macrophages. The proposed experiments will provide fundamental tools and knowledge to solve how slower protein synthesis enables bacterial survival during infection. Moreover, results will apply broadly to diverse microbes as this physiologically important chaperone is widely distributed, shares high amino acid sequence identity across species, and is required for virulence by multiple pathogens.

NIH Research Projects · FY 2025 · 2025-09

Project Summary. Myasthenia gravis (MG) is an autoimmune disorder affecting neuromuscular transmission. MG patients suffer from severe muscle weakness and increased muscle fatigability due to diminished neuromuscular signaling. MG is caused by autoantibodies that target components of the neuromuscular junction (NMJ). The most common subtype of autoantibody-mediated MG—comprising approximately 80% of the patient population—is characterized by pathogenic autoantibodies targeting the nicotinic acetylcholine receptor (AChR). There is a subset of MG patients that is termed seronegative MG (SNMG). SNMG is defined by the absence of detectable autoantibodies to the known NMJ targets. The mechanisms underlying the immunopathology SNMG are poorly understood. Consequently, the impact on these patients is considerable; treatment paradigms and outcomes are uncertain due to the lack of data associated with the disease mechanisms. In addition, SNMG patients are often excluded from clinical trials in which autoantibody positive MG patients participate. To address this knowledge gap, we will identify immune mechanisms driving SNMG. This will be achieved through the application of two high-throughput autoantibody-discovery technologies, both of which allow for the screening of exceptionally large human antigen libraries. The first (termed PhIP-seq) leverages the entire human protein catalog represented by a synthetic peptide library (peptidome), which includes all open reading frames in the human genome. The library is presented on the surface of bacteriophages as forty-nine amino acid peptides that “tile” or overlap each other. The second (termed REAP) affords screening of antigens displayed on cells as full-length domains of extracellular and membrane proteins, which are often the target of pathogenic autoantibodies. Because the antigens are expressed in live cells, protein folding into biologically relevant conformations and post-translational modifications are included. Together these approaches complement each other by collectively offering broad antigen coverage and presentation of biologically relevant epitope conformations. Carefully curated SNMG patient samples from the US, Italy and Eastern Europe will be screened. Validation of candidate antigens will be performed with independent assays used for measuring antigen-antibody binding. The second phase of this project focuses on defining the mechanisms these newly identified autoantibodies use to mediate SNMG pathology. The focus will be given to antibody characteristics including isotype and IgG subclass, followed by complement activation, receptor internalization, and interruption of receptor/channel function. We will apply in vitro assays, which we have developed, to measure these characteristics and mechanisms. Overall, this study is designed to provide detailed insights into the molecular mechanisms used by autoantibodies to facilitate the pathology of this poorly defined MG subtype. In addition to providing valuable diagnostic biomarkers, these newly defined immune mechanisms are expected to impact treatment outcomes by informing individualized and mechanism-specific applications of biological therapeutics.

NIH Research Projects · FY 2025 · 2025-09

Project summary/abstract. Current predictive biomarkers for immune checkpoint blockers have multiple limitations, and selection of patients with the highest benefit potential for immunotherapy alone or in combination with other agents is an unmet need. Tumors harboring DNA repair defects display increased sensitivity to agents targeting such pathways such as PARP1/2 inhibitors. Predictive biomarkers for such therapies rely on DNA/genomic analysis that is limited by the dependency of tumor content in samples, lack of annotation of the variants' significance in multiple DNA repair genes, and the occurrence of DNA repair alterations due to non-genomic events. Notably, immunotherapies are active in patients with malignancies harboring DNA repair defects and the efficacy of PARP inhibitors requires adaptive anti-tumor immune responses in preclinical models. This has raised interest in combining these agents. Here and through 2 complementary aims, we will validate multiplex quantitative immunofluorescence (mQIF) assays for spatially resolved measurement of key immune metrics and homologous recombination-deficiency (HRD) proteins in conventional formalin-fixed paraffin-embedded tumor biopsies. These markers are non- redundant and complementary with existing biomarkers. In Aim 1 (UH2), we will analytically validate and standardize two mQIF assays for measurement of anti-tumor immune responses (Assay #1: DAPI/CK/PD- L1/CD8/CD20) and HRD metrics (Assay #2: DAPI/CK/H2AX/RAD51/BRCA1). In Aim 2 (UH3), we will determine the pharmacodynamic and predictive biomarker role of the mQIF assays measuring anti-tumor immune responses and HRD metrics in 3 independent clinical trials using PARP inhibitors and/or PD-1 axis blockers. Together, these studies have the potential to expand the repertoire of predictive biomarkers for novel anti-cancer treatments and positively impact patients' outcomes. We have assembled a team of investigators with complementary expertise and will access unique laboratory/technical and clinical trial resources to accomplish the proposed work. The use of advanced analytical controls, cross validation of results in multiple studies and rigorous statistical definitions will support the scientific quality and success of the study.

NIH Research Projects · FY 2025 · 2025-09

Cholangiopathies are responsible for significant morbidity and mortality but remain the least understood diseases in hepatology. Cholangiocytes are the main cellular target in cholangiopathies and in response to damage or inflammation, they assume a reactive phenotype, generate pro-inflammatory mediators and cyto-chemokines, proliferate, reduce their secretory function, and further amplify the inflammatory responses. The inflammatory microenvironment in cholangiopathies is populated by several cell types that communicate with each other and with cholangiocytes. This complex cellular network is still poorly understood and exhibits disease-specific aspects that may ultimately be relevant for therapy. Among the inflammatory cells infiltrating the portal space, neutrophils have received little attention. Some disorders affecting bile ducts result in significant infiltration of neutrophils into the portal region, but the pathophysiological consequences remain unknown. The field of neutrophil biology has recently advanced in a fundamental way. There is now a recognition that some neutrophils are attracted to and then reprogrammed by tissues in an organ-specific fashion. In contrast to those in the bloodstream, these infiltrating neutrophils often live days longer in target tissues, where they go on to serve nondestructive roles such as regulation of metabolism, secretion, angiogenesis, and proliferation. Although these non-traditional roles for neutrophils have not yet been studied in cholangiopathies, in part due to difficulties (that we have overcome) in isolating neutrophils from liver in sufficient amounts for single cell transcriptomic analysis. Our preliminary evidence suggests that periductular neutrophils are largely distinct from those of neutrophils in the bloodstream. We also have generated preliminary evidence to suggest that cholangiocytes secrete cytokines/chemokines as ‘homing signals’ to attract and then reprogram neutrophils, and these homing signals may differ depending on the cholangiopathy. To make these observations, we have developed several methods for recovering neutrophils from the liver and from the biliary tree in sufficient amounts to perform scRNA-seq. Based on extensive new preliminary data, we hypothesize that neutrophils play a major role in the pathogenesis of cholangiopathies by being attracted by cholangiocytes, which reprogram the neutrophils to amplify the inflammatory response and to interact with cholangiocytes resulting in cholestasis, changes in transcriptome and altered proliferation in a condition--specific way. The nontraditional roles for neutrophils being proposed here will be paradigm-shifting in terms of how we understand the role of neutrophils in biliary diseases. Furthermore, we will advance the field of neutrophil biology as well, by providing evidence not only that transcriptomic signatures of neutrophils in the liver differ from those in the blood, that these signatures in the liver may be disease-specific and that the interactions between cholangiocytes and neutrophils are targets of potential therapeutic relevance.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Post-transcriptional modifications on mRNAs are critical for modulating protein synthesis and maintaining cellular homeostasis. mRNAs are decorated with numerous chemical modifications, including 5- methylcytosine (m5C), which is enriched in regulatory 5¢ untranslated regions (5¢-UTRs). Aberrant m5C methylation caused by overexpressed methyltransferases in cancer cells is known to stabilize specific oncogenic mRNAs, thereby promoting cell proliferation and tumorigenesis. However, how aberrant m5C methylation of 5¢-UTRs affects translation initiation and dysregulates protein synthesis remains largely unknown. Dysregulated translation of oncogenes and tumor suppressors is likely to contribute to the pathogenic effects of dysregulated methyltransferase expression in tumors. The primary objective of this proposal is to determine the regulatory role of dose-sensitive m5C modification in the early stages of translation and uncover its direct impact on ribosome recruitment to mRNA 5¢-UTRs. To study physiological m5C residues, I first purified the key mRNA modifying members of the NOL1/NOP2/SUN (NSUN) family of methyltransferases, NSUN2 and NSUN6, and verified their methyltransferase activities using known targets. Here, I will screen ~35,000 human endogenous 5¢-UTR sequences from multiple tissues, encompassing all genetic isoforms, for m5C installation by NSUN2 and NSUN6. My preliminary computational analysis indicates the presence of thousands of potential m5C targets in this 5¢-UTR pool. The overarching hypothesis of this proposal is that m5C is dose-dependently installed on 5¢-UTRs and quantitively impacts translation initiation. To test this hypothesis, I will employ an innovative high-throughput approach developed in our lab, direct analysis of ribosome targeting (DART), to quantify ribosome recruitment on UTR pools methylated with varying concentrations of NSUN2 or NSUN6. Aim 1 will identify dose-sensitive m5C targets through bisulfite sequencing, measure m5C-dependent changes in ribosome recruitment, and validate methylation-sensitive translation activity in cancer cells. Aim 2 will identify and characterize trans-acting RNA binding proteins that modulate m5C-mediated translational control. Together, the proposed work will elucidate how aberrant m5C methylation, caused by overexpression of NSUN2 and NSUN6, mediates dysregulation of translation in cancer cells. My results are expected to reveal novel and fundamental mechanisms of translation initiation in human cells and may uncover new therapeutic targets for cancers with overexpressed NSUN2 and NSUN6.

NIH Research Projects · FY 2025 · 2025-09

Epidermal differentiation disorders (EDD, a.k.a. the ichthyoses) are caused by pathogenic variants in over 50 genes and feature generalized skin scaling, thickening, and redness often associated with itch, a clinical marker of inflammation. Prior work provides evidence of T helper (Th) 17 inflammation across EDD with an immune signature resembling the common inflammatory skin disease psoriasis. Despite this, clinical response to Th17 inhibition has generally been poor or incomplete. Our lab discovered a novel genetic EDD, the erythrokeratodermia cardiomyopathy (EKC) syndrome which results from heterozygous genetic variants affecting the SR6 domain of desmoplakin. EKC features early childhood onset of inflammatory skin lesions and progressive dilated cardiomyopathy. In contrast to the majority of individuals with EDD due to other genetic causes, individuals with EKC have shown consistent and profound improvement in skin redness, inflammation and, sometimes, cardiac function, when treated with therapies inhibiting the Th17 pathway, suggesting that inflammation contributes to disease pathogenesis. We propose that EKC can serve as a model for Th17 inflammation due to genetic defects in skin barrier dysfunction, and our preliminary data shows increased innate immune activity in EKC skin and keratinocytes expressing DSP variants. We have developed unique resources that make us well-posed to study this phenomenon: skin tissue and primary cultured keratinocytes from individuals with EKC, keratinocytes engineered to stably express EKC-associated DSP variants, and a transgenic knock-in mouse model which allows us to express EKC-associated DSP variants across the entire organism or specifically in skin. We will interrogate effects on desmosomes by assessing desmoplakin molecular turnover at the cell membrane and structure and function of desmosomes using electron microscopy and dispase-based mechanical dissociation assays. In addition, we will use unbiased transcriptomic profiling to uncover cell-intrinsic innate immune signatures in cultured keratinocytes and molecular cross-talk between keratinocytes, immune cells, and stromal cells in murine skin using single cell RNA sequencing. We generated a transgenic knock-in mouse model of EKC with skin-specific expression of mutant Dsp driven by Keratin14-Cre and found that K14-DspL622P/WT mice are viable with intact barrier at birth, mirroring EKC patient presentation. Over time, K14-DspL622P/WT mice develop progressive inflammatory lesions and elevated markers of Th17 immunity, establishes the utility of this model to study Th17 inflammation due to skin barrier dysfunction. We will use flow cytometric analysis of skin tissue and plasma cytokine profiling to assess inflammatory markers in the mouse model and test how modulating the cutaneous microbiome (through both depletion and diversification of skin microbes) affects markers of Th17 inflammation. Studying the cellular determinants of innate immune activation and specific cellular interactions which drive initiation and maintenance of Th17 inflammation in EKC may elucidate mechanism underlying the pattern of Th17 polarization that is shared across EDD.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Preeclampsia (PE) affects 2-8% of pregnancies worldwide and is diagnosed as new-onset hypertension with signs of target organ damage. Women that survive PE are twice as likely to experience atherosclerosis-related conditions (coronary heart disease) leading to ischemic events (myocardial infarction and stroke) compared with women who have uncomplicated pregnancies. Epidemiological studies indicate residual risk for cardiovascular disease following PE that is not explained by pre-existing risk factors. Systemic vascular endothelial cell (EC) dysfunction and increased immune cell activation are correlated with PE and can persist sub-clinically in the years following pregnancy. Activated and dysfunctional ECs augment immune cell recruitment into the vascular wall contributing to atherosclerosis, but the effect of PE on mechanisms of atherosclerosis and the role of pre- existing risk factors remains a gap in knowledge. To understand the specific effect of PE, a well-established experimental mouse model known to elicit features seen in humans (soluble fms-like tyrosine kinase 1 (sFlt-1) adenoviral injection mid-gestation) will be studied, compared with a control pregnancy (empty vector adenovirus). Preliminary data demonstrate that mice exposed to experimental PE have 1) microvascular EC dysfunction in late gestation and 8 weeks postpartum and 2) increased number of leukocytes and T cells in atherosclerotic plaques 8 weeks postpartum, independent of plaque size, lipid content, necrotic core, or metabolic risk factors. Importantly, increased plaque inflammation contributes greatly to plaque instability and likelihood of rupture leading to ischemic events. Therefore, the hypothesis is that enhanced atherosclerotic plaque inflammation after PE is due to persistent, systemic vascular EC activation and dysfunction combined with increased proinflammatory immune cell activation and infiltration into atherosclerotic plaques. Aim 1 will test if experimental PE results in persistent EC activation, microvascular function, and large artery stiffness in atheroprone mice (LDLr-/- mice fed a high fat diet postpartum). EC activation will be assessed via immunofluorescence in mesenteric resistance arteries (microvasculature) and areas of atherosclerotic plaque development. EC dysfunction will be assessed ex vivo via wire myography, and large artery stiffness will be assessed in vivo via pulse wave velocity in late gestation, 4 weeks postpartum, and 8 weeks postpartum. Aim 2 will determine how experimental PE alters immune cell plaque inflammation using single cell RNA sequencing to determine alterations in subpopulations, cell states, gene expression, and cell-cell interactions in aortic plaques 8 weeks following PE in atheroprone mice. Completion of project aims will provide mechanisms of atherosclerosis EC dysfunction and leukocyte infiltration following PE, which can be used to investigate targeted prevention strategies and therapeutics in future work. This F32 fellowship also provides extensive training opportunities in basic science and professional development to support the applicant’s long-term goal of becoming an independent academic researcher studying female-specific cardiovascular health across the lifespan.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT One of the fundamental challenges in modern biology is to decode the functionalities of human genome sequence. Over the past decade, genome-wide association studies (GWAS) have generated a wealth of new information, including the genotype–phenotype associations in various diseases and traits. Despite clear successes in identifying novel disease susceptibility genes and in translating these findings into clinical care, GWAS has been criticized for the fact that most association signals reflect variants and genes with no direct biological relevance to phenotype. The development of large language model (LLM) has been the main driving force behind many recent breakthroughs in artificial intelligence. Research into the “genomic LLM” therefore has the potential to significantly advance our understanding of how the genetics variants lead to the changes in phenotypes by disrupting the underlying regulatory syntax of DNA. The Research Training Plan will first develop and improve the core technologies of genomic LLMs to deepen our understanding on understanding the complex regulatory mechanisms in gene regulation (Aim 1). The developed genomic LLMs will then be applied in imaging genetics studies where imaging traits are used as phenotypes (Aim 2) and the development of new machine learning (ML) approaches for Alzheimer’s disease diagnosis (Aim 3). In Aim 1, the applicant Dr. Qiao Liu will develop new genomic LLM techniques and provide biological model interpretation with special focus on how transcription factor (TF) binds DNA recognition sites in genomic regulatory regions to control genomic transcription and affect epigenomic signals in a context-specific manner. The proposed genomic LLMs will serve as solid foundation for both Aim 2 and Aim 3. In Aim 2, Dr. Liu will focus on the imaging genetics studies, which can be considered as GWAS of imaging phenotypes, for linking genetic variants/genes to structural or functional imaging features through the mediation of genomic LLMs. Genomic LLMs thus will bridge the gap between personal genetics and radiomics. In Aim 3 during the R00 phase, Dr. Liu will develop new ML approaches on AD diagnosis by considering the causal genetic-imaging-clinical pathways and leveraging the power from the genomic LLM. To succeed in these aims, a Career Development Plan is tailored to enable Dr. Liu to gain new knowledge and skills in radiomics, neuroimaging, and Alzheimer’s disease, as well as career skills through practice and coursework with the support of the outstanding mentoring team and scientific advisory committee. Stanford University is an ideal environment, providing all of the facilities needed for the proposed research and a rich interdisciplinary environment for collaborative studies. In summary, the strong mentoring team and scientific advisory committee, as well as the training plan are anticipated to fully prepare Dr. Liu to launch his independent career. The proposed studies promise to offer mechanistic insights into both genetics and radiomics, and may help uncover important genetic-imaging-clinical pathways for better understanding complex diseases.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT Abnormal development of the left-right (LR) axis during embryogenesis can manifest as a broad spectrum of severe congenital heart diseases (CHDs), a group of conditions that affects 1% of all infants and in which the cardiac structure is disrupted. The LR axis is established at the Left-Right Organizer (LRO), a transient embryonic structure conserved across several vertebrate species. The Brueckner lab has discovered that transcriptional and morphological left-right asymmetry requires motile cilia in LRO “pit cells” that generate force sensed by mechanosensing/calcium-signaling immotile cilia in LRO “crown cells.” However, how the LRO develops and the mechanisms by which transcript symmetries are first broken are not fully understood. Advancing knowledge of LRO formation and function will inform our understanding of the etiology of laterality defect-associated CHDs. The goal of this proposal is to define the cellular subpopulations that constitute the LRO and establish how they initiate LR asymmetry of the developing embryo. Based on preliminary findings, the central hypothesis of this proposal is that the mouse LRO forms from two transcriptionally dis- tinct cell populations (crown and pit) that develop further heterogeneities over time, including a newly-identified MMP21 (matrix metalloprotease 21)-positive pit cell subpopulation required to es- tablish the earliest LRO transcriptional asymmetries. To test this hypothesis, this project will utilize the mouse LRO as a model for human LR development, as evolutionary and genetic studies have revealed motile cilia as key drivers of LR asymmetry in both species. Aim 1 will investigate LRO structure and em- bryonic origins, utilizing single cell and spatial transcriptomic data, followed by in vivo validation, to identify and characterize the origins and transcriptional markers of LRO cell subpopulations. Aim 2 will investigate LRO function and expand upon the currently incomplete model of LR symmetry breaking to include the MMP21 mRNA and protein. This aim will use hybridization chain reaction in situ hybridization (HCR-FISH) and immunofluorescence (IF) experiments in mouse embryos with mutations in ciliary components and LRO genes. Long-term, this work will have both basic and translational research implications, and results from this project will provide a comprehensive view of the origins and roles of rare LRO cell populations that are essential to the proper inception of cardiac morphogenesis. Upon completion of this fellowship, the applicant will have received training in mouse genetics, early vertebrate development, scRNA-seq and spatial tran- scriptomic data generation and analysis, and advanced microscopy image analysis, as well as strengthened her skills in mentoring, scientific communication, research design, and critical thinking, thus providing her with the expertise necessary for a successful academic career in independent early developmental biology research.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT Poly (ADP-ribose) polymerase inhibitors (PARPi) are synthetically lethal in cells with homologous recombination (HR) defects, a phenotype of certain cancers. BRCA1 is one of the most commonly mutated genes in hereditary, HR-deficient cancers. Unfortunately, patients with HR-deficient cancers commonly acquire resistance to PARPi, and the mechanisms of PARPi-induced cytotoxicity are underexplored. Understanding these pathways could provide insight into how patients acquire PARPi resistance. The PARPi cytotoxic response requires cells to transit mitosis, and cells that persist through PARPi treatment (persister cells) arrest from the cell cycle. Thus, determining this mechanism of cell cycle commitment is central to understanding PARPi-induced cytotoxicity as cells face two potential fates in response to PARPi: undergo mitosis and trigger cell death or arrest from the cell cycle and persist. My preliminary data suggests that arrested persister cells have decreased levels of Lamin B1 (LMNB1, a nuclear lamina protein): loss of which is necessary and sufficient to induce cell cycle arrest in other contexts. Thus, LMNB1 downregulation may contribute to the cell cycle arrest mechanism in these persister cells. For PARPi-treated cells that undergo mitosis-dependent cell death, the pathways governing this cytotoxicity are unclear. PARPi have been shown to synergize with other drugs to induce various forms of regulated cell death (RCD) in different cell lines, suggesting that multiple forms of RCD could be triggered in BRCA1-deficient cells. A systematic investigation is needed to determine the RCD mechanisms involved in the PARPi response and the upstream regulatory pathways that initiate them. One hypothesis of a contributing regulatory pathway is cyclic GMP-AMP synthase (cGAS) / stimulator of interferon genes (STING) signaling, which canonically induces innate immune signaling in response to cytosolic DNA. cGAS/STING signaling is increased in HR-deficient, PARPi-treated cells. Higher instances of mitotic errors, including persistent DNA bridges, are also observed in response to PARPi treatment. As these structures are prone to rupture, leading to loss of nuclear envelope integrity, we hypothesize that they could be surveilled by cGAS to signal RCD in the PARPi response. The objective of this proposal is to determine the mechanisms that confer vulnerability of BRCA1-deficient cells to PARPi-induced cytotoxicity. Using BRCA1-deficient breast and ovarian cancer cell lines as models, in Aim 1 I will determine mechanism of cell cycle evasion that allows cells to persist through PARPi treatment, specifically investigating LMNB1 downregulation as a contributing pathway. In Aim 2 I will conduct an arrayed CRISPR screen targeting RCD driver genes and other regulatory pathways to determine the mechanisms of PARPi-induced cytotoxicity in cycling cells, including investigating a role for cGAS/STING signaling. Overall, this proposal will enhance our understanding of how cells evade or succumb to PARPi-induced cell death. This work could inform combination therapies with PARPi to combat PARPi resistance in patients with BRCA1-deficient cancers.

NIH Research Projects · FY 2025 · 2025-09

Project Summary/Abstract The binding of nerve growth factor (NGF) to the receptor tyrosine kinase (RTK), TrkA is critical for neuronal survival, growth, maintenance, and pain perception. Mutations in the TrkA:NGF signaling axis result in neuronal defects and pain imperception found in populations with Hereditary Sensory and Autonomic Neuropathies (HSAN). However, those with a subtype of this condition, HSAN-V, have a point mutation in NGF (NGFpainless) that preserves neurological function but show a loss of pain acuity. The molecular mechanism by which NGF regulates TrkA, and how the mutant NGFpainless alters or biases downstream signaling to guide physiological outcomes, remains elusive. I hypothesize that NGFpainless-mediated disentanglement of neurotrophic and pain signaling is regulated by the structural dynamics and/or the dimer lifetime of the TrkA:NGF-signaling competent complex, which biases downstream effectors and signaling away from pain. I will approach this hypothesis in three aims. In Aim 1, I will employ a structural approach (cryo-EM) to study intact TrkA:NGF/NGFpainless 2:2 complex to identify potential differences in structural or conformational states that may bias downstream signaling. I have already obtained high-resolution structural information for TrkA:NGF and TrkA:NGFpainless. I will also use 3D variability analysis (3DVA) to visualize the heterogeneity underlying the dynamic landscape of these complexes. In Aim 2, I will study the organization and dynamics of TrkA:NGF/NGFpainless in the context of its native membrane environment using single-molecule microscopy. Using Native-nanoBleach, a technique developed in the Bhattacharyya lab that uncovers the oligomeric distribution of protein complexes at equilibrium, I have determined that there is a decrease in the population of dimers/multimers in TrkA:NGFpainless when compared to TrkA:NGF. I will further use single-particle tracking (SPT) to study how varying NGF conditions affect dimer lifetime of the TrkA signaling complex on native, live cell membrane. In Aim 3, I will investigate any changes in phosphorylation and activation status of known downstream effectors of TrkA by quantitative western blots, targeting PLCγ1, ERK, and AKT. My preliminary data shows that there is a significant decrease in PLCγ1(pY783) in SHSY5Y neuroblastoma cells stably expressing TrkA when treated with NGFpainless versus NGF for 15 min. I will also measure these changes over a time course where I will incubate the cells with NGF variants for upto 60 min. In an alternate approach for this aim, I will use unbiased, mass spectrometry-based phosphoproteomics to identify (and quantify) changes in the global phosphorylation network regulated by the TrkA:NGF pathway when treated with NGFpainless. Together, these studies will allow me to understand the molecular basis for how NGFpainless, bearing only a single- point mutation to NGF, biases TrkA-downstream signaling away from pain perception, ultimately providing new ideas and directions to exploit this pathway either for chronic pain management or improved neurogenesis.

NIH Research Projects · FY 2025 · 2025-09

The full benefits of community engagement are yet to be realized in addiction sciences. Absent the perspectives and expertise of people with lived experience (PWLE) of substance use disorders and those who have been incarcerated, we miss the opportunity to bring rigor, relevance and reach to addiction science through the singular role that PWLE can serve in understanding the feasibility and acceptability of interventions that prevent and treat addiction and disseminating to their networks for sustainment. Our past work has shown that PWLE are critical contributors to health research, from study inception, implementation, analysis, and dissemination, yet few research institutions invest in building the capacity of PWLE to authentically contribute. Many have systematic barriers to hiring and retaining people with criminal records and addiction and to sustaining meaningful partnerships with community organizations. Built on 25 years of collective experience, the JUSTResearch Community Engaged Research Resource Center (CERRC) will offer unprecedented resources and infrastructure to build the bidirectional capacity of PWLE and academic researchers and institutions, and community stakeholders of NIDA’s Justice Community Overdose Innovation Network (JCOIN) to conduct community engaged research. JUSTResearch will be co-led by JustLeadershipUSA (JLUSA), a national organization focused on empowering formerly incarcerated individuals and Yale’s SEICHE Center for Health and Justice and Program in Addiction Medicine, which have 20+ year histories leading large community-engaged research projects in partnership with PWLE. JUSTResearch builds on our partnership co-leading health research grants and established track-record of changing policies on hiring people with criminal records and formalizing pipelines for employment in health research. The objective of JUSTResearch is to transform JCOIN research using a multi-level approach that enables authentic participation of PWLE. We will achieve this through five integrated cores: an Engagement and Support Core will convene a community advisory board to inform JCOIN science and conduct an iterative needs assessment of JCOIN hubs to maximize engagement of PWLE; a Research Capacity Building Core will build capacity of JCOIN investigators and institutions to reform hiring policies and practices and formalize hiring and faculty pipelines for PWLE; a Dissemination Core will create public resources with the Coordination and Translation Center with a focus on disseminating to broad audiences including incarcerated people; a Rapid Research Core will support research co-led by PWLE and employ systems science approaches; and an Evaluation Core will measure whether the CERRC is effective in achieving its aims. JUSTResearch will facilitate community engagement to improve the health of communities impacted by mass incarceration and addiction and build the science for future community-engaged research. This study is part of the NIH’s Helping to End Addiction Long-term (HEAL) initiative to speed scientific solutions to the national opioid public health crisis. The NIH HEAL Initiative bolsters research across NIH to improve treatment for opioid misuse and addiction.