Washington University

NIH Research Projects · FY 2025 · 2023-09

With prostate cancer (PCa) being among the most common cancers in men worldwide (estimated 1,600,000 cases, 366,000 deaths annually), the need for new biomarkers for early detection, diagnosis, monitoring, and prognosis remains urgent. Among the available management alternatives for PCa, active surveillance (AS) is recommended as an initial treatment for males with very low-, low- and favorable intermediate-risk. AS relies on serial monitoring over time to identify progression, so that the patient receives timely curative treatment, while reducing morbidities related to definite treatment delivered at time of diagnosis. However, identifying ideal candidates for AS is challenging. Despite its limited specificity, the prostate-specific antigen (PSA) is the most used test for early detection of PCa. Other factors based on biopsies such as the Gleason Group (GG), are affected by limited biopsy sampling, while the non-invasive magnetic resonance imaging (MRI) has been connected to false positives and false negatives. Finally, the implementation of molecular prognostic tests, such as Decipher in AS populations has been limited due to the lack of randomized trials using actual AS patients. Previous work suggests that PCa progression can be dependent on the interactions between extracellular matrix (ECM) proteins in the stroma with various cell types including the immune system and cancer cells. In this regard, my team of collaborators has developed clinical imaging techniques such as, diffusion basis spectrum imaging (DBSI), and matrix-assisted laser desorption ionization (MALDI) mass spectrometry, that visualize inflammatory, stromal/ECM, and cancer cell components of the tumor that could be associated with cancer progression. Therefore, I propose to leverage radiomics, a method based on data-characterization algorithms to extract imaging features, to detect patterns in pre-op MRI with the guidance of DBSI and MALDI toward optimally selecting AS candidates. My central hypothesis is that the spatial analysis of structural components in the ECM extracted from the co-registration of molecular and radiological imaging, accurately predicts tumor upgrading and upstaging in PCa. In Aim 1 I will identify a baseline radiomic signature derived from pre-op MRI to accurately predict tumor upgrading (i.e., from GG1 to GG2 or higher) by augmenting well-established biomarkers (i.e., PSA, GG, Decipher), with an exploratory SubAim co-registering DBSI and MRI to improve the prediction. In Aim 2, I will use MALDI co-registered with pre-op MRI to guide the derivation of the radiomic signature to accurately predict tumor upstaging (i.e., from T1/T2 stage to T3 or higher). This research is innovative because, to date, no distinct spatial signatures linked with the ECM and derived from co-registered molecular and radiological imaging have been associated with prediction of tumor progression in PCa. Furthermore, this K22 career transition award will provide me with the training and resources to establish an independent research program focused on developing advanced imaging and computational approaches to improve cancer diagnosis and prognostication.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY The human genome encodes tens of thousands of circRNAs, of which the translation is essential for regulating important biological functions, such as cell proliferation, differentiation, and migration. Interference with circRNA translation can lead to tumorigenesis and metastasis of cancers. It is unclear, however, how disrupting circRNA translation can cause the diseases. Identifying the regulatory components of circRNA translation can provide valuable clinical insights. However, current technologies cannot distinguish circRNAs from linear RNAs efficiently, such that the mechanism and molecular components regulating circRNA translation remain unclear. Thus, it is imperative to develop a technology that can identify the components regulating circRNA translation with higher sensitivity and specificity. I have developed a high-throughput reporter screening assay that can systematically screen and identify the RNA sequences that initiate circRNA translation specifically. Utilizing the technology, I discovered two groups of the RNA sequences that specifically drive translation on either circRNA or linear RNA, respectively, indicating that the regulatory components between linear RNA and circRNA translation are different (Chen et al., resubmitted to Science). In this proposed research, I will further adapt and apply this novel technology to (i) identify and characterize the sequence and protein components that regulate circRNA translation specifically, (ii) identify the association between genetic variation, circRNA translation, and disease, (iii) characterize circRNA translation in a tissue-dependent manner systematically, and (iv) build a non-integrative and stable gene expression platform with tunable expression level and tissue specificity. This proposed research will build the foundation of my future research as an independent researcher to investigate the regulation and the coordination between different translation machinery (cap-dependent vs. independent translation) among different RNA species (linear vs. circular RNAs). This work will be performed under the mentorship of Dr. Howard Chang at the Stanford University School of Medicine, an expert in technology development and RNA biology who has highly-successful track records of placing postdoctoral fellows into independent academic positions at leading institutions. Further training in scientific and professional skills will be achieved by utilizing the resources available through the Stanford University School of Medicine and the Office of Postdoctoral Affairs, which provide an outstanding intellectually- stimulating environment with all facilities and resources necessary for success. All proposed training will complement my previous training in RNA biology, technology development, and computer programming and facilitate my transition to an independent researcher, investigating the functions, regulations and clinical implications of circRNA.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Cervical cancer is among the most common cancer diagnoses among women, and treatment failure of standard of care chemoradiation therapy (CRT) for locally advanced cervical cancer (LACC) is as high as 30-50%. Since recurrent and metastatic diseases are not curable, there is a pressing need to identify patients at risk of treatment failure as early as possible to allow for personalized treatment, rather than after a failure and progression. While TCGA’s molecular stratification of cervical cancer using genomic data failed to associate to patient outcomes, we recently published on integrating genomic and imaging data to improve LACC risk stratification after CRT. Therefore, in this study we intend to use multi-omics data to define and validate LACC risk groups and identify group-specific treatment targets. Based on our preliminary data that indicate distinct biological mechanisms drive CRT resistance in patients with different levels of lymph node (LN) involvement at presentation, we will stratify patients by LN status to develop and validate novel radiogenomic biomarkers. Prognostic models will be developed using gene expression data from pre-treatment tumor biopsy and radiomic features from pre- treatment PET imaging data. Upstream driver and/or feature genes will be validated at the RNA and protein levels by qRT-PCR, Western blotting, and tissue microarray (TMA). One such gene identified from our preliminary data using a radiogenomic approach is nuclear factor erythroid 2–related factor 2 (NRF2), which has not been previously characterized in LACC, since it is not frequently mutated in cervical cancer. We will perform functional analysis to study NRF2 biology in LACC via clonogenic survival assay and other standard assays. In addition to pre-treatment biomarkers, we will leverage radiomic features from our time course MR images and on-treatment gene expression data to develop novel radiogenomic biomarkers to assess a patient's evolving risk of treatment failure over the course of CRT, informing adjustment of therapy at mid-treatment. The pre-treatment model will be further refined by applying deep learning to identify predictive features for CRT outcome directly from clinical PET images to inform intensified treatment from the beginning. Finally, we will apply multi-omics approaches (scRNA-seq, proteomics, metabolomics) to characterize the biology related to LACC CRT radiogenomic biomarkers. Taken together, we expect fulfillment of these aims will create a series of optimized, validated recurrence biomarkers at presentation and over the course of 6 weeks of CRT treatment, and will indicate targets for personalized alternative treatment regimens. Beyond the specific application to LACC, our proposal will generate novel methods to integrate multi-omics data to improve hypothesis-driven cancer research.

NIH Research Projects · FY 2025 · 2023-09

PROJECT ABSTRACT Nigeria has among the highest burdens of maternal morbidity and mortality, including an estimated 528 (95% uncertainty intervals: 351, 815) maternal deaths per 100,000 lives birth in 2019 (global average: 152 per 100,000). This rate far exceeds the UN Sustainable Development Target 3.1: <70 maternal deaths per 100,000 live births by 2030. Nigeria also has a large and rising burden of noncommunicable diseases, including overweight/obesity, hypertension, diabetes mellitus, and cardiovascular diseases. Unhealthy changes in dietary patterns and physical activity contribute to these adverse trends, especially during critical life stages, such as the peripartum period, infancy, and childhood. Improving maternal health behaviors and subsequent maternal cardiovascular health is a central strategy toward improving family cardiovascular health to blunt and eventually reverse the rising burden of noncommunicable diseases in Nigeria. Nigeria's National Multisectoral Action Plan for the Prevention and Control of Noncommunicable Diseases and National Task Shifting and Sharing Policy are new and relevant policy frameworks that will be leveraged to improve intergenerational cardiovascular health among Nigerians in this proposal. The objective of this study is to test effectiveness and implementation of an adapted HEALTH (Healthy Eating Active Living Taught at Home) intervention on intergenerational cardiovascular health among 1,000 pregnant women with obesity recruited during the antenatal period through a type 2 hybrid cluster randomized (1:1) trial implemented in 40 Nigerian primary healthcare centers. Using the Framework for Reporting Adaptations and Modifications-Expanded, the HEALTH intervention and its implementation will be adapted from the evidence-based, Parents as Teachers home visiting model from the US that has been used and validated by Washington University researchers, including in the NIH-funded ENRICH consortium. We will use a train-the-trainer model including certified trainers to step down training to community health extension workers and community nurse health educators. The primary effectiveness outcome will be change in maternal body weight from baseline to 18-month follow-up with >85% power to detect a between-group of difference of 2.5 kg. Secondary effectiveness outcomes include changes in maternal cardiovascular health. Exploratory outcomes include changes in infant and child growth. Safety data will be collected. RE-AIM will guide quantitative and qualitative implementation outcome assessment, and reach will be the primary implementation outcome with a >50% target of recruiting women with primary school education or less. Our team of US and Nigerian investigators at Washington University, University of Abuja, and New York University have collaborated for >5 years with support from 5 active NIH grants. We have strong support from key Nigerian stakeholders, including at local, state, and national levels. Thus, our proposal is highly feasible, efficient, likely to succeed, responsive to this funding opportunity, aligned with NIH and GACD priorities, and will be mutually beneficial to our team members through embedded capacity building.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Insulin resistance is commonly associated with obesity and is a major contributor to the development of obesity-related metabolic diseases, (e.g. nonalcoholic fatty liver disease [NAFLD] and type 2 diabetes). Most when characterized by glucose tolerance and insulin sensitivity. However, a subset of people with obesit adverse metabolic effects of excess adiposity. A better understanding of the mechanisms responsible for the differences in metabolic function between people with MHO and MUO can provide important mechanistic insights into the metabolic heterogeneity of obesity and possibly identify novel targets for therapeutic intervention. Small extracellular vesicles (sEVs, or exosomes) are produced by cells throughout the body and deliver bioactive molecules via the bloodstream to regulate metabolic processes in multiple organ systems. Studies in mice have found that sEVs released from adipose tissue modulate insulin sensitivity and contain regulatory miRNAs and bioactive lipids that alter insulin signaling. Consistent with these findings, we recently found that plasma and adipose tissue sEVs obtained from people with obesity and NAFLD impair insulin signaling in cultured myotubes, suggesting sEVs also contribute to the regulation of insulin sensitivity in people. In this proposal, we will evaluate the metabolic effects and cargo content of sEVs isolated from plasma and adipose tissue (AT) explant cultures obtained from three groups of participants. We will characterize participants based on adiposity and metabolic health: 1) metabolically healthy lean (MHL, BMI 18.5-24.9 kg/m2 and normal insulin sensitivity, defined as homeostatic model assessment of insulin resistance (HOMA-IR) <2); 2) metabolically healthy obese (MHO, BMI 30.0-44.9 kg/m2 and HOMA-IR<2); and 3) metabolically unhealthy obesity (MUO, BMI 30.0-44.9 kg/m2 and HOMA-IR>2.5). We posit that: i) sEVs from plasma and adipose tissue in people who are MUO have adverse effects on insulin action compared with sEVs from people who are MHL or MHO; ii) these metabolic differences are associated with differences in sEV content of miRNA and bioactive lipids; and iii) moderate 10% weight loss in people with MUO changes the miRNA and lipid content of plasma and adipose tissue sEVs and their effects on insulin action. The following specific aims will be addressed: 1) determine the effects of sEVs obtained from plasma and adipose tissue of MHL, MHO and MUO participants on: i) insulin action, gene expression and protein profile in cells vitro and in mice in vivo; 2) identify differences in miRNA and bioactive lipid profiles of plasma and adipose tissue sEVs obtained from MHL, MHO and MUO groups; and 3) determine whether a 10% weight loss-induced increase in insulin sensitivity in the MUO group will alter plasma and adipose tissue sEVs miRNA and lipid content and ameliorate the adverse effects of plasma and adipose tissue sEVs on insulin action in in vitro and in mice in vivo. These findings will provide the foundation for future studies in people designed to assess the potential of sEVs as a therapeutic target to prevent and treat the metabolic complications of obesity.

NIH Research Projects · FY 2025 · 2023-09

Project Summary In addition to direct killing of rapidly proliferating tumor cells, radiation therapy (RT) modifies tumor immunity. RT increases the expression and release of tumor-associated antigens, induces immunogenic cell death, and releases danger signals and chemokines that recruit immune cells to the tumor microenvironment (TME). RT can also enhance tumor killing by increasing the number of tumor-infiltrating immunostimulatory cells and inducing the expression of neoantigens. However, RT also stimulates the recruitment of other myeloid derived immune cell subsets that can have tumor permissive phenotypes. The overall balance between these immune stimulatory and suppressive effects of radiation ultimately predicts whether local tumor treatment with RT generates an effective and durable systemic anti-tumor immune response. We hypothesize that RT (and in particular SOC CRT) limits the development of long-term anti-tumor immunity in resistant tumors by disproportionately increasing the number and tumor permissive phenotypes of myeloid derived cells (MDCs) in the TME. The Washington University MicroEnvironment Tumor Effects Of Radiotherapy (METEOR) Center will enable testing of this hypothesis via a dynamic Molecular Characterization Trial with closely integrated Research Projects, Shared Resource Cores, and Cross Training Core. We will leverage our institutional expertise in genomics, proteomics, tumor metabolism and immunology to take a “deep dive” into CRT induced tumor and TME co-evolution. Our overall vision is that immunosuppressive SOC CRT associated changes in the immune TME can be further targeted to improve systemic anti-tumor immune responses after RT. Although our preliminary data implicates two immune TME cell types, macrophages and dendritic cells, our research design is intended to allow for detailed study of CRT related effects on multiple cell types within the TME. As such, our Center can adapt and incorporate new findings in other cell types during the course of funding of this grant through pilot projects and data sharing. Importantly, tumor cells and the TME are influenced by the tissue of origin of the tumor. Thus, it is important to use this approach to study multiple tumor types to establish what are the common (and tumor specific) mechanisms of CRT related immunosuppression. METEOR will thus serve as a hub to share this approach with other ROBIN Centers studying different tumor types. Since the analytic approach through our Shared Resource Cores requires only small biopsies, this will allow other Centers to send limited specimens to us for analysis. Furthermore, through our Administrative and Cross Training Cores, we will host faculty and trainees from other ROBIN centers and share with them our resources, educational materials and approach which will bring added value to the ROBIN network. As a result of this work we will be well positioned to apply for a future NIH T32 focused on training, disseminating and advancing multidisciplinary informatic partnerships to address critical research gaps in radiation oncology.

NIH Research Projects · FY 2025 · 2023-09

Project summary: The overarching goal of this project is to develop the principal investigator (PI) into an independent clinician- scientist contributing to the field of ocular autoimmunity. The PI has obtained his PhD in neuroscience, exploring the roles of the adaptive immune system in axonal injury models. He has additional clinical training in ophthalmology and subspecialty training in uveitis and ocular immunology, which forms the basis for his medical practice in the Department of Ophthalmology and Visual Sciences at Washington University in St. Louis. The five-year career development plan outlined here will utilize a combination of didactic courses, individualized training from expert collaborators, and mentorship from established investigators to enhance his knowledge base, to learn new experimental models he can continue to use in his career, and to define the research questions on which he will base the start of his independent research program. By the end of this program, he will have acquired the skills necessary to become an independent investigator. His primary mentor is Rajendra Apte, MD PhD, an R01-funded investigator who has a longitudinal track record of training early-stage investigators that have gone on to productive independent careers. He is a preeminent scholar in the field of choroidal immunity and has a strong background as a clinician scientist that will be vital for helping the PI navigate the challenges involved in transitioning to an independent lab. Dr. Apte will be supported by Rachel Caspi, PhD, the world-leading expert in experimental autoimmune uveitis, Jonathan Kipnis, PhD, the foremost expert in the adaptive immune system in border tissues, and Gregory Wu, MD PhD, a recent K08 awardee and expert on CNS autoimmunity. The PI will take advantage of his mentorship committee and the vibrant scientific environment of Washington University, one of the world’s premiere medical research institutions, to develop the unexplored topic of the adaptive immune response in the choroid. Uveitis is one of the leading causes of preventable blindness, and while much work has gone in to evaluating the effects of T cell-derived cytokines, there is a significant gap in understanding of the anatomy of the immune response including where the adaptive immune system interfaces with ocular antigens. This deficit has led to toxic and often ineffective systemic therapies as the mainstay of treatment. We show that intraocular antigens are taken up in the choroid, a tissue that has a rich adaptive immune presence. In autoimmune uveitis there is an increase of CD4+ T cells in the choroid, indicating that the choroid plays a vital role as the immune interface between the intraocular space and adaptive immune system. In this proposal, we will test the hypothesis that the adaptive immune system is activated by retinal antigens in the choroid and that this interaction plays a central role in uveitis. We will further elucidate the mechanisms contributing to immune surveillance in the choroid. Understanding the mechanisms behind this process has broad implications in the development of diagnostic testing and targeted therapy that will improve the clinical outcomes in uveitis.

NIH Research Projects · FY 2025 · 2023-09

Abstract Each day, there are an estimated 28 cervical cancer deaths in Nigeria, making cervical cancer the second most common cancer among women in the entire country. Only a minority of women eligible for screening (screen-eligible) in Nigeria (30-49 years old) regularly, have received HPV screening - an essential component of comprehensive cervical cancer prevention programs recommended by the Nigerian Federal Ministry of Health and the Society of Obstetrics and Gynecology of Nigeria (SOGON). Conventional screening that relies on Pap smears and visual inspection with acetic acid (VIA) at centralized clinics, coupled with a lack of locally relevant implementation strategies have stalled progress. Innovative strategies to decentralize screening and increase women’s ownership in this process are urgently needed. In this proposal, we will adapt an existing HPV assay that combines loop-mediated isothermal amplification (LAMP) and fast one-pot lyophilization protocol within a lateral flow assay (LFA) for the Nigerian context, and then use participatory strategies (crowdsourcing open calls and learning communities) to finalize components of a single woman-centered HPV self-test kit. Crowdsourcing open calls have a group of individuals (i.e., screen-eligible women) solve all or part of a problem, then implement selected high-quality solutions. Learning communities help participants refine and optimize solutions before evaluation or use among screen-eligible women in Nigeria. Our collaborative research team has successfully used crowdsourcing open calls and learning communities to increase HIV self- testing among Nigerian youth, providing a strong foundation for the proposed research study. Our preliminary data demonstrate that our HPV self-test prototype can reliably detect HPV 16 and 18 genotypes that account for 70% of all cervical cancer cases. Once the prototype meets stringent diagnostic and trial preparedness metrics among Nigerian women, including detection of additional common HPV genotypes (31, 35, and 52), the project will move from the initial engineering phase (Specific Aim 1) to the clinical phase (Specific Aims 2 and 3). Drawing on a design and participatory action research framework, we propose the following specific aims: (1) to adapt an HPV self-test assay for point-of-care and simultaneous detection of HPV genotypes 16, 18, 31, 35, and 52 in Nigeria; (2) to use crowdsourcing open calls and participatory learning communities among screen-eligible women to finalize a single HPV self-testing implementation strategy (3) Determine whether a final revised HPV self-testing strategy increases HPV screening among 900 screen-eligible women in 18 local government areas versus usual care using a stepped-wedge, pragmatic randomized control trial. Our study will be among the first to examine how women themselves can be prime movers in optimizing, implementing and evaluating HPV self-testing implementation strategy that incorporates their unique needs to prevent cervical cancer. Our focus on open calls and tailoring HPV services for screen-eligible women resonates with NCI, NIH, and US government strategic priorities.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY The essence of the Alzheimer’s disease (AD) phenotype is a decline in memory. Well before the onset of obvious dementia, there are changes in memory that patients and family members notice which diminish quality of life for those impacted. Therapeutic interventions that target the presymptomatic and early symptomatic stages of AD often choose a cognitive endpoint to demonstrate efficacy, but conventional memory assessments often fail to capture these subtle changes that occur early in the disease. One reason is that conventional memory assessments do not reflect how memory is relied upon in the everyday lives of participants. For example, it is very distressing for patients to experience memory lapses like forgetting first names, losing personal items, or having difficulty remembering the plot of a book or film. Conventional memory testing assesses recall at short periods, typically 30-minutes or less after learning the information, whereas in the everyday lives of patients, critical information must be recalled over much longer periods like hours, days, or even weeks to maintain quality of life. Several clinical studies have demonstrated that extending recall of newly learned information past the usual 30-minute or less delay period to much longer intervals dramatically increases the rate of decay, a concept known as accelerated long-term forgetting (ALF). We have shown that participants who carry a mutation for autosomal dominant Alzheimer’s disease (ADAD) but who are presymptomatic, perform similarly to non-carriers on conventional memory testing. Critically, when we tested mutation carriers on their long-term recall at 7 days, they had lost much more information than non-carriers, suggesting that extending the recall period may reveal important differences in memory consolidation that manifest well before the onset of noticeable dementia symptoms. An obvious challenge for measuring ALF in clinical populations is burden. Conventional testing would require multiple visits from study participants and require multiple interactions with study personnel, which is practically and financially unfeasible. The increasing ubiquity of smartphones provides an opportunity to assess cognition in populations at risk for AD, while allowing for longer-term follow-up without excessive burden or financial impact. In this study, we will develop novel ALF measures for smartphone-based administration and validate their effectiveness in presymptomatic autosomal dominant AD. Our development process will include user experience studies and clinical trial readiness audits, and the resulting application source code will be made freely available. The ultimate goal is to develop a highly sensitive, accessible, and clinically meaningful cognitive endpoint for use in international AD clinical trials, including adding ALF measures to the Dominantly Inherited Alzheimer Network-Trials Unit (DIAN-TU).

NIH Research Projects · FY 2025 · 2023-09

Project Summary/Abstract: Overall Component The Washington University Chronic Kidney Disease National Resource Center is focused on the significant problem of chronic kidney disease (CKD), which affects almost 15% of the US population and carries significant morbidity and mortality. Several scientific advances have the promise to accelerate CKD research, but many of these advances are not accessible to the kidney research community due to limited expertise and/or the need for expensive equipment. Thus, this NRC will address the fundamental challenge of providing better access to cutting-edge techniques in single cell omics, genetics, and metabolism to the kidney research community to facilitate advances in CKD research. Dr. Ben Humphreys is the overall program director, and this Center will consist of four Cores: an Administrative Core, two Biomedical Resource Cores, and a Resource Development Core. The Administrative Core will oversee distribution of funds across the Cores, communicate with the National O’Brien Consortium, manage the Summer Student Enrichment Program, and promote the involvement of early stage investigators and a diverse workforce. The Variant Validation Core, one of the Biomedical Resource Cores, investigates the pathogenicity of genetic variants of uncertain significance using CRISPR/Cas9-gene editing, in silico approaches, and tailored in vitro assays. The Metabolism Core, the other Biomedical Resource Core, provides consultation for users and access to a number of metabolic assays to interrogate changes in kidney metabolism relevant to CKD. These assays include Seahorse bioflux analysis, radioactive substrate oxidation assays of tissue ex vivo, untargeted metabolomics, and stable isotope tracer studies. The Metabolism Core will also work with the O’Brien Consortium to provide validated protocols for assays commonly performed (e.g. Seahorse assays on primary cells) and provide hands’ on training using these protocols. The Single Cell Omics Research Evolution (SCORE) Core is the Resource Development Core, which develops protocols and bioinformatics pipelines for cutting-edge techniques like split pool barcoding for single nuclei multi-omics and high resolution in-situ sequencing-based spatially resolved transcriptomics. All four Cores will work together and with the network of O’Brien National Resource Centers to make scientific advancements more accessible to the kidney research community with particular emphasis on junior investigators and development of a diverse biomedical workforce.

NIH Research Projects · FY 2024 · 2023-09

Project Summary Wheezing during preschool years is a common occurrence [1] causing significant healthcare burden [2] with frequent unscheduled physician visits, ED visits, hospitalizations, and treatment with inhaled and systemic corticosteroids along with other medications. Recurrent wheezing is often the antecedent to asthma, one of the most common chronic diseases of childhood. Though there is evidence of significant neurocognitive problems in children with other chronic health conditions such as oncological diseases, diabetes, heart disease, sickle cell, and traumatic brain injury [3], the impact of recurrent wheezing illnesses and associated treatment on the neurocognitive and emotional development of infants and preschool children has not been fully characterized. The ECHO cohort study has been collecting neurodevelopmental measures in children after the late preschool years; however, critical neurodevelopmental trajectories occur in early infancy [4]. Data suggest that these trajectories can be affected by pre-natal conditions, socioeconomic status, stress, disease severity, and/or medication use. A recent study, in non-asthmatic patients, showed that the use of systemic corticosteroids, a frequently administered treatment for wheezing illnesses, was associated with changes in brain structure [5]. Building on these findings, our overarching study objective is to understand the association of recurrent wheezing illnesses and its associated treatment on the neurocognitive and emotional development of infants and preschool children.

NIH Research Projects · FY 2025 · 2023-09

Project Summary/Abstract Dr. Mark Arcario is a cardiothoracic anesthesiologist whose long-term goal is to be an independent physician- scientist studying molecular mechanisms of allostery in ion channels. His research background is in computational biophysics of membrane proteins with a focus on the effects of lipids and lipophiles on membrane protein structure and function. He has a special interest in pentameric ligand-gated ion channels (pLGICs) and mechanisms of anesthetic action. Several endogenous lipids, including neurosteroids, phospholipids, and fatty acids, are strong allosteric modulators of pLGICs and it has been suggested that drugs, including anesthetics, exploit these innate regulatory mechanisms. It is not well-known how lipophiles, which bind at the periphery of the protein, alter channel function and equilibrium between functional states. Such a molecular mechanism is essential for the rational, structure-based design of new drugs targeting this family of channels. To address this knowledge gap, the candidate will use a combination of innovative molecular dynamics (MD) techniques to characterize state-dependent structural ensembles of prokaryotic pLGIC, ELIC (Erwinia ligand-gated ion channel), and the human 7 nicotinic acetylcholine receptor (nAChR). Aim 1 will determine how the conformation of the lipid-facing, fourth transmembrane (M4) helix varies with the functional state of the channel (i.e., resting, activated, desensitized), using computational electrophysiology methods. Aim 2 will characterize the binding site of docosahexaenoic acid (DHA), a polyunsaturated fatty acid, and how binding of DHA causes inhibition using multiscale simulation and free energy calculations. This work will yield novel insight into state-dependent dynamics of pLGICs as well as establish a molecular model of lipophilic allosteric modulation. This research will be conducted under Dr. Wayland Cheng, an expert in pLGIC structure and function with a focus on lipid binding and modulation, along with co-mentor, Dr. Grace Brannigan, an expert in computational biophysics with a focus on MD method development and application to membrane proteins. An advisory committee comprised of Dr. Alex Evers, an expert in anesthetic pharmacology, Dr. Baron Chanda, an expert in ion channel thermodynamics and gating, and Dr. Jerome Hénin, an expert in free energy calculations, will aid in the scientific development of the candidate. The research plan will allow the candidate to develop skills in free energy calculations and computational electrophysiology, which will prepare him for a career in computational biophysics of ion channels. In addition, involvement in career development activities during this award will prepare the candidate for independence by the end of the award. The Department of Anesthesiology at Washington University in St. Louis, a leader in academic anesthesiology, provides an excellent and supportive environment for ion channel research and development of physician-scientists.

NIH Research Projects · FY 2025 · 2023-09

Obesity in adults and children is associated with severe disease and poor quality of life, and disproportionately impacts rural communities and people with low incomes. Increased access to quality obesity care is an important strategy for reducing obesity and related comorbidities. The US Preventive Services Task Force (USPSTF) recommends primary care providers (PCPs) screen children and adults for obesity and offer or refer to comprehensive, intensive behavioral interventions (IBTs); however, these recommendations are rarely implemented. Lack of training for both PCPs and behavioral health providers (BHPs) is a major barrier to implementation of USPSTF-care. Other key barriers include workforce challenges, care fragmentation, clinician views on weight, limited clinic-community linkages, and patient-level socioeconomic barriers. Harnessing an upcoming Medicaid policy change, our team developed a scalable implementation strategy, Comprehensive Multidisciplinary Obesity Care (CMOC). We propose to train a collaborative team (i.e., PCPs, BHPs, Registered Dietitian Nutritionists, and community health workers) to implement USPSTF-care in Federally Qualified Health Centers (FQHCs). CMOC is an innovative approach that leverages policy supports, clinic-community linkages, and digital technology, and employs a comprehensive, multi-level training program addressing contributors to health disparities to improve weight-related outcomes and sustain impact. Using an effectiveness-implementation design with a cluster randomized controlled trial (RCT) phase and a one-way crossover phase, 20 FQHCs in urban and rural areas of Missouri including ~100 PCPs and ~6,000 benefit-eligible patients (i.e., Medicaid recipients with obesity) will be assigned to CMOC or an implementation as usual control (IAUC) condition. CMOC includes provider training in obesity care competencies (e.g., patient-centered communication, guideline-based care, awareness of contextual factors that contribute to health disparities), discipline-specific USPSTF-care, and learning collaboratives over 12 months, followed by a 12-month sustainability period. The one-way crossover phase implements CMOC in the control arm (following their participation in IAUC) over 12 months. Both phases include evaluations at 12 and 24 months post-training. To improve health outcomes among high-risk groups, our proposed study evaluates short- and long-term clinical and implementation outcomes of CMOC compared to IAUC using RE-AIM. Four aims will guide this work. Aim 1 compares patient-level effectiveness (i.e., patient relative weight change [1a] and the proportion of patients who achieve clinically significant weight loss [1b]). Aim 2 compares reach (i.e., patient treatment utilization). Aim 3 compares PCP referrals to IBT at 12 (adoption; 3a) and 24 months (maintenance; 3b), and short- and long-term changes in provider obesity care competencies (3c). Aim 4 compares implementation and service costs. In line with NIDDK Strategic Priorities, our project seeks to advance implementation of evidence-based care in communities, ultimately expanding access to promote a future without disability and disease for those most in need.

NIH Research Projects · FY 2025 · 2023-09

Project Summary: Cognitive deficits including disruptions in hippocampal-dependent memory are a hallmark of aging. Predictably, aging-associated cognitive decline is exacerbated by sleep disruptions commonly seen in the aging and elderly population. However, there is a significant lack of understanding about the mechanism behind the interconnected processes of sleep, aging and learning. One significant challenge to unraveling these mechanisms has been the lack of tools to study intracellular and extracellular signals in real time with high temporal resolution. This has made it difficult to observe the modulation of these signals alongside such dynamic processes as sleep, learning and aging. To address these challenges, our lab developed a fluorescence-lifetime based optical sensor, FLIM-AKAR, which when used in combination with a custom-built fluorescence lifetime photometry (FLiP) rig has allowed us to observe the activity of cAMP-dependent protein kinase A (PKA), an important plasticity signal that has been implicated in the formation and consolidation of sleep-dependent learning and has been shown to enhance learning in aging mice. Paring 24-hour FLiP recordings in hippocampal CA1 with simultaneous electroencephalography (EEG) and electromyography (EMG) measurements revealed a synchronized, transient activation of PKA that is associated with transitions from sleep to wake. Due to its short duration, this signal has never been observed before in a behaving animal. Thus, this study aims to explore its function on both cellular and behavioral levels and elucidate how those functions may change in aging mice. Using photoactivatable adenylate cyclase (biPAC) and perforated patch clamp, I will determine whether transient PKA activation is sufficient to cause an increase in intrinsic excitability (IE), a known function of PKA and a known cellular correlate of learning. Further, by using biPAC and photoactivatable protein kinase inhibitor peptide (PA-PKI) to bidirectionally manipulate this transient PKA signal, I aim to determine whether increasing the frequency of these transients can rescue hippocampal- dependent learning deficits in aging mice or disrupt intact hippocampal-dependent learning in adult mice. Ultimately, our findings will provide a more nuanced understanding of how PKA functions at physiologic timescales and in the context of aging, sleep, and learning. This study will also stand as an example of how taking advantage of new optical tools can bolster our understanding of how the dynamics of cell signaling relate to complex behaviors.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY This project will enhance our understanding of the humoral immune response to Klebsiella pneumoniae (Kp) and its polysaccharide antigens with the long-term goal of guiding and optimizing broad vaccine development. Kp infections, including pneumonia, urinary tract infection, and bacteremia, are sharply on the rise among hospitalized patients; CDC has declared infections with Kp and other carbapenem-resistant Enterobacteriales (CRE) demand a threat level of urgent. Beyond classical Kp typically seen in the US, emerging hypervirulent Kp strains, capable of causing liver abscess, bacteremia, and meningitis in healthy hosts, are spreading globally. This work builds on the PI's background in bacteriology, Gram-negative bacterial pathogenesis, and modeling of host adaptive immune responses, to investigate antibodies targeting Kp's polysaccharide capsule (K-type) and O-antigen. We have found that, while mice are able to produce antibodies targeting Kp capsule and O- antigen, capsule may directly interfere with O-antibody binding and killing of Kp. With our collaborators at Omniose, we have developed and are testing novel bioconjugate vaccines targeting the most prevalent K- and O-types. Bioconjugation is an alternative manufacturing process that uses recombinant E. coli strains to concurrently produce the capsule or O-antigen and an engineered carrier protein, and to enzymatically link the two. We have produced multiple K- and O-bioconjugates that have demonstrated promising efficacy in mice. As both O- and K- vaccines are under development, we will use our novel bioconjugate vaccines in murine protection experiments to determine the relative effectiveness of O-antigen or K-antigen bioconjugates against classical and hypervirulent Kp isolates. Further, we will challenge O-immunized mice with strains of closely related O-antigen structural subtypes that have not been included to date in vaccine formulations being developed commercially. Potential masking of O-antigen by capsule will be determined through mouse serum IgG ELISAs. Serum bactericidal assays (SBAs) and opsonophagocytic killing assays (OPKAs) will be developed and correlated with murine protection. Further, with bacterial mutants, complemented strains, and capsule inhibitors, we will determine the specificity of Kp O-antibody inhibition by capsule, utilizing multiple techniques including biolayer interferometry, immunofluorescence microscopy, and transmission immunoelectron microscopy. Finally, we will perform a first-ever longitudinal study of human patients with Kp infection, analyzing their sera for antibodies specific to Kp polysaccharides and their functional activity against the inciting Kp strain. At the conclusion of these studies, the relative efficacy of both K- and O-type bioconjugate vaccines will be determined, cross-protection among O-antigen subtypes will be resolved, correlates of protective immunity will be established, and mechanisms of O-antigen masking will be defined. Our results will illuminate human antibody responses to Kp infection and guide vaccine development to target this worrisome pathogen.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY/ABSTRACT: Role of APOE in endosomal processing of alpha- synuclein Dementia is among the most harmful and costly aspects of Lewy body disease (LBD) which is comprised of Parkinson disease (PD) and dementia with Lewy bodies (DLB) and shares some clinical features with Alzheimer’s disease. In particular, dementia and psychosis are often early and aggressive symptoms in patients with DLB. Pathologically, these illnesses share the feature of aggregation of misfolded forms of the protein alpha-synuclein (aSyn), termed Lewy bodies, which spread throughout multiple brain regions during the disease and are toxic to cells. In addition to Lewy bodies, patients with LBD often have amyloid plaques and neurofibrillary tangles which are hallmarks of Alzheimer’s disease, and patients with Alzheimer’s disease often have Lewy bodies in addition to plaques and tangles. The exact mechanism of how aSyn becomes misfolded and why cognitive decline is accelerated in DLB is unclear. Genetic studies point to a strong link between increased DLB risk and the APOE4 variant of the gene that encodes apolipoprotein E, another protein that is also central to Alzheimer’s disease risk. We reported that mice expressing the APOE4 version of the human APOE gene had accelerated aSyn aggregation and early death compared to other APOE genotypes. This finding is similar to the effects observed when human APOE genotypes are expressed in mouse models of Alzheimer’s disease. Our preliminary data indicate that astrocytes and microglia take up aSyn aggregates and process them through the endolysosomal pathway, which may serve as a compensatory mechanism to degrade harmful aSyn aggregates. We propose to examine the cell biological transit of aSyn aggregates through the endolysosomal pathway in astrocytes and microglia and determine if there are differences in this trafficking related to APOE genotype. We hypothesize that the APOE4 genotype impairs endolysosomal degradation of aSyn aggregates in both astrocytes and microglia, and that astrocyte expression of APOE4 in particular drives accelerated aSyn pathology leading to brain dysfunction and neurodegeneration. We will test whether this effect occurs mainly due to cell-autonomous changes within astrocytes or microglia themselves, including related to changes in gene expression in those cells, or whether it is mediated through secreted apolipoprotein E protein particles that are known to have effects by binding to receptors on both neurons and glia. The main goal of these experiments is to clarify how APOE genotype regulates endolysosomal processing of aSyn in glia and how this knowledge can be leveraged to develop novel treatments for DLB, Alzheimer’s disease, and other related dementias.

NIH Research Projects · FY 2024 · 2023-09

is a major and rapidly increasing public health concern: over 30 million individuals worldwide suffer from AD, which is projected to quadruple by 2050. AD has been reported to be the third leading cause of death in the US. With this impending global public health crisis, treatments that prevent onset or slow progression of AD are urgently needed but rarely available until the recent accelerated approval for aducenumab. Therefore, it is of great interest to identify subpopulations which benefit most from a medication when the overall treatment effect is minimum or not clinically meaningful. If such subpopulations can be identified, some of the treatments from the negative trials can be proven to at least help a portion of the AD population. In this proposal we will employ non-parametric interaction tree (IT)-based methods on mixed models for repeated measures (MMRM) and regression-based methods to identify such subpopulations. IT for MMRM builds on the assessment of the treatment-by-covariates interactions and can automatically seek subgroups of individuals in whom the treatment shows heterogeneous effects. We also explore a new and more attractive fusion penalty approach for final tree determination without any prior knowledge of grouping information. The regression-based methods aim to identify subpopulations who will benefit from AD treatment based on their characteristics, which is very flexible to make individualized treatment selection. Finally, we will develop and disseminate a user-friendly statistical software package that will enable researchers to implement these methods with ease. Our extensions will better capture individual heterogeneity in disease progression and facilitate evidence-based precision medicine in future AD studies and other research areas.

NIH Research Projects · FY 2026 · 2023-09

ABSTRACT Opioid use disorders (OUD) are responsible for a major health and socioeconomic crisis in the US, resulting in more than $500B burden on the economy and 75,673 deaths in the year leading up to April, 2021. More than 80% of OUD cases began with the use of prescription opioid painkillers, which remain in use due to their efficacy in treating severe pain. The current clinically-used analgesics target the mu opioid receptor (MOR), which also produces of liabilities of dependence and addiction leading to OUD, and potentially lethal respiratory depression. Poorly treated pain and diversion and misuse of prescription opioid drugs are key contributors to the worsening opioid epidemic. Development of new safe and effective analgesics with diminished addiction and abuse potential is desperately needed to reduce usage of MOR agonist-based analgesics which promote OUD. We propose to target the sodium site in the delta opioid receptor (DOR) as a novel mechanism to develop pain relievers devoid of the adverse effects associated with MOR agonist analgesics. We propose to use a bitopic ligand strategy, generating novel DOR agonists binding to both the conventional orthosteric site and the sodium site in the DOR. Emerging evidence suggests these novel bitopic DOR ligands produce analgesia but lack the seizure phentotype associated with first generation DOR agonists limited to targeting the orthosteric site of DOR. Our main goal is establish a relationship between DOR efficacy and potency at G-protein and arrestin signaling pathways with binding of the ligands within the sodium site. Our intial bitopic design, C6-quino, is validated by cryoEM structures suggesting that binding in the sodium site leads to partial agonism and a unique functional selectivity away from arrestin pathway signaling over known DOR agonists binding solely to the orthosteric site. To the best of our knowledge, probes with partial agonism at DOR are rare and their effects on DOR-mediated analgesia and other adverse effects are currently not well established. Our current lead has optimal in vitro ADME properties with favorable protein binding and metabolic stability, lacks the typical DOR mediated seizures and other CNS adverse effects while ameliorating allodynia in a neuropathic pain model. Our central hypothesis postulates that targeting the allosteric sodium site in conjunction with the orthostric site by diversification of DOR bitopics guided by cryoEM-enabled SAR approaches will lead to safer analgesics effective against chronic pain. Preliminary evidence suggests probes synthesized will also lack the typical side- effects associated with both DOR as well as clinically used MOR agonists. Using structure based design, we will further optimize our current lead for DOR subtype selectivity (1000x selective), in vitro potency (with DPPDE-like potency of G-protein activation) and in vivo DOR potency (seeking analgesia at ~5-10 mg/kg, s.c. or p.o.) to design our next generation bitopics. Attempts will also be made to enhance brain penetration and CNS activity by swapping the charged guanidino group. These goals will be accomplished by an interdisciplinary team with synergistic experience in medicinal chemistry, computational chemistry, structural biology and pharmacology.

NIH Research Projects · FY 2025 · 2023-09

Abstract. Lung transplantation is a well-established surgical intervention in advanced stages of the disease in pulmonary medicine. Despite improvements in surgical procedures and immunosuppressive therapeutic paradigms, the median survival rate continues to be 6 years. Literature precedents show that chronic lung allograft dysfunction (CLAD) is the most significant barrier to long-term survival of lung transplantation. However, prediction and early diagnosis of CLAD using current techniques continues to be problematic due to lower sensitivity (HRCT) and specificity (circulating biomarkers). Currently, the reference standard to detect acute rejection is a histopathological grading of transbronchial biopsies (TBBs), however, it is an invasive technique with several limitations (bleeding, inconsistent outcomes, inherent risks associated with repetitive procedures) including detection at advanced stages of the disease. Therefore, agents capable of offering non-invasive assessment of chronic rejection after transplantation are a highly desirable diagnostic nuclear medicine resource, yet continues to be an unmet need. To accomplish this objective, we have developed a new PET tracer (named as 68Ga-Galuminox), which offers noninvasive assessment of acute lung injury, and also demonstrates promising higher uptake in alveolar macrophages of mouse ex vivo lung transplant model of CLAD, and human lung recipients with a CLAD diagnosis compared to CLAD free subjects. Finally, preliminary 68Ga-Galuminox preclinical PET imaging in our mouse CLAD model has revealed (at 30 min post tail-vein injection) 2-fold higher retention in the left transplanted lung with early signs of CLAD when compared to the non-diseased (untransplanted) right lung. Notably, these observations are also consistent with the observed activation of macrophages and PMNs in CLAD lungs as measured by flow cytometry that identifies single cell Galuminox uptake by detecting its native fluorescence. Armed with this supporting information, aims of our preclinical translational RO1 are: Aim 1: Aim 1: Perform radiation dosimetry to determine human effective dose equivalent (HED), toxicology studies, and GMP validation runs for Galuminox to prepare for an IND filing; Aim 2: Perform first-in-human studies using 68Ga-Galuminox: evaluate dosimetry, biodistribution, safety, and imaging characteristics following a single injection at rest (n=8, 4 males; 4 females); and Aim 3: To evaluate performance of 68Ga-Galuminox and 18F-FDG for detection of CLAD pathogenesis in a mouse model of lung transplantation; Aim 3.1: To determine if 68Ga-Galuminox can detect early and late CLAD pathogenesis; Aim 3.2: To determine if 68Ga- Galuminox can be used to evaluate responses to CLAD treatment; and Aim 3.3: Biochemically characterize mechanism(s) of location of the 68Ga-Galuminox through cell accumulation, sub-cellular fractionation studies, pharmacokinetics, and LPS-induced inflammation either in presence or absence of mitoTempo (MTT) and Dexrazoxane (DEX) in rodents to assess its ability to serve as noninvasive molecular imaging agent for monitoring lung injury. Successful accomplishment of proposed aims could enable: a) deployment of a noninvasive redox-sensitive reporter probe as a diagnostic tool for stratification of patients with a risk factor for CLAD; b) potentially new interventional opportunities; c) enabling longitudinal studies of disease pathology; and d) monitoring therapeutic efficacy of drugs. Combined factors could significantly advance management of CLAD patients with potential detection of CLAD at earliest stages.

NIH Research Projects · FY 2024 · 2023-09

ABSTRACT New approaches to understanding BK channelopathies at the molecular level of single channels. Mutations in KCNMA1 BK potassium channels produce a wide range of channelopathies which include epilepsy, dyskinesis, autism, multiple congenital abnormalities, intellectual disability, developmental delay, axial hypotonia, ataxia, cerebral and cerebellar atrophy, bone thickening, tortuosity of arteries, malformation syndrome, and others. We suggest that the majority of studies that seek to understand the biophysical basis of these BK channelopathies have not been studying the predominant channels; we plan to do so. The majority of these channelopathies arise in individuals who are heterozygous for the mutant gene. Since BK channels are tetrameric, composed of four like subunits, mutant subunits could assemble with wild-type (WT) subunits to form an ensemble of channels with different stoichiometries. The vast majority of the ensemble channels (88%) would be heterotetrameric hybrid channels containing a varied number of mutant subunits (from one to three). However, these hybrid channels are typically overlooked, and the mutations found in channelopathy patients have usually been studied in the laboratory by expressing only the mutant subunit in a laboratory expression system. Assuming random assembly of subunits, the purely mutant channels would not represent more than 6% of the channels found in the cells of a heterozygous patient. Based on these incomplete studies many mutations were categorized into gain-of-function (GOF) or loss-of-function (LOF) categories due to the findings of enhanced or reduced activation of BK channels, respectively based on homotetrameric mutant channels. We suggest that the omission of studying the full palette of channel types present in these patients has led to a chaotic and inaccurate categorization of phenotypes by not recognizing that most aberrant channels in these patients may be hybrids which constitute the majority of the aberrant channels in heterozygous channelopathies. In Aim 1 using a combination of electrophysiological techniques including single channel analysis, we propose to show that a cell carrying one mutant and one WT KCNMA1 allele expresses an ensemble of BK channels dominated by hybrid channels assembled from both mutant and WT subunits. In Aim 2 we will determine the functional properties and gating mechanisms to determine how the predominant channel forms associated with BK channelopathies (as determined in Aim 1) alter channel activation. In Aim 3 we will test the hypothesis that some genetic variants of BK channels result in a truncated subunit that leads to a reduced amount of BK current in cells when heterozygous with WT subunits. Although these variant genes circulate in the population they are not reported to cause neurological disease when heterozygous. Nevertheless, these mutations need to be studied because reduced BK currents could confer a furtive genetic pre-disposition to neurological disease. These aims will then characterize the actions of BK variants at the molecular level of single channels for all expressed channel types, providing information key towards understanding the channelopathies.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT One of the underappreciated effect of obesity is the repression of gut innate immunity, as we recently showed in overweight/obese patients, using Paneth cell phenotype (as a surrogate for Paneth cell function) as a proof- of-concept. Wild type mice fed with western diet (WD) also developed Paneth cell defects. We further showed that WD consumption results in microbiota-mediated increase in deoxycholic acid (a secondary bile acid), which activates the FXR pathway in the ileum. FXR activation in the intestinal epithelium, as well as FXR- mediated type I interferon activation in myeloid cells, collectively trigger Paneth cell defects. The critical questions that need to be addressed before translating these findings to clinic include how individual dietary components that impact Paneth cell function, and if long-term WD consumption renders Paneth cell defects irreversible to dietary switch alone. Our long-term goal is to dissect the cellular and molecular mechanisms of how WD consumption affect the intestinal stem cell (ISC) and Paneth cell biology. These discoveries will facilitate design of trials for obese patients with gut innate immunity dysfunction. The objective of this proposal is to determine how WD consumption induces Paneth cell abnormality. The central hypothesis is that long-term consumption of dietary fructose result in Paneth cell defect due to diminished capacity of Paneth cell replenishment (by Paneth cells themselves and/or intestinal stem cells [ISC]). Our rationale is that identification of the mechanism(s) to restore Paneth cell function will offer new therapeutic opportunities for many patients, such as those with inflammatory bowel disease and graft versus host disease, of which Paneth cells play a critical role in pathogenesis. Our preliminary data suggest that dietary fructose consumption alone is sufficient to trigger Paneth cell defects, and that Paneth cell defects after long-term (≥12 months) WD consumption are not reversible by switching to standard diet. Our specific aims will test the following hypotheses: (Aim1) How dietary fructose directly induces Paneth cell defects; (Aim 2) Long-term WD consumption will impair the capacity of Paneth cells as well as ISCs to repair and replenish defective Paneth cells. Upon conclusion, we will understand the role for dietary fructose in modulating Paneth cell and ISC function. This contribution is significant since it will establish intestinal epithelial fructose catabolism and associated genes as therapeutic targets. The proposed research is innovative because we investigate how long-term exposure to poor diet on gut innate immunity, a heretofore-unexamined process. We also use state-of-the-art ISC culture system and scRNA-seq and scATAC-seq techniques to identify molecular and cellular targets that affect Paneth cell and ISC functions. Identifying the mechanisms of how diets regulate a key disease-relevant cellular phenotype will provide insight into other inflammatory disorders.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY/ABSTRACT My career goal is to develop an independent research program focused on DNA damage signaling and how it can be exploited for the treatment of cancer. The work that I propose here explores DNA damage signaling mechanisms in BRCA1 mutant cancers and the implications for PARP inhibitor response. Pathogenic BRCA1 mutations increase the risk of cancer development and impair the homologous recombination (HR) DNA damage response pathway. This HR defect confers vulnerability to DNA damaging agents, including PARP inhibitors. PARP inhibitor efficacy is limited by the development of resistance, often caused by the restoration of HR. In BRCA1 mutant cancers, PARP inhibitor resistance and HR activity can be promoted by expression of mutant BRCA1 proteins. Several types of truncated hypomorphic BRCA1 proteins have been detected, though the mechanism of localization to DNA damage and HR activity has not been established. Recently, we determined that wild-type BRCA1 recruitment to DNA damage can proceed through RNF168-dependent and -independent pathways involving the BRCA1 RING and BRCT domains. In preliminary data, we found that RNF168 depletion impairs tumor growth for some, but not all, BRCA1 mutant cancers. Additionally, we show that some cancer cells are entirely reliant on the RNF168 pathway for HR, whereas others rely on the reciprocal pathway. These findings are in line with transgenic mouse studies where different Brca1 mutant alleles produced contrasting phenotypes upon RNF168 knockout. Here, we explore the possibility that BRCA1 mutation-specific differences arise from the capacity to recruit various hypomorphic BRCA1 proteins to DNA damage. We will use cell line, patient- derived xenograft, and transgenic mouse model systems to address the fundamental question of how the RNF168 pathway contributes to tumor progression, DNA repair, and PARP inhibitor response in different BRCA1 mutant backgrounds. Specifically, we hypothesize that depletion of RNF168 in cancers expressing BRCA1 RING domain-containing hypomorphic proteins will impair tumor progression, eliminate HR activity, and restore sensitivity to PARP inhibitors. To test this hypothesis, we developed PARP inhibitor sensitive and resistant cell line and patient-derived xenograft models that express hypomorphic BRCA1 proteins. Additionally, we generated isogenic expression systems as well as transgenic mice with Rnf168 and various Brca1 mutations. Using these models, we will examine the effects of genetic depletion of RNF168 on tumorigenesis and growth, localization of BRCA1 hypomorphs and other DNA repair proteins, and response to PARP inhibition. These studies will provide mechanistic insight into the repair processes occurring in cancers and assess the potential efficacy of RNF168 pathway-targeted therapeutics. Moreover, funding of this proposal will help me to secure an independent investigator position and lay the foundation for future R01 applications to build a sustainable research program focused on DNA repair and cancer biology.

NIH Research Projects · FY 2024 · 2023-09

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by peripheral blood cytopenias, bone marrow dysplasia, and ineffective hematopoiesis. Approximately 50% of MDS, 60% chronic myelomonocytic leukemia (CMML), 20% of acute myeloid leukemia (AML) harbor heterozygous mutations in spliceosome factor genes such as SF3B1, U2AF1, SRSF2 and ZRSR2. Although many studies have shown that mutations in splicing factor genes lead to distinct patterns of aberrant splicing, no specific alternatively spliced isoform has been demonstrated to directly cause MDS. However, aberrations in splicing induced by splicing factor gene mutations create a vulnerability in MDS cells. Our group and others showed that cells harboring spliceosome gene mutations have increased sensitivity to pharmacological perturbation of the spliceosome by splicing modulator drugs. The sensitivity of spliceosome mutant cells to further splicing perturbations raises the possibility that they are vulnerable to accumulation of misspliced transcripts. A large portion of the misspliced RNAs caused by spliceosome mutations or splicing modulator treatment are nonsense mRNAs that harbor premature termination codons (PTCs). These nonsense mRNAs, which may cause deleterious effects if translated, are normally degraded by a RNA surveillance pathway called nonsense-mediated RNA decay (NMD). The prevalence of nonsense mRNAs in cancer cells with spliceosome mutations leads us to hypothesize that mutant cells will be more sensitive to NMD attenuation due to the role of NMD in the clearance of nonsense mRNAs that can be detrimental. Preliminary data from our group indicate that NMD disruption (using a SMG1 inhibitor [SMG1i]) preferentially kills cancer cells expressing different splicing factor gene mutations. This cell death is associated with the induction of R-loops and DNA damage. Building on preliminary studies, we propose to test the therapeutic potential of NMD inhibition in selective killing of spliceosome mutant cancer cells using in vivo models and define the underlying mechanism for the hypersensitivity of spliceosome mutant cells to NMD attenuation. In Specific Aim 1, we will test the therapeutic potential of NMD inhibition to selectively kill spliceosome mutant cancer cells using in vivo models. We will engraft primary mouse AML cells in congenic mice and test whether in vivo treatment with a highly specific inhibitor of SMG1 (SMG1i), the only known protein kinase that regulates the NMD pathway, can selectively kill cancer cells with spliceosome mutations. We will further establish the therapeutic potential of targeting NMD by combining SMG1i with ATR or PARP inhibitors, DNA damage repair proteins that are activated in splicing factor mutant cells. In Specific Aim 2, we will define the molecular mechanism for the sensitivity of spliceosome mutant cells to NMD attenuation. We will identify candidate NMD targets whose levels are modulated by SMG1i or mutant splicing factors, potentially nominating downstream functional targets that could be modulated for cancer treatment. Collectively, this project will nominate therapies to treat MDS and AML with splicing factor mutations.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Tear secretion (lacrimation) is an essential mechanism that lubricates our eyes and helps reduce eye irritants and microbial infections. Lacrimal deficiency is associated with many systemic or ocular diseases including primary Sjögren's syndrome, lupus, dry eyes, congenital alacrima, and viral infections. Previous studies have suggested that the superior salivatory nucleus (SSN) controls lacrimation. However, direct molecular and genetic evidence is lacking. We recently identified a highly restricted neuronal population in mouse SSN. Ablation of this neuronal population remarkably reduces lacrimation in mice, suggesting that this neuronal population controls lacrimation. To test this hypothesis, we will first determine the efferent targets and physiological properties of these SSN neurons (Aim 1). Results from these experiments will provide valuable information regarding the encoding and transmission of lacrimal signals. Second, we will investigate the role of these SSN neurons in controlling lacrimation by specific ablation or activation of this neuronal population (Aim 2). Results from these experiments will provide functional evidence for SSN control of lacrimation. Finally, we will test these SSN neurons receive sensory inputs from ocular surface and mediate reflex lacrimation (Aim 3). Results from these experiments will help define previously unrecognized connections between ocular sensory inputs and SSN, offering novel insights into the neural mechanism underlying the reflex lacrimation. In summary, our proposed research will help reveal the neural circuit for lacrimation and provide foundational knowledge for future studies of lacrimation deficiency associated with ocular or systemic diseases.

NIH Research Projects · FY 2025 · 2023-09

Hodgkin lymphoma (HL) accounts for ten percent of all lymphomas in the western world and remains a substantial clinical problem. Relapsed and refractory (R/R) HL presents a particularly critical unmet clinical need because approximately 25% of HL patients will be refractory to the standard of care or will relapse while receiving treatment, and the overall survival of R/R HL patients is ~50%. New targeted treatment options and improved risk assessment are needed to improve the therapeutic options for these patients. HL is defined by a rare malignant B cell (Hodgkin-Reed-Sternberg , HRS cells) that occupies a small fraction of the tumor microenvironment. Despite many advances in next generation sequencing technologies, the identification of somatic variants in cancers characterized by rare cell populations remains technically and analytically challenging. These obstacles have resulted in a limited number of studies that have used high throughput genome-wide technologies to characterize the genomic landscape of newly diagnosed HL, and far fewer have attempted to use these technologies to address the genomic landscape of R/R HL. Here, we address this shortfall by asking three questions: 1) Can ultra-deep genome-wide sequencing, paired with analysis and filtering strategies optimized for low VAF variants identify somatic variants driving treatment resistance in R/R HL? 2) Can we use somatic mutation status in a large cohort of newly diagnosed and R/R HL to identify genes that are predictive of response to therapy? 3) Can somatic mutations identified in genomic and/or cell free DNA describe clonal heterogeneity among HRS cells and can cell free DNA serve as biomarker of HL at diagnosis or relapse? This research will be broadly impactful because: 1) it will describe the genomic landscape of R/R HL and will identify biomarkers that are predictive of poor response to treatment; 2) it will begin to address the relationship between clonal architecture in HL, disease outcome, and prognosis; 3) it will further define cfDNA as a suitable target for HL disease monitoring By completing this proposal Dr. Felicia Gomez will gain advanced training in genomics, bioinformatics, lymphoma biology, and cancer biology in general. This proposal takes advantage of the cutting-edge genomics and cancer research at Washington University School of Medicine (WUSM). WUSM is an ideal location to complete the experiments and training described in this proposal. When Dr. Gomez transitions to independence she will continue to focus on cancer genomics and will broaden her research to address the interactions between somatic and germline variation and how this interaction affects the development and progression of lymphoma and other malignancies.