Ohio State University

NIH Research Projects · FY 2025 · 2022-08

SUMMARY/ABSTRACT Heart failure (HF) remains a major health problem and significant mortality in the United States. While defects in mitochondrial function are strongly implicated in the pathophysiology of HF, no mitochondrial-targeted therapy has been successful in the clinic to date, indicating that we still do not possess sufficient understanding of the mechanisms connecting mitochondrial dysfunction and HF development. Normal mitochondrial functions rely on maintaining the inner membrane potential (m). In the cardiomyocyte (CM), m perturbations not only directly affect ATP production, but also influence a variety of signaling pathways that modulate redox balance, Ca2+ homeostasis, and mitochondrial quality control. However, to what extent and how the loss of CM m alone impairs cardiac energetics, contractility, and electrical activity (i.e., hallmarks of HF) remains poorly defined. This is due, in part, to a lack of methods for effectively and selectively manipulating CM mitochondria in the in vivo setting. Existing pharmacological approaches to depolarize m lack both cell (CM versus non-CM) and organ (cardiac versus non-cardiac) specificity. To overcome this technical barrier, we developed an innovative mitochondrial-targeted luminoptogenetic (termed mLumOpto) technology and demonstrated that it can induce dynamic, selective CM m depolarization both in vitro and in vivo, with luciferase-luciferin pair-emitted intracellular bioluminescent illumination. The primary goal of this proposal is to utilize the mLumOpto technology to directly induce CM-specific m depolarization in intact hearts, and delineate the acute (i.e. <24 hours) and chronic (i.e. 2 weeks) impacts on cardiac functions. Our hypothesis is that CM-specific m depolarization alone is sufficient to induce adverse cardiac remodeling and HF development, which will be tested with the following Specific Aims. Specific Aim 1 will fully characterize mLumOpto-mediated m depolarization in isolated adult CMs (ACMs) in vitro, and establish luciferin dose dependency. How the mLumOpto-mediated m depolarization acutely and chronically impacts ACM function and health, respectively, will be dissected. Specific Aim 2 will determine the acute effect of CM-specific m depolarization in intact hearts. First, mLumOpto-mediated in vivo CM-specific m depolarization will be determined. Then, the acute effects of CM m depolarization on cardiac metabolism, pump function, and electrophysiology in intact hearts will be examined both ex vivo and in vivo. Specific Aim 3 will delineate the chronic effects of moderate CM m depolarization on cardiac remodeling (at gravimetric, histologic, biochemical, electrophysiological, and molecular levels) that lead to contractile dysfunction and HF development. The reversibility of CM m depolarization-induced HF will also be examined. Successful completion of this research will lead to not only an innovative technology for dynamic control of CM mitochondria in freely-moving animals, but also new findings that will lead us to novel avenues for the development and translation of future mitochondrial-targeted HF therapies.

NIH Research Projects · FY 2025 · 2022-08

Abstract: The loss of lung endothelial barrier function and endothelial cell (EC) death is a primary pathogenic feature of acute respiratory distress syndrome (ARDS). Although it appears that fundamental immune cell processes are at the core of ARDS-mediated vascular injury and dysfunction, the actual mechanism of endothelial cell death regulated by immune cells remains largely unknown. As a result there are currently no effective pharmacologic therapies. In order to develop more effective therapeutic strategies, it is therefore necessary to understand the precise mechanisms that link immune cell function to vascular dysfunction. This proposal focuses on a new molecular paradigm whereby immune cells (monocytes) drive endothelial cell death. Our extensive supportive data have lead us to formulate the overarching hypothesis that links lung endothelial injury during ARDS to the pyroptotic function of monocyte/macrophage derived GasderminD (GsdmD) encapsulated in circulating microparticles(MP) in association with purinergic receptor, P2X7. To determine the specifics of this novel pathway, we propose the following specific aims. In Aim1, we will delineate the role of monocyte derived GasderminD in mediating endothelial pyroptosis in the mechanism of ALI. We will use available murine model along with in vitro and ex vivo model of ARDS patients to study GsdmD mediated endothelial injury. In Aim2, we will determine the regulation of MP encapsulated GsdmD’s cytopathic function by monocytes. We will study the mechanism how phosphorylation of GasderminD regulates its cytopathic behavior and vulnerability to ubiquitin-mediated degradation. Finally, in Aim3, we will delineate the role of P2X7 in the mechanism of MP GsdmD mediated endothelial cell death. We will study the mechanisms of extracellular MP-associated GsdmD engagement to target cells mediated by the functional purinergic receptor P2X7. These studies will be the first to elucidate the injurious behavior of extracellular GsdmD in coordination with the purinergic receptor, P2X7 encapsulated in MPs, which may play a central role in lung injury. Execution of these studies will lay the foundation for a significant mechanistic advance regarding the cellular interplay between immune cells and endothelia that may provide opportunities for devising novel therapeutic targets that lessen the severity of lung vascular injury.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Tauopathies are a set of clinically-significant neurodegenerative disorders characterized by accumulation of fibrillar aggregates composed of tau protein within the brain. Alzheimer's disease is the most prevalent tauopathy as it accounts for the majority cases of dementia worldwide. With the prevalence of dementia expected to at least double in the next few decades, there is a desperate need to discover novel fundamental information about the aberrant tau aggregation at the root of these diseases. The recent advent of cryoelectron microscopy allowed for an unprecedented level of structural insight into the tauopathies, revealing extensive structural polymorphism among the filaments at the root of these tauopathies. This discovery has many vital implications. First, it directly suggests that distinct pathologic cellular conditions shape the tau aggregates in disease. Second, all distinct polymorphs discovered thus far retain at least one of the two key hexapeptide nucleation motifs previously shown to be vital for aggregation, implying some degree of commonality in the aggregation process that could be exploited therapeutically. Third, the presence of unique structures in each disease directly suggests the possibility of developing structure-specific chemical probes. My preliminary data demonstrates the power of structural biology in elucidating key insights into all three of these areas, and the project proposed in this fellowship application aims to expand on these results to deliver fundamental insights into tau aggregation, polymorphism, and ligand binding. Aim 1 will characterize the structural impact of multiple pathological cellular condition mimics on the structure of tau filaments. This structural analysis specifically aims to uncover novel information about the potential etiology at the root of tauopathies. Aim 2 will derive detailed mechanisms for how two in vitro inducers initiate and shape the aggregation process. This approach will uncover novel insight into how tau aggregation is triggered and identify potential therapeutic targets for disruption of aggregation. Aim 3 will characterize the driving forces behind ligand binding to specific sites on tau filaments. Such fundamental mechanistic information will unveil specifics for high-affinity ligand design and aid in the targeted development of disease-specific imaging agents. Overall, this proposal is innovative because it will deliver novel insight into multiple key aspects of tauopathies utilizing a structure-driven approach. This research is significant as it will specifically uncover information on the etiology and pathogenesis of aberrant tau aggregation and polymorphism, thus providing novel avenues for much-needed therapeutic intervention. Additionally, this research is significant because the mechanistic information derived herein will aid in the future development of disease-specific positron emission tomography tracers, in turn assisting with many clinical studies and in tracking of disease progression.

NIH Research Projects · FY 2025 · 2022-08

Obesity has been widely associated with increased cardiovascular disease (CVD) extending beyond common co-morbidities, such as dyslipidemia and diabetes, as evidenced by increased CVD risk in ‘metabolically healthy obese’ individuals. However, a full understanding of the mechanisms that drive this association remains an unmet need. Adipose tissue serves a broad role as an endocrine organ and has been shown to have a multitude of effects on cardiac physiology depending on metabolic state, adipose depot location (e.g visceral vs. subcutaneous), and primary cell type (e.g. brown vs. white). As such, the long-term goals of my laboratory are to increase our mechanistic understanding of adipose tissue-derived endocrine signaling to the myocardium and specifically how it is impacted by (1) brown adipose tissue (BAT)-mediated energy expenditure and (2) changes in adipose tissue homeostasis. To this end, we have recently identified the RNA binding protein HuR as a novel mediator of the adipose- cardiac signaling axis and have shown decreased HuR expression in subcutaneous WAT (scWAT) from obese mice and humans corresponds with decreased cardiac function. Specifically, we recently showed that adipocyte-specific deletion of HuR in mice (Adipo-HuR-/-) leads to a deficiency of BAT-mediated thermogenic metabolism, which is strongly associated with cardiovascular health, and is sufficient to induce cardiac hypertrophy and fibrosis. Preliminary data suggests that this cardiac pathology is driven by HuR-dependent adipose-derived extracellular vesicles (Ad-EVs). Our central hypothesis is that decreased HuR expression in adipose tissue contributes to cardiac pathology through disruption of adaptive thermogenesis and Ad-EV mediated endocrine signaling. Aim 1 will elucidate the mechanisms by which HuR mediates calcium cycling in BAT and the functional contribution of these HuR-dependent pathways to thermogenic metabolism. The working hypothesis is that HuR mediates thermogenesis in BAT through regulation of calcium (Ca2+) cycling. Aim 2 will delineate the functional role of HuR-dependent Ad-EVs on cardiac hypertrophy and identify the translational link between HuR activity and gene expression in human adipose tissue and cardiac function. The working hypothesis is that the loss of HuR expression in adipose tissue, either through genetic deletion or obesity, mediates a pro- hypertrophic endocrine signaling to the myocardium via Ad-EVs. This work is timely and innovative given our recent publications, the association of BAT activity with cardiovascular health in humans, and findings from Scherer and colleagues showing that large circulating EVs from adipocytes directly impact cardiomycotyes in vivo. Our results will increase our mechanistic understanding of (1) HuR as a mediator of thermogenesis via Ca2+ cycling, (2) adipose tissue signaling to the myocardium, and (3) the translational link between adipose tissue gene expression and cardiac function.

NIH Research Projects · FY 2026 · 2022-07

Employment rank within workplaces has long been known to be correlated with a wide range of health outcomes, including morbidity and life expectancy. Our proposed project will extend the research on workplace status as a determinant of health by examining the associations between measures of rank and health outcomes. To do this, we will use a novel database that links health insurance enrollment records and medical claims to administrative earnings data for more than 1.8 million workers and 50,000 firms in Utah. Using a broad set of firms will allow us to separate the impacts of financial resources from within-firm status measures. A challenge to separating correlations from causal effects is that employment rank may be endogenously determined by health status. To account for this potential endogeneity, we will construct instrumental variables based on network statistics from the labor market and/or shocks to firm organizational structure. We will use this instrumental variables design to estimate the causal effect of employment rank on health status. Finally, we will link workers to their adult children in the workforce to quantify the intergenerational persistence of health differences that operate through the workplace rank.

NIH Research Projects · FY 2025 · 2022-07

The Molecular Biophysics Training Program (MBTP) at The Ohio State University (OSU) was formed in 2017 as an organizing principle for training and interaction in molecular biophysics. OSU is a comprehensive public land grant university, one of the largest in the nation. OSU has a strong group of researchers and attracts a strong student pool in molecular biophysics, but graduate training and the research community were fragmented over several graduate programs, departments, and colleges. We have made great progress in building a unified molecular biophysics community through a shared mission of training the next generation of quantitative biomedical scientists. MBTP brings disparate groups together and creates an integrated training experience, in which students from three different graduate programs (Biophysics, the Ohio State Biochemistry Program, and the Chemistry Graduate Program) and many different undergraduate majors learn from each other and from a broader group of mentors. All trainees obtain core training in macromolecular and physical biochemistry, in the fundamentals of biophysics, and in the responsible conduct of research with the highest standard of rigor and reproducibility. This breadth ensures that trainees can communicate equally well about our most challenging biomedical problems and about the modern quantitative and molecular methods to address these, while being responsible citizens and researchers. A coordinated plan from a wide selection of elective courses ensures that students have enough depth to be successful in their research projects. A monthly workshop series and a yearly symposium provide cohesion to the program and incorporate unique training opportunities in career exploration, professional development, mentoring best practices, and continuous engagement with ethics training. Here we propose to deepen and expand this project, drawing in a large community of new young investigators in medicine and engineering as trainers, training a larger cohort of students, raising the rigor of quantitative skills, and expanding focus areas to include cryo-EM and biomedical engineering. At the same time, trainers will engage in evidence-based practices to elevate their mentoring skills. The resources invested in the program by NIH will be augmented by the institution, together allowing us to recruit and retain the strongest interdisciplinary students. Guiding principles of excellence, collaboration and interdisciplinarity are used to build on our original plan for the highest quality training, research and career development for students of molecular biophysics.

NIH Research Projects · FY 2025 · 2022-07

ABSTRACT There are an estimated 50 million Americans with chronic kidney disease (CKD), and the care provided to people with CKD and end stage kidney disease exceeds $114 billion annually. Despite these numbers, recent data indicates that there is a significant workforce shortage in the field of nephrology and urology and the total number of grants submitted to the NIH focused on kidney and urologic diseases and disorders has decreased over the past decade. This dichotomy creates a significant need to reverse the current trends and increase the number of trainees interested in pursuing a career in nephrology and urology that includes an NIDDK-related research focus. To begin to address these important issues, we propose to create a predoctoral student research program that combines the educational, clinical, and translational research expertise at The Ohio State University and Nationwide Children’s Hospital. The long-term goal of this program is to increase and diversify the number of physician-scientists and basic-scientists submitting grant applications to the NIDDK in the fields of pediatric nephrology and urology. To accomplish this goal, we propose the development of a new Student Urinary Tract Program in Education and Research (SUPER) Summer Training Program for graduate and medical students. This program will expand established research programs and partner with local resources to create new educational and research experiences promoting the understanding of kidney and urinary tract diseases. We strongly believe that creating this innovative and well-structured summer research program focused on nephrology and urology related topics will capture predoctoral student interest early in their career development - thereby increasing the potential workforce population and creating the next generation of NIDDK-research focused clinicians and scientists.

NIH Research Projects · FY 2025 · 2022-07

Heart failure is a major public health problem, affecting over 6 million Americans with a 5 year mortality rate over 40%. Heart failure has been “cured” many times in rodents, yet remains a leading cause of death in humans. This is in part, because many of the strategies so effective in rodent models, target molecules and processes that are essential for baseline physiology. Upon more rigorous testing in pre-clinical development, these strategies are proven unsafe and fail to progress into the clinic. This project uses an essential gene, BRD4, as a molecular flashlight to identify new targets that are specifically activated in pathologic conditions. The proposal will test a thus far unstudied nuclear protein, Sertad4, for its role in activating and sustaining pathologic gene expression programs in the cardiac fibroblast. Sertad4 is expressed in far fewer cell-types than many recently investigated targets that have generated considerable enthusiasm, including BRD4 (expressed in all cells). It is our hope that targeting proteins with more selective expression profiles will limit collateral damage of potential therapeutics, though no interventions are true silver bullets. Ultimately, the proposal will establish if in vivo inhibition of Sertad4 prevents fibroblast activation and preserves cardiac function following myocardial infarction. As an assistant professor, Dr. Stratton has assembled a supporting team of co-investigators and collaborators to help robustly test this hypothesis. Support for the hypothesis is found in substantial preliminary data showing that: 1) Sertad4 is essential for fibroblast activation (proliferation and myofibroblast differentiation) in response to TGF-β1 stimulation, 2) Sertad4 protein expression is elevated in human ischemic heart failure samples, 3) fibroblast Sertad4 expression is induced with TGF- β 1 stimulation in a BRD4 and p38 dependent manner (BRD4/p38 are also necessary for fibroblast activation), 4) Sertad4 is robustly expressed at sites of interstitial and perivascular cardiac fibrosis, and 5) targeting Sertad4 reduces SMAD2/3 protein expression and SMAD2/3 target gene expression. Innovative and cutting edge approaches are proposed to define how Sertad4 causes fibroblast activation, and determine if manipulating Sertad4 expression in vivo alters the course of pathologic remodeling following myocardial infarction. This project will rigorously test the ability to target Sertad4 to prevent cardiac fibrosis and heart failure, while also establishing fundamental knowledge regarding the molecular mechanisms of this novel target.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT Patients with inflammatory bowel disease (IBD) are four times more likely to acquire Clostridioides difficile infection (CDI), and experience higher mortality and excessive complications compared to the general population. The mechanism by which IBD confers susceptibility to CDI is unknown, making it impossible to implement preventative measures for IBD patients. The long-term goal is to investigate how interactions between the gut microbiota and host contribute to colonization resistance against enteric pathogens in complex diseases such as IBD. The overall objective of this application is to define the role of the gut microbiota and the microbially derived secondary bile acids (SBAs) in conferring susceptibility and contributing to the severity of CDI during intestinal inflammation caused by IBD. The central hypothesis is that inflammation-induced alterations in the gut microbiota lead to a decrease in SBAs resulting in loss of colonization resistance against C. difficile thus exacerbating this infection in IBD patients. Guided by preliminary data, this hypothesis will be tested by pursing two specific aims: 1. Determine if supplementation with the exogenous secondary bile acid ursodeoxycholic acid (UDCA) alters susceptibility and disease severity during CDI in IBD; and 2. Determine if modulation of intestinal microbiota derived bile acid composition alters susceptibility and disease severity during CDI in IBD. For Aim 1, IL10-/- colitis mice administered daily UDCA will be challenged with C. difficile. Inflammation-induced alterations in the gut microbiome, bile acid metagenome and metabolome, and host response during CDI will be defined. For Aim 2, precision gut microbiota modulation will be used to compare inflammation-induced (IL10-/- colitis) CDI susceptibility and severity in microbial ecosystems capable of synthesizing SBAs to ones that cannot. This approach is innovative because it utilizes intestinal inflammation as the sole initiator of gut microbiota and bile acid alterations to confer susceptibility to CDI. This contribution is significant because it will lead to novel non- antibiotic therapeutic and preventative interventions for IBD patients with CDI aimed at reducing morbidity, mortality, and health care costs for this patient demographic. Deciphering interactions between the gut microbiota, microbially derived SBAs, and the host may elucidate how intestinal inflammation confers susceptibility to CDI. Finally, this proposal will advance my training in shotgun metagenomics, integration of bioinformatics, murine IBD models, and rational manipulation of the gut microbiota and bile acid metabolome into hypothesis driven research. This will support my transition into an independent clinician scientist in translational and interdisciplinary infectious disease research. This work will be completed at the Ohio State University College of Veterinary Medicine under the guidance of my mentoring team with globally recognized expertise in the fields of metagenomics, bioinformatics, rational gut microbiota manipulation, host-microbe interactions, and IBD pathogenesis.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT Noise-induced hearing loss (NIHL) is one of the most prevalent hearing conditions that affects people of all ages. As a major risk factor, noise insult early in life accelerates auditory dysfunction and exacerbates hearing loss with age. Understanding the mechanisms of NIHL at early stages is crucial for the development of clinical interventions to prevent or ameliorate permanent damage of the auditory system. Pathophysiology of NIHL has been mostly reported in the cochlea, including detrimental changes in the sensory hair cells, the spiral ganglion neurons (SGN), and the cochlear synapses connecting the two. One significant finding was that cochlea synapses of the low spontaneous rate SGNs are vulnerable and can be preferentially damaged by noise, preceding the occurrence of permanent overt hearing loss. It remains unclear how such noise-induced peripheral changes link to structural and functional alterations in the central auditory system in contributing to compromised hearing perception. As the only target for all SGNs and the starting site of central auditory processing, the cochlear nucleus (CN) is expected to alter in morphology and physiology after noise insult in conjunction with selective SGN changes and impact the signal processing of the entire central auditory system. The long-term goal of this project is to elucidate the central mechanisms of NIHL in the CN by identifying noise-induced synaptopathy at the auditory nerve (AN) central synapses from different subtypes of SGNs, and clarifying the impact on the structure and function of the CN circuits. We hypothesize that AN synapses from low spontaneous rate SGNs are subject to more profound synaptopathy upon noise insult, which lead to more dramatic morphological and physiological changes in linked CN neurons with altered neural processing that contribute to NIHL. We further postulate that AN synapses from high spontaneous rate SGNs and linked CN neurons are unchanged during hidden hearing loss caused by moderate noise exposure, but damaged during overt hearing loss after traumatic noise exposure. Combining electrophysiology with immunohistochemistry in genetically modified mice, this project investigates the structure and function of identified AN synapses and neurons in CN circuits after moderate or traumatic noise exposure. In Aim1, we will identify noise-induced AN central synaptopathy both morphologically and physiologically at the giant endbulb of Held synapses from different subtypes of type I SGNs. In Aim 2, we will characterize noise-induced changes in cellular morphology and physiological properties of CN principal bushy neurons, and identify the altered CN output during NIHL. In Aim 3, we will elucidate the mechanisms of NIHL in CN inhibitory neural network by characterizing the noise-induced synaptopathy at AN bouton synapses onto D-stellate neurons and identifying the weakened inhibition onto CN bushy neurons. The outcome of this project will fill our knowledge gap on noise-induced AN central synaptopathy, clarify the linked changes in CN circuits, and ultimately elucidate the central mechanisms of NIHL in the CN.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Centrosome deregulation is associated with developmental disorders, such as microcephaly, ciliopathy, and cardiovascular disease. Despite their fundamental importance to human health, relatively little is known about the regulation of genes encoding core centrosome components, such as Pericentrin (Pcnt)-like protein (PLP), a conserved centrosome scaffold also required for ciliary function. Completion of this proposal will advance our understanding of the post- transcriptional regulation of plp mRNA. In humans, deregulation of the homologous PCNT gene results in congenital diseases, such as microcephalic osteodysplastic primordial dwarfism type II (MOPD II) and Trisomy 21. Similarly, in Drosophila, plp deregulation leads to diverse defects, including embryonic lethality, neuron dysfunction, and male sterility. The mechanisms underlying the pleiotropic phenotypes associated with plp loss are incompletely understood. plp is predicted to encode 12 mRNA variants, but what mechanisms give rise to these distinct RNA species and how their expression is spatiotemporally regulated are completely unknown. In this K99/R00 proposal, I will test the hypothesis that the PLP protein isoform expression, coupled with the post- transcriptional regulation of plp mRNA, modulates its diverse functions within different tissues. Three aims are proposed to test this hypothesis. In Aim 1, I will identify mechanisms of embryonic plp mRNA localization and translation. In Aim 2, I will explore the contribution of alternative promoter and 3’UTR usage for the generation of different plp RNA variants. In Aim 3, I will determine the expression profile of PLP protein isoforms and examine the biological function of PLP isoforms, including PLPPM, in Drosophila neuroblasts versus early embryos. The completion of this work will reveal the mechanisms of spatially and temporally distinct expression patterns of functional PLP protein isoforms in different Drosophila tissues and will improve our understanding of plp mRNA regulation, aspects of which may be deregulated in human diseases, such as neurodegenerative disorders and cardiovascular disease. Moreover, this award will provide technical and professional training in RNA- sequencing and bioinformatics, CRISPR genome editing, and advanced imaging approaches under the guidance of my expert mentors. I will follow a structured training program to enhance my professional abilities to establish and run my own successful independent laboratory.

NIH Research Projects · FY 2025 · 2022-07

Endometrial cancer (EC) demonstrates significant variation in outcomes across patient populations. Receipt of treatment in line with national recommendations is an important modifiable factor that influences EC outcomes. The National Comprehensive Cancer Network recommends surgical staging as the first step of guideline-concordant treatment and pathology factors captured from surgery inform adjuvant treatment recommendations. Our prior work demonstrates that receipt of guideline-concordant EC treatment improves survival. Therefore, observed differences in receipt of such treatment across populations is concerning and contributes to suboptimal disease outcomes. We lack a comprehensive understanding of the multilevel determinants that underlie variation in guideline-concordant EC treatment, preventing any meaningful progress in evidence-based intervention development. The extant literature has exclusively focused on individual-level factors as predictors of guideline-concordant EC treatment, which has not led to sustained improvement in care delivery. In addition, even within guideline-concordant paradigms, there can be wide variation in intensity level of recommended treatments, with markedly different impact on quality of life. The factors influencing intensity of treatment within guideline-concordant paradigms are poorly understood. Most important, there are no qualitative data to understand how patients and providers navigate decision-making regarding EC treatment, particularly in populations at increased risk of receiving non-guideline-concordant care. Absent these critical building blocks, we cannot move forward with evidence-based interventions to improve guideline-concordant EC treatment receipt. We propose to identify determinants of variation in guideline-concordant EC treatment by combining analysis of multilevel data from the Surveillance, Epidemiology, and End Results (SEER)-Medicare claims linked dataset (Aims 1-2) with in-depth interviews of women with EC and EC providers (Aim 3). Aim 1: Identify multilevel factors that predict differences in receipt of guideline-concordant EC treatment. Aim 2: Quantify variation and underlying predictors of guideline-concordant EC treatment intensity and examine associations between guideline-concordant EC treatment intensity and survival. Aim 3: Examine the multilevel dynamics that drive and constrain treatment decision-making using qualitative methods. By analyzing a high-quality, multilevel dataset and capturing first-hand accounts of treatment experiences and preferences, our team will uncover actionable factors that can be targeted to improve care delivery and outcomes for all women.

NIH Research Projects · FY 2024 · 2022-07

ABSTRACT The discovery of IDH1 and IDH2 mutations in AML, and the accompanying functional implications of the resulting neomorphic activity of these mutated enzymes, has resulted in FDA approved targeted therapies. Similarly, the changes brought about in methylation due to IDH mutations in the AML genome have ushered in treatment regimens combining venetoclax with hypomethylating agents. In this setting, where traditional chemotherapy regimens also are applied for treatment of primary diagnoses, the available outcomes data for all three therapeutic approaches should now provide clear metrics of predicted response, given the correct analytical methods. However, given a lack of direct comparison studies, no guidance exists as to which treatment choice will provide best response in IDH-mutated patients. Furthermore, emerging data about the importance of the biologic context on the response to different agents, including co-existing gene mutations and patient age, pose additional questions that need to be systematically addressed in order to provide patients with the best treatment. In order to address this imminent question, and provide data-driven treatment decision support for the ~20% of AML patients harboring IDH mutations, we are proposing a carefully designed, translational study: To directly compare the response of IDH-directed and non-IDH directed targeted therapy, we designed the first head-to-head comparison trial for older and unfit IDH-mutated patients. This long overdue study will provide information about treatment response, as well as first insights into the optimal sequence of treatments, with respect to co-existing molecular features. The trial will be complemented by correlative studies that aim to assess the utility of clonal outgrowth tracking and residual disease assessment for possible dynamic treatment adjustments (iDATA trial, Aim 1). Utilizing the AML patient collection from the Alliance for Clinical Trials in Oncology, as well as our newly established multicenter collaboration between six major US Cancer Centers, we have assembled the thus far largest cohort of 930 IDH-mutated adult AML patients, treated with standard cytotoxic chemotherapy, hypomethylating agents or IDH-directed or non-directed targeted inhibitors. Following our experience in genomic risk stratification models, we are equipped to identify markers predictive of treatment response based on treatment type and genomic context (Aim 2). Lastly, to better understand resistance and escape mechanisms to targeted and non-targeted therapies, we will leverage our large longitudinal specimen collections provide a comprehensive molecular characterization of the leukemic clones during different disease and treatment stages; including clonal and subclonal evolution, identification of clone-specific altered cellular pathways and epigenetic changes at each stage (Aim 3). We are confident that this comprehensive approach, executed by a skilled investigator team will shift current clinical practice paradigms towards data-driven and personalized treatment approaches.

NIH Research Projects · FY 2026 · 2022-06

SUMMARY Triple-negative breast cancers (TNBCs) are highly aggressive and standard cytotoxic chemotherapies (e.g. anthracycline-taxane) are the main treatment strategies in clinics. Our previous research and literature demonstrated that poly (ADP-Ribose) polymerase inhibitors (PARPi) as single agents or in combination with epidermal growth factor receptor inhibitor (EGFRi) induced a contextual synthetic lethality and reduced TNBC metastsis by inhibiting the repair of DNA damage. Our clinical trial showed that veliparib (PARPi)/lapatinib (EGFRi) achieved a 24% response rate in TNBC patients with wildtype BRCA1/2. Despite these achievements, TNBC cells often develop drug resistance to chemotherapies, have low patient response rate, and regrow after primary treatment. Novel strategies that can effectively treat TNBCs are urgently needed. Mitochondria are the powerhouse of cells and play a pivotal role in regulating cell functions, rendering them a promising oncological target. Destroying mitochondrial function, such as directly depolarizing the inner mitochondrial membrane (IMM) potential via synthesized heterologous genes, can bypass the repair of signaling transduction pathways, trigger a point-of-no-return cancer cell death, and subsequently prevent the development of drug resistance. We recently developed a mitochondrial luminoptogenetics (mLumiOpto) technology by synthesizing the heterologous light-gated channelrhodopsin in IMM and an engineered luciferase in cytoplasm, which induces IMM depolarization through opening mitochondrial channelrhodopsin with luciferase-luciferin emitted endogenous blue bioluminescence. Preliminary studies showed that mLumiOpto effectively depolarized mitochondria in TNBC cell lines representing multiple subtypes, resulted in persistent DNA damage, and significantly reduced tumor burden in three TNBC xenograft models. Applying our dual-targeted delivery vehicle, i.e. EGFR/CD276 monoclonal antibodies tagged exosome-associated adeno-associated virus (mAb-Exo-AAV), and cancer-specific promoter (cfos) in mLumiOpto achieved high TNBC specificity, functional expression, and minimal undesirable systemic toxicity. The objective of this project is to harness the combination of targeted mLumiOpto that is delivered with mAb-Exo-AAV and PARPi to eliminate TNBC cells in vivo. The hypothesis is that the combined mLumiOpto/PARPi integrates multiple anti-cancer mechanisms, i.e., IMM depolarization, DNA damage and inhibition of repair, and tumoral immunity. Specifically, large-scale dual-targeted mLumiOpto will be generated and characterized; treatment dosage and strategy will be optimized; and anti-cancer efficacy will be evaluated in TNBC primary xenograft model and distant metastatic model (Aim 1). Furthermore, the synergistic effects of mLumiOpto/PARPi will be assessed and the underlying mechanisms will be investigated in immunocompetent models (Aim 2). Finally, the metastasis reduction and heterogeneous TNBC treatment efficacy will be fully tested in metastatic and patient-derived xenograft (PDX) models, and toxicology will also be investigated (Aim 3). Successful completion of this project will provide a new strategy to treat TNBCs.

NIH Research Projects · FY 2026 · 2022-06

Project Summary This proposal aims to develop new synthetic methods that utilize electrical energy for C-H functionalization re- actions. Electrically driven C-H functionalization is significant since it eliminates superstoichiometric amounts of strong oxidants that often contribute to high costs and poor site selectivity. The innovation of the proposed work is the development of CuIII/CuII-Nu redox catalysts, where Nu represents various carbon and heteroatom-based nucleophiles. These CuIII-Nu complexes feature inverted ligand fields, where LUMO resides on the ligand instead of the Cu center. Therefore, electrochemically generated CuIII-Nu complexes can serve as tamed sources of Nu radical to enable hydrogen atom transfer and radical interception. Our method separates two-electron C-H func- tionalization processes into two parallel, single-electron oxidation events with fast electrode kinetics. As a result, the CuII/CuIII mediated C-H functionalization occurs at much lower potentials (by 0.5-2 V) than those of traditional electrochemical C-H functionalization methods, significantly improving energy efficiency and selectivity. Prelim- inary result shows selective functionalization of C(sp3)-H bonds with a wide range of simple nucleophiles that are otherwise incompatible with chemical oxidants typically used in conventional C-H functionalization reactions, For example, fluoride, which is known to cause decomposition of a wide range of oxidants, can be directly used as F source under our electrochemical condition. The only side products are readily manageable metal salts, significantly reducing the cost. Moreover, all CuIII-Nu intermediate can be isolated and characterized at low tem- peratures to reveal fundamental properties and reactivity, e.g., redox potentials, hydrogen atom transfer, radical capture, alkene addition, and other unproductive reactivity. This information is used to select suitable electro- chemical/chemical conditions, e.g., voltage, current, temperature, ligand, to rationally improve the reaction effi- ciency and selectivity.

NIH Research Projects · FY 2025 · 2022-06

PROJECT SUMMARY/ABSTRACT This career development award will provide didactic and experiential training and mentorship necessary for the candidate to become an independent and fully-funded health services researcher focused on the development and testing of health-oriented emergency department strategies to improve primary and secondary prevention of substance use disorders (SUD). The research plan focuses specifically on the epidemic of opioid use disorder (OUD), fueled by incident OUD which has been largely iatrogenic (i.e., prescribed opioids leading to OUD despite being used as directed for therapeutic purposes). Guidelines and legislation have targeted provider prescribing, but the role of the patients in therapeutic decision-making has not been addressed directly. There is an urgent and imperative need to understand factors influencing patients' motivation to seek and/or use opioids and engage them as stakeholders in the balance of pain and OUD risk. Emergency departments (EDs) have been a prime subject of opioid prescribing interventions, because they frequently initiate or continue therapeutic opioids and do so in circumstances with little provider-patient familiarity or follow-up care. The objective of this study is to measure and model associations between ED patient decision-making factors (knowledge, attitudes, beliefs, and perceptions) and patient motivation to use opioids for pain relief or avoid OUD risk. The central hypothesis is that ED patients' desire for therapeutic opioids and decision to use them when available results from potentially modifiable cognitive factors. We will conduct a mixed-methods study using a combination of focus groups, cross-sectional quantitative survey, and longitudinal follow-up of opioid seeking and therapeutic use after ED visit for acute pain to accomplish the following aims: 1) Establish a qualitative understanding of cognitive factors influencing patient motivation to use opioids for acute pain; 2) Develop the Decisions To use Opioids (DTO) survey instrument to quantify potential cognitive determinants of patient motivation to use opioids for acute pain; including 2a) Psychometric testing of the DTO, and 2b) Measure the contribution of cognitive factors to patient motivation to seek/use opioids. This will promote highly impactful advances in the field of iatrogenic OUD, by informing shared decision- making models and new behavioral interventions. The research plan is also highly complementary to the career development of an independent clinical researcher focused on the development of primary and secondary SUD prevention strategies in the ED setting, providing experience in mixed-methods, longitudinal prospective clinical research, survey validation, and predictive modeling, as well as rich preliminary data to support future investigation under the direction of a highly qualified and multidisciplinary team of mentors. The quintessential contribution of this research will be to engage the patient in helping to balance the risk of unrelieved pain with the risk of subsequent OUD.

NIH Research Projects · FY 2025 · 2022-06

ABSTRACT The majority of axons in the central nervous system (CNS) are wrapped with compact layers of myelin sheaths to ensure the rapid transmission of neuronal signals over long distances. As myelin thickness and sheath length have profound effects on conduction velocity, myelination is also crucial to the precise control of spatiotemporal activity patterns in the CNS. In the mammalian spinal cord, both descending motor and ascending sensory pathways travel over long distances, requiring fine control of conduction speed necessary for sensory-motor integration. Myelin disruption in the spinal cord after CNS trauma or disease, like multiple sclerosis, causes axonal conduction failure leading to severe impairment of neurological function. Accumulating data support the notion that neuronal activity positively regulates myelin development and also induces adaptive myelin plasticity in adulthood. However, the underlying mechanisms are still not fully understood, and no molecular mediators have been identified regulating this process. Alpha2delta1 (A2d1) subunits of voltage-gated Ca2+ channels (VGCCs) positively regulate the plasma membrane expression and the biophysical properties of VGCCs, including those controlling synaptic vesicle release. Our preliminary data suggest that A2d1 subunits positively regulate myelin development in the murine spinal cord. Building on our promising data, the proposed study aims at dissecting the neuronal- and glial-specific mechanisms underlying the role of A2d1 subunits in myelin development and repair. We propose a series of gain- and loss-of-function experiments to investigate how A2d1 subunits expressed in neurons and oligodendrocyte precursor cells positively regulate myelin development. Additionally, we will test whether manipulating A2d1 subunits effectively promotes remyelination and functional recovery after demyelinating injury. Overall, this study will shed light on the molecular mechanisms underlying activity-dependent myelin formation and plasticity and contribute to the design of translational research aimed at restoring myelin and neurological function after CNS trauma and disease.

NIH Research Projects · FY 2026 · 2022-05

Project Summary Fifteen states and the District of Columbia have legalized marijuana products for non-medical use, making them accessible to one-third of the US population. Despite having therapeutic effects, marijuana use leads to a wide range of adverse physical and mental effects. Based on approaches to regulate alcohol and cigarettes, imposing excise taxes on retail marijuana is among the most effective policies to mitigate related harmful public health consequences. However, the retail marijuana market is still in its infancy, and state and local authorities have knowledge gaps on how to best design excise tax structures (i.e., rates and bases) to reduce harms. Unlike cigarette and alcohol markets where standard products dominate, marijuana products come in different forms and various tetrahydrocannabinol (THC) levels, which consequently pose different levels of harm. Therefore, there are multiple factors to consider when setting tax structures for retail marijuana. Taxes could be based on weight, price, or potency. A weight basis has the advantage of raising prices equally to prevent switching to lower-priced products but may fail to reduce THC consumption by not targeting potency. Conversely, because more potent THC products are priced higher, price and potency bases may have the advantage of imposing higher taxes on products with higher THC levels but may fail to put effective price barriers on less potent, lower- priced products that would deter initiation. Further, marijuana prices decrease over time after legalization, calling for frequent increases in tax rates to sufficiently raise prices. It is unclear whether existing tax rates are set properly in response to this decreasing price trend. As such, which tax rates are applied to which bases will significantly alter prices and relative prices by product form and potency level, thereby influencing marijuana use and health outcomes. The tax structure design for retail marijuana is further challenged by the illegal market, which is often used as an argument against raising marijuana excise taxes. We lack empirical evidence indicating the extent to which excise tax structures drive consumers to the illegal market and how this may undermine the effectiveness of tax policies in reducing marijuana use and harms. Project goal: To evaluate how excise tax structures impact marijuana consumption and product choices among different marijuana forms and between legal and illegal products. This project will innovatively integrate state-of-the-art choice experiments and natural experiments to identify the impacts of tax structures on THC consumption and product choices. Aim 1: Examine the impact of excise tax rates for retail marijuana on marijuana use and product choices. Aim 2: Examine the impact of tax bases (weight, price, potency) on marijuana initiation and consumption. Aim 3: Evaluate the overall THC consumption and market share shifts from legal to illegal markets under different excise tax structures for retail marijuana using calibration. Results will have critical policy implications that inform how to set tax structure to mitigate harms for states contemplating excise taxation policies as they legalize, and states and localities with legal sales contemplating reforms to their current tax structures.

NIH Research Projects · FY 2026 · 2022-05

The burden of colorectal cancer (CRC) has been persistently unequal across racial/ethnic groups with mortality 30% higher among non-Hispanic Blacks (NHB) compared to non-Hispanic Whites (NHW). Moreover, the disparity remains after adjustment for important risk factors of CRC survival including tumor subsite, grade, stage at diagnosis, health insurance, and treatment utilization. The persistence of racial/ethnic CRC disparities despite attempts to account for variation in healthcare and prognostic indicators has increased focus on the role of residential environmental factors. The residential social (e.g., residential racial/ethnic composition, socioeconomic deprivation) and built environments (e.g., residential walkability, physical disorder) are conceptualized as main drivers of cancer disparities by race. Several limitations, however, currently prevent robust and translatable results, including: overreliance on neighborhood socioeconomic deprivation as a marker of exposure; lack of individual residential histories to estimate time-varying covariates, residential mobility patterns, and ‘health selection’ into neighborhoods; and underutilization of emerging technologies and cancer registry linkages that could lead to larger statistical power and novel translational targets. The purpose of this study is to utilize emerging methodologies – residential history calculation and virtual neighborhood auditing – to investigate longitudinal relationships between modifiable, residential built environment factors and CRC disparities by race and geography. Residential built environment exposure histories will be assessed through the combination of separately developed methodologies: residential history calculation of cancer cases within the New Jersey State Cancer Registry (NJCSR), and large-scale virtual neighborhood audits of >23,000 Google Street View (GSV) scenes across NJ. CRC case-specific built environment exposure histories will be constructed based on CRC cases’ residential histories and spatio-temporal models of built environment assessments repeated at multiple dates between 2009-2023 per a GSV location. Specific aims are: 1) to construct built environment exposure histories (2009-2023) of residential walkability and physical disorder for those diagnosed with CRC between 2014-2019 and test relationships between built environment factors and CRC prognostic factors (microsatellite instability testing, subsite, grade, stage at diagnosis); 2) to test relationships between time-varying built environment factors and CRC survival; and 3) to test whether relationships between race/ethnicity and CRC outcomes are mediated by time-varying built environment exposure factors of walkability and physical disorder. An opportunity exists to build a more complete characterization of CRC disparities by race and geography through integration of built environment exposure histories into cancer surveillance. Findings from these novel and rigorous methods will motivate additional registry linkages and more comprehensive epidemiologic studies, in turn, informing cancer surveillance systems, and public health interventions.

NIH Research Projects · FY 2025 · 2022-05

Abstract: The function of the lens requires the maintenance of its transparency and refractive properties throughout its lifespan but those mechanisms responsible are not well understood. While important cellular functions and pathways such as cell adhesion or GTPase signaling have long been hypothesized to play a role in preserving these functions the removal of genes associated with these cellular functions often severely disrupt the lens making the contribution of them challenging to interpret. We have overcome this barrier with the discovery that mice lacking the Arvcf gene develop bilateral, age- dependent cortical cataracts. Arvcf is a member of the p120-catenin subfamily of catenins that bind to a specific intracellular domain of cadherins and regulate Rho-GTPases and junctional protein dynamics and stability. We have additionally found that Arvcf is highly enriched within lens fiber cell junctional structures and is required for the recruitment of several proteins to the cadherin complex, normal lens refraction, preservation of fiber cell morphology, organization of the F-actin architecture, and the normal biomechanical properties of the lens. We propose three aims to address the central question: What molecular functions lie downstream of Arvcf to maintain lens transparency, refraction, cell morphology, and biomechanical properties? In the first aim we will determine which of these properties, precede, follow, or are simultaneous with cadherin complex and adherens junction instability in Arvcf deficient lenses. We will test the hypothesis that the initiating event is the reduction of cadherin complex proteins in fiber cell membranes through a combination of biophysical property measurements of whole lenses and fluorescent/electron microscopy of lens fiber cells. Our early investigations also found that Aquaporin 0 (AQP0) depends on Arvcf to associate with the cadherin complex and novel quantitative analysis of super-resolution microscopy images of interlocking protrusions demonstrated a significant reduction of AQP0 from the distal tips of these structures. Therefore, in the second Aim of this proposal we plan to test the hypothesis that Arvcf recruits AQP0 to the tips of interlocking protrusions to facilitate cell adhesion and water transport by determining whether AQP0 and the Arvcf/N-cadherin protein complex function together in adhesion and water transport. In the third aim we will test the hypothesis that altered GTPase signaling downstream of Arvcf contributes to lens function. The GTPase regulation domain of Arvcf and candidate GTPase regulatory proteins will be genetically altered to determine their contribution to Arvcf function, lens fiber cell biology, and lens transparency. Together, these project aims will identify functional mechanisms of a previously untested and important protein required for lens transparency and lens fiber cell function. Furthermore, they will elucidate mechanisms underlying the etiology of age-dependent cortical cataracts. Because of the association of murine Arvcf with cortical cataracts and the human ARVCF gene with genetic disorders, neurological diseases, and cancer, the advances made on this proposal will also have a broad impact.

NIH Research Projects · FY 2026 · 2022-05

Summary: Prenatal stress is pervasive and negatively impacts both the mother and the developing fetus; offspring bear an increased risk of aberrant neurodevelopment and psychiatric disorders. Indeed, there is a critical window in utero where neurodevelopment is vulnerable to stress-induced alterations in the intrauterine environment, including inflammation; how alterations in the maternal milieu are transmitted to the developing brain, contributing to behavioral alterations, requires scrutinizing. We have established a mouse model of prenatal stress, which in addition to altering maternal and offspring microbiota, induces inflammation in utero, including increases in the chemokine C-C motif chemokine ligand 2 (CCL2), resulting in reduced social behaviors and longstanding neuroinflammation. Furthermore, we have exciting supporting and preliminary data suggesting that normal neurodevelopment is disrupted by prenatal stress in a microbe- and CCL2-dependent manner. As a chemokine, CCL2 has a key role in both attracting myeloid cells and enhancing their inflammatory profile. Within the brain, the myeloid compartment includes microglia, the resident macrophages of the central nervous system; border-associated macrophages (BAMs), which are found in the perivascular space, choroid plexus, and meninges; and circulating monocytes. However, the effects of prenatal stress and CCL2 on the composition of and cytokine production by these subpopulations has yet to be elucidated, and is critical as cytokine signaling is important for homeostatic functions during neurodevelopment, including regulating neuro- and gliogenesis. Therefore, the goal of this R01 is to investigate the relationship between prenatal stress, brain macrophages and monocytes, and social behavior. Our model of prenatal stress will be used to test the highly novel, and integrative hypothesis that, during prenatal stress, there are alterations in the composition of brain macrophages and monocytes in the fetal brain, increasing cytokine production, influencing neurodevelopment. Next, we will determine whether cell-specific elimination of CCL2 production is able to prevent neuroinflammation and subsequent alterations in social behavior. Finally, in an exploratory aim, we will test whether alterations in the maternal microbiome are necessary and sufficient to induce the persistent neuroinflammation and concomitant reduction in social behavior in adult offspring. Our overarching hypothesis is that prenatal stress leads to fetal neuroinflammation that results in long term behavioral abnormalities by altering the composition of macrophages and monocytes in the developing brain and increasing their inflammatory profile, which is driven by disruption of maternal microbes and CCL2. This hypothesis will be tested by pursuing these aims: 1) Determining whether prenatal stress alters the composition of fetal brain macrophages and monocytes 2) Elucidating whether CCL2 drives inflammation in fetal macrophages and monocytes following prenatal stress 3) Determining whether prenatal stress-induced microbiome disruptions drive offspring neuroinflammation and reduced social behaviors.

NIH Research Projects · FY 2025 · 2022-05

Does Senescence Impair the Cardiovascular Benefits of Menopause Hormone Therapy? Atherosclerotic cardiovascular disease (ASCVD) causes approximately one-third of all deaths worldwide. The protection in women against ASCVD is reduced with aging and menopause. Menopausal hormone therapy (MHT) has not replicated this protection in postmenopausal women in clinical trials, highlighting the gap in our knowledge of the mechanisms of the protecting roles of estrogens in young women and impaired protection of MHT in aged women. The goal of this application is to investigate the mechanism by which MHT fails to reduce ASCVD events despite metabolic improvements and to define a therapeutic approach to reduce ASCVD risk in aged women. To recapitulate the physiology in postmenopausal women with MHT, mouse models of estradiol (E2) treatment with surgical menopause and atherosclerosis regression have been designed. Preliminary studies show that atherosclerosis burden under MHT was associated with blood inflammatory factor interferon gamma (IFNg) levels when hyperlipidemia was reduced. These results mirror the clinical observation that MHT could not improve postmenopausal ASCVD risk when the inflammation index is high. Aging and senescence-related cellular dysfunction may drive inflammation in the artery wall even when the blood lipid profile is normal. My overarching hypothesis is that inflammation resolution in atherosclerotic lesions is impaired by senescence-related incompetence of arterial repair in postmenopausal women with MHT. I propose that ASCVD risk will be reduced with MHT when lipid risks and inflammation in atherosclerotic lesions are resolved. I will explore this hypothesis with two Specific Aims: 1) Test the hypothesis that MHT improves lipid metabolism but does not resolve arterial senescence and atherosclerotic inflammation. 2) Test the hypothesis that correcting senescence and limiting inflammation in atherosclerotic lesions will restore the cardiovascular benefits of menopause E2 treatment. Studies proposed in this application will reveal critical mechanisms underlying why MHT fails to reverse atherosclerosis and lead to therapeutic approaches to reduce ASCVD risk in postmenopausal women. My career goal is to lead a research team focused on managing ASCVD risks. I have a strong background in lipid research and in the atherosclerosis field. The proposed project will afford me new expertise in 1) studying cellular senescence and immune cell functions in inflammation resolution in atherosclerosis regression, 2) translational science to reduce ASCVD risk by developing therapeutic methods to block inflammation in the artery wall. I have proposed a career development plan that integrates formal didactic training with a diverse hands-on mentorship committee to further refine my skills, competences, and leadership ability. It is anticipated that completion of the proposed project and training plan will place me in an ideal position to receive a tenure track faculty position.

NIH Research Projects · FY 2026 · 2022-04

Project Summary/Abstract Chronic low back pain is a debilitating disorder of significant socio-economic importance and a major gateway to opioid use. Therefore, minimally invasive non-addictive treatments that aim to address pain by targeting the underlying disease pathology are critical to improve human health and limit the growing opioid crisis. Intervertebral disc (IVD) degeneration is strongly associated with the pathophysiology of chronic low back pain; specifically extracellular matrix (ECM) breakdown, inflammation, and aberrant nerve/vascular ingrowth, all of which are significantly correlated with “Discogenic back pain” (DBP). Therefore, the overall objective of this proposal is to develop novel non-viral reprogramming-based therapies to convert degenerate nucleus pulposus (NP) and annulus fibrosus (AF) IVD cells associated with DBP, into a healthy pro-anabolic, anti-nerve/vascular phenotype, using engineered extracellular vesicles (eEVs). Our central hypothesis is that non-viral reprogramming will restore IVD structure/mechanical function, and limit nerve/vascular ingrowth associated with pain, by converting the patient’s own degenerate IVD cells into a pro-anabolic phenotype in situ. To date, clinicians do not have access to the necessary biological tools to treat IVD degeneration in patients with DBP. A critical barrier to the success of current biologic strategies involves significant logistical and regulatory challenges such as a lack of sustained drug delivery systems, poor long-term cell viability or viral reprogramming that permanently integrates with the host DNA. Our non-addictive strategy focuses on addressing these limitations. Through our recent R61 award and published work, we have shown that human NP cells can be reprogrammed towards a healthier phenotype using developmental transcription factors Brachyury or FOXF1, showing increased ECM accumulation and decreased catabolic/ inflammatory/ neurotrophic factors - all key features of a healthy IVD. Furthermore, in our mouse DBP in vivo model we have also demonstrated significant improvement in NP tissue hydration and dampened pain behaviors in animals treated with eEVs loaded with FOXF1 for up-to 12 weeks, highlighting the potential of our proposed strategy to restore structure/function of the IVD while reducing pain. However, critical gaps remain such as i) understanding the synergistic reprogramming potential of multiple developmental transcription factors in NP and AF cells, ii) how sex and age influence our strategy, and iii) evaluating therapeutic efficiency using more clinically relevant in vivo animal models that simulate the human condition and functionalized eEVs to deliver transcription factors to specific cell types within the IVD (for example, NP or AF cells). Our first aim investigates the synergistic effects of eEVs loaded with multiple transcription factors using in vitro and in vivo clinically relevant models of DBP, and quantifying efficacy of our strategy via assessment of IVD structure/function and pain markers. The second aim involves functionalizing the eEVs with ligands specific for NP or AF cells to selectively deliver cargo to degenerate NP or AF cells of the IVD in vitro and in vivo.

NIH Research Projects · FY 2025 · 2022-04

ABSTRACT Protein kinases orchestrate signal transduction pathways that are essential for most normal cellular functions. Importantly, dysregulation of protein kinase signaling is both a cause and consequence of several human diseases, especially cancer. Due to their druggability, more than seventy small-molecule therapeutics that block the ATP-binding site of kinases have been approved, mostly for oncological diseases. Since the ATP- binding site is relatively conserved, most targeted therapeutics can simultaneously inhibit on- as well as off- target kinases, impacting efficacy as well as toxicity. Remarkably, chemical proteomic profiling has now revealed that along with off-target kinases, protein kinase inhibitors can also bind and inhibit non-kinase proteins. Particularly, the mitochondria-localized heme biosynthesis enzyme, ferrochelatase (FECH) has emerged as a common target which can be inhibited by more than 10% of kinase inhibitors. How FECH inhibition influences drug responses, especially toxicities, nevertheless remains unknown. Given their widespread clinical use, there is a clear unmet need to understand the mechanistic basis of kinase inhibitor toxicities, especially related to inhibition of non-kinase proteins. In this regard, our recent study has provided the first evidence that off-target FECH inhibition by BRAF-kinase inhibitor vemurafenib contributes to renal tubular epithelial cell (RTEC) death in vitro and nephrotoxicity in vivo. However, there is a knowledge gap in our understanding of how kinase inhibitors are transported into normal cells, how the subsequent FECH inhibition drives mitochondrial dysfunction, and how these pathways are differentially regulated in males versus females. To address these questions, we have performed genome-wide RNAi and CRISPR based screens, which have provided two key insights: (i) we have identified putative mechanisms responsible for vemurafenib uptake, FECH inhibition, mitochondrial dysfunction, and RTEC cell death. (ii) We have uncovered a unique gender-specific difference in toxicity, wherein vemurafenib treatment induces a female specific FECH upregulation in RTECs imparting resistance to nephrotoxicity. In the current application we propose to utilize a suite of in vitro and in vivo chemical genetic and gene knockout approaches to further illuminate the regulatory mechanisms that govern toxicities associated with vemurafenib-induced FECH inhibition. Using this approach, in Aim 1 we will employ RTEC-specific conditional knockout mice, primary cells, and CRISPR-based knockout cell lines to examine the role of cubilin-dependent endocytosis in vemurafenib uptake, FECH inhibition, and mitochondrial dysfunction. In Aim 2 we will utilize conditional knockout mice and primary cells to examine the role of RUNX1 in female specific FECH upregulation and resistance to vemurafenib nephrotoxicity. These studies are expected to provide broad insights into the pharmacological action of kinase inhibitors in normal tissues including cellular uptake, non-kinase target inhibition, mitochondrial dysfunction, and transcriptional mechanisms underlying gender differences in toxicities.

NIH Research Projects · FY 2026 · 2022-04

Project Summary The combined impacts of type 2 diabetes mellitus (T2D) and food insecurity can be devastating. According to recent NHANES data, 19% of adults with T2D are food-insecure, and T2D prevalence is higher among those with food insecurity. Individuals with T2D and food insecurity have worse diabetes self-management and glycemic control compared to food-secure peers. While addressing food insecurity is important in T2D care, individuals with food insecurity may also have other non-medical, health-related social needs (e.g., transportation barriers, housing instability, job insecurity) that may decrease their ability to attain glucose targets and impact other diabetes-related health outcomes. Partnerships between healthcare systems and community-based organizations have emerged as a promising approach to improve diabetes control in foodinsecure populations. Given the design limitations of prior studies, there remains a critical need for randomized controlled trials (RCTs) to test the combined impact of diabetes education, food provision, and addressing unmet social needs on glycemic control in people with T2D experiencing food insecurity. Leveraging existing programs and relationships, we propose to test the combined effects of a food referral program, social needs screening and referral, and diabetes education intervention on hemoglobin A1c in a population of participants with food insecurity and T2D with A1c ³7.5% identified at OSUWMC. We plan to use a community-engaged research approach with community members and stakeholders informing the design and focus of the interventions. Aim 1: Using a pragmatic randomized controlled trial (pRCT), to test the effect of produce provision, diabetes education, and community referrals on A1c levels in individuals with T2D experiencing food insecurity. Aim 2: To assess the cost-effectiveness of each of the interventions that comprise the pRCT. Aim 3: To utilize a process evaluation to understand the contextual factors that impact the uptake, effectiveness, and sustainability of the interventions. Impact: Our proposed study aligns with the NIDDK’s interest in clinical studies addressing T2D treatment and complications in health disparities populations, as our project directly examines health system-based approaches to address non-medical, health-related social needs to improve health outcomes. Innovative solutions are warranted to combat the long-term sequelae of uncontrolled T2D combined with food insecurity that contribute to the two-fold higher all-cause mortality in T2D that disproportionality impacts lower socio-economic status populations experiencing health disparities. Food insecurity, unmet social needs, and diabetes education are critical issues that must be addressed to improve T2D treatment and care,. Our study will provide essential evidence that will support both scalable and sustainable partnerships to address these conditions.