Washington University

NIH Research Projects · FY 2026 · 2023-04

Project Summary/Abstract Neurofibromatosis (NF) encompasses a set of complex genetic disorders that affect almost every organ system and increase risk for the development of benign and malignant central and peripheral nervous system tumors. Of the three types of NF, Neurofibromatosis Type 1 (NF1) is the most prevalent occurring in approximately 1 in every 3,000 births without predilection for race, sex, or ethnicity. While NF1 is inherited in a fully penetrant autosomal dominant manner, there is wide inter-individual variability with respect to clinical features and their impact on patient morbidity. Clinical heterogeneity is a pervasive challenge for clinicians and families, as the management of children and adults with NF1 remains largely reactive, without reliable biomarkers or predictive models for early risk stratification and/or prognostic assessment at the time of diagnosis. Traditional approaches, which focus on identifying a single clinical or biological marker that can be measured and used to assess disease risk or trajectory in NF1, have achieved limited success and have hindered progress in the development of precision medicine for NF1-affected individuals. In response to these challenges, and with the opportunity to improve the care of individuals with NF1, we aim to verify and validate an alternative and generalizable approach for developing artificial intelligence (AI)-based clinical decision support tools for NF1 sub-phenotypes, implemented and evaluated in a comparative manner across two clinical sites. Our proposed project will first generate a multi-scale data set using a text-mining based clinical phenotyping algorithm to integrate and harmonize data from multiple sources such as clinical databases, structured electronic health records, and unstructured clinical notes. Secondly, we will develop AI-based pipelines capable of generating predictive models and tools to identify disease risk for three critical NF1 sub- phenotypes (OPGs, scoliosis, and ADHD). We will then evaluate the models for quantitative accuracy and clinical actionability at the point of care with the help of NF1 clinicians. Finally, we will validate these methods and models across multiple sites, so that we can better understand the challenges to generalizing and transporting such predictive models based across different healthcare systems, environments, and populations. We anticipate that the use of artificial intelligence techniques in order to study NF1-specific sub-phenotypes at two different sites will yield novel and potentially clinically-actionable and generalizable insights concerning the precision diagnosis and care of individuals with NF1, with broader applicability across a spectrum of similarly complex disease-states.

NIH Research Projects · FY 2026 · 2023-04

ABSTRACT: ECHO PROJECT Patients undergoing complex surgeries are most vulnerable during the immediate postoperative period; thus, handoffs from the OR (operating room) to ICU (intensive care unit) require seamless communication and coordination between surgical, anesthesia, and critical care teams. Postoperative handoffs are a threat to patient safety, causing ~35% of medical errors in the US. To mitigate these errors, the National Patient Safety Goal (2E) necessitated the “standardization” of handoff process and content, which resulted in adoption of information transfer checklists, handoff process-based protocols, or both. Although such strategies have improved handoff quality, our meta-analysis found that such improvements were temporary and had limited sustainability, due to the structured formats imposing “rigid” standardization with limited flexibility and support for interactive and personalized communication. Our central hypothesis is that a flexible standardization approach will lead to not only improvements in information sharing, but also improvements in shared understanding of patient risks, handoff interactivity, and handoff duration. Towards this end, we propose to develop the INTERACT (Intelligent interactive care continuity) handoff bundle, a flexible, standardized, EHR- integrated, and resilient sociotechnical intervention comprised of a: (1) telemedicine-augmented handoff process (i.e., the social component) supported by a (2) machine learning (ML)-augmented handoff report (i.e., the technical component). INTERACT underscores the importance of using a perioperative telemedicine suite as a safety net to support resilience to errors in OR-ICU handoff process and content. The ML-augmented handoff report supports personalized communication of core (i.e., standardized) and tailored (flexible) content based on predicted patient risks for postoperative complications. Aim 1 will focus on updating our current ML models for predicting risks associated with postoperative complications, based on state-of-the-art imputation and feature engineering techniques. We will enhance our model-agnostic explanation framework to support postoperative handoffs and decision-making, which will also be validated with a summative user evaluation study. Aim 2 will follow a user-centered design approach to iteratively develop and test the INTERACT bundle including handoff report design ideation, and usability testing, and lastly, the INTERACT bundle in-situ simulations. Aim 3 will adopt a Hybrid Type 1 trial design and the Care Transitions Framework to evaluate the effectiveness and implementation-potential of the INTERACT bundle. Our primary outcome is information sharing score (i.e., a measure of information completeness), while secondary outcomes include information inaccuracies, realized errors and adverse events, and ICU length of stay. With an integrated multidisciplinary approach to improving perioperative care transitions, the proposed INTERACT bundle will address the stated AHRQ FOA goals of “defragmenting information, improving communication, and assuring care team access to reliable and complete health information; and empowering care teams to improve health outcomes.”

NIH Research Projects · FY 2026 · 2023-04

Project Abstract Cancer cell mitochondria switch their metabolic phenotypes to meet the challenges of high-energy demand and macromolecular synthesis. Acute dependence of tumors on oncogenic tyrosine kinase signaling support their rapid proliferation, however, direct relevance of the kinase activity for mitochondria to support these high-energy processes remains obscure. Serendipitously, we uncovered that a non-receptor tyrosine kinase, ACK1, phosphorylates ATP synthase F1 α-subunit (ATP5F1a) at Tyr243 and Tyr246 (Tyr200 & 203 in mature protein, respectively) that increased ATP synthase activity specifically in the cancer cells. Mechanistically, ATP5F1a- phosphorylation not only excluded its binding to its physiological inhibitor, ATPase Inhibitory Factor 1 (IF1), but also created a supporting structure that extended from the bound non-catalytic nucleotide to the surface, reducing the flexibility and thereby increasing the stability of the enzyme. A new class of ACK1 inhibitor, (R)-9b reversed this process, inducing mitophagy and mitigating tumor growth. Consistently, a marked increase in ATP5F1a-phosphorylations was observed as normal prostate progressed to the malignant stage. Overall, these data provide the molecular evidence for cancer cell `mitochondrial addiction’ to Tyr-kinase indulgence, and reveals (R)-9b as a ‘mitocan’ that compromises the unique metabolic needs of cancer cells. Castration resistant prostate cancer (CRPC) remains an incurable malignancy with limited treatment options and is a significant cause of deaths in men worldwide (15). Limited efficacy and rapid development of resistance for Enzalutamide and Abiraterone, AR antagonist treatment have established a new paradigm-to achieve realistic remission, other cancer specific pathways, including metabolic must be compromised. This proposal is directed towards detailed characterization of mitochondrial metabolism modulatory properties of ACK1/ATP5F1a signaling and examine ability of (R)-9b to overcome Enzalutamide and abiraterone-resistant CRPCs. The specific aims are as follows: Specific Aim 1. Examine the mechanism by which ATP5F1a-phosphorylation regulates its activity in prostate cancer Specific Aim 2. Explore the role of ACK1/ATP5F1a signaling in prostate cancer models Specific Aim 3. Detail in vivo characterization of ACK1/ATP5F1a signaling in enzalutamide and abiraterone- resistance in mouse models of prostate cancer and patient derived xenografts (PDXs)

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY Effective treatment is an unmet and urgent need for patients with pancreatic ductal adenocarcinoma (PDAC). PDAC is characterized by mutations of the KRAS gene, which occurs in >95% of cases. However, targeting KRAS and its downstream signaling pathways, particularly the RAF-MEK-ERK mitogen-activated protein kinase (MAPK) pathway has been clinically unsuccessful due to rapid emergence of escape mechanism including autophagy. In this study, we made novel observation that MAPK inhibition (MAPKi) results in rapid and dramatic secretion of tumor necrosis factor alpha (TNF), which we found mediates both cell survival and death. Selective targeting of the pro-survival MAPKAPK2 (MK2) downstream f TNF signaling augments MAPKi-induced autophagy and cell death. To rigorously study these aspects, we have developed a 3-dimensional co-culture system to show that the anti-tumor effect of combined ERK and MK2 inhibitors is powerful enough to overcome the protection provided by adjacent cancer-associated fibroblasts (CAFs) and kill PDAC cells. We made observations that targeting MK2 induces favorable immunological changes that could potentiate checkpoint immunotherapy. The overarching goal of our proposal is to perform deeper and more comprehensive mechanistic studies to support development of novel therapeutic combinations that can be delivered to PDAC patients as clinical trials. To achieve this goal, we propose the following three Aims: 1. Aim 1: We will study the role of MK2 in autophagy in PDAC and CAFs using a new 3D spheroid culture system. We will determine the mechanism by which MAPKi-induces autophagy. Furthermore, we will identify new interacting partners of MK2 through a novel proteomic approach. 2. Aim 2: We will develop new genetic mouse models with conditional MK2-deletion to systematically dissect the role of MK2 in different cell types in PDAC progression and shaping the tumor microenvironment. 3. Aim 3: We will assess the combination of MK2 plus MAPKi and chemotherapy using a repertoire of thirty patient-derived xenograft models. We will perform additional studies using state-of-the art techniques and mouse models to develop novel immunotherapy regimens that will be rigorously tested.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY/ABSTRACT Chronic migraine is highly debilitating, poorly understood, and with limited treatment options. Although the activation of many pro-inflammatory immune cells has been shown to contribute to migraine pathophysiology, the involvement of CD3+ T lymphocytes, especially the immunosuppressive regulatory T (Treg) cells, in migraine chronification and reversal remains unknown. We will address the knowledge gap in this application. Some migraine patients exhibit numerical or functional impairment of Treg cell in the peripheral blood. In a mouse model of chronic migraine, repeated administration of nitroglycerin (NTG, a reliable trigger of migraine in patients) not only induced persistent behavioral sensitization, but also doubled the number of CD3+ T cells in the trigeminal ganglia (TG) without altering the number of Treg cells, again suggesting a loss of balance between immune activation and suppression. Repeated NTG also increased the number of TG neurons that can be activated by neuropeptides calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase- activating polypeptide (PACAP), indicating the sensitization of TG neurons. Low-dose interleukin-2 (ld-IL2) treatment, which preferentially expands and activates endogenous Treg cells, completely reversed chronic migraine-related behavioral sensitizations without altering basal nociceptive responses. Ld-IL2 also effectively reduced the number of CGRP- and PACAP-responsive TG neurons in NTG-treated mice to the control level. Mechanistically, we found that both peripheral transforming growth factor beta (TGF) and interleukin-10 (IL10) signaling are required for ld-IL2 to reverse chronic migraine-related behavioral and cellular sensitizations. In this application, we propose to test the hypothesis that neuro-immune interactions contribute to the development and resolution of chronic migraine, likely through regulating the sensitivity of TG neurons in the trigeminovascular pathway. First, we will selectively deplete CD4+ or CD8+ T cells to determine which T cell subset(s) contribute to the development and resolution of chronic headache-related sensitizations. We will then use flow cytometry to further investigate which T cell subtype(s) within CD4+ and CD8+ cells are altered by repeated NTG. Secondly, to further elucidate how Tregs and ld-IL2 reverses migraine chronification, we will examine whether ld-IL2 increases TGFβ1 and IL10 in Treg cells in TG and dura. Treg-selective gene knockout strategy and adoptive transfer of Treg cells will be employed to determine whether Treg cells are the main source of TGF1 and IL10 that mediate the effects of ld-IL2. Lastly, we will test whether repeated NTG increases CGRP and/or PACAP peptide expression in TG neurons. Conditional KO mice will be used to selectively eliminate CGRP or PACAP signaling in primary afferent neurons. We will ask whether headache chronification is entirely or partially mediated through CGRP and PACAP signaling in TG neurons. Collectively, results from this study will not only shed light on how neuro-immune interactions regulate migraine chronification and reversal, but also facilitate mechanism-based drug discovery.

NIH Research Projects · FY 2026 · 2023-04

Project Summary The endothelium responds to a multitude of chemical and mechanical factors in regulating vascular tone, angiogenesis, blood pressure and blood flow. The endothelial volume regulatory anion channel (VRAC) has been proposed to be mechano-sensitive, to activate in response to fluid flow/hydrostatic pressure and putatively regulate vascular reactivity and angiogenesis. We recently reported that the Leucine Rich Repeat Containing Protein 8a, LRRC8a (LRRC8A) is a required component of the heterohexameric complex that forms VRAC in human umbilical vein ECs (HUVECs). Endothelial LRRC8A regulates AKT-eNOS and mTOR signaling under basal conditions, and with stretch and shear-flow stimulation and is required for EC alignment to laminar shear flow. Endothelium-restricted LRRC8A KO (LRRC8A KO) mice have impaired endothelium-dependent vascular relaxation, develop hypertension in response to chronic angiotensin II infusion and exhibit impaired retinal blood flow with both diffuse and focal blood vessel narrowing in the setting of Type 2 diabetes (T2D). These data demonstrate that LRRC8a regulates AKT-eNOS, and mTOR signaling in endothelium and is required for maintaining vascular function. There remains a knowledge gap in (a) the molecular identity of specific LRRC8 heteromers that form VRAC in endothelium, (b) the molecular mechanisms that connect the endothelial LRRC8 complex to AKT-eNOS and mTOR signaling, (c) the therapeutic potential of small molecules targeting the LRRC8 complex needs to be evaluated, leading to a novel class of compounds to improve vascular function and hypertension in metabolic syndrome. We have biochemical, patch-clamp and imaging evidence that LRRC8 channel complexes are expressed and functional in lysosomes (Lyso-LRRC8) and have identified a critical channel pore mutation (R103E) that specifically disrupts LRRC8 channel activity. Given that lysosomes are signaling hubs that integrate nutrient sensing and AKT-mTOR signaling, we hypothesize LRRC8A/C channels co-regulate plasma membrane PI3K-AKT signaling and lysosome centered mTOR signaling in endothelium, and that small molecule LRRC8 complex modulators can restore dysfunctional endothelial LRRC8A/C in diabetes associated vascular disease and hypertension. To test the above hypotheses, we propose three specific AIMs that develop endothelial LRRC8 biology from molecular signaling mechanisms to proof of concept in vivo therapeutic: AIM#1: Delineate the mechanisms of plasma membrane versus lysosomal LRRC8 signaling to AKT- mTOR signaling in endothelium. AIM#2: Examine LRRC8 molecular contributions to EC function in vitro, ex vivo and in vivo AIM#3: Examine the therapeutic efficacy of small molecule LRRC8 modulators to improve vascular function and blood pressure in diabetes associated hypertension models

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY Recent advances in human genetics have unequivocally demonstrated mutations in microglia-specific genes, such as TREM2 R47H, to be some of the strongest risk factors for late-onset Alzheimer’s disease (AD). These breakthroughs point to microglia as a potential driver for AD pathology and thus a promising target for novel therapeutics. Remarkably, single-cell RNA sequencing studies have also uncovered a unique microglial subset, named disease-associated microglia (DAM), which are found to surround amyloid plaques in mouse models of AD. Compared to homeostatic microglia, DAM upregulate a cohort of signature genes, including AD risk genes TREM2 and APOE, which are also elevated in microglial subsets from human AD patients. Despite the apparent significance of DAM in AD, critical knowledge gaps exist regarding the cellular and molecular mechanisms that generate DAM as well as the functional contribution of DAM to different stages of AD pathology. The overall- objectives of this proposal are to determine the cellular origin, transcriptional regulation and subset-specific func- tion of DAM in AD. As microglial gene loci can be primed at the enhancers, microglial activation may lead to depositions of certain long-lasting epigenetic marks, which facilitate more rapid changes of gene expression upon a second hit later in life. Therefore, in theory, developmental activations, as we have observed for prolifer- ative region-associated microglia (PAM) in the normal developing white matter, may confer naïve homeostatic microglia such memory at the epigenetic level to allow their faster conversion to DAM in response to disease signals. Consistently, PAM and DAM share the same transcriptomic signature, which is presumably in turn reg- ulated by conserved transcription factors. In addition, analyses based on microglial depletion and global mutants of Trem2 and Apoe (partially controlling the DAM phenotype) have provided important hints for a possible neu- roprotective role of DAM by compacting amyloid plaques and limiting its spread. However, given the heteroge- neity of microglia and technical limitations (e.g. DAM resistant to drug depletion), the function of DAM during AD progression is still controversial. In this proposal, we will test the central hypothesis that conserved transcriptional and epigenetic mechanisms regulate DAM, which are ontogenically related to PAM and serve as the major neuroprotective microglial subset in AD amyloid pathology. Aim 1 will delineate the cellular origin of DAM in AD through genetic fate mapping and epigenetic profiling. Aim 2 will determine the transcriptional regulation of DAM in AD by transplantation of genetically modified primary microglial cells. Aim 3 will define the function of DAM in AD via microglia subset-specific manipulations. Upon completion of the proposed research, we expect to have elucidated the cellular origin, genetic and epigenetic mechanisms as well as in vivo function of the disease- associated microglia subset in AD. These results will provide mechanistic insights into selective subpopulations of microglia that may control AD progression, ultimately paving a new avenue for the development of precise microglia-based interventions to treat AD.

NIH Research Projects · FY 2026 · 2023-04

“Opioid overdose death (OOD) rates among Black Americans have increased unabated since 2015, outpacing national averages at a rate of two to one. Health disparities in the growth rate of OOD have been documented in at least 23 states, including Missouri, which has second highest rate of OOD among Black Americans: 44 per 100,000 (relative to 16 per 100,000 nationally). There is a critical need to identify and mitigate environmental determinants of health (EDOH) that drive increasing health disparities in OOD and opioid use disorder treatment utilization. One strategy that addresses EDOH—such as low geographic access to treatment and high neighborhood deprivation—is community-based outreach, which dispatches peers and community health workers to provide overdose education, naloxone distribution, and linkage to evidence-based treatment. Although these efforts can improve psychoeducation and access to treatment for Black Americans, existing outreach interventions are limited by a lack of data-driven targets. There is an urgent need to identify and disseminate geographic and environmental drivers of OOD among Black Americans to improve the efficacy of existing outreach interventions and in turn, develop data-driven solutions to health disparities affecting this population. The current K08 addresses this need by integrating geospatial information systems (GIS) technology and community based participatory research to create a digital tool designed to 1) identify current EDOH that underlie OOD among Black Americans and 2) provide data-driven targets to improve the efficacy of community-based outreach interventions. The project aims and career development plan will concurrently support Dr. Banks’ transition to an independent clinical investigator focused on the integration of technology and community engagement to improve substance use treatment among populations facing health disparities. Specific aims of the project are to: 1) develop and evaluate the predictive validity of a GIS-enabled index (the “Community Overdose Prevention Index”) to model risk for OOD based on EDOH identified and rated by community experts and 2) demonstrate initial acceptability and utility of the Community Overdose Prevention Index to guide outreach interventions aimed at reducing health disparities in OOD via interviews with peers and community health workers. Aims and related training opportunities facilitated by the rich intellectual environment at Washington University in St. Louis will support Dr. Banks’ training goals to gain expertise in digital/mobile health therapeutics, opioid use disorder treatment, and implementation science. Outcomes include the identification of EDOH that underlie OOD among Black Americans and a replicable implementation model other regions can use to identify policy and intervention targets to reduce health disparities in OOD among various populations. The research will provide the training and data necessary for an R01 application that tests the effectiveness of a mobile version of the Community Overdose Prevention Index to improve community-based prevention outcomes.”

NIH Research Projects · FY 2026 · 2023-04

ABSTRACT There is presently an urgent need to develop methodical approaches to evaluate the function of genomic vari- ants. Clinical genomic testing is growing rapidly, and with it a larger-than-ever number of genetic variants that cannot be defined as either disease-causing or benign. The problem affects clinicians and their patients, who struggle to understand and interpret molecular diagnostic reports, the implications of the results, and how to manage their patients in the absence of definitive information. Systems that involve single cells to generate high-content, high-resolution functional data are of paramount importance to solving the problem posed by var- iants of uncertain significance. Here, we propose to utilize a novel cell-based platform that uses machine learn- ing to determine the combination of morphological phenotypes that define pathogenicity. We will apply the technology to the comprehensive functional assessment of variants in IDS, the gene responsible for Hunter syndrome. The core hypothesis outlined in this proposal is that experimental data measuring the direct func- tional effects of variants will inform accurate disease risk prediction. In addition, we hypothesize that an in vitro, cell-based assay based on morphological features will more accurately detect disease compared to existing biochemical testing using artificial substrates. We will perform a functional assay in a variant library using the RaftSeq pipeline in the Buchser laboratory, where a cellular phenotype will be established and then tested us- ing a second set of variants combined with rescue experiments. The results will inform variant classification in IDS molecular testing and improve diagnosis of individuals including those identified by low iduronate-2-sulfa- tase activity on newborn screening.

NIH Research Projects · FY 2026 · 2023-04

Motivational impairments are a key feature of both psychotic and mood disorders. Decreases in motivation impair work and social function transdiagnostically, reduce quality of life, and increase public health demands. Current treatments are not sufficiently effective at reducing impairments in motivation, in part due to the need to better understand the mechanisms that give rise to these symptoms. The work in the prior round of funding provided strong evidence that abnormal effort-cost decision-making (ECDM; Effort valuation/Willingness to work in the RDoC Positive Valence System) may be a key contributor to motivational deficits in psychotic pathology (i.e., individuals with schizophrenia, schizoaffective disorder and bipolar disorder), but not among individuals with mood pathology (i.e., Major Depression). ECDM refers to calculations that individuals perform to estimate the amount of physical or cognitive "work" required to obtain a reward. Individuals with schizophrenia, schizoaffective, bipolar disorder are less motivated than healthy individuals to exert effort to obtain rewards on experimental tasks of ECDM, with associated deficits in dorsal-fontal parietal control systems, and these deficits are related to symptoms of amotivation and function in everyday life. Individuals with depression did not show such deficits on tasks of either physical or cognitive effort, despite showing clear evidence of anhedonia in everyday life. These data strongly support our hypotheses that the mechanisms that contribute to motivational impairments in psychosis differ from those in depression. Here we expand this work. In Aim 1 we will utilize novel behavioral and imaging paradigms derived from the affective science literature to assess social and non-social motivation (Consultant Sommerville, co-I Braver) to test the hypothesis that individuals with psychosis will show even greater impairment in effort allocation for social than monetary rewards, while individuals with depression will show equal impairments in both. In Aim 2, we will integrate state-of-the art neuroimaging methods (neuromelanin) to indirectly measure dopamine function (consultant Horga) to test the hypothesis that dopamine disruption will be more strongly related to indices of motivational impairments in depression versus psychosis. In Aim 3, we will incorporate innovative mobile technologies to longitudinally assess social and non-social motivated behavior and cognition (Consultant Gershon) in everyday life, testing the hypotheses that the magnitude of motivational impairments in psychosis will covary over time with cognitive impairments among individuals with psychosis, but not among individuals with depression.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer-related mortality, with most deaths attributable to metastasis and treatment failure. Unfortunately, our understanding of the pathways that underlie invasion and metastasis is incomplete, but some hints were uncovered by our recent single cell sequencing analyses which revealed that many HNSCC tumors contained cells that were neither fully epithelial nor fully mesenchymal, but were in a unique, hybrid epithelial-mesenchymal (hybrid-EM) state. The presence of hybrid-EM cells was more predictive of poor response than smoking history, and functional studies established hybrid-EM as a key driver of invasion and metastasis. Together, these observations establish hybrid-EM as a central pathway in HNSCC progression. However, hybrid-EM marker proteins are not druggable, so it remains challenging to therapeutically target this state. Cell states like hybrid-EM are regulated by a variety of mechanisms, but super-enhancers, in particular, have been identified as essential for maintaining cell identity. We therefore targeted super-enhancers in HNSCC using the BET inhibitor JQ1 and observed a reduction in HNSCC invasion of ~2-fold and a suppression of hybrid-EM. This result was encouraging, but effect size was modest due to the indirect and non-specific mode of action of JQ1. We hypothesized that if we could elucidate genes regulated by super-enhancers in HNSCC, we could identify molecular pathways that are more directly and specifically involved with invasion and metastasis and uncover more potent targets. Our initial experiments found that super-enhancers regulate cholesterol biosynthesis genes in hybrid-EM cells and that their knockdown (KD) robustly disrupts hybrid-EM phenotypes, with a >2000-fold and >8000-fold reduction in invasion upon KD of two mevalonate synthesis genes. Importantly, the protein products of many of the cholesterol genes we identified can be inhibited by commercially available small molecules. Indeed, we found that statins, which inhibit cholesterol biosynthesis, potently inhibit invasion (>10-fold reduction). These data provide strong support for our original hypothesis. We now propose to use CRISPRi to systematically investigate the role of cholesterol metabolism in invasion in HNSCC using sophisticated in vitro and in vivo models (Aim 1). In Aim 2, we will evaluate 9 small molecule inhibitors of cholesterol biosynthesis in cell lines, patient-derived organoids (PDOs), and in vivo pre-clinical models. Finally, in Aim 3, we will identify the full regulatory network of super-enhancers that control hybrid-EM, specifically focusing on Brd2-4 which are inhibited by JQ1. For these experiments, we will use single cell approaches to account for tumor heterogeneity and thereby uncover additional pathways beyond cholesterol biosynthesis that direct invasion and metastasis. Together, these experiments will provide critical insights into how super-enhancers regulate hybrid-EM, while exploring the possibility that by targeting the super-enhancer-regulated cholesterol biosynthesis pathway using existing FDA-approved drugs, we could circumvent a length drug development process and move quickly into human trials.

NIH Research Projects · FY 2026 · 2023-03

Chronic use of commonly used migraine therapies can lead to medication overuse headache (MOH). This is a paradoxical increase in severity of migraine-associated symptoms and headaches which are refractory to other treatments. Currently, the first-line treatment for MOH is drug cessation. However, during this abstinence period, patients continue to suffer from severe migraine, and a majority of MOH patients return to these medications within the first year. Targeted therapies specifically for MOH would result in better headache management and increased patient quality of life. One of the accomplishments of the previous funding cycle of this grant was to test δ opioid receptor (δOR) agonists in multiple headache models, including models of MOH. We found that δOR activation completely reversed cephalic allodynia induced by chronic medication treatment, revealing δOR agonists as a novel therapeutic strategy for MOH. In the previous funding cycle, we also performed a large scale unbiased peptidomic screen to identify overlapping mechanisms between chronic migraine and MOH. We identified pituitary adenylate cyclase activating polypeptide (PACAP) binding through PAC1 receptor as a potential link between these two disorders; and that the PACAPergic system may be distinctly involved in pain facilitation by chronic medication exposure. Upon further analysis we also found that there is high co-expression between δOR and PACAP or PAC1 in pain-processing regions, including in the periaqueductal grey (PAG) and trigeminal complex. The overall goal of this renewal is to build upon these exciting findings and determine if δOR agonists relieve migraine and MOH through inhibition of the PACAPergic system. In Aim 1, we will test G protein biased δOR agonists in novel translationally significant models of cephalic MOH and determine if they cause tolerance in this model. These studies will strengthen the evidence for drug development of δOR for MOH. In Aim 2 we will map the co-expression of δOR with PACAP and PAC1 and use biochemical and electrophysiological assays to investigate how δOR modulates PACAPergic signaling. Finally, in Aim 3 we will generate conditional knockouts of δOR in PACAP and PAC1 expressing cells, which will reveal if the behavioral effects of δOR agonists are regulated through PACAPergic signaling. The experiments proposed in this application are highly innovative and use a multidisciplinary approach. They will provide important insight on the effectiveness of δOR agonist as a therapeutic target for MOH and headache disorders more broadly and will determine if δOR agonists work through inhibition of the PACAPergic system. Further, the modulation of the PACAPergic system by δOR may be fundamental to other δOR behavioral effects, including emotional modulation and peripheral analgesia.

NIH Research Projects · FY 2026 · 2023-03

Alzheimer Disease (AD) is one of the major health problems in the US and worldwide; it is a neurodegenerative disorder that is characterized clinically by progressive dementia caused by pathological changes in brain tissue preceding clinical symptoms by 15-20 years. Clinically-accessible methods are critically needed to screen for early AD pathology and monitoring it over time, as well as for outcome measures in clinical drug trials. The goal of this grant application is to establish an MRI-based technique, Deep-Learning-Augmented quantitative Gradient Recalled Echo (DLA-qGRE), as a platform for quantitative clinical evaluation of brain tissue microstructural neurodegeneration at early preclinical stages of Alzheimer Disease (AD). DLA-qGRE is a combination of qGRE MRI technique and Regularization by Artifact REmoval (RARE) deep learning (DL) methodology, both developed by our team. qGRE data obtained from a well-characterized cohort of patients revealed the existence of brain regions with low R2t* values (Dark Matter), representing tissue essentially devoid of neurons. These data show that Dark Matter can be identified already in people with preclinical stages of AD (amyloid positive but without clinical symptoms) and also has a predictive power of future AD progression. While qGRE sequence can be implemented on any commercial MRI scanner, the data analysis currently requires hours of computing time, tempering clinical applications. To significantly accelerate and improve data analysis, as well as data acquisition, in this proposal we will use innovative RARE technique, a DL approach that explicitly accounts for the physical models of specific imaging systems and biophysical models of biological tissues. Preliminary data show that DL has a potential for reconstructing qGRE metrics in a matter of seconds with improved image quality and reduced noise. This opens opportunity for implementing DLA-qGRE as a widely available tool for clinical applications. Based on this approach, we plan to achieve the following Specific Aims: In Aim 1 we will develop DLA-qGRE data processing pipeline, compatible with MRI protocols of commercially available GRE sequences, for fast and reliable detection of microstructural pre-atrophic neurodegeneration. In Aim 2 we will optimize k-space sampling strategy for developing qGRE imaging protocol with increased isotropic resolution and simultaneously decreased MRI acquisition time. Reducing scan time will significantly help with patient comfort, be much less susceptible to motion, and reduce costs of the MRI exam. In Aim 3 we will demonstrate that in a clinical neuroradiology setting DLA-qGRE compatible with MRI protocols of commercially available GRE sequences (developed per Aim 1), and accelerated DLA-qGRE (developed per Aim 2), can reliably detect microstructural neurodegeneration in preclinical and early symptomatic AD. In Summary, successful completion of the aims of this proposal will open doors for using DLA-qGRE in clinical settings as novel and more sensitive and specific MRI-based diagnostic measure of the neurodegenerative aspects of early AD pathology as compared with current measurements of tissue atrophy.

NIH Research Projects · FY 2026 · 2023-03

Summary: Bunyaviruses (Order: Bunyavirales) are a growing and diverse family of animal and human pathogens with pandemic potential. With over 300 members and an expanding distribution of mosquito and tick vectors, these viruses are responsible for increasing outbreaks of human disease and present a significant threat to human health. Rift Valley Fever virus (RVFV) is one of the well-studied bunyaviruses and is designated as an NIAID Category A pathogen and included in the WHO’s Blueprint of Priority Diseases. The Coalition for Epidemic Preparedness Innovations (CEPI) included RVFV as part of their emerging infectious diseases vaccine program, further emphasizing the potential impact on the global health and economy. Oropouche virus (OROV) is found in South America and has caused more than 30 large epidemics resulting in over 500,000 human cases, making it the second most common arboviral disease in South America behind Dengue fever. However, the true case number is likely much higher due to Oropouche fever being misdiagnosed as Chikungunya or Dengue. A third member, La Crosse virus (LACV) is found primarily in North America and is the primary cause of pediatric viral encephalitis in the United States. Neither OROV nor LACV have been as well studied as RVFV, and thus a significant gap remains in our broad understanding of bunyavirus pathogenesis. Currently there are no approved therapeutic drugs for treatment of RVFV, OROV, or LACV disease, further highlighting the need for our proposed studies. To address this need, we conducted a genomic screen that defined several critical factors, including Lrp1, an LDL family member. In support we provide compelling preliminary data including in vitro validation in Lrp1 sufficient and deficient cells, transcomplementation studies, and direct interaction between RVFV glycoprotein Gn in vitro. We also show that inhibition of Lrp1 by endogenous ligands in vitro in multiple cell lines from evolutionarily distinct hosts, and in vivo data demonstrating the importance of Lrp1 for viral tropism and disease in mice. Here we will characterize the importance of Lrp1 for entry of multiple bunyaviruses, define molecular mechanisms, and validate the significance in vitro and in vivo. This work will be performed by highly productive and collaborative investigators with expertise in every aspect of the proposed studies, including biochemistry, viral pathogenesis, immunology, proteomics, structural biology, and virology. At completion, we expect to validate Lrp1 as pan-bunyavirus entry factor, filling a key gap in the field and to provide novel targets for therapeutic development.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY The HIV-1 reservoir is a stable pool of latently infected CD4+ T cells that rekindles viral replication even after decades of antiretroviral therapy (ART). ART alone is not curative, requiring life-long treatment for people living with HIV-1 (PLWH). The main strategies attempted so far to eliminate HIV-1-infected cells face multiple challenges, including the selection of escape variants, resistance to apoptosis, T cell exhaustion, downregulation of MHC-I by HIV-1, and localization of infected cells in immune sanctuaries. This research program has the long- term goal of developing new therapeutic approaches to eliminate or control the HIV reservoir, leading to a drug- free remission. We recently discovered that the inflammasome protein CARD8 senses the enzymatic activity of the HIV-1 protease. We demonstrated that non-nucleoside reverse transcriptase inhibitors (NNRTIs) such as efavirenz (EFV) promote dimerization of Gag-Pol and cause premature intracellular activation of protease. HIV- 1 protease cleaves CARD8 driving the formation of caspase-1-dependent inflammasome and pyroptosis. All clinical HIV-1 isolates can be sensed by CARD8 despite viral diversity because it recognizes essential protease functions. The overall objective of this application is to harness CARD8 inflammasome to develop a novel approach to enhance HIV-1 reservoir elimination independently of CTLs or antibodies and trigger cell killing through non-apoptotic cell death. The underlying central hypothesis is that EFV-induced activation of CARD8, in combination with CARD8-enhancing drugs, can clear cells with transcriptionally active proviruses, reduce viral reservoir expansion, and gradually remove proviruses integrated into euchromatin regions. The rationale for the project is that enhancing the negative selection forces will reshape the proviral landscape, accelerate its decay, and reduce viral reactivation. The central hypothesis will be tested by pursuing three specific aims: 1) Establish EFV-induced CARD8 activation and killing of expanding HIV reservoir cells upon stimulation with cognate antigens ex vivo; 2) Determine the impact of EFV on HIV-1 reservoirs in humanized mice; and 3) Investigate the impact of EFV on HIV-1 reservoirs in PLWH in vivo by studying banked samples from clinical trials. The research proposed in this application is innovative because, compared to the status quo, it focuses on a new mechanism independent from HIV-1 diversity and cell susceptibility to apoptosis. The proposed research is significant because it is expected to provide the groundwork for the development of a new curative strategy that is scalable, broadly applicable in different contexts of clinical research, and can be easily combined with other interventions aiming to achieve HIV-1 remission. At completion, the proposed work will inform future pre-clinical and clinical research on the development of new antiretroviral compounds that can potently eliminate HIV-1-infected cells by activating CARD8-sensing of HIV-1 protease activity.

NIH Research Projects · FY 2026 · 2023-03

Project Summary The primary goal of this R01 is to determine the computational and functional role of endogenous opioids in specific dorsal midbrain nuclei on motivated behaviors. The preponderance of mental illness in the United States results in tens of millions of dollars in healthcare costs. While many neuropsychiatric conditions can be dissociated based on the presence or absence of specific features, a common theme across mental illnesses is the dysregulation of affective or motivated behaviors. The endogenous opioid system is known to powerfully modulate affective and motivational neural circuits. Historically, dorsal midbrain nuclei (including ventrolateral periaqueductal gray nucleus and the adjacent dorsal raphe nucleus) have been shown to be important sites for opioid action. More recently, the lateral dorsal raphe nucleus subregion (LDRN) and nucleus accumbens were shown to be important sites in an opioid-mediated mesolimbic circuit of appetitive motivation. However, while downstream opioid activity in this LDRNaccumbens circuit specifically enhances appetitive motivation, convergent studies indicate that endogenous opioids may play a motivationally suppressive role within LDRN itself. For example, experiments in the 1980s demonstrated morphine microinjections into ventrolateral PAG/LDRN could suppress food intake. Correspondingly, pilot studies in our lab using a CRISPR-Cas9 mediated knockdown of opioid peptides oppositely facilitated food intake. Furthermore, local LDRN opioid activity appears to suppress both appetitive and aversive motivated behaviors (e.g., local opioid antagonism in LDRN increases defensive or escape behaviors). These broad, anti-motivational opioid effects suggest that dysregulation could affect a wide range of affective or motivated behaviors. Therefore, the goal of this R01 application is to determine how endogenous opioids regulate appetitive and aversive motivated behaviors in LDRN by multiplexing genetic, pharmacological, in vivo imaging, and optogenetic technologies. First, we will identify the anatomical characteristics of opioids within dorsal midbrain nuclei (Aim 1). In tandem we will test the functional localization of opioids by performing receptor selective pharmacological antagonism via wireless fluidic devices and CRISPR-Cas9 knockdown of opioid peptides. Next, we will use dual-color 1-photon endoscopic imaging to examine how local opioidergic and non-opioidergic neurons interact to encode appetitive and aversive behaviors (Aim 2). Follow up experiments will use simultaneous 1-photon imaging with cell-type selective optogenetic neuromodulation to augment or disrupt LDRN encoding and expression of motivated behaviors. Finally, we will multiplex 1-photon imaging, CRISPR-Cas9 knockdown, and cell-type specific optogenetic stimulation to determine how endogenous opioid peptides casually augment LDRN encoding and expression of motivated behaviors (Aim 3). Together, these studies may provide insights into how neuropsychiatric disorders are often characterized by affective and motivational dysregulation, and suggest specific neurochemical targets for therapeutic intervention.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY/ABSTRACT Awareness with paralysis (AWP), or unintentional awareness, is memory recall during neuromuscular blockade (NMB) and can cause catastrophic psychological harm. Our team recently demonstrated a prevalence of AWP of 3.7% in mechanically ventilated emergency department (ED) patients, 25 times higher than that observed in the operating room (OR). Therefore, there is significant rationale to examine AWP in the ED, where 300,000 patients are mechanically ventilated annually. Post-traumatic stress disorder (PTSD) develops in up to 70% of patients who experience AWP, and ~ 35% of all critically ill ventilated patients. However, it is unknown to what degree mechanically ventilated ED patients develop PTSD symptoms. Further, though the overall experience drives PTSD development, knowledge of patient-centered experiences that can be targeted to improve outcome is lacking. Our long-term goal is to improve patient-centered outcomes in mechanically ventilated patients along the ED-to-ICU interface with pragmatic and scalable interventions aimed at prevention. To that end, the overall objective of this proposal is to conduct a pragmatic, stepped wedge cluster randomized trial in five academic EDs. The central hypothesis is that by using nudges and defaults aimed at reducing ED rocuronium use, the proportion of patients experiencing AWP will be significantly reduced. In addition, we hypothesize that the psychological burden suffered by mechanically ventilated ED patients is high, and modifiable targets can be identified with a mixed-methods approach. The scientific literature and our preliminary data provide the rationale for conducting this study, and through completion of its aims we will prevent an important threat to patient safety, and develop interventions to be tested in future trials in effort to improve mental health outcomes in survivors going forward. The public health impact from this proposal resides in the fact that it can be readily implemented broadly to prevent thousands of cases of AWP annually. In addition, by targeting the ED and examining psychological outcomes through a patient-centered lens, our results are expected to have a positive public health impact by elucidating the principal pathways of long-term psychological sequelae of critical illness and clarifying the role of time-sensitive ED interventions in contributing to those outcomes. This will allow us to develop specific, targeted countermeasures to improve long-term outcomes for critical illness survivors and identify promising prevention strategies for ED implementation.

NIH Research Projects · FY 2025 · 2023-03

PROJECT SUMMARY/ABSTRACT Alcohol use disorder (AUD) continues to impose a tremendous burden on society and efficacious treatment options are severely lacking. Recently, epigenetic processes such as histone acetylation emerged as potential contributors to AUD. Acetylation of histones has been shown to facilitate DNA accessibility and gene expression. The dynamic and reversible nature of this process makes it a particularly promising potential therapeutic target. Novel evidence suggests that epigenetic regulation is dependent on metabolic state, implicating specific metabolic factors in neural functions that drive behavior (Li*, Egervari* et al, Nat Rev Mol Cell Biol 2018). Recently, our group has shown that neuronal histone acetylation is fueled by the metabolite acetyl-CoA that is produced from acetate by nuclear Acetyl-CoA Synthetase 2 (ACSS2; Mews et al, Nature 2017). As a major biological source of acetate is alcohol metabolism, I hypothesized that alcohol-derived acetate might have profound effects on the epigenetic landscape in the brain following binge drinking. Using heavy isotope labeling in mice, I showed that alcohol metabolism rapidly promotes histone acetylation in the brain by direct deposition of alcohol-derived acetyl groups onto histones in an ACSS2-dependent manner. I observed similar incorporation of alcohol-derived acetate into fetal brain, suggesting a potential role for ACSS2 during prenatal alcohol exposure. In adult mice, alcohol-induced histone acetylation led to increased expression of key neuronal genes linked to learning and memory. Strikingly, ACSS2 was required for ethanol-induced associative learning, which underlies craving and relapse after protracted periods of abstinence (Mews*, Egervari*# et al, Nature, 2019). These preliminary findings establish a direct and dynamic link between peripheral and central alcohol metabolism and brain histone acetylation with significant therapeutic potential. In this proposal, I will aim to (1) determine the importance of ACSS2 in voluntary alcohol intake and test whether ACSS2 inhibition decreases alcohol consumption in mice; (2) characterize the role of ACSS2 in prenatal alcohol exposure and in the development of fetal alcohol spectrum disorder; and (3) explore the potential relevance of this novel pathway in various brain regions that regulate different aspects of AUD. This study will make pioneering contributions to our understanding of alcohol’s effects on the brain with respect to epigenetic and metabolic processes, and has the potential to identify new pharmaceutical targets to ameliorate alcohol use disorder. In addition, the proposed training and research will greatly facilitate my transition to an independent tenured-track faculty position. I will learn a combination of computational, genomic and proteomic techniques and behavioral approaches that will help establish my niche and provide me with the skills necessary to work at the intersection of epigenetics, metabolism and alcohol neurobiology. With the acquisition of valuable skills that I describe in the training plan of this proposal, I will be in a unique position to reveal new insights into the role of epigenetic-metabolic regulation of brain function in the context of alcohol use.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer-related mortality, and most patients have poor outcomes, with a five-year survival rate that is less than 40%. The majority of deaths are attributable to metastasis and treatment failure, and unfortunately, our understanding of these pathways is incomplete. Consequently, there are no targeted therapies against these biologic processes. Recently, we performed a single cell analysis of HNSCC tumors that revealed a new transcriptional pathway – dubbed a hybrid epithelial/mesenchymal state (HEM) – as a critical driver of invasion and metastasis. This pathway is clearly distinct from the classical EMT pathway, as most key EMT transcription factors (TFs) were not expressed, with the exception of Snail2. The importance of this pathway to disease etiology was highlighted by the fact that the presence of the HEM signature in tumors was more predictive of treatment outcome than any other commonly used pathologic or demographic factor. The main driver of HEM appears to be the Snail2 transcription factor, but little is known about how Snail2 orchestrates HEM. We propose to determine the direct and indirect targets of Snail2 and assess their roles in promoting invasion and metastasis. This will reveal the proteins in the HEM pathway that promote tumor progression and thus represent targets for the rational design of therapeutics. Because HNSCC tumors are highly heterogeneous, we expect that bulk genomic approaches may not capture Snail2 targets that are activated or repressed in small subpopulations of tumor cells; yet such targets may still be highly relevant to the etiology of HNSCC progression. Therefore, we will also use a transposon-based technology known as “calling cards” that has single-cell resolution to make a comprehensive map of the transcriptional targets of Snail2 across distinct hybrid epithelial-mesenchymal cell states (Aim 1). Completion of this aim will identify the key functional targets of Snail2 and the knockdown, overexpression, and phenotyping assays that we propose will directly determine if the inhibition (or activation) of these targets blocks the effect of Snail2 on invasion and metastasis. To complement this approach, in Aim 2, we will investigate whether Snail2 acts cooperatively with other TFs and then learn the functional consequences of such cooperativity. This is important because while it has proven difficult to find small molecules that inhibit TF-DNA interactions, targeting cooperative interactions between TFs is emerging as a viable strategy for targeting oncogenic TFs. Furthermore, while these TFs cooperate with Snail2 at some loci, they likely activate other HEM genes independently or by interacting with one another, so identifying their targets will increase our knowledge of this pathway and expand the list of druggable HEM targets. Completion of these aims will provide a detailed map of a new pathway that plays a critical role in HNSCC metastasis and invasion and will identify opportunities for rational drug design and targeted therapeutics against HNSCC.

NIH Research Projects · FY 2026 · 2023-03

Project Summary/Abstract (30 lines max): Acute myeloid leukemia (AML) is an aggressive blood cancer that currently ranks as the deadliest form of leukemia in both adults and children. These unsatisfactory outcomes highlight the urgent need to develop more-effective therapies that either replace or improve the effectiveness existing chemotherapies. Metabolic pathways that regulate the synthesis and catabolism of the non-essential amino acid, serine have recently emerged as therapeutic vulnerabilities in several different types of human cancer. We and others recently discovered that certain aggressive sub-types of AML, such as those bearing MLL-rearrangements (MLLR) or internal tandem duplications of the FLT3 genes (FLT3ITD) heavily depend on serine to maintain cell cycling and the differentiation blockade. Specifically, we found that restriction of dietary serine significantly delays disease onset in a mouse model of MLLR-AML, while others have shown that chemical inhibition of serine synthesis impedes MLLR- or FLT3ITD-AML. This proposal aims to address 3 key unanswered questions: 1) How is serine utilized to support AML? Our preliminary data suggest that AML cells utilize serine to supply pools of purines and purine-derivatives such as S-adenosylmethionine (SAM), which are key anabolic precursors needed to drive cell proliferation and maintain gene expression programs, respectively. We will use a combination of metabolomics, transcriptomics and proteomics in genetically engineered mouse (GEM) and patient-derived xenograft (PDX) models of AML to precisely determine how serine is utilized to support AML. 2) What serine-regulatory enzymes support AML and why? Although several enzymes contribute to serine metabolism, we have identified MTHFD2 as a particularly important candidate in AML. Specifically, we have seen that MTHFD2 inhibition promotes the terminal differentiation of MLLR-AML cells and furthermore, MTHFD2 is the most frequently over-expressed enzyme in human cancer. We have now obtained mice bearing floxed alleles of murine Mthfd2, which we will use to determine the importance of Mthfd2 in AML and healthy hematopoiesis. Given that MTHFD2 catalyzes the incorporation of serine-derived carbons into newly synthesized purines and SAM, we will also examine how Mthfd2 deletion impacts purine and SAM levels as well as the downstream cellular processes supported by these metabolites, such as proliferation and gene expression. 3) Can we develop effective serine-targeting therapies in AML? To address this question, we will evaluate therapeutic strategies for limiting serine availability (e.g. simultaneously restricting dietary serine and serine synthesis) or chemically targeting MTHFD2 using liposomal nanoparticle technology in mouse models of AML. Collectively, the results of these studies will provide new insights into the role of serine metabolism and potentially establish a foundation for developing novel therapeutic strategies for the treatment of AML. Moreover, serine metabolism is often deregulated in many tumor settings and thus results from our proposed studies will likely have implications for other forms of human cancers.

NIH Research Projects · FY 2026 · 2023-03

Project Summary Retinitis pigmentosa (RP) is the most common form of retinal dystrophy and can be caused by mutations in any one of dozens of rod-enriched genes. The genetic heterogeneity of RP represents a major challenge for the development of effective therapies. For this reason, gene-independent treatments for RP have become a long- sought goal in vision research. In this proposal, we will test the hypothesis that knockout of the rod-specific transcription factor Nr2e3 prevents photoreceptor degeneration in multiple mouse disease models. In Specific Aim 1, we will characterize the neuroprotective effects of developmental Nr2e3 knockout in multiple models of photoreceptor degeneration, including a light-damage model and four mechanistically diverse models of RP (Pde6brd10/rd10, RhoP23H/+, Rho-/- and Cngb1-/-). We will use a combination of molecular, cellular, physiological, and behavioral assays to evaluate the efficacy and versatility of this therapeutic approach. In Specific Aim 2, we will evaluate the therapeutic potential of acute, adeno-associated virus (AAV)-delivered, CRISPR-Cas9-mediated Nr2e3 knockout in the same four mouse RP models used in Aim 1. For each model, we will evaluate the ability of acute Nr2e3 knockout to protect photoreceptors at multiple stages of degeneration. We will also compare the effects of acute Nr2e3 knockout to those of Nr2e3 overexpression, which has also been suggested to prevent degeneration. Together, these studies will test the effectiveness of Nr2e3-based reprogramming as a gene- independent therapy for RP. Finally, in Specific Aim 3, we will determine the effects of acute Nr2e3 knockout in wild-type mouse rods and identify neuroprotective factors downstream of Nr2e3. We will first compare the effects of acute Nr2e3 knockout to those of developmental Nr2e3 knockout. We will then perform RNA-seq on Nr2e3- knockout rods to generate a list of Nr2e3-downstream candidate effector genes. We will knockout or overexpress selected candidate genes, singly and in combination, in four mouse models of RP to identity those that confer a neuroprotective effect. If successful, these studies will establish Nr2e3 knockout as a novel gene-independent therapy for RP, paving the way for future studies in large-animal models of photoreceptor degeneration and for clinical studies in human patients.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY The overall objective of this proposal is to support the candidate’s career development and transition to that of an independent researcher. The outlined training plan will equip the applicant with the necessary skills to conduct innovative research in a rich, interdisciplinary, and collaborative environment, facilitating the investigation of new avenues of clinical research and trials. By utilizing multimodal imaging techniques and molecular and cellular proteomic approaches, the candidate will reinforce an already strong background in neuroimaging, learn and integrate new approaches to gain a greater holistic understanding of Alzheimer disease (AD) etiology and pathophysiology, and contribute to the success of potential new therapies for AD. β- amyloid (Aβ), one of the earliest biomarkers to accumulate during AD progression, is the most targeted factor for therapeutic intervention. However, other components, such as vascular and inflammatory/immune changes, also occur during AD progression, adding to the complexity of fully characterizing AD pathogenesis. The neurovascular unit (NVU) is relevant to the study of vascular, immune, and Aβ changes in AD, as it comprises neuronal-astrocyte signaling and the blood-brain barrier, which play a role in Aβ clearance. Age-related comorbidities in late-onset AD (LOAD) are challenging to distinguish from AD-related changes involved in the progression of the disease. This proposal aims to disconnect age- from disease-related changes to vascular and immune components and to understand the impact of these disease-related changes on anti-Aβ treatment outcomes. This will be accomplished by studying these factors in autosomal dominant AD (ADAD), a rare form of AD with a known genetic etiology and with early age of symptom onset. Previous studies utilizing proteomic approaches and imaging to assess NVU disruption have focused on LOAD populations. The goal of Aim 1 of this proposal is to assess changes in the NVU in known carriers of ADAD-related mutations and with markers of vascular changes and generate a proteomic profile of NVU changes. The goal of Aim 2 is to define the temporality and association of NVU disruption relative to other established markers of disease progression. The goal of Aim 3 is to define the influence of NVU disruption on outcomes of Aβ clearance therapies, as well as on the incidence of treatment side effects and the association with primary clinical and cognitive outcomes. Successful completion of this proposed research project will improve our understanding of the vascular- and immune-related processes involved in AD and their relationship with Aβ, the pathophysiology of AD, and their influence on treatment-related Aβ changes.

NIH Research Projects · FY 2026 · 2023-02

ABSTRACT In order to survive, grow and metastasize, tumors need the support of a favorable host environment. Local changes in normal cells in close proximity to a developing tumor can create a favorable tumor micro-environment (TME) that is necessary for tumor growth. Mesenchymal populations, including cancer-associated fibroblasts (CAFs), secrete pro- tumorigenic and immune suppressive factors, and produce extracellular matrix to create a stiffer, tumor-conducive soil. Several CAF subsets with specific functions have been identified, however, whether distinct CAF subsets derive from the same progenitor that acquire functional specificity during tumor progression or from different progenitors committed to give rise to a specific CAF population, is under investigation. We recently made the unexpected observation that cells expressing bone osteolineage marker Osterix (Osx) are present in the TME of tumors outside the bone and support tumor growth. In adult animals, Osx is expressed by committed osteoblasts (OB) and drives their differentiation. Surprisingly, we detected Osx in stromal cells, isolated from various breast cancer tumors, expressing CAF and OB makers, and in the tumor-associated stroma of patients with breast carcinomas. Co-injection of Osx+ cells with tumor cells enhance tumor growth. Importantly, Osx expression correlates with poor prognosis in breast cancer. Based on these rather unexpected findings, we hypothesize that bone-derived osteolineage Osx+ cells represent a novel subset of tumor infiltrating stromal populations contributing to tumor progression and CAF diversity. Aim 1 will determine the functional relevance of Osx+ cells in the TME and pre-metastatic sites; Aim 2 will determine the origin of Osx+ cells in the TME and pre-metastatic sites. This information will broaden the concept of cellular heterogeneity within the TME and offer a new platform for targeting the stroma with the purpose of inhibiting tumor growth.

NIH Research Projects · FY 2026 · 2023-02

Single cells infected by influenza can produce hundreds to thousands of infectious new virions. These virions spread non-uniformly, producing wide variations in the viral load per cell that are concentrated around the initial site of infection. Differences in the amount of virus that infects a particular cell can influence whether or not that cell produces new virions of its own, or if it mounts an anti-viral response. Understanding how influenza virions spread is therefore critical to understanding how infection progresses and how the host responds. The central goal of this project is to understand how genetic and biophysical features of both virus and host contribute to the spatial structure of influenza virus cellular spread, and how differences in cellular spread shape the progression of infection and the resulting cellular responses. Our prior data demonstrate that genetic and biophysical features of influenza control the way that the virus spreads at the cellular level. These features are strongly linked to three viral proteins in particular: HA, NA, and M1. The receptor-binding protein HA mediates virus attachment to naïve cells, while the receptor-destroying protein NA facilitates virus release and dissemination. The matrix protein M1 controls the shape of the virus particle and the distribution of HA and NA on the virion surface. Collectively, these proteins control the biophysical characteristics of virus particles and shape the way that virions spread throughout the host. We hypothesize that genetic mechanisms acting through these proteins, together with host factors involved in mucociliary clearance, determine the spatial pattern of viral spread and the frequency of cellular co-infection, thereby shaping the progression of disease. We will test this hypothesis through two specific aims. In Aim 1, we will use high- resolution imaging to track the spread of virions and viral infection, and we will determine how this depends on natural variations in HA, NA, and M1. Through these experiments, we will identify how these proteins collectively influence the degree of cellular co-infection that occurs during multi-cycle virus replication. In Aim 2, we will investigate how host factors involved in mucociliary clearance contribute to cellular spread of IAV, and we will determine the collective impact of viral and host factors that alter the frequency of co-infection on key infection outcomes in differentiated human airway cells. The expected outcome of this project is an improved understanding of how influenza virus surface and structural proteins contribute to intracellular aspects of viral replication by tuning the degree of co-infection that occurs during multi-cycle growth. Insights from this work will inform basic understanding of how influenza viruses navigate the host environment and will identify host and viral factors that contribute to the disparate outcomes of infection that are sometimes observed. This proposal will also introduce new tools and methodologies for investigating the spatial organization and dynamics of influenza virus infection.

NIH Research Projects · FY 2026 · 2023-02

ABSTRACT: Complex mucosal networks impact the outcome of a urinary tract infection (UTI). UTIs caused mostly by uropathogenic E. coli (UPEC) are common, highly recurrent, and a leading cause of antibiotic therapy for otherwise healthy adult women. Thus, with dire predictions of antibiotic resistance reaching a tipping point, it is imperative to better understand the mechanisms of recurrent UTIs (rUTIs) to avoid a future where ordinarily treatable infections become unmanageable. 20-30% of women have a recurrence within 6 months of their initial infection. In fact, history of UTI is an independent risk factor for subsequent UTI. Mouse models have shown that upon UPEC infection of the bladder, a long-term remodeling of the bladder mucosa occurs, the nature of which depends upon the inflammatory and infection history, which alters susceptibility to subsequent infection. This remodeling, or “memory” of a prior infection, can include i) “trained immunity” of the bladder epithelium through epigenetic reprogramming; ii) an adaptive immune response, which is sometimes protective; and iii) disruptions of the gut microbiota due to oral antibiotic therapy leading to dysbiosis. Primary epithelial stem cells cultured from bladders of mice with a history of infection recapitulate many of the reprogrammed morphologic and gene expression features present in the convalescent mouse bladder. In addition, depletion of CD4+ and CD8+ T- cells alters susceptibility to same-strain recurrence. UPEC also interact with the gastrointestinal tract (GIT) microbiota, which is directly influenced by immune functions in the GIT, such as production of the cytokine interleukin 22 (IL-22), which regulates mucin production and induces the expression of antimicrobial factors that prevent invasive colonization. The GIT microbiota in turn shapes the composition of mucus. Understanding how the GIT microbiota and mucosa work in concert to restrict UPEC colonization is therefore key to understanding UPEC's relationship with the host. This proposal seeks to investigate how a prior infection leads to trained immunity that alters the response and outcome of subsequent infections by: i) using robust mouse infection models as well as cultured primary cells to probe chromatin modifications between cell lines derived from mice with differential UTI disease histories and susceptibilities to rUTI, with particular focus on Programmed Cell Death-associated genes, as well as tumor necrosis factor alpha and cyclooxygenase-2 (Aim 1); ii) probing how prior infection shapes the formation of adaptive immunity at the bladder mucosa and how that modulates susceptibility to recurrent infection (Aim 2); and iii) identifying microbial and mucosal immune mechanisms by which the gut mucosa restricts UPEC colonization in health and dysbiosis and the roles of IL-22 and its binding partner IL-22 binding protein in regulating the microbiota and mucus quantity and quality (Aim 3). The strength of this proposal is that it seeks to understand different forms of infection memory that develop in response to an initial infection including: i) trained immunity; ii) adaptive immunity; and iii) gut dysbiosis and how these host- pathogen interactions lead to epigenetic imprints that predispose to future infections.