Weill Medical Coll Of Cornell Univ

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT – OVERALL Despite decades of research and millions of global infections and deaths, we remain without an effective vaccine against HIV. Traditional vaccine approaches have failed to induce broadly neutralizing antibodies (bnAbs), crucial for protection against diverse HIV strains. The primary goal of next-generation HIV vaccines is to stimulate B cell lineages capable of producing bnAbs. Recent successes in this realm involve structurally designed immunogens, such as the BG505 Germline-Targeting (GT1.1) HIV Envelope SOSIP trimer, which mimics native viral envelope structures and engages bnAb precursor lineages. While promising, achieving bnAb responses in plasma, even at low levels, remains unachieved in the adult populations where it has been tested. Children living with HIV exhibit faster development and more polyclonal bnAb responses compared to adults, suggesting the potential success of early-life vaccination strategies targeting bnAb responses. Our preclinical studies in nonhuman primates have indicated that infant rhesus macaques more frequently develop bnAb precursor responses following BG505 GT1.1 SOSIP trimer immunization, with robust autologous virus neutralization and early evidence of heterologous virus neutralization in plasma. This vaccine has also shown promise in adult human clinical trials in eliciting “on target” vaccine responses, without any safety concerns. While the early life immune system may offer benefits to this vaccine regimen for achieving its immunologic goals, the dosing, intervals, and potential interactions with pre-existing antibodies and other childhood vaccines is unknown, limiting the development of an optimal infant phase 1 trial. Thus, this Program aims to design and systematically test the optimal regimen of BG505 GT1.1 SOSIP trimer vaccine series as a safe and effective pediatric immunogen for eliciting lifelong bnAb responses. Specific aims include: Aim 1: Determining the optimal pediatric formulation and dose, Aim 2: Aligning immunization intervals with childhood vaccine schedules and assessment of persistence of responses into adolescence, and Aim 3: Assessing the impact of maternal antibodies and passive immunization with bnAbs on vaccine immunogenicity and efficacy. By leveraging preclinical models to assess safety, regimen, and persistence into adulthood, this Program seeks to overcome regulatory hurdles that will pave the way for optimized infant HIV vaccine trials. Successful induction of bnAbs via early-life vaccination could revolutionize HIV prevention efforts, offering hope for a future without the burden of the HIV epidemic.

NIH Research Projects · FY 2025 · 2025-08

Summary JDM R01; PIs: Pascual, V. & Wilson, P. Juvenile Dermatomyositis (JDM) is the most common pediatric idiopathic inflammatory myopathy. It typically presents with a pathognomonic rash and/or proximal muscle weakness that interferes with activities of daily living and may severely impair the patient’s quality of life. JDM is a systemic autoimmune disease as, in addition to the skin and muscle, organs such as the lungs, heart, and intestines can be involved. Characteristic immune findings include the presence of autoantibodies, increased interferon (IFN) activity and infiltration of immune cells across involved tissues. In addition, JDM is considered a “vasculopathy”, as endothelial cell loss and/or dysfunction is a pathognomonic feature of the disease. The outlook for children affected by JDM has improved in the past fifty years, but significant gaps remain around disease pathogenesis. As a result, therapy continues to rely on non-specific immunosuppression. Furthermore, sensitive, specific and minimally invasive methods to diagnose, measure disease activity and predict JDM disease course are not available. As most systemic autoimmune diseases, JDM is clinically and molecularly heterogeneous. Thus, a variety of disease phenotypes are linked to the presence of myositis-specific (MSA) autoantibodies. Differences in transitional and memory B cell and helper T cell subpopulation frequencies have been reported, but the mechanisms leading to autoantibody development are not known. We recently identified expansions of CXCR5low/neg CD27+ memory B cells and Th2 cells in JDM patient’s blood in correlation with IFN-stimulated gene (ISG) signatures and with muscle weakness. We hypothesize that these cells contribute to JDM autoreactivity and represent a valuable biomarker of disease activity (DA). Single cell (SC) transcriptional profiling of JDM PBMCs has permitted us to confirm and extend these observations and to gain insights into innate and adaptive immune alterations that correlate with DA. Here, we will leverage our preliminary cross-sectional findings through longitudinal systems-level immune monitoring of patients and in-depth characterization of MSA responses, as well as spatial analyses of involved muscle. Importantly, this project will capitalize on a very well characterized longitudinal pediatric JDM cohort, resources developed over twenty years to study the human immune system, and an exclusive group of co-investigators with complementary expertise. In order to achieve these goals, we propose the following aims: a) to assess longitudinally and at the SC level, the PBMC and antigen-specific B/T cell compartments of JDM patients stratified according to i) organ involvement, ii) autoantibody specificities, and iii) response to therapy; b) to characterize MSA responses through SC cloning, expression and repertoire analyses and test their function and pathogenic potential; and c) to map the heterogeneity of muscle resident and infiltrating immune cells using state-of-the-art spatial transcriptomic and proteomic tools. Altogether, these studies will bring important clues to understand JDM pathogenesis and disease heterogeneity and will inform novel and personalized therapeutic approaches.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY The contents of eukaryotic cells are highly dynamic, yet organized spatially and temporally. This is achieved primarily by the microtubule cytoskeleton and associated transport machinery, whose fundamental nature is highlighted by the many neurological diseases caused by mutations in them. The overarching goal of my research program is to understand how this system works at the molecular, cellular, and organismal scales. My team is highly interdisciplinary and we use in vitro biochemical reconstitution, protein engineering, single-molecule imaging, proteomics, live-cell imaging, and fungal genetics to achieve our goals. Through collaborative projects we use cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) to incorporate a structure-guided approach to understanding intracellular transport, and we develop testable quantitative physical models of transport. We have made major contributions to determining how the dynein motor works and is regulated, to developing tools and screening strategies to study bi-directional movement of cargos on microtubules, and to understanding the regulation of intracellular transport in cells. Fundamental questions that we will address here include: (1) How does the dynein motor work? Our earlier work revealed how Lis1, a protein mutated in the neurodevelopmental disease lissencephaly, interacts with dynein and regulates its mechanochemical cycle. Here, we will focus on determining the mechanistic underpinnings for how Lis1 promotes the formation of activated dynein/dynactin complexes. We will also explore a new direction—the role of RNA editing—as a previously undescribed mechanism to regulate dynein and kinesin motors. Microtubule-based motors move dozens if not hundreds of cargos. (2) How is cargo-specificity achieved? Our past work used two complementary discovery-based approaches—genetics and proteomics—to identify molecules responsible for specifying dynein’s many functions. One mechanism revealed by our past work is organelle hitchhiking, where cargos link to motors indirectly, by attaching themselves to other cargos that are directly bound to the motors. A second strategy for achieving cargo specificity is the expansion of dynein activating adaptor genes in vertebrates. However, the molecular connections between most activating adaptors and dynein’s cargo are unknown. Here, we will determine the mechanisms underlying hitchhiking and the linkages between the Hook and Ninein families of activating adaptors and their cargos. As an additional approach to understand how dynein and kinesin link to their cargos, we will visualize these connections in cells in three dimensions using cryo-electron tomography of endosomes in Aspergillus nidulans and melanosomes in Xenopus laevis melanophores, two systems where we can use exquisite genetics or chemical tools to control microtubule- based motility.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Clonal expansions are observed in a wide range of normal human tissues, as well as in cancer evolution. These outgrowths lead to clonal heterogeneity which can drive the development of treatment-resistant disease. Clones contain somatic mutations in known cancer driver genes and show evidence of positive selection. However, how these driver mutations potentiate changes to the cellular states of cells to allow clones to outcompete neighboring wild-type counterparts remains poorly characterized. So far, the identification of clonal outgrowths in normal or malignant human tissues has been mostly restricted to genotyping analysis only, as these clones often constitute a minority of cells in a sample and do not have distinguishable cell-surface markers. To address the challenge of charting clonal outgrowth during somatic and cancer evolution, we developed a range of advanced single-cell technologies that can capture multiple layers of information from a single cell. These technologies can gather information on genotypes, transcriptomes, methylomes, and protein expression. To overcome the challenge of genotyping using single-cell RNA sequencing, we developed the Genotyping of Transcriptomes (GoT) technique. With GoT, we can compare mutant and wild-type cells within the same individual to characterize the transcriptional consequences of somatic mutations. To further study the molecular mechanisms of how somatic mutations give clonal growth advantage, we have extended our multi- omics single-cell toolkit to include other modalities. As epigenetic mutations are highly frequent in cancer, we developed and applied targeted single-cell genotyping and single-cell ATAC-seq (GoT-ChA), which provides genotyping information in the context of chromatin accessibility. We aim to further expand our platform by developing single-cell methods to capture additional chromatin features, including post-translational modifications of histones (GoT-NTT), transcription factor binding (D&D-seq/GoT-D&D), and high-throughput automated joint measurement of RNA expression and whole genome sequencing (SMART-PTA). Additionally, to define clonal driver genotypes in their spatial context, we will adapt spatial transcriptomics by adding the critical feature of genotyping (GoT-Stradivari), allowing us to characterize how clone growth is affected by its interaction with the microenvironment. Our ultimate goal is to identify the underpinnings of fitness advantage in clonal outgrowth by generating multi-omic comparisons at the single-cell level between wild-type and mutant cells. The proposed comprehensive GoT toolkit will enable us to link single-cell genotypes with transcriptional, protein, epigenetic, and spatial phenotypes at high throughput. We believe that these advances will transform the study of clonal mosaicism as a harbinger of cancer and resistance to cancer therapies.

NIH Research Projects · FY 2026 · 2025-08

Project Summary/Abstract This proposal is for a five-year research career development program, focusing on the role of a key enzyme in group 3 innate lymphoid cells (ILC3s) that regulates gut physiology. Inflammatory bowel disease (IBD) is an immune-mediated inflammatory disease of the gastrointestinal tract characterized by high relapse rates, rising incidence rates, and no known cure. IBD shares common pathophysiological links with Parkinson’s disease (PD), a neurodegenerative disease with loss of dopaminergic neurons. ILC3s, the most abundant ILC in the gut, can interact with several immune and non-immune cells to promote intestinal barrier integrity, antimicrobial response, and immune tolerance. The central hypothesis is that ILC3s is equipped with a machinery to produce key factors that modulate gut inflammation and motility. The overall goal of this research is to investigate the role of these enzymes in the pathogenesis of IBD and dysmotility. To accomplish this, first, the regulation of these processes will be examined. Second, the cell-intrinsic and cell-extrinsic mechanisms by which ILC3s control gut inflammation during stress will be interrogated. Additionally, how these pathways change in human IBD will also be examined. Third, the presence of any potential links between ENS and ILC3s that control optimal gastrointestinal motility will be explored. Results of this study will delineate the role of ILC3s and associated enzymes, as well as its potential as a therapeutic target for IBD. The proposal outlines a plan to achieve my goal of becoming an expert in neuro-immune crosstalk in the context of gut inflammation and motility. Furthermore, through the proposed complementary career development plan, I will gain additional training in clinical investigation and neuroimmunology, advanced bioinformatic analysis with RNA-seq; and laboratory-based training in ILC3s biology. Throughout this research and career development activities, I will be mentored by a team lead by Dr. Gregory Sonnenberg and Dr. Timothy Wang. I am committed to a career as an independent investigator in patient-oriented translational research; and designed my training plan to acquire the knowledge and skills needed to make a meaningful and substantial contribution to the field.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a prevalent genetic disorder affecting millions worldwide, characterized by cyst formation primarily within the kidneys, leading to organ enlargement and deteriorating function. This condition often extends to the liver, posing significant clinical challenges. Traditional qualitative analysis methods are insufficient for accurate disease assessment, prognosis, and treatment guidance. Deep Learning (DL) models have shown promise in estimating Total Kidney Volume (TKV) from MRI images but face limitations in detecting early-stage disease with small cysts. This study aims to transform qualitative analysis into quantitative assessment through multi-class cyst segmentation in ADPKD. Aim 1 focuses on developing a super-resolution DL model to generate high-resolution 3D MR volumes. Multiple imaging planes (axial, coronal, sagittal) will be incorporated to enhance 3D resolution for precise biomarker calculation. Additionally, a multi-class multi-sequence DL framework will be developed for ADPKD severity assessment, which involves creating segmentation and object detection models for different cyst classes, including simple, hemorrhagic, and exophytic cysts within the kidney, liver, and pancreas. Aim 2 aims to build a prognostic model for predicting estimated glomerular filtration rate (eGFR) decline and disease progression. This will involve developing an accurate predictive model based on biomarkers extracted from high-resolution MR images, with a particular focus on cyst class information, especially hemorrhagic cysts, to improve eGFR decline forecasts and dialysis potential predictions. Aim 3 integrates patients' longitudinal data to quantify the impact of temporal dynamics on disease progression over the next 10 years, by developing a multimodal predictive model that incorporates patients' prior MRI scans and historical data. This study aims to utilize DL models to shift from qualitative to quantitative assessment of ADPKD using multi-sequence high-resolution MR images, enabling precise measurement of cyst attributes, and advancing our understanding of disease progression and treatment response for better patient outcomes.

NIH Research Projects · FY 2025 · 2025-08

Project Summary/ Abstract Alcohol use disorder poses a monumental public health challenge in society, but the mechanisms behind this disease are not fully understood. Estrogen (17β-estradiol, E2) is a steroid hormone that coordinates sex differences in brain architecture, neuronal circuitry, and behavior and has been implicated in addiction and other psychological disorders. E2 can regulate behavioral function by to binding estrogen receptors (ERs) in the brain and initiating genomic and/or rapid signaling mechanisms. Our lab has shown that female rodents in a high ovarian E2-state (proestrus) consume more alcohol compared to males and when they are in a low E2-state (metestrus). In addition, we have found that mice in proestrus exhibit decreased avoidance than those metestrus and males. In vivo infusion of ERα-specific E2 into the bed nucleus of the stria terminalis (BNST) of metestrus mice increases drinking levels to proestrus levels but does not change avoidance behaviors. These data suggest that E2 signaling in the BNST modulates neuronal activity in different ways during binge drinking compared to during anxiety. We have also demonstrated that corticotropin-releasing factor (CRF) neurons from the BNST (BNSTCRF) are critical for modulating anxiety behaviors and alcohol consumption, and robustly express ERs. Initial ex vivo electrophysiology experiments indicate that BNSTCRF neurons are a heterogeneous population, and persistently firing neurons have a more excitable phenotype present in proestrus animals compared to those in metestrus. We have also found evidence that their passive and kinetic properties exhibit E2-state dependent changes that may be contributing to higher excitability. We hypothesize that persistent firing BNSTCRF neuron excitability is being mediated through different E2 signaling mechanisms, and this is contributing to our observed behavioral effects. In Aim 1, I will use electrophysiology to parse out whether these neurons’ excitability properties are stimulated in response to rapid E2 and if there is ER-specificity in these effects. I will also test whether EtOH alters the excitability of these neurons, and if so, whether E2 status or sex plays a role in the sensitivity. In Aim 2, I will use snRNAseq to probe whether genes are differentially regulated depending on E2- state or sex, which would indicate a genomic mechanism at play. If the genes for voltage-gated ion channel genes that control excitability are differentially regulated, the effects on their corresponding currents will be queried through electrophysiology. In doing these experiments, diverse mechanisms of E2 signaling present in BNSTCRF neurons will be revealed.

NIH Research Projects · FY 2025 · 2025-08

Project Summary - Elucidating the role of the tumor suppressor KEAP1 in antitumor immunity Lung cancer is the leading cause of cancer-related deaths worldwide, with non-small cell lung cancer (NSCLC) being the most common subtype, representing approximately 85% of all diagnoses. Among the major drivers of lung cancer, about 20% of NSCLC patients exhibit loss-of-function mutations in the tumor suppressor and E3 ubiquitin ligase KEAP1. Although immune checkpoint blockade (ICB) inhibitors represent the most recent breakthrough in treating advanced NSCLC, a significant portion of patients experiences primary resistance, especially those harboring KEAP1 mutations. Our recent work describes how the loss of KEAP1 leads to the suppression of the type I interferon response, promoting lung cancer immune evasion (Marzio et al., Cell 2022). Through multi-omic profiling of NSCLC human and mouse samples, we confirmed that KEAP1-mutant NSCLCs exhibit immune-evasive features, including reduced T cell infiltration and resistance to immune checkpoint inhibitors. This analysis also identified a set of novel potential KEAP1 substrates that are overexpressed in KEAP1-mutant tumors. Among these substrates, we identified several oncoproteins that play a crucial role in the activation of the pro-inflammatory transcription factor NF-B. The goal of the proposed research is to investigate the hypothesis that the newly identified upregulated oncoproteins are bona fide KEAP1 substrates. We will employ biochemistry, molecular/cell biology, and somatic cell genetic approaches to functionally and mechanistically interrogate the contribution of these putative novel substrates in the immune-evasive phenotype observed in KEAP1-mutant NSCLCs (Aim 1). A natural corollary of this goal is to test the premise that therapeutic targeting of the validated substrate can improve the efficacy of immunotherapy for NSCLC (Aim 2). Understanding how the loss of KEAP1, along with the concomitant accumulation of its substrates, promotes immune evasion in cancer has the potential to profoundly impact our comprehension of the immune response to NSCLC and enhance the success of therapy for this tumor type. Additionally, since KEAP1 loss-of-function mutations are recurrent features in several malignancies, the significance of this proposal is evident. We anticipate that our results will present a promising opportunity to treat patients with KEAP1 alterations using novel and alternative therapeutic approaches. Completion of this work will also unveil the molecular mechanisms underlying the physiological role of KEAP1 in regulating inflammation and immunological responses during cancer development and progression, offering therapeutic opportunities for a substantial number of patients whose tumors are driven by its loss. Shaped by our longstanding dedication to the field of DNA damage response and cancer biology, this project is part of our ongoing efforts to uncover novel molecular mechanisms that contribute to malignant transformation.

NIH Research Projects · FY 2025 · 2025-08

Abstract. Type 1 diabetes (T1D) is characterized by the destruction of pancreatic β cells, which respond to signals from immune cells, leading to hyperglycemia. This study aims to grasp the mechanisms influencing T1D by investigating both intrinsic β cell factors and environmental immune cell interactions. Our interdisciplinary team combines expertise in diabetes genomics, stem cell/organoid biology, and islet biology to explore these dynamics. The preliminary studies using single-cell transcriptome, single nucleus chromatin accessibility, and single nucleus (sn) multiome profiling of human pancreatic islets from healthy and T1Daffected individuals. The same experiments were performed using human islets exposed to cytokines or virus simulating T1D conditions. Our integrative approaches identified gene regulatory elements (GREs) at diabetes GWAS. Also, we have developed tactics to differentiate human pluripotent stem cells (hPSCs) into functional vascularized-immune islet organoids containing pancreatic endocrine cells, endothelial cells and immune-like cells. Here, we will apply sn- multiomic (both short and long reads RNA-seq and ATAC-seq) profiling, hPSC-derived organoids, CRISPR- based gene editing and Cas13-based gene knockdown to systematically explore the role of intrinsic and environmental signal dynamics in T1D progression and define mechanistic network controlling β cell destruction. We propose three specific aims to advance our understanding: Aim 1: Define the cell-specific multiomic intrinsic and environmental signatures during T1D progression. We will characterize intrinsic and environmental changes in chromatin and transcriptome profiles of islets from pre-T1D, T1D, and healthy individuals, map gene/isoform expression and chromatin accessibility, and finally integrate multi-omics data to identify cellspecific quantitative trait loci (QTLs) and perform fine mapping with T1D GWAS signals. Aim 2: Decode the epigenomic network controlling β cell destruction during T1D progression. We will use massively parallel reporter assays to validate GREs at T1D GWAS loci, employ Perturb-seq and CRISPR editing to validate GREs, target genes, and their cellular phenotypes in isogenic hPSC-derived organoids. Aim 3: Determine the impact of alternative splicing on human β cell destruction. We will validate T1D-associated alternative splicing using the Xenium platform. Finally, we will examine the biological function of gene isoforms, and reverse β cell destruction in hPSC-derived organoids by targeting alternative splicing mechanisms. Our long-term goals include identifying locus-specific and network mechanisms to facilitate the development for precision medicine approaches in T1D. Key deliverables will include a comprehensive single cell triple-omic map of human islets crossing different stages of T1D progression, a molecular genetic network of intrinsic and environmental signals, and validated hPSC-derived vascularized immune-islet organoid models with T1Dassociated GRE KO, gene isoform KD, etc. These findings will pave the way for the development of novel therapeutic strategies and disease progression markers for T1D.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Proteases are pivotal players in cellular biology, acting as both guardians of homeostasis and mediators of stress responses. Their roles are indispensable to cellular function, ranging from protein turnover and immune responses to mitochondrial maintenance and beyond. Understanding the precise regulatory mechanisms of proteases provides unique opportunities to identify and exploit vulnerabilities in cancer progression. Aim 1 (F99 phase) focuses on identifying the physiologically relevant DPP9 inhibitor. DPP9 is a protease, which regulates inflammasomes—multiprotein complexes that detect danger signals and trigger pyroptosis, a lytic form of cell death. Under normal conditions, DPP9 represses the NLRP1 and CARD8 inflammasomes. Importantly, synthetic DPP9 inhibitors have been shown to activate these inflammasomes, triggering pyroptosis. However, the identity of an endogenous inhibitor remains elusive. Discovering this physiologically relevant inhibitor is crucial, as it will reveal the evolutionarily conserved danger signals that the NLRP1 and CARD8 inflammasomes evolved to sense, opening novel avenues for modulating inflammasome activation—a promising strategy for cancer immunotherapy. Preliminary data indicate that DPP9 interacts with the redox sensor KEAP1 through an ESGE motif, leading to mutual inhibition of both proteins. This interaction suppresses DPP9's catalytic activity and inhibits KEAP1's ability to sequester NRF2, stabilizing NRF2 and activating the antioxidant response system. Intriguingly, in DPP9’s native state, the ESGE motif is structurally incompatible with KEAP1 binding, suggesting that a conformational change in DPP9 is required for this interaction to occur. In Aim 1, this proposal seeks to (1) identify the stimulus that drives this conformational change, enabling KEAP1 binding and mutual inhibition, and (2) unbiasedly identify genetic regulators of DPP9 activity. Together, these studies aim to uncover the endogenous inhibitor of DPP9, define its role in inflammasome activation, and ultimately harness pyroptosis as a targeted cancer therapy. Aim 2 (K00 phase) focuses on identifying mitochondrial proteases critical for maintaining mitochondrial function under the metabolic and oxidative stresses of the tumor microenvironment. Unlike the cytosol, mitochondria lack a proteasomal degradation system and depend on proteases to prevent the accumulation of misfolded or damaged proteins, ensuring cellular adaptation and survival. In this Aim we will employ unbiased proteomics and CRISPR-based approaches to identify mitochondrial proteases and their cognate substrates that are essential for cancer cell survival. These studies will reveal how mitochondrial proteostasis enables cancer cell resilience and uncover therapeutic opportunities to selectively disrupt these processes, compromising cancer cell survival while sparing normal cells.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Embryonic development involves complex changes in cell proliferation, macromolecule synthesis, and nutrient availability, all of which impose different metabolic demands. Accordingly, cells must remodel central metabolic pathways during early development, but the metabolic requirements of early differentiation and how metabolic pathways are reprogrammed during this critical developmental window remain poorly understood. Recently, our lab demonstrated that the tricarboxylic acid (TCA) cycle, a central metabolic hub critical for energy production and provision of biosynthetic intermediates, undergoes dynamic shifts during cell state transitions. Using mouse embryonic stem cells (ESCs) as a model system, we found that naïve pluripotent ESCs, which mimic the pre-implantation epiblast, predominantly rely on the conventional configuration of the TCA cycle, in which citrate is oxidized within the mitochondria. In contrast, more committed ESCs, including those mimicking post-implantation epiblast-like states, preferentially export citrate from the mitochondria, bypassing canonical TCA cycle oxidation. Accordingly, aconitase 2 (ACO2), a key enzyme in the TCA cycle that initiates the catabolism of citrate within the mitochondria, is essential for maintaining the naïve pluripotent state but is dispensable in more differentiated cells. Notably, loss of ACO2 function in humans is linked to severe neurodevelopmental disorders, and mice with heterozygous ACO2 mutations fail to produce viable homozygous null pups. Together, these results indicate that ACO2 plays a context-specific role in early development, but what metabolic outputs of ACO2 support development and the developmental consequences of ACO2 disruption remain unknown. The goal of this project is to determine the role of ACO2 in maintaining naïve pluripotency and supporting early embryonic development. Our preliminary data indicate that ACO2 is dispensable for both energy production and generation of macromolecular precursors; rather, ACO2 is essential to prevent the accumulation of toxic citrate in highly oxidative cellular states. Aim 1 will combine genetic and pharmacologic approaches to determine whether increased citrate production in naïve ESCs induces dependence on ACO2 for citrate clearance and cell fitness. Aim 2 will reveal the developmental consequences of ACO2 loss in vivo using newly generated Aco2+/- mice. We will genotype embryos from Aco2+/- crosses to determine the stage at which Aco2-/-embryos lose viability. Based on these findings, we will isolate Aco2-/- embryos before lethality and investigate the impact of ACO2 loss on cell fate specification, proliferation, and cell death using immunofluorescence techniques. These studies will enhance our understanding of how TCA cycle outputs support early embryonic development. The training plan outlined in this proposal will be completed under the guidance of Dr. Lydia Finley and Dr. Anna-Katerina Hadjantonakis, experts in metabolism, stem cell biology, and embryology. The excellent environment and comprehensive training proposed will prepare the applicant for future success as an independent academic researcher.

NIH Research Projects · FY 2026 · 2025-07

PROJECT SUMMARY/ABSTRACT Antiretroviral therapy (ART) is highly effective against HIV-1 and has saved millions of lives. However, ART is not curative, and people living with HIV (PWH) require life-long therapy because they harbor integrated replication-competent proviruses that quickly fuel rebound when ART is discontinued. This reservoir of infected cells represents the major barrier to long-term ART-free HIV-1 control, and interventions aimed at decreasing or eliminating them remains a priority. A growing body of evidence shows that the intact, rebound-competent HIV- 1 reservoirs evolve over time in response to ongoing immunologic pressure. Importantly, reservoir composition, integration landscapes and transcriptional activity are variable among PWH. These observations highlight the need for tools to carefully characterize bona fide reservoirs and identify key drivers of persistence to tailor approaches that are more likely to effectively perturb reservoirs, and prevent the return of viremia upon ART cessation. The object of the proposed program – “INSPIRE: Innovative Strategies for Personalized Immunotherapies and Reservoir Eradication” – is to tailor a combination of immunologic approaches that disrupt latency and enhance cellular and humoral responses to effectively control or eliminate rebound competent reservoirs in vivo. The program will leverage new methods our groups developed to generate engineered and authentic reservoir CD4+ T-cells clones and engineer B cells to produce broadly neutralizing anti-HIV-1 antibodies. We will use the newly available technologies to characterize reservoirs of PWH, to define the rules governing susceptibility of silent or transcriptionally active intact proviruses to latency reversal and cell-mediated killing, and perform proof of concept experiments to suppress SHIV-1 infection long-term in the absence of ART. These technologies will be applied to categorize ‘reservoir types’ and to select interventions most likely to eliminate reservoir clones in an individual or individuals with similar ‘reservoir types’. Our program is structured in three Research Focus Areas: (1) Apply and improve platforms to isolate authentic clones of CD4+ T-cells carrying latent HIV-1 proviruses and produce engineered reporter CD4+ T-cell clones representing varied responses to bNAb therapy and use these reagents to tailor therapeutic strategies, including the selection of new clinically viable latency reserving agents, (2) Assess elimination of reservoir clones by tailored cytotoxic T lymphocytes (CTL)- and natural killer (NK)-based approaches and link to in vivo impact of broadly neutralizing antibody (bNAb) treatment, and (3) Engineer long-lasting, high-level expression of bNAbs to control established simian-human immunodeficiency virus (SHIV) infections as proof of concept towards tailored HIV-1 control.

NIH Research Projects · FY 2025 · 2025-07

Killing ~1.5 million people annually, tuberculosis (TB) remains a leading cause of death due to bacterial infections worldwide. A vaccine preventing TB in adults has yet to be developed. Control of TB thus largely depends on chemotherapy, which requires months of treatment with several drugs. Pretomanid (Pa) and delamanid (DLM) are novel nitroimidazoles that were approved for treatment of drug-resistant (DR) TB in 2014 and 2019, respectively. Combining Pa with bedaquiline (B) and linezolid (L) established to the so-called BPaL regimen, which drastically shortened treatment duration for DR-TB. Several subsequent clinical trials have confirmed the treatment shortening activity of Pa-containing regimens. Pa and DLM can kill replicating and non-replicating Mycobacterium tuberculosis (Mtb) by similar, complex mechanisms of action (MOAs) that are only partially understood. Here we propose to apply innovative genome-wide screens to identify the Mtb genes determining Pa/DLM potency in vitro and during infection. In preliminary work we identified Mtb’s major intrinsic resistance determinant for Pa/DLM, which we will analyze mechanistically using both biochemical, metabolomic and genetic techniques. Finally, we will exploit Pa’s dual mechanism of action to determine the impact of Mtb’s intrinsic Pa resistance and intrinsic Pa tolerance on treatment duration. The proposed research will be executed by a team combining expertise in mycobacterial genetics, genomics, metabolism, metabolomics and animal models with the goal to advance our understanding of Pa/DLM’s MOA, enable development of more potent nitroimidazoles and guide the design of shorter TB treatment regimens.

NIH Research Projects · FY 2025 · 2025-07

Project Summary One in every ten adults in the US suffers from Type 2 Diabetes (T2D), driven in part by an epidemic of obesity. Currently, identifying a therapy to restore the population and insulin-secreting function of beta cells is the number one unmet need in T2D. Our long-term goal is to reverse pancreatic beta cell failure to treat T2D. NAD+, an energy-sensing metabolite and redox co-factor, is implicated to play a crucial role in determining the function and survival of beta cells. Depletion of NAD, caused by inhibition of NAD synthesis or overactivation of NAD consumers, leads to impaired insulin secretion and increased cytotoxicity in beta cells. Moreover, NAD booster compounds show promises in improving insulin secretion in diet-induced obese mice. Our lab has recently discovered that dihydronicotinamide riboside (NRH), a new and potent NAD booster synthesized by us, significantly enhances insulin secretion and increases total insulin content in the pancreas of obese mice. Unlike other NAD boosters, NRH is metabolized through a previously unrecognized NAD synthesis pathway and has higher bioavailability in the pancreas. Our data suggest NRH acts by directly modulating beta cell NAD levels, replenishing NAD content in pancreatic islets reduced by obesity. Based on this knowledge, our objective is to uncover the mechanism of NAD depletion in obesity-exposed dysfunctional beta cells and identify metabolic pathways through which NAD boosters including NRH can restore their functional mass. We will use mice models of different T2D stages and human islets from healthy or T2D donors to define the correlation between beta cell NAD content and their gradual functional loss. Primary mouse and human islets, beta cell lines, and mice with different stages of beta cell dysfunction will be used to test two major aims. Aim 1 will define the changes in NAD biosynthesis and degradation pathways in beta cells under obesity-induced deterioration towards T2D. Aim 2 will use an optimized NAD boosting strategy to rescue pancreatic beta cell mass and function in T2D models. Completion of this project will provide new pathophysiological mechanisms driving beta cell deterioration due to NAD depletion. These discoveries could revolutionize therapeutic approaches, offering NAD restoration in pancreatic beta cells as new treatment strategies to not only prevent but also reverse the progression of T2D.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Solid tumors can be classified into two broad categories, T cell inflamed (hot) and non-T cell inflamed (cold) tumors. While hot tumors are characterized by a tumor microenvironment enriched in T cells, cold tumors of the immune dessert phenotype, (e.g. pancreatic and breast) lack T cell infiltration. Human cold tumors exhibit profound resistance to immune checkpoint blockade which represents a major therapeutic challenge. To design predictably effective immunotherapies for cold tumors, we must elucidate the mechanisms that control tumor- specific CD8 T cell (TST) dysfunctional states and therapeutic reprogramming. To fill this knowledge gap, I utilize clinically relevant autochthonous cold tumor models, including an oncogene-driven insulinoma model. Tracking TST differentiation longitudinally from cancer initiation to terminal endpoints, I find that TST reside in tumor draining lymph nodes (tdLN), but fail to infiltrate the pancreas, recapitulating the cold tumor immune phenotype of human insulinomas. TdLN TST express numerous inhibitory receptors (e.g. PD1, LAG3) and fail to produce effector cytokines (IFNγ, TNFα). Despite their exhausted phenotype, TST express high levels of transcription factors critical for T cell stemness and self-renewal. Strikingly, tdLN TST of do not generate differentiated progenies, which is in sharp contrast to observations in murine and human hot tumors. This poses the intriguing question whether the inability to differentiate in tdLN is a hallmark feature of TST in cold tumors. Interestingly, over the course of tumorigenesis, tdLN TST lose their ability to respond to immunotherapeutic strategies. My working hypothesis is that TST in tdLN of cold tumors fail to differentiate into effector-like TST, which is associated with the inability to egress the tdLN and infiltrate into tumors. In this application, I will leverage the power of autochthonous tumor mouse models as well as human samples, to identify the molecular and spatial factors that determine TST states in cold tumors and elucidate mechanisms of resistance and responsiveness to immunotherapeutic approaches.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Metastasis is the leading cause of solid tumor-associated death, yet our understanding of how metastasis initiating cells acquire the capacity to and colonize distant organs has remained limited, hampering development of effective treatment strategies. Large genome sequencing efforts have failed to identify metastasis-specific driver mutations in colorectal cancer (CRC), suggesting that non-genetic phenotypic plasticity is a unique feature of metastasis. We have developed an u biospecimen platform of surgically resected patient normal colon, primary CRC and metastatic tumors from 31 patients, profiled with single cell RNA-sequencing (scRNA-seq) and processed for organoid derivation for functional studies. We show that metastatic evolution across patients is characterized by reprogramming into a conserved fetal intestinal state, followed by lineage transdifferentiation into non-canonical squamous and neuroendocrine cell states that are associated with therapy resistance in patients. My preliminary data demonstrate PROX1, a transcriptional co- repressor upregulated in the fetal intestinal state, is an enforcer of intestinal lineage identity in primary tumor organoids, and its functional loss de-represses non-canonical programs during metastasis. Furthermore, PROX1 has been described to interact with subunits of nucleosome remodeling and deacetylase (NuRD) complex (LSD1 and HDAC1/2) in primary CRC to repress gene programs through epigenetic mechanisms; however, this level epigenetic regulation has yet to be explored in CRC metastasis. I hypothesize that PROX1 constrains intestinal lineage identity in CRC through repressive epigenetic mechanisms through the NuRD complex, while PROX1 dysregulation in metastasis licenses non-canonical lineage expression. I have successfully generated matched organoid pairs modified to downregulate or overexpress PROX1 to assess how PROX1 perturbation differentially alters the transcriptional and epigenetic landscape of primary and metastasis-derived organoids. In Aim 1, I will leverage these matched organoid pairs to assess how PROX1 perturbation differentially alters the transcriptional landscape of primary and metastasis-derived organoids in vitro and in vivo through mouse orthotopic intrahepatic xenograft assays. In Aim 2, I will determine the PROX1- dependent epigenetic mechanisms that restrict non-canonical differentiation and enforce intestinal lineage identity through PROX1 and NurD subunit ChIP-seq studies and chromatin landscape characterization with PROX1 perturbation. I will validate NuRD subunit interaction essentiality in enforcing a transcriptional repression through pharmacologic inhibition studies in the context of PROX1-proficient organoids. Addressing these gaps in our knowledge is of significant clinical importance will aid our understanding of metastasis to inform the development of novel treatment strategies. The work and training plan outlined in this proposal will be completed in the laboratory of Dr. Karuna Ganesh with the co-advisement of Dr. Scott Lowe at Memorial Sloan Kettering Cancer Center and will prepare me for a career as an independent physician-scientist.

NIH Research Projects · FY 2026 · 2025-07

Project Summary. (30 lines) Cancer is the 2nd leading cause of death in the US. The advent of new treatments such as immunotherapy and targeted therapies have revolutionized the fight against cancer, and when combined with surgery or chemo- therapy, often result in positive therapeutic responses. Eventually, however, most tumor cells gain the ability to resist current therapies, making it critical that we continue to define cancer causing and driving processes to increase the available arsenal of anti-cancer drugs. Given the role of metabolism and metabolic environments in cancer progression, understanding these aspects of cancer progression also has the potential to reveal new classes of cancer drugs that take advantage of cancer-associated signaling and metabolic vulnerabilities. mTOR complex 1 (mTORC1), is a protein kinase complex that senses various environmental cues and coordinates anabolic and catabolic processes to regulate cellular homeostasis. mTORC1 becomes activated when amino acids, lipids, energy sources, oxygen and growth factor levels are sufficient. Different cancer cell-associated mutations provide tumor cells with the ability to optimize usage of these regulatory components or find ways to overcome deficiencies in these mTORC1 regulators, including nutrients. Additionally, once activated it is still a mystery how mTORC1 regulates and coordinates the many processes required to promote cell growth, proliferation, migration, and survival. To better understand mTORC1 signaling, we have combined our mTORC1 phospho-proteome, proteome, interactome, metabolome and gene expression data sets, to support new discoveries. In this proposal we have outlined several goals based on these data, our recently published work and the 35 years experience of my lab, that support our long-term goals of defining mTORC1-S6K1 regulation and signaling, and for revealing new information to support efforts to kill cancer cells with activated mTORC1 signaling. Our discoveries and experience are now driving us to; (i) determine how nuclear accumulation of GSK3, upon suppression of mTORC1 signaling, regulates chromatin remodeling and DNA damage repair, nuclear events linked to biological processes altered in cancer cells, (ii) characterize a novel S6K1 effector kinase, SRPK2, and its role in mRNA biogenesis of interferon-stimulated genes and its role in therapy resistance and (iii) define a new link between essential dietary ω-6 and ω-3 fatty acids and mTORC1 activation, highlighting a potential dietary intervention as part of a treatment program for mTORC1-driven cancers . In conclusion, there’s a critical need for a greater understanding of the molecular basis of mTORC1 regulation and signaling, as well as its links to processes associated with cell growth, metabolism, survival and drug resistance. Our expectations are that successful completion of the proposed work will impact cancer biology/physiology and therapy through the identification of new therapeutic targets and biomarkers that can lead to the improve detection and elimination of cancer cells with unregulated mTORC1 signaling, estimated to occur in 70-80% of all human cancers.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY/ABSTRACT Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease and fourth leading cause of end-stage renal disease (ESRD). Hyperactivation of the cystic fibrosis transmembrane conductance regulator (CFTR) is essential for cyst formation and enlargement in ADPKD, and inhibition of CFTR is a potential therapeutic strategy. Despite its importance as a pharmacological target, the conformations transited by CFTR during physiological gating are poorly resolved, and the mechanism by which nucleotide state is communicated from the nucleotide binding domains (NBDs) to the pore 50 Å away remains obscure. Additionally, molecular mechanisms of pharmacological inhibition have been understudied despite therapeutic promise for ADPKD. Specific aim 1 seeks to determine the structural basis of allosteric communication between the NBDs and the pore using cryogenic electron microscopy (cryo-EM). Specific aim 2 seeks to determine the mechanisms of existing pharmacological inhibitors of CFTR using cryo-EM in combination with electrophysiological, biochemical, and biophysical assays. Specific aim 3 seeks to find new inhibitors of CFTR to enhance treatment of ADPKD using computational docking and medicinal chemistry coupled with high-throughput assays, electrophysiology, and mouse models of ADPKD. Successful completion of these goals will fill a gap in our understanding of the CFTR gating cycle, provide new conformations for pharmacological targeting, expand our understanding of CFTR’s pharmacological regulation, and establish new leads for treatment of ADPKD. The proposed research strategy will be completed under the mentorship of Dr. Jue Chen in the Laboratory of Membrane Biology and Biophysics at The Rockefeller University (RU) in New York City with the co-advisement of Dr. Jiankun Lyu at RU. The accompanying fellowship training plan will be completed through the Tri- Institutional MD-PhD Program of Weill Cornell Medicine, The Rockefeller University, and Memorial Sloan Kettering Cancer Center. The exceptional clinical and scientific resources of these environments will facilitate successful completion of both research and training components. The electrophysiological, biochemical, biophysical, and structural biology equipment and expertise available at The Rockefeller University is unparalleled, and trainees are encouraged to collaborate with other scientists and institutions in New York City’s rich structural biology research network. The Tri-Institutional MD-PhD Program offers a diversity of clinical, research, and extracurricular opportunities for trainees to develop the analytical, technical, communication, and mentorship skills necessary to become independent physician-scientists. Trainees are encouraged to continuously develop their clinical and scientific skills through both medical and graduate phases of the MD-PhD curriculum. This curriculum is supervised by a supportive administration that provides ample guidance for trainees to pursue productive careers promoting the health of the American public.

NIH Research Projects · FY 2026 · 2025-07

Summary: Crohn's disease (CD) affects millions of people worldwide and there is currently no cure. CD results from an interaction between a dysbiotic microbiota and genetic susceptibility variants that lead to inflammatory disease, but the cellular and microbial mechanisms underlying this are incompletely understood. Despite the emergence of new therapies for CD, most patients that require advanced therapy develop refractory disease requiring sequential therapy and / or surgery. There is therefore an urgent unmet medical need to identify the cellular and molecular mechanisms that can stratify disease and provide insight into new therapeutic strategies. Protein folding is a critical cellular process by which nascent peptides synthesized in the endoplasmic reticulum (ER) are assembled and trafficked within the cell. Disruption of ER homeostasis can lead to an ER stress response and activation of the unfolded protein response (UPR), in which various signaling pathways act to restore organelle function or trigger cell death. Seminal research has revealed a key role for the UPR in regulating proliferative and secretory intestinal epithelial cells in CD. One of the genetic variants associated with CD is the endoplasmic reticulum (ER) protein disulfide isomerase Anterior Gradient 2 (AGR2) and our cells, Crohn's expansion, leads E. IL-23-dependent the that folds, traffics, assembles cysteine-rich transmembrane receptors and intestinal glycoprotein mucins. Published data from group and others revealed that deletion of AGR2 leads to abnormalities in i ntestinal Paneth cells, goblet and stem cell proliferation resulting i n spontaneous granulomatous i ntestinal inflammation characteristic of disease. Our preliminary data reveal that bacterial dysbiosis, characterized by Enterobacteriaceae is associated with AGR2 expression i n Crohn's disease and that AGR2 deficiency in mice similarly to bacterial dysbiosis, characterized by the expansion of the CD associated pathobiont adherent-invasive coli (AIEC). Our data further reveals that AIEC are sufficient to selectively trigger epithelial cell ER stress and ileocolitis in AGR2 deficiency. Based on these preliminary data, we propose three aims to test hypothesis thatAGR2 regulates epithelial cell-specific ER stress responses that shape AIEC-host interactions in mediating ileocolitis in Crohn's disease. This research has the potential to reveal new cellular and molecular mechanisms by which AGR2 restrains ER stress to shape AIEC dysbiosis and IL-23-dependent inflammation in Crohn's disease. If successful, this research has the potential to reveal new diagnostic and therapeutic modalities for refractory Crohn's disease.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Natural Killer (NK) cells are a major component of host immunity against viral infections, rapidly responding to infected cells by secreting cytokines and lytic granules. NK cells are particularly important for controlling infections by herpesviruses, such as cytomegalovirus, which can cause significant clinical consequences in immunocompromised patients and neonates. Using a well-established mouse cytomegalovirus (MCMV) infection model, our lab and others have demonstrated that NK cells undergo dynamic metabolic and epigenetic shifts during antiviral responses to fuel their activation and effector functions. However, the mechanisms by which NK cells coordinate these metabolic and epigenetic changes are not well understood. Growing evidence indicates that central metabolites play important roles in immune cell activation by acting not only as energy sources but also as substrates for growth signaling pathways, epigenetic regulation, and effector differentiation. L-2-hydroxyglutarate (L-2HG) is an immunometabolite that has been shown to play a role in coordinating metabolic and epigenetic shifts in activated CD8+ T cells, dendritic cells, and macrophages. During activation, L-2HG accumulates and acts as a potent competitive inhibitor of ⍺KG-dependent enzymes, including histone lysine demethylases and hypoxia inducible factor prolyl hydroxylases, altering the epigenetic and metabolic state of these cells. While L-2HG is clearly important for immune cell function, the role of L-2HG in NK cells is unknown. My preliminary data indicates that NK cells limit levels of L-2HG during infection by increasing the expression of L-2HG dehydrogenase. Excess accumulation of L-2HG resulted in impaired NK cell antiviral functions. Given the impact of L-2HG on epigenetics and metabolism, I hypothesize that L-2HG accumulation is detrimental to the NK cell antiviral response by disrupting highly coordinated changes in histone methylation and by skewing the balance between glycolytic and oxidative metabolism. Using the novel mouse models of NK- specific L-2HG accumulation and depletion that I generated in collaboration with the lab of Dr. Andrew Intlekofer, I will pursue the following aims. In Aim 1, I will use in vivo and in vitro epigenetic assays to assess the impact of L-2HG accumulation on activating H3K4me3 and repressive H3K27me3 levels during NK cell responses to MCMV. In Aim 2, I will use metabolic assays to elucidate the HIF-1⍺-dependent and -independent metabolic perturbations by L-2HG in NK cells during MCMV infection. This study will define a novel metabolic-epigenetic axis in NK cells that will inform the development of improved NK cell-based antiviral therapies.

NIH Research Projects · FY 2026 · 2025-06

The human brain is a highly complex system of interconnected neurons, from which arise thought, emotion, and behavior. Neuroimaging via MRI can capture structural connectivity (SC) and functional connectivity (FC) net- works to allow insight into how behavior arises from the brain, or so-called brain-behavior mapping. Our group and others have shown that measures of the brain’s SC and FC networks and their inter-relationship can eluci- date brain-behavior relationships in health and disease/recovery. However, there is an urgent, unmet need for multi-modal connectome models that preserve inter-individual differences across the lifespan in health and dis- ease. Without such models, we cannot perform accurate brain-behavior mapping that allows probing of circuitry underlying behavior. Our long-term goal is to create computational tools for integrating metrics of and predicting behavior from brain structure and function, so we can probe circuitry underlying behavior. This project’s over- arching objective is to create and validate tools that integrate and estimate multi-modal connectome data across many individuals, with and without diagnoses, in a behaviorally relevant way. Our central hypothesis is our Kra- kencoder, which fuses and maps between multi-modal connectomes, will be more accurate and better preserve inter-individual differences compared to existing techniques, allowing more robust brain-behavior modeling. We hypothesize the Krakencoder can be used with our Network Modification (NeMo) Tool to estimate FC and SC from clinical MRI in lesioned individuals, to quantify their impact on the connectomes and probe circuitry under- lying behavior. Our hypothesis is supported by data showing i) the Krakencoder accurately maps between multi- modal connectomes in lifespan data and in lesion cohorts and ii) the NeMo Tool’s estimated SC and the Kra- kencoder’s latent space can more accurately perform brain-behavior mapping compared to other connectome metrics. Our rationale is that having an accurate model that preserves individual differences and integrates multi- modal connectomes will enable more robust brain-behavior models for use in mapping the brain’s circuitry un- derlying behavior. We will test our hypotheses via three specific aims: 1) expand the Krakencoder’s ability to accurately map between connectome flavors while preserving inter-individual differences in data from across the lifespan, 2) integrate the Network Modification Tool and the Krakencoder to estimate multi-modal connectomes and 3) probe multi-modal connectome circuitry associated with demographics and behavior. We will use brain MRI, cognitive, and behavioral data from 11 sources (total N>8000), including from healthy young adults, devel- oping and aging populations (HCP lifespan and ABCD), as well as individuals with lesions. The approach is innovative in that our work focuses on preserving inter-individual differences when combining and mapping be- tween multi-modal connectome flavors; this will increase SNR and result in more accurate brain-behavior mod- els. The proposed research is significant in that understanding of how brain anatomy and physiology relate and give rise to behavior could pave the way for novel, personalized interventions to support brain health.

NIH Research Projects · FY 2026 · 2025-06

Molecular Programs Governing Enterococcal Translocation Enterococcus faecalis is a commensal microbe residing in the gastrointestinal tract of healthy individuals. However, in hosts with dysbiosis or altered gut homeostasis, these bacteria can overgrow, breach intestinal barriers, and cause life-threatening systemic infections. This process, known as translocation, remains a largely unexplored phenomenon. Our team uncovered that E. faecalis produces a polysaccharide-rich extracellular matrix (ECM) that enables its movement across intestinal epithelial cell barriers. However, the mechanisms by which host cells perceive and respond to stress signals generated by these translocating enterococci remain elusive. Additionally, whether E. faecalis exploits specific molecular pathways in epithelial cells to facilitate its translocation remains unknown. Our project aims at addressing these fundamental gaps in knowledge. We postulate that E. faecalis induces endoplasmic reticulum (ER) stress in epithelial cells and that activation of the unfolded protein response (UPR) through this process promotes enterococcal egress from the intestine. Our study is innovative and significant because it has the potential to uncover key molecular mechanisms mediating E. faecalis transition from commensal to pathogen. Aim 1 will define how E. faecalis disrupts ER proteostasis in epithelial cells. Aim 2 will establish that E. faecalis-induced ER stress responses compromise intestinal barrier function. Aim 3 will test the hypothesis that disabling ER stress sensors in vivo thwarts enterococcal translocation in hosts experiencing antibiotic-induced dysbiosis. The proposed research will be accomplished by integrating our multidisciplinary expertise in E. faecalis pathogenesis, ER stress biology, and intestinal physiology. We have developed advanced models of translocation, crucial transgenic mice, sensitive ER stress reporter systems, and a unique understanding of the interplay between enterococci and the host UPR. The proposed studies could pave the way for new treatments that more effectively prevent or control systemic infections caused by gut-resident enterococci. Our project should also provide a strong rationale to define if other commensal bacteria exploit the UPR to cross intestinal barriers, hence expanding our mechanistic understanding of host-microbe interactions in the gut.

NIH Research Projects · FY 2026 · 2025-06

Over seven million people in the United States have acquired pathogenic mutations in their blood stem cells, known as “clonal hematopoiesis” (CH), which increases their risk for life-shortening, strokes, heart and lung disease and blood cancers. Expansion of the mutated stem cells is the root cause of these risks. While inflammation and other hematopoietic stressors can favor the mutated blood stem cells, it is not exactly clear why this is or how to block it. Much has been learned about how the cancer-causing mutations act in blood forming cells but little is known about how mutated blood forming stem cells and their progeny reorganize the local environment, or niche, in which they live. Our research has found that during periods of high blood cell demand—such as infections, bleeding, or after myelosuppressive chemotherapy—the stem cell niche, “switches on” to support stem cell expansion and increased blood cell production. Similar changes are present in CH- affected marrow. Additionally, we've discovered signals that actively “switch off” the niche when the demand for blood cells returns to normal, a process that fails in CH. The long-term goal of this research is to develop new approaches to control niche activity, keeping healthy stem cells safe and making the environment inhospitable for CH stem cells. The objective of this application is to define the mechanisms that switch the niche on and off, with the aim of blocking these mechanisms to make life hard for CH stem cells. The central hypothesis is that certain progeny of CH stem cells send signals to nearby blood vessel-lining cells in the niche to maintain the niche in an “on” configuration. Targetable signaling pathways can reduce the expansion of CH stem cells in the niche. We will complete two specific aims to test the hypothesis. Aim 1: Identify the upstream and downstream mediators(s) that normally turn off the niche and why this doesn't happen in CH. Aim 2: Determine the mechanism for turning on niche blood vessel-lining cells during stress in the presence of mutated cells. Both aims are well supported by preliminary studies and use methodologies that have already been established to be feasible. Our team has produced unique animal strains and models that allow, for the first time, genetic dissection of these processes and have developed novel ex vivo models of the niche and innovative single-cell sequencing technologies for molecular definition of the mechanisms at play. The research is innovative in its focus on how mutated blood stem cells and their progeny affect the niche under different physiologic states, challenging the view that niche functions are static and immutable. The significance of this work lies in identifying the molecular mechanisms that give malignant stem cells a competitive advantage in the niche and provides actionable therapeutic strategies to mitigate the risks for millions of people with CH.

NIH Research Projects · FY 2025 · 2025-06

Project Summary Myopathy is one of the most common manifestations of mitochondrial diseases. Although genetic and bioenergetic causes of Oxidative Phosphorylation (OxPhos) impairment are well established, there is a limited understanding of the metabolic drivers of muscle degeneration. This knowledge gap contributes to the lack of effective treatments for these disorders. Our recently published studies indicate that OxPhos defective muscle initiates an integrated systemic, multiorgan metabolic response coordinated by endocrine signals, which contributes to the pathogenesis of mitochondrial myopathy. In the COX10 KO mouse model of mitochondrial myopathy, increasing plasma levels of the myokine GDF15 activate central and peripheral neurocircuits through GFRAL signaling, which over time reduce caloric intake, induce mobilization of lipid from adipose tissue, and promote energy-consuming futile cycles in muscle. This chronic GDF15-driven fat and muscle wasting result in a cachectic phenotype which aggravates the myopathy. Therefore, we hypothesize that inhibiting the GDF15-GFRAL signaling with established anti-GDF15 and anti- GFRAL antibodies will increase caloric intake, decrease lipid mobilization and attenuate energy expenditure, thus improving body weight and muscle function. In aim 1 of this application, we will target GDF15-GFRAL signaling to prevent cachexia in pre-symptomatic COX10 KO mice. In aim 2 we will target GDF15-GFRAL signaling to treat cachexia in symptomatic COX10 KO mice. This study will establish if anti-GDF15-GFRAL Ab therapy improves mitochondrial myopathy and assess whether the Ab treatment can reverse symptoms of cachexia, setting the stage for testing anti-GDF15-GFRAL Ab therapy in human mitochondrial myopathies.

NIH Research Projects · FY 2026 · 2025-06

PROJECT SUMMARY/ABSTRACT Prostate cancer (PCa) is a clinically and molecular heterogenous disease, with biologically distinct subtypes driven by characteristic genomic alterations in both early, untreated disease and treatment resistant castration- resistant prostate cancer (CRPC). However, whether early alterations affect fidelity to prostate lineage, shape the specific resistance patterns that emerge with treatment, and the underlying mechanisms, remain unclear. We hypothesize that specific molecular features of early, untreated PCa establish distinct pathways to progression and therapeutic resistance through transcriptional control and fidelity to luminal prostate lineage. Preliminary data generated by our multi-institutional, multidisciplinary, collaborative group suggest specifically that the subclass of PCa defined by recurrent mutations in SPOP maintain strong fidelity to prostate lineage, and are therefore preferentially reliant on androgen receptor (AR) signaling and possibly resistant to conversion to AR-indifferent subtypes of CRPC. The overall objective of this proposal is to define the propensity of prostate cancers harboring SPOP mutations to progress to specific subtypes of treatment resistant CRPC, and to define the mechanisms that shape these resistance patterns. Using novel models and human prostate cancer samples, our preliminary data demonstrate that SPOP mutation reprograms AR function, altering chromatin accessibility and transcription driven by AR and making these cancers highly reliant on AR activity. In contrast, N-Myc induction combined with RB1-loss is a strong driver of AR-indifferent CRPC that functions to rewire and deactivate the AR transcriptional program. Our central hypothesis is that opposing effects on rewiring of the AR-directed epigenomic and transcriptional programs mediate the downstream impact on biology and therapeutic sensitivity shown with SPOP mutation and drivers of AR-indifferent disease. This project will elucidate the molecular details underlying these phenomena through the following Aims: 1) defining the propensity of SPOP mutant PCa to progress to AR-indifferent disease in response to specific driver alterations, 2) mechanistically, determining the contribution of FOXA1 and TRIM24 in lineage fidelity and plasticity downstream of SPOP, and 3) establishing if drivers of luminal lineage fidelity can prevent or reverse resistance to AR-targeting therapies. To accomplish this, we will leverage unique, biologically and clinically relevant model systems, innovative approaches to epigenomic and transcriptomic discovery, and data from human prostate cancer samples. This project will define the critical transcriptional processes in specific subtypes of prostate cancer and the broader applicability to treatment resistance, and provide the foundation for precision clinical trials.