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NHLBI - National Heart Lung and Blood Institute

Project summary: Cardiac fibrosis is a key pathological process contributing to heart failure, characterized by excessive deposition of extracellular matrix (ECM) that leads to increased ventricular stiffness, reduced compliance, and impaired cardiac function. Despite its central role in heart failure progression, current therapies primarily manage symptoms and do not reverse or eliminate the fibrotic burden. Targeting the activated fibroblasts responsible for ECM overproduction represents a promising therapeutic strategy. Recent advances have demonstrated that chimeric antigen receptor (CAR)-engineered immune cells can be redirected to target fibroblast activation protein (FAP), a marker of activated fibroblasts. However, the clinical application of CAR-T cells for fibrosis has been limited by significant safety concerns, including cytokine release syndrome, neurotoxicity, and long-term persistence. As an alternative, CAR natural killer (CAR-NK) cells offer a more favorable safety profile and have not been associated with these adverse effects, especially when derived from allogeneic sources. This project aims to develop universal CAR-NK cells derived from hypoimmunogenic human induced pluripotent stem cells (hiPSCs). These cells are engineered with inactivated HLA class I/II and overexpress CD47 to evade host immune recognition, allowing for off-the-shelf, allogeneic use without the need for lymphodepletion or immunosuppression. In this study, the CAR construct targets mouse FAP to enable selective elimination of activated cardiac fibroblasts. We will evaluate the therapeutic efficacy of these engineered CAR-NK cells in vitro and in vivo by cardiac fibrosis induction in NSG-SGM3-IL15 mice model. Outcomes will include fibroblast ablation, fibrosis regression, and recovery of cardiac function. This study has the potential to establish a new class of regenerative immunotherapies for cardiac fibrosis and heart failure.

OD - NIH Office of the Director

PROJECT SUMMARY Gastrointestinal acute radiation syndrome (GI-ARS) is a consequence of exposure to high doses of ionizing radiation and is characterized by extensive damage to the intestinal epithelium that leads to loss of barrier function, sepsis, and in some cases mortality. Currently there is a critical need to develop new physiologically relevant human models to study GI-ARS and to evaluate potential medical countermeasures (MCMs). This project aims to establish human intestinal organoids (HIOs) as a robust in vitro model for GI-ARS using high- content imaging approaches to assess the therapeutic potential of a microbial based MCM. Optimization of radiation dosing will be performed using a large cohort of HIOs that allow assessment of sex, age, and intestinal region on the response to radiation to be interrogated. A library of biomarkers of radiation damage will be assembled using novel screening approaches that combine multi-omics analysis, bioinformatic pipelines, and network analysis. Following biomarker identification, Cell Painting, a high content morphological profiling technique, will be implemented to characterize the cellular response to radiation treatment in a high throughput manner using a scanning disc confocal microscope and customized analyses package. The generation of detailed cellular and subcellular phenotypic profiles that associate with radiation will be used to enable rapid, quantitative assessment of therapeutic efficacy of a microbial based MCM. The MCMs to be tested are Limosilactobacillus reuteri 6475 (LR6475) organisms as a modality to deliver key growth factors necessary to promote regeneration and repair of the intestinal epithelium that target the intestinal stem cell. At the completion of the proposed studies, HIO will be validated as a translatable model for GI-ARS and a novel imaging based phenotypic screening pipeline for radiation injury and MCM evaluation will be established. This work will significantly advance efforts in radiation countermeasure development, facilitate future therapeutic screening and support preparedness for radiological emergencies.

NHLBI - National Heart Lung and Blood Institute

Hematopoietic stem cells (HSCs) are characterized by their capacity for self-renewal and the generation of all mature hematopoietic cell types within the blood and immune system. The primary objective of this research proposal is to develop novel resources that can provide a cure for a greater number of patients with lifethreatening disorders of the hematopoietic system than is currently feasible. Our goal is to enhance the availability of suitable donors for HSC transplantation (HSCT) and establish novel approaches that minimize the duration of cytopenias, the leading cause of treatment-related mortality. To address this challenge, the current research plan aims to build upon our recent discoveries that the vascular niche provides the instructional cues that enable large-scale expansion of HSCs while preserving their self-renewal capabilities. Utilizing our technologies to isolate and cultivate human primary endothelial cells (ECs), we have established young and aged bone marrow EC (BMEC) primary lines. Our preliminary data indicates that aged BMECs exhibit significantly elevated thrombospondin-1 (THBS1) and co-culture of HSCs with aged BMECs resulted in ex vivo expanded HSCs exhibiting a substantial reduction in long-term engraftment and a myeloid bias. Genetic deletion of Thbs1 or supplementation with a Thbs1 neutralizing antibody restored ex vivo expansion and function comparable to young blood products. Based on these findings, this proposal will determine whether neutralization of Thbs1 can support the function of human blood products and initiate the identification of novel angiocrine factors from a humanized in vivo BM vascular niche that support human HSC and vascular function. To this end, we will employ gold-standard assays to evaluate HSC and vascular function, as well as a novel method to humanize the murine BM vascular niche to investigate whether suppressing Thbs1 can 1) refine current ex vivo human HSC expansion protocols to enhance the quality and quantity of transplantable blood products, 2) develop novel protocols to preserve systemic vascular integrity following myelosuppressive insult, 3) enhance HSC engraftment, and 4) identify novel factors that may serve as potential therapeutic agents to improve hematopoietic and vascular function. The proposed experiments represent a sequential progression of preclinical research, ultimately aiming to investigate the transplantation of expanded long-term repopulating HSCs in patients who may benefit from allogeneic and autologous HSCTs. Successful HSC expansion and improved engraftment will ultimately expand access to HSCT for patients requiring curative HSC replacement. Accomplishing these objectives will ultimately benefit older patients who lack suitable human leukocyte antigen (HLA)-matched sibling or adult unrelated volunteer donors for HSCT, and patients requiring engineered, engraftable HSCs for the correction of genetic disorders, for whom the number of available autologous HSCs is suboptimal.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary/Abstract Protective humoral immunity is mediated by both long-lived memory B cells (MBC) and antibody secreting plasma cells (ASC). Recently it has become increasingly clear that MBC and ASC are in fact composed of functionally diverse subpopulations that can be distinguished based on unique surface marker expression, tissue localization, and the B-cell receptor (BCR) isotype expressed. During a humoral immune response, naïve B cells (nB) give rise to differentiated subsets, with the ultimate composition of the MBC and ASC pool primarily influenced by the antigen, the involvement of T cells, and the cytokine environment. Given the evidence for increased heterogeneity, it is surprising that we currently lack the genetic tools to further interrogate the complexity, molecular properties, and immunological importance of the known B cell fates. A precise phenotypic analysis of MBC and ASC subsets cannot currently be performed with current Cre-lox mouse models. Cis- regulatory elements (CEs), or enhancers, are DNA sequences that act to promote cell-type and context-specific gene expression programs. These sequences are regulated by epigenetic mechanisms, which act to control the accessibility of CEs to DNA-binding transcription factors. Over the past several years, we have characterized the epigenetic architecture that controls primary humoral immune responses to T cell dependent and independent antigens, integrated newly published datasets defining MBC subsets, and generated new preliminary data defining the CEs of ASC expressing distinct BCR isotypes. These data have revealed specific CEs that are active in defined stages of B cell differentiation, including IgA ASC and extrafollicular (EF)-MBC that arise independently of a germinal center reaction. Therefore, we hypothesize that cell-type specific CEs can be co-opted to provide precise genetic manipulation that enables functional exploration of B-cell subsets. To address this, we propose two aims designed to 1) develop new Cre recombinase tools for genetic manipulation of IgA ASC and 2) map the CEs for EF-MBC that arise during influenza infection and integrate CEs specific for EF-MBC to allow precise genetic editing of this MBC subset. These aims will utilize novel hematopoietic stem cell engineering to rapidly generate chimeric mice expressing genes of interest; therefore, bypassing the need to generate transgenic animals. Completion of these aims will provide a novel set of DNA sequences that allow for MBC and ASC specific genetic manipulation. These tools are critically important begin to derive the biology, molecular properties, and importance of the entire spectrum of B-cell differentiation to humoral immunity. If successful, it also has the potential to redefine how Cre recombinase vectors are engineered and further our understanding of CE biology in B cells and the immune system.

NIA - National Institute on Aging

PROJECT SUMMARY/ABSTRACT Neurodegenerative diseases, including Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD), exhibit neuropathological features (e.g., amyloid and p-tau accumulation) that disproportionately affect certain brain regions and subsets of cells. This selective vulnerability to neuropathological features has been recognized for decades, yet its underlying molecular mechanisms remain unclear. Long COVID, a prevalent post-pandemic condition with largely unknown disease mechanisms, is reported to exhibit p-tau pathology, linking it to neurodegeneration. Current methods, including single-nucleus RNA sequencing, are unable to integrate spatial and pathological contexts with transcriptomic profiles, limiting direct molecular characterization of disease pathology at the single cell level. Here, I propose to leverage single-cell spatial multi-omics on postmortem brain tissues to in-situ capture molecular and cellular alterations that directly linked to selective vulnerability to neuropathological features across AD, FTLD, and Long COVID at single-cell resolution—insights previously unattainable. I will also identify potential pathological modulators using functional genomics in human induced pluripotent stem cell (iPSC)-derived neurons. In this proposal, Aim1 will characterize neuropathological and transcriptomic alterations in Long COVID and compare findings to AD and FTLD. Aim2 will leverage same- slide single-cell spatial multi-omics with neuropathology phenotyping (Xenium 5K + Phenocycler-Fusion) and define the molecular-neuropathological signatures specific to amyloid, neuronal p-tau, and glial p-tau pathology in AD, FTLD and Long COVID. Aim3 will employ CRISPRi/a screening in iPSC-derived neurons to identify neuronal p-tau modifiers from signatures uncovered in Aim2. By integrating single-cell spatial multi-omics with functional genomics, this project will bridge the gap between neuropathology and molecular profiling, provide novel insights into selective vulnerability to neuropathological features and identify pathological modulators. My long-term goal is to become a physician(neuropathologist)-scientist leading a NIH-funded research laboratory focused on neurodegenerative diseases and COVID-19. This five-year mentored career development plan will provide the necessary training for my transition to independence, emphasizing expertise in single-cell spatial transcriptomics and proteomics, bioinformatics, imaging analysis, iPSC technology and CRISPR-based functional genomics. Additionally, I will deepen my knowledge of neuropathology, molecular genetics, and clinical aspects of AD, FTLD, and Long COVID. I have assembled a multidisciplinary mentorship team of distinguished physician-scientists that includes Dr. Daniel Geschwind (primary mentor), Drs. Harry Vinters and Shino Magaki (co-mentors), and advisory members Drs. Inma Cobos, Vivek Swarup, and Edward Lee. With UCLA's cutting- edge facilities, exceptional research environment, and strong clinical resources, this award will prepare me to become a competitive neuropathologist-scientist, advancing our understanding of neurodegenerative diseases and COVID-19 while informing therapeutic strategies.

NCI - National Cancer Institute

PROJECT SUMMARY Immunotherapies targeting adaptive immune checkpoints have improved cancer outcomes, but innate immune checkpoints also play crucial roles in cancer immune evasion and are promising targets for immunotherapy. The innate immune system uses "eat-me" and "don’t-eat-me" signals to regulate phagocytosis, essential for maintaining tissue homeostasis and preventing cancer. Phagocytic cells have also recently emerged as new key actors in the success of immunologically mismatched allograft transplants through human leucocyte antigens (HLA) allorecognition. Thus, identifying the molecular patterns and receptors governing phagocytosis is vital for understanding cancer clearance and transplantation. Recently published studies of the PI revealed novel functions for Vascular Cell Adhesion Molecule-1 (VCAM1) on healthy and malignant hematopoietic stem cells (HSCs). We have found that VCAM1 is highly expressed on healthy HSCs, serving as an innate immune checkpoint for entry into the bone marrow by providing a "don't-eat-me" signal in the context of major histocompatibility complex (MHC) class-I presentation. In addition, we found that leukemia cells exploit this tolerance mechanism to avoid innate immune recognition, suggesting that the VCAM1-receptor axis is a promising target for immunotherapy. However, the specific receptor mediating this interaction remains unknown. In preliminary studies, we employed proteomics and AlphaFold modeling to identify novel VCAM1 receptor candidates on phagocytic cells. Our Specific Aim 1 focuses on identifying the VCAM1 receptor promoting immune tolerance and leukemia evasion and validating its function in vitro and in vivo using mouse and human models of leukemia. Specific Aim 2 will assess the impact of inhibiting or deleting VCAM1 receptor signaling on myeloid and lymphoid leukemia cell clearance, as well as allogeneic transplantation outcomes. Successful completion of this research will advance knowledge of innate immune recognition mechanisms, identify new leukemia immunotherapy targets, and improve outcomes in stem cell transplantation. 1

NCI - National Cancer Institute

PROJECT SUMMARY In the past decade, immunotherapies have revolutionized the way many cancers are treated. By activating the immune system, these treatments enable the body's own defense mechanisms to attack cancer cells and halt tumor growth. Unfortunately, many cancers are refractory to these new therapeutics and there continues to be a desperate need for drugs with novel mechanisms of action to enhance the long-term effectiveness of cancer treatment. Type I interferons (IFN-Is) are attractive candidate drugs to fill this role since these cytokines “interfere” with the growth of tumors; indeed, manufactured IFN-Is have been used in the clinic to treat more than 10 types of cancers but the outcomes were somewhat disappointing. Due to negative feedback regulation of IFN signaling, most interferon stimulated genes (ISGs) are transiently expressed, and these feedback mechanisms ultimately suppress the anticancer effectiveness of administered IFN-Is over time. This process also limits the signaling from intrinsically produced IFN-Is, critical to the success of chemo-, radiation, and immuno-therapy. In pioneering work over many years, we unraveled the details of the negative feedback pathway and identified the ubiquitin protease family member USP18 as the central regulator driving feedback suppression of the IFN-I response. By single-cell RNA-seq analysis, we revealed that cancer stem cells are especially sensitive to USP18 depletion- triggered cell death. These novel and exciting discoveries demonstrate that targeting USP18 represents an effective and promising, yet to-date underutilized, therapeutic option for treating cancer by directly promoting immunogenic cancer stem cell death and by increasing both innate and adaptive immune responses against cancer. With the goal to discover small molecule inhibitors of the USP18-mediated IFN-I feedback loop, we conceptualized an innovative high-throughput screening (HTS) assay based on our novel IFN-I signaling biosensor cell line with CRISPR/Cas9 inserted fluorescent reporters and validated it in a pilot screen of known compounds. Since it is currently unclear which specific mechanisms for USP18 inhibition are druggable by small molecules and provide the best therapeutic window, we opted for a phenotypic discovery approach combined with target deconvolution assays. Additionally, we developed a pipeline of secondary and mechanistic assays that validates the hit compounds and provides initial insight into the targeted components of the feedback pathway. Here we propose to 1) identify inhibitors of the USP18-mediated feedback pathway through a large phenotypic HTS, 2) validate the hits in secondary assays and map their effects to the specific pathway components, and 3) evaluate the therapeutic anti-cancer potential of the final chemical probes. Successful completion of these studies will identify and validate small molecule inhibitors of USP18 that can be used to probe the therapeutic potential of the different mechanisms for inhibiting the USP18-mediated negative feedback regulation of IFN signaling. Additionally, such immunomodulators could be further development toward a new class of cancer therapeutics.

NINDS - National Institute of Neurological Disorders and Stroke

Dentatorubral-pallidoluysian atrophy (DRPLA) is a rare inherited neurodegenerative disease caused by expansion of a glutamine-coding CAG repeat in the Atrophin-1 (ATN1) gene. The role of ATN1 in the adult central nervous system (CNS) is currently unknown. However, the clinical manifestations of DRPLA lead to a constellation of symptoms that are inversely correlated to CAG-expansion (CAGEX) size, with younger individuals typically carrying the largest number of CAG repeats, experiencing the most severe disease burden. Importantly, young patients with DRPLA face poor cognitive outcomes, including developmental delay, with a prevalence of high-frequency, drug-resistant seizures and epilepsy diagnosis. The mechanisms leading to increased neuronal hyperexcitability and seizure risk in DRPLA patients are presently unknown. Thus, preclinical humanized in vitro and in vivo models of ATN1 CAGEX represent a novel platform to expediently assess innovative therapies for symptomatic seizure control and disease modification. Further, these models allow exquisite translational fidelity to define how ATN1 mutations lead to neuronal hyperexcitability. Indeed, our labs have recently identified both an increased neuronal excitability using a versatile patient-specific induced pluripotent stem cell (iPSC)-derived in vitro system and an altered seizure threshold in vivo using a novel mouse model expressing a humanized ATN1 CAG repeat expansion. In vitro studies in human patient iPSC-derived cortical neurons using live-cell calcium imaging and multi-electrode array recordings demonstrate altered neuronal network activity by manifesting increased calcium spike amplitude and hypersynchronization, both characteristics simulating epileptiform-like phenotypes. Notably, we have preliminarily demonstrated that neuronal hyperexcitability (in vitro) and seizure threshold (in vivo) can be rescued with exogenous administration of investigational ATN1- silencing antisense oligonucleotides (ASOs). Further, circadian behavior of ATN1 CAGEX mice administered the investigational ASO were normalized, indicating a disease-modifying effect in this clinically relevant rodent model of DRPLA. The present proposal thus aims to expand these pilot studies to address the mechanism behind ATN1 CAGEX-dependent spontaneous seizure risk and neuronal hyperexcitability in vitro and in vivo, and to further define the potential seizure-modifying effects of ASO infusion in the early disease course. Aim 1 will utilize patient iPSC-derived neuro-glial model to characterize the physiological and molecular mechanism of the DRPLA epileptiform and asses ASO efficacy in phenotypic rescue. Aim 2 will then expand on these in vitro studies to establish whether Atn1 CAGEX mice exhibit spontaneous recurrent seizures and define the degree to which an investigational ASO infusion influences the occurrence of these events and improves neuropathological burden. This study will deepen our understanding of the impact of ATN1 CAGEX on pathological neuronal activity and seizure risk while establishing proof-of-principle evidence that ASO intervention is a disease-modifying strategy for DRPLA, a progressive myoclonic epilepsy syndrome with no palliative or curative options.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Congenital heart disease (CHD) is a heritable, incurable group of birth defects that impact 1% of infants that is likely caused by mutations in the gene regulatory networks (GRNs) that govern heart development. However, the development of therapeutics is hindered by our incomplete understanding of the genetic etiologies underlying CHD. Discovery of the intrinsic GRNs that govern the vast array of cell types essential for heart development is the first step towards the development of therapeutic interventions. By integrating advances in artificial intelligence with genome engineering, emerging evidence from our team suggests that CHD is an oligogenic disorder driven by latent genetic interactions (GIs) between coding and noncoding variants that affect differentiation of multiple non-myocyte cell types critical for organogenesis. In preliminary studies, we employed a base-resolution neural network trained on human fetal heart chromatin accessibility data to identify noncoding variation from CHD probands from multiple cohorts, including NIH-funded programs, that is predicted to disrupt cis-regulatory element (cRE) activity suggesting these regions can act as enhancers. This analysis identified thousands of noncoding variants that are predicted to tune the regulatory activity of the cRE in discrete cell types. To overcome experimental hurdles faced by prior CHD studies that prevented broad association of genetic variants to discrete cardiac phenotypes, we next generated a time-resolved single cell multiome (RNA+ATAC-seq) that includes 200,000 cells during human induced pluripotent stem cell (hiPSC) derived cardiac organoid (cardioid) differentiation. We confirmed this system recapitulates the dynamic, nascent stages of heart development and includes cell states found in the early embryo, creating a robust model system for dissecting developmental mechanisms relevant to CHD. In this proposal, our goal is to connect machine learning tools with the cardioid differentiation model to nominate, characterize and validate complex genetic mechanisms essential for heart development and disease. First, we will employ a single-cell massively parallel reporter assay in cardioids to quantify cell type-specific effects of prioritized noncoding variants, as well as the directionality and magnitude of the variant’s effect (Aim 1). Next, we will infer novel disease-causing genes by creating a cell type and differentiation stage resolved enhancer-gene linkage atlas from our cardioid single-cell multiome data (Aim 2). Finally, we will employ naturally occurring ancestral variation from divergent source populations to identify GIs involved in heart development and disease (Aim 3). Variants uncovered in each aim will subsequently be inserted into endogenous loci in hiPSCs to identify variants that independently disrupt cardiac differentiation versus those that require perturbation of a known CHD signaling axis to perturb differentiation. This proposal rigorously integrates cutting-edge advancements in machine learning and functional genomics to elucidate fundamental GRNs implicated in cardiac differentiation, representing a crucial initial step toward understanding the genetic etiologies of CHD.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

As we age, the efficiency of the hematopoietic system declines resulting in bone marrow failure symptoms in some individuals. Myelodysplastic syndromes (MDS) are disorders primarily affecting older adults and impact around 10,000 individuals annually in the United States. MDS arises due to dysfunction in hematopoietic stem and progenitor cells (HSPCs), leading to ineffective blood cell production and an elevated risk of progression to acute myeloid leukemia (AML). The mechanisms driving the onset and progression of MDS remain poorly understood. Studying the germline and somatic genetics of inherited bone marrow failure syndromes (iBMFS) offers valuable insights into the fundamental processes required to maintain healthy hematopoiesis. While the biology of iBMFS is shaped by specific genetic defects, many of the principles uncovered are broadly relevant to HSPC regulation. Individuals with germline heterozygous loss-of-function mutations in the ATPase DEAD-box Helicase 41 (DDX41) gene tend to develop MDS later in life, resembling the sporadic form of the disease. These patients often present with high-risk MDS characteristics, such as elevated blast counts and increased risk of transformation to AML. Paradoxically, they also tend to have a more favorable overall prognosis compared to others with transforming MDS. This suggests that DDX41-mutant MDS follows a uniquely aggressive yet somewhat protective disease trajectory. We propose that studying this atypical, late-onset iBMFS can reveal broader principles governing hematopoietic fitness and dysfunction during aging. Using a germline ddx41 HET mutant zebrafish that develops age-associated MDS-like symptoms, we uncovered elevated inflammation as well as stem cell stress and quiescence gene signatures enriched in HETs. We will test the hypothesis that DDX41 mutations synergize with age-associated inflammatory signaling and/or secondary somatic mutations to exacerbate hematopoietic dysfunction. In Aim 1, we will examine how DDX41 insufficiency cooperates with age- associated cGAS activity promotes HSPC malfunctioning. In Aim 2, we will explore how co-mutations of DDX41 with CUX1 contribute to aberrant differentiation. Gaining a better understanding of this pathway may lead to the discovery of biomarkers and therapeutic strategies applicable to both hereditary and sporadic forms of MDS.

NEI - National Eye Institute

SUMMARY The optic nerve is comprised of Retinal Ganglion Cells (RGCs), which are the only neurons capable of relaying visual information from the retina to the brain. A variety of conditions contribute to ganglion cell loss, which can result in total blindness. Many therapies have been designed to protect the optic nerve, but to date no therapy has been developed that reverses damage that has already occurred. While regeneration of the optic nerve may seem ambitious, there are several species such as zebrafish that can completely regrow a damaged optic nerve, or replace lost RGCs, providing a roadmap to how this process could be reconstituted in humans. Significant progress has been made towards this goal across various mammalian systems, with particular mutations or transgenic overexpression paradigms leading to partial reconstitution of the regenerative process. These preliminary findings provide strong support that thorough characterization of the regenerative process has the potential to translate into effective therapies. However, several methodological barriers have hampered thorough understanding of RGC regeneration in zebrafish. Firstly, multiple stem cell pools are capable of producing RGCs in zebrafish, which has made it difficult to define the precise sequence of events underlying RGC production. Further, it is unclear what signals tune this regenerative response to ensure that the appropriate number and types of cells are replaced. We have generated the hypothesis that the regenerative response must be initiated by a damage signal derived from dying RGCs. We have designed a series of aims to (1) characterize the process of RGC regeneration in zebrafish with unprecedented detail, (2) identify the origins of the cues that regulate RGC regeneration, and (3) identify factors that drive RGC regeneration even in the absence of RGC loss. Together, these findings will highlight a collection of biomolecules that instruct MG to regenerate the optic nerve, which will have important translational ramifications for the treatment of Glaucoma.

NIA - National Institute on Aging

PROJECT SUMMARY/ABSTRACT The preservation of organ functions, such as eyesight and mobility, is a significant health concern in the elderly population. Organ frailty and disease progression are associated with the dysregulation of tissue homeostasis, which is typically regulated by regenerative units, consisting of adult stem cells (ASCs) and neighboring niche cells that regulate ASC function. Therefore, to better understand why regenerative functions decrease in aged individuals, uncovering how aging alters niche cell types and expression (mRNA) is critical for discovering regenerative therapeutical approaches. The potential to uncover molecular mechanisms to reverse age-related disorders prompted me to spatially profile microenvironmental niches in young, old, and regenerated tissues. I have recently developed Ex-Scope, which integrates Expansion Microscopy and Seq-Scope, a submicrometer-resolution ST (spatial transcriptomic) technology, to obtain a high-resolution multi-Omic method that represents an order of magnitude improvements over Seq-Scope. With the assistance of Dr. Guo, who has extensively worked on planarian tissues, we optimized Ex-Scope to spatially profile planarian tissue. Planarians are capable of regenerating any lost body part, but most importantly, regenerated tissues have a youthful tissue architecture; thus, making them ideal to study tissue homeostasis and rejuvenation. Using mRNA single-cell data on young, old, and regenerated planarians, as a reference dataset (obtained by Dr. Guo), we will provide spatial insight into rejuvenating mechanisms between microenvironmental niches and stem cells. Concurrent, we will demonstrate the advantageous resolution of Ex-Scope by profiling RNA granules in planarian stem cells and oocytes (young, old, and regenerated), which are compartmentalized biomolecules that regulate transcription in stem cells and the establishment of pluripotency. In aim 1) we propose to characterize RNA granules and soluble transcriptomes in planarian stem cells and oocytes, with a hypothesis that the granular structures in oocytes and ASCs would have transcriptome contents distinct from soluble cytoplasm, and 2) we propose to profile microenvironmental niches and their changes during aging and rejuvenation, with a hypothesis that aging and rejuvenation will affect cellular (single cell), tissue-level (microenvironment) and subcellular level (RNA granule) transcriptome, each of which is important for tissue function and homeostasis. We expect that the current work will give us a systematic understanding of how aging deteriorates tissue function by altering transcriptomic structure at both microscopic and macroscopic levels, and how regeneration can reverse it and rejuvenate tissue homeostasis.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY/ABSTRACT Functional dyspepsia (FD) is common, affecting 12% of adults in the United States with high morbidity (e.g., work absenteeism, malnutrition) and healthcare costs. FD symptoms most commonly include early satiation and epigastric pain, worsened by meal ingestion in the absence of clear structural etiology. Precision medicine is lacking due to the complex pathophysiology thought to underlie gut-brain axis dysfunction in FD. Identification of maintenance mechanisms is necessary to determine which existing and new treatments work for whom and why. Gut interoception—how the gut and brain communicate to sense (i.e., attend to), interpret, and integrate gut signals at both conscious and unconscious levels—may be a useful model for understanding dynamic body-to-brain (‘bottom up’) and brain-to-body (‘top down’) processing in FD. This proposal uses multi- disciplinary methods (i.e., functional magnetic resonance imaging: fMRI, resting state functional connectivity, gut connectivity, self-report). We will examine three dimensions of interoceptive processing: gastric attention, interpretation of gastric signals, and gut-brain signal integration. We will contrast gut interoception in adults with FD (n=50) to healthy controls (n=50) and a clinically relevant comparator (anorexia nervosa; n=50) to test our central hypothesis: FD is linked to neural hyper-attention to gastric signals, neural fear-based interpretation of gastric signals, and poor bi-directional gut-brain integration. First, we hypothesize FD will exhibit fasting and pre-meal neural hyper-attention to gastric cues in primary interoceptive regions (insula, anterior cingulate cortex) of the Salience Network (involved in interoception and cognitive/emotional integration). We expect hyperactivation to correlate with a trait-level gut interoceptive awareness. Second, we hypothesize that FD will show pre-and post-meal resting state hyperconnectivity in the primary hub of interoception—the mid insula— and the amygdala (primary limbic region of the Salience Network) alongside hypoconnectivity with the orbital frontal cortex (a primary food-reward region of the Salience Network). Finally, we expect FD to show greater connectivity than controls and AN between the nucleus tractus solitarius (key brain stem region involved in processing interoceptive signals) and the Salience Network, which we expect will correlate with slower gastric motility. Conceptualizing FD pathophysiology within an interoceptive framework has strong potential to advance precision medicine for FD by identifying neural mechanistic targets—hyper-attention (e.g., attention re-training), dysregulated interpretation (e.g., behavioral exposure therapy), and altered integration (e.g., vagal nerve stimulation).

NCI - National Cancer Institute

Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by rapid metastasis, limited treatment options, and worse outcomes in obesity. Yet the mechanisms linking adiposity to tumor aggressiveness remain poorly understood. Our preliminary studies in murine TNBC models and plasma from breast cancer patients identify adipocytederived large extracellular vesicles, termed adipomes (>200 nm), as potent paracrine mediators that enhance invasion, EMT-related transcriptional programs, and cellular reprogramming in both malignant EO771 cells and non-malignant MCF-10A cells. We demonstrate that lipid cargo significantly differs between adipomes derived from tumor-associated mammary fat and non-tumoral mammary fat, indicating that adipome composition closely reflects host metabolic status, tumor pathology, and disease severity. BC-Adipome exposure induces differential CpG methylation at HSPA1B regulatory loci in MCF-7 cells, and HSP70-2 expression is dramatically increased up to 85-fold in EO771 cells exposed to obese versus lean adipomes, consistent with HSPA1B’s established role in proliferation, stemness, motility, and metastatic adaptation. These findings support a mechanistic model in which obesity-modified adipomes act as metabolic-state–dependent epigenetic regulators that activate an HSPA1B-driven transcriptional program promoting TNBC progression, plasticity, and metastatic dissemination. Our central hypothesis is that obesity and tumor pathology reshape adipome composition in ways that reprogram malignant and non-malignant mammary epithelial cells to promote TNBC progression and metastasis. Aim 1 will define how adipomes derived from lean and obese adult TNBC mice regulate EMT, stemness, and transcriptional reprogramming in normal epithelial and tumor cells, identify adipome lipid and RNA cargo responsible for these effects, and map HSPA1B-associated epigenetic remodeling using syngeneic EO771/C57Bl/6 models and integrated multi-omics profiling. Because TNBC patients are highly heterogeneous and obesity-specific adipome effects are difficult to isolate in clinical cohorts, the controlled diet-induced obesity murine model provides a powerful platform to directly dissect how lean- and obese-derived adipomes reprogram epithelial and tumor cells. Aim 2 will identify circulating adipome–regulated CpG methylation signatures and transcriptomic pathways that distinguish early-stage from metastatic TNBC as a function of host BMI, using plasma adipomes from 40–50 de-identified TNBC patients to establish clinically relevant biomarkers associated with progression risk, metabolic status, and metastatic potential. This study is highly innovative because it introduces adipomes as a previously unrecognized stromal driver of TNBC aggressiveness, establishes the first mechanistic link between obesity, adipome cargo, and epigenetic reprogramming via HSPA1B, and integrates murine modeling with patient-derived adipomes to overcome clinical heterogeneity. The work is highly significant because it will define a new adipocyte–tumor communication axis, generate metabolic and epigenetic biomarkers for risk stratification, and identify therapeutic vulnerabilities targeting adipomeregulated pathways, with direct relevance for improving prognosis and treatment strategies in obese TNBC patients

NCI - National Cancer Institute

Project Summary Genetic mutations are crucial in driving cancer initiation and progression. However, while cancer is widely recognized as a genetic disease, not all cells with a high mutational burden become malignant. Conversely, some cells with a very low mutational burden can develop into cancer. This discrepancy underscores the importance of context and the tumor microenvironment in the process of tumorigenesis. Cutaneous squamous cell carcinoma (cSCC) typically arises in the stratified epithelium of the esophagus, head and neck, lungs, and skin through mutations in RAS. Previous studies in the lab have identified a population of cSCC cells, called cancer stem cells (CSCs), that are located at the tumor-stroma interface, rich in TGFβ, tumor-associated macrophages and blood vessels. My preliminary data, from a study in which I was a co-author, shows that upon oncogenic HRAS induction, normal stem cells undergo aberrant crosstalk with their neighbors that leads to a benign state with a few CSCs that then drive cSCC progression. cSCC-CSCs develop a unique gene signature that promotes malignant invasion and therapeutic resistance. I hypothesize that this signature enables cSCC-CSCs to establish the pro-tumorigenic environment for the tumor to survive and grow in. To address this concern, I plan to (Aim 1A) identify what in vivo cSCC-CSC signature genes persist when the cells are cultured under normal skin SC growth conditions and how this differs when in a more SCC- like environment (+serum and TGFβ); (Aim 1B and 1C) perform in vitro and in vivo CRISPR screens to identify which of the CSC signature genes are essential for survival independently of the niche, which depend upon prominent secreted niche factors and which are necessary only within the context of cellular/other components of the niche. With emphasis on niche-specific candidate genes, I will use rapid lentiviral genetics and multiplex immunofluorescence imaging to assess how the loss of key candidates alters the CSC niche (TME) and the mechanism these genes play in driving cancer (Aim 2). If successful, my work will represent a major advancement in our understanding of tumor progression and potentially lead to the development of new therapeutic targets.

NIEHS - National Institute of Environmental Health Sciences

PROJECT SUMMARY Vector-borne diseases such as malaria cause approximately 700,000 deaths each year. To support insect vector control, pyrethroid insecticides are widely deployed. However, the rise of resistance has reduced their effectiveness, prompting the use of combination treatments that pair pyrethroids with chlorfenapyr—an insecticide with a distinct mode of action. Evidence suggests that specific pyrethroid resistance alleles can modulate responses to chlorfenapyr, potentially driving synergistic effects when the two insecticides are combined. This interaction raises concern that similar or novel gene-by-treatment interactions could also influence human responses, complicating chemical risk assessment for genetically diverse human populations. The overall objective of this proposal is to integrate low-cost, high-throughput quantitative genetics in C. elegans with human cell culture to investigate the genes and molecular pathways that influence responses to insecticides alone and in combination. The rationale for the proposed research is that identifying the conserved genes and molecular pathways that harbor natural variants influencing responses to combined insecticide exposure can help inform risk assessment for humans. In Aim 1, we will map quantitative trait loci (QTL) that influence responses to chlorfenapyr and four pyrethroids, both individually and in combination, using wild strains and recombinant inbred lines of C. elegans. In Aim 2, we will identify specific genes and variants within QTL that cause differences in insecticide responses using experimental crosses and CRISPR/Cas9 genome-editing. In Aim 3, we will profile transcriptomic responses to insecticides in C. elegans strains with causal edits and in human HEK293 and HepG2 cells to identify conserved pathways associated with responses to combined insecticide exposure. The innovation of this proposal stems from the integration of high-throughput quantitative genetics in C. elegans with cross-species comparative transcriptomics. This approach has never been used to investigate the effects of natural variation on combined insecticide susceptibility. The proposed research is significant because it will uncover molecular mechanisms underlying natural variation in susceptibility to combined pyrethroid and chlorfenapyr exposures, and identify candidate genes that may harbor susceptibility alleles relevant to human populations. Importantly, this proposal aligns with the objectives of the AREA R15 mechanism by (1) increasing undergraduate involvement in toxicology and genetics research, and (2) strengthening the research infrastructure at the Florida Institute of Technology.

IEEE Foundation

Supports STEM education, technology literacy, and engineering innovation programs that advance technology for humanity.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Abstract The IL-17 family of cytokines is extensively studied and plays a crucial role in immunity and inflammation. While anti-IL-17A or anti-IL-17RA therapies are commonly used to treat patients with plaque psoriasis, they have been unsuccessful in treating inflammatory bowel disease (IBD) and, in some cases, have even worsened the condition. There have also been reports of new-onset IBD in individuals receiving IL-17A-neutralizing therapies. Additionally, the colonic epithelium of IBD patients often accumulates mutations that affect IL-17A signaling. These findings, counterintuitively, suggest that IL-17A responses in the gut may be beneficial. Published studies have shown that IL-17A responses in intestinal epithelial cells are critical for maintaining gut barrier integrity, supporting intestinal Th17 immune responses, regulating epithelial redox balance (via Nox1-H₂O₂), and controlling colonization by segmented filamentous bacteria (SFB). However, there remains a significant gap in understanding of the molecular mechanisms through which IL-17A promotes mucosal host defense. In addition to the epithelium and Lgr5+ intestinal stem cells (ISCs), certain intestinal mesenchymal stromal cell subsets— such as telocytes and trophocytes—also express the IL-17A receptor complex (IL-17RA/IL-17RC). At present, little is known about the role of IL-17A in these stromal subsets. Preliminary studies have identified a previously unreported function of IL-17A in activating stromal cells to induce a fetal-like reprogramming of epithelial cells. The central hypothesis of this proposal is that IL-17RA signaling in stromal cells is essential for epithelial regeneration. Aim 1 will define IL-17A-dependent regulatory pathways in specific intestinal stromal cell subsets. Aim 2 will investigate how stromal cell function supports epithelial regeneration. Aim 3 will explore stromal cell induced epithelial regeneration pathway in IBD. Impaired intestinal epithelial regeneration is a hallmark of IBD. The successful completion of these studies will have broad implications for a range of diseases, both within and beyond the gut, in which tissue regeneration is compromised.

Illinois State Board of Education

STEM education initiative grants for IL to expand science, technology, engineering, and mathematics programs in K-12 and postsecondary institutions.

ITW Foundation

Supports workforce development, STEM education, and community programs in ITW communities worldwide.

National Institutes of Health

The National Institute on Aging (NIA) intends to publish a Notice of Funding Opportunity (NOFO) to solicit applications that propose a program that supports integrative, cross-disciplinary projects aimed at scaling up mechanistic studies to understand the genomic underpinnings of the pathogenesis and progression AD/ADRD.The proposed research projects will employ interdisciplinary approaches that integrate innovative techniques to dissect the genomic drivers of AD/ADRD. These projects will leverage advanced analytical methods, including machine learning and comparative genomic analysis across multiple genetic ancestries or multiple neurodegenerative diseases, along with cutting-edge tools like genome editing, functional characterization assays, and emerging single-cell and spatial omics technologies. These studies will use sophisticated disease models, such as human stem cell-based systems, ex vivo, and in vivo models that reflect different genetic ancestries, various model organisms, or multiple neurodegenerative conditions. These cross-ancestry, cross-disease, and cross-species approaches will enable a more comprehensive investigation of the genomic drivers of neurodegeneration and neuropathogenesis. Ultimately, this initiative may lead to the identification of actionable therapeutic targets for AD/ADRD, meeting the urgent need for mechanism-driven insights that can guide precision medicine approaches for its treatment.Applications are not being solicited at this time. This Notice is being provided to allow potential applicants sufficient time to develop meaningful collaborations and responsive projects. This NOFO intends to utilize the U01 activity code. Investigators with expertise and insights into this area of aging research are encouraged to begin to consider applying for this new NOFO.

National Institutes of Health

<p style="margin-left:0in;">The National Institute on Aging (NIA) intends to publish a Notice of Funding Opportunity (NOFO) to solicit applications that propose a program that supports integrative, cross-disciplinary projects aimed at scaling up mechanistic studies to understand the genomic underpinnings of the pathogenesis and progression AD/ADRD.</p><p>The proposed research projects will employ interdisciplinary approaches that integrate innovative techniques to dissect the genomic drivers of AD/ADRD. These projects will leverage advanced analytical methods, including machine learning and comparative genomic analysis across multiple genetic ancestries or multiple neurodegenerative diseases, along with cutting-edge tools like genome editing, functional characterization assays, and emerging single-cell and spatial omics technologies. These studies will use sophisticated disease models, such as human stem cell-based systems, ex vivo, and in vivo models that reflect different genetic ancestries, various model organisms, or multiple neurodegenerative conditions. These cross-ancestry, cross-disease, and cross-species approaches will enable a more comprehensive investigation of the genomic drivers of neurodegeneration and neuropathogenesis. Ultimately, this initiative may lead to the identification of actionable therapeutic targets for AD/ADRD, meeting the urgent need for mechanism-driven insights that can guide precision medicine approaches for its treatment.</p><p style="margin-left:0in;">Applications are not being solicited at this time. This Notice is being provided to allow potential applicants sufficient time to develop meaningful collaborations and responsive projects.&nbsp;This NOFO intends to utilize the U01 activity code. Investigators with expertise and insights into this area of aging research are encouraged to begin to consider applying for this new NOFO.</p>

NIAID - National Institute of Allergy and Infectious Diseases

Abstract: Decades of research on transplantation have made it clear that the immunological barriers to engraftment are substantial. The goal of this proposal is to generate transgenic mouse models that will allow standardized and orthogonal testing of pathways important for overcoming transplantation barriers, and through this work, instruct efforts in regenerative medicine. We will generate mice that, upon Cre recombinase expression, will express modules of genes that either 1) suppress complement deposition and membrane attack complex formation; 2) express single chain peptide-β2m-MHC trimers to engage inhibitory receptors on natural killer cells; 3) express CD47 to engage the inhibitory receptor Sirpα. These animals can be used as donors for future transplantation experiments to genetically define allogeneic barriers and instruct similar edits in stem cells for regenerative medicine.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Advances in cancer treatment have led to longer survival, making late cardiovascular complications a growing concern in survivorship care. One newly recognized contributor to heart disease in cancer patients is clonal hematopoiesis of indeterminate potential (CHIP), a clonal expansion of hematopoietic stem cells due to acquired somatic mutations. CHIP has been associated with elevated risks of cardiovascular diseases (CVD) and higher all-cause mortality, independent of traditional cardiovascular risk factors. While genotoxic cancer therapies such as radiotherapy and chemotherapy have been implicated in CHIP pathogenesis, the prevalence of therapy-related CHIP and its impact on cardiovascular outcomes remain severely understudied. Furthermore, there are currently no clinical guidelines for screening CHIP in cancer patients, nor validated tools to stratify CVD risk in individuals with CHIP. The proposed study will directly address these unmet needs by defining the relationship between cancer therapies and CHIP development, evaluating the impact of therapy-related CHIP on CVD outcomes, and developing a CVD risk stratification tool tailored for cancer survivors. The research proposal leverages our Vanderbilt BioVU DNA biobank which has recently completed whole genome sequencing of >250,000 patients, including >78,000 cancer patients. The proposal consists of three aims: 1) to identify the prevalence and risk factors for CHIP in cancer patients; 2) to define and predict the impact of CHIP on CVD risk; and 3) to track the longitudinal clonal dynamics of therapy-related CHIP. Successful completion of these aims will lay the groundwork for a precision survivorship model that integrates CHIP status into a comprehensive cardiovascular risk assessment, more personalized cancer treatment strategies, and future interventional trials designed to reduce long-term CVD risk in this growing patient population. Dr. Leo Luo is a radiation oncologist and physician-scientist at Vanderbilt University Medical Center (VUMC). His research and career development will be conducted at VUMC, which offers a rich and collaborative research environment, extensive sources for early-career investigators, and an outstanding track record of successful mentorship. In addition to his primary mentor, Dr. Luo has established a research advisory committee composed of internationally recognized experts in CHIP, cardiovascular research, and bioinformatics. His structured training plan in cardio-oncology, genomic analysis, and epidemiology will develop the skills necessary for him to pursue a research program that is distinct from that of his mentors and facilitate his transition to an independent, R01-funded physician-scientist.