Search Grants — Free, No Account Required

Indiana Department of Education

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

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Project Summary/Abstract For patients with neuropathic bladder from spina bifida or spinal cord injury, the current surgery of using intestine for bladder augmentation causes high morbidity, as well as short- and long-term complications. A recent clinical trial using bioengineered bladder wall showed the feasibility and relative safety of bioengineering bladder wall but failed due to dehiscence and graft contraction from ischemia. While these grafts were engineered from synthetic matrices and autologous urothelium and muscle, they had no blood vessels. Angiogenesis from the patient’s bladder is not fast enough to prevent large graft necrosis since early imbibition and perfusion are limited to the outer perimeter of the graft. As shown in murine and porcine models, bladder vessels will connect (inosculate) with graft vessels within a few days after transplantation to the bladder and facilitate blood flow to the entire graft. Thus, engineering vessels in a large animal bladder graft and evaluating these grafts are the next and final steps before development of grafts for clinical testing. To vascularize matrix grafts, others are trying to “endothelize” grafts by soaking them with stem cells and growth factors in vitro, which creates capillary-like structures rather than organized vessels with lumens. This proposal employs a different strategy where grafts are cellularized and vascularized in vivo on the rectus muscle bed. This ensures optimum graft maturity and a healthy and functional vasculature prior to bladder transplantation. The current proposal is to build upon successes in the rodent and porcine models to design and test endothelial cell ligands to enhance endothelial adhesion and angiogenesis (Aim 1), to evaluate a novel strategy to create long, coronally-directed vessels in large grafts (Aim 2) and to then transplant these matured grafts to the bladder after partial cystectomy in pigs (Aim 3). Grafts will be analyzed grossly to determine size and histologically to determine vessel density, length, perfusion, endothelial/vascular maturity, and epithelial and stromal differentiation. Graft vessel function will be analyzed via perfusion of dyes into the blood stream. Graft function will be assessed by urodynamics to measure bladder capacity and compliance, and by standard biomechanical testing for material and viscoelastic properties. The long-term, translationally directed goals for this project are to produce and evaluate a vascularized graft in a large animal model and to develop vascularized grafts for patients with spinal anomalies or injury. Since bioengineering human bladder wall has proven feasible but not safe or efficacious due to insufficient blood supply, this project has the potential to make bioengineered bladder a realistic treatment option. The directed vascularization technologies developed in this project could be used to improve engineering of other organ tissue.

U.S. National Science Foundation

The Innovations in Graduate Education (IGE) Program is designed to encourage development and implementation of bold, new, and potentially transformative approaches to STEM graduate education training. The program seeks proposals that a) explore ways forgraduate students in STEM master s and doctoral degree programs to develop the skills, knowledge, and competencies needed to pursue a range of STEM careers, or b) support research on the graduate education system and outcomes of systemic interventions and policies. IGE projects are intendedto generate the knowledge required for the customization, implementation, and broader adoption of potentially transformative approaches to graduate education. The program supports piloting, testing, and validating novel models or activities and examining systemic innovations with high potential to enrich and extend the knowledge base on effective graduate education approaches. The program addresses both workforce development, emphasizing broad participation, and institutional capacity-building needs in graduate education. Strategic collaborations with the private sector, non-governmental organizations (NGOs), government agencies, national laboratories, field stations, teaching and learning centers, informal science organizations, and academic partners are encouraged.

NCI - National Cancer Institute

SUMMARY/ABSTRACT The Problem: Cancer cells often reside in environments deprived of growth factors and nutrients. Yet they thrive by rewiring their signaling through autocrine and paracrine “secrete-and-sense” circuits, enabling self- sustaining growth. This phenomenon, known as growth signaling autonomy, is one of the earliest recognized hallmarks of cancer and central to cancer stemness, tumor progression, and treatment resistance. However, the core molecular mechanisms driving these circuits remain poorly defined, limiting therapeutic progress. Central premise: Our data identify GIV (Gα-interacting vesicle-associated protein) as a master regulator of cancer cell signaling autonomy. GIV is a multimodular scaffold protein that integrates signaling across monomeric and heterotrimeric G proteins—elements typically studied in isolation—into a coherent, feed-forward signaling circuit that sustains EGF/EGFR-dependent growth. Endogenously expressed in many breast cancers, particularly triple-negative breast cancers (TNBCs), GIV enables cells to sustain tumor progression under nutrient- and growth factor-limiting conditions. In contrast, ER+ BCs, which often lack endogenous GIV, acquire it via intercellular transfer from stromal neighbors, highlighting a novel mode of proteomic exchange. We hypothesize that GIV promotes cancer stem cell-like states, tumor growth, and drug resistance under nutrient- and growth factor-limited conditions. GIV-dependent cancer cell signaling autonomy may also extend to neighboring GIV-deficient cancer cells via paracrine signaling, enhancing cooperative growth among heterogeneous cancer cell populations. Our team—experts in breast cancer biology, GIV signaling, and in the use of both animal and non-animal models (patient-derived organoids and tissue microarrays) alongside synthetic biology tools (cells with engineered circuits) and quantitative live-cell imaging—is uniquely positioned to test this model through integrated experimental and computational approaches. Our aims are to discover how GIV’s modular domains orchestrate key states of cancer cells driving tumor progression in the setting of: (1) intrinsic autonomy in GIV-expressing TNBCs or (2) intercellular transfer- dependent acquired autonomy in ER+BCs; and 3) establish the cooperative dynamics by which GIV-expressing autonomous cells support non-autonomous GIV-deficient neighbors in heterogeneous tumors. We leverage human organoids and tissue microarrays to preserve translational potential and ensure clinical relevance. Impact: This work will redefine cancer signaling by identifying the first mechanistic framework of secrete- and-sense growth factor autonomy within the EGF/EGFR pathway. It will also chart how a single intracellular hub (GIV) coordinates autocrine and paracrine signaling across diverse cell populations to drive tumor progression. By mechanistically linking cancer growth signaling autonomy to stemness, plasticity, tumor heterogeneity and therapeutic resistance, our findings will uncover new intervention points and provide a transformative conceptual advance in targeting signaling rewiring in breast cancer and beyond.

NHLBI - National Heart Lung and Blood Institute

Project Summary: Genetic heart disease is associated with heart failure and arrhythmias, which can lead to significant morbidity and mortality. Small animal models of genetic heart disease often fail to capture the clinically relevant features of the disease. Patient- and gene-specific therapies, such gene therapy, gene editing, and exon skipping are making their way into the clinic and are in development. In many cases, genetics has provided a clear understanding of underlying patient-specific disease mechanisms by linking a patient’s disease to a specific gene variant. However, individualized therapeutic strategies lag behind this understanding. Recognizing the limitations of animal models, the FDA Modernization Act 2.0 (2022) and subsequent FDA regulatory guidance (2024) now allow for the use of non-animal models including cell- and organoid-based models to assess therapeutic and efficacy. Patient-specific human induced pluripotent stem cells (hiPSCs) can now be readily created from patient cells such as those obtained via a standard blood draw. These cells can then be differentiated in heart-like cells to created hiPSC-derived cardiomyocytes (hiPSC-CMs), offering an platform to test personalized, genotype-specific therapies for heart disease in vitro. HiPSC-CM models can be further improved by using them to create heart-like tissues in the dish, known as engineered heart tissues (EHTs). Commercial platforms now support the creation of these tissues, and contractility measurements can be obtained non-invasively, enabling therapeutic assessment. However, arrhythmia assessments in EHTs remain limited due to the need for specialized optical equipment, the use of toxic contraction inhibitors such as blebbistatin, the need for voltage-sensitive dyes, and the terminal nature of current experimental protocols, which restrict the ability to track therapeutic effects over time. We have recently developed an electromechanically monitored EHT (emEHT) platform that enables simultaneous measurement of contractility and field potentials. This platform leverages flexible electronics technology to noninvasively and concurrently detect electrical signals and assess tissue forces. We have previously demonstrated the ability to simulate arrhythmias using electrophysiology protocols adapted from the clinic with this system. This proposal aims to develop a next-generation emEHT platform capable of simultaneously assessing action potentials and calcium transients in a non-invasive, non-terminal format through the integration of flexible electronics embedded with optical microsensors. Additionally, we aim to develop the molecular tools necessary to leverage this emEHT platform, including the stable expression of genetically encoded voltage and calcium sensors. The platform will be validated against traditional optical mapping techniques and tested using hiPSC-CMs derived from an arrhythmic form of cardiomyopathy. The development of this platform holds transformative potential for assessing arrhythmia propensity in EHTs, ultimately enabling the evaluation of the functional consequences of genotype-specific cardiovascular therapeutics in the dish.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT The three-dimensional organization of eukaryotic genomes plays a crucial role in transcriptional regulation and cellular functions. However, current genome structure models, primarily derived from genomic data, have significant limitations. They lack precise physical dimensions, fail to capture nuclear morphologies accurately, and are constrained by a resolution limit of approximately 200 kb—insufficient for studying interactions between regulatory control regions. These shortcomings hinder the use of 3D genome structures in understanding gene regulation and cellular processes. Recent advances in imaging technologies have provided powerful tools to explore 3D genome organization. In this project, we will develop a probabilistic approach to integrate genomic and imaging data to reconstruct 3D genome structures from thousands of imaged nuclei. We have three aims: (1) Develop integrative methods for inferring high-resolution single cell genome structures from sparse imaging and multi-omics data. This integration minimizes experimental biases and improves resolution and coverage by 100-fold compared to imaging alone. Our approach will offer unprecedented insights into the structural basis of gene regulation, enhancer networks, and the role of chromatin architecture in epigenetic memory formation—insights unattainable through single-cell genome-wide imaging or genomics data alone. (2) Structure-Function Mapping by analyzing the 3D regulatory architecture. We will analyze the 3D regulatory environment of genes in mouse embryonic stem cells and the reorganization of the microenvironment surrounding cell-type-specific long genes in the mouse brain cortex. For the first time, we will systematically classify genes based on their 3D regulatory microenvironment and investigate its role in gene expression. (3) We will expand our Integrative Genome Modeling (IGM) platform to incorporate imaging- based features. The platform generates a population of genome structures to reproduce the input experimental data. We will dedicate significant effort to improve user experience and enhance computational efficiency.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT The global population is aging rapidly, with the number of people over 65 expected to double by 2050. Aging alters hematopoietic stem cells (HSCs), leading to increased inflammation, immune dysfunction, and clonal hematopoiesis. These changes have been linked to hematological disorders, cardiovascular disease, and other age-related conditions. Dissecting the contribution of human HSC heterogeneity to these disorders has been accelerated by single-cell RNA-sequencing and the development of new algorithms to derive of biologically meaningful insights. These algorithms include consensus non-negative matrix factorization (cNMF) and CellAnnoTator (*CAT), which aim to identify consensus gene expression programs that shape cellular heterogeneity across datasets. However, these algorithms have not been applied to human HSC aging, and the circuitry that controls their role in aging and disease remains poorly understood. There is a here is a critical need to identify the molecular programs that drive age-associated changes in human HSCs. The long-term goal is to mitigate age-related immune dysfunction and its associated diseases. The central hypothesis is that inflammatory signaling is a principal driver of human HSC heterogeneity and that AP-1 factors define an HSC subset that expands with age. To test this hypothesis, the investigators will pursue two specific aims: 1) Identify age-associated changes in HSCs across eleven existing datasets, and 2) Determine how consensus gene expression programs shape HSC heterogeneity and relate to aging. For Aim 1, the working hypothesis is that aging HSCs exhibit consistent gene expression changes, including AP-1 activation in a subset that expands with age. Using publicly available datasets, the investigators will identify conserved gene expression and transcription factor activity changes and leverage single-cell data to quantify an aging-associated subset of inflammatory HSCs. For Aim 2, the working hypothesis is that applying cNMF and *CAT within a unified analytical framework will identify biologically meaningful gene expression programs that underlie HSC heterogeneity, including inflammatory pathways linked to aging. The investigators will identify consensus gene expression programs in HSCs, assess their association with age and inflammation, and develop an R-based computational pipeline for broader community use. The expected outcome is the discovery of programs and genes, led by AP-1, that shape heterogeneity of human HSC aging across datasets. The proposed research is innovative because it shifts from evaluating the human HSC compartment as a whole to linking a distinct HSC subset to aging and because it applies an advanced analytical framework to identify consensus gene expression programs shaping HSC heterogeneity. The significance of this research is that it will establish a foundation for predicting how HSC aging impacts immune fitness, systemic inflammation, and cardiovascular risk.

Intel Foundation

Funds STEM education and teacher development programs.

Intel Foundation

Funds STEM education programs for K-12 students, with emphasis on increasing access for underrepresented groups in technology.

NIGMS - National Institute of General Medical Sciences

Tulane University was started as the Medical College of Louisiana in 1834 and has a long track record of training physician scientist leaders in US Medicine including Dr. Michael DeBakey, Dr. Ruth L. Kirschstein, past Director of NIGMS and NIH, and Dr. Clyde Yancy, Chief of Cardiology at Northwestern University. Recognizing the need to train the next generation of physician scientists, the Dean established the Physician Scientist Program in 2002 that provides tuition support for 2 trainees per year from the Dean’s office. Over 90% of these trainees have stayed in academic research and some of the trainees have been awarded independent research grants already. Tulane has made a strong commitment to research with establishing the American Association of University (AAU) recruitment program that has led to the recruitment of several AAU scholars that have increased Tulane’s investigator initiated R01 funding by over 70% in the last five years. Moreover, President Fitts has established the Presidential Chairs of which two reside in the School of Medicine. This strategic investment has greatly increased the training opportunities for MD-PhD students. Recognizing the need to expand this program, Tulane has developed this MSTP application to take advantage of the exceptional training faculty in the School of Medicine, the School of Public Health, and the School of Science and Engineering. This program takes advantage of several pipeline programs already established at Tulane to target STEM based students in clinical and bench research and provide these undergraduates, the skillset to be competitive applicants to the MSTP program. Moreover, this program will complement the NHLBI funded R38 that focuses on the resident pipeline. The goal of TuLEAD is to train a cohort of MSTP students in innovative research in the areas of infectious disease, immunology, cardiovascular and renal physiology, and pharmacology. Aim 1: Tulane MSTP will develop a highly innovative national and local campaign to identify and encourage meritorious students to train as physician scientists and provide them with rigorous dual degree training in clinical medicine and in wet-lab or dry-lab (or both) research. Aim 2: We will train clinician-scientists with the necessary qualifications to conduct rigorous scientific research and engage in clinical and translational research across the spectrum of human disease. Training will be a through a combination of didactics, simulation, and state of the art rigorous research training. Aim 3: A key component of physician scientist development is not only learning and conducting rigorous research but to also serve as educators for the next generation. With the interaction of this program with Tulane’s undergraduate programs we well our summer research programs, MSTP trainees will also have the opportunity to serve as mentors. We have strong relationships with several undergraduate universities in New Orleans (such as Dillard University), and an established summer program for their undergraduates to work in Tulane laboratories, with current MD/PhD students mentoring one on one.

U.S. National Science Foundation

The International Research Experiences for Students (IRES) program supports international research and research-related activities for U.S. science and engineering students. The IRES program contributes to development of a diverse, globally engaged workforce with world-class skills. IRES focuses on active research participation by undergraduate and/or graduate students in high quality international research, education and professional development experiences in NSF-funded research areas. The overarching, long-term goals of the IRES program are to enhance U.S. leadership in science and engineering research and education and to strengthen economic competitiveness through training the next generation of science research leaders. IRES focuses on the development of a world-class U.S. STEM workforce through international research experiences for cohorts of U.S. students. Student participants supported by IRES funds must be citizens, nationals, or permanent residents of the United States. Students do not apply directly to NSF to participate in IRES activities. Students apply to NSF-funded investigators who receive IRES awards. To identify appropriate IRES projects, students should consult the directory of active IRES awards. All PIs, co-PIs and Senior Personnel on IRES proposals must be from U.S. based organizations. Personnel from international partners should be listed as "non-NSF funded collaborators." Guidance on information to provide for "non-NSF funded collaborators" is found in Section V.A. IRES projects engage a group of undergraduate and/or graduate students in active high-quality collaborative research, in principle at an international site with mentorship from international researchers. IRES projects must be organized around a coherent overarching intellectual theme that may involve a single discipline or multiple disciplines funded by NSF. For all IRES proposals, PIs are strongly encouraged to outline a variety of virtual, hybrid or other alternative approaches to strengthen and maintain international collaboration in addition to travel. It is expected that these approaches will extend collaboration beyond the actual international trip and strengthen IRES proposals overall.

National Park Service

Objectives of the Internships in Marine Geography Program are to: encourage a diverse group of young adults to study and pursue careers in Science, Technology, Engineering and Mathematics (STEM) fields; provide meaningful and relevant science-based internships to individuals from underrepresented groups in STEM fields; introduce program participants to science careers at NOAA; and increase relevancy, diversity, and inclusion in the NOAA workplace.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

SUMMARY Developmental gene expression is tightly regulated by the dynamic interplay of H3K4 methylation (H3K4me) and H3K27 methylation (H3K27me) associated with active and repressed genes, respectively. However, our understanding of the individual and combinatorial roles these histone modifications play in adult physiological contexts remains incomplete. To overcome these limitations, we have recently generated histone mutant transgenic tools to uncover a previously unappreciated role for H3K4me in adult hematopoiesis. Adult mice globally depleted for all forms of H3K4me via expression of an inducible histone H3 lysine-4-to-methionine (H3K4M) mutant allele succumbed to a severe loss of all major mature blood cell types. Unexpectedly however, H3K4M-expressing hematopoietic stem cells (HSCs) and most committed progenitors were present at normal numbers and persisted upon transplantation into recipient mice, suggesting that H3K4me is dispensable for the maintenance and early commitment of HSCs and progenitors but essential for the terminal maturation of progenitors. Mechanistically, we showed that H3K4me opposes the deposition of repressive H3K27me at differentiation-associated genes bivalently marked by H3K4me3 and H3K27me3 in HSCs or progenitors. Indeed, by concomitantly suppressing H3K27me in H3K4me-depleted mice with an H3K27M transgene, we could rescue the acute lethality, hematopoietic failure and gene dysregulation. Thus, our results reveal that H3K4me guides hematopoiesis by opposing repressive H3K27me at fate-instructive bivalent genes, providing the first evidence for the functional interaction between these crucial chromatin marks in mammalian tissue homeostasis. These preliminary data raise fundamental questions with clinical relevance that will be addressed in 3 complementary aims. In Aim 1, we will further define the consequences of H3K4me loss on the function of HSCs and progenitors using self-renewal and differentiation assays. Additionally, we will assess whether any observed defects are reversible upon restoration of H3K4me. In Aim 2, we will identify epigenetic regulators that mediate the H3K4M- dependent arrest and the H3K27M-dependent rescue by purifying proteins associated with H3K4M and H3K27M; measuring changes to all major histone modifications; and testing select candidates for their ability to phenocopy the effects of H3K4M and H3K27M. In Aim 3, we will dissect the molecular basis by which H3K4me/H3K27me safeguard hematopoiesis with a focus on fate-instructive cytokine receptors and transcription factors dysregulated in H3K4M mice but normalized in H3K4M/H3K27M mice. Moreover, we will investigate the contribution of other epigenetic marks to the H3K4M phenotype using DNA methylation inhibitors and a novel histone mutant library. Collectively, this proposal will leverage novel tools to probe the direct, physiological impact of two antagonizing chromatin marks on hematopoiesis. As arrested differentiation and disrupted H3K4me/H3K27me have been implicated in diverse hematological conditions, our results will elucidate the underlying mechanisms and may pave the way for novel therapeutic interventions.

NIA - National Institute on Aging

Frontotemporal dementia caused by mutations in microtubule-associated protein tau (MAPT), including the N279K mutation, is a common cause of early-onset dementia. It is neuropathologically characterized by toxic aggregation of hyperphosphorylated tau, glial activation, and neurodegeneration. The factors contributing to the disease are likely numerous and poorly understood, and no disease-modifying therapies exist for FTD. Oxidative stress (OS) occurs when a cell’s innate antioxidant system is overwhelmed by reactive oxygen species, and oxidative modifications of biological molecules have important consequences on protein, DNA, and lipid function. In particular, uncontrolled lipid peroxidation can lead to ferroptosis, a specific cell death pathway which we found to be enriched in FTD postmortem brain and may contribute to neurodegeneration. We also identified an OS and neuroinflammatory phenotype in postmortem brain from FTD patients and induced pluripotent stem cell (iPSC)-derived neurons from FTD patients. Specifically, FTD iPSC-derived neurons show upregulation of the gene secreted phoshoprotein-1 (SPP1) and its protein product osteopontin (OPN), which can activate iPSC-derived microglia in vitro. Given the centrality of OS in our FTD models and the apparent association with SPP1, this proposal seeks to investigate mechanisms of OS generation and downstream sequelae in FTD. In aim 1, I will interrogate the effects of different classes of oxidative and ferroptotic stressors on FTD MAPT N279K iPSC-derived neurons. In aim 1a I will assess cell viability and lipid peroxidation. In aim 1b I will assess tau pathology and neurite outgrowth. In aim 1c I will attempt to rescue any effects seen in aims 1a and 1b by co-treating with antioxidant and ferroptosis inhibiting compounds. In aim 2 I will characterize astrocyte-neuron crosstalk in the FTD context. First, in aim 2a I will generate iPSC-derived astrocytes from FTD MAPT N279K patients or healthy control patients and treat with OPN and assess for astrocyte reactivity. In aim 2b I will generate antioxidant response gene reporter astrocytes and treat with Ctrl or FTD neuron conditioned medium to determine the role of neuron-secreted factors in astrocyte response. Finally, in aim 3 I will explore the potential of targeting OS in FTD. I will xenotransplant FTD or Ctrl neural progenitor cells into mice forebrains and treat systemically with liproxstatin, an antioxidant and ferroptosis inhibiting compound. In aim 3a I will characterize proteins involved in these pathways as well as glial reactivity and graft survival by histology. In aim 3b I will perform snRNA-seq on micro dissected grafts to map changes in gene expression profiles in response to OS targeting.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Project Summary Gastrointestinal (GI) disorders are among the most common comorbidities in patients with Autism Spectrum Disorder (ASD). The Enteric Nervous System (ENS), composed of neurons (ENs) and glia, is crucial in regulating various aspects of gut physiology. Animal models show GI motility impairments linked to altered expression of ASD-associated genes. However, recent advancements in single-cell genomic technologies have revealed remarkable molecular diversity among ENs and highlighted significant differences in ENS gene expression patterns across species. These findings underscore the need for human-specific models to recapitulate the human ENS molecular heterogeneity and dissect the cell type-specific contribution to the GI endophenotype in ASD. Under the mentorship of Dr. Giorgia Quadrato and Dr. Jason Spence, leaders in the field of the human neural and intestinal organoids, respectively, Dr. Birtele will use a human induced pluripotent stem cell (iPSC)- derived model that includes both ENs and intestinal organoids (HIOs). Using a mix-and-match approach, patient- derived neurons co-cultured with healthy intestinal cells will isolate ENS-specific contributions to GI dysfunction. Conversely, healthy neurons cultured with patient-derived intestinal organoids will reveal non-neuronal contributions. Aim 1 (K99 phase) will study the role of SYNGAP1, a top ASD gene, in GI dysfunction. ENs will be derived from a SYNGAP1 haploinsufficient-patient derived and isogenic control iPSCs line under the mentorship of Dr. Martin Garcia-Castro, expert in neural crest differentiations. Under the guidance of Dr. Jason Spence, Dr. Birtele will generate mixed and matched ENs-HIOs. Dr. Birtele will analyze mixed and matched ENs-HIOs to determine cellular and transcriptional changes caused by SYNGAP1 haploinsufficiency. In Dr. Spence's lab, Dr. Birtele will transplant ENs and ENs-HIOs in vivo to assess GI motility and peristaltic function. Additionally, under the mentorship of Dr. Unmesh Jadhav, an expert in epigenomics and intestinal stem cells, Dr. Birtele will examine the effect of SYNGAP1 haploinsufficiency on intestinal stem cell chromatin accessibility profiles by performing single-cell ATAC-seq on mixed and matched ENs-HIOs. Given the high comorbidity of GI dysfunction across many genetic forms of ASD and the enrichment in expression of these genes in ENs, Aim 2 (R00 phase) I will perform an high-throughput screening for molecular and functional impairments in ENs cultures by applying gapmer antisense oligonucleotides (ASOs) under the guidance of Dr. Justin Ichida, leader in the field of ASOs, to knock-out 35 top ASD-associated genes.Top candidates identified in this initial screen will be validated using patient-derived lines differentiated into ENs and HIOs and cultured following the mix-and-match approach. By applying a similar pipeline of experimental procedures as in Aim1, I will compare the functional and molecular profiles of in vitro and transplanted organoids to dissect possible convergent molecular mechanisms through which ASD-associated genes contribute to GI dysfunction This research will uncover molecular mechanisms governing ENs function and provide critical insights into ASD-related GI dysfunction. 1

NHLBI - National Heart Lung and Blood Institute

Project summary Kinase inhibitors (KIs) represent critical advances in cancer treatment, yet their cardiac toxicity profiles are poorly understood. Recent clinical reports highlight significant cardiotoxicity associated with osimertinib, the sole approved therapy for EGFR T790M-positive non-small cell lung cancer (NSCLC). Approximately 3-5% of patients experience clinically significant cardiac adverse effects, including reduced left ventricular ejection fraction, heart failure, and arrhythmias, leading to treatment interruptions or discontinuation. Despite this significant clinical challenge and potential negative impact on patient survival, the underlying molecular mechanisms remain unexplored. Our preliminary studies in mouse models demonstrate early cardiac dysfunction linked to mitochondrial reactive oxygen species (mtROS) generation and increased NOX4 expression following osimertinib treatment. Clinical evidence also suggests that cardiac dysfunction, although often reversible, can seriously compromise the continuity of cancer treatment, emphasizing the urgent need for effective preventive strategies. We propose mitochondrial oxidative stress as a key driver of cardiotoxicity of osimertinib, warranting further mechanistic investigation. This research aims to elucidate the molecular mechanism of osimertinib- induced cardiotoxicity and to evaluate therapeutic strategies targeting mitochondrial dysfunction and oxidative stress through three specific aims. Aim 1 will determine the mitochondrial mechanisms underlying osimertinib-induced cardiotoxicity. Utilizing human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we will measure mitochondrial function (oxygen consumption rate, mtROS levels, mitochondrial dynamics) to validate our hypothesis that mitochondrial bioenergetics disruption drives cardiac injury. Aim 2 will assess the role of NOX4 in mediating cardiac dysfunction. We will use transgenic and knockout mouse models, specifically altering cardiac NOX4 expression, to define its contribution to osimertinib-induced oxidative stress and mitochondrial impairment. In aim 3 we will evaluate cardioprotective strategies with mitochondrial-targeted antioxidants and NOX4 inhibitors. We will test MitoQ (mtROS scavenger) and setanaxib (NOX4 inhibitor), individually and in combination, to assess their efficacy in preventing osimertinib- induced cardiac damage both in vitro and in vivo. This study addresses a critical clinical issue by uncovering novel molecular insights into KI-induced cardiotoxicity, specifically identifying mitochondrial oxidative stress and NOX4 as therapeutic targets. Our findings aim to mitigate cardiac side effects associated with osimertinib, enhancing the clinical safety and efficacy of targeted cancer therapies, and thereby enabling uninterrupted cancer treatment and improving patient outcomes.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY Splicing factors (SFs) are RNA-binding proteins that regulate alternative splicing (AS), enabling a single gene to produce a variety of mRNA transcripts and corresponding proteins. AS plays an integral role in development, cancer, and aging, and many SFs are essential for embryonic development. Therefore, SF levels must be tightly controlled to maintain proper gene expression, which can be achieved through the AS of poison exons (PEs) within their own transcripts. PEs within SF transcripts, or SF-PEs, introduce premature termination codons, triggering nonsense-mediated decay (NMD) to reduce SF protein levels, a process known as AS- NMD. Conversely, PE skipping increases SF abundance. Prior studies highlight SF-PEs as critical for cancer cell survival, but their role in non-cancerous cells remains unclear. The goal of this proposal is to determine how SF-PEs maintain SF homeostasis to modulate transcriptomes that sustain pluripotency and differentiation. Our preliminary data suggest that PEs in Srsf3 and Tra2b, two SFs linked to cancer and developmental disease, are essential for pluripotent stem cell survival and embryonic viability. However, the morphological, functional, and transcriptomic effects of PE knockout remain unclear, as does the broader role of SF-PEs in pluripotent stem cell survival. We hypothesize that SF PEs fine-tune pluripotency by buffering SF gene expression and modulating AS of target genes critical for maintaining pluripotent cell viability. Aim 1 will utilize an in vivo reverse genetics approach and long-read RNA sequencing (LR-seq) to characterize how Srsf3- and Tra2b-PEs shape mouse embryonic development. Aim 2 will investigate SF AS-NMD dynamics in vitro using a high-throughput CRISPR-based exon deletion screen to identify SF-PEs essential for iPSC viability. Conditional knockout iPSC models will be engineered to assess effects of SF-PE knockout on transcriptomes using LR-seq, SF target binding using eCLIP, and differentiation phenotypes using functional assays. Successful completion of these Aims will elucidate how SF-PEs modulate transcriptomes, safeguard cell pluripotency, and drive differentiation. This Fellowship will provide me essential training in RNA splicing, stem cell biology, genomics, and scientific communication—critical for my future career as a physician-scientist translating basic research into clinical applications.

NCI - National Cancer Institute

Project Summary PD-1 pathway targeting antibodies have improved patient outcomes in lung adenocarcinoma (LUAD). Unfortunately, most LUAD patients do not yet benefit from these therapies, and it is not clear why. Robust responses to PD-1/PD-L1 blockade require that the intratumoral CD8 T cells are in a progenitor-exhausted (TPEX) state, as TPEX cells proliferate and give rise to cytotoxic effector CD8 T cells (TEFFs). Yet, we and others have found that tumor-specific TPEX cells are primarily housed in the tumor-draining lymph node (tdLN) associated with the lung. Via migration, these cells continually replenish the tumor migration, underscoring the critical role of the tdLN as a reservoir of stem-like CD8 T cells. However, because the tdLN is the site of long- term maintenance, we hypothesize that the biology of tumor-specific TPEX and their differentiated progeny is shaped by the interactions and signals they receive in this site. Here, we propose in-depth studies on the mechanisms controlling the differentiation and maintenance of TPEX populations in the tdLN. Our proposal integrates genetically engineered LUAD models, CRISPR-based perturbations, and single-cell approaches to dissect this process. Specifically: 1. We will define how KLF2 and T-bet prevent exhaustion by repressing exhaustion-related genes (e.g., TOX) and implementing cytotoxic effector programs as T cells differentiate across the tdLN and tumor. 2. We will determine how IL-21–BATF signaling impacts on TPEX → effector CD8 T cell transitions, and the role of KLF2 in this process. We previously showed IL-21 is provided by T-follicular helper CD4 T cells in the tdLN, and we will leverage models with and without TFH responses to pinpoint how IL-21 signaling promotes CD8 T cell cytotoxicity and limits exhaustion. 3. KLF2 is transiently downregulated by TCR signals. We will determine if KLF2 downregulation is necessary for differentiation in the tdLN and the role that TCR-dependent signals play in maintaining T cell stemness in the tdLN. Our studies will investigate immune signaling pathways and transcriptional networks regulating CD8 T cells in the tdLN, elucidate mechanisms for the provision of IL-21 and its role in driving effector function, and explore the interplay between TCR and KLF2 in shaping CD8 T cell fate. These insights will shed light on immunoregulatory mechanisms that determine whether tumor-specific CD8 T cells maintain anti-tumor functions or become dysfunctional. By illuminating the biology of the tumor-specific TPEX cells in the tdLN reservoir, our goal is to identify entry points for mobilization or reprogramming through targeted interventions, to boost the efficacy of therapies against LUAD.

NIGMS - National Institute of General Medical Sciences

ABSTRACT Essentially all transcription occurs in stochastic and episodic bursts, conferring flexibility, adaptability, and diversity to otherwise identical cells. Regulating this `bursty' transcription in a timely and context-appropriate manner is key to proper development and homeostasis. Its misregulation causes an imbalance between dynamically counteracting genes and improper gene dosage compensation, often leading to various human diseases, including cancer, cardiovascular disease, metastasis, and infertility. However, molecular mechanisms underlying transcriptional burst regulation remain elusive due to the lack of proper in vivo models and precise long-term assays. Also, the results from previous studies often conflict with each other, hampering our precise understanding and therapeutic advancements. Our overarching goal is to elucidate the molecular mechanisms underpinning spatiotemporal regulation of in vivo transcriptional bursting during development, homeostasis, and disease, and discover new factors controlling its context-specificity and adaptability. Recent studies, including our work monitoring transcriptional dynamics of endogenous Notch target genes in live adult C. elegans, contradict the previous findings: the burst duration is the major parameter regulated in vivo, whereas burst frequency is the major target of regulation in vitro. What causes these discrepancies? What modulates the burst behaviors in a context-specific manner and how? To address these questions, we will use the C. elegans gonad as an in vivo transcriptional burst study model with our innovative approach, combining long- term single-molecule live RNA imaging, machine learning-based analysis and modeling, and bioinformatics to analyze burst dynamics regulation in vivo. Focusing on the burst dynamics of powerful and well-characterized Notch pathway, we will determine the precise roles of core Notch cis- and trans-regulatory elements (CREs and TREs) like promoters, enhancers, and mediators in transcriptional burst regulation both in in vivo and in vitro contexts. We will also define the novel functions of the transcriptional co-activator LAG-3 (MAML in humans) for context-specific regulation of transcriptional dynamics, focusing on its functions for biocondensate formation and chromatin modifications. Our results will fill the critical gap in knowledge about in vivo transcriptional bursting and greatly advance our understanding of transcriptional regulation and stem cell control, with the potential to discover new therapeutic targets and strategies for Notch-related diseases and infertility.

NCI - National Cancer Institute

PROJECT SUMMARY Tumor draining LNs (tdLN), are harbingers of aggressive disease, where the presence of metastases signals risk for recurrence and poor survival in melanoma patients. The tdLN basin, however, is also antigen-rich and may promote immune reinvigoration on immunotherapy. Indeed, recent neoadjuvant trials demonstrate increased efficacy when immune checkpoint blockade (ICB) is delivered prior to surgical resection, which may depend in part on the tdLN basin. Given that large-scale clinical trials failed to demonstrate the benefit of prophylactic, complete LN dissection in high-risk, LN-positive melanoma patients, there is an opportunity to consider the therapeutic potential of tdLNs as key hubs for continued tumor immune surveillance. Future progress, however, depends upon a mechanistic understanding for how anti-tumor immune surveillance in tdLNs is maintained and the impact of standard of care clinical therapy. Recent studies, both preclinical and clinical, have identified a subset of stem-like memory (TSL) cells CD8+ T cells that are produced as a function of suboptimal antigen presentation and are enriched in tdLNs. These TSL are reinvigorated upon ICB and required for response to therapy. Despite the fact that TSL are required for response to immunotherapy in mice and associated with outcome in patients, however, we lack an understanding for what might determine their differential abundance or functionality in situ. The underlying hypothesis of the proposed work is that maintaining TSL in the draining lymphatic basin will support systemic immune surveillance in patients. We therefore leverage our deep expertise in the lymphatic system, paired with new tools to track and perturb specialized T cell populations in the context of melanoma to generate mechanistic insights that can guide future strategies for clinical management of the lymphatic basin in the context of neoadjuvant therapy. We propose that understanding the mechanisms that maintain LN TSL will lead to new strategies to boost systemic immune surveillance. Successful completion of this work will aim to 1) map the differentiation trajectory of egressing CD8+ T cells as they seed draining LNs; 2) determine the dependence of TSL on lymphatic transport; and 3) define the TSL niche in mouse and human. We expect that the basic immunological insights generated here can be used to guide the application of neoadjuvant therapy in melanoma and other solid tumors. Further, this work will nominate new candidate targets or therapeutic schedules to improve local tumor control and protect against tumor recurrence and distant metastasis.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY/ABSTRACT Tregs play a crucial role in maintaining immunologic tolerance and preventing autoimmune diseases. Current treatments for these conditions often involve immunosuppressive medications, which can have harmful side effects and limited effectiveness. Our research aims to unlock new possibilities in stem cell science by manipulating the expression of the transcription factor FOXP3, the master regulator of Treg development, during T cell differentiation of induced pluripotent stem cells (iPSCs). This work seeks to understand how FOXP3 expression can be most effectively regulated during iPSC differentiation and the impact of specific approaches on T cell differentiation. I propose two specific aims to achieve this goal: Aim 1 explores the effects of introducing an exogenous source of FOXP3 on iPSC differentiation. We will examine how different levels, timing, and isoforms of exogenous FOXP3 expression influence Treg development and functionality. Aim 2 focuses on identifying and manipulating Notch signaling effectors to direct Treg lineage commitment. We will create a comprehensive gene regulatory network and employ machine learning through the Python library CellOracle to model transcription factor perturbations for candidate genes in silico. To achieve these aims, I have applied new strategies to an in vitro model of T cell development, the artificial thymic organoid (aka ATO), developed by our group. The ATO platform is currently the only in vitro system that robustly supports mature CD4+ T cell production through the developmental stages that mirror conventional thymopoiesis. I have effectively increased FOXP3 expression during iPSC differentiation in the ATO model using the following methods: constitutive overexpression via lentiviral transduction, CRISPR- Cas9 knock-in for stage-specific expression, and small molecule modulation. This multi-faceted approach allows for the mechanistic investigation of Treg development from iPSCs and will provide foundational knowledge for generating iPSC-derived Tregs as adoptive cell therapy for autoimmunity. Expected outcomes of this work include a detailed understanding of how FOXP3 expression levels and timing affect Treg development. We will also define the regulatory role of Notch signaling on FOXP3 expression for this process. This knowledge will facilitate the development of future Treg therapies, offering new hope for patients with autoimmune diseases. Our work will enhance the mechanistic understanding of iPSC differentiation into the Treg lineage and propel research in stem cell-based therapies for autoimmunity. By developing a robust platform for Treg generation from iPSCs, our project holds the potential to transform autoimmune disease treatment and advance the field of stem cell-based therapies.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY/ABSTRACT The finely tuned generation and function of regulatory T (Treg) cells are essential for maintaining the balance that allows for protective immunity while preventing harmful autoimmunity. Treg cells are heterogeneous, comprising specialized subsets that contribute to tissue repair and mediate context-specific immune responses. Despite their critical roles in essential biological processes, it remains unknown whether the subset-specific functions of Treg cells are driven by their developmental origins, transcriptional programs, or a combination of both. This unresolved challenge largely stems from two issues: the lack of unbiased means to identify mutually exclusive Treg cell subsets with distinct functions, and the absence of tools to trace their ontogeny. However, my recent discoveries have opened promising avenues for overcoming these obstacles. Using colorectal cancer models and human patient specimens, I identified that interleukin-10 (Il10) expression distinguishes two subsets of Treg cells with opposing functions: IL-10+ Treg cells, which exhibit anti-tumor properties, and IL-10– Treg cells, which promote tumor growth. Furthermore, I identified Dapl1 as a gene uniquely expressed by naïve CD4 T cells, thereby providing a definitive marker for extrathymically generated Treg cells. The overarching goal of this research proposal is to determine whether the developmental origins of IL-10– vs IL-10+ Treg cells contribute to their distinct functions, and to identify the transcriptional programs underlying these differences. This proposal tests the hypothesis that both of these subsets are of mixed developmental origins, with their distinct functions driven by differentially expressed transcription factors. Specifically, in Aim 1, using a novel Dapl1-based lineage tracing model, I will determine whether IL-10+ and IL-10– Treg cells arise from thymic or extrathymic (peripheral) origins and elucidate how these developmental pathways shape their functions. Additionally, in Aim 2, I will define the transcriptional programs that drive their subset-specific activities, by inducing Treg cell specific deletion of key regulators such as Zeb2 and Nfil3. By employing genetic mouse models, advanced single-cell analyses, and CRISPR-based screening, the proposed studies will reveal the nature of Treg cell functional heterogeneity, ultimately guiding the development of more precise immunotherapeutic strategies with major implications for public health. The proposed career development plan complements my training in cellular and molecular immunology with single-cell analysis and computational biology. I will take advantage of the extensive resources of the Memorial Sloan Kettering Cancer Center, part of the Tri-Institutional network with the Rockefeller University and Weill Cornell, as well as benefit from the mentorship of Dr. Alexander Rudensky and guidance from Advisory Committee members Dr. Christina Leslie, Dr. Ming Li, and Dr. Steven Josefowicz. By the end of the mentored phase, I will have acquired the necessary tools to conduct comprehensive studies at the intersection of immune cell heterogeneity and immune communication with the environment as an independent investigator.

NIAID - National Institute of Allergy and Infectious Diseases

Sepsis is a leading cause of death worldwide, with most patient mortality stemming from lingering immune dysfunction in sepsis survivors. A key feature of sepsis-associated immune dysregulation is monocyte exhaustion, a phenotype of paradoxical pro-inflammatory and immunosuppressive gene expression, impaired differentiation, and reduced antigen presentation. Monocyte exhaustion can persist for years after sepsis onset, a result of long-term immune memory. However, the mechanisms controlling such long-term memory remain to be elucidated. Whereas previous research has conceptualized innate immune memory through diametrically opposed mechanisms that either promote (train) or restrict (tolerize) monocyte responses, my preliminary data suggests that exhaustion represents a distinct memory state characterized by unique immune, transcriptional, and epigenetic features. Therefore, in contrast to the two-state model for innate memory, I hypothesize that innate memory represents a continuum of states driven by distinct epigenetic patterning, with prolonged, high- intensity immune stimulation leading to monocyte exhaustion in septic individuals. In Aim 1 of my proposed study, I will profile the unique transcriptional and epigenetic features defining monocyte exhaustion, as well as employ integrative modeling to determine how immune stressor strength, duration, and timing influence the establishment of distinct innate memory states. In Aim 2, given preliminary data showing genome-wide DNA hypermethylation in exhausted monocytes, I will test the hypothesis that inhibition of DNA demethylation enzyme TET2 is upstream of these epigenetic changes, and that treatment with TET agonists is a tractable therapeutic strategy to restore healthy epigenetic memory. Finally, in Aim 3, based on my recent identification of a novel DNMT3L isoform expressed in septic monocytes, I will test the altered chromatin affinity and regulatory activity of this isoform and establish its contribution to DNA methylation reprogramming during monocyte exhaustion. Completion of these proposed Aims will allow me to develop skills in new experimental techniques, including single-cell RNA sequencing, reduced representation bisulfite sequencing, in vivo mouse sepsis modeling, and cytometric arrays. Aims 1 and 3 will be pursued during the K99 mentored research phase at Virginia Tech in the laboratory of Dr. Liwu Li, an expert in the fields of monocyte biology and innate immune memory. Whereas my previous graduate studies focused on epigenetics and mammalian development, Dr. Li will provide valuable instruction as I expand into the topics of immunology and hematology. I will also pursue coursework at Virginia Tech in computational modeling of biological systems while engaging with professional development workshops covering such topics as scientific communication, mentorship, and R-series proposal development. The goal of this project is ultimately to pursue a career as an independent biomedical investigator in academic research; these studies will serve as a foundation for my own research program aimed at identifying the major molecular players responsible for establishing and maintaining innate immune memory.

NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

Abstract Polycomb group (PcG) complexes are multi-protein, evolutionarily conserved epigenetic machineries that regulate stem cell fate decisions, cell identity and early development. The PcG machinery can be divided into two major complexes: Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2). Traditionally, PcG complexes are associated with gene repression mainly via histone-modifying activities. While PRC2 catalyzes methylation on lysine 27 of histone H3 (H3K27me1/2/3) via EZH1/2, PRC1 deposits a ubiquitin group at lysine 119 of histone H2A (H2AK119ub1) via the E3-ligases RING1A/B. Interestingly, several PcG encoding genes are found to be mutated in individuals with developmental disorders. Specifically, de novo missense mutations in the genes encoding for RING1A (RING1), and RING1B (RNF2), have been found in pediatric patients with neurodevelopmental disorders. How mutations at PcG genes impair development in humans is completely unexplored. Additionally, we have discovered novel missense mutations in both genes in children with intellectual disabilities. We conducted predictive analyses using crystal structures to start understanding how these mutations affect PRC1's stability and interaction with nucleosomes. In this proposal, we will focus our efforts in one of the RNF2 mutations, which is associated with intellectual disabilities using novel knock-in ESC lines as well a new mouse model carrying a monoallelic missense mutation on RNF2. Preliminary data reveal that mutant RING1B disrupts Polycomb complex assembly, induces derepression of PRC1 and PRC2 target genes, and impaired differentiation into neurons. By ChIP-seq and mass spectrometry we will investigate chromatin occupancy and recruitment mechanisms and potential rescue strategies. Additionally, this proposal will examine how Rnf2 mutations impact hippocampal structure, and behavioral outcomes in mice. Immunohistochemistry, RNA-seq, and ATAC-seq will determine the cellular diversity and regulatory dynamics in the hippocampus, providing insights into the mutation's molecular and behavioral consequences. Overall, our proposed research aims to define the role of missense mutations in Polycomb genes in neurodevelopment in vitro and in vivo, examining epigenetic mechanisms, behavior, and neuronal architecture. This work will enhance our understanding of how missense mutations influence PRC1 function and their contribution to neurodevelopmental disorders, shedding light on the complex relationship between epigenetics and neurodevelopment. Finally, our findings could pave the way for therapeutic strategies for neurodevelopmental disorders associated with PcG mutations.