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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.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY / ABSTRACT The Eighth International FASEB Science Research Conference on Retinoids will be held August 3–6, 2026, in Niagara Falls, NY, co-located with the FASEB conference on Nutritional Modulation of Inflammation and Immunity. Originating in 1982, this meeting continues to serve as the leading international forum for advancing fundamental and translational research on vitamin A and retinoids. The 2026 program will feature major developments in established areas such as immune regulation, development, metabolic and nutritional disorders, and visual biology, while also highlighting emerging fields including epithelial and gastrointestinal health, neurodegeneration, stem cell biology, and regenerative pathways. A strong emphasis will be placed on mechanistic and translational applications of retinoid and rexinoid therapeutics, new analytical and imaging tools, and advances in the structural biology of retinoid enzymes, binding proteins, and transporters. A special co-located joint session, Retinoid and Immunity, will unite investigators from both conferences to examine the interplay between vitamin A biology, immune function, and host–pathogen interactions. The conference will provide attendees with an integrated understanding of the genetic, environmental, and microbiome-related factors that influence retinoid uptake, metabolism, and biological responses. It will convene scientists across career stages and disciplines, including biochemistry, structural biology, nutrition, cancer research, immunology, chemical biology, stem cell and regenerative medicine, and clinical research. A central mission of the meeting is to support early-career investigators through short-talk opportunities, poster spotlights, mentoring events, and meet-the-expert sessions. Selected trainee abstracts will be highlighted through oral presentations to increase visibility and promote career development. Partial NIH support is requested to sustain the conference's long-standing mission, broaden participation—especially among early-stage investigators—and continue fostering interdisciplinary progress in retinoid science.

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

Creating better therapies to prevent or inhibit deleterious immunological responses, such as in autoimmunity, is hindered by our incomplete understanding of their pathogenesis. For example, no therapeutic approach is currently capable of curing or providing durable prevention f rom autoimmune type 1 diabetes (T1D). This lack of progress largely stems f rom an incomplete understanding of the interactions among key human cellular players involved in human T1D, and of how these interactions and the environment contribute to disease-causing ef fects. Current tools, f rom animal models to basic culture dishes, are limited in providing this insight. We developed a novel alternative method (NAM) to address these limitations, by creating a three-dimensional (3D) extracellular matrix-based pancreatic tissue mimic, termed the Native Islet-Immune Cell Hydrogel Environment (NIICHE). This NIICHE platform delivers new insights into human immunological processes associated with T1D by collecting 3-D, real-time, noninvasive measurements of immune cell recruitment and engagement with targeted beta cells. With the NIICHE established as a robust, human-centric benchtop screening platform, this proposal seeks to expand its utility and capacity for further testing of the human T1D hypothesis and therapeutics. Specif ically, this R56 proposal seeks to establish the following key engineering and cellular goals. Aim 1) Integrate 3D printing methods for the addition/retrieval of cells and materials to impart spatial control and retrieval. Aim 2) Establish a protocol for the stable integration of sEC monolayer atop the NIICHE and validate visualization of extravasation through the endothelium in response to a chemokine gradient. Aim 3) Expand to additional T cell sources for use in the NIICHE platform and evaluation of the dif ferential immunogenicity of human beta cell subpopulations. Achievement of these goals will create a NAM that supports the distinct placement and retrieval of cells within the 3D matrix, as well as the capacity to distinctly interrogate the role of the endothelial barrier in immune cell recruitment. Finally, expanding our cellular repertoire to include additional T cell and beta cell sources will enhance the utility and predictive capacity of this benchtop system. Once established, this expanded platform can deliver unique insights into pathogenesis and serve as a screening tool for new therapeutic targets. Beyond the T1D focus proposed herein, we envision that this practical and highly translatable 3D platform has broad utility, as its capacity to quantitatively track and assess 3D cellular interactions, traf f icking, extravasation, and immune cell attack enables investigation of numerous immunocentric questions.

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.

NCI - National Cancer Institute

PROJECT SUMMARY Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative neoplasm that typically affects infants and toddlers. In contrast to most hematologic malignancies where RAS mutations represent late events, we and others have shown that nearly all JMML patients have initiating mutations in the RAS pathway. In the majority of patients, this is the only oncogenic mutation found in the genome. This suggests a potential opportunity to treat this disease with targeted inhibitors of RAS signaling. Indeed, we previously found that MEK inhibition (MEKi) alleviates disease in mouse models of JMML and translated this to a recently completed Phase 2 trial (NCT03190915) in children with relapsed/refractory JMML demonstrating clinical benefit of MEKi. However, only half of all patients responded, and none demonstrated molecular responses, defined by reductions in the mutant allele burden of the founding RAS mutations. We have since shown that additional RAS effector pathways (PI3K, mTOR, AKT) in addition to RAF/MEK/ERK (MAP kinase) sustain growth upon MEKi. These findings potentially explain the variable responses to MEKi observed in patients. To address these concerns, we have tested the best-in-class Pan-RAS inhibitor RMC-7977, developed by Revolution Medicines. This compound is a “molecular glue” that binds to all active RAS proteins (i.e., only in the GTP-bound state), and recruits a second protein, cyclophilin A, to sterically prevent active RAS from reaching its effectors. We have now tested RMC-7977 in primary samples from patients with JMML and in patient derived xenograft (PDX) models of JMML and acute myeloid leukemia (AML) with RAS mutations and have observed remarkable efficacy. However, the mechanisms of response and resistance to direct RAS inhibition (RASi) have not been elucidated in hematologic malignancies. We hypothesize that specific RAS genotypes including NRAS and KRAS will be more sensitive to RMC-7977 than other RAS family members including PTPN11 and NF1. We also hypothesize that the “dosage” of RAS mutations will impact sensitivity to the drug. Lastly, we hypothesize that the cell of origin in which RAS mutations arise will impact sensitivity to RASi. We will test these hypotheses using rare primary samples, PDXs and induced pluripotent stem cells. Because primary samples from infants with JMML are difficult to obtain, determinants of response to RASi cannot be adequately modeled in-vitro alone. The proposed studies will use PDX models to evaluate therapeutic efficacy, characterize mechanisms of response and resistance, and validate findings in the context of an intact hematopoietic microenvironment. These animal studies are essential for translating mechanistic discoveries into future clinical trials for children with JMML. While rare, JMML represents a genomically “simple” disease to study Pan-RAS inhibition. The results can then be applied to more common and genomically “complex” diseases like acute myeloid leukemia and myeloproliferative neoplasms that harbor RAS mutations.

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.

NIA - National Institute on Aging

Project Summary Adult tissue homeostasis depends on the tightly regulated activity of tissue-resident stem and progenitor cells. With age, this regenerative capacity declines due to impaired progenitor function, contributing to tissue dysfunction and degeneration. One of the most striking examples of this occurs in the hair follicle, a highly regenerative mini-organ that undergoes cyclical phases of growth (anagen) and rest (telogen). Aging disrupts the cycle by prolonging telogen and diminishing the proliferative output of progenitor cells, ultimately leading to follicle miniaturization and hair loss. Despite its clinical relevance, the molecular mechanisms governing progenitor cell activation and maintenance in the hair follicle remain incompletely understood. To address this gap, I performed single-cell RNA sequencing analysis, RNA velocity analysis, and immunofluorescence staining of cycling postnatal mouse skin, identifying SOX5 as a transcription factor specifically expressed in the earliest subset of activated progenitor cells at anagen onset, localized to a key structure known as the secondary hair germ (SHG). Expression then persists throughout the anagen phase within the proliferative lower matrix before becoming undetectable until the next cycle, suggesting a temporally restricted role in activating progenitor cells and guiding their commitment to a follicular lineage. Supporting this, in vitro overexpression of SOX5 in primary human keratinocytes significantly enhances proliferation, pointing to SOX5 as a central regulator of proliferative dynamics during follicular regeneration. Based on these findings, I hypothesize that SOX5 induces anagen and protects the hair follicle against aging by regulating proliferation of the hair matrix cells and directing SHG cells towards a hair follicle lineage fate. In Aim 1, I will determine whether SOX5 is required for SHG activation and sufficient to initiate early lineage specification. I will also evaluate whether SOX5 overexpression reprograms human keratinocytes toward a follicular identity. In Aim 2, I will assess the role of SOX5 in maintaining matrix proliferation and hair follicle structure during aging using a combination of ex vivo human hair follicle organ culture and a transgenic Sox5 overexpression mouse model. By elucidating how SOX5 governs progenitor cell activation and maintenance, this work may uncover therapeutic strategies to restore hair progenitor cell function in aging and hair loss disorders. More broadly, it will contribute to our understanding of how tissue-specific progenitor programs can be leveraged to counteract age-related regenerative decline.

NIA - National Institute on Aging

Summary TDP-43 pathology occurs in the majority of individuals with high Alzheimer's disease neuropathologic change (ADNC). This accumulation of cytoplasmic hyperphosphorylated aggregates of TDP-43 (pTDP-43) in neurons has been termed limbic predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC). LATE-NC occurs in similar brain regions that are affected by ADNC, however the underlying mechanisms of polyproteinopathies in the context of AD, including how they develop, if and how they interact, and the involvement of the diverse cell types of the CNS, are not well understood. We recently described a family with a pathogenic variant in the AD-associated gene SORL1 where several variant carriers underwent autopsy at the University of Washington Alzheimer's Disease Research Center. This variant, SORL1 R953C, segregated with high ADNC and a TDP-43 pathology that was characteristic for LATE- NC, but occurred in cases with much younger ages of onset. SORL1 has defined roles in endosomal trafficking and regulation of amyloid precursor protein (APP) processing, but how SORL1 in particular, and endosomal dysfunction in general, may contribute to polyproteinopathy in neurodegeneration remains to be explored. In this study we will use human induced pluripotent stem cell (hiPSC)-derived neural cells generated from SORL1 variant carriers and controls to investigate how dysfunction in endosomal trafficking and cellular stress may contribute to the accumulation, mis-localization, and phosphorylation of TDP-43. We will also test whether cells that harbor pathogenic SORL1 variants are more susceptible to modulation of TDP-43 expression. Because pathologic TDP-43 has been described in both neurons and glia, we will generate cortical neurons and astrocytes from hiPSCs for these experiments. We will perform a comprehensive characterization of endosomal pathology in post-mortem brain tissue from donors with ADNC+LATE-NC vs. ADNC or LATE-NC only. We will also analyze endosomal pathology from post-mortem samples of SORL1 variant carriers. For these studies we will use our newly established pipeline for high-resolution imaging of endosomal morphology in post-mortem tissue. Our goal in this exploratory R21 proposal is to test the hypothesis that endosomal dysfunction is a driver of TDP-43 co- pathology in AD and to develop a model of LATE-NC in a tractable, human in vitro system. Our studies will elucidate the molecular mechanisms of how dysfunction in SORL1 and endosomal pathways may lead to TDP- 43 pathology and provide a comprehensive analysis of endosomal pathology in brains of subjects with LATE-NC which, if successful, could open novel therapeutic avenues.

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

PROJECT SUMMARY Haploinsufficiency of chromatin remodeler CHD2 causes a neurodevelopmental disorder (NDD) characterized by developmental delay, intellectual disability, and epilepsy. Adjacent and upstream of CHD2 is a conserved long non-coding RNA (lncRNA) CHASERR. Deletion of CHASERR causes CHD2 overexpression and a more severe, early onset developmental disorder with significant motor and language delay, intellectual disability, and structural brain defects in humans. RNA-seq and western blot analysis of patient-specific induced pluripotent stem cells (iPSCs) and CRISPR-generated HAP1 cells have shown that CHASERR deletion increases CHD2 expression and protein levels in cis. While there is growing evidence of the role of lncRNAs in gene regulation, the mechanism of how CHASERR regulates CHD2, and the downstream consequences of too much CHD2 on global chromatin dynamics and neurodevelopment, is not well understood. Prior studies suggest that CHASERR is concentrated within its locus and binds to SPEN, a protein known to recruit HDAC3 and other chromatin remodeling proteins to repress transcription. Cleavage Under Targets and Release Using Nuclease (CUT&RUN) in HAP1s showed loss of HDAC3 occupancy at CHD2 locus in CHASERR knockout (KO) but not wildtype (WT), suggesting that the CHASERR-SPEN complex is essential to recruit HDAC3 and repress CHD2 expression. Taken together, I hypothesize that CHASERR deletion results in loss of recruitment of SPEN and other repressive proteins at the CHD2 locus, leading to a more open chromatin state permissive of increased CHD2 expression, resulting in CHD2 overproduction and global changes in chromatin dynamics. Using CRISPR-generated CHASERR KO and antisense oligonucleotide (ASO) to knockdown CHASERR in WT HAP1 cells, as well as patient-specific iPSCs and neural progenitor cells (NPCs), I will address my hypothesis with two aims. In Aim 1, I will conduct CUT&RUN on chromatin remodeling proteins (HDAC3, CHD2, EZH2) and histone modifications (H3K27ac, H3K27me3) to determine which proteins CHASERR and SPEN recruit to change local chromatin structure and repress CHD2 expression. In Aim 2, I will use a multi- omics approach with WT, ASO knockdown, and patient-derived CHASERR+/- NPCs, to understand how CHD2 overexpression affects global chromatin dynamics and transcription in a neural model. Because treatment for CHD2-related NDDs requires precise dosage control of functional CHD2, the proposed studies will help understand CHASERR’s regulation of CHD2 and its potential as a therapeutic target for CHD2 patients. Furthermore, this study will contribute to broader understandings of lncRNA biology and the biological underpinnings of childhood developmental disorders. The diverse team of mentors and the premier facilities and equipment at Northwestern will be available throughout the award period to support rigorous training to carve a successful physician-scientist career uncovering genetic mechanisms of pediatric neurological disorders.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

Retrotransposons are interspersed genomic repeats that constitute almost half of the mammalian genome. Largely residing in the heterochromatin, retrotransposons are transiently induced during early development to regulate lineage differentiation, and kept silenced in adult terminally differentiated tissues. However, in human diseases such as cancer and aging, retrotransposons often exhibit aberrantly elevated activities, whose underlying molecular trigger and functional consequences are less understood. Murine skin represents an excellent model to study retrotransposon silencing mechanisms. As our largest organ, skin harbors highly abundant, well characterized, and genetically accessible adult stem cells. Hair follicle stem cells reside in an anatomically distinct niche known as the bulge, alternating between quiescence and activation in a synchronized fashion to fuel cyclic bouts of hair growth. Over repeated insults, hair follicle stem undergo functional exhaustion, the molecular driving events of which were often unclear. In the current proposal, I plan to examine chromatin regulators that couple adult stem cell activation with retrotransposon suppression during adult skin and hair follicle regenerations. Two central heterochromatin pathways are known to silence retrotransposons: tri-methylation on histone 3 lysine 9 (H3K9), catalyzed by histone lysine methyltransferases (KMTs), and DNA cytosine methylation, catalyzed by DNA methyltransferases (DNMTs). Moreover, lineage gene expression during stem cell differentiation depends on DNA demethylation, catalyzed by the DNA demethylase ten-eleven translocation (TET). While TETs are crucial for DNA methylome remodeling in early development, their regulations of retrotransposons in adult tissues remain underexplored. My preliminary analysis of genetic models in which the endogenous retroviruses (ERVs, a type of retrotransposons), are reactivated to drive skin stem cell exhaustion and hair loss, afforded me a unique tool to tackle these questions. Specifically, my prelim data indicated that a critical signal connecting TET to H3K9 KMT and DNMT function is the post-translational modification known as O-linked-β-N-acetylglucosamine (O-GlcNAc). I hypothesize that OGlcNAc catalyzed by the OGlcNAc transferase (OGT) is essential to suppress ERVs by interacting with H3K9 KMT and DNMT in the skin. I will examine OGT-deficient skin phenotypes and O-GlcNAc changes upon ERV reactivation, and dissect the mechanisms of OGlcNAc-orchestrated ERV suppressions. Study proposed here leverage my previous training in mouse genetics, development, epigenetics, and skin biology, and are designed to further train me with the state-of-art technologies such as CRISPR and classic methodologies in biochemistry and molecular biology. My training plan and my sponsor/co-sponsor support have been tailored to further foster my critical thinking, scientific communication, leadership and career development goals within MDACC and GSBS training environment. The proposed study, if successful, will provide important mechanistic insights into retrotransposon biology in adult skin, and mature me into an independent researcher.

NIGMS - National Institute of General Medical Sciences

Project Summary Tissue stem cells are the building blocks during development, and they play a critical role in tissue regeneration and homeostasis throughout life. However, aging results in a decline in proper regulation of stem cell activity, leading to loss of homeostasis over time. Previous work in the Jones Lab demonstrated an important role for mitochondrial biogenesis and turnover in the regulation of adult stem cell behavior in Drosophila melanogaster. However, the mechanisms by which these cells maintain healthy pools of mitochondria throughout life remain unknown. I recently found that male germline stem cells (GSCs) traffic mitochondria to adjacent, somatic niche cells. Since its discovery in 2006, intercellular mitochondrial transfer has been described as a means for stem cells to maintain homeostasis under a wide range of oxidative stressors; however, transfer in vivo under physiological conditions has not been demonstrated. GSCs possess microtubule-based nanotubes that protrude to adjacent somatic niche (hub) cells and are the correct size and shape to facilitate mitochondrial transfer. Preliminary data suggest that the transfer of mitochondria between GSCs and the soma is more pronounced under stressors such as age and a high fat diet. Germline-specific knock-down of genes known to regulate mitochondrial dynamics reduced the frequency of mitochondrial transfer. Therefore, I hypothesize that GSCs utilize intercellular mitochondrial transfer to maintain a healthy pool of mitochondria throughout the life. I will use Drosophila genetics and advanced imaging techniques to test this hypothesis and characterize the specific mechanisms regulating mitochondrial transfer. Upon completion, this project will provide a better understanding of how stem cells maintain high quality organelles throughout life. In addition, my work will have strong implications for organelle-based therapies for treating age-onset diseases.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

PROJECT SUMMARY: Distal arthrogryposis (DA) is a genetic skeletal muscle disorder characterized by congenital joint contractures, muscle weakness, and reduced mobility, leading to significant morbidity. Currently, there are no FDA-approved drugs to treat DA, making physical therapy the only alternative, though often with disappointing outcomes. Despite its significant clinical impact, the precise mechanisms underlying DA's pathology remain elusive. The present grant proposal seeks to comprehensively investigate the involvement of slow myosin binding protein-C (sMyBP-C) in the pathogenesis of DA, aiming to uncover novel therapeutic targets. The overarching long-term goal of my research is to delineate the role of sMyBP- C in health and disease. sMyBP-C is a critical regulator of sarcomere structure and function in skeletal muscle. Recent studies have linked mutations in the MYBPC1 gene, which encodes sMyBP-C, with the development of DA. However, the specific molecular mechanisms by which these mutations lead to the characteristic joint contractures and muscle dysfunction observed in DA patients are not fully understood. Preliminary studies used two newly generated knock-in mouse models carrying homozygous P295L (Human P319L) and E335K (Human E359K) mutations in C2 domain of Mybpc1 gene. In these mouse models, I observed kyphosis and decreased exercise capacity at three months of age and showed increased ex vivo isometric force generation and decreased relaxation rate at low electrical stimulation. Interestingly, calcium transient and speed of relaxation were significantly reduced in the single flexor digitorum brevis fiber of both mutant mice, compared to wild-type controls. Based on these findings, my central hypothesis holds that mutations in the C2 domain of sMyBP-C disrupt the regulation of actin-myosin interaction in striated muscle in the context of force generation, calcium handling and muscle fiber type, leading to the limited movement and contractures characteristic of DA. To test this hypothesis, I will use mouse models and isogenic human induced pluripotent stem cells (hiPSC)-derived myocytes to examine how sMyBP-C mutations affect muscle function, sarcomere structure, and signaling pathways. Therefore, the primary objectives of the proposal are to (i) define the impact of MYBPC1 mutations on skeletal muscle regulation, function, and structure, (ii) determine the molecular interactions that result in hypercontraction, delayed relaxation and calcium handling, and (iii) investigate the disease progression and test two candidate drugs (myosin inhibitor and/or sarcoplasmic reticulum calcium ATPase activator) to treat the phenotypes. The outcome of this research could revolutionize our understanding of DA, establishing a direct link between sMyBP-C mutations and the molecular basis of muscle weakness and thereby provide a foundation for the development of targeted therapies aimed at restoring sarcomere function and ameliorating the clinical manifestations of DA.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY/ABSTRACT Reprogramming is crucial for cellular renewal in adult organs that lack dedicated stem cells to replace loss after injury and inflammation. Because such cell plasticity is likely to be executed by a conserved cellular program, we have begun to identify the conserved cellular-molecular features of the process of recruiting differentiated cells as progenitors. The term paligenosis has been recently coined to describe an evolutionary conserved process that a differentiated cell uses to downscale its organelle contents, activate a progenitor-like gene network, and reenter the cell cycle. The upstream triggers and molecular mechanisms initiating this regenerative program remain poorly understood. This project investigates upstream triggers of paligenosis. Using a high-dose tamoxifen injury model to induce paligenosis in zymogenic chief cells of murine stomach corpus, ultrastructural changes in the rough endoplasmic reticulum (rER) were observed during paligenosis initiation (e.g., swelling of the rER lamellae, liberation of ribosomes from rER, and overall loss of ER). This leads to the hypothesis that dynamic changes in ER are an upstream event in paligenosis. ER functioning is in part monitored by the integrated stress response with the paramount ER stress sensor being PERK, a kinase that inhibits translation of mRNA on the ribosome by phosphorylating the translation initiation protein elF2a. Phosphorylated elF2a halts global translation while upregulating a specific set of genes to restore homeostasis. Data show that high-dose tamoxifen activates the integrated stress response in paligenotic zymogenic chief cells, triggering global attenuation of protein synthesis. Preliminary data also indicate that disassembly of rER is an early paligenosis event, supporting the hypothesis that early events of paligenosis are driven by the PERK-integrated stress response pathway and the dynamic regulation and autophagy of rER. Aim 1 of this project thus seeks to detail activation of PERK over a lime course early in paligenosis in the high-dose tamoxifen injury model, and then test the PERK requirement using PERK and integrated stress response inhibitors, and Pefkllll mice crossed to chief cell-specific promoter mice. Sufficiency will be tested by inducing ER stress and by drug-induced activation of PERK. Aim 2 will detail paligenotic ER remodeling in a high-dose tamoxifen model. Using ER-phagy defective mice (Ccpgt+), the effect of ER-phagy deficiency on paligenosis will be examined. The necessity of ER-phagy receptor in initiating and regulating autophagy will also be examined in a clinically relevant context, using human gastric adenocarcinoma cell line AGS and patient-derived organoid models of normal gastric corpus and intestinal metaplasia. This fellowship project ultimately seeks to define critical upstream events that initiate cellular reprogramming during regeneration, providing new insights into ER stress signaling and ER-phagy in gastrointestinal tissue repair and disease. This fellowship also supports a mentored training plan focused on the development of skills related to project management, imaging and analytical techniques, teaching, communication, leadership, and outreach.