Grants

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY This project aims to investigate the interactions and impacts between a depleting antibody (D-aPD-1) that targets programmed death-1-expressing (PD-1+) cells and the autoimmune environment in type 1 diabetes (T1D). PD- 1+ cells are primarily effector lymphocytes that are diabetogenic in T1D.These PD-1+ cells could be a potential target for suppressing autoimmunity in T1D. Initial studies demonstrated that eliminating PD-1+ cells with a PD- 1-specific immunotoxin delayed hyperglycemia onset in NOD mice with late insulitis (L-insulitis). However, immunotoxins are not ideal for chronic disease management, leading to the development of D-aPD-1 as an alternative. Interestingly, when administered to mice with early insulitis (E-insulitis), D-aPD-1 delayed hyperglycemia onset, suggesting a protective effect. However, when given to L-insulitis mice, it unexpectedly accelerated hyperglycemia onset, indicating a pro-autoimmune response. Furthermore, unlike D-aPD-1, PD-1 immunotoxin delayed hyperglycemia onset in L-insulitis mice. These findings align with clinical observations that disease stages and therapeutic agens influences treatment outcomes, highlighting the need to elucidate the mechanisms driving these contrasting responses for better desing and application of T1D therapeutics. To address these questions, this project will investigate the hypothesis that both the immune milieu of T1D and the characteristics of PD-1+ cell-depleting agents play key roles in determining treatment outcomes. Aim 1 will uncover the immune determinants of D-aPD-1’s divergent treatment outcomes in E and L-insulitis by profiling pancreatic islets and systemic immune cells before and after D-aPD-1 treatment. We will use single-cell RNA sequencing (scRNA-seq) and flow cytometry to dentify changes in immune cell populations and pathways associated with pro- and -anti-autoimmune effects. Additionally, strategies to shift L-insulitis responses toward an E-insulitis-like profile will be explored through modulate certain immune cell types. Aim 2 will define the mechanism underlying differential responses to D-aPD-1 and PD-1 immunotoxin by comparing immune cell dynamics and gene expression changes following the treatments. We will identify key cellular mediators responsible for the pro-autoimmune response induced by D-aPD-1, while also investigating whether depleting these responsive cell populations can mitigate its adverse effects. Aim 3 will establish that Fc-free PD-1+ cell depleting agents protect L-insulitis mice from hyperglycemia. Specifically, we will assess whether the Fc-free PD-1+ depleting agents, including albumin-amended antibody-drug conjugate (A3DC) and bispecific killer cell engager (BiKE), can eliminate PD-1+ cells without triggering pro-autoimmune effects. This research could advance targeted PD-1+ cell depletion strategies, leading to safer and more effective antibody-based treatments for T1D. By addressing immune context and therapeutic design, the findings may tailor the design and application of therapeutics for T1D patients at different stages.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Metabolic dysfunction-associated steatotic liver disease (MASLD) is associated with a 4-year decrease in lifespan and is expected to become the leading cause of liver related morbidity and mortality within the next 15 years. A hallmark of MASLD is the accumulation of small (microvesicular steatosis) and large (macrovesicular steatosis) lipid droplets in the liver. However, mechanisms driving the size and zonal deposition (e.g. accumulation in periportal versus pericentral hepatocytes) of lipid droplets remains poorly understood. The carnitine palmitoyltransferase 1a (Cpt1a) enzyme facilitates long chain fatty acid oxidation, but impairments in this pathway have been associated with microvesicular steatosis and reductions in circulating polyunsaturated fatty acids (PUFAs) in humans. Therefore, this proposal aims to identify the mechanisms explaining these associations. Using a mouse model of impaired long chain fatty acid oxidation, preliminary data shows a sexually dimorphic response to liver-specific deletion of Cpt1a characterized by panlobular microvesicular steatosis in female mice while only slight periportal steatosis in male mice. In addition, female knockout mice develop exacerbated inflammation which coincides with reductions in ω3-PUFAs in the liver. Therefore, Aim 1 utilizes in- vitro mechanistic approaches to quantify lipid droplet dynamics and peroxisomal oxidation of PUFAs in response to Cpt1a-deletion in cultured hepatocytes. Aim 2 utilizes gain- and loss-of-function approaches to modulate Cpt1a expression specifically in periportal and pericentral hepatocytes across male and female mice. To assess the impact of zonal-specific Cpt1a deletion and overexpression on the progression from MASLD to metabolic dysfunction-associated steatohepatitis (MASH), livers will be used for dual spatial transcriptomics and lipidomics, and for flow cytometry to assess the immune cell profiles in the liver. Completion of these aims will yield mechanistic insight linking mitochondrial and peroxisomal oxidation to spatial lipid deposition in the liver. The proposed research reveals several innovative mechanisms that have yet to be explored, including how Cpt1a alters PUFA oxidation and lipid droplet formation across the periportal-pericentral axis. In addition, the proposed study uses innovative approaches including the use of zonal-specific gain- and loss-of-function mouse models in conjunction with next generation spatial lipidomic and transcriptomic technology to address the overall hypothesis. Strong collaborations among the University of Kentucky Arts and Sciences Imaging Center, Oncogenomics Shared Resource Facility, COCVD Analytical research Core, Biostatistics and Bioinformatics Shared Resource Facility, and the Center for Advanced Biomolecule Research at the University of Florida ensure successful completion of the proposed research by the candidate. This R01 will support the applicant in reaching his ultimate goal of becoming an independent yet collaborative, versatile PI leveraging his unique skill set in both basic and clinical research arenas to identify novel therapeutics to treat cardiometabolic disease.

NCI - National Cancer Institute

SUMMARY Anti-TROP2 antibody-drug conjugates are a new class of cancer drugs that have shown marked efficacy in treating aggressive cancers. One of these drugs, sacituzumab govitecan (SG), has been approved for treating advanced triple-negative breast cancer and hormone receptor-positive breast cancer. However, drug resistance significantly limits the rate of response to SG and the duration of the response. Identifying the resistance mechanism and developing counteracting strategies will have a significant impact on improving patient survival. However, the preclinical study of anti-TROP2 resistance mechanisms is impeded by the fact that all current human anti-TROP2 drugs do not recognize mouse TROP2 protein, and no spontaneous tumor models in immunocompetent hosts is available for anti-TROP2 studies. To overcome these barriers, we have generated a novel humanized TROP2 mouse model and generated spontaneous triple-negative breast cancer (TNBC) tumors in this model. We discovered that these tumors have heterogenous responses to anti-TROP2 ADC (SG). While some tumors showed remarkable responses to the treatment, others were completely refractory. We have established a unique collection of tumors and organoid lines that are either responsive or refractory to SG. These tumor models allow us for the first time to investigate the action of anti-TROP2 drugs in spontaneous tumors in immunocompetent preclinical models. Using these unique reagents, we will investigate the involvement of both cell-intrinsic and extrinsic mechanisms in anti-TROP2 drug resistance. These insights will open new avenues for future in-depth mechanistic investigations and developing strategies for improving response to anti-TROP2 cancer treatment.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Macrophages are innate immune cells that serve diverse roles in host defense, tissue repair, and homeostasis which are crucial to systemic physiology in all tissues. Therefore, dysregulation of macrophage function can contribute to a wide range of pathologies, including chronic inflammation, metabolic diseases, and cancer. Mitochondrial metabolites have been identified as important regulators of macrophage function with one key regulatory mode involved in post-translational modification (PTM) of cysteine residues on protein targets, which invokes changes in protein structures and functions. Within the past decade, itaconate has emerged as a crucial immunomodulatory metabolite in macrophages. Itaconate is uniquely produced in myeloid cells, most notably in macrophages, by a mitochondrial enzyme named aconitate decarboxylase 1 (ACOD1) and can directly modify protein targets in macrophages via a cysteine PTM called alkylation. However, the breadth of the cysteine targets subjected to this PTM at the proteome level in macrophages is unknown. No study to date has identified the mechanism by which ACOD1 enzyme activity is regulated. Through extensive preliminary studies, I have stoichiometrically defined the macrophage cysteine proteome undergoing itaconate-mediated alkylation by utilizing a high-throughput redox proteomic platform called mass spectrometry-based cysteine-reactive phosphate tag (CPT-MS). From this cysteine proteomic dataset, I’ve discovered a cysteine site on ACOD1 that is potently modified by itaconate. Through preliminary in vitro ACOD1 enzyme activity assays, I’ve demonstrated that the identified cysteine site is important for ACOD1 enzyme activity and that itaconate can inhibit its own production by antagonizing ACOD1 activity. Building on these preliminary data, I hypothesize that itaconate production is involved in a feedback mechanism by which itaconate alkylates crucial cysteine site(s) on ACOD1 to control enzyme activity by invoking structural changes. Using a combination of crystallography, protein biochemistry, and in vivo/in vitro CRISPR editing, I aim to 1) decipher this self-regulatory mechanism underpinning itaconate production and 2) determine the physiological importance of the ACOD1 cysteine residue identified from my CPT-MS data. The findings from this proposed work will uncover a unique mechanism of itaconate production and signaling in macrophages. Moreover, the results of this proposal will lay the foundation for the design of the first-in-class ACOD1 inhibitor/activator and other molecular effectors by targeting key cysteines to manipulate macrophage function in all macrophage-related disease contexts. Additionally, I propose a detailed training plan under this fellowship to provide me with the practical and conceptual skills that will help me complete this project and benefit my future career goals to be an independent scientist. This project involves a wide range of interdisciplinary science to which the excellent environment at Dana-Farber Cancer Institute and Harvard Medical School can provide me with the opportunity to collaborate with many experts in the fields who can give me the necessary support and training to complete my proposed aims.

NIA - National Institute on Aging

PROJECT SUMMARY Nutritional consumption of an essential lipid nutrient, the omega-3 docosahexaenoic acid (DHA), is inversely correlated with numerous diseases, yet due to its rarity in western diets an estimated 80% of the US population is nutritionally deficient in DHA. Because DHA is well documented to provide many health benefits, the exciting prospect that dietary lipid augmentation may be an effective therapeutic strategy to prevent and treat disease has been of therapeutic interest. However, effective therapeutic progress has been stymied by critical limitations in the field, including: 1) a fundamental gap in our understanding of how DHA metabolism is regulated in pre- and post-disease states; and 2) a lack of strong pre-clinical models to mechanistically test the effects of DHA metabolism on health. This research team has overcome these barriers by targeting an enzyme, long-chain acyl-CoA synthetase 6 (ACSL6), to generate a novel preclinical model with a ~50% deficit in DHA specifically in neurons independent of dietary manipulations. Preliminary data demonstrate the critical role of lipid metabolism in health evidenced by accelerated disease due to the loss of ACSL6. The team proposes to test the overall hypothesis that ACSL6-mediated DHA metabolism protects against disease. This work will (Aim 1) determine the role that DHA metabolism plays in neurological health, derived the regulatory metabolism that controls the accrual and turnover of neural DHA, and (Aim 3) test the novel hypothesis that DHA- lipid mediators in the membrane-bound state serve as a readily available pool of protective agents. (Aim 2) determine The proposed work will unmask fundamental breakthroughs, resolve long-standing unknowns, and test novel hypotheses regarding the importance of DHA metabolism in pressing aspects of health and disease.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY Understanding how genetic and neurobiological processes interact to shape variation in social behavior is a central question in biology. Most laboratory systems lack natural variation in social structure, limiting our ability to study these processes. My lab addresses this gap by studying two complementary bee systems: socially variable sweat bees, where sociality has evolved independently multiple times, and the common eastern bumble bee (Bombus impatiens), which exhibits major shifts in colony organization throughout its life cycle. The social variability encompassed by bees provides a powerful foundation to experimentally dissect the factors that modulate social behavior and to test insights across independent lineages. To this end, my lab has developed a comprehensive toolkit to study these socially variable species, including: de novo, high-quality genome assemblies, tools for behavioral quantification, and methods for high-throughput genomics and neuroimaging. Because many of the core mechanisms that modulate behavior in insects and vertebrates are highly conserved, studies in bees can provide broader insights into the biological principles that shape behavior across a wide range of taxa. Our work has identified new ways in which early-life social environments and hormone signaling can shape social behavior. Over the next five years, we will focus on three main questions: (1) What genetic and neurobiological mechanisms modulate social interactions? We will examine a sensitive window for development of the bumble bee brain and social behavior to characterize the underlying mechanisms. This work will define the timing of this window and combine transcriptomic, neurogenomic, and behavioral studies with immediate early gene profiling to map neural responses to social stimuli. (2) How does hormone signaling regulate social behavior? Our comparative genomic work identified patterns of selection on the proteins that bind and transport the insect developmental hormone, juvenile hormone (JH), and revealed that JH can act directly on the brain. We will characterize the timing of JH signaling throughout adulthood and combine hormone manipulations with behavioral, transcriptional, and neurogenetic studies to understand how this developmental hormone has been repurposed to regulate social behaviors in adults. (3) What are the molecular signatures of social plasticity? Some sweat bee species are capable of producing both solitary and social nests even within the same population. We will use genome-wide association studies and transcriptomics to identify the genetic variants and neurogenetic pathways that underlie this social flexibility. My lab’s integrative approach combines genomics, neurobiology, and behavioral studies to gain a comprehensive understanding of how social behavior emerges from the dynamic interplay of genes, brains, and the environment. Discoveries from this work will help elucidate the fundamental building blocks of sociality, shedding light on how social behavior emerges and functions across diverse organisms.

NIA - National Institute on Aging

PROJECT SUMMARY/ABSTRACT This K23 career development award aims to support Dr. Rowan Saloner in acquiring the expertise to become a leading clinical researcher focused on unraveling the neurobiological underpinnings of clinical progression in frontotemporal lobar degeneration (FTLD), Alzheimer's disease (AD), and other related dementias (ADRD), Dr. Saloner is a clinical neuropsychology postdoctoral fellow transitioning to faculty at the University of California, San Francisco, Memory and Aging Center (MAC), This K23 will focus on large-scale proteomics, clinical characterization, and bioinformatics to identify fluid biomarker signatures of FTLD and their relationship to disease progression. Through the enriched multidisciplinary training environment at the MAC, Dr. Saloner aims to accomplish the following training goals: 1) clinical integration of deep molecular phenotyping of ADRD; 2) big data/bioinformatics; and 3) scientific leadership, Dr. Saloner will translate his K23 experience into future R01 efforts to integrate high-dimensional biological tools with detailed clinical phenotyping to refine personalized biomarkers for diagnosis, prognosis, and therapeutic target engagement in ADRD. Dr. Saloner has assembled an exemplary mentorship team with expertise in neurobehavioral aging (Primary Mentor Dr, Kaitlin Casaletto ), ADRD fluid biomarkers (Co-Primary Mentor Dr. Adam Boxer), proteomics (Co-Mentor Dr, Nicholas Seyfried), biostatistics (Co-Mentor Dr. John Karnak), disease progression modeling (Collaborator Dr. Adam Staffaroni), FTLD biology (Advisor Dr. Jennifer Yokoyama), and professional development (Advisor Dr. Joel Kramer). The central rationale is that the most potent therapeutics for ADRD will modify molecular targets that change early in disease and have downstream effects on neuron loss and cognitive decline. Cutting-edge proteomic platforms that measure thousands of proteins in a single biospecimen sample have advanced understanding of AD biology in living humans. However, far less is known regarding the molecular evolution of FTLD, a common cause of young-onset dementia for which there are no effective therapies. The proposed study will leverage large-scale proteomics in cerebrospinal fluid (CSF) and plasma to identify protein signatures that precede and predict brain atrophy and cognitive decline in carriers of autosomal dominant FTLD mutations, who represent an ideal population to study early ADRD biomarkers. Antemortem CSF and plasma proteomic data in sporadic, pathology-confirmed FTLD will also be leveraged to determine the degree to which genetically-derived protein signatures translate to sporadic FTLD, which represents the majority of FTLD cases. Examining deep molecular screening tools in relation to FTLD progression strongly aligns with Actions 1.B.1 ("Expand research to identify the molecular and cellular mechanisms underlying Alzheimer's disease and related dementias") and 1.C, 1 ("Identify imaging and biomarkers to monitor disease progression") of the 2022 National Plan to Address Alzheimer's Disease. Successful completion of study aims will advance discovery of early FTLD biology in humans and identify targets for the development of urgently needed FTLD biomarkers and treatments.

NHLBI - National Heart Lung and Blood Institute

ABSTRACT Pediatric interstitial lung disease (ChILD) is a rare and devastating lung disease for which there are minimal disease modifying therapies. Often presenting in the first days of life with dyspnea and hypoxia, the clinical course of ChILD is progressive respiratory failure. A key barrier to developing therapies is the lack of preclinical modeling to interrogate the pathobiology. Many ChILD patients develop this irreversible disease from harboring mutations in the alveolar epithelial type 2 (AT2) cell-restricted Surfactant Protein gene (SFTPC) that cause protein misfolding. SFTPC-ChILD provides us the opportunity to develop preclinical models of ChILD with which to decipher the molecular mechanisms of the disease and test therapeutics. We developed a robust in vivo model of SFTPC-ChILD that recapitulates key clinical and pathologic features of the human disease. We have used this model and human induced pluripotent stem cell (iPSC)-based AT2s (iAT2s) from a SFTPC- ChILD patient harboring a misfolding SFTPC mutation to study the upstream AT2 events that initiate the disease. During normal postnatal lung development, AT2s plays a critical role in the alveologenesis needed for functional gas exchange through forming an ordered alveolar epithelium and by signaling with the developing alveolar mesenchyme. The central scientific premise for this proposal is that SFTPC-ChILD AT2s lose their critical lung development functions, and that gene-based therapies will restore these functions and protect against the SFTPC-ChILD lung phenotype. Leveraging our Sftpc-ChILD mouse and iAT2s models we will reveal how aberrant AT2s lead to ChILD. We will [Specific Aim 1] interrogate the anomalous development of the epithelium in ChILD by defining the impact of Sftpc-ChILD expression on AT2 postnatal alveologenesis and [Specific Aim 2] determine how Sftpc-ChILD AT2s orchestrate interstitial remodeling pathology through signaling with the mesenchyme. We will also [Specific Aim 3] test early postnatal gene-based therapeutic strategies to resolve Sftpc-ChILD AT2 dysfunction and ChILD pathology. When completed these studies will not only heighten our understanding on ChILD pathogenesis, but also meet our goal of establishing gene editing therapeutic approaches for a disease with limited treatments and no cures.

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

Project Summary/Abstract Epigenetic modifiers govern cell fate transition during animal development and their mutations drive multiple human congenital disorders; however, the molecular mechanisms underlying the roles of epigenetic modifiers in these normal and pathological processes remain poorly understood. It is widely believed that epigenetic modifiers function through the epigenetic marks they catalyze. Nevertheless, the discoveries of catalytic- independent role of epigenetic modifiers challenge this view, raising the question about the biological function of epigenetic marks. Mono-methylation of histone H3 at lysine 4 (H3K4me1) is a reliable mark of enhancers that shape cell identity, and its reconfiguration accompanies the differentiation of pluripotent stem cells, suggesting that the regulation of H3K4me1 plays an instructive role in cell fate transition. To examine this hypothesis, we investigated the catalytic function of LSD1 and LSD2, two paralogous histone demethylases targeting H3K4me1, in regulating gene expression during cell fate transition. Using state-of-the-art approaches such as precise genome engineering, epigenetic and transcriptomic profiling, and stem cell differentiation, we demonstrate functional synergism between the demethylase activity of LSD1 and LSD2 in regulating cellular differentiation. Based on these compelling preliminary data, here we propose to dissect the molecular mechanisms underlying how the demethylase activity of LSD1/2 regulates cell fate transition. The results generated from our proposed studies will not only reveal novel molecular mechanisms underlying the roles of H3K4me1 in gene regulation and cell fate transition, but also provide insights into understanding the pathogenesis of diseases driven by LSD1/2 loss-of-function. This research aligns with the NIH mission to advance our understanding of fundamental biological processes and contribute to knowledge relevant to developmental disorders and regenerative medicine.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

SUMMARY Necrotizing enterocolitis (NEC) is a devastating inflammatory disease that affects the intestines of premature infants. There is a major gap in our understanding of the factors that contribute to the pathophysiology of NEC, including no cure for this often deadly disease. This proposal aims to help fill the knowledge gap by defining a role of the developmentally expressed mRNA-binding protein IMP1 in intestinal mucus composition and survival during NEC. A healthy, mature mucus barrier is essential to protect the intestinal epithelium from inflammation. Our preliminary work shows that Imp1 expression promotes survival during NEC with concomitant upregulation of Qsox1, an enzyme involved in the final glycosylation step (sialyation) of mucus, which translates into the detection of less immature mucus in mice expressing Imp1 during NEC pathogenesis. Roles for IMP1 in NEC and intestinal mucus composition are not known. Understanding how Imp1 regulates Qsox1 and modulates mucus maturation could open new pathways for therapeutic development in NEC. Our long-term goal is to leverage post-transcriptional regulation of intestinal epithelial damage response to improve outcomes for infants at risk for or with NEC. The overall objective of this proposal is to delineate mechanisms of Imp1-mediated survival and intestinal mucus glycosylation. Our central hypothesis is that Imp1 promotes Qsox1 expression to enhance the mucus barrier and promote survival during NEC. Our specific aims are to 1) determine the impact of Imp1 expression on intestinal mucus composition throughout NEC pathogenesis and 2) define the requirement for Qsox1 in Imp1-mediated survival in NEC. Our approach will use Imp1 genetic mouse models combined with an experimental NEC-like intestinal injury model to define the impact of Imp1 on intestinal mucus, including glycosylation, permeability, ultrastructure, and intestinal proteomics during NEC. Key findings will be confirmed in human NEC tissue. We will define Imp1 target mRNAs in the NEC intestine and the Imp1 binding site within Qsox1 via crosslinking RNA immunoprecipitation. We will crossbreed Imp1 overexpressing and Qsox1 knockout mice to determine if Qsox1 is required for Imp1-mediated NEC survival. This research is innovative because it 1) links Imp1 to survival in NEC, 2) connects Imp1 to mucus regulation via Qsox1; 3) will define new molecular mediators of mucus composition in NEC. At the end of the project, we expect to 1) establish Imp1 as a regulator of mucus in NEC; 2) generate novel data connecting Imp1 to Qsox1 regulation and intestinal mucus; 3) use proteomics to uncover mechanisms by which Imp1 promotes survival in NEC; 4) define new pathways that could be leveraged for NEC prevention or treatment. Positive impact: This research will define a role for intestinal mucus in NEC and Imp1 as a new regulator of mucus maturation during inflammation, opening new lines of therapeutic investigation.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT Viruses are obligate intracellular parasites that cannot replicate or survive outside of a host. To produce new viral progeny, they must first gain entry into their target host cells where they hijack various cell biological processes to complete their life cycles. The overall goal of my research program is to determine the molecular mechanisms required for successful viral entry and to identify the host-pathogen interactions governing this essential step in infection. In general, viral entry requires receptor binding at the host cell surface, internalization, and subsequent transport of the virus through the endomembrane system towards its site of replication. Most viruses with DNA genomes replicate in the nuclei of infected cells and, consequently, must cross the nuclear envelope to access host DNA replication machinery. How viruses penetrate this physical barrier to enter the nucleus is a major gap in understanding that will be addressed by this research proposal. We are particularly interested in understanding how viruses with oncogenic (cancer-causing) and oncolytic (cancer-treating) potential enter cells to establish infection. Over the next five years, we will investigate the general entry mechanisms of two DNA viruses—the oncogenic Merkel cell polyomavirus (MCPyV) and the oncolytic LuIII parvovirus. Using a combination of molecular biology, biochemistry, and high-resolution microscopy techniques, this R35 proposal will determine (1) how MCPyV and LuIII target to and cross the nuclear membrane, and (2) the host factors that are required for this process. Interestingly, both viruses appear to use non-canonical nucleocytoplasmic transport mechanisms to reach the nucleus. Therefore, these research findings will increase our fundamental understanding of viral nuclear entry mechanisms to advance the diagnosis, treatment, and prevention of virus-associated disease. Moreover, the transport of molecules into and out of the nucleus is often dysregulated in unrelated human disorders, so this work also will provide additional insight into cellular nucleocytoplasmic transport mechanisms that are more broadly relevant to human health. Overall, these studies will lay the foundation for a productive and well-established research program investigating the cellular entry mechanisms of these and other DNA viruses while increasing our understanding of the basic biological pathways with which they interact.

NHLBI - National Heart Lung and Blood Institute

ABSTRACT Hematopoietic stem and progenitor cells (HSPCs), which include hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), give rise to all blood and immune cells across the lifespan. HSCs first emerge during gestation and ultimately becoming the adult hematopoietic compartment. Human studies have associated events during pregnancy such as maternal infection, diet, or exposure to microbes with increased risk of immune dysfunction in offspring; however, the mechanisms behind this are unknown. Since hematopoietic progenitors arise during gestation and seed the adult hematopoietic compartment, we hypothesize that prenatal inflammation reprograms the cellular output of distinct fetal HSPCs, thus influencing postnatal immune function. Recent data has suggested that fetal HSCs and fetal MPPs emerge independently from the intra-embryonic aorta during development. This suggests that fetal MPPs and fetal HSCs of distinct origin have specific functions and relative contributions to postnatal blood production at homeostasis and during inflammatory insults, but this has not been directly investigated. Our working hypothesis is that fetal MPPs drive the response to prenatal inflammation in order to preserve the HSC pool, shaping the postnatal hematopoietic compartment. We will test our hypothesis by integrating genetic fate mapping experiments, transplantation assays, transcriptomics, and early life infection models. Our preliminary data suggests independent emergence of fetal MPPs and fetal HSCs from the developing aorta and has revealed the first evidence of functional differences between fetal MPPs and HSCs. We have found that fetal MPPs are the first responders to Type II-IFN mediated prenatal inflammation and expand the pool of downstream myeloid cells, which remain expanded in the postnatal period. Our findings also support the idea that timing of emergence influences the functional output of fetal progenitors at steady state and in response to inflammation. The objective of this proposed work is to comprehensively examine the specific effects inflammation has on fetal HSCs and MPPs and will define the mechanisms by which prenatal inflammation shapes the adult hematopoietic system. We will determine how inflammation experienced in utero influences offspring immunity and response to postnatal immune challenges at the level of fetal HSPCs. We anticipate that the insights gained from this proposed work will help inform underlying causes of disease that may start during development.

NIGMS - National Institute of General Medical Sciences

Project summary Protein synthesis is one of the most essential functions of all cells, so it is crucial that cells generate sufficient ribosomes to support robust protein production. In order to transcribe sufficient ribosomal RNAs (rRNAs) to sustain ribosome biogenesis, eukaryotic genomes consequently harbor hundreds of copies of rRNA sequences arranged in tandemly duplicated clusters called ribosomal DNA (rDNA). Curiously, the repetitive nature of these rDNA loci makes them inherently unstable and the number of rRNA sequence copies within an rDNA locus can decrease over time. Consequently, rDNA instability is associated with organismal aging and many diseases. Still, these unstable rDNA loci are maintained throughout populations by germline mechanisms that restore lost rDNA copies to ensure sufficient rDNA is inherited at each generation. Despite the importance of this germline rDNA maintenance to indefinitely propagate the genome, the cellular processes that accomplish rDNA restoration remain unknown. Unravelling these mechanisms that neutralize rDNA instability is crucial to fully comprehending the role of rDNA instability in aging and human health, however, the repetitiveness of these loci have made them largely difficult to study in most organisms. My lab has capitalized on using Drosophila rDNA deletion mutants and their convenient phenotypes to establish a system to rapidly investigate the genetic requirements for rDNA restoration activity. Using this system we have revealed that activation of the rDNA-specific retrotransposon R2 initiates germline rDNA expansion and that this activity is regulated by mTor signaling, securing a grasp on the first valuable threads needed to unravel the processes that govern rDNA maintenance. Over the next five years, my lab will capitalize on these foundational discoveries and the uniquely tractable system we have established to reveal how mTor activity directs germline rDNA copy number maintenance. In particular, we will seek to understand how rDNA copy number is sensed and in turn affects mTor activity, and how mTor directs transcription at rDNA loci to control expression of the R2 copies that are embedded within rDNA. Accomplishing these goals revealing how rDNA maintenance is orchestrated in the Drosophila germline will enable our future studies to directly investigate the impact of rDNA instability on organismal aging and serve as an entry point to study the mechanisms that maintain more complex rDNA in other organisms.

NIGMS - National Institute of General Medical Sciences

Project Summary Cells release extracellular particles (EPs) to establish non-contact intercellular communication. EPs are packaged with biomolecules reflective of the donor cell’s status (referred to as signals) and can traverse the extracellular space to transfer these signals to neighboring or distant target cells. This EP-mediated signaling is recognized as critical for maintaining homeostasis and for disease progression, especially within the immune system, central nervous system, and tumor microenvironment. Additionally, EP-based therapeutic tools, such as drug delivery vessels, and the use of EPs for biopsy, are considered to have high potential. However, the precise regulatory mechanisms underlying EP-mediated intercellular signaling remain unclear, largely due to the heterogeneity within EPs. EPs can range in size from 30 nm to 1000 nm and may contain a variety of biomolecules, including lipids, proteins, nucleic acids, and metabolites. The specific content of individual EPs depends on multiple factors, such as their biogenesis pathways and donor cell type and status. Analyzing this heterogeneity in situ during signaling events is technically challenging, therefore, it is not clear how cells regulate the heterogeneous profile of EPs to achieve specificity during intercellular signaling. The PI’s lab uses quantitative fluorescence microscopy and spectroscopy to investigate molecular information processing at the biointerface, including the assembly and activity of biomolecules at the liquid-liquid interface, as well as imaging-based diagnostic assays with single-EP arrays. The objective of this research program is to elucidate the mechanism of EP-mediated intercellular signaling by deciphering the roles of distinct EP subtypes at the single-particle level, from their release by donor cells to their transport across intercellular physical barriers and interactions with recipient cells. The rationale of this proposal is that uncovering such fundamentals is essential for understanding EP’s biological roles under both physiological and pathological conditions. Over the next five years, the PI’s lab will address three outstanding, currently intractable questions: Q1: How are signals from the donor cell translated into the profiles of released EPs? Q2: How do EPs traverse extracellular barriers to reach recipient cells? Q3: How do the recipient cells interpret the profiles of incoming EPs? The PI and her team will address these questions through a combination of biointerface engineering, microscopy and spectroscopy, and molecular biology techniques. A network of collaborations supports this program. The impact of this project is to provide (1) opportunities for advancing EP-based therapeutic tools and diagnostic assays by improving understanding of the specificity and sensitivity in EP biology; (2) much needed techniques to investigate a variety of functional extracellular bioparticles, including synaptic vesicles and lipoproteins.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Project Summary: The gastrointestinal tract hosts a substantial microbiota reservoir that supports processes like nutrition, metabolism, immune development, and defense against pathogens. However, an imbalance in the microbiota that favors pathobionts can augment inflammation, tissue injury, and pathologies such as inflammatory bowel disease (IBD). IBD affects ~3 million people in the United States, impacting ~1 in every 100 individuals. The mechanisms governing host mucosal immune cell interactions with pathogens in IBD pathobiology are not fully elucidated. Bacterial polyphosphates are long-chain polymers (≥700 phosphate residues) with pleiotropic roles in prokaryotic metabolism, virulence, and host evasion. Recently, our group has reported that bacterial polyphosphates interfere with bone marrow-derived macrophage responses to promote lethality of Escherichia coli sepsis in mice. Emerging evidence also suggests that the IBD therapeutic, mesalamine, lowers the amount of bacterial polyphosphates in the colon and thus the colonization of the inflamed tissue. Our preliminary studies show that mice monocolonized with E. coli deficient in polyphosphate kinase (Δppk), the enzyme mediating polyphosphate synthesis, have lowered inflammation versus mice monocolonized with wild type E. coli. Notably, in experimental colitis, mice showed more weight loss, severe colitis, and reduced survival after receiving polyphosphate-exposed T cells vs non-exposed T cells from colonic lamina propria. In addition, polyphosphates decreased IL-27 expression and blocked STAT1 phosphorylation, supporting a central role for polyphosphates in altering JAK1/STAT1-dependent IL-27 signaling pathways. In metagenomic analyses, ppk was more abundant in IBD patients than controls. Here, we propose to elucidate the role of polyphosphates in exacerbating intestinal inflammation and IBD through three aims: (1) To characterize how microbiota-derived polyphosphates modulate intestinal immune cell networks with a focus on JAK1/STAT pathways (in vivo). We will also perform immunophenotyping on the effect of polyphosphates on JAK/STAT signaling pathways and IL- 12 family cytokines during intestinal inflammation through ‘omics’ approaches. (2) To characterize the effects of polyphosphates on IL-27 release and its activation of JAK/STAT signaling (in vitro). We will investigate the mechanistic effects of polyphosphates on IL-27 production and JAK1/STAT1 signaling in mononuclear phagocytes and T cells. (3) To study the involvement of polyphosphates in human diseases (IBD). For translational studies, we will characterize the abundance of ppk in blood samples and fecal metagenomics data from human IBD patients. Furthermore, we plan to investigate the therapeutic potential of polyphosphate neutralization in T cell-transfer colitis in mice. Overall, we anticipate that findings from our studies will enhance insights into the role of bacterial polyphosphates in maladaptive mucosal immune immunity, which may help define new therapeutic strategies .

NCI - National Cancer Institute

PROJECT SUMMARY Recent paradigm-shifting discoveries demonstrated that neurons are critical drivers of malignancies (cancer), leading to clinical trials to test neuromodulatory drugs in cancer patients. Many types of cancer are associated with severe pain which indicates sensory neuron hyperactivity, raising the intriguing possibility that cancer pain- associated sensory neuron activity accelerates cancer progression. Our long-term objective is to understand the role of neuronal activity in cancer pathophysiology and identify therapeutic targets to improve cancer patient survival and quality of life. Leveraging preclinical models of malignant peripheral nerve sheath tumor (MPNST), where the neoplasm develops within peripheral nerves, our preliminary data reveal that MPNST cells induce sensory neuron hyperactivity and pain, and that sensory neuron activity accelerates tumor growth. Based on these findings, our central hypothesis is that a feedforward mechanism exists between cancer pain-associated sensory neuron hyperactivity and MPNST growth. We will address the hypothesis by uncovering how MPNST cells activate sensory neurons to promote cancer pain via fibroblasts (Aim 1) and determining the mechanisms underlying pain-associated sensory neuron activity-mediated tumor progression in a neuronal subtype-dependent manner (Aim 2). To achieve these aims, we have assembled a multi-disciplinary team consisting of cancer neuroscientists (Yuan Pan, PhD and Xiaofan Guo, MD-PhD), pain researchers (Peter Grace, PhD and Andrew Shepherd, PhD), an MPNST physician-scientist (Angela Hirbe, MD-PhD), a pathologist (John Chrisinger, MD), a bioinformatician (Rajasekaran Mahalingam, PhD), biostatisticians (Chongliang Luo, PhD and Roland Bassett, PhD), and a data management and sharing expert (Robert Allaway, PhD). Together, we will leverage genetically engineered mice, mouse and human MPNST cells, human MPNST tumor specimens, and neuroscience approaches to test the hypothesis. Successful completion of these studies will uncover the mechanisms under which cancer pain-associated neuronal activity interacts with MPNST cells to drive malignancy. Findings from this study will open a new avenue for predicting prognosis and improving the survivorship and quality of life of patients with these deadly tumors.

NIA - National Institute on Aging

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder, characterized by neurofibrillary tangles, β-amyloid (Aβ) plaques, and astrocyte dysfunction. Astrocytes play critical roles in AD pathophysiology, including Aβ clearance and modulation of neuroinflammation. Thus, astrocytes are essential in the pathogenesis of AD, yet the molecular mechanisms underlying astrocyte pathology in AD are not fully understood. Robust evidence from the literature and our preliminary data in human AD tissue and an AD mouse model shows an increase in a population of astrocytes characterized by expression of the matrix-protein receptor CD44. However, the precise contributions of CD44 astrocytes to AD pathology are unknown. Our detailed initial investigations revealed an elevated spatial correlation between CD44 expression and Aβ plaques in both human AD patients and a murine AD model - suggesting a potential role for CD44 in disease initiation or progression. This proposal builds on this foundation and aims to comprehensively define the functional role of CD44 in AD disease- associated astrocyte states. Overarching hypothesis: CD44 upregulation in astrocytes drives a specific disease-associated astrocytes state and promotes AD pathology. Aim 1 will elucidate the signaling mechanisms by which CD44 alters astrocyte phenotype and function in vitro. This will involve genetic and pharmacological manipulation of CD44 cleavage and CD44 intracellular domain (ICD) signaling pathways, evaluating downstream effects on astrocyte biology, including morphology, gene expression, and functional responses. Aim 2 will investigate the impact of astrocytic CD44 on neuronal dysfunction using AD cellular models. Co-culture experiments with cortical murine and induced pluripotent stem cell (iPSC)-derived neuronal AD models will assess how astrocyte CD44 perturbations affect neuronal viability, synaptic function, and transcriptomic profiles. Leveraging single-nucleus RNA sequencing (snRNAseq), this aim will correlate in vitro findings with changes observed in AD patient brains to validate the relevance of CD44-dependent mechanisms in disease pathogenesis. Aim 3 will explore the in vivo effects of modifying CD44 in an AD mouse model, utilizing a novel astrocyte-specific Cd44 knockout mouse and targeted viral expression strategies. We will employ multiplex immunofluorescence, snRNAseq, spatial transcriptomics, and behavioral assays to quantify changes in astrocyte states, Aβ pathology, and cognitive decline. By targeting CD44 at early and late stages of pathology, this aim will determine how manipulating CD44 can mitigate or exacerbate neurodegenerative outcomes. Impact: This study will advance our understanding of CD44-mediated mechanisms in AD, proposing CD44 as a potential therapeutic target for modulating astrocyte-driven neurodegeneration and improving outcomes in AD patients.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY: CCR5 is a chemokine receptor involved in leukocyte trafficking. We identified a neuroprotective role for CCR5 in the context of West Nile virus (WNV) infection both in mice and humans. In mice, we found that mice lacking Ccr5 are significantly more susceptible to WNV-induced encephalitis compared to wild type (WT) mice. We translated these results using human cohorts, where we found that individuals that naturally lack CCR5 (CCR5∆32 homozygotes) develop more severe symptoms following infection with WNV. Furthermore, we and others have shown that this protective effect was not limited to WNV infection, but that the loss of CCR5 increased susceptibility to numerous other neurotropic flaviviruses, including tick-borne encephalitis virus (TBEV), and Japanese encephalitis virus (JEV). ZIKV is of particular concern because of its impact on pregnancy, with microcephaly and other congenital malformations, as well as fetal loss, stillbirth, and preterm birth among the possible outcomes. In preliminary data, we found that the loss of CCR5 increases susceptibility to ZIKV (a known teratogen) during maternal infections, resulting in poor pregnancy outcomes. This was also true for WNV, a virus not known to impact pregnancy. In this application, we will test the hypothesize that CCR5 is protective against flavivirus-induced congenital disease. This application will unravel the mechanisms involved in CCR5-mediated protection during maternal flavivirus infections.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY This proposal aims to elucidate how the dynamic oligomeric transitions of Copper Transporter 1 (CTR1) couple copper (Cu) homeostasis with neuronal developmental pathways. Cu is an essential micronutrient for neuronal function, and a deficiency of Cu in early life can have devastating impacts on development. We recently discovered that CTR1 can reversibly transition between trimeric and monomeric states to regulate Cu uptake. Moreover, a CTR1 mutant that fails to undergo these de-oligomerization events impairs growth factor–activated signaling pathways. These findings challenge the conventional view of CTR1 as a stable trimeric transporter and suggest that Cu-induced allosteric changes in CTR1 oligomerization directly influence neuronal health and development. Our primary objectives are to determine the mechanisms driving CTR1’s oligomeric-state transitions and to clarify how these shifts impact CTR1’s function in Cu regulation and stem cell differentiation. Using innovative single-molecule assays, such as single-molecule localization microscopy and in-cell oligomer stoichiometry assays, we will visualize and quantify CTR1’s oligomeric states in situ. Human embryonic stem cell (hESC)-derived neurons will provide a physiologically relevant model for these studies. In addition, we will conduct comprehensive proteomic and biochemical analyses to uncover key protein interaction networks that modulate CTR1 trafficking and function. This proposal aims to address two main research directions: (1) identifying the triggers behind CTR1’s transition from trimeric to monomeric forms under conditions of excess Cu, and (2) exploring CTR1’s role in stem cell differentiation, with emphasis on its interactions with Laloo and SNT1 to regulate neuronal maturation. This multidisciplinary effort will be supported by collaborations with experts in neurobiology, membrane trafficking, Cu homeostasis, and stem cell research, thereby ensuring a robust and integrative approach. The insights gained will provide significant contributions to understanding CTR1’s role in Cu regulation and cell development, as well as elucidating its broader impact on neuronal health and disease. Additionally, the methodologies developed will have broad applicability for examining the dynamics of other membrane proteins. This project is innovative for several reasons: it introduces novel single-molecule assays for in situ studies, uses a more physiologically relevant hESC model, and addresses a critical gap in our understanding of allosteric regulation mediated by CTR1 in neurons. The insights gained from this research could have far-reaching implications in neurobiology and may inform strategies for treating Cu-related neurodegenerative diseases.

NIGMS - National Institute of General Medical Sciences

Project Summary/Abstract The eukaryotic initiation factor 5A (eIF5A) is a conserved translation factor that is important for the resolution of certain ribosome stalls. eIF5A has been found to be integral for mitochondrial function from yeast to mammalian cells. Despite its recognized importance across species, the precise molecular mechanism by which eIF5A influences mitochondria is not fully understood. This gap in knowledge is especially pertinent given the established links between mitochondrial dysfunction and a range of diseases, including neurodegenerative disorders and cancer. The central objective of this project is to elucidate the mechanisms through which eIF5A depletion affects mitochondrial function, hypothesizing that such depletion leads to ribosome stalling on mitoproteins during co-translational import. This stalling is thought to trigger a mitochondrial import stress response, influencing the translation of various mitochondrial proteins and, consequently, the overall cellular health and function. By integrating gene expression reporters with advanced methodologies such as fluorescent microscopy, proteomics, CRISPR library screening, and Cryo- Electron Tomography, this research promises to offer comprehensive insights into eIF5A's role in mitochondrial health and dysfunction. The outcomes are expected to not only deepen our understanding of fundamental mitochondrial biology but also highlight potential therapeutic avenues for tackling diseases associated with mitochondrial dysfunction.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY/ABSTRACT As CD8⁺ T-cell responses are critical to immunity against cancer and cytosolic pathogens, innovative strategies to enhance cross-presentation (XP) and improve CD8⁺ T-cell responses hold great potential for advancing immunotherapies and vaccines for these diseases of major public health concern. Nevertheless, current vaccine technologies predominantly focus on inducing antibody (Ab) responses rather than boosting T- cell-mediated immunity. We have demonstrated that targeting protein antigens (Ags) to human-FcγRI (hFcγRI) can evoke CD8⁺ T-cell responses. However, a critical gap remains in fully understanding the precise role of hFcγRI in promoting XP and CD8⁺ T-cell responses. In this study, we aim to investigate the involvement of hFcγRI in XP and its impact on the ensuing CD8⁺ T-cell responses. Previously, we demonstrated that protein Ags fused to α-hFcγRI elicit CD8 T-cell responses. This prompted our hypothesis that targeting Ags to hFcγRI will promote XP of the targeted Ags leading to enhanced CD8 T-cell mediated immunity. Here we propose to test this hypothesis using a model that integrates an Ab raised against the hFcγRI – named α-hFcγRI, and a mouse strain that expresses hFcγRI on profession antigen presenting cells (APCs). Since we only use the variable fraction (Ab-Fv) of this Ab (without the Fc fraction), this approach allows selective-targeting of hFcγRI without engaging other Fc receptors (FcRs). This is unlike the approaches utilizing immune complexes (ICs) that cannot isolate the role of individual FcRs in XP, due to broad specificity of most Ab isotypes. Additionally, targeting to the activating receptor hFcγRI will induce a more potent response than the ICs - which potentially engage both activating and inhibitory receptors. Moreover, since the expression of hFcγRI is restricted to the professional APCs i.e. Macrophages (MΦs) and Dendritic cells (DCs), it is likely to reduce unwanted activation of other cell types expressing a variety of FcRs. This proposal has two major aims: 1) to investigate the role of hFcγRI-targeting in XP; and 2), to examine the role of various DC subsets in this process, in particular following intranasal delivery of the model vaccine. Overall, this study aims to fill a significant knowledge gap in understanding the role of hFcγRI in XP, which is crucial for enhancing CD8 T-cell responses of protein subunit vaccines. The findings will impact innovative vaccine strategies that effectively promote cell-mediated immunity against cancer and intracellular pathogens, while minimizing unwanted immune activation and inflammation.

NHLBI - National Heart Lung and Blood Institute

ABSTRACT Acute Respiratory Distress Syndrome (ARDS) is characterized by heightened lung inflammation and edema in the alveoli and interstitium, affecting the lung's primary function of gas exchange. The treatment for ARDS has improved over the years, but the mortality of 20-40% is still associated with ARDS, and the residual defects in lung function in survivors create a critical need for additional therapies. T regulatory cells (Tregs) are emerging as a therapeutic area of interest to facilitate lung resolution due to their reparative and anti-inflammatory properties. Previous work of my sponsor, Dr. Jason Mock, has associated a higher percentage of Tregs in the CD4+ lymphocyte population in the bronchoalveolar lavage fluid (BALF) of patients with ARDS with time to liberation from mechanical ventilation; moreover, in recent years, clinical trials for Treg cell therapy for ARDS have been initiated. However, to realize the full potential of these therapies, we must understand the specific mechanisms by which Tregs facilitate and promote resolution and repair. Our previous work identified the transcript Mmp12 as upregulated more than twenty-fold in Tregs in lungs resolving from LPS-induced ALI compared to Tregs from uninjured lungs. Mmp12's pleiotropic effects extend to the resolution of inflammation by dampening neutrophil infiltration, clearing neutrophil extracellular traps (NETs), terminating complement activation, and accelerating coagulation; however, Treg-derived Mmp12 has been largely unexplored. I hypothesize that Treg-expressed Mmp12 promotes lung resolution by cleaving proinflammatory soluble mediators and reducing the proinflammatory immune cell populations, thereby promoting optimal resolution from lung injury. Aim 1 will determine the function of Treg-derived Mmp12 in the resolution of inflammation during ALI induced by LPS or influenza. Parameters of inflammation and repair will be compared in mice with Tregs lacking Mmp12 (TregMmp12-/- mice) to mice with Tregs expressing wild-type Mmp12. Aim 2 will determine the substrates of extracellular Treg-derived Mmp12. These substrates will be identified by comparison of cleaved substrates in TregMmp12-/- and control lungs during LPS- or influenza-induced ALI using N-TAIL proteomics. Total Mmp12 protein concentration will be measured to determine the fraction of Mmp12 that Tregs contribute. The kinetics of Treg-derived Mmp12 mRNA and protein expression and of substrate cleavage by Treg-generated Mmp12 will be determined. The heterogeneity of Mmp12 expression by known Treg subsets will be assessed. These studies will not only determine the role of Treg-derived Mmp12 by identifying specific substrates that facilitate resolution but also elucidate the effects of Treg-derived Mmp12 in Treg biology and function during lung injury and repair. Through this proposal, I will (1) develop laboratory skills, (2) improve my scientific communication skills through presentations, manuscript preparation, and dissemination to the public, (3) broaden my scientific background, as well as seminars series and workshops about lung disease, the ethical conduct of research and career development.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Program Director/Principal Investigator (Last, First, Middle): Kohan, Alison B (PI); Hodges, CA (MPI) PROJECT SUMMARY Though cystic fibrosis (CF) is considered a lung disease, it presents in infancy as an intestinal disease leading to severe malnutrition and low body weight. These small intestinal symptoms are directly related to future respiratory failure and mortality rates. A major gap in the field, even with modulator therapy, is determining the mechanisms of intestinal CFTR dysfunction and how to reverse the persistent nutritional defects in patients with CF. Using a novel surgical model and primary intestinal organoids, identified fatty acid (FA) trafficking across the apical epithelial membrane as the step of lipid absorption that is inhibited by CFTR-/-. Our studies will test clinically relevant experimental diets and modulator therapy in gold-standard models of fat absorption and use patient- derived human intestinal organoids to directly quantify dietary fat absorption and chylomicron secretion. The outcome of our studies could have a measurable impact on patients with CF, especially in early life, as these studies will fill a persistent gap in knowledge about the activity of CFTR modulators in the intestine and mechanisms of fat malabsorption (in CF and in other malabsorptive or diarrheal diseases).

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY Glioblastoma multiforme (GBM) is the most aggressive and common primary brain tumor, posing significant clinical challenges despite multimodal treatments like surgical resection, chemotherapy, and radiation. The prognosis for GBM patients remains poor, primarily due to its inevitable recurrence and resistance to standard therapies. Therefore, identifying novel molecular drivers of GBM progression is essential for developing more effective treatments. Over the past decade, the isolation of glioblastoma stem cells (GSCs) has provided crucial insights into tumor initiation, maintenance, and recurrence, positioning them as a major therapeutic target. Concurrently, advances in genomic research have revealed a vast landscape of long noncoding RNAs (lncRNAs), offering new opportunities to influence gene regulation and cancer biology. Although tens of thousands of lncRNAs have been discovered, only a small fraction have been functionally characterized. Emerging evidence suggests that lncRNAs exhibit cell-type-specific expression, distinct subcellular localization, and critical roles in key oncogenic processes, including proliferation, invasion, and therapy resistance. These properties highlight their potential as both biomarkers and therapeutic targets in GBM. In this study, we propose to characterize the nuclear-retained oncogenic lncRNA, LINC01896, which is significantly upregulated in GSCs. Our preliminary data show that depletion of LINC01896 impairs GSC proliferation and stemness, emphasizing its critical role in GSC maintenance. Furthermore, higher level of LINC01896 correlates with poor overall and disease-free survival in GBM patients, suggesting its potential as both a prognostic biomarker and a therapeutic target. We will investigate how LINC01896 regulates global gene expression in GSCs and identify the molecular pathways driving GSC proliferation and stemness. Additionally, we will examine the functional impact of LINC01896 knockdown in GSCs using antisense oligonucleotides (ASOs) and evaluate these effects in preclinical 3D cerebral organoid model, which mimic the human brain's cellular complexity and tumor microenvironment. Furthermore, we will map the transcriptomic landscape of GSCs and their tumor microenvironment using single-cell RNA sequencing. Together, these studies aim to elucidate the regulatory role of LINC01896 in GSC biology and pave the way for the development of innovative lncRNA-based therapeutic strategies for GBM patients.