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NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT The following proposal outlines a 5-year career training plan that will prepare Dr. Andrew Berical to be an independent physician-scientist and leader in the field of airway epithelial functional genomics. Motile cilia are found throughout the human body, most notably on multiciliated cells (MCCs) in the conducting airway. Individuals with primary ciliary dyskinesia (PCD) have inherited variants in any one of more than 50 genes that regulate the structure or function of cilia, leading to a lifetime of chronic cough, recurrent infections and respiratory failure. Due to the complexity of the MCC molecular program and limited disease-relevant platforms, there are no targeted therapies available for PCD. An improved understanding of fundamental MCC biology and the availability of a human-based platform would have enormous implications for the PCD field. Dr. Berical’s long-term vision is to utilize pluripotent stem cell-based techniques to understand how specific genes regulate airway epithelial homeostasis and how gene variants lead to the initiation of airway diseases such as PCD, CF, asthma, COPD and IPF. Dr. Berical presents preliminary data suggesting a recently described serine-threonine kinase (STK33) has a fundamental role in the MCC developmental program. STK33 deletion results in 1) fewer MCCs, 2) fewer cilia per cell, 3) an abnormal ciliary structure and 4) reduced ciliary beat frequency. In this proposal, Dr. Berical aims to understand the mechanism by which STK33 effects the MCC molecular program to create this highly irregular phenotype. Leveraging key training opportunities through his collaborators and scientific advisory committee, he will 1) precisely characterize the STK33-dependent MCC defects using time course single cell RNA-sequencing to pinpoint when, during MCC differentiation, STK33 exerts its effect, 2) identify STK33 downstream targets and effector molecules and 3) determine the in vivo ramifications of STK33 loss on the engraftment, differentiation and function of airway epithelial cells. Following this investigation of the STK33-dependent regulation of MCC biology, Dr. Berical then expands these methods to probe the functions of a curated list of high priority ciliary kinases of unknown function. This work will provide much needed insight into the MCC molecular program and develop an essential platform for the interrogation of genes of unknown function in the airway epithelium, applicable to the genetically heterogeneous PCD, as well as other airway diseases. Dr. Berical has 80% protected time from his department to accomplish these aims under the guidance of his mentors Drs. Finn Hawkins and Darrell Kotton at the Center for Regenerative Medicine at Boston University/Boston Medical Center. He has assembled a remarkable team of advisors with diverse expertise to assist in his career development and scientific research. Dr. Berical details a comprehensive training plan that includes experiential training, didactic coursework, attendance and presentation at scientific meetings, preparation of manuscripts and acquiring additional grant support culminating in an R01. Dr. Berical has the commitment of his department to accomplish these goals and transition to an independent physician-scientist position by the end of the award.

NIH

This proposal aims to advance our understanding of clonal hemopoiesis of indeterminate potential (CHIP) in the development of atherosclerotic cardiovascular disease (ASCVD). CHIP is a recently identified acquired risk factor for ASCVD. With aging, hematopoietic stem cells accumulate mutations that can lead to a proliferative advantage resulting in CHIP. Tet Methylcytosine Dioxygenase 2 (TET2) is a commonly mutated gene in CHIP and confers a 50% increased risk for incident coronary disease. How TET2 leads to ASCVD is in humans is not well understood and there is currently no ability to assess whether a specific TET2 mutation is high-risk. The central objective of this proposal is to (1) identify TET2 mutations that are high-risk for developing ASCVD to derive a comprehensive and clinically actionable risk score calculator and (2) identify the aberrant cell states and signaling pathways among TET2 mutated immune cells in the coronary vasculature. To identify high-risk TET2 mutations, the candidate will leverage a population-scale human genetics approach in >1 million people via the Million Veteran Program (MVP). To identify aberrant cell states and signaling pathways, the candidate will deploy their novel single cell lineage tracing protocol in coronary vascular tissue followed by validation experiments via population-based human genetic association studies. The candidate's career goals are to become an independently funded physician scientist focused on developing new ways of treating ASCVD. In addition to the proposed science, the training activities outlined in the candidate's career development plan are focused on the crucial skills and experiences necessary to enable an independent research program. Combined with the direct mentorship of Ors. Brent Ferrell and Adrianna Hung, Tennessee Valley Health System Nashville VAMC represents an ideal environment for the proposed work and leverage some of the world-class strengths of the Veterans Affairs resources. The Ferrell and Hung labs have deep experience in the methods used in this proposal and are prepared to support the candidate throughout the entirety of the grant period. Overall, this VA CDA-2 proposal represents a set of innovative and timely scientific aims combined with a tractable career development plan that will meaningfully contribute to human health research and catalyze the candidate's long-term career goal of developing into an independent investigator.

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY Neural circuit development and function depends on precise interactions between neurons and glia. Astrocytes, the primary peri-synaptic glia, mediate synapse formation, stability, and function. Neuron-astrocyte crosstalk is facilitated by complex protein-protein interactions, and loss of these interactions contributes to circuit instability in many neurological disorders. Thus, understanding the mechanisms that regulate neuron-astrocyte communication is of broad clinical importance. Glycosylation is a posttranslational modification that regulates protein stability and binding through addition of sugar groups to specific amino acids. Mutation of genes in glycosylation pathways cause congenital disorders of glycosylation (CDGs), a group of monogenic disorders associated with neurological dysfunction, including epilepsy, autism, and cerebellar degeneration. The mechanisms underlying neurological dysfunction in CDGs remain unknown. Here, I focus on ALG8, an enzyme in the N-glycosylation pathway. To explore the molecular underpinnings of ALG8-CDG, I first needed to develop models that reflect the patient population. To this end, I generated a predicted null zebrafish line (alg8stl973) and human embryonic stem cell (hESC) lines with a missense mutation (p.Thr47Pro) found in ALG8-CDG patients. My preliminary data revealed a decrease in astrocyte numbers in the brains of alg8 mutant zebrafish with no change in total cells, and reduced proliferation of ALG8 mutant hESC-derived astrocytes. Moreover, in alg8stl973 fish, astrocyte morphological complexity is reduced. As astrocyte-synapse association is necessary for neuronal signaling, I hypothesize that defective glycosylation disrupts specification and maturation of astroglia, which in turn drives circuit imbalance and CDG-associated behavioral deficits. To address this hypothesis, I will leverage preexisting transgenic tools in zebrafish to label astrocytes and test whether changes in proliferation and/or cell death result in reduced astrocytes in alg8stl973 fish (Aim 1). Furthermore, I will use biochemistry and in vivo imaging to characterize how loss of alg8 impacts the glycosylation status of one key regulator of astrocyte morphogenesis: NrCam (Aim 2). Finally, as ALG8 is expressed in all neural cell types, I will use cell-type specific rescue in fish and co-culture of hESC-derived neural cells to determine which cell type(s) drive changes in astrocyte morphology and synaptogenesis in ALG8-CDG (Aim 3). My long-term goal is to define common molecular changes in brain development across distinct CDGs. Critically, various CDG subtypes result in common neurological symptoms, but the cellular and molecular underpinnings of these phenotypes are largely unknown. Similar to my preliminary findings in ALG8-CDG models, recent work indicates that astrogenesis is altered in a mouse model of MGAT5-CDG, a CDG with defective N-glycosylation. Thus, I anticipate that my findings will be broadly applicable to the CDG community and will enhance our fundamental understanding of how glycosylation shapes brain development.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary/Abstract Astroviruses are RNA viruses that commonly cause disease in humans, including gastroenteritis and fatal cases of encephalitis. Despite their broad human impact, astroviruses are understudied and many of the critical steps of the viral lifecycle are poorly characterized. There is limited understanding of host-pathogen interactions that facilitate viral replication, including the role of RNA motifs in the viral genome. Our lab was the first to cultivate astrovirus VA1 (VA1), the most common cause of astrovirus encephalitis to date. In a region overlapping with ORF2 and the 3' untranslated region, VA1 is predicted to encode a stem-loop II motif (s2m). Similar motifs with the same secondary structure have been identified in astroviruses and viruses of other viral families, with its importance in the viral lifecycle being virus-dependent. Using SHAPE-MaP on the full-length VA1 genome, we confirmed the formation of the s2m and its secondary structure. Mutagenesis of the s2m in a novel reverse genetics system for VA1 revealed the s2m to be essential. Deletion of the s2m or mutations that disrupt guanine- cytosine base pairs (GC-bp) that are critical for the secondary structure of the s2m result in virus that cannot be propagated. The mutant genomes can be rescued when complementary mutations are introduced into the s2m that restore GC-bp in the secondary structure. Mutagenesis of a position not involved in GC-bp was important but not essential for the function of the s2m. Capsid expression could not be detected from transfected genomes containing s2m mutations. Translation of capsid was also reduced by mutations of the s2m using a reporter system, and we have identified putative proteins involved in translation that may also bind to the s2m. We are now uniquely positioned to study the mechanism of action for the s2m in promoting the VA1 lifecycle using our novel tools that we have developed. Our central hypothesis is that the s2m facilitates viral translation through RNA-protein interactions, mediated by the s2m sequence, structure, and location in the genome. To test this hypothesis, we will take a combination of genetic and biochemical approaches to mechanistically understand why the s2m is essential. In other viral stem-loop structures, the loop region often serves as an important interaction site. We will define the role of the VA1 pentaloop for the function of the s2m by mutagenesis. Next, we will determine whether the function of the s2m is dependent on location in the genome. We will also assess whether the s2m must be encoded on the expressed RNA strand or if it can function independently. Using an RNA-pulldown, we have identified putative proteins that bind to the s2m that also mediate translation. We will confirm s2m-protein interactions and determine the effects of loss of function of these candidate proteins on the viral lifecycle. The findings of this project will provide important insights into the function of the VA1 s2m, address gaps in our knowledge of the VA1 lifecycle, and contribute to our larger understanding in RNA motifs in viral biology. These results will set the foundation for further dissection of the molecular biology of VA1, ultimately accelerating development of antivirals and vaccine-based approaches.

NHLBI - National Heart Lung and Blood Institute

Although squamous and mucous metaplasia are the two cardinal forms of pathologic epithelial injury response in airway disease, the cellular source and molecular mechanisms governing their formation have not been clearly defined. It has long been assumed that both forms of metaplasia arise from a common basal stem cell population biased by specific signaling factors, however, both metaplasias occur together in the same patient in the same signaling milieu. Furthermore, we have previously reported that the distal murine tracheal epithelium is predisposed to mucous metaplasia, while squamous metaplasia tends to form in the dorsal murine and human airway. In parallel, we have reported that specific hillock basal stem cells are found in dorsally located stratified squamous epithelial structures that we named hillocks. In aggregate, these findings suggest the hypothesis that squamous and mucous metaplasia arise from regionally distinct stem cell populations and that these heterogeneous stem cell populations respond differently to common pathologic signaling cascades. With regard to mechanism, high Yap signaling activity has been associated with squamous metaplasia while mucous cell differentiation requires a suppression of Yap activity. As such, we will establish the propensity of anatomically regionalized basal stem cell populations of the mouse and human airway to undergo either squamous or mucous metaplasia including (1) dorsally located murine and human hillock basal stem cells, (2) proximal and (3) distal pseudostratified murine and human basal stem cells populations. Since Yap has been directly associated with mouse and human metaplasia, we will assess the effect of temporally regulated Yap overexpression on the above stem cell populations and the consequences on both squamous and mucous metaplasia. Additionally, using ATAC-Seq and RNA-seq, we will determine the accessibility and expression of the Yap target genes that underpin the differential metaplastic propensities of the above stem cell populations. In contrast to Yap signaling, EGFR signaling activation causes both pathologic mucous and squamous metaplasia. Therefore, we will define the effects of EGFR modulation on both hillock and non-hillock pseudostratified mouse and human basal stem cells. We will also assess whether Yap overexpression will prevent EGFR-induced mucous metaplasia in distal basal stem cell populations and whether suppressing Yap will lead to diminished EGFR-induced squamous metaplasia arising from hillock basal stem cells. Finally, we provide evidence that Yap activity is dramatically upregulated following injury, but this activity subsides as injury resolves. We will define the effects of Yap modulation on the formation of the early post-injury squamous barrier epithelium and injury-associated squamous metaplasia. Understanding how heterogenous stem cell populations of the airway contribute to both squamous and mucous metaplasia and establishing how these stem cells respond to disease-associated signaling pathways will inform strategies to control pathologic metaplasia.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT Dilated cardiomyopathy (DCM) is a leading cause of heart failure, with approximately 40% of DCM cases linked to pathogenic genetic variants. Despite advances in genetic testing, there remains a major knowledge gap in how specific variants contribute to the multifaceted clinical manifestations of DCM. A notable example is a pathogenic variant in the phospholamban (PLN) gene resulting from deletion of arginine 14 (PLN-R14del). PLN induces DCM characterized by prominent ventricular arrhythmias and highly variable phenotypes, ranging from severe, early-onset disease to lifelong asymptomatic carriage. Such variability makes it challenging to establish genotype-phenotype relationships in PLN-R14del carriers, highlighting the urgent need for new technologies to bridge this gap. Advances in omics technologies are revolutionizing precision medicine. Among omics methods, top-down proteomics has emerged as a powerful technology for studying post-translational modification (PTMs), genetic variants, and splicing isoforms (collectively known as “proteoforms”). Top-down proteomics is ideally positioned for studying complex genetic diseases like PLN-R14del DCM, providing direct evidence of how genetic mutations affect proteoform compositions and linkage to function and phenotype, thereby bridging genotype-phenotype gap. Our preliminary data show that the PLN-R14del variant is associated with unique changes in cardiac proteoforms, including alterations in critical Ca2+-handling, contractile, and metabolic proteoforms, in both human patient tissue and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Hence, we hypothesize that dysregulation of Ca2+-handling, contractile, and metabolic proteoforms and the corresponding pathways contribute to variability in disease phenotypes and expressivity in PLN-R14del carriers. To test this hypothesis, we will employ a systems biology approach featuring novel multi- omics, incorporating proteomics, metabolomics and lipidomics, in combination with human clinical samples and patient-derived hiPSC-CM cellular models. Specifically, we will carry out multi-omics analysis of myocardial tissue from patients with PLN-R14del DCM, compared with genotype-negative DCM and nonfailing donor tissue as controls. The findings from the omics analyses will be further integrated with clinical data to develop patient- specific disease signatures in PLN-R14del carriers. We will also determine differences between symptomatic and asymptomatic PLN-R14del carriers through multi-omics analysis of patient-derived and isogenic control hiPSC-CMs and link them to changes in contractility/metabolism using functional assays. We will further connect the PLN-R14del variant mechanistically to proteoform alterations and their functional outcomes using gain- and loss-of-function approaches. Successful completion of the proposed study will provide new insights into the mechanisms underlying cardiac dysfunction in PLN-R14del, as well as bridge the genotype-phenotype knowledge gap in familial DCM to improve risk-stratification in variant carriers, advance our understanding of genetic diseases, and facilitate the development of targeted treatments towards precision medicine.

NIA - National Institute on Aging

Project Summary: The overarching goal of this application is to investigate cell mechanics-mediated regulation of hair follicle stem cells (HFSCs) during homeostasis and aging. We propose to study microRNA-205- mediated regulation of extracellular matrix (ECM) and actin cytoskeleton for HFSC quiescence and activation and leverage the ability of microRNA-205 (miR-205) to stimulate HFSC activation to enhance HFSC aging. MicroRNA (miRNA) is a class of small noncoding, regulatory RNAs that play important roles in mammalian development, stem cells, diseases and aging. In our preliminary studies, we have determined mechanical properties of HFSCs during homeostasis and aging. We have revealed that bulge HFSCs reside in a stiff microenvironment with high actomyosin contraction forces. In contrast, hair germ progenitors are relatively soft and undergo periodic enlargement and contraction. Notably, induction of miR-205, one of the most highly expressed miRNAs in HFSCs, downregulates many bona fide targets, which are enriched in the function of ECM, actomyosin cytoskeleton and mechanosensing. And this leads to rapid activation of HFSC cell division and promotes hair regeneration in both young and aged mice. Mechanistically, we have identified Piezo1 as a novel target of miR-205, which functions downstream of miR-205 and translates mechanical cues into a gene expression program to reinforce the mechanical properties and maintain cellular states of quiescent HFSCs. To examine the role of PIEZO1-mediated calcium influx in HFSCs, we have further developed a high-resolution intravital imaging system to accurately record calcium influx in HFSCs over an extended period of time during quiescence and activation. This allows us to quantify cumulative calcium levels and further identify transcription factors, NFATC1 and JUN (AP1), which function downstream of PIEZO1-mediated calcium influx to promote the expression of the ECM and actin cytoskeleton genes. Based on these exciting findings and promising preliminary data, we propose to further elucidate the mechanism of miR-205-mediated HFSC activation and aging through the regulation of ECM and actomyosin contraction forces (Aim 1), determine the regulation of PIEZO1-mediated mechanosensing by miR-205 (Aim 2), and leverage miR-205-induced HFSC activation to improve HFSC functions and hair growth during aging (Aim 3). Together, this application will provide new insights into the mechanisms orchestrating the mechanical properties and stem cell functions of HFSCs. By harnessing the powerful combination of live imaging, cell biology, mouse genetics, and single-cell genomics, we will establish a new paradigm for studying tissue architecture, cell mechanics and underlying mechanisms. These results will lay the foundation for leveraging noncoding, regulatory RNAs to enhance HFSC functions during aging.

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

PROJECT SUMMARY Precise regulation of cell fate specification during early embryogenesis is essential for proper tissue and organ formation, and its disruption leads to congenital malformations. However, the gene regulatory pathways controlling these early developmental decisions—particularly in humans—remain poorly understood. This proposal investigates a novel, primate-specific mechanism of cell fate control mediated by the dual-function DNA/RNA-binding protein ILF3. Identified through genome-wide screens in human pluripotent stem cells (PSCs), ILF3 is required for proper exit from pluripotency and lineage specification in human and primate—but not mouse—PSCs. Our data show that ILF3 interacts with and inhibits the RNA editing enzyme ADAR1 to limit adenosine-to-inosine (A-to-I) editing at primate-specific Alu elements, thereby preserving accurate splicing of developmental transcripts. These findings implicate ILF3 as a critical regulator of transcriptome fidelity in early primate development and introduce a novel paradigm where species-specific RNA processing fidelity serves as a developmental checkpoint. To define the developmental and mechanistic roles of ILF3, we propose three integrated aims. In Aim 1, we will use cross-species gastruloid models from human, chimpanzee, rhesus monkey, and mouse to assess ILF3's role in early lineage transitions and test whether it defines a primate- specific pathway in mammalian development. In Aim 2, we will map nascent RNA editing following acute ILF3 depletion using SLAM-seq and identify the protein domains mediating ILF3-ADAR1 interaction, linking RNA editing regulation to cell fate control. In Aim 3, we will define how ILF3 impacts RNA processing at key developmental genes by integrating splicing analysis and quantitative proteomics, uncovering direct effectors of lineage specification. Moreover, we will establish a causal link between expression of mis-edited and mis-spliced developmental regulators and proper gastruloid formation through rescue experiments. This research will uncover a previously unrecognized RNA-based regulatory mechanism controlling early primate development and provide insight into how defects in RNA editing and splicing may contribute to congenital disease. By establishing a functional framework for ILF3 in safeguarding human cell fate transitions, this work will inform future strategies for therapeutic intervention in developmental disorders, directly supporting NICHD's mission to understand and treat the origins of birth defects.

NIGMS - National Institute of General Medical Sciences

The extracellular matrix (ECM) provides essential tissue infrastructure and mechanical cues that regulates cell metabolism. Mechanotransduction is the conversion of mechanical forces from the ECM to intracellular chemical signals and plays a vital role in health and disease through cell-matrix interactions. While evidence supports a ‘mechano-metabolic link’ between mechanotransduction and metabolism, the precise pathway connecting ECM mechanical states to cellular metabolism remains poorly understood due to the ECM’s complexity, including its viscoelastic properties, which exhibit both strain-independent (linear) and strain-dependent (nonlinear) regimes. All tissues exhibit both linear and nonlinear viscoelasticity, typically reported as stiffness and strain-stiffening, respectively; however, a fundamental gap remains in understanding how these distinct mechanical properties counterbalance each other to regulate cell metabolism. Key open questions include how cells engage with nonlinear viscoelastic environments, how mechanotransduction scales with cell and tissue maturity, and how nonlinear viscoelasticity influences cellular uptake and consumption of metabolic biomolecules. To address these gaps, this proposal uses primitive and differentiated induced pluripotent stem cells (iPSCs), both as single-cell and organoid cultures, in a 3D in vitro polymeric hydrogel systems to independently present cell-accessible and cell-inaccessible nonlinear viscoelastic regimes. We combine this with material- and omics-based modeling approaches to define viscoelastic and metabolic signaling regimes. In Project 1, we will use ECM ligand-binding motifs and both primary cells and iPSCs to investigate integrin-mediated cell-matrix interactions across viscoelastic regimes, cell maturity, and tissue complexity. In Project 2, we will examine how nonlinear viscoelasticity influences cellular uptake of metabolic precursors such as lipids and apply model-based approaches to define characteristic metabolic and proteomic signatures associated with linear and nonlinear regimes. The outcomes of this proposal will advance our understanding of mechanosignaling in nonlinear viscoelastic environments and establish a mechanistic model of the mechanical regulation of cellular metabolism. The long-term goal of the lab is to develop complex in vitro models to investigate how physiological processes such as aging and pregnancy induce systemic tissue alterations that drive changes in cell-matrix interactions and mechanosignaling, ultimately influencing cell and tissue function. By uncovering a direct mechano-metabolic connection, this work will have broad implications for fundamental biology while also developing critical tools and workflows for studying cell-matrix interactions. Importantly, while this proposal focuses on fundamental biological processes, the methods and tools developed will be broadly applicable across various cell, tissue, and disease states.

Dell Technologies Foundation

Supports youth development and STEM education programs, with focus on underrepresented groups in technology careers.

Deloitte Foundation

Supports education and workforce development through grants to universities and nonprofits focused on accounting, business, and STEM.

NINDS - National Institute of Neurological Disorders and Stroke

Project Summary The brainstem relays information between the brain and the spinal cord, regulating vital autonomic functions. Like other tissues, the brainstem is vulnerable to malformation and disease. Yet, despite its importance, little is known about its development, making it challenging to understand brainstem pathology. Many of the neuron populations that regulate vital brainstem function arise from a homogenous progenitor pool that expresses the proneural transcription factor Atonal homolog 1 (Atoh1). Atoh1 is functionally relevant for driving migration, however, the mechanisms that regulate progenitor proliferation and prime cells for differentiation are unknown. This incomplete understanding of brainstem development has made it challenging to develop accurate in vitro models of the brainstem, thereby hindering studies aimed at elucidating disorders and disease. Recent transcriptomic mapping of embryonic mouse hindbrain development has revealed significant expression of Notch signaling genes. Notch signaling is a key regulator of cell fate decisions across neurogenesis, and it is known to regulate proneural genes such as Atoh1. Importantly, the development of in vitro models often relies on small molecule-based approaches that direct stem cell fate by mimicking native signaling environments. Yet, the specific role of Notch signaling in Atoh1 lineage development remains poorly defined, making it challenging to utilize this pathway to model development in vitro. This proposal will investigate how Notch signaling influences Atoh1 lineage progression by integrating computational transcriptomics, stem cell differentiation, and synthetic biology. The specific aims of this project are to: (1) define transcriptomic patterns of Notch signaling during brainstem development and predict regulatory function through in silico perturbation modeling; (2) engineer a novel multi-reporter stem cell line to visualize real-time Notch ligand dynamics during Atoh1-directed differentiation; and (3) modulate Notch activity in vitro to assess the impact of ligand induction on Atoh1 fate decisions. Together, this work will clarify how Notch signaling shapes Atoh1 lineage progression and establish tools to visualize and manipulate Notch signaling in vitro. These insights will provide foundational knowledge for improving in vitro brainstem models and for probing neurodevelopmental disorders linked to brainstem dysfunction.

NCI - National Cancer Institute

Title: Develop a human pluripotent stem cell-derived preclinical model for NUT Carcinoma Project Summary: NUT Carcinoma (NC) is a devastating cancer with no effective treatment. A deeper understanding of its oncogenesis mechanism is vital for developing treatments that improve its prognosis. Although NC cases are strongly associated with Nuclear Protein in Testis (NUTM1) fusion genes, predominantly BRD4::NUTM1 (70% of cases), their oncogenic functions have been under debate. Using one of the first two NC genetically engineered mouse models we created, we demonstrated that inducing an endogenous chromosome translocation that forms the Brd4::Nutm1 fusion gene in progenitor cells in tissues as distinctive as oral mucosa, thyroids, lungs, and pancreas can induce carcinomas recapitulating human NC. Our results provided the long-awaited proof of NUTM1 fusion genes as the oncogenes for NC. Our new GEMM provided a critical tool to deepen our understanding of the molecular mechanisms of NC oncogenesis and develop effective treatments. However, the 90 million years’ evolution distance between humans and mice posed two significant challenges for translational studies of NC using the mouse model: · Due to the evolutionary divergence of protein and sequence structure, targeting agents including CRISPR-CAS9-based gene therapy agents and NUTM1-degrading molecular glues cannot be effectively tested using mouse model. · Due to the relatively loose evolution constraints on regulatory sequences, the genetic regulatory network (GRN) controlled by the NUTM1 fusion genes could differ between the two species. This could hamper the effective identification of BRD4::NUTM1 targets for future therapeutic development. To overcome these challenges, we propose to develop a human pluripotent stem cell (hPSC) derived NC model. We will use a genetic design demonstrated in our GEMM to build human PSC cell lines for modeling NC. To gain access to the progenitor cells of respiratory epithelial tissues, from which lung NCs that account for more than 50% of reported human cases likely originate, we will use human PSC-derived teratoma in immunocompromised mice as the platform to generate NCs. We will first create and characterize the human PSC-NC model (Aim 1) and then use this model to demonstrate the BRD4::NUTM1 dependency and thus the proof-of-principle of the effectiveness of NUTM1-targeted therapy for NC. (Aim 2). Overall Impact. Our project will provide a critical human-relevant in vivo preclinical model for studying NC. It will provide a proof-of-principle demonstration of NUTM1-targeted therapy. Our study will also provide a novel generalized road plan for developing in vivo human-relevant models for fusion gene-driven cancers.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT Hematopoietic stem cell (HSC) mobilization from the bone marrow to the peripheral blood is essential for bone marrow transplants, a life-saving treatment for hematological malignancies such as leukemia, lymphoma, and multiple myeloma. Poor mobilization remains a major clinical challenge, particularly in older patients (>60 years old), who represent the majority of those diagnosed with blood cancers yet often exhibit diminished responses to mobilization treatments. Age-related changes to the bone marrow microenvironment, specifically changes in microenvironmental stiffness, are believed to contribute to these mobilization failures. It has been recently measured that the bone marrow contains unique stiffness values in each identified sub-niche and observations in age-related stiffening has been reported; however, challenges with accurately measuring these values in vivo limits our understanding on how these gradients change with age. Existing 3D bone marrow models fail to capture the nonlinear stiffness gradients observed in vivo; therefore, the long-term objective of this proposal is to improve clinical predictions of a patient’s ability to successfully mobilize HSCs for a transplant. To achieve this objective, we will engineer a heterogenous, multi-niched bone marrow model with methacryloyl gelatin (GelMA) bioinks and extrusion bioprinting technologies to decouple the effects of young and aged stiffness environments on HSC mobilization. We expect the precision and automation of this approach will more accurately recapitulate the spatially transient stiffness environments of the native sub-niches. This research will target two major knowledge gaps: 1) how nonlinear gradients and age-related changes in microenvironmental stiffness influence HSC migration and phenotype, and 2) how to improve the ability to predict a patient’s ability to mobilize HSCs for more effective and personalized transplant strategies. The overarching hypothesis of this project is that age-related stiffening is a key microenvironmental cue which restricts HSC mobilization to the peripheral blood; and the mobilization of HSCs encapsulated in in vitro biofabricated models with physiomimetic stiffnesses of young and aged bone marrow sub-niches can predict the mobilization of in vivo HSCs to the peripheral blood. I will test this hypothesis through two specific aims; 1) assess the mobilization behavior of HSCs in response to GelMA stiffness gradients, and 2) correlate in vivo mobilization behavior in young and aged mice with in vitro behavior using bioprinted bone marrow models. We expect to identify the role of transient nonlinear stiffness gradients and age-related stiffness changes on HSC mobilization behavior. Furthermore, this work will improve strategies for predicting patient-specific mobilization outcomes for patients with hematological malignancies.

NIMH - National Institute of Mental Health

Abstract Despite antiretroviral therapy (ART), HIV-associated brain injury (HABI) persists in over half of people with HIV (PWH), manifesting as chronic cognitive impairment. While HIV-1 primarily infects microglia, driving central nervous system (CNS) neuroinflammation, current preclinical models do not recapitulate the chronic, suppressed infection characteristic of the ART era. Furthermore, they do not capture complex patient genetics and multicellular, glial and neuronal, interactions in a scalable and efficient manner. To address this need for more physiologically relevant models, we propose the development of an AI-guided triculture platform comprising major CNS cell types. This platform will use induced pluripotent stem cell (iPSC)-derived microglia, astrocytes, and neurons, using both morphological profiling and other omics-based profiling to model HABI under ART suppression. AI/machine learning (ML)-driven analysis of cellular morphology, combined with multi-omic data integration, will facilitate rapid classification and prediction of microglial functional states and their impact on neuronal health. Leveraging Modulo's established triculture system, previously successful in yielding therapeutic candidates for amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) currently in Investigational New Drug (IND)-enabling studies, we will construct a scalable HABI model under ART suppression. Our objectives are to (1) develop and validate an HIV-infected, ART-suppressed triculture platform, utilizing AI/ML-driven morphological profiling to classify HABI-specific microglial states; and (2) comprehensively characterize this model through neuroinflammatory profiling, behavioral correlates, and integration with publicly available HABI patient datasets. We hypothesize that our combined computational lab-based triculture system can effectively model HABI pathophysiology under ART conditions, enabling both rapid disease state classification and identification of therapeutic targets. Through the integration of experimental and computational approaches, this platform will provide insights into HABI mechanisms and accelerate therapeutic development. We will disseminate this model to the scientific community through publication and collaboration. Connecting in vitro modeling with patient outcomes offers a powerful tool for investigating neuroimmune dysfunction in HIV and related neurological disorders. Successful implementation will yield a platform for modeling neuroHIV under ART suppression, advancing our understanding of disease mechanisms and facilitating the discovery of novel therapeutic strategies for PWH with cognitive impairment.

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

ABSTRACT Early stage pediatric faculty members, even those who have potential for success as academic investigators based upon substantial experience in basic research, will require further intensive training and mentoring in basic research to successfully embark upon an independent research career. Our proposed Child Health Research Career Development Award (CHRCDA) Program entitled “Developing Diverse Physician-Investigator Leaders for the Future of Child Health” will provide a cutting-edge research opportunities, combined with a carefully constructed mentoring and career development pathway, to enable our Scholars to emerge as leaders among the ranks of pediatric academic investigators. The basic biomedical research training community at the University of California San Diego (UCSD) has a long-established reputation of excellence, and UCSD Pediatrics now ranks in the top five in NIH research funding of all Pediatric Departments in the country. Notably, the last 5-10 years have witnessed an impressive academic expansion within of the UCSD Department of Pediatrics and the research it conducts, coupled with the formalization of its partnership with Rady Children's Hospital San Diego, the largest Children's Hospital in California. The key objectives of our CHRCDA are as follows: (1) To increase the number Pediatrician-Scientists engaged in basic research as applied to children's health; (2) to attract outstanding young pediatricians to UCSD and to facilitate their career development under the guidance of world class, established investigator-faculty mentors; and (3) to cultivate the early careers of women and minority investigators in children's health. With close input from the program pioneers in our Department, CHRCDA Scholars will participate fully in the program of UCSD National Center for Leadership in Academic Medicine (NCLAM), a longstanding and highly successful junior faculty mentoring program in UC Health Sciences that provides workshops and longitudinal mentoring in all facets required for successful advancement in academic medicine, and diversity enrichment modules including the Border Health and Doc-for-A-Day programs. All CHRCDA program faculty mentors are highly regarded scientific investigators, each leading a vibrant and cutting-edge basic or basic/translational research program of strong relevance to pediatric medicine. Research training opportunities for CHRCDA Scholars are organized into six research themes of five members each, which an integral role in the program structure, curriculum and mentorship approach: (1) Genomics, Big Data & Systems Biology; (2) Infection, Immunity & Inflammation; (3) Organ Physiology & Metabolism; (4) Neuroscience & Brain Development; (5) Human Microbiome & Child Health; and (6) Developmental & Stem Cell Biology. A guiding philosophy of this CHRCDA will be to support the greatest possible number of young physician-scientists within this Program, and an additional year of Department-funded support has been added to three years of K12 support to create a vibrant program with one new Scholar per year and four fellows total in the steady state.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

PROJECT SUMMARY Chemical vesicants such as sulfur mustard (SM) and nitrogen mustard (NM) act as alkylating agents causing severe skin injuries characterized by blistering, necrosis, and persistent pathology. Both pose significant threats to civilians and warfighters, yet no approved therapeutics exist for managing vesicant-induced skin injuries. This project addresses this critical medical gap by developing innovative multifunctional wound dressings engineered to simultaneously target cell membrane damage, chronic inflammation, and secondary infection—the primary pathological mechanisms underlying vesicant-induced skin trauma. Our therapeutic approach centers on MG53, a TRIM family protein with demonstrated efficacy in cell membrane repair and wound healing. Beyond its membrane-protective functions, MG53 enhances regenerative capacity in diabetic wounds by revitalizing hair follicle stem cell activity and exerts potent anti-inflammatory effects through NF-κB pathway modulation. Building on these mechanistic insights, we will develop transformative wound dressings that integrate a novel rapid-gelling antimicrobial hydrogel with recombinant human MG53 (rhMG53) protein. We hypothesize that this synergistic combination will dramatically accelerate wound healing and tissue regeneration following chemical vesicant exposure. The goal of this project is to engineer and optimize multifunctional wound dressings combining rapid-gelation antimicrobial hydrogel technology with rhMG53 protein for vesicant-induced cutaneous injuries. Successful completion will yield breakthrough multifunctional wound dressings with integrated tissue repair, anti-inflammatory, and antimicrobial capabilities. These shelf-stable therapeutic platforms can be strategically stockpiled and rapidly deployed as essential medical countermeasures against chemical vesicant exposure, addressing both immediate clinical needs and national security preparedness requirements.

NEI - National Eye Institute

Project Summary/Abstract Vision loss is a devastating medical problem as it can lead to reduced productivity, lower quality of life, and loss of independence. Glaucoma affects over four million Americans and 76.0 million people worldwide, making it the second leading cause of irreversible blindness globally. The disease is characterized by retinal ganglion cell degeneration with consequent loss of the axons that connect the eye to the brain and progressive damage to the optic nerve. Pharmacological and surgical interventions that lower intraocular pressure can slow or even stop retinal ganglion cell degeneration. However, many patients do not seek medical attention until the disease is advanced and others continue to experience disease progression despite treatment, ultimately resulting in the widespread loss of retinal ganglion cells and profound vision loss. Unfortunately, the human retina has minimal regenerative capacity and cannot replace lost retinal ganglion cells, making vision loss in these patients permanent. The candidate’s long-term career goals are to advance our understanding of gene regulatory networks directing retinal ganglion cell differentiation during development and to apply these insights to formulate strategies for regenerating retinal ganglion cells from dormant progenitor cells in the adult retina. The proposed career development and training plans will allow the candidate to acquire further expertise in retinal development and regeneration. By learning additional cutting-edge experimental techniques, the candidate will also enhance the scientific rigor and impact of their research program. In the first specific aim, the candidate will investigate the role of transcription factor Pou2f2 in retinal ganglion cell development using conditional gene deletion, as well as gain-of-function by in vivo electroporation of postnatal progenitors. In the second and third specific aims, the candidate will perform an in vivo screen of more than 40 candidate transcription factors to identify a combination capable of reprogramming Müller glia into retinal ganglion cells. Further studies will focus on characterizing induced retinal ganglion cell morphology, laminar position, axon extension, electrophysiology and gene expression. Adaptive optics will be used to longitudinally image the reprogramming process in vivo. Lastly, the candidate will investigate survival and circuit integration of these newly generated retinal ganglion cells in mouse models of glaucoma. Because retinal ganglion cells are widely used as a model for axonal regeneration in vertebrates, these studies have broader implications for regeneration of central nervous system neurons and pathways. The candidate will conduct the proposed research in collaboration with co-mentors Dr. Yvonne Ou and Dr. Xin Duan, and the other members of the advisory committee. Experiments will take place in Rock Hall, where the candidate has dedicated laboratory space in close proximity to all collaborators. The UCSF Department of Ophthalmology is a leading center for vision science research, providing the candidate with access to NEI P30 funded core facilities and extensive university-wide resources. The candidate will also benefit from interactions with the broader neuroscience and stem cell research communities at UCSF.

OD - NIH Office of the Director

Project Summary Accidental exposure to ionizing radiation (IR) poses a significant risk for cardiovascular morbidity, a leading cause of mortality among irradiated populations. However, the mechanisms driving long-term cardiovascular risks remain poorly understood. IR disrupts immune homeostasis, exacerbating chronic inflammation and accelerating pathological remodeling. The current multi-PI U01 proposal seeks to address this knowledge gap by developing an innovative extracorporeal system composed of human stem cell derivatives to identify biomarkers and medical countermeasures (MCMs) for radiation-induced delayed cardiac remodeling (RidCR). In Milestone 1, we will establish a 3D cardiac-immune co-culture system using iPSC-derived cardiomyocytes, endothelial cells, fibroblasts, and macrophages to simulate the immune-competent 3D microenvironment. Optimized culture conditions will be validated in 3D engineered cardiac tissues (EHTs) for physiological and inflammatory responses. In Milestone 2, we will perform multi-omics analyses on irradiated EHTs and parallel animal models to identify molecular signatures of RidCR via multi-omics and functional assessments of tissue contractility. In Milestone 3, AI/ML will integrate the generated datasets to predict candidate MCMs, which will be validated in vitro and tested in vivo using a protracted irradiation mouse model. Comprehensive evaluations will assess the efficacy of the lead MCM in mitigating RidCR.

NINDS - National Institute of Neurological Disorders and Stroke

Project summary/Abstract Variants in the ubiquitin like modifier activating enzyme 5 (UBA5) result in an ultra-rare autosomal recessive disease with neurological presentations. UBA5 patients present with infantile spasms, failure to thrive, hypotonia, developmental delay, microcephaly, intellectual deficit, loss of motor skills and seizures. Most of the patients die in childhood. Current standard of care for UBA5 deficiency is focused on managing the clinical signs with standard anti-seizure medications or surgical procedures and physical therapies, but there is no treatment. Compound heterozygous mutations in UBA5 causes impairment in a ubiquitin-like post-translational modification pathway called Ubiquitin-fold modifier 1 (UFM1). UBA5 is an E1 activating enzyme on UFM1 pathway. The role of the UBA5 and UFM1 system in the central nervous system (CNS) has not been studied. This stems from lack of a viable mammalian model for UBA5 deficiency. Our team has identified the first viable Uba5 mouse model that carries patient mutation, exhibits an overt phenotype, and recapitulates presentations of UBA5 deficiency in patients including smaller body size, motor, cognitive and gait abnormalities. Our team has postmortem tissues of UAB5 patients, their clinical course, MRI and EEG records. The first neuropathological characterization of postmortem UBA5 patient brain indicates the shared features with Uba5 mice. To determine the top adeno associated virus (AAV) vector candidate for efficacy studies in Uba5 mouse, we developed four UBA5 expressing constructs and showed their 1) efficacy in restoration of expression and function of UBA5 in UBA5 Knockout HEK293T cells and 2) durability, safety, and cell type tropism in a one-year study in wild type mouse. The top candidate, AAV9-JeT-UBA5, restored motor, cognitive and most aspect of gait abnormalities in Uba5 mouse model treated by neonatal intracerebroventricular (ICV) treatment. However, weight of treated Uba5 mice did not get normalized. We hypothesize that gradual loss of transduced cells in liver prevented long term weight gain normalization. Since the overarching goal of this project is to develop a transformational AAV gene therapy to treat our symptomatic UBA5 patient cohort at UMass Chan, we need to address the therapeutic imperfections and develop biomarkers. We will perform CNS and periphery wide gene therapy of JeT-UBA5 in pre and post symptomatic Uba5 mouse to determine the therapeutic window and feasibility of gene therapy to rescue or modify disease course. We will use 1) AAV9 capsid for combined CSF and periphery wide gene delivery in Uba5 mouse and 2) a new blood-brain barrier penetrant capsid (BI-hTFR1; interact with human Transferrin Receptor (TFRC)) for very efficient gene delivery to CNS and periphery by systemic injection. We will perform an in-depth characterization of the Uba5 mouse model and its humanized version, expressing TFRC, with clinically relevant outcomes measure (MRI and EEG) and compare them with patient findings (Aim 1). Four gene therapy approaches will be performed in Aim 2. In Aim 3 metabolomic based biomarker discovery will be performed and the best gene therapy approach to normalize transcriptomic profile of Uba5 mouse of will be determined.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary/Abstract Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that establishes lifelong latency and poses a serious threat to immunocompromised individuals and developing fetuses. A major barrier to developing effective antiviral therapies is our limited understanding of the mechanisms governing HCMV latency, due in part to challenges in modeling this process in physiologically relevant human cells. This R03 project proposes to establish adipose-derived stem cells (ADSCs) as a novel, scalable, and biologically relevant in vitro model of HCMV latency. We will define the conditions under which ADSCs support latent infection and compare their transcriptional program to established latency models using RNA sequencing. This research will advance our understanding of HCMV latency and provide a critical tool for future studies aimed at developing therapies to prevent viral reactivation in vulnerable populations.

NCI - National Cancer Institute

PROJECT SUMMARY Immunotherapies such as bispecific T-cell engagers (BiTEs) and chimeric antigen receptor (CAR) T cells have shown significant promise in treating hematologic malignancies. However, therapeutic responses vary between patients and across the different subtypes of leukemia and lymphoma. A key challenge in optimizing these therapies is the lack of in vivo preclinical models that accurately reflect both the patient's immune and cancer cell biology. Current patient-derived xenograft (PDX) models lack functional immune systems, while humanized mouse models typically involve healthy donor immune cells paired with cancer cells from a different donor. These immunologically mismatched, or allogeneic, models fail to replicate autologous immune-cancer cell dynamics and the effect the cancer microenvironment and therapy have on immune cell function. To overcome these limitations, we aim to develop innovative autologous humanized PDX models using leukemia and immune cells derived from the same patient. We will collect paired bone marrow (BM) samples from pre-B acute lymphoblastic leukemia (B-ALL) patients at diagnosis and remission. Hematopoietic stem and progenitor cells (HSPCs) from the remission BM will be expanded and transplanted into immunodeficient mice to generate humanized mice with intact immune systems. These mice will then be engrafted with diagnostic leukemia cells, creating PDX models with autologous immune and leukemia cells. In parallel, a second cohort of models will be developed using autologous peripheral blood mononuclear cells (PBMCs) and leukemia cells from the same patient. We hypothesize that these fully patient-derived autologous models will provide deeper insights into immune-leukemia interactions and enhance preclinical testing of immunotherapies. To test this hypothesis, we will pursue three specific aims. First, we will establish autologous PBMC- and HSPC- humanized models using samples from B-ALL patients with diverse genetic and risk subtypes. We will evaluate disease progression and immune responses longitudinally in these models. Second, we will assess the efficacy of a CD3/CD19 BiTE and CD19 CAR-T cells in these autologous models. Third, we will analyze the immunophenotypic and transcriptional profiles of cells from autologous models, allogeneic models, and patient samples before and after immunotherapy treatment to validate the translational relevance of the models. These studies aim to create robust, patient-specific models that can be used to test and optimize immunotherapies, ultimately improving their clinical impact in the treatment of hematologic malignancies.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT Immunotherapy has revolutionized the treatment of many tumors. However, most GBM patients have not, so far, benefited from immunotherapeutic treatment. With the goal of exploring ways to boost anti-GBM immunity, we’ve developed a B-cell-based vaccine (BVax) that consists of 4-1BBL+ B cells activated with CD40 agonism, BAFF and IFNγ stimulation. BVax migrate to key secondary lymphoid organs and are proficient at antigen cross-presentation, which promotes both the survival and functionality of CD8+ T cells. A combination of radiation, BVax, and PD-L1 blockade conferred tumor eradication in 80% of treated tumor-bearing animals. We have been successful at generating GBM patient-derived BVax that activated autologous CD8+ T cells, which shows a strong ability to kill autologous glioma cells. This demonstrates that BVax can be produced from patient’s peripheral blood. Our preliminary data obtained under the parental 5R37CA258426 proposal showed that BVax promotes the expansion of clones that differ from CD8 T cells activated by dendritic cells (DC) and the proliferation of stem-like TCF-1+ CD8 T cells. In addition, we provided solid evidence that BVax produces antibodies that react to tumor-associated antigens and inhibit tumor growth. Our central hypothesis is that the BVax have unique properties as antigen-presenting and antibody- producing cells. More specifically, BVax might present a different set of antigens to CD8 T cells. In addition, BVax monoclonal antibodies (mAbs) might have a potential therapeutic effect. This research proposal aims to deep-dive into the immune mechanisms underlying this protection and prevention of tumor growth. We will focus on two processes: antigen presentation and activation of CD8+ T-cell memory formation (Aim 1) and the characterization (sequencing and cloning) of single-BVax monoclonal Ab production (Aim 2). Overall, our study provides a novel alternative to current immunotherapeutic approaches that can be readily translated to the clinic.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT Acute myeloid leukemia (AML) remains a lethal disease despite recent therapeutic advances, including the BCL2 inhibitor venetoclax. Most patients ultimately develop relapsed or refractory disease, underscoring the urgent need for new therapies, particularly agents that synergize with venetoclax. One promising strategy is to target the integrated stress response (ISR), a conserved pathway that modulates protein synthesis through phosphorylation of eIF2á by one of four stress-sensing kinases: GCN2, PKR, PERK, or HRI. This phosphorylation reduces global cap-dependent translation while selectively increasing translation of transcripts such as ATF4, which drive adaptive or pro-apoptotic programs depending on context. Leukemia stem cells rely on chronic ISR activity to withstand metabolic stress, suggesting that further ISR activation could tip the balance toward apoptosis. Consistent with this idea, we found that venetoclax itself activates the ISR via HRI, and its efficacy in preclinical models depends on this mechanism. We discovered novel ISR modulators using our integrated platform that combines high-throughput phenotypic screening with rapid target deconvolution. Through this approach, we identified ligands of RPS23, a 40S ribosomal subunit protein, that activate the ISR through GCN2 by a mechanism distinct from venetoclax and known ribosome binders. These ligands trigger apoptosis in leukemia cells and prolong survival in aggressive AML mouse models with minimal toxicity to normal hematopoietic cells. We hypothesize that RPS23 ligands represent a novel therapeutic strategy for AML and may act synergistically with venetoclax to overcome resistance. The discovery of this ISR-inducing target, together with a bioavailable compound showing preclinical efficacy, highlights the significance and innovation of our approach and provides a strong foundation for clinical translation. To advance this therapeutic strategy, our Specific Aims will define the mechanism by which RPS23 ligands activate the ISR and evaluate their on-target toxicities and efficacy alone and in combination with venetoclax in disease-relevant models of AML. No validated in vitro or computational model currently recapitulates the integrated immune, vascular, and metabolic interactions required to evaluate therapeutic efficacy and toxicity in vivo.