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NHLBI - National Heart Lung and Blood Institute

Project Abstract The alveolar epithelium is composed of two distinct cell types—alveolar epithelial type I (AT1) cells, which facilitate gas exchange, and alveolar epithelial type II (AT2) cells, which act as progenitors for AT1 cells. Successful lung repair following alveolar injuries requires AT2 cell proliferation and differentiation into AT1 cells, a process that involves the restructuring of gene regulatory networks and cell-type specific chromatin landscapes that underly these two cell fates. A dysfunctional regenerative response has been observed in a variety of severe lung diseases, involving AT2 cells acquiring a pathologic, alveolar-basal intermediate (ABI) cell state at the expense of an AT1 fate. The mechanisms that facilitate the cell fate decisions involved in AT2 cell maintenance and differentiation are not well understood, which has resulted in a lack of effective treatments to promote alveolar regeneration. This project aims to identify and characterize regulatory, 3-dimensional “hubs” of chromatin interaction that instruct distinct alveolar epithelial cell fates, and to determine how these hubs and their associated transcription factors regulate the acquisition of healthy and disease-associated states. Using human induced pluripotent stem cell (iPSC) models of AT1- and AT2-like cells (iAT1s and iAT2s), we will apply advanced chromatin mapping techniques to identify cell-type specific enhancer-promoter interactions and to characterize chromatin hubs that potentially regulate normal AT1 and AT2 cell identity. In Aim 1, we will map these interactions in healthy iAT1 and iAT2 cells, comparing their chromatin landscapes to pinpoint regulatory hubs that we hypothesize are responsible for cell-type specific gene expression. In Aim 2, we will explore the effects of haploinsufficiency of the lung lineage transcription factor, NKX2-1, on chromatin topology of iAT2 cells, hypothesizing that reduced NKX2-1 expression disrupts normal AT2 cell identity and favors a pathological ABI state. The findings from this research will enhance our understanding of the chromatin-based mechanisms that control lung cell fate decisions and provide insights into how disruptions of chromatin organization contribute to pulmonary disease.

NIGMS - National Institute of General Medical Sciences

Project Summary/Abstract Congenital heart diseases (CHDs) are the most common birth defect, occurring approximately in 1% of live births. Over 97% of mutations associated with CHD occur within the non-coding genome, potentially disrupting transcription factor (TF) binding to regulatory DNA and dysregulating gene regulation essential for heart development. However, due to their overwhelming numbers, it has remained challenging to identify causal mechanisms between non-coding mutations and human diseases. The long-term goal of the proposed work is to describe the molecular mechanisms by which disease-associated non-coding mutations dysregulate gene expression, leading to human diseases. Herein, we propose a high-throughput biochemical strategy to study the role of CHD-associated non-coding mutations in cardiac TF binding and gene regulation. Our hypothesis is that CHD-associated non-coding mutations will impact cardiac TF- DNA binding and disrupt gene regulation necessary for proper heart development. We have leveraged genome-wide association studies (GWAS) to identify 121 CHD-associated mutations in the non-coding genome. To account for the resolution limitations of GWAS, we performed a linkage disequilibrium expansion on the CHD-associated mutations to include single-nucleotide variants from diverse ancestries, resulting in 3,232 mutations. Additionally, we included all possible alleles for each genomic variant, resulting in 12,928 permutations. In Specific Aim 1, we will measure the impact of ~12,000 single- nucleotide non-coding variants on the binding of three master cardiac developmental TFs GATA4, NKX2-5, and TBX5. We will measure binding aYinities through Single Nucleotide Polymorphism-Systematic Evolution of Ligands by EXponential enrichment (SNP-SELEX) and identify non-coding mutations that decrease or increase cardiac TF binding aYinity. Our approach will allow us to quantitatively measure TF binding aYinities for ~12,000 genomic loci in a single experiment. In Specific Aim 2, we will determine the impact of the ~12,000 CHD-associated non-coding mutations on regulatory element activity during cardiomyocyte diYerentiation. The gene regulatory activity of ~12,000 promoter and enhancer variants will be measured in human embryonic stem cells, cardiac progenitors, and cardiomyocytes by massively parallel reporter assay (MPRA). This integrated approach will train undergraduate and graduate students in high-throughput biochemistry, functional genomics, and large-scale data analysis. Completion of the proposed project will contribute to our understanding of the mechanisms of non-coding mutations in CHDs and can be extended to study gene dysregulation in multiple human diseases.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

Abstract Skeletal muscle contains a population of adult stem cells called satellite cells or muscle stem cells (MuSCs) that are responsible for regeneration after injury. MuSCs utilize gene expression programs to maintain quiescence and differentiate after injury and a key regulator of gene expression is splicing, which uniquely changes when transcripts interact with nuclear speckles. Nuclear speckles are membrane-less biomolecular condensates that phase separate proteins, RNAs and chromatin, but how these organelles regulate molecular processes in MuSCs remains unknown. Key experiments from our laboratory provide rigorous support for a role of nuclear speckles and splicing in MuSC function, which were attenuated in old age. The overarching objective of this program is to establish a systems-based approach to understand how nuclear speckles and alternative splicing contribute to MuSC programs of activation and regeneration across lifespan. In Aim 1, we will demonstrate that the loss of a nuclear speckle scaffolding protein, Srrm2, will reduce regenerative potential of muscle stem cells. In Aim 2, we will establish that increases in oxidative stress from old age attenuate nuclear speckles and RNA splicing that regulate muscle stem cell activation and repair of muscle injury. In both Aims, we will use novel transgenic animal models, sophisticated bioinformatics analysis and highly innovative molecular tools to build a comprehensive and new understanding of nuclear speckles influence on alternative splicing and stem cell activity. Successful completion of this program will advance our knowledge of fundamental cell biology for regenerative medicine, and provide a myriad set of insights across molecular, cellular and tissue scales.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY/ABSTRACT Very early onset inflammatory bowel disease (VEOIBD) is defined as disease onset at age <6 years and is associated with a more severe and aggressive disease course. Up to 10% of affected children have an underlying disease-causing monogenic immunodeficiency, such as defects in IL10 signaling, but the mechanisms driving disease in the majority of patients remain unknown. Furthermore, there are no biologic or small molecule therapies FDA-approved for use in IBD in this age group and current treatment approaches rely on empiric trials of medications approved for adult IBD. Collectively, these factors contribute to significant treatment challenges in VEOIBD and to substantial cost and morbidity stemming from delays in remission, growth failure, steroid- dependence, hospitalization, and surgical intervention. There is an urgent need to define disease mechanisms that enable biomarker-guided treatment selection in both VEOIBD and IBD more broadly. We have identified a unique subgroup of VEOIBD patients defined by markedly diminished activation of STAT3 in response to multiple cytokines, including IL10 (“STAT3-aberrant” or STAT3-abr, n=7). These patients share a clinical phenotype characterized by severe, refractory colonic disease without an identifiable monogenic basis and a blood transcriptional signature characterized by increased IL23 signaling. Notably, four of these patients have been treated with anti-IL12/23 or anti-IL23 following multiple prior medication failures, and all four achieved rapid, sustained remission, implicating IL23 as a key disease driver and therapeutic target. The overall goals of this proposal are to define the mechanisms underlying this STAT3-abr signaling state and to leverage signatures of this state as biomarker(s) of response to IL23-blocking therapies. Our central hypothesis is that excessive IL23 signaling promotes preferential activation of pro-inflammatory (e.g., IL23) STAT3-dependent programs at the expense of anti-inflammatory (e.g., IL10) STAT3-dependent programs in this STAT3-abr group and a subset of patients with later-onset disease. Aim 1 will define STAT3-dependent signaling and how IL23 modulates that signaling in STAT3-abr patients compared to controls. Aim 2 will examine epigenomic and transcriptional consequences of the STAT3-abr signaling state by examining differences in chromatin accessibility, STAT3-DNA binding, cellular composition, and gene expression in STAT3-abr patients compared to controls. Aim 3 will develop and evaluate a multimodal predictive tool that incorporates STAT3-abr clinical and transcriptional features to predict response to IL23-blocking therapy in pediatric and adult IBD cohorts. Completion of this work will mechanistically define a novel STAT3 signaling state in VEOIBD and develop a biomarker predictive of response to IL23-blocking therapies, impacting personalized medicine opportunities for IBD patients of all ages.

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.

NIGMS - National Institute of General Medical Sciences

ABSTRACT All growing cells experience DNA Replication Stress (RS, a slowing of DNA synthesis), which poses a major threat to genome integrity and viability. To safeguard against RS, stalled replication forks trigger DNA Damage Response (DDR) signaling cascades which coordinate resolution of stalled forks with cell cycle progression and other processes to ensure cellular homeostasis and survival. DDR defects can lead to severe human health consequences including stem cell dysfunction, aging, neurodegeneration, neoplasia, immune deficiencies, and developmental syndromes. While DDR signaling is clearly crucial for recovery from RS, the ways in which the many effector branches of the DDR are activated, coordinated with each other, and integrated with other cellular processes to orchestrate cell fate decisions are poorly understood. In exciting recent work we identified the RING finger E3 ligase RNF25 as a major new replication fork-localized DDR factor that is critically required for replicating cells to tolerate RS. Previous studies showed that RNF25 ubiquitylates ribosomal proteins to alleviate ribotoxicity (RNA damage). Remarkably, we demonstrated that RNF25 remediates RS independently of its canonical role in regulating ribosome function. Thus RNF25 is the first example of a `dual stress responder' that mediates responses to both genotoxicity and ribotoxicity. Our discovery of RNF25 as a major new DDR factor reveals large gaps in our knowledge of genome maintenance. The objective of this application is to define how RNF25 is regulated, and how it interfaces with other components of the DDR network and the ribotoxic stress response to alleviate RS. Our separation-of-function RNF25 mutant also provides an outstanding opportunity to determine the relative contribution of genotoxicity vs. ribotoxicity to cell fate decisions. The rationale is that we will mechanistically define a major new effector branch of the DDR (mediated by RNF25), and we will reveal how it is coordinated with other pathways to ensure genome integrity and survival. Our central hypothesis is that RNF25 critically interacts and cooperates with several important DNA repair proteins (REV7, PARPs, and MAGE-A4) to resolve RS. Our Specific Aims (SAs) are: SA1 Define how RNF25 associates with REV7 to orchestrate responses to DNA-damaging agents. SA2 Establish PARPs as mediators of RNF25 signaling in the DDR. SA3 Establish the E3 ligase cofactor MAGE-A4 as a regulator of RNF25 signaling. We will use biochemical approaches to define mechanisms by which RNF25 associates with its partner proteins. We will use unbiased screens to define genetic interactions between RNF25 and other major DDR pathways. We will use phenotypic endpoints (such as DNA synthesis at single DNA fiber resolution, cell cycle progression and viability) to define how RNF25 and its interacting proteins and pathways dictate cell fate. We propose innovative solutions to important problems such as `How do cells activate the DDR?' and `How are different branches of the DDR integrated and coordinated?'. The proposed work is significant because we will mechanistically define an important new branch of the DDR.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY/ABSTRACT Odontogenic pain, also known as toothache, is the most common form of orofacial pain across the world. Most commonly, odontogenic pain stems from inflammation of the tooth pulp following either bacterial infection or iatrogenic damage. While this pain will resolve with clinical intervention that removes the peripheral insult and dental pulp (e.g., root canal or extraction), it may be accompanied by substantial mechanical and thermal allodynia that is not alleviated by local anesthesia. We continue to lack safe approaches to achieve effective, immediate analgesia toward necessary dental treatments to resolve odontogenic pain. To inform the development of non-opioid analgesics we need fundamental knowledge of the neuronal basis and behavioral phenotypes of odontogenic pain. Mammalian teeth are highly innervated by specialized somatosensory neurons, intradental neurons, that detect damaging stimuli and initiate a reflex response to protect the teeth. However, we have yet to define mechanisms by which intradental neurons produce odontogenic pain in the context of damage and inflammation. Here, Dr. Joshua Emrick and his laboratory will investigate the neuronal basis of toothache to provide targets for future development of analgesics. The overall objective of this application is to use mouse models and powerful, cutting-edge approaches to define the neurons, circuits, and phenotypes of odontogenic pain. Aim 1 will reveal peripheral mechanisms whereby intradental trigeminal sensory neurons are activated representing a basis for pulpitis-induced pain. Aim 2 will reveal central mechanisms whereby intradental trigeminal sensory neuron inputs lead to physiological responses. Aim 3 will provide novel preclinical phenotypes of odontogenic pain in mammals to provide objective measures for future evaluation of analgesia. Upon successful completion of this project, we will have a molecularly-defined cellular origin for odontogenic pain from inflammation. Further, we will define the neurons and circuits that relay odontogenic pain in the brainstem. We also will have defined phenotypes for odontogenic pain initiated by the intradental neurons. These outcomes will provide key mechanistic insight into the origin of odontogenic pain toward the long-term development of novel non-opioid analgesics.

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.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT Defects in cellular proliferation can lead to a variety of developmental disorders as well as cancer. This dysregulation is achieved through deleterious mutations of tumor suppressor genes (TSGs). However, tumor suppressors can be non-genetically inactivated, which is difficult to detect with standard genomic techniques. While some TSG somatic mutations have been identified in hematologic malignancies, there is a critical gap in knowledge for the role of TSG silencing in leukemogenesis. This study will examine how TSGs are inactivated at the post-transcriptional level in acute myeloid leukemia (AML) and how their restoration is a novel therapeutic avenue for leukemic elimination. I found that the transcriptional repressor ZBTB7A is post-transcriptionally silenced in AML, blocking differentiation in the myeloid lineage. This is achieved through alternative polyadenylation, resulting in an isoform with a longer 3’UTR that leads to deadenylation. I was able to restore ZBTB7A expression through epigenetic targeting of upstream negative regulators as a proof of concept of TSG re-activation. During the training (K99) phase of this award, I will uncover the exact RNA-binding proteins mediating downregulation of ZBTB7A, and if this is part of a larger pro-AML regulatory network blocking differentiation. To this end, we are employing a novel long read sequencing method termed Pull-a-Long Sequencing (PL-Seq) in collaboration with Dr. Pedro Miura, to uncover the roles of ZFP36 and ELAVL family members on the transcriptional stability of ZBTB7A. ZBTB7A has two paralogs in mammals, ZBTB7B and ZBTB7C. ZBTB7B has been implicated in hematopoietic differentiation of the lymphoid lineage, and the DNA-interacting residues of its zinc fingers are highly conserved to ZBTB7A. During the independent (R00) phase, I will apply the insights and tools acquired during the training phase to identify the contribution of ZBTB7B paralogs with ZBTB7A, and ascertain if they are synergistic in mediating myeloid differentiation. In my preliminary data, I have utilized a Rosa26LSL-Cas9-eGFP mouse model to delete Zbtb7a in the murine hematopoietic system and found that loss of Zbtb7a results in hematopoietic stem and progenitor (HSPC) expansion, as well as a bias in myeloid differentiation. With the guidance of Dr. Jennifer Trowbridge, an expert in mouse modeling of the hematopoietic system, I will more fully characterize Zbtb7a hematopoietic specific knockout mice as well as Zbtb7b knockout mice. Together, this will open multiple lines of inquiry into a family of transcriptional repressors and the role of non-genetic inactivation of tumor suppressors. My proposed study will generate novel tools and testable hypotheses that will lay a strong foundation for my independent research program, where I will continue to characterize non-genetic mechanisms of AML regulation as novel means for therapeutic intervention.

NIA - National Institute on Aging

Sporadic Alzheimer’s disease (sAD) affects millions worldwide, yet the earliest molecular events that drive its progression remain poorly understood. Hallmark pathologies—amyloid-β (Aβ) plaques and tau neurofibrillary tangles (NFTs)—develop decades after subtle cellular changes have already begun. One of the strongest emerging biomarkers of preclinical AD is soluble phosphorylated tau at threonine 217 (pT217Tau), which appears in cerebrospinal fluid and plasma years before overt symptoms or brain pathology. Endosomal enlargement, a robust early abnormality linked to Aβ42 generation from amyloid precursor protein (APP), is also present in at-risk individuals. However, the spatial and temporal relationship between early-stage tau hyperphosphorylation, amyloid pathology, and endosomal changes has never been directly visualized in human or nonhuman primate brains. This gap limits the development of early diagnostic tools and therapies. The overall goal of this project is to define the earliest molecular convergence points between soluble pT217Tau, APP/Aβ42, and endosomal abnormalities across human postmortem tissue, aging nonhuman primates, and human induced pluripotent stem cell (hiPSC)–derived neurons. This multiscale approach is uniquely enabled by (1) postmortem human tissue spanning Braak stages I–VI, (2) perfusion-fixed rhesus macaque brains that preserve soluble phosphorylation states and native ultrastructure, and (3) mechanistic testing in hiPSC-derived neurons including SORL1-deficient lines, which model genetic risk for endolysosomal dysfunction. Aim 1 will define the spatial relationship between pT217Tau, endosomal enlargement, and APP/Aβ42 in early human AD stages using quantitative immunofluorescence across vulnerable (entorhinal cortex, hippocampus, dorsolateral prefrontal cortex) and resilient (primary visual cortex) regions. Aim 2 will examine age-related associations between pT217Tau, APP/Aβ42 and endosomal abnormalities in rhesus macaque cortex, applying dual-label immuno- electron microscopy to achieve nanoscale resolution of tau–endosome interactions. Aim 3 will determine whether promoting tau hyperphosphorylation in hiPSC-derived neurons is sufficient to induce endosomal abnormalities and altered APP/Aβ42 trafficking, with particular emphasis on retromer dysfunction in SORL1- deficient cells. By combining observational and experimental systems, this project will directly test the hypothesis that soluble pT217Tau—but not fibrillar tau—is preferentially associated with APP/Aβ42-containing endosomes in neurons of vulnerable cortical regions, and that this phenotype can be recapitulated in vitro. The proposed studies are highly innovative in their integration of human, nonhuman primate, and cellular models, and in their use of cutting-edge nanoscale imaging to capture disease processes at unprecedented resolution. Results will illuminate the etiology of sAD pathogenesis, identify the earliest cellular events linking tau and amyloid pathology, and reveal new targets for early intervention. Ultimately, this work has the potential to transform how we detect and treat sAD by focusing on the earliest and potentially reversible stages of the disorder.

NIGMS - National Institute of General Medical Sciences

Project Summary: Nutrients are important regulators of cell states and cell fate transitions. In this context, Vitamin C (VitC), an essential nutrient, is an important regulator of extracellular matrix (ECM) synthesis and of multiple cell fate transitions, including somatic cell to induced pluripotent stem cell (iPSC) reprogramming. However, the molecular mechanisms underlying these effects remain poorly understood. Prior studies have largely attributed VitC function to the regulation of Fe2+/α-ketoglutarate-dependent dioxygenases (αKGDDs). Yet, several studies report only minimal VitC-dependent changes in collagen hydroxylation, and our preliminary data indicate that VitC- dependent enhancement of iPSC reprogramming occurs independently of αKGDD activity. Instead, we discovered that VitC rapidly activates the oxidative pentose phosphate pathway (PPP), increasing NADPH and ribose-5-phosphate (R5P) levels, and enhances nucleotide sugar synthesis. We also found that VitC increases total ECM glycosylation, including N-linked glycosylation of collagen I (Col I) and P4HA1, the prolyl hydroxylase required for collagen secretion. We hypothesize that VitC-dependent metabolic rewiring promotes glycosylation- dependent ECM synthesis, and regulates transcriptional programs to facilitate iPSC reprogramming. The proposed research aims to understand the molecular mechanisms by which VitC regulates ECM synthesis and cell-fate transitions. AIM 1 will elucidate the molecular mechanisms of VitC-dependent metabolic rewiring and provide training in genetic manipulation and metabolite tracing techniques to enable mechanistic studies of metabolic pathway regulation. AIM 2 will determine the role of VitC-driven metabolic changes in regulating ECM synthesis, and in regulating transcriptional programs during iPSC reprogramming, and provide necessary training in single-cell RNA-seq approaches to define how metabolic and ECM changes influence transcriptional trajectories and cell fate transitions. Finally, the independent phase of this award, AIM 3, will elucidate the mechanisms by which VitC-dependent metabolic rewiring regulates ECM protein glycosylation and secretion. This work will reveal novel pathways linking nutrient-dependent metabolic rewiring to ECM remodeling and cell state regulation, with broad implications for understanding ECM-related diseases such as fibrosis and for nutrient-driven control of cell fate. The mentored phase of this award provides the opportunity to work with leaders in the fields of cell-fate regulation and metabolism, to acquire the training needed to achieve my long- term goal of becoming an independent investigator at the interface of both fields. Together, the proposed research, mentorship team, and resources provided for career development at University of California, Los Angeles, will provide the training needed to achieve the defined career goals.

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.

NIAID - National Institute of Allergy and Infectious Diseases

Multiple sclerosis (MS) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) are immune-mediated demyelinating diseases that impact close to 5 million people worldwide. White matter plaque formation leads to acute inflammation, chronic demyelination, and axonal loss. These inflammatory lesions predominantly consist of T lymphocytes and reactive microglia, which lead to the breakdown of the protective myelin sheath coating on axons and the destruction of oligodendrocytes. This degradation leads to the release of myelin oligodendrocyte glycoprotein (MOG) and other proteins expressed on the outermost later of the myelin sheath into the lesion microenvironment. With the recent explosion of biologics and immunotherapy, new treatment options have become available for MS but remain limited for MOGAD. However, while these therapies are effective at reducing the frequency of relapses, there are risks associated with long-term systemic immunosuppression and in many patients, their disease eventually progresses. Therefore, the overall goal of this proposal is to develop novel, safe, and effective treatments and strategies that can specifically modulate the immune response in MS and MOGAD. Mesenchymal stem cells (MSCs) are characterized by their immunosuppressive and regenerative properties. As such, adoptive transfer of MSCs has been investigated as an independent therapeutic option in immune-mediated diseases and was recently FDA approved in pediatric graft-versus-host disease. MSCs mediate their immunosuppressive capabilities at least partly through the secretion of immunosuppressive cytokines, such as interleukin (IL)-10. IL-10 is a potent anti-inflammatory cytokine that inhibits the synthesis of IL-2 and interferon-ɣ, two cytokines crucial for T cell development and activation. IL-10 is also reported to have neuroprotective effects by inhibiting astrocyte activation and preventing accumulation of the excitatory neurotransmitter glutamate. Previous studies have implicated that engineering MSCs with designer chimeric antigen receptors (CARs) results in enhanced trafficking to sites of inflammation and augmented immunosuppressive capabilities. This leads to our central hypothesis that engineering MSCs to express MOG-targeting CARs that secrete IL-10 (MOG-CAR-IL10) can ameliorate treatment-refractory MS and MOGAD. This hypothesis will be tested by pursuing two specific aims: Aim 1 will study trafficking of MOG-CAR-IL10 to the central nervous system in mouse models of T-cell mediated demyelination and Aim 2 will determine the immunosuppressive effects of MOG-CAR-IL10 on MS in preclinical models. By the completion of this work, we expect to have defined how MOG-CAR-IL10 interacts with T cells in MS and MOGAD and to have developed novel targeted cellular engineering therapies to ameliorate treatment-refractory MS and MOGAD more effectively. This proposal will allow me to acquire cross-disciplinary training in (i) mouse modeling, (ii) vector design and cell engineering, (iii) MSC-immune cell functional interactions, and (iv) translational science.

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.