University Of Pennsylvania

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY The goal of this proposal is to determine how cell identity, as defined by a transcriptional program, is inherited through mitosis by systematically evaluating the role of specific cis-regulatory elements and trans- acting factors. The central hypothesis is that cis-regulatory elements at gene promoters maintain transcriptional competency through mitosis while trans interactions with enhancers mediate the timely and appropriate level of gene expression during mitotic exit. This proposal will leverage how the underlying DNA sequence determines the multifaceted interactions at promoters and cell-type specific enhancers that ensure cells faithfully re- establish proper transcriptional programs. This will be evaluated in cells with a stable identity and expanded to understand how this is mediated in the context of early development, when cells undergo constant fate decisions. While it was previously thought that chromatin condensation during mitosis excluded most proteins and thereby transcription-related processes, recent studies originally emanating from the Zaret lab now indicate that transcription is active at a low level and promoters, but not most enhancers, are accessible and maintain active histone modifications [1-7]; and mitotic chromatin arms retain areas of dynamic chromatin [8]. Although genome-wide measurements of histone modifications, chromatin accessibility and chromatin organization suggest mitotic changes at various cis-regulatory elements, they are unable to garner high- resolution mechanistic insight into how mitotic memory is retained at promoters and acted upon, during mitotic exit, by enhancers. While components involved in transcription such as the TATA-binding protein TBP, the elongating form of RNAP2, and transcription factors (TFs) such as GATA1, FOXA1, and ESRRB are detected on mitotic chromatin; many of these TFs do not remain bound to their interphase enhancer targets, thus how these factors function at specific loci to transmit transcription memory through mitosis remains elusive [5-7, 9- 13]. Therefore, this proposal aims to determine how cell fate is preserved through cell division by first evaluating the functional requirement for promoters in mitotic transcription, and then the mechanisms employed by liver-specific transcription factors to mediate enhancer activation upon mitotic exit. This will be assessed in the liver-derived HUH7 human cell line, which is highly amenable to mitotic synchronization methods, and expanded to determine the role of identified factors in pluripotent and endoderm-differentiated human embryonic stem cells. This proposal will expand our understanding of the molecular mechanisms that maintain cell identity and cell fate specification, thereby improving our ability to target and modulate stem cells for therapeutic value.

NIH Research Projects · FY 2025 · 2023-08

An individual’s health decisions and knowledge stem from the norms of family, peers, and communities; media sources; and federal, state, and local policy. Many of these factors stem from a historical evolution and regional idiosyncrasies, including differences in levels of state funding, public health communications, and community health knowledge. Although there is evidence that these factors contribute to health in several domains, no past research has examined the ecology of this body of knowledge or its evolution over time. This project will investigate the interplay of these ecological factors at the state and county level to predict over-time changes in health knowledge in critical health areas, including HIV, diet, smoking, substance use, and exercise. The project will study a sample of 1500 United States residents and county-level information in the media, as well as health policies and norms. After understanding key factors affecting health knowledge, we will conduct an experiment with a new sample of participants (N = 1,000; Aim 2). Participants will be randomized to conditions that combine the key knowledge, policy, and communication factors identified in Aim 1. The project will be informed by extensive pilot data on health policy, public health communications, and health information across states and over time, as well as experience recruiting and managing large longitudinal studies. The team includes expertise in public health, psychology, medicine, communication, public policy, and economics, and will leverage the resources of the University of Pennsylvania, the Penn CFAR, and the Annenberg Public Policy Center (APPC) to conduct cutting-edge research about the determinants of health knowledge.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT This proposal describes a comprehensive 5-year mentored career development plan with the goal of training the candidate to become a leading independent physician-scientist focused on improving the diagnostic evaluation and management of pulmonary nodules (PNs) to optimize early detection of thoracic cancer and minimize unnecessary harms to patients. The candidate is currently a Post-Doctoral Research Fellow and Attending Pulmonologist at the University of Pennsylvania (Penn). The proposal builds upon Dr. Kim’s previous research training in epidemiology and biostatistics and clinical experience in thoracic oncology. PNs are commonly detected by computed tomography (CT). Lung biopsy, a highly invasive procedure, is required for a definitive diagnosis but carries significant risks and costs. Thus, clinicians face the diagnostic challenge of PN malignancy risk estimation when deciding which patients should undergo a biopsy, and which should be surveilled with repeat imaging. The overall goal of this project is to address the current inadequacy of estimating malignancy risk within the diagnostic process of PN evaluation by assessing the clinical utility and effectiveness of a radiomics-based computer-aided diagnosis (CAD) tool. This novel technology synthesizes quantitative features from raw CT imaging data invisible to the human eye and has been previously demonstrated by the candidate’s team to improve clinicians’ PN diagnostic accuracy. This project’s goal will be accomplished via three complementary specific aims. In Aim 1, a retrospective cohort study will be performed to determine the clinical utility of a CAD-based risk stratification strategy using net reclassification indices, decision curve analysis, and relative utility curves. In Aim 2, a pilot, single-center pragmatic randomized clinical trial will be conducted to compare the clinical effectiveness of a CAD-based risk stratification strategy to usual care for appropriate management of PNs, defined as biopsy or empiric treatment for malignant PNs and surveillance for benign PNs. Finally, in Aim 3, the cost-effectiveness of a CAD-based risk stratification strategy will be evaluated using decision analytic models for a simulated cohort of individuals with newly detected PNs. Dr. Kim has outlined a rigorous training plan of coursework, skills acquisition (with a focus on clinical utility analysis, clinical trial design, decision analytic modeling, and cost-effectiveness analysis), and professional career development. To realize this vision, he has assembled a distinguished, multidisciplinary mentorship and advisory team, led by his primary mentor, Dr. Anil Vachani, the Director of Clinical Research in the Section of Interventional Pulmonology and Thoracic Oncology at Penn, and co-mentor, Dr. Katharine Rendle, Deputy Director for Research at the Penn Center for Cancer Care Innovation. Penn provides an outstanding intellectual, collaborative, and supportive environment for this proposal, positioning Dr. Kim to successfully complete the aims and training plan so that he will be a competitive applicant for an R01 award to perform a multicenter comparative effectiveness trial assessing PN malignancy risk stratification strategies.

NIH Research Projects · FY 2025 · 2023-08

Project Summary The clinical and biological consequences of telomere dysfunction manifest in telomere biology disorders like dyskeratosis congenita (DC). DC is an inherited multisystem disorder characterized by degenerative phenotypes in tissues with high cell turnover, which often includes bone marrow failure and gastrointestinal (GI) disease. GI pathologies in DC patients include enterocolitis, mucosal ulceration, and malabsorption—findings that indicate a link between telomere dysfunction, intestinal regeneration, and gut barrier integrity. Thus, we seek to elucidate how telomere dysfunction impacts the homeostatic turnover of the intestinal epithelium (IE) to inform future therapeutic approaches. In the IE, intestinal stem cells (ISCs) require canonical Wnt/β-catenin pathway activity to maintain the tissue’s rapid cell turnover. Our lab discovered that Wnt activity and telomere fidelity lie in a positive feedback loop—telomerase and telomere capping genes are Wnt pathway targets, and telomere fidelity is required to sustain Wnt target gene expression. Recently, a population of mesenchymal Foxl1+ telocytes in the lamina propria directly underlying the IE has been identified as the critical niche source of Wnt ligands required for ISC proliferative self-renewal. However, in the context of telomere dysfunction, ISCs exhibit a loss of self-renewal and a broad downregulation of Wnt target gene expression, corresponding to the downregulation of Wnt ligands now known to secreted from subepithelial telocytes. The goal of this proposal is therefore to understand the consequences of telomere dysfunction on ISC niche function. Our overarching hypothesis is that telomere failure suppresses telocyte-derived niche support for ISCs, contributing to the intestinal phenotypes associated with telomeropathies. To address this hypothesis, we will utilize a novel Foxl1CreERT2-2A-tdTomato reporter mouse line that will allow for direct in situ visualization of Foxl1+ telocytes, crossed with an mTerc-/- mouse line that features genetic deletion of the telomerase RNA component (mTerc), leading to loss of telomerase activity and progressive telomere dysfunction. We seek to determine the niche factor gene expression and telomere defects in Foxl1+ telocytes in mTerc-/-::Foxl1CreERT2-2A-tdTomato mice by a combination of in situ assays and single- cell RNAseq on isolated telocytes. We will also determine the functional consequences of telocyte-specific defects on ISC self-renewal by utilizing primary organotypic co-culture assays in which primary telocytes and crypt epithelia-derived organoids from mutant mTerc-/-::Foxl1CreERT2-2A-tdTomato and control reporter mice are isolated and co-cultured. Lastly, we will determine whether wildtype donor bone marrow-derived telocytes have the ability to engraft within the lamina propria of mTerc-/- mice and rescue ISC function by restoring niche-derived Wnt activators to the ISC compartment. The findings from this proposal will elucidate how telomere dysfunction alters the ISC niche, and thus inform future therapeutic approaches aimed toward alleviating telomere dysfunction-driven disorders like dyskeratosis congenita.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Essential chromosome biology such as chromosome segregation and the preservation of genome integrity are conserved across the tree of life. Paradoxically, many proteins that support these chromosome functions are unconserved—domains and residues evolve rapidly between even closely related species. A leading resolution to this paradox posits that essential, chromosomal proteins evolve rapidly to keep pace with chromosomal regions enriched with tandemly repeating DNA sequences prone to frequent changes in array size and composition across short stretches of evolutionary time. This turnover of repetitive DNA imperils chromosome functions, triggering adaptive evolution of chromosomal proteins to restore these functions. This conceptual model of intra-genomic coevolution was proposed two decades ago, and yet the DNA repeats, the chromosomal proteins, and the vital chromosome biology sculpted by intra-genomic coevolution are largely uncharacterized. To experimental test this model, I generate an “evolutionary mismatch” between contemporary DNA repeats in Drosophila melanogaster and a fast-evolving chromosomal proteins from its closely related sister species D. simulans. To generate these mismatches, I leverage CRISPR/Cas9-mediated editing to swap native chromosomal proteins from D. melanogaster with diverged versions from D. simulans. Using this approach, my recent work demonstrates an incompatibility specifically between the D. simulans allele of the ovary-enriched chromosomal protein, MH, and the D. melanogaster-specific 359bp repeats. My findings revealed that DNA:protein coevolution is required to preserve genome integrity in the female germline. This system is now uniquely poised to reveal the chromosome biology and evolutionary consequences sculpted by coevolution. Here I integrate evolutionary, cell biology, and biochemistry approaches to investigate the chromosome biology preserved by 359bp:MH coevolution. I also probe how 359bp:MH coevolution reverberates beyond the DNA:protein interface, triggering a secondary coevolutionary process that may result in an interspecies hybrid incompatibility. Finally, I explore the pervasiveness and the consequences of evolutionary innovation at the dynamic MH gene family across the Drosophila phylogeny.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT Estimates suggest that nearly 1 in 10 hospitalized children may experience a safety or harm event, and communication breakdown is a leading contributor to patient safety and harm events. A 2010 study estimated that inefficient clinical communication cost US hospitals an estimated $12 billion annually in wasted clinician time and increased lengths of stay. The introduction of technologies such as smartphones and secure text messaging are transforming how, when, and what acute care clinicians communicate among each other. Developing an infrastructure to proactively evaluate these systems, and build their resilience, is critical. The goal of this project is to create a novel, nurse-led Pediatric Patient Safety Learning Lab with the objective of reengineering interprofessional (e.g., nurse-physician) communication work systems for pediatric acute care settings. The Learning Lab will consist of a multidisciplinary team of clinician-researchers, human factors engineers, human-centered design experts, and operational safety experts from Children's Hospital of Philadelphia and the University of Pennsylvania. We will also engage stakeholders, including bedside clinicians and families of hospitalized children, whose experience of care may be altered by changes to how clinicians communicate. We will use a systems engineering approach (Problem Analysis, Design, Development, Implement, and Evaluate) to proactively assess the interprofessional communication work system, processes, and outcomes, to identify opportunities for improvement before safety events occur. The Specific Aims of this project are to (1) Conduct a problem analysis of the interprofessional communication work system and work processes used in the care of hospitalized children; (2) Design and develop interventions to engineer a safer, more effective, and more resilient interprofessional communication work system; (3) Implement and evaluate the interventions in the clinical environment to gauge real-world impact. Our Learning Lab will be guided by two frameworks, the Systems Engineering Initiative for Patient Safety model and the Integrated Resilience Attributes Framework. The product of this Learning Lab will be a resilient system for interprofessional communication, one that can positively adapt in the face of anticipated and unanticipated changes and maintain the safety of care provided during the estimated 2.5 million pediatric hospitalizations each year.

NIH Research Projects · FY 2024 · 2023-08

Project Summary / Abstract MERS-CoV (MERS) and SARS-CoV-2 (SARS-2) are highly pathogenic coronaviruses (CoVs) that have emerged and caused public health emergencies in the past 20 years. Both of these pathogenic CoVs are betacoronaviruses, although from different lineages (merbeco and sarbeco, respectively). Like other respiratory viruses, CoVs enter the respiratory tract and establish an infection in the upper airway epithelium, where they encounter host innate immune defenses. All CoVs produce double-stranded RNA (dsRNA) as a byproduct of their replication, and this intermediate can induce three innate immune pathways in host cells: interferon (IFN) production and signaling, the protein kinase R (PKR) pathway, and the OAS/RNase L system. Studies of MERS in lower airway cell lines has shown that this virus is particularly adept at evading these dsRNA-induced innate immune pathways. This contrasts with SARS-2, which activates IFN, PKR, and RNase L pathways. Relatively little has been done to characterize the role that these pathways may play in limiting MERS and SARS-2 infection in the upper airway. Additionally, mucosal innate immune defenses such as antimicrobial peptides (AMPs) and nitric oxide (NO) that are highly expressed in the nasal epithelium have only recently been recognized as antiviral, and their role during CoV infection has yet to be characterized. Similarly, mucus production and ciliary function are primary innate immune defenses in the upper airway epithelium, and their specific roles in limiting SARS-2 and MERS infection is unclear. Interestingly, MERS and SARS-2 have different cellular tropisms in the nasal epithelium, with MERS predominantly infecting mucus-producing goblet cells and SARS-2 infecting ciliated cells, suggesting innate immune responses to these viruses may differ. I propose to use a primary nasal epithelial culture system to characterize these innate immune effector functions in the upper airway during MERS and SARS-2 infection. I hypothesize that previously underappreciated epithelial innate defenses such as AMP production, NO synthesis, and mucociliary mechanisms function to limit MERS and SARS-2 replication and spread in the nasal epithelium alongside dsRNA- induced innate immune pathways. My first aim will utilize a panel of SARS-2 and MERS recombinant viruses expressing inactive forms of important viral innate immune antagonists to characterize activation and evasion of dsRNA-induced innate immunity and downstream effects of activation of these pathways (cytokine production, cell death). My second aim will elucidate the role of ciliary and mucus function during SARS-2 and MERS infection by pharmacologically perturbing these innate processes, and will investigate the activation of and the potential inhibitory role of epithelial AMP and NO responses during MERS and SARS-2 infection. The experiments proposed will begin to characterize immune responses to pathogenic CoVs in the upper airway epithelium, the primary site of infection by respiratory viruses, with the potential to identify novel targets for antiviral therapeutics that could be effective against these and future zoonotic CoVs.

NIH Research Projects · FY 2025 · 2023-08

Abstract Neuropsychiatric disorders are characterized by distinct as well as shared clinical features that present in heterogeneous symptom profiles. Delineating the neurobiological etiology of clinical symptoms has been a key overarching aim of over 20 years of neuropsychiatric research. From the earliest studies, neuroimaging research has identified abnormalities in regional brain structure and function. However, we know that there is significant heterogeneity in brain structure and function in neuropsychiatric disorders. Imaging analytic and machine learning methods developed by our group provide the analytic approaches needed to quantify heterogeneity in neuropsychiatric disorders. Herein we leverage these methods, along with a broad international collaboration which provides a unique resource of large highly phenotyped datasets, in order to quantify heterogeneity in major depressive disorder (MDD). In the proposed project, we focus on neuroanatomy and neurofunctional connectivity in MDD. We aim to identify imaging signatures and subtypes in MDD by applying state-of-the-art harmonization, pattern analysis and machine learning methods to structural and resting state functional MRI. The analytic methods allow us to quantify the neuroanatomical and neurofunctional connectivity patterns that comprise MDD to provide powerful predictive markers at the individual level. Our goal is to arrive at a new neuroanatomical-neurofunctional (NA-NF) dimensional coordinate system in MDD (MDD COORDINATES), whereby each dimension reflects a different pattern of brain alterations, hence capturing the underlying NA-NF heterogeneity in quantifiable, replicable, and neurobiologically-based metrics. We will leverage data from our pooled cohorts consists of 4,973 adults with first episode and recurrent MDD, in a current depressive episode, that is not treatment resistant, all medication-free, and respective healthy controls. Assembling these large and powerful datasets will allow us to test our first hypothesis, namely that neuroanatomical and neurofunctional phenotypes will display high heterogeneity, which will allow us to define NA-NF dimensions of pathology. We then test the second hypothesis, namely that this heterogeneity will relate to disease-related phenotypes in MDD and different patterns of clinical outcome. Our specific aims will 1) refine and apply advanced harmonization methods in order to constructively pool and integrate this unique resource; 2) dissect the heterogeneity of the neuroanatomy and function in MDD, thereby deriving a neuroimaging- based coordinate system; 3) relate these imaging dimensions with clinical phenotypic measures, including response to treatment.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT Patients with behavioral variant frontotemporal dementia (bvFTD) develop progressive and highly distressing symptoms of social and emotional dysfunction, including loss of empathy for other people. Scientific research has struggled to understand the specific causes of empathy loss in bvFTD because empathy is difficult to measure in a way that is meaningful and not biased by other factors, such as caregiver burden, and because individuals with bvFTD have highly variable patterns of cognitive and behavioral functioning. A relatively unexplored but promising avenue of scientific inquiry is the role of autonomic nervous system (ANS) activity in empathy loss in bvFTD. The ANS is comprised of two complementary subsystems, the sympathetic and parasympathetic nervous systems, which operate together to increase and decrease levels of physiologic arousal in response to cues from the environment. Patients with bvFTD show subtle abnormalities in ANS activity that are linked to symptoms of social dysfunction, including loss of empathy, but this line of research has been hindered by reliance on traditional methods of measuring ANS output, namely hard-wired EKG and skin conductance sensors, which restrict the movement of the patient and are sensitive to motion artifacts. Recent advances in wearable smartwatch technology allow for precise, unobtrusive measurement of ANS activity with built-in motion detectors to account for changes in ANS signals that are due to movement. Smartwatch technology is well-suited for continuous measurement of ANS functioning to detect subtle abnormalities that correspond to behavioral measures of empathy in bvFTD. This project has the potential to identify specific physiologic contributions to empathy loss in bvFTD, which can be used to develop new treatments and improve early detection of social and emotional dysfunction in bvFTD.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY/ABSTRACT Obesity and eating disorders can present with uncontrolled and compulsive food seeking that is resistant to negative consequences. The goal of this proposal is to understand how consequences of food seeking actions are integrated into motivational neural circuits and describe how these circuits are modulated by homeostatic state. The mesolimbic dopamine (DA) system and the ventral tegmental area (VTA) are implicated in the control of compulsive food seeking behavior. Aversive stimuli can impact the DA system to reduce food seeking, but the impact of this stimuli is blunted when animals are hungry. Homeostatic circuits, like those downstream of agouti- related peptide (AGRP) expressing neurons, dampen aversive stimuli by suppressing neural activity in the parabrachial nucleus (PBN). The PBN provides input to the VTA, indicating that hunger may modulate the ability of aversive stimuli to impact DA signaling and compulsive behavior. I hypothesize that AGRP neurons inhibit the response of VTA projecting PBN neurons to aversive stimuli, leading to continued food seeking despite negative consequences. To test this hypothesis, I will use in vivo neuroimaging of VTA projecting PBN neurons during aversive stimuli and neurobehavioral analysis of compulsive operant food seeking behavior. In specific Aim 1, I will use microendoscopic calcium imaging to resolve the neural activity of individual VTA projecting PBN neurons during footshock, and I will determine how this neural activity changes following stimulation of AGRP neurons. In specific Aim 2, I will test the necessity of the PBN to VTA circuit in punishment-induced suppression of food seeking. Additionally, I will test whether activation of AGRP neuron terminals in the PBN is sufficient to increase food seeking despite negative consequences. Together, these experiments inform on how negative consequences impact food seeking and how this can be modulated by homeostatic circuits. Understanding this interaction will provide a novel perspective on compulsive behavior and will identify mechanisms for how compulsive behavior can develop in disease states where homeostatic circuits are disrupted. Through completing this proposal, I will gain valuable skills in in vivo neuroimaging and build my expertise in the study of operant food seeking behavior. These skills will complement my prior expertise, and significantly improve my ability to address the neural control of food-seeking behavior in my career. These technical skills, along with the training in research, mentoring, and professional development are necessary to support my success in pursuit of a career as an independent scientist studying the neurobiology of feeding.

NIH Research Projects · FY 2025 · 2023-08

Project Summary The intestinal microbiome is made up of trillions of microorganisms that inhabit the gastrointestinal tract. Metabolites produced by the microbiome reach extraintestinal tissues, and can be found in the bloodstream, and are thereby able to influence almost all tissues of the body. Interestingly, the expression of a number of genes of the gastrointestinal tract is dependent on colonization by the microbiome. In this project, we will explore the mechanisms through which the gut microbiome controls gene regulation of intestinal epithelial cells (IECs), through the study of a microbiome-modulated gene involved in IEC cell death. Salmonella enterica serovar Typhimurium (STm) is a pathogen that is adept at overcoming host defenses in order to cause disease. STm triggers an inflammatory response that benefits growth of the pathogen by taking advantage of the host response to infection. Our preliminary findings suggest that STm is able to benefit from a homeostatic microbiome- mediated cell death pathway that has yet to be described. Under steady state conditions, metabolites produced by the microbiome lead to pyroptosis of IECs, which helps maintain normal epithelial turnover. However, upon STm infection, the pathogen harnesses this pathway to elicit increased IEC pyroptosis, leading to increased STm numbers in the gastrointestinal tract. Our early results indicate loss of components of the IEC pyroptosis pathway led to lower STm luminal outgrowth and dissemination to extraintestinal organs, yet, it is unclear how STm activates IEC pyroptosis, and whether this pathway indeed leads to an increase in pyroptosis of intestinal epithelial cells during infection. Thus, I hypothesize that during infection of the gastrointestinal tract, STm takes advantage of a microbiome-controlled homeostatic IEC-specific cell death pathway to bloom to high numbers. In order to elucidate how the microbiome and STm activate IEC pyroptosis, and how this activation leads to cell death and downstream pathogen expansion within the gastrointestinal tract and in extraintestinal sites, we propose two specific aims. In AIM 1 we will assess the contribution of the gut microbiome to induction of IEC pyroptosis, by using a combination of sequencing and in vivo mouse models using conventional and gnotobiotic mice. In AIM 2 we will use mouse infection models to determine how STm elicits IEC pyroptosis during infection, and define the upstream activation pathway. Our mechanistic approach will provide a causal link between the microbiome, a host cell death pathway, and pathogen expansion. Successful completion of this proposal will identify a previously undefined IEC cell death pathway that plays a crucial role under steady state and infectious conditions. This project will additionally expand my training to include key methods and concepts in IEC biology and in the study of the microbiome. Altogether, the research and training plan proposed will facilitate a better understanding of IECs and their role in host immunity, while preparing me for a future career as an independent investigator in the field of host-microbe interactions.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Although involve PrEP case changes based, project will policies, secondary impact on condom use and HIV testing. social-behavioral their 2 MSM. PrEP These mathematical policy such recent federal and state policies targeted t o encourage PrEP (pre-exposure prophylaxis) — which (a) enabling pharmacy-based PrEP delivery 1 , (b) leveraging Medicaid program's benefits to promote (i.e., removal of prior authorization, coverage of telemedicine-delivered PrEP, and overage of targeted management) 2 , and (c) mandating zero-cost sharing 3 — have been described as the most significant that have happened with PrEP 4 – , their impact is not well understood. By implementing a theory- multi-level, longitudinal analysis of how public health policies identify policies to efficiently achieve the Ending the HIV Epidemic goals. – 13 Aim 1 will integrate prescription data, surveys, and consumer data to isolate the effects of the policies on PrEP use Aim 2 will expand our longitudinal database to include determinants of PrEP use, including attitudes, norms, and behavioral control, and explore mediating role in the relation between public health policies and PrEP uptake and persistence. Aims 1 and will mode l a) the US population, (b) MSM (men who have sex with men), and (c) Black, Hispanic, and White Aim 3 will f urther examine the effectiveness of the policies studied in Aim 1 by moving from effects on t o modeling effects on HIV transmission among MSM in general and Black, Hispanic, and White MSM. three aims will be achieved through an innovative combination of econometric, big data, tatistical, and methods. First, a difference in differences method will compare intervention areas in which a has been implemented and control areas without the policy while adjusting for unmeasured confounders as differences in culture or demographics. Second, big data analyses c 6 affect PrEP uptake and persistence, this 7 and ( s , will assess psychosocial determinants of PrEP use for different regions and investigate the role of the policies in eliciting behavioral change. Third, of important populations Award these results will inform an agent-based model of HIV transmission that will project the impact the interventions for MSM, taking into account national race/ethnic disparities. Dr. Fayaz-Farkhad has made contributions to understanding the effectiveness of interventions targeted for marginalized and is an accomplished junior researcher with ample experience in the field of HIV. This K01 will extend her trajectory with training in several key areas:(i) HIV epidemiology, (ii) the science of attitudes and behavior, (iii) health inequalities, and (iv) advanced methodological skills. provide whose project cross-cutting Behavioral The proposed K01 will training and mentorship to serve as the foundation for her career as an independent investigator research will serve to improve healthcare access for populations vulnerable to HIV infection. This is well-aligned with NIAID's strategic objectives and directly in line with the Office of AIDS Research areas of Health Disparities to reduce the incidence of HIV in priority populations and utilize and Social Sciences for HIV care.

NIH Research Projects · FY 2024 · 2023-08

Summary Despite the vast knowledge of the structure and mechanical function of mature tendons, the understanding of tenogenic cell differentiation during development and how cell types from non-tenogenic origins, such as macrophages, influence tenogenesis is limited. Tissue resident macrophages play key roles in the development of several tissues and colony stimulating factor 1 receptor (CSF1R) signaling is essential for their differentiation and survival. The source of ligands that act on CSF1R (CSF1 being the most common) often originate from adjacent resident cells. In addition to CSF1R signaling acting on the macrophages, macrophages often produce trophic factors that act on the adjacent resident cells to regulate aspects of tissue development. In exciting new data, we demonstrate that CSF1R-expressing resident macrophages are situated adjacent to CSF1-expressing tenocytes within linear arrays in the tendon fascicle from initial formation (E15.5) into adulthood, these resident macrophages rapidly accumulate to nearly 10% of the total cell population within tendons during early postnatal growth, and CSF1 produced by tenogenic cells is required for their survival. Additionally, these macrophages internalize collagen in situ, which may indicate a potential role in matrix remodeling during growth and development. Despite their relative abundance and presumed communication with adjacent tenocytes, our limited understanding of the role of resident macrophages in tendon growth and development and potential trophic signaling to tenocytes are significant gaps in knowledge. As macrophages are critical to the development and repair of numerous tissues, defining their role in tendon development will provide insight into signaling mechanisms that could be leveraged in future therapies to improve repair outcomes, which is an unmet clinical need. To address these gaps in knowledge, this proposal will define the ontogeny, distribution, and phenotypic profile of resident macrophages and establish their cross-talk with tenocytes to regulate tendon formation during growth and development. Our central hypothesis is that stable macrophage-tenocyte cross-talk exists and this communication is necessary for tendon formation. Aim 1 will define the ontogeny, abundance, and distribution of resident macrophages with respect to Csf1-expressing tenocytes and the phenotypic profile of these cells at multiple stages of growth and development. Aim 2 will then establish the cross-talk between macrophages and adjacent tenocytes and its role in tendon formation and growth. In this proposal, we will elucidate the importance of stable macrophage-tenocyte cross-talk in promoting cell differentiation and tendon formation in growth and development, thus providing new and critical insight to tendon cell biology that will inform future regenerative strategies.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Cancer cells are the organizers of their surroundings and create a tumor microenvironment (TME) favoring cell division even when oxygen and nutrients are limiting. An extreme example is pancreatic ductal adenocarcinoma (PDAC) which establishes a metabolically hostile ecosystem characterized by a hypo-vascular, severely hypoxic, and nutrient deprived stroma. Clinical trials targeting components of the PDAC stroma have not improved overall survival in unselected cohorts. Thus, while opportunities for targeting the PDAC TME exist, the development of anti-stromal therapies will require a better understanding of inter- and intra-tumoral heterogeneity. Our labs have studied two features of the PDAC stroma: the vasculature and CAFs. By analyzing multiple human datasets, and mouse models, we found that a significant portion of human PDACs (~10%) are hyper-vascular. In murine models, hyper-vascularity is associated with increased sensitivity to angiogenesis inhibitors. In addition, our preliminary studies have shown that in the more typical hypo-vascular tumors, hypoxia renders PDAC tumor cells incapable of synthesizing unsaturated fatty acids (uFAs), and therefore critically dependent upon lipids supplied by neighboring CAFs for their survival. Based on these data, we hypothesize that cancer- associated micro-vasculature and lipid secreting fibroblasts represent under-exploited, clinically relevant targets within the PDAC stroma. Here, we propose an innovative approach to delineate the cellular mechanisms by which tumor cells build and maintain critical metabolic “supply chains” and how regional differences within tumors influence nutrient utilization and vascular intravasation. Our proposal addresses both basic and translational questions and utilizes human tissue resources, implantable and genetically engineered mouse models, platforms to assess stromal geography and metabolic features, and cancer-on-chip techniques for ex vivo modeling. Our ultimate goal is to understand and manipulate the major sources from which PDAC cells derive essential nutrients (especially vital uFAs) – focusing on micro-vessels and fibroblasts. Aim 1. Determine the causes and consequences of vascular heterogeneity in PDAC Aim 2. Delineate molecular mechanisms and therapeutic opportunities underlying stromal support of lipid metabolism in PDAC

NIH Research Projects · FY 2026 · 2023-07

PROJECT SUMMARY Disorders of the esophagus are significant health problems in the U.S. and throughout the world, and esophageal cancer, of which more than 80% is esophageal squamous cell cancer (ESCC), is the 6th most common cause of cancer death worldwide. Exposure of esophageal squamous epithelial cells (keratinocytes) to injurious agents such as cigarette smoke and alcohol predisposes to ESCC, and yet malignant transformation of a single esophageal keratinocyte even in response to such stressors is rare. Thus, important cytoprotective mechanisms must exist in normal esophageal keratinocytes to respond to these insults and prevent malignant transformation of these cells. To date, these mechanisms are not well understood. Here, we propose to delineate important cytoprotective pathways in esophageal keratinocytes, focusing on the key transcriptional regulator Krüppel-like factor 5 (KLF5) and the tumor suppressor p53 in the response to physiologic stress. In normal epithelia, KLF5 functions to promote proliferation and migration and to inhibit inflammation, and in new preliminary data, we define critical functions for KLF5 and wild-type p53 in the cellular responses to exogenous stress in non-transformed esophageal keratinocytes and demonstrate that mutant p53 modulates genome-wide binding of KLF5, thereby altering the targets and pathways governed by KLF5 in this context. Our overarching hypothesis is that KLF5 and p53 are a molecular rheostat, coordinately regulating esophageal squamous epithelial responses to exogenous stressors, and that disruption of this regulation underlies defective cell repair and ESCC. To test this hypothesis, we will pursue the following interrelated Specific Aims: 1. We will define KLF5-p53 targets and function in normal keratinocytes with exogenous stress; 2. We will determine mutant p53 alterations of genome-wide KLF5 binding in homeostasis and stress; 3. We will delineate the functions of KLF5 and p53 in esophageal mucosal injury resulting from smoking and alcohol. Overall, the proposed studies provide a framework to understand the mechanisms by which normal esophageal keratinocytes respond to environmental stresses and the perturbations of these responses that underlie malignant transformation and progression in the squamous esophagus.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY About 37 million people live with human immunodeficiency virus (HIV) worldwide and about 71% of people living with HIV (PLWH) reside in low and middle income countries (LMICs). HIV enters the central nervous system (CNS) early after infection and increases the risk for mild-to-severe neurocognitive and cardiovascular disorders. Studies show that about 30 to 40% of PLWH will have neurologic complications leading to long-term motor and cognitive disability, including HIV-Associated Neurocognitive Disorders (HAND). Few studies have examined how best to provide objective assessment, diagnostic, and rehabilitative treatment tools to aid people living with disabilities as a result of HIV and HIV-stroke in LMICs. The need for these tools is great in LMICs, given the greatly limited infrastructure and healthcare resources and thus decreased access to consistent, quality rehabilitation. Our long-term goal is to develop innovative and affordable robot technologies to bridge healthcare care gaps in LMICs and leverage them to help objectively assess, diagnose and treat PLWH presenting with motor and cognitive impairment due to HAND and Stroke. In this R21 project, we leverage our affordable robot therapy system augmented with objective metrics and motor and cognitive exergames to assess PLWH in Botswana—a country with the fourth highest HIV prevalence rate (20.3%) in the world, an increasing NCD prevalence rate, and limited rehab resources. Aim 1 determines concurrent validity and reliability of using the robot therapy system to objectively ASSESS cognitive, and motor impairments in PLWH. Forty (40) PLWH and 40 HIV-negative controls will be evaluated using both clinical metrics and new robot-based metrics at two time points, at least 1-week apart. We hypothesize that robot-based metrics will have test-retest reliability as well as concurrent validity with clinical measures of motor and cognitive function that usually require rehabilitation expertise and man-power to complete. Aim 2 determines the feasibility of developing a robot-based classification of HIV-associated neurocognitive disorders (HAND). 80 PLWH without stroke will be evaluated using an augmented neuropsychological (NP) battery required to meet minimal HAND Frascati criteria and the robot assessment tasks. We will use the Frascati criteria and global deficit scoring (GDS) to classify the PLWH participants into asymptomatic neurocognitive impairment (ANI), mild neurocognitive disorder (MND) and HIV- associated dementia (HAD). We will develop a regression model using derived robot metrics and test its ability to predict HAND and GDS score. We hypothesize that the robot-based model will classify HAND with specificity and sensitivity better than HAND screening with International HIV Dementia Scale and Montreal Cognitive Assessment. The Receiver Operator Curve (ROC) will be used as empirical validation. Demographic, HIV, and lifestyle factors such as age, viral load, hypertension, diabetes, hepatitis C, and tobacco will be used to determine which increased the risk for HAND.

NIH Research Projects · FY 2025 · 2023-07

The goal of this proposal is to study how perturbation in three-dimensional (3D) genome folding alters CD4+ T cell function, mediating allergic disorders. T cell identity depends on not only the linear genome sequence that embeds millions of regulatory elements, but also the 3D chromatin architecture that orchestrates the spatial localization of the regulatory elements with their target genes. Recent advances in our understanding of nuclear organization indicate that single-nucleotide polymorphisms associated with immune-mediated diseases may impact gene regulation through altered 3D genomic structure and reorganization of large genomic regions in the disease relevant cell types. However, the link between sequence variation, cellular context, 3D genome folding, and aberrant gene expression in majority of immune-mediated complex diseases remains largely unknown. Our objective is to determine the molecular processes through which 3D genome organization in T cells is linked to allergic disorders. We formulated this objective based on four unexpected observations: (A) Our algorithmic definition of groups of densely interacting multi-enhancer elements, which we called 3D cliques, revealed that a locus harboring the Ets1 and Fli1 genes is hyperconnected in T cells. (B) This unique 3D genome architecture is conserved in human T cells coinciding with multiple polymorphisms associated to type 2 immune diseases including allergy, asthma, and atopic dermatitis. (C) We generated a novel strain of mice by deleting a non-coding sequence homologous to the allergy-associated polymorphic region in the human genome, ~250kbp downstream of the Ets1 promoter. This genetic deletion left T cell development intact but led to major defects in CD4+ T helper 1 (Th1) differentiation. Th1 cells are responsible for the control of intracellular pathogens such as bacteria and dampen Th2 responses to allergens. Hence, limited Th1 differentiation due to genetic modification of the Ets1-Fli1 3D clique may cause allergic responses. (D) We modeled the type 2 immune responses in vivo using house dust mites. In the lung tissues of mice with a deletion in the non-coding sequence in the Ets1-Fli1 3D clique, we detected a dramatic increase in allergic responses characterized by a significant accumulation of eosinophils and Th2 cells and a reduction in Th1 cells. These unpublished data provide us with compelling evidence that our engineered mouse strain is a model for understanding the role of noncoding regulatory elements and 3D genome folding in type 2 immune diseases. However, detailed cellular and molecular mechanisms through which genetic deletion in the Ets1-Fli1 locus causes overt allergic responses remain to be understood. Moreover, the generalizability of our 3D clique analysis to additional pathogenic regulatory nodes remains to be examined. This work is significant because it is the first-ever mechanistic investigation providing a connection between genome architecture and type 2 immune diseases.

NIH Research Projects · FY 2025 · 2023-07

Females are predisposed for developing systemic lupus erythematosus (SLE), but the underlying mechanisms remain obscure. Females have two X-chromosomes and equilibration of X-linked gene dosage to that of males (XY) occurs by X-Chromosome Inactivation (XCI), initiated and maintained by Xist RNA. The X-chromosome is enriched for immunity-related genes and X-linked genes are typically overexpressed in female-biased SLE, suggesting that impaired XCI maintenance contributes to SLE. Unlike other somatic cells, we found that naïve B cells lack enrichment of Xist RNA and heterochromatic marks, and that these modifications return to the inactive X (Xi) upon in vitro stimulation, which we term `dynamic XCI maintenance'. Our published work demonstrates that SLE patient naïve B cells exhibit abnormal expression of X-linked genes, suggesting a dysfunctional Xist- independent mechanism involving DNA methylation for transcriptional silencing prior to autoantigen stimulation could predispose to lupus. DNA methylation, associated with transcriptional repression, is reduced in SLE patient B cells and is an important epigenetic modification for gene silencing on the Xi. XCI is also regulated by various nuclear matrix proteins, such as hnRNPU and SAFB, which bind to Xist RNA and function to anchor Xist to the Xi or the nuclear lamina in somatic cells. Our preliminary data support the novel concept that Xist RNA transcripts are retained at the nuclear lamina by SAFB in naïve B cells, and that Xist RNA binds to hnRNPU upon B cell activation, tethering Xist RNA to the Xi to regulate transcriptional repression. We have developed a novel Xist deletion mouse model to determine how impaired XCI maintenance contributes to lupus disease, and our exciting preliminary data show that increased dsDNA levels in type I interferon induced disease. Our central hypothesis is that Xist-independent DNA methylation maintains transcriptional repression of the Xi and autosomes in naïve B cells and that upon activation, alterations in nuclear matrix protein binding promotes dynamic XCI maintenance, the dysfunction of which exacerbates Type I IFN-driven lupus disease. We will test our hypotheses with the following aims: (1) Are reductions in Xist-independent DNA hypermethylation in naïve B cells associated with aberrant gene expression in lupus? (2) How do interactions between SAFB, hnRNPU protein and Xist RNA maintain XCI in B cells and does lupus disease impair cohesin eviction by hnRNPU-Xist RNA complexes? (3) How does impaired XCI maintenance impact Type I IFN-driven lupus-like disease in female mice? IMPACT: Our novel and innovative genetic and molecular approaches will yield unprecedented mechanistic insight into how biological sex contributes to immune dysregulation of SLE disease, and will enable the identification of new molecular pathways and targets of female-biased autoimmune disease that could be amenable for therapeutic intervention.

NIH Research Projects · FY 2024 · 2023-07

Project Summary Children and young adults with metastatic Ewing sarcoma driven by oncogenic fusion transcription factor EWS- FLI1 continue to have poor outcomes. Immunotherapies using T cells, NK cells, cancer vaccines, and monoclonal antibodies are being considered for Ewing sarcoma, especially for recurrent patients. The identification of human tumor-associated antigens (TAAs) recognized by the immune system is crucial for immunotherapy. Through integration of Ewing sarcoma cell line gene expression, ChIP-seq, and normal and cancer tissue gene expression from the Genotype-Tissue Expression (GTEx), and the TCGA project, we have identified LIPI as a highly specific tumor antigen and a potential oncogene that relies on the transcriptional activity of EWS-FLI1 in Ewing sarcoma. LIPI (Lipase member I (EC:3.1.1.-) is an evolutionarily conserved protein predicted to poses a transmembrane domain and extracellular lipid hydrolase domain. The biological function of LIPI is not known but the enzymatic activity of LIPI is expected to produce lysophosphatidic acid (LPA) that potently affects several biological functions including proliferation, cell survival, and metastasis of tumor cells. We hypothesize that the LIPI is a unique biomarker and an oncogene that is expressed exclusively in Ewing sarcoma and is a potential target for cell based therapy. To our knowledge, this proposal represents the first study evaluating the role of LIPI in cellular physiology and in particular Ewing sarcoma progression and metastasis. The two specific aims of the projects are: Specific Aim 1: To elucidate the regulation and role of cell surface LIPI in Ewing sarcoma Specific Aim 2: To investigate the role of LIPI in Ewing sarcoma growth/metastasis. Following the successful completion of the proposed aims in this application, we will have evaluated the potential of LIPI as a Ewing sarcoma specific surface oncoprotein, and will have a long-term impact by establishing the strong foundation for the development of LIPI-directed immunotherapeutics against Ewing sarcoma.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY This proposal aims to develop the first potent and specific antagonists of the pro-mutagenic effects of APOBEC DNA deaminase enzymes in cells. Access to whole genome sequences has helped reveal common mutational signatures across various cancers. One such prominent mutational signature, termed SBS2, includes hypermutated clusters containing a high density of C to T/G substitutions on the same strand, a phenomenon known as kataegis. These features, in addition to the enrichment of the mutations in 5’-TC motifs, point to a causative role for APOBEC3 (A3) family enzymes. While these enzymes normally mutate and restrict retroviruses or retroelements, studies have confirmed that two family members, APOBEC3A (A3A) and APOBEC3B (A3B), have a prominent role in pathological mutagenesis targeting the host genome. The relative contributions of each enzyme remain a matter of vigorous debate, as genetic approaches specifically targeting A3A or A3B are limited by their high homology to one another and juxtaposition on the genome. Furthermore, no molecular tools currently exist that can disrupt A3 function. There is therefore a pressing need for molecular probes that can either inhibit or deplete A3 enzymes from cells. This proposal builds on the hypothesis that insights into the mechanism and substrate selectivity of A3 enzymes can be leveraged to design potent and specific antagonists. Specifically, we have demonstrated that mechanism-based inhibitor moieties can be presented in preferred secondary structures and engineered into exonuclease-resistant DNA molecules to yield potent nanomolar inhibitors of A3A. These molecules present the opportunity for facile functionalization, which can be utilized to convert classical inhibitors into molecules capable of inducing the catalytic degradation of the target APOBEC enzymes in cells, via an unprecedented combination of nucleic acid inhibitors and proteasome targeting (PROTAC) technology. Taken together, this proposal aims to fill a critical gap in the field by introducing tools to perturb APOBEC function in cells in order to reveal their underlying biology and offer a roadmap for potential therapeutics.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract: The overall goal of the University of Pennsylvania PDX Development and Therapeutics Center (UP-PDTC) is to (i) exploit the translational potential of PDX models for evaluating the response of various treatments in models with specific molecular characteristics and to (ii) work with PDXNet to enhance and extend use of PDX models to the research community with the goal to guide development of human Phase 1/2 clinical trials for human malignancies. As PDX models more faithfully reproduce human cancer than cell lines, they can contribute to the ultimate clinical implementation of cancer precision medicine. The UP-PDTC is uniquely poised to contribute to such research due to the long history of PDX research at UPENN. The UP-PDTC will comprise 4 Cores and 2 projects. The four Cores are: Administrative, PDX, Pilot Projects and Trans-Network, and a Bioinformatics Core. The Administrative Core will coordinate all UP-PDTC activities. The PDX Core builds on 15 years of experience within the Stem Cell and Xenograft Core facility of UPENN. The PDX Core currently provides over 900 mice per month to investigators at UPENN using PDX modeling for malignant and non-malignant disease. The animal facility of the PDX Core is a dedicated 6 room suite controlled by highly skilled PDX Core personnel with comprehensive infection control measures. Using these measures, the PDX Core has not had a significant infection in the colony in seven years. The Pilot Projects Core will take advantage of a growing group of collaborators to build tissue banks for hepatocellular carcinoma (HCC), clear cell renal cell carcinoma (ccRCC), breast cancer, glioblastoma, and myeloma. A major goal of the UP-PDTC is to develop and characterize new models across common and rare cancer types that can be linked with the originating patient clinical response profile and shared with PDXNet. The Bioinformatics Core takes advantage of the extensive Bioinformatics infrastructure at UPENN and will work with the Projects to more rigorously describe PDX modeling for acute myeloid leukemia (AML) and ovarian cancer. There are two projects. Project One is directed by Dr. Martin Carroll and will focus on acute myeloid leukemia. Project 1 takes advantage of one of the largest tissue banks of viable, fully annotated AML samples in the world. This tissue bank currently has over 3300 collections from over 1700 patients collected over 20 years including over 40 PDX primograft models currently available of over 100 that have been described. Dr. Carroll, working with members of the PDX Core, has a long history of developing and using the AML PDX model to define AML biology and responses to therapy. Project 2 is directed by Dr. Fiona Simpkins and takes advantage of the Ovarian Cancer Research Center Tumor BioTrust Collection. This collection is fifteen years old, also fully annotated and includes 140 well characterized PDX models. Drs. Carroll and Simpkins, have extensive experience in xenotransplantation models and in the use of xenotransplantation models to both understand disease biology and develop new therapeutic approaches for implementation in clinical trials. In their projects, they propose two discrete xenotransplantation Phase 2 studies. Dr. Carroll will test the effects of a combined therapy with Menin inhibition and KAT6A inhibition on AML differentiation and disease burden. Dr. Simpkins will study TP53 wild type clear cell ovarian carcinoma (CCOC) and low-grade serous ovarian carcinoma (LGSOC) and their response in PDX models to a “p53 stabilizer combination” (MDM2 and XPO1 inhibition). Both Dr. Carroll and Dr. Simpkins have significant experience moving pre-clinical work into human early-stage clinical trials and we anticipate that these xenotransplant phase 2 (XP2) studies will lead to molecular defined Phase 1/2 human studies. Overall, the unique resources of the UP-PDTC should enhance the use of PDX models for robust and reproducible studies that will lead to precision targeted therapeutics in humans.

NIH Research Projects · FY 2024 · 2023-07

Project Summary Intracellular bacterial pathogens such as Legionella pneumophila, the causative agent of the severe pneumonia Legionnaires' disease, are responsible for significant disease burden in the United States every year. The growing use of immunosuppressive drugs, particularly TNF blockrs, to treat autoimmune diseases comes with a significantly increased risk of infection by intracellular bacterial pathogens, including Mycobacterium tuberculosis and Legionella pneumophila. The increasing numbers of individuals who are functionally immunocompromised due to growing use of TNF blockade, combined with the growing spread of antibiotic resistance, poses a serious public health concern. Our goal is to define the mechanisms by which TNF promotes immune defense against Legionella pneumophila. This information may enable development of targeted therapeutics that promote anti- bacterial defense in immunocompromised individuals. Critically, in new preliminary studies that take advantage of our ability to dissect the interactions between Legionella and dendritic cells, we find that TNF signaling drives a Legionella effector-triggered immune response that induces apoptosis in dendritic cells, thereby restricting intracellular Legionella replication. The proposed studies will provide fundamental insight into how TNF-mediated effector-triggered immunity promotes control of intracellular Legionella replication within dendritic cells. This information may provide a basis for the development of improved therapeutics for the treatment of Legionella and other bacterial pathogens.

NIH Research Projects · FY 2024 · 2023-07

ABSTRACT Cells must integrate competing DNA repair pathways and tightly control their chromatin landscapes to maintain genomic integrity. Disruption of these control systems or defects in any one pathway result in a mutational burden with profound physiological consequences to cells and tissues. DNA repair in human cells is primarily performed by two mutually exclusive pathways governed by two different, well- characterized tumor suppressors – non-homologous end joining (NHEJ) by 53BP1 and homologous recombination (HR) by BRCA1. 53BP1 and BRCA1 are examples of intrinsically disordered proteins (IDPs) containing large stretches of low complexity amino acid sequences. 53BP1 undergoes liquid-liquid phase separation to form biomolecular condensates in vitro and at DNA lesions. Further, recent studies hint that 53BP1 condensation also plays a major role in maintaining chromatin organization. Whether BRCA1 has similar phase separation activity has not been established. In preliminary data, I show the very first evidence that BRCA1 phase separates to form biomolecular in cells and in vitro. Chromatin landscapes also play a vital role in maintaining genomic integrity. DNA damage response requires dynamic rearrangements and specific chromatin modifications to elicit rapid recruitment of repair factors. Conversely, repair factors and their complexes can also modify chromatin to drive repair programs. Despite extensive study of 53BP1 and BRCA1 tumor suppressor activities and their repair mechanisms, it remains unknown by what mechanism 53BP1 or BRCA1 condense and what role condensations plays in DNA damage response. Further, the contributions of chromatin architecture to repair pathway selection and chromatin organization within repair condensates have not been elucidated. The goal of this work is to provide me with new training and expertise to address the proposed aims and establish an independent research program. In Aim 1 (K99 phase), I will identify the specific sequence determinants that promote 53BP1 and BRCA1 phase separation and determine the contribution of protein condensates to promoting DNA repair and fidelity in repair pathway selectivity (NHEJ vs. HR). I will test the hypothesis that the magnitude of tumor suppressor condensation contributes to pathway selection. In Aim 2 (K99/R00 phase), I will define the dynamic rearrangement of chromatin in response to DNA damage and how nucleosome clustering and DNA loop compaction contribute to chromatin dynamics in 53BP1- and BRCA1-mediated repair programs. Collectively, this work will address fundamental gaps in knowledge regarding the role of phase separations in genome integrity and uncover new paradigms that underlie tumor suppressor activities.

NIH Research Projects · FY 2025 · 2023-07

Project Abstract Diffuse gliomas are fatal brain tumors that respond poorly to current immunotherapies. Using both mice models and human clinical trial data, we propose a series of experiments to identify immune biomarkers and key immune cell types that predict tumor responsiveness to therapy. These results will further our understanding of brain tumor biology and inform the future development of new treatment modalities.

NIH Research Projects · FY 2025 · 2023-07

Project Summary Merkel cell polyomavirus (MCPyV) is a ubiquitous skin infection that can cause Merkel cell carcinoma (MCC), a highly aggressive form of skin cancer. Immune suppression is one of the most important risk factors for developing MCPyV-associated MCC. MCPyV has a far greater chance to induce cancer development among immunocompromised individuals, including HIV-infected patients. However, both the MCPyV life cycle and oncogenic mechanisms remain poorly understood. The incidence of MCC has tripled over the past twenty years, but effective treatments are lacking. Therefore, a better understanding of the MCPyV life cycle and oncogenic mechanisms is needed for developing more effective treatments. In MCPyV-infected cells, the early promoter (EP) supports the transcription of early genes and plays a critical role in maintaining persistent infection. In the majority of MCCs, MCPyV DNA is clonally integrated into the cancer genome, where the EP drives the expression of viral oncogenes, large and small T antigens, to promote MCC tumor growth. MCPyV EP transcription therefore is also critical for supporting MCC oncogenesis. However, very little is known about the mechanisms that regulate MCPyV EP during either MCPyV infection or MCC development. This gap in our knowledge is largely because, until recently, the cellular tropism of MCPyV was unknown and there was a lack of a biologically relevant culture system for studying MCPyV. We recently identified human dermal fibroblast (HDF) as a natural host cell for MCPyV infection. We found that MCPyV entry is a promiscuous process, whereas its transcription is the key determinant for MCPyV host cell tropism, persistent infection, and oncogenic potential. Building on the in vitro and ex vivo infection models developed in our recent studies, we propose to discover the epigenetic mechanisms (Aim 1) as well as host cellular factors and cis-acting viral DNA elements (Aim 2) that regulate MCPyV early gene transcription. We will also apply the recently developed lipid nanoparticle (LNP) technology to abolish MCPyV oncogene transcription and obliterate MCC tumorigenesis (Aim 3). Our studies will fill a significant knowledge gap in understanding the mechanisms that regulate MCPyV early transcription during the viral life cycle and MCC tumorigenic development. Moreover, our investigation will provide important insights into the virology and oncogenic mechanism of this new human tumor virus, and identify novel targets for developing better strategies to treat the highly lethal MCC skin cancers with a rapidly rising incidence. As demonstrated by the success of COVID-19 vaccines, the highly potent LNPs have shown great promise for therapeutic applications. Therefore, the superb in vivo delivery power of LNPs affords a viable platform for translating our findings into clinical setting.