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NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT Our lab aims to understand the fundamental mechanisms of mitochondrial gene regulation to advance our understanding of metabolic adaptation and mitochondrial-associated diseases. Over the next five years, we will develop and apply cutting-edge sequencing technologies to reveal how cells regulate their energy production through mitochondrial DNA (mtDNA) packaging, expression, and replication. Mitochondria generate ATP through oxidative phosphorylation (OXPHOS) and contain their own genome, which encodes 13 core OXPHOS subunits, exists in hundreds to thousands of copies per cell, and is packaged into “nuceloids” by TFAM. The high ploidy of mtDNA has posed challenges to previous functional genomics techniques that rely on Illumina short- read sequencing, as population averaging effects obscure any relevant information. We previously developed mtFiber-seq, an approach that measures mtDNA accessibility at single-genome resolution. We discovered that most nucleoids are inaccessible but can change state during OXPHOS dysfunction or cellular generation. Many questions remain regarding how mitochondrial gene expression is regulated in response to perturbations to rewire metabolism and maintain homeostasis. The high ploidy of mtDNA allows for mechanisms unique from those in the nucleus. Using a variety of perturbations and systems, we will determine how gene expression is regulated on both short and long time scales. TFAM acts as both a repressor and activator through poorly understood mechanisms. We showed that varying TFAM levels can shift the active nucleoid population and developed an in vitro version of mtFiber-seq with reconstituted nucleoids which allowed us to understand new binding properties of TFAM and recapitulated cell culture observations. How TFAM behavior is regulated and how nucleoids shift between inactive and active states is not understood. This is likely done through TFAM post- translational modifications, but these modified PTMs remain understudied. We expect to clarify how these changes change the propensity of TFAM to repress and activate transcription and replication. We are developing an expanded mtFiber-seq approach that will allow us to simultaneously map DNA accessibility and the positions of specifically modified TFAM on single genomes. There are also many outstanding questions regarding mtDNA replication, and this process is critical for maintaining a healthy population of mtDNA. The signals controlling replication are not understood. Using mtFiber-seq with metabolic labeling, we will study differences in replicating and non-replicating populations to understand how these signals control replication and copy number. Overall, we seek to better understand the mechanisms of nucleoid regulation, and mitochondrial gene expression more broadly, by applying state-of-the-art technologies that are allowing for previously impossible levels of resolution. This work will provide fundamental insights into how cells maintain energy homeostasis and adapt to changing metabolic demands, with implications for understanding mitochondrial diseases and developing therapeutic strategies.

NIA - National Institute on Aging

SUMMARY Alzheimer’s disease (AD), the most common form of dementia, is a looming crisis that imposes huge healthcare, economic and social burden in the US. AD progression is associated with neurodegeneration while the pathogenetic mechanisms underlying neuronal death and dysfunction remain unclear. This neurodegenerative process involves complex interactions of AD hallmarks β-amyloid (Aβ) and tau with the localized inflammation contributed by glial cells. But little is known about how the cells near AD hallmarks regulate themselves and respond to the microenvironment. Recently, chromatin accessibility has been recognized as a dynamic central regulator of transcription since chromatin remodeling enables access of cis-regulatory elements, while closed chromatin regions impair the accessibility of promoters and enhances. AD-associated chromatin signatures show brain region and cell-type specificity, implicating noncoding regulatory regions within AD genetic risk loci that participate in a variety of biological pathways as well as changes in TF regulation. Another layer of cell regulation is through signaling transduction and activation of transcription factors at the protein level. The outcomes of epigenetic regulation should be better described by protein measurements since proteins predominantly represent cell identity, drug target, clinical biomarkers, signaling networks, transcriptional factors, functional readouts of proliferation, cell cycle status, metabolism regulation and apoptosis makers. However, these two layers of regulatory machinery have not been integrated before for spatially distributed heterogeneous tissue cells. The current single-cell omics tools often lack spatial information, and the spatial omics tools frequently lack single-cell resolution. Integration of single-cell spatial proteomics is particularly difficult as most state of the art can only detect a few proteins. Based on our multiplex in situ tagging (MIST) microchip technique, we have successfully measured hundreds of critical proteins in cell identification, signaling transduction and transcription from single brain cells. This technique is also compatible with chromatin accessibility assays. With that, we propose to (1) Sequentially measure functional proteome and epigenome by MIST-seq with high accuracy and spatial resolution; (2) Determine molecular signatures and regulation of cells near amyloid plaques in the early AD stages. Through the unique single-cell spatial omics technology, we will not only uncover the regulatory landscape of damaged neurons in AD onset and progression, but also reveal the contribution of the inflammatory microenvironment near Aβ plaques. The success of this project will advance our understanding of neuronal loss in the early stages of AD, assist identification of drug targets and biomarkers, and uncover the complex relationship between inflammation and neurodegeneration.

NCI - National Cancer Institute

ABSTRACT E-cigarette use (i.e., vaping) among young adults is common, and many report a strong desire to quit but difficulty in doing so. The retail environment is a key potential point of intervention whereby innovative regulations can be enacted to both encourage cessation efforts and provide support to young people seeking help with cessation. Interventions employed in the nicotine retail space may be particularly effective as they reach young people at a time when they are most susceptible to continued use (i.e., just prior to purchasing e-cigarettes or vaping products). In the current study, we propose to test two innovative policy interventions to support cessation efforts among young people using our state-of-the-art simulated vape shop experimental lab space (“The Puff Point”). The proposed policies under study include (a) availability of cessation products (nicotine replacement therapy; NRT), and (b) prominent display of cessation resources at the point of sale in the retail environment. Policies that increase access to and awareness of cessation products (e.g., NRT) and cessation resources (e.g., text-to- quit lines, cessation programs or apps) in the retail environment have the potential to positively impact cessation efforts and success in cessation among young adults. This study will utilize a sophisticated 2x2 factorial design to test the effect of both policy interventions on e-cigarette purchasing behaviors and cessation intentions. Aim 1 will assess the impact of inclusion of NRT for sale in the retail space; Aim 2 will assess the impact of prominent display of cessation support services at the point of sale; Aim 3 will assess the joint effect of both policies (i.e., the interactive effects of inclusion of NRT for sale and display of cessation literature). Participants (N=200) will include young adults (21-30 years) who report having used e-cigarettes 3 or more days per week and have purchased e-cigarettes from a vape shop at least once per month, over the last 6 months. After a remote zoom session to assess eligibility, participants will visit our simulated vape shop and make purchases under one of the four conditions. A subsample will participate in a brief qualitative interview following the purchasing task to assess perceptions of the intervention. All participants will complete both a pre-task and post-task survey to assess cessation intentions. Analyses will investigate the effect of each policy on purchases made (including number of products and total amount of nicotine in non-NRT products purchased) and cessation intentions (any intentions to quit, planned timeframe for cessation [e.g., within the next month, next 6 months, next year], and specific plans for support with cessation attempt [e.g., NRT, app). Secondary analyses will investigate changes in cessation intentions pre- to post-purchasing task and whether changes differ by experimental condition. This project addresses regulations that are easily scalable and could encourage cessation from vaping among young people. The point of sale is a key intervention point, and data are urgently needed to guide regulatory efforts to reduce nicotine use and support cessation efforts among young adults seeking to do so, to reduce the adverse public health consequences of vaping in this population.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY T cell memory is stored across heterogeneous subsets with diverse functions in both tissues and circulation. While most studies have focused on the pro-inflammatory and cytotoxic functions of memory T cells, regulatory T cells (Tregs) serve an equally important immunomodulatory role in memory responses, particularly in tissues. While specific roles for Tregs in establishing tolerance and promoting tissue homeostasis have been elucidated in mouse models, the role of human Tregs in healthy immune responses and protective immunity in vivo has been difficult to assess. Moreover, the identity and function of human Tregs in diverse tissues remains unknown. We have established an organ donor tissue resource for human immunology that has allowed us to profile antigen-specific T cells across human tissues. Through these efforts, we found that antigen-specific Tregs are substantially enriched among memory T cells that respond to antigens from multiple viruses, including SARS-CoV-2, influenza, and EBV, and are particularly enriched in lymph nodes, spleen and lungs compared to blood, bone marrow and other sites. In addition, we found that memory Tregs induce an activation program that is distinct from effector memory T cells (TEM) involving CCL17 as a novel Treg-derived cytokine not produced by TEM cells or any other T cell subset. Moreover, tissue memory Tregs exhibit clonal overlap with TEM cells within and between sites. These findings raise the possibility that memory Tregs are generated along with TEM during priming and that they share a common pre-cursor with TEM. In the proposed studies, we will pursue three aims: 1) Determine the role of antigen and tissue in memory Treg induction; 2) Define the clonal and migratory relationships (i.e. tissue distributions) between memory Treg and other memory subsets; 3) Elucidate the functional and spatial interactions of tissue Tregs with immune and structural cells in the lymph node. We will combine state-of-the-art technologies for single-cell and spatial profiling with our unique human tissue resource to elucidate mechanisms for the generation, function, and maintenance of memory Tregs in human tissues. The results from this study will be important for designing strategies to promote immunoregulation and tissue repair for protective immunity and can inform Treg-directed therapies for autoimmunity and transplantation.

Phinney Neighborhood Association

The Phinney Neighborhood Association's `Artistic Signal Box` aims to strengthen community identity and provide shared cultural landmarks by transforming functional infrastructure into compelling public art, celebrating diversity and enriching the urban environment in the PhinneyWood area (i.e. Phinney Ridge and Greenwood). The project will also provide visibility for emerging and BIPOC artists to showcase their talents and connect with the community. Nineteen artists, selected through a juried process with community input, will design artwork for 19 SDOT signal boxes. The theme, `Wander PhinneyWood,` invites artists to explore the area's uniqueness, highlighting personal and collective connections. The completed artworks will create a connected experience, encouraging exploration of local landmarks, green spaces, and businesses along the route.

Ambani Foundation

Largest Indian private foundation; funds education, healthcare, rural transformation, and arts.

Air Force Academy

The USAFA invests in an active research program for three main reasons. First and foremost, research significantly enhances the cadet learning experience. Our research is done by, for and with cadets who work alongside fellow cadets and faculty mentors. Research provides cadets with rich independent learning opportunities as they tackle ill-defined problems and are challenged to apply their knowledge and abilities.Second, our research program provides opportunities essential for faculty development. Research broadens and deepens the experience base of the faculty. This infuses current, relevant, state-of-the-art and cutting-edge applications and examples into the curriculum. This also helps our faculty remain current in their respective fields.Third, at USAFA we strive to conduct research to enhance the ability of the Air Force to perform its mission. There are ongoing research projects spanning topics as diverse as super hypersonics, cyber security, spatial disorientation, athletic performance and homeland defense. This BAA offers a vehicle for research to be performed to satisfy these three objectives, while also meeting research needs of industry counterparts/serve a public purpose. USAFA s partnerships with non-Government firms enables development in the public arena, stimulating the studies in the greater technical community. All awards issued against this BAA must serve to benefit the objectives identified above.

Air Force Academy

<p>The USAFA invests in an active research program for three main reasons. First and foremost, research significantly enhances the cadet learning experience. Our research is done by, for and with cadets who work alongside fellow cadets and faculty mentors. Research provides cadets with rich independent learning opportunities as they tackle ill-defined problems and are challenged to apply their knowledge and abilities.</p><p><br></p><p>Second, our research program provides opportunities essential for faculty development. Research broadens and deepens the experience base of the faculty. This infuses current, relevant, state-of-the-art and cutting-edge applications and examples into the curriculum. This also helps our faculty remain current in their respective fields.</p><p><br></p><p>Third, at USAFA we strive to conduct research to enhance the ability of the Air Force to perform its mission. There are ongoing research projects spanning topics as diverse as super hypersonics, cyber security, spatial disorientation, athletic performance and homeland defense. This BAA offers a vehicle for research to be performed to satisfy these three objectives, while also meeting research needs of industry counterparts/serve a public purpose. USAFA’s partnerships with non-Government firms enables development in the public arena, stimulating the studies in the greater technical community. All awards issued against this BAA must serve to benefit the objectives identified above.</p>

NIAID - National Institute of Allergy and Infectious Diseases

ABSTRACT Despite remarkable advances in HIV cure science, emerging cure candidates will likely involve trade-offs (e.g., incomplete eradication, monitoring burdens) and must compete with increasingly convenient long-acting ART; without early implementation guidance, even efficacious products may see limited uptake, particularly among the ~30–40% of people with HIV (PWH) in the U.S. who are not durably suppressed. We propose REALISE, a multidisciplinary program to define plausible cure profiles, quantify end-user preferences, and project population-level impact to inform product design and policy before market entry. Aim 1 conducts qualitative interviews with ~30 researchers and developers to delineate credible 10–20-year cure and long-acting treatment scenarios (eradication vs functional control, safety, monitoring, durability), yielding bounded “target product profiles.” Aim 2 elicits patient-centered preferences through a two-stage study: formative interviews (n=60; ≥50% not virally suppressed) to identify salient attributes; best-worst scaling (n=360 across Missouri, Georgia, and San Francisco) to prioritize attributes; and a discrete choice experiment (n=360) to quantify trade-offs versus alternative therapies, with latent class analysis to identify preference segments and estimate potential reach. Aim 3 integrates preference-based uptake from Aim 2 with Aim 1 efficacy and cost inputs in a mathematical model to estimate health impact, QALYs, net QALYs, and incremental cost-effectiveness across heterogeneous populations and Ending the HIV Epidemic jurisdictions. Innovation lies in linking cure R&D horizons to end-user preferences and transmission-dynamic outcomes, an approach that anticipates real-world use rather than retrofitting after approval. Deliverables include ranked cure attributes for product optimization, uptake projections including among unsuppressed PWH, and jurisdiction-specific value assessments to guide public health investment. By aligning cure design with what patients will accept and systems can sustain, REALISE will accelerate effective deployment of future cure strategies and maximize their contribution to Ending the HIV Epidemic. In doing so, this study advances NIH's priorities by connecting implementation science with prevention, treatment, and cure research. Using a multidisciplinary strategy to refine and extend `target product profiles,' REALISE will ensure cure development reflects patient needs and accelerate translation into real-world benefit.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT ABSTRACT Human immunodeficiency virus-1 (HIV-1) can be controlled with antiretroviral therapy (ART), but a cure remains elusive due to the persistence of latently infected reservoirs. These reservoirs remain transcriptionally silent, allowing the virus to evade immune clearance and rebound when therapy is interrupted. One proposed strategy to eliminate the latent reservoir is the shock and kill approach, which utilizes latency-reversing agents (LRAs) to reactivate viral gene expression, thereby enabling clearance by cytotoxic CD8 T cells. However, HIV accessory proteins such as Nef and Vpu downregulate HLA-I on infected cells, preventing recognition by CD8 T cells even after reactivation. IL-15 is a particular LRA of interest, along with its superagonist N-803, that can expand effector lymphocytes and enhance their cytolytic activity; however, its ability to rescue HLA-I expression on HIV-infected cells remains uninvestigated. IL-15 signals through the JAK/STAT pathway, which is negatively regulated by the non-receptor tyrosine phosphatases PTPN1 and PTPN2. Small-molecule inhibitors of these phosphatases, including 3-Hydroxy-1,2,3- benzotriazin-4(3H)-one (HODHBt) and ABBV-CLS-484 (AC-484), enhance STAT signaling, promote viral reactivation, and increase HLA-I expression on infected CD4 T cells. Preliminary data demonstrate that IL-15 can restore HLA-I expression on HIV-infected cells, and this effect is enhanced with PTPN1/2 inhibition. However, viral strains belonging to subtypes AD and D exert resistance to IL-15-mediated HLA-I rescue on HIV-infected CD4 T cells, suggesting subtype-specific mechanisms. In Aim 1, I will test whether IL-15-mediated HLA-A, -B, and -C rescue is possible when CD4 T cells are infected with viral strains isolated from the latent reservoir of people with HIV across a variety of subtypes and then assess if rescue is associated with increased recognition and killing of infected cells by HIV-specific CD8 T cells. In Aim 2, I will dissect the mechanism by which viral strains belonging to subtypes AD and D promote resistance to IL-15 signaling and whether this is associated with the viral protein Nef, the protein responsible for HLA-I downregulation. By uncovering how the IL- 15/STAT/PTPN1/2 axis regulates HLA-I expression and CD8 T cell recognition of HIV-infected CD4 T cells, this project will inform the design of cure strategies that enhance immune clearance of latent reservoirs. Completion of these studies will also provide rigorous training in immunology, virology, and translational therapeutics to prepare me for a career as an independent HIV investigator.

South End Seattle Arts Center dba ReVision Arts

ReVision Arts wants to make accessible our Rainier Valley Neighborhood Art Grant Projects and ReVision Arts Annual Please Touch Tactile Exhibit. This will include: 1) Translation and Interpretation of Neighborhood Art Grant Projects as needed. Public sub grantee events will be April to Sept. To create an accessible projects meeting ADA, we need $$ for Sign Language Interpreters for sub grantee events in the Rainier Valley. 2) Orientation on April 4th a) Provision of Interpreters for sub grantee Orientation b) Presentation regarding access and event planning 3) ReVision Arts will bring Please Touch Tactile Art Exhibit to Rainier Valley to celebrate the ADA Anniversary 2023. Attendance is free and open to all. For above events: Subgrantees mother tongue may not be English, we will provide language interpretation as requested. We want to close caption digital presentations. Digital translation is not always accurate, we need an editor /proof reader.

Refugee Women's Alliance (ReWA)

Refugee Women's Alliance will host a community carnival on April 27, 2019 from 3 pm - 6 pm at 4008 Martin Luther King Jr. Wy S. The event will showcase the creative contributions of refugee and immigrant youth and will include performances, art, and food.

Community Arts Create

Rhapsody Project instructors will collaborate with a diverse set of Seattle musicians and educators to design curriculum and deliver a weekly roots music class to 30 youth during a 10 week period between September and December of 2018 at the Hillman City Collaboratory. The project will culminate in a free community concert in December. Students will be offered a stipend for attending the classes, completing a guided independent study in the spring of 2019 and for their participation in the concert.

Rhizome (New Museum)

Grants for digital and net art projects that expand the boundaries of art, technology, and culture.

Rhode Island Foundation — Community Programs

Rhode Island Foundation — Community Programs ($1.0B in assets) supports nonprofits in RI through grants focused on education, community development, health, arts. Awards range from $5,000 to $200,000.

Rhode Island Foundation

Rhode Island Foundation ($1.0B in assets) supports nonprofits in RI through grants focused on education, arts, health, community development, environment. Awards range from $2,000 to $100,000.

Rhode Island State Council on the Arts (RISCA)

RISCA provides general operating support grants to Rhode Island arts organizations, enabling sustained programming, capacity building, and community engagement through arts and cultural activities statewide.

Rhode Island State Council on the Arts (RISCA)

Project grants support arts education initiatives in Rhode Island schools and community settings, funding artist residencies, curriculum integration, and arts programming that connects students with creative learning opportunities.

NCI - National Cancer Institute

Abstract Support is requested for a Keystone Symposia conference entitled “RNA Modifications: Bridging Biological Function and Therapeutic Potential,” organized by Drs. Michaela Frye, Schraga Schwartz, Yunsun Nam and Eckhard Jankowsky, with scientific programming input from Keystone Symposia. The meeting will take place March 9–12, 2026 at Keystone Resort in Keystone, Colorado USA. The rapid growth in RNA modification research has shown that chemical modifications of nucleotides regulate RNA metabolism and influence cell functions. Moreover, the ability of single chemically modified nucleotides to change the electrostatic charge, base pairing and stability of RNA molecules can now be used in clinical applications. This includes creating stable artificial RNA transcripts, such as mRNA vaccines or synthetic small RNA molecules, to increase or decrease the expression of therapeutic proteins. However, our understanding of the human transcriptome remains incomplete because we lack full-length RNA sequences that include all their modifications. This knowledge is crucial, as RNA modifications regulate every stage of gene expression, and their pathways are frequently dysregulated across diverse cancer types. In 2022, the first RNA modification inhibitor entered phase I clinical trials for late-stage cancer patients (ClinicalTrials.gov; NCT05584111), highlighting the translational potential of this field. This Keystone Symposia conference will convene field-leading experts to discuss the multidisciplinary aspects of RNA modification research, providing attendees with a broad overview of state-of-the-art research that is not available in other meetings or workshops centered around RNA. The conference program has been designed to highlight groundbreaking research developments, discuss current challenges and focus on therapeutic opportunities. Importantly, this conference program will explore innovative strategies to target RNA modifications in human diseases like cancer, as well as neurological and metabolic disorders. Cutting-edge therapeutic approaches, with an emphasis on the integration of advanced technologies in cancer research, will be emphasized throughout this meeting. Dynamic scientific sessions coupled with informal networking events will encourage an open exchange of emerging research concepts and directions in the field of RNA modifications and foster new collaborations.

Rochester Area Foundation

Rochester Area Foundation ($200M in assets) supports nonprofits in MN through grants focused on education, community development, health, arts. Awards range from $1,000 to $50,000.

NCI - National Cancer Institute

Prostate cancer remains the second-leading cause of cancer-related deaths in US men, mainly due to metastatic disease. Metastasis occurs most frequently in bones, thus entailing significant patient morbidity including pain, propensity to fractures and potential spinal cord compression. Moreover, the bone is a favored reservoir for undetectable disseminated tumor cells that maintain minimal residual disease and can thus critically define future patient outcomes. Despite this pressing clinical need, the mechanisms of progression to bone metastasis remain incompletely understood. Our overall goal is thus to understand the functional determinants of progression to lethal metastatic prostate cancer in order to develop more efficient therapies. Given that tumor progression and metastasis occur through multiple steps involving interactions with different benign cells and tissues, experimental models in which prostate cancer progression may be studied in a whole immunocompetent organism may help identify hitherto unappreciated mechanisms of progression. Our preliminary studies using novel mouse and human prostate cancer models show that ATAD2, an epigenetic and transcriptional regulator, is a critical mediator of metastasis (including bone) and of antitumoral immune responses. ATAD2 is progressively overexpressed during prostate cancer progression and may be an important therapeutic target because of its restricted expression in normal adult tissues as well as the presence of a potentially druggable and specific bromodomain. Furthermore, despite its widely reported association to worse survival in multiple cancer types, remarkably little is known about its functional role in metastasis. In this proposal we will determine the functional significance of ATAD2 expression for prostate cancer progression and metastasis. We will focus on its ability to modulate bone colonization and antitumoral immune responses, two critically relevant steps in the development of metastasis, and uncover the chromatin and transcriptional mechanisms through which it acts. Using state-of-the art syngeneic mouse models, ex-vivo epigenetic editing, human organoids and advanced tissue engineering technologies, our expert multidisciplinary team is uniquely poised to have a positive impact on our understanding of how tumor cells progress to lethal metastatic disease. Our studies will uncover novel mechanisms linking metastasis and immune escape, paving the way for future biomarker driven targeted therapies that may lead to durable and systemic therapeutic responses in currently incurable metastatic disease.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Project Summary Metabolic dysfunction-associated steatotic liver disease (MASLD) is an emerging health epidemic in the United States with few therapeutic options. The accumulation of lipid droplets within hepatocytes drives the pathogenesis of MASLD, yet the cellular mechanisms governing lipid storage in lipid droplets remain incompletely understood. My goal is to address this knowledge gap and advance the development of new therapies for MASLD. My preliminary work integrated genome-wide CRISPR-Cas9 screening and human genetic association studies to identify DNAJC22, a gene of unknown function, as a novel regulator of lipid droplet accumulation. Knockout of DNAJC22 dramatically reduced lipid droplet accumulation, and overexpression of DNAJC22 led to the accumulation of giant lipid droplets in hepatocytes. I also identified a predicted loss-of-function mutation in DNAJC22, p.A163T (rs146774114) that was associated with protection from MASLD across multiple human genetic biobanks. This application will explore DNAJC22 as a potential new therapeutic avenue for the treatment of MASLD. I will evaluate the cellular pathways through which DNAJC22 promotes lipid droplet accumulation and assess the impact of silencing DNAJC22 in preclinical models of MASLD. In Aim 1, I will quantify the effects of DNAJC22 overexpression, knockout, and the p.A163T variant on lipolysis, beta oxidation, and lipoprotein export pathways in human hepatoma cells and human precision-cut liver tissue slices (PCLS). In Aim 2, I will evaluate the in vivo effects of therapeutic hepatocyte- directed silencing of DNAJC22 on disease activity and fibrosis progression in two murine models of MASLD and tie these effects to alterations in neutral lipid flux. My long-term objective is to become an independent laboratory investigator that uses human genetics, functional assays, and translational disease models to define the molecular basis of MASLD. In this mentored research career development proposal, I will learn metabolic assays to dissect lipid droplet biology, acquire skills in state-of-the-art genome engineering techniques, and learn translational models of MASLD. The primary mentor of this application, Dr. Rajat Gupta, is a leader in functional genomics, and the co-mentor Dr. Jean Schaffer is a leader in MASLD and metabolism; both have exceptional track records of mentorship. A decidated advisory committee will provide training in metabolism (Dr. David E. Cohen), genome engineering (Dr. John Doench), murine MASLD models (Dr. Yury Popov), and PCLS (Dr. Gordon Jiang) and contribute scientific input and additional career guidance. This application is coupled with the full support of Beth Israel Deaconess Medical Center and the outstanding educational resources at the Broad Institute, Harvard Medical School, and affiliated institutions.

NCI - National Cancer Institute

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) has a dismal 5-year survival rate of <10%, driven in large part by a unique tumor microenvironment (TME) characterized by an intense fibrotic and inflammatory reaction, orchestrated by cancer associated fibroblasts (CAFs) and immunosuppressive immune cells. CAFs, among the most prominent cell types in PDAC, influence tumorigenesis by secreting extracellular matrix (ECM), metabolites, and cytokines. While CAFs ultimately promote cancer progression, complete removal of CAFs and their ECM from tumors has proven detrimental to patients. Therefore, understanding how to inhibit functions that promote tumorigenesis, without eliminating CAFs, is critical to develop effective targeted stromal therapies that complement standard of care regimens. Functionally, CAFs support tumor growth by inhibiting anti-tumor natural killer (NK) and CD8+ T cells and by supplying metabolites that fuel cancer cell proliferation. We recently linked glutamate/glutamine-cycling enzymes to immunosuppressive cytokine production in CAFs: ablating these enzymes restored anti-tumor immune killing of PDAC cells, while glutamate supplementation enhanced CAF immunosuppression. This led us to postulate that a CAF-derived glutamate metabolite drives immune suppression in the TME. We now show that gamma aminobutyric acid (GABA), a glutamate-derived metabolite, is present in PDAC patient tumor interstitial fluid (TIF), and that CAFs synthesize GABA de novo and express both the biosynthetic enzymes and receptors needed to produce and respond to it. Furthermore, it is known that T and NK cells possess GABA receptors, and blockade of GABA signaling in mouse models of PDAC significantly reduced tumor burden. Thus, we hypothesize that CAFs are a major source of GABA in the TME, that GABA is a driver of immunosuppression through paracrine effects on immune cells, and that GABA imparts an immunosuppressive program in CAFs, in an autocrine manner. In an innovative approach, we will address these hypotheses using a physiologically relevant 3D culturing system, state of the art murine models of PDAC, spatial transcriptomics, metabolomics, and multiplex ELISAs to uncover how gain or loss of function of GABA signaling in normal fibroblasts and CAFs alters immunosuppression and tumor growth. Murine models are essential for this study because GABAergic paracrine signaling between CAFs, cancer cells, and immune cells cannot be fully recapitulated in vitro, and syngeneic and genetically engineered PDAC models are required to assess how manipulating GABA signaling alters tumor burden and anti-tumor immunity in an intact TME. Additionally, we have a novel IRB approved protocol to study this phenomenon in patients, using patient matched plasma, TIF, and the same tissue after fluid isolation. Our long-term goal is to develop prognostic/diagnostic profiles from metabolites, cytokines, immune cell infiltrates, and target spatial transcriptomes using a novel computational pipeline that can be done as a simple blood test to find cancer early.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Project Summary/Abstract In vertebrates, self-renewing hematopoietic stem cells (HSCs) are produced from a developmental event called endothelial-to-hematopoietic transition (EHT). EHT consists of a cellular and transcriptional reprogramming that allows hemogenic endothelial cells (HECs) from a subset of embryonic arteries to leave the vessel and become blood stem cells. HSCs have the capacity to replace and restore the complete blood system upon transplant, making HSC transplant the only curative therapy available for blood diseases like leukemia and lymphoma. Given this therapeutic need, great effort has focused on the development of in vitro protocols that attempt to recapitulate the conditions of EHT for clinical expansion or de novo production of stem cells in the dish. To date none efficiently produce long-lived multipotent HSCs, suggesting that one or more developmental signals for this process remain to be defined. Mechanical forces from blood flow are an essential cue for HSC production via EHT, and the zebrafish Danio rerio provides an excellent animal model in which to study this contribution to hematopoiesis due to conserved molecular genetics of EHT in this species and the ability to observe live embryos with active circulation. Flow-driven EHT is mediated in part by the Yes-associated protein (YAP) transcription factor (TF), a transcriptional coregulator that has roles in organ growth, nutrient regulation and cell fate specification. YAP can be directed to the nucleus as a direct result of physical forces acting on the cell, but the molecular mechanisms by which this promotes EHT and HSC production are unclear. In preliminary data generated under K01 support, single-cell transcriptional analysis of wildtype, yap -/- and YAP-overexpressing HECs from zebrafish point to a role for YAP in regulating a battery of self-renewal hematopoietic TFs, cell cycling and metabolic processes. In examining these YAP gain- and loss-of-function (GOF/LOF) transcriptomes, gene module scores suggest an impaired glycolysis-to-oxidative phosphorylation rewiring in HECs. Genes related to lipid metabolism are also dysregulated by YAP perturbation and can be identified in ‘no flow’ datasets from mouse models. This R03 application will investigate the role of force-directed lipid metabolism in developmental EHT using zebrafish as a model. We hypothesize that hemodynamic forces alter lipid usage in HE to drive the metabolically intensive process of EHT. In the first aim, an unbiased approach of mass spectrometry-based lipidomic profiling will be used to quantify the abundance of lipid species in wildtype and YAP gain or loss of function (GOF/LOF) whole-embryo and sorted endothelial cell populations to determine those metabolites that are YAP-regulated (as a proxy for a major cellular transducer of mechanical force). In the second aim a candidate pathway, the secreted sphingosine-1-phosphate lipid mediator, will be studied for its role in EHT by live-imaging, chemical perturbation and state-of-the-art genome editing technologies to create tissue-specific LOF zebrafish lines. Findings from this proposal will uncover force-driven metabolic responses that might enhance production of HSCs via EHT and generate critical preliminary data to support R01 applications.