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U.S. National Science Foundation

The EDU Core Research (ECR) program offers this ECR:Core solicitation and invites proposals for fundamental research (curiosity-driven basic research and use-inspired basic research) that contributes to the general, explanatory knowledge that underlies STEM education in one or more of the three broadly conceived Research Areas: Research onSTEM Learning and Learning Environments, Research on Broadening Participation in STEM fields, andResearch on STEM Workforce Development. Within this framework, the ECR program supports a wide range of fundamental STEM education research activities, aimed at learners of all groups and ages in formal and informal settings. Fundamental researchgenerates knowledge and understanding with the potential for broad relevance. The potential implications of ECR fundamental research for improving STEM education practice may be indirect and long-term rather than direct and immediate. Moreover, whether they include basic or use-inspired basic research, all successful ECR:Core proposals focus on the advancement or refinement of foundational knowledge for STEM education. The amount of funding and duration requested in proposals submitted to the ECR:Core solicitation should align with the maturity of the proposed work and the size and scope of the empirical effort. The solicitation has three levels of funding with a range of budget sizes, and proposals may request a duration of 3 to 5 years for any level: (1)Level I proposals may request up to $500,000; (2)Level II proposals may request up to $1,500,000; (3)Level III proposalsmay request up to $2,500,000. All proposals should justify the level of funding and duration in the project description.

U.S. National Science Foundation

The EDU Core Research (ECR) program offers this ECR:Core solicitation and invites proposals for fundamental research (curiosity-driven basic research and use-inspired basic research) that contributes to the general, explanatory knowledge that underlies STEM education in one or more of the three broadly conceived Research Areas: Research onSTEM Learning and Learning Environments, Research on Broadening Participation in STEM fields, andResearch on STEM Workforce Development. Within this framework, the ECR program supports a wide range of fundamental STEM education research activities, aimed at learners of all groups and ages in formal and informal settings. Fundamental researchgenerates knowledge and understanding with the potential for broad relevance. The potential implications of ECR fundamental research for improving STEM education practice may be indirect and long-term rather than direct and immediate. Moreover, whether they include basic or use-inspired basic research, all successful ECR:Core proposals focus on the advancement or refinement of foundational knowledge for STEM education. The amount of funding and duration requested in proposals submitted to the ECR:Core solicitation should align with the maturity of the proposed work and the size and scope of the empirical effort. The solicitation has three levels of funding with a range of budget sizes, and proposals may request a duration of 3 to 5 years for any level: (1)Level I proposals may request up to $500,000; (2)Level II proposals may request up to $1,500,000; (3)Level III proposalsmay request up to $2,500,000. All proposals should justify the level of funding and duration in the project description.

U.S. National Science Foundation

ECR s Building Capacity in STEM Education Research (ECR: BCSER) supports projects that build investigators capacity to carry out high-quality STEM education research that will enhance the nation s STEM education enterprise. In addition, ECR: BCSER seeks to broaden the pool of researchers who can advance knowledge regarding STEM learning and learning environments, broadening participation in STEM fields, and STEM workforce development. Researchers institution types are particularly encouraged to submit proposals. Specifically, ECR: BCSER supports activities that enable researchers to expand their areas of expertise and acquire the requisite knowledge and skills to conduct rigorous research in STEM education. Career development may be accomplished through investigator-initiated professional development and research projects or through institutes that enable researchers to integrate methodological strategies with theoretical and practical issues in STEM education.

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY Central synucleinopathies have historically been categorized based on the type of neuropathological inclusions found in the brain at the time of autopsy, with glial cytoplasmic inclusions (GCIs) defining multiple system atrophy (MSA) and Lewy bodies (LBs) defining the Lewy body diseases – Parkinson’s disease (PD), Parkinson’s disease with dementia (PDD) and dementia with Lewy bodies. Cryo-electron microscopy (cryo- EM) studies determining the high-resolution structures of the α-synuclein (α-syn) fibrils found in these lesions have shown that misfolded α-syn adopts distinct structures, or conformations, in these two groups of disorders. This has been critical for validating the strain hypothesis, or the idea that different protein structures are associated with discrete biological consequences in a patient. However, an increasing number of studies indicate that multiple, and sometimes novel, α-syn strains can co-exist in an individual patient, complicating efforts to understand the molecular underpinnings of disease. For example, cryo-EM studies on atypical MSA patients with limbic predominant neuronal cytoplasmic inclusions resulted in the discovery of a Lewy-MSA hybrid fold of α-syn, suggesting that previously unidentified α-syn strains contribute to disease heterogeneity. This finding is consistent with our recent isolation of an undescribed α-syn strain from a PDD patient sample exhibiting biological properties that are distinct from both the MSA and LB strains. Stemming from the increasing identification of new α-syn strains in patient samples, there is a growing need to understand the specific biological consequences of each strain on disease pathogenesis. The long-term goal of our research is to use our understanding of α-syn disease biology to successfully develop diagnostics and therapeutics for synucleinopathy patients. Our objective in this application is to determine how the molecular structure of a recently isolated α-syn strain impacts the route of spread to clinical target areas in the brain, ultimately determining the clinical presentation of disease, including dementia. Building on this discovery, our hypothesis is that structurally and biologically distinct α-syn strains exhibit critical differences in disease pathogenesis in vivo. Our innovative approach will capitalize on our expertise investigating the MSA α-syn strain, which will serve as a unique opportunity to directly compare two distinct patient-derived strains to determine the effect of fibril structure on disease biology. In Aim 1, we will use serial passaging to further isolate and investigate novel α-syn sub-strains from patient sample PDD1 and will use cryo-EM to resolve the new fibril structure(s). In Aim 2, we will assess neuroinvasion following peripheral injections using the PDD1 and MSA strains to determine how structural differences in α-syn fibril structure impacts disease pathogenesis. This work is significant because we will generate the deep phenotyping needed to enable widespread identification of the novel PDD sub-strain, as well as determine the role of strain dynamics on the structure-phenotype relationship in synucleinopathies.

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

Project Summary Building a healthy adult brain requires the precise coordination of multiple developmental processes, including neural stem cell proliferation and differentiation, neuropil extension, synaptogenesis, and synaptic pruning. These processes are sensitive to genetic, environmental, and physiological conditions, and disruptions can have lasting consequences on brain structure, function, and behavior. Early-life immune activation, such as that triggered by infection, has been associated with impaired growth, cognitive deficits, and increased risk of metabolic syndrome later in life. However, the mechanisms linking immune activity during development to long- term neural outcomes remain poorly understood. This project investigates how systemic infection and innate immune signaling influence brain development and adult physiology. Neural stem cells depend on nutrient- responsive growth signaling pathways to support their proliferation and the production of neurons and glia. Preliminary data show that activation of a conserved innate immune pathway—whether through bacterial exposure or genetic manipulation—leads to reduced body and brain size and delays the reactivation of neural stem cells from quiescence. The central hypothesis is that early-life infection activates immune signaling that disrupts neurodevelopmental programs, leading to persistent changes in brain structure and function. This research will define how immune activation alters neural stem cell behavior, impacts brain growth, and shapes adult behavioral outcomes. The findings will provide insight into how developmental immune stressors influence long-term brain health and may inform our understanding of neurodevelopmental disorders associated with early-life inflammation.

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

PROJECT SUMMARY Over 1 million women living with HIV (WLWH) give birth annually. With widespread use of combination antiretroviral therapy (cART), vertical transmission has been significantly reduced, resulting in ~16 million HIV- exposed uninfected (HEU) children as of 2023. Despite being HIV negative, these children face increased risks of poor growth, infection-related mortality, and respiratory disease. These outcomes are believed to result from maternal HIV-induced inflammation and/or cART toxicity, as many antiretrovirals cross the placenta and may disrupt fetal immune development. However, distinguishing the effects of HIV versus ART is difficult in clinical studies due to challenges of studying non-HIV infected women receiving ART. Limited access to fetal tissues further hampers mechanistic insight, creating a need for translational animal models. To address this critical knowledge gap, we propose to use a rhesus macaque model of simian immunodeficiency virus (SIV) infection to investigate how maternal HIV and long-acting ART (LA-ART) affect fetal immune development. We hypothesize that despite the absence of vertical transmission, maternal SIV and LA-ART exposure dysregulates immune ontogeny in the offspring via altered hematopoiesis. A novel LA-ART regimen of FDA-approved drugs Lenacapavir (LEN) and Cabotegravir (CAB), shown to provide effective viral suppression in preliminary macaque studies, will be given bimonthly by injection to female macaques that will then undergo time-mated breeding following viral suppression. Three experimental groups will be studied: [1] SIV-infected, LA- ART treated; [2] uninfected, LA-ART treated; and [3] uninfected, untreated controls. Offspring will be delivered naturally and monitored through six months of age. Specific Aim 1 will assess how maternal SIV/LA-ART versus LA-ART alone affects infant immune maturation and function in the periphery and in tissues using flow cytometry, single-cell RNA/ATAC-sequencing, and in vitro stimulation. We will evaluate vaccine responsiveness using Varivax™ and examine B/T cell responses and receptor repertoires. Specific Aim 2 will study the impact of maternal SIV/LA-ART versus LA-ART alone on hematopoiesis in the offspring. We hypothesize that SIV/LA-ART exposure impairs differentiation and maturation of hematopoietic stem and progenitor cells (HSPCs). Bone marrow will be analyzed via flow cytometry, differentiation assays, and single-cell RNA/ATAC-sequencing. Functional HSPC capacity will be tested via transplantation into immunodeficient mice. This study uses a clinically highly relevant primate model for HIV cure research and neonatal immunity, and advanced immunological tools to uncover how maternal HIV and LA-ART exposure alter infant immune development. Findings will guide future strategies to improve immune outcomes in HEU children.

NIDCR - National Institute of Dental and Craniofacial Research

ABSTRACT Periodontitis is a common oral inflammatory condition that is epidemiologically linked to systemic disorders such as cardiovascular disease, rheumatoid arthritis, and type-2 diabetes. The relationship between periodontitis and systemic comorbidities is bidirectional, as proinflammatory diseases can also predispose to and accelerate the progression of periodontitis. Nevertheless, the factors leading to the association between periodontitis and comorbidities remains unclear. The low-grade systemic inflammation caused by periodontitis may contribute to its connection with systemic diseases. Recent studies have shown periodontitis to rewire hematopoietic stem cell (HSC) transcriptional and epigenetic profile, which is the base for trained innate immunity. Further, our own pilot data suggest that periodontitis activates HSC, inducing cell cycle entry and loss of repopulating potential in a sex-dependent way. However, the long-term effects of recurrent periodontitis on HSC function and clonal complexity remain unknown. Importantly, HSCs have limited replicative potential, and repeated acute inflammatory episodes that drive HSC proliferation may contribute to their decline. This decline is believed to be a major factor in the development of age-related hematologic diseases resulting from dysfunctional HSCs, such as clonal hematopoiesis, myeloid leukemias, and anemia. Understanding how inflammation regulates HSC fate and influences the blood system during development, aging, chronic inflammatory diseases, and hematological malignancies is crucial for uncovering the mechanistic foundations of these processes and their potential connections. In this study, we will: 1) establish a model for chronic periodontitis that mimics its long-term effects on HSCs, and 2) define the functional consequences of chronic periodontitis on HSCs, considering sexual dimorphism and local versus systemic effects. The results from this proposal will enhance our understanding of chronic inflammation's impact on HSC function. Moreover, the research outlined here will identify periodontitis as a risk factor for the development of hematopoietic pathologies. This will enable us to expand our studies to interrogate the consequences of this pathology in other tissues, both in isolation and alongside comorbidities, highlighting the importance of oral health in preventing inflammatory diseases.

NCI - National Cancer Institute

Abstract Hematopoietic stem cell transplantation (HSCT) is a life-saving treatment, yet it is associated with a multitude of acute and chronic physical and psychological sequelae. The success of this treatment and survival for patients is related to the control of adverse effects of the transplantation. While clinical and biological parameters contribute to patient recovery and survival, research suggests the important role of psychosocial determinants such as stress, mental health, coping, and physical well-being on post-transplant recovery and survival. Therefore, prevention and early detection of symptoms is a high priority in the care of these patients, and yoga therapy may be an effective treatment. Yoga therapy (YT) is an evidence-based mind-body practice recommended for cancer to relieve nausea, fatigue, pain, sleep disruption, psychosocial distress, musculoskeletal symptoms, and cognitive dysfunction, all common symptoms experienced during HSCT. Yoga has also been associated with decreased cortisol response, increased cell-mediated immunity, and decreased pro-inflammatory biomarkers, all of which may lead to improved outcomes for those undergoing HSCT. However, few studies have investigated the efficacy and implementation of YT in an inpatient setting, and, besides our successful pilot study, none in patients undergoing HSCT. This randomized, semi-blinded, multicenter trial will evaluate the efficacy and explore the implementation of YT for patients undergoing HSCT (N=300). Patients will be randomly assigned to one of two groups: 1) a Yoga Therapy group (YT) or 2) an enhanced standard care (SC) group. YT patients will participate in yoga before, during hospitalization, and after discharge. The SC group includes available services provided in a standardized manner including the use of physical therapy support and walking support. The specific aims are to: 1) Examine the efficacy of YT relative to SC on HSCT-specific QOL; 2) Examine group differences in other patient-reported outcomes including general QOL and fatigue; 3) Explore group differences in psychosocial and functional outcomes and as possible mediators including stress, mental health (depression and anxiety), sleep disturbances, growth, self-efficacy, mindfulness, and objective functional measures as well as moderators of YT including demographic factors (sex, age, etc.), baseline psychosocial factors, and medical variables (e.g., autologous vs. allogeneic transplant); and 4) Explore YT’s implementation. After the proposed efficacy trial is completed, if YT is found clinically helpful to patients undergoing HSCT, the next phase will involve undertaking a large-scale implementation and dissemination study.

NINDS - National Institute of Neurological Disorders and Stroke

ABSTRACT Lysosomal Storage Disorders (LSDs) are a diverse group of over 70 inherited metabolic diseases caused by loss-of-function mutations in lysosomal enzymes, leading to progressive accumulation of undegraded substrates in cells and tissues. Many LSDs, including Mucopolysaccharidosis Type I (MPS I, Hurler Syndrome), are life- limiting conditions with multisystem involvement and central nervous system (CNS) pathology. Current treatments such as enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) offer only partial benefit. ERT requires lifelong infusions, does not cross the blood-brain barrier (BBB), and is costly. HSCT can slow neurological decline but carries risks including graft-versus-host disease, infection, and mortality. Moreover, over 80% of LSDs still have no approved therapies, representing a significant unmet need. To overcome these limitations, we are developing a transformative platform using genetically engineered human T cells, termed Enzyme-T cells, designed to systemically and durably deliver functional lysosomal enzymes. T cells persist long-term, traffic to peripheral tissues and the CNS, and are well suited for scalable manufacturing. We engineer Enzyme-T cells using the TcBuster (TcB) transposon system, a clinical-stage non-viral gene delivery method, to stably express α-L-iduronidase (IDUA), the enzyme deficient in MPS I. Our published preclinical studies show that IDUA-T cells secrete active enzymes, cross-correct deficient cells, and are well tolerated. In the R61 phase, we will (1) engineer T cells from MPS I patients to stably express IDUA and a safety marker (EGFRt); (2) characterize enzyme secretion, immune phenotype, and genomic safety including transposon copy number and integration site analysis; (3) assess in vitro cross-correction using patient-derived cells and iPSC-derived IDUA-deficient lines; and (4) develop a quantitative systems pharmacology (QSP) model to predict biodistribution, enzyme kinetics, and dosing. In the R33 phase, we will evaluate the in vivo efficacy of IDUA-T cells in an MPS I mouse model, assessing exposure-response, tissue enzyme levels, cognitive function, and toxic glycosaminoglycan (GAG) reduction. Histopathological analysis will assess biodistribution, persistence, and safety. This multidisciplinary project combines MPS clinical expertise, advanced genome engineering, and computational modeling to develop a novel, durable, and CNS-penetrant therapy. If successful, our Enzyme-T cell platform will represent a paradigm shift in the treatment of MPS I and other LSDs with few or no effective options.

NINDS - National Institute of Neurological Disorders and Stroke

Project Summary/Abstract Medulloblastoma (MB) is the most common malignant brain tumor in children and often disseminates early within the central nervous system. Among the four molecular subgroups, Groups 3 and 4 carry the greatest metastatic burden and poorest outcomes. Although digoxin is an approved cardiac glycoside with a well-defined pharmacokinetic (PK) and safety profile and shows activity in Group 3 and Group 4 patient-derived models, translation is limited by its narrow therapeutic index and P-glycoprotein(P-gp)–mediated efflux at the blood brain barrier. We treat radiotherapy as a programmable trigger to localize drug action. Our radiotherapy-activated pro- digoxin chemotypes mask the sugar moiety with N-alkyl-4-picolinium or phenyl-azide carbonate groups to reduce transporter recognition while preserving the lactone required for target engagement; ionizing radiation cleaves the trigger in the treated field to release native digoxin, aiming to raise intratumoral exposure while keeping systemic levels within pediatric therapeutic ranges. The R61 phase will synthesize and analytically qualify two predefined pro-digoxin chemotypes and establish a stability-indicating liquid chromatography–tandem mass spectrometry method to quantify intact pro-drug, released digoxin, and scission products. Using induced pluripotent stem cell–derived brain and vessel organoid assembloids that recapitulate a functional human blood brain barrier, we will quantify radiotherapy-triggered conversion, directional permeability, and P-gp efflux to derive a dose-to-release function and a dosing-to-beam timing window. A focused pilot PK study will confirm brain exposure and lock pharmacodynamic (PD) sampling for Na⁺/K⁺-ATPase target engagement and radiation-linked biomarkers. The R33 phase will implement a preregistered two-by-two design in paired primary and recurrent Group 3 and Group 4 intracranial patient-derived orthotopic xenograft models, using volumetric magnetic resonance imaging to track burden and progression and therapeutic drug monitoring to maintain clinically relevant digoxin-equivalent troughs. Embedded regional bioanalysis will verify in-tissue conversion in irradiated brain, and a focused safety module will monitor cardiac and neurologic tolerance. Critically, R33 will deliver an integrated PK → PD → Efficacy model that links exposure and target engagement to tumor control, identifies the optimal drug-to-radiation schedule and minimal effective duration, and back-translates active mouse exposures to pediatric therapeutic drug-monitoring ranges. By coupling a human neurovascular platform, field-restricted activation chemistry, and quantitative mass spectrometry with rigorous in vivo testing, this program seeks to expand the therapeutic index for children with Group 3 and Group 4 MB and establish a generalizable path from mechanism to schedule to outcome for transporter-limited agents in pediatric neuro-oncology.

U.S. National Science Foundation

The EHR Core Research (ECR) program establishes a mechanism in the Directorate for Education and Human Resources to provide funding in foundational research areas that are broad, essential and enduring. EHR seeks proposals that will help synthesize, build and/or expand research foundations in the following core areas: STEM learning, STEM learning environments, workforce development, and broadening participation in STEM. We invite researchers to identify and conduct research on questions or issues in order to advance the improvement of STEM learning in general, or to address specific challenges of great importance. Two types of proposals are invited: Core Research Proposals (maximum 5 years, $1.5 million) that propose to study a foundational research question/issue designed to inform the transformation of STEM learning and education and Capacity Building Proposals (maximum 3 years, $300,000) intended to support groundwork necessary for advancing research within the four core areas.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary / Abstract This is a R01 application from Dr. Daniel Lingwood (PI) an Associate Professor of Medicine at the Ragon Institute of MGH, MIT and Harvard and Dr. Batista (co-PI), a Professor of Medicine at the same institution. These investigators define immunological decision making by B cells to inform vaccine design and have built humanized mouse systems that recapitulate human antibody responses. Dr. Andrew Ward (co-I) is Professor in the Department of Structural and Computational Biology at Scripps and specializes in high resolution cryoEM of antibody:antigen complexes. The application goal is universal influenza vaccine development, centered on their discovery of a human broadly neutralizing antibody (bnAb) pathway that the investigators can vaccine-elicit to protect against all influenza A viruses (IAV), the major source influenza disease and all pandemic events. Differences in N-glycosylation generally prevents antibodies from engaging the otherwise conserved hemagglutinin (HA) stem of group 1 versus group 2 IAV. To solve this issue, the investigators engineered nanoparticle immunogens that elicit cross-group IAV immunity by selectively triggering and maturing germline B cell receptors (BCRs) encoding VH1-18 QxxV class bnAbs, a rare but genetically reproducible or ‘public’ category of human pan-IAV bnAbs that accommodates N-glycan diversity on the HA stem. The investigators successfully elicit this cross-group bnAb response using a single shot within a humanized vaccine model containing the VH1-18 QxxV bnAb precursors at physiologically relevant human frequency within the naïve B cell pool. The immunogens select for key affinity enhancing mutations, including N55T in the CDRH2, a hallmark of VH1-18 QxxV bnAbs. The investigators show that N55T alone provides cross-group IAV protection by a novel antibody tilting mechanism that accommodates N-glycan diversity on the HA stem. The investigators will now test the central hypothesis that this ‘molecular switch’ endows humans with an exceptionally simple vaccine- expandable pathway for eliciting broad spectrum IAV immunity. In Aim 1, the investigators will apply their modular human vaccine model to define the number of naive VH1-18 QxxV B cells needed for pan-IAV vaccine protection; if these bnAb precursors are absent, the germline stimulating nanoparticles no longer elicit pan-IAV bnAbs, revealing a human B cell repertoire effect encoding for unprecedently broad IAV immunity. In Aim 2, the investigators will define whether their nanoparticle immunogens can co-expand multiple classes of cross-group IAV bnAbs within their human vaccine model. Critically, their engineered nanoparticle immunogens also bear germline stimulating affinity for the naïve BCRs encoding the other known classes of genetically reproducible pan-IAV bnAbs produced by humans, potentiating multiclass bnAb elicitation via pan-germline stimulation. In the Aim 3, the investigators will define how prior exposure to IAV modulates (and enhances) germline stimulation and vaccine elicitation of pan-IAV bnAbs via imprinting effects. Collectively, this proposal will exploit novel genetically hardcoded templates for eliciting cross-group IAV immunity in humans.

NIAID - National Institute of Allergy and Infectious Diseases

Abstract: A cure for HIV-1 infection, previously considered elusive, has now been documented in several individuals who underwent a hematopoietic stem cell transplant from donors with the CCR532 gene defect, providing proof-of-principle evidence that curative interventions for HIV-1 infection can be successful. However, curative approaches based on toxic allogeneic stem cell transplantation can only be used in highly selected individuals who suffer from malignant comorbidities. An alternative form of a “functional cure” for HIV-1 infection is exemplified by elite controllers, individuals who maintain undetectable levels of HIV-1 replication in the absence of antiretroviral therapy for extended periods of time. Through detailed molecular assessments of the viral reservoir profile in our previous work, we have demonstrated that HIV-1 reservoir cells in elite controllers display marked footprints of immune selection, most clearly evidenced by the selective presence of intact proviruses in heterochromatin regions. These observations strongly suggest that in elite controllers, the human immune system can recognize, target and eliminate HIV-1 reservoir cells. However, the mechanisms underlying immune targeting of the HIV-1 reservoir cell pool remain undefined. Here, we propose to address this central question in detail using a combination of advanced, high-resolution, analytic techniques in a large cohort of elite controllers that have previously been studied in our laboratory and for which a detailed characterization including the frequencies of intact proviruses annotated with corresponding proviral integration sites, is already available. In Specific Aim 1, we will deeply interrogate HIV-1 reservoir cells from elite controllers through a novel single-cell microfluidics technology designed to capture the surface phenotype and the transcriptional signature of participant-derived cells that harbor intact or defective proviruses directly ex vivo; these assays will be highly informative for unraveling specific immunological vulnerabilities of the viral reservoir cell pool, and may reveal imprints of antiviral immune activity operational in elite controllers. Specific Aim 2 will focus on using single-cell next-generation sequencing technologies to evaluate genomic, epigenetic, and epi-transcriptomic characteristics of individual HIV-1-infected cells from elite controllers to evaluate correlates of immune adaptation, and possibly, of immune evasion, in reservoir cells from elite controllers. In Specific Aim 3, we will focus on how NK cells can contribute to immune selection pressure against HIV-1 reservoir cells in elite controllers, using a pipeline of assays designed to evaluate transcriptional signatures, genomic variations, immunogenetic properties and functional characteristics of NK cells in participant-derived samples; these studies will test the hypothesis that HIV-specific T cells and NK cells represent complementary arms of antiviral immune activity that, through collaborative efforts, are inducing the remarkable clinical outcome of natural immune-mediated HIV control.

NHLBI - National Heart Lung and Blood Institute

Project Summary Respiratory viruses can acutely lead to high mortality from respiratory failure. However, respiratory viral infections also cause permanent morbidity long after recovery from the acute infection. Such chronic debilitation was best exemplified during the COVID-19 pandemic that left many patients with post-acute sequelae of SARS-CoV-2 (PASC; aka long COVID). One unfortunate consequence after a severe COVID-19 infection is the development of persistent pulmonary fibrosis (PASC-PF). However, this fibrotic remodeling of the lungs is not unique to infections by SARS-CoV-2 and has also been found with other viral infections such as influenza, MERS-CoV, SARS-CoV-1. Investigations into PASC-PF have revealed common features when compared to idiopathic pulmonary fibrosis (IPF), the archetype of progressive lung fibrosis. A fundamental feature in PASC-PF, IPF, and other types of lung fibrosis is the maladaptive repair of the lung epithelium that is an upstream driver of fibroproliferation. In particular, the type 2 alveolar epithelial cell (AT2) cells, which are the facultative epithelial stem cells of the alveoli, are exhausted and fail to properly repair the lungs after injury. Accordingly, damaged alveoli have a repair deficiency with a failure of AT2 differentiation into alveolar type 1 (AT1) cells that are necessary for lining the alveoli to facilitate gas exchange in the lungs. To that end, recent studies have found IFN-γ to be a key signaling node that is enriched in PASC-PF and IPF. Moreover, studies by our group and others have demonstrated IFN-γ blockade to reduce fibrosis and augment epithelial repair in a viral-induced lung fibrosis model. Accordingly, we have developed a central hypothesis that anti-viral immunity by CD8+ T cells directly (via IFN-γ) and indirectly (via MDM secretion of IL-1β) stalls alveolar regeneration thereby shifting the injury response toward maladaptive repair thereby activating fibroproliferative pathways. We will test the hypothesis in three aims: Aim 1. Evaluate viral-mediated maladaptive reprogramming of AT2 cell. Aim 2. Uncover the cellular and molecular mechanisms that mediate the abnormal immune-epithelial interactions contributing to chronic lung fibrosis following viral infection. Aim 3. Identify abnormal immune-epithelial interactions in human post-viral lung fibrosis . The successful completion of this study promises to elucidate the cellular and molecular etiology of lung fibrosis and have a broad impact on understanding fibroproliferative mechanisms in PASC-PF, IPF, and other forms of lung fibrosis.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Progressive familial intrahepatic cholestasis (PFIC) is a group of rare, inherited liver diseases that impair bile formation and excretion, leading to severe cholestasis, progressive liver damage, and, often, the need for liver transplantation in childhood. ATP8B1 deficiency, previously known as PFIC1, caused by mutations in the ATP8B1 gene, remains poorly understood due to the lack of suitable animal models and the rarity of the disorder. Current treatment strategies focus on reducing enterohepatic bile acid circulation but fail to prevent ongoing hepatocellular injury. The ATP8B1 protein (FIC1) maintains canalicular membrane asymmetry and modulates bile acid signaling through the farnesoid X receptor (FXR), but how its loss-of-function causes cholestasis is incompletely understood. Using patient-derived induced pluripotent stem cells (iPSCs) carrying the common ATP8B1 G308V mutation, our group has established clinically relevant hepatocyte and cholangiocyte models that recapitulate disease phenotypes. Preliminary studies reveal alterations in plasma membrane organization, FXR activation, glutamine metabolism, and cholangiocyte protective mechanisms. This proposal will test the central hypothesis that ATP8B1 G308V disrupts bile acid homeostasis and cellular resilience through impaired FXR activity, altered glutamine metabolism, and reduced cholangiocyte protection, leading to cholestasis and progressive liver pathology. Specifically, we will i) define how FIC1 deficiency and FXR suppression contribute to bile acid accumulation and hepatocyte vulnerability, ii) interrogate the role of glutamine metabolism in bile acid dysregulation and determine whether supplementation restores hepatocyte function, and iii) evaluate how restoring MUC1 expression improves cholangiocyte function and hepatobiliary homeostasis. The proposed studies will generate new mechanistic insights into ATP8B1 deficiency, provide a unique patient- derived model for studying rare cholestatic diseases, and identify molecular pathways that can be targeted for therapeutic intervention. Ultimately, this work will inform strategies not only for PFIC1 but also for other cholestatic and metabolic liver diseases of genetic origin.

NIAID - National Institute of Allergy and Infectious Diseases

The increasing application of helminth antigen-derived therapy to prevent inflammatory diseases represents a promising and innovative approach. Over the past decade, our research group has concentrated on Fh15, a recombinant molecule from the Fasciola hepatica fatty acid binding protein family, which exhibits potent antiinflammatory effects in animal models of sepsis and ulcerative colitis. We have demonstrated that a single intraperitoneal (i.p.) dose of Fh15 administered to animals one hour before or after the administration of a lethal dose of lipopolysaccharide (LPS) or live E. coli infusion significantly suppresses the cytokine storm and other inflammatory markers. Additionally, we found that a single i.p. dose of Fh15 given three times, two days apart, significantly improves the disease activity index (DAI) in a dextran sulfate sodium (DSS)-induced ulcerative colitis mouse model. These therapeutic effects correlated with reduced levels of intestinal inflammatory markers, diminished neutrophil infiltration, and lower levels of myeloperoxidase—a protein essential for neutrophil extracellular trap (NET) formation. Furthermore, we demonstrated that Fh15 can inhibit NETosis in human neutrophils and acts as a TLR4 antagonist, a receptor involved in the immune response against sepsis and ulcerative colitis, as well as in neutrophil activation and NET release. This application stems from a previous SC1 grant (1SC1AI155439-01), which resulted in one patent, approximately five publications, and the graduation of three PhD and one MS students. Our central hypothesis posits that Fh15 modulates the TLR4/NF-êB signaling pathway to suppress inflammation in animal models of sepsis and ulcerative colitis, which is associated with decreased concentrations of free extracellular DNA (eDNA) and reduced NETosis. In the aim-1 will demonstrate the relationship between the suppression of inflammatory markers induced by Fh15 in the sepsis and ulcerative colitis models and the reduction of eDNA and NETs in vivo. We will isolate free eDNA from plasma in sepsis and colitis mice treated with Fh15 and characterize the specific protein-DNA components of this eDNA through immunofluorescence staining and confocal microscopy. The results will be correlated with levels of inflammatory markers for colitis and sepsis. In the aim-2 will identify the mechanisms underlying the inhibitory effects of Fh15 on NETosis. We will isolate neutrophils from mouse bone marrow and analyze activation markers such as CD11b, CD66b, CD62L, CD64, and FcRI using flow cytometry. Additionally, we will assess the expression of TLR4, CD14, and proteins involved in the NF-êB pathway using qPCR and Western blotting. Proximity ligation assays will determine whether Fh15 directly interacts with CD14 on these cells. We will also employ proteomics and single-cell RNA sequencing to investigate changes in protein and gene expression in neutrophils and identify molecules involved in pro-inflammatory pathways that may be regulated by Fh15. The outcome of this study will provide a more comprehensive understanding of how F. hepatica molecules interact with the immune system and lay the groundwork for the development of a novel class of anti-inflammatory drugs.

NIA - National Institute on Aging

Project Summary/Abstract The US population aged 65+ is expected to increase to 23% by the year 2050. Age-related chronic inflammatory disease and infections are a significant burden on the healthcare system. Age-related changes in neutrophil function and migration play a significant role in these pathologies. A current gap in understanding mechanisms that mediate age-related changes in neutrophils limits therapeutic intervention. We have developed a scale explant assay using zebrafish across the lifespan to investigate changes in neutrophil function and migration with age in situ. Application of optical metabolic imaging (OMI) provides metabolic readouts in tissues in real time with aging. The Huttenlocher lab has pioneered genetically tractable human iPSC-derived neutrophils (iNeutrophils) to dissect signaling mechanisms that regulate neutrophils. Derivation of iNeutrophils from donors of different ages will enable investigation of these mechanisms with aging. This fellowship proposal will leverage the combined use of these techniques to disentangle the contribution of neutrophil intrinsic and environmental contributions to age-related changes in neutrophil function and migration. We hypothesize that aging alters neutrophil metabolism and function through both neutrophil intrinsic and extrinsic factors. Aim 1 will establish the zebrafish scale explant assay as a model to study aging related inflammation in epithelial tissues, canonical senescence markers, and alterations in peripheral immune cell population. Simultaneously, use of scale explant system and iNeutrophils will elucidate neutrophil metabolic changes in situ and in vitro across the lifespan. Building on our prior work showing a role for neutrophil Lyn kinase in directing migration up an H2O2 gradient, aim 2 will elucidate the role of redox-sensitive Lyn kinase in age-related neutrophil aberrant migration in situ and in vitro. We will introduce a redox-insensitive or wild-type (WT) Lyn kinase into a Lyn-deficient zebrafish. Using the scale explant assay, we will interrogate Lyn’s redox-signaling role in age-related neutrophil chemokinesis and chemotaxis in situ. Depletion of LYN and rescue with redox insensitive or WT LYN in the iNeutrophil system will elucidate the role of Lyn redox-signaling in vitro. We will interrogate how extrinsic H2O2 informs iNeutrophil chemokinesis and chemotaxis. The goal of this work is to identify key mechanisms that regulate age-related changes in neutrophil function for potential therapeutic intervention. This proposal will provide specific training in metabolic imaging, stem cell culture, and cell signaling. This project synthesizes the cell biology expertise of Dr. Huttenlocher with the metabolic and engineering expertise of Dr. Skala along with the vast scientific, medical, and translational resources available at the University of Wisconsin-Madison. This fellowship will promote the development of research, clinical, translational, mentorship, and communication skills crucial for my career as an independent physician scientist.

NIGMS - National Institute of General Medical Sciences

TITLE. Elucidating mechanisms of macropinosome formation, stabilization, and trafficking. SUMMARY/ABSTRACT Macropinocytosis, or “cell drinking,” is central to critical macrophage immune functions including wound healing, antigen presentation, and resolution of inflammation. Macropinocytosis is the non-specific and receptor independent internalization of solutes by the formation of large endocytic structures. Macropinocytosis facilitates nutrient uptake and growth in macrophages, T cells, and numerous types of cancer cells. Despite its significance, there are large gaps in the mechanistic understanding of this process. The long-term goals of this project are to identify novel mediators and cellular mechanisms of macropinocytosis. The central hypothesis is that UVRAG in cooperation with VPS34-II provides positive regulation of macropinocytosis by facilitating recruitment of effectors mediating stabilization, maturation and trafficking while PAK2 provides countervailing negative regulation. This hypothesis stems from CRISPR/Cas9 whole genome screen data produced in the applicant’s laboratory indicating that UVRAG, members of VPS34 complex II (VPS34-II), and Rab5 activators and effectors are key positive regulators of macropinocytosis. New phosphoproteome data provides a strong cross-validation of our CRISPR screens as Gapvd1/GAPex5 (Rab5 activator) and Ankfy1/Rabankyrin5 (Rab5 effector) were identified as putative downstream targets of PAK2. Rabankyrin5 and Rabenosyn5 facilitate SNARE-mediated vesicle fusion. Preliminary data confirms that PAK2 negatively regulates bulk solute uptake and could provide an explanation for the long-standing question of why Rac1 GTP-GDP cycling is required for macropinocytosis. The hypothesis will be tested by pursuing three specific aims: 1) Determine how UVRAG and VPS34-II stabilize macropinosomes following formation and facilitate transit through the cortical actin, 2) Determine the roles of GAPex5, Rabankyrin5, and Rabenosyn5 in vesicle fusion events promoting macropinosome sealing, and 3) Determine how PAK2 provides negative regulation of macropinocytosis. Under the first aim, gene editing methods already established in the applicant’s laboratory will be used to create macrophages that are deficient in UVRAG and other members of VPS34-II. Macropinosome formation, PI(3)P production, and actin dynamics will be imaged via live-cell 3D microscopy to observe spatiotemporal organization of PI(3)P and depolymerization of the cortical actin. The second aim will investigate whether PI(3)P supports macropinocytosis by recruiting GAPex-5, a Rab5 guanine exchange factor, leading to the activation of Rab5 and its effectors Rabankyrin5 and Rabenosyn5, proteins that facilitate SNARE-mediated membrane fusion. Pull-down assays will be used compare Rab5 activity and binding partners in wildtype and Gapvd1sgRNA BMDM. The third aim will investigate how kinase activity of PAK2 inhibits sealing of the macropinocytic cup by measuring Lucifer yellow uptake with or without pharmacologic inhibition of PAK2 following photoactivation of Rac1 and identify proteins downstream of PAK2 mediating PAK2 inhibition of macropinocytosis.

NIGMS - National Institute of General Medical Sciences

Project Summary The broad, long-term objectives of this application are to characterize mechanisms that allow a competitive germline stem cell (GSC) and its descendants to dominate the GSC population and cause super-Mendelian inheritance. The proposal will determine how a GSC in the Drosophila testis remodels its niche and causes the selective loss of WT neighbor GSCs. To accomplish this, the proposal will utilize immunofluorescence, genetics, RNA interference, extended ex vivo live-cell imaging, transcriptomics, chromatin labelling, and innovative assays of GSC competition and allele inheritance in F1 offspring. We will capitalize upon the powerful genetics available in Drosophila, as well as the ability to unequivocally identify the niche, GSCs, differentiating germline cells, and somatic stem cells (CySCs) and their lineage in the Drosophila testes. This proposal is supported by our published results demonstrating that (1) loss of the transcription factor Chinmo in a GSC causes the ectopic secretion of the extracellular matrix (ECM) protein Perlecan (Pcan), (2) this Pcan accumulates around the endogenous niche resulting in an ectopic ECM termed the moat within the testis lumen; (3) the moat causes the selective loss of WT neighbor GSCs, which no longer have strong adhesion with niche cells; (4) chinmo-/- GSCs remain in the resculpted niche because they upregulate ECM-binding proteins. This proposal is also supported by our unpublished results showing that Chinmo protein expression is promoted by an RNA-binding protein (RBP) in GSCs and that a ZAD-ZNF protein likely acts as a Chinmo co-factor in GSCs. In the first goal, we will determine whether clonal loss of the RBP that promotes Chinmo expression imparts that GSC with a competitive advantage. We will also determine what regulates that RBP in GSCs and test whether loss of any regulators of the RBP imparts a competitive advantage to a mutant GSC. In the second goal, we will determine whether Chinmo and the ZAD-ZNF protein work together to repress Pcan by recruiting histone methyltransferases. We will also determine how niche cells promote the ectopic Pcan produced by chinmo-/- GSCs. In the third goal, we will test the role of somatic stem cells (CySCs) in GSC competition and assess whether they push out WT neighbors GSCs. We will also use live-cell imaging to determine the types of GSC division that occur in chinmo- /- GSCs. The studies in this proposal will increase the knowledge base about GSC competition and will foster new avenues of research into mechanisms and possible treatments for human paternal age effect disorders caused by competitive spermatogonial stem cells and for tumor cells which remodel their microenvironment to benefit themselves and disadvantage WT neighboring cells.

NHLBI - National Heart Lung and Blood Institute

Project Summary: Mammalian infants exhibit immature circadian regulation: the establishment and maturation of cellular and systemic clocks occur postnatally, driven by maternal cues, feeding cycles, and light entrainment. In parallel, the neonatal heart undergoes profound structural and functional remodeling, characterized by decreased cardiomyocyte proliferation, metabolic shifts, altered expression of ion channels, enhanced electrical coupling, and changes in alternative splicing mediated by RNA-binding proteins (e.g., Rbfox1). Despite these parallel developmental processes, no molecular pathway has been determined to link the development of the circadian clock with cardiomyocyte maturation. Importantly, induced pluripotent stem cell-derived cardiomyocytes (iPSC- CMs) share a common deficiency in maturity, which is a significant limitation for their application in disease modeling, drug screening, or as cell therapy agents. Therefore, uncovering the intrinsic regulatory mechanisms of mammalian cardiomyocyte maturation has significant implications for both basic developmental biology and pre-clinical applications. We propose that a circadian clock checkpoint, comprising activators (ATF3/MEF2s) and a suppressor (NFIL3), serves as a temporal regulator of cardiac maturation during early life. The aims of this proposal are as follows: Aim 1. Determine the enhancer (Rb1en)-transcription factors circuitry (ATF3/MEF2/NFIL3) that initiates the temporal and tissue-specific expression of Rbfox1; and Aim 2. Determine the critical role for circadian clock regulatory mechanisms on cardiomyocyte maturation in vivo and hiPSC-CM maturation in vitro. These studies will establish a molecular bridge between developmental chronobiology and cardiac maturation, reveal new mechanisms of temporal regulation in postnatal heart growth, and may inform novel strategies for conditions associated with impaired cardiac maturation and enhance maturity and disease relevance of hiPSCs. Collectively these insights will reduce the burden of cardiovascular risk and advance pre- clinical applications of stem-cell-based modeling and therapeutics.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

ABSTRACT Understanding the fundamental processes that underscore hematopoiesis is necessary to generate therapeutics that can correct hematopoiesis during disease or support hematopoietic regeneration during stress. The bone marrow microenvironment, or niche, supports hematopoiesis by providing cellular and acellular cues that influence hematopoietic differentiation, hematopoietic stem cell self-renewal, and the functional integrity of the bone marrow niche itself. Inadequate or inappropriate hematopoietic differentiation can lead to disease or death by depleting hematopoietic cell populations needed for organismal survival. Additionally, commonly used therapeutics like radiation and chemotherapy cause hematopoietic cell death and damage the bone marrow niche, leaving patients in a vulnerable state of hematopoietic insufficiency. The inability to restore hematopoietic homeostasis after radiation exposure puts patients at a heightened risk for developing deadly complications, such as infection or hemorrhage. Therefore, understanding how hematopoiesis is maintained and restored is of the utmost importance. Our previous studies showed that syndecan-2 (a specific heparan sulfate proteoglycan) expressed by hematopoietic stem cells promotes long-term hematopoietic stem cell self-renewal ability by supporting quiescence. The bone marrow niche is also a rich source of proteoglycans. Our preliminary data indicate that bone marrow mesenchymal stromal cells (MSCs) also highly express syndecan-2. Genetic depletion of syndecan-2 in MSCs using transgenic mouse models caused significant hematopoietic system imbalances in the peripheral blood at steady-state and after hematologic stress. In this application, we propose to elucidate how MSC-derived syndecan-2 regulates hematopoiesis in vivo. We will use a multi-scale approach to test the function of syndecan-2 at the molecular, cellular, and systemic scales by combining transgenic knockout mouse models and in vivo injury models with high-resolution bone marrow imaging and super- resolution imaging of MSCs. We will test the role of MSC syndecan-2 in hematopoietic differentiation, growth factor organization, and signaling. Because hematopoietic demands increase during states of hematopoietic stress, we will also test the function of syndecan-2 from MSCs in hematologic and niche regeneration from radiation injury. Successful completion of these aims will define the role of MSC-derived syndecan-2 in hematopoietic homeostasis and regeneration, providing foundational knowledge needed to leverage proteoglycans to correct or boost hematopoiesis during states of imbalance or stress.

NIGMS - National Institute of General Medical Sciences

Abstract The canonical Wnt signaling pathway is a master regulator of embryonic development, stem cell renewal, and tissue regeneration. Its dysregulation drives diverse human diseases, including cancer, osteoporosis, and cardiovascular and metabolic disorders. At the center of this pathway are the co-receptors LRP5 and LRP6, which act as dynamic molecular hubs that integrate stimulatory and inhibitory cues. Despite their fundamental roles, how LRP5/6 transition between distinct conformational states and translate ligand binding into precise signaling outcomes remains poorly understood, limiting the development of targeted therapeutic strategies. This proposal aims to decode the structural and mechanistic logic of LRP5/6-mediated Wnt signaling. Leveraging cutting-edge cryo-electron microscopy, biochemistry, and cell-based functional assays, we will define how LRP5/6 dynamically remodel in response to Wnt ligands and antagonists, and how cellular factors such as pH and post-translational modifications fine-tune their activity. By linking receptor structure to function and disease-associated mutations, this work will generate a unifying framework for understanding Wnt pathway regulation and open new avenues for precisely modulating Wnt signaling in tissue regeneration and disease intervention.

NHLBI - National Heart Lung and Blood Institute

Project Summary Smoking remains a leading cause of cardiovascular disease (CVD), responsible for millions of deaths worldwide. Despite its widespread impact, the precise mechanisms linking smoking to CVD risk, particularly in relation to genetic factors, remain incompletely understood. One such genetic variation, ALDH2*2, affects approximately 540 million individuals globally and may interact with tobacco smoke to exacerbate CVD risk. However, the addictive nature of smoking, compounded by social and cultural influences, complicates efforts to reduce smoking prevalence in this population. Consequently, understanding the role of ALDH2*2 in smoking-induced CVD is crucial for advancing precision medicine for individuals affected by this genetic variation. Our previous research, which utilized induced pluripotent stem cell (iPSC)-derived endothelial cells (iPSC-ECs) from individuals carrying the ALDH2*2 variant, revealed significant endothelial dysfunction. These cells demonstrated elevated oxidative stress and inflammation, along with reduced nitric oxide production and tube formation capacity (Guo et al., Science Translational Medicine, 2023). Our recent findings further suggest that the endothelial dysfunction associated with the ALDH2*2 variant is exacerbated by exposure to cigarette smoke in both human iPSC and transgenic mouse models. Despite these findings, the specific mechanisms by which tobacco consumption exacerbates CVD risk in individuals with the ALDH2*2 variant remain unclear, impeding the development of tailored approaches for ALDH2*2 smokers. The overarching goal of our proposal is to utilize a multidisciplinary approach that integrates stem cell biology, molecular biology, toxicology, vascular physiology, and endothelial mechanobiology to elucidate the molecular mechanisms underlying ALDH2*2- and smoking- induced endothelial dysfunction. We will pursue two specific aims. In Aim 1, we will examine the ROS-FOXO1- KLF5→IL-18/IL-1β signaling axis in modulating endothelial dysfunction in both human iPSC and mouse models carrying the ALDH2*2 variant. Additionally, we will screen small molecules targeting the ROS-FOXO1-KLF5 axis in cigarette smoke-exposed ALDH2*2 iPSC-ECs to evaluate their effects on endothelial function. In Aim 2, we will examine NUP210’s interaction with the LINC complex in mediating the shear stress response in ALDH2*2- and smoking-induced endothelial dysfunction. We will utilize RNA-seq, ATAC-seq, ChIP-seq, and single-cell RNA-seq to gain mechanistic insights into how NUP210 interacts with LINC complex and regulates the H3K27me3 modification of extracellular matrix genes in response to mechanical forces. Our proposal is supported by robust preliminary data, and the successful completion of this research will identify two novel molecular mechanisms—KLF5-mediated inflammation and NUP210-mediated shear stress response—through which smoking exacerbates CVD risk in the ALDH2*2 carriers. Additionally, the study will provide insights into potential prognostic biomarkers and therapeutic targets to mitigate CVD in smokers with the ALDH2*2 allele.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT Acute myeloid leukemia (AML) is a common and aggressive hematologic malignancy with high relapse rates. Relapse is driven by chemotherapy-resistant leukemia stem cells (LSCs), and despite extensive efforts to improve treatments, the survival rate for relapsed AML is just 10%. These clinical challenges underscore an urgent need to define core mechanisms that sustain LSC survival and identify actionable vulnerabilities. Overexpression of the pro-survival protein MCL-1 is associated with poor prognosis and drug resistance in LSCs. While MCL-1 is best known for inhibiting apoptosis, its non-apoptotic functions remain poorly understood in AML and may represent therapeutic vulnerabilities. We recently discovered that MCL-1 regulates lipid metabolism by directly binding ACSL1 to promote long-chain fatty acid oxidation (FAO). Given that FAO supports LSC self- renewal and stress resistance, I hypothesize that MCL-1 confers metabolic protection in AML, independent of its well-known anti-apoptotic role. My proposed project aims to elucidate MCL-1’s non-apoptotic functions in AML metabolism and LSC maintenance. To isolate these roles, I will perturb MCL-1 in four genetically defined AML subtypes lacking BAX and BAK, rendering cells apoptosis-deficient. Preliminary data reveal that Mcl1/Bax/Bak triple knockout (TKO) AML exhibits subtype-specific growth defects compared to Bax/Bak double knockout (DKO) controls, revealing apoptosis-independent roles for MCL-1 in LSC self-renewal. I will compare TKO and DKO cells for metabolic profiles, differentiation states, and transcriptional signatures to define MCL-1-regulated programs apart from apoptosis. The second aim of my project will dissect the role of the MCL-1/ACSL1 axis in AML and achieve a mechanistic understanding of these roles. I will leverage Acsl1 conditional knockout AML models and ACSL1 mutants that modulate the MCL-1 binding interface. Using these tools, I will assess the interaction’s role in growth, FAO, self-renewal, and resistance to ferroptosis and apoptosis. Preliminary studies show that ACSL1 resists ferroptosis in AML cells, and that ACSL1 mutants which enhance or disrupt the MCL- 1/ACSL1 interaction bidirectionally alters lipid utilization. By delineating apoptosis-independent functions of MCL- 1, this work will uncover core mechanisms of AML maintenance and inform next-generation therapies to eradicate LSCs. The Opferman laboratory at St. Jude Children’s Research Hospital, with its deep expertise in MCL-1 biology, apoptosis, and hematopoietic stem cell metabolism, provides an exceptional environment to accomplish this project. Dr. Opferman and St. Jude colleagues fully support my training goals of learning techniques in cancer cell biology, designing rigorous and well-rounded experiments, scientific communication of my results, and mentorship. Core Facilities and Academic Programs Office resources will further enable rigorous execution and dissemination of my findings. Combined with support from the Ruth L. Kirschtein Predoctoral Individual National Research Service Award (F31), this training will provide an excellent launching pad toward my goal of becoming an independent academic investigator.