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NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT Title: Development of microbiome-based therapy for treatment-refractory graft-versus-host disease Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for high-risk hematological malignancies. However, complications, such as graft-versus-host disease (GVHD), continue to limit the success of allo-HSCT. The intestinal microbiome can affect the pathophysiology of acute intestinal GVHD. Recent studies suggest ileal microbiota could affect GVHD severity based on a murine model of GVHD and a clinical study showing that histological GVHD damage of the ileum rather than the colon is associated with refractoriness to treatment. However, no analyses of the small intestinal microbiome have been performed in allo-HSCT patients. Thus, we urgently need to understand the role of the ileal microbiota in epithelial regeneration to prevent treatment-refractory GVHD (TR-GVHD). In this application, we focus on two major processes of TR-GVHD: 1) insufficient suppression of alloreactive immune cells and 2) impaired tissue regeneration. We hypothesize that disrupted ileal microbiota 1) impair epithelial regeneration following GVHD damage and 2) modulate GVHD severity by influencing alloreactive immune cells. To investigate these hypotheses, we established a novel murine model of GVHD using in vivo splenic T cell depletion (SpTCD) in which GVHD is induced by bone-marrow-graft–derived T cells after SpTCD and mediated by microbiome disruption. In this model, we found that administration of Lachnospiraceae species (Lachno spp.) significantly promoted epithelial regeneration in the ileum due to luminal secondary bile acids (SBAs). We hypothesize that Lachno spp. play a key role in promoting epithelial regeneration by producing SBAs and thereby suppressing severe GVHD. Our long-term goal is to develop treatment strategies for preventing TR-GVHD induced by microbiota disruption. In Aim 1, we focus on the ileal epithelium. We propose to identify specific SBAs and SBA-mediated signaling pathways that promote epithelial regeneration after GVHD damage. In Aim 2, we will investigate the mucosal immune compartment of the ileum to uncover the mechanisms by which Lachno spp. or Lachno spp.- derived SBAs mitigate the activation of alloreactive immune cells. As a translational step, in Aim 3, we will evaluate if Lachno spp.-derived SBAs enhance the efficacy of GVHD prophylaxis with post-transplant cyclophosphamide and improve the effectiveness of corticosteroid therapy to treat GVHD. Across all Aims, our goal is to uncover novel microbiome-based strategies to prevent TR-GVHD driven by microbiome disruption.

NCI - National Cancer Institute

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

OD - NIH Office of the Director

PROJECT SUMMARY Rodent models have been foundational for biomedical discovery for decades. Yet the limitations of mice as a “universal” biomedical model are becoming increasingly apparent. Large animal models like minipigs offer a transformative opportunity to bridge this gap. Among the available minipig breeds, the naturally obesogenic Ossabaw minipig is increasingly becoming a model of choice for modeling the metabolic and chronic disease burdens that account for over 90% of U.S. healthcare costs. However, the generation of genetically engineered minipigs (GEMPs) remains inefficient and poorly scalable, limiting their utility in disease modeling and translational research. This proposal aims to overcome these limitations by establishing a precision genetic engineering (GE) and chimera generation platform using porcine induced pluripotent stem cells (piPSCs). The piPSC confer many advantages for GE efforts including the lack of senescence and maintenance over multiple generations, ease for genetic modification, potential for serial and multiplex editing, high throughput mutational screens, and contribution to soma and germline in the chimeric offspring. These attributes are eminently desirable for this animal resource. Our central hypothesis is that chimera-competent piPSC are a superior platform for programmable, high-efficiency GE and for generating functional chimeras via embryo complementation. To test this hypothesis, we propose the following two Specific Aims: Aim 1 will develop and validate a high-efficiency, site-specific genome engineering platform in Ossabaw piPSC. We will develop a high-fidelity piPSC based serine recombinases (Bxb1, PA01, KP03) platform for robust and modular GE. By targeting a universal landing pad to the pROSA26 safe harbor locus via CRISPR/Cas9, we will enable site-specific, scalable, and reproducible transgene integration, including large constructs like BACs. As a proof-of-concept, we will generate two novel models: (i) Cre-inducible dual fluorescent-PET reporter pigs for in vivo tracking and imaging, and (ii) a humanized CETP-transgenic minipig that addresses a key interspecies gap in lipoprotein metabolism and cardiovascular disease research. Aim 2 will enable somatic and germline chimerism using piPSC in lineage-deficient embryos. By modulating pluripotency state, injection parameters, and using lineage-deficient host embryos (e.g., lacking IGFR1, HHEX, PAX4, or NANOS3), we will demonstrate donor cell integration, niche occupancy, and rescue of organogenesis or gametogenesis. Donor contribution will be validated using advanced tools such as spatial transcriptomics, immunohistochemistry, and ddPCR, and functionality validated by phenotyping the founder animals. In summary, the proposal will establish and validate a high-efficiency, precision pipeline for producing GEMPs and functional chimeras. The stem cell tools and resources developed in this proposal will firmly establish Ossabaw minipigs as one of the models of choice for preclinical research.

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

PROJECT SUMMARY/ABSTRACT Development of novel contraceptive strategies is central to the mission of the Contraceptive Research Branch of the NICHD. This goal is driven by a global need for effective contraceptive methods to address: 1) the glut of unintended pregnancies (~45% of US pregnancies in 2011); 2) the high rate of elective abortion (1.15M unintended pregnancies ended in abortion in 2011 in the US); and 3) the high risk of maternal mortality (~830 women/day worldwide die due to pregnancy or childbirth complications). In a search for novel male contraceptive drug targets, we identified RBM46, which is a germ cell-specific RNA binding protein expressed by germ cells on the basement membrane of seminiferous tubules (outside the blood-testis-barrier), and is essential for spermatogenesis. Indeed, Rbm46 knockout mice are sterile and have no other phenotype, raising the distinct possibility that targeting RBM46 could lead to safe and effective male contraception by blocking spermatogenesis at the differentiating spermatogonial stage. Thus, we propose to develop drugs that target degradation of RBM46 as a means of oral, non-hormonal male contraception, which will significantly advance additional safe and reversible options for male contraception towards the clinic. Specifically, we will combine: 1) exceptional expertise in drug screening and development at UTSA and UT Health San Antonio; 2) leading expertise in male reproduction, spermatogenesis, and infertility at UTSA and ECU; 3) close proximity to one of two NIH-designated Marmoset Breeding Colonies, maintained at the Southwest National Primate Research Center; 4) growing and ongoing experience collecting and assessing marmoset sperm; 5) published experience in the use of cutting- edge single-cell genomics to assess normality of spermatogenic cell types; and 6) documented expertise with spermatogonial stem cell (SSC) transplantation. In Aim 1, we will identify small molecules that bind RBM46 and could be used to develop PROTACs. In Aim 2, we will produce initial RBM46 PROTACs and validate that they degrade the protein in vitro. In Aim 3, we will use medicinal chemistry to optimize the drug-like characteristics of top validated RBM46 PROTACs. In Aims 4 and 5, we will determine whether optimized RBM46 PROTACs induce reversible contraception in vivo using mice and marmosets, respectively. Together, these Aims are designed to advance RBM46 degradation as a novel strategy to achieve reversible, non-hormonal male contraception and provide key results to justify further preclinical investigation and eventual commercialization.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

Abstract Atopic dermatitis (AD) is a chronic skin condition characterized by T cell-driven Type 2 inflammation. The MC903- induced dermatitis model has enabled dissection of pathways driving acute AD-like disease such as keratinocyte TSLP production and Th2 signaling; however, its ability to model persistent disease states remains unexplored. Using a repeated-challenge protocol with MC903, we found that adult mice undergoing a primary bout of AD-like inflammation develop persistent, tissue-specific inflammatory memories in skin that manifest as exaggerated pathologic responses during secondary MC903 challenge. This aligns with the emerging idea that AD chronicity stems from local memories of inflammation that persist in healed lesions and exacerbate future disease flares. However, AD in childhood follows a distinct course, with a unique immune composition and lower incidence of chronic disease. We thus tested our model in neonatal mice, and despite observing similar primary response kinetics to MC903-treated adults, we strikingly saw no signs of aggravated pathology during secondary MC903 challenge. This suggests that AD-like inflammatory memory fails in early life. Characterizing the cellular and molecular mediators of these divergent skin phenotypes will be the focus of our proposed work. Our data suggests that inflammatory memory in adults is driven by the emergence of Type 1 (T1) immune features in skin such as T1 tissue-resident memory T cells (Trms), mirroring recent findings in human AD. Transcriptional profiling suggests fibroblasts help to organize these networks by supporting T cell positioning and Trm development within inflamed adult skin. Given emerging data to suggest that neonatal T cells and fibroblasts exhibit distinct inflammatory behaviors from their adult counterparts, we hypothesize that inflammatory memory is impaired in neonatal skin due to age-related differences in T cell and fibroblast function. To test this, we will first use lineage tracing to define the fates and phenotypes of adult and neonatal T cells during AD-like inflammation in developing and adult skin (Aim 1). Subsequently, we will interrogate fibroblast-T cell interactions in the atopic skin of adult and neonatal mice using in vivo profiling via scRNA-seq and immunofluorescence staining as well as in vitro functional assays involving fibroblast-T cell co-cultures. Our work is conceptually innovative and clinically relevant, especially given the growing incidence of chronic AD in adults. Completion of the proposed work will clarify the basic mechanisms by which neonatal skin escapes inflammatory memory, potentially aiding in the identification of new therapeutic interventions for chronic AD.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Metabolic-associated steatotic liver disease (MASLD) is the most prevalent chronic liver disease affecting adults and up to 40% of children with obesity. A growing body of evidence supports the role of early life stressors in the etiology of MASLD. In humans and animal models, exposure to maternal Western diet (mWD) consumption and obesity during gestation and lactation increases offspring risk for steatosis and more severe metabolic- associated steatohepatitis (MASH). MASH involves the recruitment of bone marrow derived monocytes to the liver, which give rise to macrophages (Mφ) that are unable to resolve inflammation or repair damage, accelerating liver fibrosis. Previous studies show mWD skews bone marrow (BM) derived Mφ (BMDMs) toward a proinflammatory phenotype by remodeling fetal hematopoietic stem and progenitor cells (HSPCs). Importantly, HSPC programming persists long-term, as BMDMs from juvenile offspring exposed to mWD in early life are similarly primed for inflammation by an as-yet characterized mechanism that drives inflammation long term and may contribute to ongoing fibrosis in the juvenile liver. Mitochondrial dysfunction, oxidative stress, and inflammation are hallmarks of Mφ trained immunity but crosstalk between these processes due to mWD and their impact on MASLD are not well understood. Our preliminary data shows that mononuclear cells (MNCs) from BM of 3-week-old mice exposed to mWD have decreased oxidative capacity relative to MNCs from BM of control offspring from chow-fed dams. Therefore, we hypothesize that mWD alters mitochondrial function in HSPCs during critical windows of early development to promote BMDM activation and hepatic inflammation, thereby increasing susceptibility to MASH. The overall goal of this project is to understand the mechanisms for how mitochondria are maladaptive to maternal WD during gestation and/or lactation and how specific pathways contribute to the pathogenesis of inflammation through HSPC remodeling of BMDMs. This fellowship has two aims: 1) determine the impact of mWD exposure during pregnancy and lactation on HSPC and BMDM mitochondrial metabolism in mice at weaning and 2) determine the long-term effect of mWD during gestation or lactation on hepatic Mφ populations, Mφ function, and liver fibrosis in adult offspring following WD challenge. Our approach utilizes metabolomics and proteomics, fluorometry, and respirometry to assess mitochondrial physiology and single cell RNA-sequencing to identify unique populations of hepatic Mφ, capacity for liver repair, and fibrosis in livers of adult mice from the same early life mWD exposures. Completion of this work will provide me with training in mouse research, immune cell metabolism, deeper analysis of mitochondrial physiology, bioinformatics, and use of ‘Omics technologies to use in my next steps in career development. Impact: the work from this fellowship will fill a gap in our understanding of maternal diet’s impact on the pathogenesis of pediatric MASLD.

NCCIH - National Center for Complementary and Integrative Health

PROJECT SUMMARY: Human clinical trials worldwide evaluated probiotic effects in a variety of diseases, resulting in confounding results about the therapeutic efficacy. The uncertainty of probiotic benefits stem from the vastly different patterns in diets, genetics, microbiota, and lifestyle in the human population. In addition, side effects associated with live bacteria have been reported, raising concerns about the safety of probiotics. Thus, the rationale for the mechanistic research into probiotic interactions with host diet, digestive, metabolism, and the microbial system is to identify the group of patients who may receive the most benefit from the probiotic intervention. This MPI research team, consisting of a nutrient physiologist, metabolomic biochemist and a gut biologist, has taken a chemical biology and reductionist approach, and has uncovered two probiotic metabolic pathways that regulate the host gut mucosal homeostasis and tissue repair after injury. In the first pathway, the probiotic bacterium directly produces two structural isomers of nicotinamide metabolites, one of which can be uptake by host cells and used for synthesis of nicotinamide adenine dinucleotide (NAD), a critical cellular coenzyme involved in energy production, DNA repair, and gene expression. In the second pathway, the probiotic species facilitates the degradation and removal of a serum arginine metabolite that is barrier-impairing. These pathways are interlinked as both are driven by the probiotic colonization in mice and by a tryptophan-sufficient diet and both impact the gut barrier integrity. Functional evaluations across mice, human cells and fruit fly demonstrate a conserved protection of gut barrier integrity through these mechanisms. Two aims will use stable isotope tracing to define probiotic’s metabolic impact on NAD turnover and a key NAD-dependent enzyme that has been revealed as a promising target mediating the protection; will employ fly and mouse genetics to dissect regulatory nodes and the cell types mediating the probiotic’s protection; and will utilize mouse disease models, human cell and organoid cultures to elucidate molecular basis on the regulation of intestinal stem cell renewal and epithelial healing. This research is significant as the metabolites under investigation are perturbed in inflammatory bowel disease patients, whose serum or fecal profiles of these molecules can be utilized to guide probiotic intervention. The three PIs have over a decade collaborative record with complementary expertise indispensable for this multidisciplinary project that intersects nutrition, metabolite, microbiota and gut mucosal biology.

NIA - National Institute on Aging

PROJECT SUMMARY Dementias are projected to become the most burdening group of diseases, expected to affect over 150 million people worldwide by 2050, and Alzheimer’s disease (AD) is the most common among them. Currently, over 6 million Americans are living with AD. While there have been several recently-approved drugs for AD, these drugs target amyloid beta plaques – just one facet of the disease – and have not proven effective in all patients. Non-neuronal cell types in the brain such as microglia (the brain’s immune cells) and astrocytes (star- shaped helper cells) play a critical role in disease progression but have been traditionally understudied. Microglia are known to be activated by amyloid beta plaques and they were ascribed both a protective and a detrimental role. They were shown to induce a toxic state in astrocytes. The microglia’s behavior is likely influenced by patient genetics, which might explain why the majority of AD risk genes are expressed in microglia. To determine which of the 90 known AD-associated genetic variants exert their effect through microglia, we need a better understanding of the functional links between a variant and the disease. This requires large-scale studies of diseased, human cells from genetically diverse patients, as there is a wide variety of genetic variants that can influence AD risk. This project proposes to develop an automatable protocol for the creation of human induced pluripotent stem cell (iPSC)-derived microglia (iMG) and their co-culture with primary astrocytes. Standardized co-cultures of iMGs from AD patients and primary astrocytes will allow scientists to observe how these cells interact in a diseased environment, better understand known variants, and possibly identify new risk variants. Attaining sufficient statistical power for the identification of novel variants requires many cell lines, which can only be achieved with robotic automation. This study will serve as a proof of concept to demonstrate that such co-culture systems be automated and will later be scaled up to include additional cell lines. It will furthermore help to understand how certain genetic variants contribute to AD, which might inspire new therapeutic approaches.

U.S. National Science Foundation

The Discovery Research K-12 (DR K-12) program seeks to enable significant advances in preK-12 student and teacher learning of the STEM disciplines through development, study, and implementation of resources, models, and technologies for use by students, teachers, and policymakers. Projects funded under this solicitation begin with a research question or a hypothesis about how to improve preK-12 STEM learning and teaching. Projects create or adapt and study innovative resources, models, or technologies and determine how and why implementation affects STEM learning.DR K-12 invites proposals that meet a variety of educational needs, from those that address immediate and pressing challenges facing preK-12 STEM education to those that anticipate opportunities for the future. DR K-12 especially encourages proposals that challenge existing assumptions about learning and teaching within or across STEM fields, envision needs of learners in 10-15 years, and consider new and innovative ways to educate students and teachers. Project goals, designs, and working strategies should be informed by prior research and practical experience drawn from all relevant disciplines, while focusing on concepts and skills that are central to STEM education. The DR K-12 program is primarily concerned with improving education of students and teachers in formal settings. As appropriate, the program encourages projects also to draw from knowledge and practice of learning in informal settings. While many projects supported under this solicitation will focus on exploratory development and testing of innovative ideas for some specific facet of STEM education, all proposals must explain how the work can lead ultimately to successful adoption of findings or products in the K-12 enterprise on a national scale.The DR K-12 program accepts proposals for exploratory projects, full research and development projects, and synthesis projects, as well as for conferences and workshops related to the mission of the program.

U.S. National Science Foundation

The Discovery Research PreK-12 program (DRK-12) seeks to significantly enhance the learning and teaching of science, technology, engineering, mathematics and computer science (STEM) by preK-12 students and teachers, through research and development of STEM education innovations and approaches. Projects in the DRK-12 program build on fundamental research in STEM education and prior research and development efforts that provide theoretical and empirical justification for proposed projects. Projects should result in research-informed and field-tested outcomes and products that inform teaching and learning. Teachers and students who participate in DRK-12 studies are expected to enhance their understanding and use of STEM content, practices and skills. The DRK-12 program invites proposals that address immediate challenges that are facing preK-12 STEM education as well as those that anticipate radically different structures and functions of preK-12 teaching and learning. The DRK-12 program has three major research and development strands: (1) Assessment; (2) Learning; and (3) Teaching. The program recognizes the synergy among the three strands and that there is some overlap and interdependence among them. However, proposals should identify a clear focus of the proposed research efforts (i.e., assessment, learning, or teaching) consistent with the proposal s main objectives and research questions. The program supportssix types of projects: (1) Exploratory, (2) Design and Development, (3) Impact, (4) Implementation and Improvement, (5) Syntheses, and (6) Conferences. Allsix types of projects apply to each of the three DRK-12 program strands.

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

Project Summary Faithful embryogenesis requires the precise coordination between embryonic and extraembryonic tissues. While recent advances in pluripotent stem cell (PSC)-derived partial or non-integrated embryo models have opened new avenues to dissect early human development, a significant limitation is that they lack extraembryonic lineages. Recently, a hPSCs-based blastocyst model, human blastoids that contains both embryonic and extraembryonic cells, has opened new avenues to dissect human blastocyst development. However, due to ethical and policy restrictions, projects using human blastoids are currently not funded by NIH. Consequently, there is a fundamental gap in knowledge in the molecular and cellular mechanisms of human embryogenesis, and how defects in this stage of human development contribute to pregnancy loss. To address these deficiencies, most recently we have established bovine embryo-derived stem cell lines including naïve-like embryonic stem cells (ESCs), trophoblast stem cells (TSCs), and extraembryonic endoderm stem cells (XENs), and developed an integrated model bovine blastoid by assembly of embryonic stem cells and extraembryonic stem cells, which is from a large animal species highly relevant to humans. Bovine blastoids resembled bovine blastocysts in terms of morphology, size, cell number, marker gene expression, and lineage composition and allocation, and could be robust produced with a high efficiency. Bovine blastoids represent a well-controlled cellular substrate to test hypotheses in a human-like embryo model system. The primary objective of this proposal is to use bovine blastoids as a proxy for the early steps of human development, to test key questions relevant to lineage crosstalk in vitro. We anticipate that this work will lead to an improved fundamental understanding of the key signaling events, transcriptional regulators and cell-cell interactions during human pre-implantation blastocyst development, which will help us understand the molecular and cellular contributors of early developmental anomaly in humans.

NHLBI - National Heart Lung and Blood Institute

Progressive telomere shortening, which occurs over humans' natural lifespan, is a primary molecular cause of the functional decline of stem cells in high-turnover tissues, including hematopoietic stem cells (HSCs). However, how telomere damage compromises HSCs’ functions is largely unknown. Here, we propose investigating the molecular mechanisms behind telomere damage–induced functional HSCs’ decline to uncover therapeutic strategies to ameliorate bone marrow (BM) failure disorders. In preliminary studies, we demonstrated that telomere damage does not activate programs of apoptosis or senescence in HSCs but instead induces their aberrant activation and differentiation towards the megakaryocytic lineage through the cell-intrinsic upregulation of Ifi20x/IFI16-mediated innate immune signaling response, which directly compromises HSCs’ self-renewal capabilities and eventually leads to their exhaustion. Given that HSCs’ exhaustion in the context of BM failure disorders predisposes to clonal selection, we evaluated whether telomere shortening–induced DNA damage in patients with germline mutations affecting telomere maintenance genes who developed telomere biology disorders (TBDs) is associated with clonal hematopoiesis (CH). We studied the architecture, trajectories, and impact of CH in a cohort of 207 TBD patients. CH was rare in asymptomatic patients but present in 46% of symptomatic patients and involved chromosome 1q aberrations (mainly chromosome 1q gain [Chr1q+]) and recurrent mutations in PPM1D, POT1, TERT promoter, and U2AF1S34. Compared with age-matched healthy controls, patients with TBDs had a significantly higher CH frequency, which increased with age. Regardless of allele burden, Chr1q+ and mutations in U2AF1S34 or TP53 increased the risk of developing myelodysplastic syndromes and acute myeloid leukemia. Further functional studies demonstrated that the U2AF1S34 mutation compensated for the aberrant activation of the TP53 and interferon pathways, which contribute to HSC exhaustion in patients with TBDs. These results suggest that the acquisition of U2AF1S34 mutations in TBDs compensates for the restricted cell fitness caused by germline mutations in telomere maintenance genes, which underscores the importance of understanding the molecular mechanisms of U2AF1S34 mutation–induced tumorigenesis. In this proposal, we will use innovative technologies, such as organoid and induced pluripotent stem cell systems and the MISTRG mouse model to 1) Dissect the IFI16-mediated signaling pathway under telomere attrition and determine the feasibility of targeting IFI16 in humans to rescue telomere-dysfunctional HSC function and 2) Dissect the mechanisms of U2AF1S34 mutation–induced tumorigenesis under telomere stress. The proposed study will expand our understanding of the contribution of telomere damage to HSCs’ functional decline and CH and provide new opportunities for developing strategies to improve the prevention and treatment of hematological disorders associated with telomere dysfunction.

NIDA - National Institute on Drug Abuse

PROJECT SUMMARY Opioid use disorder (OUD) is a devastating neuropsychiatric condition and a major public health crisis in the United States, with fentanyl, a synthetic opioid 100 times more potent than morphine, driving a sharp rise in overdose deaths. Although many individuals are exposed to opioids, only a subset develop OUD. This is especially evident in family studies, which estimate OUD heritability at 40–60%, suggesting that genetics shape individual risk. Genome-wide association studies (GWAS) have identified common variants associated with OUD. Over 90% of these GWAS variants are located in non-coding regions of the genome—regions that are often regulatory and whose activity varies by context (e.g., cell type, tissue, exposure). Postmortem studies have been invaluable in detecting dysregulated genes in the brains of OUD donors. However, these studies cannot distinguish inherited regulatory effects from changes caused by opioid exposure or the disease itself. For this reason, the genes and specific regulatory non-coding variants that contribute to OUD risk remain unknown. This project addresses this gap by identifying the genes that causally mediate OUD risk and by experimentally testing the effects of associated regulatory variants in human neurons exposed to fentanyl or vehicle. In Aim 1, I will apply a multi-tissue integrative Mendelian randomization framework (mintMR) to determine how inherited variation influences gene regulation (expression and DNA methylation) across human brain regions, allowing me to identify causal genes and the tissues in which they act. In Aim 2, I will use Massively Parallel Reporter Assays (MPRA) to functionally test thousands of fine-mapped non-coding variants associated with OUD, integrating these results with enhancer–gene interaction models to identify the putative target genes of regulatory variants. Together, these studies will identify the genes and brain regions that causally contribute to OUD risk and determine which non-coding OUD-associated variants affect gene regulation in unexposed and opioid- exposed human neurons. Through this project, I will receive rigorous training in computational biology, functional genomics, stem cell neuroscience, and addiction biology, preparing me for a career leading research on the molecular mechanisms of OUD.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT Induced pluripotent stem cells (iPSCs) offer a promising platform for regenerative medicine, with their ability to self-renew indefinitely and differentiate into vascular cells. However, their clinical translation in vascular diseases is limited by major challenges: the impracticality of autologous therapy, immune rejection in allogeneic transplantation, and inefficient differentiation. Allogeneic iPSC-derived cells are rapidly recognized and eliminated by the host immune system, triggering both innate and adaptive immune responses. Additionally, current differentiation protocols yield vascular cells with low and inconsistent efficiency (~20%), limiting scalability and therapeutic viability. To overcome these challenges, this study aims to engineer hypoimmunogenic iPSC-derived vascular cells that evade immune detection while efficiently integrating into host vasculature. Simultaneously, spatial transcriptomics will be utilized to map immune- vascular interactions and uncover pathways that drive immune tolerance and vascular remodeling. To overcome differentiation inefficiencies, we have developed a transcription factor-driven strategy using ETV2 and NKX3.1, enabling >95% efficiency in generating endothelial and mural progenitor cells, respectively. Their ability to integrate and enhance perfusion will be tested in ischemic hindlimb models. The project comprises three aims. First, we will engineer hypoimmunogenic vascular cells by knocking out B2M/CIITA to eliminate highly polymorphic MHC expression, preventing adaptive immune recognition and overexpressing CD47 to prevent innate immune clearance. Second, we will assess vascular integration and perfusion enhancement in ischemic models by transplanting the engineered vascular cells. Third, we will apply multiplexed spatial transcriptomics to map immune-vascular interactions, identifying pathways that regulate immune evasion and vascular remodeling. This study will provide critical insights into immune-vascular dynamics, establishing a foundation for hypoimmunogenic iPSC-based therapies that achieve long-term immune tolerance and functional vascular regeneration in ischemic diseases. The training will occur under the mentorship of Dr. Juan Melero-Martin at Boston Children's Hospital/Harvard Medical School. I will be co-mentored by an extraordinary team of scientists on my advisory committee, including Dr. Torsten Meissner and Dr. Kaifu Chen, for new training goals in immune biology, vascular biology, transcriptomics as well as career guidance. Through this training, I will acquire advanced conceptual, technical, and professional skills, preparing me for an independent research career in translational regenerative medicine.

NHLBI - National Heart Lung and Blood Institute

Abstract The human sinoatrial node (SAN) is the heart’s natural pacemaker, initiating electrical impulses that maintain rhythmic contraction and coordinate blood flow. While its role is essential, our understanding of SAN development—particularly in humans—remains limited. Sinus node dysfunction (SND), often caused by SAN cell loss or fibrosis, is a major cause of arrhythmia and the primary reason for up to 50% of artificial pacemaker implants in the US. There are no approved cellular or pharmacological treatments. Compared to chamber muscle formation, the mechanisms underlying SAN lineage specification and subpopulation diversity are poorly defined, representing a critical gap in knowledge. In preliminary studies, we used single-nucleus multiomics and spatial transcriptomics to analyze the human fetal SAN and identified candidate regulatory networks. We also developed a dual knock-in human pluripotent stem cell (hPSC) reporter line (SHOX2:eGFP; MYH6:mCherry) and established protocols to generate functional pacemaker cells, SAN organoids (Sinoids) and SAN-paced cardioids (SAN-PCOs). These tools enable real-time visualization, isolation, and functional analysis of human pacemaker cells and tissues. Three Aims are proposed to 1) identify key transcriptional regulators of SAN differentiation using inducible CRISPR interference (CRISPRi) and activation (CRISPRa) in hPSCs. Validated genes will be tested in SAN-PCO models using molecular and electrophysiological assays to assess their role in pacemaker function; 2) dissect the molecular programs guiding SAN subpopulation specification, including head, tail, and transitional zone domains. Using CRISPR-based perturbation and single-cell RNA sequencing, we will evaluate how candidate regulators shape subpopulation identity and influence SAN-cardiomyocyte interactions; and 3) assess how genetic variants associated with SND affect SAN development and function. We will generate isogenic SNP knock-in and gene knockout hPSC lines based on top candidate GWAS hits and evaluate the variant–gene–cell axis using multiomics and functional assays. By integrating single-cell genomics, gene editing, and human stem cell-derived SAN models, this study will define the molecular architecture of human pacemaker development and identify genetic mechanisms underlying SND. These findings will advance our understanding of cardiac conduction system biology and support future therapeutic strategies.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY / ABSTRACT Orofacial neuropathic pain, often stemming from somatosensory nervous system dysregulation, frequently manifests in conditions such as chemotherapy-induced neuropathy and trigeminal neuralgia. This pain commonly results in altered somatosensory experiences, including orofacial cold allodynia (pain from normally innocuous cool stimuli) and cold hyperalgesia (exaggerated pain in response to noxious cold). Current treatments often fail to alleviate these cold-sensation symptoms because the underlying cellular and molecular mechanisms for low temperature sensation are not well defined. Recent single-cell sequencing has revealed new layers of molecularly distinct somatosensory neuron populations, yet how the coding logic underlying low temperature sensation and cold-nociception is distributed across these neurons remains severely understudied. This proposal aims to delineate the neural and molecular logic underlying low temperature sensation, revealing fundamental principles essential for informing development of new strategies for cold allodynia and neuropathic pain. To accomplish this, we will apply an intersectional genetic strategy in mice, combining state-of-the-art thermoelectric cooling (TEC) probes, in vivo trigeminal (TG) functional imaging, multiplexed in situ hybridization, neural tracing, and advanced AI-based behavioral analyses. Preliminary data reveal Chrna7+ TG Aδ-nociceptors encode noxious cold sensation in teeth independent of the canonical cool receptor Trpm8, providing direct evidence that low temperature encoding is distributed across several TG classes. This proposal will 1) define molecular and neural mechanisms underlying orofacial low temperature sensation and 2) characterize behavioral outputs and central projections of low temperature encoding TG populations using optogenetics and AI-based machine learning, and mapping associated afferent brainstem nuclei.This research will launch the candidate’s independent research program aimed at elucidating low temperature encoding mechanisms throughout the periphery. This will set the stage for future directions examining higher-order circuit mechanisms of cold sensation versus pain, and how sensation is transformed in disease/pain models. This work will bring new perspectives and hypotheses towards the development of pain treatment strategies, including those for neuropathic cold allodynia and hyperalgesia. During the mentored stage, the candidate will gain valuable training in: 1) intersectional genetic techniques for neuroscience studies, 2) advanced computational analysis for calcium imaging and behavior, and 3) guidance from the formal advisory committee in lab leadership, grant writing, networking, and presentation skills. This award will set the candidate up for success in transitioning to independence and making significant discoveries towards understanding and treating pain.

NCI - National Cancer Institute

The purpose of this proposal Is to elucidate the rate-limiting non-mutational events underlying benign to malignant transition of colorectal cancer (CRC). Colon polyps are extremely common, occurring in up to 40% of adults Over 50, and are the precursors to CRC. While the causative mutations and their stepwise acquisition In this polyp-to-c:arcinoma sequence are well understood, the non-mutational mechanisms are not. In this proposal, we will use a new mouse model that emulates, for the first lime, the stepwise acquisition of mutations In the distal colon to accurately model benign-to-malignant transition In human CRC. Benign polyps are initialed via knockout of the moat commonly mutated gene in human colon polyps (Apc). The most common mutational events In advanced CRC (Kras G120, Trp53 loss) are then Induced In rare cells of established polyps wl1h spatial and temporal precision. In our preliminary studies, we have found 1hat this model accurately emulates the histopathological progression of benign-to-malignant transition of human CRC, but not in mice that are lacking T c:ells-these tumors do not progress beyond adenoma. Spatial transcriptomic (S1) analyses Of these tumors revealed that KrasG120 induces loss of signatures Of homeostatic regeneration and gain of a fetal intestinal- like stem cell state, which Is known to play a critical role In intestinal repair following injury and inflammation, KrasG120 regions within polyps were also less proliferative and depleted over time, consistent wi1h the slow cycling nature of injury-responsive stem cells in the c:olon. These findings establish that mutations, while necessary, are not sufficient to drive progression to CRC, at least when acquired In the stepwise sequence characteristic of human CRC. We hypothesize that malignant precursors in benign polyps despite having the necessary genetic mutations, require inflammatory signals to progress to cancer. These signals enable this transition by shifting the fitness landscape of the premalignant niche in favor of a fetal intestinal wound healing response over homeostatic regeneration. In Aim 1, we will leverage our innovative model to functionally interrogate the role of the fetal Intestinal state In benign-to-malignant transition by Inducing 1h18 slate via 1) wounding or non-specific T cell activation, 2) knocking out Its key transcriptional coordinator ( Yap), and 3) ablating cells in this Slate. we will use ST data from the model to define the transcriptional regulatory networks underlying this state In progressing versus non-progressing lesions. In Aim 2, we will identify rate-limiting microenvironmental factors that preferentially select for malignant precursor cells, and define the tranSC11pllonal mechanism by which these factors cooperates with, oncogenic mutations. Results will be validated against ST data we have generated on human polyps with early malignancy. Impact: completion Of !hie proposal will provide a mechanistic foundation for understanding why some benign polyps progress to cancer while most do not. This knowledge may nominate new and mare specific biomarkers for early cancer detection, as well as rate-limiting events that could be targeted fur cancer prevention.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY / ABSTRACT Hematopoietic aging is characterized by chronic inflammation, hematopoietic stem cell (HSC) dysfunction, and hematopoietic bias toward myeloid lineages, leading to less efficient immune responses and increasing incidence of hematologic neoplasia. Emerging evidence from our group and others shows that NFκB-mediated chronic inflammatory responses affect HSCs and their cellular niches in different ways. Elevated NFκB activity within HSCs leads to loss of replicative capacity, while in contrast, elevated NFκB within the bone marrow niche drives epigenomic reprogramming of HSCs and myeloid bias of their progeny. These observations lead to the central hypothesis of this proposal, that unique NFκB responses in HSCs and their niches together form a biologic buffering mechanism balancing inflammatory hematopoietic responses with HSC preservation to maintain lifelong blood production. Importantly, chronic and dysregulated inflammation is implicated in a wide range of blood disorders, yet the mechanisms governing HSC and niche cell responses remain unclear. Thus, the proposed studies are expected to open avenues of inquiry with broad relevance to hematopoietic aging, clonal hematopoiesis, myeloid neoplasia, aplastic anemia, iatrogenic myelosuppression, and more. This mentored career development award will support the advanced research training of Dr. Jennifer Chia, a hematopathologist at UCLA, to become an independent academic physician-scientist in the field of hematopoiesis. Dr. Chia received her MD and PhD degrees from the Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD program, and completed her Anatomic Pathology residency and Hematopathology fellowship at UCLA. She is now pursuing post-doctoral research training in the laboratory of Dr. Alexander Hoffmann, an internationally recognized expert in NFκB and the signaling systems that control immune cell function and fate decisions. The specific training objectives detailed in this proposal are to develop expertise in 1) inflammatory signaling and animal models of immune dysregulation; 2) HSC biology; 3) bioinformatics; and 4) the bone marrow niche. Training will be provided through mentorship by senior faculty members who are leaders in their fields, coursework, seminars, and conferences, all in the context of UCLA’s extensive resources and strong institutional commitment to the development of physician-scientists.

NIA - National Institute on Aging

Project Summary Alzheimer’s disease (AD) is a progressive neurodegenerative disease affecting more than 50 million people worldwide, but the mechanisms involved in disease development remain poorly understood. Genome-wide association studies have uncovered numerous loci associated with disease, but identifying causal variants and genes remains a challenge. Studies have revealed that AD risk variants are enriched in active enhancers of human monocytes, macrophages, and microglia, suggesting many of these variants act by disrupting gene expression specifically in myeloid cells. In addition, many genes implicated in AD risk are highly expressed in myeloid cells, strongly implicating these cell types in the etiology of AD. By integrating human genetics with epigenomic and transcriptomic datasets from human myeloid cells, we identified a myeloid cell enhancer containing an AD-associated functional variant on chromosome 11, and two putative target genes of this enhancer, embryonic ectoderm development (EED) and phosphatidylinositol binding clathrin assembly protein (PICALM). EED is an essential subunit of the polycomb repressive complex 2 (PRC2) known to function in regulation of gene expression and clearance behavior in mouse microglia, but it remains relatively unstudied in the context of AD in human cells. PICALM is an adaptor protein known to function in endolysosomal pathway and autophagy, but its role in microglia remains unknown. The overall goal of this proposal is to understand how our nominated AD risk enhancer influences the expression of its two target genes PICALM and EED, and to test the hypothesis that these two potential causal genes regulate microglia functions downstream of TREM2. In Aim 1, I will determine the role of the candidate AD risk enhancer in regulating the expression of PICALM and EED and human microglial cell function by combining CRISPR gene editing in human induced pluripotent stem cells (iPSCs), microglial differentiation protocols, and xenotransplantation methods involving direct injection of microglia precursor cells into the mouse brain. I will delete the candidate enhancer region and perform transcriptomic and epigenetic profile of edited microglia via RNAseq and ATACseq in addition to microglia functional assays. To assess the in vivo consequences of deleting the risk enhancer in microglia, I will transplant the edited microglia into the brains of wildtype and 5xFAD humanized mice and characterize transcriptomic profile of these microglia via snRNAseq analysis and perform immunohistochemistry. In Aim 2, I will investigate how PICALM and EED regulate human microglia function, namely TREM2 mediated efferocytosis, or phagocytic pathway. I will determine the subcellular localization of PICALM and EED using biochemical and imaging techniques and test the role of these two genes in microglial functions downstream of TREM2 signaling such as actin rearrangement, phagosome formation, and downstream kinase signaling. My proposed aims will help understand the mechanisms in which the causal variants and target genes drive the AD risk, and enhance our understanding of PICALM and EED in microglia and in AD.

NIAID - National Institute of Allergy and Infectious Diseases

SARS-CoV-2 protein nonstructural protein 1 (Nsp1) induces a global translation shutdown in host cells upon infection. Irrespective of mechanism, the Nsp1 imparted translation shutdown is required for efficient viral replication and to suppress the host immune response. Thus, fully understanding this protein involves understanding how it interacts with viral components and host machinery. The viral genome can escape the translational shutdown via secondary structure in its 5’ untranslated region (UTR). The first hairpin structure, stem-loop 1 (SL1), has been identified as necessary and sufficient to evade Nsp1-mediated translation shutdown. Previous reports show that host genes are suppressed differently by Nsp1. For example, translation- related genes, especially those with terminal oligopyrimidine (TOP) motifs, are translated more efficiently in the presence of Nsp1, while immune response genes are suppressed. Despite proven significant impacts of the 5’ UTR of SARS-CoV-2 and host genes, other elements remain understudied in this interaction with Nsp1. Therefore, this work examines if the 5’ UTR has other functional regions that might influence translational control and evasion of the translational shutdown. This research utilizes a recently developed method called direct analysis of ribosome targeting (DART), a high throughput method that tests the ribosome recruitment ability of thousands of 5’ UTRs. To analyze RNA features of SARS-CoV-2 and host genes that impact ribosome recruitment, a diverse pool of viral and host sequences was generated to allow thorough examination of each region of the 5’ UTR and its role in translation and evasion of host shutdown. The pool includes all known natural mutations reported in the NCBI virus sequence repository, along with systematic scanning, structural disrupting and compensatory mutations. Completing DART with and without Nsp1 will elucidate what elements facilitate translation and the evasion of Nsp1-mediated translational shutdown. The translation shutdown mechanism is thought to function through a two-pronged approach where the C-terminal domain binds the ribosome at the mRNA entry channel and sterically blocks RNAs from loading onto the ribosome, and the N-terminal domain (NTD) cleaves RNAs while bound to the ribosome, both activities preventing RNAs from being translated. However, it is unclear whether channel exclusion and cleavage are linked activities of Nsp1, or if different RNA features can mediate mRNA channel entry or escape of RNA cleavage. To address this, a high throughput cleavage experiment will be completed on pools of diverse RNAs to examine what host or viral features mediate resistance or susceptibility to cleavage, in ribosome-containing or depleted lysate. These cleavage experiments will be completed using the previously described RNA pool containing SARS-CoV-2 and immune related genes, along with another RNA pool of 24,000 sequences comprised of human genes, including translation-related sequences. This work will be instrumental for understanding the role of Nsp1 in coronavirus pathogenesis and to inform the design of future therapeutics.

NCI - National Cancer Institute

Project Summary/Abstract Acute myeloid leukemia (AML) is a lethal hematologic malignancy caused by mutations in hematopoietic stem cells. Normal karyotype AML (NK-AML) accounts for ~50% of cases and presents unique challenges due to its clinical and genetic heterogeneity. Prior research has shown that NK-AML develops from pre-existing clonal bone marrow diseases, including clonal hematopoiesis (CH) and myelodysplastic syndromes (MDS). In some CH and MDS patients, transformation to AML occurs in the absence of new mutations, indicating that epigenetic factors play a role in AML development. Recent studies by our lab using long-read DNA sequencing investigated DNA methylation as a potential source of novel contributing factors to AML development. These studies discovered thousands of discrete blocks with Allele-Specific DNA methylation (ASM) in primary AML samples that are sequence-independent and distinct from imprinted genes. These allele-specific “epimutations” are remarkably stable and are conserved in patient-matched samples obtained at presentation and relapse. Epimutations were also identified in remission samples from patients with genetic evidence of clonal disease and in single-cell clones expanded from normal hematopoietic stem cells. Furthermore, recurrent epimutations are associated with differential chromatin accessibility at genes that regulate stem cell differentiation and self-renewal, including GATA2 and genes in the HOXB cluster. Based on this evidence, we hypothesize that these allele-specific DNA methylation epimutations are clonal events that arise in normal cells and can disrupt gene regulation and be selected for during leukemogenesis. In this proposal we will investigate the role of DNA methylation epimutations in NK-AML using primary human AML samples, normal hematopoietic stem cells, and model systems via 3 aims. Aim 1 will define the landscape of recurrent epimutations in NK-AML using long-read native DNA sequencing. We will assess epimutation frequency and stability and explore their functional impact via RNA-seq, histone modification profiling, and Fiber-seq chromatin accessibility profiling. Aim 2 will use high resolution chromatin structure analysis and targeted epigenome editing to investigate the mechanisms of allele-specific gene regulation at the GATA2 locus. This aim will then test the functional consequences of allele-specific GATA2 activity by generating human AML cells with allele-specific silencing of GATA2 and engrafting them in humanized mice. These critical experiments will directly test whether epigenetic allele-specific expression of GATA2 confers a selective advantage for human AML cells in vivo. Finally, we will develop a novel targeted sequencing assay capable of detecting both mutations and epimutations and determine whether this approach improves the identification of persistent AML-related clones predictive of relapse in NK-AML patients in remission after chemotherapy. Together these studies will define the functional role of DNA methylation epimutations in AML and provide a novel molecular tool for monitoring AML patients and predicting relapse risk.

NIDA - National Institute on Drug Abuse

Neurologic complications remain prevalent in nearly 50% of people with HIV (PWH) and persist despite viral suppression with antiretroviral therapy (ART). Though the exact processes mediating HIV neuropathogenesis are not well understood, co-morbidities such as substance use disorders (SUD), which are higher in PWH compared to the general population, exacerbate neuropathogenesis of HIV and worsen outcomes. Multiple substances of misuse are reported to increase HIV replication, induce inflammatory signaling, and amplify neurodegenerative phenotypes. Thus, there is a significant need to understand the intersection between SUD and NeuroHIV to improve longitudinal care and inform the public. The overlapping effects of distinct substances of misuse on HIV pathogenesis in the CNS suggest that a common pathway may be involved through presently undefined mechanisms. All addictive substances increase extracellular dopamine in the central nervous system (CNS), which signals neurons and other nearby glial cells expressing dopamine receptors. Our lab has shown that myeloid cells such as macrophages and microglia, which are major HIV reservoirs in the brain, express dopamine receptors more D1-like receptors (D1 and D5) than D2-like receptors (D2, D3, D4). Treatment of macrophages and microglia with micromolar concentrations of dopamine increased pro-inflammatory signaling, increased viral entry, and potentiated viral secretion in vitro. We recently found that a higher D1-like to D2-like ratio is associated with a more pro-inflammatory response in microglia. Further, we showed that dopamine increases activation of nuclear factor-kappa B (NF-κB) in macrophages, and that inhibition of NF-κB can block the pro-inflammatory effects of dopamine. Together, these data suggest that dopamine-enriched brain regions, such as the cortex and striatum, may be especially vulnerable to HIV and neuroinflammation in PWH and co-morbid addiction through the action of dopamine on microglia. Therefore, the central hypothesis of this proposal is that dopamine D1-like receptor activation promotes HIV infection and NF-κB-mediated inflammation in microglia to worsen neurodegeneration. This hypothesis will be tested using human induced pluripotent stem cell (iPSC)-derived brain human cortical assembloids and several orthogonal assays to explore the dopamine-mediated pathways that modulate HIV neuroimmune pathogenesis. We will use pharmacologic activation of dopamine receptors in cortical assembloids to assess viral kinetics (Aim 1), neuroinflammation (Aim 2), and neuronal degeneration of synapses and dendrites (Aim 3). Together, these studies will significantly advance our understanding of dopamine as an immunomodulatory signaling molecule in the context of substance use and HIV, as well as expand the approaches to studying neuroimmune pharmacology using human micro-physiological systems.

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY/ABSTRACT Neurodevelopmental disorders (NDDs) affect approximately 1 in 12 children in the United States and many are caused by genetic variants in chromatin regulators. Recent work identified a new NDD caused by missense variants in the histone methyltransferase DOT1L, the sole enzyme that catalyzes histone 3 lysine 79 methylation (H3K79me). Nearly all DOT1L NDD patient variants are located within the catalytic domain of DOT1L and those tested disrupt its methyltransferase activity, implicating disrupted H3K79me as the driver of the disorder, as opposed to DOT1L’s non-catalytic functions. However, while H3K79me is strongly associated with active transcription, its downstream effects on chromatin remain unclear, especially in neurons, as no specific readers or effectors of the mark have been identified. Preliminary data suggest that H3K79me may regulate chromatin by modulating the histone variant H3.3, a critical and well-established regulator of neuronal chromatin and neurodevelopment whose disruption causes a similar NDD. H3.3 is the dominant H3 variant in neurons and is dynamically deposited and evicted at active chromatin regions to support transcriptional plasticity required for neuronal differentiation and function. Loss of H3K79me leads to global depletion of H3.3, raising the possibility that H3K79me contributes to DOT1L-associated NDDs by disrupting H3.3 chromatin dynamics. The goal of the proposed research is to define the mechanism by which H3K79me regulates H3.3 (Aim 1) and determine how H3K79me loss impacts neuronal transcription, maturation, and function (Aim 2). To isolate the specific role of H3K79me, the project will use multiple isogenic mouse embryonic stem cell lines that reduce H3K79me through distinct mechanisms and differentiate them into neural progenitor cells and post-mitotic neurons. These lines include 1) a DOT1L catalytic inactivation mutation, 2) an H3.3K79A mutation that blocks methylation of the dominant neuronal H3 variant, and 3) a DOT1L D157N variant identified in NDD patients that causes partial H3K79me loss. Aim 1 will test the hypothesis that H3K79me promotes H3.3 retention post- deposition to maintain its occupancy at active chromatin regions. Stable isotope labeling and mass spectrometry will be used to quantify the rate of H3.3 incorporation into and eviction from nucleosomes, and proteasome inhibition will test whether evicted H3.3 is degraded. CUT&RUN will identify genomic regions where H3K79me loss depletes H3.3 during neuronal differentiation. Aim 2 will test the hypothesis that H3K79me regulates synaptic gene expression and is required for the maturation and function of neurons. RNA-seq will define the genes regulated by H3K79me during neuronal differentiation, and neuronal morphology and activity analyses will define the role of H3K79me in neuronal maturation and function. This work will define the role of H3K79me in neurons and uncover the mechanism of its previously unrecognized regulation of H3.3. The findings will provide mechanistic insight into the molecular basis of DOT1L-associated NDDs and may identify chromatin pathways amenable to therapeutic interventions.

NCI - National Cancer Institute

SCIENTIFIC ABSTRACT Acute myeloid leukemia (AML) is an aggressive clonal hematologic malignancy characterized by the defects in differentiation of the myeloid lineage, heightened proliferation, and resilience to cell death. Despite recent FDA approvals of several targeted therapies including venetoclax-based regimens, most patients relapse due to the survival and expansion of residual leukemia blasts and leukemia stem cells (LSCs) evading therapy. Eradiation of these cells through novel therapeutic approaches is critical for curing AML. CD123 is a surface marker strongly expressed on AML blasts and LSCs but largely sparing normal cells including hematopoietic stem cells (HSCs). AFM28 is a novel bispecific Innate Cell Engager (ICE ®)) that in pre-clinical studies effectively depleted CD123+ leukemic cells and LSC through NK cell engagement and recently showed encouraging activity in Phase I monotherapy trial in relapsed/refractory AML. NK-cell-mediated cytotoxicity can be sensitized through BCL-2 inhibition with venetoclax (Ven). Our preliminary data demonstrate that co- targeting of CD123+ AML by NK cells and of BCL2 by Ven translates into apoptosis of both, phenotypically defined AML stem/progenitor cells and AML blasts. We hypothesize that co-targeting AML by CD123- directed NK cells and BCL-2 inhibition harnessing apoptotic machinery will elicit AML cell kill through synergistic mitochondrial apoptotic priming. We will test our hypothesis in Specific Aims: In Aim 1, we will investigate combinatorial efficacy and molecular mechanisms of co-targeting CD123+ AML by NK-cell engager and BCL2 inhibition by Ven. We will perform dynamic BH3 profiling to probe modulation of mitochondrial priming, focusing on mitochondrial membrane integrity, induction of pro-apoptotic proteins, reprogramming of mitochondrial metabolism and co-dependency on mitochondrial pathway in AML and NK cells. In Aim 2, we will first determine the safety of the combination utilizing humanized NSGS mice producing human IL15 that provides support for NK cells maintenance, engrafted with CD123+ AML cell line and treated with AMF28-NK_Ven/Aza. Next, we will study the combinatorial efficacy of AMF28-NK_Ven/Aza in vivo in patient-derived xenograft (PDX) models generated from Ven/Aza-sensitive or -resistant AML. Molecular signatures of each therapeutic arm and combinations will be determined by flow cytometry, immunochemistry, immunophenotypic profiling, methylation assays and scRNAseq. Results of this proposed work will lay the foundation and provide rationale for successfully translating the combination of potent BCL-2 inhibitor with a novel engager AFM28-directed NK cell therapy into curative targeted therapy approach for AML patients.