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NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT Squamous cell carcinoma (SCC) is one of the most common cancers worldwide, yet the early events that drive mutated epithelial stem cells toward malignancy remain poorly understood. While cellular plasticity has emerged as a key process enabling clinically normal mutation-harboring skin to acquire oncogenic potential and undergo malignant transformation, the non-genetic variables that regulate this process and ultimately drive tumorigenesis remain largely unknown. These gaps in knowledge can largely be attributed to a dearth of tools and models that capture pre-oncogenic stem cell identity landscape in intact tissues. In this project, I will investigate how the lymphatic vascular niche, increasingly recognized as a key regulator of epithelial stem cell, controls cancer stem cell fate transitions during early tumor initiation and malignant progression in skin SCC. Using deep imaging and sequencing approaches, we discovered that lymphatic vascular insufficiency predisposes stem cells to malignant transformation, while oncogenic plasticity propelling the transition from benign to metastatic carcinoma is preceded by dynamic lymphatic remodeling. The central hypothesis is that lymphatic niches evolve during tumor progression, initially supporting tolerance to oncogenic stress and later driving epigenetic rewiring that enables malignant transformation. Aim 1 will determine how lymphatic regression influences tolerance to oncogenic stress and promotes malignant transformation using in vivo lymphatic ablation models, chromatin accessibility profiling and organotypic culture systems. Aim 2 will define the molecular and spatial interactions between tumor-initiating cells and lymphatic niches during malignant progression. Leveraging enhancer-based proximity sensors, I will identify lymphatic-derived signals that promote stem cell plasticity potential. This work is expected to reveal context-dependent lymphatic cues that regulate early tumor initiation and malignant transition, while generating new tools to study vascular–stem cell interactions in vivo. These studies aim to uncover actionable pathways that can be targeted to intercept skin cancer before it becomes invasive, offering new opportunities for early intervention in a disease that affects millions. My training will take place in the Gur-Cohen Lab at UC San Diego and the Sanford Stem Cell Institute, a highly interdisciplinary environment with access to state-of-the-art tools in imaging, genomics, and computational biology. In this fellowship, I will gain advanced expertise in 3-dimensional tumor imaging, epigenomic analysis, and the development of experimental tools to interrogate the cancer stem cell interactome in real time. Under the guidance of a dedicated mentoring team, I will also strengthen my skills in scientific communication, grant writing, and mentorship. This integrated research and training plan will prepare me for an independent academic career focused on understanding how systemic signals influence stem cell fate and oncogenic potential.

NHLBI - National Heart Lung and Blood Institute

Project Summary Clonal hematopoiesis (CH) a premalignant condition affects 20% of people above age 60, increases risk for developing blood cancers like Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), and cardiovascular disease. CH-mutant clones originate in somatically-mutated hematopoietic stem cells (HSCs), with 80% of mutations occurring in epigenetic regulators, predominantly, DNMT3A. However, the molecular mechanisms that support clonal fitness remain incompletely understood. Since CH emerges with age, we reasoned that age-related changes in HSC regulation might create selective vulnerabilities that CH clones exploit for competitive advantage. Employing multiome single-cell sequencing, we recently identified zinc finger and BTB domain containing 1 (Zbtb1) as an essential transcription factor for HSC function during hematopoietic regeneration. In our preliminary analysis, while aged HSCs exhibit decreased Zbtb1 expression, DNMT3A- mutant HSCs exhibit increased Zbtb1 with hypomethylation at the Zbtb1 locus, suggesting epigenetic dysregulation drives aberrant expression. We further observed that Zbtb1 loss eliminates clonal advantage of DNMT3A-mutant HSCs. Finally, we identified direct ZBTB1 binding to the promoters of metabolic regulators, Got2 and Impdh2, as potential downstream mediators of ZBTB1 function in HSCs, and as targets in DNMT3A- mutant CH. Therefore, we hypothesize that epigenetic derepression of ZBTB1 drives clonal advantage in DNMT3A-mutant HSCs through transcriptional reprogramming of downstream targets regulating metabolic and other cellular processes essential for clonal fitness. This career development program will address two specific aims: (1) Determine the role of ZBTB1 in normal hematopoiesis and DNMT3A-mutated CH, (2) Define the epigenomic and metabolic functions of ZBTB1 in normal and DNMT3A-mutant HSCs. During the award period, the candidate, Dr. Harold Elias, MD, will conduct research at Memorial Sloan Kettering Cancer Center under the mentorship of Dr. Michael Kharas, an expert in stem cell RNA regulation, and co-mentor Dr. Marcel van den Brink, a leader in translational hematology. Both mentors have outstanding records guiding trainees to independence, complemented by advisors with expertise in epigenetics, metabolism, computational biology, and CH. He will build on his HSC biology foundation to develop critical skills for independence as an NIH-funded laboratory-based physician-scientist. His training will focus on four key areas: advanced epigenetic and metabolomics analysis, in vivo genetic screening techniques, and prime-editing optimization for in vivo CH modeling in primary human HSPCs. This comprehensive training will enable him to develop clinically relevant disease models and enhance his computational expertise in sequencing-based approaches, to generate preclinical validation data. Completion of this project will provide the candidate with the training and mentorship required to establish his academic career as an independent laboratory-based physician-scientist whose mechanistic discoveries will provide the foundation for therapeutic strategies targeting CH and its complications.

NINDS - National Institute of Neurological Disorders and Stroke

Abstract: Neurodevelopmental disorders associated with autism, intellectual disability, and cortical malformations are frequently caused by rare genetic abnormalities. Recently rare de-novo mutations in MAST1 have been identified in multiple patients with severe intellectual disability and cortical malformations. MAST1 is a microtubule- associated serine/threonine protein kinase-1 that is specifically expressed in neurons during brain development. However, little is known about the roles of MAST1 in regulating neuronal properties, and how MAST1 mutations affect the cellular and molecular mechanisms to cause neurodevelopmental deficits. The goal of this project is to elucidate the role of MAST1 in regulating neurodevelopment under normal conditions and disease states. In our preliminary experiments, we generated telencephalic brain organoids and neurons from control as well as patient and engineered stem cells with MAST1 mutations. We found microcephaly-associated size deficits in MAST1-mutated organoids due to reduced number of neurons and smaller neuropil size. We also observed elevated apoptosis, impaired neurites, and mitochondrial abnormalities in MAST1 mutated cortical neurons as compared to control neurons. We hypothesize that MAST1 regulates mitochondria trafficking in neurites and that mutated MAST1 causes neurite outgrowth deficits by disrupting mitochondria transport, ATP synthesis, and interactions with its binding partners. Collectively, these deficits lead to hyperactive mitochondria, overproduction of reactive oxygen species, neuronal apoptosis, and microcephaly-associated deficits in MAST1-mutated organoids. We will test this hypothesis in the following Specific Aims: (1) We will perform live-cell imaging on control and MAST1-mutated neurons to characterize mitochondria trafficking and ATP synthesis in axons and dendrites. (2) We will conduct structural-functional studies to determine how MAST1 interacts with mitochondria in neurons. Finally, (3) we will investigate how patient MAST1 mutations affect MAST1 interaction with its binding partners. The proposed research is significant because it is expected to substantially advance understanding of the cellular and molecular mechanisms disrupted in patient with neurodevelopmental disorders caused by MAST1 mutations and other brain disorders associated with mitochondrial abnormalities.

NCI - National Cancer Institute

PROJECT SUMMARY The main goal of the RUNX1 Research Program 10th Annual Scientific Conference and Patient Meeting is to create a dynamic convening to foster collaboration and provide opportunities for knowledge and data sharing among a diverse set of researchers, clinicians, and patients and their families for a rare disease called RUNX1 familial platelet disorder with predisposition to hematologic malignancies (RUNX1-FPD or FPDMM). Individuals with the disorder have a 35-50% lifetime risk of developing a hematologic malignancy. Of those who develop a malignancy, over 50% are diagnosed with AML. It is the only convening of its kind and continues to be an important part of deepening the communities’ understanding of the disease, of driving collaborations, and facilitating the discovery of future therapies for patients. Equally important is the opportunity for early career investigators, posts, and graduate students to present their research and receive feedback from a diverse, multidisciplinary community, a critical aspect of their training. RRP is committed to cultivating the next generation of RUNX1-FPD basic, translational, and clinical researchers. Conference participation has grown since our first conference in 2017, which had only 18 attendees. By 2021 we had over 181 attendees for the virtual conference. In 2022, we had 130 individuals - 40 patients and 90 scientists, clinicians, and genetic counselors - who attended in person in Princeton, NJ. Thanks to NCATS/NIH funding, we were able to award five (5) deserving young investigators with travel scholarships to attend the conference in 2022 and present at the poster sessions. In 2023, we returned to a virtual format with 397 registrants and a total of 219 participants. Once again, we held a poster session with four (4) young investigators.At our 2024 in-person scientific conference in Princeton, NJ we had 84 scientists, clinicians, and genetic counselors. As a result of NCI funding for the conference, we awarded travel scholarships to six (6) trainees.In 2024, we did not host a formal patient meeting alongside the scientific conference because we decided to pilot a smaller regional patient meeting in Houston to better reach patients outside of the northeast. This decision was influenced by a patient community-wide survey we conducted. Regardless, there were still seven patients and patient family members who attended the scientific conference. In 2025, our scientific conference and patient meeting will be held in September. The agenda includes research-specific sessions open to all stakeholders as well as a track dedicated to RUNX1-FPD patients and family members. The objective is to promote and drive patient-engaged research forward, with direct patient input in the development and planning of the sessions via our 23-member Research Guided by Patients Committee (RGPC). The research-focused portion of the meeting will bring together experts from the fields of hematology, immunology, oncology, cell therapy, and basic science who are committed to uncovering the mechanisms underlying the pathogenesis of RUNX1-FPD with the goal of developing therapeutic interventions that impact the bleeding and immune dysfunction issues and most importantly the predisposition to malignancy. The audience will include investigators devoted to studying RUNX1, leukemia progression, clonal hematopoiesis, gene editing, and hematopoietic stem cell transplants. It has become increasingly clear through the NHGRI-sponsored natural history study of RUNX1-FPD that immune dysfunction and inflammatory disorders are common. RUNX1-FPD includes a unique patient population where there may be a nexus between immunology and oncology based entirely on the reduced and/or altered activity of a single transcription factor, RUNX1.

NCI - National Cancer Institute

Abstract Multiple myeloma (MM) is an incurable plasma cell malignancy. Despite therapeutic advances, nearly all MM patients are expected to relapse and develop refractory disease, underscoring the need to improve the efficacy of first line therapy to prevent disease relapse. The current standard of care for newly diagnosed MM includes a myeloablative dose of the cytotoxic agent melphalan prior to autologous stem cell transplant (ASCT). Cytotoxic drugs are known to trigger therapy induced senescence (TIS) in normal cells. Several solid tumors have been shown to exhibit features of TIS following chemotherapy, and these markers in tumor cells are associated with superior progression free survival (PFS). One potential mechanism for this benefit is the activation of anti-tumor immune responses through the senescence associated secretory phenotype (SASP), which recruits and stimulates immune cells. Consistent with the findings in solid tumors, our analysis of longitudinal MM patient bone biopsies revealed a significant increase in senescence in MM cells in patients with PFS ≥2.5 years following ASCT. In contrast, patients that relapsed in <2.5 years did not exhibit increased tumor cell TIS. Additionally, patients that relapsed showed a positive correlation between MM burden post-ASCT and PFS, such that patients with the lowest plasma cell burden post-ASCT relapsed earliest. These data support that the ability of MM cells to evade apoptosis and enter a senescence-like, stable growth arrest in response to cytotoxic stress may protect against early relapse. To investigate the mechanisms underlying this phenomenon, we developed in vitro and in vivo models in which high-dose melphalan (HDM) consistently induces stable growth arrest and TIS features in human and mouse MM cell lines. Interestingly, HDM-MM cells also develop a myeloid gene signature. This myeloid gene expression was linked to functional plasticity, as HDM-MM cells cultured with osteoclast differentiation factors developed tartrate-resistant acid phosphatase (TRAcP5b) activity and multinucleation, two defining hallmarks of osteoclasts. Osteoclast-MM hybrids have previously been identified in MM patient biopsies, and our post-ASCT biopsy analysis revealed that nearly all patients had multinucleated CD138+ cells, including cells with osteoclast-like morphology on bone surfaces. Therefore, we hypothesize that TIS in MM cells activates anti-tumor immunity and drives MM-myeloid plasticity, enabling novel interactions with the bone niche that contribute to persistence and relapse. This hypothesis will be tested through the following specific aims: 1) Investigate the senescence-associated and immunological properties of MM cells that persist following cytotoxic therapy; 2) Define how TIS-myeloid plasticity supports MM cell survival and bone niche engagement. These studies will integrate our novel HDM in vitro and in vivo models with longitudinal patient biopsies. Because TIS and MM-myeloid plasticity are shaped by spatial and cellular interactions within the bone marrow microenvironment, in vivo models are essential to define how growth-arrested MM cells persist, engage the bone niche, and contribute to relapse. We expect these studies to reveal novel therapeutic strategies to target persistent, growth-arrested MM cells, ultimately preventing MM disease relapse.

NIGMS - National Institute of General Medical Sciences

We seek funding to acquire a Thermogravimetric Analyzer (TGA) with Evolved Gas Analysis (EGA) capabilities via selective sampling for GC-MS, to advance materials research and STEM education at the University of North Carolina at Pembroke (UNCP). Currently, UNCP lacks a TGA system, limiting capacity for advanced thermal analysis, reducing research productivity, and requiring reliance on external collaborations that introduce cost and delays. UNCP is a primarily undergraduate institution serving a large student population in a resource-limited region. The proposed instrument will directly support three faculty-led research initiatives—including projects led by early-career investigators—focused on biomass pyrolysis, plastics recycling, and the development of polymeric adsorbents for environmental remediation. The TGA-EGA system will enable precise measurements of decomposition temperatures, small molecule desorption profiles, and trace impurities, which are essential for characterizing complex materials. The instrument will also be integrated into upper-level chemistry courses, including organic, analytical, instrumental, and physical chemistry, providing hands-on training in thermal analysis and macromolecular characterization. These experiences will enhance preparation for careers in STEM and graduate study, strengthening the scientific workforce. The TGA-EGA will be housed in a shared research facility with broad access across departments, supporting interdisciplinary collaboration and student-faculty research. Its acquisition aligns with institutional goals to expand research infrastructure and increase access to advanced instrumentation.

Toyota USA Foundation

Supports innovative STEM education programs that develop students' skills for future careers, with emphasis on project-based learning.

NIGMS - National Institute of General Medical Sciences

Project Summary Signals that cells receive over time from a small set of pathways (e.g., BMP, Wnt, and TGFβ) shape their fate and phenotype during development, regeneration, and disease. Despite their central importance, signaling histories of individual cells are often inaccessible to direct observation, hindering quantitative analysis and obscuring their connection to eventual cell fate. This challenge is particularly pronounced in mammalian systems, where limited optical access and the constraints of size and timescale often render live imaging impractical. To address this issue, we have developed an approach to reconstruct the history of signaling activity in single cells based on endpoint fluorescence images. This is achieved by regulating CRISPR base editors to generate mutations in engineered target sites at rates proportional to the signal of interest. These mutations create a heritable record of signaling activity in the genome, which can be read out at a later time, together with the gene expression profile of the cells. Using this approach, we demonstrated that cells retain a memory of their past response level to BMP signaling for up to 18 days, providing a mechanism for long-term interactions between signals that can facilitate coordination of developmental processes over time. In this proposal, we will expand the scope and utility of our signal recording approach by extending its dynamic range to capture the broad spectrum of in vivo signal intensities and enabling simultaneous recording of the sequence and timing of two signaling pathways. We will also engineer mouse embryonic stem cells to record three key developmental pathways: BMP, Wnt, and Nodal. This will allow us to generate stem cell-derived embryo models and chimeric embryos to link cell fate and spatial organization at the onset of organogenesis with signaling activity at different time windows earlier in development. Additionally, we will investigate mechanisms that enable long-term changes in BMP responsiveness following an initial stimulation, without requiring differentiation. We will then test whether similar mechanisms exist in Wnt and Nodal pathways and assess their role in mediating long-term crosstalk between pathways. To achieve these goals, we will take an interdisciplinary approach combining gene editing, quantitative imaging, epigenomic assays, computational analysis, and generation of developmental models. The proposed goals build on my prior publications, recent preliminary data from our lab, and collaborations I have established since launching my lab. This research program will substantially advance the state of the art in molecular recording, transforming it into a technology that can be used in vivo, in mammalian systems to drive biological discovery. Our long term vision is to identify how signaling history controls cellular decision making during development, and how instructions that cells receive are coordinated over time to produce tissues with the correct number, types, and spatial arrangement of cells. Ultimately, this knowledge will inform strategies for tissue engineering, and open new avenues for understanding and treating diseases driven by dysregulated signaling.

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

Abstract In sexually reproducing organisms, the fusion of an oocyte and a sperm forms a totipotent zygote capable of generating every cell type in the body. In several model organisms such as Drosophila melanogaster, early asymmetries in the oocyte established before fertilization determine future developmental trajectories. However, the mechanisms that break symmetry and guide early cell fate biases in mammals remain poorly understood. Classical views held that mammalian embryos remain developmentally equivalent until the 16-cell stage, when inside and outside cells begin to emerge. Yet, recent studies suggest that asymmetries can arise as early as the 2-cell stage. At this early stage, both blastomeres are capable of generating all embryonic and extraembryonic lineages, but they often display reproducible biases in lineage contributions. One blastomere tends to favor epiblast fate, which gives rise to the embryo proper, while the other more often contributes to extraembryonic lineages like the trophectoderm. This phenomenon has also been observed in human embryos, implying conserved mechanisms underlying early cell fate biases. When these cells are physically separated at the 2-cell stage and cultured independently, only one consistently forms a robust inner cell mass, suggesting inherent differences in developmental potential. We have observed that in 2-cell stage mouse embryos, the cell with higher epiblast contribution correlates with the cell that inherits the sperm entry point. This research aims to identify the molecular and structural cues introduced at fertilization—such as sperm-derived components and the second polar body—that influence early fate biases in the mammalian embryo. Understanding these early symmetry-breaking events has broad implications for reproductive biology, stem cell research, and our fundamental understanding of developmental potential.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Aging leads to widespread changes in the immune system, including increased inflammation, diminished vaccine responsiveness, and greater susceptibility to infection and chronic disease. However, older adults vary significantly in their immune trajectories—some maintain robust function, while others experience immune decline. The biological mechanisms driving this heterogeneity remain poorly understood, particularly the role of upstream hematopoietic stem and progenitor cells (HSPCs), which continually regenerate the immune system throughout life. This project tests the central hypothesis that epigenetic remodeling of HSPCs contributes to immune-related hallmarks of aging and variability in these phenotypes, shaping both inflammatory set points and immune responsiveness in older adults. To address this, we leverage a well-characterized, longitudinal cohort of 62 older adults, each of whom received a different influenza vaccine over three consecutive seasons. Using our novel PBMC-PIE platform—developed to enrich and profile circulating HSPCs from cryopreserved PBMCs at single-cell resolution—we can interrogate progenitor cell states and their progeny without requiring bone marrow biopsies. This enables unprecedented, longitudinal insight into how human HSPCs are reprogrammed with age and vaccination. In Aim 1, we will identify molecular and epigenomic features in HSPCs and innate immune cells that distinguish strong from poor vaccine responders. In Aim 2, we will determine how different influenza vaccine platforms (high-dose, adjuvanted, recombinant) shape the chromatin landscape and lineage bias of HSPCs and their progeny over time. In Aim 3, we will test whether maladaptive “aged” HSPC states can be rejuvenated in vitro using candidate immunomodulatory factors identified in Aims 1–2. By linking blood-based epigenetic signatures to real-world immune outcomes, this work will reveal fundamental mechanisms of immune aging, identify biomarkers of immune resilience, and inform the development of targeted strategies to restore immune function in older adults.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY Advances in regenerative medicine continue to transform the way we treat human diseases. To maintain our prominence in this important field, this application seeks to establish a new Training Program in Developmental, Stem Cell and Regenerative Biology (DSCRB). Despite tremendous interdisciplinary strength in this area at UW-Madison, there is no programmatic training in this field. Our Training Program, which is built around a recently inauguration curriculum that provides students a PhD Minor in DSCRB, is designed to fill this gap. The Minor requires 9 class credits, which are drawn from required and elective courses offered by the 8 PhD programs from which we draw trainees; therefore, students can complete the Minor without taking additional coursework beyond the requirements of their PhD programs. While the program’s administrative home is the Department of Cell and Regenerative Biology, our 55 trainers are from 17 departments and 6 schools/colleges at UW-Madison. Trainers are selected on the relevance of their research, their training and mentorship record, and the robustness of their research program, and more junior associate trainers are mentored by a more senior trainer. We seek to attract and train a cohort of 14 predoctoral trainees, matriculating 7 trainees per year for a two-year training program, with the goal of providing rigorous intellectual, technical, and interdisciplinary training. We will engage outstanding predoctoral trainees in the second semester of their first year, immediately after research rotations are complete. At this point, each student will join a trainer’s laboratory while they continue didactic training. To build cohesion, monthly Trainee Meetings, a student-led Journal Club, and a student mentorship program will bring together participants to share their research and interact. Career development activities and ethics training will be an important focus of these meetings, emphasizing reproducible and responsible conduct of research; ethical use of animal models, stem cells, fetal tissue, and human subjects in regenerative medicine research; and how to incorporate experimental rigor. The Training Program also leverages Stem Cell and Regenerative Medicine Center (SCRMC) activities, including the SCRMC Fall Conference, and the Wisconsin Stem Cell Symposium (international symposium sponsored jointly with Promega). These symposia offer our trainees opportunities to participate in meeting organizations, host external speakers, engage in career development activities, and present their research to a broad community. The PIs of this proposal have considerable combined experience in T32 training program administration and graduate education, and there is strong institutional support. The DSCRB Training Program will provide rigorous, cutting-edge training to produce the next generation of leaders in the field, enabling breakthroughs and empowering research that will lead to transformative regenerative therapies.

NIGMS - National Institute of General Medical Sciences

This application for a Stanford University Interdisciplinary T32 Program in Regenerative Medicine will identify, through a call-for-applications, promising second-year PhD students enrolled in one of 14 Stanford Biosciences graduate training programs for funding to pursue research in mechanisms and clinical application of stem cell biology in the application of regenerative medicine approaches to human disease. Our program will support trainees whose proposed research projects align with our mission to educate future leaders in regenerative medicine. The biology of stem cells will be a central focus area and the principles that underlie regenerative medicine, such as human biology, disease mechanisms, and translation of basic science to medical advances will figure prominently in selection of participants for our training program. An established graduate program in stem cell biology will provide some of the framework for our T32 program, while Stanford’s translational Center for Definitive and Curative Medicine (CDCM) will provide value-added components of translationally-oriented laboratory research experiences, training in regulatory processes for new drugs and clinical advances, and seminars and workshops that specifically target the translation of laboratory-based discoveries to clinical applications. We will synergize with other Stanford T32s to provide elective courses for our trainees desiring more exposure to specialized topics. Our Objectives are 1: to identify the rising second-year students interested in clinical application of regenerative medicine and fund those with the highest likelihood of success in graduate school for one year with reappointment to a second year based on progress, 2: to maintain a training program that includes a rigorous basis in ethics, the reproducibility of methods, data collection, and publication in peer-reviewed journals and that provides comprehensive understanding of the biomedical enterprise, from basic discovery to the development and application of therapies, and 3: to provide individualized opportunities for our students to explore potential careers as an essential aspect of the training program. Program elements include didactic training in key areas of regenerative medicine and stem cell biology followed by research training in cutting-edge laboratories and exposure to clinical translation through CDCM-sponsored programs. Central to our plan for career development is a required internship, designed by the students, to help facilitate their successful transition into a biomedical career. Supporting the strength of our training and mentoring abilities, our past trainees have had a very high retention rate, strong publication records, and a history of successful transition to careers in academics, industry, and finance. We propose to fund 4 trainees per year for a total of 8 training slots and expect trainee outcomes to include equal numbers of academic and industry job placements. Our proposed T32 program will maintain the current high level of PhD completion rates and short time-to-degree, and expand training of PhD scientists in this novel program through focus on mechanisms of regenerative medicine and the translation of discovery science to the clinic.

NIGMS - National Institute of General Medical Sciences

Project Summary The University of Missouri-Columbia (MU) proposes to direct a Trans-disciplinary Basic Biomedical Science (TDS) T32 training program for predoctoral students in PhD programs. The mission of our training program is to facilitate the transformation of our trainees into independent scientists who are active producers of new biomedical knowledge and are adept at solving the complex problems of disease and illness that adversely affect human health. Our training program accomplishes this mission by bringing together four PhD programs including Biochemistry, Biological Sciences, Biological Engineering, and Translational Biosciences. The latter program includes parallel emphasis areas (tracks) covering specialties including microbiology & virology, gene & stem cell therapy, immunology, physiology, nutrition & exercise science, cancer biology, and epidemiology & precision health. We propose to use science identity as our organizing principle, with its composition of community belonging, self-efficacy, and achieved deliverables. Objective #1: To strengthen the safe, supportive, and nurturing nature of our community by providing training to improve the quality of the peer- and mentor-mentee relationships that are so critical to career success and satisfaction. Objective #2: To expedite fluency in the scientific method, we will institute a Critical Thinking Workshop to be followed by a new course, Biomedical Literature Colloquium, for primary literature critique, presentation, and writing. Objective #3: To promote professional communication skills and increase achieved deliverables in the form of external fellowship wins and peer-reviewed publications. Objective #4: To improve the PhD completion rate of trainees in the participating biomedical doctoral programs from the current level of 74-81% to 85%. Our active program evaluation structure will allow independent, professional assessment of trainees’ sense of science self- efficacy, science identity, perceived support, and satisfaction with mentoring relationships, all of which are predictors of academic satisfaction and persistence outcomes among predoctoral students. The TDS T32 program will implement a wide-ranging set of programmatic activities to develop the technical, operational, and professional skills that will enable our trainees to flourish in their independent scientific careers.

NIGMS - National Institute of General Medical Sciences

Project Summary Double-strand breaks (DSBs) in DNA represent one of the most severe threats to genomic integrity, with improper repair leading to mutations, chromosomal translocations, and oncogenic transformation. Cells have evolved robust pathways, including non-homologous end joining (NHEJ), homologous recombination (HR), and microhomology-mediated end joining (MMEJ), to repair DSBs. While much is known about protein-mediated DSB repair mechanisms, the role of transcript RNA in modulating repair remains poorly understood. Yet, the potential contribution of RNA molecules, particularly transcript RNA with sequence complementarity to DNA ends, remains largely unexplored. Recent discoveries from the PI’s laboratory demonstrate that RNA transcripts can bridge broken DNA ends and facilitate repair via NHEJ and MMEJ in both human and yeast systems, suggesting a conserved and fundamental role for RNA in genome maintenance. This project seeks to systematically uncover the mechanisms, regulatory proteins, and biological consequences of transcript RNA involvement in DSB repair across multiple contexts in human cells. Aim 1 establishes isogenic human embryonic kidney (HEK293T) and diploid retinal pigment epithelial (RPE-1) cell lines expressing spliced or non-spliced RNAs from the same adeno-associated virus integration site 1 (AAVS1) safe harbor locus and investigates how these transcripts modulate end-joining repair (NHEJ and MMEJ) of chromosomal DSBs induced by CRISPR/Cas9, followed by deep sequencing and bioinformatics analysis. Aim 2 identifies the key proteins and molecular processes involved in RNA-mediated end-joining repair in human cells. This goal will be achieved by inhibiting core NHEJ and MMEJ factors using chemical inhibitors and siRNAs, and by selectively reducing nascent spliced and non-spliced RNA transcripts using CRISPRi. These perturbations will define how individual proteins and transcript RNAs contribute to the efficiency, fidelity, and pathway choice of RNA-mediated end joining. Aim 3 examines whether transcript RNA differentially modulates DSB repair in exonic and intronic sequences of endogenous human genes in both HEK293T and RPE-1 cells, including breaks induced by CRISPR/Cas9 and mutagens, to understand RNA’s role in maintaining genome integrity. The significance of this work lies in establishing transcript RNA as a previously unrecognized regulator of DSB repair pathways, revealing new dimensions of genome stability control and potential therapeutic targets. The results could provide insights to improve genome editing technologies, inform strategies to reduce mutagenesis and carcinogenesis, and contribute fundamentally to understanding how RNA functions extend beyond traditional regulatory roles to directly safeguarding the genome. The outcomes will also lay groundwork for exploring RNA-mediated DNA repair in stem cells, non-dividing cells, aging models, and disease states, potentially transforming approaches to cancer prevention and therapy.

NCI - National Cancer Institute

Abstract PAX3 is an indispensable upstream regulator of normal melanocyte survival, population expansion, and migration, and these functions are subverted in melanoma proliferation, resistance to apoptosis, and metastasis. Despite significant advances in treatment, melanoma is still a deadly cancer with considerable mortality rates, and PAX3 contributes to this by supporting cancer aggressiveness, resistance to therapies, and relapse. While PAX3 is expressed in developing melanoblasts and melanocyte stem cells, downregulation of PAX3 during terminal differentiation is required. In melanoma, PAX3 expression is overexpressed, maintained, and essential for cancer progression. Melanoma cells rely on PAX3 function; indeed, a loss of PAX3 expression leads to catastrophic loss of melanoma cell growth, migration, and viability. Due to the significance of PAX3 in melanoma, it is surprising how much is unknown about PAX3 function. Classically defined as a transcription factor, only a handful of PAX3 downstream target genes in melanoma are known. Our lab and others have uncovered select downstream effector genes that can explain some but not all of the wide roles that PAX3 has on melanoma survival and progression. To uncover clues on other functions of PAX3, our laboratory performed an unbiased immunoprecipitation and mass spectroscopy (IP/MS) screen to identify PAX3 binding partners. Unexpectedly, we discovered that PAX3 interacted with several proteins involved with RNA surveillance, decay, and splicing. Further, our preliminary data support that this role in RNA regulation is independent of the transcriptional role of PAX3. The goal of this proposal is to uncover how PAX3 regulates these cell functions through downstream effector genes, by acting directly on genetic regulation or indirectly through other molecular means. To address the significant gaps in knowledge into PAX3 function, the hypothesis guiding this proposal is that PAX3 can dynamically act on downstream genes through canonical and noncanonical transcription factor functions, with outcomes modified by binding partners, tumor stage, and small molecule inhibitors. The major scientific impact of this proposal is to reveal a PAX3 dependent genomic enhancer map in melanoma connected to regulated genes, discover mechanisms for modulating this signature, and exploit these pathways as targets for therapy. The innovation of this proposal lies in a more detailed focus on PAX3, in terms of what genes are controlled and how. We propose the innovative hypothesis that PAX3 is not purely a canonical transcription factor and can function both transcriptionally and post-transcriptionally. This work is significant since the PAX3 transcriptional signature in melanoma is still undiscovered, and it is not clear if it is static or adaptive at different tumor stages or drug treatments. This work will uncover PAX3-dependent pathways driving melanoma progression and reveal potential molecular weak points that can be targeted by therapeutics. Use of mice in this proposal: The rationale for utilizing mouse models is that normal and pathological cells behave differently in vivo due to a complex microenvironment that cannot be replicated in other model systems.

NIMH - National Institute of Mental Health

Project Summary Mutations that reduce or alter the activity of repressive chromatin modifiers are frequent causes of neurodevelopmental disorders. The inaccessibility of the developing and the dynamic nature of neurogenesis so far have prevented an adequate understanding of how molecular pathologies arise downstream of the loss of specific chromatin regulators. This knowledge gap represents a hurdle for developing therapeutic interventions for neurodevelopmental disease. This proposal aims to combine targeted protein depletion with highly efficient directed differentiation regimens for human pluripotent stem cells to dissect gene regulatory functions of the autism-associated chromatin repressor EHMT1 during human cortical neurogenesis. Based on the extensive characterization of a novel, multipurpose (degradation/immunoprecipitation/visualization) degron allele, we hypothesize that interactions with cell type-specific co-factors allow EHMT1 to control the expression of stage- specific target genes during neurogenesis, resulting in the accumulation of molecular alterations and cortical neuron (CN) dysfunction when EHMT1 is lost from early development onwards. To systematically test this hypothesis, we will first determine whether molecular alterations caused by EHMT1 deficiency from earlier stages of neurogenesis onwards accumulate in CNs and to what degree dysregulated gene loci and CN function remain responsive to restoring physiological EHMT1 levels (Aim 1). We will then combine genomics, proteomics, and genetic approaches to identify how EHMT1, together with candidate recruiters and co-factors, regulates distinct gene loci at specific stages of cortical neurogenesis (Aim 2). Finally, we will expand our degron approach to dissect the functional interplay of different autism-associated H3K9 methylation regulators during human neurogenesis to identify interactions between these proteins that could be clinically exploited (Aim 3). Our experiments will determine currently unknown gene regulatory functions of disease-associated chromatin repressors at critical stages of human cortical neurogenesis. By generating mechanistic insight into how deficiencies of EHMT1 and other H3K9 methylation regulators introduce molecular pathologies in CNs, we anticipate revealing new opportunities for therapeutic interventions with specific neurodevelopmental diseases.

NEI - National Eye Institute

PROJECT SUMMARY/ABSTRACT Vision loss caused by the death of photoreceptors is a leading cause of irreversible blindness worldwide, yet therapeutic options remain limited. For this reason, the NEI’s Retinal Disease Program has identified the development of strategies for the treatment of retinal degenerations as a core program goal. Recently, several laboratories have derived photoreceptors from stem cells, making cell-replacement therapies particularly promising. Additionally, important advances have been made into manipulations that could stimulate retinal regeneration from the retinal Müller glia. The critical barrier for the success of such therapies is to understand the factors required to direct fate decisions in progenitors towards fully differentiated cell subtypes that are also capable of properly rewiring into retinal circuits. Although key transcription factors have been identified as essential for generating retinal cell classes, the target genes required to generate each retinal cell subtype are still undetermined. TBX2 is a central transcriptional regulator of all photoreceptor subtypes and is highly conserved across vertebrates. Our main hypothesis for this proposal is that retinal progenitors express TBX2 to repress the identity of photoreceptor subtypes that are not UV cones through two different mechanisms, and our main goal is to identify these two roles. Three Specific Aims are proposed: Specific Aim 1 will test the hypothesis that tbx2a and tbx2b have subdivided functions and their roles in generating photoreceptor subtypes are different. Specific Aim 2 will identify candidate factors downstream of TBX2 important for the generation of photoreceptor subtypes. Specific aim 3 will test the hypothesis that differences in Tbx2a and Tbx2b function are caused by differences in the repression domain. At the successful completion of this project, I will have identified the subdivided functions of tbx2a and tbx2b and subsequent targets to generate and maintain photoreceptor subtypes. This proposal benefits from the experimental accessibility of the retina and our deep knowledge of retinal cell types and circuits, but our approach has the potential to impact the study of other neuronal degenerative diseases. In addition to my proposed research, I designed a holistic training plan that will help me advance toward my goal of leading an independent research lab. The work planned for the F31 award period will be valuable in terms of both research and training opportunities.

U.S. National Science Foundation

TSL combines interests and resources of separate programs in the Division of Research on Learning in Formal and Informal Settings (DRL) to explore the opportunities and challenges implied by innovative visions of the future for STEM learning.?? The TSL program invites interdisciplinary teams of STEM content specialists, experts in relevant technologies, STEM formal and informal education specialists, researchers with expertise in the learning sciences, and specialists in education research and evaluation methods to submit proposals for research projects that (1) Study efficacy of existing prototypes for innovations like virtual schools, special STEM schools, and educational programs that combine opportunities of formal and informal learning resources in their communities; or (2) Design and conduct exploratory development of new potentially transformative models for STEM learning environments.?? The cross-cutting proposals will draw from work in the four primary DRL programs: Discovery Research K-12 (DR K-12), Informal Science Education (ISE), Research and Evaluation on Education in Science and Engineering (REESE), and Innovative Technology Experiences for Students and Teachers (ITEST).?? However, proposals submitted in response to this solicitation must have a scope that extends well beyond any of those programs individually.

U.S. National Science Foundation

The Transforming Undergraduate Education in Science, Technology, Engineering, and Mathematics (TUES) program seeks to improve the quality of science, technology, engineering, and mathematics (STEM) education for all undergraduate students. This solicitation especially encourages projects that have the potential to transform undergraduate STEM education, for example, by bringing about widespread adoption of classroom practices that embody understanding of how students learn most effectively. Thus transferability and dissemination are critical aspects for projects developing instructional materials and methods and should be considered throughout the project's lifetime.?? More advanced projects should involve efforts to facilitate adaptation at other sites.The program supports efforts to create, adapt, and disseminate new learning materials and teaching strategies to reflect advances both in STEM disciplines and in what is known about teaching and learning.?? It funds projects that develop faculty expertise, implement educational innovations, assess learning and evaluate innovations, prepare K-12 teachers, or conduct research on STEM teaching and learning. It also supports projects that further the work of the program itself, for example, synthesis and dissemination of findings across the program. The program supports projects representing different stages of development, ranging from small, exploratory investigations to large, comprehensive projects.

U.S. National Science Foundation

This solicitation addresses issues of translation and diffusion that arise in moving knowledge gained from fundamental learning and education research toward application in PreK-12 STEM classroom practice or leveraging knowledge derived from effective practice toward driving fundamental research. The first goal of this funding opportunity is to encourage the scientific study of theories, frameworks, and models for the translation and diffusion of knowledge, especially between fields and across contexts and levels-of-analysis (e.g., biological to cognitive/socioemotional to behavioral; individual to classroom to broader demographic variables; lab to classroom to school to district). The second goal is to advance or move specific practice, research or scientific discovery in STEM education reciprocally along the research-practice continuum. The Translation and Diffusion (TD) solicitation invites four types of proposals: Research on Translation or Diffusion proposals request funding to conduct scholarship that will advance the sciences of translation or diffusion of fundamental research knowledge toward PreK-12 formal STEM education practice by developing or refining theories, frameworks, or models (or adapting those from other domains) and conducting related research. Such proposals may also address the leveraging of effective classroom practices toward the enrichment of foundational research, constructs and models. We note that bi-directional movement across boundaries is a mutually beneficial reciprocal process. Proof-of-Concept Research proposals request funding to explore the feasibility and viability of particular knowledge or products generated from STEM education research toward advancing practice in formal PreK-12 settings (even if it is still basic or applied research and development rather than implementation). The goals are to facilitate the process by which the promise that the initial insight holds for research and practice can be realized. The outcome of such a project would lay the methodological, theoretical, empirical, design, or social foundation for conducting systematic work at the next stage of development or at the next level of analysis. Empirical and theory-building efforts to adapt initial insights from research or practice across significantly different contexts, populations, domains, and levels-of-analysis are also welcome. Synthesis proposals critically integrate the current state of knowledge on a particular topic relevant to translation and diffusion in formal PreK-12 STEM education. Such proposals should include the state of the knowledge across disciplinary communities and across relevant literatures, identify the lacunae in STEM education knowledge, and, where appropriate, lay out thenext steps for future research and development. Conference / Workshop proposals relevant to the call are also welcome.

NHLBI - National Heart Lung and Blood Institute

Project Summary/Abstract Despite surgical advancement and endovascular device innovations, endothelial injury remains a fundamental barrier in vascular surgeries, contributing to post-operative complications such as vein graft failure, restenosis, and thrombosis. Current surgical approaches offer no regenerative solution to actively protect endothelial health during or after interventions. To address this gap, we have developed a bioengineered mitochondrial therapy platform that leverages lipid-polymer-peptide surface functionalization to enable targeted mitochondria delivery, enhanced cellular uptake, and integration into the host mitochondrial network. Mitochondrial dysfunction is a key driver of impaired endothelial repair following vascular surgeries, yet mitochondrial transplantation has not been adopted clinically due to challenges in storage stability, scalability, and compatibility with surgical workflows. Our preliminary studies demonstrate successful surface engineering of stem cell–derived mitochondria with robust uptake and functional rescue in dysfunctional endothelial cells. In vivo, we show that engineered mitochondria were selectively uptake by target vessels during both intra-operative (ex vivo vein graft storage and arterial intraluminal infusion) and post-operative stages (intravenous injection) upon vascular surgeries. To further establish the translational potential of bioengineered mitochondrial therapy, we propose the following studies. Aim 1 will optimize mitochondrial source selection, storage formulation, and surface modification parameters to ensure translational feasibility. Aim 2 will validate the therapeutic efficacy and biodistribution of the optimized system in two highly clinically relevant in vivo models of open (vein graft bypass) and endovascular surgeries (balloon angioplasty). This interdisciplinary project integrates surgical sciences, bioengineering, translational medicine, and device development to establish a next-generation therapeutic approach for vascular surgeries. By advancing a procedural-compatible, device-enabled delivery system, this work directly addresses a critical need in vascular surgery by developing translatable tools and technologies to improve surgical outcomes in millions of patients with cardiovascular diseases.

NHLBI - National Heart Lung and Blood Institute

Abstract Hematopoietic stem cells (HSCs) require means to maintain genome stability to sustain a lifelong supply of blood cells. This is prominently illustrated by Fanconi Anemia (FA), which results in bone marrow failure (BMF) and increased leukemia incidence. We showed that Lnk deficiency increases HSCs in FA mutant mice and primary FA patient cells. A critical question is how LNK controls DNA replication-associated DNA damage. Mice lacking a central component of the FA pathway, Fancd2, show severely compromised HSC activity in bone marrow transplant (BMT) in vivo. Fancd2-/- HSCs and progenitors (HSPCs) cannot sustain ex vivo culture. This HSPC attrition is due to heightened DNA damage and chromosome abnormalities, causing cessation of cell proliferation and increased apoptosis. We showed that Lnk deficiency reduces DNA damage associated with replication stress in HSPCs, particularly in the FA background. Lnk deficiency suppresses single-strand DNA gaps, thus reducing ATR and p53 checkpoint activation that is linked to HSC attrition. One of key mechanisms of DNA damage tolerance in cells is translesion synthesis (TLS). TLS polymerases (Polh, Poli, Polk, and REV1) are specialized DNA polymerases that facilitate replication in the presence of DNA lesions or natural replication fork barriers that stall the replicative polymerases. Their unique ability to replicate over difficult-to-replicate regions makes them critical for maintaining genome stability. However, the complex regulation of DNA replication and repair in hematopoietic cells is not well-established. We discovered that the enhanced HSC fitness from Lnk deficiency is associated with increased TLS activity via REV1, a scaffold protein that brings multiple TLS polymerases to stressed replication forks. We thus hypothesize that Lnk deficiency promotes HSC expansion by enhancing the activity of TLS DNA polymerases to mitigate replication stress. We aim to investigate mechanisms underlying LNK regulation of TLS polymerases and their functional significance to HSC fitness in the context of FA. In Aim 1, we will investigate the role of TLS polymerases in regulating HSC function and replication stress using mice deficient for Rev1 and various TLS polymerases. In Aim 2, we will determine how LNK regulates TLS polymerases and replication factors upon replication stress in HSPCs. We will examine whether LNK impacts TLS-mediated DNA replication using TLS reporters ex vivo and in vivo. We will decipher how Lnk deficiency impacts replication fork dynamics using a combination of complementary genetics, proteomics, and pharmacological approaches. In Aim 3, we will determine the role of TLS in replication stress relief in human FA HSPCs. We will investigate whether TLS inhibition exacerbates the growth defects of human FA HSPCs and, conversely, whether overexpression of components of the TLS pathway promotes the growth of human HSPCs from healthy donors and FA patients. Our work will deepen understanding of mechanisms underpinning stem cell attrition in healthy and disease settings and may unveil new therapeutic strategies to improve HSC fitness for the treatment of FA and for stem cell-based therapy in general.

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

ABSTRACT Transposable elements (TEs) make up over half of the human genome and are increasingly recognized as key regulatory sequences of gene expression in development and disease. While cellular intrinsic transcriptional and epigenetic mechanisms controlling TE sequences are well-documented, whether and how TEs response to external microenvironmental signals, particularly mechanical forces, remains unexplored. This represents a critical gap in understanding both TE regulation and genome–environment interactions. Our preliminary data reveal that several TE families, including LTR7 from the primate-specific HERV-H family, function as Mechano-Response Enhancer Elements (MREEs) that regulate gene expression and human pluripotent stem cell fate in response to mechanical stimuli. Additional preliminary findings support our central hypothesis that TEs act as MREEs by modulating human genes and cell fate of human pluripotent stem cells through a mechanism at least partially governed by the key mechano-effector YAP, which regulates TEs’ local epigenetic activity and facilitates their long-range chromatin looping with target genes in response to mechanical changes. To test this model, we propose three specific aims: 1) determine how mechanical signals regulate the local chromatin activity of TE MREEs; 2) elucidate how mechanical cues mediate long-range chromatin interactions between TE MREEs and their distal target genes; 3) delineate the mechanism by which TE MREE modulating human embryonic stem cell fate. Our objective is to rigorously establish the novel concept that TEs function as MREEs, uncover their underlying molecular mechanisms, and assess their biological significance in regulating developmental genes and hESC fate. The expected outcome will not only shift the current paradigm of mechanobiology from protein- coding genes to non-coding regulatory elements such as TE but also improve our understanding of TE regulation and genome-environment interactions in health and disease.

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

Summary: The negative effects trauma exposure, such as those that promote PTSD, can be passed to offspring. While the environment in which affected parents raise their offspring clearly plays a role, a significant genetic component also exists. However, some of this genetic influence, typically revealed by twin studies, may stem from epigenetic inheritance; as implied by studies in male rodents, where trauma-specific changes in sperm miRNA content can lead to trauma-specific behavioral changes in all offspring. Evidence for epigenetic inheritance in humans is mainly epidemiological, but recent studies have revealed stress-induced sperm miRNA changes consistent with this idea. For example, we found that men with high Adverse Childhood Experiences (ACEs), whose children can be negatively affected, show reduced sperm levels of miR-34/449 family members; mirroring findings in mice exposed to chronic social instability (CSI) stress across generations. In mice, these sperm miRNA changes persist in preimplantation embryos post-fertilization, altering early embryonic gene expression that leads to elevated anxiety and impaired sociability in female offspring as well as reduced levels of sperm miR-34/449 in males. Our new data driving this proposal reveals another example. The degree of men’s exposure to adult trauma, as assessed by the Trauma History Questionnaire (THQ), which measures PTSD risk, correlates with the levels of miRNAs 532, 361, 375, and 491 in their sperm. The highest THQ scores are associated with 4- to 130-fold increases in these levels. In contrast, these miRNA changes do not correlate with ACE scores and miR-34/449 does not correlate with THQ score. Notably, miRNAs-532 and 375 are two of the 9 sperm miRNAs whose enhanced levels in male mice mediate how chronic variable (CV) stress leads to a suppressed HPA axis response in offspring, a trait linked to mental health disorders. This proposal investigates how elevated levels of sperm miRNAs-532,361,375 and 491 in men with high THQ scores might affect their offspring using the mouse model that previously demonstrated how elevated levels of sperm miRNAs transmit the stress-related effects of paternal CV stress across generations. Aim 1 tests how injecting THQ-associated miRNAs, at levels found in sperm of men exposed to high levels of trauma, into mouse zygotes affects the phenotypes of resulting offspring. Aim 2 will begin to reveal how these injected miRNAs lead to phenotypic changes revealed in Aim 1 by i) identifying gene expression changes induced by them in mouse blastocysts, and ii) looking for similarities to those occurring in discarded blastocysts from IVF procedures using men’s sperm with elevated THQ associated miRNAs derived from an ongoing, independently funded project. This study has the potential to: a) provide strong support for using mouse models to understand epigenetic inheritance in humans; and b) drive future research showing that a significant portion of inherited susceptibility to mental health disorders arises from epigenetic inheritance, and how to reverse it before fatherhood.