Washington University

NIH Research Projects · FY 2025 · 2020-09

ABSTRACT Preclinical imaging is widely used in cancer research to devise novel tumor detection strategies, assess tumor burden and physiology/biology, as well as to validate novel therapeutic strategies and predictive biomarkers of response to therapy. The typical imaging workflow is complex and may include multi-modality, multi-parametric imaging, multiple animals, and linked biology experiments. When multiplied by the number of animals per group in an experiment, multitudes of interventions (e.g., drugs), and the number of time-points in a longitudinal imaging protocol, the resulting datasets are vast and prohibitive to track and manage long-term. Non-trackable data results in poor reproducibility and presents obstacles for data mining and open science collaboration. To address this unmet need, we developed the Preclinical Imaging XNAT-enabled informatics (PIXI) platform. PIXI (v. 1.0.0) (available at https://www.PIXI.org/) was released for public use in February of 2024. PIXI v1.0.0 captures preclinical DICOM images and associated metadata. DICOM is not universally used nor adopted by preclinical imaging vendors as many vendors use proprietary image file formats, resulting in fragmented adoption. In Aim 1, we will extend the PIXI Server to capture non-DICOM preclinical imaging experiments, associated metadata, image-derived measurements, and linked-biology experiments to advance NCI’s precision medicine initiative (PMI). In PIXI v1.0.0, we additionally developed PIXI’s containerized application environment, integrated Jupyter notebooks to enable deployment of computational pipelines, and integrated PIXI Dashboards. As preclinical imaging data is becoming more complex with big data needs, the need for reproducible and unified analysis workflows is critical. In Aim 2, we will develop PIXI Dashboard services and computational imaging pipelines to enable visualization, processing, and analysis of preclinical imaging biomarkers and linked biology experiments, supporting NCI’s PMI objective of consistent processing and analysis of imaging biomarkers. Finally, the increasing complexity, volume, metadata needs, and big data needs of oncologic preclinical imaging workflows necessitate a domain specific preclinical imaging repository to support NIH's Policy for Data Management and Sharing (DMS). The NIH provides several domain-specific repositories for DMS, but none are dedicated for preclinical imaging nor with robust support for metadata and quality assurance needs. PIXI is uniquely positioned as a preclinical imaging DMS. In Aim 3, we will establish PIXI Center: A domain-specific data management and sharing (DMS) resource for oncologic preclinical imaging, enabling Centralized Learning in oncologic translational imaging research. The fourth aim of the proposal focuses on resource dissemination and training strategies. Overall, the renewal proposal will support the complex and growing demands in preclinical cancer imaging. We will enhance and extend PIXI’s capabilities to support additional experiments, extend metadata, develop and enable computational pipelines, and establish a much-needed domain-specific DMS, ensuring continued support, development, and wider adoption of PIXI to ultimately support NCI’s missions.

NIH Research Projects · FY 2026 · 2020-09

PROJECT SUMMARY The family of pentameric ligand-gated ion channels (pLGICs) mediate synaptic transmission in the brain and are the targets of many drugs. This project seeks to understand the structural basis of pLGIC activation by agonists and modulation by lipids. pLGICs are very sensitive to the lipid membrane environment, and therefore an accurate understanding of structure-function relationships in these channels requires consideration of the effects of lipids. Previously, we determined the activated open-channel structure of the model pLGIC, ELIC, and characterized the effect of phospholipids and fatty acids on ELIC activation and desensitization. Building upon these advancements, we will determine the structures of intermediate states of ELIC activation and the mechanism by which certain phospholipids support ELIC activity. This will be accomplished by a multidisciplinary approach using single particle cryo-EM, functional measurements in model membranes, pulsed-EPR, and molecular dynamics simulations. A major goal of this project is to perform both cryo-EM and functional analysis of pLGICs in liposomes of defined composition, thereby providing rigorous understanding into the structural basis of ion channel activation and the effects of lipids. We will also expand the project to investigate the lipid sensitivity of a human pLGIC, applying technical and biological insights gained from ELIC. The outcomes of this research are detailed structural models of pLGIC function: fundamental information that will facilitate the design of therapeutics targeting these ion channels.

NIH Research Projects · FY 2026 · 2020-08

ABSTRACT Blood diseases such as myelodysplastic syndromes (MDS) arise from hematopoietic stem cells (HSCs) that acquire genetic mutations which corrupt critical HSC functions. One of the most recurrently mutated genes in these neoplasms is the de novo DNA methyltransferase enzyme DNMT3A. However, DNMT3A mutations can occur in HSCs long before clinical presentation. Recent studies have shown that the HSC clones that predominate with age often contain mutations that are characteristic of myeloid neoplasms. This phenomenon is known as clonal hematopoiesis (CH), but only a small fraction of individuals with CH go on to develop a blood disease. This suggests that in addition to genetics, there must be other factors which act differently between individuals that select for propagation of HSCs with these mutations. Our lab is interested in identifying factors that change with age which may provide selective pressures for these mutant clones, focusing on inflammation. The prior funding period identified interferon gamma (IFNg) as an environmental stressor that selects for the outgrowth of Dnmt3a-mutant HSCs. While chronic IFNg signaling is detrimental to normal HSCs, functionality of Dnmt3a-mutant HSCs is preserved in this setting, presenting a mechanism whereby HSCs with these mutations gain clonal dominance in settings of inflammation. New data generated in our lab show that Dnmt3a-mutant HSCs are not only resistant to the detrimental effects of IFNg in vivo, but also the mutant clones produce more IFNg themselves in response to inflammation. This is associated with increased Cxcl9 expression from the mutant clones and T-cell infiltration into the BM. These observations form the scientific premise for this renewal application. We hypothesize that IFNg production by Dnmt3a-mutant clones suppresses other HSC genotypes and remodels the niche through T-cell infiltration to further reinforce their competitive advantage. We propose the following Specific Aims to investigate these questions;  Determine if IFNg production from Dnmt3a-mutant clones exacerbates their competitive advantage.  Determine if Dnmt3a-mutant clones remodel the BM niche.  Examine the cellular and molecular mechanisms by which Dnmt3a-mutant cells are hypersensitive to IFNg. The overall goal of this work is to determine the mechanisms by which inflammatory signals promote clonal expansion of Dnmt3a-mutant HSCs in the bone marrow. Approaches to eliminate or selectively inhibit emerging DNMT3A-mutant HSC clones from high-risk CH+ individuals may provide a window for intervention before the mutant cells are able to establish clonal dominance and evolve to fulminant disease.

NIH Research Projects · FY 2024 · 2020-08

The goal of this mentored career development award is to enable the candidate’s transition to independence as a physician-scientist studying the connection between plasticity, sleep and stroke. The candidate is an MD, PhD sleep neurologist with a background in engineering, human sleep electrophysiology and how it relates to brain plasticity. The award will help the candidate gain experience in basic genetic, molecular and in-vivo imaging techniques as well as an in-depth knowledge of the cellular and molecular mechanisms involved in plasticity mediated neuronal repair. This award will help the candidate to become an independent physician scientist using sleep as way to accelerate the pace of scientific discovery and its application to the care of individuals with neurological disease. This career development award brings together three experts covering the diverse fields of sleep (Landsness – PI), plasticity of brain recovery (Lee – Mentor) and optical neuroimaging (Culver – Mentor) to tackle this innovative concept and potentially open-up a new field of research. The research environment in which this career development award is proposed is outstanding. Dr. Jin Moo Lee, a translational neuroscientist and vascular neurologist with an interest in the plasticity of stroke recovery, has a long track record of both scientific and mentorship success. Dr. Joe Culver, is a long-time collaborator of the Lee lab and highly experienced in the imaging techniques the candidate will use. Finally, the Washington University neuroscience community emphasizes high quality research, career development of young faculty and collaboration, all keys to his success. An objective of this proposal is to understand the role of slow wave sleep (SWS) in repair and recovery after focal ischemic brain injury. Local SWS is critical for learning-related brain plasticity. Mechanisms involved in brain plasticity have been postulated to be necessary for successful neural repair and recovery after brain injury. Neuronal activity in somatostatinergic interneurons (SOMi) has recently been shown to critically mediate SWS. We propose to 1) determine the role of global SWS in a mouse model of brain repair following focal ischemia (photothrombosis) in somatosensory cortex and 2) locally manipulate SWS (via SOMi using chemogenetics) to determine if modulating local SWS affects cortical remapping, synaptogenesis, and sensorimotor recovery. If the hypothesis is correct, it will show that locally manipulating SWS can selectivity drive plasticity and ultimately recovery from stroke. It will also determine if SOMi might be amenable to targeting and may help shape a novel therapeutic approach to enhancing plasticity and recovery following stroke. This career development award is the ideal platform for the candidate to acquire important training in basic research techniques, deepen his understanding of the role SWS in repair and recovery of stroke, and will launch him towards an independent research career focused on using sleep to aid in care of individuals suffering from stroke.

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY Interfaces between tissues either transfer load (requiring toughness) or provide a smooth surface (requiring low friction). Fibrous interfaces are very effective at transferring load between tissues, e.g., at connective tissue-bone interfaces (“entheses”), peritoneal-mesentery interfaces, interfaces between layers of the vasculature, and the pia mater. These interfaces require toughness to resist high stresses associated with material mismatches. Surgical repair can lead to smooth interfaces becoming fibrous, (e.g., following hernia surgery) or to tough interfaces becoming weak (e.g., following tendon- and ligament-to-bone repair). In older patients with large rotator cuff repairs, for example, where the desired attachment is not reformed, up to 94% of surgical repairs fail. These challenges arise in part because the features that endow fibrous interfaces with toughness are not known. We therefore propose to develop a comprehensive modeling and experimental approach for studying the factors underlying the transition from tough to weak in a fibrous interface. Our previous work motivates the hypothesis that disorder is a key toughening feature of fibrous attachments. We will focus initially on the example of tendon attaching to bone, in which microscale disorder underlies the ordered macroscale, graded transition between the two tissues, as a foundation for studying the general problem of adhesion throughout the body. We predict that disorder enhances energy absorption by distributing failure processes and energy absorption over larger volumes of tissue. We propose this as a fundamental mechanism by which fibrous interfaces in the body transfer load effectively. We will test these ideas through two aims: (1) Identify and model the mechanisms of fibrous attachment toughening ex vivo. We will model and experimentally validate how disorder across length scales toughens the tendon-to-bone attachment. Hierarchical molecular dynamics-to- continuum models, enriched by machine learning, will be validated in vitro, in systems with nanoscale control of mineral distributions, and ex vivo, in tissue samples of fibrous attachments. (2) Identify and model the loss of fibrous attachment toughness due to pathologic settings in vivo using murine rotator cuff tendinopathy models. In both aims, nano- through milli-scale characterization will be performed to define the mechanisms driving mechanical behavior. We will test the hypothesis that pathology- induced changes at multiple length scales will predict changes in failure mode. These models and experiments will test the global hypothesis that energy absorption across hierarchies is a fundamental toughening mechanism by which fibrous interfaces resist injury level loads. Taken together, we believe that these new models of fibrous attachment will enable an understanding of how the order and complexity of fibrous attachments leads to effective attachment of tissues.

NIH Research Projects · FY 2024 · 2020-08

PROJECT ABSTRACT Nearly 40% of adolescents suffer from an anxiety disorder or major depression. These disorders usually first emerge during adolescence, and the adverse consequences of them often persist into adulthood. As such, adolescence is a critical developmental period for understanding the biological pathways related to these disorders. Poor inter-rater reliability and discriminability of anxiety and depressive disorders has been a major factor in the NIMH RDoC framework emphasizing the need for studies pairing measures of brain processes with dimensional approaches to psychiatric symptomatology. In line with this framework, the goal of this K99/R00 Pathway to Independence Award is to provide the applicant with the training necessary to identify distinct developmental neural features that are related to shared symptoms across anxiety and depression (distress), as well as neural features that are related to disorder-specific symptoms of anxiety (anxious apprehension) and depression (low positive affect). Furthermore, the applicant will require genetics training to succeed in the goal of quantifying the relative contribution of genetic influence vs. unique environmental influence on the neural features related to anxiety and depression symptomatology. In order to achieve such goals, the applicant will receive unparalleled mentorship by experts in psychopathology, genetics, and advanced neuroimaging methodologies (Drs. D. Barch, N. Dosenbach, A. Agrawal, J. Constantino, J. Luby, C. Sylvester, and D. Greene) and will have access to superb clinical, imaging, and recruitment resources at Washington University. The proposed training plan will enable the applicant to achieve several short-term goals necessary to facilitate his long-term goal of becoming an independent investigator at a Research-I University, including new training in psychopathology and genetics (twin designs). These training goals will be advanced through the proposed research. First, clinical data and functional connectivity (FC) MRI data within the large Adolescent Brain and Cognitive Development (ABCD) sample (n=11,875) will be used to identify and replicate the neural signatures (biotypes) related to transdiagnostic and disorder-specific symptoms of anxiety and depression (Aim 1). A subset of this dataset (ABCD twin dataset; n=800 twin pairs) will be leveraged to assess the heritability of these biotype/symptom relationships (Aim 2.1). Capitalizing on recent advances in FC MRI data acquisition enabling reliable quantification of individual-level FC (precision functional mapping), the applicant will quantify the genetic vs. unique environmental influence on biotype/symptom relationships, pointing towards potential unique causal pathways and unique intervention pathways (Aim 2.2). Notably, the proposed methods can be extended to other developmental neuropsychiatric disorders, setting the stage for early individualized treatment intervention. With a research program that employs multiple converging techniques and analysis methods to interrogate biomarkers of symptoms related to anxiety and depression, the applicant will continue to address research questions relevant to the NIMH throughout his independent career.

NIH Research Projects · FY 2024 · 2020-08

ABSTRACT Challenging auditory environments, e.g., classrooms, school cafeterias, playgrounds, comprise a significant portion of a child's day. Binaural hearing facilitates speech understanding in these environments where sounds and speech overlap, originate from multiple sources, and vary in level. Binaural auditory cues are necessary to understand speech and locate talkers in acoustically complex settings; however, binaural cues are degraded or eliminated for children with asymmetric hearing loss (AHL, defined as severe to profound hearing loss in one ear and mild to moderate hearing loss in the other) or single-sided deafness (SSD, defined as severe to profound hearing loss in one ear and normal or near normal hearing in the other). A cochlear implant (CI) is the only treatment that can provide hearing to an ear with severe to profound hearing loss and thus, the only opportunity for binaural hearing. Yet, children with AHL/SSD are not routinely implanted in the poor ear because the better ear benefits from amplification (AHL) or has normal hearing (SSD). Recently, the FDA granted approval to implant a subset of children with AHL/SSD; however, the approval is restrictive as it is limited to children with profound hearing loss and ≤ 5% word recognition in the poor ear. Furthermore, there is no consensus regarding CI candidacy criteria, assessment tools, or performance outcomes over time for these children. A longitudinal, prospective, multi-center clinical trial employing an FDA-approved protocol is critically needed to address deficits of children with AHL/SSD and explore treatment with a CI. Our proposed sequential, two-phase study allows for comparison of performance growth pre-implant with current hearing aid (HA) technology versus post-implant with a CI. Aim 1 obtains preliminary efficacy data in children with AHL/SSD who receive a CI in the poor ear. Post-implant performance with a CI alone is compared with pre-implant performance with a HA. Measures include word recognition and sound field detection levels. Aim 2 evaluates the effectiveness of bimodal hearing defined as a CI in the poor ear and a HA in the better ear (AHL) or a CI in the poor ear and normal hearing in the better ear (SSD). Bimodal efficacy is measured by comparing: 1) post-implant bimodal scores to pre-implant best-aided scores, 2) post-implant, bimodal scores to better ear alone scores, and 3) change in bimodal performance across time (CI phase) to change in best-aided performance over time (HA phase). Measures encompass speech recognition in noise and at soft levels, localization, and Quality of Life metrics. Essential information related to patient selection, test measures, and methodology will inform clinical management of these underserved populations. In summary, study outcomes will provide crucial evidence-based data regarding the development of binaural hearing abilities in children with AHL or SSD who receive a CI in the poor hearing ear, which is integral to the establishment of standardized treatment.

NIH Research Projects · FY 2025 · 2020-08

The Center of Regenerative Medicine (CRM) at Washington University in St. Louis supports an interdisciplinary post-doctoral T32 program focused on training in regenerative medicine (TRM T32). Breakthroughs in various bioengineering and stem cell technologies over the past decade have provided unprecedented opportunities for the development of new regenerative technologies, yet there is a lack of individuals who have received formal interdisciplinary training. This program is used to implement an integrated training structure that provides a unique, multidisciplinary experience for post-doctoral fellows in the development of new bioengineering approaches and platform technologies for applications in one of 4 research thrusts: 1) Development in the context of regeneration; 2) Cell fate engineering; 3) Biomaterials for cell and tissue engineering; and 4) Molecules, genes, and cells as regenerative therapeutics. The program involves 52 faculty mentors from 13 departments and 12 divisions, spanning the School of Medicine, the McKelvey School of Engineering, and College of Arts and Sciences at Washington University. Trainees are co-mentored by engineering and non-engineering faculty and guided by a mentoring committee. An integral aspect of this training program is the creation of a formal training plan for each individual trainee, based on a self-assessment of skills and career goals (the Individual Development Plan). Interdisciplinary training in regenerative medicine is supplemented by formal coursework and intensive workshops in complementary areas, including statistical analysis, writing, and mandatory training in research ethics, rigor, and reproducibility. We also take advantage of several long-standing and successful initiatives at Washington University to enhance the recruitment of top participants. The primary goal of this postdoctoral program is to develop highly trained individuals who can produce impactful research in regenerative medicine. Our unique interdisciplinary training approach aims to support and accelerate the discovery of transformative technologies and therapeutics for the improvement of human health.

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY Intervertebral disc (IVD) degeneration is one of the greatest contributors to low back pain, yet how the IVD can generate pain remains poorly understood. The parent grant will pursue Specific Aims to study the development of temporal and spatial changes in neuronal function and their “cross-talk” with the degenerating IVD in a mouse model of lumbar IVD degeneration. (1) We request supplement funds to support the mentorship of one post-baccalaurate trainee in research related to the parent grant to enhance her preparedness for applications to a doctoral program. We also request supplement funds to support a second pre-doctoral trainee who will bring strengths of micro-molding and iPSCs differentiation to the in vitro work proposed in the parent grant. For both trainees, we have planned independent research, scientific workshops, fellowship writing, scientific communication and national travel to make the most of this 1-year supplement period. (2) We also request supplement funds to support the goals of a PI-organized, multi-institution Rising BME Scholars Regional Conference (2022-2024). This conference is a partnership of 10 universities, mostly in the Midwest region, intended to retain Ph.D.-level research trainees in academic research careers. We request funds to evaluate 3-year conference outcomes, including job placement, success with fellowship and independent funding, experience with other scholars’ development workshops, and ratings of the conference programming. Working with professionals of an evaluation center, the conference organizers plan to publish results to illustrate a model of inter-institutional collaboration to support trainee success. We believe the process and outcomes will be of broad interest to the biomedical research community, for the potential to increase minoritized scientist representation in research. (3) Based on the premise that research mentors should use their position to advance diversity in academia, we believe an effective mentor must be an effective communicator. For this reason, funds are requested to bring a 2-day scientific communication workshop to campus, to educate 32 faculty mentors and trainees in methods of scientific communication towards the goal of strengthening their advocacy skills for research and for STEM. (4) Finally, supplemental funds are requested to bring engineering undergraduates, post-baccalaureate trainees and junior PhDs to the Annual Biomedical Research Conference for Minoritized Scientists (ABRCMS) in November 2024, and a separate cohort to the Annual Conference of oSTEM in October 2024. Completion of this work will promote retention of emerging scholars in research-focused careers, enhance the quality of faculty research mentorship, and provide critical support to trainees on the parent grant.

NIH Research Projects · FY 2024 · 2020-08

Project Summary/Abstract: This proposal describes a five-year career development program designed to support an academic, physician- scientist career. The proposed research project will capitalize on the expertise and resources available at Washington University School of Medicine, which has a strong tradition of developing physician-scientists. Dr. Timothy Ley, an expert in cancer genomics and epigenetics, and a recipient of multiple mentorship awards nationally and at Washington University, will serve as the primary research mentor. The ultimate goal of the candidate is to be an independent investigator in an academic medical center, studying epigenetic control of cutaneous carcinogenesis, and caring for patients with cutaneous malignancies. The long-term goal of this study is to define alterations in the epigenetic “state” of epidermal keratinocytes that arise during normal aging and their roles in creating age-related susceptibilities for skin cancer. Increasing age and UV light exposure are the two most prominent epidemiologic risk factors for the development of skin cancer in fair skinned populations. Recent studies have identified clonal expansions of cells harboring oncogenic mutations from clinically normal skin, suggesting that additional genetic or epigenetic events are required for transformation to skin cancer. Aging and UV light exposure not only cause DNA damage and mutations, but also have been demonstrated to cause DNA methylation changes in the skin of human patients; whether these alterations are relevant for skin cancer pathogenesis is currently unknown. Our preliminary data suggests that the DNA methylation state of epidermal cells undergoes a programmed change at specific loci in mice as they age, as do stereotypical changes in gene expression resulting in the development of a population of cells that we have named “basal aging-signature keratinocytes” (BASKs). We will explore the hypothesis that age-related epigenetic changes, including DNA methylation, may increase susceptibility to skin cancer development with the following specific aims: Aim 1: We will define the epigenetic events in murine epidermis that result from normal aging, relate them to functional changes, and assess their roles in the development of skin cancers. We will define the epigenetic changes in BASK cells, define biomarkers that allow for the purification of this population, and test the susceptibility of aged skin to KRASG12D- mediated skin tumorigenesis and the development of UVB-induced, mutated Trp53 clonal islands in the skin. Aim 2: We will define the roles of individual DNA methyltransferases for the development of age-dependent methylation states and for the neoplastic transformation of skin. Using mice conditionally deficient in Dnmt1, Dnmt3a, and/or Dnmt3b in epidermal cells, we will determine whether these enzymes contribute to age-related development of the BASK phenotype, and whether their deficiencies are relevant for KRASG12D-mediated skin tumorigenesis. If successful, insights gained from this work may allow for the creation of novel therapeutic or preventative approaches for the keratinocyte cancers, basal cell and squamous cell carcinoma.

NIH Research Projects · FY 2024 · 2020-08

Mechanosensitive mechanisms regulating cellular coordination during tissue morphogenesis and patterning Abstract: The long-term goal of this research program is to understand and identify mechanosensitive mechanisms that regulate cell-to-cell coordination of movements during normal tissue morphogenesis and patterning. Of particular interest, is the role that mechanosensitive and stretch activated proteins play in the transfer of electrical currents, ions, and second messengers between cells, as these functions are known to be critical for coordination of cellular communication within a complex tissue environment. For instance, our sense of touch, regulation of blood pressure, osmotic regulation, and balance are all regulated by mechanosensitive channels throughout the body. The importance of mechanosensitive channels is underscored by the association of many disease states with compromised mechanosensation, including atrial fibrillation, muscular degeneration, arrhythmias, polycystic kidney disease, and numerous neural diseases. Despite this, a relatively small amount is known at the level of normal, healthy individual cells about how mechanosensitive channels go from sensing force to eliciting changes in cellular signaling and/or function. Our interest therefore lies in understanding how cells assimilate ‘data’ from mechanosensitive channels to alter intra- and inter- cellular communication and coordinate individual cellular movements within tissues. To carry out this work, we plan to utilize our historic strengths in zebrafish development and tissue patterning along with sophisticated 3-dimensional in vitro tissue modeling assays to understand: 1) how mechanosensation affects intracellular signaling, particularly though the activation of transcriptional networks and altered gene expression, and 2) how mechanosensation affects intercellular signaling activities to alter patterning of tissues. We will target and utilize highly mechanosensitive cells, such as astrocytes, endothelial cells, smooth muscle cells, and epidermal cells, for our studies to understand both generalizable and cell type specific roles of mechanosensation in regulating gene expression, cellular motility, and cell-to-cell communication. These studies will provide fundamental data and cell biological knowledge to the community studying mechanosensitive channels.

NIH Research Projects · FY 2025 · 2020-07

Overall Project Abstract For the third cycle of the Intellectual and Developmental Disabilities Research Center at Washington University, we propose a next phase in a comprehensive approach to understanding, ameliorating, and/or preventing neurodevelopmental disability through translational scientific investigation at the respective levels of cell, synapse, circuit, and behavior, capitalizing upon major strengths of WUSTL in genomics, behavioral/cognitive neuroscience, and clinical-translational science. The overarching goals of our Center are as follows: (1) To sustain and evolve an integrated structure of core scientific facilities that occupy a critical niche in the scientific community, attract and support highly-qualified investigators, and facilitate high-caliber, translational research on the pathogenesis and treatment of IDDs. In this application we propose specific enhancements to each of our scientific core facilities: an expanded technical team for the Developmental Neuroimaging Core, a dedicated cellular models unit within the Model Systems Core (methods calibrated with a cross-IDDRC working group for cellular models of IDD co-led by the IDDRC@WUSTL), and a new clinical trials / natural history studies unit within the Clinical-Translational Core (CTC). The CTC will continue to facilitate the collection and interpretation of genomic, phenotypic, environmental and biomarker data across generations, and promote step-wise translation of new discoveries on risk and pathogenesis to higher-impact interventions for patients. The IDDRC@WUSTL will provide critical infrastructure for research efforts that have created synergies with other intramural and extramural Centers/Institutes, including a newly-funded in-depth longitudinal study of infants born to mothers enrolled in the March of Dimes Prematurity Research Center Cohort, the launch of a prospective replication cohort for the Infant Brain Imaging Study of Autism (IBIS), two multisite initiatives in Down Syndrome, and an NIH Autism Center of Excellence Network in gene discovery. (2) To cultivate nodes of new interdisciplinary scientific activity within the Center, in frontiers of IDD research which are critical for the derivation of higher-impact treatment and preventive intervention, along the Center’s four major themes: (i) the prevention of prematurity and its neurodevelopmental consequences; (ii) the identification of intermediate phenotypes in the development of IDD; (iii) structural and functional characterization of the developing human brain, and (iv) functional genomics relevant to IDD pathogenesis. In this cycle we will build on prior successes in cultivating a dynamic, interactive, and productive community of scientists engaged in IDD-science, challenging itself to generate and harness new knowledge toward translational advances in therapeutics and prevention. (3) To conduct a signature research project that represents a bold, critical step toward higher-impact intervention for IDD. In this project, a novel platform for standardizing multi-omic characterization of the consequences of variation in gene dosage will be implemented across dozens of isogenic cell lines, each representing haploinsufficiency in a different high-confidence IDD-related gene, to identify convergent mechanisms of IDD.

NIH Research Projects · FY 2025 · 2020-07

Over the past 30 years, the scope of systems neuroscience has expanded enormously. Functional brain imaging provided the opportunity to study the neural mechanisms of complex cognitive functions in humans. Concurrently, powerful techniques such as 2-photon microscopy, optogenetics, and high-density probes have made it possible to dissect neural circuits in animal models with unprecedented resolution. In parallel, computational approaches such as Bayesian models, dynamic systems, and deep neural networks became increasingly central to many research programs and to the field as a whole. Thus, systems neuroscience is increasingly interdisciplinary, integrating frameworks and techniques from molecular biology, neurophysiology, cognitive science, ethology, computer science, statistical physics, and more. In the face of this remarkable expansion, PhD programs face three challenges. First, the body of knowledge relevant to systems neuroscience has increased in breadth and depth. Yet, there is pressure on students to conduct research, publish, and get independent funding early in their career. This pressure translates into reducing the coursework and increasing time in the lab. Second, systems-level research is conducted in multiple departments and PhD programs. At WashU, these include the PhD programs in Neuroscience, Psychological & Brain Sciences, Biomedical Engineering – and more. Yet, students coming from different disciplines often do not speak each other’s language. Third, a successful career in science requires a sophisticated understanding of statistics and a broad portfolio of skills – writing papers and grant proposals, collaborating with colleagues with different scientific backgrounds, presenting results in scientific venues and to wider audiences, navigating the academic job market – that exceed the normal coursework. The Cognitive, Computational and systems neuroscience (CCSN) pathway was developed in response to these challenges. CCSN is an elite pathway available for graduate students in Training Years 3-4 and for post-docs in Training Years 1-2. The emphasis of CCSN is on interdisciplinary training, statistical education, and career development. CCSN students take foundational courses on cognitive science and animal behavior, systems neuroscience, and computational neuroscience. In addition, CCSN students and post-docs take semester-long courses that help them (a) learn best practices in data science and quantitative literacy and (b) develop a grant proposal aided by peer, instructor, and committee feedback. This proposal often develops into an actual NRSA application. In addition, CCSN trainees take part in Career Development activities including seminar presentations, organizing scientific events, hosting external speakers, participating in informal dinners with CCSN faculty, and community outreach events. CCSN has existed for ~20 years and has a demonstrated history of success. Here we request funds for 6 student and 2 post-doctoral fellowships. Contingent on the success of this application, WashU will provide matching funds for an additional 5 student slots.

NIH Research Projects · FY 2024 · 2020-07

Cilia (also known as flagella) are hair-like organelles protrude from the surface of most eukaryotic cells and are responsible for cellular motility, fluid flow and sensory perception. A large group of human diseases, known as ciliopathies, are caused by cilia dysfunction. The elongated shape of the cilium is supported by a highly conserved structure called the axoneme. In most motile cilia, the axoneme has a 9+2 architecture in which nine doublet microtubules (DMTs) surround a central pair of singlet microtubules (MTs). Bound periodically along the length of each DMT are a variety of MT-associated proteins and complexes that decorate the external and luminal surfaces with different periodicities (8,16, 24, 48 and 96-nm). These protein complexes are found in coherent register along the entire length of the DMT, and loss of the coherence causes impaired motility. How periodicity is established, maintained, and synchronized, especially over a long distance, has been a long-standing question in the field. Each DMT has a distinctive structure with one complete ring of A- tubule and one incomplete ring of B-tubule. How the unique architecture of the DMT is formed in vivo is still unclear. Furthermore, many axonemal components are asymmetrically distributed in both the longitudinal direction and the radial direction among the 9 DMTs. For example, 3 of the 9 DMTs contain a unique “beak” structure in the proximal B-tubule lumens. To date, the molecular components and biological functions of the beak are unknown. In this proposal, based on the identification of 33 microtubule inner proteins (MIPs) in our recent work using high-resolution cryo-electron microscopy (cryo-EM), we propose to elucidate the functions of individual MIPs during ciliogenesis using Chlamydomonas mutants. The objective of this application is to use a combination of genetic and structural approaches to investigate the architectural principles governing the assembly of DMTs and axonemes. We will focus on three key aspects of the architectural principles with the following specific aims: (1) We will identify the key proteins responsible for maintaining coherent registry between different periodicities, and investigate their mutual dependence, using Chlamydomonas mutants lacking filamentous MIPs, external coiled-coil proteins, and proteins located at interfaces between different repeat regions. (2) We will investigate the molecular mechanism governing B-tubule formation, and test two hypotheses: (i) proteins located at the MT seam, the unique site within the A-tubule, are essential for B-tubule formation; (ii) MIPs located at the outer junction (OJ) function to promote the B-tubule formation by shielding the inhibitory effects of tubulin C-terminal tails at the OJ. (3): We will identify protein components of the beak using high-resolution cryo-EM. Our preliminary data suggest that two main components are tektin filaments and SAXO proteins. We will investigate their cellular functions and relevance to human diseases using Chlamydomonas mutants and in vitro assays. Most of the proteins studied here have orthologs in human cilia; therefore, our work will provide a molecular basis for understanding the etiology of many human ciliopathies.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY Female lower urinary tract disorders, such as urinary incontinence and urinary tract infections, are common (affecting millions of women each year), costly, and sub-optimally managed. Additionally, insufficient research is performed in this area, preventing the development of new and personalized therapies and strategies to optimize care and well-being. This is, in part, because few physicians and scientists are trained to rigorously study these disorders. This T32 will help meet this need by training MD clinical fellows and PhD post-doctoral fellows to become leaders in the field of clinical outcomes research in female lower urinary tract disorders. Specifically, the aims of this Washington University School of Medicine T32 Program are to: 1) establish a rigorous recruitment and selection process to identify the most promising and diverse research-focused MD and PhD fellows; 2) provide a structured, yet flexible, environment in which trainees receive individualized mentoring and comprehensive training in female lower urinary tract disorders, research methods, the ethical conduct of research, and grant-writing; 3) provide financial support and (protected) time to allow trainees to obtain a Master’s degree in Clinical Investigation or Population Health Sciences, or relevant coursework in clinical outcomes research; 4) continue and augment a robust existing conference and didactic series in which trainees discuss current, clinical topics in female urinary tract disorders and research methods relevant to these disorders; 5) implement a robust professional and career development program tailored to enhance trainees’ transition to junior faculty status; 6) create a supportive, responsive, and flexible administrative structure to attract and nurture the most exceptional trainees, particularly women and under-represented minorities; and 7) continually evaluate and improve the Program in response to trainees’ needs. This Program will accept clinical (MD, MD/PhD) and post-doctoral PhD fellows for up to three years of research training by a mentoring team composed of at least one clinical and one methodologic (clinical outcomes) researcher. At least one of the mentors (the primary) will be a senior researcher or clinician who is established in his/her field, funded, and has an excellent mentorship track record. The Program structure is designed to foster a trans-disciplinary and collaborative culture, with the goal that these cooperative relationships will carry into the future. To that end, MD and PhD trainees will be united at weekly clinical and research conferences and in career and professional development activities. Fellows will also have the option of matriculating in a Master’s degree program to learn clinical outcomes research methods, or enhancing their existing skill set with specific graduate level courses. This program will prepare the next generation of MD and PhD researchers in female lower urinary tract disorders to perform high- quality clinical outcomes research, bringing much-needed methodologic rigor and a collaborative, trans- disciplinary approach to the most pressing questions in this field.

NIH Research Projects · FY 2024 · 2020-07

ABSTRACT Dendritic cells (DCs) play critical roles in the regulation of anti-tumor immune responses. In both mice and humans, DCs consist of distinct subsets with intrinsic differences that lead to functional specialization in the generation of immunity. Compared to mice, little is known about the functional classification of human tissue- resident DCs, and how subset distribution is altered in disease. My laboratory has published pioneering studies on the identification and functional characterization of human DC subsets with respect to their control of cellular and humoral immunity. This application focuses on our recent discovery of a new human DC subset that is delineated by CD5 expression and that we discovered initially in human skin, blood and lymph nodes. CD5+ DCs are present in both mice and humans. They efficiently polarize T cells into helper and cytotoxic responses, and their number is reduced in melanoma affected compared to healthy tissue. Preliminary data using human DCs and pre-clinical mouse models are provided to demonstrate that CD5 is a critical molecule on DCs that sustains T cell activation and tumor rejection in vivo. Although the survival of advanced melanoma patients has been extended by interventions that enhance T cell activation, the response is limited to a minority of patients. Thus, we hypothesize that harnessing the CD5 costimulatory system on DCs will contribute towards the generation of a broad T cell response and immunological memory that is required to completely eliminate melanoma cells. To address this hypothesis, we will 1) harness novel murine models developed in our laboratory to define the requirement of CD5 on either DCs or T cells in tumor immunity, anti-tumor vaccination and in shaping response to checkpoint blockade therapy; 2) determine the molecular interactions underlying the activation and anti-tumor T responses by CD5+ DCs; 3) determine whether the distribution and the function of the CD5+ DCs is restored in patients with melanoma that respond to checkpoint blockade therapy. These studies will advance our understanding of a novel mechanism by which dendritic cells function to target cancer cells and will provide a rationale for harnessing CD5+ DC biology in a therapeutic setting.

NIH Research Projects · FY 2024 · 2020-07

Project Summary The overall goal of this research is to better understand the analgesic properties of central noradrenergic systems. Emotional regulation in the face of physical injury and psychological trauma is critical to long-term survival and quality of life. Uncontrollable anxiety, anhedonia, and depression often result following periods of prolonged stress or chronic pain. Both chronic pain and stress lead to overlapping physiological adaptations such that the same tricyclic and serotonin/norepinephrine reuptake inhibitors (SNRIs) developed and used to treat depression are also effective in treating chronic pain. Therefore, norepinephrine (NE) is likely one of the key neurotransmitters regulating pain processing during stress. In this proposal we seek to define the role of NE in stress-induced modification of pain. The locus coeruleus-noradrenergic (LC-NE) system is one particular central nervous system target that holds promise for interventions in both chronic pain and stress-induced psychiatric disorders. This research focuses on understanding the mechanisms by which the LC-NE system modulates endogenous analgesia and how chronic stress affects this system. The central hypothesis of this proposal is that LC-NE neuronal activity is critical for stress-induced modulation of nociception. The first aim of this proposal will assess the role of LC-NE neurons in acute stress-induced antinociception using in vivo optogenetics and chemogenetics. The second aim seeks to understand the mechanism for the transition from acute stress-induced antinociception to chronic stress-induced pronociception. In particular, this aim seeks to determine whether repeated LC-NE stimulation from repeated stress exposure drives stress-induced pronociception. To do so we will use, using in vivo optogenetics, chemogenetics, and intersectional genetic models to remove LC-NE function during repeated restraint stress. The final aim seeks to clarify how two different models for studying chronic stress reveal opposing pain-related phenotypes. Here, we will use brain slice electrophysiology and in vivo fiber photometry to monitor LC-NE activity following these stress paradigms and in response to noxious stimuli. Together these experiments will generate previously unattainable information about LC-NE neurons and associated efferent circuitry that regulate the pain-related behaviors in response to stressors. These studies will define the role of the LC-NE system; 1) in acute stress-induced analgesia, 2) the transition to chronic stress-induced hyperalgesia, and 3) identify mechanisms by which different forms of stress alter LC-NE function and nociception. This information will be critical for translational research targeting the noradrenergic system in the treatment of pain and neuropsychiatric disorders.

NIH Research Projects · FY 2024 · 2020-07

Program Director/Principal Investigator (Last, First, Middle): Faccio, Roberta, PhD ABSTRACT Recent evidence from our lab and others indicates that the insurgence of a primary tumor leads to changes in the bone microenvironment that affect the residing hematopoietic and mesenchymal populations. These changes in the bone microenvironment occur during the early stages of tumor development, prior to any evidence of metastatic dissemination, and become more pronounced while the primary tumor progresses, even in tumors that do not metastasize to the bone. We find increased numbers of immature myeloid cells that can actively suppress T cell proliferation in bone marrow of mice bearing primary breast tumors and in newly diagnosed stage II-III breast cancer patients compared with controls. In addition to changes in immune populations, bone residing osteoblasts and osteocytes, normally involved in bone formation and regulation of bone homeostasis, respond to distal tumors by upregulating various inflammatory cytokines as well as inhibitors of the Wnt signaling pathway, such as Dkk1. Several of these bone-produced factors act locally to change the basal bone homeostasis, but can also have systemic effects that can impact, either directly or indirectly, primary tumor growth and future tumor dissemination to various sites. Wnt signaling plays important roles in bone development, hematopoiesis and cancer. Dkk1 is a Wnt/β-catenin inhibitor, known to suppress bone formation and promote bone resorption. Increased levels of Dkk1 are often observed in cancer patients and correlate with poor prognosis. We recently reported that Dkk1 is upregulatated by osteoblasts and osteocytes in the bone of mice with primary breast tumors and in cancer associated fibroblasts (CAFs) in the primary site, but at a level much lower than in the bone. Intriguingly, we found that Dkk1 exerts immune suppressive effects and its neutralization decreases primary tumor growth by reducing immature myeloid populations and enhancing T cell immune surveillance. Based on these exciting findings, our central hypothesis is that bone residing osteoblasts and osteocytes modulate primary breast cancer growth and metastatic dissemination to multiple sites by creating a systemic immune suppressive environment via production of Dkk1. Aim1. Exploring the role of the bone microenvironment during tumor progression. The goal of this aim is to determine the anti-tumor effects of Dkk1 deletion in bone versus its deletion at tumor site. Aim2. Determine the mechanism by which Dkk1 modulates immune suppression. The goal of this aim is to determine the cellular mechanism by which Dkk1 induces immune suppression. OMB No. 0925-0001/0002 (Rev. 03/16 Approved Through 10/31/2018) Page 1 Continuation Format Page

NIH Research Projects · FY 2024 · 2020-07

Abstract In patients under suppressive antiretroviral therapy (ART), HIV-1 persists in a latent state primarily in resting CD4+ T cells. Both neutralizing and non-neutralizing antibodies (nNAbs) targeting HIV-1 envelop protein can mediate killing of infected cells through antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells or antibody-dependent cellular phagocytosis (ADCP) by macrophages and other phagocytes. Since ART does not directly kill HIV-1-infected cells, antibodies can play an important role in elimination of the residual viral reservoirs. Broadly reactive neutralizing antibodies (bNAbs) that could neutralize a wide spectrum of clinical HIV- 1 isolates brought new hope to HIV cure research. There is burgeoning evidence that the Fc fragment is important to the antibody-mediated viral suppression. Moreover, the Fc fragment is indispensable for antibody- mediated cell killing because of its engagement with Fc-receptors (FcR) on the innate effector cells. It is important to evaluate antibody efficacy in tissues, especially in lymphoid tissues, because they are the major sites for HIV- 1 infection and are the most important anatomical reservoirs for HIV-1. The cell-killing functions of tissue innate effector cells are critical to antibody therapeutic effects. We compared NK cells from human tonsils or lymph nodes with those from peripheral blood, and found that vast majority of the NK cells in lymphoid tissues are immature and deficient in ADCC activity because:1) they do not express Fc-gamma receptor IIIA (CD16a); 2) they produce very little cytolytic molecules such as granzyme B and perforin. The objective of this grant is to study how to improve Fc-dependent effector cell functions to clear HIV-1 reservoirs using human tissue biopsies and humanized mouse models. We aim to: 1) develop the novel approaches to enhance ADCP activity; 2) improve maturation of NK cells in lymphoid tissues to acquire ADCC activity. Our study will provide critical implications to antibody-based HIV cure research.

NIH Research Projects · FY 2024 · 2020-07

ABSTRACT In 2019, approximately 268,600 women in the US will be diagnosed with breast cancer, making it the leading cause of cancer in women. About 25% of these cases occurred in premenopausal women. The annual incidence of breast cancer among women under age 50 has been increasing slowly since the mid-1990s, in contrast to what is observed in women aged over 50 years, where the incidence has remained stable over time. This persistent increase in the incidence among younger women indicates that new approaches to primary prevention in premenopausal women are needed. Mammographic breast density is one of the strongest risk factors for breast cancer, especially in premenopausal women, where an estimated 39% of breast cancer cases is attributable to having dense breasts. A decrease in breast density leads to a reduction in breast cancer incidence. Nevertheless, the molecular basis of mammographic breast density and the mechanisms through which dense breast increases breast cancer risk are poorly understood. A greater understanding of these mechanisms is crucial, and will uncover new biological pathways and actionable biomarkers that can be targeted to prevent breast cancer development. Metabolomics is a promising tool to provide novel insights into disease etiology, biological mechanisms, and pathways. Metabolomics has, however, not been applied to study mammographic breast density. Our pilot analyses show differences in metabolite levels between women with fatty breasts compared to women with dense breasts. Building on these novel findings, we will apply state-of-the art metabolomics platforms to 1) investigate the metabolome of mammographic breast density in premenopausal women; 2) quantify the variation in mammographic breast density explained by the metabolome; 3) determine whether the metabolome of mammographic breast density predicts breast cancer development in premenopausal women. Our overarching hypothesis is that we will leverage metabolomics to uncover the molecular mechanisms, biological pathways, as well as novel actionable biomarkers that are associated with mammographic breast density in premenopausal women. Our study population will consist of premenopausal women recruited during annual screening mammogram at the Joanne Knight Breast Health Center at the Washington University School of Medicine, St. Louis, MO. Mammographic breast density is quantitatively assessed using Volpara. Fasting blood samples are collected on the same day the women have their mammograms. The women also complete a questionnaire with detailed information on breast cancer risk factors and determinants of mammographic density. In conclusion, we will build on our exciting preliminary data to uncover novel, actionable biomarkers associated with mammographic breast density, and also breast cancer development in premenopausal women. These biomarkers could be targeted in breast cancer prevention in future studies.

NIH Research Projects · FY 2026 · 2020-07

ABSTRACT Lung transplantation (LT), the only curative therapy for end-stage lung disease, improves survival and quality of life. Scarcity of donor organs remains the predominant barrier towards wider application of LT, yet the lung utilization rate (LUR) from brain-dead donors (BDDs) is only 25%. Two predominant factors account for the low LUR: heterogeneity in assessment of organs, and uncertain impact of donor quality on long-term recipient outcomes. This leads LT programs to adopt a conservative approach, declining up to 30% of potentially acceptable lung donor offers. To address both these factors, our group developed and validated a risk score, which is highly predictive of lung utilization from BDDs (c-statistic 0.89). The lung donor (LUNDON) score is derived from a parsimonious model comprised of nine routinely collected co-variates, four of which (PaO2/FiO2 ratio, Chest X-ray, creatinine, bloodstream infection) are potentially modifiable via donor management. Moreover, the score predicts long-term survival, particularly in high acuity recipients. Our data demonstrate wide variations in LURs nationally (14-40%) indicating the urgent need for standardization of donor management and acceptance practices. In partnership with the Organ Procurement Transplant Network (OPTN), using implementation science to inform this study and leverage our findings from the ongoing work, we propose the following aims: Aim 1. To identify the determinants of implementing the LUNDON score for lung transplantation. We will employ a sequential mixed methods approach using the capacity, opportunity, and motivation (COM-B) and Theoretical Domains Framework to identify facilitators and barriers for implementing the LUNDON score. Aim 2. To evaluate the impact of the LUNDON score on donor management practices. Using SimUNet, a unique platform developed by OPTN, participants who manage lung donors will be recruited for a simulated study and provided hypothetical donor scenarios with or without knowledge of the LUNDON score. We hypothesize that knowledge of the LUNDON score will promote lung protective ventilation strategies. Aim 3. To evaluate the impact of the LUNDON score on lung utilization rates. We will conduct a simulated study via SimUNet, where LT clinicians who evaluate lung donor offers will be asked to assess clinically relevant, lung offers for hypothetical recipients with or without knowledge of the LUNDON score. We hypothesize that providing the LUNDON score will improve the LUR. Aim 4. To develop a toolkit for implementing the LUNDON score. Through an adapted premortem exercise, we will develop, refine and pilot two distinct toolkits to support the implementation of the LUNDON score at organ procurement organizations (OPOs) and LT centers, respectively. Impact: Our proposal, with strong support from the OPTN and OPOs, will establish an evidence-based approach for lung donor assessment and utilization.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY Natural genomic variants that influence cryptococcal pathogenicity Cryptococcus neoformans is a global pathogen responsible for hundreds of thousands of deaths yearly in HIV+ individuals and increasing morbidity in non-AIDS patient populations. C. neoformans strains collected from pa- tients with cryptococcal meningitis have been used to elucidate C. neoformans evolution and in efforts to corre- late disease outcome with in vitro measures such as virulence factor production or fungal growth. We know that distinct genome sequences are associated with varied clinical outcomes. However, we do not know which natural genomic variants are responsible for the differential virulence of clinical isolates. Our long-term goal is to understand the molecular mechanisms by which natural variation in the cryptococcal genome determines pathogenicity. A first step toward this goal is to identify naturally occurring variants that can be experimentally proven to influence virulence, at single-gene resolution. The next step is to characterize the impact of variants in these genes on cryptococcal biology and disease progression. We hypothesize that we can combine genetic and genomic methods with experimental follow-up to identify, validate, and characterize natu- rally occurring variants that significantly influence cryptococcal virulence. This hypothesis is supported by our compelling preliminary data, in which two distinct genetic methods implicate variants in the same genomic region as influencing infectivity. We propose to achieve our goals by pursuing three Aims: Aim 1 is to use two distinct, complementary statistical genetics methods to discover genomic regions containing naturally occurring variants that influence fungal viru- lence. Aim 2 is to further apply genetics, supported by expression analysis and mutant studies, to refine these regions and identify specific high-priority genes within them. Aim 3 is to use genome engineering to validate the influence of selected variant genes on virulence and to assess their effects on fungal gene expression, physiol- ogy, and pathogenicity in a mouse model. Applying an unbiased strategy to identify genes harboring natural variants that influence virulence will highlight new potential targets for antifungal therapy. Characterizing these variants will also pave the way for future inves- tigations of the mechanistic basis of their effects, which will elucidate key features of cryptococcal biology and pathogenesis. Our methods are applicable to other fundamental questions in cryptococcal biology, and poten- tially to other microbial pathogens. Finally, our studies will produce valuable reagents and data sets that will enhance investigations by other C. neoformans researchers.

NIH Research Projects · FY 2025 · 2020-07

PROJECT SUMMARY/ABSTRACT The rising incidence in early-onset colorectal cancer (CRC diagnosed before 50), has resulted in updated guidelines advising average-risk screening to begin at age 45. Debates centered around the substantial cost and resources of adding 21 million adults at very low risk to the screening pool, and “further personalize screening strategies” was a priority. Identifying the contributors of the rising incidence are the first steps but thus far an unmet need. Lifestyle factors that preceded and mirrored the rapid rise of early-onset CRC, including obesity, prolong sitting, and poor diet, may play a critical role. Our preliminary data support the importance of obesity and sedentary behaviors and early-onset CRC are more likely to be processed from traditional adenoma-carcinoma sequence compared to CRC diagnosed after age 65. Therefore, investigation into risk factors for early-onset advanced adenoma, the major targets of screening, will illuminate insights of colorectal carcinogenesis at younger ages. Accumulating data suggest that microbial translocation/endotoxemia, which triggers subsequent inflammation and immune response, and augmented by above-mentioned lifestyle factors, might be an emerging pathway. We hypothesized that obesity, prolonged sitting, and poor diet quality increase risk of early-onset advanced adenoma through increasing endotoxemia and inflammation, and contribute to the rise of early-onset CRC. To test these hypotheses, we will leverage lifestyle data collected throughout life course in two well- characterized prospective cohort (Nurses’ Health Study II [NHSII]) and Southern Community Cohort Study (SCCS) with archived pre-diagnostic blood, complemented by decision modeling using the Microsimulation Screening Analysis‐Colon (MISCAN‐Colon). In addition to risk factors, we will also elucidate the role of promising pharmacological agents for the prevention of early-onset CRC. We will also take a step further to conduct in- depth interviews among stakeholders to set the stage for behavioral and molecularly driven intervention trials aiming to address the unique needs and challenges in prevention and early detection. Our interdisciplinary team, led by a leader in early-onset CRC etiology, and leaders in causal inference, biomarker measurement and discoveries, decision modeling, and qualitative research, offers unparalleled expertise. This investigation will illuminate significant insights into the etiology of early-onset CRC and will be a significant step forward to optimal/personalized prevention and early detection of early-onset CRC among younger adults.

NIH Research Projects · FY 2026 · 2020-07

Cellular and Molecular Mechanisms of Germline Regeneration PROJECT SUMMARY Humans and well-established research organisms lack the ability to regenerate their reproductive cells (germ cells) and reproductive organs. Research findings from these organisms established the current view that germ cells are a distinct lineage separated from the soma; therefore, the loss of germ cells renders an organism infertile because new germ cells cannot be derived from the soma. Contradicting this widely accepted view is the fact that many organisms (e.g. hydra, flatworms, segmented worms, and sea stars) can readily regenerate germ cells. However, the cellular source of regenerated germ cells in these organisms is very poorly understood. The goal of my laboratory is to close this knowledge gap and define the cellular origins and molecular mechanisms of germ cell regeneration. Addressing this is not feasible using established research organisms like mice, fruit flies, and nematodes, because they do not regenerate germ cells. Furthermore, many of the organisms that can regenerate germ cells are not conducive to studying the mechanisms of this process because they lack transgenic tools, or their anatomies present technical challenges such as large and opaque bodies, or inaccessibility of reproductive organs. We use a segmented worm, Platynereis dumerilii, for studying germ cell regeneration. Platynereis is well-suited for this study because germ cell regeneration can be induced and is achieved quickly; transcriptome databases, a draft genome, and transgenic tools (critical for genetic lineage tracing) are available; and a small and transparent body makes it excellent for imaging. Therefore, Platynereis is a research organism that presents a rare opportunity to combine the modern techniques required to study germ cell regeneration (imaging, genetic lineage tracing, transcriptomics) in the relevant post- embryonic life stages (i.e. juveniles, adults). In this renewed funding period, we propose to focus on to themes that fall under germ cell development and regeneration in Platynereis. Theme 1 will investigate the mechanisms of germ cell development and regeneration via: a) functional genetic ablation methods, transgenesis, and tissue transplantation to identify the exact cell lineages that give rise to germ cells during development and regeneration. b) Bioinformatics approaches (e.g. single cell RNA sequencing) to identify the molecular changes taking place during reprogramming source cells into germ cells. This will allow us to obtain transcriptome trajectories over the course of regeneration and identify cell type-specific markers in the source cells, the intermediate states, and the new germ cells. Theme 2 will focus on the neurohormonal regulation of germ cell development and regeneration, which will allow us to start identifying systemic factors involved in these processes. The project will significantly contribute to our fundamental understanding of germ cell biology and the soma-germ cell distinction.

NIH Research Projects · FY 2024 · 2020-07

Abstract The host microenvironment is necessary for tumor growth and metastasis, and a major determinant of resistance to treatment and relapse. Expression of N-cadherin (Ncad), a calcium-dependent cell-cell adhesion molecule, in cancer associated fibroblasts (CAF) has been reported to favor tumor growth. Ncad is the main cadherin expressed in bone cells, where it functions in cell-cell adhesion, but also regulates signaling and differentiation. In preliminary studies we found that, contrary to expectations, ablation of the Ncad gene (Cdh2) in osteolineage cells – expressing the osteogenic marker, Osterix (Osx+) – does not affect bone engraftment of breast cancer cells; however, subcutaneous tumors grow faster and lung metastases develop earlier than in wild type littermates. We also find, unexpectedly, that Ncad is present in previously unrecognized Osx+ cells in extra-skeletal tumors. These cells have a transcriptomic profile more similar to osteogenic cells than to CAF, and favor tumor growth. Furthermore, Ncad in Osx+ cells down-regulates p38 responsive genes, a pro- tumorigenic pathway. In human breast cancer, Osx+ are an index of poor prognosis. These preliminary results demonstrate that Ncad in Osx+ cells is a negative regulator of cancer progression, an effect opposite to Ncad reported action in CAF. We contend that Ncad exerts multiple and even opposite actions on tumorigenesis depending on the cell context where it is expressed, via modulation of specific signaling pathways. Based on these preliminary data, our central hypothesis is that Ncad in pro-tumorigenic Osx+ cells restrains tumor growth by regulating signals that reprogram the tumor microenvironment. To test this hypothesis, we propose the following Specific Aims: Specific Aim 1 – Modulation of extra-skeletal tumor growth by Ncad in Osx+ cells; testing the hypothesis that Ncad in Osx+ cells restrains tumor growth; loss of Ncad in TAOC increases tumor growth and metastases in mice. Osx+ Ncad+ cells correlate with tumor grading in human breast cancer. Specific Aim 2 – Mechanisms of Ncad modulation of pro-tumorigenic signals in tumor-associate Osx+ cells; testing the hypothesis that Ncad in Osx+ cells is an upstream regulator of p38 and Pten signaling; loss of Ncad in Osx+ cells results in accentuated expression of p38-dependent pro- tumorigenic factors and decreased Pten dependent signals, leading to tumor microenvironment modification and enhanced tumorigenesis. We will use in vivo approaches, including diphtheria toxin-induced selective cell ablation, parabiosis, lineage tracking, as well as non-biased transcriptomic approaches (single cell RNAseq) to unlock the cellular and molecular mechanisms by which Ncad in extraskeletal Osx+ cells affects tumor growth and metastasis. We will also determine the clinical pathology correlates of Ncad expression in Osx+ cells in human tumors. Results of the proposed studies will lay the foundations for the development of new markers of tumor progression and/or new therapeutic strategies aimed at interrupting environmental support of cancer growth and metastasis by targeting specific cells in the tumor stroma.