Stanford University

NIH Research Projects · FY 2024 · 2020-09

Project Summary Chromatin structure and transcription regulation are essential for cellular function, and their dynamics are highly correlated both in development and in disease. However, despite decades of amazing work identifying the molecular players involved in these processes, and mapping their interactions genome-wide, we are currently unable to describe the function connecting 3D chromatin structure and transcription dynamics. This limitation stems from the fact that chromatin structure and gene expression emerge from intrinsically stochastic transitions at the single-cell level, and we are missing the critical temporal parameters associated with these transitions. Therefore, new tools to measure both chromatin structure and transcription over time in single cells are critical for understanding how the human genome is read and for predictively controlling the epigenome. Here, we propose to develop a new set of live single-cell imaging technologies to simultaneously measure changes in 3D chromatin structures and their associated dynamics of gene expression across a large range of timescales: from dynamics of individual topologically associated domains and enhancer-promoter interactions, to changes associated with stable epigenetic memory across cell cycles. For the shorter timescales (under a cell cycle), our new imaging approach combines live super-resolution microscopy of fluorescently labeled loci with end-point demultiplexing of loci identity using Optical Reconstruction of Chromatin Architecture (ORCA), in order to track and trace 3-12 points within a functional chromatin unit. This new technique, which we call live-ORCA, will allow us to measure for the first time the temporal dynamics of an entire topologically associated domain in single cells. We will use live-ORCA in conjunction with time-lapse imaging of transcriptional bursting to study the dynamics of promoter-enhancer activity throughout cell differentiation and under perturbations of the chromatin network. For the longer timescale (across multiple cell cycles), our approach will combine time-lapse microscopy of gene expression, monitoring the distance between two tagged genomic loci as a live reporter of chromatin structure, and end-point chromatin tracing of the entire gene neighborhood using ORCA. We will perform these measurements in two systems: at a highly controlled synthetic reporter where we can induce either short-term silencing or long-term epigenetic memory, and at time points in differentiation when genes commit epigenetically to a new transcriptional state. Moreover, in order to further investigate the mechanism of epigenetic inheritance, we will develop a novel microfluidic device that allows us to track changes in chromatin 3D structures across individual cell lineages. Finally, to test our quantitative understanding, we will go back and forth between these single-cell data and theoretical modelling of chromatin dynamics. This research plan will greatly advance our understanding of chromatin dynamics and its functional role in transcription regulation, while at the same time contributing a whole new set of novel imaging technologies and engineered cell lines that will serve as a jumping board for the 4D Nucleome and broader scientific community.

NIH Research Projects · FY 2025 · 2020-09

Project Summary Autism spectrum disorder (ASD) is a highly prevalent group of neurodevelopmental disorders whose treatment efficacy is limited by poor understanding of its causal molecular mechanisms. Identifying disease mechanisms in ASD involves overcoming several challenges. First, it is difficult to demonstrate causality for a given mutation, since many mutations increase risk but do not always produce ASD. Second, it is challenging to identify animal models with autism-related phenotypes that are robust across multiple genetic backgrounds. Third, limited understanding of the neuronal subtypes and circuits responsible for behavior is a barrier to studying molecular mechanisms in a disease-relevant cellular context. The proposed research meets all three of these challenges. In the F99 phase, human families with a highly penetrant form of recessive autism caused by mutations in ACTL6B, a neuronal-specific subunit of the BAF chromatin remodeling complex, were identified. Actl6b-/- mice were tested as a model for ACTL6B mutant ASD and found to exhibit autism-related behaviors on two genetic backgrounds and similar brain anatomy to the affected humans. Transcriptional analysis of Actl6b-/- cortical cultures indicated that neural activity-induced genes were de-repressed in the absence of Actl6b, even when action potentials were blocked. The elevated expression of early response genes, including AP1 transcription factors (e.g., Fos, Junb), in Actl6b-/- neurons was associated with increased chromatin accessibility at AP1 sites and activity-related transcriptional changes in late-response genes, implicating abnormal early response gene activation as a potential disease mechanism. The genomic localization of the BAF complex, AP1 transcription factors, and the NCoR complex, which interacted with BAF in cortical tissue, will be studied in wildtype and Actl6b-/- neurons to learn if altered targeting of these complexes may contribute to disease-related transcriptional changes. To gain insight into the affected neuronal circuitry, a serotonin receptor 1b (5HT1b) agonist that was shown to rescue social behavior in the 16p11 autism mouse model (PMID: 30089910) was tested and found to rescue social impairments in Actl6b-/- mice. Serotonergic neuron-specific deletion of Arid1b, the most frequently mutated BAF subunit in autism, caused social impairments in mice that could likewise be rescued with the 5HT1b agonist, indicating that BAF function in serotonergic neurons is critical for social behavior. These studies have inspired the postdoctoral (K00) research direction: to interrogate autism-related molecular mechanisms within neuronal populations that control behavior. The postdoctoral training in systems neuroscience will buoy future studies linking the functions of chromatin regulatory proteins directly to behavior. This research supports the missions of the NIH Blueprint and BRAIN Initiative by providing new tools for autism research and revealing molecular and circuit mechanisms that influence behavior.

NIH Research Projects · FY 2025 · 2020-09

Abstract MYC is the most commonly activated oncogene in human cancer. However, to date, no existing therapies directly target MYC or the MYC pathway. My goal is now to target the MYC oncogene pathway to treat human cancer. Over the last 20 years, I have gained fundamental new insights into how the MYC oncogene initiates and maintains tumorigenesis. My work has established the idea that MYC is a hallmark of cancer and that many cancers are “MYC oncogene addicted”. I have identified both tumor intrinsic and host-immune dependent mechanisms. Now, I will use these insights from lab and novel methods to develop new therapies for cancer. I was one of the first investigators to use the Tetracycline regulatory system (Tet system) to generate “conditional” transgenic mouse models to demonstrate that MYC-induced cancer is “reversible” or “oncogene addicted” (Felsher and Bishop, Molecular Cell, 1999). Since then, I have used the Tet system to make a library of oncogene driven transgenic mouse models (MYC, RAS, BCR-ABL) of T-cell acute lymphoma (T-ALL), leukemia (AML), osteosarcoma (OS), hepatocellular carcinoma (HCC), lung adenocarcinoma (LAC) and renal cell adenocarcinoma (RCC). I have used my conditional transgenic mouse model systems to not only understand how MYC and other oncogenes initiate and maintain tumorigenesis but also develop innovative methods and novel technologies to make seminal contributions in cancer research, exhibiting sustained productivity. My proposed future research is built on recent observations that have used combined RNA, ChIP and metabolomic analysis to identify that lipogenesis and CRISPR synthetic lethal screen to identify nuclear transport as examples of otherwise not known to be MYC-regulated gene pathways that when targeted can block and reverse MYC- driven cancer. Now, I propose to use my library of conditional transgenic mouse models and human PDX models to generally identify targetable genes and pathways in the MYC oncogene pathway. I will use three complimentary approaches: RNAseq, ChIPseq and DESI-MSI to identify novel vulnerabilities in MYC-driven cancers; CRISPR in vitro and in vivo synthetic lethal screens combined with CyTOF and CODEX analysis to identify targets in my MYC-driven tumor models and understand their mechanistic role in tumorigenesis; MYC function reporter systems to be able to screen for genes and therapies to target MYC-driven cancers. My proposed research program has extensive support from an interdisciplinary team of colleagues. My proposed studies will glean novel mechanistic insights for how MYC drives tumorigenesis and use these insights to develop new therapeutic targets.

NIH Research Projects · FY 2025 · 2020-09

Oral squamous cell carcinoma (OSCC) is a devastating epithelial malignancy arising from the mucosa of sites that include the oral tongue, the floor of mouth, and the buccal mucosa. Advanced disease still has a dismal 5-year survival rate of only ~50%, despite advances in surgical and radiation approaches. Immunotherapy, such as PD-1 checkpoint blockade, has shown very promising results in a number of malignancies, but in general, responses are seen only in a minority of the cases. Numerous strategies to enhance endogenous and synthetic immune-mediated rejection of tumors are under intense investigation; however, all face significant challenges pending better understanding of the interface between tumor cells and the immune system within the tumor microenvironment (TME). Areas needing clarity include (1) factors determining tumor sensitivity to immune pressure; (2) factors determining the expression of neoantigens; and (3) factors determining immune cell plasticity and “states” induced within the TME. Our research program has been investigating aspects of the tumor-immune interface. We use primary tumor samples and novel mouse models to understand how to modulate the immune response to treat human OSCC. We are specifically interested in understanding how tumor heterogeneity influences the immune response; how we can induce neoantigens on OSCC for immune targeting; and how we can enhance NK cell function to treat OSCC.

NIH Research Projects · FY 2024 · 2020-09

Machine Learning for Integrative Modeling of the Immune System in Clinical Settings In response to an immunological challenge, immune cells act in concert forming complex and dense networks. A deep understanding of these immune responses is often the first step in developing immune therapies and diagnostic tests. Multivariate modeling algorithms can simultaneously consider all measured aspects of the immune system but requires prohibitively larger cohort sizes as technological advancements increase the number of measurements (a.k.a., “Curse of Dimensionality”). To address this, we propose a series of studies to develop machine learning algorithms for comprehensive profiling of the immune system in clinical settings. Particularly, for analysis of the immune system at a single-cell-level, we will leverage the stochastic nature of clustering algorithms to produce a robust pipeline for prediction of clinical outcomes. Next, we introduce the immunological Elastic-Net (iEN) algorithm, which addresses both the curse of dimensionality and reproducibility by integrating prior immunological knowledge into the models. The cellular systems that govern immunity act through symbiotic interactions with multiple interconnected biological systems. The simultaneous interrogation of these systems with suitable technologies can reveal otherwise unrecognized crosstalk. In collaboration with several leading laboratories, we have produced multiomics datasets (including analysis the genome, proteome, microbiome, and metabolome) in synchronized groups of patients. Using these coordinated datasets, we will evaluate several algorithms for combining multiple biological modalities while accounting for the intrinsic characteristics of each assay, to reveal biological cross- talk across various systems and increase combined predictive power. Importantly, numerous population- level factors (including medical history, environmental, and socioeconomic factors) significantly impact the immune system and studies focused on homogenous patient populations often lack generalizability to other populations. To address this, we will develop machine learning strategies to integrate population-level factors directly into our immunological data. These models will objectively define subpopulations of patients and enable flexibility in the coefficients of the models (and hence, the importance of the various biological measurements) in each group. This research program will be executed using data from several biorepositories focused on various diseases. This approach will ensure generalizability of our work to previously unseen datasets and increase the long-term impact of our findings. Throughout the proposal, a major area of focus is the development of visualization and model-reduction strategies that lay the foundation for interpretation of complex models. The machine learning algorithms developed will be readily applicable to a broad range of multiomics and multicohort studies and will be available as open-source software.

NIH Research Projects · FY 2024 · 2020-09

Project Summary/Abstract This is an application for a K08 Award to Dr Jennifer Caswell-Jin, an Instructor and breast oncologist at Stanford University establishing a career in translational breast cancer genomic research. The Award will support her career development by providing training in clinical trials, biomarker development, and bioinformatic analysis of multi-omic data under the expert mentorship of Dr Christina Curtis, computational and cancer systems biologist, and Dr George Sledge, breast cancer clinical trialist and translational researcher. The proposed research focuses on the major public health problem of metastatic breast cancer, estimated to affect over 150,000 women and to cause over 40,000 deaths each year in the United States. Hormone receptor-positive (HR+) breast cancer is the most common subtype. Eight “integrative” subtypes of HR+ breast cancer have been identified based on the integration of genome-wide copy number and expression information in early-stage breast tumors. Four integrative subtypes, together comprising one-quarter of all HR+ early-stage breast cancers, exhibit a very high risk of distant metastasis; each of these subtypes is characterized by a distinct area of the genome that exhibits concomitant copy number gain and overexpression. The studies in this proposal will examine for the first time how integrative subtypes behave after metastasis, with the driving hypothesis that they may derive benefit from personalized therapeutic approaches. Aim 1 is to investigate the biology and impact of integrative subtypes in metastatic HR+ breast cancer. We will develop novel approaches to assess integrative subtypes and will learn whether they change across metastasis, whether they are associated with timing of metastasis, and whether they have differential lengths of response to standard therapies. Aim 2 is to evaluate the effects of a novel combination of targeted therapy in two integrative subtypes of metastatic breast cancer. We will perform a clinical trial that tests a targeted therapeutic approach in tumors classifying as one of two of the four high-risk integrative subtypes. Because these two subtypes are defined by focal areas of genomic alteration involving either the fibroblast growth factor receptor ligand (FGF3; integrative subtype 2) or the fibroblast growth factor receptor (FGFR1; integrative subtype 6), we hypothesize that these tumors may benefit from FGFR inhibition. Participants in this trial will receive standard endocrine therapy in combination with CDK4/6 inhibition, as well as an investigational agent that inhibits the fibroblast growth factor receptor pathway. We will also perform tumor biopsies before and during treatment to evaluate for changes that occur with this combination targeted therapy approach. Successful completion of the proposed studies will lay the groundwork for continued efforts to develop a precision oncology approach for metastatic HR+ breast cancer, with next steps to be proposed in an R01 grant application before the end of the K08 Award.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT This five-year proposal will provide a platform for Dr. Goodyer’s successful transition to an independent physician scientist investigating novel mechanisms for the prevention and treatment of cardiac rhythm disorders. Specific mentorship and training opportunities have been tailored to build on the foundation of the applicant. Dr. Goodyer will be mentored by Dr. Sean M. Wu, Associate Professor of Medicine and Pediatrics and Dr. Anne Dubin, Professor of Pediatrics, Section Chief of Pediatric Electrophysiology (EP). Additionally, an outstanding advisory team of internationally-renowned scientists has been selected, each advisor with unique experiences and skillsets in fields ranging from basic science EP to translational medicine. Dr. Goodyer’s training plan lays out a personalized program for developing his proficiency in the following key areas: 1. Use of human induced pluripotent stem cells (hiPSCs) to functionally evaluate novel cardiac conduction system (CCS) genes; 2. Small animal phenotyping skills for in vivo analyses of CCS development and function; 3. Knowledge and techniques in basic science EP; and, 4. Professional development including leadership, grant writing and science communication skills. The training plan includes experiences from Stanford courses on stem cell research (eg. STEMREM 201B: Stem Cells and Human Development), weekly seminars on cardiology and translational medicine, renowned workshops on leadership and communication as well as a tailored externship focused on advanced basic science EP techniques in the lab of advisor Dr. Chiamvimonvat, Professor of Cardiovascular Medicine at University of California Davis. These mentorship and training activities are tailored to enable the candidate to achieve specific research goals aimed at the elucidation of CCS development and function. In Aim 1, Dr. Goodyer will investigate the role of a novel, intracellular, CCS-specific gene Cpne5 (copine 5), uncovered in his recently published work in Circulation Research and associated with human heart rate variation by independent genome wide association studies. The applicant will evaluate the function of Cpne5 in conduction cells by performing in vitro loss- and gain-of-function assays using both isolated mouse and hiPSC-derived CCS cells. In Aim 2, the candidate will further investigate Cpne5 in the context of CCS development and disease in vivo by comprehensive cardiac and electrophysiological phenotyping of CRISPR-Cas9 generated Cpne5 systemic knockout mice. Finally, Aim 3 capitalizes on the applicant’s recent discovery of another previously unknown CCS-specific marker. Specifically, by targeting this cell surface marker the applicant will validate the use of a novel antibody-based optical imaging method for visualizing the CCS in human hearts ex vivo. These studies will provide a proof-of-principle for the in vivo labeling of cardiac substructures and lay the foundation for translational opportunities in the real-time visualization of the CCS during cardiac interventions to prevent accidental intraoperative damage to the CCS.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Perioperative neurocognitive disorders (PNCD) affect about 25% of patients in the period following surgery, and can persist for months or years in 10% of geriatric surgical patients. The presence of acute cognitive disturbances post-surgery increases the risk of patients eventually developing dementias such as Alzheimer's disease. Poor cognitive outcomes lead to longer hospital stays, decreased quality of life, and increased morbidity and mortality. Unfortunately, about half of elderly individuals require a surgical procedure at some time in their remaining years, and no interventions exist to prevent PNCD because the etiology is unclear. One of the main challenges in identifying the factors leading to chronic cognitive impairment is the lack of routine, comprehensive cognitive testing in the surgical care plan. This shortcoming is in part due to the lack of mobile, easy-to-use cognitive testing platforms. To establish the perioperative biomarkers contributing to cognitive decline, we will (1) integrate routine, comprehensive cognitive testing pre- and post-surgery, and (2) build a database and an analysis platform to mine this multidimensional dataset. Together, this aims to yield accurate models to pre-identify patients at risk and create targeted therapeutic interventions. We propose to build the foundational infrastructure for a precision medicine approach in geriatric surgical patients. In the R21 phase, we will build a novel comprehensive database of demographic and risk factor questionnaire responses, banked blood specimens, intraoperative electroencephalography (EEG), and inclusive cognitive testing. These data will be collected throughout the patient interaction, from the preoperative appointment through a year following the surgical procedure and available to other research teams. We will incorporate cognitive testing and collect large-scale data in the geriatric surgical setting, establishing a new precedent for subsequent multidisciplinary studies. This structure will afford us the opportunity to accurately track cognitive decline towards chronic conditions, such as dementia. In the R33 phase, we will develop an analysis platform capable of mining this multidimensional dataset. This phase will include EEG analysis and deep immune profiling using mass cytometry. Layered on these analyses we will build innovative machine learning tools to identify features and interactions contributing to both acute and chronic PNCD pathology. Our novel machine learning tools use prior knowledge to refine feature selection, thus addressing a common challenge faced by clinical research studies (having many measurements in a limited patient population), and will thus be of broad interest to other clinical research projects.

NIH Research Projects · FY 2025 · 2020-09

Project Abstract Towards Robust Multiplex Genome Engineering Beyond CRISPR-Cas9 Exemplified by the CRISPR-Cas9 system, gene-editing technology is a powerful collection of tools for probing the hidden mechanisms of human diseases by understanding and controlling the functions of human genome variants. However, existing CRISPR genome technologies have three major limitations: (1) low efficiency and lack of accuracy when making large genome modifications such as structural variants in complex diseases; (2) uncontrollable off-target effects that lead to unwanted editing and cellular toxicity; (3) variable activity and precision when performing CRISPR editing in mammalian genome across different contexts, e.g. genomic loci, cell types, and model systems. To overcome these limitations, many groups including our own have sought to develop improved CRISPR tools using experimental methods and computational techniques. Building on my previous expertise, I will work towards multiplex, robust and error- free genome engineering. My group will seek to design new microbial proteins with sequence- independent recombination and RNA-to-DNA editing capabilities (Focus 1). Then, to provide robust gene-editing tools for studying single-cell genomics, I propose to leverage versatile CRISPR designs to enable high-capacity cell barcoding to define genome dynamics at single- cell resolution (Focus 2). To validate our new tools and as initial demonstration, we will use in human cancer models, with a focus on studying the cellular dynamics that lead to tumor drug resistance through genetic perturbation (Focus 3). The ultimate goal of my lab is to enable error- free engineering of genomic variants at any sizes, with robust activities across in vitro and in vivo applications. I will use this precise toolkit to uncover the functions of long genome alterations in human diseases, a major “black box” in our genome. The success of the proposal has the promise to generate safe, reliable genome correction tools for therapeutics.

NIH Research Projects · FY 2024 · 2020-09

Project Summary The human genome encodes over 2 million DNA regulatory elements called enhancers that control gene expression in specific cell types and states. Enhancers harbor tens of thousands of genetic variants that influence risk for common diseases and traits. Each of these enhancer variants could reveal insights into the molecular mechanisms of human diseases. Yet, we have lacked tools to systematically map which enhancers regulate which genes in each of the thousands of cell types in the human body. To address this challenge, we have recently developed CRISPR tools to experimentally test thousands of enhancers in parallel, and discovered a simple computational model that can predict enhancer-gene regulation from chromatin state. These nascent technologies suggest a new strategy to map enhancers across many cell types to connect noncoding variants to target genes. Here we will develop and extend these new technologies to map and predict enhancer-gene connections at single-cell resolution. First, we will characterize how enhancer function changes across developmental trajectories, by combining our CRISPR tool with a new single-cell readout method to survey thousands of enhancer-gene connections in differentiating vascular cells. Second, we will develop a computational model that can predict enhancer-gene regulation from single-cell measurements of chromatin accessibility. Third, we will apply these tools to build maps of enhancer-gene regulation in the adult human heart, and demonstrate the utility of these maps by characterizing genetic variants associated with coronary artery disease. These technologies will enable mapping enhancer-gene regulation in many cell types, building a foundational resource for connecting noncoding genetic variants to their molecular functions. This approach will be broadly applicable to any common, complex disease. This proposal builds on the PI’s experiences in genomics and team science with the ENCODE Consortium and Variant- to-Function Initiative. This R35 Genomic Innovator Award will help the PI launch a career at the interface of human genomics and cardiovascular disease that will include significant contributions to team science efforts. The environment at Stanford University in the Department of Genetics and Children’s Heart Center is ideal for supporting these scientific and leadership roles.

NIH Research Projects · FY 2026 · 2020-09

Project Summary Certain animals, from Hydra to salamanders, can regenerate extensive portions of their bodies through a process that involves intricate coordination of diverse cell types. Despite major advances from studies of highly regenerative models, critical knowledge gaps remain in understanding how regenerative programs are switched on or off, how preexisting body structures integrate with newly formed tissues, and what determines the vast differences in regenerative capacities across animals. In this proposal, building on our prior progress, we will explore these questions by comparing species that display markedly different regenerative capacities, from the planarian Schmidtea mediterranea with its extreme regenerative ability to closely related planarian species that regenerate poorly. We will also use our newly developed transgenic and live-imaging tools to capture real-time morphodynamics in the marine flatworm Macrostomum lignano and dissect the morphogenetic tissue transformations during regeneration. Additionally, we will study the acoel worm Convolutriloba longifissura, which “farms” its photosynthetic algal symbionts to fuel regeneration. Across these systems, we will identify “regeneration suppressors” that limit regeneration during homeostasis, unravel the roles and regulation of cell turnover in controlling regeneration competence, determine whether regeneration follows an optimal morphogenetic “solution”, and uncover novel mechanisms that can support stem cell proliferation through solar power. Together, our plan integrates functional genomic analysis, new genetic tools, live-imaging, single-cell multi-omic sequencing, and mathematical modeling to illuminate how regeneration is initiated, executed, and refined. By clarifying the core design principles of regeneration across diverse organisms, we aim to advance the fundamental biological basis of regenerative medicine and inform future strategies for enhancing tissue repair while minimizing the risk of uncontrolled growth.

NIH Research Projects · FY 2024 · 2020-09

ABSTRACT The overall goal of the proposed research program is to improve our understanding of single cell biology through information maximization techniques, by applying molecular engineering and computational approaches in sequencing. Specifically, single cell sequencing is rapidly becoming the predominant method for studying human biology and disease because it removes the confounding factor of sequencing cell mixtures in bulk. However, it has major pitfalls: significant material consumption during library preparation, noisy data readouts and signal dropout, and unclear paths for data integration across datasets. The overall vision of the proposed research program is to develop a pan-omic analysis strategy that enables perpetual re-use of any single cell source material. It revolves around a hybrid molecular engineering and computational framework that is loosely inspired by principles found in computing. The experimental core of the proposed research program revolved around a new molecular technology referred to as APEX (‘Attachment- based Primer EXtension’). The major innovation of APEX is the covalent conjugation of genomic material (i.e. DNA or cDNA) to a solid phase support such as an agarose magnetic bead, followed by utilizing only polymerase- based assays for non-destructive molecular interrogation. In this project, we will focus APEX development on single cell transcriptome sequencing applications, with general applicability to genome biology. As a model system, we will utilize peripheral lymphocytes as they consist of complex subpopulations with distinct characteristics at multiple levels of omic features. The project will focus on assay development and optimization, development of bioinformatic algorithms for data integration, and scale up to large cohorts as a demonstration of the scalability of the technology.

NIH Research Projects · FY 2024 · 2020-08

NHGRI U24: ATLAS OF REGULATORY VARIANTS IN DISEASE (ARVID) PROJECT SUMMARY Genome-wide association studies (GWAS) have identified thousands of single nucleotide polymorphisms (SNPs) linked to risk of developing specific non-cancerous polygenic diseases, including ischemic heart disease, chronic obstructive pulmonary disease, Alzheimer’s dementia, type 2 diabetes, and ischemic stroke. These disease-linked SNPs concentrate in regulatory DNA active in cell types that may mediate disease risk by modulating genes (eGenes) whose expression levels may be important in pathogenesis. These disease-linked expression SNPs (eSNPs) commonly alter transcription factor (TF) DNA binding motifs, indicating they may affect regulatory DNA activity by changing gene regulator binding. This U24 proposal aims to generate a genomic resource, the Atlas of Regulatory Variants in Disease (ARVID), containing the following 3 broad categories of information: 1) the individual disease-linked human eSNPs with differential gene regulatory function in relevant cell types 2) the target genes (eGenes) that these eSNPs dysregulate and 3) the gene regulators whose DNA association such disease eSNPs alter. First, we will identify the specific functionally altered eSNPs among those linked to index SNPs identified by GWAS in the 5 widespread human diseases noted above using massively parallel reporter assays (MPRA). A resulting subset of 300 top disease risk and non-risk eSNP pairs will then be deeply characterized in isogenic cells generated by gene editing to identify directly and indirectly dysregulated target genes. This effort will produce a Genomic Compendium of a) the disease-linked eSNPs that quantitatively impact regulatory DNA function in disease-relevant cell types and of b) the eGenes for the 300 top disease eSNPs. Second, we will identify the specific gene regulators whose DNA association is altered at the 300 disease risk eSNPs above, compared to matched non-risk alleles. To do this, we will use a live-cell proteomics approach termed DNA Protein Interaction Detection (DAPID). Quantitative mass spectrometry using isobaric tagging will be complemented by quantitative chromatin immunoprecipitation (ChIP) assays using isogenic, disease-relevant cells that differ only at the single eSNP nucleotide of interest. This effort will produce a Proteomic Atlas of differential regulator binding at 300 reference-disease eSNP pairs. This NHGRI U24 will generate a genomic resource defining the DNA variants, target genes, and gene regulators involved in inherited risk for 5 common non-cancerous polygenic human diseases.

NIH Research Projects · FY 2024 · 2020-08

ABSTRACT Coronary artery disease (CAD) remains the leading cause of death in the U.S. and worldwide. Identifying genetic risk factors and uncovering the underlying biological processes will lead to the development of much needed new avenues for therapies. Decades of genetics research, especially genome wide association studies (GWAS), have led to the discovery of numerous genetic loci associated with an increased risk for CAD. However, the majority of these loci lie in non-protein-coding regions. Efforts are needed to identify causal genes associated with these loci and the underlying cellular processes and signaling pathways. Recent advances in epigenomic and transcriptomic profiling at unprecedented depth and resolution, along with targeted genome/epigenome editing provide new opportunities to identify specific genes and cellular mechanisms in CAD. This K08 career development award is designed to launch the principal investigator’s career as an independent physician scientist that utilizes complementary computational and molecular approaches to discover the mechanisms that underlies human genetic risk to cardiovascular disease and translates these findings into treatment. The principal investigator ‘s Mentor (Thomas Quertermous) is a world leader in mechanistic studies of genetic risk of atherosclerosis. The proposed training is further supplemented by an advisory committee of leaders in computational biology, genetics, and single-cell multi-omic analysis, including Michael Snyder, Erik Ingelsson, William Greenleef, and Siddhartha Jaiswal, along with formal didactic courses at Stanford University and Cold Spring Harbor. Funded by an F32, the principle investigator used a combination of in vitro and in vivo models of atherosclerosis and linked the non-coding CAD risk loci at 2q22 to ZEB2, a transcriptional repressor with a critical role in cell-state transitions. ZEB2 appears to be specifically up-regulated in phenotypically modulated smooth muscle cells (SMC) in atherosclerotic lesions, and modulates their cell-fate decisions. The proposed study will: (1) identify the causal regulatory element(s) responsible for the CAD-Risk-associated region at 2q22; (2) reveal the molecular mechanisms by which ZEB2 affects phenotypic modulation of SMC; (3) determine the cellular mechanism by which perturbation of smooth muscle cell Zeb2 expression modulates atherosclerotic lesions in vivo. The result of this study will elucidate new regulatory mechanisms that modulate atherosclerosis biology. Additionally, the principle investigator will gain the training needed to transition into an independent physician scientist focusing on translating genetic findings of human cardiovascular disease into specific mechanisms and novel therapies.

NIH Research Projects · FY 2024 · 2020-08

Project Abstract Motivation: Osteoarthritis (OA) is a painful disease that affects tens of millions of Americans, but is poorly understood, resulting in a lack of treatments. Enabling low-cost approaches for widespread study of risk factors, onset and early progression of OA will enable better understanding of OA mechanisms, treatment development, and triage of patients to different treatments based on specific disease phenotypes. Multiple systemic factors, biochemical factors, and other risk factors are associated with OA, but causes are diffi- cult to isolate and study during slow progression. Currently OA is diagnosed as joint-space narrowing using X-ray radiography, at a stage well beyond when interventions can be effective. Magnetic resonance imaging (MRI) of- fers sensitivity to morphologic and biochemical changes, but most methods are impractical for widespread clinical or research use. Usually MRI exams study only one knee, precluding the opportunity to compare knees. Sim- ilarly, biomechanics assessment typically requires numerous tests using advanced and rarely-available equip- ment and time-intensive analysis by skilled personnel, making this a challenge for widespread use. We have shown rapid, simultaneous 3D scanning of both knees with quantitative relaxometry and diffusion map- ping of connective tissues, combined with novel visualization of longitudinal change validated in a population with anterior cruciate ligament (ACL) tears. We have developed fully-automated cartilage and meniscus seg- mentation to simplify post-processing. (Our automated cartilage segmentation variability approaches that of reader-to-reader variability.) We now propose to combine MRI acquisition, reconstruction and analysis tech- niques with simple measures of kinematics into a widely applicable low-cost imaging and biomechanical test, which we will validate in subjects with ACL-injury and subjects with varying Kellgren-Lawrence grades of OA. Approach: We will begin by developing a robust 5-to-8-minute bilateral knee MRI exam, using an efficient 3D isotropic acquisition and novel deep-learning based image reconstructions. This will be followed with automated cartilage segmentation and quantitative analysis (thickness, T2, diffusion) of all 3 knee plates and automated semiquantitative scoring approaches for synovitis, bone marrow and cartilage lesions. Inertial measurement units (IMUs) will be used to measure kinematics, and gait asymmetries. We will continue our studies in ACL pa- tients to validate techniques and to develop asymmetry analyses for both imaging and biomechanical measures. Finally, in subjects with varying OA grade, we will evaluate the potential of the overall low-cost approach to relate asymmetry and longitudinal change measures to progression and OA grade. Significance: This project will develop an acquisition and analysis pipeline to quantify knee changes and left/right asymmetries that precede OA. We will characterize methods in idiopathic OA subjects and ACL- injured subjects at risk of post-traumatic OA. The very low target cost, under $120/subject, will ultimately enable widespread study of early onset and progression of different OA types, leading to earlier and better treatments.

NIH Research Projects · FY 2024 · 2020-08

Opportunity/Gap: A core tenet of the “Biodesign innovation process,” as well as other human-centered design methodologies, is the importance of innovators seeing first-hand the challenges they aim to solve. This exposure not only provides a more complete appreciation of the problem at hand, but also allows innovators to use their perspective as an “outsider” to glean unique insights that may be missed by those who experience a problem daily. It is these first-hand observations of latent needs that often seed disruptive and meaningful innovations that are vital in improving the quality and affordability of healthcare. Today, the existing undergraduate courses in Bioengineering (and otherwise) at Stanford do not include a robust needs finding curriculum or opportunities for students to directly participate in observing needs in clinical practice. Proposal: Stanford Biodesign, in partnership with the Bioengineering Department and its capstone course team, as well as with clinical collaborators across the School of Medicine, would like to guide and mentor undergraduate students in an approach to needs identification that includes direct clinical observations along with need research, need characterization, and clinical validation interviews. We believe these skills will be invaluable in preparing student innovators to pursue careers in health technology whether in academia, medicine, engineering, or business. We propose to structure this as a part-time summer program (~6-10 hours/week), targeted at rising Bioengineering seniors who are on campus for funded summer research experiences and can bring this experience to their capstone course/team in the Fall. Accordingly, we propose the following aims: (1) Develop a course curriculum for undergraduate students to learn to identify and characterize clinical needs. (2) Develop assessment tools for evaluation of student learning of the needs-identification and needs-characterization process. (3) Implement and document summer training program through iterative development and assessment.

NIH Research Projects · FY 2024 · 2020-08

Harnessing the human monocyte system to improve surgical recovery Over 30 million patients undergo a major surgery annually in the US. Patients’ recovery after surgery is highly variable and can be severely compromised by complications, such as infections, prolonged pain, and functional impairment. However, our current approaches to predict a patient’s recovery are anchored in clinical and phenotypical data and perform poorly. Surgical injury produces a multi-cellular immune response that, when dysregulated, leads to adverse surgical outcomes. Examining the human immune system in depth, in patients undergoing surgery is a logical and promising strategy to identify biological signatures for risk prediction and to reveal mechanisms that can be exploited to improve surgical recovery. Our research program utilizes the high-dimensional immune monitoring of patients undergoing surgery to identify modifiable immunological mechanisms that accurately predict a patient’s recovery. This MIRA proposal builds on our extensive translational research indicating that immune responses contained in the human Monocyte System (hMS) strongly correlate with pain resolution and functional recovery after major joint replacement surgery. We will pursue three inter-related, but non-overlapping goals focusing on the hMS: First, we will assess the phenotypic and functional dynamics of circulating monocytes in response to surgery to determine the role of the hMS in the pathobiology of surgical recovery. Second, we will interrogate the hMS before surgery to determine whether patients’ pre-surgical immune states determine the course of surgical recovery. Third, we will use a reverse translational strategy using a pre-clinical mouse model of surgery to test whether “druggable” immunological targets identified in humans can accelerate recovery. We will use the following innovative and multidisciplinary strategies: 1) high-dimensional profiling of the hMS in response to surgery using single-cell mass cytometry (including dynamic alterations in cell phenotype, homing properties and effector responses); 2) identification of cellular, epigenetic and proteomic elements of a patient’s hMS, using an integrative analytical pipeline developed by our group; 3) evaluation of new targets for selective modulation of monocyte signaling responses in a mouse model that recapitulates hallmarks of the human immune response to surgery. A major strength of this proposal is the study of trauma-related immunology in a pertinent patient population by a clinician-scientist who has effectively pioneered high-content immune monitoring techniques at the bedside. The current program focuses on pain and functional impairment after orthopedic surgery. However, we will study fundamental mechanisms that are likely shared across many acute inflammatory conditions (e.g. other surgeries, blunt trauma or traumatic brain injuries). As such, we will be able to pivot towards the integration of our findings within the broader immunologic, metabolic, and neuro-hormonal responses to injury and the evaluation of other adverse outcomes after traumatic injury, such as infections, sepsis, or neuro-cognitive impairment.

NIH Research Projects · FY 2024 · 2020-08

This is a new application for an R01 award for epidemiologist Dr. Julia Simard, at Stanford University School of Medicine, who brings an innovative lens to the field as an early stage investigator building a research program focused on adverse pregnancy outcomes in patients with systemic lupus erythematosus. For her K01 award, Dr. Simard and colleagues showed that pregnant women with lupus are a highly-medicated group, who experience more adverse outcomes, including early-onset preeclampsia, preterm delivery, infection, and stroke. In recent pilot work, Dr. Simard found that hydroxychloroquine (HCQ), which is used to manage lupus during pregnancy, may prevent preeclampsia and preterm delivery. In two populations (one US and one in Sweden), Dr. Simard also found that fewer than half of pregnant women with lupus use HCQ, despite recommendations. This has been corroborated by others as well. Dr. Simard builds upon this work in the present application via three large international populations to determine whether HCQ reduces the risk of these adverse outcomes in lupus pregnancies. Dr. Simard will partner with colleagues in Sweden, Israel, and Kaiser Permanente of Northern California’s Division of Research in the United States. These data include details on ordered and filled prescriptions, and often unavailable data on antiphospholipid antibody status and parity, two critical factors in lupus reproductive research. In aim 1, Dr. Simard will determine whether preeclampsia risk is reduced in women who use HCQ during pregnancy. In aim 2, Dr. Simard will examine preterm delivery, partitioning spontaneous from medically-indicated, to examine overall risks and medication by preeclampsia and other factors such as glucocorticoid use and gestational diabetes. In aim 3, Dr. Simard’s team will identify barriers and facilitators of HCQ use in pregnant women with lupus using an innovative mixed methods design. After evaluating patient adherence and guideline adherence by clinicians in the large US and Israeli databases, they will identify subsets less likely to adhere and examine how they differ from adherent patients. Then partnering with patients and providers, they will use these quantitative findings to identify barriers and facilitators of adherence from the patient and provider perspectives using focus groups. This aim will provide key information on HCQ use and prescription practices to improve the analytic paradigm and inform our clinical understanding HCQ use in lupus pregnancy, and provide foundational support for clinical care and possibly, a future clinical trial.

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY/ABSTRACT Alzheimer's disease (AD) is a leading cause of disability and death in the US and a major global public health problem. Time is running short if we wish to avert a global public health disaster with untold suffering, disruption of families, and severe challenges to health care systems and economies. Effectual solutions will come only from innovative research. While aging is the biggest risk factor for developing AD, it is unclear to what extent normal aging is distinct from AD and which age-related factors drive disease. Senescence is a homeostatic response, which aims to prevent the propagation of these damaged cells while they remain viable and metabolically active. Senescent-like phenotypes have been described in neurons despite neurons being post-mitotic cells and these cells may release factors that trigger senescence in surrounding glia. Senescent glia and senescent-like neurons increase in the brain with age and are thought to contribute to the loss of function associated with aging and age-related diseases like AD. Our application, entitled “Uncoupling Age- Versus Cognitive-Related Cellular Senescence in Alzheimer's Disease,” is highly responsive to the objectives outlined in the RFA-AG-20-025, by leveraging an innovative molecular imaging platform we invented at Stanford; multiplexed ion beam imaging (MIBI), in order to uncouple age- from cognitive decline-related cellular senescence. MIBI enables us to quantify, with low nanometer resolution, high-dimensional, protein-level expression patterns, single-cell (neuro/immune) interactions, and spatial localization of senescence- and AD- relevant molecules (Aim 1) in a model of healthy aging (Aim 2) and well-characterized cases of AD related cognitive impairment (Aim 3). Importantly, MIBI allows all of this to be accomplished in archival FFPE material, thus allowing retrospective analysis on a variety of existing cohorts. By creating in-depth, phenotypic cellular signatures with spatial context from our unique aging and cognitive cohorts, we will be able to provide insight for modifiable factors promoting cognitive decline by filtering those specifically associated with aging alone. In this research program, collaborative expertise in clinical neuropathology and cognitive decline, technological advancements in imaging, biochemical/molecular and cellular biology, and machine learning analytics converge in this proposed research program to address the spatio-cellular (neuro/immune, senescent) heterogeneity in non-human primate (NHP) and human models of healthy aging and AD brains. Furthermore, it will be synergistic to, and draw on expertise developed in existing infrastructure to image and organize AD clinical pathology (R01AG056287, R01AG057915, MPIs: SC Bendall, RM Angelo, TJ Montine) as well as the NIA-funded 90+ UCI cohort, control material housed in the Stanford ADRC, and NHP specimens (P50 AG047366 co-I: TJ Montine). We will reveal cellular senescent phenotypes that differentiate AD from normal age-associated senescence.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY / ABSTRACT Dengue virus (DENV) is a pathogen of high biomedical significance against which we lack effective countermeasures. Although targeted chemotherapy using combinations of direct-acting antivirals (DAAs) has proven highly successful against hepatitis C virus infection and HIV, efforts to develop analogous drugs against DENV have not been successful. The genetic diversity of DENV due to replication by an RNA-dependent RNA polymerase that lacks proofreading function presents additional challenges by making it difficult to develop vaccines and antivirals with broad-spectrum coverage of all genotypes within one viral species and facilitating the rapid development of antiviral resistance when DAAs are used as monotherapies. Recently developed methods for small molecule-induced degradation of specific proteins rely on chimeric molecules (“PROTACs,” “degronimids,” “degraders”) that have a target-specific ligand linked to a moiety that binds an E3 ubiquitin ligase (e.g., cereblon, VHL). Small molecule-binding leads to ubiquitination and proteasomal degradation of the target. This results in event-driven rather than occupancy-driven pharmacology leading to efficient removal of the target from the cell and functional ablation of all of the protein's functions. Since pharmacological activity does not require constant, stoichiometric engagement of the target, even modest affinity ligands can be effective degraders. In addition, this mechanism of action can have higher natural barriers to resistance than conventional inhibitors, as has been demonstrated in the cancer biology field. While these potential advantages are attractive for antivirals development, it remains unclear the extent to which they can be leveraged to attain significant antiviral effects. In particular, strong viral expression and localization of viral processes (and their effectors) on or near specialized membranes may limit the susceptibility of DENV and other viruses to this pharmacological strategy. Here we propose to explore whether we can successfully deploy targeted protein degradation against three essential DENV proteins: core, NS4B, and NS5. As there are currently no approved anti-DENV drugs, there is an urgent need to find new pharmacological strategies to target this virus. Starting with known inhibitors as targeting ligands for degrader development, we will develop and validate antiviral degraders. We will then use these as tools to systematically explore potential points of differentiation between degraders and conventional inhibitors in terms of affinity, potency, selectivity, duration of action and susceptibility to resistance. We will also optimize validated antiviral degraders to test the efficacy of this antiviral approach in vivo. The overall goal is to validate degradation of one or more of these targets as an antiviral strategy with high natural barrier to resistance and to advance first-in-class degraders as leads for the development of antivirals. In pursuit of this goal, we will also establish important proof of concept and the foundation for more broadly developing antiviral degraders against other viral pathogens.

NIH Research Projects · FY 2025 · 2020-07

PROJECT SUMMARY Overview: The principal mission of the Women’s Reproductive Health Research at Stanford (WRHRS) Program is to provide state-of-the-art, mentored, multidisciplinary research career development for outstanding junior clinician-scientists who will impact and improve women’s reproductive health. Thirty-six Faculty Mentors and 7 Advisory Committee members (5 internal; 2 external) representing 3 Schools, 15 Departments, and 8 Institutes at Stanford will collaborate to mentor junior faculty-level clinician-investigators (“WRHRS Scholars”) to acquire the skills and experience needed to transition into productive, independent physician-scientists able to sustain viable careers and mentor future generations. Training Program and Candidate Pool: The WRHRS Program cultivates excellence in basic, clinical, translational, data science, and epidemiology research to address the national shortage of qualified investigators in women’s reproductive health. The Program includes a structured training plan of sufficient duration to achieve independence; individualized didactic education based on skills, competencies, and needs; extensive team-based mentoring; hands-on research; and protected time with immersion in a vibrant research community. Stanford has accomplished, dedicated, and well-funded mentors, innovative curriculum, and extensive infrastructure in place to prepare WRHRS Scholars for research independence. Each Scholar will have a multidisciplinary mentor team as well as access to abundant resources and the rigorous research infrastructure of the OB/GYN Department and the University. We have an excellent pipeline of internal Scholar candidates and the means to attract candidates nationally. Two WRHRS Scholars will be supported each year of the 5-year program. Duration of support for each Scholar will average 2-4 years thereby providing training for 4 Scholars. Scholars will pursue one of six Research Focus Areas in which Stanford demonstrates exceptional strength and breadth: 1) Maternal-Child Health & Epidemiology, 2) Reproductive Biology, 3) Gynecologic Health Across the Lifespan, 4) Gynecologic Oncology, 5) Innovative Technology, and 6) Social Medicine. Scholars will develop essential academic skills and a portfolio of research projects to propel their transition to independence. Research progress and career outcomes of Scholars are evaluated on an ongoing basis by Faculty Mentors, Program leadership, and the Advisory Committee. Innovation and Impact: Our Program will catalyze growth of women’s reproductive health research in a vibrant, innovative, and collaborative environment. WRHRS is synergistic with many Stanford School of Medicine programs focused on innovation in precision medicine, global health, data science, artificial intelligence, and biotechnical innovation. Our cadre of WRHRS Scholars will be well poised to improve the reproductive health of women over the lifespan.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY Chronic metabolic dysfunction has emerged as one of the most severe medical problems worldwide, leading to increases in type 2 diabetes, insulin resistance, and cardiovascular disease. The discovery of alternative pathways to regulate whole-body glucose and energy metabolism is urgently needed to address this great medical need. Such pathways could be exploited for new therapeutic strategies to combat diabetes and insulin resistance. Using a multidisciplinary strategy combining computational, cellular, and in vivo approaches, we have recently uncovered a new adipokine from thermogenic adipose, Isthmin-1 (Ism1), that acts to promote glucose uptake in mouse and human adipocytes. The action of Ism1 requires PI3K/AKT signaling but is entirely independent of the insulin receptor. In animals rendered diabetic by high-fat diet feeding, administration of recombinant Ism1 protein or genetic elevation of circulating Ism1 improves glucose homeostasis. However, more studies are needed in order to understand the contribution of Ism1 to glucose metabolism, and to leverage this understanding for therapeutic purposes. The overall objectives in this proposal are to establish how Ism1 can control blood glucose by determining the signaling effectors and cell surface receptor that mediate the action, determine the endogenous physiological function for Ism1, and evaluate the pharmacological potential of Ism1 as a therapeutic target. In Aim 1, we will utilize biochemical, genetic, and proteomic methods to identify the signaling pathways and cell surface receptor responsible for the signaling action and glucoregulatory mechanisms of Ism1. These studies will identify Ism1’s mechanism of action and will be critical for our understanding of Ism1 signaling as an insulin-independent pathway to regulate glucose uptake. In Aim 2, we will determine the physiological function for Ism1 using our generated whole-body and adipocyte-specific Ism1 knockout mice. These studies are essential in determining the endogenous role of Ism1 in glucose metabolism. In Aim 3, we will determine the minimal requirements for Ism1 bioactivity by generating fragments, mutants, and engineered forms of Ism1. This aim will pave the way for further optimization of a polypeptide hormone as a therapeutic agent, and will be essential in understanding the effects of augmentation of this novel pathway physiology. These contributions are expected to be significant because pathways that can regulate glucose independently of insulin will open entirely new avenues to overcome insulin resistance and diabetes, which could have a significant public health impact.

NIH Research Projects · FY 2024 · 2020-07

Avoidant restrictive food intake disorder (ARFID) is a new psychiatric disorder in the Diagnostic and Statistical Manual 5 (DSM-5). ARFID has an estimated prevalence of 7.2 to 17.4 percent thus making it a significant mental health concern. ARFID is characterized by a range of dysfunctional eating behaviors including a lack of interest in eating, sensory related eating concerns (such as taste, color or texture) and a fear of adverse consequences of eating (i.e., fear of choking or vomiting). There is no evidence-based treatment for ARFID. Preliminary data from a feasibility study comparing FBT-ARFID to Usual Care (UC) provide evidence that manualized FBT adapted for patients with ARFID is feasible and effective. Recruitment and randomization averaged 1.87 participants per month over a 15 month period with an overall attrition rate of 21%, comparable to rates in fully powered studies of FBT-AN. The feasibility study also identified an efficacy signal on the difference between groups on the primary outcome (change in percent Estimated Body Weight (%EBW)) of a large effect size (ES) favoring FBT-ARFID Studies suggest that improvements in parental self-efficacy related to changing feeding and eating behaviors early in treatment is a likely mechanism of FBT for other eating disorders in youth. Our feasibility study showed a striking difference between conditions in parental self-efficacy favoring FBT-ARFID compared to UC. In addition to this promising evidence of target engagement In addition, target validation was demonstrated by the change in parental self-efficacy being significantly correlated with improvements in % EBW. Aim 1: To conduct an RCT involving children and adolescents between the ages of 6 and 12 years of age with DSM 5 ARFID and weight below 88% of EBW comparing FBT-ARFID with medical management to manualized Non-Specific Treatment UC with medical management. Treatments will be matched for time and therapist attention. We hypothesize that participants randomized to FBT-ARFID will have significantly greater change in %EBW at EOT. Aim 2: To examine early change in parental self-efficacy as a mediator of treatment effect (FBT-ARFID vs. UC on outcome). We hypothesize that positive changes due to FBT-ARFID in parental self-efficacy related to feeding behaviors using the Parents vs ARFID Scale (PvsARFID) will be associated with positive changes in %EBW at EOT. Secondarily, we will explore whether objective changes in parental re-feeding behavior is a possible mechanism of FBT-ARFID using a mediator analysis. Aim 3: To explore moderators of treatment outcome. To conduct an adequately powered study, 100 children (ages 6-12 years) will be randomized to manualized FBT-ARFID plus medical management (n=50) or manualized Non- Specific Treatment plus medical management (n=50). Assessments (blinded to treatment condition) of primary and secondary outcomes will be conducted at baseline, 1 month, 2 months, and 4 months (EOT).

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow characterized by the accumulation of immature myeloid cells defective in their maturation and function. AML affects more than 20,000 adults annually in the United States, most of them over the age of 65. Even with aggressive treatments, five-year overall survival is between 30-40%, and much lower for those over age 65. Human AML shows evidence of a hierarchical cellular organization, with a minor fraction of self-renewing leukemia stem cells (LSCs) at the apex of this hierarchy. LSCs are defined as cells that are capable of initiating the disease when transplanted into immunodeficient mice and can both self-renew by giving rise to leukemia upon serial transplantation and also partially differentiate into non-LSC bulk blasts that are unable to self-renew. The clinical significance of this leukemia stem cell model is supported by the finding that gene expression signatures of AML LSCs are independently correlated with adverse clinical outcomes. Detailed characterization of AML LSCs has demonstrated their properties of self-renewal, relative quiescence, resistance to apoptosis, and increased drug efflux that likely render them less susceptible to conventional therapies aimed at the bulk proliferative disease. Thus, the generally poor clinical outcomes in AML are attributed to chemotherapy- resistant LSCs that persist during clinical remission, eventually giving rise to relapsed disease. From a therapeutic perspective, this cancer stem cell model implies that in order to eradicate the disease and achieve long-term remissions, treatment approaches must eliminate the LSC population. Although initially described several decades ago, AML LSCs have not been rigorously purified primarily due to the extensive heterogeneity of primary human AML and limitations of available xenotransplantation models. Functional LSCs have been found to be enriched in the CD34+CD38- fraction of leukemic cells, but are also present in other immunophenotypic populations. A number of cell surface markers have been characterized on these AML LSC-enriched fractions, but none are specific for LSCs or facilitate their rigorous purification. These results have made it difficult to further characterize LSC biology and to develop methods for more specific therapeutic targeting. While the field of human AML LSCs has a rich history of investigation, many key questions remain to be addressed. Can LSCs be more rigorously identified and isolated based on cell surface marker expression? What features or programs of LSCs are associated with clinical outcomes? How do adversely prognostic LSC- associated genes regulate LSC functions? Do non-LSC blasts affect the properties of LSCs? Do AML subpopulation dynamics affect LSC properties? This proposal seeks to address these questions through the investigation of human AML LSCs based on the hypothesis that LSCs exhibit distinct functional properties and biological programs that contribute to AML pathogenesis, response to therapy, and clinical outcomes. Therefore, these LSCs represent the critical cellular target for the development of curative therapies.

NIH Research Projects · FY 2025 · 2020-07

PROJECT SUMMARY/ABSTRACT Background: Several lines of evidence suggest that insomnia contributes to emotionally distressing depressive mood symptoms through disruption of brain networks that regulate emotional functions. Of particular concern, insomnia is associated with an increased risk for suicide, even when accounting for the presence of other depressive symptoms. However, we do not yet know to what degree that the emotion regulation brain network is modified by the restoration of sleep, or whether the degree to which a sleep intervention engages these neural targets mediates reductions in depressive symptoms and suicidality. Objective: This proposal investigates the impact of a proven sleep intervention on engagement of the emotion regulation brain network as a putative mechanistic target. DESIGN/METHODS: In the R61 phase, a mechanistic trial will demonstrate feasibility and establish whether the emotion regulation brain network is modified (the target is engaged) when patients show improvements in insomnia symptoms following a proven psychosocial sleep intervention. Participants will be 70 adults experiencing elevated depressive symptoms and clinically meaningful insomnia. Depressive symptoms and insomnia will be assessed prior to, and weekly while receiving six Cognitive Behavioral Therapy for Insomnia (CBT-I) sessions across a period of eight weeks. CBT-I improves sleep patterns through a combination of sleep restriction, stimulus control, mindfulness training, cognitive therapy targeting dysfunctional beliefs about sleep, and sleep hygiene education. Emotion regulation network neural targets will be assayed prior to and following completion of CBT-I treatment. If the Go milestone criteria are met, the R33 phase (years 3-5) will include a 2-arm randomized controlled trial. We will enroll new participants (n=150) and randomize them in a 1:1 ratio to the CBT-I or to the credible control treatment for insomnia group. Participants will complete a refined measurement protocol based on the R61 phase study. Specific aims: R61 aims are to demonstrate (1) feasibility and (2) that CBT-I modifies emotion regulation network function according to pre-specified Go milestone criteria. R33 aims are to (1) confirm target engagement by testing the hypothesis that compared with an active control condition, CBT-I participants will show significant change in the emotion regulation network targets that met the Go Criteria of Study 1 in the direction of normalization, at the end of treatment, (2) examine the relationships of target engagement to treatment outcomes by study group, and (3) test whether emotion regulation network measures at baseline predict depressive symptom and suicidality reduction. IMPACT: Characterizing these associations may offer the potential to gain a deeper understanding of the neurobiological mechanisms underlying depression in the presence of insomnia. Our results will advance an evidence-based mechanistic approach to treating, and ultimately preventing, the emotionally distressing and potentially life-threatening impact of insomnia.