Stanford University

NIH Research Projects · FY 2026 · 2019-09

PROJECT SUMMARY The goal of this renewal of our successful R25 program is to continue to increase the numbers of undergraduates pursuing careers in cardiovascular medical research by providing them with a short-term mentored research experience in the laboratories of members of Stanford School of Medicine’s Cardiovascular Institute (CVI). We propose continuing the success of our existing R25 program, which was already extremely effective in its first cycle. Our program recruited 65 students from various levels of experience; all of whom intend pursue careers in cardiovascular research and medicine. Additionally, pre- and post-program evaluations showed that our R25 trainees experienced significant gains in their skills and confidence in performing research. Cardiovascular disease is the leading cause of death and disability in the US. Despite ongoing advances in clinical care and research, gaps persist in sustained engagement in research and the development of a robust workforce. Particularly for aspiring physician-scientists, challenges in recruitment, retention, and career advancement limit participation in cardiovascular research, impacting the long-term strength of the biomedical research pipeline. However, it has been shown that participation in mentored research increases the degree to which students identify as scientists and persist in academic and medical research careers. In terms of cardiovascular medicine, clinicians with research training are more likely to be aware of the environmental, genetic, and behavioral factors that impact appropriate patient diagnosis and treatment. However, because there are more undergraduates seeking research experiences than there are available positions, many undergraduate students lack the opportunity to perform independent research in an academic lab. This R25 will continue to 1) provide undergraduate students with technical training and experience in cardiovascular research; 2) provide students with cardiovascular researcher mentors and role models; and 3) build relationships between Stanford Medicine and nation-wide institutions. Stanford Medicine in general and the Cardiovascular Institute in particular are known for excellence in cardiovascular research. The Program Directors and Program Faculty involved in this R25 have ongoing state-of-the-art research programs spanning population-based, clinical, translational, and basic cardiovascular research, and they welcome the opportunity to continue to mentor R25 trainees. Together they have the mentoring expertise and the available resources to continue to support R25 trainees though our successful summer research program.

NIH Research Projects · FY 2025 · 2019-09

We propose a 2-arm, parallel group, randomized controlled trial to test the efficacy of adding two innovative “wise” social psychological interventions—growth mindset and self-affirmation—to a 6-month family-based, group behavioral intervention for weight control in children with obesity, to reduce body mass index trajectory over 12 months, compared to the behavioral intervention alone. A total of 200 10-15 year old children with obesity (BMI ≥ 95th percentile) will be randomized to the two conditions. All children will participate with their parents in a usual care six-month, family-based, group, behavioral weight control program. In addition, families randomized to the social psychological intervention condition will receive two innovative “wise” social psychological interventions—growth mindset and self- affirmation—strategically selected and timed to enhance the behavioral intervention. There remains a great need for innovative approaches to increase and sustain weight loss among children with obesity. Weight control interventions may be limited in their success in many children in part because of psychological barriers and threats they face when attempting to manage their weight. One of the most exciting recent breakthroughs in the behavioral sciences is the development of seemingly small but “wise” interventions—typically consisting of brief exercises and messages grounded in social psychological science—that produce powerful and positive long-term changes in behavior. These interventions help people flourish by altering the ways in which they think about themselves and their environments. We propose that two of these interventions with some of the strongest empirical support will be particularly useful in creating the right psychological landscape for more consistent and successful behavior change and weight loss. A growth mindset of body weight creates the vital belief that success is possible with effort and hard work. Self-affirmation helps people cope with stereotype threat by allowing them to focus on other sources of self-worth, thus freeing them from the distraction and anxiety of possibly confirming the stereotype that they won't succeed. As a result, this approach has the potential to substantially improve treatment outcomes and reduce the concurrent and future morbidities that accompany childhood obesity. Measures will be collected at baseline, 3 months, 6 months (end of the behavioral treatment), and 12 months (6 months after the end of treatment). Primary Hypothesis: Compared to children randomized to receive the usual care family-based, group behavioral weight control program alone, children randomized to receive the usual care family-based, group behavioral weight control program plus the wise social psychological interventions will have a significantly attenuated BMI trajectory over 12-months.

NIH Research Projects · FY 2025 · 2019-09

Planar cell polarity (PCP) signaling controls the polarization of cells within the plane of an epithelium, orienting asymmetric cellular structures, cell divisions and cell migration. Numerous medically important developmental defects and physiological processes in vertebrates are under control of PCP signaling, motivating considerable interest in understanding the fundamental logic and underlying mechanisms controlling PCP. Major advances toward achieving this understanding have come from leveraging of genetic approaches only possible in Drosophila, and the evident conservation of mechanism reinforces the utility of flies as a model system. I propose that the next major leaps in understanding will emerge from continued innovation in genetic approaches and from expanding the reach of these approaches by employing them in cross-disciplinary studies with technologies such as single molecule imaging and advanced biochemistry. In flies, PCP signaling controls the orientation of hairs on the adult cuticle, chirality and orientation of ommatidia in the eye, orientation of cell divisions and related processes in other tissues. Several distinct molecular modules contribute to PCP signaling. The core module acts both to generate molecular asymmetry within cells and to coordinate the direction of polarization between neighboring cells. One or more global directional modules orient core polarization with respect to tissue axes, and distinct effector modules interpret core polarity to direct morphogenetic responses. A key goal has been to understand the fundamental workings of the core PCP module. Proteins in the core module segregate within cells to form distinct complexes on opposite sides of the cell, and form intercellular bridges that signal polarity between cells. The observed behavior of this module, combined with mathematical modeling, indicates that the system likely acts as a bistable switch, breaking symmetry to produce a stable, polarized array. Positive and negative feedback necessarily underlie the function of this module, but the molecular mechanisms generating feedback are as yet unknown. Furthermore, molecular-scale asymmetry in PCP is coupled to the formation of clustering of polarity components into discrete puncta, yet the precise role of clustering in polarization is not known. Our major focus going forward will be the dissection of core PCP mechanisms by developing and employing novel genetic tools and by combining these tools with single molecule imaging and advanced biochemistry to unlock an understanding of PCP signaling that previously applied experimental approaches have so far been unable to achieve. The proposed work will deliver an unprecedented view of both the fundamental logic and the precise molecular mechanisms underlying PCP signaling as well as lay the groundwork for potential therapeutic interventions for PCP related pathologies.

NIH Research Projects · FY 2026 · 2019-09

PROJECT SUMMARY My lab discovered that NNMT is a direct GR transcriptional target gene in TNBC. I then observed relatively high NNMT expression in several aggressive patient-derived TNBC cell lines. NNMT consumes the universal methyl donor S-adenosyl methionine (SAM) for methylation of nicotinamide. High NNMT activity depletes SAM; as a result, methyltransferase targets are hypomethylated in cells with high NNMT expression. NNMT-induced DNA and histone hypomethylation have been shown to result in oncogenic gene expression in cancer cells but NNMT mechanism of action in TNBC biology remains unclear. A link between NNMT expression and mRNA hypomethylation has not previously been established as a mechanism contributing to cancer progression. N6- methyladenosine (m6A) is an abundant and reversible RNA modification in eukaryotes. Our collaborator Dr. Chuan He discovered that m6A-binding proteins mediate translational regulation by altering stability and translational efficiency of m6A-modifed mRNAs. Importantly, altered m6A mRNA methylation is implicated in the progression of several human cancers via causing changes in post-transcriptional gene expression of cancer pathways. To our knowledge, I am the first to characterize the m6A methylome of a patient-derived TNBC cell line model (MDA-MB-231): ~ 7000 m6A-modified transcripts are significantly enriched for pathways involved in cellular stress response, cell death and cell survival. In addition, I have data suggesting that NNMT activity in the MDA-MB-231 TNBC cell line results in 1) reduced m6A modification of mRNAs regulating key cancer pathways and 2) increased in vivo tumor-growth. In my dissertation research, I am testing the hypothesis that NNMT activity in TNBC cells results in 1) reduced m6A mRNA modification associated with altered protein expression of pathways mediating cellular stress response and 2) cancer stem cell-like traits associated with survival, metastatic potential and increased in vivo tumor-forming capacity. During my postdoctoral research, I aim to test whether epitranscriptomic gene expression regulates dynamic cellular phenotypes including adaptation to the changing microenvironment. I will first characterize the actively transcribed genes with polymerase ChIPseq and perform whole proteome quantification with mass spectrometry in cells exposed to distinct microenvironmental stressors (e.g. nutrient deprivation, hypoxia). I will then determine whether differential transcription of genes correlate with protein expression in different cellular states. If there is not a strong correlation, I will perform individual siRNA knockdown of all known m6A- regulatory genes and determine the effect on protein expression. I will then utilize patient-derived xenograft mouse models and the Sprague Dowley rat model of spontaneous breast cancer to determine whether the m6A-regulatory proteins are differentially expressed in distinct tumor regions with single-cell RNA sequencing.

NIH Research Projects · FY 2025 · 2019-09

SUMMARY My laboratory has been interested in the mechanisms that control the identity and the fate of cancer cells during tumor evolution, including in response to treatment. We have made important contributions to this field of research in the past decade. Our work on the retinoblastoma tumor suppressor Rb in stem cells and cancer models has identified a new function role for Rb in the control of cell identity and plasticity, which explains in part why Rb-mutant cancer cells often fail to respond to therapy. Our pioneering work on Rb-mutant small cell lung cancer (SCLC) has provided fundamental novel insights into the biology of this neuroendocrine cancer. SCLC is the most lethal form of lung cancer. Treatment options have remained virtually unchanged for the past 30 years. SCLC kills ~250,000 patients worldwide every year. As the number of heavy smokers worldwide continues to grow, SCLC will remain a major health issue this century. With unique tools to study SCLC in vivo and a highly resourceful network of collaborators, we are uniquely placed to continue to greatly impact the SCLC field by confronting key issues that few investigators address. Importantly, our research combines technically innovative approaches that will allow us to address questions about SCLC progression and maintenance that are difficult, if not impossible, to tackle using traditional human tumor-derived cell lines, previous mouse models, or cancer patient samples. We have developed rapid and accurate mouse models of human SCLC. We have used these models and patient-derived xenografts to identify the cell of origin of SCLC and biomarkers for early detection, as well as drivers of the tumorigenic phenotype of SCLC and their mechanisms of action. We have also contributed to the elucidation of the genomic landscape of mouse and human SCLC tumors. Notably, our findings have led to the implementation of clinical trials in SCLC patients. In the next 7 years, we will continue to use SCLC as a paradigm to elucidate the mechanisms that determine the identity of cancer cells, their plasticity, and their fate. We will perform these studies in the context of our recent breakthroughs investigating inter- and intra-tumoral heterogeneity in primary mouse and human SCLC tumors. Our model is that SCLC tumors, which have very few stromal cells, generate their own microenvironment to support their growth, in part through activation of Notch signaling. This intra-tumoral heterogeneity may critically contribute to the lack of response of tumors to therapies. A second major focus of our work is to elucidate the mechanisms that underlie the striking metastatic ability of SCLC to multiple organs, including the brain. We propose that the switch to a more neuronal differentiation state that accompanies the gain of metastatic ability of neuroendocrine SCLC cells is a key aspect of this high metastatic potential. We will test these ideas in vivo and ex vivo using a combination of unique genetic, molecular, and cellular approaches.

NIH Research Projects · FY 2025 · 2019-09

PROJECT SUMMARY (OVERALL) The overarching goal of this Program Project Grant (PPG) is to elucidate mechanisms of cardiotoxicity and to develop a platform to test cardiotoxicity of new cancer therapeutics. Project 1 (Moslehi) will use a combination of mouse- and cell-based models to address the central question of whether cardiotoxicity associated with ibrutinib is on- or off-target. Project 2 (Wu) will establish novel pooled “cell villages” from patients to map genetic inter-individual difference in response to kinase inhibitors to elucidate genetic susceptibility to cardiotoxicity from kinase inhibitors. Project 3 (Mercola) aims to identify mechanisms that protect against cardiotoxicity of ibrutinib using a pipeline for discovery of protective targets based on reverting epigenomic consequences of exposure to a toxic drug. All three projects will be supported by an iPSC Processing Core (Core B) (Sallam) that will facilitate a unique repository of iPSC lines from patients with ibrutinib treatment. An Administrative Core (Wu) will support all three projects and the core to facilitate a fully integrated program project and enable high- impact research. The proposed PPG and assembled group collectively introduces synergies that will significantly impact the field of cardio-oncology and make translationally actionable inroads to decrease the burden of adverse effects of cancer therapeutics.

NIH Research Projects · FY 2026 · 2019-09

Project Summary The overarching goal of my research program is to understand the dynamics of ecological systems and their influence on diseases in humans, plants, and animals to improve the health and wellbeing of humans and ecosystems. Vector-borne diseases are a growing public health problem that are intimately linked to climate, land use, and other environmental changes. Our current and future research is focused on understanding changes in vector-borne disease burdens in response to global change. Predictive approaches that mechanistically link environmental change to disease dynamics are necessary to disrupt transmission and to sustainably control outbreaks. Research in my lab uses a diverse quantitative toolkit and emerging sources of data to predict disease dynamics, uncover environmental mechanisms for transmission, attribute impacts of global change, and forecast changes in the landscape of infectious disease. In the next five years, we will build on our understanding of climatic drivers of vector-borne diseases to study the impacts of climate extremes, the limitations on and drivers of vector species range expansions, and the role of adaptive (co)evolution. We will disentangle the multidimensional effects of land use change on vector-borne diseases by combining hypothesis testing, novel causal inference methods, and geospatial datasets to uncover the land use niches for disease transmission. Our focal systems include dengue, malaria, leishmaniasis, schistosomiasis, yellow fever, Lyme disease, and their mosquito, sandfly, and tick vectors. Our work combines publicly available environmental and epidemiological data and partner-engaged research with vector control and health agencies. We apply mechanistic mathematical models, machine learning, econometric causal inference, empirical dynamic modeling methods, and other quantitative tools. We also conduct primary empirical research on mosquito and parasite thermal adaptation on the Western treehole mosquito and its ciliate parasite. We are now witnessing unprecedented impacts of anthropogenic climate and land use change alongside expansions and resurgences of vector-borne and zoonotic diseases. At the same time, expanding technological and computational capacity are accelerating the pace of infectious disease dynamics research. This is an exciting and critical time to study linkages between human and planetary health, and my research program is at the forefront of this emerging field. By discovering mechanistic linkages between environmental change and disease transmission, we can design more proactive control measures, develop policy that benefits people and the environment, and prevent and reverse the worst impacts of anthropogenic change on health and their disproportionate impacts on marginalized populations.

NIH Research Projects · FY 2025 · 2019-08

Project summary Due to advances in genome sequencing, researchers have discovered that hybridization, or genetic exchange between species, is widespread. Research over the past decade has demonstrated that hybridization is an important process that has contributed to the modern-day genomes of many eukaryotic species, including humans and our relatives. Given how recently biologists came to appreciate the importance of hybridization, there are basic unanswered questions about its genetic and evolutionary consequences. Addressing these questions from the single gene to the genomic scale is the major focus of research in my lab group. Hybridization is not simply a feature of a population's history; it can impact diverse biological processes from adaptation to disease. During the first four years of R35 funding in my lab group, we studied several fundamental genetic consequences of hybridization and developed new computational methods to study hybridization. Because the genomes of two species have been evolving is isolation, combining these genomes can have severe consequences. We used the swordtail model system developed by my lab to pinpoint the genetic interactions that breakdown in hybrids and the mechanisms through which they act. We will continue this work by building the first genome-wide map of such genetic interactions in vertebrates and by unravelling the molecular mechanisms that lead to melanoma and embryonic lethality in hybrids. My lab also studies broad scale principles governing where in the genome hybrid ancestry persists and where it is removed by selection. Our past work uncovered a key role of recombination rate and the density of conserved base pairs in shaping the dynamics of genetic exchange between species along the genome. We propose to continue this work to uncover additional processes shaping evolution after hybridization on a genome-wide scale, focusing on the role of structural rearrangements, divergence in transposable element families, and protein complexes. Together this research will help us understand the mechanisms shaping genome evolution after hybridization in species across the tree of life.

NIH Research Projects · FY 2025 · 2019-08

The Center for ELSI Resources and Analysis (CERA) was established in 2019 to develop a platform for dissemination, sharing, curation, and synthesis of ELSI research products (ELSIhub) and to convene a broad base of ELSI-interested communities to support the development of CERA resources. Building on over four years of experience and relationship-building with ELSI-relevant communities, and the knowledge-to-action (KTA) framework, we will develop processes for content development and facilitate collaboration to enhance the dissemination, uptake, and translation of ELSI research. CERA | ELSIhub will be guided by the principles of responsiveness, adaptability, engagement, rigor, transparency, and open access. Our specific aims are to: Aim 1: Develop and maintain the ELSIhub platform for researchers to share and search for ELSI research products Aim 2: Curate and synthesize ELSI research Aim 3: Facilitate new collaborations and increase uptake of ELSI research Over the past four years, we have established a solid foundation for interdisciplinary collaboration and resource development, focusing on the ELSI research community. We also conducted a comprehensive accessibility assessment of ELSIhub and CERA events and implemented a plan to broaden accessibility and develop best practices for accessibility for the ELSI research community. In the next award period, CERA will expand its focus on ELSI researchers by establishing new collaborations with genome scientists, ELSI scholars and trainees to promote interdisciplinary work in and between ELSI research and ELSI-adjacent fields of study. We will introduce the ELSIconnect program and ELSI Journal Clubs to encourage collaboration and resource sharing among a wide range of ELSI-interested groups and to crowdsource identification of high priority areas for CERA events and resources. We will explore innovative methods to assess the feasibility of responsible and valid AI-assisted curation and synthesis of ELSI research, using large language models trained on ELSI research products drawn from ELSIhub databases. We will also add an independent Evaluation Team to design and conduct program evaluation and analyze platform use.

NIH Research Projects · FY 2024 · 2019-06

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults and is associated with a dismal prognosis. Immunotherapy has demonstrated potential to generate durable antitumor activity in other types of cancer. In particular, agents that selectively target checkpoint molecules, such as anti-CTLA-4 and anti-PD-1 antibodies, have accelerated the field of cancer immunotherapy by directly combating the tumor's mechanisms of immune evasion. Notable results with these agents have already been reported in advanced melanoma, renal cell carcinoma, and lung cancer and trials are underway in GBM. Chemotherapy, which is part of the standard of care for patients with GBM, has been associated with immunosuppressive effects and with myeloablative results. Recent data from our laboratory shows that local chemotherapy may be a better alternative to systemic chemotherapy given that it avoids these untoward effects. The main goal of this proposal is to understand the main mechanisms by which GBM evades the immune system and how to thwart these mechanisms with local chemotherapy and checkpoint blockade to enhance an effective immune response against GBM. Our data demonstrates that local chemotherapy in combination with anti-PD-1 increases survival and provides an increase in memory T cells in an orthotopic glioma model and protects against tumor re-challenge. We propose to study: 1. Potential biomarkers of response in patients with GBM treated with LC and anti-PD-1 therapy as part of an ongoing clinical trial at our institution. 2. The neoantigen profile generated by LC in intracranial chemosensitive and chemoresistant murine gliomas, to determine the impact on TCR diversity and anti-tumor immune response. 3. The location and identity of APCs responsible for antigen presentation induced by LC. These data will have direct clinical relevance for the findings and can be translational into clinical trials and patient care. We expect that the data generated from these studies will provide novel insights into a previously unexplored aspect of chemotherapy and serve as a foundation for optimizing the efficacy of therapy and host immune function against GBM. The knowledge obtained from this study will undoubtedly result in better therapeutic alternatives for current unsuccessful treatment for patients with GBM.

NIH Research Projects · FY 2026 · 2019-05

PROJECT SUMMARY/ABSTRACT A class of cis-regulatory elements, called enhancers, plays a central role in orchestrating spatiotemporally precise gene expression programs during development. Perturbations in enhancer sequence or regulation can lead to disease, including congenital malformations and cancer. Furthermore, enhancer sequence divergence is emerging as an important mediator of human phenotypic variation. Key features of enhancers include their role as major sites for transcription factor (TF) and coactivator binding in the genome, as well as their ability to activate transcription over long genomic distances from their target promoters. Indeed, at some genomic loci enhancers can regulate promotes at distances of over a megabase away. Such long-range function raises a question of how is regulatory specificity within chromosomal domains containing many cis-regulatory elements accomplished to allow for precise and robust gene regulation during development and in tissue homeostasis? Proposed research program focuses on addressing fundamental knowledge gaps in enhancer biology and long- range gene regulation. We will utilize novel genomic and computational approaches to systematically probe how enhancer-promoter genomic distance, 3D contacts and inherent features of cis-regulatory elements influence transcriptional outputs, and how a broader genomic and epigenomic environment modulates promoter responsiveness to enhancers. We will complement these studies with live-cell and multiplexed DNA FISH imaging of model loci where enhancers regulate their target genes over ultra-long genomic distances. We will track movement of cis-regulatory elements and visualize nascent transcripts to capture the kinetic behavior of enhancers and promoters and its relationship with the discontinuous nature of transcription. We will introduce genetic perturbations to address how a new class of structural elements identified in our previous studies mediates robust transcriptional regulation. Finally, through precise modulation of TF and coactivator dosage at physiological levels, we will explore features that confer sensitivity or robustness of enhancers and gene expression to changes in trans-activator concentrations. Altogether, the proposed research will yield new concepts and quantitative models of long-range gene control, with broad future implications for understanding and treatment of human disease.

NIH Research Projects · FY 2025 · 2019-05

The overarching goal of this project is to understand how molecular-scale interactions at cellular adhesion complexes dictate the organization of cells and tissues. In the first portion of this project, we focus on the proteins that make up adherens junctions (AJs) and tight junctions (TJs). AJs and TJs link the cytoskeletons of neighboring cells and allow epithelial tissues to fulfill their essential function as physical barriers, respectively. Importantly, these complex molecular assemblies are exquisitely responsive to the mechanical forces generated during embryonic development and tissue repair, and in the context of diseases such as cancer and heart disease. However, only a few of the protein-protein interactions that make up these adhesion complexes have been characterized biochemically, and even less is known about the underlying mechanisms by which these structures respond to mechanical load. This lack of quantitative data presents an unavoidable roadblock in the collective effort to understand how cells build and remodel multicellular tissues. In the past funding period, we used single-molecule biophysical assays to discover multiple unanticipated mechanisms by which the proteins present in these complexes sense and respond to mechanical force. Here, we will build on these results to discover the mechanisms by which AJ and TJ proteins may act to seed larger- scale organization at the cell and tissue levels. Based on strong preliminary data, we will examine how adhesion complexes templated by αE-catenin and afadin regulate the assembly of multicellular actomyosin cables that power collective cellular motions during embryonic development and wound healing. Preliminary data likewise demonstrate that PDZ domains, a widespread class of protein domains that mediate protein- protein interactions, can exhibit striking forms of mechanosensitivity. Building on this result, we will elucidate the function of mechanosensitive PDZ-mediated interactions in controlling the assembly and dynamics of TJs, and work to discover additional forms of force sensing employed by junctional proteins. The second portion of this proposal focuses on a class of specialized cellular adhesion complexes that mediate planar cell polarity (PCP). PCP refers to the long-range, front-back polarization of cells in the tissue plane. PCP is essential in multiple developmental contexts, and aberrations in PCP signaling are a prevalent source of birth defects. Previous work shows that the core PCP components assemble into clusters at cell-cell junctions, with specific proteins asymmetrically localized to opposite sides. The molecular mechanisms that mediate the induction of this key asymmetry have remained elusive. Here, we will combine the power of Drosophila genetics with quantitative imaging approaches to test the hypothesis that multivalent protein-protein interactions within individual clusters lead to a nonlinear increase in asymmetry with cluster size. In total, work across these two areas will greatly expand our understanding of how the interactions of individual proteins within cellular adhesion complexes generate the structure and function of cells and tissues.

NIH Research Projects · FY 2025 · 2019-05

PROJECT SUMMARY/ ABSTRACT This application proposes a renewal of a Mid-career Investigator Award in Patient-Oriented research for Eleni Linos, MD DrPH, Professor of Dermatology at Stanford University. Dr. Linos’ long term career objectives are to perform rigorous research to improve the care of patients with skin disease. This renewed award will build on her previous work and accomplish the following objectives: to expand her current research portfolio to the care of elderly patients with skin disease, and the disease area of Hidradenitis Suppurativa. The mentorship goals of this renewal proposal are aligned with the research aims. The proposed studies provide hands-on research training experiences in quantitative and qualitative data collection and analysis for mentees, and will produce preliminary data for mentees' K, T, and R applications. The research aims will provide additional opportunities for mentees to develop skills in machine learning, large language models, quantitative and qualitative research, biomedical ethics, health disparities research and a new focus area of Hidradenitis Suppurativa. The new projects build directly on her previous research and leverage existing networks, collaborations and access to leaders in behavioral science and geriatrics, social media and technology. Aim 1: To describe the progression of Hidradenitis Suppurativa using a longitudinal image library from patients of all skin types. Aim 2: To describe the mental health, sleep, and quality of life burden of Hidradenitis Suppurativa. Aim 3: To identify barriers and solutions to the implementation of digital tools in the dermatologic care of patients from all backgrounds. This award will ensure that Dr. Linos has sufficient protected time to fully support her mentees. The long-term goal of this research is to expand knowledge on the care of patients with skin disease, in order to improve the quality of life and optimize dermatologic care of these patients. This K24 renewal award will provide Dr. Linos with the opportunity to expand her mentoring and research on these topics and allow her to train the next generation of dermatologic researchers so that they are equipped to improve the lives of patients with skin disease and build the research workforce of the future. Together, the Research Plan and Mentoring Plan provide a new five-year strategy that is directly relevant to the NIH and NIAMS missions of training the next generation of physician scientists to carry out patient-oriented research.

NIH Research Projects · FY 2026 · 2019-05

Glia are non-neuronal cells with diverse functions that range from forming the myelin sheath to defending the brain against infection. A major goal of our research is to use the powerful experimental advantages of zebrafish to discover new genes that are essential for the development and function of two classes of glia in the CNS, oligodendrocytes and microglia. Oligodendrocytes form myelin on axons in the CNS. After an oligodendrocyte begins to myelinate axons, it has only a short developmental window (or “critical period”) to extend new myelinating processes. Using genetic and cellular approaches in zebrafish, we have identified a number of positive and negative regulators of myelination. One of our goals is to determine how these factors control myelination during development, neural plasticity, and remyelination. In addition, we will investigate the molecular basis of the critical period. Microglia are highly motile, phagocytic glial cells in the CNS that destroy pathogens and clear debris such as apoptotic cells and damaged axons. Despite the importance of microglia in CNS health and disease, many critical questions remain to be addressed about these cells. We have conducted zebrafish mutational screens to discover essential microglial genes, and we are characterizing their functions using in vivo imaging and other approaches. The mechanistic insight gained from these studies will advance our fundamental understanding of the central nervous system, illuminate the pathways that are disrupted in diseases of the brain, and suggest avenues toward therapies for neurological disorders.

NIH Research Projects · FY 2026 · 2019-05

Project Summary/Abstract Defects in protein translation are central to multiple neurodegenerative diseases, including Alzheimer’s disease and related dementias (e.g., frontotemporal lobar dementia). An unconventional form of translation has been shown to play a role in several neurodegenerative diseases associated with nucleotide repeat diseases. In these diseases, expanded nucleotide repeats get translated in an AUG-independent manner to produce aberrant peptides that may contribute to neurodegeneration. The mechanism of this unusual form of translation, called RAN translation, has remained poorly understood. The Gitler and Puglisi laboratories have joined forces to combine biophysics and genetics approaches to elucidate the mechanism of RAN translation. We developed new single-molecule methodologies to study translation dynamics in humans and combined these with biochemical and in vivo approaches to reveal a global mechanism for RAN translation and identified potent modifiers of RAN translation, at least one of which could mitigate degeneration in several frontotemporal dementia and amyotrophic lateral sclerosis (ALS) disease models. We propose new studies to gain even deeper understanding of RAN translation. We will also expand and extend our efforts to investigate a new facet of frontotemporal dementia (FTD) pathology that we recently discovered – altered processing of the 3’ end of certain mRNAs, which are targets of the FTD and ALS disease protein TDP-43. These altered 3’ ends of the mRNAs has profound effects on the translation of the mRNAs and we have evidence that at least one of them is directly linked to FTD. Our results will build on the foundation of the prior funding period and establish a coherent dynamic mechanism of RAN translation and its regulation from initiation through protein production. We will also illuminate a new facet of TDP-43 pathology in FTD and ALS and how this impacts protein translation. More broadly, our results will provide a mechanistic foundation for understanding the interplay of translation dynamics and fidelity and protein quality control in aging and neurodegenerative diseases, including dementia.

NIH Research Projects · FY 2026 · 2019-05

PROJECT SUMMARY—ABSTRACT The combination of quantitative experimental studies and ordinary differential modeling has yielded great insight into how biological switches and oscillators can and do function. Implicit in both the experimental and theoretical approaches is the assumption that the systems in question are essentially homogeneous bags of enzymes, which of course is incorrect. Here we propose studies aimed at understanding two intriguing aspects of regulation in spatially organized cytoplasm. The first is the mechanism underpinning self-organization of the cytoplasm, and the second is the propagation of activity states through the cytoplasm via trigger waves. The main experimental system we will use is Xenopus egg extracts, which are more manipulable than intact cells, but more complex and realistic than in vitro systems. The main modeling approaches will be partial differential equations and agent based models that explicitly account for spatial dynamics. Over the next five years we plan to focus on four basic aspects of cellular organization and dynamics: 1. Self-organization. Incredibly, it turns out that crude, heterogenous interphase Xenopus egg extracts self- organize into sheets of cell-like compartments. The mechanism underpinning this self-organization is not yet understood. We plan to use inhibitors and depletions in extracts, reconstitution in vitro, and mathematical modeling to gain insight into the basis of this biological self-organization. 2. Trade-offs, speed, and homeostasis. We will use self-organized extracts to determine how basic aspects of cell physiology are affected by the crowding of the cytoplasm, initially focusing on ATP production and utilization. 3. Trigger waves. Trigger waves are self-regenerating fronts of chemical activity that can propagate without diminishing in amplitude or speed. Action potentials are trigger waves; so are calcium waves, mitotic waves, and apoptotic waves. We are examining the robustness of mitotic and apoptotic trigger waves in Xenopus egg extracts, and are testing hypotheses about the possible compartmentalization of trigger waves and bistable states. 4. Reconstitution of transcription. Under the right conditions, Xenopus egg extracts can carry out cell cycles for many hours, with DNA replication alternating with mitosis. Recently we have found that extracts appear to undergo the midblastula transition once 13-15 cell cycles have taken place. We plan to use the extract system to study the mechanism of the midblastula transition and to develop extracts as a new system for studying transcription.

NIH Research Projects · FY 2026 · 2019-05

Project Summary/Abstract The progression of cancer and infectious disease is an evolutionary process. Pathogens engage in an arms race with their hosts, and while antibiotics/antivirals have enabled us to skew the outcome of these contests, these bulwarks against contagion are being steadily eroded. Mutation and natural selection, coupled with rapid generation times and immense pathogen population sizes, provide pathogens a decisive advantage. To regain the upper hand, we must better understand the evolutionary process, to aid in the development of novel classes of antimicrobials and devise therapeutic strategies that take into account how these weapons work and how pathogens evade them. Until recently, efforts to gain a deep understanding of the adaptive process were stymied, because adaptive mutations are rare and identifying them is challenging. To solve this, we developed a system to track the evolutionary process, isolate thousands of adaptive lineages, remeasure the fitness of those lineages across many environments, and cheaply whole genome sequence hundreds to thousands of such mutants. I propose a new phase in my ambitious, integrated research program that takes full advantage of this lineage tracking system, and our discovery of Pareto fronts, which are indicative of trade-offs. We will pursue two major goals. First, we will expand our understanding of evolution in the face of abiotic selection pressures, especially those that produce adaptive trade-offs. Specifically, we will determine the role of historical contingency as it pertains to trade-offs in adaptation, the potential for trade-offs to force canalization (e.g., trapping lineages into extreme specialization), the influence that epistasis exerts on the geometry of trait space, and the extent to which adaptive constraints underlie negative and diminishing returns epistasis. Second, we will expand to contrast these findings with evolutionary outcomes under biotic selection pressures, using two new systems. To model genetic conflict and arms race dynamics within species we will use the killer yeast system. Killer yeast secrete a toxin that destroys sensitive yeast, while retaining an intracellular antitoxin. Toxin and antitoxin are both encoded by a vertically inherited virus, which itself parasitizes a second virus. We will co-evolve killer and sensitive cells and determine whether, as theory predicts, there are recurrent periods of selection and whether coevolved solutions show greater trade-offs than typically observed from abiotic selection pressures. To experimentally model arms race dynamics between species we will use crAssphage, one of the most prevalent bacteriophages associated with the human gut microbiome. The aim of these evolution experiments will be to evaluate the dynamics of phage and its bacterial host co-adaptation over trials of short- and medium-term duration. Overall, this integrated, multi-level research strategy will yield fundamental new insights into how the adaptive process is constrained under alternative forms of selection. Armed with this deeper understanding of adaptation, we will better be able to predict the likelihood of different evolutionary futures given knowledge of a genomic present and thereby gain the upper hand in our battle against human disease.

NIH Research Projects · FY 2025 · 2019-05

PROJECT SUMMARY Cardiovascular complications of cancer therapy significantly contribute to the global burden of cardiovascular diseases. Although remarkable progress has been made in understanding the genetic basis of doxorubicin- induced cardiotoxicity (DIC), we cannot predict which patients will be affected by DIC or protect patients at risk for suffering from DIC adequately. Here, we will use a novel multiplexing methodology of creating a "cell village" by pooling multiple patients' induced pluripotent stem cell (iPSC) lines in a dish to map the genetic basis of inter- individual differences in response to doxorubicin. In Aim 1, we will co-culture 100 iPSC lines in 10 distinct "cell villages," where each "cell village" contains ten independent patient-specific iPSC lines. Next, we will differentiate each "cell village" into iPSC-derived cardiomyocytes (iPSC-CMs). Finally, we will perform a single-cell multi- omics sequencing analysis of the "cell villages" to understand the impact of genetic variability on cardiomyocyte gene regulation and functions at baseline. In Aim 2, we will employ a single-cell multi-omic approach to uncover and validate the role of response eQTL in DIC prediction. We will treat iPSC-CMs in each "cell village" with doxorubicin at various doses. Next, we will perform single-cell multi-omics profiling to model the contribution of genetics to variability in responses to doxorubicin treatment. In Aim 3, we will employ 3D engineered heart tissues (EHTs) and CRISPR/cas9 genome-editing to comprehensively study the functional role of two candidate doxorubicin response genes. All in all, the proposed experiments will serve as a proof-of- principle in using the "cell village" model as a high throughput personalized drug screening platform.

NIH Research Projects · FY 2025 · 2019-04

Project Summary Type 2 diabetes (T2D) is a leading cause of death nationwide with 65% of mortality due to cardiovascular disease. The term “diabetic cardiomyopathy (T2DCM)” refers to a condition with adverse myocardial structural changes, in the absence of hypertension and vascular pathology. Although T2D and CVD are tightly intertwined, we lack a deeper understanding of T2DCM at the molecular and cellular levels. In recent years, sodium-glucose cotransporter-2 (SGLT2) inhibitors have emerged as a promising therapeutic for T2DCM, but the precise protective mechanisms in cardiomyocytes, fibroblasts, and endothelial cells which construct the cardiac microenvironment remain incompletely understood. In this multi-PI R01 renewal, our team will elucidate the mechanisms of metabolic interplay within and between cardiovascular cells which confer cardiac protection by SGLT2 inhibition. We will harness induced pluripotent stem cell (iPSC) technology to generate diabetic models of cardiovascular cell types for cellular and metabolic phenotyping of SGLT2 inhibition in vitro (Aim 1). We will construct iPSC-derived engineered heart tissues for functional phenotyping and proteomic determination of the SGLT2 inhibitor protein interactome (Aim 2). Afterward, we will validate cardioprotective mechanisms in a diabetic mouse model at single-cell resolution (Aim 3). In summary, understanding the exact role and mechanism of SGLT2 inhibition in T2DCM may contribute to finding new therapeutic modalities for the treatment of metabolic heart diseases.

NIH Research Projects · FY 2026 · 2019-03

PROJECT SUMMARY Altered cell-surface glycosylation is recognized as a hallmark of cancer. Two frequently observed cancer- associated glyco-phenotypes are hypersialylation and mucin overexpression. These cancer glycosylation patterns strongly correlate with disease aggressiveness and poor patient outcomes, but until recently their functional significance was unclear. In previous funding periods, we discovered that hypersialylation allows cancer cells to evade immune surveillance through engagement of immune inhibitory Siglec receptors. We speculate that poor responses of cancer patients to current immune therapies, including monoclonal antibodies, T cell checkpoint receptor blockade, bispecific T cell engagers and targeted cell therapies, are due to immune suppression via the Siglec/sialoglycan axis. Accordingly, we developed a new class of immune therapies comprising antibody- sialidase conjugates that degrade Siglec ligands on targeted cancer cells. In collaborative work, we found that mucin glycoproteins promote focal adhesion formation and enhance cancer cell survival and proliferation in the metastatic niche by virtue of their bulk physical properties. We also discovered that certain cancer-associated mucin glycoforms engage Siglec receptors thereby mediating immune suppression. These dual functions of cancer mucins make them attractive targets for cancer therapy. Accordingly, we made targeted mucin degraders comprising mucin-specific proteases fused to cancer antigen- binding nanobodies. Nanobody-mucinase fusions had anti-metastatic and immune potentiating activities in mouse cancer models. In the next funding period, we will build on these discoveries with three Specific Aims. In Aim 1, we will develop targeted mucin degraders based on engineered human proteases, an important step toward clinical translation. In Aim 2, we will explore a new approach to precision targeting of mucin degraders to shed and secreted mucins that are immune suppressive in circulation. Finally, in Aim 3 we will develop a new cancer immune therapy modality comprising antibody-lectin chimeras (AbLecs). These bispecific molecules simultaneously bind cancer antigens, block immune suppressive glycan ligands, and mediate immune effector functions via their Fc domains. Overall, the proposed work will set the stage for preclinical development of two new therapeutic strategies targeting the cancer glycocalyx.

NIH Research Projects · FY 2024 · 2019-01

PROJECT SUMMARY The ultimate goal of the proposed work is to develop a novel co-formulation of insulin analogues (e.g. lispro and aspart) with an amylin analogue (e.g. pramlintide) and with incretin hormones (e.g. liraglutide), to enable a transformational new treatment for diabetes constituting a true replacement therapy. The most challenging aspect of optimal glycemic control for the 1.45 million people with type 1 diabetes (T1D) in the United States is limiting large increases in blood glucose after a meal. People with type 1 diabetes do not produce the insulin required for the body to process glucose, so insulin must be replaced by daily injections. Amylin is a small peptide hormone excreted alongside insulin by pancreatic β islet cells that acts centrally to slow gastric emptying, suppress postprandial glucagon secretion, and decrease food intake, thus complementing the action of insulin to regulate blood glucose levels. Similar to insulin, amylin production is completely absent in individuals with type 1 diabetes on account of their lack of pancreatic β cells. T1D is also characterized by abnormal suppression of glucagon secretion in response to hyperglycemia. Glucagon-like peptide-1 (GLP-1) is an incretin hormone and neurotransmitter secreted from intestinal L-cells in response to nutrients to stimulate insulin and suppress glucagon secretion in a glucose-dependent manner. Long-acting GLP-1 receptor agonists (GLP-1 RAs) have become central to the treatment of diabetes and while these drugs are not yet approved for T1D patients, GLP-1 RAs appear to be well tolerated in patients with T1D and could have beneficial effects in both new onset and longstanding T1D patients. As an adjunctive therapy to insulin, GLP-1 RAs can improve glycemic control and body weight in longstanding disease while also reducing insulin requirements in T1D patients. Current administration regimens for these therapeutics are highly burdensome as they must be injected either daily or weekly or taken daily orally. A true replacement therapy, therefore, would administer both amylin and insulin simultaneously, while also delivering GLP-1 RAs. Unfortunately, Symlin (Pramlintide; AstraZeneca), the only commercial amylin analogue formulation, is formulated at pH~4 while Novolog (Aspart; Novonordisk) and Humalog (Lispro; Eli Lilly), insulin analogue formulations, are typically formulated at pH~7.4, meaning that these formulations are incompatible and must be administered in separate injections. While treatment of diabetes with separate injections of insulin and amylin analogues at mealtimes has been shown to be much more effective than insulin alone at managing diabetes, the administration of two separate injections is burdensome. We have developed a novel polymeric excipient that results in stable, ultra-fast acting insulin, to formulate insulin and pramlintide together to enable a single administration treatment mimicking endogenous hormone secretion. Additionally, we can effectively incorporate GLP-1 RAs into these formulations. This novel combination therapy will yield unprecedented postprandial glycemic control and catalyze the development of a powerful tool for the management of diabetes affording thus far unrealized therapeutic impact.

NIH Research Projects · FY 2026 · 2018-12

Abstract/Summary (Andrew Fire PI, NIGMS R35GM130366, January 2023) Our lab studies the mechanisms by which cells and organisms respond to genetic change. The genetic landscape faced by a living cell is constantly changing. Developmental transitions, environmental shifts, and pathogenic invasions lend a dynamic character to both the genome and its activity pattern. We study a variety of natural mechanisms that are utilized by cells adapting to genetic change. These include mechanisms activated during normal development and systems for detecting and responding to foreign or unwanted genetic activity. At the root of these studies are questions of how a cell can distinguish "self" vs. "nonself" and "wanted" vs. "unwanted" gene expression. Caenorhabditis elegans provides an excellent model for diverse studies of development, physiology, and gene expression, with traditional strengths of the model system in genetic and anatomical analysis combining with a highly-annotated genome and a variety of genetic and epigenetic manipulation techniques. With the variety of tools, information, and experimental questions, this system remains an attractive choice for varied studies of gene expression. C. elegans can be quite proficient at silencing foreign nucleic acid, particularly in the germline; this combined with the other readily manipulated aspects of the system provides an excellent starting point for the study of responses to foreign information. Several questions drive our research program What features allow certain RNAs to persist and propagate without encoding a replication machinery? In what circumstances are non-chromosomal inheritance processes utilized by biological systems? How do machineries that propagate non-chromosomal inheritance serve the organism? Can we adapt the underlying persistence mechanisms for experimental/therapeutic protocols aimed at sustained expression or sustained suppression?

NIH Research Projects · FY 2026 · 2018-12

PROJECT SUMMARY/ABSTRACT Targeting of oncogenic kinases with small molecule inhibitors is ow a well validated paradigm for treatment of particular cancer types. Unfortunately, resistance to ATP-competitive inhibitors typically develops due to the selection for tumor cells that harbor new mutations that induce resistance to kinase inhibitors or due to activation of by-pass signaling pathways. In addition, many tumors depend on the scaffolding function of a kinase and not on enzymatic kinase activity, thereby rendering inhibitors ineffective. For example, the pseudokinase HER3 is an obligate heterodimerization partner with EGFR and HER2 but its kinase activity is not required. In the first funding cycle, we successfully explored a fundamentally different approach to abrogate kinase signaling by developing novel small molecules that induce kinase degradation via the ubiquitin-proteasome pathway and systematically explored the degradable kinome. Here we propose to expand on this extensive fundamental work to increase coverage of the degradable kinome, further investigate the mechanism of small molecule-mediated protein degradation and expand probe development to non-degrading molecular glues. In particular, we will increase the chemical space of potential degraders to not-yet explored kinase ligand scaffolds as well as recently reported E3 ligase ligands including ligands for ligases such as IAP, MDM2, KEAP1, DCAF11, DCAF15, DCAF16, AHR, FEM1B, RNF4 and RNF114 and novel ligase attachment geometries for our previously characterized E3 ligases CRBN and VHL, and will also include the development of trivalent degrader molecules as a novel compound design approach. We have also developed new and improved global proteomics workflows to enable increased proteomic profiling of degraders in a panel of up to five different cell lineages, which will increase identified kinases from ~400 to almost 600 per compound treatment. In addition, we have developed new chemoproteomics workflows to determine ternary complex formation of glue molecules and are able to determine ubiquitination sites and profiles of degrader molecules. This extended degrader characterization data will enable us to further expand our open-source accessible degradable kinome database and will deepen our understanding of E3 ligase-dependent degradation mechanisms. We will further broaden our probe development efforts to HER3-targeting non-degrading glue molecules to investigate alternative modes of action for development of novel therapeutics. The goal of this grant application is to expand the degradable kinome database by covering an extended chemical space and expanding cellular treatment conditions, interrogate structural mechanisms of targeted protein degradation and non-degrading complexes, and assemble a HER3-targeted compound library for the development of non-degrading glue molecules as a novel therapeutic strategy to modulate HER3 activity in cancer models.

NIH Research Projects · FY 2026 · 2018-12

Abstract: Sensory hair cells are required for balance function. Vestibular hair cell degeneration causes balance dysfunction/hypofunction manifested as dizziness and vertigo. While the mammalian cochlea lacks the ability to regenerate lost hair cells, a limited degree of spontaneous regeneration occurs in the utricle, a vestibular organ detecting linear acceleration. Prior studies have shown that ATOH1 overexpression can enhance the extent of hair cell regeneration, but these regenerated hair cells fail to fully mature. In preliminary experiments, we have characterized hair cell degeneration and regeneration in the mature mouse utricle in vivo, and found that transient, rather than constitutive, overexpression of ATOH1 promotes both hair cell regeneration and maturation. The first aim of this proposal is to determine if transient ATOH1 overexpression increases hair cell regeneration and maturation. Specifically, regenerated hair cells labeled via fate-mapping are probed via histology and electrophysiology to assess bundle morphology, expression of bundle proteins, mechanosensitvity, basolateral currents, and synaptic properties including vesicle release. To gain an unbiased insight into the genetic signature of regenerated hair cells, we will use single cell RNA sequencing technologies and bioinformatic approaches to delineate the transcriptomes of ATOH1-enhanced regenerated hair cells and validate them histologically. In the second aim, we will test whether transient overexpression of ATOH1 enhances regeneration and maturation of hair cells in human utricles in vitro. We will leverage our established pipeline of human utricles from organ donors and vestibular schwannoma patients, where the latter cohort shows hair cell degeneration, a low level of spontaneous hair cell regeneration and incomplete maturation. Regenerated hair cells will be assessed histologically for bundle morphology, expression of bundle and synaptic proteins and via single cell RNA sequencing to probe transcriptomes of ATOH1-enhanced regenerated human hair cells. In summary, we will apply state-of-the art technologies (gene therapy, hair cell physiology, single cell RNA-seq, bioinformatic strategies) to study vestibular hair cell regeneration in transgenic mouse models and human utricles. We have assembled a team of experts who have worked together to collect promising preliminary data. At the end of this 5-year proposal, we will have determined whether transient ATOH1 overexpression can promote regeneration and maturation of mouse and human hair cells at the histological, electrophysiological, and transcriptomic levels.

NIH Research Projects · FY 2026 · 2018-09

Project Summary/Abstract The overall objective of this research is to gain a deeper understanding of visual processing in the macaque monkey retina, the most important animal model for understanding human vision, and to leverage that knowledge to treat blindness. The goal of the proposed work is to determine how four poorly understood retinal cell types contribute to visual processing in primates. Many of the retinal amacrine and ganglion cell types that have been characterized in non-primate retinas perform complex and specialized visual computations, extracting information such as motion direction, object orientation, and object versus background motion from visual inputs. Whether similar computations also occur in the primate retina is unclear, because most physiological studies have focused on the role of only five of the ~20 output pathways. We have identified four cell types in the macaque monkey retina that exhibit intriguing differences from the five better-studied cell types in their visual response properties. Our specific aims are to 1) determine how striking waves of activity in the network of A1 amacrine cells network shapes the light response properties of parasol ganglion cells, 2) determine how intrinsic and circuit mechanisms shape the unusual spatial and temporal response properties of broad thorny ganglion cells, and 3) determine how membrane and receptive field properties allow ON- and OFF-type smooth monostratified ganglion cells to signal distinct information to the brain from their parasol cell counterparts. At the conclusion of this work, we expect to have a deeper understanding of the visual computations performed by the primate retina and the neural mechanisms that underlie those computations. Further, this project will shed much needed light on how retinal processing in primates, and by extension humans, relates to that observed in rodents and other animal models.