Weill Medical Coll Of Cornell Univ

NIH Research Projects · FY 2025 · 2025-09

Project Summary Disruption of circadian clocks—cell-intrinsic oscillators responsible for maintaining daily physiological and behavioral rhythms—is an emerging hallmark of aging. While studies have shown that circadian clock outputs remodel or diminish with age in various tissues, the specific cellular and molecular drivers of the bidirectional relationship between aging and the circadian clock remain unknown. This project seeks to identify key cell populations whose circadian rhythms are most impacted by aging and those that are most vulnerable to loss of the circadian clock, aiming to understand how aging affects circadian rhythms across cell types and how circadian disruption influences aging at cellular and molecular levels. The main approach centers around high-throughput single-nucleus RNA sequencing of flash-frozen tissues. In Specific Aim 1, I will profile sex-matched young and aged mice sampled every four hours over two days in constant darkness, dissecting seven organs: brain, liver, heart, kidney, lung, colon, and skeletal muscle. Clustering analyses will identify cell types, and circadian genes will be characterized using the JTK_Cycle algorithm, comparing results across sex and age. I will map circadian cell-cell interactions through ligand-receptor analysis to reveal age-related shifts in intercellular communication and perform transcription factor enrichment analysis to uncover upstream regulators in cell types showing pronounced changes with aging. In Specific Aim 2, I will perform single-nucleus sequencing on wild-type young and aged mice, Bmal1 knockout mice—a genetic model of circadian disruption—and aged mice exposed to chronic jet lag—an environmental model of circadian disruption. This analysis will identify cell populations vulnerable to circadian disruption and determine whether circadian disruption-induced "early aging" reflects natural aging or represents a distinct pathology. The outcomes of this study will enhance our understanding of the interplay between circadian rhythms and aging, offering a roadmap for examining the circadian output of cell types involved in aging-associated diseases such as Alzheimer's disease, Type II diabetes, major depressive disorder, and cardiovascular disease. By uncovering the circadian rhythms of disease-associated genes and receptors, the findings could inform therapeutic timing and improve chronotherapy by aligning drug delivery with the biological rhythms of specific cell types. With the mentorship of my sponsor, co-sponsor, thesis committee, and the support of this fellowship, I am confident I will be well-prepared to pursue this project and achieve my goal of becoming a physician-scientist and an independent investigator in the field of aging biology.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY During pancreatitis, acinar cells undergo a cell fate change, known as acinar-to-ductal metaplasia (ADM). The metaplastic response can be viewed as an adaptive phenomenon, functioning to limit tissue damage through the transient loss of the acinar cell fate and repression of digestive enzyme production. However, pancreatitis is a major risk factor for the development of many pancreatic diseases. The inherent plasticity of the acinar cell creates a susceptibility to permanent acinar-to-ductal reprogramming, contributing to the formation of pancreatic neoplasia. Despite pancreatitis being a well-known source of morbidity, we do not understand the precise mechanisms governing this cell state transition. Thus, it is critical to study how acinar cell plasticity is regulated in the context of tissue injury. This will enable the identification of molecular dependencies of pancreatitis, offering new opportunities to intervene during the early disruption of pancreatic homeostasis. To understand how acinar cell plasticity is regulated, I first compared the transcriptional and chromatin accessibility landscapes of acinar, ductal, progenitor, and metaplastic cells with bulk RNA-seq and ATAC-seq. ADM has historically been described as a transdifferentiation of mature acinar cells into a “duct-like” state. My preliminary evidence suggests that ADM is a complex cell state, featuring the dedifferentiation of acinar cells to a late progenitor phenotype. Motifs belonging to the Forkhead box (Fox) family transcription factor FOXA2 were made highly accessible during ADM. FOXA2 is a pioneer factor with well-studied roles in pancreatic development, yet its contribution to adult acinar cell homeostasis is underexplored. In preliminary experiments using in vivo models of pancreatitis and conditional knockout, I have observed that FOXA2 is required for the metaplastic response to injury. Therefore, I hypothesized that FOXA2 controls acinar cell plasticity by directing the acquisition of a progenitor-like state during pancreatitis and supports eventual neoplasia by facilitating acinar- to-ductal reprogramming. I will utilize transcriptomic and epigenomic approaches to delineate how FOXA2 functions to direct lineage plasticity during pancreatitis. With in vitro and in vivo models, I will assess if FOXA2 contributes to the initiation of pancreatic neoplasia by determining if FOXA2 is required for permanent acinar-to-ductal reprogramming. Crucially, the proposed investigations will enhance my training and growth as a scientist. I will become familiar with sophisticated in vivo and in vitro models and will be exposed to cutting-edge epigenomic investigations that will help to build a strong foundation for my future career as an independent investigator.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that results from the breakdown of tolerance mechanisms in self-reactive T cells, leading to immune infiltration of the pancreas and the destruction of insulin- producing b cells. Patients with T1D require lifelong exogenous insulin treatment and often develop multiorgan dysfunction, emphasizing the need for effective treatment strategies. β cell-specific CD8 T cells are the primary pathogenic population that eliminate insulin-producing b cells in the pancreatic islets, yet intriguingly, MHC class II haplotypes confer the greatest genetic risk for T1D development suggesting a critical role of CD4 T cells in disease initiation and progression. However, many aspects of autoimmune β cell-specific CD4 T cell differentiation and function remain enigmatic, including where and how autoimmune CD4 T cell populations arise and are maintained and their exact function in driving T1D. Our lab previously identified a stem-like CD8 T cell population which self-renews and gives rise to differentiated progenies which migrate to the pancreas and eliminate b cells; the stem-CD8 T cell pool is absolutely required for sustained b cell destruction. The goal of this application is to generate a deep understanding of the phenotypic and molecular features and functional role of β cell-specific CD4 T cell populations in driving T1D and specifically CD8 T cell stemness, differentiation and function. We will leverage the non-obese diabetic (NOD) mouse model, a clinically relevant model of T1D, which shares many features with human disease to (i) assess heterogeneity and polarization/differentiation states of CD4 T cells, (ii) determine the importance of key transcription factors in regulating CD4 T cell function, and (iii) employ innovative spatial sequencing technologies to identify cell-cell interactions and signals driving β cell destruction. Our proposed studies will provide important insights into autoimmune b cell-specific T cell programming and function which could yield novel therapeutic targets for the prevention or treatment of T1D and other T cell-mediated autoimmune diseases.

NIH Research Projects · FY 2025 · 2025-09

Project Summary A hallmark of the hematopoietic system is the ability to respond to inflammatory challenges. Central to this response are hematopoietic stem and progenitor cells (HSPCs), which balance self-renewal and differentiation programs to maintain stable blood cell output. In response to acute inflammation, such as infection, pro- inflammatory cytokines activate HSPCs to rapidly augment innate immune cell production. Although initially critical for pathogen control, persistent and non-productive inflammation can drive HSPC dysregulation and stem cell loss, a key contributor to bone marrow failure syndromes, acquired neutropenia, and hematologic malignancies. Certain inflammatory stimuli, including the mycobacterium BCG, induce durable epigenetic and transcriptional reprogramming in HSPCs which is transmitted across differentiation to mature innate immune cells with enhanced effector function upon restimulation. These findings suggest HSPCs can act as a reservoir of inflammatory memory with functional consequences in differentiated immune cells, yet the cytokine signals and molecular programs that establish HSPC memory are poorly understood. This proposal aims to dissect the mechanisms through which Type II Interferon (IFNG) signaling in response to BCG establishes durable memory programs in HSPCs and their innate immune progeny cells. In Specific Aim 1, we will characterize the cellular targets of IFNG signaling within the bone marrow (BM) niche required for HSPC reprogramming, then define the contribution CD4 T lymphocytes to this response. In Specific Aim 2, we will apply single cell multi-omics and epigenomics approaches to identify the epigenetic mechanism by which IFNG signaling establishes durable memory programs in HSPCs, then trace the inheritance of these programs across differentiation to innate immune cells. This project is ideal for a physician-scientist in training, given its blend of cellular immunology and high-throughput sequencing approaches to dissect the durable impact of inflammation on the hematopoietic system, along with the potential to uncover novel therapeutic targets to address stem cell dysfunction in hematologic disease and augment innate immunity. With the mentorship of my sponsor, co-sponsor, collaborators, thesis committee, Tri-I MD-PhD program leadership, and the support of this fellowship, I will be well prepared to pursue and achieve my goal of being a physician-scientist and independent investigator.

NIH Research Projects · FY 2025 · 2025-09

Project Summary/Abstract In the United States, young women with early-stage breast cancer are increasingly choosing to have their unaffected breast removed, a procedure known as contralateral prophylactic mastectomy (CPM) or bilateral (“double”) mastectomy. The clinical benefits of CPM (e.g., no survival benefit) are minimal and there are documented harms, including an increased risk of surgical complications and the potential for negative quality of life sequelae. Additionally, the American Society of Breast Surgeons and Choosing Wisely guidelines recommend against the routine use of CPM among average risk women with unilateral breast cancer. Prior research has suggested that the risks and benefits of breast cancer surgery are not being optimally communicated and that some women have inaccurate perceptions about breast cancer risks that are likely impacting their choice of surgery. Life-stage specific factors (e.g., breastfeeding, body image) can make surgical decisions particularly complex for young women and young women report high levels of decisional conflict regarding the surgical decision. To optimally support young women making decisions about breast cancer surgery, we developed CONSYDER, a web-based decision support tool tailored to the unique concerns of young patients. In our pilot study, all women who used CONSYDER found it helped them understand the pros and cons of surgery and clarify their values around this highly preference sensitive decision. CONSYDER is innovative in its approach to decision support in that integrates supportive care resources to help manage stress and anxiety around diagnosis as part of the decision tool. These resources were valued by patients, demonstrating the acceptability of this approach that acknowledges their psychosocial needs. We are now proposing a pragmatic, stepped-wedge, multicenter trial that incorporates a Type II hybrid effectiveness-implementation design. We will test the effectiveness of CONSYDER in reducing decisional conflict before surgery among 800 young women seeing a breast cancer surgeon at Yale Smilow Cancer Center, Weill Cornell Medicine, Dana-Farber Cancer Institute, and Duke Cancer Institute. Concurrently, we will use a mixed-methods approach, including surveys and semi-structured interviews with patients and providers, to evaluate the value and implementation of CONSYDER. Implementation outcomes will include feasibility of delivery, adoption and fidelity across sites, uptake of key features, and sustainability. Our pragmatic study will test the effectiveness and implementation of an intervention designed to promote shared decision-making and reduce decisional conflict in young women with breast cancer. We expect findings from the proposed study not only to demonstrate the effectiveness of this novel intervention but also to inform the optimal delivery and broader dissemination of CONSYDER, enabling patient-centered support for the highest quality preference-sensitive decisions for young women with breast cancer facing surgery.

NIH Research Projects · FY 2025 · 2025-09

ABSTRACT Single-cell analysis has transformed biomedical research, revolutionizing our understanding of biological processes while providing invaluable insight into human physiology and disease, including cancer. However, current single-cell methodologies are largely restricted to analyzing DNA or RNA features, with analysis of the crucial regulatory protein activity layer remaining almost completely unexplored from the single-cell perspective, with current technologies only able to profile the most stable chromatin-binding factors such as histones in single cells. This has led to significant gaps in our knowledge about mechanisms of gene regulation in both normal and tumor cells, and has further prevented the development of potential precision therapies that could target pathways that are dysregulated in cancer development or progression. Therefore, the ability to assess the activity of regulatory proteins with different DNA-binding affinities, including weak or transient factors that are critical for transcription regulation, at the single-cell level would open up a new era in the field, similar to the single-cell RNA-seq revolution. To address this challenge, we will pioneer the development of new transformative methods for high-throughput single-cell mapping of DNA-binding proteins across binding affinities to define genome regulation, as well as dysregulation, in human cancer. We present Docking & Deamination plus sequencing (D&D-seq) for direct single-cell DNA footprinting, which tethers a species-specific antibody-binding nanobody to a cytosine base editing enzyme, catalyzing C-to-U edits in proximal genomic regions reflecting target binding to DNA. Combined with single-cell ATAC-seq, this approach enables high-throughput profiling of even weak factor binding to DNA in single-cells using common sequencing platforms. To develop D&D-seq for high-throughput analysis of single-cell gene regulation in cancer, we will optimize profiling across high- and low-affinity binders, and engineer a variety of molecular footprinting base editors to map multiple functional DNA:protein interactions in tumor cells. To capture gene regulation across different layers in the same cell, we will incorporate D&D-seq into common single-cell multi-omics workflows to profile transcription factor (TF) binding and chromatin accessibility together with gene expression and protein levels or histone modifications in single cells for the first time. To better assess the effects of cancer-associated somatic mutations on gene regulatory networks, we will integrate D&D-seq with our single-cell genotyping framework to identify TF binding patterns that are specific to mutant cells. Finally, to support wide adoption by the single-cell cancer genomics community, we will adapt D&D for split-and-pool barcoding, enabling high-thoughput, multimodal analysis of gene regulatory networks in hundreds of thousands of single cells without requiring specific sequencing platforms. Together, these methods will enable the direct single-cell measurement of DNA:protein interactions and TF activity in native chromatin contexts to empower novel discoveries about chromatin dynamics and transcriptional regulation in human tumor cells at unprecedented resolution, opening up entire new fields of inquiry that were unable to be pursued before.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT VEXAS (Vacuoles, E1-ubiquitin-activating enzyme, X-linked, Autoinflammatory, Somatic) is a somatically acquired X-linked disorder characterized by autoinflammatory and hematologic manifestations. First reported in 2020, VEXAS is now known to afflict ~1 in 4,269 men older than 50 years old and causes multiorgan autoinflammation and high rates of myelodysplastic syndrome (MDS). VEXAS is caused by a missense mutation in the ubiquitin-activating enzyme 1 (UBA1) gene that results in aberrant clonal expansion of mutant hematopoietic stem cells (HSPCs) and their myeloid, but not lymphoid, progeny. UBA1 is one of only two E1 enzymes that initiate the ubiquitylation cascade. The most prevalent VEXAS mutations occur at the M41T codon and impair translation initiation at Met41 of the cytosolic UBA1 isoform (UBA1b) and lead to aberrant Met67- initiated translation to generate the enzymatically impaired UBA1 isoform, UBA1c. How this switch in UBA1 isoform usage triggers VEXAS pathogenesis at the molecular and cellular levels remains unknown, owing to the lack of genetically engineered cell culture and animal models. Deploying a newly developed base-editing method, we have succeeded in recreating the most common mutation in UBA1 observed in VEXAS in primary macrophages and HSPCs. Using these systems, we have observed a hyperactive IFN-I axis and spontaneous myeloid bias of UBA1-mutant HSPCs ex vivo and in vivo. Given the links between type I interferon and hematopoiesis, these preliminary results lead us to hypothesize that UBA1 mutation may drive VEXAS pathogenesis through dysregulated interferon signaling and its consequent influence on hematopoiesis. In Aim 1, I propose to dissect the molecular mechanisms by which UBA1 mutation affects type I interferon signaling. Accordingly, we will biochemically dissect IFN-I induction pathways in VEXAS macrophages and test whether Uba1-mutant HSPCs, like their myeloid progeny, display altered IFN-I production. We will further assess the ubiquitylation status of signaling components downstream of innate immune signaling pathways in macrophages bearing a Uba1 mutation to identify mechanisms by which aberrant ubiquitylation may dysregulate IFN-I production in VEXAS. In Aim 2, I will dissect mechanisms of myeloid-biased hematopoiesis in VEXAS syndrome. Accordingly, I will interrogate whether IFN-I signaling differentially affects normal versus UBA1-mutant HSPCs ex vivo. In addition, we will evaluate enhanced myelopoiesis of UBA1-mutant HSPCs in vivo and directly test the contribution of dysregulated interferon to the myeloid-biased hematopoiesis associated with VEXAS. Collectively, our work will provide key insights into VEXAS pathogenesis using state-of-the-art genetic models and may therefore identify novel therapeutic targets in this disease of major unmet medical need.

NIH Research Projects · FY 2025 · 2025-09

Abstract Cardiac ion channels are tightly regulated—in abundance, subcellular location, turnover, and activity—to control the electrical signaling that drives heart muscle contraction. Any perturbation of this regulation can lead to arrhythmogenic sudden death. Most studies of how ion channel dysfunction contributes to pathophysiology have focused on defects of the channel pore-forming subunits. Contributions of channel auxiliary subunits are understudied and complicated by alternative or additional roles for this set of proteins. Here, we focus on fibroblast growth factor homologous factors (FHFs), proteins that bind directly to voltage-gated sodium channels (VGSCs) including the cardiac NaV1.5 channel and that are implicated in life-threatening arrhythmias. Studies on FHFs in heart have focused almost exclusively on how they regulate NaV1.5 via direct binding the channel's cytoplasmic C-terminus. The goal of this proposal is to expand knowledge of how FHFs influence cardiac physiology and how variants contribute to arrhythmias, thus providing a platform for targeted therapies. Our preliminary data uncover three unexpected aspects of FHF function in heart: 1) FHFs regulates VGSCs independent of channel interaction; 2) FHFs regulate trafficking and membrane targeting of multiple cardiac channels via control of microtubule stability (we focus on connexin43 [Cx43]); 3) FHFs affect adrenergic signaling to channels independent of ion channel interaction. We propose to define the underlying mechanisms using a novel structurally guided approach that generates an FHF incapable of binding NaV1.5 and by exploiting unbiased proteomic analyses using protein proximity labeling in cardiomyocytes in the following Aims: Aim 1: Test the hypothesis that FGF13 confers VGSC regulation by affecting local membrane cholesterol. FGF13 is the main FHF in rodent hearts. Our preliminary data offer insight into how FGF13 affects NaV1.5 via regulation of local accessible membrane cholesterol, a mechanism independent of channel binding. Among other implications this aim may explain the vulnerability of subjects with Brugada syndrome to arrhythmias during febrile illnesses. Aim 2: Test the hypothesis that FGF13 stabilizes cardiomyocyte microtubules and thus regulates ion channel trafficking through regulation of MAP4. Focusing on Cx43 trafficking, we show that ablation of FGF13 in mice destabilizes microtubules and mislocalizes Cx43 in ventricular cardiomyocytes via effects on MAP4, a key regulator of microtubules in cardiomyocytes. We will investigate the underlying mechanisms of this additional NaV1.5-binding independent effect. Aim 3: Test the hypothesis that FGF13 regulates adrenergic responses in cardiomyocytes. Exploiting an unbiased proteomic screen, we uncover that FGF13 affects adrenergic regulation of NaV1.5 and CaV1.2 Ca2+ channels. Building on our unbiased discovery platforms, we propose to determine the mechanisms by which FGF13 regulates this critical cardiomyocyte signaling pathway. Together, these Aims propose to expand an understanding of FHFs in cardiomyocyte biology and more generally uncover mechanisms for how ion channel auxiliary subunits regulate their targets.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT The Justice Community Overdose Innovation Network (JCOIN) is a coordinated effort to build the evidence base for the delivery of substance use disorder (SUD) care within the criminal-legal system. JCOIN Phase II will build upon Phase-I's success by scaling up effective interventions, evaluating implementation strategies, testing novel care models, and prioritizing interagency coordination to enhance continuity of care. However, maximizing access to evidence-based care requires simultaneously considering both the costs and benefits of alternative care models. The JCOIN-II Economic Research Resource Center (ERRC) will provide resources and evidence that support the application of health economic research to inform real-world decisions regarding SUD services within the criminal-legal system, while furthering scientific innovation in the field, and establishing a paradigm for integrating health economics into SUD policies at all levels. Specifically, the ERRC will: 1) support JCOIN-II Hubs by identifying economic research questions, and providing guidance on designing, implementing, analyzing, and translating prospective economic evaluations; 2) conduct novel and rigorous health economic research that informs complex “real-world” decisions of policymakers and stakeholders at all levels of the criminal-legal system regarding how best to utilize their limited resources to address the overdose crisis; 3) develop and disseminate tools/resources that can be readily used by practitioners and policymakers to make informed decisions about the relative budget impact of alternative strategies to care for SUD, and to inform the design of appropriate payment systems that support sustainability; and 4) provide ad hoc consultation and technical assistance on economic analyses to JCOIN-II researchers/practitioners, and to develop easily accessible educational resources on economic data collection, analysis, and considerations specific to criminal-legal contexts. The ERRC will be led by Drs. Murphy and McCollister, two health economists with vast expertise in the design and analysis of prospective economic evaluations alongside clinical trials for SUD interventions, particularly in criminal-legal settings. The MPIs have a rich history of collaboration and co-leadership, including as co-directors of: a) the Methodology Core for the NIDA-funded Center for Health Economics of Treatment Interventions for Substance Use Disorder, HCV, and HIV (CHERISH); and b) the JCOIN-I Health Economic Analytic Team (HEAT), which they established to ensure methodological rigor and harmonization in economic evaluations across JCOIN-I Hubs. We will maximize the ERRC's impact by leveraging existing partnerships with the JCOIN Coordination and Translation Center (CTC) and Methodology and Advanced Analytics Resource Center (MAARC), and forging a new partnership with the Community Engaged Research Resource Center (CERRC). Together, these centers will enhance data sharing and analysis, stakeholder engagement, and research opportunities, thereby driving the implementation of evidence-based SUD interventions and improving outcomes for individuals within the criminal-legal system. This study is part of the NIH’s Helping to End Addiction Long-term (HEAL) initiative to speed scientific solutions to the national opioid public health crisis. The NIH HEAL Initiative bolsters research across NIH to improve treatment for opioid misuse and addiction.

NIH Research Projects · FY 2025 · 2025-09

SUMMARY: Understanding the mechanisms underlying the cardioprotective and other health benefits of polyunsaturated fatty acids (PUFAs) is critical for providing sound nutritional advice. A major explanation for how PUFAs mediate their effects is by altering gene expression. Through gene expression, specific PUFAs reduce triglyceride levels and inflammation. Yet, we do not clearly understand the basis for the specificity of some, but not other fatty acids in regulating gene expression. This is a major problem for fully understanding the health implications of eating foods with complex mixtures of these different fatty acids. One of the major transcription factor families that mediates the effect of PUFAs are the peroxisome proliferator- activated receptors (PPARs). PUFAs have been found to bind to PPARs and to promoter their activation of target gene transcription. However, there has been disagreement on the effective binding affinity of PUFAs for PPARs and whether it is physiologically relevant. This has led to confusion over the role of PUFAs as PPAR agonists. However, these studies assumed that PPAR is localized to the aqueous nucleoplasm and that they interact with PUFAs in this environment. Instead, at least one PPAR protein, PPARγ, localizes to condensates, which are membraneless organelles enriched in proteins and nucleic acids. We have recently shown that these environments enrich multiple classes of lipids, including PUFAs. Additional analysis of our datasets has also demonstrated that only specific fatty acids enrich in condensates and that this correlates with their number of unsaturated bonds. In this project, we will explore a new explanation for why specific PUFAs regulate PPARγ-mediated transcription initiation more than others. Rather than different abilities to bind the PPARγ ligand binding domain, the extent to which a specific PUFA species regulates PPARγ may be determined by its ability to enrich in PPARγ condensates. Additionally, this novel form of regulation may specifically effect PPARγ-bound genes in condensates, while not affecting other PPARγ-bound genes. In Aim 1, we will determine if PPARγ agonists enrich in PPARγ-containing condensates. First, we will measure the extent to which different PUFAs and other putative PPARγ agonists enrich in PPARγ condensates in vitro. We will then determine whether a subset of PUFAs also enrich in cellular PPARγ-containing condensates using super-resolution, lipid expansion microscopy. Finally, we will determine if transcription activation by PPARγ agonists correlates with their ability to enrich in PPARγ condensates. In Aim 2, we will test whether the condensate pool of PPARγ proteins are preferentially activated by PUFAs and other PPARγ agonists. We will address this question by measuring co-activator binding to PPARγ after addition of different agonists, both in vitro and in multiple cell-base assays. Together, these two aims will establish whether specific PUFAs preferentially activate the condensate pool of PPARγ proteins.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY The proper distribution of intracellular cargo is crucial for many fundamental biological processes. Yet how cargo transport is regulated in space and time, especially within a crowded cytosol, remains poorly understood. A novel mode of molecular motor-driven transport was recently discovered by the Reck-Peterson lab in the filamentous fungus Aspergillus nidulans, where peroxisomes “hitchhike” on early endosomes and require the protein PxdA. Hitchhiking was initially discovered in filamentous fungi and was later shown to transport mRNAs, peroxisomes, and ER tubules in yeast and mammalian cells. However, the mechanism and function of peroxisome hitchhiking remains unknown. PxdA is only present in the Pezizomycotina subdivision, one of the few fungal groups that produce secondary metabolites (SMs) which have implications in human health and disease. Notable examples of SMs include penicillin, lovastatin, and mycotoxins. Filamentous fungi rely on SMs as virulence factors, signaling molecules for development, and protection from environmental stresses. While many SM biosynthetic pathways have been well-described chemically, very little is known about how SM production is mapped onto a cellular context. The long-term goal of this project is to understand how organelle hitchhiking coordinates SM production to improve the industrial production of SMs that are beneficial for human health and to identify fungal- specific targets for anti-fungal therapeutics. This project will investigate the mechanism and function of organelle hitchhiking in Aspergillus nidulans using a combination of genetics, live-cell fluorescence microscopy, as well as in vitro biochemistry and biophysics techniques. My aims are to 1) determine the molecular mechanism of organelle hitchhiking, and 2) determine the physiological function of organelle hitchhiking in filamentous fungi. Together, my work will enhance our understanding of how organelle transport and positioning in filamentous fungi regulates SM production and development.

NIH Research Projects · FY 2025 · 2025-08

The Tri-Institutional PhD Program in Computational Biology and Medicine (CBM) takes advantage of the outstanding educational and research resources of the Tri-Institutional (Tri-I) consortium in New York City – Weill Cornell Medical College (WCM), Cornell University’s medical campus; Memorial Sloan Kettering Cancer Center (MSK), comprising the basic science Sloan Kettering Institute (SKI) and research programs in Memorial Hospital (MH); and The Rockefeller University (RU) – together with Cornell University, both through its main campus in Ithaca, NY (CU-I) and its newer Cornell-Tech (CTech) campus in New York City, to train the next generation of computational biologists. The CBM provides training in the computational and quantitative approaches needed to tackle complex multidisciplinary biomedical problems and provides: coursework in both quantitative and biological sciences; research rotations to enable a well-informed thesis topic selection; mentored thesis research in one of a wide array of basic to translational laboratories; trainee Research-in-Progress seminar series to enhance program cohesion, foster fluency in relevant disciplines, and provide opportunities for scientific presentation practice; training and mentorship in performing rigorous and reproducible scientific research; an array of programmatic enrichment activities, including an annual offsite retreat which provides cross-lab and cross-campus interactions, lunches with visiting seminar speakers, and fellowship writing training; active guidance and mentoring via annual formal meetings with program co-Directors, annual thesis committee meetings that include career discussions, and annual IDPs; exposure to various career paths for successful transition into the biomedical research workforce. The Program, which is well established with a 21-year track record, has an expanding record of training success, including timely graduation and a strong record of placing graduates in research-related careers both in and outside academia. With this proposal, we are requesting 10 T32 slots (compared to 8 slots in our previous T32), which is well justified by the deep pool of highly qualified training-grant eligible applicants, large array of cutting-edge thesis research opportunities with leading faculty scientists, and the training enrichment that is inherent to an increased critical mass of students. The requested T32 funding would greatly aid the CBM program in continuing to achieve its mission of excellence in training the next generation of scientists to rigorously and reproducibly develop and apply computational and analytical methods to solve complex problems in biology and medicine and to prepare them for research-related academic and non-academic careers following timely completion of their PhD degrees.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Depression and metabolic syndrome are frequently comorbid and bidirectionally linked, yet our understanding of how metabolic dysfunction impacts features of depression is limited. Anhedonia is a core symptom of depression which encompasses deficits in reward processing including effort valuation, or the weighing of a reward’s value against the effort necessary to obtain it. Due to the intrinsic relationship between effort and energy expenditure, effort valuation represents a behavior that is both relevant to anhedonia and likely to be subject to metabolic regulation. While studies exploring this possibility are lacking, several lines of evidence indicate that the adipokine leptin, whose regulation is altered in metabolic syndrome, may serve as a metabolic input to neural circuits supporting effortful valuation. For example, previous studies have demonstrated that leptin levels correspond to adiposity and, on the basis of stored energy levels, regulate other processes tied to energy intake and expenditure, such as appetite, fertility, and thermoregulation. Further, treatment of mice with chronic corticosterone (CORT) induces effort valuation deficits while concomitantly increasing leptin levels and inducing a leptin resistant phenotype that mirrors metabolic syndrome. Notably, CORT’s effects on effortful valuation are associated with reduced activity in the anterior cingulate cortex (ACC), a brain region required for effort valuation that contains a sparse population of excitatory neurons that express the leptin receptor (Lepr). Lastly, this putative function of leptin is supported by our preliminary data which shows a CORT-induced downregulation of Lepr expression in layer 2/3 excitatory neurons in the ACC, an effect that is reversed after treatment with ketamine, a rapid-acting antidepressant. This result indicates a link between the CORT-induced hyperleptinemia and local changes in ACC leptin signaling. Based on these data, we hypothesize that ACC leptin signaling, through changing the activity of Lepr+ neurons, supports effortful reward seeking and mediates the effects of CORT- induced metabolic dysfunction on effort valuation. In this proposal, we will first fully characterize the extent and time course of CORT’s metabolic effects using metabolic chambers. This will then allow appropriately timed viral manipulations of Lepr levels in the ACC neurons of transgenic mice, which will be used to determine whether altered ACC leptin signaling is necessary and sufficient for the effects of CORT on effortful reward seeking behavior. Next, we will use transgenic mice and viral expression of a fluorescent calcium indicator to determine the effect of leptin signaling on the spontaneous activity of Lepr+ neurons in both control and CORT mice. Finally, we will evaluate the role of Lepr+ neurons in supporting effortful reward seeking by recording and manipulating their activity with fiber photometry and optogenetics, respectively, in behaving mice engaged in an effort valuation task. Completion of the research proposed here will determine the role of ACC leptin signaling on effort valuation, thereby improving our understanding of mechanisms mediating the relationship between metabolic dysfunction and hedonic behavior.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Regulatory T cells (Tregs) play an essential role in maintaining immune homeostasis by preventing autoimmunity, modulating inflammation, supporting tissue repair, and ensuring balanced immune responses. These functions are orchestrated by cell-extrinsic signals that drive transcriptional programs necessary for Treg functional outputs. Recent studies have characterized the effects of various cell-extrinsic signals on Tregs one at a time. However, due to the diversity, scale, and context-dependent effects of cell-extrinsic signals, comprehensive understanding of how complex in vivo environmental signals shape Treg functions is lacking. A systematic understanding of how Tregs interpret cell-extrinsic signals will yield novel insights into basic mechanisms of immunoregulation, enable precise manipulation of Treg functions in pathological conditions, and inform the design of novel therapeutic strategies in settings of autoimmune and inflammatory diseases and cancer. This project aims to comprehensively characterize how cell-extrinsic signals shape Treg functions across distinct inflammatory environments. In Specific Aim 1, Perturb-seq will be used to, in one pooled experiment, profile transcriptional changes induced by genetic knockout of hundreds of Treg cell surface receptors in the context of systemic polytypic (type 1, 2, 3) autoimmunity and tissue inflammation (Aim 1a). Computational analyses of these data will identify shared and unique transcriptional programs elicited by diverse cell-extrinsic signals and categorize their functional relevance (Aim 1b). The effects of relevant newly identified signaling pathways on Tregs will be validated in follow-up experiments (Aim 1c). In Specific Aim 2, the context-dependent effects of Treg signaling pathways in archetypal inflammatory environments (type 1, 2, and 3) will be explored using mouse models of airway infections (influenza, Nippostrongylus brasiliensis, Aspergillus fumigatus). We will identify inflammatory environment driven variation in ligand and receptor expression for novel signaling pathways identified in Aim 1 (Aim 2a) and generate mice with Treg-specific receptor deficiencies to interrogate context- dependent effects of signaling pathways on Tregs in these infection models (Aim 2b). Context-dependent effects on Treg functional outputs will be assessed through analyses of immune responses to infection and tissue damage in these models (Aim 2c). Overall, this project will leverage innovative high-throughput approaches and diverse mouse models of infection to comprehensively elucidate how cell-extrinsic signaling environments influence Treg phenotype and function in context-dependent manners. The depth of understanding provided by these studies will facilitate development of clinical approaches for precise modulation of Tregs with significant therapeutic implications. Furthermore, this proposal is tailored to a physician-scientist in training as it focuses on a cell type with demonstrated clinical relevance and profiles cell-extrinsic signal effects that can be readily modulated therapeutically.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY KRAS is the most frequently mutated oncogene in lung adenocarcinoma (LUAD) and colorectal cancer (CRC). In the US alone, cancers carrying KRAS mutations account for almost 100,000 deaths per year. New mutant- selective and pan-KRAS inhibitors have recently emerged as a potent and effective strategy to block KRAS- mediated oncogenic signaling, however early clinical signs suggests that drug resistance is not only possible, but a common outcome to KRAS-targeted treatment. Defining the mechanism/s underlying acquired resistance to KRAS targeted therapies will drive more effective treatment stratification for patients and guide the development of combination or second-line therapies to prevent or reverse drug resistance. Drug resistance can arise through both genetic changes and non-genetic cellular adaptations that either reduce drug exposure or bypass a tumor's dependence on the drug target. Using matched patient biopsies from a recent KRAS inhibitor clinical trial, and KRAS-driven pre-clinical model systems, we identified the induction of inflammatory gene expression as a conserved response to KRAS inhibition that emerges prior to the known resistance drivers. In parallel, through the use of CRISPR base editing (BE) screens, we identified a subset of cancer-associated mutations that enable resistance to KRAS inhibition and are associated with elevated inflammatory signaling. Finally, our data show that targeting inflammatory mediators JAK and/or TBK1 can reduce the emergence of KRAS resistance. Together with published prior work, our data suggest model whereby resistance to KRAS inhibition can be triggered by a cell intrinsic inflammatory transcriptional programs – induced by drug exposure - that promote progression to previously described drug-resistant states. In AIM 1 we will determine whether the induction of an inflammatory transcriptional response in cancer cells following KRAS inhibition is the trigger that drives the genesis of lineage reprogramming and drug resistance. Further, we will directly test whether targeting key signaling nodes in the drug-induced inflammatory response can prevent lineage reprogramming and/or drug resistance. In AIM 2 we will use focused cancer-associated BE screens and BE-PerturbSeq profiling to identify those genetic changes that promote an enhanced inflammatory response and/or lineage reprogramming and determine whether multiple distinct cancer-associated mutations converge on a common cellular response to drug treatment. In particular, we will determine whether mutations in CIC, SMAD4, and TGFBR2 that are enriched in drug resistant populations, drive resistance through the engagement of specific inflammatory signaling programs. Together, work in this proposal aims to provide mechanistic understanding of how specific genetic and non- genetic changes drive resistance to KRAS inhibition. This work will help define the patient populations and tumor context where KRAS inhibitors are most effective and catalyze the development of combination strategies to prevent drug resistance and improve outcomes for patients with KRAS mutant cancer.

NIH Research Projects · FY 2025 · 2025-08

The overarching aim and long-term goal of my research is to: improve identification, intervention, and prevention of elder mistreatment among persons living with Alzheimer’s disease and related dementias (ADRD) by: (1) mentoring and supporting the next generation of researchers and (2) advancing patient-oriented research in this field. Elder mistreatment (EM) is common, with older adults with ADRD at much higher risk, and is associated with adverse health outcomes. Unfortunately, EM is dramatically under-recognized and underreported. An assessment by a healthcare provider may represent the only contact outside the family for many older adults experiencing EM, particularly older adults with ADRD. Therefore, clinicians have a unique opportunity to identify suspected EM and initiate intervention, and an urgent priority exists to improve EM detection and response in healthcare settings. My research and that of collaborators and mentees addresses this pressing need. My colleagues and I have already used innovative approaches to identify injury patterns and forensic biomarkers specific to physical EM. We have also successfully linked EM cases to Medicare claims data, describing patterns of healthcare utilization among these older adults. I have also led the development and implementation of the Vulnerable Elder Protection Team (VEPT), a first-of-its-kind ED/hospital-based multi-disciplinary consult service to assess and provide care to potential EM victims. Much more research is needed, though, and few junior researchers are currently focused in this area. Recruiting, mentoring, and supporting the next generation of elder mistreatment researchers is critical. Also, as victims are particularly likely to receive care in an Emergency Department (ED), research focused on improving identification of and intervention for elder mistreatment is essential to optimize geriatric ED care. I will leverage my expertise, experience, datasets, clinical programs, and collaborative relationships to mentor promising researchers in elder mistreatment and geriatric emergency medicine. Through protected time and training in this K24, I will mentor the next generation of researchers in these areas. My research aims are to: (1) identify differences in electronic health record information between older adults with ADRD experiencing EM presenting to the ED in comparison with non-exposed older adults with ADRD, including developing and validating a machine learning algorithm to predict EM exposure, (2) describe rates and patterns of health care utilization of older adults with ADRD experiencing EM identified using Medicare claims data before and after initial EM diagnosis in comparison to controls, and (3) use focus groups with primary care clinic leaders and team members to explore how to optimally integrate a novel screening tool for neglect in older adults with ADRD into workflow in a primary care setting. These projects complement NIA-funded research in my patient-oriented research lab. Each will advance EM research and improve detection and response in healthcare while also offering many opportunities for mentees to contribute and to take a leadership role in related work.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Surgery is very common and risky for older adults. Nearly 25% of aging patients lose independence after surgery, and 13% die within 1 year, 3x higher than expected mortality without surgery and 3x higher than younger patients getting surgery. This trend is highly concerning for public health since the fraction of US adults older than 65 years continues to grow and there is no standardized way to incorporate aging into risk stratification for surgery. Despite the fact that chronological age (time since birth) is a known risk factor for postoperative outcomes, it is an insufficient proxy for health in older adults. Restricting surgical care based upon chronological age alone is imprecise and potentially harmful since it can exclude people who may benefit from surgery. As an alternative, molecular estimates of age, termed “biological age”, provide a quantitative, modifiable, and biologically-based estimate of health in older adults and are not simply a reflection of underlying comorbidities. Biological age can even distinguish disease- and mortality-risk in people of the same chronological age and comorbidities. Despite extensive evidence for the clinical impact of biological age, there is a significant knowledge gap: we have no understanding of how biological age impacts surgical outcomes. The proposed research aims to: (1) determine the effect of preoperative biological age on 1-year postop mortality; and (2) evaluate preoperative biological age as a predictor of loss of independence after surgery. This study will be conducted using the UKBiobank, a large prospective cohort study (N=502,649) that uniquely integrates molecular, clinical, questionnaire, and mortality data, to investigate surgical risk related two independent metrics of biological age relevant to surgical recovery – immunologic age and metabolic age. This project will be the first to test the hypothesis that preoperative biological age is associated with various patient- centered surgical outcomes, even when correcting for well-known medical, surgical, and sociodemographic risk factors. The contribution of our project will be significant because it will give us a way to distinguish biological risk for surgery in aging adults who are currently clinically indistinguishable. This will help guide shared decision-making between patients and surgeons before surgery. The successful completion of this project opens the door to targeting biological age with pharmacologic and non-pharmacological interventions to improve postoperative outcomes.

NIH Research Projects · FY 2026 · 2025-08

Project Summary Despite advancements in the therapeutic targeting of amyloid beta (Aβ), Alzheimer's disease (AD) remains a persistent clinical challenge. Neuroinflammation, an early AD marker, emphasizes the need to comprehend and regulate crosstalk between the central nervous system (CNS) and the immune system. Beyond microglia, CNS-infiltrating leukocytes play a role, yet the mechanisms governing their brain entry and antigenic priming remain elusive. Recent findings highlighting the calvarial bone marrow as a source of these leukocytes suggest that regulating the calvarial marrow environment may be a crucial, overlooked element in AD pathogenesis. However, the skeletal cells responsible for forming this environment remain unknown. Our prior research identified two distinct skeletal stem cell (SSC) populations in the calvarium, essential for calvarium formation but incapable of marrow formation. Building upon this foundation, we recently discovered a novel SSC type with the capacity to support hematopoiesis within the calvarium. Disrupting the function of this SSC in mice suppressed calvarial marrow formation, leading to a 70% reduction in leukocytes. Notably, this SSC deficiency coincided with diminished immune cell infiltration and microglial abundance in the brain of an AD mouse model. Within the calvarium, these SSCs orchestrate the creation of a unique marrow environment, characterized by the presence of previously unidentified cellular elements, including previously unrecognized antigen-presenting cells not found in other skeletal marrow regions. Driven by these findings, we will investigate the critical role of these marrow-forming SSCs in establishing a unique marrow niche and understanding its influence on AD progression, as outlined in the following aims: In Aim 1, we will determine the role of newly identified calvarial SSC in CNS immunopathology by genetically manipulating this SSC and assessing the impact on the progression of AD models. We will further determine whether treatment with FDA-approved osteoporosis drugs targeting this stem cell impacts AD progression and thereby offer a rapid path for clinical translation of our findings. In Aim 2, we will determine how the unique cellular characteristics of the calvarial marrow environment, orchestrated by these SSCs, modulate AD progression. In particular, we will investigate how a population of calvarial antigen-presenting cells we have newly identified in conjunction with this SSC contributes to AD pathogenesis. Lastly, in Aim 3, we will determine if the calvarial marrow properties observed in mice translate to humans. This will involve a combined approach using autopsy and surgical specimens to identify the human counterparts of these novel calvarial SSCs and other unique calvarial features, such as the presence of calvarial the specialized calvarial antigen-presenting cells identified above in mice. Altogether, this project will establish the major new concept that a specific new calvarial stem cell regulates immune function in the brain, thereby providing new therapeutic opportunities to slow the progression of neurodegenerative diseases of aging.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT In 2023, adolescents and young people accounted for approximately 25% of new HIV infections globally. The period after weaning but before sexual debut represents a window of relatively low HIV acquisition risk, providing an opportune time for administering a multi-dose vaccine regimen to elicit protective immunity prior to adolescence. The success of a protective HIV vaccine will rely on the induction of broadly neutralizing antibodies (bnAbs). Given the potential for rapid bnAb development in children living with HIV, vaccines targeting B cell evolution towards bnAbs may be more effective in early life. Germline-targeting immunization strategies have shown great potential by using a series of priming, shaping, and polishing immunogens to activate bnAb precursor B cells and guide them towards potent and broad neutralization. While these priming immunogens effectively engage bnAb precursors, identifying appropriate strategies to drive bnAb evolution remains a challenge. The overall goal of this project is to identify an optimal prime-boost strategy to guide antibody maturation towards neutralization breadth in the setting of immune maturation. We hypothesize that an early life HIV immunization strategy incorporating multivalent HIV env immunogens will drive efficient priming and maturation of B cell responses towards neutralization breadth. Our hypothesis will be investigated under the following aims: 1) Compare sequential versus simultaneous administration of germ-line targeting immunogens for induction of CD4bs and V2 apex bnAb precursors; 2) Determine whether a multivalent prime- boost strategy will better drive the development and maturation of bnAb lineages towards neutralization breadth.; and 3) Define the evolution of vaccine-elicited B cell responses and mAbs following immunization with bnAb B cell lineage designed immunogens through a structure-to-sequence approach. The proposed studies will help establish a prime-boost regimen to evolve bnAb B cell lineages throughout early childhood towards neutralization breadth prior to adolescence. These studies will establish early life as an optimal time for beginning a sequential HIV immunization strategy and inform the next phase of bnAb germline-targeting immunization strategies for evaluation in preclinical and human clinical trials.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT HIV remains a critical global health issue, particularly in Sub-Saharan Africa, where infection rates remain high, and a significant pediatric burden persists. Whilst antiretroviral therapies exist, adherence remains an issue, and the need for a cure remains urgent. A major barrier to a cure is the HIV reservoir, which persists under suppressive treatment. The HIV reservoir consists of clonally expanded populations of infected CD4 cells, which can remain latent for extended periods of time, thereby allowing the virus to evade immune responses and treatment. Clonal expansion can occur over time under suppressive treatment due to various mechanisms including homeostatic proliferation, antigenic stimulation, and CTL resistance. Furthermore, the CD4 reservoir, under suppressive antiretroviral treatment, is a rare population of cells that are difficult to isolate and culture. Early in my postdoctoral career, I developed a methodology to isolate pure CD4 HIV reservoir clones from people living with HIV on suppressive antiretroviral treatment. From this, I was able to isolate pure CD4 HIV reservoir clonal populations and culture them ex vivo. I was able to begin phenotypically, genotypically, and functionally characterizing these clones using flow cytometry based functional assays and CITE-Seq. This method development has laid the foundation for this proposal in optimizing CD4 HIV clonal reservoir isolation and establishing a system to interrogate clone characteristics and to describe the clonal landscape, whilst pursuing functional assays that will inform mechanisms of clone persistence. CD4 HIV reservoir clones can now be analyzed for intrinsic mechanisms of resistance to CTL-mediated killing via flow cytometry functional assays including proliferation assays, CTL-mediated killing assays, and latency reversal agent assays. These experimental efforts will inform clone elimination efforts through enhancement of autologous cytotoxic T cells (CTLs). In Aim 1 I hypothesize that clonal CD4 HIV reservoir isolation methodology will need to be adapted based on the phenotypic characteristics of each unique HIV reservoir to enable successful isolation of pure clonal populations. In Aim 2 I propose priming of autologous CTLs will improve CD4 HIV reservoir elimination through CTL-mediated killing by a slow clearing of active or partially active reservoir clones. And in Aim 3 I propose extending this research into Subtype C HIV populations and pediatric populations, who I hypothesize will be more difficult to isolate CD4 HIV reservoir clones from given the reduced reservoir size in pediatric populations infected with HIV. I propose novel approaches to obtain pure clonal populations of CD4 T cells carrying HIV proviruses and phenotypic, genotypic, and functional characterization of clones. This study will enhance our understanding of the HIV reservoir by providing a unique opportunity to study the HIV reservoir landscape in pure infected clonal populations.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Mycobacterium tuberculosis (Mtb) is one of the world’s leading causes of death from infection. Curative chemotherapeutic regimens exist, but they are long, often toxic and plagued by emergence of drug resistance. Macrophages are the major cellular locale for Mtb in the host before formation of largely acellular cavities. Adjunctive therapies could potentially speed the cure of tuberculosis by improving the ability of macrophages to kill Mtb. Within macrophages, Mtb secretes an enzyme, protein kinase G (PknG), that enters the macrophage cytosol, where it blocks fusion of lysosomes with phagosomes and promotes degradation of proteins that help activate macrophage mycobactericidal mechanisms. Targeted protein degradation (TPD) is a promising new therapeutic avenue. Compounds that mediate TPD differ from active site inhibitors by working catalytically and therefore sub-stoichiometrically with respect to the target. TPD as a form of anti-mycobacterial chemotherapy has been introduced for model targets in non-tuberculous mycobacteria, but not yet demonstrated for an Mtb protein in its native state. Here we propose to exploit the macrophage’s ubiquitin-proteasome system to degrade PknG as it has been secreted by Mtb and has entered the macrophage cytosol by synthesizing and testing proteolysis-targeting chimeras (PROTACs) that link a PknG inhibitor to binders of the E3 ubiquitin ligases cereblon and von-Hippel Lindau.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a prevalent genetic disorder affecting millions worldwide, characterized by cyst formation primarily within the kidneys, leading to organ enlargement and deteriorating function. This condition often extends to the liver, posing significant clinical challenges. Traditional qualitative analysis methods are insufficient for accurate disease assessment, prognosis, and treatment guidance. Deep Learning (DL) models have shown promise in estimating Total Kidney Volume (TKV) from MRI images but face limitations in detecting early-stage disease with small cysts. This study aims to transform qualitative analysis into quantitative assessment through multi-class cyst segmentation in ADPKD. Aim 1 focuses on developing a super-resolution DL model to generate high-resolution 3D MR volumes. Multiple imaging planes (axial, coronal, sagittal) will be incorporated to enhance 3D resolution for precise biomarker calculation. Additionally, a multi-class multi-sequence DL framework will be developed for ADPKD severity assessment, which involves creating segmentation and object detection models for different cyst classes, including simple, hemorrhagic, and exophytic cysts within the kidney, liver, and pancreas. Aim 2 aims to build a prognostic model for predicting estimated glomerular filtration rate (eGFR) decline and disease progression. This will involve developing an accurate predictive model based on biomarkers extracted from high-resolution MR images, with a particular focus on cyst class information, especially hemorrhagic cysts, to improve eGFR decline forecasts and dialysis potential predictions. Aim 3 integrates patients' longitudinal data to quantify the impact of temporal dynamics on disease progression over the next 10 years, by developing a multimodal predictive model that incorporates patients' prior MRI scans and historical data. This study aims to utilize DL models to shift from qualitative to quantitative assessment of ADPKD using multi-sequence high-resolution MR images, enabling precise measurement of cyst attributes, and advancing our understanding of disease progression and treatment response for better patient outcomes.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Proteases are pivotal players in cellular biology, acting as both guardians of homeostasis and mediators of stress responses. Their roles are indispensable to cellular function, ranging from protein turnover and immune responses to mitochondrial maintenance and beyond. Understanding the precise regulatory mechanisms of proteases provides unique opportunities to identify and exploit vulnerabilities in cancer progression. Aim 1 (F99 phase) focuses on identifying the physiologically relevant DPP9 inhibitor. DPP9 is a protease, which regulates inflammasomes—multiprotein complexes that detect danger signals and trigger pyroptosis, a lytic form of cell death. Under normal conditions, DPP9 represses the NLRP1 and CARD8 inflammasomes. Importantly, synthetic DPP9 inhibitors have been shown to activate these inflammasomes, triggering pyroptosis. However, the identity of an endogenous inhibitor remains elusive. Discovering this physiologically relevant inhibitor is crucial, as it will reveal the evolutionarily conserved danger signals that the NLRP1 and CARD8 inflammasomes evolved to sense, opening novel avenues for modulating inflammasome activation—a promising strategy for cancer immunotherapy. Preliminary data indicate that DPP9 interacts with the redox sensor KEAP1 through an ESGE motif, leading to mutual inhibition of both proteins. This interaction suppresses DPP9's catalytic activity and inhibits KEAP1's ability to sequester NRF2, stabilizing NRF2 and activating the antioxidant response system. Intriguingly, in DPP9’s native state, the ESGE motif is structurally incompatible with KEAP1 binding, suggesting that a conformational change in DPP9 is required for this interaction to occur. In Aim 1, this proposal seeks to (1) identify the stimulus that drives this conformational change, enabling KEAP1 binding and mutual inhibition, and (2) unbiasedly identify genetic regulators of DPP9 activity. Together, these studies aim to uncover the endogenous inhibitor of DPP9, define its role in inflammasome activation, and ultimately harness pyroptosis as a targeted cancer therapy. Aim 2 (K00 phase) focuses on identifying mitochondrial proteases critical for maintaining mitochondrial function under the metabolic and oxidative stresses of the tumor microenvironment. Unlike the cytosol, mitochondria lack a proteasomal degradation system and depend on proteases to prevent the accumulation of misfolded or damaged proteins, ensuring cellular adaptation and survival. In this Aim we will employ unbiased proteomics and CRISPR-based approaches to identify mitochondrial proteases and their cognate substrates that are essential for cancer cell survival. These studies will reveal how mitochondrial proteostasis enables cancer cell resilience and uncover therapeutic opportunities to selectively disrupt these processes, compromising cancer cell survival while sparing normal cells.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Cancer metastasis is a complex, multistep process that requires cells to adapt and survive in stressful conditions. These adaptive responses are carried out by proteins within the cell. Many proteins can play different roles based on their context, as has been demonstrated for STimulator of INterferon Genes (STING). STING has a well characterized role in innate immunity and has been proposed to activate an anti-tumor immune response early in disease progression. However, recent work shows that its chronic activation in cancer cells can drive metastasis. In addition to its role in the Type 1 interferon response, STING also has putative roles in ER stress response signaling and the NF-kB pathway. An explanation for how STING plays these roles is pleiotropy, meaning one gene can cause multiple, unrelated phenotypes. It is unclear how these roles are mediated and how these roles change in different contexts such as during cancer progression. My central hypothesis is that STING adopts adaptive functions mediated through binding partners that interact with distinct functional domains. The 2 specific aims of my project are (1) to map the emergent downstream functions of STING and determine if these are mediated by binding partners, and (2) probe one pro-metastatic STING variant (K337X) to understand how binding partners that stabilize or degrade STING influence its function. To quantify residue-specific functions of STING in a high-throughput manner, my lab has generated a saturation mutagenesis library of STING with each variant distinguished by a DNA encoded barcoded. We have performed scRNA-seq screens on the variant library with the natural agonist of STING (cGAMP) to quantify downstream transcriptional effects with each variant. I am now coupling select variants identified in the screen with functional fluorescent reporters to classify and validate variants able to perform downstream interferon, ER stress, and NF- kB functions. I will then use proteomics to identify if binding partners are responsible for differences in variant function. We have also performed 2 metastasis assay screens with the library and identified one striking pro- metastatic variant, K337X. I am following up on 2 specific binding partners that are known to either stabilize or promote degradation of STING to test if the context of STING, meaning binding partners available, rather than specific clinical mutations could be responsible for its emergent functions. I will study these functions of STING in 2 mouse models of chromosomally unstable triple negative breast cancer cell lines. The long-term goal of this work is to better understand the mediators of STING’s functions so that its dysregulation in cancer can be better understood and more effectively targeted with therapeutics.

NIH Research Projects · FY 2025 · 2025-08

Abstract. Type 1 diabetes (T1D) is characterized by the destruction of pancreatic β cells, which respond to signals from immune cells, leading to hyperglycemia. This study aims to grasp the mechanisms influencing T1D by investigating both intrinsic β cell factors and environmental immune cell interactions. Our interdisciplinary team combines expertise in diabetes genomics, stem cell/organoid biology, and islet biology to explore these dynamics. The preliminary studies using single-cell transcriptome, single nucleus chromatin accessibility, and single nucleus (sn) multiome profiling of human pancreatic islets from healthy and T1Daffected individuals. The same experiments were performed using human islets exposed to cytokines or virus simulating T1D conditions. Our integrative approaches identified gene regulatory elements (GREs) at diabetes GWAS. Also, we have developed tactics to differentiate human pluripotent stem cells (hPSCs) into functional vascularized-immune islet organoids containing pancreatic endocrine cells, endothelial cells and immune-like cells. Here, we will apply sn- multiomic (both short and long reads RNA-seq and ATAC-seq) profiling, hPSC-derived organoids, CRISPR- based gene editing and Cas13-based gene knockdown to systematically explore the role of intrinsic and environmental signal dynamics in T1D progression and define mechanistic network controlling β cell destruction. We propose three specific aims to advance our understanding: Aim 1: Define the cell-specific multiomic intrinsic and environmental signatures during T1D progression. We will characterize intrinsic and environmental changes in chromatin and transcriptome profiles of islets from pre-T1D, T1D, and healthy individuals, map gene/isoform expression and chromatin accessibility, and finally integrate multi-omics data to identify cellspecific quantitative trait loci (QTLs) and perform fine mapping with T1D GWAS signals. Aim 2: Decode the epigenomic network controlling β cell destruction during T1D progression. We will use massively parallel reporter assays to validate GREs at T1D GWAS loci, employ Perturb-seq and CRISPR editing to validate GREs, target genes, and their cellular phenotypes in isogenic hPSC-derived organoids. Aim 3: Determine the impact of alternative splicing on human β cell destruction. We will validate T1D-associated alternative splicing using the Xenium platform. Finally, we will examine the biological function of gene isoforms, and reverse β cell destruction in hPSC-derived organoids by targeting alternative splicing mechanisms. Our long-term goals include identifying locus-specific and network mechanisms to facilitate the development for precision medicine approaches in T1D. Key deliverables will include a comprehensive single cell triple-omic map of human islets crossing different stages of T1D progression, a molecular genetic network of intrinsic and environmental signals, and validated hPSC-derived vascularized immune-islet organoid models with T1Dassociated GRE KO, gene isoform KD, etc. These findings will pave the way for the development of novel therapeutic strategies and disease progression markers for T1D.