Icahn School Of Medicine At Mount Sinai

NIH Research Projects · FY 2026 · 2025-08

PROJECT SUMMARY Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene are the primary cause of cystic fibrosis (CF) and are associated with many other genetic diseases. Thus, CFTR is a compelling therapeutic target for treating various CFTR-related disorders. Genome editing techniques can correct the defective genes at their native locus and permanently restore the gene function, potentially offering a one-time curative treatment for CF. However, given that over 1,700 CF-causing mutations have been identified in the CFTR gene, mutation- specific genome editing methods, such as base editing, may not be suitable for correcting the wide range of CFTR mutations. To address these limitations, we propose to develop a more generalizable genome editing strategy for CFTR correction. Because most CFTR mutations occur within the exons or at the exon-intron boundary, we propose to replace the frequently mutated exons with their wild-type counterpart. Therefore, a single set of gene editing agents can correct all the mutations within the same exon. Prime editing (PE) employs an extended guide RNA (pegRNA) as a template for a conjugated reverse transcriptase to incorporate desired edits into the targeted genomic site. Recently, we devised a paired prime editing strategy to program the replacement of target genomic sequences without requiring a DNA donor in vitro and in vivo. Building upon this work, the goal of this proposal is to develop paired prime editing-based sequence replacement strategies to correct a broad spectrum of CFTR mutations, regardless of the mutation types or locations. We hypothesize that replacing the defective CFTR exons with corrected ones will effectively restore CFTR protein expression and function in vitro and in vivo. In the proposed research, we will utilize a donor-free sequence replacement strategy to replace individual CFTR exons without creating double-stranded DNA breaks or requiring a DNA donor (Aim 1). To further expand the editing scope and efficiency of the paired prime editing approaches in inserting or replacing large DNA sequences, we will develop a paired PE-based homology-independent insertion strategy and apply it to correct CFTR mutations caused by large deletions or occurred in the large exons (Aim 2). Finally, we will explore the therapeutic applications of the newly-established paired prime editing-mediated gene correction strategy by delivering the genome editing agents to the lungs of a CF mouse model (Aim 3). Successful completion of this project will result in a gene therapy strategy tailored for CF and provide a universal gene correction framework for treating genetic diseases of high allelic heterogeneity.

NIH Research Projects · FY 2025 · 2025-08

Project Summary: It is widely appreciated that erythropoiesis serves as an excellent model for studying modifications in chromatin organization and resultant gene expression patterns within identical cells. This process involves significant chromatin reshuffling and a loss of topologically associating domains, ultimately leading to global transcriptional silencing upon terminal erythroid differentiation. Despite these insights, the precise structural changes underpinning the distinct gene expression seen in terminally differentiated erythroid cells remain enigmatic. Therefore, there is an urgent need to elucidate the detailed molecular mechanisms governing chromatin architecture modifications during erythroid terminal differentiation, to enhance our understanding of erythropoiesis-related diseases, particularly anemia. The overall objective of the proposed research is to decipher the roles and interactions of erythroid-specific transcription factors and chromatin structural proteins during erythropoiesis, defining how they lead from cellular expansion to differentiation. Our central hypothesis posits that erythroid-specific transcription factors and architectural proteins direct modifications in chromatin organization integral to erythroid terminal differentiation. To this end the specific aims of this proposal seek to combine functional studies with state-of-the-art-multi-omics approaches to: 1) Test the functional role of architectural proteins during terminal differentiation in mouse erythropoiesis 2) Identify and characterize chromatin re-organization events during terminal differentiation 3) Identifying the molecular mechanisms underlying the 3D chromatin changes during erythropoiesis

NIH Research Projects · FY 2025 · 2025-08

Summary: Human cytomegalovirus (CMV) is a β-herpes virus with high seroprevalence rates of 60-90% within the population that can spread through bodily fluids, organ transplants, and from mother to fetus via the placenta. Virus proliferation significantly increases the morbidity and mortality of immunocompromised individuals, such as newborns, organ transplant recipients, AIDS patients, and the elderly. Approximately 30,000 solid organ transplant and 23,000 bone marrow transplants operations are performed in the U.S. every year. CMV is the leading cause of birth defects, affecting ~1% of newborns giving rise to ~30,000 new cases of CMV infection reported annually in the US. While several drugs have been approved by the FDA for the treatment of CMV infections, including ganciclovir, foscarnet, letermovir, and recently maribavir, these compounds were found to exhibit high frequencies of drug resistance and severe side effects, including bone marrow toxicity, gastrointestinal disruption, and nephrotoxicity. Given the large number of patient populations at risk for CMV- associated diseases and the estimated cost to treat CMV in the US ($4.4 billion/year by the National Academy of Sciences), novel therapeutic strategies to treat CMV-associated diseases is needed. The development of novel therapeutics that target different steps of the viral life cycle to limit virus propagation and spread would provide therapeutic for treating CMV-related diseases. We developed a high-content screening assay using a CMV AD169 reporter virus to screen >112,000 compounds with Microbiotix to identify inhibitors that block the early stage of infection. One compound, MBXC-4302, is a N-arylpyrimidinamine (NAPA) that exhibited potent anti-CMV infection activity (IC50 ~3 µM), limited cytotoxicity (CC50 >100µM), favorable in vitro ADME properties, and a responsive structure activity relationship (SAR). The preliminary SAR analysis identified the analogs with an improved selective index. Our general hypothesis is that the NAPA compounds represent novel CMV entry inhibitors which can be developed into effective novel CMV therapeutics to prevent virus proliferation and spread. To evaluate our hypothesis, we plan to complete the following Aims: 1) Characterize the mechanism of action and target of NAPA compounds; 2) Optimize the NAPA series through SAR-driven analog generation; 3) Evaluate NAPA compounds to limit virus proliferation using in vitro models; and 4) Evaluate pharmacokinetics, tolerability, and efficacy of prioritized NAPA analogs in proliferation studies in vivo We plan to develop 1-2 lead NAPA analogs as effective CMV therapeutics that inhibit CMV dissemination as monotherapy or combination therapy with FDA approved CMV drugs to prevent virus-associated diseases by limiting viral load.

NIH Research Projects · FY 2025 · 2025-08

Respiratory infections like those caused by influenza A virus (IAV) represent a prominent cause of morbidity and mortality globally. Outside of its well-characterized effects in the lung, IAV infection accelerates cardiovascular disease, leading to an approximate six-fold increase in the likelihood of myocardial infarction (MI). This strongly suggests an essential pulmonary-cardiac immunological crosstalk during infection. Despite these observations, very little is known mechanistically as to how IAV disrupts cardiac homeostasis, damages the heart and promotes cardiovascular disease pathogenesis. Using a combination of innovative mouse models of respiratory infection, myocardial infarction (MI) and heart failure (HF), Dr. Jeffrey Downey will comprehensively probe the hypothesis that IAV translocates from the lung to the heart and induces an antiviral response that damages the heart, leaving it more susceptible to disease. Elucidating how exactly IAV accesses and impacts the heart will not only unravel a novel pulmonary-cardiovascular immunological axis, but will also serve to address an unmet clinical need in countering infection-induced cardiovascular events. This proposal is composed of three aims. In the first aim, we will study the effects of pulmonary IAV infection on the heart, building on preliminary data that show (1) elevated troponin in humans and mice and (2) impaired heart function in mice after infection. We will determine which cells become infected and examine how the virus causes damage, expanding on our preliminary data that suggest direct type I interferon (IFN-I) signaling on cardiomyocytes causes the decline in cardiac function. In the second aim, we will investigate how underlying infection alters the pathogenesis of MI and influences its severity using a unique approach that combines our IAV mouse model with two types of MI. We will moreover genetically target IFN-I signaling in cardiomyocytes and utilize cutting-edge CRISPR/Cas9 technologies to minimize MI risk following infection. Finally, in aim three during the independent phase, Dr. Downey will interrogate the bidirectional influence of anti-IAV immunity on the development and progression of HF, by testing if established HF promotes susceptibility to IAV and if previous infection accelerates HF. Collectively, these studies will greatly expand our understanding of inter-organ inflammatory communication during infection and disease, while also providing greatly needed therapeutic targets. Dr. Downey will primarily conduct this research within the Cardiovascular Research Institute at the esteemed Icahn School of Medicine at Mount Sinai under the primary mentorship of Dr. Filip Swirski, a world-renowned investigator of cardio-immunology, and co-mentorship by Dr. Michael Schotsaert, a leader in virology, microbiology, and vaccinology. Dr. Downey has additionally assembled an elite Advisory Committee comprised of Dr. Mandy Van Leent, a pioneer in non-invasive multiparametric imaging and immunotherapy, and Dr. Deepak Bhatt, the leading expert in interventional cardiology and cardiovascular clinical trials. Together, these mentors will help Dr. Downey foster his own successful research program and facilitate his career as an independent biomedical investigator.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY DNA methylation (5mC) is a critical epigenetic mechanism in hematopoiesis. Down syndrome (DS), or trisomy 21 (T21), predisposes individuals to unique conditions of hematopoietic malignancies, such as the preleukemic condition Transient Abnormal Myelopoiesis (TAM) and Myeloid Leukemia associated with Down Syndrome (ML- DS). To enhance our understanding of the role of 5mC in these conditions, our project aims to map 5mC dynamics in conjunction with chromatin accessibility and RNA expression at single-cell resolution across these critical stages of DS hematopoiesis. Aim 1 focuses on the fetal stage, employing our innovative single-cell multi- omic sequencing technology, SHARE-ME-seq, to explore highly enriched hematopoietic stem cells (HSCs) in T21 fetal liver. By capturing over 1 million single-cell profiles, this aim will profile samples from ten donors between 16-23 weeks of gestation, allowing us to capture the epigenetic landscape during the crucial period of HSC expansion. The data generated will enable us to establish a detailed molecular timeline, revealing how 5mC influences HSC self-renewal and predisposition towards preleukemia. Aim 2 extends this study to postnatal stages, where we will examine primary patient samples from TAM and ML-DS stages. Here, we will apply the same SHARE-ME-seq technology to discern how specific 5mC patterns evolve from the preleukemic TAM stage to full-blown ML-DS. By integrating concurrent analyses of chromatin accessibility and gene expression, this aim will characterize the role of epigenetic modifications in driving the progression of DS-associated leukemia. Additionally, histone modification profiling through CUT&Tag will be conducted to identify active, repressed, or poised enhancers and promoters, providing deeper insights into the chromatin dynamics and lineage priming that contribute to leukemogenesis in DS. These comprehensive and innovative approaches are designed to uncover the interactions between DNA methylation, chromatin accessibility, and gene expression in DS- associated hematopoiesis. By elucidating these mechanisms, our study aims to identify new therapeutic targets and improve intervention strategies for hematologic malignancies in DS, ultimately enhancing the prognosis and quality of life for individuals affected by this condition. The data generated will serve as a high-quality reference, informing future research and therapeutic development tailored to DS individuals.

NIH Research Projects · FY 2025 · 2025-08

Engineering Metal Ion-Immunity Crosstalk for Metalloimmunotherapy Metal ions play essential roles in numerous immune processes through their unique structural, catalytic, and regulatory interactions with immune sensors, ion transporters, enzymes, and signaling pathways. Emerging studies have demonstrated that metal ions could modulate various immune processes for disease treatment. This lays the foundation for a new class of immunotherapy, which we dubbed “metalloimmunotherapy”. However, a comprehensive understanding of metal ion-immunity crosstalk and effective strategies for delivering metal ions to immune cells in vivo is still lacking in this field. The overarching goal of my lab is to understand how metal ions interact with immune system and to leverage advanced medicinal strategies to deliver metal ions to immune cells, modulating the immune system towards immunostimulatory or anti-inflammatory states for disease therapy. In this proposal, we aim to address the key knowledge gaps and technical challenges by systematically interrogating the metal ion-immunity interactions and developing specialized metal ion delivery systems to activate or regulate immune responses. In Part I, we identified extensive modulatory effects of metal ions on various immune pathways through a systematic high-throughput screening. Next, we will use gut as a window to investigate such metal ion-immunity crosstalk in vivo and validate our findings. This will elucidate the specific roles of metal ions in regulating gut immune cell functions and determine how these interactions contribute to gut diseases, including colon cancer and inflammatory bowel diseases (IBDs). In Part II, we proposed to engineer metal ion-immunity crosstalk to activate immune system for effective treatment of advanced colon cancer. We will develop novel coordination nanoparticles and hydrogels for effective delivery of metal ions and immune activators to amplify anti-tumor immune response. We will further establish the mechanism of action and improve the efficiency based on rational design. In Part III, we proposed to engineer metal ion-immunity crosstalk to regulate immune system in gut for treating IBDs. We will introduce two coordination nanoparticle-based strategies to target pathogenic immune cells in gut, inhibiting multiple IBD-related inflammatory processes and restoring immune balance. Successful completion of the proposed studies will provide groundbreaking insights of metal ion- immunity crosstalk and lay out a new framework for advancing metalloimmunotherapy. We will uncover previously unknown metal ion-immune interactions and their roles in health and disease. Furthermore, we will address key delivery challenges and biological gaps, paving the way for breakthrough treatments for advanced cancers and severe inflammatory or autoimmune diseases.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Glioblastomas (GBM) are aggressive, primary malignant brain tumors that are devastating for patients and caregivers due to high symptom burden, early physical and cognitive decline, caregiver support needs, and existential distress stemming from life expectancy from diagnosis on the order of months. Palliative care, defined by the National Quality Forum as “patient and family-centered care that optimizes quality of life (QOL) by anticipating, preventing, and alleviating suffering” improves QOL and reduces physical and psychological symptom burden for patients with serious illness. Caregivers of patients who receive palliative care have reduced distress, anxiety, and depression. Multiple oncological societies recommend that palliative care be integrated alongside usual oncology care within 3 months of diagnosis into the treatment plan of all aggressive cancers, including GBM. Yet in patients with GBM, palliative care referrals occur close to death, if at all. This care gap is likely due to the neurologic and neuropsychiatric symptoms, cognitive deficits, and accumulation of disability early in the disease course that are characteristic of GBM and differentiate the palliative care needs of patients with GBM from those in other advanced cancers. Clinicians specializing in palliative care (SPC) typically have limited training in neurology and neuro-oncology, leading neuro-oncologists to manage palliative care needs themselves (primary palliative care; PPC). However, time constraints and the prioritization of cancer-directed therapies limit PPC capacity. Because SPC consultation provides more advanced, comprehensive palliative care delivery, a model that expands PPC capacity and promotes earlier SPC referral is needed. Rita “Caroline” Crooms, MD, MPH, developed this proposed career development award to generate new knowledge about feasibility and acceptability of a novel model that embeds a PPC care navigator and a SPC clinician into a neuro-oncology practice. Using the Consolidated Framework for Implementation Research to inform successful intervention design, Dr. Crooms’ aims are: 1) analyze clinical trajectories in GBM and other high-grade gliomas to identify events (e.g. hospitalization) and patient characteristics (i.e., age, comorbidities) suggestive of higher palliative care needs to guide timing and content of intervention activities; 2) convene a stakeholder advisory board of patients, caregivers, and neuro-oncology and SPC clinicians to finalize the intervention protocol and implementation processes; and 3) pilot test the refined intervention in the neuro-oncology practice of a large, academic health system. Dr. Crooms with her expert mentors/advisors designed a career development plan to enhance her research skills and facilitate successful completion of the proposed work. This plan includes training on longitudinal data analysis; stakeholder engagement; and consensus and clinical trial methodology. This work will provide key preliminary data for Dr. Crooms to conduct a future R01 multisite clinical trial of the intervention to evaluate its impact on QOL in GBM – and advance her long-term goal to optimize palliative care delivery for people with brain tumors and their caregivers.

NIH Research Projects · FY 2025 · 2025-08

Abstract Inflammatory Bowel Disease (IBD) is a complex genetic disorder marked by dysregulated gut immune responses to environmental factors, influenced by numerous genetic variants. Affecting over 0.7% of Americans and millions worldwide, IBD's genetic and molecular underpinnings remain incompletely understood. Although genome-wide association studies (GWAS) have successfully identified hundreds of IBD- associated loci, the prioritization of regulated genes and their functional relevance in disease-relevant cells remain unclear. This project seeks to fill these gaps by utilizing a large-scale CRISPRi Perturb-seq screen in primary human monocytes, a key cell type in IBD pathogenesis.The specific aims of this proposal are to: (1) elucidate the transcriptional consequences of IBD candidate genes in monocytes from diverse populations using a GWAS-guided Perturb-seq approach, and (2) functionally characterize non-coding variants from East Asian (EAS) IBD-associated loci. This innovative approach will identify key regulatory pathways and gene programs involved in monocyte and macrophage function, and highlight potential ancestry-specific differences. Furthermore, this study will improve understanding of how non-coding IBD-associated variants shape immune responses by linking these variants to their target genes. In the mentored K99 phase, the research goal is to map the genotype-phenotype landscape of IBD candidate genes in human monocyte-derived macrophages and systematically link GWAS-nominated variants to their target genes. During the R00 phase, the focus will shift to exploring the genetic effects on macrophage differentiation and immune responses within their native tissue environment using in vivo Perturb-seq, with particular emphasis on genes identified during the K99 phase as key regulators of macrophage plasticity, and functionally characterizing select non-coding variants to gain mechanistic insights into gene regulation. The short-term career goal is to complete the proposed research, develop a multi-pronged pipeline for GWAS guided functional study, and receive multidisciplinary training under the mentorship of Dr. Cho and a diverse advisory committee. The long-term career goal is to lead a research lab focused on understanding how genetic variants contribute to dysregulated immune responses in chronic inflammatory diseases, ultimately guiding the development of more precise and personalized therapeutic strategies. The collaborative and resource-rich environment of the New York area provides an ideal setting for achieving these goals.

NIH Research Projects · FY 2025 · 2025-08

Project Summary / Abstract In this project, the investigators propose to study and validate the enzyme succinyl-CoA:glutarate-CoA transferase (SUGCT) as a novel pharmacological target for the treatment of glutaric aciduria type 1 (GA1). GA1 is an autosomal recessive inborn error of lysine, hydroxylysine and tryptophan degradation. Patients can present with brain atrophy and macrocephaly, and may develop dystonia after acute encephalopathic crises that lead to striatal degeneration. The disorder is caused by a deficiency of the enzyme glutaryl-CoA dehydrogenase (GCDH), which leads to the accumulation of neurotoxic metabolites such as 3-hydroxyglutaric acid. GA1 is considered a treatable disorder and therefore included in newborn screening programs in many countries. However, current treatment consists of dietary intervention, carnitine supplementation, and emergency treatment which requires intense efforts from both caregiver, patient and clinical team. It must be meticulously maintained, but even so neurological disease still develops in 25% of patients, with these negative outcomes reflecting historical health inequities and social determinants of disease. Thus, the treatment needs of GA1 patients are unmet. Development of pharmacological therapies, however, is hampered by limited understanding of pathophysiological mechanisms. The biochemical phenotype of the GA1 mouse model is similar to that of human disease. High lysine exposure of GA1 mice is necessary to induce a clinical phenotype resembling human GA1, but the susceptibility is variable and a significant number of mice remain asymptomatic. Although the genetic background of the mouse model is known to be one of the main modulating factors, the underlying molecular mechanism has previously remained unknown. The investigators have now found that SUGCT, an enzyme directly involved in the metabolism of glutaryl-CoA, is polymorphic between relevant mouse strains, which explains the difference in vulnerability to high lysine exposure. Based on published and preliminary data, the investigators hypothesize that pharmacological SUGCT inhibition is a novel therapeutic option for GA1. The overall objective of this R01 proposal is to further validate SUGCT as a novel therapeutic target for the treatment of GA1 and to develop selective small molecule SUGCT inhibitors to provide pre-clinical proof-of- concept. In order to reach this objective, the investigators propose the following three specific aims. In AIM 1, the investigators will establish the functions of SUGCT through biochemical, cell line and structural biology studies. In AIM 2, the investigators will validate SUGCT as a drug target in GA1 mice through the generation of whole body and tissue-specific Sugct KO mice. In AIM 3, the investigators will identify SUGCT inhibitors through a high-throughput small molecule screen (HTS) and further validate current in-hand hits and new ones through medicinal chemistry. The ultimate goal of this project is to better understand the pathophysiology of GA1 and provide a novel path toward finding pharmacological treatment options for affected patients.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT An estimated 1,700,000 people worldwide suffer from an intracerebral hemorrhage (ICH) every year. Recent presentation of StrokeNet Site Survey data revealed that the top two “Acute Research Priorities” were the “Medical and Surgical Treatment of ICH” (StrokeNet National Investigator Meeting on October 9th, 2023, Toronto). The ENRICH trial demonstrated clinical benefit for surgical evacuation of lobar hemorrhages within 24 hours but did not find benefit for basal ganglia hemorrhages (BGH). Deep ICH, often due to hypertension, is the most common ICH subtype (up to 69% in some studies), particularly in under-represented racial/ethnic populations. Amongst 35-75 year olds, African Americans have twice the annual incidence of deep ICH than whites. BGH represents half of all deep ICH. There is an unmet need for effective treatments for this devastating disease. ENRICH's lack of efficacy in BGH may be secondary to the large craniotomy required by the ENRICH approach (a 13.8-15.8 mm dissection path through the brain) or the time to surgery (median 16.8h (IQR: 11-21h), as literature suggests earlier evacuation may result in better outcomes. We hypothesize that a minimally invasive endoscopic clot evacuation technique, called SCUBA, is an attractive and logical approach to improve outcomes when performed early (within 8-16h) or ultra-early (<8h) after BGH onset. A total of 300 subjects with spontaneous BGH will be randomized to either SCUBA endoscopic evacuation or standard non- surgical medical therapy. Our specific aim is: To evaluate the utility of SCUBA in BGH patients with LKW-to- randomization time ≤16h, and to determine whether confirmatory study in either <8h, 8-16h, or both cohorts is indicated. The primary efficacy endpoint is UW-mRS at 180 days. Successful study completion will provide crucial safety and feasibility information for early/ultra-early BG ICH evacuation using SCUBA. A successful treatment for BGH would have major global public health significance.

NIH Research Projects · FY 2026 · 2025-08

Project Summary / Abstract Substance use disorders (SUD) are a major public health concern in the United States, with an estimated 40 million Americans struggling with at least one SUD in 2020. Despite the tremendous progress made in addiction neuroscience, there exists a major disconnect in our understanding of craving and addictive decision-making, which represent two key interacting features at the heart of all compulsive disorders. Addictive decision-making can directly lead to suboptimal outcomes for afflicted individuals and are frequently studied using value-based decision-making paradigms. Craving, in contrast, represents the highly subjective urge to ingest drugs and can persist even after long periods of abstinence. In laboratory studies, craving is often studied with cue reactivity paradigms; yet it is still debatable how cue reactivity paradigms relate to real-life cravings or addictive behaviors. As such, the overarching goal of this proposal is to identify the computational and neurophysiological mechanisms underlying the interplay between craving and decision-making, by leveraging recent advances in computational psychiatry and human intracranial neuroscience and using cannabis craving as a test case. In Aim 1, we will identify a generalizable computational algorithm underlying the craving-choice interplay by deploying a paradigm that assesses craving and decision-making across two independent samples of cannabis users (online: n=1,000; in-person: n=100). We will implement a joint modeling approach to account for the bidirectional relationship between cannabis craving and addictive decision-making. We hypothesize that a) craving will amplify sensitivity (i.e. learning rate) to cannabis-specific prediction errors (CPEs); b) trial-wise cannabis value and CPE signals will simultaneously modulate cannabis craving; and c) the bidirectional relationship between cannabis craving and CPE will be modulated by the severity of cannabis dependence.. In Aim 2, we will investigate the temporal dynamics of the insula-orbitofrontal cortex (OFC) circuitry underlying the craving-choice relationship. We will conduct intracranial recording in participants who are implanted with epilepsy monitoring electrodes (n=15) in these brain regions. We hypothesize that a) high-frequency activity (HFA, 70- 200Hz) in OFC will encode drug-specific PEs and HFA in the insula will encode craving intensity; and b) fast bidirectional information flow between OFC and insula will represent the mutual influence between value-based choices and craving. In Aim 3, we will explore the causal influence of OFC/insula activity on craving and decision- making by carrying out targeted intracranial stimulation of OFC/insula. If successful, findings from this project could provide a formal, neurocomputational account for the “spiral of addiction” that results from mutually reinforcing relationship between cannabis craving and addictive choices. Furthermore, this framework can be applied to a wide range of addictive and compulsive disorders, as craving and maladaptive choices are commonly observed across substance and behavioral addictions. These results might also provide proof-of- principle for future neurostimulation treatments for these disorders.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Obstructive sleep apnea (OSA) affects arounds 24 million Americans and increases cardiovascular (CV) morbidity and mortality. The primary treatment for OSA is continuous positive airway pressure (CPAP) therapy, but CPAP usage has not been shown to reduce CV events in clinical trials. One major limitation to our understanding of the OSA-CV disease link is the current diagnostic and prognostic marker of OSA: the apnea- hypopnea index (AHI). AHI does not well predict individual CV disease risk, and reducing AHI to normal levels does not improve CV disease outcomes. Thus, better CV risk prediction tools are needed for OSA patients. Further, being at high risk does not always translate to significant benefits from treatment. However, there is currently no way of forecasting which OSA patients will receive the most benefit, or harm, from CPAP therapy. Patients with suspected OSA undergo a comprehensive sleep study, known as a polysomnogram, either at home or in a lab. Polysomnograms collect up to 20 channels of data, including heart rate, muscle movements, and brain activity; but nearly none of this data is used in clinical practice and what data is used is often compressed into summary statistics (e.g., average heart rate over 8 hours). The goal of this proposal is to use a novel artificial intelligence technique known as transformer-based neural networks, or transformers, to analyze the multimodal, longitudinal data available from a polysomnogram in order to better predict CV risk and treatment response in OSA patients. To accomplish this, we will leverage data from existing diverse epidemiological datasets (Aim 1) and randomized clinical trials testing CPAP versus usual care on CV disease outcomes (Aim 2) to fine-tune our pre-trained sleep specific transformer model. We will compare our risk prediction (Aim 1) and treatment response (Aim 2) tools to clinical metrics such as the AHI to demonstrate their improved predictive utility. Our approach is innovative in its use of cutting-edge artificial intelligence and estimation of treatment heterogeneity techniques. Transformers have become a foundational and transformative technology in artificial intelligence due to their exceptional ability to handle sequential data and capture complex patterns. Their influence on the artificial intelligence landscape is profound, and they continue to drive innovations and improvements in artificial intelligence research and technology. Further, our proposal has high significance given the prevalence of OSA, and the lack of available tools to predict CV risk and CPAP treatment response specifically in OSA patients. In particular, a tool that could predict whether an OSA patient will actually receive benefit from CPAP would revolutionize the field. Our approach will improve clinical risk prediction, treatment guidelines, and patient outcomes, as well as possibly extract novel health-relevant features of sleep for future clinical applications and mechanistic insight into OSA, CV disease, and the link between the two, significantly impacting the field of sleep medicine.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT The purpose of this K23 career development award is to prepare Dr. Renny to become an independent physician-investigator focused on improving care for youth with substance use through emergency department (ED)-based behavioral interventions and use of technology to facilitate interventions and linkages more broadly throughout health systems. In her K23 research project, Dr. Renny will address the critical deficit of research on evidence-based approaches to link youth who use substances with needed outpatient care after their ED visit. She will develop and pilot test a pediatric emergency department-initiated program, the Pediatric Emergency Department-initiated Support program To Link Care (PEDS-TLC) for youth with substance use. PEDS-TLC will link youth who report monthly or more substance use on the Screening to Brief Intervention (S2BI) screening tool (“higher risk for a SUD”) with outpatient care (adolescent medicine or addiction treatment, depending on severity of use) to receive further treatment after their PED visit. In Aim 1, Dr. Renny will adapt and integrate into the electronic health record (EHR) an evidence-based brief negotiated interview (BNI) and change plan for youth at higher risk of a SUD to facilitate linkage to care. Following the ADAPT-ITT framework, and with the assistance of a key stakeholder board and pilot testing by youth, Dr. Renny will adapt the BNI and change plan to be age- and setting-specific, including a more detailed action plan and motivational interviewing theory-based post-PED messaging (e.g., appointment reminders, action plan reminders) as part of the PEDS-TLC program. Once all components of PEDS-TLC are finalized, the action plan and interim messaging will be integrated into the EHR through the EPIC MyChart Care Companion application to assist with transition to outpatient care. In Aim 2, Dr. Renny will conduct a pilot randomized controlled trial (RCT) of the PEDS-TLC program versus standard care to examine feasibility, acceptability, and short-term efficacy of linkage to care and also substance use reduction at 30- and 90-day follow-up. During the K23 award period, Dr. Renny will build on her experience and skills as a pediatric emergency medicine physician, medical toxicologist, and healthcare researcher to gain new expertise in: (1) adapting behavioral interventions for youth with substance use; (2) engaging stakeholders in research; (3) usability testing of information technology (IT) interventions; and (4) designing, executing, and analyzing behavioral intervention trials for youth. She will receive in-depth mentorship from a multi-disciplinary team of experts in ED-based behavioral health interventions, adolescent substance use, stakeholder engagement, IT and digital health, clinical trials, and statistical analysis. The products of this K23 project will provide Dr. Renny with a well- designed, innovative program (PEDS-TLC) and critical preliminary data that informs the anticipated effect size and sample size needed for a subsequent NIH R01 RCT of PEDS-TLC.

NIH Research Projects · FY 2025 · 2025-08

PROGRAM SUMMARY The p53 protein has been extensively studied for its tumor suppressor activity in human cancer. Missense mutation of the TP53 gene is a common event in tumors. This contrasts with other tumor suppressors where deletion or loss of expression is more prevalent. Studies in genetically engineered mice have shown that knock- in of particular tumor-derived p53 mutations results in an altered tumor spectrum often accompanied by metastasis. Since this is not typically seen in p53-null mice, it has been proposed that mutant p53 not only has loss of tumor suppressor activity but in some cases gains oncogenic activity as well. Different tumor mutants of p53 behave in distinct manners in such assays adding to the complexity of the findings. In this Program led by James Manfredi, four integrated and significant Projects will address key aspects of mutant p53 and its control of the metastatic phenotype. Project 1, "Systematic studies of mutant p53 allelic variation", led by Drs. Scott Lowe (Memorial Sloan Kettering Cancer Institute) and Francisco Sanchez-Rivera (Massachusetts Institute of Technology) will perform high-throughput cellular and molecular phenotyping of mutant TP53 alleles. Project 2 led by Dr. Wei Gu (Columbia University), "Mechanisms of the hot-spot mutant p53R175H in tumor metastasis". centers on a novel protein-protein interaction involving mutant p53 that is selective for 175H. Project 3, “Defining the roles of mutant p53 and Mdm2 in invasion and metastasis”, led by Drs. Anil Rustgi and Carol Prives (Columbia University), emphasizes the study of the biological roles of mutant p53 as well as the Mdm2 protein in promoting invasion and metastasis. Project 4 led by Drs. James Manfredi and Emily Bernstein (Icahn School of Medicine at Mount Sinai), "Mechanisms of transcriptional regulation by mutant p53" will examine transcriptional mechanisms for mutant p53 oncogenesis. One Shared Resource Core will support this effort. Core 1 (Leader: Emily Bernstein, Ph.D., Epigenomics of mutant p53) will perform essential next generation sequencing analyses with a specific focus on mutant p53 and the p53-associated cistrome and transcriptome. Targeting tumor suppressors as a therapeutic strategy has been challenging since it by its nature needs to rely on restoration of wild-type functions. The notion that mutant p53 has gain-of-function activity, most likely driven by protein-protein interactions raises the intriguing possibility that this oncogenic activity may eventually be targeted. Given the frequency of p53 mutation in human cancer, this is an exciting possibility.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Influenza A virus (IAV) infects over 30 million people annually throughout the United States. Those with severe IAV infection often develop secondary bacterial coinfection by Staphylococcus aureus (SA), leading to acute lung injury (ALI) with high mortality. Though antiviral and antibiotic therapies are available, increasing rates of resistance among IAV and SA has rendered these therapies increasingly ineffective. Thus, new understanding of how IAV promotes secondary SA infection is needed to develop novel treatment approaches. Since the critical site of IAV-SA coinfection is lung alveoli, this F31 project focuses on studies that use alveolar epithelial cells and intact alveoli of live lungs to define the molecular mechanisms by which IAV inhibits alveolar defense to promote SA coinfection. Reports indicate that IAV induces both dephosphorylation and ubiquitination of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ion channel that drives alveolar wall liquid (AWL) secretion, a major alveolar lung defense. The hypothesis of this F31 project is that protein phosphatase 2A (PP2A) dephosphorylates CFTR to cause CFTR ubiquitination in IAV-exposed alveolar epithelial cells, leading to loss of AWL secretion and alveolar SA retention. Aim 1 seeks to define the role of PP2A in IAV- induced CFTR dephosphorylation and ubiquitination using murine lung epithelial-12 cells and cultured primary mouse and human alveolar type 2 (AT2) cells isolated from mouse and human lungs by flow cytometry. PP2A will be inhibited by pharmacologic and genetic approaches. The effect of PP2A inhibition on CFTR dephosphorylation and ubiquitination will be determined by immunoblot or mass spectrometry, which will also provide an opportunity to define the specific amino acid sites that are dephosphorylated and ubiquitinated in IAV infection. Aim 2 seeks to determine the effect of alveolar cell type-specific PP2A deletion on IAV-induced AWL inhibition and alveolar SA retention using transgenic mice that harbor inducible PP2A deletion in alveolar type 1 (AT1) or AT2 cells. Confocal imaging of live, intact lung alveoli will be used to define the role of PP2A in IAV- induced inhibition of AWL secretion and SA retention. Proof-of-concept experiments using wild-type mice will support the transgenic mouse experiments. The long-term goals of this F31 project are to gain new insights into IAV-SA pathogenesis in lung alveoli and, if the preliminary findings bear out, to identify PP2A as a novel therapeutic target. Importantly, this project will provide outstanding training and career development toward the establishment of an independent research career. Specific training goals include the generation of a new first- author publication, acquisition of critical technical and scientific skills, and preparation for high-quality post- doctoral research fellowship training.

NIH Research Projects · FY 2025 · 2025-08

SUMMARY Dysregulation of neurogenesis during brain development can impact neuron numbers, subtypes, and connectivity, leading to a range of neurodevelopmental disorders. Our understanding of neuronal differentiation is still limited, largely centered on growth factors, morphogens, and lineage-specific transcription factors, while other fundamental aspects of cell biology such as plasma membrane properties and cytoskeleton mechanics are overlooked. Emerging evidence reveals a critical role of membrane-related electrical processes in regulating non-excitable cells such as neuroprogenitor cells (NPC) during corticogenesis. This R21 proposal explores the previously under-studied inner membrane surface charge as critical regulator of morphological transformation and neuronal lineage commitment. It is well known that anionic phospholipids are distributed asymmetrically between the inner and outer leaflets of the plasma membrane. This is maintained by an ATP-dependent process that leads to an overall negative surface charge of the inner membrane, known as zeta potential. This local potential is different from the Nernst potential, a more global electrophysiological transmembrane potential generated by diffusible ions. While the Nernst potential is well characterized in excitable cells such as neurons, the inner leaflet surface charge and the associated mechano-electrical coupling is under-studied. The asymmetric distribution of phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP2) in the inner leaflet can influence plasma membrane stability via cytoskeletal interactions; they also serve as signaling hubs for many downstream effectors. Our recent study revealed that NPC stiffness and cortical actin network can affect the timing of neuronal differentiation. Reducing cell stiffness and cortical actin can accelerate neuronal differentiation, likely through altered membrane surface charge, thus highlighting a novel link between mechano- electrical coupling at plasma membrane and neuronal differentiation. Here, we will test the central hypothesis that regulation of the inner membrane surface charge is critical during neurogenesis by affecting the organization of cortical actin and signaling cascades associated with membrane phospholipids. In Aim 1, we will use live-cell imaging, advanced fluorescent probes, and patch-clamp recordings to characterize the temporal and spatial distribution of inner leaflet surface charges, phospholipid composition, and cortical actin rearrangement in correspondence to morphological transformation during neurite outgrowth and neuronal differentiation. In Aim 2, we will manipulate membrane surface charge using our newly developed molecular actuators (ACTU- or ACTU+) to either increase or decrease the negative charge at the inner membrane surface and assess the impact on neuronal differentiation, maturation, survival, and functionality. In summary, this R21 proposal explores mechano-electrical regulation of neurogenesis, which will provide insights into neurodevelopmental disorders and improve disease modeling.

NIH Research Projects · FY 2025 · 2025-08

Summary Multiple myeloma (MM) is a hematologic cancer characterized by abnormal plasma cells in the bone marrow, affecting 0.76% of the U.S. population with over 35,000 new cases annually; despite being historically considered incurable, recent treatment advances now achieve a median progression-free survival of 41 months. Diagnostic imaging has evolved significantly, with Whole Body MRI (WB-MRI) critical for initial staging and monitoring of MM, where WB diffusion-weighted imaging (WB-DWI) excels in lesion detection and treatment response assessment. These imaging techniques are complemented by the Dixon SPINE protocol, which measures high fat fractions (FF) associated with deeper responses in MM patients, and the assessment of sarcopenia and bone marrow density (BMD), which are crucial but underexplored indicators of overall survival and disease progression. Advanced deep learning algorithms are being developed to enhance the segmentation and quantification of these imaging biomarkers, aiming to create predictive, multi-modal AI models to improve MM patient management and outcomes. This project aims to create deep learning models to enhance the management of MM, focusing on automating the quantification of disease burden and developing predictive models for patient outcomes. Aim 1 aims to develop automated segmentation tools using WB-MRI to identify key biomarkers. such as bone lesions, FF, sarcopenia, and BMD, for assessing disease progression. Aim 2 builds on the biomarkers identified in Aim 1, integrating them with clinical, biological, and genetic factors to dynamically assess patient outcomes, including treatment response and overall survival, following the standards of the International Myeloma Working Group (IMWG). This aim involves developing longitudinal AI models that could revolutionize personalized medicine by predicting disease trajectories and treatment responses. Aim 3 involves a prospective study to validate the created tools and predictive models in real clinical settings. In summary, the goal of this project is to develop and validate advanced AI-driven tools for automated disease burden quantification, establishing biomarkers in multiple myeloma, and for creating multi-modal AI models to predict patient outcomes on longitudinal data. The significance of this work lies in its potential to dynamically and quantitatively enhance prognostic assessments and monitoring for multiple myeloma patients, leading to more precise and effective management strategies.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Histone variant dysfunction has profound consequences for tumor cells; however, their role in the tumor microenvironment (TME) remains unclear. Using an autochthonous, immunocompetent BRAFV600E/PTEN- deficient primary melanoma model, I showed that mice devoid of macroH2A (dKO) develop larger tumors, accompanied by accumulation of immunosuppressive monocytes and depletion of functional cytotoxic T cells. This compromised anti-tumor response stems from intrinsic upregulation of inflammatory genes, including Ccl2, Cxcl1 and Il6, in cancer-associated fibroblasts (CAFs), whose frequency and activation increase in the absence of macroH2A. dKO CAFs also downregulate myofibroblast-associated genes, raising the possibility that macroH2A impacts fibroblast polarization during tumor progression, skewing the balance between inflammatory (iCAFs) and myofibroblast (myCAF) subtypes. Mechanistically, this novel function for macrohistones in the melanoma TME involves 3D chromatin organization, as we found that macroH2A loss leads to increased looping between inflammatory genes and active enhancers. We hypothesize that epigenetic modulation of CAFs could direct their phenotype, in turn affecting immune cell activity in the melanoma TME. I aim to define how CAF hyper-activation in the absence of macroH2A shapes tumor initiation and the ensuing immune response. To this aim, I will leverage primary CAF cultures derived from human melanoma (PDMCAFs). I will first characterize how transcription of macroH2A genes is regulated in PDMCAFs to give rise to variable macroH2A levels. Next, I will establish to what extent PDMCAF cultures represent the functional CAF subtypes present in vivo in melanoma. Third, I will determine if macroH2A epigenetically regulates inflammatory gene loci in human cells by coordinating chromatin looping. Finally, I will test if macroH2A deficiency skews fibroblast polarization towards an inflammatory vs. myofibroblastic phenotype in CAFs during tumor progression. Therapeutic approaches that leverage the TME, such as immunotherapy, cure a subset of otherwise deadly melanomas. However, CAF plasticity hampers their efficacy and can have both tumor-promoting and restraining effects. The published study I led was the first to demonstrate a role for a histone variant in modulating the inflammatory properties of the melanoma stroma. Addressing whether similar mechanisms affect CAF plasticity in human melanoma is particularly timely, as epigenetic deregulation may promote a "cold" immune microenvironment. Therefore, this proposed project will make an important contribution to understanding how epigenetic deregulation within the TME could potentially impact melanoma initiation, response to therapy, progression, recurrence, and/or dormancy.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Food allergy, an incurable condition affecting ~10% of the US population, is a departure from immune homeostasis where aberrant antibody response, microbial dysbiosis, and local and systemic symptoms are well-reported. With exposure to culprit foods, individuals with food allergy experience symptoms including oral swelling, hives, vomiting, and anaphylaxis. The oral mucosa is the first interface between ingested food antigens and the immune system. Our studies have revealed differences in food-specific antibody levels in saliva between food allergic children and those who are only food sensitized. We have shown that allergen- specific antibody profiles in saliva can predict food allergy threshold, severity, and organ-specific symptoms. We have also found that the saliva microbiome and metabolome differ in children with and without food allergy. Building on this work, we aim to study the origins of food allergy and propose to characterize the early-life trajectory of the oral environment as food allergy arises. To date, there has been no study of the oral environment as children acquire food allergy. Our central hypothesis is that the development of food allergy is associated with distinct trajectories during the first three years of life in oral mucosal immunology, oral microbial communities and metabolites, and their cross-talk with systemic factors. We will leverage longitudinal samples and existing data from the NIH/NIAID Systems Biology of Early Atopy (SunBEAm) study, a multi-center birth cohort of 2,500 children from across the US who have undergone extensive longitudinal phenotyping, including doctor-supervised food challenges for food allergy assessment. We have been leading and working on SunBEAm since it began. To address our first hypothesis that the inception of food allergy is associated with a distinct oral mucosal immunologic trajectory, we will characterize the development of allergen-specific antibodies and cytokine milieu in saliva during the first 3 years of life and identify saliva antibody and cytokine predictors of food allergy inception (Aim 1). To address our second hypothesis that children who attain food allergy host different oral microbial communities and metabolites, we will profile the saliva microbiome and metabolome during the first 3 years of life and chart microbial and metabolite dynamics associated with food allergy acquisition (Aim 2). Our third hypothesis is that directional cross-talk between the oral and systemic environments is associated with food allergy development. We will build temporal networks and employ causal methods to integrate the multi-dimensional oral data generated by this study with multi-omic systemic data that we have in hand for the same participants to identify directional relationships between the oral and systemic environments over time and their causal impact on food allergy onset (Aim 3). This study will generate unprecedented findings on the immunologic, microbial, and metabolomic trajectories of the early-life oral environment and their relationship with food allergy development. Findings could inform food allergy prevention and biomarkers using non-invasive saliva samples.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Development of metastatic castration resistant prostate cancer (mCRPC) is dictated by multiple signaling pathways that regulate apoptosis, the androgen axis, anoikis, epithelial-mesenchymal transition (EMT), cell plasticity, invasion and angiogenesis. Disruption of mechanisms underlying these processes and the phenotypic changes they drive within the tumor microenvironment (TME), is critical to overcoming therapeutic resistance and tumor recurrence in patients with advanced disease. The overall goal of this proposal is to determine the mechanisms that link conversion of EMT to mesenchymal-epithelial transition (MET) and anoikis to and progression to advanced therapeutically-resistant prostate cancer. We developed novel agents that target tumor epithelial and endothelial cells that induce anoikis (by inhibiting the AKT pathway) and prevent metastasis by reducing tumor cell adhesion to the extracellular matrix (ECM) and inhibit invasion in vivo. Our hypothesis is that intracellular re-programming of EMT and anoikis within the TME, promotes prostate tumor invasion to lymph nodes and tumor recurrence after prostatectomy in patients. Three Specific Aims will be addressed to gain knowledge of the mechanisms underlying anoikis resistance and lymph node invasion during prostate cancer progression. In Specific Aim 1, we hypothesize that TGF-β-mediated EMT is linked to anoikis resistance, and EMT to MET conversion facilitates anoikis induction and therapeutic response in mCRPC. Studies will determine mechanisms by which EMT programming facilitates prostate cancer cell configuration to a phenotype responsive to drug-induced anoikis in in vitro. Specific Aim 2 will identify the functional significance of extracellular vesicles (EVs) cargo in prostate cancer progression. We hypothesize that EVs contribute to anoikis resistance and tumor invasion through cargo dissemination. We will characterize EMT effectors in prostate cancer cells and EVs via multi-omics, contributing to a primed-to-die phenotype in pre-clinical models of advanced prostate cancer at different stages of tumor progression and correlate changes in EVs cargo with invasive properties. Specific Aim 3 will establish predictive signatures of lymph node invasion and prognostication in prostate cancer patients. We hypothesize that intercellular communications between primary prostate tumor cells and lymph nodes leads to cancer cell invasion, and EVs act as extracellular conveyors to facilitate lymph node invasion and metastatic spread. This Aim will establish new predictive profiles of molecular and phenotypic effectors in prostate tumors and in EVs in clinical progression of prostate cancer. The proposed studies will provide new insights into the mechanisms programming resistance to anoikis and EMT in the prostate TME in vitro and in vivo. The work will be of translational impact in: (a) defining the therapeutic value of anoikis-agents in conferring vulnerability in treating mCRPC; (b) establishing the role of EVs as contributors to lymph node invasion during tumor progression; and (c) identification of profiles (signatures) of lymph node invasion and tumor recurrence in prostate cancer patients with advanced disease.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY The biology underpinning the preponderance of Alzheimer’s disease (AD) in postmenopausal women has until recently remained unclear. While declining estrogen levels have been implicated, there is a clear correlation, in post–menopausal women, between cognitive decline and high levels of the pituitary glycoprotein, follicle–stimulating hormone (FSH). We have discovered FSH as a target for several aging disorders––namely, osteoporosis, obesity, and AD. Inhibiting FSH action, either genetically in Fshr–deficient mice or using FSH– blocking antibodies reduces body fat, increases bone mass, and from our newest and most exciting results, prevents AD in mice [Nature, 2022, PMID: 35236988]. The deleterious effect of FSH on cognition is mediated through abundant FSH receptors (FSHRs) in several AD–vulnerable brain regions, including the granular cell layer of the dentate gyrus, pyramidal layer, and cortical layer V. Activation of molecules downstream of brain FSHRs, namely C/EBPb and arginine endopeptidase, in AD–prone 3xTg mice results in neuropathology and memory loss, while Fshr downregulation in hippocampal neurons attenuates the FSH–induced AD phenotype. Here, we seek to develop, validate, and study newly engineered adeno–associated viruses (AAVs) that carry a cell–specific enhancer to enable gene knockdown in Fshr–positive neurons. We hypothesize that high– efficiency, AAV–mediated Fshr knockdown in Fshr–positive, AD–vulnerable neurons in the granular layer of the dentate gyrus, pyramidal layer, and cortical layer V will reduce AD–like neuropathology and memory loss. The R61 Pilot Phase will consist of studies focused on creating and validating two enhancer–AAV constructs–– containing either a neuron–specific enhancer, mscRE4, or Fshr distal enhancers consisting of evolutionarily conserved regions (ECRs)––packaged in one of 8 existing brain–permeant AAV capsids. As a readout of enhancer–driven gene expression, we will study the expression of YFP or mCherry, respectively, in Fshr–positive cells. Once we establish a novel enhancer–AAV toolkit, the R33 Implementation Phase will focus on studying the extent of Fshr knockdown by the AAV–enhancers in AD–vulnerable neurons. Importantly, we will ask the question whether Fshr downregulation in these neurons translates into an attenuation of FSH–induced neuropathology and memory loss. Our studies represent the first attempt at using an innovative AAV–based strategy to interrogate a brain–resident pituitary hormone receptor in relation to its role in the pathogenesis of AD. Carrying different cargos, our new toolkit could be deployed to probe additional pathogenic mechanisms of AD, thus laying the groundwork for multiple therapeutic options.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Understanding the timing and regulation of metabolism during development is crucial for identifying the biochemical pathways that drive brain growth. The importance of metabolism during brain development is evidenced for example by changes in mitochondrial activity, which, when disrupted, result in neurological deficits at specific age ranges. Despite this evidence, the metabolic pathways that predominate during key stages of development remain largely unknown. For the past two decades, metabolic modeling based on 13C isotopic tracing has been successfully used to quantify the activity of metabolic pathways in the brain, yet these efforts have mostly focused on the mature brain, leaving critical gaps in our understanding of metabolism during development. This project seeks to fill these gaps by tracking, quantifying, and localizing key metabolic pathways that regulate brain growth. Given its fundamental role in energy production and biosynthesis, we will focus in this project on central carbon metabolism, a network of metabolic pathways that spans from glucose uptake to its oxidation in the Krebs cycle. Preliminary data from the wildtype mouse brain indicate that glucose is primarily metabolized through glycolysis until postnatal day 15 (P15), after which oxidative metabolism becomes dominant. Additionally, during the first 10 postnatal days, the propionate-to-succinyl-CoA pathway serves as a major biosynthetic hub, transitioning to pyruvate carboxylation thereafter. Based on these findings, we hypothesize that there are two critical metabolic switches during mouse brain development: (1) a shift from glycolytic to oxidative metabolism and (2) a biosynthetic switch from the propionate-to-succinyl-CoA pathway to pyruvate carboxylation. To test this hypothesis, we propose three specific aims: (1) to determine the onset and duration of these metabolic transitions in the developing mouse brain, (2) to quantify the rates at which metabolic transitions occur, and (3) to localize the cell types where these pathways operate. This project is significant because it will pinpoint critical periods during brain development when metabolism undergoes pivotal changes, representing potential windows of vulnerability that could predispose to neurodevelopmental disorders. This proposal is innovative in its combination of the latest advancements in mass spectrometry analysis of 13C enriched metabolites with mathematical modeling to analyze and localize metabolism in the developing brain for the first time. Our long-term goals following successful completion of this project are twofold: (1) to expand this project by analyzing metabolic pathways beyond central carbon metabolism during neurodevelopment, and (2) to apply this approach to mouse models and patients with neurometabolic diseases. The knowledge gained from this project is also expected to benefit the broader scientific community, as we aim to make this platform open source to further the understanding of the role of metabolism during neurodevelopment in both health and disease.

NIH Research Projects · FY 2025 · 2025-08

Program Summary This Program Project grant will use multiple model systems and approaches to address the pathogenic mechanisms associated with neural disease in the Congenital Disorders of Glycosylation (CDG), with the long- term goals of elucidating how glycosylation regulates neural function. Nearly all identified CDG types have neurological involvement, but the molecular and cellular basis for these phenotypes is poorly understood. This limited understanding of the pathogenic mechanisms has greatly impeded therapy development. The overall Program is organized into three Research Projects, a Research Core, and an Administrative Core. The three Research Projects will utilize zebrafish, Drosophila and human neural models, with each model system bringing distinct advantages to the Program. The overall Program is structured to allow mechanisms identified in one Project to be investigated and further elaborated in the other systems, and to permit the collective and diverse expertise of the team to be actively shared across projects. Our combined and integrated expertise will create a Program with far greater impact than any of the individual Projects alone. We will focus on multiple CDG (PMM2-CDG, PIGA-CDG, ALG13-CDG), caused by pathogenic variants in the early steps of related biosynthetic pathways, with devastating neurologic phenotypes. These disorders of N-linked and glycosylphosphatidylinositol (GPI)-anchored glycosylation share many neurological phenotypes including ataxia, intellectual disability, and seizures. How defects in these two distinct glycosylation pathways converge to cause these common phenotypes is a major question addressed by this P01. Research Project 1 will leverage zebrafish models for PMM2-CDG and PIGA-CDG to define how defects in protein glycosylation and sugar phosphate metabolism impact neurons and glia during cerebellar development. Research Project 2 utilizes Drosophila to determine the cell-type and mutation-specific contributions to neurological disease in PIGA-CDG and PMM2-CDG, and to identify genetic modifiers of both disorders. Research Project 3 will utilize PMM2-CDG and ALG13-CDG human cortical organoids to investigate specific hypotheses on how altered glycosylation and metabolism impact neuronal excitability and network activity. The Research and Administrative Cores will provide key proteomic/glycoproteomic support, and scientific, logistical and fiscal oversight, respectively for all three Research Projects. This P01 brings together the demonstrated expertise of seven investigators at five different institutions, and is led by two PD/PIs with demonstrated leadership ability and clinical/basic science research expertise in CDG. The specific aims of the Overall Program, which represent the key scientific directions of the P01 are: 1) to investigate the specific cell types (neuron vs. glia) that drive CDG neural phenotypes, 2) to define the molecular basis of disease-associated neurological phenotypes, and identify the sensitive glycoproteins and pathways responsible for these phenotypes, and 3) to perform functional analyses of identified genetic modifiers of CDG disease pathogenesis.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY A major barrier for treating solid tumors are myeloid cells such as monocytes, macrophages, and neutrophils with diverse immunoregulatory functions in the tumor microenvironment (TME). These cells are mostly derived from hematopoietic-origin precursors in the bone marrow (BM) and expand in the TME due to tumor-driven myelopoiesis. This atypical expansion of myeloid lineage provides a steady supply of pathogenic mono- macrophages in the TME and contributes to immunosuppression as well as therapy resistance. Future myeloid-targeting immunotherapies will need to target this tumor-BM axis and limit pathogenic myelopoiesis to durably reshape the TME and enable anti-tumor activity. To do so, we need a clearer understanding of genetic and epigenetic changes that occur in BM myeloid progenitors upon tumor inflammation that ultimately influences their survival, mobilization, and metabolism. My K99 proposal addresses this important question and aims to study the epigenetic regulation and priming of key regulatory pathways in tumor-educated BM myeloid progenitors. My postdoctoral work has demonstrated that activation of the NRF2 oxidative stress response is an important maladaptation in cancer-infiltrating macrophages, driving pro-survival pathways to sustain immunosuppressive activity in the TME. Based on exciting preliminary data in mouse and human, I hypothesize that tumors influence the chromatin state of BM myeloid progenitors, priming NRF2 activity to rewire metabolism, poise cytoprotection, and limit inflammatory gene activation. As part of my K99 proposal, I will assess how NRF2 influences chromatin loci in tumor-educated BM progenitors (Aim 1A) and effects metabolic adaptations (Aim 1B), using chromatin assays such as CUT&RUN and ATAC sequencing with functional metabolic readouts via probe-based cytometry. These studies lay the groundwork for my R00-phase work on the regulation of reactive myelopoiesis in response to chemotherapy. Clinicians have long recognized that cytotoxic chemotherapies severely impact BM and drive a myeloid-biased rebound linked to treatment resistance and tumor relapse. I seek to understand the long-term changes triggered by chemotherapy-induced oxidative stress and what role the resultant myeloid expansion plays in tumor immune escape and therapy resistance. In my R00, I will establish an independent program around reactive myelopoiesis in cancer therapy resistance, initially focusing on the role played by oxidative stress in chemotherapy-induced myeloid rebound in pancreatic cancer (Aim 2). I will utilize longitudinal biospecimens from pancreatic cancer (PDAC) patients undergoing standard-of-care chemotherapy to interrogate oxidative stress response using targeted cytometry and chromatin. I will exploit mouse models of chemo-refractory PDAC to study the specific contribution of BM oxidative stress to relapse. Building on my research plan, resources, training, and advisory expertise at Mount Sinai, I will advance the field of tumor-associated myelopoiesis and help develop better, more durable myeloid- targeting immunotherapy strategies that work in synergy with existing therapies for cancers such as PDAC.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Almost every person in the US has detectable levels of per- and polyfluoroalkyl substances (PFAS) in their blood. PFAS are a large class of >4,000 chemicals, known as toxic “forever chemicals”, that contribute to worse cardio-metabolic health, decreased immune functioning, and cancer risk. Focusing on total exposure to PFAS as a chemical class, rather than on individual PFAS chemicals, has been emphasized for regulatory purposes and clinical biomonitoring. However, the field lacks a standardized way to quantify cumulative PFAS exposure burden, which is a key obstacle for advancing PFAS risk assessment and epidemiological findings. Different studies and laboratories measure different sets of PFAS biomarkers. Over time, there is regrettable substitution, in which some PFAS chemicals are phased out of use by industry and substituted with other PFAS chemicals that exert similar health impacts. Our research team is at the forefront of developing a common, standardized scale to quantify PFAS exposure burden, building off of ESI PI Shelley Liu’s R03 and K25. We introduced novel applications of item response theory (IRT), traditionally used to create standardized scales for high-stakes educational testing, and demonstrated how it could be applied to PFAS biomarker data. We developed the 2017-2018 US PFAS exposure burden calculator (R/Shiny app) based on nationally representative data for 2017-2018. We showed that IRT enables us to set a standardized scale that makes full use of all available PFAS biomarker data, even if different studies measure different sets of PFAS biomarkers, so that exposure burden scores are on the same scale across studies, enabling cross-study harmonization. In Aim 1, we will extend our preliminary calculator to additional years (1996-2022) and extend to the peri-natal and early life stages, using nationally representative NHANES data and data from 6 US birth cohort studies for life stages not available in NHANES (HOME Study, Healthy Start, BBC, GenC, Project Viva, CIOB), total N=20,911. We will create personalized exposure burden metrics to account for exposure source heterogeneity due to diet/lifestyle habits, using differential item functioning analysis and mixture IRT; and account for time trends/period effects due to regrettable substitution using IRT vertical scaling. We will create a point-and-click PFAS burden calculator with software maintenance, active user tracking and dissemination. In Aim 2, we will develop new methods to estimate PFAS burden scores for emerging high-dimensional PFAS-omics data, generated from advances in non-targeted high-resolution mass spectrometry exposure science. We will create novel multi-dimensional IRT methods with mixed item response functions for continuous, zero-inflated data, yielding subscores and overall score to PFAS-omics data. Simulations and applications will be based on n=4,188 participants with PFAS-omics data from the Sister Study. Over the course of this R01, we expect that >1,000 studies on PFAS epidemiology with be published; our common, standardized scale of PFAS exposure burden will enable harmonization, pooled analyses, meta-analyses, biomonitoring and report-back.