Weill Medical Coll Of Cornell Univ

NIH Research Projects · FY 2024 · 2020-09

Project Summary/Abstract Tau neurofibrillary tangle (NFT) deposits are a characteristic hallmark of Alzheimer's disease and their appearance correlates closely with cognitive decline and disease progression. Mutations in tau cause frontotemporal dementia, establishing a critical role for tau in the etiology of neurodegeneration and dementia. In Alzheimer's disease, tau becomes hyperphosphorylated, likely leading to its release from microtubules and thereby facilitating its subsequent assembly into pathological aggregates. Other tau post-translational modifications (PTMs) such as acetylation are also implicated in disrupting tau-microtubule interactions and promoting tau aggregation. However, considerable evidence suggests that mature tau fibrils found in NFTs are not the species that cause neuronal death, and instead that oligomeric intermediates formed during the conversion of tau from a monomer to a highly ordered fibril are the toxic species. While recent breakthroughs have provided high-resolution structures of brain-derived tau aggregates, the structures of tau oligomers remain largely unknown. In aim 1 of this proposal, we will determine the structure of a novel membrane- induced toxic tau oligomer that we recently discovered using a combination of cutting edge solid-state NMR and ESR spectroscopy. By generating the first detailed structure picture of any toxic tau oligomeric species, we will advance our understanding of the interactions that stabilize tau oligomers and make possible structure function studies of their formation and their toxicity. The details of how PTMs influence tau interactions with microtubules or other interaction partners remain poorly understood. Recently, our collaborators discovered a novel tau PTM, lysine-succinylation, which occurs specifically in Alzheimer's brains but not in control brains and promotes tau aggregation, suggesting that it may contribute to disease development. In aim 2 of this proposal, we will determine the effects of this novel PTM on the functional interactions of tau with microtubules, as well as with unassembled tubulin and cell membranes. We will compare these effects to those of lysine-acetylation, which has been shown to be a key mediator of tau function and toxicity. We will employ a combination of direct and saturation transfer NMR methods using tau peptides, tau fragments and full-length tau isoforms. Our in vitro measurements will be correlated with and will inform studies on how these PTMs affect tau interactions in model cells and cultured neurons. The results may provide alternative functional tau targets for disease intervention, important given the challenges associated with targeting amyloid aggregates and aggregation cascades. In addition to perturbing functional interactions of tau, PTMs may also directly influence the formation and structure of tau aggregates. In aim 3 of this proposal, we will investigate the effects of lysine-succinylation and acetylation on membrane-induced tau oligomer formation, structure and toxicity. Given the potential role of existing and novel tau PTMs in disease, this information may be useful for the development of novel tau-targeted therapeutics.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT Prostate cancer is the most commonly diagnosed malignancy in U.S. men. There are approximately 1 million prostate biopsy (Bx) performed annually in the U.S. Almost all Bx are performed as an office based procedure in under 15 minutes. The precision of Bx has improved over the last decade with the introduction of MRI guidance/targeting of suspicious lesions within the prostate. However, significant limitations remain with this approach, including a significantly increasing risk of post-Bx infection. This arises because more than 97% of all prostate Bx are performed via a transrectal (TR) approach that introduces rectal bacteria with each pass of the Bx needle into the sterile urinary tract. The current risk of post-TR Bx infection, even with antimicrobial prophylaxis, is high at approximately 7% overall with 3% (30,000 men) requiring hospitalization annually. Transperineal (TP) Bx is an alternate approach that eliminates the direct introduction of bacteria from the rectum to the prostate. This approach, which is perfomed without antimicrobial prophylaxis, instead passes the Bx needle through the perineal skin and pelvic floor. TP Bx has not been widely adopted for several reasons. Historically, it has been considered too painful for patients in the clinic and thus was traditionally performed under general anesthesia. The added time, inconvenience and cost has limited its national adoptance. Second when TR Bx was initially adopted over 40 years ago, antibiotic resistance of rectal flora was not a challenge. Beyond the potential for in-office TP Bx to significantly reduce or eliminate Bx infections, TP Bx may also improve cancer detection as studies of TP Bx (performed under general anesthesia) demonstrate higher detection rates for prostate cancer, particularly for anterior zone tumors, compared to TR Bx. This is notable as anterior tumors are difficult to sample with TR Bx. Anterior tumors are also twice as likely to occur in African American men. In fact, our research demonstrates that some of the outcomes disparities in African American men may stem from an underdiagnosis of anterior prostate cancers. Although TR Bx is used widely, it is associated with a significant and increasing risk of Bx infections due to growing antibiotic resistance, highlighting the urgent need for a safer alternative approach to prostate Bx. We have refined a TP Bx approach under local anesthesia with MRI-targeting/guidance without the need for antibiotic prophylaxis. We hypothesize that TP MRI targeted Bx will: (1) largely eliminate post-Bx infections and costly hospitalizations for urosepsis; (2) be performed in the office with similar discomfort and non-infectious complications compared to TR MRI targeted Bx; and (3) have significantly better detection of prostate cancer. A multi-center randomized controlled trial will be conducted to evaluate in-office TP MRI targeted vs. TR MRI targeted Bx, the current gold standard. This has transformative impact to change current standard of practice. The investigators have a track record for collaboration. The environment comprises 4 high-volume, SPORE funded centers of excellence that serve diverse populations.

NIH Research Projects · FY 2026 · 2020-09

Microbiomes are diverse ecosystems, critical for ecological studies and human health, consisting of numerous species across known or unexplored domains of life. Metagenomic sequencing is essential for studying microbiomes due to the difficulty of culturing many organisms, addressing the challenge of 'unknown unknowns' in community composition. Accurate metagenome reconstruction is necessary to explore the interplay between microbiome composition and functional capacity. Current bioinformatics tools require validation using comprehensive datasets paired with precise reference standards. We propose the "Microbiome-in-a-Bottle" (MIAB) project to create benchmark materials for metagenomics, similar to the Genome-in-a-Bottle (GIAB) initiative for human genomes. MIAB will curate high- confidence isolate genomes, integrate multiple sequencing technologies (Illumina short-reads, Hi-C, PacBio and Oxford Nanopore long-reads, and LoopSeq and TELL-Seq synthetic long-reads). Our goal is to provide high-quality benchmark sets for tool validation and we will also release raw sequencing data. My lab will also continue novel method development for metagenome assembly in the next 5 years. Existing one-click metagenomics pipelines simplify workflows but face challenges with inflexible parameters, reliance on dependencies, and lack of customization. Manual curation and visualization are crucial for refining analyses and improving result accuracy. Scalability, reproducibility, and user-friendly yet flexible solutions are needed for diverse metagenomics research applications. One of our lab's major goals over the next five years is to extend and maintain CAMP (Core Analysis Modular Pipeline), a modular ecosystem for high-throughput metagenomics. We plan to enhance it using Artificial Intelligence (AI), particularly large-language models. Our lab has been at the forefront of AI model development and its applications in genomics and various biomedical domains in recent years, and we will leverage this expertise for the success of this project. My group has collaborated with the MetaSUB Consortium in recent years. MetaSUB collected 5,000 metagenomic samples from public transit surfaces in 60 cities worldwide, providing valuable insights into the diversity of urban microbiomes. However, the evolutionary forces shaping these communities remain unexplored. As part of a new project in our lab, we plan to investigate this over the next five years. We will apply various models to analyze and compare datasets from several major cities with extensive sampling, including NYC, London, and Hong Kong.

NIH Research Projects · FY 2024 · 2020-08

Abstract Since 2010, clinical medicine and public health have benefited from a rapid surge of clinical research on chronic diseases using data from electronic health records (EHRs). However, while millions of patient records are included in large EHR networks, they are not population-representative random samples, a constraint which has restrained their utility for population health research. The non-representative nature of patients represented in EHR data also poses a major challenge when performing cross-site validation of EHR-based findings, as study findings tend to reflect the unique characteristics of populations served by specific health care systems. We propose to perform an integrated secondary data analysis of three unique datasets: 1) the Health and Retirement Survey (HRS, begun in 1992 and ongoing) that has nationally representative health interview data for over 20 years, as well as biomarkers, physical assessment information, prescription drug data, and claims linkages including Medicare D drug claims; 2) the New York University Langone Health EHR data (NYU-CDRN, 2009 to now) including demographics, vitals, diagnoses, lab results, prescriptions and procedures; 3) the New York City Clinical Data Research Network (NYC-CDRN) which is an EHR network that comprises 20 NYC healthcare institutions, including the NYU-CDRN, with longitudinally linked data on over 12 million patient encounters under a Common Data Model; and 4) Veterans Affairs Ann Arbor Healthcare System (VAAAHS) Corporate Data Warehouse (CDW), which provides an important complement to the NYC-CDRN patient population when assessing our method’s reproducibility and generalizability for the rural patient population in care. We will leverage these four datasets to support three strands of questions on EHR-based risk predictions: 1) assessing its utility for population inference, 2) developing individualized absolute risk predictions, and 3) assessing its reproducibility and cross-site validation. We will predict risk of subsequent incident cardiovascular disease (CVD) in older patients (age 50 and older) with type 2 diabetes (T2DM). Broader use of these methods will be generally applicable to other diseases outcomes. To achieve these objectives, our study will 1) develop and validate EHR phenotyping and diagnosis time algorithms against gold standard chart review (Aim 1); 2) assess the population-generalizability of EHR-based risk estimation models by comparing with cohort-based risk estimation models and develop EHR bias adjustment methods for population inference (Aim 2); 3) develop methods for EHR-based individualized absolute risk prediction (Aim 3), and establish the developed methods via cross-site validation (Aim 4).

NIH Research Projects · FY 2024 · 2020-08

Cognitive impairments, including memory loss, are prevalent in the elderly and patients with neurodegenerative disease. However, the exact causes of aging-related cognitive impairments are uncertain, and effective prevention and treatment options are limited. Increasing evidence implicates astrocytic-neuronal interactions in a wide range of normal and pathophysiological processes, including memory loss and neurodegeneration. However, the exact mechanisms by which astrocytes may contribute to disease-related cognitive impairments are not known. Transactive response DNA-binding protein 43 kDa (TDP-43) is associated with diverse aging- related neurodegenerative disorders and its dysfunction correlates with cognitive decline in humans. Recent studies suggest that glial TDP-43 plays important roles in the brain and its dysfunction might contribute to disease pathogenesis. In support, mutant TDP-43 can cause cell-autonomous impairments in isolated astrocytes and its astrocytic expression in animal models causes behavioral deficits and premature mortality, suggesting that astrocytic TDP-43 is essential for brain function and its dysregulation can cause disease. Despite these intriguing findings, the roles of astrocytic TDP-43 in cognitive decline and astrocytic-neuronal interactions are not known. Our preliminary studies suggest that astrocytic TDP-43 dysregulation occurs in human cases with Alzheimer’s disease and causes memory loss in transgenic mouse models. In addition, our results implicate astrocytic TDP- 43 in regulating glial and neuronal gene expression, astrocytic-neuronal interactions, and neuronal plasticity. However, these effects and causal links between astrocytic TDP-43 dysregulation and neuronal activities linked to memory require further investigation. Here, we will investigate how astrocytic TDP-43 dysfunction affects the brain in common dementias by defining its roles in hippocampus-dependent memory (Aim 1), gene expression, neuronal activities (Aim 2), and astrocytic-neuronal signaling mechanisms (Aim 3). In these studies, we will test novel hypotheses that astrocytic TDP-43 dysregulation alters astrocytic-neuronal chemokine signaling and specific aspects of glutamatergic transmission and neural plasticity. We will use a combination of advanced molecular and cellular approaches in transgenic mice and cell cultures to target and probe specific cell populations and brain regions. These studies are poised to reveal novel TDP-43-linked mechanistic cascades, advance our understanding of how astrocytic-neuronal interactions contribute to cognitive decline, and identify novel therapeutic strategies that reduce TDP-43-linked deficits in diverse disorders.

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY Candidate: My career goal is to improve the wellbeing of older adults with multiple chronic conditions and polypharmacy by developing and rigorously testing rational strategies to improve medication prescribing practices; and to become an independent clinician investigator and national leader in Geriatric Cardiology. This area is particularly relevant for me as a heart failure cardiologist, as I care for older adults who almost universally have multiple chronic conditions and polypharmacy. My track record as an emerging leader in this area is evidenced by multiple lead- and senior-author manuscripts, 4 grants as the Principal Investigator including a GEMSSTAR, my position as the Founding Director of the Weill Cornell Heart Failure with Preserved Ejection (HFpEF) Program for the Aging, and my role as the Chair of the American College of Cardiology Geriatric Cardiology Section Early Career Professionals Working Group. Mentors and Environment: This project will be conducted under the mentorship of 4 well-funded investigators who will contribute their complementary expertise in Geriatrics (Mark Lachs), Geriatric Cardiology (Mathew Maurer), Implementation Science (Monika Safford), and N-of-1 trials (Ian Kronish). Advisors in deprescribing, shared decision making, and biostatistics will provide additional content expertise to ensure the success of the proposed project and catalyze my career development. This project will take place within the supportive environment of Weill Cornell Medicine, which has demonstrated a deep commitment to my development as a clinician-investigator for the past several years. Mentored Research Project: Despite its role as an integral part of patient-centric and goal-concordant prescribing practice, deprescribing is seldom incorporated into clinical practice due to several barriers. To improve patient-centered medication management, there is a need to develop processes that can overcome these barriers. The objective of this proposal is to determine whether N- of-1 trials—as a pragmatic patient-centered approach to medication optimization that can overcome key barriers of deprescribing— can lead to increased patient confidence regarding the decision to continue or discontinue a medication. To achieve this, I will: 1) determine key features of a feasible and pragmatic protocol for deprescribing N-of-1 trials using a stakeholder-engaged iterative design approach; and 2) determine the preliminary effectiveness of an adapted N-of-1 protocol on patient decision confidence and deprescribing, and 3) determine the facilitators and barriers of implementing an adapted N-of-1 protocol into real-world clinical practice by conducting a type-1 hybrid effectiveness-implementation trial. I will achieve these aims by using ß- blocker in heart failure with preserved ejection fraction (HFpEF) as a prototype for older adults with multimorbidity and polypharmacy. This proposal will inform a future multicenter randomized controlled trial focused on improving patient-reported outcomes, and provide a formative opportunity for me to obtain the knowledge and skills necessary to become a leading researcher on patient-centered medication management.

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY The overall objective of this proposal is to integrate large-scale VA and non-VA data across New York, Chicago and Florida to study the risk of suicidal ideation, suicide attempts, suicide and accidental opioid overdose deaths in Veterans receiving chronic opioid therapy (COT).

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY Hepatitis C virus infection (HCV) is the most important cause of chronic infectious disease death in the US, with over 2 million people infected and nearly 20,000 dying annually. The greatest burden; occurs among “baby boomers,” born 1945-1965 (75% of those infected). HCV disproportionately affects racial/ethnic minorities (25% of those infected are Blacks; Black mortality is 87% and Hispanic mortality 43% higher than among Whites), persons with HIV disease and injection drug users. Direct-acting antiviral (DAA) therapy recently became available; it can cure >95% of people with HCV, but only half have been diagnosed and a much smaller proportion treated. CDC’s National Viral Hepatitis Action Plan (NVHAP) calls for identification and treatment of all those infected, but access for racial/ethnic minorities is problematic, due to costly medicines, stigmatization of those with HCV, variable access to specialists, inconsistent provider awareness of need to diagnose and treat, and variable coverage of treatment, particularly by state Medicaid plans. We propose to use an exploratory sequential mixed methods design to analyze HCV testing and treatment patterns among US baby boomers, tracking the diffusion of DAA therapy nationally and across demographic groups since initial licensing in 2014, and to conduct qualitative work and surveys of patients and physicians to explore factors contributing to disparities in care. The Specific Aims are: 1) To use national and state-level publicly-administered health insurance claims data to describe variations in HCV screening and treatment among the baby boomer cohort by a) examining differences in HCV screening rates across race/ethnicity, community socioeconomic status (cSES), and primary care utilization; b) evaluating the time between HCV diagnosis and antiviral treatment initiation among individuals with a new diagnosis of HCV, by race/ethnicity and cSES; and c) evaluating time between FDA approval of the first DAA HCV treatment, and initiation of DAA treatment for the prevalent population of baby boomers with chronic HCV, by race/ethnicity and cSES; and, informed by these findings 2) To assess the contribution of patient and provider factors to shaping disparities in diagnosis and treatment of HCV by adapting existing measures and/or developing novel measures, and administering these to samples of HCV patients and providers, by a) using patient focus groups and participatory feedback to adapt measures of stigma and discrimination in health care and other settings, knowledge and beliefs about HCV and its treatment, and barriers to care (competing needs, depression, drug use, access, patient costs, distance to care), then administering them to diverse HCV patients in two US regions; and b) developing measures of HCV knowledge, attitudes, experiences and practices among physicians, then administering them to primary care, gastroenterology, hepatology, and infectious disease physicians in two US regions. We will share study instruments and results with community partners, who will be very involved in all phases of the research, and with other key stakeholders and investigators, to facilitate the development and evaluation of interventions to address the problems identified in this study.

NIH Research Projects · FY 2024 · 2020-08

Abstract. The goal of this R61/R33 proposal is to carry out a phase IA/IB clinical study of AAVrh.10hFXN (a serotype rh.10 adeno-associated virus coding for human frataxin) to treat cardiac manifestations of Friedreich’s ataxia (FA), the most common inherited ataxia. FA is a fatal, presently, untreatable disorder. Most cases result from intron variants in the frataxin (FXN) gene; when inherited from both parents, there is resulting haploinsufficiency of FXN gene expression. While progressive neurologic disease limits mobility, cardiomyopathy is responsible for substantial morbidity and 60% of deaths secondary to progressive heart failure and arrhythmias. Cardiac MRI (CMR) data from our group and others demonstrate that FA-associated cardiomyopathy initially manifests with increased left ventricular (LV) myocardial mass (a potentially reversible phenotype) prior to development of myocardial fibrosis (irreversible damage). AAVrh.10hFXN, the therapeutic gene transfer vector to be used in the proposed human study, is a nonhuman primate-derived serotype rh.10 capsid with a constitutive promoter driving the normal human frataxin cDNA. AAVrh.10hFXN will be administered intravenously, a vector and route which in experimental animal models effectively delivers genes to the heart. Based on our preclinical efficacy data in two murine models in which intravenous AAVrh.10hFXN reverses the consequences of FA cardiomyopathy, together with extensive safety data, we are ready to initiate a phase IA/IB clinical trial with the following aims. R61 aim 1. Prepare and submit an Investigational New Drug package and gain approval from the FDA and other regulatory groups (Institutional Review Board, Biosafety) to initiate a phase IA/IB clinical trial. Milestone. Full regulatory approval to initiate parts A and B of the clinical trial, enroll the 1st subject in part A. R61 aim 2 and milestone. Manufacture clinical grade AAVrh.10hFXN for the part A (safety/dose-ranging) clinical trial. R33 aim 3. Carry out the part A (safety/dose-ranging) trial to determine the maximum tolerable dose of AAVrh.10hFXN therapy for the cardiac manifestations of FA. Milestone. Initiate and complete assessment of n=9 individuals (3 doses, 3 each), with an audited final report. R33 aim 4 and milestone. Manufacture clinical grade AAVrh.10hFXN for part B (safety/preliminary efficacy) clinical trial. R33 aim 5. Carry out the part B safety/preliminary efficacy study at the highest tolerable dose from part A. Milestone. Complete assessment of n=15 individuals, with an audited final report. Given that genetic variance is responsible for many forms of cardiomyopathy and that existing treatments are limited, this study offers a potential therapeutic paradigm shift to reverse cardiac phenotype and thus improve clinical outcomes for a broad range of patients with genetically mediated cardiomyopathies at risk adverse LV remodeling and its devastating clinical consequences.

NIH Research Projects · FY 2024 · 2020-07

Epithelial to mesenchymal transition (EMT) has been enthusiastically proposed as an essential mechanism for tumor metastasis, since the EMT-associated features such as migration, invasion, resistance to apoptosis and stemness properties, adequately meet the requirements for metastasis. Taken the challenges of tracing the EMT process in vivo, we developed a strategy of using a mesenchymal-specific Cre-mediated switch of fluorescent markers in a multiple-transgenic mouse (MMTV-PyMT:Fsp1-Cre:Rosa26mT/mG, Tri-PyMT). Surprisingly, we found that lung metastases were predominantly composed of pre-EMT RFP+ tumor cells exhibiting epithelial phenotypes under normal conditions. Importantly, the post-EMT tumor cells did exhibit resistance to chemotherapy, significantly contributed to recurrent lung metastases after chemotherapy. These findings pointed to the complexity of EMT contributions in tumor progression and revived vigorous discussions in the community. Given the transient, reversible and dynamic natures of the EMT process, and concerns about the Tri-PyMT model, we proposed new lineage tracing models to study the roles of EMT in metastasis and chemoresistance. Aim 1. To explore the contributions of EMT mechanism in tumor metastasis and chemoresistance by using Snail-CreERT2 mediated EMT lineage tracing model. The Fsp1-Cre mediated Tri-PyMT model may not be sufficient to report all EMT events. Metastatic cells could undergo EMT by activating distinct EMT transcription factors (TFs) such as Snail. Therefore, we have established a Snail-CreERT2–mediated EMT lineage tracing model. In-depth analyses will be performed to clarify the roles of EMT in metastasis and chemoresistance with this model. Aim 2. To explore the dynamic changes of EMT statuses in human breast cancer metastasis and chemoresistance. EMT reporter cell lines (MDA-MB-231:Vim/RFP and BT-474:ECAD/GFP cells) carry knockin fluorescent tags within EMT marker genes (Vimentin and E-cadherin, respectively). Unlike the Cre-mediated models, the fluorescence expression in these cells is quantitative and reversible, which allows us to analyze the dynamic changes of EMT status with and without chemotherapy in human breast cancer models. Aim 3. To assess the evolutionary lineages of metastasis-initiating cells and the involvement of EMT mechanism via genetic barcoding models. In addition to using EMT markers, we will genetically barcode the Tri-PyMT cells using the homing-CRISPR technique. This model will allow us to depict the evolutionary trajectories from primary tumor cells to individual metastases, and determine the origins of the metastasis (pre- EMT vs. post-EMT cells). Further, we will develop genetic barcoding mice (MARC:CRISPR:PyMT) for lineage tracing of spontaneous metastatic cells and assessing the contributions of the EMT program to metastasis. Impact: Resolving the controversies in the field will not only improve our mechanistic understanding of tumor metastasis but also provide novel targets/opportunities in combatting the deadly disease.

NIH Research Projects · FY 2025 · 2020-07

ABSTRACT Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is an unexplained multisystem/multisymptom disorder characterized by severe and debilitating fatigue for which there are currently no validated diagnostic tests nor accepted therapies. The present proposal focuses on exploring a naturally occurring and widely available dietary supplement, N-acetylcysteine (NAC) – a prodrug for in situ synthesis of the primary intracellular antioxidant, glutathione (GSH) – as a highly promising treatment for ME/CFS. Driving this exploratory clinical trial are recent compelling preliminary findings by the applicants which showed that 4 weeks of daily supplements of 1800mg of NAC (a) alleviated a significant in vivo brain GSH deficit in patients with ME/CFS as measured directly with proton magnetic resonance spectroscopy (1H MRS) (b) ameliorated ME/CFS symptoms and (c) decreased the levels of plasma markers of oxidative stress in ME/CFS patients while eliciting no changes in healthy controls. The significance of these novel findings is that they represent the clearest and most direct evidence to date that NAC has the ability to spur in situ synthesis and restoration of brain GSH levels, and that GSH regulates its own synthesis through a "feedback inhibition" mechanism whereby GSH synthesis is turned on or off depending whether tissue GSH levels are low or normal, respectively. The main objective of the present proposal is to further investigate through a double-blind, placebo-controlled, randomized clinical trial, the mechanism(s) of in situ GSH synthesis control by administering different doses of NAC or placebo only to ME/CFS patients shown by 1H MRS to have a significant cortical GSH deficit at baseline. We postulate that this narrow requirement for a GSH deficit at baseline will represent a cohort selection refinement that would identify patients who would be most likely to show cortical GSH elevations in response to NAC treatment because GSH synthesis would not be feedback- inhibited. The expectation is that if successfully completed, the proposed study would advance our understanding of the mechanism(s) of action of NAC in in vivo cortical GSH synthesis, confirm oxidative stress as a viable treatment target for dietary NAC, and establish changes in 1H MRS measures of cortical GSH and in plasma markers of oxidative stress as biomarkers of treatment target engagement and of therapeutic response and, importantly, suggest optimal experimental conditions (cohort selection criteria, NAC dose) that would be suitable for use in future full-scale efficacy clinical trials of NAC for treatment of ME/CFS or other conditions in which redox imbalance has been postulated.

NIH Research Projects · FY 2025 · 2020-07

The Molecular Biophysics Training Program (MBTP) at the Weill Cornell Graduate School (WCGS) is built on a decades-long history of biophysics research and training at the two research centers comprising WCGS - Weill Cornell Medicine (WCM) and the Memorial Sloan-Kettering Cancer Center (MSKCC) located across the street from each other. MBTP will bring together biophysics trainees from two WCGS graduate programs: the Biochemistry & Structural Biology (BSB) program, part of the umbrella Biochemistry, Cell and Molecular Biology (BCMB) program and the Physiology, Biophysics and Systems Biology (PBSB) program. Union of the biophysics trainees in these two programs is a natural fit given that biophysics is inherently multidisciplinary – bringing together biology, chemistry, physics, computational biology, and mathematics. MBTP will provide: 1) outstanding learning and mentoring for predoctoral trainees in molecular biophysics; 2) world-class research opportunities; 3) an interactive and interdisciplinary cohort of students and faculty to foster scientific exchange and collaboration; 4) strong foundation in performing rigorous and reproducible scientific research and 5) knowledge of and exposure to various career paths for successful transition into the biomedical research workforce. The formation of the MBTP program is the latest in a nearly three-decade maturation of synergistic connections between WCM and MSKCC.

NIH Research Projects · FY 2024 · 2020-07

Role of BCL10 somatic mutations in lymphomagenesis and response to BCR-targeted therapies ABSTRACT Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy and a molecularly heterogenous disease. Two recent genomic profiling studies of large DLBCL patient series subclassified these patients in five distinct genomic groups. Both studies essentially agreed in their classification and described a previously unnoticed subtype reminiscent of Marginal Zone Lymphoma (MZL), namely C1 or BN2 lymphoma. This C1/BN2 subtype is characterized by frequent translocations of BCL6 and activating mutations of NOTCH2 and NF-κB signaling genes. Among the latter, 30% of the patients displayed BCL10 mutations, which are rare in other DLBCL subtypes (<2%) but relatively common in MZL (8%). In fact, BCL10 is critical for MZ B-cell development and its overexpression mediates hyperproliferation and eventually lymphomas of MZ origin. However, the effect of BCL10 mutations on lymphomagenesis has not been studied. BCL10 forms a high order complex (CBM) with CARD11 and MALT1, also lymphoma oncogenes. This complex serves as a docking platform for recruitment and activation of other proteins leading to NF-κB activation. BCL10 somatic mutations in DLBCL can be classified in: CARD domain missense and C-terminus truncating mutants. BCL10 CARD mediates CARD11-BCL10 and BCL10-BCL10 interactions while BCL10 C-terminal domain mediates BCL10-MALT1 interaction. In preliminary studies, both classes of mutants accelerate BCL10 polymerization, rewire complex structure and composition and, induce constitutive activation of NF-κB mediated transcription and MALT1 protease activity. We hypothesize that CARD and C-terminal mutations induce gain-of-function and drive lymphomagenesis by activating CBM complex activity and its downstream signaling pathways including NF-κB and that they will do so through distinct molecular mechanisms. Based in our preliminary results, we predict that: i) BCL10 gain-of- function mutations will enhance CBM complex activity by disrupting BCL10 auto-inhibitory structure through distinct molecular mechanisms based on specific biochemical effects of CARD missense or C-terminal truncating mutations; ii) this will cause acceleration of lymphomagenesis in cooperation with NOTHC2 activating mutations, and iii) BCL10 gain-of-function mutations will confer resistance to classical BCR pathway kinase inhibitors such as Ibrutinib (BTK inhibitor), thus requiring targeting downstream proteins such as MALT1 inhibitors or alternative pathways. Our goals for this proposal are to elucidate the molecular mechanism by which specific BCL10 somatic mutations classes alter the high order molecular structure of the CBM complex, to determine how this impacts MZ B-cell growth and survival to cause lymphomas, and to leverage this information to design of novel therapeutic approaches for C1/BN2 lymphomas.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY/ ABSTRACT The proposed K23 Patient-Oriented Research Career Development Award is designed to provide the candidate with the conceptual knowledge and technical skills needed for a career of an independent investigator focused on the engagement of behavioral and neural network targets during personalized psychotherapies for mid- and late depression. The candidate will conduct her training and research activities at the ALACRITY Center of the Weill Cornell Institute of Geriatric Psychiatry. The proposal is based on the premise that identifying specific behavioral and neural network targets can guide development of streamlined interventions with potential for broad scalability and reach. “Engage”, a streamlined therapy for late-life depression, whose principal intervention is “reward exposure”, may change Positive Valence Systems (PVS) functions. Preliminary studies by the candidate show that early increase in resting state functional connectivity (rsFC) of PVS structures during “Engage” predicts increased behavioral activation. Additionally, compared to solitary pleasant activities, exposure to rewarding social interactions during “Engage” leads to greater increase in behavioral activation and reduction of depression severity. Finally, a machine learning analysis conducted by the candidate showed that low perceived social support is the strongest predictor of poor response early in psychotherapy. These findings are in line with animal and human studies demonstrating the protective role of social rewards. Based on these observations, the candidate developed “Engage-S”, a social-reward based version of “Engage”, aimed to increase exposure to meaningful social interactions with others. The training study proposes to examine whether social reward exposure in “Engage-S” enhances PVS abnormalities and improves mid- and late-life depression. The participants will be 60 middle-aged and older adults (age ≥ 50) with major depression who will be randomized to 9-weeks of “Engage-S” or to a Symptom Review and Psychoeducation (SRP) comparison condition. During treatment, we will examine target engagement of the PVS with rsFC, a behavioral activation scale (BADS), and performance on a novel social reward paradigm at baseline, 5 weeks, and 9 weeks. We will use computational methods to identify neuroimaging and behavioral profiles associated with treatment response. The training plan consists of formal courses, structured tutorials, and hands-on methodological training that will offer the candidate knowledge and skills in: 1) Functional neuroanatomy of depression and aging; 2) Use of fMRI to assess target engagement during psychotherapies for mid- and late-life depression; 3) Computational modeling for the identification of clinical and neuroimaging predictors of treatment response that can be used to personalize psychotherapy; and 4) Generate preliminary data for an R-series experimental therapeutics target engagement application.

NIH Research Projects · FY 2025 · 2020-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. The Tri-Institutional PhD Program in Chemical Biology (TPCB) is an innovative graduate program that provides students with comprehensive training at the interface of chemistry, biology, and medicine. TPCB is unique in that it is offered jointly by three premier research centers with adjacent campuses in the heart of New York City: Memorial Sloan Kettering Cancer Center, The Rockefeller University, and Weill Cornell Medical College. This stand-alone, direct admission graduate program was established in 2001 as one of the first in the world to focus specifically on training the next generation of chemical biology researchers. Over the last 23 years, TPCB has established itself as a leading graduate program in chemical biology, with 109 PhD graduates and 53 current students (36 training grant-eligible, TGE). Students come from both chemistry and biology backgrounds and the program combines quantitative chemical training with deep insights into forefront problems in biomedical research. The program works with each student individually to ensure they have the resources necessary to thrive. All years, URG vs All Students 34 / 189 All years, URG vs TGE 32 / 105 Current, URG vs All Students 16 / 53 Current, URG vs TGE 15 / 36 = 41.6% The curriculum comprises two chemical biology courses created specifically for TPCB students, additional core and elective courses, a seminar course, lab rotations, student Research-in-Progress seminars, annual student-organized Tri‑I Chemical Biology Symposium and TPCB Student Career Development Retreat, and the Sanders Tri‑I Chemical Biology Seminar Series. Students receive extensive career development training and formal ongoing instruction in Responsible Conduct of Research and Rigor & Reproducibility. Students have opportunities to conduct PhD thesis research with 58 dedicated faculty mentors working across the spectrum of chemical biology with >$1M average annual funding. Students move seamlessly between the three campuses and have access to the state-of-the-art research facilities and rich scientific resources on all three campuses, providing extensive opportunities to pursue fundamental and translational research and to engage in multidisciplinary collaborations. Importantly, TPCB students are deeply involved in the organization and design of the training experience, and the program has evolved continuously in response to student feedback. As a result, TPCB students have been exceptionally productive, publishing over 580 scientific papers (average 5.4 papers/graduate), graduating in an average of 5.5 years. TPCB alumni have continued onto successful careers across the tenure track (15%), biotech (34%), pharma (8%), other labs (12%), and research-related careers (30%). TPCB receives strong support from the graduate schools but is still unable to admit all of the qualified candidates who apply. Thus, this NIGMS Chemistry–Biology Interface T32 training grant will enable further growth and enhancement of this successful training program and its broader impacts on the scientific community.

NIH Research Projects · FY 2025 · 2020-07

PROJECT SUMMARY/ABSTRACT The burden of America's opioid crisis has heavily fallen on children, a vulnerable population increasingly exposed to opioids in utero or childhood through parental opioid use (POU). POU exposure in utero may lead to a newborn experiencing neonatal opioid withdrawal syndrome, while POU exposure in childhood may lead to a child experiencing maltreatment and family separation due to parental drug overdose mortality, parental institutionalization, or foster care placement. Life course theory postulates that early life adversity, especially in utero and early childhood, may lead to lifelong physical and mental health, substance use, behavioral, and socioeconomic problems. This K01 Mentored Research Scientist Development Award application proposes a training and research plan that will support Dr. Angélica Meinhofer on a path towards independence, focused on elucidating the impact of exposure to POU in early life (in utero up to age 8) on mental health disorders, chronic conditions, infectious diseases, injuries, and healthcare utilization in early and middle childhood. The training plan supplements Dr. Meinhofer's prior expertise in opioid use disorders, health economics, and policy evaluation with training in (1) child health with a life course perspective, (2) behavioral health systems and services for children and families, (3) epidemiology and biostatistics methods, and (4) complex data management and linkage algorithms. Dr. Meinhofer will achieve the proposed training objectives with a combination of formal coursework, workshops, and hands-on experience, as well as the mentorship of Dr. Bruce Schackman, Dr. Yuhua Bao, Dr. Katherine Keyes, and Dr. Rachel Dunifon. Drawing from the Medicaid Analytic eXtract (MAX) linked with other datasets and combining a longitudinal, population-based study design with difference-in-differences and propensity score methods, Dr. Meinhofer will use the knowledge and skills acquired through these training activities to achieve the following aims: (1) Estimate the association between exposure to opioids in utero and physical and mental health, and healthcare utilization in early childhood; (2) Estimate the association between exposure to parental drug overdose mortality in early childhood and physical and mental health, injuries, and healthcare utilization in early and middle childhood; and (3) Estimate the association between exposure to parental opioid use disorders medication treatment in early childhood and physical and mental health, injuries, and healthcare utilization in early and middle childhood. Understanding how early life exposure to POU may affect offspring outcomes over the life course provides a strong foundation upon which clinicians and policymakers can design a more proactive, coordinated, family-centered, and overall more effective agenda. The proposed K01 Award will provide Dr. Meinhofer with the resources, training, and mentoring needed to become an R01-funded independent investigator leading a multidisciplinary research program to inform policies for improving the wellbeing of children and families affected by parental substance use.

NIH Research Projects · FY 2023 · 2020-06

ABSTRACT: Heart failure continues to be a leading cause of mortality and morbidity. Understanding the basic mechanisms of heart regeneration in humans and stimulating them would improve the lives of many patients. Proliferation of heart muscle cells (cardiomyocytes) is essential for heart development and regeneration. After birth, the proliferative potential of cardiomyocytes declines, but neonatal mammalian hearts are thought to maintain the ability to regenerate until cardiomyocytes undergo permanent cell cycle arrest, establishing a barrier for regeneration. Strategies for stimulating cardiomyocyte proliferation are under development and would be most effective when initiated before cardiomyocytes withdraw permanently from the cell cycle. However, the point at which cardiomyocyte proliferation ceases in humans is controversial; some studies place this point before birth, some in the first year after birth, and our data demonstrate cardiomyocyte proliferation extending well into childhood, i.e., the first 10 – 20 years of life. This picture is further complicated by our published and unpublished results suggesting that infants with congenital heart disease (CHD) have decreased cardiomyocyte proliferation. Resolving how age and heart disease alter the temporal pattern of cardiomyocyte cell cycle withdrawal is an essential problem in cardiac biology. Improved understanding of cardiomyocyte proliferation will be critical for developing new regenerative strategies to prevent and treat heart failure in patients with cyanotic CHD, the most common birth defect affecting 0.3% of newborns. We will determine when cardiomyocyte proliferation decreases in pediatric patients by investigating two leading cardiac diseases: tetralogy of Fallot (ToF), the most common form of cyanotic congenital heart disease (CHD), which affects 0.3% of newborns, and dilated cardiomyopathy (DCM), the most common cause for heart transplantation in the pediatric age group. Our central hypothesis is that cardiomyocyte proliferation shows an age- and disease-dependent decline. We will enroll neonates, infants, and children to quantify cardiomyocyte proliferation. We have formed a research team consisting of pediatric cardiologists and cardiac surgeons, pathologists, and basic scientists. The critical element of our approach is the use of a highly innovative method in which we will label patients with innocuous thymidine carrying stable isotope markers (15N-thymidine and 2H- thymidine). Cells in S-phase of the cell cycle incorporate thymidine into their DNA, and their offspring retain the label for at least 6 cell divisions. We will visualize the isotope in post-surgical tissue samples with an innovative method: Multi-isotope imaging mass spectrometry (MIMS) to quantify cardiomyocyte proliferation and differentiation. Preliminary data from our first study patient with ToF provide first-in-human results and validate our approach.

NIH Research Projects · FY 2024 · 2020-06

Alpha-synuclein (aSyn) pathology is linked to synucleinopathies including Parkinson's disease and Lewy body dementia, but the underlying disease mechanisms remain poorly understood. The prevalent viewpoint has emerged that aggregation of aSyn triggers neuropathology through a gain-of-toxic-function mechanism, and approaches to eliminate aSyn represent an active area of research for treatment. Yet, aSyn aggregation may also endanger neurons by removing aSyn from synaptic vesicles (its physiologically relevant intracellular location) and thereby causing loss-of-function. Through its synaptic vesicle-bound state, aSyn regulates synaptic vesicle trafficking, and chaperones SNARE-complex assembly to maintain neurotransmitter release. Thus, removing aSyn from neurons may not be protective, but detrimental. The objective in this application is to determine the impact of synaptic vesicle-binding of aSyn on aSyn function and neuron survival, using rationally designed variants of aSyn that stabilize synaptic vesicle-binding. The central hypothesis is that stabilizing binding of aSyn on synaptic vesicles reduces aSyn toxicity and pathology. Guided by strong preliminary data, this hypothesis will be tested in three specific aims: 1) Determine the effect of increased synaptic vesicle-binding of aSyn on SNARE-complex assembly; 2) Assess the effect of increased synaptic vesicle-binding of aSyn on synaptic vesicle cycling; and 3) Test if increased synaptic vesicle-binding of αSyn rescues neurotoxicity and pathology in vivo. Under the first aim, SNARE-complex assembly will be quantified in vivo and in vitro, using cell biological and biochemical techniques. Under the second aim, αSyn multimerization, synaptic vesicle pools and clustering, and synaptic vesicle cycling will be quantified, using cell biological, biochemical and biophysical techniques. Under the third aim, mouse models will be generated by stereotactic injections of lentiviral vectors into the substantia nigra of aSyn knockout mice to assess effects of mutant aSyn variants on αSyn-induced toxicity and pathology, using behavioral assays on mice and biochemical, histological and ultrastructural analyses on injected brains. The study is expected to show improved aSyn function and delayed pathology upon stabilization of synaptic vesicle-binding of αSyn. This research is innovative because it 1) tests the novel hypothesis that stabilizing synaptic vesicle-bound αSyn reduces aSyn pathology, 2) creates new tools to study function and dysfunction of αSyn, and 3) uses a multidisciplinary approach to test our hypothesis from single molecules and cellular systems to live mice. This work is significant, because it will 1) clarify the importance of synaptic vesicle-binding of aSyn for neuron function, 2) provide new insights into the molecular mechanism of synaptic vesicle-binding of αSyn, 3) uncover the contribution of loss-of-function of aSyn to disease pathogenesis, and 4) have translational importance for the development of new treatment strategies aimed at stabilizing synaptic vesicle-bound αSyn.

NIH Research Projects · FY 2024 · 2020-05

PROJECT SUMMARY Kaposi's sarcoma (KS) is the most common cancer globally in people living with HIV, and among the most common cancers in Sub-Saharan Africa, and is caused by infection by the Kaposi sarcoma herpesvirus (KSHV, also called HHV-8). This virus also causes primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). While PEL is rare, it is an aggressive malignancy with few therapeutic options. KSHV-associated diseases are difficult to model because this virus is species-specific, it does not transform cells in in culture, in vitro infection frequently leads to a mixture of latent and lytic viral gene expression, and related animal viruses do not cause the same pathologies. Furthermore, KS lesions are composed of a mixture of cells that include latently KSHV-infected spindle cells and a mixed inflammatory infiltrate that includes numerous CD8+ and CD4+ T cells, plasma cells, macrophages, and mast cells. While substantial attention has been given to the histogenesis of the spindle cells, the immune infiltrates in KS lesions have only been superficially and incompletely described. The overarching goal is this application is develop preclinical in vitro, ex vivo and in vivo models of KSHV-associated diseases, including KS, MCD and lymphoma. To model KS, we will apply observations from human lesions, and include the immune elements of this disease. This will be accomplished through the following specific aims: 1) conditional expression of major latency transcript genes in immunocompetent mice; 2) examine the tumor immune environment in KS lesions in patients and test the role of major immune subsets in mice; and 3) engineer synthetic, in vitro and ex vivo Kaposi sarcoma-like tissue niches for controlled growth of healthy and diseased primary endothelial cells. We will examine the effects of first line therapeutic approaches, targeted therapy and immunotherapy in these models to validate them for preclinical use.

NIH Research Projects · FY 2026 · 2020-05

Project Summary Synapses are fundamental to nervous system function and information processing in the normal and pathological brain. As highly dynamic biological structures, they display a broad range of activity and plasticity. A detailed picture of the molecular interactions occurring within a synapse is required to understand how synaptic protein dynamics ultimately shape activity in the nervous system in health and disease. In this proposal, we investigate Munc13, Munc18, and the neuronal SNAREs, highly conserved proteins that are crucial for proper synaptic function. Mice lacking any one of these core proteins die at birth, and there are numerous neurodegenerative and psychiatric disorders associated with defects in the function of these proteins. For instance, mutations in the human Munc13 ortholog (Unc13A) are associated with ALS, myasthenia, microcephaly, and severe autism. Mutations in the Munc18 ortholog (STXBP1) are associated with epileptic encephalopathies. We propose to study the molecular mechanisms underlying Munc13 and Munc18 function at the synapse using a unique and powerful combination of in vivo and in vitro approaches including physiology, quantitative imaging, behavioral assays, genetics, and protein/lipid biochemistry. Much of the detailed mechanistic work on the core proteins of synaptic transmission has been performed in vitro. To move forward on these molecular models, we need an in vivo testing platform that allows precise manipulations of the core machinery in their native environment with endogenous expression levels. To this end, we have built a collection of C. elegans mutants in the core machinery of synaptic transmission including the SNARE proteins, UNC-13, UNC-18, and CPX-1 (worm orthologs of Munc13, Munc18, and complexin, respectively) using CRISPR Cas9 gene editing to avoid over-expression issues that have plagued structure/function studies in the past. By combining various mutations using double and triple mutants, we are investigating detailed mechanistic questions in vivo using endogenous proteins. This step is essential in furthering our understanding of the molecular mechanisms underlying synaptic transmission. In addition, advances in protein structure prediction have enriched our hypotheses for how the core machinery operates as a functional unit, so we are also exploring novel interactions between these proteins with the goal of expanding the current models for the molecular underpinnings of neurotransmitter release. The experiments proposed here will provide new insights into the mechanisms that control neurotransmitter release, its modulation, and use-dependent plasticity in the brain.

NIH Research Projects · FY 2025 · 2020-05

Project Summary/Abstract Intrinsically disordered proteins and protein regions (IDPs and IDRs) lack stable tertiary structure but retain biological function. Understanding the structure/function relationships of IDPs/IDRs presents a significant challenge because of their variable and dynamic nature. Recently, an increased awareness and recognition of the prevalence and roles of IDPs and IDRs in membrane trafficking and organization has emerged. The primary goal of the proposed research is to advance our understanding of how the dynamic and highly variable structure of IDPs mediates their functions in membrane trafficking and organization. A key aspect of IDP function in membrane trafficking and organization involves direct IDP-membrane interactions, which occur in conjunction with disorder-to-order transitions, or in the absence of protein ordering. Formation of membrane- binding amphipathic helices (AHs) is a common example of the former, but the mechanisms underlying and regulating the formation, stability, specificity and function of such membrane-associated AHs remain poorly understood. Factors that govern membrane binding by IDRs that remain disordered in the bound state are even less well understood. A major area of proposed research centers on delineating mechanisms for these types of IDP-membrane interactions using proteins such as alpha-synuclein, metabotropic glutamate receptors and complexins as models via a combination of in vitro characterization of structure and dynamics and in vivo functional assays. Another emerging aspect of IDP function is their ability to mediate formation of condensates or membraneless organelles. Recently, it has been demonstrated that IDR-containing membrane-binding proteins can form cytosolic condensates that sequester and organize intracellular reservoirs of membrane vesicles. The mechanisms that regulate the ability of condensates to interact with and organize membranous vesicles or compartments have barely been explored and represent second major focus of this proposal. The primary model system for these efforts will be the clustering of membrane vesicles mediated by the protein synapsin, and the regulation of this clustering by IDPs/ IDRs such as synucleins and rab proteins. Efforts will also include investigating the role of condensate formation in the organization of tubulo-vesicular organelles such as the endocytic recycling compartment. These systems will be characterized using structure/function analyses combining in vitro characterization of condensate formation and vesicle recruitment/release with in situ and in vivo functional studies. Achieving the goals of this proposal will serve to advance our understanding of different mechanisms that underlie the roles of IDPs and IDRs in the regulation of membrane trafficking and organization. By focusing on specific models with physiological significance, namely factors governing vesicle exocytosis, organellar contact sites, cell signaling and the formation of clustered vesicular structures in neurons and other cell types, the results will make a significant impact on specific fields as well as broaden our general understanding of how protein disorder contributes to the organization of cellular membranes.

NIH Research Projects · FY 2026 · 2020-04

Inflammatory bowel disease (IBD) affects millions of people worldwide, causing significant morbidity. Despite improvements in medical therapy, nearly one-third of IBD patients develop refractory disease requiring hospitalization or surgery. The long-term goal of this proposal is to identify the cellular and molecular mechanisms underlying disease in order to design targeted therapies that are safer and more effective. Group 3 innate lymphoid cells (ILC3s) play a central role in the pathophysiology of IBD. As the major producers of IL- 22, ILC3s play a critical role in promoting mucosal healing in IBD2. Emerging data, however, has revealed that ILC3s can function as a double-edged sword, driving tissue inflammation in mouse models of colitis and IBD. In addition to these innate effector functions, recent studies have revealed that MHCII+ RORt+ ILCs can act as antigen presenting cells (APCs) which limit T cell responses against commensal bacteria and promote regulatory T cells. The objective of this proposal is to define the molecular and cellular mechanisms that regulate these heterogenous functions of ILCs in IBD. Our published and preliminary data identify TNF-like cytokine 1A (TL1A) as central regulator of these heterogenous function of ILC3s. TL1A is highly expressed in human colonic tissue during IBD and variants in TNFSF15, the genetic locus that encodes TL1A, confer higher risk for more aggressive disease complications. While early studies showed a pathogenic role for TL1A in driving inflammatory T cell responses, subsequent work from our own group and others have revealed a direct role for TL1A in regulating ILCs. Using RNA-seq of TL1A-stimulated intestinal ILC3, our preliminary data reveal that TL1A signaling robustly and specifically induce expression of the transcription factor Bhlhe40. Bhlhe40 is a member of the basic helix- loop-helix TF family, which is a key regulator of cytokine production by macrophages and T cells. Bhlhe40 is required for inflammation and promotes immune responses in autoimmunity, transplantation, and cancer. Single cell data identified Bhlhe40 in intestinal ILCs, but the potential role of Bhlhe40 in ILC3 is unknown. Using new genetic mouse models and human biopsy samples, this proposal will test the hypothesis that Bhlhe40 in ILC3s is critical in shaping innate immunity to in inflammatory colitis as well as coordinating mucosal cellular responses to maintain antigen specific tolerance in the intestine. The expected outcomes of these aims are to identify a new role for Bhlhe40 in shaping innate effector functions of ILC3s and revealing a central role for intestinal ILC3s in coordinating cellular immunity to enforce intestinal tolerance. If successful, these findings will uncover new mechanistic targets for regulating ILC3s in IBD.

NIH Research Projects · FY 2026 · 2020-04

PROJECT ABSTRACT The overarching goal of our proposed research program is to develop a discovery pipeline that will enable identification of transcriptional codes for engineering tissue-specific endothelial cells (ECs) for therapeutic organ regeneration of heart, lung and blood. Therapies for organ regeneration promises unlimited access to the replacement tissues. However, despite breakthroughs in uncovering the molecular underpinnings of organ morphogenesis and organoid technology, translation of regenerative medicine to the clinic has confronted with hurdles. These bottlenecks are in part due to the lack of understanding as to how niche cells coordinate organ repair. Specifically, contribution of vascular niche cells that supply regenerative signals has not been realized. This R35 application builds upon the novel proposition that poor healing after organ damage is due to the dysfunction and loss of the tissue-specific ECs. This programmatic proposal examines the hypothesis that reconstitution of stem cells in injured organs is dependent on the pro-regenerative angiocrine signals supplied by tissue-specific vascular niche ECs. We have shown that organotypic ECs by deploying defined angiocrine factors support lung, cardiac, hepatic and hematopoietic regeneration. Thus, ECs perform actively as dynamic, tissue-specified niche cells critical for tissue homeostasis and repair. To test this and to set up the stage for therapies, we have engineered adaptable mouse, nonhuman primate and human ECs by transducing the transduction factor (TF) ETV2 into adult mature ECs (R-VECs) and differentiating human induced pluripotent stem cells (iPSCs) into generic fetal-like ECs (iVECs) that could inform on the pathways that induce organotypic TFs. These adaptive iVECs and R-VECs will be cocultured with heart, lung, and blood organoids in vitro or infused in vivo in mice undergoing organ repair to identify the induction of organotypic TFs in these cells. The educated iVECs and R-VECs will be recovered and subjected to RNA profiling and de novo motif discovery to identify induced tissue-specific TF(s). The identified TFs will be overexpressed or knocked down in ECs, to validate their function in sustaining organotypic and angiocrine profile for organ repair. We anticipate that transplantation of organotypic ECs will promote long-lasting tissue repair without provoking tumorigenesis or fibrosis. We have initiated FDA-approved human clinical trials to examine the safety and efficacy of allogeneic generic EC infusion for hematopoietic recovery. As a follow up, we intend to assess the contribution of R-VECs or iVECs-derived from nonhuman primates to regeneration in the pigtail macaque monkeys with the intention of translating the potential of organotypic ECs to clinic. The expected outcomes of the proposed research are identification of molecular signals and transcriptional determinants of tissue-specific vascular and angiocrine heterogeneity. Goals of this proposal fit with the mission of NHLBI R35 award to develop innovative regenerative discovery pipeline to promote safe and efficacious treatments for cardiac, pulmonary and blood maladies.

NIH Research Projects · FY 2025 · 2020-03

PROJECT SUMMARY/ABSTRACT In order to maintain the compact structure of chromatin yet ensure access and functionality when required, eukaryotic genomes utilize multiunit chromatin remodeling complexes such as BAF, to enable dynamic binding of transcription factors to DNA. Being so instrumental in genome regulation, it is not surprising that BAF-complex genes are the most frequently affected by somatic mutations in cancer, in 20% of all patients and in 23% of diffuse large B cell lymphoma (DLBCL). However, the mechanism by which BAF promotes malignant transformation and lymphomagenesis is unclear. Based on our initial analysis, the BAF complex seems to be an important regulator of germinal center B cells, DLBCL cell-of-origin. We hypothesize that BAF enables chromatin accessibility for factors involved in germinal center B cell differentiation and prevents activated B cells from staying in the tumorigenic state of rapid cycling. To investigate the underlying mechanism, we will (Aim 1) define the biological role and mechanism of action of BAF in the normal humoral immune response. To this aim, we will use computational and experimental methods in genomics to determine BAF-complex composition, BAF genomic binding and BAF-dependent changes in chromatin accessibility in primary germinal center B cells with inactivating BAF mutations found in lymphoma patients. Furthermore, we will determine the role of BAF in nucleosome mobility and positioning in germinal center cells using a novel computational approach. The Melnick lab has discovered that regulation of nuclear architecture plays a critical role in germinal center B cell biology and that perturbation of factors involved in nuclear topology leads to lymphoma. However, these factors, such as the cohesin complex, are rarely mutated in lymphoma. By investigating changes in nuclear topology associated with binding of the BAF-complex, we will test if this discrepancy is explained by BAF mutations that might carry out these architectural functions. Furthermore, we will (Aim 2) determine the role of BAF in the initiation and clonal evolution of lymphoma and other tumors through effects on chromatin plasticity. We hypothesize that BAF complex exerts its function by globally inducing nucleosome mobility and exposing transcription factor motifs. Once a mutation in a BAF subunit occurs and the general fluidity of nucleosomes is lost, nucleosomes might be preferentially locked in an unfavorable chromatin position. This might lead to stochastic activation of malignant programs. To address this question, we plan to expand our computational approach to model changes in chromatin stiffness within cancers affected by BAF mutations using publicly available data. Furthermore, we will establish a simple-to-use parallel single-cell transcriptome and chromatin accessibility assay and apply it to lymphoma tumors from our BAF mutant mouse models. Taken together, the proposed project will provide insights into the mechanism of BAF-mediated formation of lymphoma. In case our findings support the hypothesis of BAF being the master regulator of tumor suppression in B cells, we will be able to identify novel therapeutic targets for patients with BAF mutations and further classify those tumors.

NIH Research Projects · FY 2025 · 2019-12

In the previous award period, we identified a critical function for the histone variant H3.3 and its phosphorylation in the rapid transcription of inflammatory genes across immune cells and especially in macrophages and germinal center B cells. Surprisingly, we also discovered that H3.3 expression (encoded by the two identical paralogs H3f3a and H3f3b) is dynamically regulated during inflammation in both human and mouse. With these genes among the most differentially expressed in inflammatory and disease states, it led us to predict that their modulation may act as a tunable regulator of immune responses. In support of this, we find that H3.3 levels are rate-limiting for stimulation-induced transcription and that inflammatory gene transcription titrates with graded expression of H3.3. Here, we test the hypothesis that H3.3 dosage acts as a rheostat for stimulation induced transcription across immune cells and their activation states. In Aim 1 we will define the impact of H3.3 dosage on inflammatory gene transcription, immune cell function, and defense to infection, using genetic mouse models with varying levels of H3.3 (from 0 to 4 copies) and competitive mixed bone marrow chimeras, in vitro transcriptomic and epigenomic studies, and carefully selected in vivo infection models (Listeria monocytogenes, Aspergillus fumigatus, and Plasmodium chabaudi). With these approaches, we will define the impact of H3.3 titration and its expression from the H3f3a and H3f3b loci on immune cell differentiation, activation, and pathogen control. We will identify which genes, and functional categories of genes, are most sensitive to H3.3 dosage. To understand the regulatory logic of H3.3 expression tuning, we will identify the gene enhancers and transcription factors that control cell-type-specific and stimulation- responsive expression of H3f3a and H3f3b. We apply innovative computational prediction of enhancers from single cell combined transcriptomic and epigenomic datasets and have identified putative enhancers at the H3f3a and H3f3b loci that we will functionally assess using genetic mouse models and in vitro enhancer perturbation assays (CRISPRi). In Aim 2 we will dissect the molecular mechanisms by which H3.3 regulates transcription, including its function in RNA polymerase II pause-release, co-transcriptional splicing, and establishing the histone post-translational modification landscape. To understand H3.3-dependent transcription processes we apply precision run-on sequencing (PRO-seq) and chromatin immunoprecipitation with exonuclease digestion (ChIP-exo) to map RNA polymerase II and other transcription regulatory factors at nucleotide resolution, and we quantify histone post-translational modifications that occur on or require H3.3. Finally, in Aim 3 we apply an innovative H3.3 mutagenesis screen to identify functional residues and modifications on H3.3. This screen will be performed in primary macrophages, enabling us to assess the impact of each mutation on inflammatory gene expression and cell activation, and will generate a first-of-its- kind functional roadmap of histone modifications in mammalian cells.