Weill Medical Coll Of Cornell Univ

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY Loss of hearing is a prevalent sensory pathology in the United States that affects over 30 million people. A significant proportion of deafness is attributed to sensorineural hearing loss, which often involves the damage of afferent nerve fibers which relay auditory information from the mechanosensitive hair cells of the inner ear to the brain. The restoration of physiologic hearing would require the regeneration of afferent fibers into the sensory epithelium of the cochlea, followed by the reinnervation of appropriate hair cell targets. Nerve regeneration studies in humans and other mammalian models are lacking due to the limited accessibility of the inner ear. The zebrafish lateral line system, composed of superficial fluid-flow detecting hair cells and afferent nerve fibers, offers a simple and accessible model of nerve regeneration. In this model, there are likely various paracrine, juxtacrine, and neuron-autonomous signaling mechanisms working in coordination to guide axon pathfinding and target selection. Aim 1 of this proposal will determine the molecular cues expressed by target sensory hair cells to guide reinnervation by regenerating afferent axons of the lateral line. Following transection of the lateral line nerve, hair cells from the zebrafish will be isolated at multiple timepoints. In these hair cells, the expression changes of canonical and non-canonical molecular cues that may be used to attract axonal growth cones will be quantified through transcriptome sequencing. Aim 2 will investigate the neuronal bias for reinnervation of developmentally related hair cell populations. Although studies suggest that neurons retain a memory for their original hair cell targets, how this memory is established or maintained is unknown. A transgenic imaging technique will be used to label and trace clonal populations of regenerating axons following transection of the lateral line nerve. It is hypothesized that neurons prefer reinnervating hair cells that arose from a shared sensory placode during development. Aim 3 will reveal changes in the local physical environment of the regenerating nerve to allow entry of individual afferent fibers into their target organ. By imaging transgenic fish with fluorescently labeled Schwann cells and collagen, changes will be shown in Schwann cell tracts and the epithelial basement membrane to permit entry of individual axons into the zebrafish neuromast, which contains target hair cells. It is hypothesized that physical gaps form in Schwann cell and basement membrane layers in close proximity to denervated hair cells to allow passage of regenerating axons branching off the main nerve bundle. Together, these studies will elucidate the mechanisms governing afferent nerve regeneration and the reinnervation of hair cells, which may provide insight towards restoring hearing in the deafened human cochlea. These studies will be carried out under the direct mentorship of Dr. A. J. Hudspeth at The Rockefeller University and within the supportive environment of the Tri-Institutional MD-PhD Program in New York City.

NIH Research Projects · FY 2026 · 2023-06

Why some people develop Multiple Sclerosis and others do not, despite similar genetic risk and quantities of circulating autoreactive lymphocytes, is not known. Our long-term goal is to identify environmental triggers of MS, define the molecular and cellular basis of their action, and in doing so, propose new diagnostic tools and therapeutic targets. The objectives of this proposal are to determine mechanistically how Clostridium perfringens epsilon toxin (ETX) and Bordetella pertussis toxin (PTX) overcome CNS immune privilege to trigger autoimmunity in the context of myelin autoreactive lymphocytes and to understand why ETX causes lesions to develop in the forebrain, cerebellum, brainstem, and spinal cord in contrast to PTX where lesions are more commonly localized to the spinal cord. The central hypothesis of this project is that ETX and PTX trigger CNS autoimmunity by inducing critical dysfunction at CNS barriers necessary for entry of pathogenic lymphocytes. The central hypothesis will be tested by pursuing two aims: 1) Determining the effect of cell specific deletion or introduction of the ETX receptor MAL (Myelin and Lymphocyte Protein) in active immunization models of experimental autoimmune encephalomyelitis (EAE), compare the neuroanatomical location, phenotype, and activation state of immune infiltrates between PTX- and ETX-induced EAE, and explore the effect of ETX on human lymphocytes, and 2) Determine the genes induced and suppressed in CNS-endothelial cells by ETX and PTX and define their function in overcoming CNS immune privilege through loss-of-function strategies. We will pursue these aims using an innovative combination of targeted genetic mutations to isolate cellular and molecular targets of ETX required to induce disease. We will use confocal microscopy, immunohistochemistry, high dimensional flow cytometry, and unbiased sampling of the entire CNS to compare the effects of ETX with PTX on immune phenotype, demyelination, and neuroanatomic localization of lesions. To determine toxin induced genes functioning to overcome CNS immune privilege, we will apply a combination of unbiased mRNA profiling techniques to CNS endothelial cells isolated from different neuroanatomic regions, advanced bioinformatics to define relevant gene modules, immunohistochemistry to validate localization of these induced proteins within individual post-capillary venules, and conditional loss-of-function mutations in endothelial cells to determine function. The rationale underlying this proposal is that completion will define the role by which a toxin, clinically associated with MS, functions in the multi-step process of autoimmunity, and will identify key molecular targets that can be tested therapeutically. This work will also help establish an experimental model that has greater clinical relevance to MS and more closely resembles MS neuropathology than experimental autoimmune encephalomyelitis models reliant on pertussis toxin.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY Atopic disease is a major public health problem affecting up to 20% of the world’s population, with rising prevalence in recent decades. It is associated with fatal reactions (food allergy), and with significant psychosocial and financial burden for children and families. Environmental and in utero exposures are known risk factors, but little is known about the mechanisms through which exposures may confer risk. One of these known exposures is maternal antenatal anxiety, which has been associated both with elevated markers of allergy risk (such as levels of Immunoglobluin E (IgE)) and with atopic diagnosis in children. Maternal antenatal anxiety has also been linked to immune dysregulation in both mothers and children, and elevated innate immune activity in children at birth may be a key marker of future allergic risk – yet no study has attempted to link dyadic maternal and neonatal immune dysregulation to allergy risk in the same mother-child pairs. In this prospective, longitudinal, observational study, we propose to follow 200 mother-child pairs (132 at risk for clinically significant anxiety and 68 healthy controls) from the second trimester of pregnancy until infants reach 12 months of age. We hypothesize that women with clinically significant antenatal anxiety will demonstrate elevated immune activity compared to healthy controls, that stimulated cells and gene expression from umbilical cord blood in these pregnancies will similarly show evidence of increased immune activity, and that maternal and child immune activity will be associated with markers of allergy risk in children. We will measure immune cell populations and cytokines produced by stimulated cells in mothers and children; will conduct methylation analyses of immune-related genes in the cord blood of children (focusing on known biomarkers of future allergic risk); will measure atopic dermatitis in children; and will measure IgE to common allergens and maternal report of food allergic reactions in children. In addition, we will observe mother-child interactions surrounding feeding to account for elements of the postnatal environment that may contribute to elevated child allergic risk. Our thorough and rigorous experimental plan will help to illuminate pathways that contribute to the association between maternal antenatal anxiety and child allergy risk, and may lead to novel interventions to ameliorate symptoms and risk in both mother and child.

NIH Research Projects · FY 2026 · 2023-06

Abstract. This is an ancillary study to SOURCE (SPIROMICS Study of Early COPD Progression, NHLBI HL144718), an observational study of the early manifestations of chronic obstructive pulmonary disease (COPD). Based on the knowledge that the small airway epithelium (SAE) is the earliest site of the pathology of COPD, the focus of this ancillary proposal is to characterize the disordered SAE differentiation in SOURCE participants and correlate this biologic dysfunction with the abnormal early COPD small airway clinical phenotypes characterized in SOURCE. We will capitalize on the availability of resources from SOURCE for use in our study, including: detailed clinical phenotyping of small airway structure and function, with serum, lavage fluid, SAE cytopreps, SAE RNA, and SAE basal stem/progenitor cells obtained by bronchoscopy from n=80 individuals with early COPD ages 30-55, and n=20 normal controls. As an additional control, we will provide parallel clinical data and biologic samples from our BioBank n=10 healthy smokers. Based on our observations that in early COPD, SAE basal stem/progenitor cells have a decreased capacity to form a normal differentiated SAE, preliminary data demonstrating that BMP4 and SPDEF are upregulated in the SAE in early COPD smokers and that BMP4 induces squamous metaplasia and SPDEF induces goblet cell hyperplasia, the proposed studies will characterize the role of BMP4 and SPDEF in the pathogenesis of the disordered small airway differentiation in early COPD. We hypothesize that early COPD is characterized, in part, by SAE upregulation of BMP4 and SPDEF which, in turn, contribute to the clinical abnormalities of the small airways that characterize early COPD. The results will help determine if BMP4 and SPDEF and/or their driver genes and/or downstream signaling pathways are potential targets for future therapeutic intervention to reverse/prevent the small airway abnormalities that characterize early COPD. We propose 3 specific aims. Aim 1: To characterize the disordered SAE in early COPD and evaluate the hypothesis that SAE basal cells (BC) from individuals with early COPD have a reduced capacity to differentiate into a normal SAE as assessed in vitro using air-liquid interface cultures. Aim 2: To examine the hypothesis that the disordered SAE BC differentiation in early COPD is caused, in part, by SAE overexpression of BMP4 and SPDEF, resulting in activation of their respective receptor downstream signaling pathways and consequent abnormal SAE differentiation of squamous metaplasia and goblet cell hyperplasia. Aim 3: To test the hypothesis that diminished small airway epithelial BC differentiation and BMP4- and SPDEF- mediated biologic parameters correlate with the clinical parameters of small airway structure and function in the same SOURCE participants with early COPD.

NIH Research Projects · FY 2025 · 2023-06

Black and Hispanic adults suffer disproportionately from obesity and obesity-related conditions (e.g., Hispanic/White ratio: diabetes (1.7) compared to their white counterparts. While behavioral weight loss (WL) interventions are the first line in treating obesity, they have mixed outcomes. Most fail to make a persistent impact, especially in specific racial/ethnic subgroups. For example, Black women lose 50% less weight than their white counterparts, even when enrolled in fully powered, well-designed behavioral WL trials. One plausible reason for these sub-optimal results is a focus on behavior change at the individual level alone while neglecting critical social and environmental forces. Social networks have been increasingly shown to influence health behaviors, yet they have seldom been harnessed in studies targeting weight loss, especially among racial/ethnic minorities. In response to PAS-20-160 Small R01s for Clinical Trials Targeting Diseases within the Mission of NIDDK, this application will compare the effects of a social-network enhanced lifestyle intervention (hereafter termed "ROBUST" Reducing OBesity Using Social Ties) to an individual level lifestyle intervention (control) on modifying multiple network-level barriers to weight loss. We will randomly enroll 132 Black or Hispanic adults with obesity (BMI > 30 kg/m2) and invite up to two social network members of participants in the ROBUST arm to a 24 –week multi-competent lifestyle intervention. We will evaluate whether the ROBUST intervention not only addresses individual-level behaviors (i.e., healthy eating, increased physical activity) but also: 1) reduces social undermining as well as changes perceived health norms by activating communal coping - a behavioral process that involves thinking, communicating, and acting as if a health risk (i.e., Type 2 diabetes) is shared; and 2) dampens the harmful effects of increased interpersonal conflict on weight by teaching participants how to induce a positive affect and self-affirming mindset which we have shown in a previous trial (NHLBI-U01HL07843) prevents unwanted weight gain. Participants in the control arm will receive the same number of lifestyle sessions as those randomized to ROBUST. But, their social network will not be directly engaged in the study. We hypothesize that the ROBUST intervention will satisfy all a priori feasibility/acceptability criteria for recruitment, retention, and study conduct. Additionally, the ROBUST intervention will result in positive changes in multiple barriers to behavior change, leading to a more significant proportion of participants experiencing improvements in diet, physical activity, and weight loss at 24 weeks compared to the control group. If proven effective, the preliminary data gathered from this small RO1 will support a fully powered RCT that will result in an optimized multi-level obesity intervention that impacts critical clinical endpoints (i.e., BMI, insulin resistance, and hemoglobin A1c) in a population at very high risk of poor outcomes related to obesity and its metabolic sequelae.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY Glioblastoma (GBM) is a devastating brain tumor disease with a median overall survival of approximately 15 months. GBM patients die because of the constant ability of GBM to acquire resistance mechanisms against anti-cancer therapies, therefore leading to an inevitable tumor recurrence. Radiation therapy (RT) is a pivotal modality for improving overall survival of GBM. However, GBM invariably recurs, which suggests that RT is eliciting or exacerbating mechanisms of resistance in GBM. Identifying and overcoming the contributing factors involved in GBM resistance is a major challenge in Radiation Oncology. GBM metabolism and its role in immune evasion emerges as a RT-induced resistance mechanism in GBM. Specifically, we have preliminary data indicating that irradiated GBM cells reprogram their metabolism towards the generation of fatty acids. Such metabolic reprogramming after RT is impairing the innate immune recognition and systemic anti-tumor immunity elicited by RT. More precisely, we have preliminary evidence that fatty acid synthesis is inhibiting nucleic acid sensing-dependent interferon type I (IFN-I) responses and is promoting immunosuppressive signals such as the programmed-death-1 (PD-1) and the programmed-death ligand 1 (PD-L1). As a consequence, cancer cell-intrinsic IFN-I will not be released in response to RT. This ultimately limits anti-tumor immune response against GBM by precluding infiltration effector T cells into the GBM microenvironment. We have recently demonstrated that cancer cell-intrinsic IFN-I response is an essential step to convey immunogenicity of an irradiated tumor. Consequently, by increasing energy supply, limiting innate immunity and increasing immunosuppression, RT-induced fatty acid synthesis is likely to be a major GBM resistance mechanism that not only impacts RT response of GBM but also provides means to evade immune recognition. In this application, we propose to test the novel and innovative hypothesis that fatty acid metabolism induced by RT controls immune escape and GBM survival. Successful completion of this proposal will define how fatty acid synthesis facilitates GBM immune evasion and provide pre-clinical evidence for fatty acid inhibitors as a novel approach to restore the immunogenicity of irradiated GBM.

NIH Research Projects · FY 2026 · 2023-06

PROPOSAL SUMMARY There are critical vaccine barriers to eliciting cytotoxic CD8 T cells against intracellular pathogens. Current vaccine technologies have yielded limited success for protection against infections with intracellular pathogens like tuberculosis, malaria, and HIV where CD8 T cells prevent and control infection. Licensed vaccines generate mostly neutralizing or opsonizing antibodies, and their efficacy is contingent on a stable antigenic profile. Some adjuvants like alum elicit helper type 2 CD4 T cells, but CD8 T cell immunity has been difficult to achieve. CD8 T cells can target conserved internal microbial components that are more difficult for pathogens to mutate. The unparalleled potency, cross-protective immunity, and immunological memory mediated by CD8 T cells underscores the urgency of developing CD8 T cell vaccines. To elicit CD8 T cell immunity, an adjuvant needs to induce MHC presentation of the antigens present in the vaccine formulation by dendritic cells (DC), potent antigen-presenting cells that prime naïve CD8 T cells. The MHC class I presentation of exogenous antigens such as vaccine components by DC takes place through cross-presentation. Understanding the mechanisms that regulate DC cross-presentation is thus critical for designing adjuvants that elicit strong CD8 T cell immunity. Our published and unpublished work has shown that Toll-like receptors (TLR), which detect microbes and alert the immune system, positively regulate DC cross-presentation. When studying the regulation of cross-presentation, it is important to consider the route of antigen internalization into DC. Depending on the size of the internalized antigen, internalization can be through phagocytosis (for particles that are >1µm in diameter) or endocytosis (<1µm in diameter). We found that the TLR-dependent regulation of cross-presentation is different for endocytic and phagocytic antigens. The common component that dictates the efficiency of cross-presentation to CD8 T cells is correct subcellular trafficking of MHC-I molecules to sites of internalized antigen. For phagocytic antigens, TLR signals control the traffic of MHC-I molecules from endosomal recycling compartments (ERC) in DC specifically to phagocytic antigens such as from bacteria or infected dying cells. For endocytic antigens, we found that a distinct TLR signaling machinery is involved, which controls endocytic antigen cross-presentation to CD8 T cells and traffics MHC-I molecules to endocytosed antigen from a cellular source other than the ERC. Using a variety of validated and complementary approaches, we will investigate TLR-regulated mechanisms of endocytic antigen cross-presentation and subcellular MHC-I trafficking to endocytosed antigens. We will elucidate how the distinct TLR mechanisms that regulate endocytic antigen cross-presentation impact protective circulating and tissue-resident CD8 T cell memory elicited by vaccination. We will use prototype subunit vaccines formulated with adjuvant/TLR ligand combinations that engage and boost DC endocytic antigen cross-presentation. Deciphering regulatory mechanisms of endocytic antigen cross-presentation will directly impact the design of effective CD8 T cell vaccines to clinically relevant old and new pathogens including those with pandemic potential.

NIH Research Projects · FY 2026 · 2023-06

Abnormal and potentially life-threatening blood clotting is seen in other severe infections, such as SARS- CoV-2, MERS, and H1N1 influenza. Platelets are produced by megakaryocytes. Enhanced megakaryopoiesis is found in severe COVID-19. Increased megakaryopoiesis is commonly found in autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus (SLE). Hypercoagulation also accounts for a significant percentage of mortality and morbidity in cancer patients. However, the regulation of megakaryocyte differentiation and function, however, remains poorly understood. The gut microbiome shapes tissue homeostasis beyond the gut through release of metabolites or microbial ligands. The link between the gut microbiome and megakaryocyte function is poorly understood. Our preliminary data demonstrate that fiber-fermenting gut bacteria that generate short-chain fatty acids (SCFAs) downregulate the ACE2 receptor, the entry receptor for SARS-CoV-2 infection and transmission; SCFA treatment led to reduced viral burdens in both mice and hamsters following infection with SARS-CoV-2 or a pseudovirus expressing the spike protein of SARS-CoV-2. In addition, our work uncovered a potentially novel function of SCFAs in limiting the coagulation response via the Sh2b3-Mpl axis to modulate megakaryopoiesis and platelet turnover. This proposal aims to define the role of the gut microbiome in the regulation of megakaryocyte maturation and function at steady state and in viral infection. This goal will be accomplished with the following 3 Specific Aims: 1. Define the role of the gut bacteria in steady-state maturation and function of megakaryocytes; 2. Interrogate the role of the gut microbiome in megakaryocyte maturation and function in viral infection; 3. Define the role of SCFAs in megakaryocyte maturation and function in homeostasis and viral infection. We will decipher the SCFA-Sh2b3-Mpl axis in the regulation of megakaryopoiesis and platelet turnover at steady state in viral infection, and explore the benefit of using SCFA-producing gut bacteria or pectin fiber to control megakaryocyte response in viral infection. This work will uncover a link between the gut microbiome and megakaryocyte response at steady state and in viral infection. Our findings will potentially identify specific gut commensal bacteria or metabolites that, either directly or indirectly, modulate the maturation of megakaryocytes and function; this knowledge can be leveraged in the development of therapeutics to treat uncontrolled megakaryopoiesis in various disease settings. These bacteria and metabolites of interest, including SCFAs as our data supported, could be additionally used as biomarkers to predict the risk of excessive megakaryopoiesis.

NIH Research Projects · FY 2026 · 2023-06

Project Summary/Abstract Prostate cancer arises as an androgen driven disease and therefore androgen receptor (AR) targeting therapies have been a major focus of prostate cancer treatment. Lineage lose mechanism cancer histologic transformation from an AR-positive prostate adenocarcinoma to an AR-negative small cell carcinoma that expresses neuroendocrine markers, often referred to as neuroendocrine prostate cancer (NEPC). NEPC is clinically aggressive and prognosis is poor. Therefore, effective treatment for NEPC patients remains an unmet clinical need. A thorough molecular understanding of NEPC progression is needed for the development of effective treatments for this lethal disease. Although NEPC tumors arise clonally from prostate adenocarcinoma and share genomic alterations, there is significant epigenetic deregulation during the transformation process. However, mechanistically, we still do not know how these epigenetic alterations arise and how best to leverage these alterations as a therapeutic opportunity. Our preliminary and published data from in vivo, in vitro and ex vivo models (NEPC-patient-derived organoids) suggest that the N-Myc transcriptome and cistrome is androgen-dependent and drives epithelial plasticity and the acquisition of clinically-relevant, NEPC molecular program and a reprogramming of the epigenome. Most recently, based on data from a new genetically engineered mouse model (GEMs), we found that N-Myc induction synergizes with RB1 loss to deregulate DNA methylation readers, writers (e.g. DNMT1 and DNMT3B) and erasures (e.g. TET1). Interestingly, we and others have shown that specific molecular or pharmacological interventions can revert NEPC phenotype to a luminal—more clinically manageable—adenocarcinoma phenotype. Our over-arching hypothesis, which is based on our published and preliminary data, is that specific molecular alterations (e.g. MYCN induction/RB1 loss) in prostate cancer cells drive lineage plasticity through epigenetic reprogramming (i.e., DNA methylation) as a mechanism of resistance to anti-AR therapy and this leads to transformation to NEPC. To address this hypothesis, we will employ patient-derived organoids and xenograft and novel genetically engineered mouse models to elucidate the role and specificity of DNMTs/TET1 in establishing the NEPC-related DNA methylation program (Aim 1), characterize the upstream regulation of DNMTs expression in the progression to NEPC (Aim 2) and to assess the therapeutic potential of DNMTs inhibition alone or in combination with AR targeted therapy to block the transition to or maintenance of NEPC (Aim 3). Successful completion of these Aims will provide unique insights into NEPC development, identify key and potential targetable mediators of lineage plasticity, and provide rationale for future clinical strategies to target the underlying epigenetic mechanisms that drive the transition from prostate adenocarcinoma to NEPC. plasticity, a process by which differentiated cells their identity and acquire alternative lineage programs, has recently been identified as an emerging of resistance to targeted therapies in several cancer types including prostate cancer prostate this plasticity can manifest as . For

NIH Research Projects · FY 2025 · 2023-05

The overarching mission of the Center for Social Capital (SoCa) is to bring together academic institutions, community-based organizations, and a unique group of stakeholders to improve cancer risk and outcomes. New York City (NYC) is a region of extremes with some of the richest and poorest living in close proximity. These extremes have profound impact on health with major disparities in life expectancy with cancer driving these differences as the number 1 and 2 cause of premature mortality and overall mortality, respectively.  NYC is one of the most heterogenous in the U.S., with approximately 2/3 non-White, 1/3 foreign-born, and at least 1/5 living below the Federal poverty line across four counties with persistent poverty. With such a heterogenous population, come challenges with respect to implementing cancer control programs to meet the needs of all. Using a community-engaged approach, we identified the top three structural determinants that exerted the greatest barriers to early detection and treatment of cancer in communities of persistent poverty were; 1) financial burden; 2) low health literacy, and 3) community/ social context (i.e., lack of social support/cohesion, stigma, discrimination).   NYC has a long history of migration and immigration, which has resulted in racially/ethnically segregated communities. Residential segregation concentrates disadvantages in minority communities by limiting social, economic, and educational opportunities and resources while concentrating poverty in these communities. However, for some segments of the population, especially new immigrants, residing in a highly segregated community has positive health effects through co-ethnic social support networks also known as “ethnic enclaves” and resource availability. Hence the constructs of social capital and social cohesion may play a significant role in mediating the relationship between residential segregation and negative health outcomes, such as cancer incidence and mortality. Our mission as a Center is to improve cancer risk and outcomes in persistent poverty census tracts throughout NYC by promoting multi-generational health.  Specifically, we aim to: Develop a rich interdisciplinary, and collaborative partnership with community organizations that will infuse stakeholder-engaged research methods and will build sustainable approaches for cancer control (Aim 1); Conduct two complementary projects that focus on novel interventions in persistent poverty communities aimed at two structural determinants of health—education and health care—in which the interventions aim to increase social capital among youth in school settings, and patient navigators in Federally Qualified Health Centers as a means to alleviate disparate cancer risks and outcomes (Aim 2); and leverage capacity across four geographically separate groups of persistent poverty census tracts in NYC to cultivate the next generation of investigators and develop sustainable Core infrastructure to achieve improved cancer health outcomes (Aim 3).

NIH Research Projects · FY 2025 · 2023-05

Project Summary/Abstract Richter’s syndrome (RS) is a critical complication of up to 10% of chronic lymphocytic leukemia (CLL) patients, which develops as an aggressive transformation into a diffuse large B cell histology, and is mostly refractory to existing therapies. RS pathogenesis remains largely unknown and cellular and mouse models for molecular studies are limited. To address this challenge, Dr. ten Hacken has developed novel human-genetics inspired mouse models through CRISPR-Cas9 multiplexed B-cell editing, recapitulating CLL transformation into RS. Already through her preliminary studies, Dr. ten Hacken demonstrated how selected mutational co-occurrences facilitate disease transformation, and are associated to distinct transcriptional changes and therapeutic vulnerabilities—work that is presently near completion. Dr. ten Hacken is now planning to introduce a new set of mutations in genes involved in epigenetic programming of B cells, which were identified as putative RS drivers in recent human genomic analyses. In Aim 1, Dr. ten Hacken will introduce epigenetic drivers in mice to assess the impact of the selected alterations (and their combinations) on CLL transformation. As part of this Aim, Dr. ten Hacken will also assess the transcriptional and genetic faithfulness of these models to human disease. In Aim 2, Dr. ten Hacken will functionally characterize the modeled gene mutations, while dissecting changes in the epigenetic landscape underlying transformation of CLL into RS. Epigenetic dependencies identified through these studies will be cross-compared with human RS datasets and validated in human primary samples with similar genetic make-ups. In Aim 3, Dr. ten Hacken will perform in vitro and in vivo preclinical testing of a panel of agents (comprehensive of chemotherapy and novel targeted agents) in order to design mutation (or co- mutation) specific treatment strategies. To carry out the proposed work, Dr. ten Hacken has enlisted collaborators who are experts in computational biology, systems immunology, mouse pathology, molecular pharmacology, biostatistics, functional genomics and epigenetics. Dr. ten Hacken has outlined a 3-year career development plan that will allow her to foster her personal professional development (including leadership, grant writing, negotiation and communication skills), and to gain additional scientific training in bioinformatics and biostatistics. Dr. ten Hacken’s independent research program will be focused on translational research in hematological malignancies, with the longer-term objective of undertaking clinical correlative research and functional genomic analyses of other lymphoid and myeloid malignancies. Through her proposed work, Dr. ten Hacken anticipates to contribute 2 high-impact manuscripts within the award term. She will present yearly at international conferences, and will be ready for her first R01 submission towards the end of Year 2. The proposed studies are expected to provide critical insight into the biology and natural history of Richter’s syndrome, and the mouse models Dr. ten Hacken is developing will represent useful tools to dissect pathogenic mechanisms and test novel treatment strategies for this largely incurable malignancy.

NIH Research Projects · FY 2025 · 2023-05

Abstract Addressing Surgical Disparities at the Root: Improving Diversity in the Surgical Workforce The recent COVID-19 pandemic, racially motivated murders, and subsequent protests underscore what we already know--that racial disparities in medicine run far deeper than patient outcomes alone and must be addressed at all levels. Racial and gender disparities in surgical outcomes and satisfaction are well documented. From a pipeline perspective, surgery struggles to maintain and promote underrepresented minority (UIM) and women residents and faculty. Efforts to improve diversity in the workforce overtime have not kept pace with the increased diversity in our patient populations. There is evidence that improving diversity in the surgical workforce can improve the quality and outcomes of care for UIM and women patients. The proposed study involves a team of interdisciplinary investigators with complimentary expertise and a strong record of research in the topic area collaborating with multiple stakeholder societies. Our objective is to reduce disparities in surgical care using a novel, transdisciplinary, multi-institutional deviance approach to characterize disparities in the surgical workforce, set best practice guidelines, and develop a pilot intervention. Our central hypothesis is that by using deviance methodology we can identify best practices in retention and promotion of women and minority faculty and trainees in surgery that can be used to help increase diversity in the work force and ultimately patient quality of care. We will perform a secondary data analysis to measure diversity among surgical faculty across the U.S.; identify programs with the best and worst records of promotion and retention of UIM and women faculty; and document the range of promotion and retention among these groups at academic medical centers to identify predictors of successful diversity efforts Using AAMC data combined with ABS data and qualifying as well as certifying examination data, we will describe the sociodemographic profile of surgical trainees and current attrition rates for surgical residents in the U.S., identifying those programs with the best and worst records of graduating residents. We will utilize data on cultural competency and bias assessment surveys collected from our target programs as well as focus groups with the National Medical Association, the Association of Women Surgeons and the Historically Black Medical Colleges and Universities and in-depth, qualitative interviews with program administrators, UIM and women trainees, faculty, and department and division heads at programs that have been successful - or struggled at - retaining and promoting UIM and women faculty to study best and worst practices and organizational characteristics. Finally, we will take predictors from our quantitative analysis, themes from our qualitative analysis, and coordinate a Delphi panel of academic leaders and patient advocacy groups to create a set of best practice guidelines and develop a pilot study to test at poorly performing programs. By defining best practices for retention and promotion of residents and faculty, we can develop best practices and test these to help improve diversity, equity, and inclusion in the academic surgical workforce.

NIH Research Projects · FY 2025 · 2023-05

PROJECT SUMMARY/ABSTRACT The E2F transcriptional program, which controls cell cycle commitment and progression in proliferating cells, is upregulated following DNA damage in neurons. Neuronal DNA damage and cell cycle dysregulation are features of neurodegeneration that have also been associated with psychiatric diseases. Although the function of E2F during proliferation has been extensively studied, the role of E2F in nonproliferating cells, such as in neurons, has received less attention. Emerging evidence indicates that E2F plays a role in DNA damage repair, independent of its role in cell cycle entry. This role may be especially important in neurons, which are uniquely vulnerable to DNA damage. Although E2F induction has been linked to resolution of DNA damage in bulk-cell preparations of neurons, such bulk analyses fail to account for potentially confounding heterogeneity within samples. Single cell analysis is necessary to examine the relationship between E2F activity and DNA damage and to link E2F dynamics to functional outcomes in the same cells. The Meyer Lab specializes in single-cell analysis of high-throughput microscopy data, and the lab has recently developed a fluorescent biosensor of E2F activity for this purpose. Quantitative microscopy using live- and fixed-cell readouts of E2F activity and DNA damage will allow signaling history to be mapped to cell fate outcomes at single-cell resolution in thousands of cells. My hypothesis is that in postmitotic neurons, E2F is reversibly activated to drive DNA repair without DNA replication following sublethal DNA damage; however, if DNA damage activates E2F beyond the threshold for S-phase entry, E2F induces DNA replication and apoptosis. By characterizing how the E2F program is regulated in neurons and how it contributes to DNA damage repair, this study will support the identification of potential treatment targets for preserving genetic integrity in brain disorders characterized by genotoxic stress. This research project represents an important component of my training for a career as an independent investigator, employing high-throughput single-cell methods to study the biology of complex diseases. My long- term goal is to become a physician-scientist, practicing as a psychiatrist while also running a basic science lab in an academic hospital. The plan outlined in this proposal along with the mentorship of my sponsor, thesis committee, and the leadership of the Tri-I MD- PhD program, will help me achieve these career aspirations.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY (ABSTRACT) The use of cannabis for recreation and medicinal purposes is disproportionately high among people living with HIV (PLWH) and nearly half of cannabis using PLWH are estimated to be at risk for cannabis use disorder. Yet, whether cannabis is therapeutic or detrimental on the central nervous system (CNS) of PLWH remains controversial, highlighting the need for well-controlled studies generating reproducible data from specific cannabinoids, brain regions, and CNS cell types. Our research has shown that cell-type specific epigenetic patterns relate to HIV-associated cognitive impairment in PLWH, more frequent or recent cannabis use may reduce myeloid inflammation and impact brain structure in PLWH, and our recent single cell studies of the CNS in PLWH revealed distinct CSF microglia-like cells expressing CD204 in PLWH and ongoing HIV viral transcription in cerebrospinal fluid cells despite ART. Prior research has shown neurogenic brain regions including the subventricular zone of the lateral ventricles and hippocampus are of high relevance to persistent HIV infection and cannabinoid exposures. However, critical gaps in understanding neurogenic brain regions at single cell level in the setting of HIV and cannabinoid exposures remain. We are leveraging brain tissues from an established oral dosing model of cannabidiol and THC in a nonhuman primate (NHP) model of HIV, application of new single cell technologies permitting the simultaneous profiling of gene expression and open chromatin from the same cell (10X Genomics Multiome: RNA+ATAC) in brain tissues, a new single cell assay capable of measuring multiple histone modifications, and pharmacological profiling of current ART regimens and cannabinoid levels in brain tissues. Our central hypothesis is a therapeutic role of cannabinoids in ameliorating HIV neuropathogenesis in the CNS by enhancing the proliferation and survival of neural progenitor cells and immature neurons and reducing glial inflammation. To identify cell types, epigenetic cell states, and gene pathways relevant to neuropathogenesis, viral persistence, and cannabinoid exposures, we are harnessing 180 brain tissue samples from an established oral administration of either cannabidiol (CBD) or Δ9- tetrahydrocannabinol (THC) in NHP and single cell assays (10X Genomics single nucleus multiome and a new single cell assay developed at the NYGC capable of measuring the genome-wide presence of multiple histone modifications and protein-DNA binding sites). Moreover, accompanying single cell data will be generated from conserved neurogenic brain regions of human postmortem brain tissues from 40 donors based on HIV status (+/-) and cannabis exposure (+/-). We will also explore the frequency of single cells in neurogenic regions of the brain that are infected and impacted by cannabinoids by harnessing a bioinformatics pipeline that detects both viral transcripts and transposase accessible provirus. This project will generate comprehensive single cell datasets in NHP and humans to improve our understanding of the cross talk between HIV and cannabinoids in neurogenic regions of the brain and has high programmatic priority to goals of the SCORCH program expansion.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY/ABSTRACT Malignancy is the leading cause of mortality amongst people living with HIV (PLWH). Of these malignancies, Non-Hodgkin lymphoma (NHL) is the most common hematological neoplasm affecting PLWH. Despite the introduction of highly active antiretroviral therapy (ART), PLWH are 10-20 times more likely to develop NHL than those without HIV. Epstein Barr Virus (EBV) is an oncogenic virus that is associated with the lymphomas most commonly observed in PLWH. Active HIV infection can lead to Acquired Immunodeficiency Syndrome (AIDS) in untreated people, and it is known that AIDS promotes the development of EBV+ lymphomas, termed AIDS- defining cancers (ADCs), the effect of host factors, including endogenous retroviruses on lymphomagenesis in immunocompetent PLWH treated with ART, remains to be explored. Work in the Cesarman and Nixon labs has shown that EBV and HIV each hijack host cell signaling pathways upon infection to induce the expression of endogenous retroelements, including as human endogenous retroviruses (HERV). To determine the contributions of these viruses on lymphomagenesis, we will generate data from in vitro lymphoma models to test the effect of EBV and HIV proteins on the expression of endogenous retroviral oncoproteins. Additionally, we will perform bulk and single-cell sequencing of Diffuse Large B Cell Lymphoma (DLBCL) from PLWH to determine the changes in cell populations of the tumor microenvironment (TME). We hypothesize that there will be unique populations of EBV infected B cells with pre-malignant transcriptomic signatures that correlate with dysregulated HERV expression, including expression of the HERVK(HML2) Np9 oncogene. In Aim 1, we will determine the impact of EBV latency III LMP2A and extracellular HIV p17 variants on the expression of the Np9 oncogene. We will perform stepwise molecular and cellular studies to determine the translational impact of exogenous viral infection on the expression of this endogenous retroviral oncogene. We will additionally perform multi-omic analysis to identify viral and host factors relevant to the transcriptional machinery of virus-mediated Np9 oncogene expression. In Aim 2, we will use our novel established single cell pipeline to determine locus-specific endogenous retroviral transcription of DLBCL samples from PLWH. Preliminary data from our work in the Nixon lab strongly suggests that we will be able to use the pipeline that we have developed to identify and quantify these HERV transcripts from RNA-sequencing data. We will assess the relative cell populations of samples that are EBV+ to those that are EBV- and determine the differences in host gene, retroviral and viral transcripts. Additionally, we intend to validate these single cell findings with bulk RNA-sequencing from a larger cohort of EBV+/- fresh frozen tissue of DLBCL from PLWH.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY G protein-coupled receptors (GPCRs) are finely tuned signaling molecules that are central to diverse neurophysiological processes and serve as major drug targets for neurological and psychiatric disorders. A major form of GPCR regulation occurs through the action of multifunctional β-arrestins (β-arrs) which bind to activated receptors to drive functional desensitization, control receptor trafficking, and initiate G protein-independent signaling cascades. Despite progress, our mechanistic understanding of GPCR/β-arr coupling is limited and based on a small number of prototypical family A GPCRs. It is critical to improve our understanding of GPCR/β- arr coupling by unraveling the biophysical basis and biological consequences of variability between different GPCR subfamilies and subtypes, as well as between different pharmacological ligands. The metabotropic glutamate receptors (mGluRs) form an eight-member family of family C GPCRs with a unique architecture consisting of large extracellular, ligand binding domains that mediate constitutive dimerization. Due to their roles in synaptic neuromodulation, mGluRs have emerged as drug targets for neurodevelopmental, neurodegenerative, and psychiatric disoders, as well as cancers. However, it has been difficult to harness mGluRs therapeutically because of a lack of understanding of their basic signaling and regulatory properties. Deciphering the mechanisms of mGluR regulation is particularly challenging since this subfamily is dramatically expanded by heterodimerization and is targeted by a broad panel of orthosteric and allosteric ligands with distinct properties. Until recently, mGluR/β-arr coupling has been poorly characterized, but we recently found that a subset of mGluRs is capable of robust β-arr coupling while others are highly resistant, providing another dimension of molecular diversity to this GPCR family. We will build on our recent findings with a battery of structural, biophysical, and cell-based measurements, to understand the underlying mechanisms and physiological consequences of mGluR/β-arr coupling. In aim 1, we will develop and harness a single molecule imaging assay to define the determinants and stoichiometry of mGluR/β-arr complex formation and then use electron microscopy (negative stain, cryo-EM) to solve high resolution structural snapshots of mGluR/β-arr complexes. In aim 2, we will use in vitro and live cell biophysical assays to define the basis of mGluR C-terminal domain and transmembrane core coupling to β-arrs across subtypes and probe the effects of distinct ligand types and heterodimerization on mGluR/β-arr coupling. In aim 3, we will investigate the trafficking and functional consequences of mGluR/β-arr coupling using high- resolution optical and proximity proteomics techniques in both cell lines and cortical neurons, with a focus on presynaptic signaling and trafficking. Together, this project will provide a full picture of the regulation of mGluRs by β-arrs, providing a deeper understanding of this critical receptor family and gaining broader insight into GPCR/β-arr coupling.

NIH Research Projects · FY 2025 · 2023-05

ABSTRACT (30 lines) Neonatal seizures occur once per 1000 live births and are associated with subsequent development of epilepsy, cerebral palsy, and intellectual disability. Most (85%) are acute symptomatic seizures (i.e., due to an acute injury). Roughly one-quarter of survivors will subsequently develop epilepsy (i.e., chronic unprovoked seizures) after a months-to-years delay. We propose to address three questions in the management of acute symptomatic neonatal seizures. Our central hypothesis is that optimizing the clinical management of this vulnerable population will reduce the development of epilepsy. First, what is the best second-line anti-seizure medication (ASM) for acute neonatal seizures? Our preliminary data suggest the two most used ASMs, levetiracetam (LEV) and phenytoin/fosphenytoin (PHT), have roughly equivalent effectiveness. We will conduct in-depth chart abstraction (780 neonates at 18 centers) and apply modern statistical techniques to compare their effectiveness for short-term (seizure cessation) and long-term (epilepsy by age 2) outcomes. Demonstration of equivalence would favor the use of LEV, given its tolerability and safety profile. Second, if neonates with seizures (age 0-28 days) continue ASM therapy in infancy (age 1-12 months), does ASM selection alter the risk to develop infantile spasms syndrome (ISS)? Our preliminary analyses found an association of oxcarbazepine (OXC) use with ISS, in alignment with recent work demonstrating that voltage- gated sodium channel blockade can induce epileptic spasms in mice (using tetrodotoxin) and may increase the risk for ISS in human infants (case reports and single-center data). This finding needs confirmation before recommending avoidance of OXC in infants, particularly given the effectiveness of OXC and other voltage- gated sodium channel blockers for some monogenetic epilepsies that begin in the first year of life. We will confirm this finding in 587 infants. Third, after discharge from the neonatal intensive care unit, which infants will develop epilepsy? We propose to validate our published epilepsy prediction rule in 1800 neonates for subsequent use as enrollment criteria for epilepsy prevention trials. We will conduct this work using the Pediatric Epilepsy Learning Health System (PELHS), a consortium of US academic pediatric epilepsy programs that work collaboratively to improve outcomes for children with epilepsy through cycles of electronic health record (EHR) collection and analysis. This rigorous set of studies will generate much-needed evidence to support treatment decisions in a vulnerable population, in alignment with the 2020 Epilepsy Research Benchmarks, including II-3 (biomarkers), II-5 (interventions to prevent epileptogenesis), and III-4 (predict, prevent seizures). The proposal is aligned with a long-standing goal of clinical neuroscience research: to predict and prevent epilepsy in high-risk individuals. The results will directly inform clinical care for neonates with acute symptomatic seizures as well as lay the foundation for epilepsy prevention trials.

NIH Research Projects · FY 2026 · 2023-05

Project Summary/Abstract COVID-19 has proven to be a metabolic disease resulting in adverse outcomes disproportionally afflicting individuals with diabetes or obesity. Patients infected with SARS-CoV-2 and hyperglycemia suffer from longer hospital stays, increased need for mechanical ventilation and mortality compared to those without hyperglycemia. We found that insulin resistance rather than beta cell failure is the predominant cause of hyperglycemia in acute COVID-19. The insulin sensitizing hormone adiponectin is diminished in the circulation of COVID-19 patients compared to controls. Furthermore, we demonstrate that SARS-CoV-2 can directly infect adipocytes. Importantly, we find replicating virus in adipose tissues of both autopsy samples from COVID-19 patients and in mouse and hamster experimental models of SARS-CoV-2 infection. Together these data suggest that SARS-CoV-2 triggers adipose tissue dysfunction to drive insulin resistance and adverse outcomes in acute COVID-19. In this proposal, we seek to follow up on these studies and assess the mechanisms driving adipose tissue dysfunction in acute and recovered models of COVID-19. We will pursue the following specific aims: 1. Assess the impact of acute SARS-CoV-2 infection on glucose homeostasis in obese and non-obese mice. 2. Map the spatiomolecular interactions and dissect the molecular mechanisms of SARS-CoV-2 infection in adipose. 3. Determine the long-term glycometabolic consequences of SARS-CoV-2 infection. The overall goal of these studies is to assess how COVID-19 can drive adipose tissue dysfunction and hyperglycemia and will shed light on novel targets to combat metabolic complications induced by COVID-19.

NIH Research Projects · FY 2026 · 2023-05

ABSTRACT End-stage liver disease is associated with morbidity and mortality often requiring a liver transplant. Dysfunction and capillarization of liver sinusoidal endothelial cells (LSECs) could contribute to impaired hepatic repair and cirrhosis. Thus, uncovering the mechanisms by which LSECs acquire their specialized functions to support hepatic repair could enable the development of therapies for scar-free liver repair. We have shown that activation of Id1 and Cxcr7 in LSECs induce angiocrine factors, that guide hepatic regeneration without fibrosis. However, the mechanism by which LSECs acquire their pro-hepatic functions is unknown. We show that while transcription factors (TFs) Fli1 and Erg dictate the vascular fate and homeostasis of LSECs (Gomez- Salinero JM, et al, Nature Cardiovascular Research, 2022), induction of TF c-Maf specifies the phenotype and regenerative functions of LSECs (Gomez-Salinero JM, et al, Cell Stem Cell, 2022). Induction of c-Maf in generic human endothelial cells (ECs) switches on liver-specific LSEC signatures and angiocrine factors supporting hepatocyte functionality. In mice in which c-Maf is deleted in adult ECs, recovery from CCl4 results in fibrosis and LSECs regression to arterial cell fate. Thus, we hypothesize that maintenance of LSEC vascular cell fate requires constitutive Fli1 or Erg expression, sustaining LSECs homeostatic functions. Induction of c- Maf enforces specialized pro-regenerative functions and interactions of LSECs with hepatocytes, Kupffer and stellate cells that prevents stress-induced LSEC arterialization promoting hepatic repair without fibrosis. To this end, we have reprogrammed human generic ECs to an adaptable tubulogenic state. These Reset-Vascular Endothelial Cells (R-VECs) self-assemble into a 3D vascular network, transporting human blood and arborizing hepatocytes (Palikuqi B et al. Nature, 2020). The vascularized hepatic aggregates with stellate and Kupffer cells within scalable and perfusable microfluidic devices establish a human Hepatic-on-VascularNet platform, enabling study of physiologically adaptive cross-talk between human hepatocytes and LSECs. This hypothesis will be tested by performing these Aims: Aim 1: Define the mechanism by which hierarchical Fli1 and Erg expression through c-Maf induction sustains specialization of LSECs at steady state, during hepatic regeneration and after CCl4 induced liver injury. AIM 2: Uncover the contribution of c-Maf expressed in the Kupffer cells that by enforcing and sustaining LSEC vascular attributes regulate hepatic homeostasis during liver regeneration and CCl4 induced liver injury. AIM 3: Employ the human Hepatic-On-VascularNet platform to uncover the mechanism by which c-Maf is induced functionally in human liver ECs to form specialized LSECs. Determine whether human c-Maf induced LSECs (iLSECs) can restore hepatic regeneration. Specifically, the role of infusing human iLSECs in restoring hepatic repair post-acetaminophen (APAP) injury without provoking fibrosis will be assessed. These studies will uncover the molecular determinants of human LSECs and allow strategies to employ iLSECs to sustain its pro-regenerative and anti-fibrotic attributes for liver repair.

NIH Research Projects · FY 2026 · 2023-04

SUMMARY (30 lines max) The vast majority of cancer mortality is due to metastases. A major unmet need in oncology is how to predict and prevent metastatic progression. To address this need, it is imperative we advance our understanding of how tumor cells acquire metastatic capability. Epithelial-to-mesenchymal transition (EMT) is a normal physiologic process in wound healing and development by which polarized epithelial cells undergo biochemical, metabolic, and epigenetic changes and convert to a mesenchymal phenotype, characterized by enhanced migratory and invasive capacity. Cancers co-opt this reprogramming to acquire metastatic ability. Understanding how tumor cells undergo EMT is critical to unraveling how cancer becomes metastatic. Historically, studies have primarily focused on genetic mutations or gene expression changes that trigger EMT and metastasis. But recent work has suggested that non-genetic factors such as metabolites can promote cancer progression, suggesting unexplored areas of cancer biology. To gain a deeper understanding of EMT and capture the sequential changes necessary for cancer to become metastatic, we performed proteomics, metabolomics and transcriptomics at multiple time points as cells underwent EMT. Our multifaceted approach revealed rich new insights. We found that propionate metabolism was dysregulated during EMT. Indeed, we observed increased propionyl-CoA and methylmalonic acid (MMA), a dicarboxylic acid by-product of propionate metabolism, during early EMT. To test the functional effect of elevated MMA, we treated lung and breast cancer cells with MMA. Strikingly, we found that MMA triggered EMT and enhanced migratory and invasive capacity. Prior to our discovery, little was known about propionate metabolism and MMA other than propionate metabolism is dysregulated in rare inborn errors of metabolism collectively referred to as “methylmalonic acidemias.” Some recent studies have also shown that MMA increases in serum with age and is linked to age-related, all-cause mortality. To our knowledge, our findings are the first to demonstrate that propionate metabolism plays a role in EMT and cancer cell progression. This led us to study how propionate metabolism is altered during EMT. Our long-term goals are to define how this pathway is regulated by multiple inducers of metastasis and at multiple points in the pathway, and to define how MMA produced by cancer cells can also influence the tumor microenvironment. Our proposed studies will address a critical need for a greater understanding of the molecular basis of metastasis. Our expectations are that successful completion of the proposed work will impact cancer biology by shifting the paradigm and highlighting critical non-genetic factors that drive cancer progression. Consequently, our work will identify new biomarkers and therapeutic targets that can be exploited to prevent and inhibit metastases.

NIH Research Projects · FY 2026 · 2023-04

Project Summary/Abstract Humans have approximately 40 trillion cells and ~ 0.1% of them divide every day for tissue maintenance, wound repair, and pathogen defense. Such cells include stem, progenitor and differentiated cells that typically spend most of their time in a non-proliferative state (quiescence, G0) but when stimulated can undergo one or more rounds of cell division (proliferation). This fundamental decision between quiescence and proliferation is made in the G1 phase of the cell cycle. Importantly, along with making the decision to proliferate, cells must load sufficient origins of replication (origin licensing) during G1 to replicate their DNA without error during S phase. How cells make the decision to enter quiescence is not fully understood. Critical questions of how E2F and APC/CCdh1 activities are temporally integrated to coordinate the regulation between quiescence and proliferation and the licensing of origins of replication also remain unanswered. The Meyer lab specializes in the use of single-cell analysis of live-cell imaging data. By utilizing recently developed fluorescent reporters for key cell cycle proteins, the lab can answer various biological questions with very high resolution. This project will employ the use of fluorescent activity reporters for CDK1/2, APC/CCdh1, CRL4Cdt2 and E2F to understand signaling dynamics of S phase entry, G0 entry and origin licensing in G1. The goal of this proposal is to compare the dynamic synergy between E2F activity and APC/CCdh1 activity in quiescent and cycling single cells and identify how cells maintain an origin licensing period. The objective of this proposal is to show the existence of two S phase entry signaling pathways controlled by synergy between E2F and APC/CCdh1 activity in single cells and how this synergy controls origin licensing and quiescence entry. My central hypothesis is that the temporal interplay between E2F and APC/CCdh1 activities is the primary regulator of the decision between proliferation and quiescence and, ensures proper origin licensing to prevent DNA damage in S phase. I plan to test this hypothesis with the following specific aims: 1. Understand the interplay between E2F and APC/CCdh1 activities in regulating the proliferation-quiescence decision. 2. Determine the function of E2F and APC/CCdh1 activity timing in origin licensing and DNA replication fidelity. The successful completion of this project is expected to show the multifaceted roles of APC/CCdh1 and E2F synergy. This project will resolve a historic enigma of how cells faithfully coordinate licensing and DNA replication by utilizing the remarkable natural heterogeneity that exists in cell populations. Further, the completion of this project will provide crucial insights into how cancer cells may evade chemotherapies that target DNA replication by entering a dormant quiescent state and how healthy cells are able to maintain quiescent populations for tissue repair and growth.

NIH Research Projects · FY 2025 · 2023-04

Project Summary Currently, most nanotechnology cancer therapies focus on the treatment of primary tumors, but it is important to leverage the potential of nanomedicine to combat cancer spread at each stage of the metastatic process. Lung metastasis is a highly aggressive, complex, and heterogeneous disease. There is no effective treatment for metastatic lung tumors and chemotherapy is the only option to prolong patients’ clinical prognosis. Alternative strategies, including targeted therapy and immunotherapy have been proposed, but they failed to successfully treat metastatic lesions. There is an urgent need to accelerate progress toward curing lung metastases and reduce patients’ mortality. Our goal is to develop a new therapeutic approach that carries more drugs to the metastatic lung tumors and retains on-site to release a broad-spectrum antitumor medication. In this project, we propose to use peptide-based nanofiber (pNFP6) with preferential lung-targeting properties to overcome the barrier of selective drug delivery to metastases. The pNFP6 is innovative as multiple nanofibers can rearrange into a large interfibril network to prolong the local retention and offer a long-term treatment. The nanofiber technology will be combined with ionizing radiation therapy to enhance the drug post-delivery antitumor efficacy. Our central hypothesis is that the combinatorial therapy will cooperatively and synergistically inhibit the disease progression leading to an effective treatment of lung metastases. For proof-of-principle studies, we will use pNFP6 to carry and deliver doxorobucin (Dox), a standard cytotoxic agent and radiosensitizer. The nanofibers will favor the drug accumulation and retention on-site while radiotherapy will promote the overall anticancer effect through direct tumor cell killing and radiation-mediated immunogenicity. The spatiotemporal-controlled drug release will be essential to ensure the therapeutic success. To establish the potential of this antimetastatic multiplexed approach, two specific aims will be pursued: (1) evaluate the local drug release and its impact on the therapeutic efficacy; and (2) define the therapeutic and survival benefit of Dox-pNFP6 when combined with radiation therapy. To achieve Aim 1, we will synthesize a panel of Dox-loaded pNFP6 analogues using different cleavable linkers sensitive to tumor microenvironment stimuli to release the drug. We will study the in vivo drug delivery, release, and tumoral uptake using Light Sheet Fluorescence Microscopy and MALDI-imaging. and identify the optimal release mechanisms in response to metastatic lung tumors. To complete Aim 2, we will assess the therapeutic efficacy (tumor inhibition and survival benefit) and toxicity profile of Dox-pNFP6 combined with radiation therapy in several animal models bearing metastatic lung tumors. The treatment outcomes will be compared to free Dox and Doxil, the FDA-approved liposomal formulation of Dox. We will also investigate the molecular and immune pathways activated by this new therapeutic strategy to better understand the mechanisms responsible for the enhanced anticancer activity. Successful completion of this project will provide an effective therapeutic solution with clinical impacts on the treatment and management of lung metastases.

NIH Research Projects · FY 2025 · 2023-04

PROJECT SUMMARY Medical care in the United States is increasingly delivered in health systems—which the Agency for Healthcare Research and Quality has defined as organizations with at least one hospital and one physician group providing comprehensive care that operate through common ownership or joint management. In the past decade, health system acquisition of physician practices has increased markedly; more than half of U.S. physicians are now affiliated with one of 637 health systems. Prior research has found that vertical integration can lead to higher commercial health care prices, but has uncertain effects on quality, spending, and utilization for Medicare beneficiaries. Little is known about how health system expansion influences care for medically complex Medicare patients, a distinct population that has high health needs and frequent interactions with the medical system. The long-term goal of the proposed research is to determine whether the growth of health systems improves care for medically complex patients and what health system strategies and characteristics contribute to their success or failure in doing so. The overall objective of this project is to provide health care leaders and policymakers with an understanding of whether and how health systems change the delivery and quality of care for medically complex Medicare beneficiaries, with a focus on patients in acquired primary care practices. The central hypotheses are that, on average, health system acquisition of practices improves care for medically complex patients, but that improvement varies across health systems. These hypotheses will be tested by pursuing three specific aims: (1) determine the impact of health system acquisition of primary care practices on utilization, spending, and quality for medically complex patients (overall and for three subpopulations); (2) examine variation by health system in spending and quality changes for medically complex patients at acquired practices, and identify characteristics of systems that most improve their care; and (3) understand how the experiences of physicians and the efforts of health system leaders differ at systems that improve care for medically complex patients at acquired practices from those that do not. This project will use Medicare claims data and qualitative data collected by the researchers. Methodologically, it will employ rigorous quasi-experimental approaches including difference-in-differences analyses, as well as in- depth, semi-structured interviews with health system leaders and practicing physicians. The proposed research is innovative, in the applicants' opinion, because it fills critical gaps about whether, which, and how health system acquisitions improve care for medically complex patients. It will also build on AHRQ's prior investment in developing a Compendium of U.S. Health Systems, and make an expanded database publicly available for other researchers to use. The project is significant because it will provide rigorous evidence on the impact of vertical integration across a range of quality, utilization, and spending outcomes and will identify strategies that may improve the design of health systems to create a higher value medical system.

NIH Research Projects · FY 2025 · 2023-04

SUMMARY Clonal outgrowths are observed across a wide range of normal human tissues. Clones harbor somatic mutations in known cancer and other driver genes, and show evidence of positive selection. Nevertheless, how these driver mutations alter the cellular states of cells to allow clones to outcompete wildtype counterparts remains poorly understood. To date, efforts to chart clonal outgrowths in normal tissues have been largely limited to genotyping. This is due to the fact that clones often affect a minority of cells in a sample, without distinguishing cell surface markers or morphological features. To address this challenge, we developed an array of multi-omic single-cell technologies that are capable of capturing multiple layers of information (e.g., genotypes, transcriptomes, methylomes, protein expression) from the same single cells. Moreover, we addressed the specific challenge of genotyping in scRNA-seq in single cells at high throughput by developing genotyping of transcriptomes. Importantly, this technology turns the admixture of mutant and wildtype cell from a limitation to an advantage, enabling the direct comparison of mutant (“winner”) and wildtype (“loser”) cells within the same individual. Capitalizing on our experience with single-cell technology development, we aim to extend the multi-omics single-cell GoT (Genotyping of Targeted loci) toolkit to allow to interrogate how somatic mutations lead to clonal growth advantage. First, we will develop and enhance our targeted single-cell genotyping in the context of chromatin accessibility (GoT-ChA). This technology critically performs genotyping from DNA directly, obviating limiting dependencies on mutated loci gene expression. Thus, it can be applied to extracted nuclei, critical for the SMaHT initiative. We will build on GoT-ChA using nanobody tethered transposases to jointly profile somatic mutations and histone modifications in single nuclei (GoT-EpiM). To capture transcriptional changes together with somatic mutation genotyping and chromatin accessibility, we will further use transposition of mRNA:cDNA hybrid in GoT-ChA-RNA. Finally, we will leverage recent advances that use antibodies tagged with oligonucleotides to capture mutated loci, chromatin and intra-nuclear proteins such as transcription factors (GoT-ChA-Pro). In aim 2, we will collaborate with genomic characterization centers to apply these technologies to primary human samples to define how clonal mutations in normal tissues alter chromatin, histone modifications, transcriptomes and protein abundance profiles to yield clonal outgrowth. Our overarching goal is to invoke multi-omic comparisons at the single-cell level between wildtype and mutant cells to comprehensively identify the underpinnings of fitness advantage in clonal outgrowth. The proposed comprehensive GoT toolkit will enable to link, at high throughout single-cell genotypes with transcriptional, protein, and epigenetic, with important implication in the study of clonal mosaicism as a harbinger of cancer, as well as other human health outcomes.

NIH Research Projects · FY 2025 · 2023-04

PROJECT SUMMARY Inflammatory bowel disease (IBD) arises when gut microbes elicit inappropriate immune responses in the intestine, leading to chronic inflammation and tissue damage. As cases rise worldwide, it is important to identify the pathways that restrain these responses and promote mucosal healing. Critically, group 3 innate lymphoid cells (ILC3s) are a recently identified cell type which is enriched in the healthy intestine and becomes dysregulated during IBD, colon cancer and other chronic inflammatory disorders. Further, mouse models have revealed fundamental roles for ILC3s in controlling immune tolerance, immunity, and inflammation in the intestine. Despite these advances, the mechanisms by which ILC3s sense and respond to the intricate milieu of signals present in the intestinal mucosa to coordinate intestinal health, impact inflammation, or promote tissue healing remains unclear and represents an important area of future investigation. In new preliminary data, I have unexpectedly determined that ILC3s are uniquely enriched in expression of a cell surface heparan sulfate proteoglycan receptor, syndecan-4, a pathway linked to wound healing, cell migration/adhesion, and control of growth factor signaling. Further, I identified a cellular and molecular mechanism regulating syndecan-4 on ILC3s and discovered that syndecan-4 becomes dysregulated in experimental mouse models of intestinal inflammation or human IBD. Finally, mice lacking ILC3-specific syndecan-4 show greater tissue damage and susceptibility to intestinal inflammation. This provokes a novel hypothesis that syndecan-4 is a critical pathway impacting ILC3 biology, host-microbiota homeostasis, and tissue repair during intestinal health and inflammation. The fundamental focus of this research proposal is to test this hypothesis and define the regulation and functional significance of ILC3-specific syndecan-4 by employing novel mouse models, innovative technical approaches, and translational studies. It is expected that the results from the two aims of this proposal will reveal crucial signaling mechanisms for inflammatory responses in the intestine that may have therapeutic potential for the treatment of IBD.