University Of Pennsylvania

NIH Research Projects · FY 2025 · 2022-09

Alzheimer's Disease (AD) is the most common cause of dementia, affecting nearly 6 million Americans and continuing to increase in prevalence with the aging population. AD causes progressive memory loss and cognitive impairment that can eventually lead to the total inability to perform daily functions. The brain is the most cholesterol-rich and lipid-diverse organ in the body and depends on tight regulation of lipid metabolism and transport to maintain proper neural signaling transduction and cognitive function. Several large GWAS have associated the ABCA7 gene locus with AD, with variants that are predicted to have reduced function associated with increased AD risk. ABCA7 is a member of the ATP-binding cassette transporter subfamily A (ABCA), with well described functions as membrane phospholipid (PL) translocases and mediators of cholesterol and lipid efflux from cells. The most characterized ABCA family member is ABCA1, which has been extensively characterized regarding its role in cellular phosphatidylcholine (PC) and cholesterol efflux to extracellular acceptors apolipoproteins apoA1 and apoE. ABCA7 is highly homologous to ABCA1, suggesting that ABCA? mediates transport of certain lipids from inside to outside the cell in response to acceptors like apoA1 and apoE. However, the specific lipids transported by ABCA7 and the mechanisms by which it modulates AD risk have not been established. GWAS of plasma metabolites identified a significant association of the ABCA7 gene locus with certain ceramide species. Lipidomic profiling of Abca7-/- mouse brain revealed dysregulation in several lipid classes, including ceramides, which coincided with impaired cognitive functions, suggesting a functional role of these lipids in memory and cognition. While ABCA1 is also expressed in the brain, the cellular expression of ABCA1 and ABCA 7 is different, and genetic variation at ABCA1 does not carry the same risk of AD at the population level. Therefore, it is hypothesized in this proposal, that ABCA7, in contrast to ABCA1, translocates and promotes cellular efflux of specific lipid species in specific brain cell types in an AD-protective manner, and that reduced ABCA? activity leads to cellular accumulation of toxic lipid species, contributing to neural cell dysfunction and AD. To test this hypothesis, the following research aims are proposed: 1) Determine the specific lipid species affected by deletion of ABCA7 (compared with ABCA1) in three different iPSC-derived brain cells (neurons, microglia, and astrocytes), and establish the effect of ABCA 7 deletion on AD-relevant functions of iPSC­ derived brain cells; 2) Identify ABCA7 protein domains that differentiate ABCA7 from ABCA1 with regard to specificity of lipid translocase activities; 3) Characterize the functional effects of naturally­occurring ABCA7 coding variants that are significantly associated with AD.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract: Castleman disease (CD) describes a group of rare and poorly understood hematologic disorders that share characteristic histopathologic alterations, lymphadenopathy, and systemic inflammation but vary in etiology, symptomatology, treatments, and outcomes. Approximately 2,000 individuals of all ages are diagnosed with CD each year in the US. Unicentric CD (UCD) involves one region of enlarged lymph nodes and typically milder inflammatory symptoms; the three currently-recognized subtypes of multicentric CD (MCD) result from different etiologies but involve progressive episodes of systemic inflammatory symptoms and life-threatening cytokine- driven multi-organ dysfunction, such as the liver, kidneys, and bone marrow. The underlying mechanisms and therapeutic targets are not well understood. While various treatments are often tried for CD, systematic evaluations of these treatments have not been performed, the optimal treatments for each subtype are not known, and biomarkers to identify patients likely to respond to certain treatments are needed. New treatment approaches are also needed, but no validated, patient-centric treatment response criteria or biomarkers exist to consistently evaluate promising treatment approaches in clinical trials. Further, clinical data had not been centralized and no evidence-based diagnostic or treatment guidelines existed until recently. To address these barriers, we established an international longitudinal natural history study of CD in 2016 through a 5-year collaborative partnership between the Castleman Disease Collaborative Network, Janssen Pharmaceuticals, and the University of Pennsylvania (lead institution). Developed by a team of physicians, researchers, and patients, ACCELERATE utilizes an innovative patient-powered study design whereby CD patients in the US and globally self-enroll online and our study team obtains and systematically extracts complete medical record data into a central database. The use of common data standards, rigorous data extraction protocols, and expert adjudication of each case ensure that the data is of high quality and interpretability. We have enrolled over 500 CD patients and collected extensive, longitudinal data on approximately one-half that have been leveraged to characterize the spectrum of CD, support the development of evidence-based treatment guidelines, and advance translational research. Despite these advances, significant unmet needs remain for the majority of CD patients who do not have an effective FDA-approved therapy. Unfortunately, our 5-year funded study has ended. We are seeking funding to leverage the data collected from our 5-year collaborative partnership, build upon infrastructure from this multi- stakeholder collaboration, and continue enrollment and data collection to identify clinically-meaningful patient subtypes, clinical outcome measures, and novel treatment approaches. Our proposed studies have the potential to transform care for CD patients, overcome barriers to therapeutic product development, and establish a model for rare disease natural history studies.

NIH Research Projects · FY 2025 · 2022-09

The prevailing approach to precision cancer medicine relies on genetic profiling of patients, followed by identification of the malignant gene product, and delineation of the mechanisms of that protein product in causing disease. As a result, much of the future of precision oncology is built on the hope of tailoring therapeutic interventions based on diagnostic technologies that acquire complex genomic and transcriptomic data. Despite the focus on cancer genetics, the unique functional capabilities acquired by normal cells during tumor development are driven by the aberrantly activated tumor cell proteome that arises not only from gene mutations but also from epigenetic reprogramming, post-translational alterations, or rewiring of signaling pathways. Unfortunately, integrating traditional measurements of protein biochemistry that reflect tumor cell biology and the therapeutics to which a tumor would respond into clinical decision-making for cancer patients is challenging due to the uniqueness of each protein and limitations in existing technologies. Thus, our proposal focuses on mechanism-based cancer research at the interface of chemistry and cancer biology to develop quantitative approaches that evaluate dynamic changes in the proteome in order to characterize unique features of tumor biology with the long-term goal of motivating novel targeted therapies. Specifically, we aim to establish an innovative new development and discovery platform termed Probe Enabled Activity Reporting (PEAR) for tumor proteome profiling by leveraging chemical biology approaches to understand the molecular complexity of proteomic changes necessary for tumor cell function, as well as cellular adaptations to cancer therapy. The foundation of our bedside-to-bench and back again approach is rooted in the hypothesis that novel chemical probe reactomes exist in cancer cells themselves and changes in the reactome profile in response to cancer therapeutics will reflect alterations in protein function that drive cancer cell adaptations and thus, would be ideal for new treatment modalities in the future. In interconnected and interdisciplinary discovery and elucidation modules, we will utilize state-of-the-art patient derived cancer models to both visualize and identify the protein targets of chemical biology probes in pre- and post-treatment with the hypothesis that the differential reactomes will be indicative of proteomic liabilities, therapeutic response, and unique aspects of tumor cell biology. The major outcomes from investing in PEAR for tumor proteome profiling to enable therapeutic development will be development of methodology to visualize reactive targets, identification of treatment induced reactive targets and establishing their functional relevance, and unraveling unique tumor cell biology based on a novel compartmentalized reactive target method. Taken together, our proposal will establish and validate novel concepts and methodologies that can be applied across the broad spectrum of solid tumors and as an extension, holds the potential to provide fundamental insights into tumor biology and transform precision oncology by providing a platform to improve existing paradigms for drug discovery.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT The overall goal of this application is to elucidate the mechanism of Lys63 (K63)-linked polyubiquitination of extracellular signal-regulated kinases ERK1 and ERK2, and to determine the contribution of this novel post-translational modification to tumorigenesis. ERK1 and ERK2, which share high structural and functional similarities, are downstream effectors of a mitogen-activated protein kinase (MAPK) cascade that dictates cellular behavior and cell fate decisions in response to a wide range of intracellular signals. The ERK signaling pathway is the primary mitogenic pathway in mammalian cells. It is aberrantly upregulated in a large fraction of human tumors due to prevalence of mutations in the upstream signaling components, and reactivation of ERK is also the most common mechanism for resistance to drugs that target these upstream components. Developing a therapy that is applicable for the wide range of tumors driven by a hyperactive ERK pathway and that can circumvent drug resistance remains a major challenge in cancer research. To contend with this challenge requires a comprehensive understanding of how ERK is specifically and efficiently activated within the MAPK cascade. The current knowledge of ERK activation is largely limited to their phosphorylation by the upstream kinase MEK. In our preliminary studies, we have found that ERK is conjugated to K63-linked polyubiquitin chains, and that this post-translational modification correlates with ERK activation. Furthermore, we have identified the tripartite-motif protein TRIM15 as a ubiquitin ligase, and the tumor suppressor CYLD as a deubiquitinating enzyme (DUB), that may dynamically regulate ERK ubiquitination. Here we will test the central hypothesis that K63 ubiquitination of ERK is critical for the specific and efficient activation of these kinases and that dysregulation of this post-translational modification contributes to the pathogenesis and therapeutic resistance of tumors. We propose three specific aims. First, we will characterize the role of TRIM15 and CYLD in K63 ubiquitination of ERK and define how their interactions with ERK are regulated by mitogenic stimuli. Second, we will elucidate the function and mechanism of K63 ubiquitination in the activation of ERK. Third, we will determine the role of TRIM15 and CYLD in tumorigenesis and therapeutic resistance. Collectively, these aims will address fundamental issues in ERK biology and oncogenic signaling, and will provide valuable information for the development of effective therapies for the plethora of human tumors that are driven by a hyperactive ERK signaling pathway.

NIH Research Projects · FY 2025 · 2022-09

The number of medical conditions for which the results of genetic testing change the medical management of patients is exponentially increasing. However, a minority of eligible patients receive genetic testing, despite the implications for downstream care. System- (methods to identify eligible patients and return results), clinician( e.g., knowledge, limited workforce), and patient- (e.g. , concerns about costs and adverse effects) level barriers foster uncertainty and a tendency to rely on the status quo - failing to use genomic information to guide medical care. Implementation science methods and frameworks are ideal for addressing this practice gap, especially those that consider multi-level barriers and the role of human decision-making in contexts with uncertainty. Our team has built the infrastructure to address system-barriers to delivering genetic testing across our health system - an integrated system within the electronic health record (EHR) that enables direct ordering and resulting of genetic tests as structured data - now with multiple requests for dissemination. Our team also is using behavioral economics as an implementation science framework to improve healthcare by using nudges (EHR defaults, patient priming) to overcome clinician and patient barriers, concurrently addressing health disparities (e.g., higher practice gaps among racial minorities). Merging these areas, we propose a highly innovative project that will evaluate, for the first time, the use of nudges to clinicians (EHR defaults for either: 1) referring to genetics clinic or 2) ordering for genetic testing) and/or nudges to patients (communication to prime patients about the benefits of genetic testing prior to appointment). In Aim 1, we will develop electronic phenotyping algorithms for 10 clinical conditions, which will drive diagnosis-specific genetics referral and testing; we will refine our nudges working with a Stakeholder Advisory Council. In Aim 2, we will conduct a hybrid type 3 implementation study, using a cluster randomized design with 228 clinicians (physician, Advanced Practice Practitioners) as the unit of randomization (N= 120 clusters) and 16,500 patients with one of the 10 conditions to examine the impact on the rate of genetic testing of: the patient priming nudge, the two clinician nudges, combining the patient and each of the clinician nudges, vs. a generic best practice alert (BPA) (no clinician or patient nudge). We will examine patient (e.g., race), clinician (e.g., specialty), and system (e.g. , community vs. academic center) moderators of nudge effects on genetic testing rate and assess an effectiveness outcome (rate of clinician action following identification of a pathogenic variant). In Aim 3, we will engage in systematic methods to disseminate our EHR integration of genetic testing, EHR-based algorithms, and other materials and systems built for the clinical trial through Epic, PheKB, NHGRl's AnVIL, and GitHub. Our study will be immensely impactful, as it will yield a novel, effective, and transferrable EHRbased infrastructure that enables the sustainable delivery of genomic medicine, greatly advancing the field.

NIH Research Projects · FY 2024 · 2022-09

Learning from Hospital Preparedness during COVID: Chronically Under-Resourced Nurses and Patient Safety This study will evaluate how hospital nurses weathered the COVID-19 public health emergency, whether and to what extent hospital nurse resources (staffing, work environment, Magnet designation) buffered nurses from poor outcomes (such as burnout) during the pandemic and facilitated recovery 3 years after the onset of the COVID emergency, and the extent to which patient outcomes, safety, quality, and value of care indicators paralleled changes in nurse outcomes and hospital nurse resources over the study period. We will accomplish these objectives by leveraging already existing data from over 33,000 hospital nurses in 244 hospitals in New York and Illinois, [Wave 1 data collected just before COVID (Dec 2019-Feb 2020); Wave 2 collected 1 year after COVID onset] and by conducting primary data collection of repeat measures [Wave 3 to be collected 3 years after COVID onset (Oct 2022-Dec 2022)]. Each Wave includes repeated measures of nurse outcomes (e.g., burnout, job dissatisfaction, intent to leave job), hospital nurse resources (staffing, work environment, Magnet), measures of patient safety and quality of care, including items from the AHRQ Patient Safety Culture survey. These cross-sections of data will be linked with contemporaneous (1) patient-level data from CMS MedPAR Medicare to study risk-adjusted patient outcomes among patients hospitalized for common medical, surgical, and COVID diagnoses; (2) Hospital Compare data to evaluate hospital-level measures of patient satisfaction and healthcare value (Medicare spending per beneficiary), (3) American Hospital Association data for considering organizational features of hospitals, and (4) publicly available COVID hospitalization data to account for variation in COVID burden across hospitals. In combination, we will have 3 cross-sections of data from 244 hospitals (with fluctuating nurse and patient populations) just before, 1 year and 3 years after the onset of the COVID emergency. Our analytic approach uses multi-level nested (hierarchically-related) linear and logistic regression models (with interaction terms). The COVID emergency offers a unique opportunity to make a major advance in our scientific understanding of the potentially causal relationships between nurse outcomes and patient outcomes, which have until now largely only been rigorously evaluated in the cross- section. The tremendous shock imposed by the COVID emergency, combined with our propitiously timed data, enable us to evaluate how the pandemic impacted hospital nurses and what hospital factors contribute to a more favorable recovery in the years following the COVID emergency. Together, this evidence will inform high- impact actionable policy and organizational solutions for building and sustaining safe, high value healthcare systems that can endure future public health emergencies and thrive during ordinary times.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Delays and missed opportunities for timely treatment contribute significantly to disparities in cervical cancer mortality in low- and middle-income countries (LMICs) compared to high-income countries (HICs). Cervical cancer is one of the most common female cancers globally, with approximately 90% of cases and deaths occurring in LMICs, particularly those with high rates of HIV. This global disparity is partly driven by successful efforts in HICs to implement evidence-based practices focused on early detection and timeliness of care. In Botswana, a LMIC with an extremely high prevalence of HIV and cervical cancer, we identified substantial delays in cancer care from diagnosis to treatment, driven by a myriad of individual- and system-level barriers. To date, most of the implementation and cancer control research in Botswana and other LMICs has focused on prevention and screening, with limited focus on treatment following diagnosis of HIV-associated malignancies. As such, there is a critical need to identify effective strategies to ensure timely care, and to understand contextual factors that shape the response to strategies. Without this fundamental knowledge, cervical cancer will remain a public health crisis in Botswana and other LMICs. To help fill this critical gap, this study will test the effectiveness of adaptive strategies on timely treatment adoption using a Sequential Multiple Assignment Randomized Trial (SMART) design and evaluate contextual mechanisms contributing to the success or failure of each adaptive strategy using qualitative comparative analysis. The adaptive strategies are designed to target individual- and system-level determinants identified in our preliminary data, including delayed communication of results, individual and structural barriers to accessing treatment, and suboptimal care coordination between referring and cancer treatment clinics, and are supported by systematic evidence of the effectiveness of nudge strategies in clinical care. The primary implementation outcome will be adoption, defined as the initiation of treatment within 90 days. Secondary implementation outcomes include fidelity (i.e., completion of recommended treatment), reach, acceptability, implementation costs, and cancer and HIV-related clinical outcomes. The rationale for the study is that enhancing coordination, communication, and navigation through centralized outreach will both increase timely treatment adoption and be scalable and sustainable after the project is completed. This innovative study responds directly to the call by the National Cancer Institute to develop and test implementation strategies in cancer control in LMICs. Furthermore, the highly efficient design enables the comparison of different adaptive strategies within one study, helping to advance an understanding of the minimum level of intervention needed to improve and sustain cancer control in lower resource settings. If successful, these strategies can be easily translated to address other areas of cancer control. The long-term goal of this project is to decrease cervical cancer mortality in LMICs by developing and implementing effective and sustainable strategies.

NIH Research Projects · FY 2025 · 2022-09

Asthma, a chronic disease that manifests as airway hyperresponsiveness to specific environmental stimuli, affects over 20 million American adults. Disparities in adult asthma prevalence, severity, and death in the U.S. are well known but few approaches have significantly decreased them. Studies of real-world populations such as those derived from Electronic Health Records (EHRs) are invaluable to guide the design of personalized care strategies because they capture a large number of diverse and vulnerable people. Additionally, EHR data can be leveraged to identify geospatial areas where people are at peak risk of a condition by studying the geographic distribution of affected patients. We have identified individual- and area-level factors that are associated with asthma using EHRs linked to rich and diverse sources of social, economic, and environmental variables, and we have developed methods to appropriately extract information from EHR data that are heterogeneously available across patients and reduce bias in the analysis of these imperfect data. This proposal will identify sub-groups of adults with asthma who share common patterns of demographic, clinical, social, and environmental exposure characteristics using EHR data augmented with data on social, economic, and environmental factors, which will enable the design of effective precision strategies to reduce asthma exacerbations. Our aims are to: 1) develop and validate natural language processing (NLP) algorithms to extract social, occupational and allergy information from EHR notes; 2) determine geospatial areas that have increased asthma exacerbation risk using spatial generalized linear mixed models and identify risk factors in these areas; and 3) use Bayesian hierarchical clustering techniques to identify asthma sub-phenotypes that will form the basis of a clinical decision support tool that offers precision care strategies. This project will result in the creation of a tool that can assist in the care of adults with asthma based on actionable risk factors and motivate public health strategies to mitigate the burden of asthma in specific regions. Our novel data integration approaches, sub-phenotyping methods, and software developed will have broad applicability for the study of any condition using EHR or other real-world data.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Traumatic brain injury (TBI) affects millions of individuals annually resulting in disrupted neuronal circuitry, persistent neurological deficits, and increased susceptibility to secondary infections. Following TBI, the peripheral immune system (PIS) cells contribute to subsequent neuroinflammation and exacerbate neuropathology by homing to the injured brain, associating with micropathological features, and releasing inflammatory factors. Additionally, following TBI, the injured brain releases damage signals into the blood which alters PIS homeostasis and functionality. Indeed, these adjustments to the PIS can result in chronic immunodeficiency, reduced tissue regenerative capacity, impaired neurological outcomes, and an increased mortality rate. However, the mechanisms and outcomes of how these two organ systems affect one another after trauma has never been investigated in a clinically relevant model of diffuse TBI. Therefore, I propose to quantify the liming, extent, and location of the infiltrating PIS in the brain after TBI, to investigate TBI-induced changes to PIS functionality at baseline and after a clinically relevant immune challenge, and to fabricate a therapeutic treatment strategy that will employ cells of the PIS to modulate TBI-induced neuroinflammation. Specifically, immunomodulatory microparticles will be loaded into infiltrating immune cells and these autologous microparticle-loaded cells will be administered intravenously after TBI. Thereafter, the therapeutic efficacy of these cells will be quantified by characterizing the extent of infiltration, effects on the PIS, and distribution of neuropathology. To complete this work, I propose to utilize a high-fidelity preclinical porcine model of closed-head diffuse TBI - which is the most clinically relevant model of TBI biomechanics in use today - along with comprehensive and quantitative PIS characterization. I hypothesize that infiltrating immune cells will localize with micropathological features, the innate and adaptive PIS will exhibit chronic immunosuppression after TBI, and that neuroinflammation will be mitigated when infiltrating immune cells are loaded with immunomodulatory microparticles. Information gained from this proposal will develop a translationally-relevant treatment strategy for TBI that could improve care of affected individuals and inform basic science questions about neuro-immune interactions. Importantly, this research can only be completed at the University of Pennsylvania and VA Medical Center because of unique resources, equipment, and institutional environment that is not available anywhere else in the world. During this career development award, I will have access the injury device that induces the porcine closed-head diffuse TBI, equipment and assays for comprehensive PIS characterization, immunomodulatory microparticle fabrication, and large animal facilities. This career development award will offer a unique training opportunity, answer basic scientific questions, develop translational immunomodulatory tools, and foster committed mentorship that will cultivate a specialized research niche on neuro-immune interactions that will allow me to transition into an independent researcher.

NIH Research Projects · FY 2025 · 2022-09

The autonomic nervous system has an important role in the pathogenesis of cardiac arrhythmias thus providing a critical opportunity for therapeutic intervention. Modulation of the autonomic nervous system has been attempted through numerous means including surgical sympathectomy, catheter-based ablation procedures, and transcutaneous approaches. Although autonomic innervation has been shown to have a significant effect on arrhythmogenicity, the complex network of interactions and the optimal strategies for interrupting this network are inadequately characterized. To further the field, this research will integrate established principles and techniques from neuroscience to study the role of autonomic neuromodulation in the treatment of cardiac arrhythmias. The investigation will focus on the ability of repetitive transcutaneous magnetic stimulation (TcMS) to modulate synaptic strength of autonomic cardiac innervation. The feasibility of this non-destructive and non-invasive technology is supported by Dr. Markman’s recent work published in JAMA targeting the cervical sympathetic chain with an inhibitory TcMS protocol in patients with ventricular tachycardia (VT) storm. The proposed research plan aims to improve understanding of the cardiac effects of autonomic neuromodulation, and to assess the efficacy of TcMS in patients with VT storm. Aim 1 seeks to characterize the cardiac electrophysiological effects of autonomic neuromodulation by invasively measuring conduction properties as well as levels of catecholamines and inflammatory cytokines before and after neuromodulation. This will develop critical tools for characterizing neural-cardiac interactions, allowing definitive assessment of their complex relationship. Aim 2 seeks to characterize the cardiac electrophysiological effects of autonomic neuromodulation by invasively measuring conduction properties as well as levels of catecholamines and inflammatory cytokines before and after neuromodulation. In combination, the findings from these aims will yield critical information regarding the mechanistic characterization of complex cardiac-neural pathways and help establish the role of a novel method of neuromodulation. In addition, this research will promote critically important continued interdisciplinary collaboration between neuroscientists and cardiovascular medicine. Dr. Markman, an early career investigator and a fellow in cardiac electrophysiology, has a long-term goal of establishing an independent research program in autonomic neuromodulation focused on mechanistically defining the complex neural circuits involved in cardiac arrhythmias. These research aims are part of a comprehensive training plan and will be supervised by a mentorship and advisory team consisting of national leaders in arrhythmia and neuroscience research and will guide his transition to an independently funded research career.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Transcranial magnetic stimulation (TMS) is currently approved by the FDA for the treatment of depression, obsessive compulsive disorder, and smoking cessation. Despite evidence that TMS improves symptoms by modulating brain connectivity, the few published studies that have measured brain connectivity before and after neuromodulatory TMS have been population-, dose-, and pattern-specific, with connectivity effects that are limited in scope to a handful a priori regions of interest. Accordingly, there is a critical need for generalized, comprehensive model that explains how functional brain connectivity changes at the whole-brain level following neuromodulatory TMS. Therefore, the objectives of this grant are to 1) develop a model using whole- brain estimates of the TMS-induced electric (e)-field to predict changes in resting state functional connectivity following neuromodulatory TMS, and 2) validate this model in a large cohort of healthy volunteers receiving multiple doses of either intermittent or continuous theta burst stimulation (iTBS and cTBS, respectively). Our central hypothesis is that changes in functional connectivity will vary systematically with the current density at the cortex, operationally defined using e-field modelling. We have pilot data suggesting that the variability in pre-post rsFC changes following TMS can be predicted using estimates of the current density at the cortex with a medium to large effect size. Our approach will be to measure rsFC in healthy volunteers before and after each of 3 doses (5 sessions/dose; 600 pulses/session) of iTBS or cTBS. Stimulation will be delivered to the left dlPFC, and targeting will be individualized based on fMRI data collected during the Sternberg working memory paradigm. Our primary outcome measure will be the percent of variability in pre-post rsFC accounted for by our model. Our rationale for this approach is that by collecting resting state data pre and post these doses of iTBS and cTBS, we will be able to quantify the effect of pattern (i.e. cTBS vs. iTBS) and dose (i.e. number of pulses) on functional connectivity changes. This work is innovative because it uses a novel application of e-field modelling to predict changes in rsFC data following TMS administration.

NIH Research Projects · FY 2025 · 2022-09

Abstract: Immunocompromised HIV-positive patients have serious complications with opportunistic oncogenic viral infections that can lead B-cell lymphomas. Epstein-Barr virus (EBV) and Kaposi’s sarcoma associated virus (KSHV) are two human oncogenic gammaherpesviruses associated with B-cell lymphomas either individually or as co-infections. EBV-associated B-cell lymphomas are established as latency III infection with the major latent genes expressed as well as the small non-coding RNAs. EBV transformed B cells drive latency III, also seen in HIV associated EBV-positive lymphomas. EBV is also associated with other lymphomas including Burkitt’s lymphoma, Hodgkin’s and non-Hodgkin’s lymphoma, and post-transplant and AIDS associated lymphomas in immunocompromised HIV-patients. EBV also efficiently transforms human primary B-cells in vitro, into immortalized lymphoblastoid cell lines (LCLs). These nascent transformed B cells express latent genes, one of which is the Epstein-Barr nuclear antigen EBNA3C, essential for immortalization of B-cells. EBNA3C regulates cellular and viral gene expression through interaction with transcription repressors, and complexes of the mammalian cell cycle which include CyclinA, and components of the SCF proteosome degradation pathway. Our long term goal is to determine the role of EBNA3C in reprogramming viral and infected cell genomes through interactions with the tumor suppressor Rb and the regulatory consequences of these interactions as related to cell survival, cell cycle regulation and proliferation. We will investigate the mechanism of Rb regulation through specific post- translation modifications after infection by EBV, which includes phosphorylation and acetylation important for targeted ubiquitination. We will determine if enhanced phosphorylation/acetylation of Rb occurs through recruitment of CyclinD/Cdk4/6 complexes by EBNA3C important for cell cycle progression. This results in loss of Rb through ubiquitination which leads to cell and viral genome reprogramming by activation of the cellular E2F pathway, cell cycle progression, increased survival and malignant transformation. These studies will examine the role of EBNA3C in regulating the Rb/CyclinD/E2F network important for B-cell immortalization with implications for novel insights into KSHV and EBV contributions to latency III lymphomas in HIV patients.

NIH Research Projects · FY 2025 · 2022-09

7. Project Summary/Abstract Mycosis Fungoides/Cutaneous T Cell Lymphoma (MF/CTCL) is a rare, indolent Non-Hodgkin's Lymphoma (NHL), without a known cure, that presents as skin patches, plaques, tumors, or erythroderma with significant morbidity and impact on health-related quality of life (HR-QoL). Effective early treatment improves HR-QoL and may prevent disease progression. As per current NCCN guidelines, many skin-directed therapies (SDTs) are used for the treatment of early stage MF/CTCL including topicals, ultraviolet (UV) phototherapy, and radiation therapy. None have been FDA-approved for first-line therapy and most are used off-label. Given the chronic, recurrent nature of MF/CTCL, most patients require repeat courses of therapy and trying other therapies. The use of several SDTs are limited by skin irritation/dermatitis and long-term cumulative toxicity. Given this, there is an urgent need for additional SDTs for CTCL with fewer side effects. Topical hypericin is non-mutagenic, not systemically absorbed, activated by noncarcinogenic visible light, selectively taken up by tumor cells in the skin (up to 10-fold) and induces mitochondrial apoptosis. Phase 1, 2 and 3 clinical studies in MF/CTCL have demonstrated safety and efficacy. A recent multicenter, randomized, placebo-controlled, blinded Phase 3 study, enrolled 169 patients with Stage IA, IB or IIA CTCL and administered treatments twice weekly for at least 6 weeks (Cycle 1). In Cycle 1, 116 subjects received SGX301 to 3 index lesions with an overall response rate (ORR) of 16% compared to placebo ORR 4% (n=50, p=0.04). In Cycle 2 the ORR in patients receiving SGX301 for both cycles (12 weeks) was 40% (p<0.0001 vs placebo or 6 weeks of treatment). SGX301 treatment is safe and well tolerated, with potential favorable long-term safety given its mode of action. Although the trial showed efficacy, the dosing regimen was inflexible and short duration vs other skin-directed therapies where peak ORR can take 4-24 months of treatment. The current proposal will study SGX301 efficacy/safety utilizing a continuous treatment schedule for up to 1 year in an open label, multicenter clinical trial of 50 patients over 6 sites. The Specific Aims are: 1: Define optimal duration of SGX301 therapy to maximal ORR in early stage MF/CTCL patients on a “real world” treatment schedule up to 12 months. 2: Define safety profile during treatment. 3: Assess efficacy of SGX301 in disease subtypes and different skin lesions (patch vs plaque). 4: Assess genomic changes in skin/peripheral blood (utilizing high throughput sequencing [HTS-PCR] and targeted next generation sequencing [NGS]) and apoptosis markers (utilizing immunohistochemistry) as correlates of clinical response to SGX301 therapy.

NIH Research Projects · FY 2025 · 2022-09

The mammalian germline must be reprogrammed to facilitate proper development. This reprogramming, which includes the erasure of DNA methylation and histone modifications, ensures the establishment of gamete- appropriate epigenetic patterns and minimizes the transmission of epimutations to offspring. While much of the genome undergoes replication-coupled passive DNA demethylation, a critical role for Ten-eleven Translocation (TET) family enzymes, specifically TET1, has been demonstrated for active demethylation of genomic sequences such as imprinting control regions (ICRs) and germline-specific genes. The proposed work will use newly developed mouse strains and sequencing technologies to test the hypothesis that iterative oxidation and noncatalytic functions of TET1 are required for DNA methylation erasure and reprogramming of the mouse genome, including ICRs and meiosis-specific genes, during germline and somatic development. TET enzymes can catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Oxidized 5mC bases, particularly 5hmC, can play independent epigenetic roles in somatic tissues including the brain, but are most significantly thought to function as DNA demethylation intermediates. The distinctive demethylation pathways supported by 5hmC versus 5fC/5caC have confounded efforts to decipher the precise mechanistic role for TET1. Yet further challenges are posed by potential non-catalytic roles for TET1, which is known to interact with chromatin modifying enzymes. Published work and our preliminary data suggest that a role for catalytic and non-catalytic TET1 activities for demethylation, but the mechanism, timing and target sequences remain incompletely understood. Thus, we propose to address (1) whether iterative oxidation to 5fC/5caC is required for reprogramming, (2) whether TET1 has a noncatalytic reprogramming role, and (3) what sequences require various TET activities. Specific Aim 1 will examine the precise role of TET1 in reprogramming at ICRs and genome-wide in primordial germ cells (PGCs). We have engineered mice that either stall 5mC oxidation at 5hmC (Tet1v) or lack catalytic function (Tet1hxd) and will test their effects on DNA methylation reprogramming using our new technology which resolves 5mC and 5hmC, and profile associated chromatin dynamics during PGC development. Our preliminary data using the new Infinium Mouse BeadChip suggest that the Tet1 mutant mice sperm have non-overlapping aberrant patterns of DNA modification. Thus, Specific Aim 2 will assess the epigenomic and phenotypic consequences of Tet1 mutations in homozygous mutant gametes and the offspring that arise from these gametes. Finally, Specific Aim 3 will determine the epigenomic and phenotypic consequences of Tet1 stalling and catalytic mutations in homozygous mutant adult and aging mice. This work will enable an assessment of the role of TET enzymes in genome reprogramming, dissecting the requirement of noncatalytic activity and iterative oxidation by TET enzymes.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Higher brain functions include the ability to learn expectations about the world, update those expectations appropriately when given new sensory information, and use those continually updating expectations to guide behavior. How neural circuits implement these flexible information-processing dynamics is not known. We propose a novel research project that, consistent with the goals of the BRAIN initiative, uses innovative, methodologically integrated approaches to understand how activity pattens in a specific circuit in the primate brain support flexible updating used for behaviorally relevant decisions. The circuit includes two main components with known properties relevant to our proposed studies. The first component is the dorsolateral prefrontal cortex (dlPFC), which includes neurons that encode ongoing processing of expectations and sensory evidence in working memory. The second component is the locus coeruleus (LC)-norepinephrine (NE) neuromodulatory system, which can affect working-memory representations in the dlPFC. However, it is not known whether and how LC-NE modulations of working-memory representations in dlPFC contribute to flexible decision-making. Building on our previous work on flexible decision-making and effects of the LC-NE system on neural information processing, we propose and test the hypothesis that LC-mediated NE release in dlPFC governs how dlPFC neural populations flexibly combine learned expectations held in working memory with incoming sensory information to form decisions that guide behavior. We test this hypothesis by training monkeys on a novel task that allows us to quantify how learned expectations and new sensory information are combined in a flexible, context-dependent manner to make saccadic decisions. We then elucidate the underlying circuit mechanisms, via three Aims that each leverage an innovative set of approaches. Aim 1 is to measure how LC and dlPFC activity at a single-neuron resolution relates to flexible decision-making. Aim 2 uses multiple techniques, including electrical microstimulation for temporal specificity and chemogenetics for pathway specificity, to test for causal roles of temporally specific firing patterns of LC->dlPFC projections on flexible decision-making. Aim 3 uses computational modeling to relate LC-dlPFC circuit properties (including NE-mediated changes in neuronal gain) to computational principles that support flexible decision-making. Each Aim alone provides new insights into correlative, causal, and computational contributions of the LC-dlPFC circuit to flexible decision-making. Taken together, these studies provide a novel, unified view of how the LC- PFC circuit performs critical computations that flexibly combine expectations with evidence to inform decisions.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Chengcheng Jin, Ph.D Neutrophils are the most abundant immune cells in human blood. They are multi-functional innate myeloid cells that play key roles in pathogen infection, tissue repair, as well as cancer. As a main composition of the tumor- associated immune cells in multiple cancer types, neutrophils have emerged as a critical player to promote cancer progression via diverse mechanisms, such as mediating tissue remodeling, driving local inflammation, suppressing anti-tumor T cells. However, no viable strategy is currently available to target neutrophils for cancer therapy. This reveals fundamental questions and challenges: are tumor-associated neutrophils (TAN) distinct from normal blood neutrophils? Do all the neutrophils in the TME function identically and carry out the broad range of tumor-promoting activities? Is it possible to selectively target the tumor-promoting neutrophils without impairing those essential for protecting us from bacterial infection? Our vision is to develop an in-depth and broad understanding of transcriptional and epigenetic reprogramming of neutrophils in the tumor microenvironment (TME). This will reveal novel regulatory mechanisms unique to tumor-promoting neutrophils that can serve as targets of precision cancer immunotherapies while preserving immune surveillance in healthy tissues. Our strategy is to take an integrated approach that leverages the unique expertise and knowledge that we have established in genetically engineered mouse models. Specifically, we will (1) combine phenotypic, transcriptional and chromatin profiling of neutrophils in different TME at the single-cell level, (2) apply fate mapping and spatial transcriptomics to reveal the neutrophil dynamics in TME, (3) establish and utilize novel genetic perturbation tools to identify and functionally validate key regulators of neutrophil function in cancer. By analyzing the tissue/tumor-associated neutrophils from different microenvironment, we have identified distinct neutrophil subsets that are induced by different components of the TME. Therefore, our overall hypothesis is that specific factors in the tumor microenvironment such as the local microbiota and tissue-resident immune cells, as well as the genetic makeup and immunogenicity of cancer cells may differentially regulate the neutrophils. Our goal is to identify cell-extrinsic factors from the TME that reprogram neutrophils to functionally discrete subsets. Meanwhile, we will apply novel techniques to track TANs and dissect neutrophil-intrinsic pathways that direct their functional diversification in cancer. Our study will provide a blueprint for transcriptional control of neutrophil responses in cancer and opens possibilities for stage/gene/environment-specific therapeutic modulation of neutrophil function in cancer. Furthermore, the conceptual and technological advances generated here will build the foundation for future investigations into neutrophils in additional cancer types and beyond, shedding light on pathways and molecules that can serve as novel therapeutic targets to manipulate neutrophils for treating cancer and other diseases.

NIH Research Projects · FY 2025 · 2022-09

Chimeric antigen receptor T (CAR-T) cells have produced unprecedented results in blood cancers, but clinical responses in solid tumors are rare due to the detrimental effects of the hostile, immunosuppressive tumor microenvironment (TME) and the use of patient derived, dysfunctional T cells adversely affected by advanced disease state and previous chemotherapy. Invariant Natural Killer T cells (iNKTs) are a distinct lineage of CD1d-restricted T lymphocytes with natural tissue (and tumor) tropism, direct cytolytic effect on CD1d+ cancer cells and tumor associated macrophages and adjuvant effects on endogenous anti-tumor immunity. Unlike conventional T cells, allogeneic iNKTs mediate a robust graft-versus tumor effect without inducing graft-versus-host disease, thus eliminating the need for gene editing to maintain tolerance. Moreover, preclinical studies in mouse models suggest that CAR-enhanced iNKTs can eradicate solid and solid-like hematological tumors where CAR-T cells fail. We hypothesize that allogeneic CAR-engineered iNKTs will overcome the barriers to successful CAR-therapy and provide a powerful off-the-shelf universal platform with curative potential for solid tumors. Yet, unsolved questions related to allo CAR-iNKT safety, optimal preconditioning regimens to promote persistence, cell dose and therapeutic efficacy in solid tumors remain. Furthermore, while healthy human donors for allogeneic iNKT clinical trials are randomly selected, cells generated from different donors have different immunomodulatory capacities that may affect their engraftment and survival and it is currently unknown which products are “best” for adoptive cell therapy (ACT). These questions cannot be adequately addressed in mice due to the dissimilarity between murine and human iNKT cells. In contrast, our preliminary studies demonstrate that canine and human iNKT cells share remarkable phenotypic and functional similarities and can be CAR engineered and expanded to clinical scale for trial use. Here we will use immunocompetent pet dogs with spontaneous osteosarcoma (OSA), which is remarkably similar to pediatric OSA, to advance allogeneic IL-13Rα2-targeting CAR-iNKT cells into the human clinic. We will first characterize CAR-iNKT cells from different donor dogs and generate a master cell bank of canine alloCAR-iNKT products with different immunomodulatory capacities. Next, we will address the safety and effects of 3 strategically designed pre-conditioning regimens, including a combination of cytotoxic chemotherapies, low dose total body irradiation and a clinical grade iNKT glycolipid agonist, RGI-2001, on allo-CAR-iNKT engraftment and persistence in dogs with metastatic OSA. Finally, we will determine the maximum tolerated dose of alloCAR-iNKT cells using an accelerated dose escalation trial design and evaluate their effects on the TME, systemic immunome and disease free interval in dogs with appendicular OSA. This work addresses pivotal questions for the advancement of allogeneic-CAR-iNKT cells in a clinically relevant canine cancer “model” to accelerate clinical use of this promising “off the shelf” approach in human patients with solid tumors.

NIH Research Projects · FY 2025 · 2022-09

Acute myeloid leukemia (AML) is a disease of blocked differentiation in which blasts fail to mature and proliferate continuously. Differentiation therapy, which aims to reactivate latent maturation programs and induce cell cycle exit, is curative in the promyelocytic (APL) AML subtype, but not in other AML subtypes. Epigenetic factors help sustain the differentiation block, and inhibitors of regulators such as the LSD1, BRD4, and DOT1L has recently been shown to re-activate myeloid differentiation programs in selected AML models. However, these inhibitors generally do not achieve terminal differentiation and disease remission. Accordingly, there is significant need to identify more regulators of the AML differentiation block and to test whether their inhibitors can induce terminal differentiation when used individually or in combination regiments. To identify novel regulators of AML differentiation, we recently performed a chromatin-focused CRISPR sgRNA screen using gain-of- differentiation as a readout. This screen identified the H3K9 histone acetyltransferase KAT6A as a key driver of AML differentiation arrest, and mechanistic work showed that KAT6A and the H3K9ac histone binding protein ENL closely cooperate to active promoters of AML oncogenes. We confirmed that both genetic (CRISPR) and small molecule inhibition of KAT6A markedly induces differentiation and reduces proliferation most commonly in MLL-rearranged (MLL-r) AMLs, and also in selected MLL-wild type (MLL-WT) AMLs. Further, KAT6A inhibitors synergize with inhibitors of either LSD1 or DOT1L to induce near-terminal differentiation and fully halt proliferation in vitro. This proposal has three goals: First, we will determine the mechanisms by which KAT6A and ENL are recruited to chromatin and activate transcription. We will identify the protein domains in KAT6A and the MOZ complex it resides in that are responsible for its binding to chromatin at MLL-AF9 targets and non- MLL-AF9 targets. We will also identify any transcription factors interacting with KAT6A and ENL and test their effect on recruitment of the KAT6A-ENL module to chromatin. Our second goal is to test the therapeutic potential of targeting KAT6A, individually or in combination with LSD1 or DOT1L inhibitors, in genetically-defined AML mouse models. We will employ an Mll-Af9 model and a Dnm3a/Flt3-ITD model and test the effect of inhibitor treatment schemes on disease progression and overall survival. We will also test the effect of inhibitor treatments on normal hematopoiesis. Our third goal will be to test the response of clinical AML patient samples to inhibition of KAT6A, individually or in combination with LSD1 or DOT1L inhibitors. We will perform drug response assays in MLL-r and MLL-WT samples in vitro using OP9 feeder layer culturing methodology, and will also perform PDX transplant models and test the response to KAT6A and LSD1 or DOT1L inhibitors in vivo.

NIH Research Projects · FY 2024 · 2022-09

Thrombocytopenia absent radius (TAR) syndrome is a rare congenital disorder that causes absence of the radii, reduced numbers of mature megakaryocytes (MKs), and thrombocytopenia. TAR is caused by mutations in the RBMBA gene, resulting in reduced mRNA expression of RBMBA and levels of its encoded protein, Y14, in patient platelets. Since Y14 has no known roles in MK biology, it is currently not understood how deficiencies in this protein contributes to a MK phenotype without affecting other hematopoietic lineages. Previous studies of Y14 depletion have identified a role for Y14 in apoptosis and cell cycle regulation, but it is unclear whether this is the mechanism responsible. Both the postnatal emergence of the thrombocytopenia in TAR and the known differences in MKs derived from primitive or definitive progenitor cells suggest that definitive MKs may present a more severe phenotype and thus be a more insightful model. By modeling this disease in vitro using patient-derived induced pluripotent stem cells (iPSCs) and isogenic corrected lines, we can assess the effects of TAR on pure cell populations to observe lineage- and developmental stage-specific changes without influence from the compensatory feedback mechanisms that regulate blood cell generation in vivo. Overall, we hypothesize that Y14 depletion in TAR syndrome impairs maturation of definitive MKs more severely than primitive MKs through altered cell cycle and apoptosis regulation, and it does not affect the development of other blood lineages. Aim 1 of this proposal will determine the specific characteristics of MKs that is altered due to Y14 depletion during primitive and definitive differentiation. Aim 1A will evaluate aspects of MK maturation and functionality to determine the specific MK phenotype, and Aim 1 B will determine if reduced Y14 alters apoptosis and cell cycle progression in MKs as a potential mechanism for this phenotype. Using RNA-seq, we will detect differential expression of genes related to these pathways or identify any novel targets with the potential to contribute to the MK defect. Aim 2 will address the MK specificity of TAR by comparing consequences of Y14 depletion in MK differentiation to erythroid and myeloid differentiations. Aim 2A will discern whether the hematopoietic lineages regulate Y14 RNA or protein levels differently. Aim 28 will use cell proliferation and lineage-specific surface marker expression to detect potential defects in erythroid or myeloid development. We will also determine whether cell cycle and apoptosis regulation are altered in these other lineages as well as any additional pathways that are identified in Aim 1 B. This will be the first study to directly compare the regulation of cell cycle, apoptosis, and MK maturation during primitive and definitive hematopoiesis and test whether these models have the potential for divergent disease phenotypes. The results of this study will not only elucidate the mechanism of TAR syndrome in MKs, but its insight into MK biology at different stages of development will have important implications for improving current in vitro disease models and tailoring therapeutics to distinct tissue systems to reduce human disease.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract Dr. Kim is enrolled in a uniquely combined periodontics and Doctor of Science in Dentistry (DScD) program at the University of Pennsylvania School of Dental Medicine. Under the mentorship of Dr. Boesze- Battaglia and support from consultants, Dr. Kim will investigate cytolethal distending toxin (Cdt) mediated modulation of phagocytic function. This topic is important in the field of periodontology as it will advance our understanding of the pathophysiological mechanism by Cdt produced by A. actinomycetemcomitans (Aa) modulates local host defense regarding localized aggressive periodontitis (LAP). Dr. Kim is committed to a career in academics, which will be greatly enhanced by the educational, technical and career development training opportunities afforded by the K08 Award. The established specialty/DScD program at Penn will serve as an important stepping stone for Dr. Kim's long-term goal to eventually emerge as an independent researcher studying the pathophysiological mechanism of bacterial and host immune response in an effort to develop adjunctive/alternative therapeutics for periodontitis. Among many possible bacteria that can cause periodontitis, Aa was known as the etiology for LAP. Our current understanding is that Aa is a critical pathogen providing a suitable environment for other pathogens and cause LAP. Among different types of toxins produced by Aa, Dr. Kim's project focus on Cdt produced by Aa which causes cell cycle arrest, apoptosis in T cells and upregulates pro-inflammatory cytokines in macrophages by phosphoinositide 3-kinases blockade. Cdt acts as a phosphatidylinositol-3,4,5-triphosphate phosphatase and causes phosphatidylinositol (PI) pool imbalance which is crucial for not only the cellular response but phagosome degradation. Dr. Kim's overarching hypothesis is that Aa Cdt-mediated disruption in macrophage phagosome processing leads to Aa survival contributing to disruption of local host defense. In Aim1, Dr. Kim will investigate phagosome maturation with effector proteins and phago-lysosome fusion regarding Cdt-mediated PI pool imbalance in macrophage. In Aim 2, Dr. Kim will study effect of Cdt on macrophage and survivability of Aa by using wild type and Cdt deficient mutant Aa. Furthermore, Dr. Kim will investigate the pro-inflammatory and oxidative stress in relation to survivability of Aa. For aim 3, Dr. Kim will investigate synthetic secoisolariciresinol diglucoside (LGM2605), potent free radical scavenger, antioxidant, and anti-inflammatory agent, in mitigating Aa mediated inflammation and bone loss via in vivo and in vitro. Collectively, these studies will further our understanding of the mechanisms underlying the role of Aa in the evasion of phagocytosis and early events in microbial dysbiosis. Moreover, we will expand future therapeutic strategy for LAP with a potential anti- inflammatory agent.

NIH Research Projects · FY 2025 · 2022-09

The primary mission of the coordinating center (CC) at the University of Pennsylvania is to facilitate the performance of immunotherapy clinical trials in dogs with cancer within the K9CIN (previously known as the Pre- medical Cancer Immunotherapy Network for Canine Trials (PRECINCT)) and to determine the suitability of dogs with spontaneous cancer to study immunomodulatory agents and combination therapies to inform human clinical trials. The CC will achieve these goals by supporting a highly coordinated clinical trials network of participating U01 sites and will work in consultation with U01 investigators, the network's Steering Committee, representatives from the Comparative Oncology Program (COP) at NCI and an External Advisory Committee to provide expert services in project management and research technology, data management, and biostatistical and bioinformatic support. Specifically, PRECINCT's CC will provide comprehensive project oversight, supervise all project management and regulatory compliance activities, and coordinate site management for all aspects of trial projects. Through the CC, U01 investigators will have ready access to research services and standard operating procedures (SOPs) for clinical and immunological monitoring and collection of data, reagents and biospecimens that will facilitate the performance of clinical research across the network. Patient accrual will be tracked and where necessary, cross-site accrual will be coordinated within the network to expedite enrollment. The CC will assist in designating supplemental funds to U01 awardees to support intra-network collaborative projects identified by a chosen scientific review panel for funding. We will continue to build infrastructure and expand the network and associate memberships will be encouraged to achieve the critical mass necessary to build working groups to advance PRECINCT's mission and build inter-network collaborations with IOTN, PI-DDN and PACMEN. We will extend PRECINCT's Data Management System to collect, store and share all clinical and correlative data amongst the U01 sites and with the COP directors and Steering Committee, through the development of a cloud-based workspace that will facilitate intra-network collaborative projects and we will perform data quality control and re-formatting required to enable ready upload to the Integrated Canine Data Commons (ICDC). We will build a specimen and reagent registry that will facilitate sharing and tracking of resources and serve to ensure their most efficient use within the network. This together with development of a cloud-based workspace, will foster collaborations between U01 researchers. We will provide biostatistical and bioinformatic support for study design and data analysis to ensure that clinical trials performed within the network are strategically designed and appropriately powered to achieve meaningful clinical and laboratory data results in the most expedite way. Together, the activities and expertise provided by the CC will ensure that reliable and reproducible datasets emerge from these studies which aim to provide essential insight for future translational application to human patients.

NIH Research Projects · FY 2025 · 2022-09

Cherenkov imaging incorporating surface imaging for TSET Abstract Whole-body skin electron radiotherapy has been clinically demonstrated to be effective to treat mycosis fungoides. However, due to setup positioning uncertainties and potential patient movements during total skin electron therapy (TSET), dose delivered to the patient skin tissue can deviate from dose prescription. Cherenkov emission from tissue has recently been demonstrated, providing a mapping related to the radiation delivery to skin tissue. The signal is optimally captured by time-gated intensified cameras, synchronized to the linear accelerator pulses, allowing rejection of the majority of background room light, and providing real time video of each radiotherapy treatment with high dose rates. The implementation of Cherenkov imaging offers an excellent technology to detect abnormalities in the treatment, which would otherwise go unnoticed. This proposal seeks to advance this technology as a verification tool through a clinical trial to monitor the daily treatment delivery of TSET patients and develop proper corrections that can be applied to the acquired signal to ensure it is quantitatively accurate in documenting delivered skin dose. Three of the most dominant factors, which alter the linearity between dose and Cherenkov signal, are the corrections for perspective direction, tissue curvature, and tissue optical properties. These important corrections are quantified in this pilot study of TSET, in partnership with DoseOptics LLC, the company that developed the Cherenkov imaging technology for radiation verification, to perfect technology for daily monitoring of radiation delivery. We will compare the dosimetry accuracy of Cherenkov imaging by comparing measurements in-vivo at 9 locations using OSLD and Scintillator detectors. In addition, we will perform measurements in optical phantoms of known tissue optical properties at TSE treatment conditions and Monte-Carlo simulation studies to quantity the three correction factors. We will also develop techniques to overlay skin dose obtained from corrected Cherenkov image to patient-specific surface anatomy based on surface 3D body contour data obtained before TSE treatment to assess the actual skin dose distribution for daily TSE treatment and perform a comprehensive comparison of dose distribution based on MC-based treatment planning system. Taking together, this project will advance on the most compelling systems for radiotherapy imaging in decades. The core of the project is combined technology systems, testing the utility in the setting of TSET.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY My long-term career goal is to tackle neurodegeneration as an academic researcher. Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), an Alzheimer's Disease Related Dementia (ADRD), are both fatal neurodegenerative disorders characterized by neuronal loss. ALS is primarily characterized by motor impairments stemming from loss of motor neurons, whereas the main symptoms of FTD include changes in personality, behavior, and language stemming from loss of cortical neurons in the frontal and temporal lobes. ALS and FTD exist on a disease spectrum, where some patients present with features of both diseases. A molecular hallmark shared by almost all ALS patients and approximately half of FTD patients is the pathological aggregation of the RNA-binding protein TDP-43. It has recently been established that the solubility of TDP-43 is increased by its binding to RNA. My preliminary data utilizing in vitro aggregation assays with purified TDP-43 indicate that missense mutations in TDP-43 can alter the ability of RNA to prevent aggregation of TDP-43. Based on these findings, I hypothesize that aberrant aggregation of TDP-43 in ALS/FTD models is due to alterations in TDP-43:RNA interactions. and that directly manipulating the RNA interactions of TDP-43 can rescue disease phenotypes. The goal of this proposal is to assess if differences in the RNA interactions of TDP-43 are present in disease by 1) examining the ability of short RNAs to prevent aggregation of disease-linked TDP-43 variants in vitro and 2) determining if there are alterations in the RNAs that TDP-43 binds to in ALS/FTD patient-derived neurons versus control neurons. The proposal also aims to 3) determine if administration of short RNAs that bind TDP-43 can rescue disease phenotypes in ALS/FTD patient-derived neurons. The proposed experiments will provide critically lacking information on the interactions between TDP-43 and RNA in disease as well as strategies to target these interactions therapeutically. The proposed experiments will substantially contribute to my training, allowing me to gain expertise in new techniques such as electron microscopy, crosslinking immunoprecipitation (CLIP), and sequencing data analysis. My training environment will foster success for the proposal, combining the biochemical expertise of the Shorter laboratory and the exceedingly collaborative environment at the University of Pennsylvania and within the Neuroscience Graduate Group. These studies will facilitate both my scientific and career goals by supporting my evolution into an academic researcher who develops therapeutics for patients with neurodegenerative disease.

NIH Research Projects · FY 2024 · 2022-09

The innate immune system provides protection against bacterial pathogens by initiating a highly conserved cell death response that promotes pathogen clearance following the detection of pathogen-mediated perturbations. The executioner caspases-3 and -7 (Casp3/7) are activated by the initiator caspase-8 (Casp8) to induce apoptosis, an immunologically silent form of cell death. Conversely, pyroptosis, mediated by caspase-1 (Casp1), is highly inflammatory, and is associated with inflammasome activation, lytic plasma membrane pore formation by Gasdermin D (GSDMD), and secretion of IL-1 family cytokines. Yersinia pseudotuberculosis (Yp) is one of the three human pathogens in the Yersinia genus along with Yersinia enterocolitica and Yersinia pestis, the causative agent of plague. Yp utilizes a conserved type Ill secretion system to inject virulence factors, known as Yersinia outer proteins (Yops) into the host cell cytosol, to facilitate infection. While Yops are important for bacterial virulence, they also enable the host to detect the presence of the bacteria and generate an immune response. In particular, YopJ blocks inflammatory gene expression, triggering a pathway of host cell death mediated by Casp8 and involving activation of Casp1 that is independent of all known Casp1 activation regulators. Surprisingly, bulk assays show that Casp8 activation in response to Yp infection induces activation of both apoptotic Casp3/7 and pyroptotic Casp1, posing the question of how an individual cell might be simultaneously undergo two distinct forms of cell death. Surprisingly, my new preliminary microscopy analysis reveals that individual cells display morphological features of either apoptosis or pyroptosis, suggesting that individual cells undergo distinct cell fate choices masked by bulk population-based analyses. Intriguingly, Casp3 negatively regulates GSDMD, providing a built-in negative regulatory mechanism to limit pyroptosis. Intriguingly, individual Yp-infected cells vary in their levels of YopJ injection and Casp3 cleavage, suggesting that variability in YopJ injection enables cells to integrate levels of Casp3 and -1 activation, thereby regulating the choice between apoptosis and pyroptosis. These data and my preliminary findings provoke the hypothesis that Casp8 mediates a novel form of Casp1 activation, and the relative balance of Casp8-dependent Casp3/Casp1 activation in individual cells determines apoptotic and pyroptotic fates in response to immune signaling blockade. In this fellowship, I aim to use powerful single-cell-based approaches to dissect the regulation of single-cell fates during infection with Yp. In Aim 1, I will define the mechanism of Caspase-1 activation during Yersinia infection, which we previously demonstrated occurs through a pathway independent of known inflammasome components. In Aim 2, using innovative single cell-based caspase reporters, I will dissect the role of YopJ and test whether levels of YopJ injection determine the cell death pathway choice in individual cells. Understanding the molecular basis for the regulation of cell death during Yp infection will provide novel insights into anti-bacterial host defense and facilitate the development of host-directed approaches to combat infections.

NIH Research Projects · FY 2025 · 2022-09

Abstract Despite cancer impressive strides in conventional small molecule therapeutics and novel cancer immunotherapies, continues to be a devastating disease. Certain cancers are refractory to available therapies, and patients' responses show large individual variability. Cancer involves striking dysregulation of epigenetic pathways, with pharmacological approaches targeting chromatin regulators in the clinic and under development. There is also profound involvement of metabolic pathways in cancer, including fascinating nuclear-localized metabolic enzymes. These paradigm-shifting pathways represent an entirely new avenue for targeted therapies, with the potential to directly and specifically modulate cancer-related gene expression programs. However, our understanding of epigenetic mechanisms in cancer, especially as they connect to nuclear metabolic pathways, remains in its infancy. Our history of groundbreaking research revealing new chromatin biology and uncovering genomics and proteomics of the transcription factor p53, both in its role as a crucial tumor suppressor and as a ruinous oncogene, underpins our proposed novel directions. In addition, we have new findings of a chromatin-localized role of a cancer-linked metabolic enzyme directly “fueling” a histone modifying enzyme. Utilizing this background, in this revised proposal, we propose to investigate novel epigenetic regulation and its intersection with nuclear metabolism, using a variety of normal and cancer cell lines, as well as translational mouse models of cancer. We will investigate pivotal developmental- and disease-relevant DNA regulatory elements, called enhancers, which our published findings expose as crucial in p53 function, but which remain understudied in both wildtype and mutant p53 function. In addition, we have recently illuminated a with will speckles chromatin, expression. enzyme wholly novel and unanticipated mechanism of wildtype p53 associating membrane-less bodies, called nuclear speckles, to traffic p53's gene targets for enhanced expression; we dive deeply into the underlying mechanisms for wildtype and oncogenic p53, and explore alterations of in cancer. Other recent findings implicate a nuclear metabolic-epigenetic axis to coordinate, directly at metabolic enzyme production of cofactors with chromatin enzyme function to activate gene We will unravel mechanisms underlying this organization, identify new examples of metabolic coordination with epigenetic enzymes, and determinewhether the nuclear metabolic-epigenetic axis is critical to cancer. Crucially, we have developed an inhibitor targeting ACSS2, a nuclear metabolic enzyme, which presages potential new advancement in therapy. Taken together, our combined focus on roles and interactions of epigenetics and metabolism related to cancer promises to delineate novel mechanisms involved in tumor formation. This paradigm-shifting, multidisciplinary work will bridge separate but related areas of cancer biology and is thus ideal for this broad funding mechanism. Critically, results from our research will guide much-needed future treatments and significantly benefit patient populations.