University Of Pennsylvania

NIH Research Projects · FY 2024 · 2018-07

PROJECT SUMMARY/ABSTRACT This application seeks renewal of support for the University of Pennsylvania’s (Penn’s) postdoctoral training program in health services research (HSR). The program’s overall goal is to train investigators to conduct independent, rigorous, and high-impact HSR and thereby improve the quality, safety, efficiency, and equity of health care organization, financing, and delivery. Our program leverages Penn’s well-established institutions and resources dedicated to producing high-quality and high-impact HSR, including top-tier educational programs, outstanding faculty and mentorship, a robust research infrastructure, and a strong institutional commitment to HSR. Addressing the complex challenges facing the nation’s healthcare system requires diverse perspectives and partnerships across multiple academic disciplines. In recognition of this, we have designed our post-doctoral training program for both clinicians seeking to become clinician-researchers by acquiring HSR skills, as well as PhD-trained economists seeking to apply their methodological expertise to the field of HSR. Our program not only develops these competencies for both trainee groups, but also, by linking clinician- researchers with PhD-trained postdocs in Penn’s collaborative and interdisciplinary HSR training environment, we aim to develop leaders who can build diverse and inclusive investigator teams that combine disciplinary and institutional expertise. Our T32 program will continue to be administered by Penn’s Perelman School of Medicine, which houses one of our training program’s key educational curricula—the Master of Science in Health Policy Research—and will closely collaborate with Penn’s Leonard Davis Institute for Health Economics (LDI), which runs the second key training program component—LDI’s postdoctoral program for health economists. The Perelman School and LDI have a long and robust history of successful collaborations. This program will also continue to draw on additional faculty expertise and mentoring from Penn’s Wharton School, Penn’s School of Nursing, and the Children’s Hospital of Philadelphia. Together these entities are well suited to provide the highest quality training environment to both clinician-researchers and PhD-trained economists seeking to become independent and influential health services researchers. Thus, our HSR training program capitalizes on Penn’s unique strengths and long history in fostering cutting-edge research across its schools and disciplines. Our program’s graduates will have the necessary skills to address the health care delivery system’s most pressing problems and make health care organization and delivery higher quality, safer, more efficient, and equitable.

NIH Research Projects · FY 2025 · 2018-07

The goal of this Midcareer Investigator Award in Patient-Oriented Research is to enhance Dr. Scott Halpern’s ability to mentor students, residents, post-doctoral fellows, and junior faculty in developing and testing strategies to improve fairness in the enrollment and analysis of randomized clinical trials (RCTs) among patients with acute and chronic respiratory failure. During his initial K24, Dr. Halpern completed 3 scientific aims that help augment the efficiency of such RCTs, and 3 career development goals that made him a more effective mentor. Milestones completed during the initial K24 include his primarily mentoring 7 new individuals to receive NIH K awards and 8 mentees to earn 14 R01 or equivalent independent research grants. This renewal K24 proposes new scientific aims that will improve the conduct and analysis of respiratory failure RCTs while helping Dr. Halpern develop new skills in training more junior mentors, building infrastructures that catalyze successful mentorship, and developing and applying fair models to predict outcomes and guide care for patients with acute and chronic respiratory failure. The new scientific aims of this K24 renewal will leverage the resources and patients enrolling in several new initiatives of Dr. Halpern’s Palliative and Advanced Illness Research (PAIR) Center. Aims 1a and 1b will test the hypotheses that using mobile patient recruitment strategies and behavioral economic approaches to framing consent decisions will improve the representativeness of respiratory failure RCTs. These aims will leverage parallel insights being developed through his recently established Behavioral Economics to Transform Trial Enrollment Representativeness (BETTER) Center, funded by the American Heart Association to promote more representative enrollment in cardiovascular RCTs. Dr. Halpern will also lead qualitative interviews among patients with acute and chronic respiratory failure engaged in prospective cohort studies led by several of his mentees. Aims 2a and 2b will use patient-level data from 15 RCTs completed by the PAIR Center or NHLBI’s ARDSNet and PETAL Networks to determine whether the 7 predictive models most commonly used for risk adjustment in respiratory failure RCTs perform fairly across cohorts defined by demographic characteristics. These latter aims will provide essential insights for Dr. Halpern’s team’s future efforts to develop more accurate, consistent, and balanced models. Renewal of this K24 award would enable Dr. Halpern to quell growth in his administrative responsibilities and thus (1) maintain the time he currently commits to mentoring, (2) attend meetings to learn from more senior mentors and research leaders and to train his junior faculty mentees to develop their own mentoring skills, and (3) grow his national impact on POR mentoring by supporting his training in research leadership and growth of the “Junior Faculty Visiting Professor Program” he established under his original K24.

NIH Research Projects · FY 2026 · 2018-07

Abstract Eukaryotic genomes encode genetic information in their linear sequence, but appropriate expression of their genes requires chromosomes to fold into complex and spatially distinct three-dimensional structures. Recent advances in genomic-based approaches have uncovered a hierarchy of DNA interactions, from small chromatin loops that connect genes and enhancers to larger chromosomal domains and nuclear compartments. However, despite the remarkable conservation of these organizational features and their impact on gene function, we have a very limited understanding of how chromosomes are spatially partitioned, functionally packaged, and relatively positioned in the nucleus. Technical limitations have also hindered our ability to ask questions regarding cell-to-cell variability and the relationship between chromatin folding, positioning, and function at single cell resolution. Our previous studies involved the development of two technologies that use fluorescent in situ hybridization (FISH) to interrogate chromosome positioning at single-cell resolution. Our goal is to build on this work and use these tools to elucidate how chromosomal segments find each other and then form stable interactions within cells. To this end, we have developed a rapid and precise method for identifying candidates involved in chromosome interactions. We now propose to employ this technology to isolate novel architectural proteins, which will be followed by a battery of genomic and in situ-based assays to characterize the candidates. Collectively, the studies proposed here will uncover novel molecular mechanisms underlying nuclear organization, providing a new avenue to study how chromatin folding and positioning is established and inherited, and how dysfunctional organization contributes to disease.

NIH Research Projects · FY 2025 · 2018-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. The overall objective of this training program is to identify, motivate, and train the next generation of neuroscientists and neuroengineers in Computational Approaches to the Neuroscience of Audition and Communication (CANAC). This objective maps elegantly onto the 2023-2027 NIDCD Strategic Plan and its Theme Areas. In particular, this training program acts at the interface between (a) Theme 1’s Goals #1 and #2, which identify and characterize different cell populations in both peripheral and central regions and the neural circuits involved in sensory processing and (b) Theme 2’s Goal #3 to develop in silico (computer) models to enable insights into normal and disordered function. These Theme Areas require not only rigorous experimental manipulations but also sophisticated computational theory to understand the generated data and to make testable predictions for future science. As such, we propose to continue our T32 training program in order to train the next generation of scientists, who are grounded in the experimental neuroscience of auditory and communication systems and versed in theory and computation. A unique aspect of our training proposal is its integrative philosophy, which leverages a highly collaborative and cross-disciplinary approach to auditory and communication science fostered by faculty on the Penn campus: predoctoral students will master techniques from a variety of traditional fields to become independent investigators vested with skills in both experimental and computational neuroscience. Our program curriculum includes core and elective courses designed to achieve this breadth of knowledge. Fundamental to the goals of our program, all trainees receive cross-disciplinary training: each trainee will be formally co-mentored by two faculty members, one whose expertise is computational and another whose expertise is in the experimental neuroscience of auditory and communication systems. We have devised a sophisticated evaluation team to keep track of progress and outcomes, and plan a comprehensive training program, including coursework, a reading group, journal clubs, seminar series, and an annual retreat. Accepted trainees will receive two years of funding. We will instruct all trainees in the responsible conduct of research. Together, these activities comprise an integrative, cross-disciplinary training program that will develop a talented pool of trainees to become long- term leaders in the field of auditory and communication neuroscience.

NIH Research Projects · FY 2026 · 2018-06

Project Summary/Abstract: Human herpesvirus(HHV)-8-negative, idiopathic multicentric Castleman disease (iMCD) is a deadly hematologic illness involving polyclonal lymphoproliferation and multiple organ system dysfunction. iMCD is diagnosed in approximately 1,500 individuals annually in the USA; 35% die within 5 years. Limited options exist beyond cytotoxic chemotherapies for the 66% of patients refractory to interleukin-6 (IL-6) blockade with siltuximab; relapse is common. The etiology, pathological cell types, and dysregulated signaling pathways are poorly understood. Improved understanding of disease mechanisms is necessary to identify new treatments. In the previous funding cycle, we demonstrated that iMCD patients display increased mTOR signaling in lymph nodes and activated T cells in circulation. Inhibiting mTOR signaling with sirolimus led to a decrease in activated T cells and clinical improvement in a subset of patients. However, the signals leading to increased mTOR activation and alternative pathways involved in treatment-refractory patients remain unknown. We propose that dysregulated cytokine signaling could be responsible for the increased mTOR signaling in immune cells and hyperinflammation in iMCD. Our recent preliminary data suggest that T cells from iMCD patients in remission display increased pS6 expression, a read-out of mTOR activation, upon stimulation with Type I interferon (IFN-I) and IL-6 compared to healthy controls. Given that IFN-I and IL-6 signal through JAK1 and JAK2, we hypothesized that JAK1/2 inhibition could abrogate the increased mTOR activation induced by IL-6 and IFN- I. Indeed, in vitro JAK1/2 inhibition by ruxolitinib abrogated the increased mTOR activation in iMCD patient T cells. Based on these in vitro results and proteomics data indicating enrichment of JAK-STAT3 signaling across iMCD patients, we administered ruxolitinib to a highly treatment refractory and critically ill pediatric iMCD patient, who has been in complete remission for 24 months, 20-fold longer than her previous average remission duration. We hypothesize that JAK1/2 signaling is a central mediator of mTOR activation and iMCD pathogenesis, JAK-mediated hypersensitivity to cytokine stimulation is the mechanistic basis, and ruxolitinib interrupts iMCD by inhibiting JAK1/2, T cell activation, and mTOR. In Aim 1, we will study the activation of JAK1/2 and mTOR signaling in iMCD patient samples during flare. In Aim 2, we will rigorously evaluate how iMCD patient T cells respond to cytokine stimulation with IFN-I and IL-6 in vitro and establish the mechanistic link between JAK1/2 and mTOR signaling. Based on our preliminary data, we hypothesize that IRS1 phosphorylation downstream of JAK1/2 can lead to PI3K/AKT and mTOR activation. Aim 3 outlines a mechanistic study of ruxolitinib in iMCD patients to investigate JAK1/2 signaling in vivo. The proposed studies will advance understanding of dysregulated signaling pathways and cell types in iMCD and may lead to a new treatment paradigm for iMCD and related disorders. Using clinical and discovery data to uncover a novel use for an existing drug is critical to identify therapies for the 95% of rare diseases with no FDA-approved therapy.

NIH Research Projects · FY 2025 · 2018-06

Females are predisposed for developing systemic lupus erythematosus (SLE), but the underlying mechanisms remain obscure. Chronic inflammation is also a feature of SLE, and the majority of SLE patients have elevated type I interferon (IFN) levels and increased expression of interferon signature genes. B cells from female SLE patients exhibit aberrant expression of X-linked immunity-related genes, including TLR7, TASL, IRAK1, TIMP1, and BTK, suggesting that dysregulation of X-linked gene expression may contribute to the female bias of this disease. While Type I IFN has pleiotropic effects on the immune system, it also upregulates TLR7 expression, raising the intriguing possibility that chronic inflammation from elevated type I IFN further exacerbates dysfunctional X-linked gene expression to promote disease in female SLE patients. Females have two X- chromosomes and equilibration of X-linked gene dosage to that of males (XY) occurs by X-Chromosome Inactivation (XCI), maintained by Xist RNA, heterochromatic modifications, and a compact 3D chromosome architecture. We have shown that activated B cells from SLE patients and a female-biased mouse model of lupus (NZB/W F1) have reduced accumulation of Xist RNA and heterochromatic marks on the Xi, and abnormal expression of some X-linked genes in naïve and activated B cells. Impairments with XCI maintenance, using a B cell specific Xist deletion (Xist cKO) mouse model, revealed that female Xist cKO mice injected with pristane have elevated autoantibody production and increased numbers of germinal center B cells (GCs) and age- associated B cells (ABCs). Notably, type I IFNs can trigger rapid rearrangement of 3D chromosome compartments that govern gene expression and prior studies revealed that CD4+ T cells from SLE patients have widespread 3D chromosome architecture changes that was associated with differential gene expression, including that of TLR7. Based on these data, our central hypothesis is that chronic inflammation that accompanies SLE alters the enrichment of heterochromatic histone marks and spatial organization of the Xi in B cells, resulting in abnormal X-linked gene expression of TLR7, TASL and other X-linked immunity genes. We will test our hypotheses with the following aims: (1) How does chronic inflammation from elevated Type I IFN impact Xi gene expression, heterochromatic epigenetic modifications across the Xi, and Xi spatial organization in mouse B cell subsets in a lupus-like disease model? (2) Is there aberrant biallelic transcription of X-linked genes, altered enrichment of active and silent epigenetic modifications, and disrupted 3D architecture of the X chromosome in circulating B cells from SLE patients with high vs low type I IFN levels? IMPACT: Our novel and innovative genetic and molecular approaches will yield unprecedented mechanistic insight on the influence of chronic inflammation on the epigenetic mechanisms of XCI maintenance, and will enable the identification of new molecular pathways and targets of female-biased autoimmune disease that could be amenable for therapeutic intervention.

NIH Research Projects · FY 2025 · 2018-06

PROJECT SUMMARY Association cortex undergoes protracted development throughout childhood and adolescence. This extended window of association cortex plasticity is understood to enhance executive and socioemotional functioning, whereas experiences that diminish plasticity—such as environmental adversity—confer risk for psychopathology. At present, the biological origins of prolonged association cortex plasticity in humans remain under characterized, precluding a mechanistic understanding of how neurodevelopmental malleability interacts with the environment to foster either resilience or psychiatric vulnerability. Animal studies of cortical plasticity have identified maturational increases in inhibitory neurotransmission and cortical myelination as two key biological regulators of plasticity. As inhibition and myelination increase and the murine cortex transitions from plastic to mature, intrinsic cortical activity transitions from widespread and synchronized (producing high amplitude neural recordings) to suppressed and sparse — producing low amplitude recordings. This development-linked shift in intrinsic activity amplitude thus provides an animal model-informed, functional readout of local circuit plasticity. We recently leveraged this functional marker in humans and found that declines in the amplitude of intrinsic fMRI fluctuations (termed fluctuation amplitude) were coupled to the maturation of cortical myelin and temporally unfolded along a hierarchical, sensorimotor-association cortical axis (Sydnor et al., Nature Neuroscience 2023). Here we will map the normative progression of developmental plasticity from sensory to association cortex and link precocious reductions in association cortex plasticity to transdiagnostic overall psychopathology. Building upon our initial work examining plasticity measures in one cross-sectional dataset, we propose to generalize our findings to two additional cross-sectional datasets (the HCP-Development and the Healthy Brain Network; total n=6,530) and map within-participant change using the ABCD study (n=11,563). These datasets will allow us to comprehensively map the development of our functional measure of plasticity (Aim 1) and link it to both the development of a major plasticity restricting factor (intracortical myelin; Aim 2). Next, we will determine whether lower socioeconomic status (SES) is associated with accelerated closure of plasticity in association cortex (Aim 3), and finally delineate links to trans-diagnostic overall psychopathology (Aim 4). This research program capitalizes upon a highly successful first project period (>60 publications) and robust preliminary data and a highly cohesive team of UPenn investigators with expertise in neurodevelopmental psychopathology and neuroinformatics (Satterthwaite), network science and machine learning (Bassett), imaging statistics (Shinohara), psychometrics (Moore), and developmental psychology (Mackey). Together, this innovative proposal will provide compelling evidence that development of association cortex plasticity is critical for transdiagnostic psychopathology.

NIH Research Projects · FY 2026 · 2018-05

Human betacoronaviruses induce a wide spectrum of respiratory disease. MERS-CoV (MERS) and SARS-CoV- 2 (SARS-2), cause severe lethal pneumonia, while SARS-2 can also cause mild to asymptomatic disease in some individuals. In contrast, infections with the “common” respiratory human beta-CoV OC43 are largely limited to the upper respiratory tract. With only a few drugs that show promise against human CoVs, it remains imperative to develop therapeutics for current and future emergent human CoVs. For this, we need to identify and understand the host-virus interactions common to all or different among these viruses. A major focus of our lab has been on the double-stranded (ds)RNA induced host responses to beta-CoVs: interferon production and signaling; oligoadenylate synthetase-ribonuclease L and protein kinase R (PKR), that are both antiviral and pro- inflammatory. We have shown that the conserved CoV replicase encoded nsp15 endoribonuclease (Endo)U reduces dsRNA accumulation and activation of all three pathways while subgenera specific accessory proteins add to the antagonism. Merbeco (MERS-CoV) and embeco (OC43) viruses shut down these pathways effectively; in contrast, the sarbecovirus, SARS-2, replicates in respiratory derived cells despite activation of all three. Preliminary data suggest that downstream effects of RNase L on inflammation and cell death may be more impactful during SARS-2 infection than the modest antiviral effects. PKR and PKR-like ER kinase (PERK), both kinases of the integrated stress response, when activated, phosphorylate protein synthesis initiation factor eIF2a, leading to protein synthesis attenuation, providing an important control point in infection. Activation of PERK also initiates a pathways of the unfolded protein response (UPR) to ER stress, which produces antiviral effects and inflammation. We found that MERS and OC43 infection suppress phosphorylation of eIF2a by preventing PKR activation and through a PERK pathway feedback loop, dephosphorylating eIF2a. In contrast SARS-2 promotes p-eIF2a through both PKR and PERK, but maintains viral protein synthesis. This may occur in part via the CoV replicase nsp1 protein, which promotes selective attenuation of host, but not viral, protein synthesis. Our overall hypothesis is that betacoronaviruses of each subgenus have evolved unique ways to interact with host responses to optimize replication and spread and that these differences influence pathogenic outcomes. We propose to determine: the role of the conserved CoV EndoU and subgenera specific accessory proteins in antagonizing host innate response pathways; the impact of OAS-RNase L activation on viral replication, inflammation and cell death; and the effects of host PKR/PERK and viral nsp1 on control of protein synthesis and virus replication. Our overall goal is to understand the similarities and differences in host interactions among beta-CoVs of three subgenera. Our findings may identify novel therapeutic interventions targeting host or viral proteins and indicate whether they may work as pan-CoV therapies or be unique to each beta-CoV subgenus, and thus help prepare for future CoV outbreaks.

NIH Research Projects · FY 2026 · 2018-05

SUMMARY We propose to determine i) the specific effects on eukaryotic translation of posttranscriptional modifications (PTMs) of mRNA and tRNA and ii) how readthrough of premature termination codons (PTCs) is modulated by downstream sequence context and stimulated by translation readthrough inducing drugs (TRIDs). In so doing we will contribute to the development of new therapeutic approaches for treatment of diseases which are linked to PTMs and PTCs. Our approach is to apply a reconstituted in vitro system, denoted PURE‐LITE, to elucidate mechanisms of elongation and termination using a combination of ensemble and single molecule approaches. Such studies can aid in interpreting the results of related cellular and clinical studies and suggest new directions to pursue. Our Specific Aims are: 1. Determine the effects of mRNA and tRNA posttranscriptional modifications on elongation and termination. m6A, pseudoU (,inosine (I) and m5C are among the most common PTMs of eukaryotic mRNA. There currently are no direct measurements of how these PTMs affect cognate elongation, misreading, readthrough and termination. We will determine which modifications have the most pronounced effects on these processes and identify which steps during elongation and termination are responsible for these effects, which are the most likely to be involved in altering translational functions in cells. We will prepare mRNAs containing modifications in sense or nonsense codons, pair these modified codons with cognate and near‐ cognate tRNAs for the three elongation processes, and compare the rates and stoichiometries of elongation with those obtained with the corresponding unmodified mRNAs and tRNAs. tRNAs will be selected to examine how different modifications of nucleotides 34 and 37 of the anticodon loop affect assay results. Similar studies for termination will compare modified and unmodified mRNAs. 2. Determine the effects on readthrough of UGA codons of downstream sequence context and TRID termination inhibitors. There is strong evidence that UGA PTCs are the most susceptible to readthrough and that sequences immediately downstream from a UGA PTC influence readthrough efficiency. Our results have demonstrated that ataluren, the only TRID currently approved for treating patients with a PTC disease, acts exclusively by competitively inhibiting release factor complex (RFC). Ataluren shows variable results in stimulating readthrough, as measured in cell‐based assays and clinical trials. We hypothesize that this variability may be a consequence of how PTC downstream sequence affects RFC activity. We will test this hypothesis by determining how closely ataluren inhibition of RFC as a function of downstream sequence correlates with ataluren stimulation of readthrough activity. Such tests will be repeated with ataluren added in combination with agents which stimulate readthrough by a mechanism orthogonal to that of ataluren, potentially leading to synergistic results. The most effective combinations will also be tested in cellular assays. In parallel experiments we will seek to identify TRID termination inhibitors more potent than ataluren, via high throughput screening with a plate reader assay, virtual screening and medicinal chemistry. These parallel efforts should improve the treatment of PTC‐based diseases and the selection of patients most likely to benefit from such treatments.

NIH Research Projects · FY 2026 · 2018-05

Molecular motors drive the active transport of organelles along the cellular cytoskeleton. Organelle transport is critically important in neurons, cells that extend axons reaching up to 1m in length. Axons have limited capacity for biosynthesis and degradation, thus axonal transport is required to supply newly synthesized proteins and organelles and to remove aging proteins and dysfunctional organelles. Accumulating evidence supports a cargo-specific model for axonal transport, in which the opposing activities of kinesin and cytoplasmic dynein motors are regulated by a distinct complement of regulatory proteins including scaffolding proteins and activating adaptors. We are interested in the mechanisms that regulate the transport of key organelles including mitochondria, autophagosomes, and synaptic vesicle precursors. We are also interested in the mechanisms that lead to site-specific delivery, such as the targeting of newly synthesized synaptic components to presynaptic sites along the axon. We hypothesize that this delivery is dependent on the localized regulation of cytoskeletal dynamics and organization, which directly affect the initiation and termination of cargo motility. Finally, we are interested in the mechanisms by which molecular motors and cytoskeletal dynamics actively remodel organelle membranes, leading to tubulation, fission and fusion. We tackle these questions using the synergistic approaches of live cell imaging and in vitro reconstitution with single molecule resolution. We will continue to focus on three major goals. Goal 1: Understanding the integrated regulation of organelle transport. Each type of organelle transported along the axon has a distinct pattern of motility that directly relates to its function. We seek to understand the specific mechanisms involved, focusing on essential axonal cargos, such as mitochondria and autophagosomes, testing the model that the cargo-specific, integrated regulation of motors allows for sustained transport over long time scales and distances. In Goal 2, we seek to understand the localized regulation of organelle dynamics within defined axonal zones, such as the delivery of synaptic vesicle precursors to presynaptic sites along the axon. These zones exhibit distinct patterns of cytoskeletal organization and cytoskeletal dynamics. We are interested in the mechanisms that enhance the rate-limiting step of transport initiation and control cargo delivery/retention at specific sites of cellular need. And in Goal 3, we will study organelle remodeling driven by molecular motors and/or cytoskeletal dynamics. Organelles such as mitochondria undergo dramatic remodeling via mechanisms including fission and fusion. We hypothesize that molecular motors and cytoskeletal filaments provide an adaptable toolbox that can be specifically tuned to regulate dynamic organelle morphology. Together, these approaches will provide important new insights into organelle dynamics in neurons. As deficits in axonal transport lead to neurodegeneration, we hope that our progress may provide new opportunities for targeted and effective therapeutic approaches.

NIH Research Projects · FY 2026 · 2018-03

Project Summary The Food and Drug Administration's approval of Luxturna marked a new era in the fight to cure blindness caused by inherited retinal degenerations (IRDs). However, despite an approved treatment for RPE65- mediated disease, there are still over 280 molecularly distinct, currently untreatable IRDs that cause vision loss from progressive death of the rod, cone, and retinal pigment epithelial (RPE) cells. Further, treatment with Luxturna does not fully restore vision to normal levels in RPE65-mediated disease, particularly for foveal cone vision, and there is variability between patients in the amount of vision gained post-treatment. Numerous therapeutic approaches, including gene therapies similar to Luxturna, are under development for IRDs. To maximize chances of success, it is critical to understand the natural sequence of degeneration in each disease, both to optimize the timing and retinal location of applied therapies as well as to enable precise evaluation of whether the therapies had an effect. We will use multi-modal adaptive optics scanning light ophthalmoscopy (AOSLO) to simultaneously observe the cone inner segment (IS) and waveguiding outer segment (OS) mosaics and RPE mosaics to identify AO structural phenotypes in IRDs. We will also use AO microperimetry and optoretinography to test photoreceptor function in the same IRD patients. Finally, we will measure the AO structural and functional responses to Luxturna gene therapy for RPE65-mediated disease and AAV2.hCHM gene therapy for choroideremia. Based on our preliminary and published data, we hypothesize that cone function correlates with structural phenotype, cones exhibit dysfunction prior to their structural loss, gene therapy will slow or halt structural degeneration, and gene therapy will reverse photoreceptor dysfunction at retinal locations where cells are structurally intact but functionally compromised. The information gained through our studies will enable cellular scale assessment of the safety and efficacy of gene therapy and the will validate AO cellular scale outcome measures for use in future clinical trials.

NIH Research Projects · FY 2026 · 2018-03

Despite progress over the past 50 years in lowering the rate of smoking, tobacco use remains the leading cause of premature death in the US. To ensure the sustained and impactful research dedicated to addressing tobacco use, rigorous inter-disciplinary mentoring programs are needed to prepare the next generation of researchers committed to patient-oriented research (POR) in this area. I have led a successful research program focused on testing methods to improve the use and effectiveness of treatments for tobacco for 21 years. For the first cycle of my K24, my primary mentorship goal was to train in mentorship and to formalize and enhance the mentoring activities that I had been engaged in from 2001-2017 into a structured, consistent, and comprehensive mentoring program in tobacco POR for students, fellows, and junior faculty. With K24 support over the past 4 years, I provided training to 21 new mentees, yielding 50 papers with mentees and 7 papers under review or in revision (>230 total by mentees) and 32 grants, including 5 Ks and 11 R01s, 3 as a New Investigator PI, compared to 25 mentees, 34 publications with mentees, and 2 Ks and 2 R01s in the preceding 17 years. For the first cycle of the K24, my primary research goal was to augment my tobacco POR by building expertise in implementation science through training and by pilot testing use of the nicotine metabolite ratio (NMR) – a genetically-informed biomarker of nicotine metabolism rate – for personalizing tobacco treatment as an implementation strategy to increase use of tobacco treatments. K24 support over the past 4 years, led to six new grants in tobacco and implementation science, 4 focused on testing the NMR in clinical practice: HIV care, primary care, and in-patients (R01 CA243914; U54 GM104941; P50 CA244690; R01 HG012670; SAP#4100083101; R01 DA056050). I leveraged this research in implementation science to augment my mentoring by serving as faculty on two national implementation science training programs – NCI's Training Institute for Dissemination and Implementation Research in Cancer and Washington University at St. Louis's Institute for Implementation Science Scholars – and by serving as co-chair for the Cancer Center Cessation Initiative's Implementation Science Working Group. In this renewal, I will: 1) sustain the mentoring program in tobacco and implementation science POR that I established with the first K24 cycle, using a structured, comprehensive, and individualized approach for an additional 15 mentees; and 2) augment our tobacco and implementation science POR by conducting a pilot study to develop messages informed by behavioral economics for HIV+ patients and infectious disease clinicians that are related to intention to refer for (clinician) or engage with (patient) tobacco use treatment and then test the impact of these messages integrated into the electronic health record as implementation science strategies to increase actual referral for and engagement in tobacco treatment in the HIV clinical setting. This renewal will ensure that I continue to provide the mentoring needed to build the next generation of scientists to address tobacco use and to continue to grow my impactful research in tobacco control and implementation science.

NIH Research Projects · FY 2025 · 2017-09

Project Summary: Glioblastomas (GBM) are among the most lethal of all human cancers, with a median survival of around one year. Vasogenic edema is a universal problem for GBM patients, and corticosteroids are commonly used perioperatively to control cerebral edema and are frequently continued throughout subsequent treatment, notably radiotherapy (RT), for the amelioration of side effects. Vasogenic edema is a significant cause of morbidity in patients with both primary and metastatic brain tumors, both from the direct impact of edema and from indirect effects related to the requirement for chronic corticosteroid use to manage it. In general, the mechanism of edema formation is still unclear. We have shown that dexamethasone (DEX) administration significantly compromises RT efficacy, together with emerging new therapies that augment the immune response to treat brain tumors. Although we and others have identified antibodies and agents targeting vascular endothelial growth factor (VEGF) at high anti-cancer doses that effectively reduce edema, their use is associated with increased infiltration of tumor growth-promoting macrophages and development of resistance. Our preliminary and published work identified the pro-inflammatory IL-1β from blood-born monocyte/monocyte-derived macrophages (MDM) as a downstream target of DEX in exerting its anti-edema effect. We have shown that targeting IL-1β is as sufficient as DEX in reducing edema but does not compromise RT efficacy. In contrast to anti-VEGFA antibody treatment, targeting IL-1β does not induce increased infiltration of tumor-promoting TAMs; on the contrary, it decreases the number of tumor-promoting TAMs and reduces the number of exhausted CD8+ T-cells. This application thus aims to use three distinct genetically engineered mouse models of GBM that exhibit significant differences in TAMs and anatomical structures of the brain-blood barrier (BBB), together with human primary GBM patient-derived xenograft models, in combination with cell type specific IL-1β ablation strategies to determine detailed mechanisms of IL-1β effects on vascular permeability and edema formation in GBM. The proposed studies will provide new mechanistic insights into the fundamental cellular and molecular biology of IL- 1β in glioblastoma. The proposed studies test whether targeting IL-1β is a promising novel anti-edema therapy for heterogeneous GBM with dual efficacy –against edema (in our renewal application) and neoplastic growth that we demonstrated in our ongoing R01.

NIH Research Projects · FY 2024 · 2017-09

Scientific and medical data are growing exponentially, in terms of size, complexity, and sheer number of datasets. The challenge lies in how to best tap this plethora of data, to build explanatory and predictive models of living systems, and to discover the biomarkers that will lead to improved medical diagnoses and treatments. The critical bottleneck lies in software support for enabling discovery. The next generation software platform for scientific discovery will require a focus on sustainability, security, user-experience, and accessibility. The Blackfynn Platform directly addresses these needs, enabling team science for big data. Our platform provides a single seamless point of data access and management for multi-modal data. It enables rapid viewing and search, integration and linking of data across modalities, followed by analysis via custom or standardized tools. Our commitment to the SPARC program is focused on three main thrusts: - Develop a cloud-based scientific data management platform tailored to the needs of SPARC investigators. Blackfynn will expand its platform to support the specific needs of the SPARC investigators and develop infrastructure to integrate with deliverables from the SPARC SIM-CORE and MAP-CORE. - Drive collaborative efforts with the other SPARC cores and investigators and promote interoperability and seamless integration of systems. Blackfynn will develop processes and tools to support coordination within the SPARC program. We will create an open architecture and enhance our platform to foster collaboration and tool interoperability. - Develop a sustainable ecosystem for the neuroscience community outside and beyond the SPARC initiative. Efforts in this project will be lost without a clear strategy to expand use of the selected platform(s) beyond the SPARC effort. We will work with the SPARC team to develop solutions to guarantee sustainability and develop an open data exchange platform that can be used by anyone to find and access public data hosted on the Blackfynn platform at cost.

NIH Research Projects · FY 2025 · 2017-09

Abstract The broad, long-term objective of this project concerns the development of novel statistical methods and computa- tional tools for statistical and probabilistic modeling of human microbiome and shotgun metagenomic data motivated by important biological questions and experiments. As we move to the next phase of microbiome research, it has become increasingly evident that lack of methods suitable for analyzing such large-scale microbiome data has emerged as a bottleneck to effectively understand the functions and dynamics of microbiota. There is a pressing need to develop statistical and computational methods for large-scale shotgun metagenomics data analysis in order to accelerate in- novations in microbiome data science. This project aims at narrowing this gap by developing new statistical models, novel inference procedures, and fast computational algorithms. The specific aims of the current project focus on two important aspects of microbiome data analysis: (1) developing new statistical methods and fast computational algo- rithms for phylogenomic-based analysis of metagenomic sequencing data in large-scale human microbiome studies; (2) developing statistical methods and inference procedures for quantifying and comparing the potential energy land- scape and stability of microbial communities. Under each of these two broad aims, several related statistical methods will be developed to address the key questions of how to perform phylogenomics-based microbiome analysis and how to quantify and link microbial community stability to disease risk and progression. These problems are all motivated by the PI's close collaborations with Penn investigators on metagenomic studies of Crohn disease, childhood obesity and disease progression among patients with chronic kidney disease (CKD)). Specifically, this project will develop meth- ods for phylogenomics-based association analysis using a set of universal marker genes, phylogenetic-Ising models and change-point phylogenetic-Ising models for assessing the microbial community energy landscape and stability, and time-invariant Ising models for understand consensus taxon-taxon interactions based on longitudinal microbiome studies. The new methods can be applied to both 16S rRNA and shotgun metagenomic sequencing data and will ideally facilitate the identifications of microbial composition, community stability and microbial networks underlying var- ious complex human diseases and biological processes. The project will also investigate the robustness, power and efficiencies of these methods and compare them with existing methods. Finally, this project will develop practical and feasible computer programs for the implementation of the proposed methods, and for the evaluation of the performance of these methods through extensive simulations and analysis of various on-going microbiome studies through the PI's collaborations with Penn physicians and biologists. All programs developed under this grant and detailed documenta- tion will be incorporated into our current software and made available free-of-charge to interested researchers.

NIH Research Projects · FY 2026 · 2017-09

Cell migration is a critically important process in key biological events such as tissue morphogenesis, immune response, and cancer metastasis. Directional migration of multiple cell types depends on the dense actin network that rapidly forms at the cell leading edge and facilitates its protrusion via polarized elongation of the actin filaments. Our published studies revealed two novel interconnected determinants of the function of actin at the cell leading edge: actin's nucleotide coding sequence and actin arginylation. Using integrated approaches that combine protein biochemistry, cell biology, and mouse transgenesis, work from my lab demonstrated that arginyltransferase (ATE1), the enzyme that arginylates proteins, specifically regulates the function of actin during cell migration and contributes to virtually every physiologic process involving long-range migration and tissue remodeling in mice. These studies drive my research program, which aims to characterize the novel mechanisms of actin regulation by nucleotide coding sequence and arginylation. Our recent data show that N-terminal arginylation of the leading edge actin is a dynamic event that exhibits a rapid response to extracellular stimuli and is essential for maintaining cell migration speed. Moreover, arginylation is highly specific to the ubiquitous and essential β-actin isoform but not to the closely homologous γ-actin in the same cell types. Remarkably, this specificity is determined at the nucleotide level by the mRNA coding sequence, which is responsible for the differential translation rates of different actin isoforms, exerting downstream effects on their folding rates and co-translational ubiquitination. This novel actin regulatory mechanism targets incorrectly arginylated actin isoforms for degradation and ensures that only the fast­ accumulating β-actin becomes arginylated in cells. Thus, actin arginylation at the cell leading edge is a tightly regulated process that is genetically encoded in its nucleotide sequence, suggesting that arginylation is the primary level of actin regulation that occurs prior to any other actin-dependent event. Uncovering the essential steps of this regulation in actin function and coordination of cell migration in vivo constitutes my long-term research goal.

NIH Research Projects · FY 2025 · 2017-09

We extend our structural mechanistic studies into eukaryotic transcription to determine the functions of the conserved and essential Mediator complex during assembly and disassembly of the RNA polymerase II (pol II) transcription pre-initiation complex (PIC) and in the divergent transcription that arises from pol II promoters. Building on our recent success in developing highly efficient in vitro reconstituted transcription system, we will leverage this system to isolate and dissect a series of PIC-Mediator complexes representing a range of states, from initiation, to early elongation, to complexes that might function on divergent promoters. We will address the molecular mechanisms underlying transitions between these states through structural and biochemical means involving cryo- EM, cross-linking and mass spectrometry (XL-MS), and multi-wavelength analytical ultracentrifugation.

NIH Research Projects · FY 2026 · 2017-09

PROJECT SUMMARY DNA packaging into chromatin mediates chromosome segregation, telomere protection, and genome integrity, among other essential, conserved cellular processes. However, many chromatin proteins are strikingly unconserved—domains and residues evolve rapidly between even closely related species. This paradox of conserved, chromatin-dependent functions supported by fast-evolving chromatin proteins suggests that some essential cellular processes require recurrent innovation. The biological significance of this paradox is poorly understood, and yet aberrant chromatin packaging is a hallmark of cancer, infertility, and aging. We hypothesize that the pervasive rapid evolution of chromatin proteins is driven by the exceptionally rapid evolution of DNA repeats, including transposons and DNA satellites. Specifically, we hypothesize that chromatin proteins and DNA repeats antagonistically coevolve: repetitive DNA evolution imperils essential chromatin functions, triggering reciprocal evolution of chromatin proteins to restore these essential chromatin functions. Repeated bouts of DNA repeat evolution and chromatin protein adaptation result in exquisitely coevolved, species-specific components of the genome. To probe this model of antagonistic coevolution, we conduct evolution-guided functional analysis: we swap into a focal genome a diverged chromatin protein from a closely related species, generating an "evolutionary mismatch" between the contemporary DNA repeats of one species and a contemporary chromatin protein of another species. Upon swapping a diverged version of a transposon-silencing protein into Drosophila melanogaster, we triggered transposon hyper-proliferation and a consequent loss of genome integrity. Upon swapping a diverged version of a DNA satellite-associated protein into D. melanogaster, we similarly triggered a profound loss of genome integrity but through a distinct pathway. Having successfully defined the genome components engaged in antagonistic coevolution during the current funding period, we are now poised to unravel the molecular mechanisms by which DNA repeats imperil essential chromatin biology and the molecular mechanisms by which chromatin proteins mitigate these threats. Leveraging our two established systems, we will probe the evolutionary and functional diversification of two vital chromatin-mediated pathways shaped by coevolution: 1) the regulation of chromatin accessibility at genomic regions vulnerable to transposon insertions and 2) the resolution of DNA entanglements enriched in DNA satellite arrays. Our published and preliminary data also propel our investigations of how antagonistic coevolution reverberates beyond the two embattled parties, triggering secondary coevolutionary dynamics that preserve protein:protein interactions among multiple host chromatin proteins. Using transgenics, cell biology, biochemistry, and classical genetics, we will elucidate the otherwise invisible hazards of DNA repeat evolution and determine how adaptive evolution shapes and reshapes chromatin to preserve vital genome functions.

NIH Research Projects · FY 2026 · 2017-09

ABSTRACT Ants exhibit highly evolved eusocial behaviors including stark division of labor among female castes, where the queen carries out all reproduction and worker castes forage for food and defend the colony. Interestingly, and of great relevance to aging research, sterile workers are shorter-lived, with variable lifespans between distinct castes. Reproductive queens are long-lived, with lifespans differing three to ten-fold between queen and worker. Remarkably, the genomes of the sterile and reproductive castes are nearly identical, and thus differences in lifespan and behavior arise from non-genetic mechanisms. We investigate two species of ants, each with advantages for study of mechanisms linking aging with complex social behavior. In Harpegnathos saltator, loss or removal of the queen leads to altered behavior in the workers, with antennal dueling and eventual ascendance of workers to reproductive status. From a longevity perspective, the induced reproductive caste exhibits four-fold longer lifespan, thus providing a simple experimental switch to uncover important causality underlying aging. In Camponotus floridanus, there are two distinct worker castes, forager and soldier, with the soldier exhibiting a two-fold longer lifespan than the forager. These behaviors are programmed early in life, but exhibit plasticity during aging. Intriguingly, these castes can be experimentally reprogrammed from soldier-to-forager, thus providing a second paradigm to study the relationship of behavior to aging. Our overall premise is that genomic, epigenomic, and proteomic regulation—all hallmark foundational causes of aging—are at the heart of caste-differentiated lifespan disparities and relationship to caste behavior. We thus propose to utilize ants to investigate the epigenetic and physiological basis of the dramatic lifespan differences between reproductive and distinct worker castes. In H. saltator we have evidence in the long-lived reproductive caste for two mechanisms extending lifepan. First, we detect increased expression of a unique HSF (Heat Shock Factor) providing proteomic protection and longer lifespan via upregulation of the Heat Shock Response transcriptional pathway. Second, we find increased expression of a unique Ago2 (Arogonaut) that binds miRNAs that specifically target for destruction certain mRNAs that lower lifespan in short-lived workers. In C. floridanus we find that distinct chromatin-based epigenetic mechanisms are central to foraging, which is an age-linked behavior, and we can manipulate these pathways to reprogram soldier caste to forage. In the proposed research we will investigate these causal mechanisms, and then manipulate lifespan with a combination of genetic and epigenetic approaches to promote these mechanisms. The ant model system provides an exceptional opportunity to integrate social behavior with aging, and to uncover key epigenetic processes underlying universal aging pathways. Results from the research will provide fundamental knowledge about control of lifespan that can be translated to more sophisticated mammals.

NIH Research Projects · FY 2026 · 2017-09

The seemingly straightforward function of the centromere in directing chromosome segregation is difficult to reconcile with multiple complexities of the underlying molecular machinery, particularly rapid evolution of both centromere DNA and proteins and seemingly redundant pathways linking the DNA to spindle microtubules. This project focuses on centromere drive as a key to unlocking centromere complexity. Selfish centromere DNA sequences bias their transmission to the egg in female meiosis, while centromere proteins evolve to suppress fitness costs of drive while maintaining essential centromere functions. Our recent work determined how selfish centromeres interact with spindle microtubules to bias their segregation. We developed mouse model systems exploiting natural variation in mouse centromere DNA, defined tubulin detyrosination as the key post-translational modification creating meiotic spindle asymmetry, showed that microtubule-destabilizing proteins act as drive effectors exploited by selfish centromeres, established an integrated model for both drive and suppression, and sequenced Murinae genomes for molecular evolution analyses to identify rapidly evolving centromere proteins. Our progress represents crucial steps towards understanding the centromere drive conflict but leaves key gaps in our understanding of drive and suppression and centromere protein evolution, which are addressed in this proposal. First, we will determine how selfish centromeres interact with an asymmetric spindle to bias their segregation. Our previous findings suggest a hypothesis that we will test by manipulating microtubule destabilizing activities at centromeres in live cells, using chemical optogenetic approaches that we developed. Second, we will test whether genetically different centromeres differentially recruit centromere proteins, a central but untested component of the centromere drive theory. Using hybrid mouse zygotes with divergent maternal and paternal centromere satellite DNA sequences as a model system, we will determine if rapidly evolving centromere protein interact differentially with different centromere DNA sequences. Third, we will test for reproductive fitness costs associated with functional differences between centromeres, taking advantage of our hybrid mouse model systems in which paired homologous chromosomes in meiosis have divergent centromeres. Fourth, we will test the concept that centromere proteins have evolved to suppress costs due to functional differences between centromeres, which has been the most challenging part of the drive theory to address experimentally. With tractable experimental systems, a mechanistic model for drive and suppression, and molecular evolution analyses of centromere proteins in place, we will address this challenge by testing whether recurrent changes in rapidly evolving centromere proteins have functional implications consistent with our model. Overall, by investigating centromeres in the context of genetic conflict, this project represents a unique contribution to studies of chromosome segregation and inheritance, with broad consequences for reproductive biology and chromosome evolution.

NIH Research Projects · FY 2025 · 2017-08

OVERALL PROJECT SUMMARY / ABSTRACT The long-term goals of this renewal P01 are to develop next generation immunotherapy with chimeric antigen receptor (CAR) T cells and to translate this research into new therapies with curative potential for patients with blood cancer. The CAR developed at our center was the first cell and gene therapy to ever receive approval from the FDA, initially for refractory/relapsed pre-B cell acute lymphocytic leukemia (ALL) in 2017 and lymphoma in 2018. However, multiple myeloma (MM), acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) remain as the major unmet medical need in blood cancers. Our central hypothesis is that therapies with combination of CAR T cells and advanced forms of human genome editing will enable this powerful therapy to reach a broader spectrum of patients with blood cancer. We have brought together a cadre of exceptional investigators from multiple disciplines who have collaborated and published together for many years. Each disease-focused project will be led by recognized authorities in the field. To achieve our goals, we have three Projects that build on progress during the previous funding period and will continue to coordinate closely with essential shared resource cores. In Project 1, we will determine the clinical and immunological impact of treating patients on two clinical trials: (i) CAR T cells targeting CD19 will be tested with genetic disruption of CD5, CTLA- 4 and TET2 to address CLL and lymphoma, which is lack of sustained effector CAR T function in these patients. In AML, the central problem in CAR T cell therapy is the lack of a known surface antigen that is present on AML but lacking from normal hematopoiesis. The goal of Project 2 is to open a wide therapeutic window for AML by genetically-modifying normal marrow to make it resistant to killing by anti-AML CAR T cells, and delivering potent anti-leukemic CAR T cells specific for CD45. Engineered HSC that are genetically edited to install a hematopoietic system facilitating non-toxic therapy with these potent CAR T cells will be developed. In Project 3, the overall hypothesis is that anti-myeloma efficacy will be maximized by (i) testing the efficacy of marrow- derived CAR T compared to current standard of care blood-derived CAR T (ii) improving persistence of BCMA CAR T with orthogonally mutated IL-2/IL-2R technology and mRNA/lipid-nanoparticle vaccine technology to overcome suboptimal persistence and efficacy of current BCMA T cells. The Scientific and Administrative Cores for this P01 are essential for our progress including provision of project management for collaboration and biostatistics, clinical safety and monitoring, and fiscal support (Core A), a GMP facility for manufacture of cells and identification of new binders for CAR targets (Core B), and a state-of- the-art platform for GLP analysis to provide high dimensional data of the samples generated in all Projects (Core C). Our renewal application has the potential for paradigm-shifting impact to transform the lessons of CAR T for ALL into meaningful efficacy against all hematologic malignancies, solid cancers and provides direction for extending beyond cancer to autoimmune disorders.

NIH Research Projects · FY 2025 · 2017-08

PROJECT SUMMARY/ABSTRACT The Pennsylvania Animal Diagnostic Laboratory System (PADLS) at the University of Pennsylvania’s New Bolton Center offers comprehensive veterinary diagnostic services in the areas of pathology, microbiology, molecular diagnostics, and toxicology, as fully accredited by the American Association of Veterinary Laboratory Diagnosticians (AAVLD) and a first-tier laboratory member of the Veterinary Laboratory Investigation and Response Network (Vet-LIRN). Safeguarding the animals and citizens of the Commonwealth of Pennsylvania against threats to animal health and food safety, the laboratory serves as a critical Eastern United States resource, making it an excellent fit for this Vet-LIRN project proposal. Our laboratory is experienced in the analysis of a variety of sample and matrix types, including but not limited to animal samples, environmental samples, vermin, water, animal drug products, and animal and human food products and ingredients such as grain, meat, fish, and milk. We will use our laboratory’s veterinary diagnostic experience, expertise, and infrastructure to accomplish the work described for this project. In addition to addressing the need for added laboratory capacity in the event of a large-scale outbreak or incident involving animal food, drug-related illnesses, or other large-scale emergency events requiring surge capacity testing, the PADLS New Bolton Center Laboratory can further strengthen the Vet-LIRN through diagnostic activities that better enable early detection of emerging events involving national food safety and security and facilitate the rapid responses that can minimize harm and best protect both human and animal health. Our laboratory provides comprehensive veterinary diagnostic services to the Pennsylvania and the surrounding region through federal, state, and local collaborations. Some analyses are part of normal surveillance activities or routine necropsies, while others are associated with cases of excessive, unexpected, or otherwise unexplainable animal losses or illness that may potentially pose a risk to animal health and/or animal or human food safety. The laboratory adheres to AAVLD’s “Requirements for an Accredited Veterinary Medical Diagnostic Laboratory”, meeting specific requirements pertaining to all aspects of the laboratory and its activities. AAVLD also requires that our laboratory has a Quality System that documents policies, systems, programs, and procedures. Due to the varied nature of the sample matrices we receive we are constantly improving and refining our analytical methods in order to ensure their sensitivity and specificity. We are confident in our ability to continue to support Vet-LIRN in this project due to our present qualifications and experience and a proven history of successful Vet-LIRN collaborations through both the infrastructure and method development grant programs.

NIH Research Projects · FY 2025 · 2017-08

Molecular and Cellular Mechanisms of Copper-Dependent Nutrient Signaling and Metabolism PROJECT SUMMARY/ABSTRACT Akin to organic nutrients, such as oxygen, lipids, amino acids, and carbohydrates, the transition metal copper (Cu) is an essential dietary nutrient for normal physiology and development. Decades of research highlight the physiological and disease associated consequences of disrupting homeostatic mechanisms that ensure proper Cu acquisition, storage, and distribution to Cu-dependent enzymes. However, phenotypes associated with alterations in Cu availability cannot be fully explained by the limited number of enzymes that traditionally harness the redox potential of Cu as a catalytic cofactor. Recent discoveries in Cu biology have revealed direct Cu binding at non-catalytic sites within signaling molecules that modulate cell proliferation via the protein kinases MEK1/2, lipid metabolism via the phosphodiesterase PDE3B, and nutrient recycling via the autophagic kinases ULK1/2. The emergence of this new paradigm in nutrient sensing and protein regulation has established that Cu is a critical mediator of intracellular signaling, provided evidence for the existence of molecular mechanisms for sensing changes in Cu abundance, and expanded the contribution of Cu to cellular processes necessary for adaptation to nutrient scarcity. This grant proposal will focus on the intersections between Cu homeostasis, nutrient signaling, and metabolism by examining the interplay between mechanisms of Cu-sensing necessary for cellular energy homeostasis and evaluating the necessity of Cu for metabolic flexibility under nutrient and oxygen stress. Specifically, we will build on our novel findings from the past 5 years by elucidating mechanisms of: i) Cu-controlled autophagy-lysosomal biogenesis and function, ii) Cu-mediated metabolic flexibility via direct control of glycolytic flux, and iii) interconnectivity between mitochondrial Cu transport and cytosolic nutrient sensing signaling pathways necessary for metabolism. By launching these three interconnected focus areas, we will increase our fundamental knowledge of the molecular and cellular features of Cu-dependent enzymes and cellular processes and enable therapeutic targeting of Cu-dependent disease vulnerabilities.

NIH Research Projects · FY 2025 · 2017-08

PROJECT SUMMARY The translation of research from the bedside to real-world practice utilizing disciplines including disease epidemiology, biostatistics, comparative effectiveness research, evidence synthesis, decision modeling, quality improvement, and implementation science is essential to improve the health of populations. The NIH, FDA and other organizations have increasingly recognized the importance of this broader sense of translational research. However, the pool of clinician investigators trained to perform such research is limited, particularly in the fields encompassed by the mission of the NIDDK. Training in these disciplines requires the same rigor as traditional “wet-bench” research; and undergraduate exposure and training in public health and epidemiology are relatively uncommon, leading to a significant underrepresentation of these fields in graduate training. To address these needs, in 2017 we established the Undergraduate Clinical Scholars Program (UCSP), an innovative undergraduate research opportunity (URO) at the University of Pennsylvania. The specific aims of the UCSP are two-fold: Aim1. To foster a strong interest among talented undergraduates in human clinical research with a focus on digestive, pancreatic, liver, kidney, and diabetes research; and Aim 2. To establish durable mentoring relationships between talented undergraduates interested in pursuing clinical research and supportive faculty mentors. The cornerstone of the UCSP is an intensive clinical- research experience that entails close interaction with and mentorship by a UCSP faculty member. UCSP students also complete a structured curriculum in epidemiology and biostatistics that is enhanced with a statistics laboratory and lectures/discussion groups in ethics, scientific writing and pathways to medical school admissions (MD and combined degree programs). The in person program culminates with a formal research symposium where students present and defend their work. The UCSP infrastructure provides administrative support to help sustain the mentorship relations after the summer program. The highly competitive program receives approximately 500 applicants per year. To date, we have had 28 UCSP students. Students have been involved in 13 peer-reviewed publications, received a number of honors including 2 Marshall Scholarships, and 8 students are currently in medical school with another 8 applying this year. In aggregate, this innovative URO has successfully provided superb faculty, exceptional scientific resources and exciting intellectual environment for trainees to be exposed to and expand their knowledge and interest in clinical and health services research with a key outcome to motivate trainees in clinical research careers with a focus on digestive, pancreatic, liver, kidney and endocrine related research.

NIH Research Projects · FY 2025 · 2017-08

Project Summary Renal cell carcinoma is among the ten most prevalent malignances in the United States, exhibiting an increased incidence in both men and women since 2001. The most common kidney cancer subtype is “clear cell” renal cell carcinoma (ccRCC), which accounts for ~75% of all cases. For early-stage disease, surgical resection of ccRCCs can be curative, although survival drops significantly for advanced, metastatic cancers. Multiple therapies are now available to ccRCC patients, including anti-angiogenic VEGF/receptor tyrosine kinase inhibitors, immune checkpoint blockade, mTORC1-based drugs, and a novel HIF-2a inhibitor. However, not all patients respond to these treatments and five-year relapse rates now approach 40%, and the majority of these cases develop metastases. Importantly, ccRCCs lack common oncogenic mutations observed in other human cancers, including PI3K, PTEN, TP53, and KRAS, which hinders successful treatment using corresponding targeted therapies. Instead, we have generated copy number variation, transcriptomic, and metabolomic data to identify multiple metabolic pathways that are universally altered in ccRCC tumors. These include loss of the gluconeogenic enzyme fructose-1,6-bisphosphate 1 (FBP1) and urea cycle enzymes, including argininosuccinate synthetase 1 (ASS1), argininosuccinate lyase (ASL), and arginase 2 (ARG2). Finally, ccRCCs exhibit unusually high numbers of lipid droplets, organelles which store triglycerides and cholesterol esters and a hallmark of this disease. By delineating the molecular consequences of these universal metabolic changes, we have developed new therapeutic strategies to target most patients diagnosed with this kidney cancer subtype. Moreover, our findings have been extended to other cancers such as hepatocellular carcinoma (HCC) and soft tissue sarcoma (STS) which appear to engage in highly similar metabolic reprogramming. Our data demonstrate that “senolytics” like ABT-263 could be deployed for the treatment of HCC, whereas ITX-5061, an inhibitor of the HDL cholesterol transporter SCARB1, may be effective for treating ccRCC. The results are paradigm-shifting in that understandable skepticism remains regarding the utility of “drugging” cancer metabolism, considering the metabolic heterogeneity, plasticity, and redundancy observed in various cancers. However, our results using autochthonous in vivo tumor models provide a rationale for deeper exploration. Ongoing and future work will investigate how consistent metabolic adaptations within the tumor parenchyma impact stromal components, such as fibroblasts and immune cells, based on an arsenal of complementary in vitro and in vivo models, that include novel autochthonous HCC and STS mouse models and ccRCC and HCC patient derived xenografts and organoids. A principal conceptual innovation of our recent work is the demonstration that multiple metabolic networks are consistently altered (~100%) in genetically diverse cancers like ccRCC, HCC, and STS, and the identification of novel, highly feasible therapeutic strategies.