Wistar Institute

NIH Research Projects · FY 2025 · 2016-09

Project Summary Bioinformatics Facility, supported by the Cancer Center Support Grant (CCSG) awarded to the Wistar Institute, collaborates with the vast majority of cancer center members on multiple research projects. My role as a Scientific Director of the Bioinformatics Facility is to be engaged on all steps of many of those research programs. The institute-wide involvement in cancer-related studies allows me to develop comprehensive data analyses pipelines applicable across multiple programs, as well as to re-use result elements, including databases, summarized and annotated datasets and other integrative resources, in analyses customized to particular studies. In combination with project-specific research efforts, which in turn contribute to the existing knowledgebase, the integrative approach provides a robust scientific environment for applying methods of computational biology to various cancer-related studies as demonstrated in the application.

NIH Research Projects · FY 2025 · 2010-07

Project Summary Human telomeres are specialized chromatin structures uniquely programmed to coordinate cellular division with genomic integrity, thus providing a senescence barrier to cancer-cell evolution. Telomere structural maintenance and genome sensing functions require extensive epigenetic signaling and higher-order chromatin dynamics that are poorly understood. Our previous studies have revealed the important roles of telomere encoded repeat RNA (TERRA) in the coordinate regulation of telomere chromatin structure, DNA replication and repeat-length maintenance. We have found that TERRA expression can be regulated by subtelomeric binding of chromatin organizing factor CTCF and tumor suppressor protein p53 in response to DNA damage stress. TERRA is also regulated by histone assembly factors DAXX and ATRX that are implicated in the Alternative Lengthening of Telomeres (ALT) through recombination. New preliminary data indicates that DAXX and ATRX are also required for p53 binding to subtelomeres in response to DNA damage, suggesting a coordinate regulation of these factors in telomere chromatin control and TERRA expression. In Aim 1, we investigate the role of DAXX and ATRX in regulating telomeric and subtelomeric chromatin accessibility that is critical for p53 binding, TERRA expression, and telomere DNA repeat stability. TERRA is also known to interact with shelterin component TRF2. In Aim 2, we propose to investigate the interaction of TERRA with the TRF2 amino terminal GAR domain, and how this further regulates telomeric DNA replication, chromatin structure, and chromosome conformation. Finally, in Aim 3, we will investigate how TERRA and telomeric chromatin can be processed into vesicular bodies in response to telomeric stress to generate extracellular inflammatory signals. We propose that exosomal TERRA and telomeric chromatin serve as telomere-specific Alarmins that are important in cellular senescence and tumor microenvironment signaling. These studies will provide an integrated analysis of telomere transcription and chromatin regulation, with stress response pathways that are important in human cancer and related inflammatory disease. Central Hypothesis: Telomeres are specialized epigenetic elements that sense and signal replication stress to control cell proliferative capacity and senescence. TERRA represents an important telomere-repeat generated molecule that functions in chromatin structure, DNA replication and repair, and DNA damage signaling. Characterization of normal and pathogenic functions of TERRA will provide important insight into the mechanisms driving genetic instability in cancer and related genetic instability syndromes.

NIH Research Projects · FY 2025 · 2009-09

Project Summary Mounting evidence from our laboratory and others suggests that a lysosomal degradation pathway, called autophagy, plays a central role in orchestrating inflammation and cancer biology. Recently, our laboratory generated UVRAG (UV irradiation resistance associated gene) mutant mice that show normal levels of basal autophagy but are deficient in stimulus-induced autophagy. With this mouse model, we demonstrate that autophagy dysregulation could exacerbate inflammation and promote spontaneous cancers. However, major gaps still exist in our understanding of intricate relationship between autophagy, inflammation and cancer, including 1) what are the mechanisms by which inadequate autophagy perpetuates inflammasome response and drives inflammation-associated pathologies? and 2) how does basal autophagy suppression affect tissue homeostasis and promote cancer susceptibility? This project will fill these gaps by focusing on two Specific Aims, including 1) investigating molecular mechanism of autophagy dysfunction on inflammatory signaling and inflammation-associated pathologies; and 2) identifying molecular mechanism of autophagy inhibition in Wnt/b- catenin signaling activation and spontaneous tumorigenesis. These aims will be addressed using multidisciplinary innovative approaches that integrate state-of-the-art genetic, biochemistry, single cell analysis, and physiological assays in cells, 3D organoid culture, and mice with targeted mutations in genes related to UVRAG function and autophagy deregulation. Together, we anticipate that these studies will elucidate the mechanisms underlying the intricate dialog between imbalance of autophagy, uncontrolled inflammation, and spontaneous tumorigenesis, thereby providing important new insights into the functional repertoire of autophagy and facilitating the development of much-needed new strategies for the treatment of inflammatory pathologies and cancer, particularly those associated with autophagy defects.

NIH Research Projects · FY 2025 · 2006-04

Abstract: Kaposi’s Sarcoma (KS)-Associated Herpesvirus (KSHV) is a human g-herpesvirus responsible for KS, pleural effusion lymphoma (PEL), multlicentric Castleman’s disease (MCD) and KSHV inflammatory cytokine syndrome (KICS). KS and PEL are HIV-AIDS-defining malignancies that occur at high frequency during AIDS, but also after treatment with ART especially in endemic regions. KSHV oncogenesis is driven by the complex life cycle of KSHV involving long-term latent infection of B-lymphocytes, reactivation and persistent infection of lymphatic endothelial and mesenchymal stem cells, and immune evasion. KSHV encoded nuclear antigen LANA is expressed in all KSHV-associated tumors and is essential for maintenance of the viral episome during latent infection in proliferating cells. KSHV LANA is also thought to contribute directly to viral oncogenesis and pathogenesis through interactions with tumor suppressor proteins and rewiring of host gene expression programs. We have previously shown that LANA contributes to the viral DNA replication, epigenetic regulation and higher-order structure of KSHV episomes. We have also identified chromatin organizing factors, such as CTCF and cohesins, as key regulatory factors in the control of latent gene expression and restriction of lytic reactivation. More recently, we have explored KS tumor transcriptomics and drug sensitivity assays to identify metabolic pathways implicated in the regulation of LANA and KSHV-infected cell survival. We now propose to advance each of these findings and continue our long-term effort to understand the role of LANA in episome maintenance, latency regulation, and oncogenesis. Specifically, we will investigate (1) the role of LANA in regulating latency-associated DNA replication and genome integrity through a potential intrinsic tyrosine recombinase activity that regulates replication termination and genome decatenation at the viral terminal repeats, (2) the role of LANA and cellular chromatin organizing factors DAXX, CTCF and cohesin in forming higher-order nuclear bodies that protect and organize the viral episome to maintain stable genomes and gene expression patterns in latently infected cells, and (3) how onco-metabolic stress disrupts LANA function and KSHV latency to drive tumorigenesis. Together these studies provide an integrated framework to further advance our knowledge of KSHV infection and latency, and provide new opportunities for therapeutic intervention in KSHV- associated disease.

NIH Research Projects · FY 2026 · 2005-06

Abstract In this proposal, support is requested for years 20-24 of an application that has been devoted to the study of hypomorphic variants of the TP53 tumor suppressor. Hypomorphs are defined as germline missense mutations that result in partially functional p53 protein. This is in contrast to fully inactivating p53 mutations such as those that occur in Li Fraumeni syndrome (LFS). To summarize the differences between LFS and hypomorphs: whereas 80% of individuals with LFS develop cancer by the age of 10-15, approximately 40% of hypomorph carriers develop cancer by the age of 35-40. Several years ago, we hypothesized that studying p53 hypomorphs, which predispose to cancer but retain significant p53 function, might yield finely-honed insight into the key activities and target genes for p53-mediated tumor suppression. In this funding cycle this hypothesis has paid off, starting with our analysis of the Y107H hypomorph. We created a mouse model for Y107H and find that these mice have a significantly elevated risk for cancer. We used CRISPR/Cas9 to create two clones of Y107H in the HCT116 background and identified the epigenetic modifier PADI4 as a gene that fails to be transactivated by Y107H. Notably, we find that PADI4 is the single gene that fails to be transactivated by any of six different cancer-associated p53 hypomorphs. PADI4 is an enzyme that converts arginine to citrulline on histones and other proteins, thus reducing charge and influencing protein localization and protein-protein interaction. We find that PADI4 is itself tumor suppressive, and that the ability of PADI4 to suppress xenograft tumors requires an intact immune system. In this funding cycle, we will explore two exciting new findings: PADI4 binds to p53 and citrullinates it on the C terminus. Induction of PADI4 causes relocalization of p53 toward binding sites for ETS transcription factors. We find that induction of PADI4 causes increased interaction of p53 with the ETS family member ELF2. We also find that p53 and PADI4 induction leads to a relocalization of citrullinated histone H3 to the cytosol. In this revised competitive renewal, we will determine the mechanism whereby PADI4 and citrullination impact p53 transcriptional function (Aim 1). This aim will take advantage of our unique engineered cell lines and mouse models containing p53 hypomorphic variants, as well as p53 and PADI4 knockout mice. In Aim 2 we propose to dissect the downstream signaling pathway of cytosolic citrullinated histone H3, and to determine the relevance of this pathway to tumor suppression by p53. Because p53 transactivates PADI4 in a tissue-specific manner, Aims 1 and 2 will employ mouse models in order to determine the cell/tissue type where p53 and PADI4 cooperate in transactivation (Aim 1) and extrusion of citrullinated histone H3 (Aim 2). Toward these goals, we have accrued a team of experts in genomics (Gardini), innate immune signaling (Shinde and Zhang), Proteomics (Tang), protein citrullination (Thompson), and p53 family function (p63, p73, Flores). We are excited to continue these studies, as they provide unique insight into p53 function and tumor suppression.

NIH Research Projects · FY 2026 · 1997-04

PROJECT SUMMARY – OVERALL Rated Exceptional at both the 2013 (impact score, 19) and 2018 (impact score, 14) renewals of the Cancer Center Support Grant (CCSG), and receiving a two-year MERIT extension from the National Cancer Institute (NCI) in 2022, the Wistar Cancer Center continues an exponential path of research excellence. Leveraging a deep-rooted culture of collaboration, a nearly complete equivalence between The Wistar Institute and the Cancer Center, and a unified leadership structure where an experienced Cancer Center Director is also Institute President and Chief Executive Officer, the Cancer Center functions as a catalyst for innovation in basic and translational cancer research with broad societal impact. An unprecedented institutional commitment of over $81 million during the last five years enabled the recruitment of 17 new Cancer Center members at all academic ranks, transformed the technological and scientific capabilities of all 8 CCSG-supported Shared Resources, and strategically realigned Cancer Center Programs for maximal transdisciplinary collaboration with the launch of a new Genome Regulation and Cell Signaling (GRCS) Program. The Wistar Cancer Center of 2025 comprises 32 members and has a total funding base of $14,610,429. Peer-reviewed, cancer-related funding stands at $12,120,630 (83%), of which $6,765,960 is from the NCI translating into a strong cancer focus of 55%. As collaboration is a foundational hallmark of the Cancer Center, 77% of peer-reviewed, cancer-related funding and 37% of discovery publications are the product of collaboration. Currently, Wistar is the only NCI-designated “basic” Cancer Center to contribute to two Specialized Programs of Research Excellence (SPORE), three NCI program project grants, two NCI consortium grants and twenty-nine multi-investigator R01 awards (22 from the NCI, 76%). Building on these advances, the Cancer Center achieved unique societal impact during the last CCSG budget period. Groundbreaking discoveries in viral oncogenesis, mitochondria-induced tumor plasticity, cancer-associated neoepitopes, and therapeutic HPV vaccination advanced to first-in-human oncology clinical trials, the partnership with the Helen F. Graham Cancer Center brought cutting-edge cancer research to the community, and life-changing ‘work-study’ programs transformed the paradigm of workforce development, continued education and public-private partnership in the region. Against this backdrop, a 2021-2026 Bold Science // Global Impact strategic plan charts a new vision for the Cancer Center. Backed by over $67 million raised in the most successful philanthropic capital campaign ever, the largest facility and faculty expansion in Wistar’s 130-years history has been launched with the creation of a new, cancer-focused Center for Advanced Therapeutics, and the addition of ~40,000 square feet of new, state-of-the-art laboratory space. In the next five years, an expanded Wistar Cancer Center will redefine mechanisms of oncogenesis, target tumor-driving pathways from the bench to the clinic and unravel tumor immune circuits through multidisciplinary collaboration, trans-institutional partnership, and fostering the next generation of leaders in cancer research.

NIH Research Projects · FY 2025 · 1976-07

Project Summary The overarching goal of this longstanding “Training Program in Basic Cancer Research” is to provide the fundamental skills necessary for our trainees to succeed in outstanding careers in biomedical cancer research. Toward that goal we provide them with superior training in the conduct of cutting edge research, combined with a deep appreciation of the clinical challenges that doctors and patients face. To achieve this goal, we have created a unique Training program, now entering its 45th year, entitled the Training Program in Basic Cancer Research (TP-BCR). Support is requested for five postdoctoral fellows, and three predoctoral fellows from the University of Pennsylvania Perelman School of Medicine, program in Biomedical Graduate Studies. Located at the heart of the University of Pennsylvania campus, Wistar offers a superb research environment, recently rated “Exceptional” at the most recent CCSG renewal in 2018. We have crafted a training program that combines unique sub-programs entitled “Bench to Bedside Training” and a “Science Entrepreneurs” program, thus offering our trainees exposure to clinicians and clinical problems in cancer research, unique insight into the business of science and intellectual property, and a robust curriculum that includes workshops in teaching, writing, and grant preparation. Our 26 faculty mentors tackle diverse questions in cancer, most have long-standing successful training records, and all are well-funded, with an average of $890,000/year/mentor (DC). Our current trainee pool consists of 79 trainees, which includes 24 predoctoral trainees (13 TGE) and 55 postdoctoral trainees (20 TGE). There is an average of four TGE applicants for each proffered slot, thus allowing us to place the most highly qualified trainees in this program. All slots have been continuously filled for the entire funding period, and our trainees continue to be highly productive, with 4.7 manuscripts and 2 first author manuscripts per trainee averaged in the past three cycles, and 98% of our trainees continuing in research-related or research-intensive careers. Taken together, this long-standing program, which is the only T32 centered at The Wistar Institute, will provide the fundamental skills necessary for our trainees to flourish in successful independent careers in cancer research.