Author: The Wistar Institute

Lieberman studies how certain viruses establish a long-term latent infection that can lead to cancer or autoimmune disorders.

Lieberman joined The Wistar Institute in 1995 as an assistant professor. He earned his bachelor’s degree in chemistry from Cornell University and a doctorate in pharmacology/virology from The Johns Hopkins University School of Medicine, which was followed by a postdoctoral fellowship at the University of California, Los Angeles.

Lieberman Chairs the Program in Gene Expression and Regulation at the Ellen and Ronald Caplan Cancer Center at The Wistar Institute. In 2010, Lieberman became the first director of The Wistar Institute Center for Chemical Biology and Translational Medicine. Using the advanced screening technologies of Wistar’s Molecular Screening Facility, the Center enables scientists to identify and characterize new molecules and compounds that hold the most promise for developing into therapeutic drugs for cancer and other diseases.

The Lieberman Laboratory

Research in the Lieberman laboratory centers on understanding how the cancer-associated viruses persist in a latent state and increase the risk of cancer and autoimmune disorders. EBV and KSHV establish latent infections that are associated with several human malignancies, including Burkitt’s lymphoma, nasopharyngeal carcinoma, Hodgkin’s disease, and post-transplant lymphoproliferative disorder for EBV, and Kaposi’s Sarcoma for KSHV. EBV has also been implicated in multiple sclerosis and other autoimmune disorders.

Lieberman and his team found that oncogenic viruses can interact with cellular proteins that regulate telomeres—the repetitive DNA sequences found at the ends of chromosomes. Telomeres protect chromosomes from loss of genetic information, and a similar process is thought to preserve the virus during latency. The Lieberman laboratory worked out several biochemical pathways that control the stability, replication, and gene expression patterns of the latent virus. New research also focuses on the epigenetic controls of latent viruses and human telomeres, and how interactions between viruses and telomeres may induce a malignant transformation of the infected cell.

Research in the Lieberman laboratory centers on understanding how the cancer-associated viruses, like Epstein-Barr virus (EBV) and Kaposi’s Sarcoma Associated Herpesvirus (KSHV), persist in a latent state and increases the risk of cancer cell evolution. EBV and KSHV establish latent infections that are associated with several human malignancies, including Burkitt’s lymphoma, nasopharyngeal carcinoma, Hodgkin’s disease, and post-transplant lymphoproliferative disorder for EBV, and Kaposi’s Sarcoma for KSHV.

The researchers have recently found that viral DNA replication and maintenance is regulated by interactions with cellular telomere binding proteins. Telomeres are the repetitive DNA sequences found at the ends of chromosomes. Telomeres protect chromosomes from loss of genetic information, and a similar process is thought to preserve the virus during latency. The Lieberman research team has worked out several biochemical pathways that control the stability, replication, and gene expression patterns of the latent virus. They have found that changes in viral chromatin structure alters the cancer-risk associated with latent infection.

The Lieberman lab continues to study EBV and KSHV genome maintenance proteins, EBNA1 and LANA, respectively. These proteins bind to the viral OriP, but they also bind to the cellular chromosome at unknown sites. The Lieberman lab has identified the cellular chromosome binding sites for both EBNA1 and LANA in latently infected B-lymphocytes. LANA was found to bind to host genes involved in gamma-interferon signaling and LANA may antagonize STAT1/STAT3 binding to host genes important for MHC peptide presentation and processing. EBNA1 may promote higher order structures, including interchromosome linkages that may promote translocations similar to those observed in Burkitt’s lymphoma.

The role of chromosome architecture and higher-ordered structure is also important for genome maintenance. The Lieberman lab has studied the role of chromatin architecture proteins CTCF and cohesins in regulating viral genome structure and gene expression during latent infection. They have shown that CTCF and cohesins mediate long-distance interactions that are important for control of gene expression and maintenance of a stable latent infection. Loss of genome architecture leads to a change in gene expression and a transition from a circular to linear viral genome.

Maintenance of telomere structures that maintain the ends of linear chromosomes is also important for human genome stability. The Lieberman lab is studying the chromatin structure of telomeres and the expression of a telomere repeat-containing non-coding RNA, termed TERRA. They have shown that TERRA is overexpressed in highly proliferating cells in human and mouse cancers. The TERRA form nuclear aggregates in cancer cells in mouse models of medulloblastoma, and TERRA RNA levels were highly over-expressed in human ovarian cancer biopsies. The regulation and function of TERRA expression, and its role in regulating telomere length and stability are the focus of future research.

The Lieberman laboratory is also pursuing the development of small molecule inhibitors of the EBV encoded origin binding protein EBNA1. The laboratory is collaborating with structural biologists and medicinal chemists to advance hits into lead compounds for testing in animal models of EBV lymphomagenesis. These small molecules will be considered for further development as inhibitors of EBV-associated malignancies.

Liang’s research explores basic mechanisms underlying fundamental cellular processes in inflammation, infection, and cancer, broadly focusing on autophagy, organelle homeostasis, genomic stability, membrane trafficking, and virus-host interaction.

Liang obtained her M.D. degree from Qingdao University School of Medicine, China, and her Ph.D. degree in genetics from State University of New York (SUNY) at Stony Brook. She received postdoctoral training in tumor virology at Harvard Medical School in the Department of Microbiology and Molecular Genetics. She established her laboratory in 2009 in the Department of Molecular Microbiology and Immunology, University of Southern California, Keck School of Medicine in Los Angeles, where she was promoted to tenured associate professor in 2015. Liang joined The Wistar Institute as a professor in 2020.

The Liang Laboratory

Autophagy (literally “self-eating”) is the natural, regulated mechanism cells use to digest and remove unwanted components. This process influences diverse aspects of cell homeostasis and constitutes a barrier against malignant transformation.

The Liang lab identified a novel autophagy pathway controlled by the tumor suppressor gene UVRAG (UV-radiation Resistance-Associated Gene) that also plays a direct role in DNA repair and chromosomal stability. The group studies autophagy and related pathways in leukemia, colorectal cancer, melanoma pathogenesis and therapy resistance, and in viral persistency.

Zhang studies the role of immune cells called macrophages in tumor growth and metastasis in the abdominal cavity.

Zhang received his B.S. in microbiology and immunology from Shandong University, China, and a Ph.D. in biochemistry and molecular biology from the University of Oklahoma Health Sciences Center. He completed his postdoctoral training in the Department of Pathology and Immunology of Washington University School of Medicine and joined The Wistar Institute in 2021 as an assistant professor.

The Zhang Laboratory

Macrophages are currently considered as highly heterogenous and plastic with different developmental origins and microenvironmental signatures.

The Zhang lab focuses on understanding how macrophages regulate tumor growth and metastasis in the peritoneal space. We use single-cell sequencing, ATAC sequencing, large-scale imaging, multiphoton intravital imaging, novel genetic mouse models, and patient samples to understand how different subsets of macrophages function differently in mice and in humans. Our long-term goal is to develop novel macrophage-based immunotherapies to treat peritoneal cancers.

Current projects in the lab include:

1. Heterogeneity and origins of macrophages during peritoneal carcinomatosis.
2. Activation of macrophages against tumor progression.
3. Tissue-specific functions and microenvironment of peritoneal macrophages.

Weiner directs a translational molecular immunology research team focused on creating novel immunotherapy approaches for disease prevention and treatment using synthetic nucleic acid technology. Accomplishments of the team and collaborators include the first clinical studies of DNA vaccines, with a focus on advances in gene optimization and electroporation (EP)-mediated DNA delivery. Their work has revitalized the field, rapidly and safely moving new advances into human studies. These include the world’s first Zika vaccine, the first MERS vaccine, an advanced Ebola Vaccine, and a novel HIV vaccine, among others. Additionally, the Weiner laboratory has helped to develop immunotherapy approaches that are currently in clinical testing for HPV-associated cancer, prostate and other cancers. The first clinically efficacious therapeutic DNA vaccine for HPV cervical intraepithelial neoplasia CIN) has moved into a licensure trial (REVEAL). Weiner and his lab have received several awards/honors for their accomplishments, including the Vaccine Industry Associations Outstanding Academic Research Laboratory (2015 & 2016), being named one of the Top 20 Translational Research Laboratories of the Year (Nature Biotechnology 2016, 2017 & 2018) and the 2014 Stone family Award for Cancer Research. Weiner was named one of the nation’s top 40 most influential vaccine scientists in 2014, received the 2011 NIH Directors Translational Research Award and is an elected fellow of the American Association for the Advancement of Science since 2011 and a fellow of the International Society for Vaccines, for which he served as president from 2018 to 2020. Weiner is an avid trainer, advisor and advocate for students, fellows and junior faculty as he is highly committed to developing the careers of young scientists.

Weiner received his B.S. in biology from Stony Brook University, N.Y., and his M.S. in biology from the University of Cincinnati. He then earned a Ph.D. in developmental biology with a focus on molecular immunology from the University of Cincinnati, College of Medicine. Weiner joined the University of Pennsylvania as a research fellow in the Department of Pathology and Laboratory Medicine, where he rose through the ranks to become Professor. He held a second appointment from The Wistar Institute from 1990 to 1993. At Penn, he served as co-chair of the Tumor Virology Program of the Abramson Cancer Institute and as chair of the Gene Therapy and Vaccine Training Program.

The Weiner Laboratory

The Weiner laboratory represents one of the pioneering research teams in the field of DNA vaccines and immunotherapies. The lab has published more than 420 scientific papers, chapters and reviews, including many seminal papers in the DNA vaccine and synthetic nucleic acids field, and is credited with generating more than 70 patents. Along with collaborators, the Weiner Lab was the first to move DNA vaccines to human clinical studies, establishing their initial safety and immunogenicity. The team also helped to develop the new field of nucleic acid-encoded antibodies, or dMAbs. More than a dozen experimental clinical therapies and vaccines have been developed from research from the Weiner laboratory, including the first Zika vaccine in clinical trials, the first MERS vaccine, a novel Ebola vaccine as well as novel immunotherapy for HPV-associated cancer and precancer, and a novel immunotherapeutic vaccine for glioblastoma. Other notable reports from the Weiner lab include the first DNA vaccine studied for HIV and for immunotherapy of cutaneous T-cell lymphoma and the early development of DNA-encoded genetic adjuvants, including IL-12.

The Zika virus has become a global health concern due to its rapid spread and concerning link to microcephaly in infants. The Weiner lab has received funding from the Bill & Melinda Gates Foundation for a collaborative project with Inovio Pharmaceuticals and HUMABS BioMed to develop DNA encoded monoclonal antibodies (DMAbs) against Zika. DMAbs are injected into the muscle and the human body is used as its own bioreactor to produce protective antibodies. Working with highly effective antibodies for which sequences were provided by HUMABS, the Weiner lab is optimizing DMAbs for high levels of expression, binding and neutralization. Once these DMAbs are shown to be protective in mice, they will be tested in nonhuman primates and eventually prepared for clinical trials with the assistance of Inovio.

Funded by the Bill & Melinda Gates Foundation, this study addresses the possibility of achieving vaccinal effect by co-delivering DNA-encoded monoclonal antibodies (DMAbs) and synthetic DNA vaccines to prevent HIV infection.

Funded by the Bill & Melinda Gates Foundation, the focus of this study is to develop new synthetic DNA vaccines encoding designed circumsporozoite protein (CSP) antigens to provide a new generation advanced CSP component for a prophylactic malaria vaccine.

The Weiner lab has a long history of working on DNA vaccine for human immunodeficiency virus (HIV). The lab is currently a part of a multi-institutional group working on developing a prophylactic HIV vaccine under a five-year Integrated Preclinical/Clinical AIDS Vaccine Development Program grant from the National Institutes of Allergy and Infectious Disease (NIAID). Working with Inovio Pharmaceuticals, Duke University, Emory University, University of Massachusetts, and the National Institutes of Health, the group aims to build on the success of previous generations of DNA vaccines in order to induce strong cellular and humoral responses. The main focus of the program is inducing broad responses against the diverse HIV surface protein, Env.

The Weiner lab has teamed up with investigators at the University of Pennsylvania to develop a novel vaccine to prevent cancer development in high-risk patient populations. This includes individuals who carry mutations in the BRCA1 or BRCA2 genes and are susceptible to the development of breast, pancreatic and ovarian cancer. This novel vaccine encodes the tumor antigen TERT, which is highly expressed in tumor cells, and is particularly high in tumor samples from patients with mutations in DNA damage repair pathways, such as the BRCA1/BRCA2 pathway. The lab developed this vaccine and is currently working to improve efficacy using combination therapies with immune checkpoint blockade antibodies in mouse models. In parallel, a clinical trial is being conducted for high-risk patients in remission after adjuvant therapy for TERT DNA vaccine with or without IL-12 immune plasmid adjuvant. This project is funded by a Breakthrough Science Team Award through the Basser Research Center for BRCA at the University of Pennsylvania.

Funded by the Department of Defense (DoD), this study will characterize a novel vaccine targeting follicle stimulating hormone receptor (FSHR) in ovarian cancer.

The objective of this study is to support improvements in the immunogenicity and durability of seasonal influenza vaccines, and the development of innovative influenza vaccine approaches that provide robust, durable, broadly protective mucosal and systemic anti-influenza immunity (“universal influenza vaccines”). Research will support iterative vaccine design based on detailed immunologic assessment of influenza vaccine candidates through preclinical animal studies, early phase clinical trials and healthy volunteer human challenge studies, to advance the most promising vaccine candidates into phase 1/2 clinical trials.

Translational Platform Program encompassing cGMP manufacturing and clinical development of DNA vaccine candidates against both Lassa virus and MERS coronavirus, with funding from the Coalition for Epidemic Preparedness Innovations (CEPI).

Funded by the Coalition for Epidemic Preparedness Innovations (CEPI), Wistar will support the development of a synthetic DNA vaccine and nanoparticles against Wuhan coronavirus 2019-nCoV. Successful characterization of the blocking antibody will permit us to explore monoclonal antibody production, engineer the monoclonal antibody into the synthetic DNA-based DMAb platform, and engineer into the nanoparticle platform as potential vaccines and immunotherapeutics against the Wuhan Coronavirus 2019-nCoV.

This NIH-funded study addresses Novel DNA-encoded monoclonal antibodies (DMAbs) for control of antimicrobial tesistant (AMR) Pseudomonas aeruginosa infection.

This NIH-funded study addresses development of a multivalent, DNA vaccine-mediated protection against Tuberculosis.

Villanueva studies the molecular signaling pathways that become deregulated in melanoma with the goal of identifying suitable targets for therapy, particularly for tumors with limited therapeutic options.

Villanueva received her Bachelor of Science degree in biology at Universidad Peruana Cayetano Heredia in her native Peru. She then enrolled in the graduate program at University of Miami School of Medicine and she earned a Ph.D. in Molecular Cell and Developmental Biology. She pursued postdoctoral training at the University of Pennsylvania School of Medicine where she began her research on melanoma. Villanueva joined The Wistar Institute as a postdoctoral fellow in the Herlyn laboratory, and was later appointed assistant professor in the molecular and cellular oncogenesis program.

Meet the Villanueva Lab Team

The Villanueva Laboratory

The Villanueva laboratory is actively studying the molecular mechanisms mediating drug resistance in melanoma, aiming at designing effective therapies that will overcome it. The lab has extensively investigated the role of the RAF/MEK and PI3K/mTOR pathways as therapeutic targets and the mechanisms underlying resistance to inhibitors that block these signaling cascades. More recently, the team is focusing on identifying new targets or vulnerabilities that can be therapeutically exploited in NRAS mutant melanoma, a tumor type in dire need of new treatments.

The focus of the Villanueva lab is to identify novel targets to overcome drug resistance in melanoma.

The Villanueva lab has developed pre-clinical models that show how melanoma gains resistance to BRAF and MEK inhibitors. Using these models, they demonstrated that melanoma cells treated with RAF inhibitors bypass the effects of the drugs by reactivating the MAPK pathway and/or activating alternative signaling pathways, including RTKs, PI3K/mTOR and STAT3. For example, the lab identified a novel MEK2 mutation that, together with BRAF amplification, confers resistance to RAF and MEK inhibitors. Based on these findings, the team tested combination therapies to overcome drug resistance.

NRAS is a poorly characterized RAS family member, and the biology of NRAS mutant tumors remain inadequately understood. There are very limited treatment options for patients carrying NRAS mutations, which are present in more than 25 percent of all melanomas. As targeting NRAS directly has thus far not been possible, the aim of the lab is to eradicate this type of tumors by blocking critical RAS effectors or pathways that are essential for tumor survival. The team has identified several non-oncogene dependencies that are critical for survival of melanoma cells including BRD4, TERT(*) and the ribosomal serine/threonine kinase S6K2. The lab is investigating the role of these dependencies in melanoma and evaluating the impact of blocking their activity on tumor initiation, maintenance and survival using 3-D organotypic spheroids, patient-derived xenograft (PDX) models and syngeneic mouse models.

Watch the animation below to learn more about our strategies to combat NRAS mutant melanoma.

Tempera’s research focus is on the epigenetic mechanisms underlying Epstein Barr virus (EBV) infection to identify new viral functions that can be targeted as novel therapeutic approaches for treating EBV-associated cancers.

Tempera obtained his B.Sc. and Ph.D. degrees from University of Rome “La Sapienza”, Italy. He was a postdoctoral fellow at Wistar and established his laboratory at the Fels Institute for Cancer Research and Molecular Biology at Temple University, where he was promoted to associate professor. In 2020, Tempera returned to Wistar as an associate professor in the Gene Expression & Regulation Program.

The Tempera Laboratory

Approximately 90 percent of the world population is infected with EBV and carries the virus in a silent state for life. Although EBV infection is asymptomatic in most cases, in some people it can cause infectious mononucleosis. In immunocompromised individuals, such as in HIV-infected persons and transplant recipients, EBV can cause B-cell transformation and malignancies including Burkitt’s lymphoma, nasopharyngeal carcinoma, and Hodgkin’s and non-Hodgkin’s lymphomas. EBV-associated cancers are still treated with chemotherapy, even though they have a specific pathogenic cause, but various attempts are being made to target EBV directly and develop EBV-specific therapies.

One approach to control EBV infectivity is by changing the expression of viral genes. The Tempera lab studies how epigenetics contributes to regulating the gene expression patterns adopted by EBV during latency. They utilize their expertise in genomics and genome-wide data analysis to better understand the link between the three-dimensional structure of chromosomes, chromatin composition and gene expression during EBV latency.

Learn more about the members of the Tempera Lab

EBV-induced malignancies have been challenging to target, in large part because EBV establishes a latent infection with complex and dynamic gene expression patterns. These patterns are referred to as “latency types” and can adapt to diverse host cell types and immunological responses. This dynamic gene expression allows EBV to escape eradication by immune surveillance and persist as a long-term, latent infection. In a series of studies, our group has explored the role of epigenetics in the regulation of EBV latency.

Although a role for epigenetic modifications in EBV has been appreciated for decades, our results over the past years have yielded surprising and important insights into its mechanism. We found that different EBV latency programs correlate with different epigenetic states of the viral episome, and we identified CTCF as the primary cellular factor that regulates these epigenetic patterns. Overall our work shows that:

• The epigenetic regulation of EBV is a dynamic process involving both histone modifications and DNA methylation;
• The CTCF cellular factor is critical for the maintenance of the viral chromatin state of EBV;
• CTCF regulates the viral promoters.

These findings defined the important function of CTCF as a chromatin organizer of the EBV viral genome.

Our team has explored the three-dimensional organization of the EBV genome during latency. Since CTCF promotes chromatin loops in the human genome, we tested the possibility that CTCF acts in a similar way on the EBV genome. By combining 3C assay, ChIP-seq and EBV genetic methods, our group has shown that:

• The EBV genome adopts alternative chromosome conformations during latency;
• CTCF is the critical cellular factor that mediates chromatin loop formation in EBV;
• The viral enhancer at the Ori P region of EBV serves as a “chromatin hub” regulating viral promoters through chromatin loops;
• The disruption of EBV three-dimensional organization impairs viral gene expression.

Our results have important biological implications since they demonstrate for the first time that viral genomes can also regulate their function by adopting different three-dimensional configurations.

We discovered that PARP1 can interact with the EBV genome. PARP1 is a host enzyme that post-translationally modifies proteins through the transfer of ADP-ribose from NAD+ onto acceptor proteins. The role of PARP1 in DNA damage and apoptosis is well characterized; however, in recent years, PARP1 has also been implicated in chromatin modification, transcriptional regulation and inflammation.

Evolutionary analyses have identified other PARP family proteins that likely have evolved a role in host-virus conflicts. Indeed, a body of evidence suggests that PARP1 may serve as a stress sensor — a function that would be especially relevant in regulating herpesvirus lytic reactivation. CTCF is also PARylated by PARP1, subsequently altering its insulator function.

Based on the established role of CTCF in EBV latency, the functional interactions of PARP1 and CTCF, and because CTCF and PARP1 are both implicated as viral restriction factors in other herpesviruses, our group has studied the role of PARP1 in regulating the EBV epigenome.

We have shown for the first time that LMP1 signaling affects important chromatin-modifying enzymes, specifically PARP1. PARP1 regulates global gene expression by relaxing the chromatin structure and inhibiting the accumulation of repressive histone marks.

We discovered that inhibiting PARP1 suppresses malignant transformation in vitro and represses the expression of previously identified LMP1 genetic targets. Thus, our team has explored the hypothesis that PARP1 activity plays an important role in EBV-induced oncogenesis. Current projects in our laboratory aim at establishing PARP inhibitors as a novel target for EBV-induced cancers and determining the pathogenic mechanisms involved in EBV-mediated tumor initiation.

Our group discovered that inhibition of PARP1 activity dramatically changes the expression levels of hundreds of genes, including genes involved in cancer. We found that increased levels of the Polycomb Repressive Complex 2 (PRC2) catalytic subunit EZH2 are responsible for some effects caused by PARP inhibition.

We reported for the first time that:

• PARP1 and EZH2 occupancy negatively correlate across the genome;
• PARP1 can directly modify EZH2;
• PARylation alters the enzymatic activity of EZH2.

Based on these data, our team has explored the hypothesis that PARP1 and PARylation play an important and underappreciated role in EZH2 activity and inhibitors of PARP can alter PRC2-mediated gene repression. We are working to establish PARP1 and PARylation as a novel mechanism of EZH2 regulation and determine mechanisms and functional relevance of PARP-mediated EZH2 inhibition.

Tang applies cutting-edge mass spectrometry-based proteomics to a wide range of biomedical projects to facilitate discoveries that will yield novel insights that are not biased by prior knowledge and have the potential to lead to new scientific directions.

Tang received his formal training in biochemistry and molecular biology through earning his Ph.D. from the Institute of Molecular and Cell Biology in Singapore. In 2000, Tang joined The Wistar Institute as a postdoctoral fellow in the laboratory of Dr. David Speicher and subsequently became the managing director of the Proteomics and Metabolomics Shared Resource. He was promoted to assistant professor in 2022.

The Tang Laboratory

Proteomics contributes to a better understanding of the molecular basis of diseases at the systems biology level, and has the potential to identify new therapeutic targets and potential diagnostic and prognostic biomarkers. The Tang laboratory collaborates with other researchers using state-of-the-art high-resolution mass spectrometry and related experimental strategies to investigate proteome changes and protein posttranslational modifications associated with cancers, such as melanoma, prostate, and ovarian cancers, and other diseases. For some studies, proteomics results are combined with other omics technologies – particularly metabolomics and lipidomics – to provide a more complete picture of the molecular contributors to diseases.

The Tang laboratory is actively involved in multiple collaborative projects that focus on defining disease-related cellular mechanisms and discovering therapeutic targets of diseases. These projects involve diverse experimental strategies that include global proteome profiling, quantification of disease biomarkers, characterization of protein post-translational modifications, identification of protein interactomes, and global polar metabolite and lipid profiling. Results from these analyses have provided insights into mechanisms underlying different cancers such as melanoma, prostate cancer, and ovarian cancer, as well as identified putative biomarkers for disease states.

The Tang laboratory is also involved in improving proteomics technologies in key areas including:

• Chemical crosslink-MS. Chemical crosslinking combined with mass spectrometry (MS) analysis is a powerful method to study protein-protein interaction networks and to obtain valuable structural information from protein complexes. Both traditional non-cleavable and MS-cleavable cross-linkers can be used for identification of protein-protein interaction sites, but MS-cleavable crosslinkers are advantageous because of their ability to generate distinguishing fragment ions during MS/MS that greatly improve identification of crosslinked peptides and crosslinked sites. These diagnostic fragment ions will also reduce the search space during data analysis, allowing the crosslinkers to be used in whole proteome labeling studies. We are interested in developing a robust and reliable workflow for efficient identification of crosslinked sites on proteins using MS-cleavable cross-linkers, such as DSSO and DSBU.
• MS-based glycomics and glycoproteomics. Glycosylation is one of the most abundant post-translational modifications (PTMs) in mammalian cells and is crucial for a wide range of biofunctions. Aberrant glycosylation of proteins has been linked to various diseases, including cancers. The major strength of MS-based analyses is the isolation and fragmentation of analytes to obtain structural information. MS-based glycomics typically consists of the following steps: glycan release by either PNGase F treatment (for N-linked glycans) or β-elimination (for O-linked glycans), glycan enrichment using solid phase separation techniques, glycan derivatization, and LC-MS/MS identification. Global profiling of released glycans has been used to distinguish healthy and disease states. However, information on the protein carriers of glycans and residue site localization is lost after glycan release. The more powerful MS-based glycoproteomics approach that we plan to focus on involves structural analysis of glycopeptides. Protein extracts are proteolytically digested followed by glycopeptide enrichment with subsequent LC-MS/MS analysis. Since the glycan is left intact on the peptide, this method allows the identification of the glycosylated proteins and quantitation of the glycan structures on the glycoproteins.

Showe’s research interests focus on using genomics-based approaches and big data to better understand disease processes with an emphasis on the immune system and cancer. Her laboratory has actively contributed to the development of genomics and bioinformatics at The Wistar Institute. Showe is the scientific director of the Genomics shared resource and associate director of the Center for Systems and Computational Biology. Her group has been active in implementing new methods for the generation and analysis of large and complex datasets.

Showe received her bachelor’s degree in biology from Wilkes College (PA) and spent three years at the Salk Institute before receiving her master’s degree in developmental biology from Temple University. After obtaining her Ph.D. in Biology from the University of Pennsylvania, she moved to Europe to conduct her postdoctoral studies at the Biozentrum der Universitat, Basel, Switzerland. She was a research assistant professor at the University of Pennsylvania and an assistant professor at Children’s Hospital of Philadelphia before joining The Wistar Institute in 1983. She became a full professor in 2007.

The Showe Laboratory

The Showe laboratory applies a variety of omics approaches to answer complex biomedical questions in the context of the interactions between the immune system and cancer, with the goal of developing novel tools for early diagnosis and markers of therapy response, survival and relapse. Through an extensive network of collaborations with other laboratories at Wistar and several other institutions, the lab’s research addresses a diversified group of diseases, including lung cancer, glioblastoma multiforme and cutaneous T cell lymphoma. They also have several long-term collaborative efforts at Wistar on HIV and influenza, and an international collaboration on multiple sclerosis with the University of Nottingham. The lab’s diverse interests are further evidenced by collaboration with the University of Fairbanks to understand how hibernating black bears and Alaskan ground squirrels modify gene expression in various tissues during hibernation. She developed the first bear transcriptome microarray in collaboration with the Fairbanks group.

The Showe lab has been a pioneer in the development of cancer biomarkers from blood. In 2009, they published novel findings showing how mononuclear white blood cells (PBMC) from lung cancer patients contain a 29-gene, tumor-relevant gene signature that accurately predicts whether a lung nodule detected by CT scan is benign or malignant. They also demonstrated that information in blood gene expression patterns could predict patient survival and inform further treatments. In moving these studies forward to a clinical application, the lab has adopted a simplified and standardized blood sample collection using commercially available PAXgene RNA stabilizing tubes and successfully moved their assay from the developmental microarray platform to the to the clinically approved Nanostring nCounter platform, (Kossenkov et al, 2019). The technology has been patented and is in development for commercialization.

Ongoing effort: As part of the NCI Early Detection Network, the lab is developing new methods to detect and diagnose cancers early, when they are more easily and successfully treated, and new methods to predict recurrence/survival after treatment. This is a collaborative study with investigators at The University of Pennsylvania, Roswell Park, NYU, Temple University, The Helen F. Graham Cancer Center, NYU Medical Center, Meridian Health, N.J. and the Barzilai Medical Center, Israel.

Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of non-Hodgkin lymphomas with characteristics of “skin-homing” T lymphocytes. The most common forms of CTCL are the skin-associated mycosis fungoides (MF) and a more aggressive leukemic form, Sezary Syndrome (SS). SS has been the focus of the laboratory studies. Early interest in this cancer was based on the observation that patients were severely deficient in the production of interleukin 12, a cytokine originally identified and characterized at The Wistar Institute, and on availability of a unique collection of patients being treated at the University of Pennsylvania Department of Dermatology. Based on initial studies, Showe was awarded one of the first National Cancer Institute Director’s Challenge grants to develop molecular diagnostics for CTCL using microarray platforms. This study led to the identification of a small number of genes that could detect SS by microarray or Q-RTPCR. The lab also identified some new specific cellular defects and a signature of poor prognosis that was independent of stage or circulating malignant cell numbers.

Ongoing studies: in collaboration with clinicians at the University of Pennsylvania (Alain Rook and Ellen Kim), Columbia University (Susan Bates) and NCI (Richard Piekarz), present work focuses on understanding mechanisms of therapeutic response both in vitro and in vivo to combined treatments such as toll-receptor agonists and interferon as well as histone deacetylase inhibitors. Combining data from gene and microRNA expression produced using microarrays and next-generation sequencing, the lab is defining parameters that distinguish responders from non-responders and markers of residual disease that can be monitored to detect potential recurrences before they are clinically evident. Using single cell analysis, the team is probing malignant cell heterogeneity and therapeutic response.

Glioblastoma multiforme (GBM) is the most common and severe form of primary malignant brain cancer. GBM is an aggressive cancer with no effective treatments and the median survival time is only 15 months from diagnosis. GBM is a heterogenous tumor having multiple disease subtypes, so it is important for clinicians to understand how these various GBM subtypes respond to emerging therapies that may one day be useful to glioblastoma patients.

Building on collaborative studies initiated with Ramana Davuluri and Donald O’Rourke, the Showe lab is developing a diagnostic platform to help classify GBM tumor subtypes to match to more efficacious treatments.

In a long-term collaboration with the lab of Luis J. Montaner at Wistar, the Showe lab applies functional genomic approaches to understand mechanisms of immune evasion in HIV infections and explore new ways to manage current HIV-1 infections, including the possibility of using immune-mediated control of virus infection upon interrupting drug therapy. Additional studies have focused on developing biomarkers to assess secondary infections, such as tuberculosis, in the presence of an active HIV infection. Showe is a co-Investigator on the BEAT-­HIV Delaney Collaboratory grant to continue these studies.

In collaboration with Cris Constantinescu at The University of Nottingham, UK, the Showe laboratory is examining gene expression profiles in blood and tissue samples from multiple sclerosis patients involved in clinical trials with immuno-modulatory drugs. The aim is to determine whether it is possible to identify gene expression patterns that correlate with responsiveness and non-responsiveness to therapy and to better understand disease initiation and progression.

Studies in collaboration with Hildegund C.J. Ertl at Wistar have examined gene expression changes in young and aged populations as a function of flu vaccination to try to understand the poorer responses to vaccinations in general and in the elderly population in particular. In addition, potential effects of race and timing of repeated vaccination on response were examined. Additional studies using mouse models have explored protocols that increase vaccine response with the potential benefit of improving vaccine efficacy in the growing elderly population.

Shinde is an immunologist with interest in characterizing key factors in the tumor microenvironment and gut microbiome that contribute to the refractory nature of cancer and target them for therapies.

Shinde obtained his D.V.M. from Nagpur Veterinary College, India, and his Ph.D. in immunology from the Augusta University. Before joining Wistar as the first/inaugural Caspar Wistar Fellow, he trained as a postdoctoral fellow in the Tumor Immunotherapy Program at the Princess Margaret Cancer Center in Toronto, Canada.

The Shinde Laboratory

The tumor microenvironment (TME), consisting of stroma, immune cells and extracellular matrix, is a key determinant of cancer initiation, progression and resistance to therapies. Understanding the heterogeneity of the TME and the molecular mechanisms contributing to immune responses is critical to enhance immunotherapy and prevent/overcome resistance. Macrophages are major components of the TME and regulate inflammation, angiogenesis, extracellular matrix remodeling, and T-cell suppression. Alterations in the metabolism of macrophages affect their function. Research in the Shinde lab focuses on characterizing the molecular mechanisms underlying metabolic plasticity in macrophages during tumorigenesis.

Commensal microbiota residing in the gut can also modulate the TME and affect tumor progression and response to therapy. Host, dietary and environmental factors contribute to changes in the microbiome. The lab is also interested in characterizing the healthy/symbiotic or disease-modulating/dysbiotic microbiome and its crosstalk with immunometabolism during disease development.

The first project in our laboratory seeks to understand how host metabolism impacts disease progression and therapy resistance in pancreatic cancer. Tumor metabolism in pancreatic cancer is known to be dysregulated both in neoplastic and in tumor microenvironment (TME) cells, significantly altering immune responses. Metabolic profiling has identified increased plasma levels of branched-chain amino acids (BCAA) in a variety of disorders including pancreatic ductal adenocarcinoma (PDAC). Our new work shows that transcriptional regulators of BCAA oxidation, such as Krüppel-like factors (KLF), are linked to the phenotype of tumor-associated macrophages (TAMs). This project seeks to elucidate the functional implications of KLF biology and BCAA catabolism in TAMs for driving tumor progression and resistance to therapy.

The second project in our lab investigates the connections between gut microbial metabolic pathways and therapy response. Immune responses can be altered by metabolic processes occurring outside the TME, for example, by metabolites produced by gut bacteria. Recent studies show that particular species of gut bacteria influence PDAC outcomes and response to therapy and do so by altering innate and adaptive immunity; however, the specific mechanisms are not clear. This project seeks to identify the metabolic pathways by which the gut microbiome influences antitumor immune responses, impacting PDAC burden and therapy response.

Our studies will have potential clinical impact providing insights into previously unsuspected targets for cancer therapy. More broadly, we expect our findings to shed light on how diet, host metabolism and gut microbiome are linked in shaping immune function and therapy response.

Schug is interested in investigating metabolic adaptation in cancer cells through cell biology, biochemistry, liquid chromatography-mass spectrometry (LC-MS)-based lipidomics, and metabolomics.

After completing his B.S. in biology from Saint Joseph’s University, Schug continued his studies in Philadelphia and earned a Ph.D. in molecular cell biology from Thomas Jefferson University. In 2008, he began his postdoctoral studies at the Beatson Institute in Glasgow, U.K. Schug joined The Wistar Institute in 2016 as an assistant professor. He also holds adjunct faculty positions in the Department of Systems Pharmacology and Translational Therapeutics at the University of Pennsylvania and the Department of Biochemistry and Molecular Biology at Drexel University.

The Schug Laboratory

Alterations in the acquisition and metabolism of nutrients are now firmly recognized as hallmarks of cancer development. Many, if not all, oncogenes and tumor suppressor genes induce metabolic reprogramming in cancer cells through changes in the regulation of enzymes and transporters. These changes are necessary for cancer cells to meet the combined biomass and energy demands for growth and are only satisfied by increased capture and synthesis of cellular building blocks such as sugars, fats, and proteins. In addition, cancer cells often invade other tissues where the availability of certain nutrients is drastically different or grow so quickly that the blood supply, and the accessibility to oxygen and other nutrients that comes with it, becomes scarce. During these conditions of nutrient stress, many cancer cells will adapt and use alternative available resources to survive. As part of this process, we also study the interactions between cancer cells and tumor infiltrating immune cells. The lab seeks to better understand the competition for nutrients in the tumor microenvironment through the study of diet, immunometabolism, and tumor metabolism using LC-MS based approaches.

The Schug laboratory is interested in identifying and therapeutically targeting the metabolic changes that arise during the development of cancer and metastasis. They combine cell biology, biochemistry, metabolomics, lipidomics, and genomics to uncover novel metabolic vulnerabilities in cancer. These targets are then further developed for use as effective therapeutic strategies to improve cancer patient treatment.