Wistar Institute

NIH Research Projects · FY 2026 · 2023-04

Glioblastoma (GBM), the most aggressive and lethal form of brain cancer, is characterized by a profound immunosuppressive microenvironment (TME) that restricts the effects of promising immunotherapies. Therefore, there is a pressing need to develop more effective interventions to overcome this mechanism of resistance. Tumor associated macrophages (TAMs) are a mixture of monocyte-derived macrophages (MDM) and microglia (MG), and they are instrumental for the maintenance of the immunosuppressive state of GBM. However, there are no effective approaches to overcome the immunosuppressive activity of TAMs in GBM, mainly due to an incomplete understanding of TAM regulatory functions. Our long term-goal is to dissect targetable metabolic and molecular mechanisms regulating TAM functions in the context of GBM; as these discoveries will facilitate novel therapies to target immunosuppression and improve the dismaying outcome of GBM patients. A recent study demonstrated that TAM are major consumers of glucose and maintain a robust glucose metabolism in the TME. However, it has not yet been determined how GBM supports the adaptation to glucose metabolism in TAMs and the functional consequences of this adaptation also remain elusive. Endoplasmic reticulum (ER) stress activation is associated with the malignant progression of glioma and with the infiltration of anti-inflammatory macrophages. PKR-like ER kinase (PERK), a critical ER stress sensor, was found to be significantly activated in human glioma tissues, and its inhibition altered ATP/lactate production by glioma cells. Our preliminary data expanded these findings indicating that MDM demonstrated highest glucose avidity among MG and neoplastic cells in GBM tumors, and PERK was strongly activated in GBM infiltrating GLUT1+MDM. Contrary to MG, MDM exhibited potent immunosuppressive activity. GLUT1+MDM were the only contributors to the suppressive activity associated with MDM in GBM tumors. GBM-derived factors primed activation of PERK signaling in MDM, which correlated with metabolic reprogramming resulting in high glycolysis, immunosuppressive functions, histone lactylation, and no change in histone acetylation. Based on our crucial observations, we hypothesize that a PERK-driven perturbation of glucose metabolism in MDM governs their immunosuppressive functions via lactate-derived lactylation of histone lysine residues. We will test this hypothesis through the following aims: Aim1: to elucidate underlying mechanisms of how PERK governs glycolysis in MDM in GBM tumors; Aim2: to define glucose-driven epigenetic modifications that regulates immunosuppressive programs in MDM; Aim3: to investigate the therapeutic potential of an epigenetic targeting approach to modulate the functions of TAMs in GBM. The proposed studies are highly innovative because they will elucidate a previously uncharacterized link between ER stress and glucose metabolism that regulates the activity of TAMs via epigenetic mechanisms. Our proposal will provide a mechanistic rationale for the development of novel therapies to target immunosuppressive TAMs and enhance the efficacy of immunotherapy in GBM patients.

NIH Research Projects · FY 2026 · 2023-04

Project Summary This submission represents a collaborative application from Maureen Murphy PhD at the Wistar Institute in Philadelphia and Kara Maxwell MD/PhD from the University of Pennsylvania Perelman School of Medicine. The goal is to study the function of a novel p53 mutation that was first discovered in several highly cancer- prone families seen in Dr. Maxwell’s clinic at the Hospital of the University of Pennsylvania. This variant, G334R in p53 protein (G331R in mouse p53) was found in nine different families with multiple cancers in multiple generations, including cousins with pediatric adrenal tumors; the latter are a hallmark of cancer- predisposing mutations in TP53. Moreover, we find that several tumors from these patients show loss of heterozygosity for p53. We show that the G334R variant is impaired for oligomerization, and for the induction of a small subset of p53 target genes, several of which are themselves tumor suppressors (PCLO, PLXNB3, and others). Our functional analyses of the G334R variant suggest that this is not a traditional Li Fraumeni mutant; that is, a mutation that completely inactivates p53 function. Rather, our functional data are most consistent with G334R being a p53 “hypomorph”, or a genetic variant that shows impaired, but not completely inactive, function. Specifically, 1) G334R possesses some ability to suppress colony formation in tumor cells, though it shows less ability than wild type p53; 2) G334R is fully capable of transactivating the overwhelming majority of p53-regulated genes, but is defective in the transactivation of approximately two dozen p53 target genes; 3) the peak incidence for breast cancer in G334R individuals is between the ages of 35-55, while most cases of Li Fraumeni occur between the ages of 20-40, and sporadic breast cancer peaks between the ages of 55- 75. The overarching goal of this proposal is to identify the mechanism(s) whereby G334R is impaired for function. In a completely unexpected finding, we show that there are some activities of the G334R hypomorph that are shared with two other cancer-predisposing p53 hypomorphs, P47S and Y107H. These activities include a heightened propensity to misfold into a conformation that is specific for mutant p53, as well as increased sensitivity to glutaminase inhibitors. We also show that using RNA sequencing data and machine learning approaches, we have identified a 42-gene signature that is predictive of a p53 hypomorph, and can distinguish a p53 hypomorph from WT p53 or a benign variant with 100% accuracy. The proposed research will help us better understand tumor suppression by p53, and to identify other carriers of hypomorphic genetic variants of p53.

NIH Research Projects · FY 2026 · 2022-12

Overall Summary A protective vaccine for HIV is arguably the most important prevention strategy to end the global HIV pandemic. The RV144 trial demonstrated a correlation between protection and envelope (Env) specific serum IgG antibodies. However, there was a notable lack of HIV-specific neutralizing antibodies (nAbs) and T cell responses. The DNA platform has several advantages, it is (1) simple to design, (2) can co-deliver molecular adjuvants, (3) can deliver complex structural antigens, and (4) is safe and well tolerated, even after numerous boosts. In our previous IPCAVD program we developed DNA-launched nanoparticle (DLNP) vaccines targeting Env which induced potent and neutralizing immunity in vivo. We have also developed DNA-launched native-like trimer (DL-NLT) immunogens including the clinically relevant BG505 MD39 trimer (Xu et al. Nat. Comm 2022). Protein nanoparticles are difficult to manufacture and poorly stimulate CD8+ T cells. We reported that in vivo assembled nanoparticles drive extremely rapid and strong B and T cell immunity. Functional antibody responses require help from germinal center (GC) follicular helper T cells (TFH). In previous clinical trials with HIV antigens, we observed increased B and T cell responses in the presence of plasmid-encoded IL-12 (pIL-12). Work from members of this team has demonstrated that co-delivery of adenosine deaminase (ADA-1) and plasmid-encoded IL-21 (pIL-21) can enhance antibody induction in mice in a GC-dependent fashion. Here, we combine the DLNP and DL-NLT platforms to generate DNLPs bearing stabilized NLTs focusing immune responses on apex and CD4bs B cell lineage-targeting Envs (DNLP-ACE). We will characterize humoral and cellular immunity to these constructs in the presence of molecular IL-12 with and without additional GC-targeting adjuvants, in mouse and non-human primate vaccination studies. The overarching hypothesis of this project is that DLNP-ACE immunogen technology combined with the co-delivery of genetic adjuvants is a novel approach for the development of HIV-1 vaccines that promotes robust, durable, broad, and neutralizing antibody responses, and supports effector T cell function in vivo A key innovation of this project will be to integrate the selected DNLP-ACE HIV vaccine constructs in a dual expressing plasmid construct with next generation noninvasive intradermal skin electroporation (EP) devices which promote functional CTL and humoral immune responses. The clinical development plan for this IPCAVD is directed by a leading HIV vaccine development organization, Inovio Pharmaceuticals (INO), which has expertise in development of synthetic DNA vaccines and in vivo electroporation delivery. Inovio will oversee clinical grade production of 2 plasmid constructs at a state- of-the-art cGMP plasmid manufacturing facility. Inovio’s processes have passed rigorous international regulatory reviews and have been used in dozens of human clinical trials in the U.S., Europe and Asia, including multiple Phase II and in Phase III studies.

NIH Research Projects · FY 2026 · 2022-11

Project Summary HIV-1 vaccines that can elicit broadly neutralizing antibodies (bnAbs) are a primary goal. To date, it has been demonstrated that a single bnAb lineage (VRC01-class) can be specifically activated in human trials. Antibodies from this trial do not neutralize HIV-1 and heterologous sequential immunization is thought to be required to develop bnAbs. Heterologous sequential immunization has been employed to generate bnAbs in pre-clinical models. While VRC01-class antibodies isolated from infected individuals can be quite broad, the limit of breadth for VRC01-class antibodies induced by these types of vaccination strategies in people is unknown. Therefore, we are developing an alternative bnAb lineage targeting vaccine (VH1-46 class) through advanced protein engineering approaches and assessment in newly generated human immunoglobulin knock-in mice harboring VH1-46 germline antibodies. Further, we are pursuing innovative approaches to develop a dual bnAb lineage targeting vaccine which could synergize with current VRC01-class vaccines. Individual lineage targeting vaccines may not succeed at fully maturating these lineages, thus severely limiting the neutralization breadth and ultimate effectiveness of these vaccines. Dual lineage targeting may be critical for success of the first bnAb- eliciting HIV-1 vaccine.

NIH Research Projects · FY 2025 · 2022-09

Project Summary In December 2019 a novel coronavirus, named sudden acute respiratory syndrome coronavirus-2 (SARS-CoV- 2). SARS-CoV-2 rapidly spread around the globe causing a pandemic disease termed coronavirus disease of 2019 (COVID-19). There have been more than 80 million infections and close to two million deaths from COVID- 19 to date. SARS-CoV-2 is the third beta coronavirus of zoonotic origin to cause human epidemics. It is similar to, but distinct from, Middle East respiratory syndrome coronavirus (MERS-CoV) and sudden acute respiratory syndrome virus-1 (SARS-CoV-1), both of which have caused outbreaks this century. While several candidate vaccines for SARS-CoV-2 have recently received emergency use authorization, the longevity of vaccine-induced responses, the continued emergency of mutation within SARS-CoV-2 strains, and the disproportionate morbidity and mortality among elderly patient populations present continued challenges to control of SARS-CoV-2. Thus, vaccine modalities which can address these challenges for SARS-CoV-2 vaccines and allow for targeting of multiple potentially pandemic coronaviruses simultaneously are greatly needed. Innovative vaccines which can develop broad immunity against known and newly emergent human coronavirus is a key goal in the field. The effects of antigen epitope diversity, density, valency, duration of antigen availability, and adjuvant- induced cytokine environment on the potency and breadth of vaccine-induced Reponses remains unclear. Nanoparticle vaccine formulations allow the ability to manipulate these variables. We have generated self- assembling synthetic DNA-launched nanoparticle vaccines (DLNPs) which displayed increased immunogenicity compared to matched synthetic DNA launched monomer vaccines or protein-in-adjuvant formulations. We determined that synthetic DNA launched nanoparticles increased both cellular and humoral responses. Recombinant nanoparticle vaccines are thought to mediate their increased immunogenicity by persisting in the lymph nodes for extended periods compared to protein antigens, promoting enhanced antigen presentation by follicular dendritic cells and increasing germinal center formation and humoral immunity. Cell-mediated responses to nanoparticle vaccines are less well understood but similar mechanisms may be at play. We will capitalize on the novel in vivo assembling synDLNP platform we have created to manipulate these variables and determine their effects on acute and long-term responses to CoV antigens in young and aged models.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ ABSTRACT Infectious diseases are serious and recurrent health threats. Particularly concerning are viruses with the capacity to mutate and generate de novo diversity in short periods of time. These viruses adapt to new hosts and environments, and continuously escape from the host anti-viral immune response. Preventative vaccines are highly desirable; however, no defined guidelines exist for the design of efficacious vaccines against rapidly mutating viruses such as HIV-1, influenza, or the current SARS-CoV2, with multiple different circulating variants. Despite their high diversity, viral variants present conserved regions that are essential for viral fitness and infectivity. These conserved epitopes are their Achilles heels, and the focus of antibody-based vaccine design efforts. A vaccine against a highly mutating virus should elicit an antibody response that specifically targets the conserved regions of the virus, as it would recognize and neutralize the broad diversity of its variants. Significant efforts in the field have focused on engineering viral immunogens to make their conserved epitopes more available for antibody recognition. Unfortunately, targeting antibody responses to specific conserved epitopes of interest is incredibly challenging. Complex antigens, such as viral spike proteins, elicit polyclonal responses dominated by antibodies to non-conserved epitopes. These antibodies have no potential to broadly neutralize the virus, and also interfere with the maturation of broadly protective antibodies in the germinal centers. Aiming to elicit broadly neutralizing antibodies (bNAbs) against a conserved epitope of HIV-1, we recently designed and evaluated a new HIV-1 Envelope (Env)-based priming immunogen, which elicited bNAb-like antibodies against a conserved epitope of Env in wild type mice and macaques; despite this achievement, these antibodies showed no neutralization activity against HIV-1, suggesting that additional immunization would be required to induce bNAbs. Nevertheless, further immunization in macaques elicited a polyclonal antibody response of only limited potency and breadth, dominated by antibodies to non-conserved epitopes of Env. Based on these observations, I hypothesize that reducing interfering antibody responses to non-conserved viral epitopes, and tracking the antibody responses with potential to become bNAbs, will pave the path towards bNAb development and inform vaccine design efforts. In this proposal, we will design and evaluate a novel strategy to modulate epitope immunodominance, which in addition, will allow us to record and track the history of antigen-B-cell interactions in vivo. The proposed technology will be used to customize the immunodominance properties of complex antigens in order to direct the antibody response to the epitopes of interest. In addition, we will use our new technology to barcode B cells responding to multiple immunizations, track their fates and record their history of antigen encounters. This groundbreaking technology will provide very valuable information to elucidate the mechanisms governing the B cell responses to vaccination and infection, and will significantly contribute to establish guidelines for vaccine design.

NIH Research Projects · FY 2025 · 2022-09

Project Summary / Abstract Our proposal focuses on the development of novel Mdm2-targeted proteolysis targeting chimeras (PROTACs) that efficiently degrade Mdm2 in p53 mutant and deficient cancer cells and are expected to target p53- independent functions of Mdm2. Our lead compounds bind with high affinity, degrade Mdm2, kill cancer cells that lack functional p53, and are efficacious in vivo. We have also demonstrated safety and tolerability in vivo, synthetic tractability, metabolic stability, and suitable in vivo exposure in mouse pharmacokinetic studies for in vivo efficacy evaluation. The tumor suppressor p53, a transcription factor, has an essential role in the prevention of human cancer. In the absence of cellular stress, the p53 protein is maintained at low levels due to its binding to Mdm2, an E3 ubiquitin ligase. However, half of all human cancers have inactivated p53 by mutation or deletion. To test if Mdm2 is required for the survival of cancer cells that lack p53 we inducibly deleted Mdm2 in primary murine p53-null lymphoma and sarcoma cells. Mdm2 loss resulted in apoptosis in vitro and in vivo in p53-null cancers, with significantly reduced tumor burden and increased survival benefit. We and others have reported that Mdm2 has p53-independent functions by binding and regulating other proteins, such as the p53 family member, p73, and Nbs1 in the Mre11-Rad50-Nbs1 DNA break repair complex. p73 has a homologous N-terminal transactivation domain to p53 which binds in the same site on Mdm2. Additionally, unlike p53, p73 is rarely inactivated in human cancers. Since Mdm2-p53 inhibitors are not responsive to tumors with inactivated p53, we pursued a PROTAC approach and provide preliminary data to confirm their ability to kill p53 mutated or deficient cancer cells. Our hypothesis is that the degradation of Mdm2 via Mdm2-targeted PROTACs will kill cancers with mutant or deleted p53 by activating the p53-independent activities of Mdm2. We will test this hypothesis with two Specific Aims. Aim 1 will focus on expanding the characterization of our lead Mdm2 targeting compounds and evaluate these in in vivo xenograft models. Aim 2 will be lead optimization of two Mdm2 PROTACs that killed p53 mutant and deleted cancers. The goal of these aims is to have a characterized, effective, and potent Mdm2 PROTAC for clinical evaluation for the treatment of p53-inactivated cancers.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Aberrations in chromatin structure can be a causal mechanism of numerous human disease and developmental syndromes. R-loops are RNA containing chromatin structures that are deregulated in numerous developmental disorders and cancers. In most cases the underlying mechanisms and the functional significance of R-loop deregulation and whether they are causal to specific disease is not clear. Activity dependent neuroprotective protein (ADNP) is a homeodomain containing transcriptional repressor that is critical for neuronal differentiation and neurodevelopment. ADNP mutations cause ADNP syndrome, a condition characterized by intellectual disability and features of autism spectrum disorder. ADNP is upregulated during neurodevelopment. ADNP contains zinc finger motifs and a homeodomain, both of which can bind nucleic acids. ADNP syndrome mutations result in protein products that lack the homeodomain, underscoring the importance of this region to ADNP function. However, the precise contributions of the zinc fingers and homeodomain to ADNP localization and function in gene expression during neuronal differentiation is not well understood. Our preliminary data have uncovered a novel role for ADNP in the resolution of R-loops. Biochemical assays revealed that ADNP resolves R-loops – in a homeodomain dependent manner. ADNP also suppresses R-loops in vivo, as deletion of ADNP in mouse embryonic stem cells (mESCs) resulted in R-loop accumulation specifically at ADNP target sites, but not at sites not bound by ADNP. Last, ADNP-deficient mESCs and mESCs exclusively expressing an ADNP mutant lacking the homeodomain fail to differentiate into neural progenitor cells (NPCs), indicating an essential role for ADNP and its homeodomain in neuronal differentiation. These results suggest a potentially novel mechanism – R-loop resolution – for ADNP, and possibly other homeodomain-containing proteins, in gene regulation, which can link R-loop dysfunction to numerous disorders and diseases caused by mutations in homeodomain proteins. Based on our preliminary data, we hypothesize that ADNP drives neuronal differentiation by suppressing R-loops; as a corollary, we propose that resolving R-loops that accumulate upon ADNP loss may rescue gene expression programs to enable neuronal differentiation. We propose two aims to test our central hypothesis. In Aim 1, we will elucidate the molecular basis of R-loop resolution by ADNP by identifying roles of the ADNP protein domains and their interactions with nucleic acids. In Aim 2, we will elucidate how ADNP suppression of R-loops influences neuronal differentiation.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY While several therapies have been approved for melanoma, there are limited treatment options for NRAS mutant (NRASmut) tumors, which account for ~30% of all melanomas. NRASmut tumors are extremely aggressive and are associated with poor patient survival. These types of tumors are highly resistant to available targeted therapies and are poorly responsive to immunotherapies. Therefore, there is an urgent unmet need to identify novel targets and effective therapies to help this large population of melanoma patients who do not respond to currently available treatments. Our goal is to identify critical vulnerabilities that can be targeted to offset drug resistance in NRAS mutant melanoma. Oncogenic NRAS activates both the MAPK and PI3K pathways. However, inhibiting either pathway alone is barely effective in patients and co-targeting both pathways leads to unacceptable toxicities in patients. We previously reported that BRAF-mutant melanomas resistant to BRAF and MEK inhibitors (MAPKi-R) have sustained activation of the ribosomal protein S6 kinase. We have now discovered that MAPKi-R NRASmut melanomas rely on the S6K2 isoform for survival. Selective S6K2 blockade, in the context of active S6K1, perturbs redox and lipid metabolism, triggering lethal lipid peroxidation in NRASmut melanoma cells that are resistant to MAPK inhibition. Based on our preliminary findings, we postulate that S6K2 controls metabolic and redox homeostasis, and melanoma cell survival, thereby constituting a novel and promising therapeutic target. As S6K is a node of convergence of the MAPK and PI3K/mTOR pathways, we further posit that selectively blocking S6K2 can overcome resistance to MAPKi mediated by broad molecular mechanisms that rely on these pathways. In this project we will define the mechanism whereby S6K2-dependent lipid and redox homeostasis contributes to drug resistance and promotes survival of MAPKi-R melanoma. Furthermore, we will exploit the dependency of melanoma on S6K2 to offset MAPKi resistance. Our proposed strategy, which is significantly different from MAPK/PI3K inhibition, will enable functional precision by targeting a convergent subnetwork representing a vulnerability selectively in tumor cells. We anticipate that our studies will provide a mechanistic framework to inform the design of therapeutic strategies targeting S6K2 directly, or alternatively, S6K2 specific effector pathways and improve the outcomes of NRASmut melanoma patients and possible other types of RAS-mutant tumors.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY ATRX is an epigenetic factor that is mutated in ATRX syndrome. ATRX is required for the maintenance of multipotent neuroprogenitor cells (NPCs), particularly as these cells initiate differentiation programs in the brain. Mutations that cause ATRX syndrome cluster within two domains: the ATRX PHD finger domain, and the helicase domain. These mediate interactions with histones, and remodel chromatin, respectively. Interestingly, mutations in the PHD finger are associated with severe intellectual disability and psychomotor impairment, while mutations in the helicase domain often manifest with milder neurodevelopmental delays but more severe genital abnormalities13. The basis for this genotype-phenotype correlation has never been investigated. ATRX has well- established roles in molecular processes that are crucial for normal brain development including histone variant H3.3 deposition and Polycomb repressive complex 2 targeting for epigenetic silencing. It also has a poorly understood role in regulating CTCF, a critical genome architectural protein that is essential for development. Our preliminary data indicate PHD finger and helicase mutations of ATRX differentially regulate CTCF localization and may underlie genotype-phenotype correlations. Our preliminary data has uncovered a genome-wide role for ATRX in CTCF localization. We found that ATRX knock down (KD) in mouse embryonic stem cells (mESCs) results in CTCF accumulation at many genomic sites, including both imprinted and non-imprinted loci. We also discovered that ATRX interacts with ADNP and DNMT3L, both of which can prevent CTCF binding. Using CRISPR/Cas9, we have generated isogenic ESCs where the endogenous Atrx allele has been replaced by point mutants found in ATRX syndrome patients. We show that mutations in the PHD finger cause significant impairment of NPC differentiation, while mutations in the helicase domain cause more subtle differentiation delays. Our preliminary findings and novel reagents uniquely position us to interrogate the consequence of ATRX mutations, both for genome organization through CTCF, and for the ability of ESCs to differentiate into NPCs. Based on our preliminary data, we propose to test the hypothesis that distinct ATRX domains regulate specific chromatin processes that impinge to different extents on the function of CTCF. In Aim 1, we will investigate the consequence of ATRX syndrome mutations to gene expression and genome organization during neuronal differentiation. In Aim 2, we will decipher the mechanisms by which ATRX regulates CTCF localization.

NIH Research Projects · FY 2026 · 2022-04

Project Summary/Abstract Notch1 signaling is an important mediator of stem cell self-renewal and therapeutic resistance, and the most prevalent oncogene (~60%) in T-cell acute lymphoblastic leukemia (T-ALL) - an aggressive neoplasm of T cell progenitors that affects both children and adults. Although current intensive chemotherapies can suppress the disease, they come at the cost of serious side effects and are insufficient to eliminate Notch1-driven leukemic cells. One in five children and one in two adults with T-ALL do not survive due to either unresponsive or relapsed disease. Efforts to target oncogenic Notch1 with small-molecule inhibitors have been hampered by their inherent cytotoxicity. Overcoming these difficulties will require improved understanding of the oncogenic mechanisms controlled by Notch1 and a better appreciation of the genes and pathways that regulate Notch1- driven leukemogenesis as potential targets of T-ALL therapy. Through Drosophila studies and the generation of mouse models for T-ALL, we have discovered that, T-ALL-associated Notch could be degraded by an unconventional endo-lysosomal module through a physical interaction with the autophagic tumor suppressor UVRAG, which reshapes Notch activity and resultant Notch-dependent cellular response. Thus, the central hypothesis of this proposal is that the endo-lysosomal titration of Notch activity by UVRAG represents a unique mechanism governing Notch1 before proteolytic processing, and that disruption of this regulatory module impacts T-cell homeostasis and contributes to T-ALL. Specifically, we propose experiments to comprehensively dissect the molecular mechanism of UVRAG-mediated endo-lysosomal inhibition of Notch1 in T-ALL. Furthermore, we will elucidate the unequivocal impact of this mechanism on the self-renewal and stemness of leukemia-initiating cell function in human T-ALL primary samples. Finally, we will use the mouse models to test the concept that boosting this mechanism could restore Notch homeostasis and achieve sustained T-ALL remission. These aims will be addressed using multidisciplinary innovative approaches that integrate state-of-the-art genetic, biochemistry, high-resolution imaging, and physiological assays in cells and transgenic mouse models. We now bring within this proposal a collaboration of world-wide leaders in T-ALL pathology and molecular biology along with clinicians and pathologists. Our use of patient-derived T-ALL samples will maximize the relevance of our findings for eventual translation to T-ALL patients in the clinic. Overall, this project will lead to an in-depth understanding of Notch1-driven leukemogenesis, and provides a critical trajectory for the development of optimal anti-leukemia strategies against this aggressive lymphoid malignancy.

NIH Research Projects · FY 2026 · 2022-01

The long-term goal of this R01 is to understand how epigenetic mechanisms control Epstein-Barr Virus (EBV) latency and carcinogenesis. EBV latent infection is associated with a diverse spectrum of epithelial and lymphoid malignancies. The highly adaptive nature of EBV infection to various host cells and environments suggests that it exploits fundamental cellular processes of dynamic gene regulation. EBV is known to adapt various gene expression programs, termed latency types, in different host cell and tumor environments. These latency types and viral gene expression patterns are determined by epigenetic factors ranging from nucleosome positioning, histone modifications, CpG DNA methylation, transcription factor occupancy, and chromosome conformation. The mechanisms regulating viral and host DNA epigenetic controls are not fully understood but are critical for understanding viral latency and oncogenesis in diverse cell types. We have been investigating the process through which EBV establishes and regulates the epigenetic program of both viral and host genomes. In the previous funding cycles, we identified the viral tegument protein BNRF1 as a binding partner of DAXX-histone H3.3 complex and showed that this interaction is required for viral chromatin assembly and gene expression during the early, pre-latent phase of infection. We have identified viral and cellular transcription factor binding sites for EBNA1, EBNA2, CTCF, cohesin (RAD21), EBF1, RBP JK and chromosome conformations that change during the establishment of latency and correlate with different latency types. We have assayed chromatin accessibility and RNA expression changes during the multiple stages of EBV-induced B-cell immortalization to correlate gene expression with chromatin architecture. We have also found that viral and host DNA methylation programming depends on viral EBNA2 and vmiRNAs that coordinately regulate TET2 expression and cytosine hydroxymethylation and demethylation. We now propose to further advance these studies to better understand the role of epigenetic mechanisms in the control of EBV latency and oncogenicity. We will test the overarching hypothesis that EBV reprograms host epigenetic mechanisms to enable viral genome persistence and transcriptional plasticity that drives EBV-associated oncogenesis.

NIH Research Projects · FY 2025 · 2021-12

PROJECT SUMMARY Research Plan: An impaired hematopoietic differentiation process underlies bone marrow malignancies like leukemia, but we still lack the mechanistic understanding of the sequence of regulatory events that misleads the differentiation process. Since epigenomic regulatory patterns are major features of leukemic development, understanding the chromatin dynamics of a failed (malignant) hematopoietic differentiation process can help define the molecular basis of leukemia. A prerequisite to such an understanding is a framework that allows investigation of the progressive changes in the activity of the regulatory elements (RE) during hematopoietic differentiation. Single-cell CUT&Tag (scCUT&Tag) technology is well-suited for such studies as RE activity through histone modification profiles can be investigated in a lineage-specific manner. However, poor understanding of the cell-type-specific histone modification patterns makes the task challenging. To overcome this challenge, we designed scCUT&Tag-pro which allows simultaneous measurement of cell-surface protein and in-silico integration of gene-expression and chromatin accessibility. I will leverage this novel multimodal framework to investigate the RE and progressive changes in their activity during hematopoiesis. First, I will define a multimodal reference mapping framework for mouse hematopoiesis. This framework will allow me to integrate multiple histone modification profiles onto one reference and compare the chromatin states of the RE between a wild type (WT) and mouse model with loss of function in histone methyl transferase (HMT) (Aim 1). Second, since HMTs regulate transcription through the interaction network of RE. I will define a chromatin state aware map that dynamically links REs across developmental trajectories. I will use this framework to investigate the changes in the interaction of REs due to HMT loss (Aim 2). Third, since the transcriptional state of a cell emerges from the underlying gene regulatory network (GRN), I will integrate single-cell gene expression data with histone modification profiles and extend it to define a chromatin state aware model of GRN. I will compare the WT and HMT loss experiments and define the differential GRN (Aim 3). Altogether, this research proposal seeks to pioneer the computational methods for the integrated analyses of multimodal single-cell histone modifications and systematically dissect progressive changes in the system-level function of the regulatory circuits that misleads hematopoietic differentiation using mouse models with conditional HMT loss of function in the hematopoietic compartment. I have developed a 5-year career development plan to meet my goal of becoming an independent investigator in the multi-disciplinary field of computational cancer biology. The mentorship committee will also provide me the guidance in my research and academic job search. Given the excellent the outstanding record of training multiple independent scientists, New York Genome Center provides me an ideal environment to attain my scientific career goals.

NIH Research Projects · FY 2026 · 2021-12

Project Summary The prominent change in the myeloid compartment in cancer is the expansion of pathologically activated immature myeloid cells with the potent ability to suppress immune responses – myeloid-derived suppressor cells (MDSC). In tumor-bearing mice, the total population of MDSC consists of three groups of cells: the most abundant (>75%) immature, pathologically activated neutrophils (PMN-MDSC); less abundant (<20%) population of pathologically activated monocytes (M-MDSC); and small (<5%) population of early myeloid precursors. In the tumor microenvironment MDSC are more immunosuppressive than in peripheral lymphoid organ. However, the mechanism of this phenomenon remains rather elusive. The gaps in our knowledge is in understanding the mechanisms regulating the function of MDSC in tumors and specific requirements for their targeting. In this proposal we will test the hypothesis that there are distinct populations of MDSC in tumors. These populations can be defined by specific markers and most importantly, have different sensitivity to ferroptotic cell death which determines their functional activity. We will test the concept that targeting ferroptosis in PMN-MDSC in cancer may have functional consequences for immune responses. The goal of this project is to uncover the mechanisms regulating myeloid cell function in tumors and to develop novel approaches to the regulation of immune responses in cancer. We propose the following Specific Aims: (1) To identify the mechanism of ferroptosis-mediated immune suppression induced by PMN-MDSC in tumors; and (2) To investigate therapeutic potential of targeting ferroptosis in PMN-MDSC.

NIH Research Projects · FY 2025 · 2021-09

Project Summary – Overall This Wistar/UPenn Skin SPORE represents a highly successful and longstanding collaboration. Immune checkpoint inhibition has revolutionized melanoma therapy to the point where every high-risk melanoma patient will be treated at some point with these agents. However, many major questions remain on how best to use these immune therapeutics. Project 1 will address the unmet need to find an effective biomarker to select patients for single agent versus combination immunotherapy. Many patients start treatment with ipilimumab and nivolumab, when they may have responded to anti-PD-1 antibody (Ab) alone, exposing these patients unnecessarily to the toxicity of combination checkpoint inhibition. Project 1 builds on a fundamental discovery made through our Developmental Research Program (DRP) that exosomal PD-L1 is an immunosuppressive factor secreted by melanomas. We propose rigorous clinical utility studies designed to demonstrate this blood- based measurement as a highly sensitive and specific predictive biomarker for anti-PD-1 antibody (Ab)-based therapy. Project 2 will address a second unmet need for a safer and effective combination regimen that promises to be effective in anti-PD-1 Ab refractory patients. Based on extensive preclinical data and a new molecular target in the autophagy pathway, we have developed a clinical trial of combined anti-PD1 Ab and autophagy inhibition, a new strategy for reprogramming tumor-associated macrophages to enhance the efficacy of T cell killing. Project 3 fills a major gap in the treatment of early disease by conducting a clinical trial with anti-PD1 Ab in Stage IIB/C melanoma patients. Besides in-depth characterization of the immune response, the Project’s preclinical studies will lead to new strategies for enhancing the immune stimulatory capacity of dendritic cells in the tumor microenvironment. These three highly translational Projects are supported by longstanding Cores that have a proven track record of adapting to the rapidly changing needs of melanoma and non-melanoma skin cancer researchers. Each Project was chosen by the current SPORE leadership for its potential for significance, impact and innovation. Together, they have the potential to advance therapeutically exploitable biological insights into new, clinically important therapies of patients with melanoma. Funding from the SPORE has provided us with important advantages, including a mature, collective, translational mindset, an efficiently functioning tumor bank, and a highly evolved framework of collaboration between The Wistar Institute and UPenn. The SPORE has allowed us to bolster horizontal and vertical collaborations with academic and industry partners throughout the world. The Career Enhancement Program and DRP have enabled transition to new leadership, have formed the three Projects proposed, and have allowed our research to reach into other cancers of the skin including SCC, CTCL and Merkel Cell carcinoma. These programs will continue to be supported robustly by strong institutional support from both Wistar and UPenn. Funding of this SPORE will bring new advances from the bench to the bedside and fulfill our overall mission of improving survival for skin cancer patients.

NIH Research Projects · FY 2025 · 2021-08

Summary Current HIV curative strategies have proven insufficient to eradicate viral reservoirs or prevent viral rebound after antiretroviral therapy (ART) cessation. The unifying hypothesis for the BEAT-HIV Collaboratory is that through a better mechanistic understanding of HIV latent reservoirs and host factors governing viral control and reactivation, long-term viral remission or eradication of HIV will be achieved by combination immunotherapy inclusive of bNAbs, adoptively transferred immune cells, and nanoparticle therapies. We will test this hypothesis by pursuing three highly interconnected research focus areas. The first aim will seek to understand epigenetic status of intact proviruses, extrinsic/intrinsic factors affecting proviral reactivation and expression, and novel host mechanisms for post-treatment control of HIV. The second aim will develop strategies for long-term control in the absence of ART by use of DNA-delivered anti-HIV bNAbs and eCD4Ig in combination with optimized tissue-based CD8 T-cell- and NK cell-mediated responses. The third aim will develop a combination nanotherapy and immunotherapy strategy to eradicate viral reservoirs. All aims will be supported by a clinical biorepository (blood and tissue), CD34+ or patient-derived xenograft humanized mice, non-human primate (NHP) models, and a clinical trial development group as a link to the ACTG. Community engagement will advance education and a socio-behavioral sciences and ethics focus by leveraging a >25-year relationship with the local HIV community thereby ensuring partnership with stakeholders. Central administration of resources will ensure achievement of high impact milestones, study team communications, and yearly goal- oriented resource allocation and/or redistribution as informed by advances in the field. As an established Collaboratory, we bring together diverse expertise, innovation, and industry partners to develop and test novel strategies to advance an HIV cure and/or durable viral control in the absence of ART under a single common multi-investigator, multi-industry team. Studies within the three interconnected aims together with a strong community engagement plan will lay the groundwork for future clinical trials that will integrate new knowledge gained by the BEAT HIV-1 Collaboratory to eradicate or functionally cure HIV infection.

NIH Research Projects · FY 2024 · 2021-08

Project Summary Viral infections are known to produce double-stranded RNA (dsRNA), a molecule that is not present at high levels in uninfected host cells. This property of dsRNA is exploited by cells to sense viral infection and deploy anti-viral countermeasures. While DNA viruses produce viral mRNA molecules that look identical to cellular RNA, many DNA viruses are thought to produce dsRNA due to the process of symmetrical gene transcription of both strands of DNA. When we looked for the presence of dsRNA during adenovirus (AdV) infection using modern antibody-based techniques we found no evidence of dsRNA production, directly countering the existing dogma. Considering many DNA viruses encode antagonists of cellular dsRNA-sensing pathways, this directly calls into question the relevance of dsRNA sensing during DNA virus infection. While wildtype AdV did not produce detectable dsRNA, viral mutants which can no longer splice their own transcripts efficiently saw robust accumulation of dsRNA within the nucleus. Furthermore, these dsRNA-producing mutants activated cytoplasmic sensors of dsRNA such as PKR and RNaseL. The use of mutant viruses provides a unique opportunity to assess host responses to dsRNAs derived from DNA virus infection. Still, the question of how these nuclear dsRNAs are detected by cytoplasmic sensors remains unanswered. By completion of this mentored career development award I will gain training in RNA sequencing, quantitative mass spectrometry, and the bioinformatics approaches to analyze both. In the mentored phase I will continue my training with AdV, a relatively simple virus that provides powerful tools to understand regulation and sensing of DNA virus derived nuclear dsRNA. In the independent phase I will utilize herpes simplex virus (HSV- 1), a complex virus able to exert control over dsRNA-sensing pathways, as a model virus to study exploitation of dsRNA for viral gene regulation. This proposal will reveal the binding partners and localizations of DNA virus derived dsRNA as well as new strategies in which viruses exploit host cell gene regulatory machinery. In Aim 1 I will determine the localization and binding partners of viral dsRNA using immunoprecipitation coupled to next generation sequencing and mass spectrometry. These experiments will determine how nuclear dsRNA leads to activation of cytoplasmic sensors, as well as how AdV interacts with and blocks these novel pathways. In Aim 2 I will determine how HSV-1 regulates its own viral gene expression using the nuclear retention of overlapping viral transcript pairs that form dsRNA. The outcome of these experiments will reveal a new mechanism for viral gene regulation with broad implications for all herpesviruses. The outstanding training environment at CHOP and the University of Pennsylvania, coupled with the excellent advisory committee I have assembled, will greatly facilitate my research during the mentored phase as well as launch my career with the skills necessary to transition to an independent faculty position studying how host cells sense the RNAs generated by DNA viruses.

NIH Research Projects · FY 2025 · 2021-06

Project Summary EBV latent infection is responsible for ~200,000 new cancers per year. To date, there are no EBV- specific therapeutic agents that selectively and efficaciously treat EBV-positive tumors. All known EBV tumors consistently express one viral nuclear protein, EBNA1, that is required for maintaining the EBV genome and promoting infected cell survival. We have developed highly selective, drug-like small molecules that bind EBNA1 and block its ability to bind DNA, maintain EBV genomes, and promote host-cell survival. Here we propose to better understand the mechanism through which disruption of EBNA1 DNA binding leads to tumor growth inhibition, and use this information to identify rational combinatorial agents to enhance chemotherapeutic efficacy. We propose to enhance the potency of the first generation EBNA1 inhibitors by attaching proteasome targeting molecules (PROTACS) to selectively target EBNA1 for degradation. Finally, we will take advantage of new mechanistic data revealing that EBNA1 functions as an OriP-specific endonuclease and resolvase. We propose to develop new structure and mechanism-based inhibitors of EBNA1 that can increase potency necessary for highly efficacious cancer therapy. By integrating these strategies to understand the growth arrest response of EBNA1 inhibition (aim 1) to better develop rational approaches for combinatorial therapies (aim 2) and develop next generation molecule with structure/mechanism based drug design principles (aim 3), we will advance EBNA1 inhibitors for the treatment of EBV-associated malignancies and related-diseases. We will test the overarching hypothesis that EBNA1 is an effective target for small molecule inhibitors to treat EBV cancers. The major goal of this proposal is to understand the tumor cell response to EBNA1 inhibition and to enhance efficacy of EBNA1 inhibitors to treat EBV-associated cancers more efficaciously. The team associated with this proposal has the unique expertise and strong collaborative history to execute the aims of this proposal. Collectively, these investigations will provide fundamental insights into how EBNA1 functions at the molecular level and will lay the foundation for the development of new strategies to treat EBV cancers.

NIH Research Projects · FY 2025 · 2021-05

Epstein-Barr Virus (EBV) reprograms host cell gene expression and metabolism during the establishment of latency and the immortalization of B-lymphocytes. The regulatory mechanisms coordinating this reprogramming with EBV latency reflect important events in viral oncogenesis, yet remain poorly understood. We have found that key viral regulators of EBV latency, including the major tegument protein BNRF1 and the EBV Nuclear Antigen EBNA1 coordinate key aspects of purine metabolism during establishment of latency and immortalization of primary B-cells. In this R01, we focus on how EBV reprograms purine metabolic gene expression, and how purine metabolites contribute directly to EBV tumorigenesis. One clue to this coordinate regulation is provided by the viral-encoded tegument protein BNRF1 that shares extensive structural similarity to the purine biosynthetic enzyme FGARAT (also called PFAS) and functions in viral chromatin assembly during primary infection. Orthologues of BNRF1 are found in all gamma herpesviruses, including KSHV ORF75, and share the common function of disarming components of the PML-nuclear body (PML-NB) and its anti-viral functions. We have previously shown that BNRF1 interacts with the histone H3.3 chaperone DAXX and displaces its interaction with the ATP-dependent SNF2-like helicase ATRX to enable selective expression of latency-specific viral genes during primary infection. However, it has not yet been shown how the viral FGARAT homology domain is linked to cellular purine biosynthesis and/or signaling. Using metabolomics mass spectrometry, we provide new preliminary data indicating that the purine biosynthetic pathway is among the most significantly perturbed by EBV during B-cell immortalization. Integrating gene expression (RNA-Seq), chromatin accessibility (ATAC-Seq), and EBNA1-DNA binding to host chromosome (ChIP-Seq), we identified cellular metabolic genes, including adenine deaminase (ADA), adenosine kinase 4 (AK4), and purinergic receptors P2RY8 and P2RX5 as direct targets of EBNA1 transcriptional regulation during EBV immortalization. We now propose to investigate the mechanisms by which EBV senses and reprograms purine metabolism and how purinergic signaling regulates establishment of EBV latency and host cell transformation. We will test the central hypothesis that EBV coordinately regulates cellular purine metabolism with viral and cellular gene expression during the B-cell immortalization process, and that purinergic signaling is critical for viral latency and oncogenesis. Specifically, we will investigate how EBV regulates expression of purine metabolic genes during primary infection (Aim 1), elucidate how purine metabolism impacts the establishment of EBV latency (Aim 2), and investigate the role of purine metabolism and signaling in B-cell immortalization, immune signaling, and EBV-induced tumorigenesis (Aim 3). These studies will advance our understanding of basic mechanisms coordinating gene expression with metabolism and identify new targets for therapeutic intervention in viral latency and viral-associated cancers.

NIH Research Projects · FY 2025 · 2021-05

Project Summary/Abstract The ultimate goal of this mPI proposal is to address a fundamental gap in knowledge on the role of acetyl-CoA metabolic reprogramming in regulating cyclin E-high ovarian cancer DNA damage response, transformation, and response to therapy. The results from these studies could have a significant impact on the treatment of the ~20% of high grade serous ovarian cancer (HGSOC) patients with high cyclin E expression, which are resistant to emerging PARP inhibitor therapies due to proficiency in homologous recombination (HR)-mediated DNA repair. This research plan focuses on assessing the experimentally and mechanistically determining the spaciotemporal metabolic reprogramming of acetyl-CoA on histone hyperacetylation and enhancement of HR-mediated DNA repair and whether this pathway can be targeted in cyclin E-high HGSOC patients in combination with emerging PARP inhibitor therapies to obtain a synthetic lethality and sustained therapeutic response. The proposed studies are based on our preliminary findings that glucose-derived acetyl-CoA is upregulated in cyclin E-high cells, acetyl-CoA is spatially regulated in the cytoplasm and nucleus, and cyclin E-high cells display hyperacetylation of histones known to be involved in HR repair. In line with these data, we will explore two overarching scientific aims: 1) quantitatively dissect acetyl-CoA metabolic reprogramming in cyclin E-high HGSOC and its contribution to HR-mediated DNA repair; and 2) to determine whether acetyl-CoA mediated epigenetic changes contributes to ovarian tumorigenesis and therapeutic response. The completion of the scientific aims of this proposal will not only provide new mechanistic insights into the interplay between the acetyl-CoA-mediated metabolic-epigenetic axis during ovarian tumorigenesis, but will also establish targeting this axis as a strategy to improve therapeutic outcome for HGSOC patients with high cyclin E. The proposed research is of high impact because the mechanistic underpinning of these pathways has the potential to transform the management of HGSOC patients with high cyclin E. As PARP inhibitors are being developed for many cancer types, studies will have far-reaching implications for identifying novel strategies to inhibit HR-mediated DNA repair and develop future cancer therapeutics strategies for a wide range of patients.

NIH Research Projects · FY 2025 · 2021-01

We discovered that a human TP53 gene variant at amino acid 47 codes for a “hypomorphic” p53 protein. This hypomorph p53 variant (P47S) retains most but not all of its functions and has been linked to changes in innate and adaptive immunity. Macrophages containing the P47S variant are defective in ferroptosis, M2 polarized, and show more productive infections by H. pylori (Hp) and other bacteria. Macrophage adaptive response is essential for eliminating Hp infection and is suppressed in chronic infections that lead to gastritis. Unbiased WT and P47S macrophage proteomics revealed marked differences in Liver X Receptor activation, arginase II activity, inflammation, iron transport, and antibacterial defense machinery which regulate immune response and directly affect outcomes of bacterial infections. Although p53 is known to regulate immune response, we are the first to discover the exact genetic variant causing the effect on innate immunity. In this project, we aim to reduce exacerbated Hp infection damage associated with the P47S SNP by in-depth mechanistic and therapeutic study. Our human studies will give translational relevance to this project. We will test ~500 Hp seropositive individuals for the prevalence of P47S and other SNPs. Then we will focus on improving macrophage response, Hp clearance, and reduce chronic gastric dysplasia in P47S mice using Liver X Receptor (LXR) agonists. Moreover, we will generate a genome-wide map of macrophage binding of LXR to ‘fine tune’ its activity on macrophage activation and eliminate undesired effect of hepatic steatosis. Since Hp is found both extracellularly and within macrophages, specific depletion of immune cells will be used to distinguish the role of macrophages from other tissue environment in Hp pathogenesis. These studies will be validated by bone marrow swaps between WT and P47S mice. We will prioritize and test pathways associated with LXR such as arginase pathway (Arg2, Slc7a2), iron transport (Tfrc, Slc40a1, Fth1) and innate immune proteins (TLR3 and TLR7) as novel therapeutic targets. Lastly, we will test macrophage regulatory factors (NOTCH1 and mTOR), energy metabolism (switching from ox-phos to glycolysis by Metformin) and polyamine pathway (DFMO-polyamine synthesis inhibitor) in fine tuning LXR agonist induced reversal of anti-inflammatory polarization in P47S mice, clearance of Hp infection, and reduction of gastric dysplasia. This proposal will provide translational relevance by associating the P47S SNP to increased Hp prevalence and pathogenesis while providing several therapeutic avenues to improve clearance of Hp infection and gastritis.

NIH Research Projects · FY 2025 · 2020-04

Project Summary/Abstract The ultimate goal of this proposal is to address a fundamental gap in knowledge on the role of the cell cycle inhibitor p16 in regulating pro-tumorigenic metabolism. The results from these studies could have a significant impact on the treatment of melanoma patients, of which ~30-40% have downregulation or deletion of p16. This research plan focuses on experimentally and mechanistically determining the role of p16 loss in pro-tumorigenic nucleotide metabolism and whether this pathway can be targeted in p16-low melanomas alone or in combination with mutant BRAF inhibitors to obtain a sustained therapeutic response. The proposed studies are based on preliminary findings that loss of p16 expression upregulates the newly-identified ATR-mTORC1 signaling axis to increase nucleotide metabolism through the pentose phosphate pathway, and modulation of this pathway is a metabolic vulnerability for p16-low cancer cells. In line with these data, we will explore two overarching scientific aims: 1) to mechanistically dissect the ATR-mTORC1 pathway downstream of p16 loss in melanomagenesis and determine the contribution of pro-tumorigenic nucleotide metabolism to the observed phenotypes; and 2) to elucidate whether targeting the ATR-mTORC1 pathway in combination is synergistic in p16-low melanomas alone or in combination with mutant BRAF inhibitors. The completion of the scientific aims of this proposal will not only provide new mechanistic insights into the interplay between metabolism and the cell cycle during tumorigenesis, but will also establish targeting the novel ATR-mTORC1 axis as a strategy to improve therapeutic outcome for melanoma patients with low p16 expression. The proposed research is of high impact because the mechanistic underpinning of these pathways has the potential to transform the management of melanomas with low p16. As p16 is altered in ~50% of all human cancers, these studies will have far-reaching implications for identifying metabolic vulnerabilities and developing future cancer therapeutic strategies for a wide range of patients.

NIH Research Projects · FY 2026 · 2018-04

Epigenetics is crucial for regulating the Epstein Barr Virus (EBV) different latent gene expression programs, referred to as latency types. It has recently been discovered that these different latent programs are regulated by changes in a complex network of histone modifications, chromatin 3D structure, and epigenetic factors. However, it is still unknown what cellular signals trigger these epigenetic changes and what mechanisms couple these signals to chromatin-dependent viral gene regulation during latency. Our long-term goal is to understand how epigenetics regulates EBV latency. Cellular metabolism drives epigenetic changes as chromatin-modifying factors relay their activity on specific metabolites. Thus, metabolic changes can be coupled with transcriptional changes through epigenetic means. In the previous funding period, we identified a potential mechanism that could link NAD+ metabolism to viral EBV gene expression: PARP1 regulation of EBV chromatin; the activity of PARP1 relies on NAD+ availability. We demonstrated that: 1) EBV infection activates PARP1, and 2) PARP1 forms a complex with CTCF that controls the EBV genome 3D genome structure. Here we expand on these observations and report that in order to regulate CTCF functions and maintain an open chromatin state across the EBV genome, PARP1 may need to interact with NMNAT1. NMNAT1 is a critical nuclear enzyme for NAD+ biosynthesis in the nucleus, and an essential one for supporting PARP1 activation. We are currently confirming that NAD+ depletion alters chromatin composition and CTCF occupancy across the viral genome, eliciting cytotoxicity in EBV+ B cells. We have already identified the NuRD repressive chromatin complexes and the macroH2A1.1, a histone variant that binds NAD metabolites, as part of the mechanism that couples NAD+ levels with transcriptional changes of the EBV genome through PARP1 and CTCF. We have done this using proteomics, ChIP, and NAD+ depletion. Based on this data, we hypothesize that NAD+ metabolism influences EBV latency through PARP1-mediated chromatin changes in viral transcription programs.

NIH Research Projects · FY 2025 · 2017-09

Project Summary The Proteomics and Metabolomics Shared Resource (PMSR) provides a comprehensive set of proteomics and metabolomics assays to the Wistar Cancer Center, Fox Chase Cancer Center at Temple University, and Sidney Kimmel Cancer Center at Thomas Jefferson University as a primary goal. Resources of the PMSR are also available to investigators in other Cancer Centers and academic institutions as a secondary goal. Dr. Tang’s role as the PMSR Managing Director is to support NCI-funded cancer research projects by providing expert consultation and state-of-the-art technologies that operate at maximum performance at affordable costs to cancer investigators and other biomedical researchers. The Managing Director will assist in experimental design, perform MS data analyses as needed, and assist in the biological interpretation of results. The Managing Director will also devote substantial effort to optimizing and implementing new methods, update analytical and data analyses methods, and update instrumentation to ensure each project is performed using state-of-the-art methodologies. This is critical because instrumentation, software, and analytical strategies continue to evolve rapidly, and most current and anticipated future projects involve very challenging proteomics and metabolomics problems. Proteomics projects will include: 1) in-depth, global quantitative comparisons of exosomes, secretomes, cell lysates, tissues and biological fluids; 2) quantitative comparisons of post-translational modifications; and 3) LC-MS/MS analysis of isolated protein complexes with and without chemical crosslinking. Quantitative data will be obtained either using label-free quantitation of integrated MS ion currents, or using stable isotopes such as SILAC or TMT isobaric tag labeling. Metabolomics projects will include quantifying the steady state levels of polar metabolites, lipids and fatty acids, and 13C isotope tracer analysis. Representative specific plans for future development of new analytical approaches include: 1) improving characterization of protein palmitoylation with identification of specific modification sites; 2) de novo peptide sequencing for identification of HLA peptides; 3) an alternate approach to ubiquitome enrichment; and 4) absolute quantitation of gut metabolites. In addition, Dr. Tang will implement additional new or improved methods that are likely to be needed in future cancer projects. These proteomics and metabolomics analyses will contribute to critical data required to identify biomolecular targets, as well as generate hypotheses that are vital for the success of the cancer-related projects described in this application.

NIH Research Projects · FY 2025 · 2017-09

Project Summary – Overall The overarching goal of this Patient Derived Xenograft (PDX) Development and Trial Center (T-PDTC) is to develop functional precision combination therapies that can be translated into clinical trials to overcome drug resistance and produce to long-term responses improving the outcomes of melanoma patients. The melanoma treatment landscape has radically improved in the past decade due to availability of new immune- and targeted- therapies available. Targeted therapies using BRAF and MEK inhibitors have been approved for patients with BRAFV600E/K mutations, which are present in ~50% of cutaneous melanomas. These treatments elicit clinical responses in ~80% of BRAFV600 mutant patients. However, most patients eventually progress. Additionally, there are no effective targeted therapies for patients whose tumors harbor wild-type BRAF. Thus, there is an urgent unmet clinical need to develop efficacious treatments to prevent or overcome resistance to current FDA- approved therapies. To facilitate the development of new therapeutic strategies that can be translated into clinical trials, we have developed a broad collection of PDX models that reflects the clinical, histological, and genetic heterogeneity of melanoma. Our collection of >500 PDX models represents one of the largest collections for any human malignancy. Our initial studies have demonstrated that our PDX collection recapitulates the molecular heterogeneity observed in patients. This collection also includes a subset of PDX established from patients with intrinsic and acquired resistance to targeted- and immune-therapies and rare melanoma subtypes. These efforts have generated a robust pre-clinical resource to develop, refine, and prioritize new functional precision combinatorial therapies for melanoma patients. This T-PDTC constitutes a multi-disciplinary and multi- institutional Program focused on the use and continued expansion of our melanoma PDX collection and organoids to identify new therapeutic combination approaches that will fill important clinical gaps. The Program consists of two research projects and three Cores from a team that has worked extremely well together over the last five years, publishing high-impact collaborative papers. The Research Projects are designed to develop functional precision combination therapies for the most challenging types of melanomas: those that are resistant to current therapies and tumors that lack BRAF-mutations (BRAFWT). We will map the molecular landscape of our melanoma PDXs and organoids by integrating DNA, RNA, and protein data to nominate combination therapies matching their molecular profile. We will develop and implement mechanism-based preclinical trials of drug combinations in our large set of molecularly characterized PDXs. To do this, we will focus on NCI- Investigational New Drug (IND) agents to advance melanoma precision therapy, while offsetting drug resistance and producing durable responses. We expect to gain new mechanistic knowledge about the biology of drug resistant melanoma that will lead to improved and durable precision therapies. We expect to identify biomarkers to select tumors for specific treatments and provide data-driven recommendations for early phase clinical trials.