A National Cancer Institute-funded Program Project led by Rolf Renne, Ph.D., has discovered several key similarities in how microRNAs and long noncoding RNAs in three herpesviruses drive tumor development. The team’s success in leveraging resources in computing, immunology and bioinformatics to understand how the latent herpesviruses cause cancers has led to renewed support for a translational phase over the next five years.
Renne, a professor in the UF College of Medicine’s department of molecular genetics and microbiology, has made several seminal findings on the role of microRNAs in Kaposi’s-sarcoma herpesvirus, or KSHV. He is collaborating on the project with Scott Tibbetts, Ph.D., an immunologist and professor in the department of molecular genetics and microbiology at UF who studies the murine herpesvirus 68, or MHV68, a naturally occurring pathogen in mice that can cause lymphomas, and Erik Flemington, Ph.D., a bioinformatics expert and professor at Tulane who studies the Epstein-Barr virus, or EBV.
Operating in three cores through a funding mechanism that fosters collaboration, the team began the project with a $6.5 million grant in 2017. The NCI has now renewed the team’s funding for another five years and increased the award to $7.5 million.
“Our original hypothesis was that we could learn so much more about the role of noncoding RNAs if we studied them in more than one virus at the same time,” said Renne, associate director of basic sciences at the UF Health Cancer Center. “We showed that we can use a mouse model to learn more about the human viruses, which we could not do before. Now, we can ask the question: ‘Is this true in humans too?’ Our ultimate goal is to find vulnerabilities of these tumors related to noncoding RNAs, which could lead to a novel therapeutic strategy targeting oncogenic RNAs.”
The team’s achievements in the first phase include:
- Using a modified Crosslinking and Sequencing of Hybrids protocol, or qCLASH, the team identified high-confidence miRNA targetomes across all three viruses, linked miRNA targeting to immune evasion and cancer hallmark phenotypes (proliferation, migration, angiogenesis, glucose metabolism and aberrant splicing) and revealed that a significant number of miRNA targets are shared among all three viruses.
- Becoming the first to demonstrate deregulation of host long noncoding RNA expression in gamma-herpesvirus infected cells of lymphoid and endothelial origin and identifying cross-talk between viral miRNAs and host cellular long noncoding RNAs. This novel paradigm of regulating gene expression was also demonstrated for host miRNA/long noncoding RNA interactions.
- Becoming the first to discover a new class of EBV- and MHV68-encoded circular noncoding RNAs. The group and others also reported the discovery of KSHV circular RNAs.
- Revealing that short and long noncoding RNAs are the predominant viral gene products detectable in EBV+ Burkitt’s lymphoma and gastric cancer, a finding that is notably different from cell lines grown in vitro. This noncoding RNA-centered gene expression strategy may contribute to efficient immune evasion in vivo, and the team believes that transcriptome analysis of patient samples will be crucial to identify long noncoding RNA-dependent pathogenesis for both EBV- and KSHV-associated AIDS malignancies.
- Using the MHV68 in vivo system, demonstrating the first in vivo phenotype for a gamma-herpesvirus long noncoding RNA and further demonstrating that the long-studied EBV EBER1 noncoding RNA shares this function.
- Developing innovative technologies. One is qCLASH, an assay developed in 2018 by Renne’s lab to identify microRNA targetomes. It has been applied to all three projects and resulted in published data on all three. The technology has now expanded for use in other cancers at UF and beyond, with more than 18 papers published on the technology. The other technology is a bioinformatics tool called TRIMD that allows researchers to analyze RNAseq data from many different sources. This has created the first high-density transcriptomes for EBV and MHV68, with KSHV in the works.
To date, the team’s results from the first funding cycle have been reported in 20 research publications and five review articles, including nine that were coauthored by either two or three project leaders.
“The nature of these projects is to accelerate research, and we have done that tremendously,” Renne said. “There are not many Program Projects in the world — this is a very special funding mechanism. If we had stayed in our silos and done these experiments independently without the opportunity to constantly compare our data, we would not have been able to create our new hypothesis.”
“If we had stayed in our silos and done these experiments independently without the opportunity to constantly compare our data, we would not have been able to create our new hypothesis.”Rolf Renne, Ph.D.
Structured for success
The structure of the NCI P01 Program Project grant, the only such multi-investigator grant at the Cancer Center, has been key to the project’s success by developing cores, or shared resources, that all three projects use. Renne leads the administrative core at UF, providing project oversight with the support of research coordinator Dorci Nance, M.A. He also runs the virus recombination core at UF, which made more than 100 viruses in the first funding cycle. Flemington leads a viral RNA-sequencing and bioinformatics core at Tulane, which facilitates high-speed computing for all three projects.
In the new phase, the team will shift from strictly in vitro models for KSHV and EBV to new studies that use human tumor samples, humanized mouse models and xenograft mouse models. The group has added a fourth core for clinical samples and tumorigenesis, which Tibbetts will direct.
“A major thrust for the next phase is the expanded development of in vivo tumorigenesis models using each of the viruses,” Tibbetts said. “The renewal provided funding for a new clinical sample and tumorigenesis core, which will allow us to greatly expand the translational side of our studies examining the function of noncoding RNAs in gammaherpesvirus malignancies. Not only will we be examining clinical samples from advanced human tumors, but we will also be able to test the function of specific noncoding RNAs in the genesis and progression of tumors in an in vivo setting. Our in vivo tumorigenesis studies with the murine virus already provided strong preliminary data that helped us secure renewal funding, so we are very excited to see what expanded murine tumorigenesis studies and our new translational experiments with EBV and KSHV tumors will reveal in the coming years.”
With the new core, the team has also recruited new experts. AIDS malignancy samples will be provided by Suki Subbiah, M.D., who directs the HIV Core Tumor Biorepository at Louisiana State University, and by a Kaposi’s sarcoma clinic in South Africa. Additional oral Kaposi’s sarcoma lesions will be provided by Don Cohen, D.M.D., M.B.A., M.S., who directs the UF Oral and Maxillofacial Pathology Laboratory. The team has recruited Genevieve Crane, M.D., Ph.D., a hematopathologist and expert in AIDS malignancies at the Cleveland Clinic, to support detailed analyses of the samples. Lauren McIntyre, Ph.D., professor in UF’s department of molecular genetics and microbiology, has also been recruited to provide biostatistics and additional bioinformatics support.
The investigators have prioritized constant communication between the cores. They monitor progress through an array of tools, including regular progress reports, monthly video conferences, a dedicated Program Project website, real-time data exchange through secure servers, and live screensharing.
“The P01 has been a fantastic bridge between UF and Tulane and we have a highly productive, synergistic relationship,” Flemington said. “The constant communication helps me think in new and different ways about how we approach EBV, and I hope that has gone in the other direction as well.”
“The P01 has been a fantastic bridge between UF and Tulane and we have a highly productive, synergistic relationship.”Erik Flemington, Ph.D.
Tibbetts agreed, noting the value of a collaboration in which each group not only works on a different virus, but also brings a different expertise and viewpoint to the table.
“The Program Project really does what it is supposed to do — it provides funding for truly synergistic work among multiple labs that helps us to more rapidly advance our common goals,” he said.
Another key component of the P01 mechanism is internal and external oversight committees with international experts who review data, provide feedback and ensure quality. The committees participate in the program’s annual retreats, which provide project leaders, core directors, core managers and advisory board members an opportunity to assess progress and make adjustments.
External members include:
- Britt Glaunsinger, Ph.D., a professor and HHMI investigator in the department of molecular and cell biology at UC Berkeley
- Eric Johannsen, M.D., associate professor of medicine at the University of Wisconsin
- Charles Rice, Ph.D., professor of virology at the Rockefeller University, who received the 2020 Nobel Prize in Physiology or Medicine for his work discovering the hepatitis C virus.
- Mathias Munschauer, head of the independent Helmholtz Young Investigator Group at the Helmholtz Institute for RNA-based infection research, who has been added for the new phase and is an expert in systems and RNA biology.
Internal members include Maurice Swanson, Ph.D., a prominent RNA biologist and professor in the UF department of molecular genetics and microbiology, and Alfred Lewin, Ph.D., an emeritus professor in that department and an experienced RNA biology and RNA structure researcher.
Thanks to an additional $225,000 per year from UF, the group has created a dedicated training component in all cores. Trainees from UF travel to Tulane for annual sessions on bioinformatics and trainees at Tulane travel to UF for recombinant virus training. The cores also hold regular hands-on workshops.
These sessions have provided trainees ranging from undergraduates to postdocs with in-depth education that they may not receive in more traditional bioinformatics classes given the challenges specific to herpesvirus research. Trainees learn the importance of good experimental design and become adept in cutting-edge techniques. They also have the chance to participate in the annual retreats.
“These training activities are unique to our Program Project because it’s something that is not required by the NCI, but it is highly innovative,” Renne said. “We believe that this component will have a lasting impact on AIDS malignancies research by training a generation of highly competent virologists and cancer researchers.”
The team is also planning the first international conference based on the project, tentatively set for 2023 and titled “Noncoding RNAs in viral disease.” The goal of the interactive symposium is to bring together members of all three labs, the full external and internal advisory boards, and renowned RNA biologists from around the world to present and discuss diverse aspects of RNA biology, with a special emphasis on noncoding RNAs functioning at the virus-host interface.