Program Project Grant

Retrovirus Models of Cancer: National Institutes of Health and National Cancer Institute Funded Program Project Grant

Principal Investigator: Patrick Green, PhD

An investigative team of Center for Retrovirus Research faculty has been awarded a $9.1 million program project grant (PPG) from the National Cancer Institute to investigate retroviral models of cancer (years 16-20; 2020-2025). The ultimate goal of this integrated multidisciplinary and multi-institutional Program Project Grant is to utilize the human T-cell leukemia virus type 1 (HTLV-1) T-cell immortalization model to gain understanding of the microenvironmental, cellular, and viral factors that lead to progression to leukemia, and with this knowledge, to identify unique targets for diagnosis and treatment of HTLV-1 infection and adult T-cell leukemia (ATL) and related leukemia/lymphoma. The grant includes three integrated projects and three cores.

PPG Investigator headshot photos

Purpose: mechanisms of retrovirus-mediated disease

The PPG is designed to foster synergistic interactions between the laboratories and characterize mechanisms that define retroviral exploitation of the cellular environment during HTLV-1 replication and disease progression. Concurrently each laboratory has key interrelated goals to define events that control the transition of lymphocytes from activation through immortalization and transformation.  

The specific aims are:

Aim 1: To characterize the actions of the HTLV-1 hbz mRNA and HBZ protein on human T-cells related to cell dysfunction, establishment of viral latency, the interrelationship with CTCF function and hbz effects, and the impact of hbz on microenironment.

Aim 2: To determine if and how retrovirus-induced cellular changes can exploit unique microenvironments that contribute to viral latency, paraneoplastic disease syndromes and ATL.

 Aim 3: To discover new therapeutic targets against retrovirus infections, retroviral-induced lymphoma and their paraneoplastic disease syndromes.

Program Projects

Preliminary data indicates that hbz is multifunctional, playing important roles in both the RNA and protein forms throughout infection and establishment of latency. The hbz gene product is expressed early after viral infection in an animal model, and hbz mRNA is expressed in all ATL tumors. We hypothesize that cellular protein interaction with hbz mRNA 2o structure affects T-cell signaling pathways important for viral persistence and cellular proliferation; these cellular proteins may affect hbz mRNA stability and function. Our new proteomics data has identified, and we further validated, multiple distinct T-cell proteins that bind hbz mRNA. In vitro approaches will be used to identify the hbz mRNA interactive region and determine the functional role of cellular binding proteins in cell proliferation. We will use siRNA and knockout studies targeting binding proteins to evaluate hbz function in the absence of these cellular proteins. In vivo approaches will be used to determine the contribution of the hbz mRNA and its interactive proteins to virus persistence and tumor formation. We have shown that HTLV-1 lacking HBZ protein expression is attenuated as early as 1 week post infection in a rabbit model of persistent infection. Data from our group and others indicated that HBZ not only counteracts HTLV-1 Tax by disrupting Tax-mediated transcription and targeting NFkB (p65) for degradation, but also blocks senescence of the infected cell and enhances survival. We provide novel results indicating that CRISPR targeting of hbz reduces cell proliferation in culture. Our new proteomics data has identified, and we further validated, multiple distinct cellular proteins that bind HBZ protein. In vitro approaches, including protein interaction mapping, cellular proliferation assays, and cellular transformation assays, will be utilized to identify the HBZ interactive region and the functional role of key cellular binding proteins in HTLV-1 pathobiology. In vivo approaches will be used to determine the contribution of these HBZ interactive proteins to the establishment of persistence, infected cell survival, and tumor formation. Results from both Aim 1 and 2 will provide a platform for planning of long-term experiments to use targeted gene editing to disrupt hbz functional domains key to pathogenesis and disease.

The specific aims are:
Aim 1: To dissect the mechanism(s) of action of hbz mRNA and determine its contributions to the pathogenic process. Hypothesis: Cellular protein interactions with hbz mRNA 2o structure translates to cell signaling pathways important for viral persistence and cellular proliferation.

Aim 2: To dissect the mechanism(s) of action of HBZ protein and determine its contributions to HTLV-1 pathobiology. Hypothesis: HBZ protein interacts with novel cellular proteins that contribute to the multifunctional behavior and survival of T-cells.

ATL develops in 2-5% of people infected with HTLV-1, and is an aggressive malignancy of helper T-cells, associated with hypercalcemia and osteolytic lesions. ATL’s unique relationship to bone (long latency in the bone marrow, bone invasion, osteolytic lesions, and hypercalcemia) makes it an ideal model to dissect and identify the critical factors that support tumor development and progression in bone. This model will also shed light on mechanisms of other late recurring and bone-tropic tumors including multiple myeloma, breast and prostate cancer. We found that the HTLV-1 tax viral oncogene is critical to both ATL development and osteolytic bone destruction through non-cell autonomous effects on bone resorbing osteoclasts (OC). We and others have found that hbz caused elevated expression of Wnt5a by CD4+ T-cells from ATL patients and by ATL patient T-cell and HTLV-1 infected cell lines. Wnt5a promotes bone resorption, as well as bone formation. The role of Wnt5a is unknown in the context of ATL or HTLV-1 infection in animal models, but it is a candidate to explain the mixed osteolytic/osteoblastic tumors observed after direct intratibial injection of ATL cell lines. Both isotypes (L and S) of Wnt5a will be studied. To design appropriate therapies, we need to determine if these effects are a direct effect of Wnt or possibly a down-stream effect. This will be a collaboration between Project 1.
Heparanase (HPSE) is highly expressed on CD4+ T-cells from ATL patients compared to control T-cells. It is also upregulated when Hbz protein is expressed in both mouse CD4+ T-cells and human Kit-225 T-cells. HPSE is a secreted enzyme that can act in the extracellular matrix of bone to degrade heparan sulfate side chains. It is also taken up by lysosomes, in an autocrine and paracrine fashion (into tumor and bone cells), where it can affect autophagy, exosome release and exosome cargo. HPSE also leads to syndecan-1 release, which has prometastatic and proangiogenic effects. Independent of its enzymatic activity, HPSE can affect tumor cell signaling by promoting Akt activation and proliferation, a pathway active in ATL. Inhibition of HSPE enzymatic activity in one myeloma model reduced tumor burden, and expression of HPSE in similar models increased both RANKL and DKK1 expression by OBs. We find that overexpression of HPSE in the ATL cell line MT2 increased RANKL expression as well. Given its pleiotropic effects in the bone microenvironment, we hypothesize that HPSE may function as a master regulator of ATL/HTLV-1 bone disease, affecting both ATL cells and the microenvironment.

The specific aims are:
Aim 1: To determine the role of Wnt5a, regulated by HTLV-1 oncogene hbz, in modulating the bone microenvironment during ATL development and progression. Hypothesis: Hbz expression in transformed ATL cells reprograms the bone microenvironment and thereby affects tumor progression and paraneoplastic bone disease.

Aim 2: To determine the role of heparanase (HPSE) in modulating the bone microenvironment during ATL development and progression. Hypothesis: Hbz up regulates heparanase expression in transformed ATL cells, reprograms the bone microenvironment and thereby affects HTLV-1 latently infected preneoplastic T-cell, tumor progression and paraneoplastic bone disease.

In support of Project 3, we used an infectious molecular clone of HTLV-1, designated pACH, to confirm the report that the integrated HTLV-1 genome contains a single CTCF-BS. An infectious molecular clone of HTLV-1 with a mutation in the CTCF-BS, designated pACH∆CTCF, was produced and further confirmed that the mutations did not affect overlapping reading frames p12/p8, p30, and HBZ. Moreover, ACH∆CTCF viruses immortalized CD4+ cells with similar kinetics, and efficiency as wild-type virus. These cell lines were utilized for preliminary humanized mouse experiments. Since CTCF is important for establishment of latency, or reactivation from latency for many herpesviruses, we asked whether CTCF regulated HTLV-1 latency. Although HTLV-1 is clinically latent in infected individuals for many decades, there is very little known about viral latency for HTLV-1 or other retroviruses, other than HIV-1. These studies made use of sublines of Jurkat T-lymphoid cells stably transfected with five copies of the Tax-response element containing the enhancer-less HTLV-1 promoter regulating the tdTomato gene (tdTom) (Jet cells). JET-cells were used to identify, sort (using tdTOM expression) and select out clones carrying a single full-length provirus that produce infectious virus after induction with PMA and ionomycin. Using this approach, we produced 293, 259, and 307 clonal infected JurkaT-cell lines with ACH, ACHp12stop, and ACH∆CTCF viruses, respectively. This resulted in 108, 107, and 72 cell lines, respectively, with no detectable tdTom background, but above background levels with stimulation with PMA and ionomycin. From these cell lines, 19, 24, and 10 cell lines had a full-length integrated HTLV-1 genome, and 8, 9, and 3 cell lines produced infectious virus, respectively. These findings suggest that CTCF binding to HTLV DNA is important in the establishment of latency, possibly due to more limited number of proviral integration sites that are compatible with productive or latent HTLV-1 infection.

The specific aims are:
Aim 1: Effect of CTCF on HTLV-1 latency. Hypothesis: CTCF binding to the HTLV provirus regulates establishment of latency by regulating the level of viral expression that is optimal for lymphocyte proliferation and survival.

Aim 2: Effect of CTCF on HTLV-1 replication and transformation in humanized mice. Hypothesis: CTCF regulates tumorigenesis in infected humanized mice by optimizing levels of virus replication and/or effects on tumorigenesis.

Aim 3: Effect of CTCF on ATL cell line proliferation in mice. Hypothesis: Effects of CTCF on ATL cell proliferation in mice are mediated by binding the HTLV-1 genome, DNA looping, with associated changes in DNA methylation and marks of chromatin activation that are associated with hbz expression.

Support Cores

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Director: Patrick Green, PhD: Co-Investigators: Amanda MacFarlane, PhD; Soledad Fernandez, PhD; Lianbo Yu, PhD

The overall goals of the Administrative and Biostatistics Core are to:

  • Provide a structure to facilitate effective interactions toward accomplishment of the aims of this Program Project and specifically will foster and promote the goals of the program and each component by providing scientific direction, administrative support, oversight, integration of program activities and ensuring operational efficiency
  • Provide biostatistics and other statistical support for the program.

In this manner, close coordination will be effected with Project 1 (OSU), Project 2 (WUSTL), Project 3 (WUSTL), Virus Vector Core (OSU) and Animal Core (OSU). In addition, Administrative/Biostatistics Core will be the focal point of interactions with the NCI Program Officer.

The core has the following specific aims:
Aim 1: Provide research direction by setting the research agenda to utilize the HTLV-1 T-cell immortalization model to gain understanding of the microenvironmental, cellular, and viral factors that lead to progression to leukemia, and with this knowledge, to identify unique targets for diagnosis and treatment of HTLV-1 infection and adult T-cell leukemia and related leukemia/lymphoma.

Aim 2: Ensure operational efficiency for Program components by providing centralized grant administration, information dissemination, budget data processing, and exchange of information and services.

Aim 3:  Promote integration of the Research Projects and Cores to promote interaction between the two primary universities represented by the investigators, and relevant external entities.

Aim 4: Provide biostatistics support for the three Projects and the two Cores of the P01 Program.

Director: Kristine Yoder, PhD

This core will support the individual projects of the PPG. HTLV-1 infected cells can be difficult to transfect making lentiviral vectors key to experimental success. Lentiviral vectors may be used for overexpression or shRNA reduced expression of genes of interest. CRISPR/Cas9 lentiviral vectors will be used to prevent expression of host genes or for homology directed repair when coupled with a donor DNA for homologous recombination. This research group is the first to propose CRISPR/Cas9 genome editing to disable the HTLV-1 hbz gene and prevent disease progression. Integrase defective lentiviral vectors (IDLV) are part of an unbiased method to quantify the off-target editing of CRISPR guide RNAs (gRNA). Finally, the core is developing a CRISPR/Cas9 vector with inducible, self-limiting Cas9 expression.

The specific aims are:

 Aim 1: Provide custom retroviral vectors to the PPG labs.

Aim 2: Design, validate, and optimize CRISPR/Cas9 vectors.

Aim 3: Develop an inducible, self-limiting HTLV-1 CRISPR/Cas9 vector.

Director: Stefan Niewiesk, DVM, PhD; Co-Investigator: Krista LaPerle, DVM, PhD

The overall goal of the Animal core is to support the development and use of the rabbit model, and various applications of the NSG mouse model within this PPG. The guiding principles for the core are efficient planning and testing of recombinant viruses, genetically modified ATL cell lines, and continued development of humanized mice as a translational tool for understanding cancer development and drug testing. The core works closely with the OSUCCC Comparative Pathology Shared Resource which will provide comparative pathology assessment of tissues from experimental animals.  

The specific aims are:

Aim 1: Testing mutant viruses in the rabbit model for viral growth and induction of antibodies.

Aim 2: Testing of transfected ATL cell lines in a transplantation mouse model.

Aim 3: Testing mutant virus in the induction of lymphoproliferative disease in humanized mice.

Aim 4: Histological analysis of infected organs from rabbits and mice.