Program Project Grant

The study of retroviruses has led to important discoveries in basic cell biology including cell signaling, regulation of gene expression, cellular transformation, and cancer development. Our collaborative work within this P01 focuses on human T-cell leukemia virus type 1 (HTLV-1), which is the infectious cause of adult T-cell leukemia/lymphoma (ATLL) and the neurodegenerative disease HAM/TSP. Disease progression by HTLV-1 has been attributed to the viral gene Tax. However, we and others have provided strong evidence that another viral gene, termed Hbz, plays a critical role throughout infection, establishment of latency, and finally in the malignant process. In the past funding cycle, we identified the importance of Hbz in HTLV-1 pathobiology through gene editing. These results support the utility of future Hbz-targeted therapeutic mechanisms for the treatment of HTLV-1-associated diseases. We also expanded our proposed work through P01 collaborations to develop an envelope (Env) mRNA lipid nanoparticle (LNP) vaccine. mRNA vaccine technology has recently emerged as a safe, effective, and scalable approach to combat infectious diseases and cancer, offering several advantages over traditional vaccine design and production. The HTLV-1 Env glycoprotein is immunogenic and required for entry of target cells – making it a favorable target for vaccine development. Our initial Env mRNA-LNP vaccine candidate is immunogenic and protective in our rabbit model of infection. However, a majority of ATLL patients do not express Env (or Tax), whereas Hbz is the only viral gene consistently expressed throughout infection and disease development. Taken together, this suggests that an effective vaccine against HTLV-1 and subsequent disease development should target both Hbz and Env. Our overall hypothesis is that an HTLV-1 mRNA-LNP vaccine (Hbz, Env) can be developed to elicit neutralizing antibody (nAb) and T-cell responses that prevent infection and/or HTLV-1-mediated disease. This highly integrated proposal has three Aims and several Project and Core collaborations.

• Aim 1 will identify the optimal immunogenic Hbz and Env mRNA vaccine using different vaccine candidates. We will utilize in vitro approaches to examine relative efficacy (expression, localization, conformation, effects on cell signaling) of each vaccine candidate. We will also use in vivo approaches to assess and compare vaccine immunogenicity (Ab, nAb, T-cell responses) in HLA-transgenic mice (Project 3, Animal Core B).
• Aim 2 will measure the effectiveness of Hbz and/or Env mRNA vaccine protection. Studies in rabbits will measure vaccine-induced Ab, nAb, T-cell responses, protection against viral challenge, and treatment of viral infection (Project 3, Animal Core B, Genomics Core C). Studies in HLA-transgenic humanized mice will be used to measure vaccine protection against and treatment of disease (Project 2, Animal Core B, Genomics Core C). We will also characterize genomic phenotype of cells that escape vaccine protection (Genomics Core C).
• Aim 3 will assess the importance of nAb and/or CD8T cells on protection and vaccine response in animals (Animal Core B, Genomics Core C).

We expect our work will accelerate vaccine development against immune targets that are critical for HTLV-1 preneoplastic and tumor cell survival and help further characterize the HTLV-1 immune response.
 

Adult T cell leukemia/lymphoma (ATLL), a malignancy of HTLV-1 infected T cells, stands out from other acute leukemias in its frequent association with hypercalcemia and propensity to form localized lymphomatous osteolytic lesions. Osteoclasts are directly responsible for the degradation of bone that releases calcium. In the previous funding period, we demonstrated that patient derived ATLL cells (ATLL-PDX) and a subset of HTLVinfected T cell lines release small extracellular vesicles (sEV) with powerful osteoclastogenic effects in vitro and in vivo. We have found that the proteome of osteolytic sEV is enriched for metabolic pathways; in concert, the transcriptional signature of osteoclasts exposed to these sEV reflect a metabolic shift, demonstrating enhanced glycolysis and hypoxia, features of a tumorigenic microenvironment. We hypothesize that crosstalk between HTLV+T and osteoclasts provides a tumorigenic microenvironment impacting HTLV+T cell fate. Previous work, including our own, has focused primarily on the ability of transformed ATLL cells to enhance the formation and activation of osteoclasts. This proposal’s focus on HTLV-1 infected T cells prior to transformation and the feedback on osteoclasts and other cells in bone, is novel. 

The specific Aims are:

• Aim 1: To define impact of the bone microenvironment on HTLV-1 infected T cells via Notch and TGFβ. In this aim, we examine the signals from bone, resulting from crosstalk with HTLV+T, that impact the fate of HTLV+T cells. To do this, we will profile gene expression and the epigenetic state of HTLV+T cells residing in bone from our mouse model, compared to extraskeletal site.
• Aim 2: To define the role of osteoclasts in remodeling of the bone microenvironment by HTLV-1 infected T cells and their sEV. We will implant HTLV+T or their sEV into bones of mice, with or without osteoclast blockade, and perform detailed analysis of bone tissue by morphological and molecular analysis. We will also determine the role of serum amyloid A, released by OC, and mir155 and mir21 present in osteolytic sEV. 

The successful completion of this project will provide detailed information about cellular and molecular interactions that may be therapeutically targeted to reduce the transition to ATLL or used to identify risks for progression.
 

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATLL), an aggressive lymphoproliferative malignancy refractory to therapy. Our previous work showed that chromatin insulator, CTCF, binds HTLV-1 DNA at a single site (vCTCF-BS), and regulates virus gene expression in an integration site dependent manner through effects on DNA methylation and histone modifications. In addition, we showed that a viral enhancer in the 3’ portion of the genome is important for expression of the antisense viral oncogene hbz. 

The current project will define the role of epigenomic regulation by these key regulatory elements in the following aims:

• Aim 1: To determine the combined effects of vEnhancer and vCTCF-BS in HTLV-1 persistence and disease examining viral gene expression and T-cell immortalization in culture and in rabbits and in humanized immunodeficient mice. A multi-omic approach will examine proviral and T-cell receptor clonality in comparison to primary ATLL samples.
• Aim 2: To determine the role of chromatin insulators in HTLV-1 infection, replication, and gene expression comparing binding sites and effects on DNA looping of CTCF, cohesin, and other interactive proteins in HTLV-1 reactivation from latency, and in primary infected clinical samples. Effects on cohesin kockdown on virus gene expression will also be examined. Transcriptional and post-transcriptional effects will be assessed by short and long read RNA sequencing.
• Aim 3: To determine the impact of epigenetics on HTLV-1 pathogenesis. Effects of vCTCF-mutation will be examined in CD4+ and CD4+CD8+ lymphocytes, and in ATLL tumor samples, as well as the ability of an exogenous looping protein to reconstitute the effects of vCTCF-BS mutation.

We expect that knowledge obtained from these studies can guide epigenomic therapeutic approaches for ATLL.