Human T-cell Leukemia Virus Type 1 Tax Research Articles

PCBP1 interacts with the HTLV-1 Tax oncoprotein to potentiate NF-κB activation.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma. The HTLV-1 Tax constitutively activates nuclear factor-κB (NF-κB) to promote the survival and transformation of HTLV-1-infected T cells. Despite extensive study of Tax, how Tax interacts with host factors to regulate NF-κB activation and HTLV-1-driven cell proliferation is not entirely clear. Here, we showed that overexpression of Poly (rC)-binding protein 1 (PCBP1) promoted Tax-mediated IκB kinase (IKK)-NF-κB signaling activation, whereas knockdown of PCBP1 attenuated Tax-dependent IKK-NF-κB activation. However, Tax activation of HTLV-1 long terminal repeat was unaffected by PCBP1. Furthermore, depletion of PCBP1 led to apoptosis and reduced proliferation of HTLV-1-transformed cells. Mechanistically, PCBP1 interacted and co-localized with Tax in the cytoplasm, and PCBP1 KH3 domain was indispensable for the interaction between PCBP1 and Tax. Moreover, PCBP1 facilitated the assembly of Tax/IKK complex. Collectively, our results demonstrated that PCBP1 may exert an essential effect in Tax/IKK complex combination and subsequent NF-κB activation, which provides a novel insight into the pathogenetic mechanisms of HTLV-1.

Mouse Models for HTLV-1 Infection and Adult T Cell Leukemia.

Adult T cell leukemia (ATL) is an aggressive hematologic disease caused by human T cell leukemia virus type 1 (HTLV-1) infection. Various animal models of HTLV-1 infection/ATL have been established to elucidate the pathogenesis of ATL and develop appropriate treatments. For analyses employing murine models, transgenic and immunodeficient mice are used because of the low infectivity of HTLV-1 in mice. Each mouse model has different characteristics that must be considered before use for different HTLV-1 research purposes. HTLV-1 Tax and HBZ transgenic mice spontaneously develop tumors, and the roles of both Tax and HBZ in cell transformation and tumor growth have been established. Severely immunodeficient mice were able to be engrafted with ATL cell lines and have been used in preclinical studies of candidate molecules for the treatment of ATL. HTLV-1-infected humanized mice with an established human immune system are a suitable model to characterize cells in the early stages of HTLV-1 infection. This review outlines the characteristics of mouse models of HTLV-1 infection/ATL and describes progress made in elucidating the pathogenesis of ATL and developing related therapies using these mice.

Primary cells from patients with adult T cell leukemia/lymphoma depend on HTLV-1 Tax expression for NF-κB activation and survival

Adult T cell leukemia/lymphoma (ATL) is an aggressive malignancy secondary to chronic infection with human T cell leukemia virus type 1 (HTLV-1). The viral oncoprotein Tax initiates T cell transformation through activation of critical cellular pathways, including NF-κB. Unexpectedly, Tax protein is not detectable in most ATL cells, in contrast to the HTLV-1 HBZ protein which antagonizes Tax effects. Here, we demonstrate that primary ATL cells from patients with acute or chronic ATL express very low levels of Tax mRNA and protein. Critically, survival of these primary ATL cells is dependent on continued Tax expression. Mechanistically, Tax extinction results in reversal of NF-κB activation, P53/PML activation and apoptosis. Tax drives interleukin-10 (IL-10) expression and recombinant IL-10 rescues the survival of tax-depleted primary ATL cells. These results demonstrate the critical role of continued Tax and IL-10 expression for the survival of primary ATL cells, highlighting their relevance as therapeutic targets.

Tax-targeted dendritic cell vaccine therapy for long-term remission of adult T-cell leukemia-lymphoma

Adult T-cell leukemia-lymphoma (ATL) is a highly aggressive peripheral T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1) infection occurring in approximately 5% of patients after prolonged latent period. ATL relapses within a short period despite its transient response to multiagent chemotherapy and the prognosis is extremely poor due to anticancer drug resistance and immunodeficiency. Although novel agents with different mechanisms, such as molecular targeted agents, have improved the prognosis, the number of cured patients remains limited. Hematopoietic stem cell transplantation resulted in long-term remission, whereas its indication is limited due to treatment-related mortality. As most ATL patients are of advanced age, development of a lesser toxic treatment is necessary. Therefore, we developed a novel therapeutic dendritic cell vaccine targeting the HTLV-1 Tax antigen. The safety profile has been confirmed in a pilot and phase I clinical studies, and a promising long-term clinical efficacy has also been obtained. This novel vaccine is a noninvasive, long-lasting therapy for ATL and can potentially be extended to different applications for low-grade ATL and high-risk HTLV-1 carriers.

The endogenous HBZ interactome in ATL leukemic cells reveals an unprecedented complexity of host interacting partners involved in RNA splicing.

Adult T-cell leukemia/lymphoma (ATL) is a T-cell lymphoproliferative neoplasm caused by the human T-cell leukemia virus type 1 (HTLV-1). Two viral proteins, Tax-1 and HBZ play important roles in HTLV-1 infectivity and in HTLV-1-associated pathologies by altering key pathways of cell homeostasis. However, the molecular mechanisms through which the two viral proteins, particularly HBZ, induce and/or sustain the oncogenic process are still largely elusive. Previous results suggested that HBZ interaction with nuclear factors may alter cell cycle and cell proliferation. To have a more complete picture of the HBZ interactions, we investigated in detail the endogenous HBZ interactome in leukemic cells by immunoprecipitating the HBZ-interacting complexes of ATL-2 leukemic cells, followed by tandem mass spectrometry analyses. RNA seq analysis was performed to decipher the differential gene expression and splicing modifications related to HTLV-1. Here we compared ATL-2 with MOLT-4, a non HTLV-1 derived leukemic T cell line and further compared with HBZ-induced modifications in an isogenic system composed by Jurkat T cells and stably HBZ transfected Jurkat derivatives. The endogenous HBZ interactome of ATL-2 cells identified 249 interactors covering three main clusters corresponding to protein families mainly involved in mRNA splicing, nonsense-mediated RNA decay (NMD) and JAK-STAT signaling pathway. Here we analyzed in detail the cluster involved in RNA splicing. RNAseq analysis showed that HBZ specifically altered the transcription of many genes, including crucial oncogenes, by affecting different splicing events. Consistently, the two RNA helicases, members of the RNA splicing family, DDX5 and its paralog DDX17, recently shown to be involved in alternative splicing of cellular genes after NF-κB activation by HTLV-1 Tax-1, interacted and partially co-localized with HBZ. For the first time, a complete picture of the endogenous HBZ interactome was elucidated. The wide interaction of HBZ with molecules involved in RNA splicing and the subsequent transcriptome alteration strongly suggests an unprecedented complex role of the viral oncogene in the establishment of the leukemic state.

NF-κB-Induced R-Loops and Genomic Instability in HTLV-1-Infected and Adult T-Cell Leukemia Cells.

Human T-cell leukemia virus type 1 (HTLV-1) is a human delta retrovirus that causes adult T-cell leukemia/lymphoma (ATL) in 3–5% of the infected population after decades of clinical latency. HTLV-1 Tax is a potent activator of IKK/NF-κB and a clastogen. While NF-κB activities are associated with cell survival and proliferation, constitutive NF-κB activation (NF-κB hyperactivation) by Tax leads to senescence and oncogenesis. Until recently, the mechanisms underlying the DNA damage and senescence induced by Tax and NF-κB were unknown. Current data indicate that NF-κB hyperactivation by Tax causes the accumulation of a nucleic acid structure known as an R-loop. R-loop excision by the transcription-coupled nucleotide excision repair (TC-NER) endonucleases, Xeroderma pigmentosum F (XPF), and XPG, in turn, promotes DNA double-strand breaks (DSBs). NF-κB blockade prevents Tax-induced R-loop accumulation, DNA damage, and senescence. In the same vein, the silencing of XPF and XPG mitigates Tax senescence, while deficiency in either or both frequently occurs in ATL of all types. ATL cells maintain constitutively active NF-κB, accumulate R-loops, and resist Tax-induced senescence. These results suggest that ATL cells must have acquired adaptive changes to prevent senescence and benefit from the survival and proliferation advantages conferred by Tax and NF-κB. In this review, the roles of R-loops in Tax- and NF-κB-induced DNA DSBs, senescence, and ATL development, and the epigenetic and genetic alterations that arise in ATL to reduce R-loop-associated DNA damage and avert senescence will be discussed.

HTLV-1 activates YAP via NF-κB/p65 to promote oncogenesis

Adult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) infection. HTLV-1 exerts its oncogenic functions by interacting with signaling pathways involved in cell proliferation and transformation. Dysregulation of the Hippo/YAP pathway is associated with multiple cancers, including virus-induced malignancies. In the present study, we observe that expression of YAP, which is the key effector of Hippo signaling, is elevated in ATL cells by the action of the HTLV-1 Tax protein. YAP transcriptional activity is remarkably enhanced in HTLV-1-infected cells and ATL patients. In addition, Tax activates the YAP protein via a mechanism involving the NF-κB/p65 pathway. As a mechanism for this cross talk between the Hippo and NF-κB pathways, we found that p65 abrogates the interaction between YAP and LATS1, leading to suppression of YAP phosphorylation, inhibition of ubiquitination-dependent degradation of YAP, and YAP nuclear accumulation. Finally, knockdown of YAP suppresses the proliferation of ATL cells invitro and tumor formation in ATL-engrafted mice. Taken together, our results suggest that p65-induced YAP activation is essential for ATL pathogenesis and implicate YAP as a potential therapeutic target for ATL treatment.

Autophagy in Human T-Cell Leukemia Virus Type 1 (HTLV-1) Induced Leukemia.

Viruses play an important role in the development of certain human cancers. They are estimated to contribute 16% to all human cancers. Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus to be discovered and is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive T-cell malignancy with poor prognosis. HTLV-1 viral proteins interact with mechanisms and proteins present in host cells for their own benefit, evading the immune system and promoting the establishment of disease. Several viruses manipulate the autophagy pathway to achieve their infective goals, and HTLV-1 is not the exception. HTLV-1 Tax viral protein engages NF-κB and autophagy pathways prone favoring viral replication and T cell transformation. In this review we focus on describing the relationship of HTLV-1 with the autophagy machinery and its implication in the development of ATLL.

The E3/E4 ubiquitin conjugation factor UBE4B interacts with and ubiquitinates the HTLV-1 Tax oncoprotein to promote NF-κB activation.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), and the neurological disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax protein persistently activates the NF-κB pathway to enhance the proliferation and survival of HTLV-1 infected T cells. Lysine 63 (K63)-linked polyubiquitination of Tax provides an important regulatory mechanism that promotes Tax-mediated interaction with the IKK complex and activation of NF-κB; however, the host proteins regulating Tax ubiquitination are largely unknown. To identify new Tax interacting proteins that may regulate its ubiquitination we conducted a yeast two-hybrid screen using Tax as bait. This screen yielded the E3/E4 ubiquitin conjugation factor UBE4B as a novel binding partner for Tax. Here, we confirmed the interaction between Tax and UBE4B in mammalian cells by co-immunoprecipitation assays and demonstrated colocalization by proximity ligation assay and confocal microscopy. Overexpression of UBE4B specifically enhanced Tax-induced NF-κB activation, whereas knockdown of UBE4B impaired Tax-induced NF-κB activation and the induction of NF-κB target genes in T cells and ATLL cell lines. Furthermore, depletion of UBE4B with shRNA resulted in apoptotic cell death and diminished the proliferation of ATLL cell lines. Finally, overexpression of UBE4B enhanced Tax polyubiquitination, and knockdown or CRISPR/Cas9-mediated knockout of UBE4B attenuated both K48- and K63-linked polyubiquitination of Tax. Collectively, these results implicate UBE4B in HTLV-1 Tax polyubiquitination and downstream NF-κB activation.

Rapid and label-free electrochemical DNA biosensor based on a facile one-step electrochemical synthesis of rGO-PPy-(L-Cys)-AuNPs nanocomposite for the HTLV-1 oligonucleotide detection.

Human T cell leukemia virus type 1 (HTLV-1) as the first human retrovirus is currently a serious endemic health challenge. Despite the use of assorted molecular or serological assays for HTLV-1 detection, there are several limitations due to the lack of a confirmatory test that may affect the accuracy of the results. Herein, a novel label-free biosensor for the detection of HTLV-1 Tax gene has been reported. An electrochemical facile ecofriendly synthesis method has been demonstrated based on a synthesis of nanocomposite of reduced graphene oxide, polypyrrole, and gold nanoparticles (rGO-PPy-(l-Cys)-AuNPs) deposited on the surface of screen-printed carbon electrode. Electrochemical techniques were used to characterize and study the electrochemical behavior of the rGO-PPy-(l-Cys)-AuNPs, which exhibited a stable reference peak at 0.21V associated with hybridization forms by applying the differential pulse voltammetry. The designed DNA biosensor presented a wide linear range from 0.1fM to 100µM and a low detection limit of 20 atto-molar. The proposed biosensor presented in this study provides outstanding selectivity, sensitivity, repeatability, and reproducibility.

Inefficient Tax-Specific T-Cell Responses in Mice after Syngeneic Transplantation with tax-Transgenic Mouse-Derived Adult T-Cell Leukemia Cells.

Adult T-cell leukemia (ATL) is induced by chronic latent infection with human T-cell leukemia virus type 1 (HTLV-1). HTLV-1 Tax is an oncogenic factor that can be targeted by host T-cell responses. However, the expression of Tax in vivo is little in ATL cells and the impact of Tax-specific T-cell responses on ATL progression remains unclear. In the present study, we examined Tax-specific T-cell responses in C57BL/6 mice after syngeneic transplantation with tax-transgenic mouse-derived ATL (mATL) cells. We first confirmed that cellular tax cDNAs are mostly maintained and detectable in the spleen three weeks after mATL cell transplantation. However, mATL cell transplantation did not induce significant Tax-specific T-cell responses. Mice immunized with DNA and adenovirus vectors expressing Tax exhibited Tax-specific CD4+ T-cell responses but showed no enhancement of the responses or reduction in cellular tax cDNA levels after mATL cell transplantation. This study provides an animal model for analyzing the interaction between ATL cells and host immune responses as well as indicates the limited impact of Tax-specific T-cell responses on the proliferation of ATL cells.

Effects of interferon inducible protein 16 (IFI16) on viral protein and pro-inflammatory cytokine expression in human T-cell leukemia virus type 1 (HTLV-1)-positive T cells

Objective To investigate the effects of interferon inducible protein 16 (IFI16), a cytosolic DNA sensor, on the expression of human T-cell leukemia virus type 1 (HTLV-1) proteins and pro-inflammatory cytokines in adult HTLV-1-positive T cells. Methods IFI16 expression in different HTLV-1-positive T cell lines was detected by immunoblot assay. Specific siRNA targeting the IFI16 gene was constructed and the gene silencing efficiency was detected by immunoblot assay. Expression of HTLV-1 Tax protein at mRNA and protein levels was respectively detected by real-time PCR and immunoblot assay after knocking down the expression of IFI16 in HTLV-1-positive T cells with siRNA. Expression of interferon (IFN)-α, IFN-γ, tumor necrosis factor (TNF)-α, Tax and Env were detected by real-time PCR. Results Compared with the HTLV-1-negative T cell line Jurkat, IFI16 expression was enhanced in the HTLV-1-positive T cell lines MT2, MT4 and C8166. Tax expression was increased, while that of IFN-α, IFN-γ and TNF-α was decreased in MT2 and MT4 cells after silencing the expression of IFI16 with siRNA. Conclusions IFI16 expression was increased in HTLV-1-positive MT2 and MT4 cells. Meanwhile, IFI16 promoted the production of interferon and pro-inflammatory cytokines and inhibited the expression of HTLV-1 proteins. Key words: Interferon inducible protein 16 (IFI16); Human T-cell leukemia lymphoma virus type 1 (HTLV-1); Interferon; T lymphocyte; TNF-α

Conflicting effects of poly(ADP-ribose) polymerase inhibitor on cell-mediated and virion-mediated HTLV-1 infection

Adult T-cell leukemia and human T-cell leukemia virus type 1 (HTLV-1) – associated myelopathy/tropical spastic paraparesis, which develop after HTLV-1 infection, are difficult to cure. In particular, the mode of HTLV-1 propagation is not well understood. Poly (ADP-ribose) polymerase-1 is reported to be a co-activator of HTLV-1 Tax protein; however, the effects of polyADP-ribosylation on infectivity of HTLV-1 have not been fully clarified. We studied the effects of a PARP inhibitor on two modes of HTLV-1 transmission: through cell adhesion between MT-2 cells (an HTLV-1-infected cell line) and uninfected cells and through virus particles produced by HTLV-1-producing c77 cells. Although the PARP inhibitor decreased HTLV-1 infection through cell adhesion, it increased HTLV-1 infection through virion production and caused apoptosis of HTLV-1-infected cells. Thus, careful consideration is required for clinical application of PARP inhibitors in HTLV-1 patients.

Association Between HTLV-1 Genotypes and Risk of HAM/TSP

Human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurological disorder presenting with spastic paraparesis, sphincter dysfunction, and mild sensory disturbance in the lower extremities, which develops in a small minority of HTLV-1-infected individuals. HTLV-1-specific T cells are efficiently activated through dedicated human leukocyte antigen-mediated mechanisms, a process considered deeply involved with its pathogenesis. It has been reported that the lifetime risk of developing HAM/TSP differs between ethnic groups, and there is an association between HTLV-1 tax gene subgroups (i.e., tax subgroup-A or -B), which correspond to HTLV-1 “cosmopolitan subtype 1a subgroup A (i.e., transcontinental subgroup)” and “cosmopolitan subtype 1a subgroup B (i.e., Japanese subgroup),” respectively, and the risk of HAM/TSP in the Japanese population. These findings suggest that a given host’s susceptibility to HAM/TSP is deeply connected with both differences in genetically determined components of the host immune response and HTLV-1 subgroup. Therefore, it is crucial for ongoing work to focus on developing novel treatments and preventative approaches for HAM/TSP. In this review, based on an overview of the topic and our latest research findings, the role of the HTLV-1 subgroup on the effects of virus–host interactions in the pathogenesis of HAM/TSP is discussed.

HTLV-1 Tax-1 interacts with SNX27 to regulate cellular localization of the HTLV-1 receptor molecule, GLUT1.

An estimated 10–20 million people worldwide are infected with human T cell leukemia virus type 1 (HTLV-1), with endemic areas of infection in Japan, Australia, the Caribbean, and Africa. HTLV-1 is the causative agent of adult T cell leukemia (ATL) and HTLV-1 associated myopathy/tropic spastic paraparesis (HAM/TSP). HTLV-1 expresses several regulatory and accessory genes that function at different stages of the virus life cycle. The regulatory gene Tax-1 is required for efficient virus replication, as it drives transcription of viral gene products, and has also been demonstrated to play a key role in the pathogenesis of the virus. Several studies have identified a PDZ binding motif (PBM) at the carboxyl terminus of Tax-1 and demonstrated the importance of this domain for HTLV-1 induced cellular transformation. Using a mass spectrometry-based proteomics approach we identified sorting nexin 27 (SNX27) as a novel interacting partner of Tax-1. Further, we demonstrated that their interaction is mediated by the Tax-1 PBM and SNX27 PDZ domains. SNX27 has been shown to promote the plasma membrane localization of glucose transport 1 (GLUT1), one of the receptor molecules of the HTLV-1 virus, and the receptor molecule required for HTLV-1 fusion and entry. We postulated that Tax-1 alters GLUT1 localization via its interaction with SNX27. We demonstrate that over expression of Tax-1 in cells causes a reduction of GLUT1 on the plasma membrane. Furthermore, we show that knockdown of SNX27 results in increased virion release and decreased HTLV-1 infectivity. Collectively, we demonstrate the first known mechanism by which HTLV-1 regulates a receptor molecule post-infection.

Astute strategies of HTLV-1 with driven viral genes

Human T-cell leukemia virus type 1 (HTLV-1) is the world's first retrovirus with pathogenicity to cause adult T-cell leukemia-lymphoma (ATL) and chronic inflammatory diseases,such as HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and HTLV-1 uveitis. As the virological characteristic, HTLV-1 can transmit efficiently only through cell-to-cell contact. Spread of infection and viral persistence is ingeniously driven by several viral genes as exemplified by HTLV-1 bZIP factor (HBZ) and tax. After the infection, the virus promotes proliferation and immortalization of the infected cells with acculturating immunophenotype into effector/memory T cells. In addition, HBZ enhances expression of co-inhibitory receptors on the surface of infected cells, potentially leading to suppression of host immune responses. These viral strategies can also result in unforeseen by-product, the pathogenicity of HTLV-1-associated diseases. In this review, with recent progress of HTLV-1 researches, we focus on astute regulation systems of the viral genes developed by HTLV-1.

Diversity of cell phenotypes among MT-2 cell lines affects the growth of U937 cells and cytokine production.

We previously reported the diversity of structure and integration sites of human T-cell leukemia virus type 1 (HTLV-1) provirus among different MT-2 cell lines. This raised the question as to whether cell phenotypes also differed among MT-2 cell lines. The influence of two different MT-2 cell lines (MT-2J and MT-2B) on the growth of the promonocytic leukemia cell line, U937, was investigated. Protein levels and mRNA expression of cytokines were also investigated. In addition, Western blot analysis of HTLV-1 regulatory proteins, Tax and HBZ, was also performed. Culture supernatant from MT-2B, but not MT-2J, cells showed marked suppressive effects on U937 cell growth. MT-2B showed high tumor necrosis factor (TNF)-α, TNF-β, and interferon (IFN)-γ both in protein levels of the culture supernatant and mRNA levels of the cells. Analysis using recombinant cytokines indicated that the suppressive effects of MT-2B were due, at least in part, to high levels of TNF-β and its synergic effects with IFN-γ in the culture supernatant. Protein levels of HTLV-1 Tax and HBZ were higher in MT-2B than those in MT-2J cells. These molecules have been reported to affect the cytokine production of HTLV-1 infected cells; therefore, the difference in these molecules may have accounted for the differences in cytokine production between MT-2J and MT-2B cells. Furthermore, because MT-2 cells showed a large variation of integrated HTLV-1 proviruses as well as cell phenotypes, it is important to exercise caution in the assessment and interpretation of experimental data from MT-2 cells.

In Situ Hybridization of HBZ and Tax in FFPE Samples from ATLL Patients and Its Association with Clinicopathological Characteristics

IntroductionAdult T-cell leukemia/lymphoma (ATLL) is a mature T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). Many studies on HTLV-1-related mRNA including HTLV-1 bZIP factor (HBZ) and Tax have been performed, mainly using cell lines, patient-derived cells, and mice. However, there are scant data concerning HTLV-1-related mRNA in formalin-fixed, paraffin-embedded (FFPE) tissue samples. We detected HBZ and Tax mRNA on FFPE tissue samples using in situ hybridization (ISH), and investigated the association with clinicopathological characteristics.Materials and methodsEighty-seven biopsy samples from newly diagnosed ATLL patients were examined. The same samples were used in a previous study (Miyoshi et al. Blood, 2016). Use of patient materials and clinical information was approved by the Ethical Committee of Kurume University, in accordance with the Declaration of Helsinki.ISH was performed on FFPE tissue samples using RNAscope 2.5 HD Reagent Kit-BROWN (Advanced Cell Diagnostics, Hayward, CA). HBZ-and Tax-specific probes were used. MT-4 (HTLV-1 immortalized cell line) and Jurkat (T-cell acute lymphoblastic leukemia cell line) were used as positive and negative controls, respectively. Dot-like signals were counted at high magnification (40 diameters) on 10 randomly selected fields and the number of signals per 1,000 ATLL cells were calculated. The results were reviewed by two experienced hematopathologists. High expression was indicated when more than the median value of HBZ or Tax signals was stained. The antibodies for immunohistochemistry targeted CD4, CD30, Ki-67(MIB-1), CCR4, FoxP3, GATA3, IRF4, HLA class I, β2-microglobulin (β2M), PD-1, and PD-L1.Clinicopathological characteristics of ATLL patients were compared by Fisher's exact test (2-sided), Mann-Whitney's U test, and Spearman's rank correlation analysis. Overall survival was estimated by the Kaplan-Meier method and compared by the Log-rank test. P< .05 was considered statistically significant. EZR ver. 1.32 was used for all statistical analyses.ResultsIn the histogram and scatter plot of HBZ-ISH and Tax-ISH, the median values of HBZ signals and Tax signals were 806/1000 ATLL cells (range 0.4 - 4013.1) and 5.0/1000 ATLL cells (range 0.1 - 891.2), respectively (Figure 1). Representative samples of HBZ-ISH and Tax-ISH are presented in Figure 2A and 2B, respectively.Notably, the high-expression group of HBZ displayed significant reductions in skin lesions (P = .025), Ann Arbor stage (P = .021), and peripheral blood involvement (P = .028). The high-expression group of Tax displayed significant increases in lactate dehydrogenase activity (P = .0020), splenomegaly (P = .0070), CD 30 (P = .015), and PD-1 in tumor-infiltrating lymphocytes (PD-1_TIL; P <.0010).Weak but significant positive correlation was observed between HBZ and IRF4 (ρ = .33; P = .029), between HBZ and PD-L1 (ρ = -. 28; P = .010), between Tax and CD4 (ρ = .41; P = .0074), between Tax and CD30 (ρ = .26; P = .016), between Tax and GATA 3 (ρ = .26; P = .014), between Tax and PD-1 in ATLL cells (ρ = .25; P = .0074), between Tax and PD-L1 in ATLL cells (ρ =. 32; P = .0026), and between Tax and PD-1_TIL (ρ = .37; p = .0026) (Figure 3). There was almost no correlation between HBZ and Tax (ρ = .052; P = .63). There were no significant differences between HBZ or Tax and OS (Log-rank P = .21 and .95, respectively).ConclusionThe RNA scope assay is useful for detecting low expressing mRNA in FFPE tissue samples. Our data suggest that ISH of HBZ and Tax can detect differences in clinicopathological characteristics of ATLL patients. In clinical and cell samples, HBZ and Tax might be involved in the pathogenesis of ATLL.DisclosuresNo relevant conflicts of interest to declare. [Display omitted] DisclosuresAsano:Takeda: Honoraria; Chugai Pharma: Honoraria; Celgene: Honoraria.

Dynamic Changes of Chromatin Structure and Transcriptome By Transient Expression of HTLV-1 Tax in ATL Cells

Adult T-cell leukemia-lymphoma (ATL) is a fatal malignancy of CD4+ T cells, which is caused by human T-cell leukemia virus type 1 (HTLV-1). HTLV-1 encodes two oncogenic viral factors, Tax and HTLV-1 bZIP factor (HBZ) in the sense and antisense of the provirus respectively. HBZ is constitutively expressed in infected cells and plays critical roles for cell proliferation. Tax potently promotes viral replication and activates various oncogenic signaling pathways in HTLV-1 infected cells; however, Tax expression is generally suppressed in infected cells owing to its high immunogenicity. Therefore, the dynamics of Tax expression and contribution of oncogenic transformation by Tax in HTLV-1 infected cells have not been well elucidated. Recently, we have reported that Tax is transiently expressed in a small subpopulation of ATL cells, which induces drastic changes in the host transcriptional profile (Mahgoub M., et al. Proc Natl Acad Sci USA. 2018). Moreover, the single cell analysis showed that expression of anti-apoptotic and NF-kB related genes were upregulated in Tax-expressing cells compared to Tax-non-expressing cells. HBZ expression was increased in Tax-non-expressing cells than Tax-expressing cells, suggesting that HBZ may regulate cell proliferation in a different time phase from Tax. On the other hand, precise mechanisms for regulation of host gene expression by transient Tax expression are not well known. In this study, we analyzed structural change of host chromatin accompanied by transient Tax expression to clarify the transcriptional dynamics of host genes in ATL cells.ATL cell lines (MT-1 and KK-1), which express EGFP under the control of Tax, were used to sort Tax-expressing and -non-expressing cells by a cell sorter, and each population was subjected to H3K27ac ChIP-seq and ATAC-seq analyses. DNA libraries were quantified and sequenced on Illumina NextSeq 500 (single-end) for ChIP-seq, and on Illumina Hiseq 4000 (paired-end) for ATAC-seq, respectively.H3K27ac ChIP-seq data showed that the peaks correlated with Tax expression were observed in many genetic loci including NF-kB related genes. Among Tax-expressing MT-1 cells, H3K27ac were highly enriched super-enhancers, such as IL2RA, TRAF3, TNFRSF8, PHF13 loci, compared to Tax-non-expressing cells. There were more H3K27ac-marked active enhancers in Tax-expressing cells than Tax-non-expressing cells across the entire region, suggesting that global structural change may occur in Tax-expressing cells.Using the data of ATAC-seq, pathway analyses were performed by GREAT. This analysis revealed that the pathways associated with immune activation, such as Toll-like receptor signaling, TNFR2 signaling, IL-2 signaling, and Th1/Th2 differentiation pathways were significantly enriched in Tax-expressing cells. Analysis of genome-wide distribution of motifs in open chromatin region, were carried out with HOMER, and result showed that motifs of NF-kB, and AP-1 family (e.g., JunB, Fra1, Fra2) were significantly enriched in Tax-expressing cells compared to Tax-non-expressing cells, which is consistent with the results of pathway analyses of RNA-seq. The genome-wide motif footprinting analysis was performed using Protein interaction quantitation (PIQ) methods. It was found that motifs of NF-kB, and AP-1 family (e.g., JunB, Fra1, Fra2) were occupied in large number of regions and showed higher DNA accessibility in Tax-expressing cells. In contrast, motif of CTCF was likely occupied in large number of regions and exhibit higher DNA accessibility in Tax-non-expressing cells. Importantly, mRNA levels of NF-kB and AP-1 transcription factors were significantly higher in Tax-expressing than Tax-non-expressing cells.These findings suggest that structural changes of NF-kB and AP-1 family recognition sites, and activation of pathways related to these factors could trigger global transcriptional changes in host genome by transient Tax expression, which is the critical event for persistent infection of HTLV-1 and leukemogenesis of ATL. DisclosuresMatsuoka:Bristol Myers Squibb: Research Funding.

Distinct gene expression signatures induced by viral transactivators of different HTLV-1 subgroups that confer a different risk of HAM/TSP

BackgroundAmong human T cell leukemia virus type 1 (HTLV-1)-infected individuals, there is an association between HTLV-1 tax subgroups (subgroup-A or subgroup-B) and the risk of HAM/TSP in the Japanese population. To investigate the role of HTLV-1 subgroups in viral pathogenesis, we studied the functional difference in the subgroup-specific viral transcriptional regulators Tax and HBZ using microarray analysis, reporter gene assays, and evaluation of viral-host protein–protein interaction.Results(1) Transcriptional changes in Jurkat Tet-On human T-cells that express each subgroup of Tax or HBZ protein under the control of an inducible promoter revealed different target gene profiles; (2) the number of differentially regulated genes induced by HBZ was 2–3 times higher than that induced by Tax; (3) Tax and HBZ induced the expression of different classes of non-coding RNAs (ncRNAs); (4) the chemokine CXCL10, which has been proposed as a prognostic biomarker for HAM/TSP, was more efficiently induced by subgroup-A Tax (Tax-A) than subgroup-B Tax (Tax-B), in vitro as well as in unmanipulated (ex vivo) PBMCs obtained from HAM/TSP patients; (5) reporter gene assays indicated that although transient Tax expression in an HTLV-1-negative human T-cell line activated the CXCL10 gene promoter through the NF-κB pathway, there was no difference in the ability of each subgroup of Tax to activate the CXCL10 promoter; however, (6) chromatin immunoprecipitation assays showed that the ternary complex containing Tax-A is more efficiently recruited onto the promoter region of CXCL10, which contains two NF-κB binding sites, than that containing Tax-B.ConclusionsOur results indicate that different HTLV-1 subgroups are characterized by different patterns of host gene expression. Differential expression of pathogenesis-related genes by subgroup-specific Tax or HBZ may be associated with the onset of HAM/TSP.