HBZ Expression Research Articles

Targeted zinc-finger repressors to the oncogenic HBZ gene inhibit adult T-cell leukemia (ATL) proliferation.

Human T-lymphotropic virus type I (HTLV-I) infects CD4+ T-cells resulting in a latent, life-long infection in patients. Crosstalk between oncogenic viral factors results in the transformation of the host cell into an aggressive cancer, adult T-cell leukemia/lymphoma (ATL). ATL has a poor prognosis with no currently available effective treatments, urging the development of novel therapeutic strategies. Recent evidence exploring those mechanisms contributing to ATL highlights the viral anti-sense gene HTLV-I bZIP factor (HBZ) as a tumor driver and a potential therapeutic target. In this work, a series of zinc-finger protein (ZFP) repressors were designed to target within the HTLV-I promoter that drives HBZ expression at highly conserved sites covering a wide range of HTLV-I genotypes. ZFPs were identified that potently suppressed HBZ expression and resulted in a significant reduction in the proliferation and viability of a patient-derived ATL cell line with the induction of cell cycle arrest and apoptosis. These data encourage the development of this novel ZFP strategy as a targeted modality to inhibit the molecular driver of ATL, a possible next-generation therapeutic for aggressive HTLV-I associated malignancies.

CARD11 mutation and HBZ expression induce lymphoproliferative disease and adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1). In addition to HTLV-1 bZIP factor (HBZ), a leukemogenic antisense transcript of HTLV-1, abnormalities of genes involved in TCR-NF-κB signaling, such as CARD11, are detected in about 90% of patients. Utilizing mice expressing CD4+ T cell-specific CARD11(E626K) and/or CD4+ T cell-specific HBZ, namely CARD11(E626K)CD4-Cre mice, HBZ transgenic (Tg) mice, and CARD11(E626K)CD4-Cre;HBZ Tg double transgenic mice, we clarify these genes’ pathogenetic effects. CARD11(E626K)CD4-Cre and HBZ Tg mice exhibit lymphocytic invasion to many organs, including the lungs, and double transgenic mice develop lymphoproliferative disease and increase CD4+ T cells in vivo. CARD11(E626K) and HBZ cooperatively activate the non-canonical NF-κB pathway, IRF4 targets, BATF3/IRF4/HBZ transcriptional network, MYC targets, and E2F targets. Most KEGG and HALLMARK gene sets enriched in acute-type ATL are also enriched in double transgenic mice, indicating that these genes cooperatively contribute to ATL development.

Histone Deacetylase Inhibitors Abolish HBZ Protein Expression and Induce Cell Death in HTLV-1 Related Adult T-Cell Leukemia-Lymphoma

Background:Adult T-cell leukemia-lymphoma (ATL) is a malignancy with dismal prognosis caused by HTLV-1. ATL is incurable by conventional drugs. Histone deacetylase (HDAC) inhibitors (HDIs) are broadly active anti-neoplastic agents that can be cautiously exploited in the treatment of viral related lymphomas. HTLV-1 usually establishes lifelong latency in human CD4+ T-cells, which in turn propagate and persist as result of the multifaceted functions of HTLV-1 basic zipper factor (HBZ) through various signaling pathways that promote growth and proliferation, and block apoptosis and cell senescence. All ATL cells harbor at least one copy of latent intact HTLV-1 provirus in most cases, and unlike Tax, all tumors express HBZ from the negative 3‘ proviral strand via SP-1 activity. The HTLV-1 promoter is negatively regulated by HDACs at the 5‘ long terminal repeat (LTR), and by HBZ, which disrupts Tax positive interaction with the transcriptional activator p300/CBP, thus blocking viral transcription. Therefore, treatment of HTLV-1 infected cells with HDIs can induce expression of Tax protein, which is highly immunogenic, and which could promote immune-mediated clearance of ATL cells in vivo.Methods:Low-passage IL-2 dependent ATL cell lines established from patients with acute type ATL and freshly isolated ATL cells were treated with several HDIs (VPA, sodium butyrate, vorinostat, belinostat, panabinostat, and mocetinostat) at various doses and time points. Apoptosis was measured by annexin V and FACS analysis. Expression of HBZ protein, Tax, acetyl and total histone 3 subunits (H3), BIM, caspase 3, caspase 9, Jun B, c-Jun, Jun D, and Sp-1 were measured by Western blots before and after HDI treatment. HBZ and Tax mRNA expression was measured by quantitative RT-PCR. SP-1 binding to HTLV-1 specific oligonucleotide consensus sequence was measured by gel shift assay using nuclear protein extracts before and after HDI treatment.Results:HDIs sequentially increased acetylated H3 levels, blocked HBZ protein, upregulated Tax expression, stabilized BIM, and induced cleavage of pro-caspases 9 and 3 (intrinsic apoptotic pathway), resulting in apoptosis of ATL cells. HDIs tested (VPA and belinostat) blocked expression of Jun-B, which is known to be upregulated by HBZ, while no significant effects were observed on c-Jun and Jun D protein levels. HDIs tested (VPA, belinostat, and panabinostat) did not significantly alter spliced-HBZ RNA levels or affect SP-1 binding to HBZ promoter-specific oligonucleotide sequence, suggesting a post-translational mechanism may contribute to abrogation of HBZ in ATL cells.Conclusions:Our data demonstrate that HDIs target ATL cells in part by abrogating HBZ protein, which could negatively impact the longevity of ATL and HTLV-1 cells in vivo. Long-term clinical use of HDIs might be beneficial for eliminating residual ATL cells after definitive treatment. A pilot clinical trial testing belinostat-based consolidation therapy for aggressive leukemic ATL types is currently ongoing at our institution (available at Clinicaltrials.gov , NCT02737046). DisclosuresNo relevant conflicts of interest to declare.

Dual cytoplasmic and nuclear localization of HTLV-1-encoded HBZ protein is a unique feature of adult T-cell leukemia

Adult T-cell leukemia-lymphoma (ATL), is a highly malignant T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1), characterized by poor prognosis. Two viral proteins, Tax-1 and HTLV-1 basic-zipper factor (HBZ) play important roles in the pathogenesis of ATL. While Tax-1 can be found in both the cytoplasm and nucleus of HTLV-1 infected patients, HBZ is exclusively localized in the cytoplasm of HTLV-1 asymptomatic carriers and in patients with the chronic neurologic disease HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HBZ is only localized in the nucleus of ATL cell lines, suggesting that the nuclear localization of HBZ can be a hallmark of neoplastic transformation. In order to clarify this crucial point, we investigated in detail the pattern of HBZ expression in ATL patients. We made use of our monoclonal antibody 4D4-F3, that at present is the only reported reagent, among the few described, able to detect endogenous HBZ by immunofluorescence and confocal microscopy in cells from asymptomatic carriers, HAM/TSP and ATL patients. We found that HBZ is localized both in the cytoplasm and nucleus of cells of ATL patients irrespective of their clinical status, with a strong preference for the cytoplasmic localization. Also Tax-1 is localized in both compartments. As HBZ is exclusively localized in the cytoplasm in asymptomatic carriers and in non-neoplastic pathologies, this finding shows that neoplastic transformation consequent to HTLV-1 infection is accompanied and associated with the capacity of HBZ to translocate to the nucleus, which suggests a role of cytoplasmic-to-nuclear translocation in HTLV-1- mediated oncogenesis.

Nanomicellar Curcumin Supplementation Improves the Clinical Manifestations of HAM/TSP Patients.

HTLV-1 infection causes a chronic, progressive, demyelinating, neuroinflammatory disease called HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Treatment of HAM/TSP patients which have high levels of proviral load and pro-inflammatory markers is a challenge for clinicians. Therefore, we aimed to investigate the immunomodulatory, anti-inflammatory, and antiviral effects of curcumin in HAM/TSP patients. In this study, 20 newly diagnosed HAM/TSP patients (2 men and 18 women) were enrolled and evaluated for clinical symptoms, HTLV-1 proviral load, Tax and HBZ expression, neopterin serum concentration, and complete blood count (CBC) before and 12weeks after treatment with nanomicellar curcumin (80mg/day, orally). Clinical symptoms such as the mean Osame Motor Disability Score and Ashworth Spasticity Scale Score were significantly improved after the treatment (P=0.001 and P=0.001). Sensory symptoms such as pain and paresthesia were significantly decreased in all of the patients (P=0.001). Furthermore, urinary disorders, including urinary frequency, incontinence, and the feeling of incomplete bladder emptying, were significantly improved (P=0.001, P=0.003, and P=0.03). However, the mean HTLV-1 proviral load (P=0.97) and CBC were similar, whereas Tax, HBZ, and neopterin levels tend to increase after the treatment (P=0.004, P=0.08, and P=0.04). Results suggest that curcumin can safely improve the clinical symptoms of HAM/TSP patients but has no observable positive effects on the HTLV-1 proviral load, Tax, and HBZ expression. Therefore, prolonged use or the use of curcumin with antiviral agents in addition to clinical signs and symptoms can reduce the HTLV-1 proviral load and the expression of functional viral factors such as Tax and HBZ.

CARD11 Mutation Induces Oligoclonal Expansion of T-Cells, and Accelerates ATL Development in Combination with HBZ

Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell lymphoma that develops in about 5% of human T-cell leukemia/lymphoma virus 1 (HTLV-1) carriers. In addition to viral oncogenes, namely tax and HTLV-1 bZIP factor (HBZ), gene mutations, highly enriched for T-cell receptor (TCR)-NF-kB signaling, should be involved in the development of ATL. Among gene mutations, mutation in CARD11, a cytoplasmic scaffolding protein required for TCR-induced NF-kB activation, was detected in 24% of ATL patients. Here we generated a mouse model for conditional expression of a human ATL-derived CARD11(E626K) gain-of-function mutant, and demonstrated CARD11 activation induced oligoclonal expansion of T-cell and infiltration to many organs. We also showed that expression of HBZ accelerated mutant CARD11-induced lymphoproliferative diseases. We introduced a human Card11(E626K) into the mouse genome at the ROSA26 locus. After crossing with CD4-Cre Tg, CARD11(E626K)CD4-Cre mice was obtained. In CD4+ cells from CARD11(E626K)CD4-Cre, the amount of cleaved BCL-10 and NF-kBp65 increased compared with those in WT CD4+cells, confirming the activation of NF-kB. About half of CARD11(E626K)CD4-Cre mice died on or after 6 months after birth. At 6 months, leukocytosis was observed in CARD11(E626K)CD4-Cre, and accordingly the number of CD4+ cells cells was about 1.43 times greater than those in WT mice. The most affected organ in CARD11(E626K)CD4-Cre mice was lung. Alveolar septum was thickened by infiltrated cells at 6 months, and worsened subsequently. CD3+ T-cell accumulated around capillary blood vessels, and had high proliferation indices (>50%), as assayed by Ki-67 staining. CARD11(E626K)CD4-Cre mice developed lymphadenopathy (4/8 mice (50%) at 6M and 4/6 mice (66.7%) at 12M). Normal lymph node (LN) architecture was barely preserved, and medullary sinus was expanded with CD3+ T-cell. Some of them were positive for FoxP3, and had moderate proliferation indices (25%-50%). Among CD4+ T-cell, the proportion of naive T-cell (Tn) decreased, and that of effector/memory T-cell (Tem) and regulatory T-cell (Treg) increased compared to WT LNs. The proportion of Treg in CD4+ T-cell from LN of 6M- and 12M-old CARD11(E626K)CD4-Cre mice was 25% and 40%, respectively, which values were much larger than the normal range as 10-15%. We next examined the clonality of CD4+ cells in spleen and swollen LNs from CARD11(E626K)CD4-Cre mice. The clonality of the TCR repertoires of 20 individual Vb gene famines from Vb1-20 was assessed by a PCR. The clonality assay using the TCR repertoires exhibited an oligoclonal pattern in 4 of 5 splenic CD4+ cells, and 1 of 2 LN CD4+ cells from CARD11(E626K)CD4-Cre mice. To assess the effect of HBZ constant expression on CARD11(E626K)CD4-Cre mice, we generated HBZ Tg, in which HBZ cDNA was expressed under the CD4 promoter. Similar to the previous report (by Satou et al.), our HBZ Tg showed increased number of Tem, destroyed architecture of lung such as thickened alveolar septum by infiltrated cells and decreased alveolar space by edema, and the lymphadenopathy after 12M (66.7%). We then crossed CARD11(E626K)CD4-Creand HBZ Tg, and obtained the compound mice. The compound mice caused more aggressive lymphoproliferative diseases compared with CARD11(E626K)CD4-Cre mice. Most of compound mice died within 8M. At 6M, architecture of lung, kidney, spleen, and LN was destroyed. In lung, alveolar space of lung was scarcely observed caused of T-cell invasion. Alveolar septum was thickened with infiltrated cells, and CD3+ cells accumulated around capillary blood vessel. Some of them were positive for FoxP3, and indicated moderate proliferation indices (25-50%). T-cell invasion was also observed in kidney. Lymphadenopathy was detected in 6 of 9 (66.7%) with completely destroyed architecture, increment of the proportion of Fox3+ cells, and moderate proliferation indices (25-50%). The clonality assay using the TCR repertoires exhibited an oligoclonal pattern in 4 of 4 splenic CD4+ cells, and 2 of 2 LNs CD4+ cells from compound mice. These results suggest that CARD11 mutant-induced NFkB activation and constant HBZ expression may have similar effects, such as T-cell infiltration into organs and LN adenopathy, and that the simultaneous occurrence of both may have additive effects. Disclosures Sugiyama: Chordia Therapeutics, Japan.: Current Employment. Morishita:Chordia Therapeutics Inc.: Current Employment, Current equity holder in private company. Shimoda:Perseus Proteomics: Research Funding; PharmaEssentia Japan: Research Funding; AbbVie Inc.: Research Funding; Astellas Pharma: Research Funding; Merck & Co.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Kasei Medical: Research Funding; Japanese Society of Hematology: Research Funding; The Shinnihon Foundation of Advanced Medical Treatment Research: Research Funding; Celgene: Honoraria; Shire plc: Honoraria; Novartis: Honoraria, Research Funding; Takeda Pharmaceutical Company: Honoraria; Bristol-Myers Squibb: Honoraria.

Human T-cell lymphotropic virus HBZ and tax mRNA expression are associated with specific clinicopathological features in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is caused by human T-cell leukemia virus type 1 (HTLV-1). HTLV-1-associated mRNA, including HBZ and tax, is deeply involved in the pathogenesis of ATLL. Using 88 ATLL tissue samples, we performed in situ mRNA analysis of HBZ and tax, and investigated its association with clinicopathological characteristics of ATLL. The median value of HBZ signals (/1000 ATLL cells) was 795.2 (range: 0.4-4013.1) and of tax signals (/1000 ATLL cells) was 5.1 (range: 0.1-891.2). The low-expression HBZ group displayed significant increase in the number of skin lesion (P = 0.0283). The high-expression tax group displayed significant increase in the number of PD-1-positive tumor-infiltrating lymphocytes (P < 0.0001). In addition, we identified patients with very high-expression of tax signals (400 or more signals/1000 ATLL cells). These patients displayed significant reductions in the expression of HLA class I (P = 0.0385) and β2M (P = 0.0124). Moreover, these patients displayed significantly poor overall survival (median survival time [MST] 7.7 months, 95% confidence interval [CI] [4.7-NA]), compared with the survival in patients with less than 400 tax signals (MST 22.6 months, 95% CI [13.7-41.7]) (P = 0.0499). These results suggest that Tax-mediated treatment of ATLL should be performed carefully in the high-expression tax group. More detailed studies could elucidate the clinicopathological significance of HBZ and tax mRNA expressions in ATLL.

Human T-Cell Leukemia Virus Type 1 (HTLV-1) bZIP Factor Upregulates the Expression of ICAM-1 To Facilitate HTLV-1 Infection.

Human T-cell leukemia virus type 1 (HTLV-1) causes multiple pathological effects, ranging from a form of leukemia to a spectrum of inflammation-mediated diseases. These diseases arise from one or several infected CD4+ T cells among thousands acquiring proliferation and survival advantages and ultimately becoming pathogenic. Given the low incidence of HTLV-1-associated diseases among carriers, such cellular evolutionary processes appear to occur rarely. Therefore, infectious spread of HTLV-1 within the T-cell population may be one underlying factor influencing disease development. Free HTLV-1 virions are poorly infectious, so infection of T cells relies on direct contact between infected and target cells. Following contact, virions pass to target cells through a virological synapse or cellular conduits or are transferred to target cells within an extracellular matrix. Lymphocyte functioning antigen 1 (LFA-1) on the surface of the target cell engaging with its ligand, ICAM-1, on the surface of the infected cell (effector cell) initiates and stabilizes cell-cell contact for infection. We found that stable expression of an HTLV-1 accessory protein, HTLV-1 bZIP factor (HBZ), in Jurkat T cells increases homotypic aggregation. This phenotype was attributed to elevated ICAM-1 expression in the presence of HBZ. Using a single-cycle replication-dependent luciferase assay, we found that HBZ expression in Jurkat cells (used as effector cells) increases HTLV-1 infection. Despite this effect, HBZ could not replace the critical infection-related functions of the HTLV-1 regulatory protein Tax. However, in HTLV-1-infected T cells, knockdown of HBZ expression did lead to a decrease in infection efficiency. These overall results suggest that HBZ contributes to HTLV-1 infectivity.IMPORTANCE Human T-cell leukemia virus type 1 (HTLV-1) causes a variety of diseases, ranging from a fatal form of leukemia to immune-mediated inflammatory diseases. These diseases occur rarely, arising from one or a small subset of virally infected cells infrequently evolving into a pathogenic state. Thus, the process of HTLV-1 cell-to-cell transmission within the host helps influence the probability of disease development. HTLV-1 primarily infects T cells and initially spreads within this cell population when virally infected T cells dock to uninfected target T cells and then transfer HTLV-1 virus particles to the target cells. Here we found that the viral protein HTLV-1 bZIP factor (HBZ) promotes infectivity. HBZ accomplishes this task by increasing the surface abundance of a cellular adhesion protein known as intercellular adhesion molecule 1 (ICAM-1), which helps initiate and stabilize contact (docking) between infected and target T cells. These results define a novel and unexpected function of HBZ, diverging from its defined functions in cellular survival and proliferation.

A-104 Tracing the intracellular journey of HTLV-1 HBZ during infection: From asymptomatic carriers to HAM/TSP ending to ATL: A one-way ticket?

HTLV-1 is the etiological agent of Adult T cell Leukemia (ATL) and of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) neurologic disorder. Although substantial progress has been made in understanding some cellular and molecular mechanisms underlying the pathology of the neoplastic and neurological diseases, still the intimate reason why some HTLV-1 infected patients develop HAM/TSP and others progress toward the neoplastic state are largely elusive. Two viral proteins, Tax-1 and HBZ, play an important role in both diseases. For example, Tax-1, the crucial transactivator needed for viral replication, has been implicated in the initiation but not in the maintenance of neoplastic process since leukemic cells may not express it. On the other hand, HBZ seems to be expressed throughout the infection and, importantly, is always expressed in leukemic cells. Nevertheless, the precise mechanism through which HBZ participates to initiation and/or maintenance of leukemic state is still unclear, as it is unclear the association of HBZ expression with initiation and/or maintenance of the inflammatory syndrome leading to HAM/TSP. Studies of this aspect have been partially prevented by the lack of suitable reagents to detect endogenous HBZ protein during the various phases of HTLV-1 infection and disease development. The recent isolation of a specific monoclonal antibody against HBZ, the 4D4-F3 mAb, by our group has allowed to precisely quantify and determine the subcellular distribution of the viral protein. We have demonstrated that HBZ, previously considered an exclusive nuclear protein, is mainly localized in the cytoplasm of PBMCs from HAM/TSP patients. Here, the analysis of a larger panel of patients has added new relevant informations on this point. PBMC from 13 HTLV-1 infected asymptomatic carriers (AC), 14 HAM/TSP and 4 ATL patients were analysed. Expression and subcellular distribution of endogenous HBZ and Tax-1 proteins were assessed by confocal microscopy with the 4D4-F3 and A51-2 mAbs, respectively. HBZ expression was confined to the cytoplasm of PBMC of HAM/TSP patients, appearing as discrete dots. The percentage of HBZ-positive cells ranged between 1% and 10% of total PBMC with the exception of one negative patient. Tax-1 was expressed in all HAM/TSP cases analyzed, ranging from 2% to 15% of total PBMC. Tax-1 was preferentially localized in the cytoplasm with fewer speckle-like dots in the nucleus. Interestingly, only in 1 case co-expression of HBZ and Tax-1 was observed in the same cells. In this unique case, however, although 8% and 15% of the cells were positive for HBZ and Tax-1, respectively, only 1% of cells co-expressed the 2 viral proteins. Of particular interest was the finding that PBMC of 6 out of 13 AC expressed HBZ (1%–4% of the cells) and again with an exclusive cytoplasmic localization. Tax-1 was expressed in 10 out of 13 cases (1%–11% of the cells). Only in 2 AC, HBZ and Tax-1 were found to be co-expressed in a very limited proportion of cells. The study of 4 distinct cases of ATL demonstrated, instead, that HBZ protein was expressed in the vast majority of the leukemic cells and importantly with an exclusive nuclear localization. Interestingly, none of these ATL cases were found to express Tax-1 protein. These results extend our previous observation on the dichotomy of HBZ localization between HAM/TSP and ATL, pointing to the exclusive either cytoplasmic or nuclear localization in the 2 diseased states, respectively. Moreover, they show a rather selective expression in distinct cells of either HBZ or Tax-1. The unprecedented observation that in AC, HBZ is expressed only in the cytoplasm strongly suggests a progressive modification of HBZ localization during the disease states associated to HTLV-1infection. Future studies will clarify whether the distinct HBZ intracellular localization is simply a marker or a causative event of disease evolution.

HTLV-1-host interactions on the development of adult T cell leukemia/lymphoma: virus and host gene expressions

BackgroundAdult T-cell leukemia/lymphoma (ATLL) is a lymphoproliferative disorder of HTLV-1-host interactions in infected TCD4+ cells. In this study, the HTLV-1 proviral load (PVL) and HBZ as viral elements and AKT1, BAD, FOXP3, RORγt and IFNλ3 as the host factors were investigated.MethodsThe study was conducted in ATLLs, HTLV-1-associated myelopathy/tropical spastic paraparesis patients (HAM/TSPs) and HTLV-1-asympthomatic carriers (ACs). The DNA and mRNA from peripheral blood mononuclear cells were extracted for gene expression assessments via qRT-PCR, TaqMan assay, and then confirmed by western blotting.ResultsAs it was expected, the HTLV-1-PVL were higher in ATLLs than ACs (P = 0.002) and HAM/TSP (P = 0.041). The HBZ expression in ATLL (101.76 ± 61.3) was radically higher than in ACs (0.12 ± 0.05) and HAM/TSP (0.01 ± 0.1) (P = 0.001). Furthermore, the AKT1 expression in ATLLs (13.52 ± 4.78) was higher than ACs (1.17 ± 0.27) (P = 0.05) and HAM/TSPs (0.72 ± 0.49) (P = 0.008). However, BAD expression in ATLL was slightly higher than ACs and HAM/TSPs and not significant. The FOXP3 in ATLLs (41.02 ± 24.2) was more than ACs (1.44 ± 1) (P = 0.007) and HAM/TSP (0.45 ± 0.15) (P = 0.01). However, RORγt in ATLLs (27.43 ± 14.8) was higher than ACs (1.05 ± 0.32) (P = 0.02) but not HAM/TSPs. Finally, the IFNλ3 expression between ATLLs (31.92 ± 26.02) and ACs (1.46 ± 0.63) (P = 0.01) and ACs and HAM/TSPs (680.62 ± 674.6) (P = 0.02) were statistically different, but not between ATLLs and HAM/TSPs.ConclusionsThe present and our previous study demonstrated that HTLV-1-PVL and HBZ and host AKT1 and Rad 51 are novel candidates for molecular targeting therapy of ATLL. However, high level of RORγt may inhibit Th1 response and complicated in ATLL progressions.

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.

The Human T-Cell Leukemia Virus Type 1 Basic Leucine Zipper Factor Attenuates Repair of Double-Stranded DNA Breaks via Nonhomologous End Joining.

Adult T-cell leukemia (ATL) is a fatal malignancy of CD4+ T cells infected with human T-cell leukemia virus type 1 (HTLV-1). ATL cells often exhibit random gross chromosomal rearrangements that are associated with the induction and improper repair of double-stranded DNA breaks (DSBs). The viral oncoprotein Tax has been reported to impair DSB repair but has not been shown to be consistently expressed throughout all phases of infection. The viral oncoprotein HTLV-1 basic leucine zipper (bZIP) factor (HBZ) is consistently expressed prior to and throughout disease progression, but it is unclear whether it also influences DSB repair. We report that HBZ attenuates DSB repair by nonhomologous end joining (NHEJ), in a manner dependent upon the bZIP domain. HBZ was found to interact with two vital members of the NHEJ core machinery, Ku70 and Ku80, and to be recruited to DSBs in a bZIP-dependent manner in vitro We observed that HBZ expression also resulted in a bZIP-dependent delay in DNA protein kinase (DNA-PK) activation following treatment with etoposide. Although Tax is reported to interact with Ku70, we did not find Tax expression to interfere with HBZ:Ku complex formation. However, as Tax was reported to saturate NHEJ, we found that this effect masked the attenuation of NHEJ by HBZ. Overall, these data suggest that DSB repair mechanisms are impaired not only by Tax but also by HBZ and show that HBZ expression may significantly contribute to the accumulation of chromosomal abnormalities during HTLV-1-mediated oncogenesis.IMPORTANCE Human T-cell leukemia virus type 1 (HTLV-1) infects 15 million to 20 million people worldwide. Approximately 90% of infected individuals are asymptomatic and may remain undiagnosed, increasing the risk that they will unknowingly transmit the virus. About 5% of the HTLV-1-positive population develop adult T-cell leukemia (ATL), a fatal disease that is not highly responsive to treatment. Although ATL development remains poorly understood, two viral proteins, Tax and HBZ, have been implicated in driving disease progression by manipulating host cell signaling and transcriptional pathways. Unlike Tax, HBZ expression is consistently observed in all infected individuals, making it important to elucidate the specific role of HBZ in disease progression. Here, we present evidence that HBZ could promote the accumulation of double-stranded DNA breaks (DSBs) through the attenuation of the nonhomologous end joining (NHEJ) repair pathway. This effect may lead to genome instability, ultimately contributing to the development of ATL.

HTLV-1 HBZ Viral Protein: A Key Player in HTLV-1 Mediated Diseases.

Human T cell leukemia virus type 1 (HTLV-1) is an oncogenic human retrovirus that has infected 10–15 million people worldwide. After a long latency, 3–5% of infected individuals will develop either a severe malignancy of CD4+ T cells, known as Adult T-cell Leukemia (ATL) or a chronic and progressive inflammatory disease of the nervous system designated Tropical Spastic Paraparesis/HTLV-1-Associated Myelopathy (HAM/TSP). The precise mechanism behind HTLV-1 pathogenesis still remains elusive. Two viral regulatory proteins, Tax-1 and HTLV-1 bZIP factor (HBZ) are thought to play a critical role in HTLV-1-associated diseases. Tax-1 is mainly involved in the onset of neoplastic transformation and in elicitation of the host’s inflammatory responses; its expression may be lost during cell clonal proliferation and oncogenesis. Conversely, HBZ remains constantly expressed in all patients with ATL, playing a role in the proliferation and maintenance of leukemic cells. Recent studies have shown that the subcellular distribution of HBZ protein differs in the two pathologies: it is nuclear with a speckled-like pattern in leukemic cells and is cytoplasmic in cells from HAM/TSP patients. Thus, HBZ expression and distribution could be critical in the progression of HTLV-1 infection versus the leukemic state or the inflammatory disease. Here, we reviewed recent findings on the role of HBZ in HTLV-1 related diseases, highlighting the new perspectives open by the possibility of studying the physiologic expression of endogenous protein in primary infected cells.

Single-cell heterogeneity and cell-cycle-related viral gene bursts in the human leukaemia virus HTLV-1

Background: The human leukaemia virus HTLV-1 expresses essential accessory genes that manipulate the expression, splicing and transport of viral mRNAs. Two of these genes, tax and hbz, also promote proliferation of the infected cell, and both genes are thought to contribute to oncogenesis in adult T-cell leukaemia/lymphoma. The regulation of HTLV-1 proviral latency is not understood. tax, on the proviral plus strand, is usually silent in freshly-isolated cells, whereas the minus-strand-encoded hbz gene is persistently expressed at a low level. However, the persistently activated host immune response to Tax indicates frequent expression of tax in vivo. Methods: We used single-molecule RNA-FISH to quantify the expression of HTLV-1 transcripts at the single-cell level in a total of >19,000 cells from five T-cell clones, naturally infected with HTLV-1, isolated by limiting dilution from peripheral blood of HTLV-1-infected subjects. Results: We found strong heterogeneity both within and between clones in the expression of the proviral plus-strand (detected by hybridization to the tax gene) and the minus-strand ( hbz gene). Both genes are transcribed in bursts; tax expression is enhanced in the absence of hbz, while hbz expression increased in cells with high tax expression. Surprisingly, we found that hbz expression is strongly associated with the S and G 2/M phases of the cell cycle, independent of tax expression. Contrary to current belief, hbz is not expressed in all cells at all times, even within one clone. In hbz-positive cells, the abundance of hbz transcripts showed a very strong positive linear correlation with nuclear volume. Conclusions: The occurrence of intense, intermittent plus-strand gene bursts in independent primary HTLV-1-infected T-cell clones from unrelated individuals strongly suggests that the HTLV-1 plus-strand is expressed in bursts in vivo. Our results offer an explanation for the paradoxical correlations observed between the host immune response and HTLV-1 transcription.

The effect of HTLV-1 virulence factors (HBZ, Tax, proviral load), HLA class I and plasma neopterin on manifestation of HTLV-1 associated myelopathy tropical spastic paraparesis

BackgroundPrevious studies have suggested debatable roles of Tax and HBZ gene expression in the pathogenesis of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). In this study, HTLV-1 and host interactions in the manifestation of HAM/TSP were evaluated. MethodsA cross-sectional study was conducted on 33 HAM/TSP patients and 38 HTLV-1 asymptomatic carriers (ACs). HTLV-1-Tax, HBZ gene expression, and proviral load (PVL) were assessed using the quantitative real-time PCR (TaqMan), host plasma neopterin level, and HLA-I, and the clinical manifestation were evaluated. ResultsThe HTLV-1 PVLs in HAM/TSP and ACs were 306±360.741 copies/104 PBMCs and 250.98±629.94 copies/104 PBMCs, respectively; the PVL was higher in HAM/TSP than that in ACs (p=0.004). HTLV-1 Tax and HBZ expression in HAM/TSP was higher than that in ACs, wherein only the Tax expression was statistically significant (p=0.039). In contrast to Japanese HTLV-1-infected subjects, HLA-A*02, HLA-A*24, HLA-Cw*08, and HLA-B*5401 did not exhibit preventive effects for HAM/TSP manifestation.The plasma neopterin level was significantly higher in HAM/TSPs than that in ACs; furthermore, there was a strong significant correlation between plasma neopterin and PVL (R=0.76, p=0.001).Moreover, there were significant correlation between urinary disturbances and haematological indices, including the RBC count (R=−0.61, p=0.01) and Hematocrit (Ht) index (R=−0.75, p=0.002), and between mobility disturbances with Tax expression (R=−0.58, p=0.02) and WBC counts (R=−0.54, p=0.04), and finally, a significant association was found between the sensory disturbances and PVL (p=0.05). ConclusionOverall, HTLV-1 PVL and Tax may be the valid predictors of disease development, and the neopterin level may be a valid predictor of disease progression. In addition, Tax and neopterin are more helpful than PVL for the monitoring of HTLV-1-infected patients.

Abstract 3285: HTLV-1 viral oncogene Hbz induces leukemia with osteolytic bone involvement in mice

Abstract Adult T-cell leukemia/lymphoma (ATL) develops in a subset of patients infected with the HTLV-1 virus. Most ATL patients become refractory to chemotherapy and have a median survival time of 6 months. Although uncommon in hematologic malignancies, 80% of ATL patients develop osteolytic lesions and hypercalcemia of malignancy. Bone resident and metastatic tumors release paracrine factors that modulate the bone microenvironment to facilitate disease progression and decrease survival. HTLV-1 encodes 2 viral oncogenes, Tax and Hbz. Tax is critical to ATL development and regulates tumor growth and proliferation in part through trans-activation of NFκB and CREB. We have previously shown Tax expression driven by the Granzymbe B promoter is sufficient for the development of leukemia/lymphoma with osteolytic lesions and hypercalcemia. We and others have shown that Tax alters the expression of paracrine factors that modulate the bone microenvironment through effects on bone forming osteoblasts (OB) and bone resorping osteoclasts (OC). Tax is expressed in early lymphocyte transformation with low expression in advanced ATL. HBZ is expressed early in lymphocyte transformation and throughout ATL progression. We hypothesize that in ATL cells, HTLV-1 viral oncogenes Tax and Hbz cooperate to modulate bone metabolism in a paracrine manner to enhance ATL tumor growth and progression. Mice with Granzyme B driven Hbz expression (T and NK cells) develop leukemia/lymphoproliferative disease in lymph nodes correlating with increased spleen weight. We found that lymphoproliferative disease is also present in the bone marrow. Hbz mice have decreased trabecular bone at 18 months by microCT and radiographic analysis. These data suggest Hbz can alter bone metabolism. Future studies will define the effects of Hbz on bone formation, OB and OC specific effects and tumor progression. Understanding HTLV-1 oncogene modulation of the bone microenvironment will uncover critical pathways in tumor/bone cross talk enabling the development of novel targeted therapies for ATL patients. Citation Format: Alison Esser, Dan Rauch, Jingyu Xiang, John Harding, Nicole Kohart, Patrick Green, Stefan Niewiesk, Thomas Rosol, Lee Ratner, Katherine Weilbaecher. HTLV-1 viral oncogene Hbz induces leukemia with osteolytic bone involvement in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3285.

Absolute quantification of HTLV-1 basic leucine zipper factor (HBZ) protein and its plasma antibody in HTLV-1 infected individuals with different clinical status.

BackgroundHuman T cell leukemia virus type 1 (HTLV-1) basic leucine zipper factor (HBZ), which is encoded by a minus strand mRNA, is thought to play important roles in the development of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). However, a comprehensive analysis of HBZ, including mRNA and protein expression, humoral immunoreactivity against HBZ, and HTLV-1 proviral load (PVL), in HTLV-1-infected individuals with different clinical status has not been reported previously.ResultsIn this study, using novel monoclonal antibody-based in-house enzyme-linked immunosorbent assay systems, we report the absolute quantification of HBZ protein and its plasma antibody in clinical samples from HTLV-1-infected individuals with different clinical status. The data were compared to both HBZ mRNA levels and PVL. The results showed that plasma anti-HBZ antibody was detectable only in 10.4 % (5/48) of asymptomatic carriers (ACs), 10.8 % (13/120) of HAM/TSP patients, and 16.7 % (7/42) of ATL patients. HBZ protein was detected in three out of five patients with acute ATL, but was not detected in patients with HAM/TSP (0/10) or ACs (0/4). Thus, an antibody response to HBZ was not associated with the PVL or the expression of HBZ (both at the mRNA and protein levels) or the clinical status of the infection.ConclusionsThe present results emphasize the extremely low expression and immunogenicity of HBZ in natural HTLV-1 infection. However, there is a possibility that the low but distinct expression of HBZ protein in PBMCs is associated with the survival of HTLV-1-infected cells and the development of ATL.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0263-z) contains supplementary material, which is available to authorized users.

Permissive Sense and Antisense Transcription from the 5' and 3' Long Terminal Repeats of Human T-Cell Leukemia Virus Type 1.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus, and, as such, its genome becomes chromosomally integrated following infection. The resulting provirus contains identical 5' and 3' peripheral long terminal repeats (LTRs) containing bidirectional promoters. Antisense transcription from the 3' LTR regulates expression of a single gene, hbz, while sense transcription from the 5' LTR controls expression of all other viral genes, including tax. Both the HBZ and Tax proteins are implicated in the development of adult T-cell leukemia (ATL), a T-cell malignancy caused by HTLV-1 infection. However, these proteins appear to harbor opposing molecular functions, indicating that they may act independently and at different time points prior to leukemogenesis. Here, we used bidirectional reporter constructs to test whether transcriptional interference serves as a mechanism that inhibits simultaneous expression of Tax and HBZ. We found that sense transcription did not interfere with antisense transcription from the 3' LTR and vice versa, even with strong transcription emanating from the opposing direction. Therefore, bidirectional transcription across the provirus might not restrict hbz or tax expression. Single-cell analyses revealed that antisense transcription predominates in the absence of Tax, which transactivates viral sense transcription. Interestingly, a population of Tax-expressing cells exhibited antisense but not activated sense transcription. Consistent with the ability of Tax to induce cell cycle arrest, this population was arrested in G(0)/G(1) phase. These results imply that cell cycle arrest inhibits Tax-mediated activation of sense transcription without affecting antisense transcription, which may be important for long-term viral latency. The chromosomally integrated form of the retrovirus human T-cell leukemia virus type 1 (HTLV-1) contains identical DNA sequences, known as long terminal repeats (LTRs), at its 5' and 3' ends. The LTRs modulate transcription in both forward (sense) and reverse (antisense) directions. We found that sense transcription from the 5' LTR does not interfere with antisense transcription from the 3' LTR, allowing viral genes encoded on opposite DNA strands to be simultaneously transcribed. Two such genes are tax and hbz, and while they are thought to function at different times during the course of infection to promote leukemogenesis of infected T cells, our results indicate that they can be simultaneously transcribed. We also found that the ability of Tax to induce cell cycle arrest inhibits its fundamental function of activating viral sense transcription but does not affect antisense transcription. This regulatory mechanism may be important for long-term HTLV-1 infection.

Next-Generation Sequencing Reveal Proviral Genome and Transcriptome in Adult T-Cell Leukemia/Lymphoma

Background:Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell neoplasm caused by human T-cell leukemia virus type-1 (HTLV-1) retrovirus infection. As for its pathogenesis, viral products, such as Tax and HBZ, play indispensable roles and their oncogenic mechanisms have been extensively studied. Recently, we have performed an integrated genetic study of a large number of ATL cases and revealed the entire landscape of somatic mutations, copy number alterations, and gene fusions in ATL. However, the detailed analysis of HLTV-1 integration using next-generation sequencing has not been performed so far. In this study, combining whole-genome and RNA sequencing data, we delineated the effect of HTLV-I integration on viral and cellular transcription.Patients and Methods:We performed WGS and RNA-seq for 48 and 57 ATL cases, respectively. All the analyses of the sequencing data were performed using our in-house pipelines. We analyzed HTLV-1 proviral genomic structure and the effect of HTLV-1 integration on viral and cellular transcription.Results:A cardinal feature of ATL genome is HTLV-1 integration, which was precisely located in all the cases analyzed by WGS. Multiple proviral integration sites were detected in 12 cases (total, 62 HTLV-1 integrations sites). The provirus integration was clonal in the architecture inferred from somatic mutations, and apparently randomly integrated into the host genome as previously reported. Within the HTLV-1 genome, frequent 5' proviral segment (gag/pol/env loci) deletions and/or sense gene (gag/pol/env/tax/rex/p13/p30) mutations were observed, which seem to cause defective viral replication/production, whereas HBZ gene was maintained in all the cases. RNA-seq revealed that HTLV-1 integration in ATL cells was associated with aberrant transcription. In general, viral transcripts were predominantly derived from the antisense strand, whereas sense transcription was largely suppressed, leading to global silencing of the sense genes. Especially, in contrast to the ubiquitous HBZ expression (antisense strand), tax expression (sense strand) was almost completely lost in all but one case, which exceptionally exhibited high expression of both tax and HBZ. Strikingly, in most cases, the antisense transcripts were not terminated in 5'-long terminal repeat (LTR), but read through into the juxtaposed cellular genome, extending into up to 50 kb downstream therefrom (read-through transcript). Moreover, in 11 sites of intragenic proviral integration, aberrantly spliced fusion transcripts were observed between LTR and the affected gene, and more commonly associated with antisense (n = 9) than sense (n = 2) integration, accompanied by upregulated cellular gene expression. In other cases (n = 3), fusion transcripts were also generated between HBZ and an exon of highly expressed cellular gene adjacent to the integration site. These results indicate the potential significance of antisense transcription and aberrant fusion transcripts with host genome sequences during ATL development. Although the precise role of these novel aberrant antisense transcripts remains unknown, antisense transcripts containing the LTR region has been implicated in NF-κB activation, which is a hallmark of ATL pathogenesis.Conclusion:In summary, combining WGS and RNA-seq data, we demonstrated the global silencing of sense-oriented viral transcripts (including Tax) and the predominance of aberrant antisense-directed transcription, which often involved cellular gene expression, including aberrant fusion transcripts between host and viral genomes (read-through and aberrantly spliced fusion transcripts). These results suggest that antisense transcription and abnormal virus-host fusion transcripts play pivotal roles in the pathogenesis of ATL. DisclosuresTobinai:Gilead Sciences: Research Funding. Miyazaki:Kyowa-Kirin: Honoraria, Research Funding; Celgene Japan: Honoraria; Sumitomo Dainippon: Honoraria; Chugai: Honoraria, Research Funding; Shin-bio: Honoraria.

Triple Therapy with Prednisolone, Pegylated Interferon and Sodium Valproate Improves Clinical Outcome and Reduces Human T-Cell Leukemia Virus Type 1 (HTLV-1) Proviral Load, Tax and HBZ mRNA Expression in Patients with HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis.

Considering that there is no effective treatment for human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis, this study aimed to assess the impact of triple combination therapy-interferon-α, valproic acid, and prednisolone-on clinical outcomes, main HTLV-1 viral factors, and host anti-HTLV-1 antibody response. HTLV-1 proviral load (PVL), and HBZ and Tax mRNA expression levels were measured in peripheral blood mononuclear cells of 13 patients with HTLV-1-associated myelopathy/tropical spastic paraparesis before and after treatment with 180 μg pegylated interferon once a week, 10-20 mg/kg/day sodium valproate, and 5 mg/day prednisolone for 25 weeks using a TaqMan real-time polymerase chain reaction assay. Furthermore, anti-HTLV-1 titer, Osame Motor Disability Score, Ashworth spasticity scale, and urinary symptoms (through standard questionnaire and clinical monitoring) were assessed in patients before and after the treatment. HTLV-1 PVL and HBZ expression significantly decreased after the treatment [PVL from 1443 ± 282 to 660 ± 137 copies/10(4) peripheral blood mononuclear cells (p = 0.01); and HBZ from 8.0 ± 1.5 to 3.0 ± 0.66 (p < 0.01)]. Tax mRNA expression decreased after the treatment from 2.26 ± 0.45 to 1.44 ± 0.64, but this reduction was not statistically significant (p = 0.10). Furthermore, anti-HTLV-1 titer reduced dramatically after the treatment, from 3123 ± 395 to 815 ± 239 (p < 0.01). Clinical signs and symptoms, according to Osame Motor Disability Score and Ashworth score, improved significantly (both p < 0.01). Urinary symptoms and sensory disturbances with lower back pain were reduced, though not to a statistically significant degree. Although signs and symptoms of spasticity were improved, frequent urination and urinary incontinence were not significantly affected by the triple therapy. The results provide new insight into the complicated conditions underlying HTLV-1-associated diseases.