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Significance HTLV-1 manifests many diseases, which cause morbidity and mortality in 5∼10% of infected individuals, including the fatal adult T cell leukemia/lymphoma (ATLL) and debilitating myelopathy (HAM/TSP). However, the rarity of these diseases had made it prohibitory to conduct large-scale prospective observational studies. This work enabled calculating the standard mortality ratio of HAM/TSP patients and also identified ATLL as one of the major causes of death among these patients. We also identified the features that lead HAM/TSP patients to develop ATLL: having dominant clonal expansion of HTLV-1–infected cells with ATLL-associated somatic mutations. Furthermore, this manuscript describes genomic changes occurring in HAM/TSP patients at the actual time of their ATLL transformation., Human T cell leukemia virus 1 (HTLV-1) causes the functionally debilitating disease HTLV-1–associated myelopathy/tropical spastic paraparesis (HAM/TSP) as well as adult T cell leukemia lymphoma (ATLL). Although there were concerns that the mortality of HAM/TSP could be affected by the development of ATLL, prospective evidence was lacking in this area. In this 5-y prospective cohort study, we determined the mortality, prevalence, and incidence of ATLL in 527 HAM/TSP patients. The standard mortality ratio of HAM/TSP patients was 2.25, and ATLL was one of the major causes of death (5/33 deaths). ATLL prevalence and incidence in these patients were 3.0% and 3.81 per 1,000 person-y, respectively. To identify patients at a high risk of developing ATLL, flow cytometry, Southern blotting, and targeted sequencing data were analyzed in a separate cohort of 218 HAM/TSP patients. In 17% of the HAM/TSP patients, we identified an increase in T cells positive for cell adhesion molecule 1 (CADM1), a marker for ATLL and HTLV-1–infected cells. Genomic analysis revealed that somatic mutations of HTLV-1–infected cells were seen in 90% of these cases and 11% of them had dominant clone and developed ATLL in the longitudinal observation. In this study, we were able to demonstrate the increased mortality in patients with HAM/TSP and a significant effect of ATLL on their prognosis. Having dominant clonal expansion of HTLV-1–infected cells with ATLL-associated somatic mutations may be important characteristics of patients with HAM/TSP who are at an increased risk of developing ATLL.

We recently took advantage of the universal expression of cell adhesion molecule 1 (CADM1) by CD4+ cells infected with HTLV-1 and the downregulation of CD7 expression that corresponds with the oncogenic stage of HTLV-1-infected cells to develop a flow cytometric system using CADM1 versus CD7 plotting of CD4+ cells. We risk-stratified HTLV-1 asymptomatic carriers (AC) and indolent adult T-cell leukemia/lymphoma (ATL) cases based on the CADM1+ percentage, in which HTLV-1-infected clones are efficiently enriched. AC and indolent ATL cases were initially classified according to their CADM1+ cell percentage. Follow-up clinical and flow cytometric data were obtained for 71 cases. In G1 (CADM1+ ≤ 10%) and G2 (10% < CADM1+ ≤ 25%) cases, no apparent clinical disease progression was observed. In G3 (25% < CADM1+ ≤ 50%) cases, five out of nine (55.5%) cases progressed from AC to smoldering-type ATL. In G4 (50% < CADM1+ ) cases, the cumulative incidence of receiving systemic chemotherapy at 3 years was 28.4%. Our results indicate that the percentage of the CD4+ CADM1+ population predicts clinical disease progression: G1 and G2 cases, including AC cases, are stable and considered to be at low risk; G3 cases, including advanced AC cases and smoldering-type ATL cases based on the Shimoyama criteria, are considered to have intermediate risk; and G4 cases, which are mainly indolent ATL cases, are unstable and at high risk of acute transformation.

Human T-cell leukemia virus type 1 (HTLV-1) mainly infects CD4+T-cells and induces chronic, persistent infection in infected individuals with some progressing to develop adult T-cell leukemia/lymphoma (ATL). Whilst HTLV-1 alters cellular differentiation, activation and survival, it is unknown whether and how these changes contribute to malignant transformation of infected T-cells. In this study, we used single-cell RNA-Seq and TCR-Seq to investigate T-cell differentiation and HTLV-1-mediated transformation processes. We analyzed 87,742 single cells from peripheral blood of 12 infected and 3 uninfected individuals. Using multiple independent bioinformatic methods, we demonstrated that naïve T-cells dynamically change into activated T-cells including infected cells, which seamlessly transitioned into ATL cells characterized by clonally expanded, highly-activated T-cells. Notably, the more activated ATL cells are, the more they acquire Treg signatures. Intriguingly, HLA class II genes were uniquely induced in infected cells, further upregulated in ATL cells and was induced by viral protein Tax. Functional assays revealed that by upregulating HLA class II, HTLV-1-infected cells can act as tolerogenic antigen presenting cells (APCs) to induce anergy of antigen specific T-cells. In conclusion, our study revealed the in vivo mechanisms of HTLV-1-mediated transformation and immune escape at single-cell level.Graphical Abstract

Subclonal genetic heterogeneity and their diverse gene expression impose serious problems in understanding the behavior of cancers and contemplating therapeutic strategies. Here we develop and utilize a capture-based sequencing panel, which covers host hotspot genes and the full-length genome of human T-cell leukemia virus type-1 (HTLV-1), to investigate the clonal architecture of adult T-cell leukemia-lymphoma (ATL). For chronologically collected specimens from patients with ATL or pre-onset individuals, we integrate deep DNA sequencing and single-cell RNA sequencing to detect the somatic mutations and virus directly and characterize the transcriptional readouts in respective subclones. Characteristic genomic and transcriptomic patterns are associated with subclonal expansion and switches during the clinical timeline. Multistep mutations in the T-cell receptor (TCR), STAT3, and NOTCH pathways establish clone-specific transcriptomic abnormalities and further accelerate their proliferative potential to develop highly malignant clones, leading to disease onset and progression. Early detection and characterization of newly expanded subclones through the integrative analytical platform will be valuable for the development of an in-depth understanding of this disease., Characterising the clonal architecture of Adult T-cell leukemia-lymphoma (ATL) remains crucial. Here, the authors develop a capture-based sequencing panel and use deep DNA and single cell RNA sequencing and report distinct genomic and transcriptomic features associated with subclonal evolution.

Approximately 10–20 million of Human T-cell leukemia virus type-1 (HTLV-1)-infected carriers have been previously reported, and approximately 5% of these carriers develop adult T-cell leukemia/lymphoma (ATL) with a characteristic poor prognosis. In Japan, Southern blotting has long been routinely performed for detection of clonally expanded ATL cells in vivo, and as a confirmatory diagnostic test for ATL. However, alternative methods to Southern blotting, such as sensitive, quantitative, and rapid analytical methods, are currently required in clinical practice. In this study, we developed a high-throughput method called rapid amplification of integration site (RAIS) that could amplify HTLV-1-integrated fragments within 4 h and detect the integration sites in > 0.16% of infected cells. Furthermore, we established a novel quantification method for HTLV-1 clonality using Sanger sequencing with RAIS products, and the validity of the quantification method was confirmed by comparing it with next-generation sequencing in terms of the clonality. Thus, we believe that RAIS has a high potential for use as an alternative routine molecular confirmatory test for the clonality analysis of HTLV-1-infected cells.

Glypican-1 (GPC1) protein in exosomes was recently identified as a biomarker for the early detection of pancreatic ductal adenocarcinoma (PDAC). Immunohistochemical analyses and in vitro assays were conducted to assess the usefulness of GPC1 as a PDAC biomarker, to reveal the biological role of GPC1 in pancreatic carcinogenesis, and to ascertain the regulation mechanism of GPC1. An aberrant overexpression of GPC1 protein which is usually absent in normal pancreatic duct, was a widespread marker across the full spectrum of human PDAC precursors, PDAC, and pancreatic cancerous stroma. In intraductal papillary-mucinous neoplasms (IPMNs), GPC1 tended to be positive in gastric-type IPMN. KRAS mutations were found in all GPC1-positive IPMN cases and in one-third of GPC1-negative IPMN cases. In pancreatic cell lines, GPC1 depletion caused remarkable inhibition of cell growth and migration, suggesting its oncogenic roles. GPC1 depletion upregulated the molecules associated with cell cycle arrest in pancreatic cell lines. Furthermore, KRAS and ecotropic viral integration site 1 (EVI1) oncoprotein upregulated GPC1 expression. In a clinical cohort, GPC1 overexpression was not correlated with pancreatic cancer prognosis. Taken together, these findings suggest the necessity of establishing a threshold of GPC1 value for detecting pancreatic malignancy because GPC1 is overexpressed even in low-grade PDAC precursors which do not always become malignant. Our study also reveals a new aspect of pancreatic carcinogenesis: KRAS and EVI1, two important molecules in early phases of pancreatic carcinogenesis, positively regulate GPC1 expression and likely promote pancreatic carcinogenesis.

Patients with adult T-cell leukemia (ATL) exhibit a poor prognosis and overall survival rate when treated with standard chemotherapy, highlighting the continued requirement for the development of novel safe and effective therapies for human T-cell leukemia virus type 1 (HTLV-1)-related diseases. In this study, we demonstrated that MK-2048, a second-generation HIV-1 integrase (IN) inhibitor, potently and selectively kills HTLV-1–infected cells. Differential transcriptome profiling revealed significantly elevated levels of gene expression of the unfolded protein response (UPR) PKR-like ER kinase (PERK) signaling pathway in ATL cell lines following MK-2048 treatment. We also identified a significant downregulation in glucose regulated protein 78 (GRP78), a master regulator of the UPR in the CD4+CADM1+ HTLV-1–infected cell population of primary HTLV-1 carrier peripheral blood mononuclear cells (PBMCs) (n = 9), suggesting that HTLV-1–infected cells are hypersensitive to endoplasmic reticulum (ER) stress-mediated apoptosis. MK-2048 efficiently reduced proviral loads in primary HTLV-1 carrier PBMCs (n = 4), but had no effect on the total numbers of these cells, indicating that MK-2048 does not affect the proliferation of HTLV-1–uninfected PBMCs. MK-2048 specifically activated the ER stress–related proapoptotic gene, DNA damage-inducible transcript 3 protein (DDIT3), also known as C/EBP homologous protein (CHOP), in HTLV-1–infected but not uninfected cells of HTLV-1–carrier PBMCs. Our findings demonstrated that MK-2048 selectively induces HTLV-1–infected cell apoptosis via the activation of the UPR. This novel regulatory mechanism of the HIV IN inhibitor MK-2048 in HTLV-1–infected cells provides a promising prophylactic and therapeutic target for HTLV-1–related diseases including ATL.

The retrovirus HTLV-1 integrates into the host cellular DNA, achieves persistent infection, and induces human diseases. Here we demonstrate that viral DNA-capture-seq is useful to characterize HTLV-1 provirus in naturally virus-infected individuals, providing comprehensive information about the proviral structure and the viral integration site. We analyzed peripheral blood from 98 naturally HTLV-1-infected individuals and found that defective proviruses were present not only in patients with leukemia, but also in those with other clinical entities. We further demonstrated that infected clones with defective-type exhibited higher degree of clonal abundance than those with full-length type. The frequency of defective-type in HTLV-1-infected humanized mice was lower than that in infected individuals, indicating that defective proviruses were rare at the initial phase of infection but preferentially selected during persistent infection. These results demonstrate robustness of viral DNA-capture-seq for HTLV-1 infection and shed light on its potential application for other virus-associated cancers in human.

Extensive studies on metastasis-associated proteins, S100A4 and MTA1, have been carried out for over two decades, but correlation of both proteins remains obscure. Here we show evidence for the correlation in angiogenesis. First, silencing of each protein by siRNA-mediated knockdown in mouse endothelial MSS31 cells resulted in the inhibition of tube formation. Unexpectedly, the knockdown of MTA1 affected not only its own expression but also the expression of S100A4, whereas silencing of S100A4 did not affect the MTA1 expression. Additionally, non-muscle myosin IIA (NMIIA) phosphorylation, which was partly controlled by S100A4, was found to be upregulated by knockdown of both proteins in MSS31 cells. Moreover, cycloheximide treatment of MSS31 cells revealed that the rate of S100A4 degradation was accelerated by MTA1 knockdown. This finding, together with our observation that cytoplasmic MTA1, but not nuclear MTA1, was colocalized with S100A4, suggested the involvement of MTA1 in S100A4 stability. The direct in vivo angiogenesis assay showed that both protein siRNAs provoked a significant inhibition of new blood vessel formation induced by angiogenic factors, indicating their anti-angiogenic activities. Treatment of human pancreatic tumor (PANC-1) xenograft in mice with mMTA1 siRNA resulted in tumor regression via suppression of angiogenesis in vivo, as also observed in the case of human prostate cancer xenograft treated with mS100A4 siRNA. Taken together, these data led us to conclude that the MTA1-S100A4-NMIIA axis exists in endothelial cells as a novel pathway in promoting tumor vascular formation and could be a target for suppressing tumor growth and metastasis.

Human T-cell leukemia virus type 1 (HTLV-1), the first reported human oncogenic retrovirus, is the etiologic agent of highly aggressive, currently incurable diseases such as adult T-cell leukemia–lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 proteins, including Tax and HBZ, have been shown to have critical roles in HTLV-1 pathogenicity, yet the underlying mechanisms of HTLV-1-driven leukemogenesis are unclear. The frequent disruption of genetic and epigenetic gene regulation in various types of malignancy, including ATL, is evident. In this review, we illustrate a focused range of topics about the establishment of HTLV-1 memory: (1) genetic lesion in the Tax interactome pathway, (2) gene regulatory loop/switch, (3) disordered chromatin regulation, (4) epigenetic lock by the modulation of epigenetic factors, (5) the loss of gene fine-tuner microRNA, and (6) the alteration of chromatin regulation by HTLV-1 integration. We discuss the persistent influence of Tax-dependent epigenetic changes even after the disappearance of HTLV-1 gene expression due to the viral escape from the immune system, which is a remaining challenge in HTLV-1 research. The summarized evidence and conceptualized description may provide a better understanding of HTLV-1-mediated cellular transformation and the potential therapeutic strategies to combat HTLV-1-associated diseases.

Homeostatic proliferation (HSP) is a major mechanism by which long-lived naïve and memory CD4+ T cells are maintained in vivo and suggested to contribute to the persistence of the latent HIV-1 reservoir. However, while many in vitro latency models rely on CD4+ T cells that were initially differentiated via T-cell receptor (TCR) stimulation into memory/effector cells, latent infection of naïve resting CD4+ T cells maintained under HSP conditions has not been fully addressed. Here, we describe an in vitro HSP culture system utilizing the cytokines IL-7 and IL-15 that allows studying latency in naïve resting CD4+ T cells. CD4+ T cells isolated from several healthy donors were infected with HIV pseudotypes expressing GFP and cultured under HSP conditions or TCR conditions as control. Cell proliferation, phenotype, and GFP expression were analyzed by flow cytometry. RNA expression was quantified by qRT-PCR. Under HSP culture conditions, latently HIV-1 infected naïve cells are in part maintained in the non-dividing (= resting) state. Although a few HIV-1 provirus+ cells were present in these resting GFP negative cells, the estimated level of GFP transcripts per infected cell seems to indicate a block at the post-transcriptional level. Interestingly, neither TCR nor the prototypic HDAC inhibitor SAHA were able to reactivate HIV-1 provirus from these cells. This lack of reactivation was not due to methylation of the HIV LTR. These results point to a mechanism of HIV control in HSP-cultured resting naïve CD4+ T cells that may be distinct from that in TCR-stimulated memory/effector T cells. This work was supported by Grants from the Ministry of Health, Labor and Welfare in Japan for AIDS Research and from the Japan Agency for Medical Research and Development, AMED (YT-Y). JM and AM were funded by a grant from the Spanish Ministry of Economy and Competitiveness and FEDER (Grant no. SAF2013-46077-R).

The retrovirus human T-cell leukemia virus type 1 (HTLV-1) integrates into the host genome and persists for the lifetime of the host. There are tens of thousands of different infected clones in a HTLV-1 carrier and each clone can be identified by its unique viral integration site. Only about 5% of infected people develop the hematological malignancy, adult T-cell leukemia-lymphoma (ATL). However, it is unclear how a certain infected clone, among various different ones, is selected as a malignant clone. It has been reported that viral integration alters transcripts of the cellular host genes adjacent to the integration site, even generating truncated or virus-host chimeric transcripts. Because each infected clone has a unique viral integration site, each clone possibly has unique virus-host chimeric transcripts, which were not present in the host before infection. Therefore, we hypothesized that the integrated provirus generates virus-host chimeric transcripts that may play a role in the clonal selection of the HTLV-1-infected cell. We previously reported HTLV-1 DNA-capture-seq using biotinylated DNA-probes for the viral genome, to increase the sensitivity and efficiency of viral-sequences detection. In this study, we used HTLV-1 RNA-capture-seq for PBMCs samples from ATL patients to test the hypothesis in a highly sensitive manner. The results showed the presence of chimeric transcripts in 19 out of 30 ATL patients. We next quantified the abundance of chimeric transcripts by droplet digital PCR, and found that the expression levels of chimeric transcripts were similar to those of viral RNAs containing splice junction of HBZ, in 5 of 19 chimeric transcripts positive ATL cases, although the levels varied among different ATL cases. To identify the whole sequences of the chimeric transcripts, we performed Oxford Nanopore sequencing. This approach revealed that the HTLV-1 provirus generates various splicing chimeric transcripts with the host genes in both viral sense and antisense orientations. The transcriptional start site of most of the sense chimeric transcripts was the R region of the 5'- or 3'-long terminal repeats (LTRs) in the proviral sequences, indicating that the chimeric transcripts were generated using the viral promoters because the LTRs work as a promoter for the viral transcripts. Given the structure of the chimeric transcripts with the viral promotors, the expression of the fused host genes could be enhanced by generating the chimeric transcripts. We evaluated the mRNA expression of the fused host genes of the chimeric transcripts by RNA-seq, and the results correlated with those obtained by ddPCR. To clarify the impact of viral integration on the clonal expansion, we analyzed HTLV-1-infected Jurkat cells. The clonality analysis of infected cells by HTLV-1 DNA-capture-seq showed that some infected clones were remarkably expanded for 4-6 months culture. We also confirmed that some of them harbored virus-host chimeric transcripts by HTLV-1 RNA-capture-seq. This study revealed the expression levels and the structures of virus-host chimeric transcripts in ATL patients. We are currently investigating the functional role of chimeric transcripts in the clonal proliferation of infected cells in vitro. Disclosures Uchimaru: Daiichi Sankyo Co., Ltd..: Research Funding. Kimura:Novartis: Honoraria, Research Funding; Ohara Pharmaceutical Co.: Research Funding.

Nonsense-mediated mRNA decay (NMD) is an essential and conserved cellular mRNA quality control mechanism. RNA signals to express viral genes from overlapping open reading frames potentially initiate NMD, nevertheless it is not clear whether viral RNAs are sensitive to NMD or if viruses have evolved mechanisms to evade NMD. Here we demonstrate that the genomic and full-length mRNAs of Human-T-cell Leukemia Virus type-I (HTLV-1), a retrovirus responsible for Adult T-cell Leukemia (ATL), are sensitive to NMD. They exhibit accelerated turnover in NMD-activated cells, while siRNA-mediated knockdown of NMD-master-regulator, UPF1, promotes enhanced stability of them. These effects on RNA stability were recapitulated by a reporter construct encoding the HTLV-1 translational frameshift signal of gag-pol. In agreement with the RNA stability, viral protein expression from the integrated provirus was inversely correlated with cellular NMD activity. We further demonstrated that the viral RNA-binding protein, Rex, approves the stability of viral RNA by inhibiting NMD. Significantly, Rex establishes a general block to NMD, as both NMD-responsive reporter transcripts and natural host-encoded NMD substrates were stabilized in the presence of Rex. Thus, we suggest that Rex not only stabilizes viral transcripts, but also perturbs cellular mRNA metabolism and host cell homeostasis via inhibition of NMD.

Molecular abnormalities involved in the multistep leukemogenesis of adult T-cell leukemia (ATL) remain to be clarified. Based on our integrated database, we focused on the expression patterns and levels of Ikaros family genes, Ikaros, Helios, and Aiolos, in ATL patients and HTLV-1 carriers. The results revealed profound deregulation of Helios expression, a pivotal regulator in the control of T-cell differentiation and activation. The majority of ATL samples (32/37 cases) showed abnormal splicing of Helios expression, and four cases did not express Helios. In addition, novel genomic loss in Helios locus was observed in 17/168 cases. We identified four ATL-specific short Helios isoforms and revealed their dominant-negative function. Ectopic expression of ATL-type Helios isoform as well as knockdown of normal Helios or Ikaros promoted T-cell growth. Global mRNA profiling and pathway analysis showed activation of several signaling pathways important for lymphocyte proliferation and survival. These data provide new insights into the molecular involvement of Helios function in the leukemogenesis and phenotype of ATL cells, indicating that Helios deregulation is one of the novel molecular hallmarks of ATL.

Although human T cell leukemia virus type I (HTLV-I) is undoubtedly involved in the immortalization and leukemogenesis of infected cells, mechanistic underpinnings of its molecular pathophysiology in long latent period of Adult T-cell leukemia (ATL) remain to be elucidated. One of the most significant recent advances in biomedical research has been the discovery of small noncoding RNAs designated microRNA (miRNA), which affect the field of virology including HTLV-1 research. Mounting evidence indicates that viruses use these miRNAs to manipulate both cellular and viral gene expression. Viral infection also can exert a profound impact on the cellular miRNA expression profile. Some studies have demonstrated that some deregulations of miRNA are involved in the pathogenesis of HTLV-1. Furthermore, global analyses of ATL patient samples have provided a conceptual progress that Polycomb family induces miR-31 silencing, resulting in overexpression of NF- kappaB inducing kinase (NIK) following NF-kappaB activation. Given that miRNAs act as pleiotropic molecules essential in all cellular events, deregulation of miRNA signature caused by HTLV-1 infection strongly involves the imbalance of molecular network of lymphocytes. Recognition and understanding of the widespread molecular applicability of miRNAs will increasingly have much effect on the development of novel strategies to treat the HTLV-1-associated diseases. Here we discuss our current knowledge of viral miRNAs and virally influenced cellular miRNAs and their relationship to ATL.

Biological robustness is exposed to stochastic perturbations, which should be controlled by intrinsic mechanisms; the promiscuous signaling network without appropriate alleviation is the true nature of cancer cells. B cell receptor (BCR) signaling is a major source of gene expression signature important for B cell. It is still unclear the mechanism by which the expression of functionally important genes is continuously deregulated in malignant lymphomas. Using RISC-capture assay, we reveal that multiple BCR signaling factors are persistently regulated by microRNA (miRNA) in human B cells. Clinical samples from patients with diffuse large B-cell lymphoma (DLBCL, n = 83) show loss of an essential miRNA set (miR-200c, miR-203, miR-31). Conventional screening and RISC profiling identify multiple targets (CD79B, SYK, PKCβII, PLCγ1, IKKβ, NIK, MYD88, PI3K class I (α/β/δ/γ), RasGRP3); signaling network habitually faces interference composed by miRNA group in normal B cells. We demonstrate that simultaneous depletion of the key miRNAs enhances translation of the multiple targets and causes chronic activation of NF-κB, PI3K-Akt and Ras-Erk cascades, leading to B cell transformation. This study suggests that compensatory actions by multiple miRNAs rather than by a single miRNA ensure robustness of biological processes.

HTLV-1 Tax deregulates signal transduction pathways, transcription of genes, and cell cycle regulation of host cells, which is mainly mediated by its protein-protein interactions with host cellular factors. We previously reported an interaction of Tax with a histone methyltransferase (HMTase), SUV39H1. As the interaction was mediated by the SUV39H1 SET domain that is shared among HMTases, we examined the possibility of Tax interaction with another HMTase, SMYD3, which methylates histone H3 lysine 4 and activates transcription of genes, and studied the functional effects. Expression of endogenous SMYD3 in T cell lines and primary T cells was confirmed by immunoblotting analysis. Co-immuno-precipitaion assays and in vitro pull-down assay indicated interaction between Tax and SMYD3. The interaction was largely dependent on the C-terminal 180 amino acids of SMYD3, whereas the interacting domain of Tax was not clearly defined, although the N-terminal 108 amino acids were dispensable for the interaction. In the cotransfected cells, colocalization of Tax and SMYD3 was indicated in the cytoplasm or nuclei. Studies using mutants of Tax and SMYD3 suggested that SMYD3 dominates the subcellular localization of Tax. Reporter gene assays showed that nuclear factor-κB activation promoted by cytoplasmic Tax was enhanced by the presence of SMYD3, and attenuated by shRNA-mediated knockdown of SMYD3, suggesting an increased level of Tax localization in the cytoplasm by SMYD3. Our study revealed for the first time Tax-SMYD3 direct interaction, as well as apparent tethering of Tax by SMYD3, influencing the subcellular localization of Tax. Results suggested that SMYD3-mediated nucleocytoplasmic shuttling of Tax provides one base for the pleiotropic effects of Tax, which are mediated by the interaction of cellular proteins localized in the cytoplasm or nucleus.

Previous reports indicate that nuclear factor (NF)-kappaB regulates induction of human immunodeficiency virus type 1 (HIV-1) gene expression in latently infected cells. However, the role of NF-kappaB in cells with active HIV-1 replication is not well understood. In this study, we examined the effect of a new NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on HIV-1 replication in a human T cell line and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PHA-PBMCs). We further explored the mechanism of DHMEQ-mediated inhibition of HIV-1 replication. DHMEQ inhibited HIV-1 replication in HIV-1-infected Molt-4 and PHA-PBMCs. DHMEQ inhibited constitutive NF-kappaB activity in HIV-1-infected PHA-PBMCs and HIV long terminal repeat promoter activity driven by tumor necrosis factor (TNF)-alpha and the trans-activator Tat. The single-round assay using vesicular stomatitis virus-pseudotyped virus in the human T cell line M8166 indicated that DHMEQ treatment resulted in decreased integration of HIV-1 provirus into the host genome and decreased HIV-1 expression. These results indicate that NF-kappaB regulates early events as well as the initial and accelerated expression of HIV-1 in its life cycle. Therefore, we conclude that NF-kappaB is a molecular target for controlling active HIV-1 replication.

Quantitative PCR (qPCR) analysis of human T-cell leukemia virus type 1 (HTLV-1) was used to assess the amount of HTLV-1 provirus DNA integrated into the genomic DNA of host blood cells. Accumulating evidence indicates that a high proviral load is one of the risk factors for the development of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, interlaboratory variability in qPCR results makes it difficult to assess the differences in reported proviral loads between laboratories. To remedy this situation, we attempted to minimize discrepancies between laboratories through standardization of HTLV-1 qPCR in a collaborative study. TL-Om1 cells that harbor the HTLV-1 provirus were serially diluted with peripheral blood mononuclear cells to prepare a candidate standard. By statistically evaluating the proviral loads of the standard and those determined using in-house qPCR methods at each laboratory, we determined the relative ratios of the measured values in the laboratories to the theoretical values of the TL-Om1 standard. The relative ratios of the laboratories ranged from 0.84 to 4.45. Next, we corrected the proviral loads of the clinical samples from HTLV-1 carriers using the relative ratio. As expected, the overall differences between the laboratories were reduced by half, from 7.4-fold to 3.8-fold on average, after applying the correction. HTLV-1 qPCR can be standardized using TL-Om1 cells as a standard and by determining the relative ratio of the measured to the theoretical standard values in each laboratory.

Purpose: Cell adhesion molecule 1 (CADM1), initially identified as a tumor suppressor gene, has recently been reported to be ectopically expressed in primary adult T-cell leukemia–lymphoma (ATL) cells. We incorporated CADM1 into flow-cytometric analysis to reveal oncogenic mechanisms in human T-cell lymphotrophic virus type I (HTLV-I) infection by purifying cells from the intermediate stages of ATL development. Experimental Design: We isolated CADM1- and CD7-expressing peripheral blood mononuclear cells of asymptomatic carriers and ATLs using multicolor flow cytometry. Fluorescence-activated cell sorted (FACS) subpopulations were subjected to clonal expansion and gene expression analysis. Results: HTLV-I–infected cells were efficiently enriched in CADM1+ subpopulations (D, CADM1posCD7dim and N, CADM1posCD7neg). Clonally expanding cells were detected exclusively in these subpopulations in asymptomatic carriers with high proviral load, suggesting that the appearance of D and N could be a surrogate marker of progression from asymptomatic carrier to early ATL. Further disease progression was accompanied by an increase in N with a reciprocal decrease in D, indicating clonal evolution from D to N. The gene expression profiles of D and N in asymptomatic carriers showed similarities to those of indolent ATLs, suggesting that these subpopulations represent premalignant cells. This is further supported by the molecular hallmarks of ATL, that is, drastic downregulation of miR-31 and upregulation of abnormal Helios transcripts. Conclusion: The CADM1 versus CD7 plot accurately reflects disease progression in HTLV-I infection, and CADM1+ cells with downregulated CD7 in asymptomatic carriers have common properties with those in indolent ATLs. Clin Cancer Res; 20(11); 2851–61. ©2014 AACR.

Epigenetic program is a molecular basis of cellular identity and functions. We have shown the transcriptome (Cancer Cell, 2012) and the underlying histone methylation patterns (Blood, 2016) of adult T-cell leukemia-lymphoma (ATL). The epigenetic landscape of ATL is defined as "genome-wide H3K27me3 accumulation". Similar accumulation has been detected in multiple cancers, particularly in hematological malignancies. Relief of the cumulative methylation may restore the aberrant transcriptome to ideal expression signature, leading to a promising treatment. Due to variation characteristics, EZH2 is recognized as a druggable target. However, extrinsic regulation of methylation pattern is still challenging without knowledge of molecule(s) and their combination that needs to be targeted to reprogram the epigenome in anticipation of synthetic lethality. To redefine the conventional concept, we first examined expression and global occupancy patterns of H3K27 methyltransferases EZH1 and EZH2. EZH2 expression was low in CD4+ T cells and significantly high in ATL. In contrast, basal EZH1 was very high in mature lymphocytes compared with undifferentiated lineages and also high in ATL. ChIP-on-chip analysis revealed significant changes in distributions of both EZH1 and EZH2 in primary ATL cells, which caused abnormal H3K27me3 accumulation. Importantly, over 80% of H3K27me3 are specifically induced by either EZH1 (28.9%) or EZH2 (14.4%), or by both (39.3%). Moreover, they have differential functions by regulating specific targets; EZH2 suppresses various transcription regulators, establishing complex gene regulatory network. EZH1 would rather directly induce H3K27me3 at genes involving functional processes such as lymphocyte activation. We detected EZH1 and EZH2 in functional polycomb repressive complex 2 (PRC2) in ATL and DLBCL cells. Unexpectedly, EZH1/2 were co-localized with H3K4me3 at a small population of the lymphoma genome. The "EZH1/2 + H3K4me3" non-canonical targets such as cell cycle activators were highly expressed in ATL. We next compared functions of EZH1/2. Single depletion of EZH1 or EZH2 suppressed T- and B-lymphoma cell proliferation, which supports their unique functions. The broad reduction of H3K27me3 was achieved by EZH2 inhibition; however, genome-wide ChIP demonstrated that EZH2 inhibition or depletion triggered strong compensatory action of EZH1, followed by rebound accumulation of H3K27me3 at many functional genes in EZH1+ lymphoma cells. We found that a transcription factor YY1 affects PRC2 recruitment and EZH1 compensation after EZH2 inhibition. Double depletion of EZH1+EZH2 or YY1+EZH2 surmounted the epigenetic homeostasis. In addition, depletion of EZH1 significantly enhanced effects of EZH2 inhibitors in ATL and DLBCL cells (5.47~205-fold). These data indicate requirements for the aberrant epigenome, namely, fluctuating EZH2 (upregulation or mutation) and underlying stable EZH1. We have succeeded in developing an unprecedented EZH1/2 dual-inhibitor (IC50 9.0 nM (EZH1); 10 nM (EZH2)). The new drug successfully inhibited genome-wide EZH1/2 occupancies without compensation, resulting in effective H3K27me3 reduction and gene reactivation compared with the single inhibitor. Of note, the dual-inhibitor suppressed expression of the non-canonical EZH1/2 + H3K4me3 target genes. Besides H3K27me3 reprogramming, EZH1/2 inhibition could modulate genes through H3K4me3 regulation and indirect influence on the secondary regulatory network and signaling pathways such as NF-kB. The EZH1/2 dual-inhibitor significantly blocked primary ATL cell survival and in vivo tumor growth of ATL and DLBCL models. Furthermore, we evaluated drug efficacy in various models of hematological malignancies including ATL, DLBCL (EZH2 WT and mutant), Burkitt lymphoma, and PTCL, which are all originated from EZH1+ differentiated lymphocytes. All of them showed higher sensitivity against the EZH1/2 inhibitor compared with the EZH2 solo inhibitor (5.2~200-fold). We did not detect strong adverse effects in normal T cells. These integrated molecular and phenotypic analyses provide a novel concept "EZH1+EZH2 dual-targeting" and demonstrate validity of the new drug in the treatment of malignant lymphomas. Taking these results, a Phase I clinical trial against T- and B-cell non-Hodgkin lymphomas including ATL is now under way in Japan. Disclosures Yamagishi: Daiichi Sankyo Co., Ltd.: Research Funding. Honma:Daiichi Sankyo Co., Ltd.: Employment. Adachi:Daiichi Sankyo Co., Ltd.: Employment. Ohsugi:Daiichi Sankyo Co., Ltd.: Research Funding. Utsunomiya:Daiichi Sankyo Co., Ltd.: Speakers Bureau. Tsukasaki:Daiichi Sankyo Co., Ltd.: Consultancy; Takeda: Research Funding. Tobinai:Zenyaku Kogyo: Honoraria; Takeda: Honoraria, Research Funding; Mundipharma KK: Honoraria, Research Funding; Kyowa Hakko Kirin: Research Funding; Celgene: Research Funding; Daiichi Sankyo Co., Ltd.: Consultancy; SERVIER: Research Funding; Ono Pharmaceutical: Research Funding; Eisai: Honoraria, Research Funding; Chugai Pharma: Research Funding; Abbvie: Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; HUYA Bioscience: Honoraria. Araki:Daiichi Sankyo Co., Ltd.: Employment.

Clinical results achieved in 100 cases of aortic valve replacement with the Omniscience (O-S) valve during the period from 1980 to 1985 as well as 100 cases of aortic valve replacement with the Omnicarbon (O-C) valve during the period from 1985 to 1989 were studied. Concomitant surgical procedures including mitral valve replacement were performed in 63 patients in the O-S group and 67 patients in the O-C group. Cumulative follow-up in the two groups was carried out for a total of 559 and 273 patient-years, respectively. The overall 4-year actuarial survival rate was 82% +/- 3.8% in the O-S group and 89.5% +/- 3.2% in the O-C group, the corresponding rates for patients undergoing isolated aortic valve replacement being 82.9% +/- 4.2% in the O-S group and 91.9% +/- 3.5% in the O-C group. The overall 4-year actuarial event-free rate with respect to thromboembolic complications was 88.8% +/- 3.3% in the O-S group and 94.4% +/- 2.8% in the O-C group, as compared with the corresponding rates of 89.2% +/- 3.6% in the O-S group and 95.9% +/- 2.8% in the O-C group for patients undergoing isolated aortic valve replacement. The overall rate of valve-related complications, including thromboembolism, anticoagulant-related hemorrhage, perivalvular leak, infection, and structural failure, was 78.8% +/- 4.2% in the O-S group and 89.3% +/- 3.5% in the O-C group (p less than 0.05), and for isolated aortic valve replacement, 79.7% +/- 4.5% in the O-S group and 89.6% +/- 4.1% in the O-C group.(ABSTRACT TRUNCATED AT 250 WORDS)