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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.

Although global H3K27me3 reprogramming is a hallmark of cancer, no effective therapeutic strategy for H3K27me3-high malignancies harboring EZH2(WT/WT) has yet been established. We explore epigenome and transcriptome in EZH2(WT/WT) and EZH2(WT/Mu) aggressive lymphomas and show that mutual interference and compensatory function of co-expressed EZH1 and EZH2 rearrange their own genome-wide distribution, thereby establishing restricted chromatin and gene expression signatures. Direct comparison of leading compounds introduces potency and a mechanism of action of the EZH1/2 dual inhibitor (valemetostat). The synthetic lethality is observed in all lymphoma models and primary adult T cell leukemia-lymphoma (ATL) cells. Opposing actions of EZH1/2-polycomb and SWI/SNF complexes are required for facultative heterochromatin formation. Inactivation of chromatin-associated genes (ARID1A, SMARCA4/BRG1, SMARCB1/SNF5, KDM6A/UTX, BAP1, KMT2D/MLL2) and oncovirus infection (HTLV-1, EBV) trigger EZH1/2 perturbation and H3K27me3 deposition. Our study provides the mechanism-based rationale for chemical dual targeting of EZH1/2 in cancer epigenome., 論文

Both natural viral infections and therapeutic interventions using viral vectors pose significant risks of malignant transformation. Monitoring for clonal expansion of infected cells is important for detecting cancer. Here we developed a novel method of tracking transgene integration sites. RAISING (Rapid Amplification of Integration Sites without Interference by Genomic DNA contamination) is a sensitive, inexpensive alternative to established methods. Its compatibility with Sanger sequencing combined with our CLOVA (Clonality Value) software is critical for those without access to expensive next-generation sequencing. To model our method, we analyzed samples from 698 patients infected with the retrovirus HTLV-1, which causes adult T-cell leukemia/lymphoma (ATL). We defined a clonality value identifying ATL patients with 100% sensitivity and 95.3% specificity, and our preliminary longitudinal analysis suggests this may also be useful for ATL risk assessment. We anticipate future studies will confirm the broad applicability of our technology, especially in the emerging gene therapy sector.

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.

Latently infected T lymphocytes are an important barrier toward eliminating a persistent HIV infection. Here we describe an HIV-based recombinant fluorescent-lentivirus referred to as “rfl-HIV” that enables to analyze sense and antisense transcription by means of fluorescence reporter genes. This model virus exhibited similar transcriptional and functional properties of the antisense transcript as observed with a wild type HIV, and largely facilitated the generation of latently-infected T cells clones. We show that latently-infected cells can be divided into two types, those with and those without antisense transcription. Upon addition of latency reversal agents, only the cells that lack antisense transcripts are readily reactivated to transcribe HIV. Thus, antisense transcripts may exhibit a dominant suppressor activity and can lock an integrated provirus into a non-reactivatable state. These findings could have important implications for the development of strategies to eradicate HIV from infected individuals. This work was supported by grants from Japan Society for the Promotion of Science (JSPS KAKENHI #15H06877 for MK-I, #JP17K08800 for KT), ViiV Healthcare Japan Research Grant 2015 (MK-I), Grants-in-Aid from the Ministry of Health, Labour and Welfare (H24-AIDS-008 to YT-Y) and Japan Agency for Medical Research and Development (AMED #JP17fk0410305h0103 to YT-Y and #JP18fk0410003 to KT). MK-I received Fellowships from Japan Foundation for AIDS Prevention and JSPS Oversea Research Fellow Program. AM and JM were supported by a grant from the Spanish Ministry of Economy, Industry and Competitiveness and FEDER grant no. SAF2016-75505-R (AEI/MINEICO/FEDER, UE) and through the “María de Maeztu” Program for Units of Excellence in R&D (MDM-2014-0370).

The present review introduces recent outstanding progress pertaining to Enhancer of zeste homolog 2 (EZH2), especially regarding its mode of action as a master regulator of chromatin, and provides molecular-based evidence for targeting EZH2 in cancer therapy. We discuss the active development of small molecules targeting the enzymatic activity of EZH2/polycomb repressive complex 2 (PRC2).Genetic, transcriptional, and posttranscriptional dysregulation of EZH2 is frequently observed in many cancer types. EZH2 promotes tumorigenesis by altering the expression of numerous tumor suppressor genes. Furthermore, the executive molecular processes initiated by EZH2, such as NF-κB activation, microRNA silencing, tumor immune evasion, and noncanonical transcription regulation, appear to be the fundamental characteristics of each cancer. Systematic investigations have suggested coordinated regulation of the cancer epigenome wherein antagonistic complexes of both polycomb and SWI/SNF are involved. Frequent loss-of-function mutations in epigenetic factors, such as ARID1A, SMARCA4, SMARCB1, BAP1, and KDM6A, are likely to elicit the EZH2/PRC2-addicted situation. Our comprehensive understanding encourages the development of advanced strategies for the appropriate manipulation of the cancer epigenome. Moreover, a couple of small molecules that can effectively inhibit the enzymatic activity of EZH2/PRC2 have been translated into early-phase clinical trials.The EZH2-mediated epigenome and subsequent transcriptome define cellular identity. Effective and specific strategies for the manipulation of EZH2/PRC2 may lead to the development of more precise cancer medicines.

One of the hallmarks of cancer, global gene expression alteration, is closely associated with the development and malignant characteristics associated with adult T-cell leukemia (ATL) as well as other cancers. Here, we show that aberrant overexpression of the Ellis Van Creveld (EVC) family is responsible for cellular Hedgehog (HH) activation, which provides the pro-survival ability of ATL cells. Using microarray, quantitative RT-PCR and immunohistochemistry we have demonstrated that EVC is significantly upregulated in ATL and human T-cell leukemia virus type I (HTLV-1)-infected cells. Epigenetic marks, including histone H3 acetylation and Lys4 trimethylation, are specifically accumulated at the EVC locus in ATL samples. The HTLV-1 Tax participates in the coordination of EVC expression in an epigenetic fashion. The treatment of shRNA targeting EVC, as well as the transcription factors for HH signaling, diminishes the HH activation and leads to apoptotic death in ATL cell lines. We also showed that a HH signaling inhibitor, GANT61, induces strong apoptosis in the established ATL cell lines and patient-derived primary ATL cells. Therefore, our data indicate that HH activation is involved in the regulation of leukemic cell survival. The epigenetically deregulated EVC appears to play an important role for HH activation. The possible use of EVC as a specific cell marker and a novel drug target for HTLV-1-infected T-cells is implicated by these findings. The HH inhibitors are suggested as drug candidates for ATL therapy. Our findings also suggest chromatin rearrangement associated with active histone markers in ATL.

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.

SummaryConstitutive NF-κB activation has causative roles in adult T cell leukemia (ATL) caused by HTLV-1 and other cancers. Here, we report a pathway involving Polycomb-mediated miRNA silencing and NF-κB activation. We determine the miRNA signatures and reveal miR-31 loss in primary ATL cells. MiR-31 negatively regulates the noncanonical NF-κB pathway by targeting NF-κB inducing kinase (NIK). Loss of miR-31 therefore triggers oncogenic signaling. In ATL cells, miR-31 level is epigenetically regulated, and aberrant upregulation of Polycomb proteins contribute to miR-31 downregulation in an epigenetic fashion, leading to activation of NF-κB and apoptosis resistance. Furthermore, this emerging circuit operates in other cancers and receptor-initiated NF-κB cascade. Our findings provide a perspective involving the epigenetic program, inflammatory responses, and oncogenic signaling.

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.

We have previously reported induction of transcriptional gene silencing (TGS) of HIV-1 by short hairpin RNA (shRNA) expressed in MOLT-4 cells. The shRNA (termed shPromA) targets the highly conserved tandem NF-kB binding sequences of the HIV-1 promoter. Recent articles have reported that TGS mediated by promoter-targeted siRNAs was exclusively the result of sequence non-specific off-target effects. Specifically, several mismatched siRNAs to the target promoter sequences were reported to also induce significant TGS, suggesting TGS was a consequence of off-target effects. Here, following extensive investigation, we report that shPromA induces sequence specific transcriptional silencing in HIV-1 infection in MOLT4 cells, while four shRNA variants, mismatched by 2-3 nucleotides, fail to suppress viral replication. We confirm similar levels of shRNA expression from the U6 promoter and the presence of processed/cleaved siRNAs for each construct in transduced MOLT-4 cells. HIV-1 sequence specific shPromA does not suppress HIV-2, which has an alternate NF-kB binding sequence. As a result of the unique sequence targeted, shPromA does not induce down-regulation of other NF-kB driven genes, either at the mRNA or protein level. Furthermore, we confirmed shPromA does not have sequence non-specific off-target effects through unaltered expression of CD4, CXCR4, and CCR5, which are used for viral entry. Additionally, shPromA does not alter PKR, IFN levels, and three downstream mediators of IFN-a response genes. Our data clearly shows that shPromA achieved highly specific TGS of HIV-1, demonstrating that effective TGS can be induced with minimal off-target effects.

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.

Adult T-cell leukemia-lymphoma (ATL) shows global gene expression alterations that confer cellular characteristics and unfavorable prognosis. However, molecular mechanisms of the sustained expression changes are largely unknown, because there is no study addressing the relationship between landscapes of the gene expression and epigenetic modifications. Here, we analyzed ATL epigenome and integrated it with transcriptome from primary ATL cells and those from corresponding normal CD4(+)T cells to decipher ATL-specific "epigenetic code" that was critical for cell identity. We found that polycomb-repressive complex 2 (PRC2)-mediated trimethylation at histone H3Lys27 (H3K27me3) was significantly and frequently reprogrammed at half of genes in ATL cells. A large proportion of the abnormal gene downregulation was detected at the early stage of disease progression and was explained by H3K27me3 accumulation. The global H3K27me3 alterations involved ATL-specific gene expression changes that included several tumor suppressors, transcription factors, epigenetic modifiers, miRNAs, and developmental genes, suggesting diverse outcomes by the PRC2-dependent hierarchical regulation. Interestingly, a key enzyme, EZH2, was sensitive to promiscuous signaling network including the NF-κB pathway and was functionally affected by human T-cell leukemia virus type I (HTLV-1) Tax. The Tax-dependent immortalized cells showed H3K27me3 reprogramming that was significantly similar to that of ATL cells. Of note, a majority of the epigenetic silencing has occurred in leukemic cells from indolent ATL and also in HTLV-1-infected T cells from asymptomatic HTLV-1 carriers. Because pharmacologic inhibition of EZH2 reversed epigenetic disruption and selectively eliminated leukemic and HTLV-1-infected cells, targeting the epigenetic elements will hold great promise in treatment and prevention of the onset of ATL and HTLV-1-related diseases.

Quantitative PCR (qPCR) for human T-lymphotropic virus 1 (HTLV-1) is useful for measuring the amount of integrated HTLV-1 proviral DNA in peripheral blood mononuclear cells. Many laboratories in Japan have developed different HTLV-1 qPCR methods. However, when six independent laboratories analyzed the proviral load of the same samples, there was a 5-fold difference in their results. To standardize HTLV-1 qPCR, preparation of a well-defined reference material is needed. We analyzed the integrated HTLV-1 genome and the internal control (IC) genes of TL-Om1, a cell line derived from adult T-cell leukemia, to confirm its suitability as a reference material for HTLV-1 qPCR. Fluorescent in situ hybridization (FISH) showed that HTLV-1 provirus was monoclonally integrated in chromosome 1 at the site of 1p13 in the TL-Om1 genome. HTLV-1 proviral genome was not transferred from TL-Om1 to an uninfected T-cell line, suggesting that the HTLV-1 proviral copy number in TL-Om1 cells is stable. To determine the copy number of HTLV-1 provirus and IC genes in TL-Om1 cells, we used FISH, digital PCR, and qPCR. HTLV-1 copy numbers obtained by these three methods were similar, suggesting that their results were accurate. Also, the ratio of the copy number of HTLV-1 provirus to one of the IC genes, RNase P, was consistent for all three methods. These findings indicate that TL-Om1 cells are an appropriate reference material for HTLV-1 qPCR.

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.

Despite prolonged antiretroviral therapy, HIV-1 persists as transcriptionally inactive proviruses. The HIV-1 latency remains a principal obstacle in curing AIDS. It is important to understand mechanisms by which HIV-1 latency is established to make the latent reservoir smaller. We present a molecular characterization of distinct populations at an early phase of infection. We developed an original dual-color reporter virus to monitor LTR kinetics from establishment to maintenance stage. We found that there are two ways of latency establishment i.e., by immediate silencing and slow inactivation from active infection. Histone covalent modifications, particularly polycomb repressive complex 2 (PRC2)-mediated H3K27 trimethylation, appeared to dominate viral transcription at the early phase. PRC2 also contributes to time-dependent LTR dormancy in the chronic phase of the infection. Significant differences in sensitivity against several stimuli were observed between these two distinct populations. These results will expand our understanding of heterogeneous establishment of HIV-1 latency populations.

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.

The molecular hallmarks of adult T cell leukemia (ATL) comprise outstanding deregulations of signaling pathways that control the cell cycle, resistance to apoptosis, and proliferation of leukemic cells, all of which have been identified by early excellent studies. Nevertheless, we are now confronted the therapeutic difficulties of ATL that is a most aggressive T cell leukemia/lymphoma. Using next-generation strategies, emerging molecular characteristics such as specific surface markers and an additional catalog of signals affecting the fate of leukemic cells have been added to the molecular hallmarks that constitute an organizing principle for rationalizing the complexities of ATL. Although human T cell leukemia virus type 1 (HTLV-1) is undoubtedly involved in ATL leukemogenesis, most leukemic cells do not express the viral protein Tax. Instead, cellular gene expression changes dominate homeostasis disorders of infected cells and characteristics of ATL. In this review, we summarize the state of the art of ATL molecular pathology, which supports the biological properties of leukemic cells. In addition, we discuss the recent discovery of two molecular hallmarks of potential generality; an abnormal microRNA (miRNA) pattern and epigenetic reprogramming, which strongly involve the imbalance of the molecular network of lymphocytes. Global analyses of ATL have revealed the functional impact of crosstalk between multifunctional pathways. Clinical and biological studies on signaling inhibitory agents have also revealed novel oncogenic drivers that can be targeted in future. ATL cells, by deregulation of such pathways and their interconnections, may become masters of their own destinies. Recognizing and understanding of the widespread molecular applicability of these concepts will increasingly affect the development of novel strategies for treating ATL.

Background Recent evidence proposes a novel concept that mammalian natural antisense RNAs play important roles in cellular homeostasis by regulating the expression of several genes. Identification and characterization of retroviral antisense RNA would provide new insights into mechanisms of replication and pathogenesis. HIV-1 encoded-antisense RNAs have been reported, although their structures and functions remain to be studied. We have tried to identify and characterize antisense RNAs of HIV-1 and their function in viral infection. Results Characterization of transcripts of HEK293T cells that were transiently transfected with an expression plasmid with HIV-1NL4–3 DNA in the antisense orientation showed that various antisense transcripts can be expressed. By screening and characterizing antisense RNAs in HIV-1NL4–3-infected cells, we defined the primary structure of a major form of HIV-1 antisense RNAs, which corresponds to a variant of previously reported ASP mRNA. This 2.6 kb RNA was transcribed from the U3 region of the 3′ LTR and terminated at the env region in acutely or chronically infected cell lines and acutely infected human peripheral blood mononuclear cells. Reporter assays clearly demonstrated that the HIV-1 LTR harbours promoter activity in the reverse orientation. Mutation analyses suggested the involvement of NF-κΒ binding sites in the regulation of antisense transcription. The antisense RNA was localized in the nuclei of the infected cells. The expression of this antisense RNA suppressed HIV-1 replication for more than one month. Furthermore, the specific knockdown of this antisense RNA enhanced HIV-1 gene expression and replication. Conclusions The results of the present study identified an accurate structure of the major form of antisense RNAs expressed from the HIV-1NL4–3 provirus and demonstrated its nuclear localization. Functional studies collectively demonstrated a new role of the antisense RNA in viral replication. Thus, we suggest a novel viral mechanism that self-limits HIV-1 replication and provides new insight into the viral life cycle.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T cell leukemia/lymphoma and is refractory to currently available combination chemotherapy. The unfavorable prognosis results from an inadequate understanding of how diseases are caused and maintained in human T-cell leukemia virus type I (HTLV-1)-infected individuals. To date, direct comprehensive analyses of leukemic cells have identified the intrinsic molecular hallmarks of ATL. Among these, polycomb group (PcG)-mediated epigenetic disruption has been known to be a crucial characteristic of ATL (Yamagishi et al., Cancer Cell, 2012). However, no attempt has been made to determine the global epigenomic status explaining the deregulated gene expression pattern specific to ATL. In this study, we performed integrative analyses of epigenome (n=3) and transcriptome (n=58) of primary ATL patient cells and corresponding normal CD4+ T cells to decipher the ATL-specific 'epigenetic-code' that was critical for cell identity. We found that PcG-mediated tri-methylation at histone H3Lys27 (H3K27me3) was significantly and frequently reprogrammed at over half of genes (53.8%) in ATL cells, the pattern of which appears distinct from other cancer types and PcG-dependent cell lineages such as ES cells and peripheral T lymphocytes. Large proportion of the abnormal gene downregulation occurred at an early stage of disease progression and was explained by the H3K27me3 accumulation. The global H3K27me3 alterations were involved in determination of key genes such as miR-31, CADM1, EVC1/2, CDKN1A, and NDRG2, which are essential for ATL cell survival and other cellular characteristics. In addition, PcG generated diverse outcomes by the remote regulation of a broad spectrum of gene regulators, including various transcription factors, miRNAs, epigenetic modifiers, and developmental genes. Thus, the emerging epigenomic landscape is a fundamental characteristic of ATL. Although EZH2 mutations conferring gain-of-function were undetected in ATL (0/50; 0%), EZH2 level was significantly upregulated and inversely correlated with H3K27me3 targets, indicating that the global alteration of H3K27me3 mark depends on the abundance of EZH2 and other core components of the polycomb repressive complex 2 (PRC2). We found that EZH2 was sensitive to promiscuous signaling network including NF-kB pathway and was functionally affected by HTLV-1 Tax through both NF-kB activation and direct association. The Tax-dependent immortalized cells showed H3K27me3 reprogramming that was significantly similar to that of ATL cells. Of note, the majority of epigenetic silencing has occurred in leukemic cells from indolent type ATL and also in HTLV-1-infected T cells from asymptomatic HTLV-1 carriers. Collectively, our results unveiled that epigenetic reprogramming arises at an early stage of ATL development. Tracing the epigenetic marks and expression patterns in samples obtained from patients of various diagnostic categories, as well as in other biologically relevant models has supported the efficacy and relevance of targeting PRC2. Relief of the cumulative methylation may restore the aberrant transcriptome to ideal expression signature, permitting favorable treatments. According to the expression profiling, EZH2 may represent the first-choice as a druggable target. In addition, peripheral T cells highly express EZH1 that compensates for the EZH2 functions. We found that simultaneous depletion of the two H3K27me3 writers EZH1 and EZH2 significantly diminished cellular H3K27me3 level and dramatically inhibited ATL cell growth compared with single depletion, suggesting that the compensatory actions of EZH1/2 may be critical for ATL. To selectively eliminate the ATL and HTLV-1-infetced populations, we have developed a novel EZH1/2 dual-inhibitor that shows strong inhibitory effects for both of EZH1 and EZH2. Treatment with the new drugs showed significant inhibitory effects on the ATL cell survival derived from patients. Interestingly, the dual-inhibitor reversed the epigenetic disruption and selectively eliminated leukemic and immortalized cells from HTLV-1 infected individuals. Collectively, this approach will achieve potent and selective synthetic lethality by targeting the regulators of H3K27me3 in aggressive and indolent ATL cells, as well as in clonally expanded infected cells, improving medical care and the prevention of disease onset. Disclosures Yamagishi: Daiichi Sankyo Co., Ltd.: Research Funding. Honma:Daiichi Sankyo Co., Ltd: Employment. Adachi:Daiichi Sankyo Co., Ltd: Employment. Araki:Daiichi Sankyo Co., Ltd.: Employment. Watanabe:Daiichi Sankyo Co., Ltd.: Research Funding.

B-cell receptor (BCR) signaling is a major source of gene expression signature important for B cell survival, functions, and development. The integrated signaling amplitude should be equilibrated; when chronically activated by genetic perturbations or other mechanisms, BCR signaling has been accepted as a stem in the pathogenesis of malignant lymphoma/leukemia. The promiscuous signaling network without appropriate alleviation is an essential property of cancers. Diffuse large B-cell lymphoma (DLBCL) with aggressive phenotypes often associates with BCR signaling activation and epigenetic abnormalities, both of which are well correlated with lymphoma subtypes and disease progression. However, the relationship of the two fundamental characteristics is unknown. It is also still unclear the mechanism by which the expression of functionally important genes is continuously deregulated. microRNAs (miRNAs) are an emerging class of intrinsic buffering molecules that have diverse functions in mainly post-transcriptional regulation through miRNA-RISC (miRISC) formation. Using optimized RISC-capture assay, we revealed that multiple BCR signaling factors were persistently regulated by miRNA in human B cells. Clinical samples from newly diagnosed patients with DLBCL (n=83) showed epigenetic loss of an essential miRNA set (miR-200c, miR-203, miR-31). Conventional screening and RISC profiling identified multiple targets (CD79B, SYK, PKCbII, PLCg1, IKKb, NIK, MYD88, PI3K class I (a/b/d/g), RasGRP3); the miRNA-orchestrated overlapping interactome suggested multiple regulatory windows in signaling pathways, which implied their compensatory roles. The shared components within the interconnected signaling pathways were under constant surveillance by the miRNAs. We demonstrated that simultaneous depletion of the key miRNAs enhanced translation of the multiple targets and caused seamless signaling crosstalk among canonical/noncanonical NF-kB pathways, PI3K-Akt-mTOR and Ras-Erk cascades, and downstream of BCR and BAFF receptors. In addition to the miRNA loss, mTOR activation was also positively correlated with signaling amplitude, offering the possibility of selective therapy targeting the translation machinery. The defenseless signaling crosstalk appeared to be a prerequisite for lymphoma development because the essential miRNA set were functionally lost in all tested lymphoma samples. Surprisingly, a common epigenetic mechanism was identified. Polycomb-mediated H3K27me3 accumulation and reciprocal H3K4me3 loss were frequently observed at the miRNAs loci in primary DLBCL samples. We found that lymphoma-associated deregulation (mutation and/or expression change) of EZH2 and MLL2 cooperated to cause the sustained signaling activities of NF-kB, Ras-Erk, PI3K-Akt and mTOR pathways through miRNA silencing en masse. Of note, the perturbation of histone-modifying polycomb and trithorax groups resulted from variations of pooled miRNAs and signaling integrity. The identified coherent circuit may be a source of pathological robustness and phenotypic convergence. Most malignant B cell clones have some kind of genetic lesions. In particular, recurrent variations have been identified within signaling cascades, where miRNA-formed cooperative gene interference (CGI) could buffer the signaling noise. We re-established some experimental models that mimicked the signaling cascade observed in clinically established malignant clones. We experimentally confirmed the buffering roles of the key miRNAs against genetic mutation/alteration of CARD11, A20, MYD88 and PTEN. Reciprocally, coordinated reduction of the functional miRNAs promoted biological processes caused by genetic perturbations. It is therefore conceivable that systematic redundancy of miRNA is inherently required for robustness against genetic alterations that are acquired in lymphoma evolution. We illustrate an example of a biological masterplan comprised of signaling pathways, compensatory actions of multi-layered miRNAs, translation regulation and epigenetic mechanisms. Malignant cell opts for epigenetic reprogramming to stabilize mutation-driven processes and to surmount the demarcation threshold of transformation. Reversing the plastic characteristics of epigenetic alterations is expected in future clinical settings. Disclosures Yamagishi: Daiichi Sankyo Co., Ltd.: Research Funding. Watanabe:Daiichi Sankyo Co., Ltd.: Research Funding.

Although crucial roles of microRNA have begun to emerge, detailed studies with ATL patients have not been achieved. Using 40 primary ATL samples and 22 samples of normal CD4+ T-cells, we determined the microRNA signatures of ATL and revealed loss of miR-31, which has recently been reported as a metastasis-associated miRNA. All ATL cases invariably showed undetectable or very low levels of miR-31, clearly implying that miR-31 loss is involved in ATL development. As a novel miR-31 target gene, we identified NF-κB inducing kinase (NIK) that plays central roles in noncanonical signaling and constitutive activation of NF-κB in various cancers, including ATL. Restoration of miR-31 downregulated the levels of NIK and NF-κB activity, resulting in reduction of malignant phenotypes, containing proliferative index, anti-apoptosis, and chemotaxis in ATL cells. Furthermore, lentivirus-introduced miR-31 could induce strong apoptosis in primary tumor cells freshly isolated from ATL patients, indicating pivotal functions of miR-31 as a tumor suppressor. Global copy number profiling demonstrated that 21 cases out of 168 (12.5%) have genomic loss of 9p21 containing miR-31 region. Furthermore, expression profiling and ChIP assay showed requirement of overexpression of histone methyltransferase in epigenetic suppression of miR-31 and aberrant NF-κB activation in primary ATL cells. Knockdown of methyltransferase complex restored the miR-31 expression and consequently inhibited NIK-dependent NF-κB cascade. These findings illustrated that genetic and epigenetic abnormalities link to NF-κB activation through the loss of miR-31. Considering aberrant epigenomics associated with cancers, the emerging relationship provides us a conceptual advance in understanding the broad-acting oncogenic signaling.

Epigenetic regulations globally determine gene transcription. Recent studies have revealed that expression changes and genetic mutations of epigenetic factors cause epigenetic imbalance in cancers. We previously reported that aberrant expression of Polycomb repressive complex 2 (PRC2) components causes constitutive NF-κB activation through silencing of miR-31 in ATL cells. However, the underlying mechanisms by which the epigenetic imbalance is induced and maintained remain to be elucidated. Here, we conducted ChIP-on-chip and transcriptome analyses of ATL and normal CD4+ T cells and found that the epigenetic reprogramming closely associates with ATL specific gene expression signature. Leukemic cell-specific silencing of cell cycle regulator, CDNK1A, was correlated with H3K27me3 level. In addition, orchestrated loss of microRNAs and multiple transcription factors appears to be mediated by the epigenetic mechanism. EZH2 upregulation and H3K27me3 accumulation were found in HTLV-1-infected populations derived from ATL patients as well as asymptomatic carriers. Furthermore, EZH2 inhibition blocked Tax-dependent cell growth and its immortalization in vitro. Intriguingly, the Tax-triggered immortalizing cells partially mimicked the methylation pattern observed in ATL cells, suggesting that the epigenetic alterations are closely involved in immortalization of infected cells and disease progression. In parallel, we found that over expression of PRC2 core components was induced by active signaling cascades including NF-κB in ATL cells. Collectively, our results suggest a coherent positive feedback mechanism comprised of PRC2 and NF-κB signaling, which stabilizes the epigenetic rearrangement and phenotypic outcomes in HTLV-1 infected cells. Since pharmacological inhibition of EZH2 selectively killed ATL and HTLV-1 infected cells in ex vivo culture, targeting the epigenetic elements will hold great promise in treatment and prevention of ATL and HTLV-1-related diseases.

ATL is a highly aggressive T-cell neoplasm caused by HTLV-1. We have recently demonstrated that EZH2, a catalytic enzyme of histone H3K27 methylation, is overexpressed in ATL cells, which contributes to persistent NF-B activation by repressing a tumor-suppressive miRNA, miR-31. This suggests that epigenetic abnormalities are closely associated with the molecular hallmarks of leukemic cells. However, the mechanisms by which epigenetic disruption is caused and sustained in HTLV-1 infected cells remain to be clarified. In the present study, we first found that Tax directly interacts with EZH2 in HTLV-1 infected cells. Given that epigenetic state is directly affected by cell lineage-dependent transcription factors and genetic background, we assessed the functional interconnection between HTLV-1 Tax and the histone methyltransferases in an appropriate model. Using a lentivirus vector, we introduced tax gene into peripheral blood mononuclear cells (PBMCs) and CD4+ T-cells from healthy donors and established Tax-transduced T-cells. In this model, we observed overexpression of EZH2, aberrant accumulation of H3K27 trimethylation, and epigenetic silencing of p21 cip1/waf1 ,a ll of which have been observed in primary ATL cells. In addition, EZH2 inhibition reduced the viability of Tax-transduced T-cells. Our results strongly suggest that Tax epigenetically affects gene expression through its interaction with EZH2, and that Tax-dependent epigenetic abnormalities may be involved in determining the molecular characteristics of HTLV-1-infected cells as well as ATL cells. Since epigenetic marks are potentially reversible, development of genuine epigenetic-targeted drugs will hold great promise in treatment of HTLV-1-related diseases.

The molecular hallmarks of ATL comprise outstanding deregulations of signaling pathways that control cell cycle, apoptosis resistance, and proliferation of leukemic cells. Using integrative analyses of primary ATL cells, we discovered unique molecular characteristics of ATL (Yamagishi et al., Cancer Cell, 2012). Genetic and epigenetic disruption leads to numerous gene expression alterations that dominate disorders of homeostasis and characteristics of ATL. In particular, a novel tumor suppressor miR-31 is completely lost in all ATL cases, leading to constitutive NF-kB activation via NIK overexpression. Polycomb family directly involves in the silencing of miR-31, providing a novel interconnection between epigenetic reprogramming and NF-kB pathway. In addition, we recently unveiled molecular mechanisms how Polycomb-dependent epigenetic perturbation is abnormally sustained in HTLV-1 infected and leukemic cells. We discuss the recent discovery of molecular hallmarks of potential generality, an abnormal miRNA pattern and epigenetic reprogramming, which strongly involve the imbalance of the molecular network of lymphocytes. Because epigenetic marks are potentially reversible, development of genuine epigenetic-targeted therapy drugs holds great promise in HTLV-1-related diseases. Furthermore, we also introduce additional signaling pathways affecting leukemic cell fate. Pathway analysis based on our comprehensive dataset and biological studies suggest breaking down of the essential signaling pathways such as Hedgehog and p38 pathways, which support the biological properties of ATL. Because these organized principles may be directly associated with the clinical traits of ATL, targeting the one outstanding hallmark or co-targeting of multiple molecular hallmarks in mechanism-guided combinations will result in more effective and durable therapies for aggressive ATL.