Dai Fujikawa

Epigenetic Heterogeneity in HIV-1 Latency Establishment

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.

HTLV-1 Tax disrupts the host epigenome by interacting with a Polycomb group protein EZH2

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.

Epigenetic deregulation of Ellis Van Creveld confers robust Hedgehog signaling in adult T-cell leukemia.

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.

Select gp120 V2 domain specific antibodies derived from HIV and SIV infection and vaccination inhibit gp120 binding to alpha 4 beta 7.

The GI tract is preferentially targeted during acute/early HIV-1 infection. Consequent damage to the gut plays a central role in HIV pathogenesis. The basis for preferential targeting of gut tissues is not well defined. Recombinant proteins and synthetic peptides derived from HIV and SIV gp120 bind directly to integrin α4β7, a gut-homing receptor. Using both cell-surface expressed α4β7 and a soluble α4β7 heterodimer we demonstrate that its specific affinity for gp120 is similar to its affinity for MAdCAM (its natural ligand). The gp120 V2 domain preferentially engages extended forms of α4β7 in a cation -sensitive manner and is inhibited by soluble MAdCAM. Thus, V2 mimics MAdCAM in the way that it binds to α4β7, providing HIV a potential mechanism to discriminate between functionally distinct subsets of lymphocytes, including those with gut-homing potential. Furthermore, α4β7 antagonists developed for the treatment of inflammatory bowel diseases, block V2 binding to α4β7. A 15-amino acid V2 -derived peptide is sufficient to mediate binding to α4β7. It includes the canonical LDV/I α4β7 binding site, a cryptic epitope that lies 7–9 amino acids amino terminal to the LDV/I, and residues K169 and I181. These two residues were identified in a sieve analysis of the RV144 vaccine trial as sites of vaccine -mediated immune pressure. HIV and SIV V2 mAbs elicited by both vaccination and infection that recognize this peptide block V2-α4β7 interactions. These mAbs recognize conformations absent from the β- barrel presented in a stabilized HIV SOSIP gp120/41 trimer. The mimicry of MAdCAM-α4β7 interactions by V2 may influence early events in HIV infection, particularly the rapid seeding of gut tissues, and supports the view that HIV replication in gut tissue is a central feature of HIV pathogenesis.

HIV vaccine candidate activation of hypoxia and the inflammasome in CD14 + monocytes is associated with a decreased risk of SIV mac251 acquisition

Qualitative differences in the innate and adaptive responses elicited by different HIV vaccine candidates have not been thoroughly investigated. We tested the ability of the Aventis Pasteur live recombinant canarypox vector (ALVAC)–SIV, DNA–SIV and Ad26–SIV vaccine prime modalities together with two ALVAC–SIV + gp120 protein boosts to reduce the risk of SIVmac251 acquisition in rhesus macaques. We found that the DNA and ALVAC prime regimens were effective, but the Ad26 prime was not. The activation of hypoxia and the inflammasome in CD14+CD16− monocytes, gut-homing CCR5-negative CD4+ T helper 2 (TH2) cells and antibodies to variable region 2 correlated with a decreased risk of SIVmac251 acquisition. By contrast, signal transducer and activator of transcription 3 activation in CD16+ monocytes was associated with an increased risk of virus acquisition. The Ad26 prime regimen induced the accumulation of CX3CR1+CD163+ macrophages in lymph nodes and of long-lasting CD4+ TH17 cells in the gut and lungs. Our data indicate that the selective engagement of monocyte subsets following a vaccine prime influences long-term immunity, uncovering an unexpected association of CD14+ innate monocytes with a reduced risk of SIVmac251 acquisition.Distinct monocyte subsets associate with different outcomes of SIV vaccines.

Molecular hallmarks of adult T cell leukemia: miRNA, epigenetics, and emerging signaling abnormalities

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.

Inhibition of Tunneling Nanotube (TNT) Formation and Human T-cell Leukemia Virus Type 1 (HTLV-1) Transmission by Cytarabine

The human T-cell leukemia virus type 1 (HTLV-1) is highly dependent on cell-to-cell interaction for transmission and productive infection. Cell-to-cell interactions through the virological synapse, biofilm-like structures and cellular conduits have been reported, but the relative contribution of each mechanism on HTLV-1 transmission still remains vastly unknown. The HTLV-1 protein p8 has been found to increase viral transmission and cellular conduits. Here we show that HTLV-1 expressing cells are interconnected by tunneling nanotubes (TNTs) defined as thin structures containing F-actin and lack of tubulin connecting two cells. TNTs connected HTLV-1 expressing cells and uninfected T-cells and monocytes and the viral proteins Tax and Gag localized to these TNTs. The HTLV-1 expressing protein p8 was found to induce TNT formation. Treatment of MT-2 cells with the nucleoside analog cytarabine (cytosine arabinoside, AraC) reduced number of TNTs and furthermore reduced TNT formation induced by the p8 protein. Intercellular transmission of HTLV-1 through TNTs provides a means of escape from recognition by the immune system. Cytarabine could represent a novel anti-HTLV-1 drug interfering with viral transmission.

HTLV-1-mediated epigenetic pathway to adult T-cell leukemia-lymphoma

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.

EZH2 dependent epigenetic landscape in adult T cell leukemia and Tax immortalized cells

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.