Adult T-cell Leukemia Patients Research Articles

Increased H19/miR-675 Expression in Adult T-Cell Leukemia Is Associated with a Unique Notch Signature Pathway.

The Notch pathway is a key cancer driver and is important in tumor progression. Early research suggested that Notch activity was highly dependent on the expression of the intracellular cleaved domain of Notch-1 (NICD). However, recent insights into Notch signaling reveal the presence of Notch pathway signatures, which may vary depending on different cancer types and tumor microenvironments. Herein, we perform a comprehensive investigation of the Notch signaling pathway in adult T-cell leukemia (ATL) primary patient samples. Using gene arrays, we demonstrate that the Notch pathway is constitutively activated in ATL patient samples. Furthermore, the activation of Notch in ATL cells remains elevated irrespective of the presence of activating mutations in Notch itself or its repressor, FBXW7, and that ATL cells are dependent upon Notch-1 expression for proliferation and survival. We demonstrate that ATL cells exhibit the expression of pivotal Notch-related genes, including notch-1, hes1, c-myc, H19, and hes4, thereby defining a critical Notch signature associated with ATL disease. Finally, we demonstrate that lncRNA H19 is highly expressed in ATL patient samples and ATL cells and contributes to Notch signaling activation. Collectively, our results shed further light on the Notch pathway in ATL leukemia and reveal new therapeutic approaches to inhibit Notch activation in ATL cells.

IPSC-Derived CD4 T Cell Generation and Investigation of CD4/CD8 T Cell Lineage Choice

iPSC-derived functionally rejuvenated antigen-specific cytotoxic T lymphocytes generated from exhausted CTLs show promising antitumor effect towards refractory tumors. Currently, we are preparing for clinical trials using iPSC-derived CTLs for EBV-associated lymphomas. These iPSC-derived T cells (iPSC-Ts) all generated into CD8 T cells, and stable generation of CD4 single positive (SP) T cells from iPSCs has never been accomplished without enforced CD4 gene transduction even though many research groups have challenged to do so. Even when an iPSC is established from a CD4 T cell clone, differentiating this iPSC does not generate into CD4 T cells, but in fact it generates into CD8 T cells. Therefore, we aimed to investigate why iPSC-Ts only differentiate into CD8T cells, and not into CD4T cells. We focused on adult T cell leukemia (ATL), because HTLV-1 infected CD4 T cells clonally expand. We reprogrammed CD4T cells obtained from acute-type ATL patients into iPSCs (ATL-iPSCs), then differentiated them into T cells using our established method. Interestingly, ATL-iPSCs from different donors all successfully differentiated into CD4 SP T cells (ATL-iPSC-Ts). Upon characterization of ATL-iPSC-Ts we confirmed that they showed FOXP3 positive naive regulatory T cell (Treg) phenotype. To investigate the function of these cells, we performed 51Cr-release assay. The cytotoxicity of HTLV-1 Tax-specific CTLs (Tax-CTLs) with or without ATL-iPSC-Ts against primary ATL cells was measured. The percentages of ATL cells lysed by Tax-CTLs was 34 ± 3.2%, whereas by the addition of ATL-iPSC-Ts, the cytotoxicity decreased to 23.4 ± 1.0% revealing the functionally suppressive effect of ATL-iPSC-Ts. In order to further understand the mechanism of CD4/CD8 T cell lineage choice, and discover the key regulators needed for generation of CD4 SP T cells from iPSCs, we performed single cell RNA-sequencing (scRNA-seq) on ATL-iPSC-Ts. As a control we used healthy donor (HD) CD4 SP T cell clone-derived iPSCs (HD-iPSCs), which differentiated into CD8 T cells (HD-iPSC-Ts [CD8 +]). We identified several genes expressed at a significantly higher level on HD-iPSC-Ts (CD8 +) than on ATL-iPSC-Ts (CD4 +). We presumed these genes to be essential for CD8 T cell differentiation. Therefore, we used CRISPR/Cas9 technology to knock out the respective gene in HD-iPSCs and confirmed if these iPSCs could differentiate into CD4 SP T cells, instead of CD8 T cells. This resulted in successful generation of CD4 SP T cells. We confirmed that these CD4 SP T cells were FOXP3 negative and were not Tregs. On the other hand, overexpression of this gene on ATL-iPSCs differentiated into CD8 SP T cells, instead of CD4 T cells. In conclusion, our work represents the first successful generation of natural CD4 SP T cells from iPSCs. The gene we identified appears to be a pivotal regulator of CD4/CD8 T cell lineage choice in our iPSC-T differentiation system. While more investigation is required, our method shows promise towards facilitating stable generation of CD4 T cells for T cell therapy.

Establishment of a Cynomolgus Macaque Model of Human T-Cell Leukemia Virus Type 1 (HTLV-1) Infection by Direct Inoculation of Adult T-Cell Leukemia Patient-Derived Cell Lines for HTLV-1 Infection.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). However, the precise mechanisms leading to HTLV-1 chronic infection and the onset of the diseases have remained unclear, and effective vaccines for inhibiting the infection and the progression of pathogenesis have therefore not been developed. The use of a nonhuman primate (NHP) model is thought to be important for revealing the mechanisms of the progressive status and for the development of prevention procedures. In this study, we developed a cynomolgus macaque (CM) model of HTLV-1 infection by direct intravenous inoculation of HTLV-1-producing cells derived from ATL patients. The cell line used for infection, ATL-040, was selected as the most infectious one in our cell line library. CMs inoculated intravenously with 1 × 108 ATL-040 cells per animal became persistently infected with HTLV-1, as shown by the HTLV-1 provirus load (PVL) in peripheral blood mononuclear cells and HTLV-1-specific antibodies (2/2 animals). One CM inoculated intravenously with 1 × 107 ATL-040 cells did not have detectable PVLs despite the fact that anti-HTLV-1 antibodies were maintained for more than 2 years. Furthermore, immunological approaches, including CD8+ T cell depletion prior to infection (3/3 animals) and intrathecal inoculation (3/3 animals), led to increased proviral loads in the cynomolgus monkeys. The present method and the cynomolgus monkey model of HTLV-1 infection will be beneficial for immunological and virological studies on HTLV-1 aiming at the development of anti-HTLV-1 prophylactic vaccines and therapy drugs. IMPORTANCE HTLV-1 was discovered in the 1980s as the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, the precise mechanisms leading to HTLV-1 chronic infection and the onset of the diseases still remain unidentified. Thus, no effective vaccines to inhibit the infection and the progressive of pathogenesis have been developed. The use of appropriate animal models is essential for understanding HTLV-1 infection and pathogenesis. In order to establish a new nonhuman primate model for studies on HTLV-1 infection, cynomolgus monkeys were infected with HTLV-1 under a variety of experimental conditions. Our method, using a cell line generated from an ATL patient as a source of HTLV-1, was able to establish HTLV-1 infection in monkeys with a 100% success rate. This cynomolgus macaque model of HTLV-1 infection will contribute to the elucidation of HTLV-1 infection and its associated disease development.

Particularities of Neurological Manifestations in Adult T-Cell Leukemia/Lymphoma: Need for a Multidisciplinary Approach-A Narrative Review.

ATL is a rare but a highly aggressive T-cell neoplasm associated with human T-cell leukemia virus-1 (HTLV-1) infection. Human T-cell lymphotropic virus type-1 (HTLV-1) is a oncogenic retrovirus responsible for the development of adult T-cell leukemia (ATL), but also for other non-malignant diseases, such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 has a higher prevalence in Japan, the Caribbean, South America, intertropical Africa, Romania, and northern Iran. ATL patients can have an extensive spectrum of neurological manifestations. Numerous factors can be implicated, such as central nervous system infiltrates, neurolymphomatosis, complications to medication or allogeneic stem cell transplantation, HAM/TSP, infections, metabolic disturbances. The neurological complications are not always easy to recognize and treat. Thus, this review underlines the necessity of a multidisciplinary approach in ATL patients with neurological symptomatology.

A protective role of HTLV-1 gp46-specific neutralizing and antibody-dependent cellular cytotoxicity-inducing antibodies in progression to adult T-cell leukemia (ATL).

Human T-cell leukemia virus type-1 (HTLV-1) establishes a long-term persistent infection in humans and causes malignant T-cell leukemia, adult T-cell leukemia (ATL). HTLV-1-specific cytotoxic T lymphocytes have been suggested to play a major role in the immunosurveillance of HTLV-1-infected T cells. However, it remains unclear whether HTLV-1-specific functional antibodies are also involved in the host defense. To explore the role of antibodies in the course of HTLV-1 infection, we quantitated HTLV-1-specific neutralizing and antibody-dependent cellular cytotoxicity (ADCC)-inducing antibody levels in plasma from asymptomatic carriers (ACs) and ATL patients. The levels of neutralizing antibodies, as determined by a syncytium inhibition assay, were significantly lower in acute and chronic ATL patients than in ACs. The levels of ADCC-inducing activity were tested using an autologous pair of HTLV-1-producing cells and cultured natural killer (NK) cells, which showed that the ADCC-inducing activity of IgG at a concentration of 100 µg/ml was comparable between ACs and acute ATL patients. The anti-gp46 antibody IgG levels, determined by ELISA, correlated with those of the neutralizing and ADCC-inducing antibodies. In contrast, the proviral loads did not correlate with any of these antibody levels. NK cells and a monoclonal anti-gp46 antibody reduced the number of HTLV-1 Tax-expressing cells in cultured peripheral blood mononuclear cells from patients with aggressive ATL. These results suggest a protective role for HTLV-1 neutralizing and ADCC-inducing antibodies during the course of HTLV-1 infection.

Dimethyl Fumarate Induces Apoptosis via Inhibiting NF-κB and STAT3 Signaling in Adult T-cell Leukemia/Lymphoma Cells

Adult T-cell leukemia (ATL) is a peripheral T lymphocytic malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) infection. Despite treatment that includes novel agents that have been developed, most of ATL patients relapse and acquire multidrug resistance. As a result, the creation of newer agents is critical. Dimethyl fumarate (DMF) has several effects in cancer cells, including cell signaling, proliferation and cell death. However, its antitumor effects on ATL cells remain unknown. In this study, we looked at DMF's antitumor effects on ATL cells. We examined the effects of DMF on proliferation and apoptosis using the trypan blue exclusion assay and annexin V/propidium iodide staining in HTLV-1-infected and transformed T-cell lines, MT-1 and MT-2 cells. We also evaluated the effects of DMF on the nuclear factor-kappa B (NF-κB) and signal transducers and activators of transcription 3 (STAT3) signaling pathways and anti-apoptotic proteins by immunoblotting. DMF inhibited proliferation and induced apoptosis in MT-1 and MT-2 cells by activating poly ADP-ribose polymerase (PARP). Furthermore, DMF inhibited the constitutive activation of both canonical and non-canonical NF-κB pathways in MT-2 cells and the non-canonical NF-κB pathway in MT-1 cells. DMF also inhibited the constitutive tyrosine phosphorylation of STAT3 and the expression of anti-apoptotic proteins, c-IAP2 and survivin in both cells. These results indicate that DMF inhibits proliferation and induces apoptosis in HTLV-1-infected and transformed T-cells by suppressing NF-κB and STAT3 signaling pathways. DMF should be investigated further as a novel agent for ATL.

540A simulation to evaluate HTLV-1 screening for pregnant women in prevention of adult T-cell leukemia

Abstract Background In Japan, the nation-wide screening program of pregnant women for HTLV-1 infection was launched in 2010 to prevent mother-to-child transmission. It is difficult to assess the effect of the screening program on adult T-cell leukemia (ATL) prevention by observational study because ATL develops after long-term (about 50 years) latency in HTLV-1 carriers. Thus, we assessed the effect of the screening using computer simulation. Methods We constructed our model that simulates lifetime history of the children born to HTLV-1 carrier mothers. Lifetime risks of ATL among the children in unscreened group and screened group were estimated employing Monte Carlo simulation. We developed three scenarios for the risk estimation of ATL in screened groups, each making different assumptions for the choice of feeding methods (formula feeding, short-term breast-feeding, and long-term breast-feeding). Results Our simulation revealed that lifetime risk of ATL among the children born to HTLV-1 carrier mothers was 1.19 % in unscreened group and 0.21 to 0.27 % in screened groups. Conclusions In Japan, it is estimated that there were 1,363 pregnant women with HTLV-1 infection in 2016 (Sekizawa 2018). From this figure, the estimated annual number of ATL patients prevented by the screening is 12.6 to 13.5. Key message HTLV-1 screening in pregnant women for one year is estimated to reduce the number of ATL among their children by about 13 in Japan.

Blockade of IL-2 and IL-15 Gamma Chain Receptor Signaling Decreases Leukemic Cell Viability in T-Cell Large Granular Lymphocyte Leukemia and Adult T-Cell Leukemia

IntroductionInterleukin-15 (IL-15) and IL-2 are important cytokines that drive the progression of many T-cell malignancies such as T-cell Large Granular Lymphocyte Leukemia (T-LGLL) and HTLV-1 derived Adult T-cell Leukemia (ATL).T-LGLL is characterized by clonal expansion of cytotoxic T cells and is associated with a constitutively active JAK/STAT pathway controlled by cytokines including IL-15 and IL-2. A computational systems approach identified IL-15 as a master regulator of T-LGLL survival and patients have increased IL-15/IL-15Ra serum levels. Cytokine signaling leads to increased phosphorylation and activation of STAT proteins. T-LGLL cells exhibit STAT3 or STAT5 mutational activation in some cases and constitutive activation in all patients. T-LGLL treatments are not selective or curative, and the commonly utilized immunosuppressive agents are not effective in all patients.ATL is an aggressive T-cell disorder that also exhibits aberrant IL-2 and IL-15 cytokine signaling. Tax, identified as an essential oncogene in the disease, is associated with increased cytokine and cytokine receptor expression. ATL patients have high IL-2Ra gene expression and PBMCs from chronic/smoldering ATL patients can depend on autocrine production of cytokines for proliferation. Patients with ATL have poor prognosis and current cytokine-targeting therapies have not proven to be efficacious.The gc receptor is shared by the gc-family cytokines including IL-2, -4, -7, -9, -15 and -21. BNZ-1 (formerly known as BNZ132-1-40) is a pegylated peptide designed to specifically bind the gc receptor to block IL-2, IL-15 and, to a lesser extent, IL-9 cytokine signaling. Because these cytokines are vital to the survival of T cells in T-LGLL and ATL, these diseases represent ideal models to test the hypothesis that treatment of leukemic cells with BNZ-1 will result in reduced proliferation and viability.MethodsThese studies utilized a human T-LGL cell line (TL1), PBMCs from T-LGLL patients and ATL patients, and human IL-15 transgenic NSG mice injected with the ED40515(+) ATL cell line expressing luciferase. In vitro assays were performed after an overnight cytokine withdrawal and 20-minute pretreatment with BNZ-1 prior to addition of cytokine. Leukemic mice were divided into 3 treatment groups; BNZ-1 (40 mg/kg i.v. 2x per week for 4 weeks), JAK1/2 inhibitor Ruxolitinib (Ruxo) (50 mg/kg/day s.c. by pump for 28 days) and PBS control. Tumor burden was measured through bioluminescence imaging (Xenogen IVIS).ResultsOur results demonstrate that in vitro treatment of TL1 cells with BNZ-1 inhibits IL-2 and IL-15 mediated expansion and viability. BNZ-1 impaired the growth of these cytokine-dependent cells to the level observed in cells without exogenous cytokine supplementation. In addition, BNZ-1 was able to drastically block IL-15 and IL-2 induced phosphorylation events, specifically STAT1, STAT3, STAT5, ERK and Akt. The compound blocked IL-15 mediated viability and signaling more effectively than that mediated by IL-2.The BNZ-1 mediated decreased viability of PBMCs from T-LGLL patients was observed in the presence of either IL-2 or IL-15 stimulation and regardless of STAT3 mutational status. A potent reduction of IL-2 and IL-15 mediated STAT1, STAT3, STAT5 and ERK phosphorylation was also observed in T-LGLL PBMCs with BNZ-1 treatment. Proliferation of PBMCs from ATL patients was inhibited by 58 to 80 percent by the addition of either the antiIL-2Ra antibody daclizumab or and the combination treatment of daclizumab, anti-IL-15 and anti-IL-9. BNZ-1 at 10 µM inhibitedproliferation by 60 to 80 percent.Leukemic burden in the ATL tumor bearing mice was reduced by both BNZ-1 and Ruxo when compared to controls. The BNZ-1 treatment group showed comparable efficacy to that observed in the Ruxo group. Furthermore, BNZ-1 therapy was associated with lower levels of IL-2Ra, which reflects reduced tumor burden at 4 and 7 weeks when compared to that observed with the PBS group.In summary, BNZ-1 shows promise as a potential therapy for T-LGLL and ATL. Results from this study support its advancement in clinical trials in the treatment of T-LGLL and ATL patients. DisclosuresTagaya:Bioniz Therapeutics: Employment. Azimi:Bioniz Therapeutics: Employment.

Arsenic Trioxide and Interferon-Alpha Eradicate Adult T Cell Leukemia Initiating Cells through PML/p53 Senescence and Activation of Innate Immunity

Adult T cell leukemia (ATL) is an aggressive chemo-resistant T cell malignancy secondary to chronic HTLV-I infection. The viral oncoprotein Tax initiates ATL in transgenic mice. We previously showed that arsenic trioxide (As) and interferon-α (IFN) combination, known to trigger proteasome mediated Tax degradation and apoptosis in ATL cells, cures murine ATL through clearance of leukemia initiating cells (LIC). We also showed that the triple combination of As, IFN and zidovudine is highly effective in the treatment of chronic ATL patients, inducing a shift from an immunosuppressive Treg/Th2 profile at diagnosis (high IL-10; low IL2; low IFN-γ) towards a Th-1 immunocompetent profile (high IFN-γ; high IL2; low IL-10).To dissect the molecular basis of As/IFN-induced ATL LIC eradication, primary ATL SCID mice were treated with As/IFN for 3 days. Spleen derived ATL cells were injected into secondary recipient SCID mice left untreated and sacrificed on a weekly basis. Whereas, secondary mice derived from untreated primaries succumbed from ATL between 3 and 4 weeks after transplantation, we observed a biphasic growth intersected by a spontaneous regression phase in untreated secondary mice transplanted from As/IFN treated primary mice. This process was associated with telomere shortening, DNA damage, activation of the PML/P53 axis, and senescence in tumor cells. Interestingly, injection of spleen derived ATL cells from secondary SCID sacrificed before ATL senescence, into untreated tertiary and quaternary SCID, resulted in normal ATL development, indicating that senescence of ATL cells in secondary SCID mice resulted in a change of the microenvironment that culminated in ATL cell apoptosis and loss of ATL LIC activity. Furthermore, injection of spleen derived ATL cells from untreated secondary SCID sacrificed before or after ATL senescence, into untreated tertiary NSG mice, resulted in normal ATL development. These NSG mice, contrary to SCID mice, lack natural killer (NK) cells and have an impaired macrophage activity. In agreement with these results, injection of spleen derived ATL cells from treated primary SCID into untreated secondary NSG mice, resulted in normal ATL development. RT-PCR and ELISA analysis of sorted CD25 positive (ATL) or negative (microenvironment) cells revealed treatment-induced upregulation of MIP1, RANTES, and IFN-γ in the CD25 negative fraction in untreated secondary SCID, following ATL senescence, and concomitant with apoptosis and loss of LIC activity. Conversely, and as previously seen in treated ATL patients, IL-10 expression was rapidly and dramatically downregulated in the CD25 positive fraction in untreated secondary SCID mice. Addition of the proteasome inhibitor PS-341 to AS/IFN treatment of primary mice reverted all above results, presumably indicating the role of As/IFN-induced Tax degradation in the observed phenotypes.Collectively, these results demonstrate that As/IFN-induced, PML/p53 dependent, senescence of ATL cells resulted in activation of the innate immunity that culminated in apoptosis and loss of ATL LIC activity. The striking similarity of these results with patients' data suggests a critical role for innate immunity in ATL cure. DisclosuresHermine:Novartis: Research Funding; Hybrigenics: Research Funding; Celgene: Research Funding; AB Science: Equity Ownership, Honoraria, Patents & Royalties, Research Funding; INatherys: Equity Ownership, Research Funding.

Short‐term cultured autologous peripheral blood mononuclear cells as a potential immunogen to activate Tax‐specific CTL response in adult T‐cell leukemia patients

Activation of CD8+ Tax‐specific CTL is a new therapeutic concept for adult T‐cell leukemia (ATL) caused by HTLV‐1. A recent clinical study of the dendritic cell vaccine pulsed with Tax peptides corresponding to CTL epitopes showed promising outcomes in ATL patients possessing limited human leukocyte antigen (HLA) alleles. In this study, we aimed to develop another immunotherapy to activate Tax‐specific CTL without HLA limitation by using patients’ own HTLV‐1‐infected cells as a vaccine. To examine the potential of HTLV‐1‐infected T‐cells to activate CTL via antigen presenting cells, we established a unique co–culture system. We demonstrated that mitomycin C‐treated HLA‐A2‐negative HTLV‐1‐infected T‐cell lines or short‐term cultured peripheral blood mononuclear cells (PBMC) derived from ATL patients induced cross–presentation of Tax antigen in co–cultured HLA‐A2‐positive antigen presenting cells, resulting in activation of HLA‐A2‐restricted CD8+ Tax‐specific CTL. This effect was not inhibited by a reverse transcriptase inhibitor. IL‐12 production and CD86 expression were also induced in antigen presenting cells co–cultured with HTLV‐1‐infected cells at various levels, which were improved by pre–treatment of the infected cells with histone deacetylase inhibitors. Furthermore, monocyte‐derived dendritic cells induced from PBMC of a chronic ATL patient produced IL‐12 and expressed enhanced levels of CD86 when co–cultured with autologous lymphocytes that had been isolated from the same PBMC and cultured for several days. These findings suggest that short‐term cultured autologous PBMC from ATL patients could potentially serve as a vaccine to evoke Tax‐specific CTL responses.

FHIT as a biomarker for early screening of adult T-cell leukemia.

Adult T-cell leukemia (ATL) is an incurable leukemia deriving from human T-cell leukemia virus (HTLV-I) infected cells. In our most recent study, we discovered that methylation of the tumor suppressor, fragile histidine triad gene (FHIT), exists in the majority of acute and chronic ATL patients. Methylation was seen in non-tumorigenic cells, in cells with low levels of HTLV-I integrated DNA, in longitudinal samples from HTLV-I carriers, in a percentage of HTLV-I carriers, and in direct descendants of ATL patients. Overall, this suggests that FHIT methylation is likely present in patients, prior to HTLV-I infection, and predisposes HTLV-I carriers to ATL development. In this commentary we discuss the importance of developing diagnostic tools for the early detection of FHIT methylation and the possibility that prior FHIT methylation may predispose any individual to the development of cancer.

Constitutive Activation of NF-κB in Primary Adult T-Cell Leukemia Cells

AbstractHuman T-cell leukemia virus type I (HTLV-I) is an etiologic agent of adult T-cell leukemia (ATL). The viral protein Tax induces the activation and nuclear translocalization of transcription factor NF-κB, which is proposed to play a crucial role in the transformation of T cells by HTLV-I. However, the HTLV-I genes including Tax are not expressed significantly in primary leukemic cells from ATL patients. In this study, we examined the basis for NF-κB activation in freshly isolated leukemic cells from ATL patients. We found that leukemic cells from ATL patients, like HTLV-I–infected T-cell lines, display constitutive NF-κB DNA binding activity and increased degradation of IκBα (an inhibitor of NF-κB). Whereas the NF-κB binding activity in Tax-expressing T-cell lines consisted mostly of p50/c-Rel, fresh ATL samples contained p50/p50 and p50/p65 heterodimers. One T-cell line derived from ATL leukemic cells, TL-Om1, displayed constitutive NF-κB activity, as well as enhanced degradation of IκBα, despite the lack of detectable Tax expression. Interestingly, the NF-κB in TL-Om1 consists of p50/p50 and p50/p65 like that in fresh primary leukemic cells. Our results suggest that activation of NF-κB occurs through a Tax-independent mechanism in leukemic cells of ATL patients, possibly due to differential NF-κB subunit activation.

Enhanced efficacy of JAK1 inhibitor with mTORC1/C2 targeting in smoldering/chronic adult T cell leukemia

Adult T-cell leukemia (ATL) is an aggressive T-cell lymphoproliferative malignancy of regulatory T lymphocytes (Tregs), caused by human T-cell lymphotropic virus 1 (HTLV-1). Interleukin 2 receptor alpha (IL-2Rα) is expressed in the leukemic cells of smoldering/chronic ATL patients, leading to constitutive activation of the JAK/STAT pathway and spontaneous proliferation. The PI3K/AKT/mTOR pathway also plays a critical role in ATL cell survival and proliferation. We previously performed a high-throughput screen that demonstrated additive/synergistic activity of Ruxolitinib, a JAK1/2 inhibitor, with AZD8055, an mTORC1/C2 inhibitor. However, effects of unintended JAK2 inhibition with Ruxolitinib limits it therapeutic potential for ATL patients, which lead us to evaluate a JAK1-specific inhibitor. Here, we demonstrated that Upadacitinib, a JAK-1 inhibitor, inhibited the proliferation of cytokine-dependent ATL cell lines and the expression of p-STAT5. Combinations of Upadacitinib with either AZD8055 or Sapanisertib, mTORC1/C2 inhibitors, showed anti-proliferative effects against cytokine-dependent ATL cell lines and synergistic effect with reducing tumor growth in NSG mice bearing IL-2 transgenic tumors. Importantly, the combination of these two agents inhibited ex vivo spontaneous proliferation of ATL cells from patients with smoldering/chronic ATL. Combined targeting of JAK/STAT and PI3K/AKT/mTOR pathways represents a promising therapeutic intervention for patients with smoldering/chronic ATL.

Evaluating mRNA expression of tax, B chain of PDGFand PDGF-β receptorsas well as HTLV-I proviral load in ATL patients and healthy carriers.

Adult T-cell leukemia (ATL) is a life-threatening malignant neoplasm of CD4+ T cells resulted from human T-cell leukemia virus type I (HTLV-I). Tax1 protein of HTLV-I can induce malignant proliferation of T-cells bymodulating the expression of growth factors such as platelet-derived growth factor (PDGF). Here, we aimed to investigate the proviral load (PVL) of HTLV-I in ATLand also to evaluate the mRNA expression of B chain of PDGF and PDGF-β receptors in ATL patients and HTLV-I-infected healthy carriers. To this end, peripheral blood mononuclear cells (PBMCs) were isolated by using Ficoll-Histophaque density centrifugation. The mean of HTLV-I PVL in ATL patients (42,759 ± 15,737 copies/104 cells [95% CI, 9557-75962]) was significantly (p = .01) higher than that in healthy carriers (650 ± 107 copies/104 cells [95% CI, 422-879], respectively. The HTLV-I PVL in ATL patients exhibited a significant correlation with PBMC count (R = .495, p = .001). The mRNA expression of Tax, B chain of PDGF, and PDGF-β receptor genes was significantly higher in healthy carriers than in patients with ATL. In conclusion, the expression of the canonical PDGFβ and its receptor, and their correlation with Tax expression cannot be a suitable indicator and/or prognostic factor for progression of ATL in HTLV-I carriers.

HTLV-1 viral oncoprotein HBZ contributes to the enhancement of HAX-1 stability by impairing the ubiquitination pathway.

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that causes adult T-cell leukemia (ATL). The viral protein HTLV-1 basic leucine-zipper factor (HBZ), which is constitutively expressed in all ATL patient cells, contributes toward the development of ATL; however, the underlying mechanism has not been elucidated yet. Here, we identified HS-1-associated protein X-1 (HAX-1) as a novel binding partner of HBZ. Interestingly, HAX-1 specifically associated with HBZ-US, but not HBZ-SI, in the cytoplasm. HBZ suppressed the polyubiquitination levels of HAX-1 protein by inhibiting the association HAX-1 with F-box protein 25 (FBXO25), which is a member of the SCF E3 ubiquitin ligase complex, and promoted the stabilization of HAX-1 levels. In fact, the protein levels of HAX-1 were significantly increased in HTLV-1 infected and the overexpressing HBZ in uninfected T-cell lines. Enhanced HAX-1 correlated well to suppression of caspase 9 processing, suggesting that HBZ may contribute to the enhancement of antiapoptotic function for HAX-1. Our results revealed a role for HBZ on HAX-1 stabilization by abrogating the ubiquitination-mediated degradation pathway, which may play an important role in understanding the potential mechanisms of HTLV-1 related pathogenesis.

Interaction of perforin and granzyme B and HTLV-1 viral factors is associated with Adult T cell Leukemia development.

Objective(s):Human T cell leukaemia virus type 1 (HTLV-1) is associated with adult T cell leukaemia (ATL), a malignant lymphoproliferative disease that infects CD4 T cells. It is not clear why the majority of HTLV-1-infected individuals remain asymptomatic carries (ACs) and a minority develop ATL. Cellular immune response has a critical role in ATL and destroys malignant and HTLV-1-infected cells. Perforin and granzyme have important functional roles in apoptosis and destruction of infected cells. In the present study we examined the role of perforin and granzyme in ATL patients and ACs.Materials and Methods:Peripheral blood mononuclear cells (PBMCs) were isolated from ATL patients and ACs by using Ficoll-hypaque density centrifugation. RNA was extracted and cDNA was synthesized. A real-time PCR TaqMan method was designed and optimized for evaluation of perforin, granzyme, tax, and HBZ gene expression. HTLV-1 proviral load (PVL) was quantified in patients with ATL and ACs.Results:The mRNA expression of tax and HBZ was significantly higher in ATL patients than ACs (P=0.011 and P=0.0001,respectively). The HTLV-1 PVL was higher in ATL patients compared to with AC group (P=0.015). There was a significant increase in perforin gene expression in ACs compared with ATL patients (P=0.002). Furthermore, the expression of granzyme was also higher in ACs compared with ATL patients, and significant differences were observed between the two groups (P=0.036).Conclusion:Low expression of perforin and granzyme in ATL patients seems to influence the efficiency of CTL function and destruction of HTLV-1-infected cells, which might contribute to the disease pathogenesis.

IL-2/IL-2 Receptor Pathway Plays a Crucial Role in the Growth and Malignant Transformation of HTLV-1-Infected T Cells to Develop Adult T-Cell Leukemia.

T cells infected with human T-cell leukemia virus type 1 (HTLV-1) transform into malignant/leukemic cells and develop adult T-cell leukemia (ATL) after a long latency period. The tax (transactivator from the X-gene region) and HBZ (HTLV-1 bZIP factor) genes of HTLV-1 play crucial roles in the development of ATL. The process and mechanism by which HTLV-1-infected T cells acquire malignancy and develop ATL remain to be elucidated. Constitutive expression of interleukin-2 (IL-2) receptor α-chain (IL-2Rα/CD25), induced by the tax and HBZ genes of HTLV-1, on ATL cells implicates the involvement of IL-2/IL-2R pathway in the growth and development of ATL cells in vivo. However, the leukemic cells in the majority of ATL patients appeared unresponsive to IL-2, raising controversies on the role of this pathway for the growth of ATL cells in vivo. Here, we report the establishment of 32 IL-2-dependent T-cell lines infected with HTLV-1 from 26 ATL patients, including eight leukemic cell lines derived from five ATL patients, while no T-cell lines were established without IL-2. We have shown that the IL-2-dependent ATL cell lines evolved into IL-2-independent/-unresponsive growth phase, resembling ATL cells in vivo. Moreover, the IL-2-dependent non-leukemic T-cell lines infected with HTLV-1 acquired IL-2-independency and turned into tumor-producing cancer cells as with the ATL cell lines. HTLV-1-infected T cells in vivo could survive and proliferate depending on IL-2 that was produced in vivo by the HTLV-1-infected T cells of ATL patients and patients with HTLV-1-associated diseases and, acts as a physiological molecule to regulate T-cell growth. These results suggest that ATL cells develop among the HTLV-1-infected T cells growing dependently on IL-2 and that most of the circulating ATL cells progressed to become less responsive to IL-2, acquiring the ability to proliferate without IL-2.

Functional Analysis of Aberrantly Spliced Caspase8 Variants in Adult T-Cell Leukemia Cells.

The genetic and molecular alterations responsible for leukemogenesis and progression of HTLV-infected adult T-cell leukemia (ATL) have not been fully clarified. Previously, we reported that various genes are not only overexpressed but also abnormally spliced in ATL cells. Here, we identified various CASP8 transcript variants in PBMCs from a smoldering-type ATL patient, which encode aberrant truncated caspase 8 (Casp8) isoforms. Among those, we focus on the three transcript variants, CASP8L (including the first 136 bp of the intron 8 between exon 8 and exon 9), CASP8-ΔE4 (without the exon 4), and CASP8-ΔE7 (without the exon 7), because they encode isoforms, Casp8L, Casp8-ΔE4, and Casp8-ΔE7, respectively, without the C-terminal catalytic domains. In this study, we conducted in vitro characterization and functional analysis of those mutant Casp8 isoforms to clarify their changed functions compared with the wild-type (WT)-Casp8. We demonstrated that these abnormal Casp8 isoforms showed lower ability to induce apoptosis than WT-Casp8 due to their dominant-negative interactions with WT-Casp8, which impair WT-Casp8 homodimerization that is essential for induction of apoptosis. Moreover, Casp8L and Casp8-ΔE7, which have only two death-effector domains, significantly activated NFκB by forming filament-like structures, which probably function as scaffolds for the IKK complex formation. In view of increasing levels of these abnormal CASP8 transcripts in primary PBMCs from HTLV-1 carriers and patients with ATL, we propose a possibility that overexpression of those Casp8 mutants, with lower proapoptotic activities and higher NFκB-activating functions than WT-Casp8, may be one of the molecular abnormalities causing malignant transformation and growth of ATL cells. IMPLICATIONS: We describe naturally occurring CASP8 transcription variants in PBMCs from patients with ATL, which encode truncated Casp8-mutant isoforms with lower proapoptotic activities and higher NFκB-activating functions compared with WT-Casp8.

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

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

H-103 Clinical landscape of adult T-cell leukemia: Its impacts on hematology, virology, and immunology

Prof. Takatsuki and his colleagues reported adult T-cell leukemia (ATL) as the distinct clinical entity based on the clinical features including the presence of leukemic cells with characteristic nuclear shape (flower cells), skin lesion, hypercalcemia and frequent opportunistic infections, and mature T-cell immunophenotyped (Uchiyama T, et al, Blood, 1977). Importantly, they already predicted that ATL is caused by the pathogen since the birth places of ATL patients accumulated in the southwestern Japan (Kyushu and Okinawa). In 1980, Prof. Gallo and his colleagues reported the first human retrovirus, human T-cell leukemia virus (HTLV), from a patient with cutaneous T-cell lymphoma who was later diagnosed as ATL. Thereafter, it was established that HTLV causes ATL. Since Prof. Gallo found another human retrovirus, the first human retrovirus was named human T-cell leukemia virus type 1 (HTLV-1). Detection of anti-HTV-1 antibody enables us to identify HTLV infected individuals, which discloses unique distribution of HTLV-1carriers in Japan and the world. In addition, serological studies disclosed clinical landscape of ATL. Based on clinical features, ATL is classified to 4 clinical subtypes: acute, chronic, lymphoma-type and smoldering types (Yamguchi K, et al, Blood, 1983). In addition, the familial clustering of ATL cases was prominent, suggesting that genetic or virological factors are involved in development of ATL. Serological analyses found that HTLV-1 mainly transmits from male to female in horizontal transmission. Another clinical feature of ATL is the frequent spontaneous remission, which suggests immunological mechanism. Early studies of HTLV-1 focused on Tax, which not only transactivates viral genes transcription from the plus strand, but also influences cellular genes. However, Prof. Takatsuki's group thought that clinical ATL samples contain clue of leukemogenic mechanisms by HTLV-1. We found that Tax was not expressed in half of ATL cases, and 3 mechanisms of inactivation of Tax expression: deletion of 5′ long terminal repeat (LTR), DNA methylation of 5′LTR, and genetic changes of the tax gene (nonsense mutation, deletion and insertion), indicating that Tax is not necessary for maintenance of ATL (Tamiya S, et al, Blood, 1996). However, pX region and 3′LTR remain intact in all ATL cases, indicating that this region is critical for ATL. Thereafter, we showed that HTLV-1 bZIP factor (HBZ), which is encoded by the minus strand, plays critical roles for pathogenesis by HTLV-1. ATL is the first human disease caused by retrovirus. Tremendous efforts on HTLV-1 research by many researchers contribute to studies of next human retrovirus, human immunodeficiency virus (HIV). Thus, discovery of ATL in association with identification of causative human retrovirus leads to great contribution to medical science.