ATL Cells Research Articles

In Situ Hybridization of HBZ and Tax in FFPE Samples from ATLL Patients and Its Association with Clinicopathological Characteristics

IntroductionAdult T-cell leukemia/lymphoma (ATLL) is a mature T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). Many studies on HTLV-1-related mRNA including HTLV-1 bZIP factor (HBZ) and Tax have been performed, mainly using cell lines, patient-derived cells, and mice. However, there are scant data concerning HTLV-1-related mRNA in formalin-fixed, paraffin-embedded (FFPE) tissue samples. We detected HBZ and Tax mRNA on FFPE tissue samples using in situ hybridization (ISH), and investigated the association with clinicopathological characteristics.Materials and methodsEighty-seven biopsy samples from newly diagnosed ATLL patients were examined. The same samples were used in a previous study (Miyoshi et al. Blood, 2016). Use of patient materials and clinical information was approved by the Ethical Committee of Kurume University, in accordance with the Declaration of Helsinki.ISH was performed on FFPE tissue samples using RNAscope 2.5 HD Reagent Kit-BROWN (Advanced Cell Diagnostics, Hayward, CA). HBZ-and Tax-specific probes were used. MT-4 (HTLV-1 immortalized cell line) and Jurkat (T-cell acute lymphoblastic leukemia cell line) were used as positive and negative controls, respectively. Dot-like signals were counted at high magnification (40 diameters) on 10 randomly selected fields and the number of signals per 1,000 ATLL cells were calculated. The results were reviewed by two experienced hematopathologists. High expression was indicated when more than the median value of HBZ or Tax signals was stained. The antibodies for immunohistochemistry targeted CD4, CD30, Ki-67(MIB-1), CCR4, FoxP3, GATA3, IRF4, HLA class I, β2-microglobulin (β2M), PD-1, and PD-L1.Clinicopathological characteristics of ATLL patients were compared by Fisher's exact test (2-sided), Mann-Whitney's U test, and Spearman's rank correlation analysis. Overall survival was estimated by the Kaplan-Meier method and compared by the Log-rank test. P< .05 was considered statistically significant. EZR ver. 1.32 was used for all statistical analyses.ResultsIn the histogram and scatter plot of HBZ-ISH and Tax-ISH, the median values of HBZ signals and Tax signals were 806/1000 ATLL cells (range 0.4 - 4013.1) and 5.0/1000 ATLL cells (range 0.1 - 891.2), respectively (Figure 1). Representative samples of HBZ-ISH and Tax-ISH are presented in Figure 2A and 2B, respectively.Notably, the high-expression group of HBZ displayed significant reductions in skin lesions (P = .025), Ann Arbor stage (P = .021), and peripheral blood involvement (P = .028). The high-expression group of Tax displayed significant increases in lactate dehydrogenase activity (P = .0020), splenomegaly (P = .0070), CD 30 (P = .015), and PD-1 in tumor-infiltrating lymphocytes (PD-1_TIL; P <.0010).Weak but significant positive correlation was observed between HBZ and IRF4 (ρ = .33; P = .029), between HBZ and PD-L1 (ρ = -. 28; P = .010), between Tax and CD4 (ρ = .41; P = .0074), between Tax and CD30 (ρ = .26; P = .016), between Tax and GATA 3 (ρ = .26; P = .014), between Tax and PD-1 in ATLL cells (ρ = .25; P = .0074), between Tax and PD-L1 in ATLL cells (ρ =. 32; P = .0026), and between Tax and PD-1_TIL (ρ = .37; p = .0026) (Figure 3). There was almost no correlation between HBZ and Tax (ρ = .052; P = .63). There were no significant differences between HBZ or Tax and OS (Log-rank P = .21 and .95, respectively).ConclusionThe RNA scope assay is useful for detecting low expressing mRNA in FFPE tissue samples. Our data suggest that ISH of HBZ and Tax can detect differences in clinicopathological characteristics of ATLL patients. In clinical and cell samples, HBZ and Tax might be involved in the pathogenesis of ATLL.DisclosuresNo relevant conflicts of interest to declare. [Display omitted] DisclosuresAsano:Takeda: Honoraria; Chugai Pharma: Honoraria; Celgene: Honoraria.

North American ATLL has a distinct mutational and transcriptional profile and responds to epigenetic therapies

AbstractAdult T-cell leukemia lymphoma (ATLL) is a rare T cell neoplasm that is endemic in Japanese, Caribbean, and Latin American populations. Most North American ATLL patients are of Caribbean descent and are characterized by high rates of chemo-refractory disease and worse prognosis compared with Japanese ATLL. To determine genomic differences between these 2 cohorts, we performed targeted exon sequencing on 30 North American ATLL patients and compared the results with the Japanese ATLL cases. Although the frequency of TP53 mutations was comparable, the mutation frequency in epigenetic and histone modifying genes (57%) was significantly higher, whereas the mutation frequency in JAK/STAT and T-cell receptor/NF-κB pathway genes was significantly lower. The most common type of epigenetic mutation is that affecting EP300 (20%). As a category, epigenetic mutations were associated with adverse prognosis. Dissimilarities with the Japanese cases were also revealed by RNA sequencing analysis of 9 primary patient samples. ATLL samples with a mutated EP300 gene have decreased total and acetyl p53 protein and a transcriptional signature reminiscent of p53-mutated cancers. Most importantly, decitabine has highly selective single-agent activity in the EP300-mutated ATLL samples, suggesting that decitabine treatment induces a synthetic lethal phenotype in EP300-mutated ATLL cells. In conclusion, we demonstrate that North American ATLL has a distinct genomic landscape that is characterized by frequent epigenetic mutations that are targetable preclinically with DNA methyltransferase inhibitors.

A genetic IFN/STAT1/FAS axis determines CD4 T stem cell memory levels and apoptosis in healthy controls and Adult T-cell Leukemia patients

ABSTRACTAdult T-cell leukemia (ATL) is an aggressive, chemotherapy-resistant CD4+CD25+ leukemia caused by HTLV-1 infection, which usually develops in a minority of patients several decades after infection. IFN + AZT combination therapy has shown clinical benefit in ATL, although its mechanism of action remains unclear. We have previously shown that an IFN-responsive FAS promoter polymorphism in a STAT1 binding site (rs1800682) is associated to ATL susceptibility and survival. Recently, CD4 T stem cell memory (TSCM) Fashi cells have been identified as the hierarchical cellular apex of ATL, but a possible link between FAS, apoptosis, proliferation and IFN response in ATL has not been studied.In this study, we found significant ex vivo antiproliferative, antiviral and immunomodulatory effects of IFN-α treatment in short-term culture of primary mononuclear cells from ATL patients (n = 25). Bayesian Network analysis allowed us to integrate ex vivo IFN-α response with clinical, genetic and immunological data from ATL patients, thereby revealing a central role for FAS -670 polymorphism and apoptosis in the coordinated mechanism of action of IFN-α. FAS genotype-dependence of IFN-induced apoptosis was experimentally validated in an independent cohort of healthy controls (n = 20). The same FAS -670 polymorphism also determined CD4 TSCM levels in a genome-wide twin study (p = 7 × 10−11, n = 460), confirming a genetic link between apoptosis and TSCM levels. Transcriptomic analysis and cell type deconvolution confirmed the FAS genotype/TSCM link and IFN-α-induced downregulation of CD4 TSCM-specific genes in ATL patient cells.In conclusion, ex vivo IFN-α treatment exerts a pleiotropic effect on primary ATL cells, with a genetic IFN/STAT1/Fas axis determining apoptosis vs. proliferation and underscoring the CD4 TSCM model of ATL leukemogenesis.

HSP90 inhibitor 17-AAG plays an important role in JAK3/STAT5 signaling pathways in HTLV-1 infection cell line HUT-102

目的通过体外实验探索成人T细胞白血病（ATL）细胞内JAK/STAT信号通路的异常持续激活与HSP90蛋白的相关性。方法使用不同浓度17-烯丙胺-17-脱甲氧格尔德霉素（17-AAG）作用于HTLV-1感染ATL细胞株HUT-102，CCK8法检测细胞增殖，流式细胞术检测细胞凋亡，RT-PCR检测JAK3基因mRNA表达，Western blot检测JAK3、STAT5、p-STAT5、HSP90蛋白表达。结果HUT-102细胞株内HSP90蛋白表达量显著高于正常人外周血单个核细胞（P<0.05）。17-AAG能显著抑制HUT-102细胞增殖，17-AAG的浓度在0~2 000 nmol/L时，细胞存活率随着给药浓度的提高逐渐下降，17-AAG浓度在2 000~8 000 nmol/L范围时，细胞存活率差异无统计学意义（P>0.05）。细胞凋亡实验中，未刺激组细胞凋亡率为（9.23±0.67）%，刺激组凋亡率随着17-AAG浓度增加而显著增加，浓度2 000 nmol/L时凋亡率达到（87.82±2.46）%；但在不同浓度（0、250、500、1 000 nmol/L）抑制剂刺激HUT-102细胞24 h后，JAK3 mRNA的表达无明显变化。结论HTLV感染的ATL细胞株HUT-102细胞中，JAK3激酶作为HSP90的客体蛋白被异常活化，导致其下游信号蛋白p-STAT5异常持续性激活。17-AAG能显著下调JAK3、p-STAT5蛋白表达，抑制HUT-102细胞增殖、诱导凋亡。

Functional Impairment of Co-Inhibitory Receptors Promotes T-Cell Proliferation in HTLV-1 Associated Adult T-Cell Leukemia Cells

IntroductionAdult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1). It has been reported that co-inhibitory receptors such as programmed cell death 1 (PD-1) and T cell immunoglobulin and ITIM domain (TIGIT) are highly expressed on ATL cells and HTLV-1 infected cells. However, ATL cells and HTLV-1 infected cells proliferate regardless of their high expression. Although it has been known that HTLV-1 bZIP factor (HBZ), which is constitutively expressed in ATL cells and HTLV-1 infected cells, promotes T cell proliferation, the detailed mechanisms remain unclear. In this study, we found that HBZ promotes T-cell proliferation by interfering the suppressive function of co-inhibitory receptors.MethodsWe analyzed T-cell proliferation of HBZ transgenic (HBZ-Tg) mice that specifically express HBZ in CD4+ T cells, and expression of co-inhibitory and co-stimulatory molecules on ATL cells and CD4+ T cells of HBZ-Tg mice. Furthermore, the function of TIGIT and PD-1 was studied using HBZ-transduced murine CD4+T cells. The co-localization of SHP-2 and PD-1 in the presence of HBZ was analyzed by immunoprecipitation and confocal microscope. The immunoprecipitation and confocal microscope were also used to analyze interaction between HBZ and THEMIS and HBZ localization in the presence of THEMIS.ResultsAlthough HBZ promotes T-cell proliferation, we found that some co-inhibitory receptors, TIGIT and PD-1, were highly expressed on CD4+ T cells of HBZ-Tg mice and ATL cells. As mechanisms, HBZ upregulated transcriptions of these genes. Based on these observations, we hypothesized that HBZ impairs suppressive functions of TIGIT and PD-1 while it increases expression of these co-inhibitory receptors. To address this question, we analyzed the suppressive activity of TIGIT and PD-1 in the presence of HBZ. We transduced HBZ by the retrovirus vector into primary murine T cells and evaluated the proliferation after stimulated TIGIT or PD-1 with anti-CD3 and its ligand. As a result, TIGIT and PD-1 did not inhibit T-cell proliferation in the presence of HBZ, indicating that HBZ impairs the suppressive function of TIGIT and PD-1. Both TIGIT and PD-1 possess SHP-2, a tyrosine phosphatase, binding domains in its cytoplasmic tail, ITIM or ITSM motif. Therefore, we next studied whether HBZ influences the interaction between PD-1 and SHP-2. Tyrosine phosphorylation of PD-1 was induced with pervanadate and then SHP-2 recruitment and PD-1/SHP-2 co-localization were investigated. HBZ inhibited recruitment of SHP-2 to the ITSM motif of PD-1. Indeed, phosphorylation of SHP-2 was decreased in CD4+T cells of HBZ-Tg mice and HBZ-transduced murine primary T cells. Furthermore, function of SHP-2 to dephosphorylate ZAP-70 and CD3-zeta was suppressed in the presence of HBZ. These data showed that HBZ inhibited recruitment of SHP-2 to ITSM motif of PD-1 and suppressed its inhibitory function. Next, we examined how HBZ inhibits the interaction between PD-1 and SHP-2. HBZ did this by interacting with THEMIS, which forms a complex with Grb2 and SHP-2. Moreover, HBZ hindered the interaction between THEMIS and Grb2. In general, THEMIS is localized in the cytoplasm, whereas it has been reported that HBZ is localized in the nucleus. When we expressed THEMIS or HBZ, THEMIS existed in the cytoplasm (50 of 50 cells: 100%) whereas HBZ was mainly localized in the nucleus (67 of 74 cells: 90.5%). Interestingly, when both proteins were expressed in the same cells, HBZ changed its localization to cytoplasm (28 of 79 cells: 35.4%) and co-localized with THEMIS. These findings suggest that THEMIS changes the localization of HBZ from nucleus to cytoplasm. Thus, HBZ functions not only in the nucleus but also in the cytoplasm by interacting with host factors. Since THEMIS is expressed only in T-lineage cells, inhibition of the suppressive effects of co-inhibitory receptors by HBZ accounts for how HTLV-1 induced proliferation only T cells in vivo.ConclusionsOur findings demonstrated that HBZ promotes T-cell proliferation upon TCR stimulation by impairing the suppressive signal of co-inhibitory receptors. This study presents the first evidence of mechanisms how HBZ attenuates the inhibitory signals and promotes T-cell proliferation. DisclosuresNo relevant conflicts of interest to declare.

Mogamulizumab for ATLL in Clinical Practice

IntroductionAdult T-cell leukemia/lymphoma (ATLL) is an aggressive peripheral T cell neoplasm that is resistant to conventional chemotherapy and carries a poor prognosis. The effect of mogamulizumab, an immunoglobulin (Ig) G1 monoclonal antibody targeting CCR4 for ATLL cells, was reported in a previous phase 2 study in which mogamulizumab monotherapy was evaluated in relapsed ATLL patients. The overall response rate (ORR), median progression-free survival (PFS) and median overall survival (OS) were 50%, 5.2 and 13.7 months, respectively. It was not stated whether these values were derived in the real world or in clinical practice. Here we evaluate the clinical impact of mogamulizumab treatment in CCR-4-positive aggressive ATLL patients in clinical practice.Patients and methodsWe retrospectively analyzed 101 CCR-4-positive ATLL patients who received at least one cycle of mogamulizumab infusion between March, 2012 and April, 2016 in 7 facilities in Miyazaki prefecture, an HTLV-1 endemic area in Southwestern Japan. The ORR, PFS, OS and adverse effects (AEs) were evaluated. We next compared OS in patients with at least one course of mogamulizumab therapy with that in historical control patients without mogamulizumab therapy.ResultsOf the 101 patients, 92 were evaluable for treatment response, survival and AEs. The median age was 70 years old (range; 45 to 90), and 52 patients (51%) were more than 70 years old. According to Shimoyama's criteria, 66 patients were classified as acute type, 32 as lymphoma type, and 3 as chronic type. All 3 chronic-type ATLL patients had at least one unfavorable risk factor. Of the 101 patients, 96 had refractory or relapsed ATLL when mogamulizumab treatment was started, and the prior treatments consisted of VCAP-AMP-VECP, CHOP, DeVIC or CHASE therapy, with an average of 2 courses. In the 5 remaining cases, mogamulizumab was administered as the initial therapy for ATLL. Mogamulizumab was administered as monotherapy in 87 cases (86%), and as combination therapy with other drugs in 14 cases (14%).The ORR was 37%, including a complete remission rate of 19%. The median PFS and OS were 1.8 and 4.2 months, respectively. Among the 101 patients treated with mogamulizumab, only 26 (26%) fulfilled the inclusion criteria of the phase 2 clinical study. Among patients who met those inclusion criteria, the median PFS and OS were 6.0 and 8.4 months, respectively. The use of mogamulizumab improved OS in clinical practice. The median OS of patients receiving mogamulizumab therapy was 12 months, whereas that of patients who did not receive mogamulizumab in the historical cohort was 8.4 months. Hematologic toxicity and skin rash were the most common AEs, and both were manageableConclusionMogamulizumab therapy showed clinically meaningful activity in ATLL patients, with an acceptable toxicity in clinical practice. DisclosuresNo relevant conflicts of interest to declare.

Dysregulation of c-Myb Pathway by Aberrant Expression of Proto-oncogene MYB Provides the Basis for Malignancy in Adult T-cell Leukemia/lymphoma Cells.

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive human T-cell malignancy induced by human T-lymphotrophic virus-1 (HTLV-1) infection. The genetic alterations in infected cells that lead to transformation have not been completely elucidated, thus hindering the identification of effective therapeutic targets for ATL. Here, we present the first assessment of MYB proto-oncogene dysregulation in ATL and an exploration of its role in the onset of ATL. We investigated the expression patterns of MYB splicing variants in ATL. The molecular characteristics of the c-Myb-9A isoform, which was overexpressed in ATL cells, were examined using chromatin immunoprecipitation and promoter assays. We further examined the biologic impacts of abnormal c-Myb overexpression in ATL using overall c-Myb knockdown with shRNA or c-Myb-9A knockdown with morpholino oligomers. Both total c-Myb and c-Myb-9A, which exhibited strong transforming activity, were overexpressed in ATL cells in a leukemogenesis- and progression-dependent manner. Knockdown of either total c-Myb or c-Myb-9A induced ATL cell death. c-Myb transactivates nine genes that encode essential regulators of cell proliferation and NF-κB signaling. c-Myb-9A induced significantly stronger transactivation of all tested genes and stronger NF-κB activation compared with wild-type c-Myb. Our data demonstrate that c-Myb pathway overactivation caused by unbalanced c-Myb-9A overexpression is associated with disorders in cellular homeostasis and consequently, accelerated transformation, cell proliferation, and malignancy in ATL cells. These data support the notion of the c-Myb pathway as a promising new therapeutic target for ATL. Clin Cancer Res; 22(23); 5915-28. ©2016 AACR.

WRN-targeted therapy using inhibitors NSC 19630 and NSC 617145 induce apoptosis in HTLV-1-transformed adult T-cell leukemia cells.

BackgroundHuman T-cell leukemia virus type 1 (HTLV-1) infection is associated with adult T-cell leukemia/lymphoma (ATLL), a lymphoproliferative malignancy with a dismal prognosis and limited therapeutic options. Recent evidence shows that HTLV-1-transformed cells present defects in both DNA replication and DNA repair, suggesting that these cells might be particularly sensitive to treatment with a small helicase inhibitor. Because the “Werner syndrome ATP-dependent helicase” encoded by the WRN gene plays important roles in both cellular proliferation and DNA repair, we hypothesized that inhibition of WRN activity could be used as a new strategy to target ATLL cells.MethodsOur analysis demonstrates an apoptotic effect induced by the WRN helicase inhibitor in HTLV-1-transformed cells in vitro and ATL-derived cell lines. Inhibition of cellular proliferation and induction of apoptosis were demonstrated with cell cycle analysis, XTT proliferation assay, clonogenic assay, annexin V staining, and measurement of mitochondrial transmembrane potential.ResultsTargeted inhibition of the WRN helicase induced cell cycle arrest and apoptosis in HTLV-1-transformed leukemia cells. Treatment with NSC 19630 (WRN inhibitor) induces S-phase cell cycle arrest, disruption of the mitochondrial membrane potential, and decreased expression of anti-apoptotic factor Bcl-2. These events were associated with activation of caspase-3-dependent apoptosis in ATL cells. We identified some ATL cells, ATL-55T and LMY1, less sensitive to NSC 19630 but sensitive to another WRN inhibitor, NSC 617145.ConclusionsWRN is essential for survival of ATL cells. Our studies suggest that targeting the WRN helicase with small inhibitors is a novel promising strategy to target HTLV-1-transformed ATL cells.

Adult T-Cell Leukemia and Retinoid

Adult T-cell leukemia/lymphoma (ATL/ATLL) is an aggressive lymphoid disease caused by human T-cell leukemia virus type 1 (HTLV-1). It is reported that retinoid suppressed the proliferation of malignant cells including ATL cells. In this study, we showed the mechanism of retinoid action for ATL cells. We observed that NF-kB transcriptional activity as well as cell proliferation decreased in HTLV-1-positive T-cell lines by treatment with retinoid. Further, we observed that retinoid reduced HTLV-1 proviral DNA. Interestingly, retinoid significantly inhibited reverse transcriptase (RT) activity similar to azidothimidine (AZT) on HTLV-1-positive T-cell lines. Therefore, AZT was inhibitory of proviral DNA load but not NF-kB transcriptional activity on HTLV-I, however retinoid was inhibitory of both NF-?B and proviral DNA on HTLV-1. Furthermore, we showed cellular senescence in HTLV-I positive T-cell lines and in primary ATL cells obtained from acute ATL patients. The number of senescent cells significantly increased in the HTLV-I positive T-cell lines after treatment with retinoid, but not in the HTLV-I negative ones. These results indicated that retinoid could have three roles, as a NF-?B inhibitor, as a RT inhibitor and as a facilitating cellular senescence.

Polycomb-dependent epigenetic landscape in adult T-cell leukemia

AbstractAdult 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.

Recurrence of Psoriasis Vulgaris Accompanied by Treatment with C-C Chemokine Receptor Type 4 (CCR4) Antibody (Mogamulizumab) Therapies in a Patient with Adult T cell Leukemia/ Lymphoma: Insight into Autoinflammatory Diseases

Adult T cell leukemia / lymphoma (ATL) is one of the most aggressive hematological malignancies caused by human T-lymphotropic virus type-I (HTLV-1). Mogamulizumab is a new defucosylated humanized monoclonal antibody agent which targets C-C chemokine receptor type 4 (CCR4) expressed occasionally on the surface of ATL cells. However, adverse events such as drug eruptions have also been highlighted, at least in part, via the dysfunction of regulatory T cells (Tregs). We herein report a pronounced recurrence of systemic psoriasis vulgaris accompanied by the treatment of mogamulizumab in a patient with ATL. Pathological examinations may suggest a mechanistic link between the recurrence of autoinflammatory diseases and anti-CCR4 antibody therapies.

Carbonic Anhydrase IX Promotes the Tumorigenicity of Adult T-Cell Leukemia/Lymphoma-Derived Cells in NOG Mice

Carbonic anhydrase IX (CA9) is a membrane-associated carbonic anhydrase that regulates pH through its extra-cellular catalytic domain and thereby fosters tumor cell survival, growth, and invasion. The expression of CA9 is induced by hypoxia via HIF-1α. CA9 overexpression is known to be closely associated with aggressive behaviors and poor prognoses in various types of human solid tumors. In particular, CA9 is a critical mediator of the expansion of breast cancer stem cells in hypoxic niches by sustaining the mesenchymal and 'stemness' phenotypes of these cells. However, as for hematological malignancies, there are few reports suggesting a relation between its tumorigenecity and CA9. Here we demonstrated that CA9 plays a crucial role in promoting the tumorigenicity of the ATL-derived cells.Recently we established a highly tumorigenic subline, ST1-N6, from an ATL-derived cell line, ST1, by serial transplantation in immunodeficient NOG mice. ST1-N6 cells developed subcutaneous tumors under a shorter duration and smaller cell number than parental ST1 cells. DNA microarray gene expression analysis revealed an upregulated expression of CA9 in ST1-N6 cells as compared with ST1 cells. After serial sorting of ST1 cells by CA9 expression, we obtained ST1-CA9high and ST1-CA9low sublines. Like ST1-N6 cells, ST1-CA9high cells showed stronger tumorigenicity than ST1-CA9low or parental ST1 cells. Knockdown of CA9 expression with shRNAs suppressed the tumorigenicity of ST1-CA9high cells, whereas ST1 cells introduced with CA9 significantly showed enhanced tumorigenicity. Another ATL-derived cell line, TL-Om1 also showed increased tumorigenicity in NOG mice when introduced with CA9. These observations indicate that CA9 contributes to promoting the tumorigenic potential of ATL-derived cells in xenotransplantation.To investigate CA9 expression on fresh ATL cells, we prepared peripheral blood from six acute ATL patients, and tested for quantitative expression of CA9 in sera of six ATL patients and three healthy donors by ELISA. The amounts of serum CA9 were 31.1~95.4 pg/ml in the healthy donors, while they were 175.6~1, 971.6 pg/ml in the ATL patients. We next prepared ATL tissues from nine ATL patients, and two of them contained a few CA9high cells, which were cells expressing CD25, suggesting that a part of ATL lymphoma cells expressed CA9. These results suggested that the amount of serum CA9 tends to be higher in ATL patients than healthy donors.Based on these findings, we suppose that CA9 plays an important role in the malignant development of ATL. DisclosuresNo relevant conflicts of interest to declare.

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.

Anti-CCR4 antibody activates virus specific immune response in STLV-1 infected Japanese monkey

Transgenic mouse model is useful to study pathogenesis of viral genes like tax and HBZ. However, it is impossible to study the host immune responses by these transgenic mice. We used simian T-cell leukemia virus type 1 (STLV-1) infected Japanese monkeys (JMs) to analyze host immune responses after treatment by anti-CCR4 monoclonal antibody (mAb). In the previous study, we reported that anti-CCR4 mAb treatment in STLV-1 infected JMs significantly reduced CCR4 positive cells and STLV-1 proviral load (PVL). In this study, we investigated the long-term effect (48 weeks) on PVL and immune responses to STLV-1 Tax (sTax) and bZIP factor (SBZ) after the anti-CCR4 mAb treatment. STLV-1 PVL in the JMs was kept at the lower level than 0 week even 48 weeks later. To further investigate the antiCCR4 mAb induced antiviral effect, we analyzed regulatory T (Treg) cells and sTax and SBZ specific T-cell responses. Treg cells, which were CCR4 positive, were rapidly reduced after the treatment. The number of activated Treg cells (most functional population) was severely suppressed. Furthermore, anti-CCR4 mAb treatment of infected CD4 T cells from the JMs enhanced engulfment by phagocytes, which likely enhances antigen-presentation. Treated JMs showed spontaneous activation of cytotoxic T-lymphocytes (CTLs) to sTax and SBZ. However, the immune response to other antigen was not enhanced. In consistent with these data, the enhanced T-cell responses (anti-Tax and HBZ) were also observed in anti-CCR4 mAb treated ATL patients. Taken together, anti-CCR4 mAb induces CTLs to sTax/Tax and HBZ/SBZ by two different mechanisms: reduced Treg cells and enhanced antigen presentation by antibodydependent cell-mediated phagocytosis. These effects of anti-CCR4 mAb induce prolonged suppression of PVL, which might enable long-term control of ATL and HTLV-1 infected cells.

Novel small-molecule SIRT1 inhibitors induce cell death in adult T-cell leukaemia cells.

Adult T-cell leukaemia/lymphoma (ATL) is an aggressive T-cell malignancy that develops after long-term infection with human T-cell leukaemia virus (HTLV)-1. The identification of new molecular targets for ATL prevention and treatment is desired. SIRT1, a nicotinamide adenine dinucleotide+ -dependent histone/protein deacetylase, plays crucial roles in various physiological processes, including aging and apoptosis. We previously reported that ATL patients had significantly higher SIRT1 protein levels than healthy controls. Here, we demonstrate that two novel small-molecule SIRT1 inhibitors, NCO-01/04, reduced cell viability and enhanced apoptotic cells in peripheral blood monocyte cells of patients with acute ATL, which has a poor prognosis. NCO-01/04 also reduced the cell viability with DNA fragmentation, Annexin V-positive cells, and caspase activation. However, a caspase inhibitor did not inhibit this caspase-dependent cell death. NCO-01/04 enhanced the endonuclease G level in the nucleus with loss of the mitochondrial transmembrane potential, which can promote caspase-independent death. Interestingly, NCO-01/04 increased the LC3-II-enriched protein fraction, indicating autophagosome accumulation as well as autophagy. Thus, NCO-01/04 simultaneously caused caspase activation and autophagy. These results suggest that NCO-01/04 is highly effective against ATL cells in caspase-dependent or -independent manners with autophagy, and that its clinical application might improve the prognosis of patients with this fatal disease.

T memory stem cells are the hierarchical apex of adult T-cell leukemia

AbstractAdult T-cell leukemia (ATL) is a peripheral CD4+ T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). Despite several investigations using human specimens and mice models, the exact origin of ATL cells remains unclear. Here we provide a new insight into the hierarchical architecture of ATL cells. HTLV-1–infected cells and dominant ATL clones are successfully traced back to CD45RA+ T memory stem (TSCM) cells, which were recently identified as a unique population with stemlike properties, despite the fact that the majority of ATL cells are CD45RA–CD45RO+ conventional memory T cells. TSCM cells from ATL patients are capable of both sustaining themselves in less proliferative mode and differentiating into other memory T-cell populations in the rapidly propagating phase. In a xenograft model, a low number of TSCM cells efficiently repopulate identical ATL clones and replenish downstream CD45RO+ memory T cells, whereas other populations have no such capacities. Taken together, these findings demonstrate the phenotypic and functional heterogeneity and the hierarchy of ATL cells. TSCM cells are identified as the hierarchical apex capable of reconstituting identical ATL clones. Thus, this is the first report to demonstrate the association of a T-cell malignancy with TSCM cells.

A Gain-of-Function CCR4 Mutations in Adult T-Cell Leukemia/Lymphoma (ATLL) Enhance the Chemotactic Abilities and PI3K/AKT Activation

High expression of CC chemokine receptor 4 (CCR4) has been identified as a hallmark gene in ATLL, an aggressive peripheral T-cell neoplasm. CCR4 is a chemokine receptor, which has a critical role in immune cell trafficking including Th2, T-reg and skin-homing memory T-cells. CCR4 ligands, CCL17 and CCL22, were produced in lymph nodes and skin from dendritic cells, macrophages and Langerhans cells. Most ATLL cases express surface CCR4 (90%) and infiltrate to lymph nodes and skin. These observations suggest that CCR4 could have a role in ATLL biology, but it is still unclear whether dysregulation of CCR4 function contributes to ATLL pathogenesis.We performed RNA-Seq for two primary ATLL cases and discovered recurrent non-sense mutations in CCR4. Though an extended analysis using Sanger sequencing, CCR4 mutations were detected in 14/53 ATLL samples (26%) and consisted exclusively of nonsense or frameshift mutations that truncated the coding region at C329, Q330 or Y331 in the carboxy-terminus. We noticed that the location of the CCR4 mutations in ATLL were reminiscent of mutations affecting the chemokine receptor CXCR4 in the WHIM syndrome, a human immunodeficiency disease. Most CXCR4 mutations in the WHIM syndrome are nonsense or frameshift, resulting in carboxy-terminal truncation of the protein and conferring a gain-of-function phenotype with respect to chemotaxis towards the CXCR4 ligand SDF1. We therefore hypothesized that mutant CCR4 isoforms might enhance chemotaxis of the affected cells to CCR4 ligands.Chemotaxic assay using 32Db, a mouse myeloid cell line, and ED40515(+), an ATLL cell line, clarified that the ectopic expression of CCR4-Q330* enhanced the chemotactic ability of the transduced cells toward CCL17 and CCL22 rather than CCR4-WT transduced cells with statistical significance. To understand the mechanism of this enhanced chemotactic ability, we studied the change in surface CCR4 levels after CCL22 exposure in CCR4-WT-and CCR4-Q330*-reconstituted ED40515(+) cells. Compared with CCR4-WT, CCR4 internalization in CCR4-Q330*-reconstituted cells was significantly impaired. Thus, the ATLL CCR4 mutants impair desensitization by ligand, which likely contributes to the enhanced chemotaxis of cells bearing these mutants.We explored the influence of the ATLL CCR4 mutants on PI(3) kinase (PI3K)-dependent activation of AKT since it has been reported that binding of CCL22 to CCR4 activates AKT in CEM leukemic T-cells and in human Th2 cells. CCR4-Q330*-reconstituted ED40515(+) showed strong activation of AKT with CCL22 ligation compared with CCR4-WT-reconstituted cell. The AKT activation was cancelled with pan-PI3K inhibitor, BKM120, indicating that CCR4-mediated AKT activation was PI3K dependent.Lastly, we tested whether the acquisition of CCR4 mutations by ATLL cells imparts a selective growth advantage relative to cells with wild type CCR4. CCR4-Q330*-reconstituted cells had a selective growth advantage in the presence of CCL22, supporting at least in part the hypothesis that CCR4 mutation are able to provide the affected cells a positive selection pressure through CCL22 ligation and contributes to ATLL pathogenesis.We discovered for the first time recurrent somatic mutations in CCR4 in ATLL. CCR4 mutations were detected in 14/53 ATLL samples (26%) and consisted exclusively of nonsense or frameshift mutations that truncated the coding region at C329, Q330 or Y331 in the carboxy-terminus. Functionally, the CCR4-Q330* was a gain-of-function since it increased cell migration towards the CCR4 ligands CCL17 and CCL22, in part by impairing receptor internalization. This mutant enhanced PI(3) kinase/AKT activation following receptor engagement by CCL22 in ATLL cells, and conferred a growth advantage in in vitro cultures. Our findings provide a rationale to test whether inhibition of CCR4 signaling might have a therapeutic potential for patients with ATLL. DisclosuresNo relevant conflicts of interest to declare.

WS3-4 - New Treatment Modalities for Atl

ATL is a distinct malignancy of Treg/TH2 cells with caused by HTLV-1. ATL still has a worse prognosis than other T-cell malignancies in general. The diversity in clinical features and prognosis of patients with this disease has led to its subtype-classification into acute, lymphoma, chronic, and smoldering types. In case acute, lymphoma or unfavorable chronic subtypes (aggressive ATL), and favorable chronic or smoldering subtypes (indolent ATL), intensive chemotherapy followed by allogeneic stem cell transplantation and watchful waiting until disease progression has been recommended, respectively in Japan. A retrospective analysis suggested that the combination of interferon alpha and zidovudine was promising for the treatment of ATL, especially for leukemic subtypes. There is no plateau with an initial steep slope and subsequent gentle slope in the survival curves for aggressive and indolent ATL treated with chemotherapy and watchful waiting, respectively, although the prognosis is much better in the latter (MST; 1 year vs. 5 years), suggesting the need of refinement of subtype-classification. Defucosylated humanized anti-CCR4 monoclonal antibody (mogamulizumab), which showed strengthened ADCC activity as compared to usual Ab in preclinical analysis, was approved for the treatment of relapsed/refractory ATL based on the results of phase I and II studies, with response rate of about 50% and manageable toxicities including moderate to severe skin reaction, in Japan. Recently, the results of the subsequent randomized phase II study of chemotherapy +/- mogamulizumab for untreated patients with aggressive ATL was reported. This combination is anticipated because the former was more effective for ATL cells in lymphnode than those in peripheral blood and the latter was vice versa. The combination of mLSG15 plus mogamulizumab was well tolerated and produced a higher CR rate. Several new agent trials for ATL are ongoing and in preparation after translational research, including histone deacetylase inhibitors, a purine nucleoside phosphorylase inhibitor, a proteasome inhibitor and lenalidomide.

Preclinical efficacy of the synthetic retinoid ST1926 for treating adult T-cell leukemia/lymphoma

AbstractAdult T-cell leukemia/lymphoma (ATL) is an aggressive neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). The HTLV-1 oncoprotein Tax plays an important role in ATL pathogenesis. ATL carries a poor prognosis due to chemotherapy resistance, stressing the need for alternative therapies. Here, we investigate the preclinical efficacy of the synthetic retinoid ST1926 in ATL and peripheral T-cell lymphomas. Clinically achievable concentrations of ST1926 induced a dramatic inhibition of cell proliferation in malignant T-cell lines and primary ATL cells with minimal effect on resting or activated normal lymphocytes. ST1926 induced apoptosis, DNA damage, and upregulation of p53 proteins in malignant T cells, whereas it caused an early downregulation of Tax proteins in HTLV-1–positive cells. In murine ATL, oral treatment with ST1926 prolonged survival and reduced leukemia cell infiltration, white blood cell counts, and spleen mass. In spleens of ST1926-treated animals, p53 and p21 proteins were upregulated, poly (ADP-ribose) polymerase was cleaved, and Tax transcripts were reduced. These results highlight the promising use of ST1926 as a targeted therapy for ATL.

Alternative splicing signatures discriminate ATL cells from untransformed CD4+ counterparts deriving from HTLV-1 infected individuals

The clonal expansion and malignant transformation of HTLV-1 infected CD4+ T-cells have been linked to the reprogramming effects of HTLV-1 on host transcriptional profile. Coupled to transcription, alternative splicing (AS) is a post-transcriptional process that plays critical role in the complexity of transcriptome and splicing abnormalities frequently occur in cancer. To examine whether AS modifications associate with HTLV-1-associated leukemogenesis, we compared the exon expression profiles of ATL cells with that of CD4+ T-cell clones obtained by limited-dilution cloning of PBMC deriving from HTLV-1 carriers. 3 ATL cells and 12 untransformed infected clones clustering in infected, uninfected, PHA-stimulated or unstimulated CD4+ T cells were compared for exon RNA content using Exon Chip Human microarray. Hierarchical clustering analysis identified 12516 alternative spliced events (3642 genes) that clearly separated ATL samples from the 4 untransformed phenotypes mentioned above. In contrast, the exon content of 1539 genes differed between untransformed infected and uninfected T-CD4+ cells. Overall, less than 5% alternatively spliced genes were found differentially expressed at the transcriptional level. Microarray data were confirmed for 18 AS events using exon specific RT-PCR analysis. Pathway analysis of alternatively spliced genes (3642) in ATL cells revealed new AS-based pathways for p53 signaling, cell cycle and DNA replication while those of untransformed infected CD4+ T-cells were enriched in pathways for cellular movement and DNA repair. These findings unveil a new layer of complexity in the interplay between HTLV-1 and host cell gene expression machinery in which AS might play a central role in tumor initiation and promotion.