Comprehensive high-throughput meta-analysis of differentially expressed microRNAs in transcriptomic datasets reveals significant disruption of MAPK/JNK signal transduction pathway in Adult T-cell leukemia/lymphoma

Abstract Background Human T-lymphotropic virus 1 (HTLV-1) infection may lead to the development of Adult T-cell leukemia/lymphoma (ATLL). To further elucidate the pathophysiology of this aggressive CD4+ T-cell malignancy, we have performed an integrated systems biology approach to analyze previous transcriptome datasets focusing on differentially expressed miRNAs (DEMs) in peripheral blood of ATLL patients. Methods Datasets GSE28626, GSE31629, GSE11577 were used to identify ATLL-specific DEM signatures. The target genes of each identified miRNA were obtained to construct a protein-protein interactions network using STRING database. The target gene hubs were subjected to further analysis to demonstrate significantly enriched gene ontology terms and signaling pathways. Quantitative reverse transcription Polymerase Chain Reaction (RTqPCR) was performed on major genes in certain pathways identified by network analysis to highlight gene expression alterations. Results High-throughput in silico analysis revealed 9 DEMs hsa-let-7a, hsa-let-7g, hsa-mir-181b, hsa-mir-26b, hsa-mir-30c, hsa-mir-186, hsa-mir-10a, hsa-mir-30b, and hsa-let-7f between ATLL patients and healthy donors. Further analysis revealed the first 5 of DEMs were directly associated with previously identified pathways in the pathogenesis of HTLV-1. Network analysis demonstrated the involvement of target gene hubs in several signaling cascades, mainly in the MAPK pathway. RT-qPCR on human ATLL samples showed significant upregulation of EVI1, MKP1, PTPRR, and JNK gene vs healthy donors in MAPK/JNK pathway. Discussion The results highlighted the functional impact of a subset dysregulated microRNAs in ATLL on cellular gene expression and signal transduction pathways. Further studies are needed to identify novel biomarkers to obtain a comprehensive mapping of deregulated biological pathways in ATLL.

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Enhancer profiling identifies critical cancer genes and characterizes cell identity in adult T-cell leukemia

Blood ◽

10.1182/blood-2017-06-792184 ◽

2017 ◽

Vol 130 (21) ◽

pp. 2326-2338 ◽

Cited By ~ 26

Author(s):

Regina Wan Ju Wong ◽

Phuong Cao Thi Ngoc ◽

Wei Zhong Leong ◽

Alice Wei Yee Yam ◽

Tinghu Zhang ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

T Cell Activation ◽

Gene Expression Analysis ◽

Cell Activation ◽

Leukemia Cells ◽

Cancer Genes ◽

Cell Leukemia ◽

Adult T Cell Leukemia ◽

Key Points

Key Points Enhancer profiling combined with gene expression analysis identifies CCR4 and TIAM2 as critical cancer genes in ATL. Super-enhancers are enriched at genes involved in the T-cell activation pathway in ATL, reflecting the origin of leukemia cells.

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Assessment of HTLV-1 proviral load, LAT, BIM, c-FOS and RAD51 gene expression in adult T cell leukemia/lymphoma

Medical Microbiology and Immunology ◽

10.1007/s00430-017-0506-1 ◽

2017 ◽

Vol 206 (4) ◽

pp. 327-335 ◽

Cited By ~ 6

Author(s):

Samaneh Ramezani ◽

Abbas Shirdel ◽

Houshang Rafatpanah ◽

Mohammad Mehdi Akbarin ◽

Hanieh Tarokhian ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

Proviral Load ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Adult T Cell Leukemia ◽

Rad51 Gene

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Interleukin-10 gene expression in adult T-cell leukemia

Blood ◽

10.1182/blood.v88.3.1035.bloodjournal8831035 ◽

1996 ◽

Vol 88 (3) ◽

pp. 1035-1045 ◽

Cited By ~ 55

Author(s):

N Mori ◽

PS Gill ◽

T Mougdil ◽

S Murakami ◽

S Eto ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

Cell Lines ◽

Interleukin 10 ◽

Jurkat Cells ◽

Leukemic Cells ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Nf Kappa B ◽

Adult T Cell Leukemia

We studied the serum levels of interleukin-10 (IL-10), in patients with adult T-cell leukemia (ATL) caused by human T-cell leukemia virus type I (HTLV-I) infection. Elevated IL-10 levels were observed in 33 of 45 patients with ATL. Fresh leukemic cells from ATL patients as well as HTLV-I-infected T-cell lines MT-2, SLB-1, and C10/MJ expressed IL-10 mRNA by reverse transcription-polymerase chain reaction analysis, whereas IL-10 mRNA was not detected in normal peripheral mononuclear cells and an uninfected T-cell line Jurkat. IL-10 protein was also detected in the culture medium of leukemic cells from ATL patients as well as these HTLV-I-infected cell lines, and in the extracellular fluids of ATL patients. Interestingly, MT-4 cells, which did not express Tax although transformed by HTLV-I, did not express IL-10 at either the mRNA or protein level. To elucidate the role of the HTLV-I encoded transactivator Tax in IL-10 gene expression, Jurkat cells were transfected with a Tax expression plasmid. In transiently transfected Jurkat cells, endogenous IL-10 mRNA expression was induced by Tax. Stably transfected Jurkat cell lines expressed IL-10 mRNA and secreted IL-10 protein into the culture medium. The nuclear factor (NF)-kappa B pathway is a target for Tax transactivation. We treated MT-2 cells with phosphorothioate antisense oligonucleotides to the p65 subunit of NF- kappa B. A reduction in the expression of p65 was accompanied by a reduction in IL-10 gene expression and IL-10 production. We showed that the IL-10 kappa B-like sites ( kappa B1,-2,034 to -2,025; kappa B2, - 1,961 to -1,952; kappa B3, -452 to -443) specifically formed a complex with NF-kappa B-containing nuclear extract from MT-2 cells and that NF- kappa B bound with the highest affinity to the kappa B2 element (kappa B2 > kappa B3 > kappa B1). These data suggest a general role for NF- kappa B activation in the induction of IL-10 gene transcription. Activation of IL-10 in HTLV-I-infected cells may contribute to the pathology associated with HTLV-I infection.

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Novel Cellular Immunotherapy Using Allogeneic Vγ9/δ2-T Cells Gene-Modified to Express HTLV-1 P40Tax-Specific TCR for the Treatment of Adult T Cell Leukemia

Blood ◽

10.1182/blood-2019-122412 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3216-3216

Author(s):

Hiroshi Fujiwara ◽

Satoshi Okumura ◽

Yoshihiro Miyahara ◽

Linan Wan ◽

Isao Tawara ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Line ◽

Research Funding ◽

Cellular Immunotherapy ◽

Cell Leukemia ◽

Adult T Cell Leukemia ◽

Healthy Donors

Background: Adult T-cell leukemia/lymphoma (ATL) is a refractory peripheral T-cell malignancy caused by human T-lymphotropic virus type 1 (HTLV-1) infection. Although only allogeneic hematopoietic stem cell transplantation (allo-HSCT) displaying the graft-vs. ATL (GvATL) can bring durable remission, allo-HSCT is largely ineligible for newly diagnosed ATL patients due to disease aggressiveness and advanced age-related conditions. Thus, a novel treatment with safety and efficacy instead of allo-HSCT still remains an unmet need, and a cellular immunotherapy using TCR or CAR gene-modified immune cells exerting GvATL could be such an option. However, to generate those effector cells from autologous T cells of heavily pre-treated ATL patients faces many obstacles. To circumvent those hurdles, an employment of unconventional allogeneic Vγ9/δ2-T cells which are potentially free from the risk of GVHD could provide greater treatment opportunity for ATL patients due to highly extended donor availability. Taking above, here, we have newly devised an adoptive immunotherapy using a novel HTLV-1 p40Tax-specific TCR gene-modified allogeneic Vγ9/δ2 T cells against ATL. Methods: After written informed conscent, we firstly established novel HLA-A24 restricted TCR-α/β genes from HTLV-1 P40Tax301-309 (SFHSLHLLF)/HLA-A24 tetramer-positive peripheral CD8+T-lymphocytes of ATL patinets in durable remission using a single cell cloning method. Then, we confirmed that T cells gene-modified with these TCR-α/β genes exerted the epitope-specific and HLA-A24-restricted responses. Next, in order to achieve highly stable expression of this TCR-α/β heterodimer on gene-modified Vγ9/δ2-T cells, we newly developed a retroviral vector co-expressing TCR-α/β and CD8 α/β genes using self-cleaving P2A and E2A peptides. Using this vector, allogeneic Vγ9/δ2-T cells from healthy donors numerously expanded with high purity in our novel culture system were subjected to gene-transfer to express relevant TCR α/β complex. Thereafter, we asssessed target-reactive cytokine production and cytocidal activity mediated by those gene-modified allogeneic Vγ9/δ2-T cells both in vitro and in vivo. Finally, we additionally assessed a potential risk of GVHD using intravenous administration of another TCR gene-modified Vγ9/δ2 T-cells in vivo. Results: To start with PBMCs from healthy donors, allogeneic Vγ9/δ2-T cells were stably multiplied greater than thousandfold with a quite high purity (≥95%) using our novel bisphosphonate derivative PTA (tetrakis-pivaloyloxymethyl2-(thiazole-2-ylamino) ethylidene-1,1-bisphosphonate) combined with both 25 ng/ml of IL-7 and IL-15 in culture for 8 to 10 days. The stable expression of introduced TCR α/β heterodimer on Vγ9/δ2-T cells were successfully achieved by co-expression of CD8 α/β molecule. Those gene-modified Vγ9/δ2-T cells successfully recognized target peptide (SFHSLHLLF) in an HLA-A24 restricted fashion, and similarly demonstrated a cytocidal activity both in vitro and in vivo against HLA-A24 positive HTLV-1 infected cell lines (TL-Su and ILT#Hod), but not HLA-A24-negtive/Tax-positive cell line ILT#37 or HLA-A24-positve/Tax-negative cell line ATN-1. Furthermore, intravenously administered those TCR gene-modified Vγ9/δ2-T cells quickly and durably eradicated luciferase-gene modified TL-Su cells, but not ATN-1 cells in xenografted immunodeficient (NOG) mice, examined by in vivo imaging system. Finally, infused HLA-A2 restricted and NY-ESO-1 specific TCR (G50) gene-modified Vγ9/δ2-T cells exerted durable antitumor activity without causing GVHD using NOG mice xenografted with HLA-A2 positive melanoma cell line cells (NW-MEL-38). Conclusions: Our preclinical observations here obviously demonstrated the potential utility of TCR-α/β gene-modified allogeneic Vγ9/δ2-T cells for the treatment of ATL without causing GVHD. Further studies regarding biological behaviors of HTLV-1 Tax specific TCR-α/β gene-modified allogeneic Vγ9/δ2-T cells following target recognition in vivo are warranted, however, based on these lines of evidence and currently conducting assessments using clinical samples, we are planning to launch a novel clinical trial, particularly focusing on the applicability of HLA partially matched relative donors, as the source of gene-modified allogeneic Vγ9/δ2-T cells, which could highly extend the donor availability. Disclosures Fujiwara: BrightPath Biotherapeutics, Co.,Ltd.: Other: member of the department endowed by BrightPath Bio. Okumura:BrightPath Biotherapeutics, Co.,Ltd.: Other: member of the department endowed by BrightPth Bio.. Miyahara:BirghtPath Biotherapeutics, Co., Ltd.: Other: member of the department endowed by BrightPath Bio.. Wan:BrightPath Biotherapeutics, Co., Ltd.: Other: member of the department endowed by BrightPath Bio.. Tawara:Astellas Pharma: Research Funding; Ono Pharmaceutical: Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding. Shiku:BrightPath Biotherapeutics, Co., Ltd.: Other: Chair of the department endowed by BrightPath Bio..

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DNA Microarray Analysis of Stage Progression Mechanism in Adult T-Cell Leukemia.

Blood ◽

10.1182/blood.v104.11.2042.2042 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2042-2042

Author(s):

Young Lim Choi ◽

Kunihiro Tsukasaki ◽

Yasuyuki Onimaru ◽

Yasuaki Yamada ◽

Shimeru Kamihira ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

Dna Microarray ◽

Acute Stage ◽

Molecular Signature ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Adult T Cell Leukemia ◽

Stage Progression

Abstract Adult T-cell leukemia (ATL) is an intractable malignancy of peripheral CD4-positive T cells, in which human T-cell leukemia virus type I plays a pathological role. Clinical course of ATL can be subdivided into several phases; indolent “smoldering” and “chronic” stages, and aggressive “acute” stage. Individuals at the former usually undergo a stage progression toward the latter within several years, and ATL cells at the acute stage are highly refractory to current chemotherapeutic reagents. Molecular mechanisms underlying this stage progression are poorly understood yet. DNA microarray enables us to quantitate the mRNA amount for tens of thousands of genes simultaneously, and may provide novel insights into the pathogenesis as well as the stage progression mechanism of ATL. However, since the proportion of ATL cells within mononuclear cells (MNCs) of peripheral blood (PB) varies among different stages, it would be desirable to purify and directly compare ATL cells for an accurate profiling of gene expression. Given the fact that most PB MNCs are occupied by ATL cells in both chronic and acute stages, we purified CD4-positive T cells from PB of ATL individuals at chronic (n = 19) or acute (n = 22) stage. As a normal control, surface marker-matched T cells were purified from PB of healthy volunteers, and stimulated in vitro or not with PHA (n = 3 for each). A total of 47 specimens were thus subject to DNA microarray analysis with Affymetrix HGU133 A&B array sets, measuring the transcriptional level of ~33,000 human genes. Unsupervised hierarchical clustering based on the whole genes indicated that normal CD4-positive T cells, irrespective of PHA stimulation, had a molecular signature distinct from that of CD4-positive ATL cells, while the acute and chronic stages of ATL were not clearly separated from each other. Combination of a t-test (Welch’s ANOVA, P<0.001) and an effect size selection (>150 U) identified 46 genes, expression of which contrasted the two clinical stages of ATL. Correspondence analysis of such stage-associated genes also demonstrated visually that both phases of ATL have a distinct molecular signature. Additionally, a very high accuracy was obtained by an artificial neural network in a trial of gene expression-based stage diagnosis. Further, we tried to screen, from our data set, novel “acute stage-specific gene markers”, resulting in the identification of a gene for a cytokine receptor. Intriguingly, the serum concentration of its ligand protein was elevated in ATL patients, especially at the acute stage. This autocrine loop for cell growth would be an interesting candidate for the transforming events which triggers the stage-progression in ATL, and also be a candidate for the targets of novel ATL therapies.

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Functional Interactions of Ets1 with Viral and Cellular Proteins Mediate Ets1’s Effects on Transcription and the Pathogenesis of Adult T-Cell Leukemia/Lymphoma.

Blood ◽

10.1182/blood.v106.11.2608.2608 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2608-2608

Author(s):

Scott D. Gitlin ◽

Matthew D. Morse

Keyword(s):

Gene Expression ◽

T Cell ◽

Dna Binding ◽

Transcriptional Activity ◽

Viral Proteins ◽

Cellular Proteins ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Adult T Cell Leukemia ◽

Carboxy Terminal

Abstract Members of the ets gene family have been implicated in the pathogenesis of a variety of hematologic malignancies. The Ets1 proto-oncogene is a DNA-binding, sequence-specific transcriptional activator of several cellular and viral gene promoters, including the long terminal repeat (LTR) of the human T-lymphotropic virus type I (HTLV-I). Ets1 has the ability to transform both erythroid and myeloid progenitor cells and has been implicated in the formation of certain leukemias. In our studies to understand adult T-cell leukemia/lymphoma (ATL), we have previously shown that Ets1 cooperatively interacts with HTLV-I Tax1 to synergistically transactivate the HTLV-I LTR and cellular promoters. We hypothesize that Ets1’s transcriptional effects on viral and cellular gene expression contributes to the development and manifestations of ATL. The interaction between Ets1 and Tax1 raises the possibility that Ets1 plays a role in the transformation of HTLV-I-infected T-lymphocytes and in the clinical manifestations of ATL. However, little is known about how Ets1 regulates gene expression and participates in transformation events. Electrophoretic mobility shift assays demonstrate that although Ets1 can bind to specific DNA elements, binding to the HTLV-I LTR and other promoters is not necessary for Ets1’s transcriptional activity when Tax1 is present. Deletion mutagenesis of Ets1 identifies that the amino terminal, central and distal carboxy terminal domains of Ets1 individually increase transcription from the HTLV-I LTR. The DNA-recognition domain-containing proximal carboxy terminal region of Ets1 inhibits HTLV-I LTR transcription. Only wild type Ets1 and a protein consisting of the entire carboxy terminus bind to the HTLV-I LTR. Transcriptional activity of truncated Ets1 proteins is not dependent on their LTR-binding ability. The synergistic Ets1/Tax1 interaction appears to require the involvement of additional cellular proteins, including the cAMP response element binding protein (CREB). In transient transfections of CREB-deficient cells, functional CREB cooperatively interacts with Ets1 to transactivate the HTLV-I LTR. In the presence of CREB, Tax1 has an additive effect on Ets1 transactivation. CREB appears to interact with specific Ets1 domains in mediating transcriptional activity and may involve an NF-kB pathway. SP1 transactivation of the HTLV-I LTR is independent of Ets1 or Tax1. Transient transfection of SP1-deficient cells reveals that SP1 transactivation of the HTLV-I LTR is independent of Ets1 or Tax1. Ets1 has no transcriptional activity on the HTLV-I LTR in the absence of SP1. These results demonstrate that: (1) Ets1 contains multiple functional domains that likely interact in mediating transcription from the HTLV-I LTR and which are not dependent on DNA binding; (2) Ets1 requires the presence of specific cellular and/or viral proteins to mediate its activities; and (3) HTLV-I Tax1, CREB, possibly NF-kB, and perhaps other cellular proteins mediate Ets1’s transcriptional activity. Further elucidation of Ets1 interactions with cellular and viral proteins is important for understanding Ets1’s role in cellular and disease processes.

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