Adult T-cell Leukemia Cell Lines Research Articles

Extracellular CD30 and ADAM10/17 Regulate Brentuximab Vedotin-Induced Cell Death in an Adult T-Cell Leukemia Cell Line

Adult T-cell leukemia/lymphoma (ATL) is a human T-cell leukemia virus 1 (HTLV-1)-induced malignancy of mature T lymphocytes with poor outcomes. Brentuximab vedotin (BV), which is an anti-CD30 antibody conjugated with monomethyl auristatin E, is included in the treatment of CD30-positive ATL, but there is no useful therapeutic marker for BV. Soluble CD30 (sCD30) in serum is increased in aggressive-type ATL at diagnosis, but the effects of extracellular CD30 on BV-induced cell death in ATL remain unclear. Similarly, a disintegrin and metalloproteinase (ADAM) 10 and 17 have CD30 sheddase activity in anaplastic large cell lymphoma, but this activity is unknown in ATL. The present study showed that sCD30 concentrations were related to BV activity in ATL-related cell lines. Extracellular vesicles (EVs) such as exosomes containing CD30 also inhibited BV activity. Additionally, the knockdown of ADAM10/17 significantly reduced sCD30 concentrations with increased cell death by BV. Our results suggest that ADAM10/17 are involved in sCD30 production in ATL. Moreover, endogenous extracellular CD30, such as sCD30 shed by ADAM10/17, and CD30-positive EVs may be responsible for the BV-induced cell death. Correctively, extracellular CD30 concentrations, including EV, may be useful as biomarkers for BV therapy in ATL.

Mouse Models for HTLV-1 Infection and Adult T Cell Leukemia.

Adult T cell leukemia (ATL) is an aggressive hematologic disease caused by human T cell leukemia virus type 1 (HTLV-1) infection. Various animal models of HTLV-1 infection/ATL have been established to elucidate the pathogenesis of ATL and develop appropriate treatments. For analyses employing murine models, transgenic and immunodeficient mice are used because of the low infectivity of HTLV-1 in mice. Each mouse model has different characteristics that must be considered before use for different HTLV-1 research purposes. HTLV-1 Tax and HBZ transgenic mice spontaneously develop tumors, and the roles of both Tax and HBZ in cell transformation and tumor growth have been established. Severely immunodeficient mice were able to be engrafted with ATL cell lines and have been used in preclinical studies of candidate molecules for the treatment of ATL. HTLV-1-infected humanized mice with an established human immune system are a suitable model to characterize cells in the early stages of HTLV-1 infection. This review outlines the characteristics of mouse models of HTLV-1 infection/ATL and describes progress made in elucidating the pathogenesis of ATL and developing related therapies using these mice.

Targeted zinc-finger repressors to the oncogenic HBZ gene inhibit adult T-cell leukemia (ATL) proliferation.

Human T-lymphotropic virus type I (HTLV-I) infects CD4+ T-cells resulting in a latent, life-long infection in patients. Crosstalk between oncogenic viral factors results in the transformation of the host cell into an aggressive cancer, adult T-cell leukemia/lymphoma (ATL). ATL has a poor prognosis with no currently available effective treatments, urging the development of novel therapeutic strategies. Recent evidence exploring those mechanisms contributing to ATL highlights the viral anti-sense gene HTLV-I bZIP factor (HBZ) as a tumor driver and a potential therapeutic target. In this work, a series of zinc-finger protein (ZFP) repressors were designed to target within the HTLV-I promoter that drives HBZ expression at highly conserved sites covering a wide range of HTLV-I genotypes. ZFPs were identified that potently suppressed HBZ expression and resulted in a significant reduction in the proliferation and viability of a patient-derived ATL cell line with the induction of cell cycle arrest and apoptosis. These data encourage the development of this novel ZFP strategy as a targeted modality to inhibit the molecular driver of ATL, a possible next-generation therapeutic for aggressive HTLV-I associated malignancies.

Gamma-secretase inhibitor does not induce cytotoxicity in adult T-cell leukemia cell lines despite NOTCH1 expression

BackgroundActivated mutations in NOTCH1 are drivers of T-cell type acute lymphoblastic leukemia/lymphoma. The γ-secretase inhibitor (GSI), which suppresses the function of NOTCH1, is expected to be a molecular-targeted agent. NOTCH1 is also expressed in other malignant neoplasms. We aimed to determine the function of NOTCH1 expression and the effects of GSI on adult T-cell leukemia/lymphoma (ATL) caused by long-term human T-cell leukemia virus type I (HTLV-1) infection.MethodsWe analyzed the expression of NOTCH1 in six ATL- and HTLV-1-infected cell lines and investigated the influence of activated NOTCH1 (i.e., the cleaved form of NOTCH1) together with GSI on cell proliferation.ResultsActivated NOTCH1 found in ATL- and HTLV-1-infected cell lines was undetectable after incubation with GSI, regardless of Tax expression (HTLV-1-coded protein). Whole-exome sequencing revealed that activated NOTCH1 mutations were undetectable in six ATL- and HTLV-1-infected cell lines, regardless of abundant NOTCH1 expression. Moreover, GSI did not suppress the growth of ATL cell lines.ConclusionsThese findings suggested that NOTCH1 protein is constitutively activated but is likely a passenger during NOTCH1-mutation-negative ATL cell proliferation.

New halogenated C15 acetogenins from Okinawan sea hare Aplysia dactylomela

Two new C15 acetogenins 1 and 2, along with four known sesquiterpenes isolaurenisol (3), laurokamurene D (4), itomanol (5), and acetylelatol (6), were isolated from sea hare Aplysia dactylomela. The structures, including the relative stereochemistries, of 1 and 2 were determined based on spectroscopic evidence. Finally, the in vitro cytotoxicities of the six compounds against S1T cells, an adult T-cell leukemia cell line, were evaluated.

Impact of anti-diabetic sodium-glucose cotransporter 2 inhibitors on tumor growth of intractable hematological malignancy in humans

Under the dysfunction of mitochondria, cancer cells preferentially utilize both glycolytic and pentose phosphate pathways rather than electron transport chains to desperately generate adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH), classically recognized as the Warburg effect. Based on this background, the present study tested the hypothesis that anti-diabetic sodium-glucose cotransporter 2 (SGLT2) inhibitors would exert a tumor-suppressive impact on intractable human hematological malignancies via the modulation of glucose metabolism within cells and cell cycles. The level of mRNA for SGLT2 was remarkably elevated in leukemic cells from patients with adult T-cell leukemia (ATL), one of the most intractable blood cancers in humans, and as well as in two kinds of ATL cell lines (MT-1 and MT-2). Two kinds of SGLT2 inhibitors, Luseogliflozin and Tofogliflozin substantially suppressed the proliferation of MT-1 and MT-2 cells in both adherent and anchorage-independent culture conditions. Such a suppressive effect on tumor cell growth was reproduced by Luseogliflozin in leukemic cells in peripheral blood from patients with ATL. In MT-2 cells, both of SGLT2 inhibitors considerably attenuated glucose uptake, intracellular ATP levels, and NADPH production, resultantly enhancing cell cycle arrest at the G0/G1 phase. From the standpoint of metabolic oncology, the present study suggests that SGLT2 inhibitors would be a promising adjunctive option for the treatment of the most intractable human hematological malignancies like ATL.

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.

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.

Activation of PERK-ATF4-CHOP pathway as a novel therapeutic approach for efficient elimination of HTLV-1–infected cells

AbstractPatients with adult T-cell leukemia (ATL) exhibit a poor prognosis and overall survival rate when treated with standard chemotherapy, highlighting the continued requirement for the development of novel safe and effective therapies for human T-cell leukemia virus type 1 (HTLV-1)-related diseases. In this study, we demonstrated that MK-2048, a second-generation HIV-1 integrase (IN) inhibitor, potently and selectively kills HTLV-1–infected cells. Differential transcriptome profiling revealed significantly elevated levels of gene expression of the unfolded protein response (UPR) PKR-like ER kinase (PERK) signaling pathway in ATL cell lines following MK-2048 treatment. We also identified a significant downregulation in glucose regulated protein 78 (GRP78), a master regulator of the UPR in the CD4+CADM1+ HTLV-1–infected cell population of primary HTLV-1 carrier peripheral blood mononuclear cells (PBMCs) (n = 9), suggesting that HTLV-1–infected cells are hypersensitive to endoplasmic reticulum (ER) stress-mediated apoptosis. MK-2048 efficiently reduced proviral loads in primary HTLV-1 carrier PBMCs (n = 4), but had no effect on the total numbers of these cells, indicating that MK-2048 does not affect the proliferation of HTLV-1–uninfected PBMCs. MK-2048 specifically activated the ER stress–related proapoptotic gene, DNA damage-inducible transcript 3 protein (DDIT3), also known as C/EBP homologous protein (CHOP), in HTLV-1–infected but not uninfected cells of HTLV-1–carrier PBMCs. Our findings demonstrated that MK-2048 selectively induces HTLV-1–infected cell apoptosis via the activation of the UPR. This novel regulatory mechanism of the HIV IN inhibitor MK-2048 in HTLV-1–infected cells provides a promising prophylactic and therapeutic target for HTLV-1–related diseases including ATL.

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.

H-104 ATL, ADF and human virus research

Prehistory of ATL Discovery and ADF/TRX. More than 40 years have passed since our initial description of peculiar cases of adult-onset leukemia with abnormal cells having multi-convoluted nuclei and T cellproperties, frequent in the southern regions of Japan in the early 1970s. In the initiation of Adult T-Cell Leukemia (ATL) research, both of my mentors Drs. Kimishige Ishizaka and Kiyoshi Takatsuki have been involved. Dr. Kiyoshi Takatsuki was a kind of hero of our medical school in B cell malignancy multiple myeloma and Heavy Chain Diseases. Dr. Kimishige Ishizaka, Discoverer of Immunoglobulin E (IgE) reaginic antibody, and Dr. Takatsuki had been working in the field of Immunoglobulin and myeloma proteins with great pioneers including late H Kunkel, and EF Osserman. Before my first description of ATL Cases with Dr. Takatsuki and the active instruction of late Dr. Toru Masuda in the Institute for Virus Res (IVR) of Kyoto Univ. returning from the Late Dr. L Herzenberg in Stanford Univ., I had been involved in the study of peculior myeloma-related disease, POEMS Syndrome/Takatsuki Disease/Crow-Fukase Syndrome. The POEMS Syndrome are characterized by polyneuropathy, organomegaly, endocrinopathy, myeloma protein, and skin changes. Drs. Y. Nishitani and Late M. Fukase in Kyoto Univ. also contributed in this Takatsuki Disease/Syndrome research. Indeed, Dr. Takatsuki was a kind of hero of our medical school in B cell malignancy multiple myeloma and HeavyChain Diseases, before ATL and HTLV. Retrospectively, the study of adult T cell leukemia (ATL) and the related virus HTLV-I was a forerunner for all of human retrovirology, in which AIDS and the related retrovirus HIV were identified a few years later in the 1980s. I remember the great contribution of late Dr. Yohei Ito, who had initiated the cooperative interaction between Kyoto and Dr. Gallo in NCI in 70's. Using the anti-TAC monoclonal antibody generated by the late Dr. Takashi Uchiyama, my close colleague, during his stay in Dr. T. A. Waldmann's laboratory in NIH Bethesda, a cDNA encoding IL-2Ra chain was cloned by our group in Kyoto and by Waldmann's group in Bethesda. Abnormal IL-2Ra chain expression and the IL-2 dependency of ATL cell lines greatly contributed to the study of leukemogenesis of ATL. A new soluble factor named ADF/ATL-derived factor was also detected in ATL cell lines. Dr. Michiyuki Maeda greatly contributed establishing many ATL cell lines with his green thumb. After years of study, ADF proved to be a first human counterpart of thiol-related oxido-reductase thioredoxin/TRX, which opened the field of redox regulation of cell signaling involved in a variety of diseases. Quite interestingly, TRX-family member protein, GIF/MIF involved in the IgE regulation was characterized by the group of Dr. K. Ishizaka who have moved from J. Hopkins Univ. Baltimore to La Jolla, LIAI. Close interaction among Drs. Kimishige Ishizaka, Kiyoshi Takastuki and T. A. Waldmanns before ATL and HTLV-I study was an essential base for our initiation of ATL research with late Takashi Uchiyama and many other close colleagues. I remember Dr. Gallo's helpful comment on the importance of the 3 Letter naming of ATL and ADF. In the therapeutic directions of ATL, monoclonal antibody seems to be still struggle: (1) Anti-Tac IL-2Ra Chain Therapies have been geared by TA Waldmann Group with many coworkers. However, the therapeutic effectiveness may be limited; (2) Anti-CCR4 mAbs applied to ATL cases by Dr. R Ueda Group in Japan is therapeutically effective. However high frequency of severe side effect namely Stevens-Johnson Syndrome, Toxic Epidermal Necrolysis (SJS/TEN) have been reported. I may recall great contribution of late Drs. Takashi Uchiyama, Mitsuru Tsudo, Kazumasa Usami in Kyoto Univ. and Thomas Tursz of IGR Paris in our early ATL and ADF research. This paper is also dedicated for late Dr. Kimishige Ishizaka, discoverer of IgE and my great mentor close to Dr. Kiyoshi Takatsuki.

IL-2 and IL-15 blockade by BNZ-1, an inhibitor of selective γ-chain cytokines, decreases leukemic T-cell viability.

Interleukin-15 (IL-15) and IL-2 drive T-cell malignancies including T-cell large granular lymphocyte leukemia (T-LGLL) and HTLV-1 driven adult T-cell leukemia (ATL). Both cytokines share common γ-chain receptors and downstream signaling pathways. T-LGLL is characterized by clonal expansion of cytotoxic T cells and is associated with abnormal JAK/STAT signaling. ATL is an aggressive CD4+ T cell neoplasm associated with HTLV-1. T-LGLL and ATL share dependence on IL-2 and IL-15 for survival and both diseases lack effective therapies. BNZ-1 is a pegylated peptide designed to specifically bind the γc receptor to selectively block IL-2, IL-15 and IL-9 signaling. We hypothesized that treatment with BNZ-1 would reduce cytokine mediated proliferation and viability. Our results demonstrated that in vitro treatment of a T-LGLL cell line and ex vivo treatment of T-LGLL patient cells with BNZ-1 inhibited cytokine mediated viability. Furthermore, BNZ-1 blocked downstream signaling and increased apoptosis. These results were mirrored in an ATL cell line and in ex vivo ATL patient cells. Lastly, BNZ-1 drastically reduced leukemic burden in an IL-15-driven human ATL mouse xenograft model. Thus, BNZ-1 shows great promise as a novel therapy for T-LGLL, ATL and other IL-2 or IL-15 driven hematopoietic malignancies.

Inhibition of proliferation of adult T-cell leukemia cells by celastrol

We studied the effect of celastrol on the proliferation and apoptosis of adult T-cell leukemia (ATL) cells. After treating adult T-cell leukemia cell lines with different concentrations of celastrol, we analyzed the cell proliferation by MTT and colony formation assays. Flow cytometry was conducted to detect cell apoptosis by Annexin V-FITC/PI staining. Western blotting and dual-luciferase reporter assay were done to study the mechanism how celastrol suppressed the growth of adult T-cell leukemia cells. Celastrol could significantly inhibit the proliferation of adult T-cell leukemia cells, and induce apoptosis of ATL cells. With the increase of the concentration of celastrol, the ratio of Bax/Bcl-2 protein was up-regulated. The Caspase-3/7 protein was cleaved and activated after treatment with celastrol. Moreover, the expression of HTLV-1-encoded viral protein Tax was significantly inhibited in the celastrol treated cells. Taken together, these results indicated that celastrol effectively inhibited the proliferation of adult T-cell leukemia cells by regulating the expression of Bcl-2 family protein, and induced cell apoptosis by activating Caspase dependent pathway. In addition, celastrol could inhibit the expression of viral protein Tax. This study will provide an experimental basis for the clinical application of celastrol in the treatment of adult T-cell leukemia.

Complete Sequences of the Human T-Cell Leukemia Virus Type 1 Proviral Genomes from Newly Established Adult T-Cell Leukemia Cell Lines in Oita Prefecture, Japan.

ABSTRACTWe report two complete proviral genome sequences of human T-cell leukemia virus type 1 (HTLV-1) isolated from the peripheral blood specimens of acute type adult T-cell leukemia (ATL) patients in Oita Prefecture, Japan.

An activating mutation of interferon regulatory factor 4 (IRF4) in adult T-cell leukemia

The human T-cell leukemia virus-1 (HTLV-1) oncoprotein Tax drives cell proliferation and resistance to apoptosis early in the pathogenesis of adult T-cell leukemia (ATL). Subsequently, probably as a result of specific immunoediting, Tax expression is down-regulated and functionally replaced by somatic driver mutations of the host genome. Both amplification and point mutations of interferon regulatory factor 4 (IRF4) have been previously detected in ATL., K59R is the most common single-nucleotide variation of IRF4 and is found exclusively in ATL. High-throughput whole-exome sequencing revealed recurrent activating genetic alterations in the T-cell receptor, CD28, and NF-κB pathways. We found that IRF4, which is transcriptionally activated downstream of these pathways, is frequently mutated in ATL. IRF4 RNA, protein, and IRF4 transcriptional targets are uniformly elevated in HTLV-1-transformed cells and ATL cell lines, and IRF4 was bound to genomic regulatory DNA of many of these transcriptional targets in HTLV-1-transformed cell lines. We further noted that the K59R IRF4 mutant is expressed at higher levels in the nucleus than WT IRF4 and is transcriptionally more active. Expression of both WT and the K59R mutant of IRF4 from a constitutive promoter in retrovirally transduced murine bone marrow cells increased the abundance of T lymphocytes but not myeloid cells or B lymphocytes in mice. IRF4 may represent a therapeutic target in ATL because ATL cells select for a mutant of IRF4 with higher nuclear expression and transcriptional activity, and overexpression of IRF4 induces the expansion of T lymphocytes in vivo.

Development of sugar chain-binding single-chain variable fragment antibody to adult T-cell leukemia cells using glyco-nanotechnology and phage display method.

Adult T-cell leukemia (ATL) is an intractable blood cancer caused by the infection of human T-cell leukemia virus type-1, and effective medical treatment is required. It is known that the structure and expression levels of cell surface sugar chains vary depending on cell states such as inflammation and cancer. Thus, it is expected that the antibody specific for ATL cell surface sugar chain would be an effective diagnostic tool and a strong candidate for the development of an anti-ATL drug. Here, we developed a stable sugar chain-binding single-chain variable fragment antibody (scFv) that can bind to ATL cells using a fibre-type Sugar Chip and phage display method. The fiber-type Sugar Chips were prepared using O-glycans released from ATL cell lines. The scFv-displaying phages derived from human B cells (diversity: 1.04 × 108) were then screened using the fiber-type Sugar Chips, and an O-glycan-binding scFv was obtained. The flow cytometry analysis revealed that the scFv predominantly bound to ATL cell lines. The sugar chain-binding properties of the scFv was evaluated by array-type Sugar Chip immobilized with a library of synthetic glycosaminoglycan disaccharide structures. Highly sulphated disaccharide structures were found to have high affinity to scFv.

A New Guaiane-type Sesquiterpenoid from a Bornean Soft Coral, Xenia stellifera

A new guaiane-type sesquiterpenoid, 10β- O-methyl-1αH,5αH-guaia-6-en-4β-ol (1) along with two known compounds, 10- O -methyl alismoxide (2) and alismoxide (3) were isolated from a population of Bornean soft coral Xenia stellifera. The structure of this metabolite was elucidated based on spectroscopic data such as NMR and HRESIMS. These compounds were evaluated for their biological activity against adult T-cell leukemia cell line.

MAGI-1 expression is decreased in several types of human T-cell leukemia cell lines, including adult T-cell leukemia.

Membrane-associated guanylate kinase with inverted orientation protein 1 (MAGI-1) is a cytoplasmic scaffold protein that interacts with various signaling molecules; it negatively controls the cell growth of various types of cells and positively controls cell-cell interaction. In T cells, MAGI-1 has been shown to inhibit Akt activity through its interaction with PTEN and MEK1. In this study we found that MAGI-1 expression is decreased in multiple (9 out of 15) human T-cell leukemia cell lines, including adult T-cell leukemia (ATL), T-cell acute lymphoblastic leukemia and chronic T-cell lymphocytic leukemia. The overexpression of MAGI-1 protein in a MAGI-1-low ATL cell line reduced cellular growth. While the overexpression of MAGI-1 protein in a MAGI-1-low ATL cell line reduced the Akt and MEK activities, the knockdown of MAGI-1 in a MAGI-1-high ATL cell line augmented the Akt and MEK activities. Collectively, the findings of the present study suggest that the decreased expression of MAGI-1 in human T cells contributes to the development of several types of T-cell leukemia, partly through the stimulation of the Akt and MEK pathways.

NOTCH1 Activation Depletes the Pool of Side Population Stem Cells in ATL.

HTLV-I infection is associated with the development of adult T-cell leukemia (ATL), a malignancy characterized by a high rate of disease relapse and poor survival. Previous studies reported the existence of side population (SP) cells in HTLV-I Tax transgenic mouse models. These studies showed that these ATL-like derived SP cells have both self-renewal and leukemia renewal capacity and represent Cancer Stem Cells (CSC)/Leukemia-Initiating Cells (LIC). Since CSC/LIC are resistant to conventional therapies, a better characterization is needed. We isolated, sorted and characterized SP cells from uncultured PBMCs from ATL patients and from ATL patient-derived cell lines. We then identified several specific signaling pathways activated or suppressed in these cells. Expression of viral gene HBZ and Tax transcriptional activity was also investigated. Using gamma-secretase inhibitor (GSI, Calbiochem) and stably transduced ATL cell lines expressing TET-inducible NOTCH 1 intracellular domain (NICD), we characterized the role of activated NOTCH 1 in the maintenance of the SP cells in ATL. Our studies confirm the existence of SP cells in ATL samples. These cells demonstrate lower activation of NOTCH1 and Tax, and reduced expression of STAT3, β-catenin/Wnt3 and viral HBZ. We further show that PI3K and the NOTCH1 signaling pathway have opposite functions, and constitutive activation of NOTCH1 signaling depletes the pool of SP cells in ATL-derived cell lines. Our results suggest that in ATL, a balance between activation of the NOTCH1 and PI3K signaling pathway is the key in the control of SP cells maintenance and may offer therapeutic opportunities.

Development of Engineered T Cells Expressing a Chimeric CD16-CD3ζ Receptor to Improve the Clinical Efficacy of Mogamulizumab Therapy Against Adult T-Cell Leukemia.

Mogamulizumab (Mog), a humanized anti-CC chemokine receptor 4 (CCR4) mAb that mediates antibody-dependent cellular cytotoxicity (ADCC) using FcγR IIIa (CD16)-expressing effector cells, has recently been approved for treatment of CCR4-positive adult T-cell leukemia (ATL) in Japan. However, Mog failure has sometimes been observed in patients who have accompanying chemotherapy-associated lymphocytopenia. In this study, we examined whether adoptive transfer of artificial ADCC effector cells combined with Mog would overcome this drawback. We lentivirally gene-modified peripheral blood T cells from healthy volunteers and ATL patients expressing the affinity-increased chimeric CD16-CD3ζ receptor (cCD16ζ-T cells). Subsequently, we examined the ADCC effect mediated by those cCD16ζ-T cells in the presence of Mog against ATL tumor cells both in vitro and in vivo cCD16ζ-T cells derived from healthy donors killed in vitro Mog-opsonized ATL cell line cells (n = 7) and primary ATL cells (n = 4) depending on both the number of effector cells and the dose of the antibody. cCD16ζ-T cells generated from ATL patients (n = 3) also exerted cytocidal activity in vitro against Mog-opsonized autologous ATL cells. Using both intravenously disseminated model (n = 5) and subcutaneously inoculated model (n = 4), coadministration of Mog and human cCD16ζ-T cells successfully suppressed tumor growth in xenografted immunodeficient mice, and significantly prolonged their survival (P < 0.01 and P = 0.02, respectively). These data strongly suggest clinical feasibility of the novel combined adoptive immunotherapy using cCD16ζ-T cells and Mog for treatment of aggressive ATL, particularly in patients who are ineligible for allogeneic hematopoietic stem cell transplantation. Clin Cancer Res; 22(17); 4405-16. ©2016 AACR.