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•UCK2, a pyrimidine salvage enzyme, was overexpressed in HTLV-1–infected T cells and supported cell proliferation in ATL leukemogenesis.•Azacitidine-resistant cells with UCK2 loss adapted to pyrimidine salvage dysfunction by increasing de novo pyrimidine biosynthesis.

Adult T-cell leukemia-lymphoma (ATL) is an aggressive hematological malignancy of CD4+ T cells transformed by human T-cell lymphotropic virus-1 (HTLV-1). Most HTLV-1-infected individuals are asymptomatic, and only 3% to 5% of carriers develop ATL. Here, we describe the contribution of aberrant DNA methylation to ATL leukemogenesis. HTLV-1-infected T-cells and their uninfected counterparts were separately isolated based on CADM1 and CD7 expression status, and differentially methylated positions (DMPs) specific to HTLV-infected T cells were identified through genome-wide DNA methylation profiling. Accumulation of DNA methylation at hypermethylated DMPs correlated strongly with ATL development and progression. In addition, we identified 22 genes downregulated because of promoter hypermethylation in HTLV-1-infected T cells, including THEMIS, LAIR1, and RNF130, which negatively regulate T-cell receptor (TCR) signaling. Phosphorylation of ZAP-70, a transducer of TCR signaling, was dysregulated in HTLV-1-infected cell lines but was normalized by reexpression of THEMIS. Therefore, we hypothesized that DNA hypermethylation contributes to growth advantages in HTLV-1-infected cells during ATL leukemogenesis. To test this idea, we investigated the anti-ATL activities of OR-1200 and OR-2100 (OR21), novel decitabine (DAC) prodrugs with enhanced oral bioavailability. Both DAC and OR21 inhibited cell growth, accompanied by global DNA hypomethylation, in xenograft tumors established by implantation of HTLV-1-infected cells. OR21 was less hematotoxic than DAC, whereas tumor growth inhibition was almost identical between the 2 compounds, making it suitable for long-term treatment of ATL patient-derived xenograft mice. Our results demonstrate that regional DNA hypermethylation is functionally important for ATL leukemogenesis and an effective therapeutic target.

Adult T-cell leukemia-lymphoma (ATL) is an aggressive hematological malignancy of CD4+T cells transformed by human T-cell lymphotropic virus-1 (HTLV-1). Most HTLV-1–infected individuals are asymptomatic, and only 3% to 5% of carriers develop ATL. Here, we describe the contribution of aberrant DNA methylation to ATL leukemogenesis. HTLV-1–infected T-cells and their uninfected counterparts were separately isolated based on CADM1 and CD7 expression status, and differentially methylated positions (DMPs) specific to HTLV–infected T cells were identified through genome-wide DNA methylation profiling. Accumulation of DNA methylation at hypermethylated DMPs correlated strongly with ATL development and progression. In addition, we identified 22 genes downregulated because of promoter hypermethylation in HTLV-1–infected T cells, including THEMIS, LAIR1, and RNF130, which negatively regulate T-cell receptor (TCR) signaling. Phosphorylation of ZAP-70, a transducer of TCR signaling, was dysregulated in HTLV-1–infected cell lines but was normalized by reexpression of THEMIS. Therefore, we hypothesized that DNA hypermethylation contributes to growth advantages in HTLV-1–infected cells during ATL leukemogenesis. To test this idea, we investigated the anti-ATL activities of OR-1200 and OR-2100 (OR21), novel decitabine (DAC) prodrugs with enhanced oral bioavailability. Both DAC and OR21 inhibited cell growth, accompanied by global DNA hypomethylation, in xenograft tumors established by implantation of HTLV-1–infected cells. OR21 was less hematotoxic than DAC, whereas tumor growth inhibition was almost identical between the 2 compounds, making it suitable for long-term treatment of ATL patient-derived xenograft mice. Our results demonstrate that regional DNA hypermethylation is functionally important for ATL leukemogenesis and an effective therapeutic target.

Hypomethylating agents (HMA) are commonly used to treat high-risk myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML).Decitabine (DEC), one of the HMAs, is S phase-specifically incorporated into DNA to exert its effect, and sustained plasma concentration may increase its efficacy (Saunthararajah et al. J Clin Invest. 2015). Therefore, we developed OP-2100, an orally bioavailable prodrug of DEC. It showed equal efficacy to conventional HMAs in various blood cancer mouse models and exhibited a favorable safety profile in healthy mice (Watanabe et al. Blood. 2020; Ureshino et al. Mol Cancer Ther. 2021). In addition, it exhibited a sustained-release pharmacokinetic (PK) profile when orally administered to monkeys. In a phase I study, OP-2100 monotherapy showed the expected PK profile on patients with relapsed or refractory higher-risk MDS and CMML, and we reported the preliminary result.

The combination of venetoclax (VEN), an inhibitor of the anti-apoptotic factor BCL2, with the hypomethylating agent (HMA) decitabine (DEC) is the current frontline standard therapy for elderly patients with acute myeloid leukemia (AML). Despite the success of VEN/HMAs in inducing remission in AML, significant proportion of patients remain refractory to the combination therapy and others relapse after initial response.

Green tea is a popular world-wide beverage with health benefits that include preventive effects on cancer as well as cardiovascular, liver and Alzheimer’s diseases (AD). This study will examine the preventive effects on AD of a unique aroma of Japanese green tea. First, a transgenic Caenorhabditis elegans(C. elegans)CL4176 expressing human β-amyloid peptide (Aβ) was used as a model of AD. A hexane extract of processed green tea was further fractionated into volatile and non-volatile fractions, named roasty aroma and green tea aroma fractions depending on their aroma, by microscale distillation. Both hexane extract and green tea aroma fraction were found to inhibit Aβ-induced paralysis, while only green tea aroma fraction extended lifespan in CL4176. We also found that green tea aroma fraction has antioxidant activity. This paper indicates that the green tea aroma fraction is an additional component for prevention of AD.

Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature CD4+T cells caused by human T-cell lymphotropic virus type 1 (HTLV-1)-induced T-cell transformation. Following infection with HTLV-1, it takes several decades for HTLV-1 carriers to develop ATL. The prognosis of ATL remains poor despite several new agents are approved in the last few years. Recently, it has been noted that epigenetic abnormalities both DNA methylation and tri-methylation at histone H3Lys27 (H3K27me3) contribute to ATL leukemogenesis. Here, we investigated the effect of combination treatment with DNA demethylating agents (azacytidine (AZA), decitabine (DAC), and OR-2100 (OR21), which is silylated derivative of decitabine) and inhibitors of enhancer of zeste homolog 2 (EZH2) (EPZ-6438 and DS-3201b) which catalyze trimethylation of H3K27, in ATL. The combination of DAC and OR21 but not AZA with EZH inhibitors exhibited synergistic anti-ATL effects in vitroand in vivoconcomitant with DNA demethylation and reduction of H3K27me3. The combination induced gene expression reprogramming. Dual-specificity phosphatase 5 (DUSP5), an ERK-specific phosphatase, was identified as a key molecule that mediated the inhibitory effect of combination treatment by inactivating the ERK signaling pathway. DUSP5 was downregulated by DNA methylation and H3K27me3 accumulation in the promoter region in HTLV-1–infected cells from ATL patients during ATL leukemogenesis. The present results demonstrate that dual targeting of aberrant DNA and histone methylation synergistically suppresses tumor cell growth by restoring DUSP5, and the dual targeting of aberrant DNA and histone methylation is a feasible therapeutic approach for ATL.

By using Acoustic Doppler Current Profiler (ADCP) measurements with the four round-trips method to remove diurnal/semidiurnal tidal currents, the detailed current structure and volume transport of the Tsushima Warm Current (TWC) along the northwestern Japanese coast in the northeastern Japan Sea were examined in the period September–October 2000. The volume transport of the First Branch of the TWC (FBTWC) east of the Noto Peninsula was estimated as approximately 1.0 Sv (106m3/s), and the FBTWC continued to flow along the Honshu Island to the south of the Oga Peninsula. To the north of the Oga Peninsula, the Second Branch of Tsushima Warm Current and the eastward current established by the subarctic front were recombined with the FBTWC and the total volume transport increased to 1.9 Sv. The water properties at each ADCP line strongly suggested that most of the upper portion of the TWC with high temperature and low salinity flowed out to the North Pacific as the Tsugaru Warm Current. In the north of the Tsugaru Strait, the volume transport of the northward current was observed to be as almost 1 Sv. However, the component of the TWC water was small (approximately 0.3 Sv).By using Acoustic Doppler Current Profiler (ADCP) measurements with the four round-trips method to remove diurnal/semidiurnal tidal currents, the detailed current structure and volume transport of the Tsushima Warm Current (TWC) along the northwestern Japanese coast in the northeastern Japan Sea were examined in the period September–October 2000. The volume transport of the First Branch of the TWC (FBTWC) east of the Noto Peninsula was estimated as approximately 1.0 Sv (106m3/s), and the FBTWC continued to flow along the Honshu Island to the south of the Oga Peninsula. To the north of the Oga Peninsula, the Second Branch of Tsushima Warm Current and the eastward current established by the subarctic front were recombined with the FBTWC and the total volume transport increased to 1.9 Sv. The water properties at each ADCP line strongly suggested that most of the upper portion of the TWC with high temperature and low salinity flowed out to the North Pacific as the Tsugaru Warm Current. In the north of the Tsugaru Strait, the volume transport of the northward current was observed to be as almost 1 Sv. However, the component of the TWC water was small (approximately 0.3 Sv).

In the southwestern part of the Okhotsk Sea, oceanographic and sea-ice observations on board the icebreaker Soyawere carried out in February 1997. A mixed layer of uniform temperature nearly at the freezing point extending down to a depth of about 300 m was observed. This is much deeper than has previously been reported. It is suggested that this deep mixed layer originated from the north (off East Sakhalin), being advected along the shelf slope via the East Sakhalin Current, accompanied with the thick first-year ice (average thickness 0.6 m). This vertically uniform winter water, through mixing with the surrounding water, makes the surface water more saline (losing a characteristic of East Sakhalin Current Water) and the water in the 100–300 m depth zone less saline, colder, and richer in oxygen (a characteristic of the intermediate Okhotsk Sea water). The oceanographic structure and a heat budget analysis suggest that new ice zone, which often appears at ice edges, can be formed through preconditioning of thick ice advection and subsequent cooling by the latent heat release due to its melting.

We developed a system to measure the internal profile of small diameter hole. The minimum measurable diameter is 0.14 mm and the maximum measureable depth is 2 mm. The system can measure the roundness, straightness, cylindricity, and roughness. The main difficulty to measure the small diameter hole is in the relative positioning and aligning between hole and sensor probe. We discuss the principle of alignment, and also the calibration method for the sensor with fine fiber probe.

The purpose of this thesis is to clarify the frameworks of Japan's public policies toward distribution systems and marketing, and to analyze changes in these policies and their impacts on business. Inasmuch as the momentum provided by the SII talks has spawned a series of measures, the genereal drift of administrative policy has shifted dramatically in favor of deregulation and efforts to strengthen the nation's antimonopoly legislation. This paper is based not only on literature that is presented in the references, but also on much literature in Japanese and on primary data from private and public sectors that are not included in the references.

Kamachi: Ohara Pharmaceutical Co.: Research Funding. Ureshino:OHARA Pharmaceutical Co.: Research Funding. Yoshida:OHARA Pharmaceutical Co., Ltd.: Research Funding. Kurahashi:Ohara Pharmaceutical Co.: Employment. Watanabe:Ohara Pharmaceutical Co.: Research Funding. Okada:Japan Agency for Medical Research and Development: Research Funding; Bristol-Myers Squibb: Research Funding. Kimura:Ohara Pharmaceutical Co.: Research Funding; Novartis: Honoraria, Research Funding.

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disease induced by t(9;22)(q34;q11) translocation. The prognosis of patients with CML has dramatically improved since tyrosine kinase inhibitors (TKIs) were introduced, and recent studies show that approximately 40 to 50 % of CML patients achieved deep molecular response (DASISION and ENESTnd trial) within several years. However, therapeutic options for patients with CML who are resistant for TKIs are limited. Besides BCR-ABL kinase domain mutation, somatic mutations associated with epigenetic gene alteration (e.g. TET, DNMT3A) are reportedly involved in TKI resistance and disease progression. Thus, we investigated the efficacy of DNA demethylating agents in CML. Azacitidine (AZA) and decitabine (DAC), currently available as DNA demethylating agents, have low bioavailability for oral administration because they are easily degraded by cytidine deaminase. Then, we are developing a novel demethylating agent, OR21, with possible oral absorbability as a prodrug of DAC. In vivoanalysis using cynomolgus monkeys, the area under the curve of DAC after intraduodenally administration of DAC (1.5mg/kg, 6.6µmol/kg) or OR21 (2.25mg/kg, 6.6µmol/kg) was 0.01 µM·h, 0.298 µM·h respectively, indicating OR21 have high oral absorbability.

Watanabe: OHARA Pharmaceutical Co., Ltd.: Research Funding. Ureshino:OHARA Pharmaceutical Co., Ltd.: Research Funding. Kurahashi:OHARA Pharmaceutical Co., Ltd.: Employment. Fukuda-Kurahashi:OHARA Pharmaceutical Co., Ltd.: Employment. Kimura:OHARA Pharmaceutical Co., Ltd.: Research Funding.

Ureshino: OHARA Pharmaceutical Co., Ltd.: Research Funding. Watanabe:OHARA Pharmaceutical Co., Ltd.: Research Funding. Kurahashi:OHARA Pharmaceutical Co., Ltd.: Employment. Kurahashi:OHARA Pharmaceutical Co., Ltd.: Employment. Ureshino:OHARA Pharmaceutical Co., Ltd.: Research Funding.

Aberrant DNA methylation profiles in various types of cancer have highlighted the importance of DNA methylation in human carcinogenesis and opened the prospect of targeting aberrant DNA methylation with demethylating agents as a therapy for cancer. Adult T-cell leukemia-lymphoma (ATL) is an aggressive hematological malignancy derived from CD4 (+) T-cells transformed by human T-cell lymphotropic virus-1 (HTLV-1). Although it has been estimated that 20 million people are infected with HTLV-1 worldwide, most HTLV-1 infected individuals have no symptoms and only 3-7% of HTLV-1 positive carriers develop ATL in their life time. HTLV-1 infected cells undergo multistep leukemogenesis and there are four clinical subtypes of ATL, smoldering type, chronic type, acute type, and lymphoma type. It takes several decades to acquire aggressive phenotypes through the accumulation of genetic and epigenetic abnormalities. In this study, we aimed to elucidate the contribution of aberrant DNA methylation to ATL leukemogenesis and the anti-ATL effects of DNA demethylating agents.

Myelodysplastic syndromes (MDS) is a clonal disorder caused by mutation in hematopoietic stem cells (HSCs), including several somatic mutations and aberrant DNA hypermethylation, which can induce silence of tumor-suppressor genes and lead to the pathogenesis and/or progression of MDS. Thus, reducing methylation with the aim of genes reestablishing the expression of silenced tumor-suppressor genes represents one promising approach for the treatment of MDS. Azacytidine (AZA) and/or decitabine (DAC), two DNA methyltransferase (DNMT) inhibitors currently available for the treatment of MDS, have dramatically improved the prognosis of MDS. However, these DNMT inhibitors must be administered intravenously or subcutaneously, necessitating that patients must visit a hospital every day for treatment. Thus, oral DNMT inhibitors are desired. OR21 is a novel and potentially oral-absorbable DAC prodrug with an oral absorbability of +2.14 (Log P value).

ABSTRACTAlthough the anti-CCR4 antibody mogamulizumab (moga) shows striking antitumor activity against adult T cell leukemia (ATL), it can also cause fatal immunological pathology such as severe skin rash and graft-versus-host disease, which might be attributed to depletion of CCR4+regulatory T cells. We previously showed that next generation sequencing enables precise analysis of the T cell receptor (TCR) repertoire, and we here used the technique to reveal the immunological dynamics in moga-treated ATL patients. Treatment with moga resulted in remarkable reduction or elimination of clonal cells, and enhanced reconstitution of non-tumor polyclonal CD4+T cells and oligoclonal CD8+T cells. Interestingly, cutaneous T cells infiltrating moga-related skin rashes did not share the same major clones in peripheral blood, which minimizes the possibility of cross-reaction. Thus, deep sequencing of the TCR can reveal the immune reconstitution of moga-treated ATL and provides powerful insights into its mode of action.

To understand the regulation of Helicobacter pylori (H. pylori)-associated gastric carcinogenesis, we examined the effect of B-cell translocation gene 2 (BTG2) expression on the biological activity of Tipα, an oncoprotein secreted from H. pylori. BTG2, the human ortholog of mouse TIS21 (BTG2/TIS21), has been reported to be a primary response gene that is transiently expressed in response to various stimulations. Here, we report that BTG2 is constitutively expressed in the mucous epithelium and parietal cells of the gastric gland in the stomach. Expression was increased in the mucous epithelium following H. pylori infection in contrast to its loss in human gastric adenocarcinoma. Indeed, adenoviral transduction of BTG2/TIS21significantly inhibited Tipα activity in MKN-1 and MGT-40, human and mouse gastric cancer cells, respectively, thereby downregulating tumor necrosis factor-α (TNFα) expression and Erk1/2 phosphorylation by reducing expression of nucleolin, a Tipα receptor. Chromatin immunoprecipitation proved that BTG2/TIS21inhibited Sp1 expression and its binding to the promoter of the nucleolin gene. In addition, BTG2/TIS21expression significantly reduced membrane-localized nucleolin expression in cancer cells, and the loss of BTG2/TIS21expression induced cytoplasmic nucleolin availability in gastric cancer tissues, as evidenced by immunoblotting and immunohistochemistry. Higher expression of BTG2 and lower expression of nucleolin were accompanied with better overall survival of poorly differentiated gastric cancer patients. This is the first report showing that BTG2/TIS21inhibits nucleolin expression via Sp1 binding, which might be associated with the inhibition of H. pylori-induced carcinogenesis. We suggest that BTG2/TIS21is a potential inhibitor of nucleolin in the cytoplasm, leading to inhibition of carcinogenesis after H. pylori infection.