Daisuke Koyama

Promoter methylation confers kidney-specific expression of the Klotho gene

The aging suppressor geneKlotho is predominantly expressed in the kidney irrespective of species. Because Klotho protein is an essential component of an endocrine axis that regulates renal phosphate handling, the kidney‐specific expression is biologically relevant; however, little is known about its underlying mechanisms. Here we provide in vitro and in vivo evidence indicating that promoter methylation restricts the expression of the Klotho gene in the kidney. Based on evolutionary conservation and histone methylation patterns, the region up to –1200 bp was defined as a major promoter element of the human Klotho gene. This region displayed promoter activity equally in Klotho‐expressing and ‐nonexpressing cells in transient reporter assays, but the activity was reduced to ~20% when the constructs were integrated into the chromatin in the latter. Both endogenous and transfected Klotho promoters were 30–40% methylated in Klotho‐nonexpressing cells, but unmethylated in Klotho‐expressing renal tubular cells. DNA demethylating agents increased Klotho expression 1.5‐ to 3.0‐fold in nonexpressing cells and restored the activity of silenced reporter constructs. Finally, we demonstrated that a severe hypomorphic allele of Klotho had aberrant CpG methylation in kl/kl mice. These findings might be useful in therapeutic intervention for accelerated aging and several complications caused by Klotho downregulation.—Azuma, M., Koyama, D., Kikuchi, J., Yoshizawa, H., Thasinas, D., Shiizaki, K., Kuro‐o, M., Furukawa, Y., Kusano, E. Promoter methylation confers kidney‐specific expression of the Klotho gene. FASEB J. 26, 4264–4274 (2012). www.fasebj.org

A Tet-On Inducible System for Controlling CD19-Chimeric Antigen Receptor Expression upon Drug Administration

On-target/off-tumor effects can be problematic if the antigen target for CAR T-cell therapy is also expressed on normal tissue. An inducible CAR expression strategy was used that controlled CAR expression and shows promise in discriminating between tumor and B-cell expression of CD19. T cells genetically modified with a CD19 chimeric antigen receptor (CD19CAR) are remarkably effective against B-cell malignancies in clinical trials. However, major concerns remain regarding toxicities, such as hypogammaglobulinemia, due to B-cell aplasia or severe cytokine release syndrome after overactivation of CAR T cells. To resolve these adverse events, we aimed to develop an inducible CAR system by using a tetracycline regulation system that would be activated only in the presence of doxycycline (Dox). In this study, the second-generation CD19CAR was fused into the third-generation Tet-On vector (Tet-CD19CAR) and was retrovirally transduced into primary CD8+ T cells. Tet-CD19CAR T cells were successfully generated and had minimal background CD19CAR expression without Dox. Tet-CD19CAR T cells in the presence of Dox were equivalently cytotoxic against CD19+ cell lines and had equivalent cytokine production and proliferation upon CD19 stimulation, compared with conventional CD19CAR T cells. The Dox(+) Tet-CD19CAR T cells also had significant antitumor activity in a xenograft model. However, without Dox, Tet-CD19CAR T cells lost CAR expression and CAR T-cell functions in vitro and in vivo, clearly segregating the “On” and “Off” status of Tet-CD19CAR cells by Dox administration. In addition to suicide-gene technology, controlling the expression and the functions of CAR with an inducible vector is a potential solution for CAR T-cell therapy–related toxicities, and may improve the safety profile of CAR T-cell therapy. This strategy might also open the way to treat other malignancies in combination with other CAR or TCR gene–modified T cells. Cancer Immunol Res; 4(8); 658–68. ©2016 AACR. See related Spotlight by June, p. 643.

Overexpression of the shortest isoform of histone demethylase LSD1 primes hematopoietic stem cells for malignant transformation.

Recent investigations indicate that epigenetic regulators act at the initial step of myeloid leukemogenesis by forming preleukemic hematopoietic stem cells (HSCs), which possess the increased self-renewal potential but retain multidifferentiation ability, and synergize with genetic abnormalities in later stages to develop full-blown acute myeloid leukemias. However, it is still unknown whether this theory is applicable to other malignancies. In this study, we demonstrate that lysine-specific demethylase 1 (LSD1) overexpression is a founder abnormality for the development of T-cell lymphoblastic leukemia/lymphoma (T-LBL) using LSD1 transgenic mice. LSD1 expression is tightly regulated via alternative splicing and transcriptional repression in HSCs and is altered in most leukemias, especially T-LBL. Overexpression of the shortest isoform of LSD1, which is specifically repressed in quiescent HSCs and demethylates histone H3K9 more efficiently than other isoforms, increases self-renewal potential via upregulation of the HoxA family but retains multidifferentiation ability with a skewed differentiation to T-cell lineages at transcriptome levels in HSCs. Transgenic mice overexpressing LSD1 in HSCs did not show obvious abnormalities but developed T-LBL at very high frequency after γ-irradiation. LSD1 overexpression appears to be the first hit in T-cell leukemogenesis and provides an insight into novel strategies for early diagnosis and effective treatment of the disease.

Proteasome inhibitors exert cytotoxicity and increase chemosensitivity via transcriptional repression of Notch1 in T-cell acute lymphoblastic leukemia

The Notch signaling pathway has been recognized as a key factor for the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), because of the high incidence of activating mutations of Notch1. Notch inhibition could serve as a new treatment strategy for T-ALL; however, the attempts to perturb Notch signaling pathways have been unsuccessful so far. In this study, we found that proteasome inhibitors exert cytotoxic effects on T-ALL cells with constitutive activation of Notch1 to a similar extent as myeloma cells. The proteasome inhibitor bortezomib repressed the transcription of Notch1 and downstream effectors including Hes1, GATA3, RUNX3 and nuclear factor-κB (NF-κB) (p65 and p50), coincided with downregulation of the major transactivator Sp1 and its dissociation from Notch1 promoter. Overexpression of the Notch1 intracellular domain (NICD) significantly ameliorated bortezomib-induced cytotoxicity against T-ALL cells. Drug combination studies revealed that bortezomib showed synergistic or additive effects with key drugs for the treatment of T-ALL such as dexamethasone (DEX), doxorubicin and cyclophosphamide, which were readily abolished by NICD overexpression. The synergy of bortezomib and DEX was confirmed in vivo using a murine xenograft model. Our findings provide a molecular basis and rationale for the inclusion of proteasome inhibitors in treatment strategies for T-ALL.

The Novel Orally Active Proteasome Inhibitor K-7174 Exerts Anti-myeloma Activity in Vitro and in Vivo by Down-regulating the Expression of Class I Histone Deacetylases

Background: Orally active proteasome inhibitors with novel mechanisms of action are in high clinical demand. Results: K-7174, a homopiperazine derivative with a unique mode of proteasome inhibition, exhibits anti-myeloma effects via transcriptional repression of class I histone deacetylases. Conclusion: K-7174 is an orally active proteasome inhibitor with a unique mechanism of action. Significance: K-7174 may compensate for conventional proteasome inhibitors in cancer treatment. Bortezomib therapy is now indispensable for multiple myeloma, but is associated with patient inconvenience due to intravenous injection and emerging drug resistance. The development of orally active proteasome inhibitors with distinct mechanisms of action is therefore eagerly awaited. Previously, we identified homopiperazine derivatives as a novel class of proteasome inhibitors with a different mode of proteasome binding from bortezomib. In this study, we show that K-7174, one of proteasome inhibitory homopiperazine derivatives, exhibits a therapeutic effect, which is stronger when administered orally than intravenously, without obvious side effects in a murine myeloma model. Moreover, K-7174 kills bortezomib-resistant myeloma cells carrying a β5-subunit mutation in vivo and primary cells from a patient resistant to bortezomib. K-7174 induces transcriptional repression of class I histone deacetylases (HDAC1, -2, and -3) via caspase-8-dependent degradation of Sp1, the most potent transactivator of class I HDAC genes. HDAC1 overexpression ameliorates the cytotoxic effect of K-7174 and abrogates histone hyperacetylation without affecting the accumulation of ubiquitinated proteins in K-7174-treated myeloma cells. Conversely, HDAC inhibitors enhance the activity of K-7174 with an increase in histone acetylation. These results suggest that class I HDACs are critical targets of K-7174-induced cytotoxicity. It is highly anticipated that K-7174 increases the tolerability and convenience of patients by oral administration and has the clinical utility in overcoming bortezomib resistance as a single agent or in combination with HDAC inhibitors.

Dexamethasone palmitate successfully attenuates hemophagocytic syndrome after allogeneic stem cell transplantation: macrophage-targeted steroid therapy

Hemophagocytic syndrome (HPS) induced by uncontrolled macrophage activation and subsequent graft failure is a frequent and prominent complication after allogeneic stem cell transplantation (allo-SCT), a cause of severe morbidity and death, and a therapeutic challenge. Liposome-incorporated dexamethasone, dexamethasone palmitate (DP), shows greater efficacy against macrophages as compared to dexamethasone sodium phosphate (DSP). Based on our findings that DP achieves significantly larger decrease than DSP on the viability of primary human macrophages compared in vitro, we tested the effects of DP in patients with HPS. A decrease in number of macrophages in the bone marrow and prevention of engraftment failure were observed in all patients without any severe complications. In conclusion, these data provide a rationale for testing DP as a first-line treatment for patients with HPS after allo-SCT.

Clinical significance of hemophagocytosis in BM clot sections during the peri-engraftment period following allogeneic hematopoietic SCT

The effects of macrophage activation on the outcome of allogeneic hematopoietic SCT (allo-HSCT) have yet to be fully examined. A total of 70 adult patients who received a first allo-HSCT for hematological diseases were studied. We counted the number of hemophagocytic cells in BM clot sections on day +14±7, and analyzed its impact on subsequent outcome. In all, 23 patients were diagnosed as having increased numbers of hemophagocytic cells (HP group), whereas 47 were not (non-HP group). The HP group was not associated with an increased incidence of acute or chronic GVHD, but was associated with worse hematopoietic recovery than the non-HP group. The 2-year OS for the HP group and the non-HP group was 30 and 65% (P<0.01), respectively, and 2-year non-relapse mortality was 48% and 27% (P<0.01), respectively. Multivariate analysis confirmed that the HP group was associated with a lower OS (hazard ratio (HR)=2.3; 95% confidence interval (CI), 1.0–5.4; P=0.048) and higher non-relapse mortality (HR=4.0; 95% CI, 1.6–9.9; P<0.01). The HP group had higher incidences of death due to graft failure (P<0.01) and endothelial complications, such as sinusoidal obstruction syndrome and transplant-associated microangiopathy (P=0.01). Macrophage activation is a previously unrecognized complication with negative impact on outcome of allo-HSCT.

Purine Analog-Like Properties of Bendamustine Underlie Rapid Activation of DNA Damage Response and Synergistic Effects with Pyrimidine Analogues in Lymphoid Malignancies

Bendamustine has shown considerable clinical activity against indolent lymphoid malignancies as a single agent or in combination with rituximab, but combination with additional anti-cancer drugs may be required for refractory and/or relapsed cases as well as other intractable tumors. In this study, we attempted to determine suitable anti-cancer drugs to be combined with bendamustine for the treatment of mantle cell lymphoma, diffuse large B-cell lymphoma, aggressive lymphomas and multiple myeloma, all of which are relatively resistant to this drug, and investigated the mechanisms underlying synergism. Isobologram analysis revealed that bendamustine had synergistic effects with alkylating agents (4-hydroperoxy-cyclophosphamide, chlorambucil and melphalan) and pyrimidine analogues (cytosine arabinoside, gemcitabine and decitabine) in HBL-2, B104, Namalwa and U266 cell lines, which represent the above entities respectively. In cell cycle analysis, bendamustine induced late S-phase arrest, which was enhanced by 4-hydroperoxy-cyclophosphamide, and potentiated early S-phase arrest by cytosine arabinoside (Ara-C), followed by a robust increase in the size of sub-G1 fractions. Bendamustine was able to elicit DNA damage response and subsequent apoptosis faster and with shorter exposure than other alkylating agents due to rapid intracellular incorporation via equilibrative nucleoside transporters (ENTs). Furthermore, bendamustine increased the expression of ENT1 at both mRNA and protein levels and enhanced the uptake of Ara-C and subsequent increase in Ara-C triphosphate (Ara-CTP) in HBL-2 cells to an extent comparable with the purine analog fludarabine. These purine analog-like properties of bendamustine may underlie favorable combinations with other alkylators and pyrimidine analogues. Our findings may provide a theoretical basis for the development of more effective bendamustine-based combination therapies.

Suitable drug combination with bortezomib for multiple myeloma under stroma-free conditions and in contact with fibronectin or bone marrow stromal cells

Several clinical trials have demonstrated the effectiveness of bortezomib in combination with various anti-myeloma agents; however, no definitive information is available regarding drugs best suited for use in combination with bortezomib. Using isobologram analysis, we investigated the combined effects of bortezomib with four key anti-myeloma drugs (melphalan, cyclophosphamide, doxorubicin and lenalidomide), which represent components of major bortezomib-based regimens with corticosteroids, in three myeloma cell lines (U266, RPMI8226 and KMS-12BM) under various conditions. Melphalan showed the best performance with bortezomib under all culture conditions tested (liquid culture, on fibronectin-coated plates, and co-culture with bone marrow stromal cells), whereas cyclophosphamide was antagonistic with bortezomib especially in the presence of stromal cells. Doxorubicin showed additive effects under stroma-free conditions and in contact with fibronectin, but was rather antagonistic in the presence of stromal cells. In contrast, lenalidomide exerted the most favorable effect with bortezomib in contact with stromal cells. Consistent with these results, caspase-3 was activated more strongly by melphalan than by other agents in combination with bortezomib. Moreover, bortezomib-induced up-regulation of CHOP was readily enhanced by lenalidomide in contact with stromal cells. The present findings may provide fundamental information for the selection of bortezomib-based regimens for myeloma patients.

Alkylating agents induce histone H3K18 hyperacetylation and potentiate HDAC inhibitor-mediated global histone acetylation and cytotoxicity in mantle cell lymphoma

Alkylating agents induce histone H3K18 hyperacetylation and potentiate HDAC inhibitor-mediated global histone acetylation and cytotoxicity in mantle cell lymphoma