Tomonari Takemura

The FOXM1 transcriptional factor promotes the proliferation of leukemia cells through modulation of cell cycle progression in acute myeloid leukemia

FOXM1 is an important cell cycle regulator and regulates cell proliferation. In addition, FOXM1 has been reported to contribute to oncogenesis in various cancers. However, it is not clearly understood how FOXM1 contributes to acute myeloid leukemia (AML) cell proliferation. In this study, we investigated the cellular and molecular function of FOXM1 in AML cells. The FOXM1 messenger RNA (mRNA) expressed in AML cell lines was predominantly the FOXM1B isoform, and its levels were significantly higher than in normal high aldehyde dehydrogenase activity (ALDH(hi)) cells. Reduction of FOXM1 expression in AML cells inhibited cell proliferation compared with control cells, through induction of G(2)/M cell cycle arrest, a decrease in the protein expression of Aurora kinase B, Survivin, Cyclin B1, S-phase kinase-associated protein 2 and Cdc25B and an increase in the protein expression of p21(Cip1) and p27(Kip1). FOXM1 messenger RNA (mRNA) was overexpressed in all 127 AML clinical specimens tested (n = 21, 56, 32 and 18 for M1, M2, M4 and M5 subtypes, respectively). Compared with normal ALDH(hi) cells, FOXM1 gene expression was 1.65- to 2.26-fold higher in AML cells. Moreover, the FOXM1 protein was more strongly expressed in AML-derived ALDH(hi) cells compared with normal ALDH(hi) cells. In addition, depletion of FOXM1 reduced colony formation of AML-derived ALDH(hi) cells due to inhibition of Cdc25B and Cyclin B1 expression. In summary, we found that FOXM1B mRNA is predominantly expressed in AML cells and that aberrant expression of FOXM1 induces AML cell proliferation through modulation of cell cycle progression. Thus, inhibition of FOXM1 expression represents an attractive target for AML therapy.

Transcriptional Repression of Cdc25B by IER5 Inhibits the Proliferation of Leukemic Progenitor Cells through NF-YB and p300 in Acute Myeloid Leukemia

The immediately-early response gene 5 (IER5) has been reported to be induced by γ-ray irradiation and to play a role in the induction of cell death caused by radiation. We previously identified IER5 as one of the 2,3,4-tribromo-3-methyl-1-phenylphospholane 1-oxide (TMPP)-induced transcriptional responses in AML cells, using microarrays that encompassed the entire human genome. However, the biochemical pathway and mechanisms of IER5 function in regulation of the cell cycle remain unclear. In this study, we investigated the involvement of IER5 in the cell cycle and in cell proliferation of acute myeloid leukemia (AML) cells. We found that the over-expression of IER5 in AML cell lines and in AML-derived ALDHhi (High Aldehyde Dehydrogenase activity)/CD34+ cells inhibited their proliferation compared to control cells, through induction of G2/M cell cycle arrest and a decrease in Cdc25B expression. Moreover, the over-expression of IER5 reduced colony formation of AML-derived ALDHhi/CD34+ cells due to a decrease in Cdc25B expression. In addition, over-expression of Cdc25B restored TMPP inhibitory effects on colony formation in IER5-suppressed AML-derived ALDHhi/CD34+ cells. Furthermore, the IER5 reduced Cdc25B mRNA expression through direct binding to Cdc25B promoter and mediated its transcriptional attenuation through NF-YB and p300 transcriptinal factors. In summary, we found that transcriptional repression mediated by IER5 regulates Cdc25B expression levels via the release of NF-YB and p300 in AML-derived ALDHhi/CD34+ cells, resulting in inhibition of AML progenitor cell proliferation through modulation of cell cycle. Thus, the induction of IER5 expression represents an attractive target for AML therapy.

Down-regulation of Thanatos-associated protein 11 by BCR-ABL promotes CML cell proliferation through c-Myc expression.

Bcr‐Abl activates various signaling pathways in chronic myelogenous leukemia (CML) cells. The proliferation of Bcr‐Abl transformed cells is promoted by c‐Myc through the activation of Akt, JAK2 and NF‐κB. However, the mechanism by which c‐Myc regulates CML cell proliferation is unclear. In our study, we investigated the role of Thanatos‐associated protein 11 (THAP11), which inhibits c‐Myc transcription, in CML cell lines and in hematopoietic progenitor cells derived from CML patients. The induction of THAP11 expression by Abl kinase inhibitors in CML cell lines and in CML‐derived hematopoietic progenitor cells resulted in the suppression of c‐Myc. In addition, over‐expression of THAP11 inhibited CML cell proliferation. In colony forming cells derived from CML‐aldehyde dehydrogenase (ALDH)hi/CD34+ cells, treatment with Abl kinase inhibitors and siRNA depletion of Bcr‐Abl induced THAP11 expression and reduced c‐Myc expression, resulting in inhibited colony formation. Moreover, overexpression of THAP11 significantly decreased the colony numbers, and also inhibited the expression of c‐myc target genes such as Cyclin D1, ODC and induced the expression of p21Cip1. The depletion of THAP11 inhibited JAK2 or STAT5 inactivation‐mediated c‐Myc reduction in ALDHhi/CD34+ CML cells. Thus, the induced THAP11 might be one of transcriptional regulators of c‐Myc expression in CML cell. Therefore, the induction of THAP11 has a potential possibility as a target for the inhibition of CML cell proliferation.

JmjC-domain containing histone demethylase 1B-mediated p15Ink4b suppression promotes the proliferation of leukemic progenitor cells through modulation of cell cycle progression in acute myeloid leukemia

The histone demethylase JHDM1B has been implicated in cell cycle regulation and tumorigenesis. In addition, it has been reported that JHDM1B is highly expressed in various human tumors, including leukemias. However, it is not clearly understood how JHDM1B contributes to acute myeloid leukemia (AML) cell proliferation. In this study, we investigated the cellular and molecular function of JHDM1B in AML cells. In AML cell lines and AML‐derived ALDHhi (high aldehyde dehydrogenase activity)/CD34+ cells, the levels of JHDM1B mRNA were significantly higher than in normal ALDHhi/CD34+ cells. Reduction of JHDM1B expression in AML cells inhibited cell proliferation compared to control cells, through induction of G1 cell cycle arrest, an increase in the p15Ink4b mRNA and protein expression. JHDM1B mRNA was overexpressed in all 133 AML clinical specimens tested (n = 22, 57, 34, and 20 for M1, 2, 4, and 5 subtypes respectively). Compared to normal ALDHhi/CD34+ cells, JHDM1B gene expression was 1.57‐ to 1.87‐fold higher in AML‐derived ALDHhi/CD34+cells. Moreover, the JHDM1B protein was more strongly expressed in AML‐derived ALDHhi/CD34+ cells from compared to normal ALDHhi/CD34+ cells. In addition, depletion of JHDM1B reduced colony formation of AML‐derived ALDHhi/CD34+ cells due to induction of p15Ink4b expression through direct binding to p15Ink4b promoter and loss of demethylation of H3K36me2. In summary, we found that JHDM1B mRNA is predominantly expressed in AML‐derived ALDHhi/CD34+ cells, and that aberrant expression of JHDM1B induces AML cell proliferation through modulation of cell cycle progression. Thus, inhibition of JHDM1B expression represents an attractive target for AML therapy.

Reduction of Raf Kinase Inhibitor Protein Expression by Bcr-Abl Contributes to Chronic Myelogenous Leukemia Proliferation

Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The Ras/Raf-1/MEK/ERK pathway is constitutively activated in Bcr-Abl-transformed cells, and Ras activity enhances the oncogenic ability of Bcr-Abl. However, the mechanism by which Bcr-Abl activates the Ras pathway is not completely understood. Raf kinase inhibitor protein (RKIP) inhibits activation of MEK by Raf-1 and its downstream signal transduction, resulting in blocking the MAP kinase pathway. In the present study, we found that RKIP was depleted in CML cells. We investigated the interaction between RKIP and Bcr-Abl in CML cell lines and Bcr-Abl+ progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of RKIP and reduced the pERK1/2 status, resulting in inhibited proliferation of CML cells. Moreover, RKIP up-regulated cell cycle regulator FoxM1 expression, resulting in G1 arrest via p27Kip1 and p21Cip1 accumulation. In colony-forming unit granulocyte, erythroid, macrophage, megakaryocyte, colony-forming unit-granulocyte macrophage, and burst-forming unit erythroid, treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced RKIP and reduced FoxM1 expressions, and inhibited colony formation of Bcr-Abl+ progenitor cells, whereas depletion of RKIP weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl+ progenitor cells. Thus, Bcr-Abl represses the expression of RKIP, continuously activates pERK1/2, and suppresses FoxM1 expression, resulting in proliferation of CML cells.

Small GTPase RAB45-mediated p38 activation in apoptosis of chronic myeloid leukemia progenitor cells

Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. BCR-ABL transforming activity is mediated by critical downstream signaling pathways that are aberrantly activated by tyrosine kinases. However, the mechanisms of BCR-ABL anti-apoptotic effects and the signaling pathways by which BCR-ABL influences apoptosis in BCR-ABL-expressing cells are poorly defined. In this study, we found that treatment with ABL kinase inhibitors or depletion of BCR-ABL induced the expression of RAB45 messenger RNA and protein and induced apoptosis via reduction of mitochondrial membrane potential and p38 activation in CML cell lines and BCR-ABL(+) progenitor cells from CML patients. Overexpressed RAB45 induced the activation of caspases-3 and -9 and reduced the expression of Survivin, XIAP, c-IAP1 and c-IAP2 in CML cells. Moreover, in colony-forming cells derived from CML-aldehyde dehydrogenase(hi)/CD34(+) cells, treatment with ABL kinase inhibitors induced RAB45 expression and reduced mitochondrial membrane potential, resulting in inhibited colony formation of Bcr-Abl(+) progenitor cells. The overexpression of RAB45 significantly decreased colony numbers and induced apoptosis through the activation of caspases-3 and -9. Furthermore, the overexpression of RAB45 increased the phosphorylation levels of p38, resulting in the induction of apoptosis and inhibition of proliferation of CML progenitor cells. Our results identify a new signaling molecule involved in BCR-ABL modulation of apoptosis and suggest that RAB45 induction strategies may have therapeutic utility in patients with CML.

Double-Expressor Lymphoma Is Associated with Poor Outcomes after Allogeneic Hematopoietic Cell Transplantation

Double-expressor lymphoma (DEL) is a diffuse large B cell lymphoma that exhibits co-expression of MYC and BCL2 proteins by immunohistochemistry. Patients with double-expressor lymphoma have a poor prognosis after standard chemoimmunotherapy or after high-dose chemotherapy with autologous transplantation, but the prognostic impact of DEL after allogeneic hematopoietic cell transplantation has not been well characterized. We retrospectively analyzed 60 consecutive patients with de novo diffuse large B cell lymphoma or transformed follicular lymphoma who underwent allogeneic transplantation at our center and had available immunohistochemistry data. Thirty-seven patients (62%) had DEL. The 2-year progression-free and overall survival rates were lower in patients with DEL than in those without DEL (20% versus 78%; overall P <.001 and 46% versus 77%; overall P = .016, respectively). The cumulative incidence of disease progression at 2 years was higher in patients with DEL (60% versus 13%; overall P = .005). The cumulative incidence of nonrelapse mortality did not differ statistically in the 2 groups. Even in patients with DEL and chemosensitive disease at transplantation, the 2-year progression-free survival rate was only 27% due to early disease progression. Multivariate analysis showed associations between DEL and increased risks of progression-free survival events (hazard ratio [HR], 4.58; 95% confidence interval [CI], 2.07-10.2; P <.001), overall mortality (HR, 2.29; 95% CI, 1.03-5.09; P = .042) and disease progression (HR, 3.60; 95% CI, 1.38-9.44; P = .009). Patients with DEL had poor outcomes after allogeneic transplantation. Innovative strategies are needed to improve outcomes in this population.

Beneficial impact of low-dose rabbit anti-thymocyte globulin in unrelated hematopoietic stem cell transplantation: focusing on difference between stem cell sources

Recently, Lee SJ et al. [1] showed that unrelated bone marrow transplantation (uBMT) was favored because patients who underwent BMT had less long-term complications such as chronic graft-versus-host disease (GVHD) than those who underwent unrelated peripheral blood stem cell transplantation (uPBSCT). In order to prevent longterm complications, it is crucial to establish a strategy to effectively prevent severe chronic GVHD after PBSCT without compromising overall survival (OS). Although anti-T lymphocyte globulin (ATG) might be an option to prevent chronic GVHD, there is still controversy regarding the benefit of ATG [2, 3]. The use of high ATG doses can lead to profound depletion of donor-derived T cells, which can increase the risk of viral infection and attenuate graft-vs.-leukemia effects. Since T-cell profiles vary significantly among stem cell sources, the optimal ATG dose might also differ. We previously reported that use of low-dose ATG-Thymoglobulin (ATG-T; median dose, 1.5 mg/kg) was associated with a lower incidence of severe/refractory chronic GVHD and superior GVHD-free, relapse-free survival (GRFS) in unrelated hematopoietic stem cell transplantation (HSCT), and 98% of patients received uBMT in this study [4]. To evaluate the impact of low-dose ATG-T on clinical outcomes in unrelated HSCT including both uBMT and uPBSCT, we performed a single-center, retrospective analysis of 232 patients with hematological disorders who received unrelated HSCT at the National Cancer Center Hospital from 2012 to 2016 (uBMT, n= 199; uPBSCT, n= 33). This study was approved by the Institutional Review Board of the National Cancer Center, Tokyo, Japan. In this cohort, all patients in the ATG-T group received rabbit ATG (Thymoglobulin; Genzyme-Sanofi, Lyon, France). Acute and chronic GVHD and GRFS were defined according to previously published criteria [5–7]. Chronic lung dysfunction was diagnosed with computed tomography and a respiratory function test according to Bacigalupo’s report [8]. The probability of OS and GRFS were calculated using the Kaplan–Meier method, and groups were compared using the log-rank test. The cumulative incidences of NRM, relapse, acute GVHD, chronic GVHD, and viral infection were calculated by Gray’s method. In the competing risk models for GVHD and viral infection, relapse and death before these events were defined as competing risks. In the competing risk models for NRM, relapse was defined as competing risk. The variables that were evaluated in these analysis were as follows: patient age at transplant (age ≥ 40 vs. age< 40), patient gender (male vs. female), ECOG performance status (0–1 vs. 2–4), disease status (CR vs. non-CR), disease risk index (DRI) (low vs. intermediate vs. high vs. very high) [9], hematopoietic cell transplantation-comorbidity index (HCT-CI) score (0 vs. 1–2 vs. ≥3) [10], intensity of the conditioning regimen (myeloablative conditioning [MAC] vs. reduced-intensity conditioning [RIC]), ATG as GVHD prophylaxis (yes vs. no), HLA disparity assessed by allele typing of HLA-A, B, C, and DRB1 in the GVH-direction (none vs. 1 allele vs. * Shigeo Fuji fujishige1231@gmail.com

Abstract 5053: Depletion of PHLPP1 and 2 by Bcr-Abl promotes CML cell proliferation

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC [Objective] PHLPP1 and 2 (pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase 1 and 2) is identified as the dephosphorylation enzyme of Akt, the same as that of PP2A of the dephosphorylation enzyme of Akt, and regulate the cell-growth signal of Akt through a dephosphorylation of the phosphorylated Akt (p-Akt). Previously, we reported the expression of PHLPP1 and 2 were suppressed in CML cell lines. In this study, we investigated the the CML and its progenitor cell proliferation through the phosphorylation of Akt by depletion of PHLPP1 and 2. [Method] CML cell lines (K562, Meg01, SHG3) and the CML clinical specimen (n= 10) were used for this research. The changes in the expression of PHLPP1 and 2 by treatment with Abl kinase inhibitors (STI571, ANM107 and BMS354825) or knock down with Bcr-Abl gene were evaluated by RT-PCR method. The influence on the expression of PHLPP1 and 2 in AML cell line transfected with Bcr-Abl also evaluated. CML progenitor cells derived from CML patients separated by ALDH activity, and the expression of PHLPP1 and 2 were investigated by quantitative RT-PCR method. p-Ser473 Akt1, 2 and 3 by Abl kinase inhibitor or knock down with PHLPP1 and 2 were measured, and the influence on the effect of cell growth inhibition by MTT assay. The expression of PHLPP1 and 2 and colony formation in colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM), colony forming unit-granulocyte, macrophage (CFU-GM) and burst forming unit-erythroid (BFU-E) derived from Bcr-Abl positive hematopoietic progenitor cells also evaluated. [Result] The expression of mRNA and protein of PHLPP1 and 2 were increased by treatment with Abl kinase inhibitor or Bcr-Abl knock down by siRNA in CML cell lines and AML cell line trasfected with Bcr-Abl gene. Moreover, p-Ser473 Akt 2 and 3 were decreased according to the expression of PHLPP1, and also p-Ser473 Akt1 and 3 according to the expression of PHLPP2. The Abl kinase inhibitors induced the expression of PHLPP1, 2 and the reduction of p-Ser473 Akt isoforms. The Abl kinase inhibitors inhibited the CML cell proliferation via the depletion of PHLPP1 and 2. In Bcr-Abl positive progenitor cells, the expression of PHLPP1 and 2 was increased, and colony formation was suppressed by Abl kinase inhibitor and by Bcr-Abl siRNA. The colony formation in progenitor cells knocked down PHLPP1 and 2 were decreased when treated with Abl kinase inhibitors. [Conclusion] These results suggest that Bcr-Abl might promote CML cell proliferarion through continuous phosphorylation of p-Ser473 Akt1, 2 and 3 by suppression of PHLPP1 and 2, and that the induction of PHLPP1 and 2 may be effective to regulate the cell proliferation in CML cells. It may be also that the induction of expression of PHLPPs has the potential possibility as the targets on the regulation of cell proliferation in Bcr-Abl positive progenitor cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5053.

Abstract 5060: Downregulation of THAP11 by Bcr-Abl promotes CML cell proliferation through c-Myc expression

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC [Background and Purpose] THAP11 (thanatos-associated protein 11) is one of the zinc-dependent, sequence-specific DNA-binding factors, which regulate cell proliferation, apoptosis and cell cycle. It has been reported that THAP11 is ubiquitously expressed in normal tissues and frequently downregulated in several tumors. In CML cells, we found that THAP11 expression was inhibited. We investigated the mechanisms of the suppression of THAP11 and its function in CML cell proliferation. [Methods] The cells used in this study were human CML cell lines, K562 and Meg01 cells. Primary CML cells were obtained from the bone marrow of CML (CP) patients. Human normal mononuclear cells (MNCs) were isolated from bone marrow of healthy volunteers after obtaining informed consents. For analysis of THAP11 mRNA expression, quantitative RT-PCR was performed in all cell lines treated with Abl kinase inhibitors (STI571, AMN107, and BMS354825). For proliferation analysis and the expression of c-Myc in CML cells, MTT assays, western blot and cell cycle analysis were performed in all cell lines transfected with Bcr-Abl siRNA, THAP11 siRNA, or THAP11 cDNA respectively. Moreover, THAP11 expression, c-Myc expression, and the colony counts of CFU-GEMM, CFU-GM, and BFU-E were analyzed in CML stem/progenitor cells transfected with Bcr-Abl siRNA or treated with Abl kinase inhibitors. [Results] In CML cell lines treated with Abl kinase inhibitors or transfected with Bcr-Abl siRNA, the expressions of THAP11 and c-Myc mRNA and protein were significantly increased compared to untreated cells. On the other hand, in CML cells transfected with the THAP11 cDNA, the c-Myc expression was suppressed. The overexpression of THAP11 induced G1 cell cycle arrest through p27 and p21 accumulation, and inhibited the CML cell proliferation. Moreover, in CML stem/progenitor cells obtained from patients with CML, the transfection with Bcr-Abl siRNA or treatment with Abl kinase inhibitors increased the expression of THAP11 mRNA and protein, and decreased the c-Myc expression and the counts of CFU-GEMM, CFU-GM and BFU-E. [Conclusion] Our results demonstrated that the Bcr-Abl suppressed the expression of THAP11, the depletion of THAP11 induced c-Myc expression, and induced the proliferation of CML cells through cell cycle progression. Moreover, induction of THAP11 expression inhibited the proliferation of CML stem/progenitor cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5060.