Takaaki Konuma

Dependency on the polycomb gene Ezh2 distinguishes fetal from adult hematopoietic stem cells

Polycomb-group (PcG) proteins are essential regulators of hematopoietic stem cells (HSCs). In contrast to Bmi1, a component of Polycomb repressive complex 1 (PRC1), the role of PRC2 and its components in hematopoiesis remains elusive. Here we show that Ezh2, a core component of PRC2, is essential for fetal, but not adult, HSCs. Ezh2-deficient embryos died of anemia because of insufficient expansion of HSCs/progenitor cells and defective erythropoiesis in fetal liver. Deletion of Ezh2 in adult BM, however, did not significantly compromise hematopoiesis, except for lymphopoiesis. Of note, Ezh2-deficient fetal liver cells showed a drastic reduction in trimethylation of histone H3 at lysine 27 (H3K27me3) accompanied by derepression of a large cohort of genes, whereas on homing to BM, they acquired a high level of H3K27me3 and long-term repopulating capacity. Quantitative RT-PCR revealed that Ezh1, the gene encoding a backup enzyme, is highly expressed in HSCs/progenitor cells in BM compared with those in fetal liver, whereas Ezh2 is ubiquitously expressed. These findings suggest that Ezh1 complements Ezh2 in the BM, but not in the fetal liver, and reveal that the reinforcement of PcG-mediated gene silencing occurs during the transition from proliferative fetal HSCs to quiescent adult HSCs.

3-Deazaneplanocin A is a promising therapeutic agent for the eradication of tumor-initiating hepatocellular carcinoma cells.

Recent advances in stem cell biology have identified tumor‐initiating cells (TICs) in a variety of cancers including hepatocellular carcinoma (HCC). Polycomb group gene products such as BMI1 and EZH2 have been characterized as general self‐renewal regulators in a wide range of normal stem cells and TICs. We previously reported that Ezh2 tightly regulates the self‐renewal and differentiation of murine hepatic stem/progenitor cells. However, the role of EZH2 in tumor‐initiating HCC cells remains unclear. In this study, we conducted loss‐of‐function assay of EZH2 using short‐hairpin RNA and pharmacological inhibition of EZH2 by an S‐adenosylhomocysteine hydrolase inhibitor, 3‐deazaneplanocin A (DZNep). Both EZH2‐knockdown and DZNep treatment impaired cell growth and anchorage‐independent sphere formation of HCC cells in culture. Flow cytometric analyses revealed that the two approaches decreased the number of epithelial cell adhesion molecule (EpCAM)+ tumor‐initiating cells. Administration of 5‐fluorouracil (5‐FU) or DZNep suppressed the tumors by implanted HCC cells in non‐obese diabetic/severe combined immunodeficient mice. Of note, however, DZNep but not 5‐FU predominantly reduced the number of EpCAM+ cells and diminished the self‐renewal capability of these cells as judged by sphere formation assays. Our findings reveal that tumor‐initiating HCC cells are highly dependent on EZH2 for their tumorigenic activity. Although further analyses of TICs from primary HCC would be necessary, pharmacological interference with EZH2 might be a promising therapeutic approach to targeting tumor‐initiating HCC cells.

The polycomb group gene product Ezh2 regulates proliferation and differentiation of murine hepatic stem/progenitor cells

BACKGROUND & AIMS Polycomb group proteins initiate and maintain gene silencing through chromatin modifications and contribute to the maintenance of self-renewal in a variety of stem cells. Among polycomb repressive complexes (PRCs), PRC2 initiates gene silencing by methylating histone H3 lysine 27, and PRC1 maintains gene silencing through mono-ubiquitination of histone H2A lysine 119. We have previously shown that Bmi1, a core component of PRC1, tightly regulates the self-renewal of hepatic stem/progenitor cells. METHODS In this study, we conducted lentivirus-mediated knockdown of Ezh2 to characterise the function of Ezh2, a major component of PRC2, in hepatic stem/progenitor cells. RESULTS Loss of Ezh2 function in embryonic murine hepatic stem/progenitor cells severely impaired proliferation and self-renewal capability. This effect was more prominent than that of Bmi1-knockdown and was partially abrogated by the deletion of both Ink4a and Arf, major targets of PRC1 and PRC2. Importantly, Ezh2-knockdown but not Bmi1-knockdown promoted the differentiation and terminal maturation of hepatocytes, followed by the up-regulation of several transcriptional regulators of hepatocyte differentiation. CONCLUSIONS Our findings indicate that Ezh2 plays an essential role in the maintenance of both the proliferative and self-renewal capacity of hepatic stem/progenitor cells and the full execution of their differentiation.

Role of the polycomb group proteins in hematopoietic stem cells.

Polycomb group (PcG) proteins play a role in the transcriptional repression of genes through histone modifications. Recent studies have clearly demonstrated that PcG proteins are required for the maintenance of embryonic as well as a broad range of adult stem cells, including hematopoietic stem cells (HSCs). PcG proteins maintain the self‐renewal capacity of HSCs by repressing tumor suppressor genes and keep differentiation programs poised for activation in HSCs by repressing a cohort of hematopoietic developmental regulator genes via bivalent chromatin domains. Enforced expression of one of the PcG genes, Bmi1, augments the self‐renewal capacity of HSCs. PcG proteins also maintain redox homeostasis to prevent premature loss of HSCs. These findings established PcG proteins as essential regulators of HSCs and underscored epigenetics as a new field of HSC research. In this review, we focus on the role of PcG proteins in the epigenetic regulation of the self‐renewal capacity and multipotency of HSCs.

Unrelated Cord Blood Transplantation after Myeloablative Conditioning in Adults with Acute Myelogenous Leukemia

We analyzed the disease-specific outcomes of adult acute myelogenous leukemia (AML) patients treated with unrelated cord blood transplantation (CBT) after myeloablative conditioning. Between August 1998 and February 2008, 77 adult patients with AML were treated with unrelated CBT. All patients received 4 fractionated 12 Gy total body irradiation (TBI) and chemotherapy as myeloablative conditioning. The median age was 45 years, the median weight was 55 kg, the median number of nucleated cells was 2.44 x 10(7)/kg, and the median number of CD34-positive cells was 1.00 x 10(5)/kg. All patients received a single and HLA mismatched cord blood unit. The cumulative incidence of neutrophil recovery at day 50 and platelet recovery at day 200 was 94.8% and 91.7%, respectively. A higher CD34-positive cell dose was associated with faster hematopoietic recovery. The cumulative incidence of grade III to IV acute graft-versus-host disease (aGVHD) and extensive-type chronic GVHD (cGVHD) was 25.1% and 28.6%, respectively. With a median follow-up of 78 months, the probability of event-free survival (EFS) at 5 years was 62.8%. The 5-year cumulative incidence of treatment related-mortality (TRM) and relapse was 9.7%, 25.8%, respectively. In multivariate analyses, the risk factor identified for event free survival (EFS) was disease status and cytogenetics. These results suggest that unrelated CBT after myeloablative conditioning could be safely and effectively used for adult patients with AML.

Lis1 regulates asymmetric division in hematopoietic stem cells and in leukemia

Cell fate can be controlled through asymmetric division and segregation of protein determinants, but the regulation of this process in the hematopoietic system is poorly understood. Here we show that the dynein-binding protein Lis1 is critically required for hematopoietic stem cell function and leukemogenesis. Conditional deletion of Lis1 (also known as Pafah1b1) in the hematopoietic system led to a severe bloodless phenotype, depletion of the stem cell pool and embryonic lethality. Further, real-time imaging revealed that loss of Lis1 caused defects in spindle positioning and inheritance of cell fate determinants, triggering accelerated differentiation. Finally, deletion of Lis1 blocked the propagation of myeloid leukemia and led to a marked improvement in survival, suggesting that Lis1 is also required for oncogenic growth. These data identify a key role for Lis1 in hematopoietic stem cells and mark its directed control of asymmetric division as a critical regulator of normal and malignant hematopoietic development.

β-Arrestin2 mediates the initiation and progression of myeloid leukemia

β-Arrestins were initially discovered as negative regulators of G protein-coupled receptor signaling. Although β-arrestins have more recently been implicated as scaffold proteins that interact with various mitogenic and developmental signals, the genetic role of β-arrestins in driving oncogenesis is not known. Here we have investigated the role of β-arrestin in hematologic malignancies and have found that although both β-arrestin1 and -2 are expressed in the hematopoietic system, loss of β-arrestin2 preferentially leads to a severe impairment in the establishment and propagation of the chronic and blast crisis phases of chronic myelogenous leukemia (CML). These defects are linked to a reduced frequency, as well as defective self-renewal capacity of the cancer stem-cell population, in mouse models and in human CML patient samples. At a molecular level, the loss of β-arrestin2 leads to a significant inhibition of β-catenin stabilization, and ectopic activation of Wnt signaling reverses the defects observed in the β-arrestin2 mutant cells. These data cumulatively show that β-arrestin2 is essential for CML disease propagation and indicate that β-arrestins and the Wnt/β-catenin pathway lie in a signaling hierarchy in the context of CML cancer stem cell maintenance.

Allogeneic stem cell transplantation for hepatosplenic gammadelta T-cell lymphoma.

Hepatosplenic gammadelta T-cell lymphoma (HSTCL) was first described by Farcet et al. in 1990 [1]. Most cases of HSCTL occur in young men. Patients typically present with hepatosplenomegaly and bone marrow infiltration with resultant cytopenias but comparatively little lymphadenopathy. The prognosis of HSCTL is poor with reported median survivals of 8 – 16 months and few instances of longterm disease free survival with conventional chemotherapy [2,3]. Recently, a limited number of cases treated with allogeneic SCT for HSTCL have been reported [2 – 15]. We previously reported a patient of HSTCL successfully treated with allogeneic bone marrow transplantation (BMT) from an HLAidentical sibling [6]. In this report, we provide an update of this patient with 7-years follow-up and review the literature for allogeneic SCT for HSTCL. A 23-year-old Japanese male was admitted to our hospital in June 1999 with abdominal distension, malaise, and night sweats. As previously described, the patient was diagnosed with hepatosplenic gamma/delta T-cell lymphoma and treated with intensive B-NHL86 protocol chemotherapy. After the two courses of chemotherapy, he achieved complete remission (CR). Thereafter, he received allogeneic BMT in August 1999 from an HLAidentically matched younger sister. The conditioning regimen consisted of four fractionated 12-Gy total body irradiation (TBI) on Day 79 and Day 78 and high-dose etoposide (60 mg/kg), which was administered as 24-h continuous intravenous infusion on Day 74. Graft-versus-host disease (GVHD) prophylaxis consisted of intravenous cyclosporine and short-term methotrexate. He had evidence of grade II acute GVHD of the skin and gut, which required no steroid treatment. Chimerism evaluation assessed by fluorescent in situ hybridization (FISH) using a mixture of X and Y chromosome-specific probes revealed complete donor chimerism of bone marrow cells on Day þ28. Because of high risk of disease relapse after transplantation, cyclosporine was tapered rapidly and finished on Day þ70. Thereafter, mild chronic GVHD of the liver and skin developed at 4 months after BMT, requiring treatment with oral cyclosporine for 8 months. At 5 years post-BMT, bone marrow examination revealed complete donor chimerism by sex mismatched FISH. Seven years after BMT, the patient is alive and free of disease. Treatment modalities for HSTCL have considerable heterogeneity [3], including splenectomy, corticosteroids, purine analogue, anthracycline containing regimens such as CHOP (cyclophosphamide, hydroxydaunomycin, vincristine and prednisone) or CHOP-like regimen, second or third generation aggressive lymphoma regimen such as IEV (ifosphamide, epirubicin and etoposide) or modified MACOP-B (methotrexate, etoposide instead of adriamycin, cyclophosphamide, vincristine, predonisone and bleomycin), alemtuzumab and autologous and allogeneic stem cell transplantation. However, such treatments have limited efficacy and the vast majority of patients will die from progressive disease. Although allogeneic SCT, which is the only potentially curative therapy, has been attempted to treat

Bmi1 Confers Resistance to Oxidative Stress on Hematopoietic Stem Cells

Background The polycomb-group (PcG) proteins function as general regulators of stem cells. We previously reported that retrovirus-mediated overexpression of Bmi1, a gene encoding a core component of polycomb repressive complex (PRC) 1, maintained self-renewing hematopoietic stem cells (HSCs) during long-term culture. However, the effects of overexpression of Bmi1 on HSCs in vivo remained to be precisely addressed. Methodology/Principal findings In this study, we generated a mouse line where Bmi1 can be conditionally overexpressed under the control of the endogenous Rosa26 promoter in a hematopoietic cell-specific fashion (Tie2-Cre;R26StopFLBmi1). Although overexpression of Bmi1 did not significantly affect steady state hematopoiesis, it promoted expansion of functional HSCs during ex vivo culture and efficiently protected HSCs against loss of self-renewal capacity during serial transplantation. Overexpression of Bmi1 had no effect on DNA damage response triggered by ionizing radiation. In contrast, Tie2-Cre;R26StopFLBmi1 HSCs under oxidative stress maintained a multipotent state and generally tolerated oxidative stress better than the control. Unexpectedly, overexpression of Bmi1 had no impact on the level of intracellular reactive oxygen species (ROS). Conclusions/Significance Our findings demonstrate that overexpression of Bmi1 confers resistance to stresses, particularly oxidative stress, onto HSCs. This thereby enhances their regenerative capacity and suggests that Bmi1 is located downstream of ROS signaling and negatively regulated by it.

Human herpesvirus 6 variant B infection in adult patients after unrelated cord blood transplantation.

Human herpesvirus 6 var1iant B (HHV-6B) infection was studied in 23 adult patients who underwent cord blood transplantation (CBT). HHV-6B DNA was detected by quantitative polymerase chain reaction analysis after CBT in the sera from 15 patients (65%) at day 14 or 15 (week 2), from 16 patients (70%) at day 21 or 22 (week 3), and from 3 patients (13%) at day 28 or 29 (week 4). HHV-6B DNAemia was found in none of the 20 patients examined at day 7 or 8 (week 1). The overall incidence of HHV-6B DNAemia reached 87% (20 of 23 patients). This incidence was much higher than after unrelated bone marrow transplantation (19%, P < .0001). In CBT patients, positive HHV-6B DNAemia at week 3 was significantly associated with early skin rash (88% versus 14%, P < .005) and grade II–IV acute graft-versus-host disease (aGVHD) (69% versus 14%, P < .05). In contrast, positive HHV-6B DNAemia at week 2 was associated with neither skin rash nor aGVHD. Prospective large-scale studies are needed to determine the role of HHV-6 infection in CBT patients.