High expression of ABCG2 induced by EZH2 disruption has pivotal roles in MDS pathogenesis.

Both proto-oncogenic and tumor-suppressive functions have been reported for enhancer of zeste homolog 2 (EZH2). To investigate the effects of its inactivation, a mutant EZH2 lacking its catalytic domain was prepared (EZH2-dSET). In a mouse bone marrow transplant model, EZH2-dSET expression in bone marrow cells induced a myelodysplastic syndrome (MDS)-like disease in transplanted mice. Analysis of these mice identified Abcg2 as a direct target of EZH2. Intriguingly, Abcg2 expression alone induced the same disease in the transplanted mice, where stemness genes were enriched. Interestingly, ABCG2 expression is specifically high in MDS patients. The present results indicate that ABCG2 de-repression induced by EZH2 mutations have crucial roles in MDS pathogenesis.

Protease-activated receptor-1 inhibits proliferation but enhances leukemia stem cell activity in acute myeloid leukemia.

Eradication of leukemia stem cells (LSCs) is the ultimate goal of treating acute myeloid leukemia (AML). We recently showed that the combined loss of Runx1/Cbfb inhibited the development of MLL-AF9-induced AML. However, c-Kit+/Gr-1- cells remained viable in Runx1/Cbfb-deleted cells, indicating that suppressing RUNX activity may not eradicate the most immature LSCs. In this study, we found upregulation of several hemostasis-related genes, including the thrombin-activatable receptor PAR-1 (protease-activated receptor-1), in Runx1/Cbfb-deleted MLL-AF9 cells. Similar to the effect of Runx1/Cbfb deletion, PAR-1 overexpression induced CDKN1A/p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1 deficiency also prevented leukemia development induced by a small number of MLL-AF9 leukemia stem cells (LSCs) in vivo. PAR-1 deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose-dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there were a large number of LSCs, while a small number of LSCs required PAR-1 for their efficient growth. Mechanistically, PAR-1 increased the adherence properties of MLL-AF9 cells and promoted their engraftment to bone marrow. Taken together, these data revealed a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML.

UBASH3B/Sts-1-CBL axis regulates myeloid proliferation in human preleukemia induced by AML1-ETO.

The t(8;21) rearrangement, which creates the AML1-ETO fusion protein, represents the most common chromosomal translocation in acute myeloid leukemia (AML). Clinical data suggest that CBL mutations are a frequent event in t(8;21) AML, but the role of CBL in AML1-ETO-induced leukemia has not been investigated. In this study, we demonstrate that CBL mutations collaborate with AML1-ETO to expand human CD34+ cells both in vitro and in a xenograft model. CBL depletion by shRNA also promotes the growth of AML1-ETO cells, demonstrating the inhibitory function of endogenous CBL in t(8;21) AML. Mechanistically, loss of CBL function confers hyper-responsiveness to thrombopoietin and enhances STAT5/AKT/ERK/Src signaling in AML1-ETO cells. Interestingly, we found the protein tyrosine phosphatase UBASH3B/Sts-1, which is known to inhibit CBL function, is upregulated by AML1-ETO through transcriptional and miR-9-mediated regulation. UBASH3B/Sts-1 depletion induces an aberrant pattern of CBL phosphorylation and impairs proliferation in AML1-ETO cells. The growth inhibition caused by UBASH3B/Sts-1 depletion can be rescued by ectopic expression of CBL mutants, suggesting that UBASH3B/Sts-1 supports the growth of AML1-ETO cells partly through modulation of CBL function. Our study reveals a role of CBL in restricting myeloid proliferation of human AML1-ETO-induced leukemia, and identifies UBASH3B/Sts-1 as a potential target for pharmaceutical intervention.

Posttranslational modifications of RUNX1 as potential anticancer targets.

The transcription factor RUNX1 is a master regulator of hematopoiesis. Disruption of RUNX1 activity has been implicated in the development of hematopoietic neoplasms. Recent studies also highlight the importance of RUNX1 in solid tumors both as a tumor promoter and a suppressor. Given its central role in cancer development, RUNX1 is an excellent candidate for targeted therapy. A potential strategy to target RUNX1 is through modulation of its posttranslational modifications (PTMs). Numerous studies have shown that RUNX1 activity is regulated by PTMs, including phosphorylation, acetylation, methylation and ubiquitination. These PTMs regulate RUNX1 activity either positively or negatively by altering RUNX1-mediated transcription, promoting protein degradation and affecting protein interactions. In this review, we first summarize the available data on the context- and dosage-dependent roles of RUNX1 in various types of neoplasms. We then provide a comprehensive overview of RUNX1 PTMs from biochemical and biologic perspectives. Finally, we discuss how aberrant PTMs of RUNX1 might contribute to tumorigenesis and also strategies to develop anticancer therapies targeting RUNX1 PTMs.

Male predominance among Japanese adult patients with late-onset hemorrhagic cystitis after hematopoietic stem cell transplantation.

Late-onset hemorrhagic cystitis (LHC) after hematopoietic stem cell transplantation (HSCT) is mainly caused by viral infections. We retrospectively analyzed the records of 141 Japanese adult patients who underwent a first allogeneic HSCT from 1995 to 2002. In all, 19 patients developed LHC a median of 51 days after HSCT. Adenovirus (AdV) was detected in the urine of 10 LHC patients, of whom eight had AdV type 11. Five of the six available serum samples from these patients were also positive for AdV type 11, but the detection of AdV in serum was not associated with a worse outcome. Male sex and the development of grade II-IV acute graft-versus-host disease were identified as independent significant risk factors for LHC. Male predominance was detected in LHC after HSCT, as has been previously shown in children with AdV-induced acute HC. The detection of AdV DNA in serum did not predict a poor outcome.

Reverse seroconversion of hepatitis B virus after hematopoietic stem cell transplantation.

Hepatitis B virus (HBV) reactivation in patients previously positive for hepatitis B surface antibody (HBsAb), so-called reverse seroconversion, has been considered to be a rare complication after hematopoietic stem cell transplantation (HSCT). We experienced two patients who developed reverse seroconversion among nine who were HBsAb positive and Hepatitis B core antibody (HBcAb) positive before HSCT; one after autologous bone marrow transplantation (BMT) and another after allogeneic peripheral blood stem cell transplantation (PBSCT). We reviewed the literature and considered that reverse seroconversion of HBV after HSCT is not uncommon among HBsAb positive recipients. The use of corticosteroids, the lack of HBsAb in donor, and a decrease in serum HBsAb and HBcAb levels may predict reverse seroconversion after HSCT.

Evi1 defines leukemia-initiating capacity and tyrosine kinase inhibitor resistance in chronic myeloid leukemia.

Relapse of chronic myeloid leukemia (CML) is triggered by stem cells with a reconstituting capacity similar to that of hematopoietic stem cells (HSCs) and CML stem cells are a source of resistance in drug therapy with tyrosine kinase inhibitors (TKIs). Ecotropic viral integration site 1 (EVI1), a key transcription factor in HSC regulation, is known to predict poor outcomes in myeloid malignancies, however, incapability of prospective isolation of EVI1-high leukemic cells precludes the functional evaluation of intraindividual EVI1-high cells. Introduction of CML into Evi1-internal ribosomal entry site (IRES)-green fluorescent protein (GFP) knock-in mice, a versatile HSC-reporter strain, enables us to separate Evi1-high CML cells from the individual. Evi1-IRES-GFP allele models of CML in chronic phase (CML-CP), by retroviral overexpression of BCR-ABL and by crossing BCR-ABL transgenic mice, revealed that Evi1 is predominantly enriched in the stem cell fraction and associated with an enhanced proliferative as well as a leukemia-initiating capacity and that Evi1-high CML-CP cells exhibit resistance to TKIs. Overexpressing BCR-ABL and NUP98-HOXA9 in Evi1-IRES-GFP knock-in mice to model CML in blast crisis (CML-BC), in which Evi1-high cells turned to be a major population as opposed to a minor population in CML-CP models, showed that Evi1-high CML-BC cells have a greater potential to recapitulate the disease and appear resistant to TKIs. Furthermore, given that Evi1 heterozygosity ameliorates CML-CP and CML-BC development and that the combination of Evi1 and BCR-ABL causes acute myeloid leukemia resembling CML-BC, Evi1 could regulate CML development as a potent driver. In addition, in human CML-CP cases, we show that EVI1 is highly expressed in stem cell-enriched CD34+CD38-CD90+ fraction at single-cell level. This is the first report to clarify directly that Evi1-high leukemic cells themselves possess the superior potential to Evi1-low cells in oncogenic self-renewal, which highlights the role of Evi1 as a valuable and a functional marker of CML stem cells.

EVI-1 interacts with histone methyltransferases SUV39H1 and G9a for transcriptional repression and bone marrow immortalization.

The ecotropic viral integration site-1 (EVI-1) is a nuclear transcription factor and has an essential function in the proliferation/maintenance of haematopoietic stem cells. Aberrant expression of EVI-1 has been frequently found in myeloid leukaemia as well as in several solid tumours, and is associated with a poor patient survival. It was recently shown that EVI-1 associates with two different histone methyltransferases (HMTs), SUV39H1 and G9a. However, the functional roles of these HMTs in EVI-1-mediated leukemogenesis remain unclear. In this study, we showed that EVI-1 physically interacts with SUV39H1 and G9a, but not with Set9. Immunofluorescence analysis revealed that EVI-1 colocalizes with these HMTs in nuclei. We also found that the catalytically inactive form of SUV39H1 abrogates the transcriptional repression mediated by EVI-1, suggesting that SUV39H1 is actively involved in EVI-1-mediated transcriptional repression. Furthermore, RNAi-based knockdown of SUV39H1 or G9a in Evi-1-expressing progenitors significantly reduced their colony-forming activity. In contrast, knockdown of these HMTs did not impair bone marrow immortalization by E2A/HLF. These results indicate that EVI-1 forms higher-order complexes with HMTs, and this association has a role in the transcription repression and bone marrow immortalization. Targeting these HMTs may be of therapeutic benefit in the treatment for EVI-1-related haematological malignancies.

Pbx1 is a downstream target of Evi-1 in hematopoietic stem/progenitors and leukemic cells.

Ecotropic viral integration site-1 (Evi-1) is a nuclear transcription factor, which is essential for the proliferation/maintenance of hematopoietic stem cells (HSCs). Aberrant expression of Evi-1 has been frequently found in myeloid leukemia, and is associated with a poor patient survival. Recently, we reported candidate target genes of Evi-1 shared in HSCs and leukemic cells using gene expression profiling analysis. In this study, we identified Pbx1, a proto-oncogene in hematopoietic malignancy, as a target gene of Evi-1. Overexpression of Evi-1 increased Pbx1 expression in hematopoietic stem/progenitor cells. An analysis of the Pbx1 promoter region revealed that Evi-1 upregulates Pbx1 transcription. Furthermore, reduction of Pbx1 levels through RNAi-mediated knockdown significantly inhibited Evi-1-induced transformation. In contrast, knockdown of Pbx1 did not impair bone marrow transformation by E2A/HLF or AML1/ETO, suggesting that Pbx1 is specifically required for the maintenance of bone marrow transformation mediated by Evi-1. These results indicate that Pbx1 is a target gene of Evi-1 involved in Evi-1-mediated leukemogenesis.

AML1-Evi-1 specifically transforms hematopoietic stem cells through fusion of the entire Evi-1 sequence to AML1.

The t(3;21) chromosomal translocation seen in blastic crisis of chronic myeloid leukemia and secondary leukemias results in a formation of a chimeric protein AML1-Evi-1, which suppresses wild-type AML1 function. Loss of AML1 function causes expansion of hematopoietic progenitor cells, whereas it is not sufficient for the development of leukemia. To identify essential mechanisms through which AML1-Evi-1 exerts full leukemogenic potential, we introduced AML1-Evi-1 and its mutants in murine bone marrow cells, and evaluated their transforming activities by colony replating assays. The transforming activity of AML1-Evi-1 was lost when any of the known functional domains of Evi-1 was deleted from the chimeric protein, and forced expression of Evi-1 did not transform the AML1-deleted bone marrow cells. Unlike the MLL-ENL and AML1-ETO leukemia-related chimeric proteins, AML1-Evi-1 could transform only the hematopoietic stem cell fraction. Moreover, AML1-Evi-1-transformed cells show a cell-marker profile distinct from that of the cells transformed by AML1-ETO, which also suppresses AML1 function. Thus, leukemogenic activity of AML1-Evi-1 may be due to activation of molecular mechanisms distinct from those activated by MLL-ENL or AML1-ETO in the hematopoietic stem cell fractions.