Maintenance of the hematopoietic stem cell pool in bone marrow niches by EVI1-regulated GPR56.

Acute myeloid leukemia with high ecotropic viral integration site-1 expression (EVI1(high) AML) is classified as a refractory type of leukemia with a poor prognosis. To provide new insights into the prevention and treatment of this disease, we identified the high expression of EVI1-regulated G protein-coupled receptor 56 (GPR56), and the association of high cell adhesion and antiapoptotic activities in EVI1(high) AML cells. Knockdown of GPR56 expression decreased the cellular adhesion ability through inactivation of RhoA signaling, resulting in a reduction of cellular growth rates and enhanced apoptosis. Moreover, in Gpr56(-/-) mice, the number of hematopoietic stem cells (HSCs) was significantly decreased in the bone marrow (BM) and, conversely, was increased in the spleen, liver and peripheral blood. The number of Gpr56(-/-) HSC progenitors in the G0/G1-phase was significantly reduced and was associated with impaired cellular adhesion. Finally, the loss of GPR56 function resulted in a reduction of the in vivo repopulating ability of the HSCs. In conclusion, GPR56 may represent an important GPCR for the maintenance of HSCs by acting as a co-ordinator of interactions with the BM osteosteal niche; furthermore, this receptor has the potential to become a novel molecular target in EVI1(high) leukemia.

Clinical significance of CADM1/TSLC1/IgSF4 expression in adult T-cell leukemia/lymphoma.

Cell adhesion molecule 1 (CADM1/TSLC1) was recently identified as a novel cell surface marker for adult T-cell leukemia/lymphoma (ATLL). In this study, we developed various antibodies as diagnostic tools to identify CADM1-positive ATLL leukemia cells. In flow cytometric analysis, the percentages of CD4(+)CADM1(+) double-positive cells correlated well with both the percentages of CD4(+)CD25(+) cells and with abnormal lymphocytes in the peripheral blood of patients with various types of ATLL. Moreover, the degree of CD4(+)CADM1(+) cells over 1% significantly correlated with the copy number of the human T-lymphotropic virus type 1 (HTLV-1) provirus in the peripheral blood of HTLV-1 carriers and ATLL patients. We also identified a soluble form of CADM1 in the peripheral blood of ATLL patients, and the expression levels of this form were correlated with the levels of soluble interleukin 2 receptor alpha. Moreover, lymphomas derived from ATLL were strongly and specifically stained with a CADM1 antibody. Thus, detection of CD4(+)CADM1(+) cells in the peripheral blood, measurement of serum levels of soluble CADM1 and immunohistochemical detection of CADM1 in lymphomas would be a useful set of markers for disease progression in ATLL and may aid in both the early diagnosis and measurement of treatment efficacy for ATLL.

Sumoylation of MEL1S at lysine 568 and its interaction with CtBP facilitates its repressor activity and the blockade of G-CSF-induced myeloid differentiation.

MEL1 (MDS1/EVI1-like gene 1/PRDM16), which was identified as a gene near the chromosomal breakpoint in t(1;3)(p36;q21)-positive human acute myeloid leukemia cells, belongs to the PRDI-BF1-RIZ1 homologous (PR) domain (PRDM) family of transcription repressors. The short form of MEL1 (MEL1S), which lacks the PR-domain at the N-terminus, is the main form expressed in t(1;3)(p36;q21)-positive acute myeloid leukemia cells. The overexpression of MEL1S blocks granulocyte colony-stimulating factor (G-CSF)-induced myeloid differentiation in interleukin-3-dependent murine myeloid L-G3 cells. In this study, we show that treatment with the histone deacetylase inhibitor trichostatin A abolished the blockade of myeloid differentiation in L-G3 cells overexpressing MEL1S. The expression of MEL1S containing mutated CtBP-interacting motif (CIM) in L-G3 cells still blocked the myeloid differentiation induced by G-CSF. We found that the small ubiquitin-related modifier (SUMO) motif (SM) at lysine 568 (VKAE) adjacent to the CIM was necessary to obtain the maximum transcriptional repressor activity of MEL1S. L-G3 cells expressing MEL1S, and bearing mutated CIM and SM differentiated into granulocytes in response to G-CSF; this indicated that both the SUMO modification at lysine 568 and CtBP binding were required for MEL1S-mediated transcriptional repression and blockade of differentiation, which might be relevant for the process of leukemogenesis.

CD52 as a molecular target for immunotherapy to treat acute myeloid leukemia with high EVI1 expression.

Ecotropic viral integration site 1 (EVI1) is an oncogenic transcription factor in human acute myeloid leukemia (AML) with chromosomal alterations at 3q26. Because a high expression of EVI1 protein in AML cells predicts resistance to chemotherapy with a poor outcome, we have searched for molecular targets that will treat these patients with AML. In this study, we determined that CD52, which is mainly expressed on lymphocytes, is highly expressed in most cases of AML with a high EVI1 expression (EVI1(High)). CAMPATH-1H, a humanized monoclonal antibody against CD52, has been used to prevent graft-versus-host disease and treat CD52-positive lymphoproliferative disorders. Here, we investigated the antitumor effect of CAMPATH-1H on EVI1(High) AML cells. CAMPATH-1H significantly inhibited cell growth and induced apoptosis in CD52-positive EVI1(High) leukemia cells. Furthermore, CAMPATH-1H induced complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity against CD52-positive EVI1(High) leukemia cells. After an intravenous injection of CAMPATH-1H into NOD/Shi-scid/IL-2Rγ;null mice with subcutaneous engraftment of EVI1(High) leukemia cells, tumor growth rates were significantly reduced, and the mice survived longer than those in the phosphate-buffered saline-injected control group. Thus, CAMPATH-1H is a potential therapeutic antibody for the treatment of patients with EVI1(High) leukemia.

Downregulation of ZEB1 and overexpression of Smad7 contribute to resistance to TGF-beta1-mediated growth suppression in adult T-cell leukemia/lymphoma.

Zinc-finger E-box binding homeobox 1 (ZEB1) is a candidate tumor-suppressor gene in adult T-cell leukemia/lymphoma (ATLL). ZEB1 binds phosphorylated Smad2/3 to enhance transforming growth factor-beta1 (TGF-beta1) signaling. In addition to downregulation of ZEB1 mRNA, we found overexpression of inhibitory Smad, Smad7, in resistance of ATLL cells to growth suppression by TGF-beta1. A protein complex of Smad7 and histone deacetylase constantly bound to the promoter region of TGF-beta1 responsive genes with the Smad-responsive element (SRE) to inhibit TGF-beta1 signaling; however, ectopic expression of ZEB1 reactivated TGF-beta1 signaling by binding to Smad7 and recruiting the Smad3/p300 histone acetyltransferase complex to the promoter after TGF-beta1 stimulation in ATLL. Conversely, because ZEB1 mRNA was detected in the late stages of T-cell development, we used CTLL2 cells with ZEB1 expression, a murine peripheral T-cell lymphoma, and found that a complex of Smad3, Smad7 and ZEB1 was bound to the SRE of the p21(CDKN1A) promoter after the induction of Smad7 by TGF-beta1 treatment. Because the duration of TGF-beta1-induced transcriptional activation of PAI-1 and p21 was shortened in shZEB1-expressing CTLL2 cells, ZEB1 may have a role in enhancing TGF-beta1 signaling by binding not only to Smad3 but also to Smad7 in the nucleus. Altogether, these results suggest that both ZEB1 downregulation and Smad7 overexpression contribute to resistance to TGF-beta1-mediated growth suppression in ATLL.

Biochemical identification of Xenopus Pumilio as a sequence-specific cyclin B1 mRNA-binding protein that physically interacts with a Nanos homolog, Xcat-2, and a cytoplasmic polyadenylation element-binding protein.

Translational activation of dormant cyclin B1 mRNA stored in oocytes is a prerequisite for the initiation or promotion of oocyte maturation in many vertebrates. Using a monoclonal antibody against the domain highly homologous to that of Drosophila Pumilio, we have shown for the first time in any vertebrate that a homolog of Pumilio is expressed in Xenopus oocytes. This 137-kDa protein binds to the region including the sequence UGUA at nucleotides 1335-1338 in the 3'-untranslated region of cyclin B1 mRNA, which is close to but does not overlap the cytoplasmic polyadenylation elements (CPEs). Physical in vitro association of Xenopus Pumilio with a Xenopus homolog of Nanos (Xcat-2) was demonstrated by a protein pull-down assay. The results of immunoprecipitation experiments showed in vivo interaction between Xenopus Pumilio and CPE-binding protein (CPEB), a key regulator of translational repression and activation of mRNAs stored in oocytes. This evidence provides a new insight into the mechanism of translational regulation through the 3'-end of mRNA during oocyte maturation. These results also suggest the generality of the function of Pumilio as a translational regulator of dormant mRNAs in both invertebrates and vertebrates.