ALBERT

Description: Chromosomal abnormalities, such as translocation, mutation or deletion, are central to the pathogenesis of human cancers. Recently, several transcription factors have been isolated as genes responsible for leukemia from the region surrounding chromosomal breakpoints, which are implicated in the regulation of normal hematopoiesis. Among on them, ecotropic viral integration site-1 (Evi1) is a transcription factor activated by retroviral integration in murine leukemias and chromosomal rearrangements in human leukemias. Evi1 is a zinc finger transcription factor and contains two separated DNA-binding domains. It was reported that Evi1−/− embryos die at approximately E10.5, exhibiting widespread hypocellularity and hemorrhaging. However, the role in normal hematopoiesis or authentic target genes of Evi1 has not been elucidated. Here, we show that Evi1 is predominantly expressed in hematopoietic stem cells (HSCs) in embryos and adult bone marrows, and Evi1−/− embryos are markedly decreased in numbers of HSC. One embryo-equivalent cells from E9.5 P-Sp of Evi1+/+, Evi1+/− and Evi1−/− embryos (Ly5.2) were transplanted into a busulfan-conditioned newborn recipient (Ly5.1). At 2 months posttransplant, donor-derived Ly5.2(+) cells could be detected in the peripheral blood of the recipients that received P-Sp cells from the Evi1+/+ and Evi1+/− but not from the Evi1−/− embryos. Thus, Evi1 is critical for the generation of HSCs in the P-Sp. Both Evi1−/− embryos and yolk sac showed marked retardation in the organization of the vascular system, particularly in vascular remodeling, compared with controls. Using an in vitro P-Sp culture analysis, we found normal in vitro differentiation of endothelial cells in Evi1−/− P-Sp cultures but defects in their in vitro network formation, which is normally promoted by Ang-1 secreted from developing HSCs in P-Sp cultures. HSCs from adult bone marrow or HSCs from E9.5 wild type embryos rescued defective angiogenesis in Evi1−/− embryos. The fine vascular network coincided with the region where HSCs formed a colony. Their round morphology confirmed that exogenous adult HSCs did not differentiate into elongated endothelial cells. We showed that recombinant Ang-1 alone restored the defective angiogenesis in Evi1−/− embryos to a wild type level. It is suggested that the defect in hematopoietic cells induced defective angiogenesis in Evi1−/− embryos mediated by Ang-1. Notably, mRNA expression of GATA-2, which is essential for proliferation of definitive HSCs, was profoundly reduced in Evi1−/− embryos. Analysis of the GATA-2 promotor revealed that Evi1 directly binds to the 5′ upstream region of the GATA−2 exon and positively regulates its promoter activity in vitro and in vivo. Restoration of GATA-2 expression dramatically rescued the defective expansion of Evi1−/− embryos HSCs in vitro. Our results reveal that GATA-2 is a critical in vivo target for Evi1 and indicate hierarchical regulation of the HSC pool by transcriptional regulators.

Description: Transferrin receptor 1(TfR1) is a type II transmembrane glycoprotein regulating the intracellular uptake of iron and is involved in cell growth, proliferation and survival. TfR1 is highly expressed on malignant cells, including those of hematologic malignancies. Therefore, TfR1 may be an attractive target for therapeutic monoclonal antibodies. We generated a panel of fully-human, anti-TfR1 monoclonal antibodies and evaluated the anti-tumor effects of these antibodies both in vitro and in vivo. The results led to the selection of TSP-A74, an antibody with potent in vitro and in vivo anti-tumor activity, for further evaluation in several hematologic malignancy models. First, the efficacy of TSP-A74 was evaluated in acute myeloid leukemia (AML) models. Two AML cell lines, Kasumi-1 and HL-60, were subcutaneously inoculated in severe combined immunodeficiency (SCID) mice. After the tumors were grown to a size of 150 mm3, TSP-A74 was administrated intravenously (IV) once weekly for 4 weeks at doses of 0.4, 2 and 10 mg/kg and 1, 3 and 10 mg/kg for the Kasumi and HL60 xenograft models, respectively. TSP-A74 demonstrated complete tumor regression in these two xenograft models at 10 mg/kg and complete tumor growth suppression in the Kasumi model at 2 mg/kg. Even at the low dose of 1 mg/kg, TSP-A74 demonstrated tumor growth inhibition (TGI) of 60% in the HL60 model. Next, the anti-tumor efficacy of TSP-A74 was assessed in an acute lymphoblastic leukemia (ALL) model. The ALL cell line, CCRF-CEM, was engrafted into SCID mice intravenously. After 3 days, TSP-A74 was administrated IV at a dose of 10 mg/kg once weekly for 4 weeks. The control mice (n=10) rapidly developed leukemia and none survived at 42 days after leukemia cell engraftment. However, 7 of 10 (70%) mice treated with TSP-A74 survived to 179 days after engraftment when the study was terminated. Finally, the efficacy of TSP-A74 was evaluated in non-Hodgkin's lymphoma subcutaneous xenograft models. TSP-A74 produced complete regression of established tumors in the SU-DHL-2 (diffuse large B-cell lymphoma) xenograft model at a dose of 3 mg/kg and tumor growth inhibition of 100 % in the HH (cutaneous T cell lymphoma) xenograft model at a dose of 10 mg/kg. These results indicate that the human anti-TfR1 monoclonal antibody, TSP-A74, could be a new therapeutic candidate for hematologic malignancies. Disclosures Zhang: Perseus Proteomics Inc.: Employment. Nomura:Perseus Proteomics Inc.: Employment. Aikawa:Perseus Proteomics Inc.: Employment. Sudo:Perseus Proteomics Inc.: Employment. Morishita:Perseus Proteomics Inc.: Research Funding.

Description: Oncogenic mutations confer on cells the ability to propagate indefinitely, but whether oncogenes alter the cell fate of these cells is unknown. Here, we show that the transcriptional regulator PRDM16s causes oncogenic fate conversion by transforming cells fated to form platelets and erythrocytes into myeloid leukemia stem cells (LSCs). Prdm16s expression in megakaryocyte-erythroid progenitors (MEPs), which normally lack the potential to generate granulomonocytic cells, caused AML by converting MEPs into LSCs. Prdm16s blocked megakaryocytic/erythroid potential by interacting with super enhancers and activating myeloid master regulators, including PU.1. A CRISPR dropout screen confirmed that PU.1 is required for Prdm16s-induced leukemia. Ablating PU.1 attenuated leukemogenesis and reinstated the megakaryocytic/erythroid potential of leukemic MEPs in mouse models and human AML with PRDM16 rearrangement. Thus, oncogenic PRDM16s expression gives MEPs an LSC fate by activating myeloid gene regulatory networks.

Description: Mantle cell lymphoma (MCL) has been mostly incurable, and there is an urgent need to identify targetable molecules for development of a more effective treatment strategy. Bromodomain and extraterminal domain (BET) proteins associate with acetylated histones and facilitate transcription of target genes, and bromodomain-containing 4 (BRD4), a member of BET proteins, recruits the P-TEFb complex to genomic lesions in chromatin and thereby activates RNA Pol II at specific promoter sites of target genes. In addition, super-enhancers have been recognized as regulatory regions with a high level of acetylated histones, mediator complexes and BRD4, and super-enhancers in cancer cells are enriched at oncogenes. Recent studies have shown that BRD4 promotes expression of pivotal molecules in disease development, maintenance and progression in various cancers, including lymphoma. Given, we in this study examined the effect of BRD4 inhibition on human MCL-derived cell lines, Jeko-1, JVM-2, MINO and Z138, and performed broad screening of BRD4-regulated molecules using genome-wide approaches to identify therapeutic targets for MCL. As the results, treatment with a BRD4 inhibitor I-BET151 for 72 h showed a dose-dependent inhibitory effect on cell proliferation in all four cell lines, with half maximal inhibitory concentrations (IC50s) of 15.6 nM, 3.6 nM, 2.6 nM and 3.0 nM in Jeko-1 cells, JVM2 cells, MINO cells and Z138 cells, respectively, which was accompanied by G1/S cell cycle arrest and the induction of apoptosis. Next, we performed comprehensive gene expression profile (GEP) analysis for JVM2 and Z138 cells with or without I-BET151 treatment, and BRD4 chromatin immunoprecipitation sequencing (ChIP-Seq) in JVM2 cells treated with 10 nM I-BET151 or DMSO. Accordingly, GEP analyses revealed that more than 600 genes were commonly upregulated by more than 1.5-fold and downregulated by less than 0.67-fold, respectively, in JVM2 and Z138 cells treated by I-BET151, while ChIP-Seq showed that 7988 BRD4-binding regions were dysregulated by I-BET151, with most of these sites in enhancer regions, and 547 BRD4-binding regions were characterized as super-enhancers. Integrated analysis using the Reactome Pathway Database and the results of GEP and ChIP-Seq showed that a series of genes involved in the B cell receptor (BCR) signaling pathway and IKZF-MYC axis are regulated by BRD4 in MCL cells. To confirm whether each BRD4 target contributes to survival and proliferation of MCL cells, we focused on several candidate targets: the BCR pathway, IKZF and MYB. However, ibrutinib, a Bruton kinase inhibitor, suppressed cell growth in only two of the four cell lines (MINO and JVM2) in a dose-dependent manner, while lenalidomide, an inhibitor of the IKZF family, did not affect cell survival, despite its potency in decreasing IKZF1 and IKZF3 proteins. MYB silencing using shMYB did not decrease cell proliferation in any of the four MCL cell lines. In conclusion, our study disclosed that BRD4 regulates transcription of multiple genes by binding to enhancer region, partly involving super-enhancers and multiple known pathways, such as BCR signaling and the IKZF-MYC axis, which play essential roles in survival of MCL cells. While the efficacy of single targeting of BCR-signaling, IKZF, or MYB was limited, I-BET151 concomitantly inactivated the BCR pathway and IKZF and had a high growth inhibitory efficacy in MCL cells. These results suggest that simultaneous targeting of multiple molecules involved in the BCR pathway and IKZF-MYC axis may overcome resistance to ibrutinib and/or lenalidomide in MCL, and that BRD4 inhibitors are promising candidates for MCL treatment. Disclosures Kuroda: Chugai Pharma: Honoraria, Research Funding. Taniwaki:Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.,: Research Funding; Astellas Pharma Inc,: Research Funding.

Description: Recurrent translocation t(10;11) has been reported to be associated with acute myeloid leukemia (AML). Recently, two types of chimeric transcripts, MLL-AF10 in t(10;11)(p12;q23) andCALM-AF10 in t(10;11)(p13;q14), were isolated. t(10;11) is strongly associated with complex translocations, including invins(10;11) and inv(11)t(10;11), because the direction of transcription of AF10 is telomere to centromere. We analyzed a patient of AML with t(10;11)(p11.2;q23) and identified ABI-1 on chromosome 10p11.2, a human homolog to mouse Abl-interactor 1 (Abi-1), fused with MLL. Whereas the ABI-1 gene bears no homology with the partner genes of MLL previously described, the ABI-1 protein exhibits sequence similarity to protein of homeotic genes, contains several polyproline stretches, and includes asrc homology 3 (SH3) domain at the C-terminus that is required for binding to Abl proteins in mouse Abi-1 protein. Recently, e3B1, an eps8 SH3 binding protein 1, was also isolated as a human homolog to mouse Abi-1. Three types of transcripts of ABI-1 gene were expressed in normal peripheral blood. Although e3B1 was considered to be a full-length ABI-1, the MLL-ABI-1fusion transcript in this patient was formed by an alternatively spliced ABI-1. Others have shown that mouse Abi-1 suppresses v-ABL transforming activity and that e3B1, full-length ABI-1, regulates cell growth. In-frame MLL-ABI-1 fusion transcripts combine the MLL AT-hook motifs and DNA methyltransferase homology region with the homeodomain homologous region, polyproline stretches, and SH3 domain of alternatively spliced transcript of ABI-1. Our results suggest that the ABI-1 gene plays a role in leukemogenesis by translocating to MLL. © 1998 by The American Society of Hematology.

Description: Chromosome translocations involving band 12p13 are known to be involved in a variety of hematologic malignancies, some of them resulting in rearrangement of the ETV6/TEL gene. Applying the fluorescence in situ hybridization (FISH) method, we found a cryptic translocation t(12;15)(p13;q25) in an adult acute myeloid leukemia (AML) patient. Hybridization with cosmid probes showed that the ETV6 gene was rearranged in this translocation. A patient-specific cDNA library was screened with ETV6 cDNA, and a novel fusion transcript was identified between the ETV6 andTRKC/NTRK3 gene located on 15q25. TRKC is a receptor tyrosine kinase that is activated by neurotrophin-3 (NT-3). It is known to be expressed broadly in neural tissues but not in hematologic cells, so far. ETV6-TRKC chimeric transcript encoded the pointed (PNT) domain of the ETV6 gene that fused to the protein-tyrosine kinase (PTK) domain of the TRKC gene. Two types of fusion transcript were determined, one that included the entire PTK domain of TRKC and the other in which the 3′-terminal 462 bp of TRKC was truncated within the PTK domain. Western blot analysis showed the expression of both chimeric proteins of 52 and 38 kD in size. Our results suggest that chimeric PTK expressed in the leukemic cells may contribute to cellular transformation by abnormally activating TRK signaling pathways. Moreover, this is the first report on truncated neurotrophin receptors associated in leukemia.

Description: DNA methylation plays critical roles in the development and differentiation of mammalian cells, and its dysregulation has been implicated in oncogenesis. This study was designed to determine whether DNA hypomethylation-associated aberrant gene expression is involved in adult T-cell leukemia (ATL) leukemogenesis. We isolated hypomethylated DNA regions of ATL cells compared with peripheral blood mononuclear cells from a carrier by a methylated CpG-island amplification/representational difference analysis method. The DNA regions identified contained MEL1, CACNA1H, and Nogo receptor genes. Sequencing using sodium bisulfite-treated genomic DNAs revealed the decreased methylated CpG sites, confirming that this method detected hypomethylated DNA regions. Moreover, these hypomethylated genes were aberrantly transcribed. Among them, MEL1S, an alternatively spliced form of MEL1 lacking the PR (positive regulatory domain I binding factor 1 and retinoblastoma-interacting zinc finger protein) domain, was frequently transcribed in ATL cells, and the transcriptional initiation sites were identified upstream from exons 4 and 6. Transfection of MEL1S into CTLL-2 cells conferred resistance against transforming growth factor β (TGF-β), suggesting that aberrant expression of MEL1S was associated with dysregulation of TGF-β-mediated signaling. Although Tax renders cells resistant to TGF-β, Tax could not be produced in most fresh ATL cells, in which MEL1S might be responsible for TGF-β resistance. Our results suggest that aberrant gene expression associated with DNA hypomethylation is implicated in leukemogenesis of ATL. (Blood. 2004;103:2753-2760)

Description: We have identified a novel gene MEL1 (MDS1/EVI1-like gene 1) encoding a zinc finger protein near the breakpoint of t(1; 3)(p36;q21)-positive human acute myeloid leukemia (AML) cells. Here, we studied the structure, expression pattern, and function of MEL1 in leukemia cells. In this study, we have identified 3 transcription start sites, 1 in exon 1 and 2 in exon 2, and 2 kinds of translation products, 170 kDa (MEL1) and 150 kDa (MEL1S). Notably, the 150-kDa band of MEL1S was detected mainly in the t(1;3)(p36;q21)-positive AML cells. By immunoblot analysis and proteolytic mapping, it is suggested that the 150-kDa band of MEL1S in the leukemia cells is translated from the internal initiation codon ATG597 in exon 4 and is mostly lacking the amino-terminal PR domain of MEL1. By the cyclic amplification and selection of targets (CASTing) method for identifying consensus sequences, it was shown that the consensus sequences of MEL1 were included in 2 different consensus sequences for DNA-binding domain 1 and 2 (D1-CONS and D2-CONS) of EVI1. In reporter gene assays, MEL1S activated transcription via binding to D2-CONS; however, the fusion of MEL1 or MEL1S to GAL4 DNA-binding domain (DBD) made them GAL4 binding site–dependent transcriptional repressors. Moreover, overexpression of MEL1S blocked granulocytic differentiation induced by granulocyte colony-stimulating factor (G-CSF) in interleukin-3 (IL-3)–dependent murine myeloid L-G3 cells, while MEL1 could not block the differentiation. Thus, it is likely that overexpression of the zinc finger protein lacking the PR domain (EVI1 and MEL1S) in the leukemia cells is one of the causative factors in the pathogenesis of myeloid leukemia.

Description: Leukemia cells survive and proliferate under conditions of metabolic stress by acquiring mutations that increase energy metabolism. Here, we aimed to identify a specific metabolic inhibitor and examine transcription factor-enhanced changes in energy metabolism by refractory leukemia cells. Overexpression of Ecotropic Virus Integration site 1 protein homolog (EVI1) in adults and children with mixed lineage leukemia-rearrangement acute myeloid leukemia (MLL-r AML) has a very poor prognosis. We focused on metabolic reprograming of MLL leukemia cells expressing EVI1, since the metabolic relationship between MLL and EVI1 is unclear. We used an extracellular flux analyze to examine metabolic changes during leukemia development in a mouse model of MLL-r AML expressing high levels of EVI1 (EVI1+). To examine whether EVI1 regulates energy metabolism in MLL-rearranged leukemia cells, we used transgenic mice expressing EVI1 (TG) in LSK and GMP cells model in which AML is driven by the MLL-AF9 oncogene. We measured oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using a flux analyzer. TG MLL-AF9 mice showed a significantly higher basal and capacity of OCR than WT MLL-AF9 mice ex vivo. EVI1+ cells showed accelerated oxidative phosphorylation (OXPHOS) prior to activation of glycolysis, and higher dependency on glutamine as an energy source. To identify the metabolic pathways regulated by EVI1, we performed capillary electrophoresis time-of-fight mass spectrometry-based metabolome profiling of WT and TG MLL-AF9 leukemia cells. We found significant differences between the cells in terms of the amounts of metabolites derived from the glycolytic and TCA cycles. Fructose 1,6-bisphosphate and lactate were up-regulated in TG MLL-AF9 cells, implying activation of glycolysis. Moreover, the amounts of fumarate and malate (metabolites of the TCA cycle) were significantly higher in TG MLL-AF9 cells. EVI1 played a role in glycolysis as well as driving expression of genes engaged in the tricarboxylic acid cycle. Next, we tested whether pharmacological inhibition of glycolysis and glutaminolysis suppresses MLL-AF9. L-asparaginase (ASP) [which catalyzes hydrolysis of asparagine (Asn) and glutamine (Gln) to asparatic acid or glutamic acid, respectively] markedly suppressed proliferation of TG MLL-AF9 cells, EVI1highAML cell lines. To examine the therapeutic potential of ASP in vivo, we treated secondary recipients of TG MLL-AF9 AML cells with ASP or control (vehicle), beginning 5 days post-transplantation. Mice then received intraperitoneal injections (five times per week) of distilled water or ASP (1000 U/kg). ASP led to a significant reduction in the number of GFP+ AML cells in the peripheral blood and increased the survival of recipient mice. Next, we examined an AML xenograft model. Two groups of NOG mice were injected subcutaneously with UCSD/AML1 cells and then treated with ASP or control. ASP -treated mice showed a significant reduction in the growth of AML tumors. Overall, these findings indicate that ASP -mediated inhibition of OXPHOS is a potential treatment for AML. We clarified that increased glutamine dependency by MLL-r AML cells showing high EVI1 expression makes them sensitive to ASP. We found that the energy advantage of AML cells is acquired via transcription factor-mediated activation of mitochondrial metabolism, leading to a poor prognosis. Furthermore, we show that new therapeutic options can be identified by examining the energy-based metabolic characteristics of leukemia cells. Disclosures No relevant conflicts of interest to declare.