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The genetic basis of leukemogenesis in adults with B-cell acute lymphoblastic leukemia (B-ALL) is largely unclear, and its clinical outcome remains unsatisfactory. This study aimed to advance the understanding of biological characteristics, improve disease stratification, and identify molecular targets of adult B-ALL. Adolescents and young adults (AYA) (15 to 39 years old, n = 193) and adults (40 to 64 years old, n = 161) with Philadelphia chromosome-negative (Ph−) B-ALL were included in this study. Integrated transcriptomic and genetic analyses were used to classify the cohort into defined subtypes. Of the 323 cases included in the RNA sequencing analysis, 278 (86.1%) were classified into 18 subtypes. The ZNF384 subtype (22.6%) was the most prevalent, with 2 novel subtypes (CDX2-high and IDH1/2-mut) identified among cases not assigned to the established subtypes. The CDX2-high subtype (3.4%) was characterized by high expression of CDX2 and recurrent gain of chromosome 1q. The IDH1/2-mut subtype (1.9%) was defined by IDH1 R132C or IDH2 R140Q mutations with specific transcriptional and high-methylation profiles. Both subtypes showed poor prognosis and were considered inferior prognostic factors independent of clinical parameters. Comparison with a previously reported pediatric B-ALL cohort (n = 1003) showed that the frequencies of these subtypes were significantly higher in AYA/adults than in children. We delineated the genetic and transcriptomic landscape of adult B-ALL and identified 2 novel subtypes that predict poor disease outcomes. Our findings highlight the age-dependent distribution of subtypes, which partially accounts for the prognostic differences between adult and pediatric B-ALL.

The developmental pathway for human megakaryocytes remains unclear, and the definition of pure unipotent megakaryocyte progenitor is still controversial. Using single-cell transcriptome analysis, we have identified a cluster of cells within immature hematopoietic stem- and progenitor-cell populations that specifically expresses genes related to the megakaryocyte lineage. We used CD41 as a positive marker to identify these cells within the CD34+CD38+IL-3RαdimCD45RA- common myeloid progenitor (CMP) population. These cells lacked erythroid and granulocyte-macrophage potential but exhibited robust differentiation into the megakaryocyte lineage at a high frequency, both in vivo and in vitro. The efficiency and expansion potential of these cells exceeded those of conventional bipotent megakaryocyte/erythrocyte progenitors. Accordingly, the CD41+ CMP was defined as a unipotent megakaryocyte progenitor (MegP) that is likely to represent the major pathway for human megakaryopoiesis, independent of canonical megakaryocyte-erythroid lineage bifurcation. In the bone marrow of patients with essential thrombocythemia, the MegP population was significantly expanded in the context of a high burden of Janus kinase 2 mutations. Thus, the prospectively isolatable and functionally homogeneous human MegP will be useful for the elucidation of the mechanisms underlying normal and malignant human hematopoiesis.

MEF2D fusion (M-fusion) genes are newly discovered recurrent gene abnormalities that are detected in approximately 5% of acute lymphoblastic leukemia (ALL) cases. We previously found that the loss of the micro RNA target site in wild-type MEF2D gene by translocation led to strong expression of M-fusion protein in ALL cells by evasion from micro RNA and that M-fusion protein inhibited the transcriptional activity of PAX5, a B-cell differentiation regulator, in a dose-dependent manner. These findings prompted us to explore drugs that induced proteolysis of M-fusion protein as possible therapeutic agents for M-fusion-positive ALL. We developed a high-throughput screening system to find compounds that reduced protein expression level of MEF2D. The expression vector of the fusion protein of N-terminal half of MEF2D (MEF2D N) and luciferase (MEF2D N-Luc) was stably transfected to 293T cells (MEF2D N-Luc/293T). Stable transfectant of the expression vector of luciferase was also established (Luc/293T). We could easily measure protein expression level in these cells by luciferase assay. We screened 3766 compounds with known pharmaceutical activities with this system and selected staurosporine, a multi-kinase inhibitor, as a possible proteolysis-inducer of MEF2D. Staurosporine strongly reduced the luciferase value in MEF2D N-Luc/293T but not in Luc/293T (Figure 1A). Staurosporine induced proteolysis of MEF2D-HNRNPUL1 (M-H) and MEF2D-DAZAP1 (M-D) in M-fusion-positive ALL cell lines within 6 h. Proteolysis of M-fusion proteins were inhibited not by MG-132, a proteasome inhibitor, but by Z-VAD FMK, a caspase inhibitor, indicating that these proteolyses were caspase-dependent (Figure 1B). Consistent with this, Z-VAD-FMK blocked apoptosis by staurosporine in M-H positive ALL cell lines . We confirmed the cleavage of M-H by caspase 3 and caspase 7 in vitro and identified the cleavage site (Figure 1C). Furthermore, staurosporine demonstrated stronger cytotoxic effect on M-fusion-positive ALL cell lines than M-fusion-negative ones (Figure 1D). These results indicated that staurosporine induced apoptosis of M-fusion-positive ALL cells through caspase-dependent proteolysis of M-fusion protein at least in part. Luciferase-based proteolysis screening provided a novel strategy for the development of anti-cancer drugs. Figure legends Figure 1. A. Staurosporine strongly reduced the luciferase value in MEF2D N-Luc/293T but not in Luc/293T. MEF2D N-Luc/293T were treated with 3766 compounds (2uM each) for 24 h. Then luciferase assays were performed to estimate the amount of MEF2D N-Luc protein. Top 15 compounds which reduced the relative luciferase value were selected for the second screening where compounds were added to MEF2D N-Luc/293T and Luc/293T, then we estimated their effect on the expression of MEF2D N-Luc and luciferase. Results of the second screening were plotted on a scattergram, on which the relative luciferase value in Luc/293T and MEF2D N-Luc/293T were set on the Y-axis and X-axis, respectively. Relative luciferase values are relative values to those of control cells treated with vehicle (DMSO). Staurosporine and K252a, an analog of staurosporine, were selected as hit compounds which reduced MEF2D N-Luc but not luciferase. B. Staurosporine reduced the expression of M-H and M-D, which inhibited by Z-VAD FMK. M-H-positive ALL cell lines, Kasumi-7 and Kasumi-9 and a M-D-positive ALL cell line, TS-2 were treated with 1uM staurosporine, 20uM Z-VAD-FMK, or both for 6 h. Cells were lysed and subjected to immunoblotting with indicated antibodies. C. Caspase-3 and caspase-7 cleaved M-H in vitro. M-H and its mutants with aspartate substitutions at possible caspase cleavage sites were synthesized with [35S]-methionine-labeled in vitro translation and were incubated with purified caspase-3 or -7. Cleaved fragments were resolved on SDS-PAGE and visualized by autoradiography. Mutant 4 was resistant to the cleavage by both caspases. D. Staurosporine demonstrated stronger cytotoxic effect on M-fusion-positive ALL cell lines than M-fusion-negative ones. ALL cell lines were treated with staurosporine at the indicated doses for 24 h. Cell viabilities were measured using MTT assay. Figure 1 Disclosures Kiyoi: Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Honoraria; Astellas Pharma Inc.: Honoraria, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Eisai Co., Ltd.: Research Funding; Bristol-Myers Squibb: Research Funding; Daiichi Sankyo Co., Ltd: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Perseus Proteomics Inc.: Research Funding.

The optimal treatments for relapsed acute promyelocytic leukemia (APL) remain equivocal. We conducted a phase 2 study to evaluate the efficacy and feasibility of a sequential treatment consisting of induction and consolidation with arsenic trioxide (ATO), peripheral blood stem cell (PBSC) harvest after high-dose cytarabine chemotherapy, and autologous hematopoietic cell transplantation (HCT). Between 2005 and 2009, 35 patients (26 with hematologic and 9 with molecular relapse) were enrolled. Induction therapy resulted in complete remission in 81% of those with hematologic relapse, and most patients became negative for PML-RARα after the first ATO consolidation course, but 4 remained positive. Administration of the second ATO consolidation course further decreased the transcript levels in 3 patients. In total, 25 patients proceeded to PBSC harvest, all of whom successfully achieved the target CD34+ cell doses, and 23 underwent autologous HCT with PML-RARα-negative PBSC graft. Posttransplant relapse occurred in 3 patients, and there was no transplant-related mortality. With a median follow-up of 4.9 years, the 5-year event-free and overall survival rates were 65% and 77%, respectively. These findings demonstrate the outstanding efficacy and feasibility of the sequential treatment featuring ATO and autologous HCT for relapsed APL. This study was registered at http://www.umin.ac.jp/ctr/ as #C000000302.

Although survival rate for children with Acute Lymphoblastic Leukemia (ALL) now exceeds about 90%, the outcome of adult patients with ALL is extremely poor. These differences might be attributed to the lack of insights into pathogenesis and clinical behavior of adult-ALL. Gross chromosomal alterations including chromosome translocations and aneuploidy are considered as early events in ALL and constitute disease subtypes. To identify chromosome translocations underlying adult with Ph-negative B-ALL, we performed RNA-seq analysis on RNA from individuals with B-ALL who had been treated on the Japan Adult Leukemia Study Group (JALSG) ALL202-O protocol (n = 149). We successfully identified chromosome translocations in 100 patients (67.1%). ZNF384 fusions were most frequently detected in 30 patients (20.1%) and they had wide range of fusion partners. DUX4- and MEF2D- fusions were also recurrently found in 7 (4.7%) and 9 (6.0%) patients, respectively. Chromosome translocations activating kinase and cytokine receptor were found in 25 patients (16.8%) with Ph-like gene expression profile. These alterations were almost completely mutually exclusive indicating these are likely to be primary genetic events. For simplicity, here we define (1) fusions involving ZNF384, DUX4, MEF2D, CEBP and PAX5 as well as TCF3-PBX1 and ETV6-RUNX1 as Transcription Factor fusions (TF fusions; 49% of patients), (2) fusions involving CRLF2, JAK2, PDGFRB, EPOR and ABL as Kinase/cytokine-receptor Activating fusions (KA fusions; 15%) and (3) non-recurrent fusions or the absence of fusions/aneuploidy as B-others (30%). First, we analyzed impact of the patient age on types of fusion genes, based of combined data of ALL202-O cohort, childhood B-ALL cohort (Lilljebjörn H, et al. 2016: n = 189) and ALL202-U cohort (Yasuda T, et al. 2016: n = 54). We found that incidence of ZNF384-, CEBP- fusions and B-others increases as patients age, whereas ETV6-RUNX1 and PAX5 fusions were more prevalent in younger patients, exhibiting negative association with age. DUX4 fusions and TF fusions were most prevalent in Adolescent and Young Adult (AYA) generation. JAK2-, PDGFRB-, EPOR- and KA- fusions were positively correlated with age. Next, we analyzed association between patient survival and types of fusions. In Japanese adult B-ALL cohort (ALL202-O and ALL202-U cohort), we observed ZNF384-, DUX4- fusions and TCF3-PBX1 were associated with better disease-free survival than B-others. Furthermore, when combined, MEF2D- (n = 14), CEBP- (n = 4), PAX5- fusions (n = 2) and ETV6-RUNX1 (n = 2) exhibited significantly better disease-free survival than B-others, indicating TF fusions were associated with an improved outcome. In contrast, KA fusions were associated with poorer disease-free survival than B-others. KMT2A fusions were comparable with B-others regarding to patient disease-free survival. These results allowed us to develop a prognostic schema to identify three distinct risk profile groups, based on types of fusion genes and cytogenetics (Table1); favorable-risk (5-year rate of disease-free survival 67.4%), intermediate-risk (5-year rate of disease-free survival 42.5%) and adverse-risk (5-year rate of disease-free survival 9.6%). This prognostic schema predicted the outcome independently of age, sex and methotrexate dose in multivariate analysis (p < 0.001). In conclusion, we promoted a better understanding of the genetic basis of adult B-ALL by focusing on fusion genes. Each chromosome translocations were closely associated with age. ZNF384-, KA fusions and B-others were characteristic for older-adult patients (40-65 years old) with B-ALL. We clearly demonstrated specific primary chromosome abnormalities are strong prognostic marker. Functional properties of primary genetic events (TF fusions vs. KA fusions) might be a key determinant of biological characteristics and clinical outcome. Disclosures Kiyoi: Novartis Pharma K.K.: Research Funding; Celgene Corporation: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sanofi K.K.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Phizer Japan Inc.: Research Funding; Eisai Co., Ltd.: Research Funding. Naoe:Astellas Pharma Inc.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding.

ZNF384 fusion (Z-fusion) genes are recently identified recurrent fusion genes of B-acute lymphoblastic leukemia (ALL) and cause differentiation block of B-cells; however, its molecular mechanisms have yet to be clarified. Common structural character of Z-fusion proteins is that fusion partners are fused to the N-terminal end of full-length ZNF384 (Figure 1A), suggesting that protein-fusions confer specific transcriptional targets on ZNF384. We searched Z-fusion-specific transcriptional targets that could cause differentiation block of B-cells by analyzing the data of gene expression profile of 54 primary B-ALL samples containing 9 Z-fusion positive ALL. We selected ID2 and SALL4 as potential targets. Both genes were expressed markedly higher in Z-fusion-positive ALL. ID2 acts as an inhibitor of E2A, B cell differentiation regulator, and SALL4 plays essential roles in maintaining pluripotency of embryonic stem cells. In the luciferase assays, EP300-ZNF384 (E-Z) and SYNRG-ZNF384 (S-Z) showed stronger transcriptional activities on the promoters of these genes than wild-type ZNF384 (Wild-Z). The introduction of E-Z or S-Z into 293T cells and THP-1 cells induced mRNA expression of these genes more strongly than that of Wild-Z (Figure 1B). We identified Z-fusion binding sites in the promoters of these genes. DNA binding abilities of Z-fusions to these sites were not stronger than that of Wild-Z in electro mobility shift assay. GST-pull down assay showed that E-Z associated with EP300 more strongly than Wild-Z (Figure 1C). Consistent with this, co-expression of EP300 enhanced the transcriptional activity of E-Z better than that of Wild-Z (Figure 1D). These results indicated that ID2 and SALL4 were Z-fusion-specific transcriptional targets and that the high affinity to EP300 was responsible for the strong transcriptional activity of Z-fusions. Our results shed a new insight into the molecular mechanisms of leukemia development by Z-fusions. Figure legends Figure 1. A. Schematic presentation of structures of Wild-Z and Z-fusion proteins. B. Introduction of Z-fusion genes enhanced the mRNA expression of ID2 and SALL4. Wild-Z and Z-fusion genes were introduced to THP-1 cells by nucleofection. Twenty-four hours after gene introduction, the mRNA expression of ID2 and SALL4 were quantified by RQ-PCR and potted on bar charts as relative values to the mRNA expression in the control cells. The expression of ID2 and SALL4 were shown in the left and right bar charts, respectively. C. E-Z associated with HAT more strongly than Wild-Z. Glutathione beads attached with Glutathione S transferase (GST) or GST-fused HAT were incubated with Wild-Z or E-Z synthesized in vitro with [35S]-Methionine labeling. Wild-Z or E-Z associated with GST-HAT were visualized with autoradiography (left panel), quantified, and plotted on the bar charts (right panel). Of note, the quantified association were adjusted for the ratio of the number of methionine containing in Wild-Z and E-Z, 12 to 45, and plotted on the bar charts. D. Co-expression of EP300 enhanced the transcriptional activity of E-Z better than that of Wild-Z. Luciferase assay were performed with or without co-expression of EP300 and the relative value to the control were plotted on the bar charts. Disclosures Naoe: Astellas Pharma Inc.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding. Kiyoi:FUJIFILM Corporation: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Celgene Corporation: Research Funding; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Sanofi K.K.: Research Funding.

To examine the efficacy of intensified maintenance chemotherapy, we conducted a prospective multicenter trial in adult patients with newly diagnosed acute promyelocytic leukemia treated with all-trans retinoic acid and chemotherapy. Of the 302 registered, 283 patients were assessable and 267 (94%) achieved complete remission. Predicted 6-year overall survival in all assessable patients and disease-free survival in patients who achieved complete remission were 83.9% and 68.5%, respectively. A total of 175 patients negative for PML-RARα at the end of consolidation were randomly assigned to receive either intensified maintenance chemotherapy (n = 89) or observation (n = 86). Predicted 6-year disease-free survival was 79.8% for the observation group and 63.1% for the chemotherapy group, showing no statistically significant difference between the 2 groups (P = .20). Predicted 6-year survival of patients assigned to the observation was 98.8%, which was significantly higher than 86.2% in those allocated to the intensified maintenance (P = .014). These results indicate that the intensified maintenance chemotherapy did not improve disease-free survival, but rather conferred a significantly poorer chance of survival in acute promyelocytic leukemia patients who have become negative for the PML-RARα fusion transcript after 3 courses of intensive consolidation therapy.

Diffuse large B-cell lymphoma (DLBCL) comprises molecularly distinct subgroups such as activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCLs. We previously reported that CD5(+) and CD5(-)CD10(+) DLBCL constitute clinically relevant subgroups. To determine whether these 2 subgroups are related to ABC and GCB DLBCLs, we analyzed the genomic imbalance of 99 cases (36 CD5(+), 19 CD5(-)CD10(+), and 44 CD5(-)CD10(-)) using array-based comparative genomic hybridization (CGH). Forty-six of these cases (22 CD5(+), 7 CD5(-)CD10(+), and 17 CD5(-)CD10(-)) were subsequently subjected to gene-expression profiling, resulting in their division into 28 ABC (19 CD5(+) and 9 CD5(-)CD10(-)) and 18 GCB (3 CD5(+), 7 CD5(-)CD10(+), and 8 CD5(-)CD10(-)) types. A comparison of genome profiles of distinct subgroups of DLBCL demonstrated that (1) ABC DLBCL is characterized by gain of 3q, 18q, and 19q and loss of 6q and 9p21, and GCB DLBCL is characterized by gain of 1q, 2p, 7q, and 12q; (2) the genomic imbalances characteristic of the CD5(+) and CD5(-)CD10(+) groups were similar to those of the ABC and GCB types, respectively. These findings suggest that CD5(+) and CD5(-)CD10(+) subgroups are included, respectively, in the ABC and GCB types. Finally, when searching for genomic imbalances that affect patients' prognosis, we found that 9p21 loss (p16(INK4a) locus) marks the most aggressive type of DLBCL.

Abstract 1703 The majority of acute promyelocytic leukemia (APL) cases are characterized by the presence of a PML-RARA fusion gene. In a small subset, RARA is fused to a different partner including PLZF, NPM1, NuMA, STAT5b, PRKAR1A and FIP1L1. Here we identified a novel RARA fusion transcript, BCOR-RARA in a t(X;17)(p11;q12) variant of APL. The patient was a 45-year-old man. Although the patient was clinically responsive to ATRA, repeated standard chemotherapy with ATRA did not effect a cure. The bone marrow promyelocytes had unique morphologic features, including rectangular and round cytoplasmic inclusion bodies. They were more granular than those of AML M2 but less granular than the classical t(15;17) APL. Flow cytometric analysis revealed strong expression of CD13, CD33, CD56, weak expression of CD11c and lack of HLA-DR and CD7. The karyotype analysis detected a novel chromosomal translocation described as 45,-Y, t(X;17)(p11.2;q12)[19]/ 46,XY[1]. FISH analysis indicated one intact and two split signals of RARA and two intact signals of PML. To amplify unknown chimeric fusion transcripts, we performed 5'-RACE. The sequence revealed that BCL6 co-repressor, BCOR cDNA from exons 9 to 12 to be fused to RARA exon 3. By RT-PCR, we confirmed full length chimeric fusion transcripts spanning from the start codon to 4,948 nt of BCOR cDNA (NM_001123384) fused to RARA cDNA from exon 3 to the stop codon. The chimeric cDNA had an in-frame codon from BCOR through RARA, creating a 1,931 amino acid fusion protein. One of the consistent features in all known RARA fusion partners is self-association. To determine whether BCOR-RARA self-associate, we performed co-immunoprecipitation assays. These results showed that BCOR-RARA is able to self-associate both through the region of BCOR-S and the ankyrin repeat domain of BCOR. In addition, BCOR-RARA associated with BCL6. RXR recruitment is a critical determinant of transforming potential of oligomeric RARA fusion proteins. To investigate how BCOR-RARA associates with RARE in vitro, we performed EMSA. These results showed that BCOR-RARA/RXRA complex associates with RARE in an alternative manner compared to RARA and PML-RARA. Deregulation of RARA transcriptional activations has a central role in pathogenesis of APL. Therefore, we evaluated ATRA-induced transcriptional activation of 4× RAREs with a reporter assay in HepG2 cells. Without ATRA, BCOR-RARA repressed the reporter activity. With addition of ATRA, BCOR-RARA induced transcriptional activation very weakly. Subsequently, we evaluated dominant-negative effects of the samples in the RARA/RXRA pathway. In contrast to BCOR, BCOR-RARA clearly inhibited ATRA-induced RARA transcriptional activation as well as PML-RARA. Furthermore, we asked which domains are sufficient for the dominant-negative effects with the deletion mutants. The results indicated that the region spanning from 999 to 1,409 aa of BCOR-RARA has pivotal roles in the dominant-negative effects. Correct protein function is highly dependent on intracellular localization. To investigate subcellular localization of BCOR-RARA, we performed immunofluorescence analysis in 293T cells. In BCOR-RARA-expressing cells, BCOR-RARA localized as two patterns; (I) diffusely in the nucleus as well as PML-RARA in 82% of the cells, (II) diffusely in the nucleus and aggregately in the cytoplasm in 18% of the cells. The subcellular localization of BCOR-RARA was clearly distinguishable from the punctuate pattern as shown in the nucleus of BCOR-expressing cells. Moreover, co-immunofluorescence analysis between BCOR-RARA and BCL6 indicated that the subcellular localization of BCOR-RARA/BCL6 is distinct from BCOR/BCL6. BCOR-RARA was found to possess common features with other RARA fusion proteins. These included: (I) the same break point in RARA cDNA; (II) self-association; (III) RXRA is necessary for BCOR-RARA to associate with the RARA responsive element; (IV) action in a dominant-negative manner on RARA transcriptional activation; (V) aberrant subcellular relocalization. It should be noted that there was no intact BCOR found in the 45,-Y,t(X;17)(p11;q12) APL cells because they featured only a rearranged × chromosome. These results highlight essential features of pathogenesis in APL in more detail. BCOR appears to be involved not only in human congenital diseases but also in a human cancer. Disclosures: No relevant conflicts of interest to declare.

Transcription factor GATA-2 is implicated in the survival and growth of multipotential progenitors. Here we report that the promyelocytic leukemia zinc finger (PLZF) protein can interact with GATA-2 and can modify its transactivation capacity. Fanconi anemia zinc finger (FAZF), a PLZF-homologous protein that has been variously described as ROG (repressor of GATA), and TZFP (testis zinc finger protein) also interact with GATA-2. The zinc finger region of GATA-2 is required for binding to PLZF and FAZF, but distinct interfaces on the PLZF and FAZF molecules mediate the interaction, suggesting that GATA-2 activity is controlled by these 2 homologous proteins through distinct mechanisms. GATA-2 can also physically associate with the PLZF-RARα fusion protein generated by the t(11;17) chromosomal translocation associated with acute promyelocytic leukemia (APL). Functional experiments showed that this interaction has the capacity to render GATA-dependent transcription responsive to treatment with a combination of all-trans retinoic acid and the histone deacetylase inhibitor trichostatin A (TSA). This combination of drugs has been shown to stimulate the terminal differentiation of leukemic t(11;17)-associated APL blasts, raising the possibility that GATA target genes may be involved in the molecular pathogenesis of APL.

The aberrant regulation of B-cell receptor (BCR) signaling allows unwanted B cells to persist, thereby potentially leading to autoimmunity and B-cell malignancies. Casitas B-lineage lymphoma (Cbl) proteins suppress BCR signaling; however, the molecular mechanisms that control Cbl function in human B cells remain unclear. Here, we demonstrate that CIN85 (c-Cbl interacting protein of 85 kDa) is constitutively associated with c-Cbl, Cbl-b, and B-cell linker in B cells. Experiments using CIN85-overexpressing and CIN85-knockdown B-cell lines revealed that CIN85 increased c-Cbl phosphorylation and inhibited BCR-induced calcium flux and phosphorylation of Syk and PLCγ2, whereas it did not affect BCR internalization. The Syk phosphorylation in CIN85-overexpressing and CIN85-knockdown cells was inversely correlated with the ubiquitination and degradation of Syk. Moreover, CIN85 knockdown in primary B cells enhanced BCR-induced survival and growth, and increased the expression of BcLxL, A1, cyclin D2, and myc. Following the stimulation of BCR and Toll-like receptor 9, B-cell differentiation- associated molecules were up-regulated in CIN85-knockdown cells. Together, these results suggest that CIN85 is required for Cbl-mediated regulation of BCR signaling and for downstream events such as survival, growth, and differentiation of human B cells.

Self-renewal activity is essential for the maintenance and regeneration of the hematopoietic system. The search for molecules capable of promoting self-renewal and expanding hematopoietic stem cells (HSCs) has met with limited success. Here, we show that a short isoform (AML1a) of RUNX1/AML1 has such activities. Enforced AML1a expression expanded functionally defined HSCs, with an efficiency that was at least 20 times greater than that of the control in vivo and by 18-fold within 7 days ex vivo. The ex vivo–expanded HSCs could repopulate hosts after secondary transplantations. Moreover, AML1a expression resulted in vigorous and long-term (> 106-fold at 4 weeks) ex vivo expansion of progenitor cell populations capable of differentiating into multilineages. Gene expression analysis revealed that AML1a expression was associated with up-regulation of genes, including Hoxa9, Meis1, Stat1, and Ski. shRNA-mediated silencing of these genes attenuated AML1a-mediated activities. Overall, these findings establish AML1a as an isoform-specific molecule that can influence several transcriptional regulators associated with HSCs, leading to enhanced self-renewal activity and hematopoietic stem/progenitor cell expansion ex vivo and in vivo. Therefore, the abilities of AML1a may have implications for HSC transplantation and transfusion medicine, given that the effects also can be obtained by cell-penetrating AML1a protein.

Oligo-array comparative genomic hybridization (CGH) and gene-expression profiling of natural killer (NK)–cell neoplasms were used in an effort to delineate the molecular pathogenesis involved. Oligo-array CGH identified two 6q21 regions that were most frequently deleted (14 of 39 or 36%). One of these regions included POPDC3, PREP, PRDM1, ATG5, and AIM1, whereas the other included LACE1 and FOXO3. All genes located in these regions, except for POPDC3 and AIM1, were down-regulated in neoplastic samples, as determined by gene-expression analysis, and were therefore considered to be candidate tumor-suppressor genes. A20 and HACE1, the well-known tumor-suppressor genes located on 6q21-23, were included as candidate genes because they also demonstrated frequent genomic deletions and down-regulated expression. The Tet-Off NK cell line NKL was subsequently established for functional analyses. Seven candidate genes were transduced into Tet-Off NKL and forced re-expression was induced. Re-expression of FOXO3 and PRDM1 suppressed NKL proliferation, but this was not the case after re-expression of the other genes. This effect was confirmed using another NK cell line, SNK10. Furthermore, genomic analyses detected nonsense mutations of PRDM1 that led to functional inactivation in one cell line and one clinical sample. PRDM1 and FOXO3 are considered to play an important role in the pathogenesis of NK-cell neoplasms.

We report here the results of polymerase chain reaction (PCR) for bcr- abl transcript and clinical details derived from 64 chronic myelogenous leukemia (CML) patients after allogeneic bone marrow transplantation (BMT). A total of 139 samples (2 to 220 weeks after BMT) were analyzed and bcr-abl transcript was detected in 99 samples from 52 patients. Patients were defined as bcr-abl early negative (EN) if they had > or = 1 negative PCR result < or = 1 year post-BMT (n = 13), and bcr-abl late positive (LP) if they had > or = 1 positive PCR result > or = 1 year post-BMT (n = 21). Among LP patients, only two patients had hematologic/cytogenetic (clinical) relapse. Another 19 LP patients remained in clinical remission 7 to 130 weeks after positive analysis for bcr-abl transcript, including 5 patients who had persistent bcr-abl transcript detectable even 2 years after BMT. To estimate the relationship between clinical data and residual bcr-abl transcript, EN patients are compared with LP patients. However, no clinical data studied were significantly associated with the persistent PCR positivity. If only patients in chronic phase are compared, the t-test showed significant correlation between leukocyte count just before BMT and sustained bcr-abl transcript (P < .05). These results suggest that PCR positivity is frequently observed in CML patients who sustain clinical remission after BMT, without being predictive of imminent clinical relapse. Tumor burden at the time of BMT may play an important role in the latency of bcr-abl positivity after BMT.

The constitutive activation of nuclear factor-κB (NF-κB) has been implicated in tumorigenesis of lymphoid malignancies. We have previously shown that chromosome 6q was frequently deleted in ocular marginal zone B-cell lymphoma and identified TNFAIP3/A20, a negative regulator of NF-κB pathways, as the primary target for 6q deletion. In the study reported here, we extended the analysis to other subsets of non-Hodgkin lymphomas and found that A20 is frequently deleted in mantle cell lymphoma and diffuse large B-cell lymphoma. Importantly, A20 promoter methylation or gene mutation is also frequently detected in these lymphomas, raising the possibility that inactivation of A20 may be involved in lymphomagenesis. To address this question, we conducted overexpression experiments in lymphoma cell lines with A20 deletion and down-regulated expression of A20 with an siRNA technique in Epstein-Barr virus–infected lymphoblastoid cell lines. These experiments found that overexpression of A20 induced apoptosis and silencing of A20 was associated with resistance to apoptosis and enhanced clonogenicity. The cells with down-regulated A20 exhibited enhanced NF-κB activities, which may account for the observed effects. These results indicate that our study provides a novel insight into molecular mechanisms leading to lymphoma and that specific targeting of NF-κB pathways may be advantageous for treatment.

PAX5 is a transcription factor required for B-cell development and maintenance. We previously showed that PAX5-PML, a fusion gene found in acute lymphoblastic leukemia (ALL), dominant negatively inhibited PAX5 transcriptional activity. Reported data including ours revealed that PAX5 fusion proteins had possible oncogenic ability; however, leukemogenicity of PAX5 fusion genes and other PAX5 mutations in mice model has not been clarified, yet. Here we demonstrated leukemia development in mice by introducing PAX5-PML. Pro B cells derived from mouse fetal liver were transfected with PAX5-PML expression vector and transplanted into mice. All 8 transplanted mice died with pro B ALL from day 63 to 158. Leukemic cells could be serially transplanted and mice died more rapidly with repetition (Figure A). Among the target genes transcriptionally activated by PAX5, expressions of BLNK, Fcer2a, and CD72 were significantly repressed in leukemia cells but repression of CD19 and CD79a were mild, suggesting the importance of down regulation of these genes for differentiation block. We compared mRNA expression profile between leukemia cells and normal pro B cells and gene set enrichement analysis (GSEA) identified candidates for second hits for development of leukemia. We analyzed the mechanism of the selective repression of CD19, Fcer2a, and BLNK and the significance of the second hit candidates, using a cell line established from leukemia cells of the third transplanted mouse. The results will show the meeting. Figure 1 Figure 1. Disclosures Sugimoto: Otsuka Pharmaceutical Co., Ltd: Employment. Naoe:Zenyaku Kogyo: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Kyowa Hakko Kirin Co. LTD: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Novartis Pharma,: Research Funding; Bristol-Myers Squibb: Research Funding; Otsuka Pharmaceutical Co. LTD: Research Funding; FUJIFILM Corporation: Research Funding. Kiyoi:Zenyaku Kogyo: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Kyowa Hakko Kirin Co. LTD.: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Bristol-Myers Squibb: Research Funding; FUJIFILM Corporation: Research Funding.

Introduction: Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of non-Hodgkin lymphomas noted for their poor prognosis. Their molecular pathogenesis has not been entirely elucidated. We previously found that primary thyroid T-cell lymphoma (PTTL) is a distinct entity among heterogeneous PTCLs and that this disease is characterized by the genomic loss of 6q24 (Br J Haematol., 161:214-223). In this study, we extended the analysis to other types of PTCLs and performed functional assays to identify causative genes located on 6q24. Methods: Focusing on chromosome 6q loss, we reexamined previous comparative genomic hybridization data from 267 PTCL cases comprising 6 PTTLs, 51 PTCLs-not otherwise specified (NOS), 62 adult T-cell leukemias/lymphomas, 35 natural killer (NK)-cell lymphomas, 39 angioimmunoblastic T-cell lymphomas (Genes Chromosomes Cancer, 46:37-44), and 74 anaplastic large cell lymphomas (Br J Haematol., 140:516-526). Gene expression levels were determined by using published gene expression profiling (GEP) data (GSE6338 and GSE19069) and quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Subsequently, we established Tet-Off cell lines belonging to several lineages (6 T-cell lines, 1 NK-cell line, 4 B-cell lines, 1 myeloid cell line, and 3 epithelial cell lines) for functional analyses. Results: Genomic loss of 6q24 was observed in 8% (n = 267) of PTCL cases, and it occurred most frequently in PTTL cases (67%; n = 6). All the genomic losses were heterozygous; homozygous loss of this region was not observed in our analysis. The smallest region of deletion, observed in a PTTL case, was considered the minimal common region (MCR) of 6q24 loss. The MCR contained 2 known coding genes, STX11 and UTRN. Combined GEP data and quantitative RT-PCR analyses showed that the expression of STX11, but not UTRN, was markedly lower in PTCL than in normal T-cells. We therefore regarded STX11 as the most probable candidate gene located in 6q24. Syntaxin 11, encoded by STX11, is a t-SNARE protein that plays a role in binding vesicles to cell membranes, and alteration of STX11 in the germline causes familial hemophagocytic syndrome type 4. To further evaluate genomic alteration of STX11, mutation analysis was performed on PTCL-NOS and PTTL cases as well as T-cell lines, for which adequate DNA was available. This revealed STX11 mutations in 2 cases (1 PTCL-NOS case and 1 T-cell line). Wild-type STX11 expression suppressed the proliferation of T-cell lines bearing genomic alterations at the STX11 locus only, and it did not show suppressive effects on other lineage cell lines (Fig. 1). Expression of STX11 induced cellular apoptosis in the cell line, although the number of apoptotic cells induced was relatively small. Interestingly, expression of a novel STX11 mutant (p.Arg78Cys), observed in a T-cell line, did not exert suppressive effects on the induced cell lines suggesting that there was a loss-of-function mutation (Fig. 2). Finally, we evaluated the clinical impact of STX11 alteration in PTTL and PTCL-NOS cases where data were available. This showed that PTCL-NOS cases with genomic alterations of STX11 tended to have a poorer prognosis than those without (Fig. 3; P = 0.069). Conclusion: In the present study, we examined the MCR of 6q24 loss and showed that STX11 acts as a tumor suppressor gene in PTCLs only. These findings provide a novel approach for understanding the molecular pathogenesis of PTCLs, and they may contribute to the future development of new drugs for the treatment of PTCLs. Disclosures No relevant conflicts of interest to declare.

In the chronic phase of chronic myeloid leukemia (CML-CP), leukemic stem cells do not necessarily depend on the BCR-ABL tyrosine kinase activity for their growth and survival and thus resistant to tyrosine kinase inhibitors (TKIs). In this study, we aimed to identify the initial progenitor population that is getting switched on BCR-ABL growth signaling and tried to elucidate the underlying molecular mechanisms of BCR-ABL dependent cell growth. We thus intensively analyzed the involvement status of CML clones in each developmental stage at diagnosis. To identify the hematopoietic stem or progenitor cell stage that is responsible for CML clone expansion, bone marrow cells from 13 newly-diagnosed CML-CP patients were analyzed by FACS, and purified stem/progenitor populations were tested for the t(9;22) involvement by FISH. Gene expression signature of each purified population was also evaluated by cDNA microarray. CD34+CD38- HSC fraction was markedly diminished in all CML-CP patients compared to healthy volunteers ( Gene expression analysis of stem/progenitor populations in CML patients revealed that IRF8 and GFI1, transcription factors playing critical roles in myeloid differentiation and cell proliferation, were down-regulated specifically in CMPs as compared with that in normal controls (Figure 2). In order to substantiate the role of IRF8 and GFI1 in CML pathogenesis, we used a CML mouse model established by enforced retroviral expression of BCR-ABL. As in analysis of CML patients, BCR-ABL expressing CMPs but not stem/multipotent progenitor cells acquired growth advantage over normal counterparts. Importantly, the expression of IRF8 and GFI1 became undetectable after BCR-ABL transduction in the expanding CMPs. Our observations revealed that, in CML-CP hematopoiesis, BCR-ABL dependent cell proliferation initiates at the CMP stage, and is accompanied with the down–regulation of IRF8 and GFI1. Because IRF8 knockout mice develop myeloproliferative disorders, and because CMPs expand in GFI1 null mice, the attenuation of these molecules could be downstream effector of BCR-ABL dependent myeloid cell growth. Taken together, the reactivation of these molecules might be useful to develop alternative therapeutic strategies for CML-CP, for example, with TKI-resistant BCR-ABL mutants. Disclosures: Miyamoto: Kyushu University Hospital: Employment.

Abstract 768 In murine hematopoiesis, hematopoietic stem cells (HSCs) and multi-potent progenitors (MPPs) have been identified within the LSK (Lin− Sca-1+ c-kit+) fraction of bone marrow cells (Morrison SJ and Weissman IL Immunity 1994). We have proposed that the first commitment step at the myeloid versus lymphoid bifurcation occurred outside the LSK fraction, where myeloid or lymphoid lineage-committed progenitors such as common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs) are prospectively isolated (Akashi et al Nature 2000). However, recent studies revealed that the initial commitment step might occur within LSK fraction. Adolfsson et al showed that a fraction of LSK cells expressing Flt3 at a high level have lost megakaryocyte/erythroid (MegE) potential and were largely primed for granulocyte/monocyte (GM) and lymphoid cells. This population was named lymphoid-primed multipotent progenitors (LMPPs) (Adolfsson et al Cell 2005). In our hands, by utilizing mice harboring a fluorescent reporter for GATA-1 transcription factor, we demonstrated that the initial upregulation of GATA-1 transcription factor was observed within MPP population, which was defined as CD34+ LSK fraction, and GATA-1+ MPPs were capable of generating only myelo-erythroid cells but lacked lymphoid potential (Arinobu et al Cell Stem Cell 2007). This result strongly suggested that the initial myelo-erythroid commitment occurs at the MPP stage. To isolate the earliest myelo-erythroid LSK progenitors in normal mice without utilizing the GATA-1 reporter system, we conducted expression profiling of GATA-1+ MPP population by cDNA microarray analyses and following validation by FACS analyses. We found that a cell-surface antigen CD41 was specifically expressed at a high level in GATA-1+ MPP cells, indicating that CD41hi MPP cells might be the corresponding population to GATA-1+ MPP cells. CD41hi MPPs gave rise exclusively to GM and MegE colonies in vitro, but no lymphoid colonies were observed even under lymphoid-inducible conditions such as co-culture with OP9 or OP9-DL1 stromal cell layer. In vivo, CD41hi MPPs showed differentiation potential into GM cells, erythroid cells and platelets, lacking B or T lymphoid cell-producing potential in congenic transplantation assay. While the GM cell-reconstitution potential of LMPPs and CMPs peaked around day10 and day4 after transplantation, respectively, CD41hi MPPs had their peak around 3 weeks and relatively long-lasting reconstitution potential, presumably reflecting their immaturity. To further evaluate differentiation potential more directly and quantitatively, CD41hi MPPs with LMPPs or CMPs were injected together into one individual recipient mouse. In this head-to-head competitive assay system, CD41hi MPPs generated a plenty of mature GM cells, whose numbers were nearly ten times of those produced from the original CMPs or LMPPs in a reconstitution setting. Thus CD41hi MPPs possess potent restricted-lineage differentiation potential into GM and MegE lineages. In addition, the gene expression analysis by single-cell quantitative real-time PCR and cDNA microarray revealed that the CD41hi MPP population expresses both GM- and MegE-affiliated genes, but not lymphoid genes, reflecting their lineage restriction. More importantly, each single CD41hi MPP cell expresses either one or both of GATA-1 and PU.1 at a low level, suggesting that the promiscuous expression of these transcription factors might play a critical role in myelo-erythroid lineage commitment (Miyamoto et al Developmental Cell 2002). Furthermore, to evaluate the functional importance of CD41hi MPP population, we created the systemic infectious model mice, mimicking the severe peritonitis. CD41hi MPP pool as well as GMP expands dramatically in response to an increased demand for GM cells, while LMPP does not; thus, CD41hi MPPs compose physiologically important pathway in myelo-erythropoiesis. Accordingly, CD41hi MPP population might represent the earliest branch point for myelo-erythroid development, which resides upstream of the conventional CMP. Based on these data, we propose to redefine the true CMP as CD41hi MPP population. This CD41hi earliest myelo-erythroid progenitor should be useful to investigate the molecular mechanisms for hematopoietic lineage fate decision. Disclosures: No relevant conflicts of interest to declare.