ALBERT

Description: 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.

Description: Transcription factor AML1/RUNX1, initially isolated from the t(8;21) chromosomal translocation in human leukemia, is essential for the development of multilineage hematopoiesis in mouse embryos. AML1 negatively regulates the number of immature hematopoietic cells in adult hematopoiesis, while it is required for megakaryocytic maturation and lymphocytic development. However, it remains yet to be determined how AML1 contributes to homeostasis of hematopoietic stem cells (HSCs). To address this issue, we analyzed in detail HSC function in the absence of AML1. Notably, cells in the Hoechst 33342 side population fraction and c-Kit-positive cells in the G0 cell cycle status were increased in number in AML1-deficient bone marrow, which suggests enrichment of quiescent HSCs. We also found an increase in HSC number within the AML1-deficient bone marrow using limiting dilution bone marrow transplantation assays. Thus, the number of quiescent HSCs is negatively regulated by AML1, loss of which may result in accumulation of leukemic stem cell pool in AML1-related leukemia. To identify mechanisms through which functional loss of AML1 exerts leukemogenic potential, we focused on the AML1-Evi-1 chimeric protein, which is generated by the t(3;21) chromosomal translocation and disturbs the normal function of AML1. We introduced AML1-Evi-1 and its mutants into murine bone marrow cells, and evaluated hematopoietic cell transformation by colony replating assays. The transforming activity of AML1-Evi-1 was impaired when any of the major functional domains of AML1-Evi-1 was lost. Moreover, overexpression of Evi-1 could not transform AML1-deleted bone marrow cells, suggesting that fusion of AML1 and Evi-1, rather than AML1 suppression and Evi-1 overexpression, is essential for AML1-Evi-1 leukemogenesis. Intriguingly, among the hematopoietic progenitor cell fractions, AML1-Evi-1 could transform only the uncommitted, immature hematopoietic cells, which contrasts with MLL-ENL, a chimeric protein generated in t(11;19) leukemia. AML1-Evi-1 transformed cells show a surface marker profile different from that of the cells transformed by AML1-MTG8/ETO, another leukemic gene product that also perturbs AML1 function. These results provide a valuable clue to a distinct mechanism determined by the Evi-1 moiety in the AML1-Evi-1 leukemogenesis and to a role of AML1 loss in the self-renewal of leukemic stem cells.

Description: The Notch1-RBP-Jκ and the transcription factor Runx1 pathways have been independently shown to be indispensable for the establishment of definitive hematopoiesis. Importantly, expression of Runx1 is down-regulated in the para-aortic splanchnopleural (P-Sp) region of Notch1- and Rbpsuh-null mice. Here we demonstrate that Notch1 up-regulates Runx1 expression and that the defective hematopoietic potential of Notch1-null P-Sp cells is successfully rescued in the OP9 culture system by retroviral transfer of Runx1. We also show that Hes1, a known effector of Notch signaling, potentiates Runx1-mediated transactivation. Together with the recent findings in zebrafish, Runx1 is postulated to be a cardinal down-stream mediator of Notch signaling in hematopoietic development throughout vertebrates. Our findings also suggest that Notch signaling may modulate both expression and transcriptional activity of Runx1.

Description: B-cell acute lymphoblastic leukemia (B-ALL) carrying DUX4 fusions is a novel cluster of B-ALL. DUX4 fusions are generated from insertions of wild- type (WT) DUX4, mainly into the IGH locus.The translocation replaces the 3′ end of the WT DUX4 coding region with a fragment of IGH or another gene, producing DUX4 out-of-frame fusion proteins devoid of the C terminus of WT DUX4. Usually, WT DUX4 is expressed in germ cells in testis, while its expression is epigenetically repressed in somatic tissues. Recently, it is identified to plays a critical role in transcriptional programs at the cleavage of human fertilized egg. In B-ALL, DUX4-IGH (D-I) is shown to be essential for leukemic transformation; however, little is known about the mechanistic basis. Here in this study, we extensively investigated the biological effects of D-I. First, we assessed the role of D-I using in vitro cell culture assays with human cord blood (CB) CD34+ cells. Introduction of D-I significantly caused retention of the CD34+ cell population compared with the mock vector, even though it failed to preferentially promote differentiation toward B cell lineage in vitro. To analyze the epigenetic and transcription control by D-I, we performed chromatin immunoprecipitation coupled with sequencing (ChIP-seq) using cell lines. In NALM6, a B-ALL cell line carrying D-I, a subset of D-I binding sites is accompanied by H3K4me3 and H3K27ac. We also assessed the histone modification status in Reh cells, a B-ALL cell line without DUX4 fusions, and observed that active histone marks are detected after binding of ectopically expressed D-I. Nevertheless, RNA sequencing of NALM6 and Reh overexpressing D-I showed minimal activation of genes near the D-I binding sites compared with those of NALM6 overexpressing WT DUX4. WT DUX4 is known to preferentially bind and activate repeat elements, especially human endogenous retroviral (HERV) elements in embryonic cells. NALM6 cells overexpressing WT DUX4 showed a drastic increase in the expression of HERV elements, while NALM6 and Reh overexpressing D-I did not. The expression of HERV elements was not altered by D-I in all the genomic regions, and we did not observe increased expression of HERV elements in patient leukemia samples with DUX4 fusions as well. Furthermore, Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) showed that chromatin status was not affected by the binding of D-I at the D-I bound HERV element, indicating that transcriptional and insulating ability of WT DUX4 in these areas are lost in D-I. Next, we performed ATAC-seq using NALM6 cells, comparing the status between pre- and post- D-I knockdown. Genomic areas with decreased ATAC signal after knockdown of D-I are enriched in D-I binding sites, and ATAC signal was increased when we compared the status between pre- and post- induction of D-I in Reh cells. Through the immunoprecipitation of endogenous D-I in NALM6 cells, we identified SWI/SNF complex elements as binding partners of D-I, further highlighting the chromatin opening ability of D-I. Motif analysis of the genomic areas with decreased ATAC signal after knockdown of D-I identified only DUX4 motif as a significant motif, suggesting that D-I is not apparently cooperating with other transcription factors. On the other hand, ATAC signal was increased in substantial genomic areas after knockdown of D-I, and motif analysis identified SPI1, TCF3, and EBF1 motifs. Integrated analysis of transcriptome data also supports the idea that transcription factors related to B cell differentiation are repressed in the presence of D-I, and derepressed after knock down of D-I. Despite the attenuated transcriptional activity, B-ALL carrying DUX4 fusions manifests a characteristic expression pattern. D-I binding sites are not always relevant to the gene areas with increased transcriptions. Therefore, we compared the genomic areas where ATAC signal is raised by D-I, and genes whose expression is affected by D-I. We identified genes with ATAC signal change both in NALM6 cells with D-I knockdown and in Reh cells with D-I induction. We identified D-I binding in some of these genes, and the pharmacological inhibition of one of the genes caused cell death in NALM6 cells in vitro and in vivo, suggesting that this gene is the genuine target of D-I. In summary, our study elucidated the detailed difference of function between WT DUX4 and DUX4-IGH, and demonstrated the ability of DUX4-IGH as a chromatin modulator. Disclosures No relevant conflicts of interest to declare.

Description: Familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML) is an autosomal dominant disorder and is characterized by inherited thrombocytopenia and a lifelong risk of development of hematological malignancies. Although inherited RUNX1 mutation is the cause of thrombocytopenia, additional genetic events may be responsible for the tumor development as only 40 % of FPD/AML patients develop leukemia. Because of the rarity of this disorder, underlying mechanisms for malignant transformation in FPD/AML have not been elucidated. Thus we conducted a nationwide survey in Japan to collect samples and make a diagnosis of patients with familial thrombocytopenia or hematological malignancies. As a result, 56 pedigrees were extracted, and seven pedigrees with RUNX1 mutation were diagnosed as having FPD/AML, in which eight out of 14 patients had developed hematological malignancies. To systematically identify additional genetic alterations, we utilized whole exome sequencing in two patients with FPD/AML who had RUNX1_F303fsX566 mutation and developed MDS (Patient 1) or myelofibrosis (Patient 2) followed by AML. We identified 12 and 10 somatically acquired nonsynonymous mutations in these patients, respectively. Intriguingly, the two patients had common CDC25C mutation at codon 234 (D234G). Further genomic screening of other pedigrees revealed that four out of eight FPD/AML patients who developed hematological malignancy harbored somatic mutations in CDC25C (CDC25C_D234G in three patients and CDC25C_H437N in one patient). Recurrent CDC25C mutation in FPD/AML with subsequent hematological malignancy implies that it forms common genetic foundation of transformation in this disease. CDC25C is a phosphatase that prevents premature mitosis in response to DNA damage at the G2/M checkpoint, while it is constitutively phosphorylated at Ser216 throughout the interphase by c-TAK1. When phosphorylated at Ser216, CDC25C binds to 14-3-3 protein, which sequestrates CDC25C in the cytoplasm and inactivates it. In all of the mutated forms of CDC25C that we found in FPD/AML, their binding capacity with c-TAK1 and 14-3-3 protein was reduced, resulting in decreased phosphorylation status of CDC25C at Ser216. As a consequence, those CDC25C mutants led to enhanced mitosis entry, which was exaggerated by radiation-induced DNA damage. These results demonstrate that CDC25C mutation results in disruption of DNA checkpoint machinery. It is known that FPD/AML-associated RUNX1 mutations evokes DNA damage and induces cell cycle arrest in hematopoietic cells, suggesting that the DNA checkpoint mechanism is activated in the presence of those types of RUNX1 mutation. We found, however, that introduction of CDC25C mutation results in the marked enhancement of mitosis entry in spite of co-expression of RUNX1 mutation in Ba/F3 cells. Thus, premature mitosis by loss of DNA checkpoint mechanisms in the presence of mutated CDC25C may contribute to malignant transformation of RUNX1-mutated cells. Interestingly, analysis of clonal evolution during leukemic transformation revealed that a clone defined by CDC25C mutation was dominant in the early phase of disease progression in both patients, which supports the idea that CDC25C mutation is associated with establishment of the founder clone during the leukemic progression of FPD/AML. In Patient 1, the founder clone with CDC25C mutation acquired FAM22G and COL9A1 (group 1) mutations, followed by occurrence of GATA2 and LPP (group 2) mutations to become a dominant clone in the AML phase, whereas another subclone of the founder defined by CHEK2 and DTX2 (group 3) mutations regressed. Similar hierarchical progression was observed in Patient 2. An additional single cell genomic sequencing of bone marrow cells from Patient 1 in the AML phase revealed that group 1/2 mutations and group 3 mutations were mutually exclusive, which supports our predicted model. Collectively, these results indicate that somatic mutation in CDC25C is a recurrent event in the early phase of leukemic progression of FPD/AML, which induces premature mitosis and genetic instability in hematopoietic cells with germline RUNX1 mutation. Disclosures: Usuki: Alexion Pharmaceuticals, Inc.: Speakers Bureau. Kurokawa:Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Research Funding.

Description: Transcription factor AML1 (also called Runx1), which was initially isolated from the t(8;21) chromosomal translocation frequently found in the acute myelogenous leukemia FAB M2 subtype, is essential for the development of multilineage hematopoiesis in mouse embryos. By analyzing conditional AML1 knockout mice, we have previously shown that AML1 negatively regulates the number of immature hematopoietic cells defined as lineage-negative, CD34− Sca-1+ c-Kit+ (34KSL) cells in adult hematopoiesis, while it is required for megakaryocytic maturation and lymphocytic development. The former is a significant observation because an increase in hematopoietic stem/progenitor cells due to defective AML1 function may be closely related to the development of human leukemia. In support of this is the fact that mice in which leukemic chimeric protein AML1/ETO is expressed in hematopoietic cells are subject to myeloproliferative disease and develop leukemia after additional mutation. However, it has remained yet to be determined how AML1 contributes to homeostasis of hematopoietic stem cells (HSCs). To address this issue, we analyzed in detail HSC function in the absence of AML1. Notably, cells in the Hoechst 33342 side population (SP) fraction are increased in number in AML1-deficient bone marrow, which suggests enrichment of quiescent HSCs. We quantitatively evaluated HSCs by bone marrow transplantation assays using limiting dilution and found a significant increase in HSC number within the AML1-deficient bone marrow. These results indicate that the number of quiescent HSCs is negatively regulated by AML1, loss of which may result in accumulation of leukemic stem cell pool in AML1-related leukemia.

Description: Acute myelogenous leukemia 1 (AML1; runt-related transcription factor 1 [Runx1]) is a member of Runx transcription factors and is essential for definitive hematopoiesis. Although AML1 possesses several subdomains of defined biochemical functions, the physiologic relevance of each subdomain to hematopoietic development has been poorly understood. Recently, the consequence of carboxy-terminal truncation in AML1 was analyzed by the hematopoietic rescue assay of AML1-deficient mouse embryonic stem cells using the gene knock-in approach. Nonetheless, a role for specific internal domains, as well as for mutations found in a human disease, of AML1 remains to be elucidated. In this study, we established an experimental system to efficiently evaluate the hematopoietic potential of AML1 using a coculture system of the murine embryonic para-aortic splanchnopleural (P-Sp) region with a stromal cell line, OP9. In this system, the hematopoietic defect of AML1-deficient P-Sp can be rescued by expressing AML1 with retroviral infection. By analysis of AML1 mutants, we demonstrated that the hematopoietic potential of AML1 was closely related to its transcriptional activity. Furthermore, we showed that other Runx transcription factors, Runx2/AML3 or Runx3/AML2, could rescue the hematopoietic defect of AML1-deficient P-Sp. Thus, this experimental system will become a valuable tool to analyze the physiologic function and domain contribution of Runx proteins in hematopoiesis.