Leukemogenesis In Acute Myeloid Leukemia Research Articles

Acquired somatic mutations of isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) in preleukemic disorders.

Mutations of isocitrate dehydrogenase isoform 1 and 2 (IDH1 and IDH2) genes have been identified in glioblastoma and acute myeloid leukemia (AML). However, little is known about the molecular alterations of IDH genes in preleukemic disorders with a propensity to transform to AML. We performed polymerase chain reaction-denaturing high performance liquid chromatography (PCR-DHPLC) followed by direct sequencing to detect IDH mutations in 237 patients with myeloproliferative neoplasms (MPNs; n=108), myelodysplastic syndrome (MDS; n=22), paroxysmal nocturnal hemoglobinuria (PNH; n=41), and aplastic anemia (AA; n=66). No IDH1 R132 and IDH2 R172 mutations were identified in the entire cohort, whereas IDH1 G105G allele was detected in 4/108 MPN (3.70%), 2/22 MDS (9.09%), and 2/41 PNH (4.88%) patients. Three IDH2 R140Q mutations were found in 2/108 MPN (1.85%) and 1/22 MDS (4.54%) patients, while one IDH2 G145G allele was found in 0.92% (1/108) of MPN patients. Overall, our data suggest that IDH mutations are rare in the preleukemic disorders and may not be the major initial step in AML leukemogenesis.

DNA methylation-mediated epigenetic silencing of miR-720 contributes to leukemogenesis in acute myeloid leukemia

To investigate the expression level and regulation mechanism of miR-720 as well as the association of miR-720 expression with leukemia biological characteristics. Expression and promoter methylation of miR-720 were determined by quantitive PCR and pyrosequencing in 38 patients with AML and 20 normal controls. Lentivirous-mediated miR-702 overexpression was constructed in AML cell line kasumi-1. The cell proliferation, apoptosis, cycle, colony formation, migration and P53-mediated apoptosis pathway were determined. AML patients showed significantly lower miR-720 expression compared with normal controls (0.69±0.09 vs 3.00±0.46, P<0.01); The methylation level of miR-720 promoter region in AML patients were significantly higher than normal controls [(75.56±2.35)% vs (47.65±2.78)%, P<0.01]. miR-720 overexpression in kasumi-1 cells induced significantly increased cell apoptosis (P=0.017), elevated apoptosis sensitivity to etoposide (P=0.004), and reduced cell proliferation (P<0.01). miR-720 overexpression also induced reduced colony formation (P=0.005), cell cycle arrest in G(1)/G(0) phase and decreased migration ability in kasumi-1 cells. In addition, overexpression of miR-720 significantly induced increased cell apoptosis-related proteins including P53 and Bax, and activation of NF-κB signal transduction pathway. After kasumi-1 cells were treated with 1uM decitabine for 48 hours, miR-720 promoter methylation reduced significantly, and miR-720 expression significantly increased. The expression of miR-720 in AML patients reduced significantly, and DNA methylation-mediated epigenetic silencing of miR-720 contributed to maintain the malignant characteristics of AML.

GATA2 mutations in patients with acute myeloid leukemia-paired samples analyses show that the mutation is unstable during disease evolution.

Recently, mutations of the GATA binding protein 2 (GATA2) gene were identified in acute myeloid leukemia (AML) patients with CEBPA double mutations (CEBPA (double-mut)), but the interaction of this mutation with other genetic alterations and its dynamic changes during disease progression remain to be determined. In this study, 14 different missense GATA2 mutations, which were all clustered in the highly conserved N-terminal zinc finger 1 domain, were identified in 27.4, 6.7, and 1% of patients with CEBPA (double-mut), CEBPA (single-mut), and CEBPA wild type, respectively. All but one patient with GATA2 mutation had concurrent CEBPA mutation. GATA2 mutations were closely associated with younger age, FAB M1 subtype, intermediate-risk cytogenetics, expression of HLA-DR, CD7, CD15, or CD34 on leukemic cells, and CEBPA mutation, but negatively associated with FAB M4 subtype, favorable-risk cytogenetics, and NPM1 mutation. Patients with GATA2 mutation had significantly better overall survival and relapse-free survival than those without GATA2 mutation. Sequential analysis showed that the original GATA2 mutations might be lost during disease progression in GATA2-mutated patients, while novel GATA2 mutations might be acquired at relapse in GATA2-wild patients. In conclusion, AML patients with GATA2 mutations had distinct clinic-biological features and a favorable prognosis. GATA2 mutations might be lost or acquired at disease progression, implying that it was a second hit in the leukemogenesis of AML, especially those with CEBPA mutation.

The leucine twenty homeobox (LEUTX) gene, which lacks a histone acetyltransferase domain, is fused to KAT6A in therapy‐related acute myeloid leukemia with t(8;19)(p11;q13)

The monocytic leukemia zinc finger protein KAT6A (formerly MOZ) gene is recurrently rearranged by chromosomal translocations in acute myeloid leukemia (AML). KAT6A is known to be fused to several genes, all of which have histone acetyltransferase (HAT) activity and interact with a number of transcription factors as a transcriptional coactivator. The present study shows that the leucine twenty homeobox (LEUTX) gene on 19q13 is fused to the KAT6A gene on 8p11 in a therapy-related AML with t(8;19)(p11;q13) using the cDNA bubble PCR method. The fusion transcripts contained 83 nucleotides upstream of the first ATG of LEUTX and are presumed to create in-frame fusion proteins. LEUTX is known to have a homeobox domain. Expression of the LEUTX gene was only detected in placenta RNA by RT-PCR, but not in any tissues by Northern blot analysis. The putative LEUTX protein does not contain any HAT domain, and this is the first study to report that KAT6A can fuse to the homeobox gene. The current study, with identification of a new partner gene to KAT6A in a therapy-related AML, does not elucidate the mechanisms of leukemogenesis in KAT6A-related AML but describes a new gene with a different putative function.

Acute Myeloid Leukemia With Isolated Trisomy 13 Is a Genetically Homogenous Entity With a High Frequency Of Mutations In Genes Encoding Components Of The Splicing Machinery and Extremely Poor Prognosis

Acute myeloid leukemia (AML) with isolated trisomy 13 (AML+13) is rare and frequently associated with FAB M0 morphology. The clinical course is not well characterized but according to the ELN classification of intermediate prognosis. Eighty to one-hundred percent of patients (pts) with AML+13 carry mutations in the RUNX1 gene. Over-expression of FLT3 (located on chromosome 13 [chr 13]) due to the additional gene copy on the third chr 13 was proposed as a mechanism of leukemogenesis in AML+13 (gene dosage hypothesis).We set out to characterize the clinical course of AML+13 pts and elucidate their molecular background using whole exome sequencing, targeted resequencing and gene expression profiling. We identified 23 pts with AML+13 enrolled in a multicenter trial of the German AML Cooperative Group (AMLCG-1999) and compared this group to 386 pts without +13 who were classified in the ELN Intermediate-II genetic category. All pts received intensive induction chemotherapy. There was no significant difference in age, white blood cell or platelet count between the two groups. However, LDH levels were significantly (p=.01) lower in the AML+13 group while bone marrow blast percentage was significantly higher (p=.04). Twelve AML+13 pts (52%) reached complete remission, but all relapsed. Relapse-free and overall survival were inferior in the AML+13 group compared to other ELN Intermediate-II pts (median RFS, 9 vs 15 months, p=.01; median OS, 7 vs. 13 months, p=.03).Remission samples from two AML+13 pts were available as normal control for exome sequencing. Using SureSelect human all exon target enrichment (Agilent) followed by 80bp paired-end sequencing on an Illumina GAIIx platform, we were able to identify non-synonymous leukemia-specific mutations affecting, among others, RUNX1, ASXL1, PTPN11 and CEBPZ.Genes identified by exome sequencing and a panel of genes recurringly mutated in AML were studied by targeted amplicon resequencing in all AML+13 pts with available material (16/23; Figure). As described before, a high incidence of RUNX1 mutations (75%) was identified. In addition, we detected mutations in spliceosome components in 14/16 (88%) of AML+13 pts, including SRSF2 codon 95 mutations in 13/16 pts (81%). One patient without SRSF2 mutation showed a mutation in SF3B1. Moreover, recurring mutations were found in ASXL1 (44%) and BCOR (25%), and were associated with RUNX1 and SRSF2 mutations. Interestingly, both pts without mutations in the splicing machinery had mutations in DNMT3A, which were also mutually exclusive with mutations in RUNX1 or ASXL1. Two pts carried mutations in CEBPZ suggesting that CEBPZ is a novel recurringly mutated gene in AML. [Display omitted] To further characterize this genetically homogenous subgroup, we compared gene expression profiles of 9 pts with AML+13 with 509 AML pts without +13. We identified 678 (up-regulated 492; down-regulated 186) probe sets as significantly deregulated. Only 59 (8.7%) of these probe sets were localized on chr 13, but of those, 55 were up-regulated and only 4 were down-regulated. Up-regulated probe sets on chr 13 included FOXO1, FLT3 and RB1. The strongest down-regulated probe set on chr 13 belonged to the tumor suppressor gene SPRY2, which is a negative regulator of receptor tyrosine kinases. Gene set enrichment analysis showed significant deregulation of gene sets associated with regulation of transcription and nuclear transport.In summary, our study is the first to show that AML+13 is significantly associated with inferior OS and RFS compared to other intermediate-risk cytogenetic abnormalities in a homogeneously treated cohort. Furthermore, we present evidence that AML+13 leukemias are a genetically quite homogenous subgroup. AML+13 is not only associated with a high rate of RUNX1 mutations but also with mutations in SRFS2, ASXL1 and BCOR. The incidence of mutations in SRSF2 in AML+13 is the highest of any AML subgroup reported so far. In addition, our gene expression data show a homogenous expression profile associated with AML+13. The striking association of a few recurring mutations in AML+13 suggests a biological relationship with synergistic lesions during leukemogenesis. While mutations in RUNX1, ASXL1 and up-regulation of FLT3 were previously reported as markers of poor prognosis in AML, the combination of these lesions might be responsible for the extremely poor outcome of AML+13. Disclosures:Krebs:Illumina: Honoraria. Greif:Illumina: Honoraria.

Role of Epigenetic Readers in Pathogenesis and Therapy of Acute Leukemias

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with a dismal survival rate of less than 30 percent and is thus a disease where novel therapeutics are urgently required. AML is highly heterogeneous at both the molecular and clinical level. However, recurrent themes of altered transcriptional and epigenetic control are evident from sequencing and mechanistic studies. Recently, critical epigenetic regulators have surfaced as mediators of leukemogenesis and potential therapeutic targets in AML and other hematological malignancies. We and others have recently demonstrated that the Bromodomain and Extra Terminal (BET) protein family of transcriptional adapter proteins are required for transformation by MLL-fusion proteins, where they facilitate the localization of aberrant transcriptional complexes to chromatin at genes critical for leukemogenesis. Furthermore, we and others have demonstrated that small molecule inhibitors, such as I-BET151 and JQ1, of the protein-protein interactions between these proteins and acetylated lysine residues in histone tails are effective in pre-clinical models of MLL leukemias. We have recently extended studies of BET inhibition to other forms of AML using I-BET151 and found a range of sensitivities across a panel of AML cell lines and patient samples with common recurrent mutations. These cell lines also demonstrate a common cellular response to MLL-fusion leukemias, with induction of apoptosis and G1/0 arrest, suggesting that similar transcriptional programs may be targeted. In further investigating this, we identified a common transcriptional program downregulated in sensitive leukemia subtypes. In addition, using diagnostic gene expression profiles, we also demonstrate that this program can classify AML patients, suggesting that the program may provide predictive biomarkers and potentially identify those patients most likely to respond to BET inhibitions. Focusing on the most common mutation in AML, mutations of the nucleophosmin gene (NPM1c mutations), we present evidence to suggest that wild-type NPM1 has an inhibitory influence on BRD4, which is relieved upon NPM1c mutation and cytosplasmic dislocation. NPM1c mutation allows upregulation of the core transcriptional program facilitating leukemia development, and this program is abrogated by I-BET therapy. Finally, we demonstrate the efficacy of I-BET151 in human cell lines, a unique murine model, and in primary patient samples of NPM1c AML. Taken together, we have used BET inhibitors as a laboratory tool to dissect out mechanisms of aberrant transcriptional control in AML. Furthermore our pre-clinical evaluation supports clinical trials of BET inhibitors across multiple AML subtypes and identifies potential predictive biomarkers of sensitivity and response to inform these studies. Disclosures:No relevant conflicts of interest to declare.

Mutations Of Genes Regulating DNA Methylation Are Common In MLL Partial Tandem Duplication AML and DNMT3A Mutations Are Associated With Adverse Outcome

Abstract Background and purpose Abnormalities of genes regulating DNA methylation have been described in acute myeloid leukemia (AML). MLL protein is a transcriptional regulator and governs proper hematopoiesis through its histone methyltransferase activity. AML with partial tandem duplication of MLL (MLL-PTD) was associated with an unfavorable prognosis. The cooperating roles of MLL-PTD with other mutated genes regulating DNA methylation have not been comprehensively studied in AML. We aimed to determine the prevalence and clinical impact of mutations of DNA methylation regulators in AML with MLL-PTD. Materials and methods Bone marrow samples from 98 AML patients with MLL-PTD were analyzed for gene mutations of TET2, DNMT3A, IDH1 and IDH2. MLL-PTD was screened by RT-PCR and confirmed by real-time quantitative PCR assays. The mutational analysis was performed with PCR assays followed by direct sequencing for TET2 (whole coding exons 3–11) and IDH1/2 (hotspots exon 4). For the detection of DNMT3A mutations, the PCR products amplified for entire coding exons 2 to 23 were first screened with denaturing high-performance liquid chromatography followed by direct sequencing for the abnormal profiles. Results The frequency of TET2, IDH1, IDH2 and DNMT3A mutations in AML patients with MLL-PTD was 17.0% (16/94), 10.2% (10/98), 18.4% (18/98), and 31.6% (31/98), respectively. Taken together, 61.1% of patients with MLL-PTD had at least one mutated gene of DNA methylation regulators. TET2, IDH1 and IDH2 mutations were mutually exclusive with each other whereas DNMT3A mutations frequently co-existed with other DNA methylation modifiers:TET2 (n=8), IDH1 (n=5) and IDH2 (n=4). No differences were observed between the mutation status of the DNA methylation modifiers and clinico-hematologic features of patients with MLL-PTD except that TET2 (P=0.012) and DNMT3A (P=0.024) mutations were associated with older age. Of the 55 MLL-PTD patients who received standard chemotherapy, IDH2 mutation was associated with a lower complete remission rate (25.0% vs 67.8%, P=0.018), while DNMT3A mutations conferred an inferior event-free survival (0.0 vs 6.8 months, P=0.027) and overall survival (6.0 vs 11.5 months, P=0.032). In multivariate analysis, older age (P=0.008) and DNMT3A mutations (P=0.049) were independent adverse factors for overall survival. The crosstalk between MLL-PTD and genes involving DNA methylation in the leukemogenesis of AML warrants further investigation. Conclusions Gene mutations involving DNA methylation frequently co-existed in AML patients with MLL-PTD, especially DNMT3A mutations which conferred a poor outcome. Our study demonstrated the importance of genetic alterations involving DNA methylation in the pathogenesis of MLL-PTD AML and provided potential epigenetic-targeted therapy. Grant support The work was supported by NHRI-EX93-9011SL, NSC95-2314-B-195-001, NSC96-2314-B-195-006-MY3, NSC97-2314-B-182-011-MY3 and MMH-E-101-09. Disclosures: No relevant conflicts of interest to declare.

Aberrant Mer receptor tyrosine kinase expression contributes to leukemogenesis in acute myeloid leukemia

Acute myeloid leukemia (AML) continues to be extremely difficult to treat successfully, and the unacceptably low overall survival rates mandate that we assess new potential therapies to ameliorate poor clinical response to conventional therapy. Abnormal tyrosine kinase activation in AML has been associated with poor prognosis and provides strategic targets for novel therapy development. We found that Mer receptor tyrosine kinase was over-expressed in a majority of pediatric (29/36, 80%) and adult (10/10, 100%) primary AML patient blasts at the time of diagnosis, and 100% of patient samples at the time of relapse. Mer was also found to be expressed in 12 of 14 AML cell lines (86%). In contrast, normal bone marrow myeloid precursors expressed little to no Mer. Following AML cell line stimulation with Gas6, a Mer ligand, we observed activation of prosurvival and proliferative signaling pathways, including phosphorylation of ERK1/2, p38, MSK1, CREB, ATF1, AKT and STAT6. To assess the phenotypic role of Mer in AML, two independent short-hairpin RNA (shRNA) constructs were used to decrease Mer expression in the AML cell lines Nomo-1 and Kasumi-1. Reduction of Mer protein levels significantly increased rates of myeloblast apoptosis two to threefold in response to serum starvation. Furthermore, myeloblasts with knocked-down Mer demonstrated decreased colony formation by 67–87%, relative to control cell lines (P<0.01). NOD-SCID-gamma mice transplanted with Nomo-1 myeloblasts with reduced levels of Mer had a significant prolongation in survival compared with mice transplanted with the parental or control cell lines (median survival 17 days in parental and control cell lines, versus 32–36 days in Mer knockdown cell lines, P<0.0001). These data suggest a role for Mer in acute myeloid leukemogenesis and indicate that targeted inhibition of Mer may be an effective therapeutic strategy in pediatric and adult AML.

Aberrant Mer receptor tyrosine kinase expression contributes to leukemogenesis in acute myeloid leukemia

Aberrant Mer receptor tyrosine kinase expression contributes to leukemogenesis in acute myeloid leukemia

LR11 Plays a Critical Role in Leukemogenesis of Acute Myeloid Leukemia by Regulating Adhesion and Homing Ability to Bone Marrow.

Abstract 2409 Introduction:Membrane-anchored uPAR plays a role in regulating the engraftment and mobilization of murine hematopoietic stem/progenitor cells (HSPCs) to bone marrow (BM). Acute myeloid leukemia (AML) patients with higher proportions of uPAR-positive cells posed higher relapse risk after chemotherapy. LR11 (also called SorLA or SORL1) is a regulator of uPAR function through complex formation with uPAR., and the LR11 expression is highly induced in AML cells, and circulating soluble form of LR11 (sLR11) levels are significantly increased in AML patients and ameliorated by chemotherapy (Sakai et al. 2012). Although LR11 is over-expressed in AML, the role of over-expressed LR11 in AML leukemogenesis remains unknown. Recently we found that LR11 mRNA expression levels are drastically increased in murine transformed granulocyte macrophage progenitors (GMPs) with MLL-AF9 fusion gene. Here, we show the pathological significance of LR11 expressions in the progression of AML using mouse leukemia model. Materials and Methods:Stably LR11-knockdown (LR11-KD) U937 cells were generated by transfection of shRNA expression vector specific for LR11. For generation of mouse AML model, the GMPs transformed by MLL-AF9 (MA9-GMPs) or human leukemic cells were transplanted into syngeneic mice or immune-deficient mice, respectively. For colony replating assays, MA9-GMPs were cultured in methylcellulose medium and colonies were counted and pooled, and then 1×104 cells were replated in the same medium. For cell growth assays, 5th round MA9-GMPs were transferred to liquid culture. An adhesion assay was performed using human mesenchymal stromal cells (MSC)-coated plates. Results:U937 cells expressed high level of LR11 mRNA and protein, but there was no difference in cell growth between control and LR11-KD U937 cells in vitro. We transplanted LR11-KD or control U937 cells into recipient mice, and monitored the percent of peripheral blood (PB) leukemia cells by flow cytometry. All the recipient mice transplanted with control U937 cells were died of leukemia by 3 weeks after transplantation, whereas the survival of mice transplanted with LR11-KD U937 cells were prolonged significantly (the median survival: 17.5 days for control cells vs. 28 days for LR11-KD cells, Figure. 1). The percent of leukemia cells in PB of the mice transplanted with LR11-KD U937 cells on day 13 after transplantation were significantly lower than those of mice transplanted with control U937 cells (1.2 ± 1.1% vs. 10.2 ± 5.8%, respectively). We next examined the effect of LR11 on the malignant phenotype by cell growth assay and colony replating assay using the MA9-GMPs from Lr11+/+ and Lr11−/− mice. There was no difference in the cell growth and colony forming ability between Lr11+/+ and Lr11−/− MA9-GMPs. However, the donor chimerism of mice transplanted with Lr11−/− MA9-GMPs was significantly lower than that of mice transplanted with Lr11+/+ MA9-GMPs (the percentage of chimerism on day 28 and on day 35 after transplantation: 54.6 ± 2.1% and 66.3 ± 11.0% for Lr11+/+ donor cells vs. 0.7 ± 2.1% and 0.5 ± 0.1% for Lr11−/− donor cells). We finally examined the cell motility analysis. In the adhesion assay, the attached cell numbers to MSC-coated plates were significantly reduced to 0.7-fold in the LR11-KD U937 cells compared with control U937 cells. In homing assay, we transplanted LR11-KD U937 cells and control cells into recipient mice. The number of LR11-KD U937 cells in recipient BM 16 hours after transplantation was significantly decreased compared with those of control cells (0.011 ± 0.004% for control cells vs. 0.003 ± 0.001% for LR11-KD cells, Figure 2). Conclusion:These data showed that LR11 plays a critical role in the pathogenesis of AML by enhancing the cell adhesion and homing ability to BM. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Expression of CEBPA is reduced in RUNX1-mutated acute myeloid leukemia

Expression of CEBPA is reduced in RUNX1-mutated acute myeloid leukemia

Heritable polymorphism predisposes to high BAALC expression in acute myeloid leukemia

Overexpression of the brain and acute leukemia, cytoplasmic (BAALC) gene is implicated in myeloid leukemogenesis and associated with poor outcome in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia patients. Additionally, high BAALC expression occurs in glioblastoma, melanoma, and childhood gastrointestinal stroma tumors, suggesting an oncogenic role for BAALC. However, the mechanisms underlying the deregulated expression are unknown. We hypothesized that a common heritable genetic feature located in cis might account for overexpression of BAALC in an allele-specific manner. By sequencing the genomic region of BAALC we identified nine informative single nucleotide polymorphisms (SNPs) and tested them for a possible association with BAALC expression levels. We show that BAALC overexpression occurs in the presence of the T allele of SNP rs62527607[GT], which creates a binding site for the activating RUNX1 transcription factor in the BAALC promoter region. The mechanism is demonstrated experimentally in vitro using luciferase reporter assays and electrophoretic mobility shift assay (EMSA) analysis. The association of high BAALC expression with the T allele and its correlations with RUNX1 expresser status are shown in vivo in a test set (n = 253) and validation set (n = 105) of samples from cytogenetically normal AML patients from different populations. Thus, we identify a heritable genomic feature predisposing to overexpression of an oncogene, thereby possibly leading to enhanced AML leukemogenesis. Our findings further suggest that genomic variants might become useful tools in the practice of personalized medicine.

The Clinical Features and Prognostic Impact of DNMT3A Gene Mutation in Japanese Patients with De Novo AML

Abstract 2537 Background and Aims:Several gene mutations were found in acute myeloid leukemia (AML) and were expected to be used as prognostic factors. The nucleophosmin 1 (NPM1) and CCAAT/enhancer-binding protein alpha (CEBPA) gene mutations are associated with a favorable outcome and lack of a transplant benefit, but FMS-like tyrosine kinase 3 (FLT3) mutations leading to an internal tandem duplication (ITD) are associated with adverse outcome. Recently the DNA methyltransferase 3A (DNMT3A) gene mutation was identified by whole-genome sequencing in patients with AML. DNMT3A encodes for the enzyme DNA (cytosine-5)-methyltransferase 3A and belongs to the family of other methyltransferases like DNMT1 and DNMT3B. These enzymes are involved in adding methyl groups to the cytosine residue of CpG dinucleotides and thus play an important role in epigenetic regulation of genes, but the mechanism of DNMT3A mutation -associated leukemogenesis was still unknown. About clinical impact of DNMT3A mutation, several groups reported that DNMT3A mutation have been associated with adverse outcome. We analyzed clinical features and prognostic impact of DNMT3A mutation in patients with Japanese patients with AML. Methods:We retrospectively analyzed 188 cases of de novo AML treated at Nippon Medical School Hospital and its affiliated facilities from 2000 to 2010. We analyzed 188 samples at initial presentation and 70 samples at relapse. Mutation analyses were performed for FLT3ITD by PCR amplification, and NPM1, CEBPA, and DNMT3A mutations by direct sequence. To validate sequencing results, PCR products were inserted into the pCR2.1-TOPO vector using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA). Recombinant plasmids isolated from 8 to 12 white colonies were sequenced. Results:The median age was 53.0 years (range, 17–87 years) with 62.1% being males. Patients were followed-up for 0.03–126.1 months after initial presentation, with a median of 19.9 months. DNMT3A mutations were detected in 35 cases (18.6%) at initial presentation, and 12 cases (17.1%) at relapse. Most frequently, codon R882 located in exon 23 was mutated, and 32 cases (91.4%) of these mutations were located within methyltrasferase domain.We evaluated the correlation of clinical and genetic characteristics with DNMT3A mutations. Age (p=0.2884), sex (p=0.6964), and platelet count (p=0.9940) were not significantly different in AML patients with and without DNMT3A mutations. However the frequency of higher white blood cell count (p=0.0001), M5 in FAB classification (p=0.0018), and intermediate risk karyotype (p=0.0032) in patients with DNMT3A mutations were significantly higher than those in patients without them. Also, patients with DNMT3A mutations had a mutation in NPM1 (p<0.0001) and FLT3ITD (p=0.009) more frequently.We next assessed the prognostic impact of DNMT3A mutations. For total cohort of patients with AML, complete remission rate and rates of relapse free survival (RFS) at 5 years were not statistically different between AML patients with and without DNMT3A mutations. However the overall survival (OS) at 5 years in patients with DNMT3A mutation (11.0%) was significantly lower than those in patients without them (28.9%) (p=0.0005). Among the intermediate risk karyotype or FLT3ITD negative AML patients, RFS at 5 years were not statistically different between AML patients with and without DNMT3A mutations, but OS at 5 years in patients with DNMT3A mutation (intermediate risk karyotype: 12.6%, Flt3ITD negative: 11.6%) was significantly lower than those in patients without them (intermediate risk karyotype: 23.9%, p=0.0231, FLT3ITD negative: 30.9%, p=0.0046). Conclusions:This study shows that DNMT3A mutation is an important adverse prognostic factor among intermediate risk karyotype or FLT3ITD negative AML patients. Recently TET2 and Isocitrate Dehydrogenase (IDH) 1/2 gene mutations in de novo AML were reported. These genes including DNMT3A play an important role in epigenetic regulation of genes such as methylation etc, and mutations of these genes may be associated with leukemogenesis of AML. Now we are analyzing TET2 and IDH1/2 mutations among same our AML cohort, and we will show the prognostic impact of TET2, IDH1/2 and DNMT3A mutations in patients with de novo AML. Disclosures:No relevant conflicts of interest to declare.

Signal Transducer and Activator of Transcription 3 Promotes Leukemogenesis

Abstract 1395We have previously shown that constitutive activation of the signal transducer and activator of transcription (STAT) 3 protein is associated with worse outcome in patients with acute myeloid leukemia (AML). To clarify the role of STAT3 signaling in AML leukemogenesis, STAT3 shRNAmirs were introduced into the AML cell line HEL that carries the Janus kinase 2 with V617F mutation leading to constitutive tyrosine phosphorylation and activation of STAT3 and STAT5. Clonal line of HEL cells expressing STAT3 shRNAmir or control shRNA were generated and then transduced with a lentiviral virus harboring a constitutively expressed firefly luciferase gene. Engraftment of these cells was evaluated in a disseminated xenograft non-obese diabetic severe immunocompromised mouse model. When tested in vitro, HEL STAT3 knock-down (HEL-STAT3KD) cells showed normal STAT5 expression and activity but had lower proliferation rates than control cells, PKC-bII and Pim1 were down-regulated, and activation of ERK1/2 was increased. Trans-signaling by hyper-IL-6 enhanced STAT3 phosphorylation proportional to STAT3 level. In vivo imaging of luciferase activity demonstrated significant delay in engraftment of STAT3KD cells compared with controls (Figure). To the best of our knowledge, this is the first demonstration that STAT3 knockdown delays leukemia cell engraftment, that STAT3 signaling is crucial for AML leukemogenesis and should promote STAT3-tailored therapeutic targeting. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

HoxA10 Influences Protein Ubiquitination by Activating Transcription of ARIH2, the Gene Encoding Triad1

HoxA10 is a homeodomain transcription factor that is maximally expressed in myeloid progenitor cells. An increase in HoxA10 expression correlates with poor prognosis in human acute myeloid leukemia (AML). Consistent with this scenario, HoxA10 overexpression in murine bone marrow induces a myeloproliferative neoplasm that advances AML over time. Despite the importance of HoxA10 for leukemogenesis, few genuine HoxA10 target genes have been identified. The current study identified ARIH2, the gene encoding Triad1, as a HoxA10 target gene. We identified two distinct HoxA10-binding cis elements in the ARIH2 promoter and determined that HoxA10 activates these cis elements in myeloid cells. Triad1 has E3 ubiquitin ligase activity, and we found that HoxA10-overexpressing myeloid cells exhibited a Triad1-dependent increase in protein ubiquitination. Therefore, these studies have identified the regulation of protein ubiquitination as a novel function of Hox transcription factors. Forced overexpression of Triad1 has been show previously to inhibit colony formation by myeloid progenitor cells. In contrast, HoxA10-overexpressing myeloid progenitor cells exhibited increased proliferation in response to low doses of various cytokines. We found that Triad1 knockdown further increased cytokine-induced proliferation in HoxA10-overexpressing cells. Therefore, these studies have identified a HoxA10 target gene that antagonizes the overall influence of overexpressed HoxA10 on myeloproliferation. This result suggests that the consequences of HoxA10 overexpression reflect a balance between the target genes that facilitate and antagonize proliferation. These results have implications for understanding the mechanisms of leukemogenesis in AML with Hox overexpression.

The Crosstalk Between the Matrix Metalloprotease System and the Chemokine Network in Acute Myeloid Leukemia

Matrix metalloproteinases (MMPs) comprise a large family of zinc-dependent endopeptidases, which are best known for their ability to degrade essentially all components of the extracellular matrix (ECM). By breaking down ECM, MMPs may remove physical barriers, thus allowing cells to migrate and potentially invade other tissues. Recent evidence, however, shows that the proteolytic activities of MMPs also affect several fundamental physiological processes. Primary human acute myeloid leukemia (AML) cells often show constitutive release of several MMPs and chemokines, and there seems to be a crosstalk between the MMP system and the chemokine network. Firstly, the nuclear factor-κB (NF-κB) system represents a common regulator at the transcriptional level both for MMPs (e.g. MMP-1 and MMP-9) and for the constitutive release of several chemokines (CCL2-4/CXCL1/8) by primary human AML cells. Secondly, the crosstalk at the molecular level probably includes MMP-mediated structural alteration and activation of constitutively released chemokines involved in AML cell migration (e.g. CXCL12) and stimulation of bone marrow angiogenesis (e.g. CXCL8). Thirdly, at a functional level the two systems interact because the chemokine network plays a role in similar physiological processes as the MMPs, including AML cell proliferation and migration and local regulation of angiogenesis. Both the chemokine system and MMPs are currently being evaluated as targets in anti-angiogenesis/cancer therapy and may also have potential therapeutic implications in AML. This review introduces the different members of the MMP family and describes their interactions with the chemokine network and the possible involvement of MMPs together with chemokines in leukemogenesis and chemosensitivity in AML.

Clinical Relevance of Changes of ROS/Peroxiredoxin 1 (Prx1) and Glutathione Peroxidase 1 (Gpx1) Level In AML Primary Cells before and after Induction Chemotherapy

AbstractAbstract 4376 Background:Previously, we had reported that antioxidants (ie, Prx & catalase) were down regulated at the time of diagnosis but those levels were restored to the level of normal individuals as ones achieved CR with signal transduction inhibitors on primary cells of chronic myeloid leukemia(Blood 114: Abstract 1114, 2009). Meanwhile it is plausible that primary cells of acute myeloid leukemia may have different biologies comparing to chronic myeloid leukemia in terms of the regulation of ROS/Prx's and Gpx1. In this study, we investigated the changes of ROS/Prx's and Gpx1 level in the primary cells at the time of diagnosis and at complete remission of AML. In addition, we studied the level of differentiation and changes of ROS/Prx's by ATRA on NB4 cell line simultaneously to study the potential correlation between differentiation and changes of ROS/Prx's and Gpx1. Method:Pairs of bone marrow sample at the diagnosis and at hematologic remission status of AML patients were evaluated for detection of intracellular ROS using dichlorodihydrofluorescein diacetate (DCFH-DA) and immunoblot assay for the expression of Prx1-6 and Gpx1. We studied the entire process of differentiation by treatment with ATRA on NB4 cell line in vitro in time dependent manner for 4 days. Changes of ROS/Prx's and Gpx1 were evaluated and the level of differentiation was confirmed by Wright's stain simultaneously for 4 days. Result:In bone marrow samples of AML patients, intracellular ROS levels at the time of diagnosis were significantly higher than those of remission status samples. However, the expression of Prx1 and Gpx1 were increased as well at the time of diagnosis. With induction chemotherapy(3+7), elevated ROS/Prx1 and Gpx1 level came down to the level of normal volunteer bone marrow donors. In NB4 cells, intracellular ROS level were being reduced as time went on after All-trans-retinoic acid (RA) treatment showing progressive differentiation for 4 days. The level of differentiation by RA treatment was confirmed by Wright's stain in time dependent manner. Expression of Prx1 and Gpx1 were decreased after RA treatment by Western blot analysis as well. The activities of NADPH oxidase and Rac1-GTP, which are an essential component of the ROS producing NADPH oxidase, decreased in RA treated NB4 cells as time elapsed compared with control NB4 cells. Conclusion:Our data suggested that leukemogenesis of acute myeloid leukemia could be related with increased expression of ROS/Prx1 and Gpx1. Elevated level of expression on ROS/Prx1 and Gpx1 were down to the levels of BM of normal individuals as ones achieve complete remission. These findings are quite opposite with the previous observation on CML suggesting different biology in terms of ROS/Prx1 and Gpx1 dysregulation in AML. Interestingly, differentiation processes using RA in NB4 cells were correlated with decreased the expression of Prx1 and Gpx1 in time dependent manner. These data suggest the potential usefulness of differentiation agents(ie, RA and others) in AML treatment via modulation of ROS/Prx1 and Gpx1. Disclosures:No relevant conflicts of interest to declare.

Expression Patterns of Hoxa4 and Meis1 Genes Are Regulated by Promoter Hypermethylation and When Combined Predict Survival in AML.

INTRODUCTION: Acute myeloid leukemia (AML) is a heterogeneous disease with varying survival rates depending mostly upon the molecular phenotype of the single leukemic clone. The most powerful predictor for the outcome of the individual patient is the cytogenetic profile at the time of diagnosis, dividing the patients into good, intermediate and adverse prognostic group. However, given that 40–60 percent of patients exhibits a normal karyotype and are assigned to an intermediate prognostic group, identification of biologic parameters, which either alone or in combination, predict disease outcome more precisely are needed. We have previously performed a gene expression profiling study (Grubach et al, Eur J. Hematol. 2008 Apr 10. [Epub ahead of print]) on a series of Polycomb, Hox and Meis genes expressed in hematopoietic cells. AIM: Based on the finding that HOXA4 could be used as a predictor for outcome in AML patients with a normal karyotype, we hypothesized that combining the gene expression of the HOXA4 gene and co-factor MEIS1 might unravel a leukemogenic impact in other cytogenetic prognostic groups (Grimwade et al. Blood. 1998 Oct 1;92(7):2322–33). In addition, given that epigenetic events might contribute to the regulation of these genes, we determined whether promoter hypermethylation of CpG islands in the promoter regions were of relevance to the expression levels of HOXA4 and MEIS1.MATERIALS & METHODS: Diagnosis samples from 248 AML patients were analyzed by RQ-PCR for expression levels of HOXA4 and MEIS1. 157 of these patients were further analyzed for promoter hypermethylation of the same genes by bisulphite treatment of DNA followed by methylation-specific melting curve analysis (MS-MCA).RESULTS: When combining the gene expression levels of HOXA4 with MEIS1 into the three main groups (low HOXA4/low MEIS1, low HOXA4/high MEIS1 and normal-high HOXA4/high MEIS1; (the latter pooled to enable statistical calculations)), clear differences in overall survival were found (Fig. 1). Thus, within the group of patients exhibiting low levels of HOXA4 transcript, those with a high expression of MEIS1 had a significantly worse outcome than those having low MEIS1 expression (p=0.025). Importantly, in a multiparameter regression analysis, the prediction was independent of the cytogenetic grouping, of mutations in NPM1 and FLT3 genes, WBC and age. Given the efficacy of demethylating therapy, we also considered the mechanism of HOXA4 and MEIS1 gene regulation. Thus, when promoter methylation of HOXA4 and MEIS1 in 157 patients was investigated, we found that 15 % of the patients had hypermethylation of the promoter region of MEIS1 and 77% of the patients showed hypermethylation of HOXA4. Importantly, a significant correlation for both of the genes between the expression level and methylation state was observed (MEIS1, p=0.001 and HOXA4, p=0.007).CONCLUSION: The altered expression levels of HOXA4 and MEIS1 in AML reflect, at least partly, an epigenetic regulation by virtue of promoter hypermethylation. The level of transcripts of HOXA4 and MEIS1 seem to contribute to the leukemogenesis in AML and can serve as independent prognostic variables regardless of their cytogenetic and molecular background. [Display omitted]

Activation of p53 Signaling Is Synergistically Enhanced by Bcl-2 Inhibition through Induction of Noxa and Bak/Bax Heterodimers Resulting in Apoptosis of AML Stem Cells

Disruption of Mdm2-p53 interaction by the small molecule Nutlin-3a activates p53 signaling and induces apoptosis in acute myeloid leukemia (AML) (Kojima et al., Blood 2005). ABT-737 binds to the hydrophobic BH3-binding groove of the Bcl-2 family members Bcl-XL, Bcl-2, Bcl-w but not Mcl-1 and exerts its pro-apoptotic function by preventing anti-apoptotic Bcl-2 family members from sequestering activating BH3 proteins (Konopleva et al., Cancer Cell 2006). We have recently reported that Nutlin-3a and ABT-737 induced Bax conformational change and mitochondrial apoptosis in AML cells in a strikingly synergistic fashion (Kojima et al., Cell Cycle 2006). In this study, we investigated molecular mechanisms of this synergism. Nutlin-3a and ABT-737 synergistically induced apoptosis in OCI-AML3 and MOLM13 cells with wt-p53, but not in p53-null HL-60 cells or in OCI-AML3 stably transfected with p53 shRNA. Both, Nutlin-3a and ABT-737 induced Noxa protein levels resulting in Noxa/Mcl-1 heterodimers and therefore inactivation of Mcl-1. Lentiviral transfection of MOLM13 cells with Noxa shRNA protected cells from ABT-737/Nutlin-induced apoptosis. As reported by us, ABT-737 disrupted Bcl-2/Bak leading to formation of pro-apoptotic Bak/Bax heterodimers. Despite increased p53 levels there was no change in p53/Bcl-2 or p53/Bak association; however, combined exposure of cells to Nutlin-3a/ABT-737 induced massive Bak/Bax heterodimers and facilitated direct binding of p53 to Bax. HCT-116 cells deficient in Bax were largely protected from pro-apoptotic effects of the Nutlin-3a/ABT-737 combination. Taken together, these findings indicate that in leukemic cells p53-dependent apoptosis is executed through pro-apoptotic Bcl-2 family proteins, which are also targeted by BH3 mimetics. Bcl-2 is critical for AML leukemogenesis (Padua et. al., ASH 2008) and is overexpressed in AML stem cells. In primary AML samples, the Nutlin-3a/ABT-737 combination synergistically depleted CD34+CD38−CD123+ AML stem cells (combination index 0.5). Since high Bcl-2 and increased Bcl-2/Bax ratios are associated with poor clinical response in AML, the combination strategy proposed here may have considerable therapeutic promise in targeting AML stem cells.

Down-regulation of Notch-1 expression decreases PU.1-mediated myeloid differentiation signaling in acute myeloid leukemia

The Notch receptor-mediated signaling pathways control cell fate in many types of organisms including neurogenesis, myogenesis and hematopoiesis in mammalian species. During normal hematopoiesis, Notch-1 promotes myeloid differentiation through up-regulation of the transcriptional factor PU.1. We therefore speculated that down-regulation of Notch-1 expression might be involved in the leukemogenesis of acute myeloid leukemia (AML). Here we investigated Notch-1 expression and its association with PU.1-mediated differentiation signaling in AML. The transcriptional level of Notch-1 and PU.1 was evaluated in 6 AML cell lines and 54 AML patient samples using real-time PCR analysis, and Western blot analysis of Notch-1, PU.1 and one of its downstream targets, the M-CSF receptor (MCSFR), was performed to test for confirmation. A significant decrease in the transcription levels of Notch-1 was noted in AML cell lines and patient samples, and decreased Notch-1 protein expression in AML was confirmed by Western blotting. Down-regulation of Notch-1 expression was associated with a decrease in PU.1/MCSFR expression in AML. Co-immunoprecipitation experiments showed that partial disruption of the Notch-1/PU.1 complex was noted in AML cells. No detectable mutation of Notch-1 (ANK, PEST) and PU.1 (PEST, DBD) was noted by PCR-single-strand conformation polymorphism (SSCP) assay. These results suggest that down-regulation of Notch-1 expression decreases PU.1/MCSFR expression and disrupts the Notch-1/PU.1 complex, which may impede the PU.1-mediated myeloid signaling and contribute to the leukemogenesis of AML.