Increased DNA methylation of Dnmt3b targets impairs leukemogenesis.

The de novo DNA methyltransferases Dnmt3a and Dnmt3b are of crucial importance in hematopoietic stem cells. Dnmt3b has recently been shown to play a role in genic methylation. To investigate how Dnmt3b-mediated DNA methylation affects leukemogenesis, we analyzed leukemia development under conditions of high and physiological methylation levels in a tetracycline-inducible knock-in mouse model. High expression of Dnmt3b slowed leukemia development in serial transplantations and impaired leukemia stem cell (LSC) function. Forced Dnmt3b expression induced widespread DNA hypermethylation inMyc-Bcl2-induced leukemias, preferentially at gene bodies.MLL-AF9-induced leukemogenesis showed much less pronounced DNA hypermethylation upon Dnmt3b expression. Nonetheless, leukemogenesis was delayed in both models with a shared core set of DNA hypermethylated regions and suppression of stem cell-related genes. Acute myeloid leukemia patients with high expression of Dnmt3b target genes showed inferior survival. Together, these findings indicate a critical role for Dnmt3b-mediated DNA methylation in leukemia development and maintenance of LSC function.

AML1/ETO induces self-renewal in hematopoietic progenitor cells via the Groucho-related amino-terminal AES protein.

The most frequent translocation t(8;21) in acute myeloid leukemia (AML) generates the chimeric AML1/ETO protein, which blocks differentiation and induces self-renewal in hematopoietic progenitor cells. The underlying mechanisms mediating AML1/ETO-induced self-renewal are largely unknown. Using expression microarray analysis, we identified the Groucho-related amino-terminal enhancer of split (AES) as a consistently up-regulated AML1/ETO target. Elevated levels of AES mRNA and protein were confirmed in AML1/ETO-expressing leukemia cells, as well as in other AML specimens. High expression of AES mRNA or protein was associated with improved survival of AML patients, even in the absence of t(8;21). On a functional level, knockdown of AES by RNAi in AML1/ETO-expressing cell lines inhibited colony formation. Similarly, self-renewal induced by AML1/ETO in primary murine progenitors was inhibited when AES was decreased or absent. High levels of AES expression enhanced formation of immature colonies, serial replating capacity of primary cells, and colony formation in colony-forming unit-spleen assays. These findings establish AES as a novel AML1/ETO-induced target gene that plays an important role in the self-renewal phenotype of t(8;21)-positive AML.

Inhibitor of cyclin-dependent kinase (CDK) interacting with cyclin A1 (INCA1) regulates proliferation and is repressed by oncogenic signaling.

The cell cycle is driven by the kinase activity of cyclin·cyclin-dependent kinase (CDK) complexes, which is negatively regulated by CDK inhibitor proteins. Recently, we identified INCA1 as an interaction partner and a substrate of cyclin A1 in complex with CDK2. On a functional level, we identified a novel cyclin-binding site in the INCA1 protein. INCA1 inhibited CDK2 activity and cell proliferation. The inhibitory effects depended on the cyclin-interacting domain. Mitogenic and oncogenic signals suppressed INCA1 expression, whereas it was induced by cell cycle arrest. We established a deletional mouse model that showed increased CDK2 activity in spleen with altered spleen architecture in Inca1(-/-) mice. Inca1(-/-) embryonic fibroblasts showed an increase in the fraction of S-phase cells. Furthermore, blasts from acute lymphoid leukemia and acute myeloid leukemia patients expressed significantly reduced INCA1 levels highlighting its relevance for growth control in vivo. Taken together, this study identifies a novel CDK inhibitor with reduced expression in acute myeloid and lymphoid leukemia. The molecular events that control the cell cycle occur in a sequential process to ensure a tight regulation, which is important for the survival of a cell and includes the detection and repair of genetic damage and the prevention of uncontrolled cell division.

Profiling of histone H3 lysine 9 trimethylation levels predicts transcription factor activity and survival in acute myeloid leukemia.

Acute myeloid leukemia (AML) is commonly associated with alterations in transcription factors because of altered expression or gene mutations. These changes might induce leukemia-specific patterns of histone modifications. We used chromatin-immunoprecipitation on microarray to analyze histone 3 lysine 9 trimethylation (H3K9me3) patterns in primary AML (n = 108), acute lymphoid leukemia (n = 28), CD34(+) cells (n = 21) and white blood cells (n = 15) specimens. Hundreds of promoter regions in AML showed significant alterations in H3K9me3 levels. H3K9me3 deregulation in AML occurred preferentially as a decrease in H3K9me3 levels at core promoter regions. The altered genomic regions showed an overrepresentation of cis-binding sites for ETS and cyclic adenosine monophosphate response elements (CREs) for transcription factors of the CREB/CREM/ATF1 family. The decrease in H3K9me3 levels at CREs was associated with increased CRE-driven promoter activity in AML blasts in vivo. AML-specific H3K9me3 patterns were not associated with known cytogenetic abnormalities. But a signature derived from H3K9me3 patterns predicted event-free survival in AML patients. When the H3K9me3 signature was combined with established clinical prognostic markers, it outperformed prognosis prediction based on clinical parameters alone. These findings demonstrate widespread changes of H3K9me3 levels at gene promoters in AML. Signatures of histone modification patterns are associated with patient prognosis in AML.

5-azacytidine enhances efficacy of multiple chemotherapy drugs in AML and lung cancer with modulation of CpG methylation.

The DNA methyltransferase (DNMT) inhibitory drugs such as 5-azacytidine induce DNA hypomethylation by inhibiting DNA methyltransferases. While clinically effective, DNMT inhibitors are not curative. A combination with cytotoxic drugs might be beneficial, but this is largely unexplored. In the present study, we analyzed potential synergisms between cytotoxic drugs and 5-azacytidine in acute myeloid leukemia (AML) and non-small cell lung cancer (NSCLC) cells. Lung cancer and leukemia cell lines were exposed to low doses of 5-azacytidine with varying doses of cytarabine or etoposide for AML cells (U937 and HL60) as well as cisplatin or gemcitabine for NSCLC cells (A549 and HTB56) for 48 h. Drug interaction and potential synergism was analyzed according to the Chou-Talalay algorithm. Further analyses were based on soft agar colony formation assays, active caspase-3 staining and BrdU incorporation flow cytometry. To identify effects on DNA methylation patterns, we performed genome wide DNA methylation analysis using 450K bead arrays. Azacytidine at low doses was synergistic with cytotoxic drugs in NSCLC and in AML cell lines. Simultaneous exposure to 5-azacytidine with cytotoxic drugs showed strong synergistic activity. In colony formation assays these synergisms were repeatedly verified for 5-azacytidine (25 nM) with low doses of anticancer agents. 5-azacytidine neither affected the cell cycle nor increased apoptosis. 450K methylation bead arrays revealed 1,046 CpG sites in AML and 1,778 CpG sites in NSCLC cells with significant DNA hypomethylation (24-h exposure) to 5-azacytidine combined with the cytotoxic drugs. These CpG-sites were observed in the candidate tumor-suppressor genes MGMT and THRB. Additional incubation time after 24-h treatment led to a 4.1-fold increase of significant hypomethylated CpG-sites in NSCLC cells. These results suggest that the addition of DNA demethylating agents to cytotoxic anticancer drugs exhibits synergistic activity in AML and NSCLC. Dysregulation of an equilibrium of DNA methylation in cancer cells might increase the susceptibility for cytotoxic drugs.

Maintenance of leukemia-initiating cells is regulated by the CDK inhibitor Inca1.

Functional differences between healthy progenitor and cancer initiating cells may provide unique opportunities for targeted therapy approaches. Hematopoietic stem cells are tightly controlled by a network of CDK inhibitors that govern proliferation and prevent stem cell exhaustion. Loss of Inca1 led to an increased number of short-term hematopoietic stem cells in older mice, but Inca1 seems largely dispensable for normal hematopoiesis. On the other hand, Inca1-deficiency enhanced cell cycling upon cytotoxic stress and accelerated bone marrow exhaustion. Moreover, AML1-ETO9a-induced proliferation was not sustained in Inca1-deficient cells in vivo. As a consequence, leukemia induction and leukemia maintenance were severely impaired in Inca1-/- bone marrow cells. The re-initiation of leukemia was also significantly inhibited in absence of Inca1-/- in MLL-AF9- and c-myc/BCL2-positive leukemia mouse models. These findings indicate distinct functional properties of Inca1 in normal hematopoietic cells compared to leukemia initiating cells. Such functional differences might be used to design specific therapy approaches in leukemia.

Allogeneic transplantation versus chemotherapy as postremission therapy for acute myeloid leukemia: a prospective matched pairs analysis.

PURPOSE: The majority of patients with acute myeloid leukemia (AML) who achieve complete remission (CR) relapse with conventional postremission chemotherapy. Allogeneic stem-cell transplantation (alloSCT) might improve survival at the expense of increased toxicity. It remains unknown for which patients alloSCT is preferable. PATIENTS AND METHODS: We compared the outcome of 185 matched pairs of a large multicenter clinical trial (AMLCG99). Patients younger than 60 years who underwent alloSCT in first remission (CR1) were matched to patients who received conventional postremission therapy. The main matching criteria were AML type, cytogenetic risk group, patient age, and time in first CR. RESULTS: In the overall pairwise compared AML population, the projected 7-year overall survival (OS) rate was 58% for the alloSCT and 46% for the conventional postremission treatment group (P = .037; log-rank test). Relapse-free survival (RFS) was 52% in the alloSCT group compared with 33% in the control group (P < .001). OS was significantly better for alloSCT in patient subgroups with nonfavorable chromosomal aberrations, patients older than 45 years, and patients with secondary AML or high-risk myelodysplastic syndrome. For the entire patient cohort, postremission therapy was an independent factor for OS (hazard ratio, 0.66; 95% CI, 0.49 to 0.89 for alloSCT v conventional chemotherapy), among age, cytogenetics, and bone marrow blasts after the first induction cycle. CONCLUSION: AlloSCT is the most potent postremission therapy for AML and is particularly active for patients 45 to 59 years of age and/or those with high-risk cytogenetics.

No association of the A260G and A386G DAZL single nucleotide polymorphisms with male infertility in a Caucasian population.

BACKGROUND: The human DAZ gene family includes two autosomal genes, BOULE and DAZL, and a Y-chromosomal DAZ gene cluster. All are RNA-binding proteins and assumed to be master regulators of germline gene expression. We have investigated the impact of two DAZL polymorphisms, located at nucleotide positions 260 (SNP 260) and 386 (SNP 386), on the fertility of Caucasian men. These single nucleotide polymorphisms (SNPs) have been described previously to be associated with spermatogenic failure. METHODS: Blood samples were collected and genomic DNA was extracted from 165 normozoospermic men and 202 oligo- or azoospermic patients, of whom 28 displayed an AZFc deletion. The frequencies of A or G allelic variants in SNP 260 and 386 were analysed via TaqMan allelic discrimination assays. In both cases, the A to G transition leads to a threonine to alanine change. RESULTS: A total of 24.2% of the controls showed a heterozygous nucleotide variant (AG) for the SNP 260 and the remaining 75.8% were homozygous for A. In the AZFc-deleted group, this distribution was significantly different, with 39.3% for AG, 57.1% for AA and 3.6% for GG. However, the increased heterozygosity was not correlated with sperm counts and morphology. The patients without deletions displayed a similar allelic pattern to the controls (24.1% AG/75.9% AA). For SNP 386, only the AA nucleotide variant was found in all subjects studied and in no case was the previously described heterozygous AG variant found. CONCLUSION: In a selected Caucasian population, the DAZL SNP 386 is completely absent and SNP 260 is not associated with spermatogenic failure and therefore does not represent a molecular marker for genetic diagnosis of male infertility.