Digital Restriction Enzyme Analysis of Methylation (DREAM) by Next Generation Sequencing Uncovers Epigenetic Disturbances In Acute Myelogenous Leukemia.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3635-3635
Author(s):  
Frank Neumann ◽  
Jean-Pierre Issa ◽  
Yue Lu ◽  
Marcos R Estecio ◽  
Rong He ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Cell Lines ◽  
Cpg Islands ◽  
Leukemia Cell ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Cpg Sites ◽  
Leukemia Cell Lines ◽  
Normal Controls

Abstract Abstract 3635 DNA methylation is a key epigenetic mark affecting the configuration of chromatin and the potential for gene expression. Disorganization of DNA methylation contributes to the development of leukemia. There is a need for high resolution, quantitative and cost effective methods to investigate changes of methylome in leukemia. To achieve this goal, we have recently developed a digital restriction enzyme analysis of methylation (DREAM) for quantitative mapping of DNA methylation at approximately 50,000 CpG sites across the whole genome (Jelinek et al., ASH 2009, abstract 567). The method is based on creating distinct DNA signatures at unmethylated or methylated CCCGGG sites by sequential restriction digests of genomic DNA with the SmaI and XmaI endonucleases and on resolving these signatures by massively parallel sequencing. Using the DREAM method, we have analyzed DNA methylation in bone marrow cells from 2 patients with AML, 3 samples of white blood cells from healthy adults and 2 myeloid leukemia cell lines (K562 and HEL). The first patient (Pt#1) was a 72 year-old male with AML transformation of the myelodysplastic syndrome (MDS). He had 32% blasts in the bone marrow and a complex karyotype. He had received lenalidomide treatment only. The second AML patient (Pt#2) was a 28 year-old male suffering from a relapse of an AML FAB M1. The bone marrow showed 87% of blasts and a complex karyotype. The patient was heavily pretreated with daunorubicin, ara-C, etoposide, 6-thioguanine, dexamethasone and l-asparaginase. Neither of the patients received demethylating drugs. Using typically 2 sequencing lanes per sample and paired-end reads of 36 bases on the Illumina Gene Analyzer II platform, we acquired 20–38 (median 33) million sequence tags per sample; of these, 7–17 (median 12) million were mapped to SmaI/XmaI sites unique in the human genome. With a threshold of minimum 20-fold coverage, we obtained quantitative information on the DNA methylation level of 39,603-53,312 (median 44,490) CpG sites associated with 8,939-10,735 (median 9,517) genes. In general, methylation was largely absent within CpG islands (CGI). The CpG sites most protected from methylation were in CGI and within 1 kb from gene transcription start sites (TSS). These regions were represented by 13,474 CpG sites. Focusing our analysis on these CpG sites, methylation >10% was detected only in 268 sites in normal controls (1.9%). The numbers of sites with methylation >10% were significantly higher (P<.0001, chi-square test) in both AML patients: 397 sites in Pt#1 (2.9%) and 2,143 sites in Pt#2 (15.6%), respectively. Leukemia cell lines mirrored the pattern of CGI hypermethylation seen in primary AML cells. Methylation >10% in CGI within 1 kb from TSS was observed at 2,331 sites (17.0%) in K562 and at 2,484 sites (18.1%) in HEL. Differential hypermethylation in AML patients affected 906 genes, including multiple genes previously shown to be methylated in cancer, such as CDKN1B, FOXO3, GATA2, GATA4, GDNF, HOXA9, IGFBP3, SALL1 and WT1. Methylated genes were significantly enriched in canonical pathways affecting embryonic stem cell signaling, Wnt-beta-catenin signaling and pluripotency suggesting an important role in AML stem cells. In contrast to CGI, it is known that CpG sites outside of CpG islands (NCGI) are generally fully methylated in normal cells. We analyzed 11,220 NCGI sites that were >1 kb from gene TSS. Methylation >90% was observed at 5,217 (46%) sites in normal controls, in 5,380 sites (48%) in Pt#1, while only in 1,873 sites (17%) in Pt#2 (P<.0001). Leukemia cell lines also showed this NCGI hypomethylation with only 1,422 (13%) fully methylated sites in K562 and 4,200 sites (37%) in HEL. Thus, significant degrees of hypomethylation in NCGI were observed in Pt#2, and in K562 and HEL cell lines, but not in Pt#1. In conclusion, high resolution quantitative mapping of DNA methylation changes in leukemia is feasible using the DREAM method. Relatively small alterations in DNA methylation observed in the MDS/AML Pt#1 contrasted with extensive hyper and hypomethylation found in Pt#2 with relapsed AML M1. Our results illustrate the complexity and diverse extent of DNA methylation changes in leukemia. Disclosures: No relevant conflicts of interest to declare.

Cancer Drivers Affected by Aberrant DNA Methylation in MDS and AML

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1716-1716 ◽  
Author(s):  
Jaroslav Jelinek ◽  
Shoudan Liang ◽  
Frank Neumann ◽  
Rong He ◽  
Yue Lu ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Research Funding ◽  
Cpg Islands ◽  
Enzyme Analysis ◽  
Epigenetic Mark ◽  
Aberrant Methylation ◽  
Healthy Controls ◽  
Cpg Sites ◽  
Normal Controls ◽  
Hypermethylated Genes

Abstract Abstract 1716 Cytosine methylation is an epigenetic mark affecting accessibility of DNA to transcription. Cancer is associated with hypermethylation in CpG islands (dense clusters of CpG sites frequently present around gene transcription starts) and hypomethylation of sparse CpG sites outside CpG islands. Complex changes of DNA methylation in leukemia permanently disturb epigenetic regulation and participate in leukemogenesis. To characterize epigenetic aberrations in myeloid neoplasms, we analyzed DNA methylation in 16 patients with myelodysplastic syndrome (MDS), 7 patients with acute myeloid leukemia (AML) and 5 healthy controls. Using Digital Restriction Enzyme Analysis of Methylation, we quantified DNA methylation at CpG dinucleotides within approximately 40,000 CCCGGG restriction sites across the genome. We analyzed methylation differences between healthy controls and patients with MDS and AML. CpG sites within CpG islands (CGI sites) are typically not methylated in normal tissues. We found 18,738 CGI sites with methylation <5% in normal controls. MDS and AML patients showed heterogeneous hypermethylation >20% in these sites, ranging from 5 to 2720 (median 186) hypermethylated sites in individual patients. The median number of hypermethylated CGI sites was 146 in MDS and 1234 in AML patients. Altogether, we found 5069 CGI sites corresponding to 2183 genes differentially hypermethylated in MDS or AML. GpG sites outside CpG islands (NCGI sites) are generally methylated. We found only 3262 NCGI sites unmethylated (<5% methylation) in normal controls. Hypermethylation pattern of these NCGI sites in individual MDS and AML patients was similar to that of CGI (r=0.85), with 5–388 (median 38) sites hypermethylated over 20%. Altogether, we found 848 NCGI sites corresponding to 629 genes hypermethylated. Hypermethylation affecting both CGI and NCGI sites was found in 273 genes. In order to identify potential drivers in the plethora of methylation changes, we compared the hypermethylated genes with the Sanger Institute “Cancer Consensus” listing 457 genes. The list of 2539 hypermethylated genes contained 74 genes (3%) from the cancer list (51 in CGI, 10 in NCGI and 13 in both CGI and NCGI). Next we analyzed hypomethylation events in MDS and AML. We found 10,509 CpG sites (1210 CGI, 9299 NCGI) with methylation level >80% in normal controls. Methylation levels <30% in MDS and AML patients were observed at 1–439 (median 23) sites. Hypomethylation affected mostly NCGI sites and the numbers of sites hypomethylated in individual patients positively correlated with hypermethylation at CGI sites (r=0.39). The total of 1153 hypomethylated sites corresponded to 777 genes. Twenty-two genes (3%) were present on the cancer list. Six genes (CBFA2T3, FGFR3, FLI1, MLLT1, PHOX2B and PRDM16) showed both hyper and hypomethylation in different parts of the gene when compared to normal controls. Interestingly, translocations involving 5 of these genes have been reported in blood malignancies. The number of ‘cancer’ genes affected by epigenetic events in individual patients was 1–29 (median 8) in MDS and 2–44 (median 20) in AML. In summary, we have detected tens to thousands of CpG sites with aberrant methylation in MDS and AML patients. Our data suggest that approximately 3% of DNA hypermethylation and hypomethylation events are potential drivers in the leukemogenic process in MDS and AML. DNA methylation changes were detected in 90 genes (13%) of the 457 cancer gene list. Our findings thus support the importance of epigenetics in leukemia. Disclosures: Neumann: Sanofi-Aventis: Employment. Issa:GSK: Consultancy; SYNDAX: Consultancy; Merck: Research Funding; Eisai: Research Funding; Celgene: Research Funding; Celgene: Honoraria; Novartis: Honoraria; J&J: Honoraria.

3D Genomics of Acute Meyloid Leukemia Reveals the Imbalance between DNA Methylation Canyon Interactions and Leukemic Specific Enhancer Network Interactions

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 45-45
Author(s):  
Xiaotian Zhang ◽  
Xue Qing David Wang ◽  
Haley Gore ◽  
Pamela Himadewi ◽  
Fan Feng ◽  
...  
Keyword(s):  
Dna Methylation ◽  
High Resolution ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Chromatin Organization ◽  
Data Set ◽  
Hematopoietic Stem ◽  
Leukemia Cell Lines ◽  
Hyperacetylated Domains ◽  
Primary Leukemia

Changes in 3D chromatin organization like enhancer hijacking are believed to the driver for disease development like leukemia. Here we performed high-resolution HiC assays on primary acute myeloid leukemia (AML) samples and cell lines to dissect the abnormal 3D chromatin organization in AML. Our data set covers 5 AML samples and 3 AML cell lines. This dataset includes the common genetic abnormalities in AML: MLL-rearrangement, NPM1 mutation, RUNX1 mutation, and IDH1/TET2 mutations. We have recently generated high-resolution map for normal human hematopoietic stem cells (HSC) (Zhang et al. Mole Cell. 2020). In comparison with the HSC 3D chromatin organization, we found TADs and loops are very stable in both primary leukemia samples and cell lines. Less than 5% of all TADs in HSC fuse in AML, mimicking the enhancer hijacking scenario. These fusion events do not cause the gene expression changes of genes in the fused TAD. Interestingly, in TET2 or IDH1 mutated AML blast, two-fold more TAD fusion events occurred in primary AML blast in comparison with RUNX1 and MLL-r leukemia, with a loss in the CTCF sites on the TAD fusion break point. We previously found in HSC, the Polycomb marked DNA methylation Canyons (DMC) form multi-Mb size long-range interactions. DMC interactions in general decrease in primary AMLs. AMLs with IDH1 or TET2 mutations shows the biggest reduction in DMC interactions. Hypermethylation in the DMCs is observed in the AML samples with IDH1/2 or TET2 mutations, suggesting DNA methylation level in DMCs controls DMC 3D interactions directly. In leukemia cell lines, the DMC interactions almost disappear, with further hypermethylation in DMCs. Compared with normal HSC, we found in AML, the AML-specific H3K27ac marked regions form leukemia specific loops and transcription stripes in both cell lines and primary samples. Particularly in MLL-r primary leukemias, we found broad H3K27ac covered, hyperacetylated domains (10kb to 200kb). 22 such hyperacetylated domains were identified and associated with leukemogenic genes such as SATB1, ZEB2 and HOXA. All these domains formed distinct 3D micro TAD in the MLL-r primary leukemia in comparison with the HSPC, and CTCFs are not located at the border of these domains. Taken together, suggest active leukemia specific transcription created new 3D genomic interactions which is independent of cohesion-CTCF mediated loop extrusion. Interestingly, in HOXA cluster, we found a geneless DMC 1.3MB upstream of HOXA switched from Polycomb binding site to active enhancer site in the leukemia cells. By applying CRISPR/Cas9 editing, we found this canyon is essential for survival of HOXA high expressing leukemia cell lines like OCI-AML3 and MV4:11. In summary, we found the 3D chromatin organization in human leukemia significantly alters in two opposite way 1. The significant loss of Polycomb marked DMC interactions caused by the DNA hypermethylation and 2. The leukemic specific hyperacetylated domains form its own distinct micro TAD and stripes in the 3D chromatin organization. Disclosures No relevant conflicts of interest to declare.

Aberrant DNA Methylation of the Src Tyrosine Kinase Hck Is a Frequent Event in Human Leukemia and May Predict for Poor Prognosis in Adult Acute Lymphocytic Leukemia (ALL).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1542-1542
Author(s):  
Koyu Hoshino ◽  
Hui Yang ◽  
Claritsa Santos-Malave ◽  
Blanca Sanchez-Gonzalez ◽  
Guillermo Garcia-Manero
Keyword(s):  
Dna Methylation ◽  
Protein Expression ◽  
B Cell ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Human Leukemia ◽  
Expression Levels ◽  
Leukemia Cell Lines ◽  
Aberrant Dna Methylation

Abstract Aberrant DNA methylation of promoter-associated CpG islands is a frequent phenomenon in human leukemias, and in particular in adult ALL. Hck is a member of the Src family of tyrosine kinases, and functionally is located downstream of BCR-ABL signaling in chronic myelogenous leukemia (CML). Hck expression is limitedly to myeloid cells and B cell lymphocytes. Although some evidence indicates that Hck is required for malignant transformation and apoptosis, its role in leukemia is not fully understood. Here we analyze the role of aberrant DNA methylation of Hck in leukemia cell lines and patients. Using BLAT, we first identified the presence of a canonical CpG island in the near proximity of the transcription start site of HcK. To detect and measure DNA methylation, we designed a combined bisulfite restriction PCR assay. Using this assay, we found that Hck was methylated in 13 out of 23 hematopoietic and 8 out of 10 non-hematopoietic cell lines, but not in the bone marrow from 6 healthy individuals. We subsequently studied Hck expression by real-time PCR using GAPDH expression as an internal control. Hck expression was lower (dCT = −14.2± 3.6) in 7 Hck methylated cell lines than in 8 Hck unmethylated ones (dCT= −9.0± 3.5), p=0.017. All the cell lines studied were of myeloid or B cell origin. We then treated the Raji cell line with the hypomethylating agent 5-aza-2-deoxycytidine (DAC). DAC treatment resulted in partial hypomethylation of Hck and in an increment of Hck expression (dCT: −19.37 to −8.47). Subsequently, the effects of DAC treatment on Hck protein expression levels were analyzed using Western blot. These experiments showed a strong correlation between hypomethylation, gene re-expression and protein expression levels. These data therefore indicates that DNA methylation is an important aberrant regulator of Hck expression in leukemia cell lines. Based on the relevance of these findings, we then analyzed the frequency of Hck methylation in patients with leukemia. Using a cut-off of 10%, Hck was found to be methylated in 15 out of 44 (34%) patients with ALL, 9 out 23 pts (39%) with CML, and 3 out 10 pts (30%) with AML. Of importance, the density of Hck methylation was significantly higher in patients with ALL (mean 11.3%; range 0–76) compared to those with CML(5.2%; range 0–12) or AML ( 7.5%, range 0–14), p=0.02. Hck methylation was not associated with a B cell phenotype or the presence of the Philadelphia chromosome in patients with ALL. Nine ALL pts out of 15 with Hck methylation had died compared to 7 out 29 unmethylated (total ALL group n=34). Median survival had not been reached for the group of patients with no Hck methylation (n=29) compared to 116 weeks for those with Hck methylation (n=15) (p=0.08). All pts had been treated with hyperCVAD based chemotherapy. These data indicates that Hck methylation is a frequent phenomenon in human leukemia that maybe associated with a worse prognosis in ALL and suggests that Hck has a tumor suppressor like function in these disorders.

Analysis of Class I and II Histone Deacetylase Fails To Identify a Human Leukemia Specific Expression Profile.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2130-2130 ◽  
Author(s):  
Guillermo Garcia-Manero ◽  
Andy S. Quesada ◽  
Shirisha Maddipoti ◽  
Shaoqing Kuang ◽  
Weigang Tong ◽  
...  
Keyword(s):  
Clinical Trials ◽  
B Cells ◽  
B Cell ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Expression Profiles ◽  
Hdac Inhibitors ◽  
Leukemia Cell ◽  
Leukemia Cell Lines ◽  
Normal Controls

Abstract Histone deacetylase (HDAC) inhibitors are being developed clinically for the treatment of leukemia. Because HDACs are composed of a large number of different proteins, and substrate specificity may differ among different HDAC inhibitors, it is important to understand if human leukemias are characterized by specific HDAC expression patterns. To study this, we have analyzed using real-time PCR and Western blots, all major class I and II HDACs in normal marrow controls (N=13, including 10 CD19+ B cell specimens), leukemia cell lines (N=25), samples from patients with AML (N=6), MDS (N=12), CLL (N=10) and human samples (N=6) obtained pretreatment and sequentially from patients with leukemia treated with two different phase I clinical trials of HDAC inhibitors: MGCD0103 and vorinostat. In general, normal controls were characterized by low levels of HDACs 1 to 10, although normal CD19+ B cells exhibited a significant increased expression of HDAC1 and 5. In leukemia cell lines, HDAC 1, 2 and 3 were expressed at higher levels than 4 to 10 but there were no differences between leukemia cell lines and normal controls or B cells. HDAC mRNA expression was not modified by cell proliferation or treatment with HDAC inhibitors. No specific HDAC expression profiles were detected in primary human AML or MDS samples. In contrast, CLL primary samples were characterized by an overexpression of HDAC 1,3,5 and 10, although this pattern was not significantly different than that of normal CD19+ B cells. Sequential analysis of human samples obtained from patients treated with two different HDAC inhibitors, vorinostat or MGCD0103 on two different clinical trials, did not affect expression profiles in patients with MDS or AML. Overall, mRNA expression results correlated with protein levels. In summary, our results indicate that AML or MDS are not characterized by a leukemia specific HDAC expression profile but that B cells and B cell leukemia are characterized by a significant overexpression of HDAC 1. This could explain the activity observed with HDAC inhibitors in B cell malignancies and serve as the bases for clinical studies of HDAC inhibitors in CLL.

Genome-Wide Identification of Aberrant Promoter Associated CpG Island Methylation in Acute Lymphoblastic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2127-2127
Author(s):  
Shao-qing Kuang ◽  
Weigang Tong ◽  
Hui Yang ◽  
Mathew K. Lee ◽  
Zhi-Hong Fang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
B Cell ◽  
Cell Lines ◽  
Cpg Island ◽  
Trichostatin A ◽  
Methylation Status ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Leukemia Cell Lines ◽  
Aberrant Dna Methylation

Abstract Aberrant DNA methylation is a common molecular feature of both pediatric and adult ALL. Specific methylation patterns predict for poor prognosis (Shen et al Blood 2004), and reactivation of epigenetically inactivated molecular pathways results in induction of leukemia cell death (Kuang et al. Oncogene 2007). Until now most studies of methylation in ALL have been based on arbitrary gene selection methods. To overcome this limitation and to study hundreds of promoter CpG islands simultaneously, we have developed a method that combines MCA (Methylated CpG Island Amplification) with either RDA (Representational Difference Analysis) or the Agilent Promoter Microarray platform. With these methods differentially methylated DNA treated with bisulfite is generated after mixing tester DNA (in our case DNA from de novo refractory Ph negative and MLL negative ALL patients) with driver DNA (normal B cell controls) and using specific restriction enzymes and several rounds of PCR. DNA fragments thus generated are either cloned (RDA) or labeled and spotted on the Agilent Array. Using this technology, that can potentially interrogate up to 17K promoters, we have identified 932 promoters targets of aberrant DNA methylation in poor risk ALL from patients that cannot be currently identified by standard molecular methods (Ph and MLL negative). The genes associated with these promoters are distributed through the human genome but an overrepresentation of methylated promoters located in chromosomes 3, 9, 11 and 19 was detected. Using molecular pathway clustering analysis, 404 of these genes are grouped together in 29 specific functional pathways. We have validated the methylation of 31 of these 923 genes by bisulfite pyrosequencing. Of these, 27 (87%) were confirmed to be hypermethylated in 23 human leukemia cell lines but not in normal controls (N=15). Methylation status analysis of these 27 genes allowed for the segregation of T cell versus B cell leukemia cell lines. Fifteen of these genes (GIPC2, RSPO1, MAGI1, CAST1, ADCY5, HSPA4L, OCLN, EFNA5, MSX2, GFPT2, GNA14, SALL1, MYO5B, ZNF382 and MN1) were also frequently hypermethylated in primary ALL samples. Expression analysis of 6 of these genes (GIPC2, MAGI1, ADCY5, HSPA4L, OCLN and GNA14) in leukemia cell lines further confirmed methylation associated gene silencing. Treatment of methylated/silenced cell lines with 5′-aza-2′-deoxycytidine and trichostatin A resulted in gene re-expression, further confirming the role of DNA methylation in their silencing. In summary, we have identified in excess of 900 targets of aberrant DNA methylation in ALL. The study of the epigenetically suppressed pathways represented by these genes should allow us to further understand the molecular pathogenesis of ALL and develop new prognostic biomarkers for patients with Ph and MLL negative disease.

Targeting the Mnk Pathway Enhances Chemotherapy-Induced Antileukemic Responses.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1617-1617
Author(s):  
Jessica K Altman ◽  
Heather Glaser ◽  
Amanda Redig ◽  
Antonella Sassano ◽  
Martin S Tallman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Map Kinase ◽  
Cell Lines ◽  
Negative Feedback ◽  
Peripheral Blood ◽  
Leukemia Cell ◽  
P38 Map Kinase ◽  
Human Leukemia ◽  
Inhibitory Effects ◽  
Leukemia Cell Lines

Abstract Mnk kinases (mitogen-activated protein kinase [MAPK]-interacting kinases) are downstream effectors of Map kinase pathways, including the MEK/Erk and the p38 Map kinase signaling cascades. We have previously shown that Mnk kinases and the p38 Map kinase pathway are activated in a negative feedback regulatory manner during treatment of cells with arsenic trioxide, and that molecular or pharmacological inhibition of their activation enhances arsenic trioxide-dependent apoptosis and antileukemic responses (J Biol Chem. 283:12034–42, 2008, and Cancer Res. 66:6763–71, 2006). We examined the activation status of Mnk kinases in response to treatment of AML cells with chemotherapy and the function of these kinases in the generation of antileukemic responses. The human AML cell lines U937, K562, or MM6 were treated with cytarabine, in the presence or absence of a commercially available pharmacological Mnk-1 inhibitor (Calbiochem); and the phosphorylation of Mnk and its downstream effector, eIF4E, were assessed. Treatment with cytarabine increased phosphorylation of Mnk and eIF4E. The cytarabine-dependent eIF4E phosphorylation was blocked when human leukemia cell lines were treated with the pharmacologic Mnk inhibitor, indicating that Mnk regulates eIF4E activity. Such phosphorylation was also found to be defective in Mnk1/Mnk2 double knockout mouse embryonic fibroblast (MEF) cells, as compared to wild-type MEFs. Importantly, cytarabine-induced apoptosis was strongly enhanced in Mnk1−/− Mnk2−/− MEFs, as compared to Mnk1+/+Mnk2+/+ MEFs. To define the role of Mnk kinases in the generation of chemotherapy-induced antileukemic responses, human leukemia cell lines and bone marrow or peripheral blood mononuclear cells from patients with AML were used in clonogenic assays in methylcelluose to determine the effects of Mnk inhibition in the cytarabine-mediated leukemic progenitor (CFU-L) growth. The Mnk inhibitor potentiated the inhibitory effects of cytarabine on U937-derived CFU-L colonies and bone marrow or peripheral blood-derived CFU-L from 3 patients with AML. Interestingly, combinations of the Mnk inhibitor with the mTOR inhibitor, rapamycin, also resulted in more pronounced inhibitory effects on CFU-L colony formation than each agent alone. Altogether, these findings demonstrate that the Mnk pathway is activated during treatment of AML cells with cytarabine and that such activation occurs in a negative feedback regulatory manner to counteract the antileukemic effects of cytarabine. They also raise the possibility that targeting Mnk kinases may provide a novel approach to enhance the effects of chemotherapy on AML cells in vitro and possibly in vivo.

Altered Patterns of DNA Methylation on Chromosomes from Leukemia Cell Lines: Identification of 5-Methylcytosines by Indirect Immunodetection

1998 ◽  
Vol 103 (2) ◽  
pp. 101-109 ◽  
Author(s):  
Mustapha Bensaada ◽  
Héléne Kiefer ◽  
Gérard Tachdjian ◽  
Jean Michel Lapierre ◽  
Valère Cacheux ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Leukemia Cell Lines

Classifying Leukemias Based on Epigenetic Alterations.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2123-2123
Author(s):  
Jaroslav Jelinek ◽  
Marcos R.H. Estecio ◽  
Kimie Kondo ◽  
Rong He ◽  
Jiri Zavadil ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Lymphoid Cell ◽  
Myeloid Leukemia ◽  
Cpg Island ◽  
Leukemia Cell ◽  
Epigenetic Alterations ◽  
Leukemia Cell Lines ◽  
Lymphoid Cell Lines ◽  
Myeloid Leukemia Cell

Abstract Acute leukemia is caused by alterations of blood-forming stem cells leading to uncontrolled growth and diminished capacity to differentiate into mature functional blood elements. Beside genetic changes, epigenetic alterations are increasingly recognized as important events in the pathogenesis of leukemia. Cytosine methylation in CpG islands at gene transcription start regions can cause heritable gene silencing and have the same functional effects as inactivating mutations. Hundreds of genes may become epigenetically silenced in leukemia. While many of the methylated genes are not expressed in blood cells, silencing of genes critically important for control of stem cell self-renewal, proliferation, differentiation, and/or survival can contribute to the malignant phenotype. We used a genome-wide method to identify methylated genes by hybridizing a CpG island microarray with amplicons obtained by the methylated CpG island amplification technique (MCAM). We analyzed 10 leukemia cell lines with different cellular origin (myeloid cell lines KG1, KG1a, HEL, K562, and TF1; T lymphoid cell lines CEM and JTAg; and B lymphoid cell lines ALL1, BJAB, and Raji). On average, 266 genomic loci were found to be hypermethylated in these cell lines, ranging from 56 (KG1) to 483 loci (Raji), reinforcing the idea of extensive epigenome alteration in leukemia. Unsupervised hierarchical clustering showed distinct methylation pattern in the cell lines of lymphoid origin versus myeloid leukemia cell lines and a GM-CSF-dependent erythroleukemia cell line TF-1, justifying the use of methylation markers for uncovering of tumor-specific pathways of gene inactivation. There was a striking difference in the number of hypermethylated genes between two closely related myeloid leukemia cell lines: KG1 (56 methylated loci) and its undifferentiated variant KG1a (225 methylated loci). cDNA microarray analysis showed that deoxy-azacitidine treatment induced expression of genes differentially methylated in KG1a (DKKL1, GBX, HIVEP3, KCNAB1, KIAA1102, NAV2, NEIL1, and RAX) but not in KG1 cells where these genes were unmethylated. Finally, we used bisulfite PCR followed by pyrosequencing analysis to quantitatively measure DNA methylation of several genes detected by MCAM. Ongoing analyses of bone marrow samples from leukemia patients showed hypermethylation of the following genes: GDNF (in 4/22 [18%] AML and 7/20 [35%] ALL patients), HAND2 (in 5/22 [23%] AML and 7/20 [35%] ALL patients), HIVEP3 (in 9/22 [41%] AML and 6/20 [30%] ALL patients), MPDZ (in 2/6 [33%] AML and 15/20 [75%] ALL patients), and NEIL1 (in 2/20 [10%] AML and 1/12 [8%] ALL patients). Mapping of DNA methylation abnormalities may detect epigenetic markers important for leukemia classification and prognosis. Identification of pathways frequently silenced by DNA methylation may also suggest new targets for specific therapy.

Dual Epigenetic Control of CCAAT/Enhancer Binding Protein α (C/EBPα) Expression in Acute Myeloid Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2116-2116 ◽  
Author(s):  
Bjoern Hackanson ◽  
Kristi L. Bennett ◽  
Romulo M. Brena ◽  
Jinmai Jiang ◽  
Katie Maharry ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Binding Protein ◽  
Leukemia Cell ◽  
Epigenetic Alterations ◽  
Leukemia Cell Lines ◽  
Enhancer Binding Protein ◽  
Acute Myeloid

Abstract CCAAT/enhancer binding protein α (C/EBPα) is a master regulator for myeloid differentiation. As a potential tumor suppressor, its loss of function through mutations and posttranslational mechanisms has been thoroughly investigated in acute myeloid leukemia (AML). Recently, we have demonstrated the importance of epigenetic alterations in deregulating C/EBPα expression patterns in lung cancer and head and neck squamous cell carcinoma (HNSCC). In this study we investigated the role of DNA methylation and other epigenetic factors in the regulation of C/EBPα in AML using 94 patient samples and 7 leukemia cell lines. A comprehensive and quantitative DNA methylation analysis of C/EBPα’s large CpG island using MassARRAY (Sequenom©) technology and BioCOBRA identified a distinct and densely methylated upstream promoter region (−1423 bp to −1121 bp in relation to the transcription start site) in 20% (19 of 94) of AML patient samples and in five of seven leukemia cell lines, while the core promoter remained unmethylated. This aberrant DNA methylation pattern was associated with two generally prognostically favorable cytogenetic subgroups: inv(16)(p13q22) and t(15;17)(q22;q21). While DNA methylation levels in normal bone marrow samples (NBM) were very low (median: 0%, range: 0%–5%) when compared to NBM, we observed significantly higher DNA methylation levels in the inv(16) and t(15;17) cytogenetic subgroups, with median DNA methylation levels of 29% (range: 1% to 81%) and 5% (range: 1% to 94%), respectively (p&lt;0.05). However, using semi-quantitative RT-PCR, we could not detect a significant correlation between DNA methylation of C/EBPα and its expression in the inv(16) subgroup. Interestingly, while epigenetic-targeting compounds induced up-regulation of C/EBPα mRNA in vitro, C/EBPα protein was diminished. This unexpected scenario was postulated to be compatible with the presence of a microRNA targeting C/EBPα mRNA. Using a computational microRNA prediction approach and functional studies, we show that C/EBPα mRNA is a target for microRNA-124a. We additionally demonstrate that microRNA-124a is frequently silenced by epigenetic mechanisms in AML, becomes upregulated following epigenetic treatment and targets the C/EBPα 3′-untranslated region (3′UTR). In this way, C/EBPα protein expression is reduced in a posttranscriptional manner. Our results indicate that epigenetic alterations of C/EBPα are a frequent event in AML and that epigenetic treatment can result in down-regulation of a key hematopoietic transcription factor.

Genome-Wide DNA Methylation Profile of CLL with 17p del Allowed Identification of Multiple Epigenetically Inactivated Molecular Pathways with Prognostic Value in Human Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 492-492
Author(s):  
Wei-Gang Tong ◽  
William G. Wierda ◽  
Neby Bekele ◽  
Shao-Qing Kuang ◽  
Michael J. Keating ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Cpg Island ◽  
Trichostatin A ◽  
Methylation Status ◽  
Cpg Islands ◽  
Human Leukemia ◽  
Dna Hypomethylation ◽  
Line P ◽  
Normal Controls

Abstract Aberrant DNA methylation of multiple promoter associated CpG islands is a very prevalent phenomenon in human leukemias. Data from our laboratory indicates that methylation profiling allows the identification of leukemia patients with different risk and prognosis. Despite the advances in the understanding of the molecular biology of CLL, few studies of DNA methylation have been performed in CLL. In the current study, we have developed a new assay combining MCA (Methylated CpG island Amplification) with the Agilent promoter CpG array to identify simultaneously hundreds of abnormally methylated CpG islands in CLL. To perform this, we compared DNA from two CLL patients with 17p del (tester) with that of CD19+ B cells from two age-matched controls (driver). We identified 280 promoter CpG islands differentially methylated in CLL compared to normal controls. Most of these genes are located on chromosomes 19 (16%), 16 (11%), 17 (10%) and 11 (9%). We also performed interaction pathway and functional analysis of these 280 genes using the online Ingenuity Pathway Analysis tools. The initial analysis divided these genes into 25 functional networks, with the majority of genes fall into top 10 networks. The major functions of genes in these interaction networks involve cancer, organ development, cell death, drug metabolism, DNA replication and repair. We validated 22 of these genes (ADCY5, R-spondin1, LHX1, GALGT2, TFAP2C, ING1, SOX11, SOX14, SALL1, LTBP2, APP, DXL1, DLX4, KLK10, BCL11B, NR2F2, FAM62T, HAND2, BNC1, SPOCK, Prima1 and MLL1) in samples from 78 CLL patients and 10 age-matched normal controls. The characteristics of the 78 patients are: median age 59 (range 39–79), male 70%, Rai stage 0–II/III–IV (83%/17%), IgVH unmutated 49%, ZAP-70 positive 33%. Our results indicate that most of the genes identified by the array are frequently hypermethylated in CLL patients compared with healthy controls. Methylation frequency ranged from 20%–100% in CLL patients. Expression analysis of four selected genes (LHX1, GALGT2, TFAP2C and Prima1) in human leukemia cell lines and CLL patient samples by real-time PCR further confirmed methylation associated gene silencing, and treatment of these cell lines with hypomethylating agent 5-aza-2′-deoxycitidine with or without the HDAC inhibitor Trichostatin A resulted in gene re-expression and induction of DNA hypomethylation. We also analyzed the association of methylation status of these genes with IgVH mutation status, ZAP70 expression and patient survival. Unmutated IgVH was associated with increased methylation levels of LINE (p<0.0001), which is a marker for global gene methylation and SALL1 (p=0.00008). Expression of ZAP-70 (>20%) was associated with increased methylation levels of LINE (p<0.00001), MLL (p=0.02) and SALL1 (p=0.048). Further analysis showed that methylation status of LINE (p=0.007), SALL1 (p=0.019), ADCY5 (p=0.021), R-spondin1 (p=0.002) and APP (p=0.002) correlated with survival. In conclusion, our studies indicate that MCA/promoter array technique allows the identification of large number of promoter CpG islands aberrantly methylated in CLL and also the identification of novel tumor suppressors and signaling pathways that could be important in the tumorigenesis of CLL and other hematological malignancies.

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