Eph Receptor Tyrosine Kinases and Ephrin Ligands Are Epigenetically Inactivated in Acute Lymphoblastic Leukemia and Are Potential New Tumor Suppressor Genes in Human Leukemia.

Abstract The Eph (erythroprotein-producing hepatoma amplified sequence) family receptor tyrosine kinases and their ephrin ligands (ephrins) are involved in a variety of functions in normal cell development and cancer. We have identified several members of this family as potential targets of aberrant DNA methylation using Methylated CpG Island Amplification (MCA) / DNA promoter microarray technology. This is of importance as there are no prior reports of potential Eph receptor or Ephrin epigenetic inactivation in human leukemia. To further investigate the role of Eph receptor and ephrin family genes in leukemia, we have analyzed their DNA methylation status in a panel of 23 leukemia cell lines and 65 primary ALL patient samples. Aberrant DNA methylation of 9 of these genes (EPHA4, EPHA5, EPHA6, EPHB2, EPHB3, EPHB4, EphrinA5, Ephrin B2, and EphrinB3) was detected in multiple leukemia cell lines but not in normal samples by bisulfite pyrosequencing. In ALL patient samples, the frequencies of DNA methylation detected in the promoter regions of these genes ranged from 23% to 87% for EPHA4, EPHA5, EPHA6, EPHB2, EPHB3, EPHB4, EphrinA5, Ephrin B2, and EphrinB3. Expression analysis of 3 of these genes (EPHA5, EPHB4 and Ephrin B2) in leukemia cell lines by real-time PCR further confirmed methylation associated gene silencing. Treatment of methylated/silenced cell lines with DNA methyltransferase inhibitor 5′-aza-2′-deoxycytidine resulted in gene re-expression. Forced overexpression of EPHB4 using a lentivirus transduction system in Raji cell lines resulted in decreased cell proliferation and adhesion-independent cell growth, as well as in an increase in staurosporine induction of apoptosis. In addition, EPHB4 overexpression resulted in a significant downregulation of phosphorylated Akt pathway but had no effect on mitogen-activated protein kinase pathway. In summary, we describe for the first time the epigenetic suppression of Ephrin receptors and their ligands in human leukemia, indicating that these genes may be potential tumor suppressors in leukemia. Targeting of these pathways may result in the development of new potential therapies and biomarkers for patients with ALL.

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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 ◽

10.1182/blood.v104.11.1542.1542 ◽

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.

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

Blood ◽

10.1182/blood.v110.11.2127.2127 ◽

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.

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