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.

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.

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

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2128-2128 ◽  
Author(s):  
Shao-qing Kuang ◽  
Zhi-Hong Fang ◽  
Gonzalo Lopez ◽  
Weigang Tong ◽  
Hui Yang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Eph Receptor ◽  
Leukemia Cell Lines ◽  
Ephrin Ligands ◽  
Aberrant Dna Methylation

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.

scholarly journals The Oligodendrocyte Lineage Transcription Factor 2 (OLIG2) Is Epigenetically Regulated in AML and Suppresses Leukemia Cell Growth

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3540-3540
Author(s):  
Arzu Yalcin ◽  
Marlon Kovarbasic ◽  
Mahmoud Abdelkarim ◽  
Gregor Klaus ◽  
Julius Wehrle ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factor ◽  
Cell Growth ◽  
Protein Expression ◽  
Mrna Expression ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Inverse Correlation ◽  
Leukemia Cell Lines ◽  
The Impact

Abstract Introduction: DNA methylation differences between normal and cancer tissue that result in differential expression of genes are a hallmark of acute myeloid leukemia (AML). DNA methylation mediated silencing of specific genes, especially transcription factors, can provide a growth advantage for malignant cells. Global DNA methylation analyses have not only led to a better understanding of AML subgroups and the impact of epigenetic aberrations in leukemogenesis, but also to the identification of new epigenetically regulated genes. We and others have recently identified the oligodendrocyte lineage transcription factor 2 (OLIG2) as differentially methylated in AML cell lines compared with normal bone marrow cells. Aim of the study: With the hypothesis that OLIG2, which is not expressed in normal hematopoiesis, may play a role in cancerogenesis as shown for acute lymphoblastic leukemia (Lin et al., Cancer Res. 2005) and malignant glioma (Mehta et al., Cancer Cell 2011), we sought to further dissect the impact of OLIG2 in AML, implementing functional studies and primary samples. Results: First, in a cohort of 93 AML patients, we could confirm previous results by Kröger et al. (Blood 2008) that OLIG2 is differentially methylated: using pyrosequencing, 37 patients (39.8%) showed methylation levels > 25% (range (r): 26-79%) in the 5 CpG containing amplicon of the OLIG2 promoter region, independent of cytogenetic subgroup. In a small subset of 13 patients where expression-data was available, an inverse correlation between OLIG2 DNA methylation and mRNA expression was significant (r2=0.55, p<0.005). This observation was further supported by a highly significant inverse DNA methylation/mRNA expression correlation in 10 leukemia cell lines (r2=0.74, p< 0.002). Moreover, we could demonstrate that this inverse correlation held also true for OLIG2 protein levels in cell lines with strong expression in THP-1 and NB-4, moderate expression in HL-60 and HEL and no expression in U937, KG-1A, PL-21, Kasumi-1, K-562 and Jurkat. Interestingly, while CD 34+ cells from two healthy donors and 10 out of 12 AML patients where protein was available, showed no protein expression, OLIG2 was expressed in 2 patients, both bearing the translocation t(15;17). This corresponds well to OLIG2 expression of cell line NB-4, which also harbours t(15;17). Treatment of non-expressing cell lines PL-21 and U937 with 200 nM 5-aza-2'-deoxycytidine led to robust re-expression of OLIG2, both on mRNA and protein level, strongly implicating DNA methylation as a silencing mechanism in a subset of AML. To investigate the relationship between OLIG2 expression and AML cell growth we used a siRNA transient knock-down in OLIG2 expressing cell lines THP-1 and NB-4. While OLIG2 protein expression measured via densitometry could be strongly reduced to 38% and 45% from pre-treatment levels in THP-1 and NB-4 cells, respectively, no change on cell viability or cell growth was detected. However, stable over-expression of OLIG2 using the lentiviral-vector pLeGO-iG in Kasumi-1 cells, led to a significant growth-inhibition of 32.2% (r: 27.0-37.3%) after 5 days and a 47.7% (r: 30.7-64.6%) increase of apoptotic cells (Annexin-V-staining) as compared to control-vector transfected cells. This negative effect on cell proliferation supports our presumption that OLIG2 could act as a growth-regulator in a subgroup of AML. This could be caused by a direct interaction between OLIG2 and a cell cycle regulator or a transcription factor complex. Conclusion: We show that OLIG2 (I) is in part epigenetically regulated via DNA methylation in AML, resulting in an inverse correlation between DNA methylation and expression; (II) can be re-expressed upon demethylating treatment in cell lines, therefore making it an attractive biomarker to study in AML patients treated with demethylating agents; (III) shows antiproliferative activity in leukemia cell lines and thus should be further studied as a potential tumor suppressor in AML. Disclosures Lübbert: Cephalon / TEVA: Travel support Other.

XmAb™ Fc Engineered Anti-CD19 Monoclonal Antibody with Enhanced In Vitro Efficacy Against Lymphoma and Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2335-2335
Author(s):  
Eugene Zhukovsky ◽  
Seung Chu ◽  
Matthew Bernett ◽  
Sher Karki ◽  
John Richards ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Affinity Maturation ◽  
Effector Function ◽  
Lymphocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Cell Surface Receptor ◽  
Leukemia Cell Lines

Abstract CD19 is a pan-B cell surface receptor expressed from early stages of pre-B cell development through terminal differentiation into plasma cells. It is an attractive target for cancers of lymphoid origin since it is expressed on the vast majority of Non-Hodgkin Lymphoma (NHL) as well as different types of leukemia, including those (e.g. pediatric ALL) lacking CD20 expresion. The majority of NHL patients will relapse after the current treatment regimen of chemotherapy combined with rituximab (CHOP-R) despite major improvements in response rates and progression free survival. Thus salvage regimens with novel non-cross resistant antibody therapies are warranted. Here we present the characterization of a novel Fc-engineered and humanized anti-CD19 antibody (XmAb™ CD19) that was generated using our XmAb™ antibody engineering technologies. This novel antibody is highly cytotoxic against a panel of lymphoma and leukemia cell lines as well as primary cancer cells. The main features of this antibody are: increased affinity for Fc gamma receptors (FcgR), improved effector function, and significantly increased antitumor potency. Humanization and affinity maturation technologies were applied to this antibody in order to: decrease immunogenicity, increase affinity, and increase stability of the engineered antibody. Since internalization is expected to impact a naked antibody’s effector functions, we assayed its internalization rate using Eu-labeled XmAbCD19 and observed an unexpectedly low rate of internalization. Therefore, we proceeded to investigate several direct and indirect (Fc-mediated) mechanisms of antibody-mediated cytotoxicity. The potency of XmAbCD19 in antibody-dependent cell-mediated cytotoxicity (ADCC) increased 10- to 100-fold relative to a native/non Fc-engineered version (CD19-IgG1) of the antibody in a screen of 16 NHL and leukemia cell lines (chronic lymphocytic leukemia [CLL], B-cell acute lymphoblastic leukemia [B-ALL], hairy cell leukemia [HCL], follicular lymphoma [FL], mantle cell lymphoma [MCL], chronic myelogenous leukemia [CML], and Burkitt’s lymphoma [BL]). ADCC potency (EC50) and efficacy (% Lysis) of the Fc-engineered anti-CD19 antibody were superior to that of rituximab: in CLL - 10- and 1.5-fold higher, in B-ALL - 10- and 100-fold higher, and in HCL - 6- and 1.2-fold higher, respectively; unlike XmAbCD19 native CD19-IgG1 mediated little ADCC. Moreover, XmAbCD19 mediated potent ADCC of primary patient-derived ALL cells that was also significantly increased in potency and efficacy relative to rituximab and CD19 IgG1. Furthermore, XmAbCD19 demonstrated 50-fold increased antibody-dependent cellular phagocytosis (ADCP) relative to CD19-IgG1. Finally, XmAbCD19 exhibited robust anti-proliferative activity that was 10-fold more potent than that of rituximab. In order to facilitate preclinical toxicology studies with XmAbCD19, we assayed its cross-reactivity with non-human primate (NHP) species CD19 (cynomolgus and rhesus monkeys). In contrast to the parental murine antibody, the affinity-optimized humanized XmAbCD19 reacted with B cells from both NHP species. In summary, our data suggest that our anti-CD19 Fc variant antibody engineered for increased effector function is a promising next-generation immunotherapeutic for a variety of leukemias and lymphomas.

Epigenetic Inactivation of Notch Signaling Target Genes HES in B Cell Acute Lymphoblastic Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3372-3372
Author(s):  
Shaoqing Kuang ◽  
Patrick Zweidler-McKay ◽  
Hui Yang ◽  
Zhi Hong Fang ◽  
Weigang Tong ◽  
...  
Keyword(s):  
Dna Methylation ◽  
T Cell ◽  
B Cell ◽  
Notch Signaling ◽  
Cell Lines ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cell Lines ◽  
Cell Acute Lymphoblastic Leukemia

Abstract The Notch signaling pathway has been implicated in multiple functions during normal hemato-lymphoid development. It also plays critical roles in T-cell leukemogenesis through influencing T-cell proliferation, differentiation and survival. In contrast, we have previously reported a tumor suppressor role in B-cell leukemias, where Notch signaling leads to growth inhibition and apoptosis. The Notch target genes Hairy/Enhancer of Split (HES1-7) encode transcriptional repressors with basic helix-loop-helix (bHLH) domains. Functional and phenotypic analyses of some of the HES family members have been reported, however, expression and epigenetic regulation of the HES family in leukemia is largely unknown. Using Methylated CpG Island Amplification (MCA) / DNA promoter microarray, we identified several HES family genes as hypermethylated in B cell acute lymphoblastic leukemia (B ALL). We further investigated the comprehensive methylation profiles of HES family genes in a panel of leukemia cell lines and ALL patient samples by bisulfite pyrosequencing. Aberrant DNA methylation of HES2, HES4, HES5 and HES6 was detected in most B ALL cell lines including B-JAB, RS4:11, REH, Raji and Ramos but not in normal B cell controls. In contrast, in T cell leukemia cell lines such as Molt4, PEER, T-ALL1 and J-TAG, these genes were generally unmethylated. In B ALL patient samples, the frequencies of DNA methylation in the promoter regions of these genes were 25% for HES2, 50% for HES4, 76% for HES5 and 71% for HES6. Expression analysis of HES4, HES5 and HES6 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 HES gene re-expression. Finally, forced re-expression of HES5 and HES6 in methylation silenced Rs4 and REH cell lines inhibited cell growth. These results suggest that the Notch/HES signaling pathway is epigenetically-inactivated in B ALL. These data support the role of the HES family as tumor suppressors in pre-B ALL and establish epigenetic modulation as a novel mechanism of Notch pathway regulation. We anticipate that therapies capable of activating Notch/HES signaling may have therapeutic potential in B cell leukemias.

Sign in / Sign up

Export Citation Format

Share Document