Attenuation of Apoptosis Stimulating Protein of p53-1 (ASPP1) Associates with Therapy Failure in Acute Myeloid Leukemia (AML)

Abstract ASPP1 belongs to a family of p53-binding proteins and enhances apoptosis by stimulation of p53-transactivation of selected proapoptotic target genes. It is preferentially expressed in hematopoietic stem cells (HSC) and together with p53 preserves the genomic integrity of the HSC pool. Consequently, attenuated expression of ASPP1 which is linked to methylation of the promoter region has been associated with malignant transformation and development of acute lymphoblastic leukemia and lymphomas. We now provide evidence that ASPP1 is highly altered in AML suggesting a role in leukemogenesis as well as therapy response. ASPP1 mRNA and protein expression levels of freshly isolated native patient samples (68) and healthy bone marrow donors (29) were determined by qRT-PCR and western immunoblotting. Statistical analyseswere performed. To explore implications of attenuated ASPP1 levels with regard to apoptosis induction and proliferation, ASPP1-expressing leukemia cell lines (MOLM14, Jurkat), native patient blasts or native bone marrow donor samples were stably silenced using a retroviral shRNA approach. Vice versa, ASPP1 was stably overexpressed in AML cell lines expressing per se low ASPP1 levels. Expression was thereby confirmed by qRT-PCR and western blotting. XTT viability and annexin V-based apoptosis assays were performed using standard chemotherapeutics in comparison to empty vector controls. Decitabine was used as an epigenetic sensitizer via hypomethylation of the promoter region. ASPP1 mRNA expression was found to be frequently and highly statistically significantly (p=0.001) attenuated in AML. Low ASPP1 mRNA levels thereby translated into attenuated protein expression. Retroviral ASPP1-interference lead to perturbed proliferation capacities (up to 3-fold increase) and attenuated apoptosis upon standard chemotherapeutics in leukemia cell lines as well as native leukemia blasts. As expected, overexpression of ASPP1 resulted in significantly attenuated proliferation and higher induction of apoptosis in all tested cell lines and patient blasts. Intriguingly, epigenetic therapy using the hypomethylating agent decitabine resulted in upregulation of ASPP1 expression in leukemia cells with originally low basal ASPP1 levels as confirmed by qRT-PCR and western blotting. Consequently, decitabine pretreatment sensitized these patient samples towards chemotherapy with a favorable proapoptotic overall efficacy compared to chemotherapy alone. Our results demonstrate that dysfunctional regulation of ASPP1 expression likely contributes to the biology of leukemogenesis and to primary therapy resistance in a subgroup of patients with acute leukemia and seems to be linked to hypermethylation. Prospective clinical studies are warranted to evaluate the roleas a biomarker for risk stratification in leukemia patients and for monitoring therapy responses. Disclosures No relevant conflicts of interest to declare.

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ARHGAP21 Is Overexpressed in Bone Marrow of Acute Leukemia Patients and Interacts with Focal Adhesion Kinase (FAK) in Hematopoietic Cells.

Blood ◽

10.1182/blood.v108.11.4450.4450 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4450-4450

Author(s):

Fabiola Traina ◽

Sheila M.B. Winnischofer ◽

Luciene Borges ◽

Mariana Lazarini ◽

Carolina L. Bigarella ◽

...

Keyword(s):

Bone Marrow ◽

Cell Adhesion ◽

Acute Leukemia ◽

Mrna Expression ◽

Focal Adhesion Kinase ◽

Cell Lines ◽

Western Blotting ◽

Focal Adhesion ◽

Leukemia Cell ◽

Leukemia Cell Lines

Abstract ARHGAP21 is a new protein recently described by us and characterized as a Rho-GTPase activating protein (Rho-GAPs), a negative regulator of RhoGTPase signaling pathways. RhoGTPases mediate many aspects of cell biology, including proliferation, apoptosis, survival, adhesion and actin cytoskeleton dynamics. Recently, ARHGAP21 was identified as a new component of cell-cell junctions that controls α-catenin recruitment and is able to interact with α-catenin, ARF1 and ARF6, important proteins in the cytoskeleton assembly and adherent junctions. Cell-to-cell adhesion is one of the major factors that restrict cellular invasion and metastasis in malignant diseases. In the attempt to verify the role of ARHGAP21 in leukemogenesis, we aimed to verify the expression level of ARHGAP21 mRNA and protein in bone marrow samples from adults with diagnosis of acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL). In addition, we attempted to verify a possible interaction of ARHGAP21 with proteins involved in cell adhesion, as Focal adhesion kinase (FAK). FAK is a non-receptor tyrosine kinase, and coordinates signals from integrins, cytokines, growth factor receptors, and oncogenes. In AML cells, FAK has been found to be overexpressed and associated with enhanced blast migration, increased cellularity, and poor prognosis. The National Ethical Committee Board approved the study and informed-written consent was obtained from all subjects. Quantitative real-time PCR analysis was performed using specific primers to ARHGAP21 transcript, normalized by β-actin control. ARHGAP21 mRNA expression was found to be significantly higher in AML (n = 37) and ALL (n = 9) samples when compared with normal hematopoietic cells (n= 5) (medians; AML: 3.01 vs 0.25, P < 0.0001; ALL: 4.47 vs 0.25, P = 0.0010; Mann-Whitney test). ARHGAP21 mRNA expression showed a positive correlation with bone marrow blast cell counts (Spearman test, P = 0.0362). Western blotting analysis showed higher ARHGAP21 expression in acute leukemia cell lines: Jurkat, Molt-4, K562 and HL60 in comparison to normal peripheral blood mononuclear cells (PBMC). Immunoprecipitation and Western blotting assays using anti-ARHGAP21 and anti-FAK antibodies detected the interaction between ARHGAP21 and FAK in protein extracts of Jurkat cells and normal PBMC. Pull down assays using three different FAK-GST fusion proteins: C-terminal (residues 687–1054), FERM domain (residues 60–349) and catalytic domain (residues 390–696) in protein cell lysates of HL-60 and normal PBMC showed that ARHGAP21 is associated with the C-terminal region of FAK. In conclusion, our data show that ARHGAP21 is overexpressed in AML and ALL cells and is associated with FAK in leukemia cell lines and in normal PBMC. These findings give rise to the hypothesis that ARHGAP21 may be involved in leukemogenesis, aiming this gene as a candidate for anti-tumor therapy.

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c-kit expression in human megakaryoblastic leukemia cell lines

Blood ◽

10.1182/blood.v83.8.2133.2133 ◽

1994 ◽

Vol 83 (8) ◽

pp. 2133-2144 ◽

Cited By ~ 27

Author(s):

ZB Hu ◽

W Ma ◽

CC Uphoff ◽

H Quentmeier ◽

HG Drexler

Keyword(s):

Growth Factors ◽

Cell Lines ◽

Leukemia Cell ◽

Northern Blotting ◽

Lymphoma Cell ◽

Mrna Levels ◽

Bryostatin 1 ◽

Rt Pcr ◽

Megakaryoblastic Leukemia ◽

Leukemia Cell Lines

Abstract A panel of 164 continuous human leukemia-lymphoma cell lines was analyzed for expression of c-kit using Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). The c-kit transcripts were detectable in cell lines assigned to the myeloid (in 7 of 29 by Northern blotting and in 4 of 8 by RT-PCR), monocytic (in 1 of 24 by Northern blotting and in 3 of 6 by RT-PCR), erythroid (in 6 of 8 by Northern blotting and in 5 of 5 by RT-PCR), and megakaryoblastic (in 10 of 10 by Northern blotting) lineages, c-kit expression was not seen by Northern blotting or RT-PCR analysis in any of the 93 lymphoid leukemia, myeloma, or lymphoma cell lines. Treatment of four megakaryoblastic cell lines with protein kinase C activators (phorbol ester 12-O-tetradecanoylphorbol 13-acetate and Bryostatin 1) led to terminal differentiation as assessed by morphologic alterations, changes in the surface marker profile, and growth arrest. These effects were associated with enhanced c-kit mRNA expression. Exposure to all- trans retinoic acid down-regulated c-kit mRNA levels, while simultaneously causing morphologic alterations in all four cell lines. Stimulation with growth factors (interleukin-3, granulocyte macrophage- colony stimulating factor, and insulin-like growth factors I and II), used to assess any role of c-kit in proliferative processes, did not lead to significant upregulation or downregulation of c-kit expression. The finding of constitutive and high expression of c-kit mRNA in all megakaryoblastic leukemia cell lines and its modulation by various reagents might further contribute to the understanding of megakaryopoietic proliferation, differentiation, and leukemogenesis.

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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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Stimulation of TNF-α Production from Monocytes by Anti-Moesin Antibodies in the Serum of Patients with Aplastic Anemia: A Novel Mechanism of Myelosuppression by Autoantibodies.

Blood ◽

10.1182/blood.v108.11.976.976 ◽

2006 ◽

Vol 108 (11) ◽

pp. 976-976

Author(s):

Hiroyuki Takamatsu ◽

Xingmin Feng ◽

Xuzhang Lu ◽

Tatsuya Chuhjo ◽

Katsuya Okawa ◽

...

Keyword(s):

Cell Lines ◽

Western Blotting ◽

Blood Cells ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Surface Proteins ◽

Healthy Individuals ◽

Monocytic Leukemia ◽

Leukemia Cell Lines ◽

Tnf Α

Abstract Although aplastic anemia (AA) is a T-cell mediated disease, recent studies have revealed the presence of antibodies (Abs) specific to proteins derived from hematopoietic progenitor cells in the serum of AA patients. It is as yet unclear whether these auto-Abs play some roles in the pathophysiology of AA. We previously demonstrated that Abs specific to moesin, a membrane-cytoskeleton linker protein in the cytoplasm, were detectable in approximately 37% of AA patients. Some reports identified moesin-like molecules on the surface of blood cells such as T cells and macrophages. It is therefore conceivable that anti-moesin Ab in AA patients may bind these immune cells and modulate hematopoietic function of AA patients. To test these hypotheses, we first studied the expression of moesin on various types of blood cells using monoclonal Ab specific to moesin (clone 38/87). Flow cytometry detected the expression of the protein recognized by anti-moesin Ab on T cells and monocytes from healthy individuals, acute monocytic leukemia cells lines including U937 and THP-1, and an acute T-lymphoblastic leukemia cell line, Molt-4, but failed to detect the molecule on CD34+ cells from healthy individuals and myeloid leukemia cell lines as well as B-lymphoblastic leukemia cell lines. Treatment of THP-1 cells with phorbol 12-myristate 13-acetate (PMA)/lipopolysaccharide (LPS) augmented the expression level of moesin. To confirm the expression of the moesin-like protein on the surface of monocytic leukemia cell lines, Molt-4 and THP-1 were treated with sulfo-NHS-SS-biotin, and the cell surface proteins were isolated with avidin-fixed column, and were subjected to Western blotting and peptide mass fingerprinting. Western blotting with anti-moesin monoclonal Abs showed two clear bands of proteins (75 kD and 80 kD); an amino acid sequence compatible with moesin was confirmed in the protein eluted from the 80 kD band. Next, we purified anti-moesin Abs from AA patients’ sera using affinity chromatography with recombinant moesin protein. Western blotting showed binding of the serum-derived Abs to a fraction of surface proteins of Molt-4, U937 and THP-1. When THP-1 cells were incubated in the presence of PMA and LPS with 5 αg/ml of control IgG or anti-moesin Abs derived from an AA patient’s serum, TNF-α production from THP-1 cells stimulated by anti-moesin Abs was 1.9–2.3 times as much as that from the control culture depending on the concentration of LPS. Incubation of THP-1 cells in the presence of monoclonal anti-moesin Abs showed the similar augmentation of TNF-α production. These results indicate that anti-moesin Abs may be involved in the suppression of hematopoiesis of AA patients by stimulating TNF-α production from monocytes.

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Targeting the Mnk Pathway Enhances Chemotherapy-Induced Antileukemic Responses.

Blood ◽

10.1182/blood.v112.11.1617.1617 ◽

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.

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RASSF2 Functions As a Tumor Suppressor in t(8;21) AML Via Promotion of Apoptosis

Blood ◽

10.1182/blood.v124.21.3590.3590 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3590-3590

Author(s):

Samuel A Stoner ◽

Russell Dekelver ◽

Miao-Chia Lo ◽

Dong-Er Zhang

Keyword(s):

Gene Expression ◽

Tumor Suppressor ◽

Cell Lines ◽

Hematopoietic Cells ◽

Leukemia Cell ◽

Leukemia Cells ◽

Mrna Levels ◽

Human Cd34 ◽

Leukemia Cell Lines ◽

Cd34 Cells

Abstract The t(8;21) chromosomal translocation is one of the most common chromosomal translocations associated with acute myeloid leukemia (AML), found in approximately 12% of de novo AML cases. The majority of these cases are classified as FAB-subtype M2 AML. The t(8;21) results in the stable fusion of the AML1 (RUNX1) and ETO (RUNX1T1) genes. The AML1-ETO fusion protein is composed of the N-terminal portion of AML1, which includes the DNA-binding Runt-homology domain, and nearly the full-length ETO protein. The primary accepted mechanism by which AML1-ETO promotes leukemia development is through the aberrant recruitment of transcriptional repression/activation complexes to normal AML1 target genes. Therefore, the identification of individual genes or biological pathways that are specifically disrupted in the presence of AML1-ETO will provide further molecular insight into the pathogenesis of t(8;21) AML and lead to the possibility for improved treatment for these patients. We identified RASSF2 as a gene that is specifically downregulated in (2-4 fold) in total bone marrow of t(8;21) patients compared to non-t(8;21) FAB-subtype M2 AML patients by analyzing publicly available gene expression datasets. Similarly, using a mouse model of t(8;21) AML we found Rassf2 mRNA levels to be nearly 30-fold lower in t(8;21) leukemia cells compared to wild-type Lin-Sca-cKit+ (LK) myeloid progenitors. Gene expression analysis by RT-qPCR in leukemia cell lines confirmed that RASSF2 mRNA levels are significantly downregulated (8-10-fold) in both Kasumi-1 and SKNO-1 t(8;21) cell lines as compared to a similar non-t(8;21) HL-60 cell line and to primary human CD34+ control cells. In addition, expression of AML1-ETO in HL-60 or CD34+ cells results in a decrease in RASSF2 mRNA expression, which further suggests that RASSF2 is a target for regulation by AML1-ETO. Assessment of published ChIP-seq data shows that AML1-ETO binds the RASSF2 gene locus at two distinct regions in both primary t(8;21) AML patient samples and in the Kasumi-1 and SKNO-1 cell lines. These regions are similarly bound by several important hematopoietic transcription factors in primary human CD34+ cells, including AML1, ERG, FLI1, and TCF7L2, implicating these two regions as important for the regulation of RASSF2 expression during blood cell differentiation. Overexpression of RASSF2 in human leukemia cell lines using an MSCV-IRES-GFP (MIG) construct revealed that RASSF2 has a strong negative effect on leukemia cell proliferation and viability. The overall percentage of GFP-positive cells in MIG-RASSF2 transduced cells markedly decreased compared to MIG-control transduced cells over a period of 14 days. This effect was primarily due to significantly increased apoptosis in the RASSF2 expressing cell populations. Similarly, we found that expression of RASSF2 significantly inhibits the long-term self-renewal capability of hematopoietic cells transduced with AML1-ETO in a serial replating/colony formation assay. AML1-ETO transduced hematopoietic cells were normally capable of serial replating for more than 6 weeks. However, AML1-ETO transduced cells co-expressing RASSF2 consistently had reduced colony number and lost their ability to replate after 3-4 weeks. This was due to a dramatically increased rate of apoptosis in RASSF2 expressing cells. RASSF2 is reported to be a tumor suppressor that is frequently downregulated at the transcriptional level by hypermethylation in primary tumor samples, but not healthy controls. Here we have identified RASSF2 as a target for repression, and demonstrated its tumor suppressive function in t(8;21) leukemia cells. Further insights into the molecular mechanisms of RASSF2 function in AML will continue to be explored. Disclosures No relevant conflicts of interest to declare.

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RUNX1/CBFβ Dosage Is Critical for MLL Leukemias Development

Blood ◽

10.1182/blood.v124.21.2187.2187 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2187-2187

Author(s):

Xiaomei Yan ◽

Yoshihiro Hayashi ◽

Xinghui Zhao ◽

Aili Chen ◽

Yue Zhang ◽

...

Keyword(s):

Protein Expression ◽

Cell Lines ◽

Research Funding ◽

Leukemia Cell ◽

Down Regulation ◽

Leukemia Cell Lines ◽

Flanking Regions ◽

The Impact

Abstract Transcription factors RUNX1/CBFβ play critical roles in hematopoiesis. Both of them are frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. We have previously shown that MLL, RUNX1, and CBFβ interact and form a regulatory complex to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFβ activity remains unknown. To determine the impact of MLL fusion protein on RUNX1/CBFβ, we introduced either MLL, MLL-BP (longer N-terminal Flag-tagged MLL construct which contains CXXC domain; 1-1406), or MLL-fusions together with RUNX1, CBFβ, or both RUNX1 and CBFβ into 293T cells. MLL-BP and MLL fusions significantly decreased RUNX1 levels compared with controls (empty vector and MLL). CBFβ protein was mildly decreased by MLL-BP and MLL-fusions when expressed alone. However, when CBFβ was co-expressed with RUNX1, it was significantly decreased compared with controls. The expression levels of RUNX1 and CBFβ proteins in LSK cells from Mll-Af9 knock-in mice were significantly lower than those from wild-type (WT) mice. To confirm these findings in human acute myeloid leukemia (AML), we measured the expression of RUNX1 and CBFβ at both mRNA and protein levels in various leukemia cell lines. The expression levels of RUNX1 and CBFβ proteins were significantly decreased in AML cells with MLL fusion and MLL partial tandem duplication (MLL-PTD) compared with those in AML cells without MLL aberrations. MLL fusions still have CXXC domain. In MLL-PTD, the CXXC domain is duplicated. Our data showed that RUNX1 protein is not only down-regulated by MLL fusion proteins, but also by MLL-BP. Thus, to determine which region is involved in the down-regulation of RUNX1, we introduced a series of MLL deletion mutants into 293T cells and measured RUNX1 protein expression. MLL deletion mutants without CXXC domain had no effect on RUNX1 stability. The construct which contains point mutations in CXXC domain also lacked the ability to reduce RUNX1 expression. Furthermore, overexpression of only CXXC domain and flanking regions could down-regulate RUNX1 protein expression. These results suggest that MLL fusion proteins and the N-terminal MLL portion of MLL fusions down-regulate RUNX1 and CBFβ protein expression via the MLL CXXC domain and flanking regions. To understand the impact of RUNX1/CBFβ down-regulation on hematopoietic stem and progenitor cells (HSPCs), we generated RUNX1+/–/CBFβ+/– mice as a hypomorph model. The percentage of bone marrow (BM) LSK cells from RUNX1+/–/CBFβ+/– mice was significantly increased compared with that from WT mice. Using BM cells from these mice, we performed in vitro CFU assay and in vivo bone marrow transplantation (BMT) assay. BM cells from RUNX1+/–/CBFβ+/– mice provided more colonies in CFU assay compared with those from WT mice. To determine whether restoration of RUNX1 could repress the MLL mediated leukemogenesis, we retrovirally overexpressed WT RUNX1 in BM cells from Mll-Af9 knock-in mice. Using transduced BM cells, we performed in vitro CFU assay and in vivo BMT assay. RUNX1 overexpressed Mll-Af9 (Mll-Af9/RUNX1) cells underwent terminal differentiation after 2 times replating, while control vector transduced Mll-Af9 (Mll-Af9/Control) cells could still be replated more than 4 times. All the recipient mice transplanted with Mll-Af9/Control cells developed AML. In contrast, all the recipient mice transplanted with Mll-Af9/RUNX1 never develop AML. Furthermore, when we treated MLL leukemia cell lines with DOT1L inhibitor (EPZ-5676), RUNX1 protein levels in these MLL leukemia cell lines were significantly increased 48 hours after the treatment in comparing with controls treated with DMSO. However, there was no significant mRNA expression level change of RUNX1within 48 hours. Future studies are needed to fully understand the mechanism of whether this increasing RUNX1 protein level by DOT1L inhibitor is through blocking CXXC domain and flanking regions mediated degradation. In conclusion, MLL aberrations down-regulate RUNX1/CBFβ via their CXXC domain and flanking regions. Down-regulation of RUNX1/CBFβ plays critical role for MLL mediated leukemia development. Targeting RUNX1/CBFβ levels allows us to test novel therapies for MLL leukemias. Disclosures Mulloy: Celgene: Research Funding; Seattle Genetics: Research Funding; Amgen: Research Funding; NovImmune: Research Funding.

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MicroRNA (miRNA)-29b Targets DNMT3A and B and Induces Re-Expression of the Hypermethylated ESR1 and p15 Genes in Acute Myeloid Leukemia (AML).

Blood ◽

10.1182/blood.v110.11.718.718 ◽

2007 ◽

Vol 110 (11) ◽

pp. 718-718

Author(s):

Ramiro Garzon ◽

Shujun Liu ◽

Muller Fabbri ◽

Zhongfa Liu ◽

Jiuxia Pang ◽

...

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Cell Lines ◽

K562 Cells ◽

Luciferase Reporter ◽

Aberrant Methylation ◽

Mrna Levels ◽

Down Regulation ◽

Functional Link ◽

Qrt Pcr

Abstract Aberrant DNA methylation has been shown to play an important role in AML by silencing structurally normal genes important for hematopoiesis. MiRNAs are small non-coding RNAs that down-regulate gene expression. Although aberrant methylation of miRNAs has been reported, it is not known whether miRNAs themselves can modulate DNA methylation. To address this question, we tested the functional role of miRNAs that are predicted to target both DNA methyltransferase 3A and 3B (DNMT3A and B), in AML cells. We cloned the 3′UTR of both DNMT3s into a luciferase reporter and co-transfected K562 cells with scrambled or sense sequences of each of the miRNAs predicted to interact with DNMT3A and B. We found that miR-29b significantly repressed luciferase activity of both DNMT3A and B constructs (42% and 67%, respectively) compared with the controls. Transfection of the pre-miR-29b, but not of the scrambled, resulted in a marked reduction of the DNMT3A and B mRNA levels measured by qRT-PCR in MV4-11 cells (80% and 90%, respectively) and in K562 cells (23% and 67% respectively).We also observed a significant down-regulation of DNMT3s protein by immunoblotting in K562 cells transfected with miR-29b relative to the controls. These results were then validated in AML patients. Both miR-29b and DNMT3B expression levels were measured by qRT-PCR in bone marrow samples obtained from 12 AML patients with primary disease. We found that miR-29b expression was inversely correlated to DNMT3B mRNA levels (Pearson coefficient R=−0.66; P=0.03). Functionally, we showed that the mRNA expression of p15 and ESR1, that are silenced through promoter hypermethylation in AML, were increased in K562 and MV4-11 cells transfected with miR-29b compared with the controls. ESR1 was 1.9 and 1000 fold upregulated in miR-29b-tranfected K562 and MV4-11cells, respectively and p15 was 11.6 fold upregulated in miR-29b-tranfected MV 4–11 cells. Interestingly, we observed that the expression of miR-29b-1, which is silenced in K562 cell lines, was 6.5 fold up-regulated following treatment with 2.5 uM decitabine, a DNA hypomethylating nucleoside compared with untreated controls. Consistent with these results, we also showed 5.6 fold increase in miR-29b-1 expression in post-treatment CD34+ selected bone marrow blasts from AML patients (n=3) who received decitabine (20 mg/m2/day x 10 days) on a clinical protocol (OSU 0336) compared with pretreatment baselines. These results were consistent with the methylation analysis of CpGs up to 30kb upstream from the 5′ encoding regions of the miR-29b-1 precursors in the same patients. Using MassArray (sequenom) technology, we showed that all the CpGs were highly methylated (95 to 100%) in pre-treatment baseline samples and these methylation levels decreased by 20% after decitabine treatment. These preliminary data provide a functional link between miRNAs and aberrant epigenetics by suggesting that: miR-29b targets DNMT3A and B; miR-29b is silenced in AML cell lines and patient primary blasts and its expression is restored in vitro and in vivo by ypomethylating agents; 3) increase of miR-29b levels results in DNMTs down-regulation and re-expression of otherwise hypermethylated genes. Altogether, there data support a rationale for developing novel miRNA-based therapeutic strategies that alone and in combination with other hypomethylating agents may target aberrant epigenetics in AML.

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

Blood ◽

10.1182/blood.v116.21.3635.3635 ◽

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.

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CREB: A Key Regulator of Normal and Neoplastic Hematopoiesis

Advances in Hematology ◽

10.1155/2009/634292 ◽

2009 ◽

Vol 2009 ◽

pp. 1-8 ◽

Cited By ~ 18

Author(s):

Salemiz Sandoval ◽

Martina Pigazzi ◽

Kathleen M. Sakamoto

Keyword(s):

Bone Marrow ◽

Transgenic Mice ◽

Cell Lines ◽

Myeloid Leukemia ◽

Cellular Proliferation ◽

Leukemia Cell ◽

Cellular Transformation ◽

Camp Response Element Binding ◽

Lymphoid Leukemia ◽

Leukemia Cell Lines

The cAMP response element-binding protein (CREB) is a nuclear transcription factor downstream of cell surface receptors and mitogens that is critical for normal and neoplastic hematopoiesis. Previous work from our laboratory demonstrated that a majority of patients with acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) overexpress CREB in the bone marrow. To understand the role of CREB in leukemogenesis, we examined the biological effect of CREB overexpression on primary leukemia cells, leukemia cell lines, and CREB overexpressing transgenic mice. Our results demonstrated that CREB overexpression leads to an increase in cellular proliferation and survival. Furthermore, CREB transgenic mice develop a myeloproliferative disorder with aberrant myelopoiesis in both the bone marrow and spleen. Additional research from other groups has shown that the expression of the cAMP early inducible repressor (ICER), a CREB repressor, is also deregulated in leukemias. And, miR-34b, a microRNA that negative regulates CREB expression, is expressed at lower levels in myeloid leukemia cell lines compared to that of healthy bone marrow. Taken together, these data suggest that CREB plays a role in cellular transformation. The data also suggest that CREB-specific signaling pathways could possibly serve as potential targets for therapeutic intervention.

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