Expression Profile of Transcription Factor ELK-1 and ELK-1 Target Genes on Lymphoma-Leukemia Cell Lines

Abstract Abstract 4649 MicroRNAs(miRNAs) are short non-coding RNAs that are involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs. Recent studies revealed that genetic exchange of miRNAs between cells can be accomplished through microvesicles(MVs),which are small exosomes/vesicles of endocytic origin shed from the surface membranes of activated platelets and normal or malignant cells. Generally, MVs are enriched in various bioactive molecules of their parental cells, such as proteins, DNA, mRNA and miRNAs. So, exploration of changes of the miRNAs profiles of MVs derived from leukemia cells will contribute to a better understanding of the pathogenesis of leukemias and then provide valuable suggestions about how to diagnose, prevent and control leukemia. In the present study,we used Agilent miRNA microarray to analyze the miRNAs of MVs derived from three different leukemia cell lines (K562, Nalm-6 and Jurkat) and their cell counterparts, and normal plasma derived MVs as normal control. The significantly differentially expressed miRNAs between test group and normal control group were determined by t-test(P<0.05 and Fold Change>2).The putative target genes of the differentially expressed miRNAs were predieted by Targetscan. We observed that 125 miRNAs (93 up-regulated and 32 down-regulated) were determined to be differentially expressed in MVs derived from three leukemia cell lines compared with their normal control counterparts. Four miRNAs including miR-1290, miR-1268,miR-1246 and miR-1305 were found to be hundreds fold alteration. In addition, let-7f, miR-26a, miR-26b and miR-223 were significantly under expressed. We next found that MVs derived from lymphocytic leukemia cell lines(Nalm-6 and Jurkat) shared 100 miRNAs of 888 miRNAs, 99 upregulated and only one miRNA downregulated. Meanwhile, 44 miRNAs were just altered in Nalm-6-MVs, 9 miRNAs are just altered in Jurkat-MVs. Moreover, we found 22 miRNAs were only altered in K562-MVs, only one miRNAs(miR-191) altered in lymphocytic-MVs. We also found that MVs miRNA profiles were significantly different, compared the MVs derived from three leukemia cell lines with their cell counterparts. K562 cells and K562 MVs shared 112 miRNAs. 122 miRNAs were only altered in cells and 77 miRNAs were only altered in their MVs. Similarly, Nalm-6 cells and nalm-6 MVs shared 154 miRNAs, and 9 miRNAs were only altered in cells and 102 miRNAs were only altered in their MVs. Jurkat cells and Jurkat MVs shared 111 miRNAs, and 15 miRNAs were only altered in cells and 95 miRNAs were only altered in their MVs. The results of real-time qRT-PCR consisitanted with that observed in the microarray assays. We identified target genes of these differently expressed miRNAs by TargetScan, and retrievaled recent literature on the properties and biogenesis of these aberrant miRNAs and their potential role in cancer progression. Differentially expressed miRNAs include known oncomirs (e.g miR-96) as well as miRNAs that were not previously universally associated with cancer. Specific examples include let-7f,miR-191 and miR-21, which were consistently down regulated and miR-223 which is consistently up regulated in cancer, in the context of our cohort. Furthermore, miR-1246 mediates the functions of p53 family members, and BCL-2,KRAS may be target of miR-1305. In summary,we firstly used the miRNA microarray to seek for miRNAs with differential expression in MVs derived from K562, Nalm-6 and Jurkat cells. Predicting the putative target genes of the miRNAs with bioinformatic softwares may play a foundation for further studies exploring the biomolecular mechanism of oncogensis and development of leukemia and searching for related biomolecular markers of diagnosis and treatment in leukemia. Disclosures: No relevant conflicts of interest to declare.

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Transcription factor expression in B-cell precursor-leukemia cell lines: preferential expression of T-bet

Leukemia Research ◽

10.1016/j.leukres.2004.12.010 ◽

2005 ◽

Vol 29 (7) ◽

pp. 841-848 ◽

Cited By ~ 3

Author(s):

Akira Harashima ◽

Yoshinobu Matsuo ◽

Hans G. Drexler ◽

Ayumi Okochi ◽

Ryuichi Motoda ◽

...

Keyword(s):

Transcription Factor ◽

B Cell ◽

Cell Lines ◽

Leukemia Cell ◽

Cell Precursor ◽

Preferential Expression ◽

Leukemia Cell Lines ◽

Factor Expression ◽

Transcription Factor Expression

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Activation of C/EBPa Myeloid-Specific Transcription Factor By NAMPT/SIRT1-Triggered Deacetylation

Blood ◽

10.1182/blood.v126.23.2199.2199 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2199-2199

Author(s):

Bardia Samareh ◽

Masoud Nasri ◽

Inna Zimmer ◽

Olga Klimenkova ◽

Leonie Keller ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Transcription Factor ◽

Cell Lines ◽

Myeloid Leukemia ◽

Target Genes ◽

Leukemia Cell ◽

Myeloid Differentiation ◽

Granulocytic Differentiation ◽

Acute Myeloid ◽

Myeloid Leukemia Cell

Abstract Previously, we described new mechanism of G-CSF-triggered granulocytic differentiation of hematopoietic stem cells (HSCs) via activation of the enzyme Nicotinamide Phosphorybosyltransferase (NAMPT) leading to NAD+ production and activation of NAD+ -dependent protein deacetylase sirtuin 1 (SIRT1). We found, that upon stimulation of HSCs with NAMPT, SIRT1 bound to the key myeloid transcription factor C/EBPα followed by transcriptional induction of C/EBPα target genes G-CSFR and G-CSF and granulocytic differentiation. In the present work we investigated the mechanism of NAMPT/SIRT1-triggered deacetylation of C/EBPα. We found that C/EBPα is acetylated at the position Lys 161, which is evolutionarily conserved. Lys 161 is localized in the transactivation element III (TE-III) of the transactivation domain (TAD) of C/EBPα protein, which is responsible for recruitment of SWI/SNF and CDK2/CDK4. Western blot and DUOLINK analysis using rabbit polyclonal antibody specifically recognizing acetyl-Lys 161 of C/EBPα revealed predominantly nuclear localization of acetylated C/EBPα protein in acute myeloid leukemia cell lines NB4 and HL60 as well as in primary HSCs. Induction of myeloid differentiation of HSCs by treatment with G-CSF as well as ATRA-induced differentiation of NB4 cells resulted in the deacetylation of C/EBPα. NAMPT inhibition in NB4 and HL60 cell lines using specific inhibitor FK866 led to the dramatically elevated levels of acetylated C/EBPα and reduced amounts of total C/EBPα protein, which was in line with diminished mRNA expression of C/EBPα target genes (G-CSF, G-CSFR and ELANE). Interestingly, treatment of acute myeloid leukemia cell line HL60 with NAMPT or transduction of HL-60 cells with NAMPT-expressing lentiviral construct induced myeloid differentiation of these cells even without addition of ATRA. This was in line with time- and dose-dependent increase of total C/EBPα protein levels upon NAMPT treatment. Therefore, NAMPT overcomes transcriptional repression of C/EBPα in HL-60 cells by activation of positive CEBPA autoregulation. Taken together, we described a new mechanism of regulation of C/EBPα activities in hematopoiesis and leukemogenesis by its post-translational modification via NAMPT/SIRT1-triggered de-/acetylation. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v124.21.3540.3540 ◽

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.

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Identification of Meis1 as a Target of CREB in Myeloid Leukemogenesis.

Blood ◽

10.1182/blood.v108.11.1945.1945 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1945-1945

Author(s):

Samuel D. Esparza ◽

Deepa Shankar ◽

Stanley Nelson ◽

Kathleen Sakamoto

Keyword(s):

Bone Marrow ◽

Transcription Factor ◽

Cell Lines ◽

Myeloid Leukemia ◽

Leucine Zipper ◽

Target Genes ◽

Bone Marrow Cells ◽

Mrna Level ◽

Leukemia Cell ◽

Self Renewal

Abstract The cyclic AMP response element binding protein, CREB, is a leucine zipper transcription factor that induces the expression of genes that regulate proliferation and survival. We previously demonstrated that CREB expression was increased in bone marrow cells from patients with acute leukemia. Transgenic mice that overexpress CREB in myeloid cells develop a myeloproliferative/myelodysplastic syndrome after one year. Although CREB has been implicated in the development of leukemia, the mechanisms by which CREB overexpression leads to a malignant phenotype remain elusive. To identify CREB target genes, we performed microarray analysis with RNA from two CREB overexpressing cell lines, K562 and NFS60. Both murine (NFS60) and human (K562) myeloid leukemia cell lines were stably transfected with CREB under the control of the myeloid-specific HMRP-8 promoter. Meis1, an important transcription factor in hematopoiesis, was found to be upregulated 40-fold in CREB overexpressing cells compared to controls. In the development of granulocytes, Meis1 expression decreases as cells differentiate. Similar to CREB, Meis1 is expressed in CD34+ hematopoietic cells but not in CD34- cells. Meis1 expression has been reported to promote self-renewal of stem cells and inhibit G-CSF-dependent differentiation into granulocytes. We hypothesized that CREB overexpression may lead to persistent expression of Meis1, increased self-renewal, and a block in differentiation of primitive hematopoietic progenitor cells, thereby contributing to leukemogenesis. We first examined Meis1 expression in CREB overexpressing K562 and NFS60 cells. Significantly higher levels of Meis1 were detected in Western blot analysis with lysates from CREB overexpressing K562 cells compared to parental cells. In addition, the degree of CREB expression correlated with the degree of Meis1 expression when evaluated at the protein level. Experiments performed using the NFS60 cell line showed similar results. To investigate Meis1 expression in primary myeloid leukemia cells, bone marrow from patients with acute myeloid leukemia (AML) were analyzed for CREB and Meis1 expression. Samples from three of the four patients had elevated CREB expression. Patients whose cells had the highest levels of CREB also had highest expression of Meis1. The one patient with minimal CREB expression showed no detectible levels of Meis1. Evaluation of cell lines and primary cells using quantitative real time PCR is underway to determine potential differences in CREB and Meis1 expression at the mRNA level. Chromatin immunoprecipitation will also be performed to determine whether Meis1 is a direct target of CREB. Our data demonstrate that CREB overexpression correlates with overexpression of Meis1 in both cell lines and primary AML cells. These results suggest that CREB and Meis1 are co-regulated in AML cells and may contribute to the leukemia phenotype.

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

Blood ◽

10.1182/blood.v112.11.3372.3372 ◽

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.

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MiR-34b Promoter Methylation and Regulation of CREB Expression In Myeloid Transformation

Blood ◽

10.1182/blood.v116.21.538.538 ◽

2010 ◽

Vol 116 (21) ◽

pp. 538-538

Author(s):

Elena Manara ◽

Emma Baron ◽

Alessandra Beghin ◽

Claudia Tregnago ◽

Emanuela Giarin ◽

...

Keyword(s):

Cell Lines ◽

Promoter Methylation ◽

Myeloid Leukemia ◽

Target Genes ◽

Fetal Liver ◽

Leukemia Cell ◽

Leukemia Cell Lines ◽

Myeloid Leukemia Cell

Abstract Abstract 538 The cAMP response element binding protein (CREB) is a nuclear transcription factor downstream of various stimuli and is critical for the pathogenesis of leukemia. CREB overexpression promotes abnormal proliferation, cell cycle progression, and clonogenic potential in vitro and in vivo. We found that CREB deregulation in Acute Myeloid Leukemia (AML) is due to both genomic amplification and aberrant miRNA expression. CREB has been shown to be a direct target of the microRNA, miR-34b. The inverse correlation between CREB and miR-34b expression has been described in myeloid leukemic cell lines. Mir-34b restoration reduced CREB levels and leukemia proliferation in vitro. One reason for the lower expression of miR-34b in myeloid leukemia cell lines is the hypermethylation of its promoter. Our goal was to characterize the role of miR-34b in AML progression using primary cells and mouse models. We also studied the regulation of miR-34b expression in cells from patients with AML and myelodysplastic syndromes (MDS). Primary AML cells transiently overexpressing miR-34b had decreased clonogenicity, as well as increase in apoptosis (9.9 vs. 25.5%, p<0.001). Primary leukemia cells from AML patients (n=3) treated with the demethylating agent 5-aza-2′-deoxycytidine showed a rise in miR-34b expression after 16 hours (RQ=7±2.6). We also observed a concomitant decrease in CREB protein expression and its target genes. In vivo, miR-34b overexpression resulted in decreased CREB expression and suppression of leukemia growth in flank tumor models with HL-60 and K562 cells injected into NOD-SCID IL-2receptor gamma null (NSG) mice, measured by bioluminescence and tumor volume (n=10 per group). These results demonstrated that miR-34b is an important tumor-suppressor through downregulation of CREB. We next investigated miR-34b expression in a large series of AML patients (n=118), a group of MDS patients (n= 49), and healthy bone marrows (HL-BM) (n=17) by quantitative PCR. Our results demonstrated lower miR-34b expression in blast cells from AML patients at diagnosis compared to HL-BM. The lower miR-34b expression in AML patients correlated with elevated CREB levels, similar to myeloid leukemia cell lines. The expression levels of miR-34b in bone marrow from MDS patients were intermediate between AML patients and HL-BM. These results suggest that miR-34b regulates CREB and is involved in the evolution of MDS to AML. In an effort to understand the mechanism of miR-34b downregulation in primary AML and MDS BM cells, miR-34b promoter methylation was determined using MS-PCR in both patient cohorts. The miR-34b promoter was found to be methylated in 65% (78/118) of AML patients at diagnosis, while it was unmethylated in all MDS samples (49/49). In particular, 3 MDS patients that evolved to AML had miR-34b promoter hypermethylation exclusively at the onset of AML. We further tested this hypothesis by downregulating miR-34b in primary HL-BM and fetal liver cells by using both oligonucleotides and a lentiviral transduction. An increase in CREB mRNA and several CREB target genes (for example cyclin B1, cyclin E2, p21) was observed. Moreover, the cell cycle profile demonstrated increased numbers of cells in S phase compared to negative controls. Methylcellulose colony formation was also increased in HL-BM and fetal liver cells transduced with a miR-34b inhibitor compared to controls (197 vs. 101, p<0.001). Therefore, we conclude that miR-34b promoter methylation is critical for the pathogenesis of AML through regulation of CREB-dependent pathways. Disclosures: Sakamoto: Abbott Laboratories, Inc.: Research Funding; Genentech, Inc.: Research Funding.

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Myeloid Leukemia Cells With MLL partial Tandem Duplication Are Sensitive To Pharmacological Inhibition Of The H3K79 Methyltransferase DOT1L

Blood ◽

10.1182/blood.v122.21.1256.1256 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1256-1256 ◽

Cited By ~ 4

Author(s):

Michael W.M. Kühn ◽

Michael Hadler ◽

Scott R. Daigle ◽

Chun-Wei Chen ◽

Amit U. Sinha ◽

...

Keyword(s):

Cell Lines ◽

Target Genes ◽

Leukemia Cell ◽

Leukemia Cell Line ◽

Hoxa Cluster ◽

Histone 3 ◽

Leukemia Cell Lines ◽

Equity Ownership

Abstract Genetic alterations of the mixed-lineage leukemia (MLL) gene are commonly identified in acute leukemias. In acute myeloid leukemia (AML), a partial tandem duplication (PTD) of MLL occurs in about 5-10% of AML patients and is associated with adverse prognosis. The mutation leads to an in-frame duplication of exons 5 to 11 resulting in the production of an aberrant MLL protein. Unlike chromosomal rearrangements of MLL, this mutation does not affect the functional histone 3 lysine 4 (H3K4) methyltransferase domain. However, AMLs carrying a MLL-PTD and MLL-rearranged leukemias share some common characteristics, such as overexpression of HOXA-cluster genes and dysregulated epigenetic functions. Recently, leukemias with various MLL-translocations have been shown to be dependent on the histone 3 lysine 79 (H3K79) methyltransferase, DOT1L, and are sensitive to EPZ004777, a recently described selective small-molecule DOT1L inhibitor. EPZ-5676, a DOT1L-inhibitor with improved potency and drug-like properties, has recently been identified and is currently under clinical investigation. To evaluate the therapeutic potential of DOT1L-inhibition in MLL-PTD positive leukemia cells, we assessed the effect of EPZ004777 on the MLL-PTD containing leukemia cell lines MUTZ-11 and EOL-1. In vitro treatment with EPZ004777 over a 14-day period resulted in dramatic reduction of cell proliferation compared to DMSO vehicle control in both cell lines beginning 7 days after the start of treatment. Similar results were obtained for MOLM-13, a leukemia cell line harboring a MLL-translocation, but not for HL-60, a non-MLL-rearranged leukemia cell line. To further investigate whether these findings reflect a selective response to EPZ004777 or non-specific drug toxicity, we first explored the genome-wide H3K79 dimethylation (H3K79me2) profile using chromatin immunoprecipitation (ChIP) followed by next generation sequencing in untreated MUTZ-11 cells. Across the HOXA-cluster locus, we detected a similar H3K79me2 distribution pattern as previously reported in MLL-rearranged leukemias. Further analysis of H3K79me2 in MUTZ-11 and EOL-1 cells after treatment with the inhibitor showed profound suppression of those marks as assessed by western blot and ChIP-PCR. Subsequent global gene expression analysis revealed concurrent downregulation of HOXA-cluster and other MLL-target genes after 7 days of DOT1L inhibition. To investigate the effect of EPZ004777 on the MLL-PTD positive cells in more detail, we analyzed cell differentiation and apoptosis upon a 10-day exposure to the compound. As previously described for EPZ004777-sensitive MLL-rearranged leukemias, drug treatment resulted in increased expression of CD11b and morphological changes consistent with myeloid cell differentiation. We further observed apoptotic cell death after EPZ004777 treatment as measured by an increase in the percentage of Annexin V positive cells and cleaved Caspase 3 protein compared to vehicle controls. In order to determine the effect of DOT1L inhibition in vivo, we tested the recently identified DOT1L-inhibitor, EPZ-5676, for its ability to inhibit leukemia growth in a subcutaneous EOL-1 xenograft model in immunocompromised rats. Similar to what we observed in vitro, continuous intravenous administration over 21 days led to substantial dose-dependent inhibition of tumor growth, abrogation of H3K79me2, and concurrent downregulation of selected MLL-target genes. Thus, we demonstrate unexpected sensitivity of MLL-PTD containing leukemia cell lines to the DOT1L inhibitors EPZ004777 in vitro and EPZ-5676 in vivo. These data suggest that patients with myeloid malignancies carrying this particular mutation might benefit from treatment with therapeutic approaches that target DOT1L. Disclosures: Daigle: Epizyme, Inc: Employment, Equity Ownership. Olhava:Epizyme Inc.: Employment. Pollock:Epizyme Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Armstrong:Epizyme Inc.: Has consulted for Epizyme Inc. Other.

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FOXM1 Binds Nucleophosmin in AML and Confers Resistance to Chemotherapy

Blood ◽

10.1182/blood.v126.23.2467.2467 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2467-2467 ◽

Cited By ~ 3

Author(s):

Irum Khan ◽

Maryam Zia ◽

Marianna Halasi ◽

Peter Gann ◽

Sujata Gaitonde ◽

...

Keyword(s):

Transcription Factor ◽

Cell Line ◽

Cell Lines ◽

Leukemia Cell ◽

Bone Marrow Biopsies ◽

Empty Vector ◽

Leukemia Cell Lines ◽

Foxm1 Expression ◽

Increased Sensitivity ◽

Oncogenic Transcription Factor

Abstract Background: The nucleo-cytoplasmic shuttle protein nucleophosmin (NPM1) is mutated in 50% of cytogenetically normal AML cases (CN-AML) resulting in the nuclear export of this protein and a favorable prognosis. The mechanism by which mutated NPM1 (NPM1mut) confers this advantage in CN-AML is unclear. We have previously demonstrated that FOXM1, an oncogenic transcription factor, co-localizes with NPM1 in cancer cells and NPM-binding is required for sustaining the level of FOXM1. In the NPM1mut AML cell line OCI-AML3, as detected by immunofluorescence, NPM1 and FOXM1 co-localize in the cytoplasm where FOXM1 is transcriptionally inactive. We hypothesize that improved outcomes in the subset of NPM1mut AML is partly attributable to the cytoplasmic relocalization and functional inactivation of FOXM1. Methods: Patient samples were identified retrospectively using the University of Illinois Cancer Center Registry (IRB #: 2013-1245). Clinical, laboratory and molecular data were collected by chart review. Formalin fixed paraffin embedded bone marrow biopsies were retrieved to prepare slides. Patients with favorable or unfavorable karyotypes were excluded and only biopsies with >70% blasts were included. We performed a 2-target staining protocol with fluorescent antibodies to visualize NPM1 and FOXM1 in the same cells using the Vectra multispectral tissue imaging system. This system combines machine learning capability (inForm® software) with acquisition of spectral data from each pixel in the image, so that multiple targets can be quantified in the same image. Leukemia cell lines with stable knockdown of FOXM1 were generated using lentiviral transduction of HL60 cells (NPM1wt) transduced with PLKO.1-shFOXM1 or PLKO.1-empty vector. In parallel, OCI-AML3 cells (NPM1mut) were transduced with pBabe control or pBabe-FOXM1 expressing retrovirus to induce overexpression of FOXM1. Results: FOXM1 is a well-characterized oncogenic transcription factor. In this study we examined its role in AML. In a series of AML cell lines, cellular fractionation followed by immunoblotting revealed strong nuclear expression of FOXM1 in HL-60 and KG1, both of which express the wild-type NPM1 gene. In comparison the OCI-AML3 cell line, carrying the mutated NPM1 gene, showed predominantly cytoplasmic expression of FOXM1 where it is inactive. To better understand the role of FOXM1 in mediating chemoresistance in AML, we inhibited FOXM1 expression in the HL60 leukemia cell line. Stable knockdown of FOXM1 in this cell line resulted in increased sensitivity to the chemotherapeutic agent cytarabine compared to empty vector transduced cells. This was demonstrated by increased caspase-3 cleavage as detected by immunoblotting and decreased cell viability by Trypan blue exclusion after 24 hour exposure to the drug. We then proceeded to validate our observations of the FOXM1-NPM interaction in primary AML samples. Using quantitative data generated from the Vectra imaging of 20 AML pre-treatment bone marrow biopsies we show a strong correlation between the nuclear: cytoplasmic ratios of FOXM1 and NPM, Pearson correlation co-efficient r=0.87, which is indicative of cellular co-localization. When the patients are stratified in 2 groups based on NPM1 mutation status, mean cytoplasmic expression of FOXM1 is 2-fold higher in NPM1mut patients (36% versus 18%). Conclusion: We demonstrate that FOXM1, an oncogenic transcription factor is predominantly cytoplasmic and thereby inactive when NPM1 is mutated in AML. We show the functional significance of FOXM1 in mediating chemoresistance in this disease using AML cell lines. Using a novel quantitative imaging modality we establish for the first time the cellular co-localization of FOXM1 and NPM1 in AML primary blast cells. Studies are ongoing to manipulate FOXM1 expression in additional leukemia cell lines and xenograft models of AML to confirm our findings that FOXM1 inhibition in AML leads to increased sensitivity to chemotherapy. Disclosures No relevant conflicts of interest to declare.

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