scholarly journals High Expression of Mir-9 down-Regulates the Poor Outcome Prognosticator ERG and Associates with Reduced Relapse-Rates in Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1575-1575 ◽  
Author(s):  
Heike Weidner ◽  
Marius Bill ◽  
Laura Schmalbrock ◽  
Madlen Jentzsch ◽  
Laura Kloss ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Transcription Factor ◽  
Myeloid Leukemia ◽  
Empty Vector Control ◽  
Empty Vector ◽  
The Poor ◽  
Relapse Rates ◽  
Acute Myeloid

Abstract In acute myeloid leukemia (AML) high expression of the transcription factor ERG (ETS related gene) is associated with dismal outcome. The mechanisms that regulate differential ERG expression remain to be fully elucidated. MicroRNAs (miRs), small RNAs that are able to regulate gene expression, have emerged as important players in AML. We hypothesized that part of the differential expression of ERG is mediated by miRs. In silico prediction tools identified three putative miR-9 binding sites (BS) in the 3'-untranslated region (UTR) of ERG. First, we determined the expression levels of ERG & miR-9 in eight leukemia cell lines (i.e. KG1a, K562, THP-1, MV4-11, EOL1, NB4, OCI-AML3 & ME1) & found an inverse correlation between ERG & miR-9 expression (rank correlation -0.90). The cell line KG1a had the highest ERG & low miR-9 expression, and was therefore used for miR-9 overexpression experiments. In these cells miR-9 overexpression decreased ERG expression at the mRNA level to 82±7% (P=.079) & at the protein level to 72±14% (P=.005) after 12 hours (h) compared to empty vector control transfected cells. Next, we tested the activity of the three putative miR-9 BS in the 3'-UTR of ERG using luciferase assays. 12 h after cotransfection of HEK-293T cells with a miR-9 overexpression vector & an appropriate luciferase vector containing two of the putative BS (BS1 & BS2) from the 3'-UTR of ERG, we found the luciferase activity reduced to 52±4% (P=.023). Mutation experiments showed BS1, but not BS2 to be essential for this activity. The insertion of BS3 into the luciferase vector had no effect on reporter gene expression. Thus miR-9 most likely regulates expression of the transcription factor ERG by directly binding to BS1 in its 3'-UTR. To test if a differential expression of miR-9 is also of functional significance in AML, we first analyzed its impact on cell proliferation. Overexpression of miR-9 led to decreased proliferation rates in KG1a cells compared to control vector treated cells. After 5 days, the relative cell count was 133±21% vs. 241±67% in the miR-9 overexpressing cells compared to empty vector expressing cells, respectively. Next, we determined if this difference was based on a higher apoptosis rate. An Annexin V staining revealed no significant difference between the apoptotic threshold of miR-9 overexpressing (21%) and empty vector cells (21%) after 24 h. However, a subsequent cell cycle analysis demonstrated a higher percentage of miR-9 overexpressing cells in the G2/M phase, (39±2%) compared to the empty vector control treated cells (31±3%) after 24 h (P=.084), indicating that the cell cycle is slowed or stopped during cell division. Since miR-9 targets the poor prognostic marker ERG & higher miR-9 expression led to decreased proliferation & reduced cycling in AML cells in vitro we speculated that the differential miR-9 expression would also impact the outcome of AML patients (pts). Mature miR-9 is derived from three precursor molecules of which pre-miR-9-1 & pre-miR-9-3 are known to be expressed in hematopoietic cells. We assessed the pre-miR-9-1 expression of bone marrow by real-time PCR in 131 AML pts (median age 64 [range 22 – 75] years) with favorable (n=4, 3%), intermediate (n=90, 69%), adverse (n=33, 25%), or unknown (n=4, 3%) cytogenetic risk (according to the Medical Research Council [MRC] Classification) who received a RIC-HCT. The median follow-up was 4 years. The pre-miR-9-1 expression levels were normalized to ABL to define high & low pre-miR-9-1 expressers by the median expression of all AML pts. At diagnosis, high pre-miR-9-1 expresser status associated with a lower white blood count (P=.065) and lower % of peripheral blasts (P=.108) by trend. Furthermore, pts with high pre-miR-9-1 expression were more likely to be NPM1 wild-type (P=.047) & FLT3-ITD negative (P=.020). Pts with high pre-miR-9-1 had a lower probability of relapse (P=.048). In conclusion, miR-9 targets & regulates expression of the poor outcome predictor ERG. Overexpression of miR-9 led to decreased proliferation and a pause in AML cell cycling. Furthermore, high pre-miR-9-1 expression associated with reduced relapse rates in AML. Thus a pharmacologically induced expression of miR-9 in AML blasts may improve outcomes of AML pts. Disclosures No relevant conflicts of interest to declare.

MicroRNA-29a Is An Oncogene in Acute Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 683-683
Author(s):  
Christopher Y. Park ◽  
Yoon-Chi Han ◽  
Govind Bhagat ◽  
Jian-Bing Fan ◽  
Irving L Weissman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Hematopoietic Stem Cells ◽  
Mirna Expression ◽  
Myeloid Leukemia ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Abstract microRNAs (miRNAs) are short, non-protein encoding RNAs that bind to the 3′UTR’s of target mRNAs and negatively regulate gene expression by facilitating mRNA degradation or translational inhibition. Aberrant miRNA expression is well-documented in both solid and hematopoietic malignancies, and a number of recent miRNA profiling studies have identified miRNAs associated with specific human acute myeloid leukemia (AML) cytogenetic groups as well as miRNAs that may prognosticate clinical outcomes in AML patients. Unfortunately, these studies do not directly address the functional role of miRNAs in AML. In fact, there is no direct functional evidence that miRNAs are required for AML development or maintenance. Herein, we report on our recent efforts to elucidate the role of miRNAs in AML stem cells. miRNA expression profiling of AML stem cells and their normal counterparts, hematopoietic stem cells (HSC) and committed progenitors, reveals that miR-29a is highly expressed in human hematopoietic stem cells (HSC) and human AML relative to normal committed progenitors. Ectopic expression of miR-29a in mouse HSC/progenitors is sufficient to induce a myeloproliferative disorder (MPD) that progresses to AML. During the MPD phase of the disease, miR-29a alters the composition of committed myeloid progenitors, significantly expedites cell cycle progression, and promotes proliferation of hematopoietic progenitors at the level of the multipotent progenitor (MPP). These changes are manifested pathologically by marked granulocytic and megakaryocytic hyperplasia with hepatosplenomegaly. Mice with miR-29a-induced MPD uniformly progress to an AML that contains a leukemia stem cell (LSC) population that can serially transplant disease with as few as 20 purified LSC. Gene expression analysis reveals multiple tumor suppressors and cell cycle regulators downregulated in miR-29a expressing cells compared to wild type. We have demonstrated that one of these genes, Hbp1, is a bona fide miR-29a target, but knockdown of Hbp1 in vivo does not recapitulate the miR-29a phenotype. These data indicate that additional genes are required for miR-29a’s leukemogenic activity. In summary, our data demonstrate that miR-29a regulates early events in normal hematopoiesis and promotes myeloid differentiation and expansion. Moreover, they establish that misexpression of a single miRNA is sufficient to drive leukemogenesis, suggesting that therapeutic targeting of miRNAs may be an effective means of treating myeloid leukemias.

The Role of TWIST1 Gene Expression and Hypoxic Microenvironment in Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3839-3839
Author(s):  
Emilia Carolina Malafaia ◽  
A. Mario Marcondes ◽  
Ekapun Karoopongse ◽  
Daniele Serehi ◽  
Maria de Lourdes L. F. Chauffaille ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemia Cells ◽  
Twist1 Expression ◽  
Disease Biology ◽  
Acute Myeloid

Abstract TWIST1, a basic helix-loop-helix (bHLH) transcription factor, plays a critical role in mesodermal development and organogenesis. Overexpressed TWIST1 has been thoroughly related to epithelial-mesenchymal transition (EMT) in solid tumors (QIN Q et al., 2012) and has been described as an emerging risk factor in hematological neoplasms (MERINDOL et al., 2014). . Many questions remain to be addressed concerning to the role of TWIST1 in acute myeloid leukemia (AML). The understanding of TWIST1 in leukemia cells and its interaction with microenvironment can offer new insights in regards to disease biology and therapeutic targets for patients with AML. Objectives: 1) to evaluate the role of stroma contact and hypoxia in TWIST1 expression in myeloid cell lines. 2) To evaluate the functional impact of overexpressing TWIST1 on KG1a and PL21 cells. 3) To evaluate TWIST1 expression in primary cells of AML patients. Methods: In order to mimic bone marrow microenvironment, myeloid cells were co-cultured with mesenchymal HS5 cell line and PO2 1% was established with Smart -Trak¨ 2 (Sierra Instruments, Inc.) equipment. Quantitative mRNA was determined using TaqMan¨ Universal Master Mix (Applied Biosystems, Foster City, CA) and 3-step standard cycling conditions with sequence-specific primer TWIST1 normalized to the expression of β-actin. KG1a and PL21 cells were transduced with lentivirus vector carrying e-GFP ("enhanced green fluorescence protein") for stable expression of TWIST1. Transduced cells were sorted by FITC fluorochrome and then verified through western blot analysis with TWIST1 antibody. For quantification of apoptosis, cells were labeled with PE-conjugated antibody using annexin V-phycoerythrin and propidium iodide (BD Biosciences, USA). DAPI (4',6- diamidino-2-phenylindole dihydrochloride) was used to stain DNA and determine cell cycle information . Apoptosis and cell cycle were analyzed by FACS -Becton Dickinson Canto II (BD Biosciences). Statistical analysis was assessed with unpaired t test. Results: Hypoxia induced TWIST1 mRNA expression in OCIAML3, PL21, KG1a and ML1 cell lines (fold-increased 46.3, 29.8, 12.9 and 2.3 respectively). Cells expressing endogenous TWIST1 protein (OCIAML3 and ML1) showed resistance to apoptosis in a hypoxic microenvironment (normoxia versus hypoxia: OCI/AML3, 22.6 % vs 11.7% and ML1, 29.8% vs. 7.5%) in contrast, cells not expressing endogenous TWIST1 protein (KG1a and PL21) went to apoptosis in the same conditions. Thus, overexpressing TWIST1 in KG1a and PL21 induced apoptosis protection in hypoxia (KG1a unmodified vs. modified: 17.6 ± 6.3 vs. 2.8 ± 6.3, p=0.04; PL21 unmodified vs. modified: 26.9 ± 10.9 vs. 3.2 ± 0.6, p=0.04) (fig 1). We found increased TWIST1 mRNA levels in bone marrow samples of 23 AML patients (3.88 ± 1.59) compared with 5 healthy controls (0.54 ±0.25) (p= 0.02) (fig 2). Patients in the highest tertile of TWIST1 expression did not show differences in percentage of blasts in bone marrow and complete remission after treatment compared with patients in low and middle tertile. Conclusion: Our data suggest TWIST1 gene expression protects acute myeloid leukemia cells from apoptosis in a hypoxic microenvironment. Moreover, our results showed increased expression of TWIST1 in AML patients. Thus, TWIST1 is a potential gene involved in leukemogenesis and should be further explored to understand disease biology and potential therapeutic targets. Disclosures No relevant conflicts of interest to declare.

scholarly journals Convergent organization of aberrant MYB complex controls oncogenic gene expression in acute myeloid leukemia

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Sumiko Takao ◽  
Lauren Forbes ◽  
Masahiro Uni ◽  
Shuyuan Cheng ◽  
Jose Mario Bello Pineda ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Transcription Factor ◽  
Myeloid Leukemia ◽  
Human Cancers ◽  
Co Activation ◽  
Alternative Transcription ◽  
Transcription Factor Complexes ◽  
Oncogenic Gene ◽  
Acute Myeloid

Dysregulated gene expression contributes to most prevalent features in human cancers. Here, we show that most subtypes of acute myeloid leukemia (AML) depend on the aberrant assembly of MYB transcriptional co-activator complex. By rapid and selective peptidomimetic interference with the binding of CBP/P300 to MYB, but not CREB or MLL1, we find that the leukemic functions of MYB are mediated by CBP/P300 co-activation of a distinct set of transcription factor complexes. These MYB complexes assemble aberrantly with LYL1, E2A, C/EBP family members, LMO2, and SATB1. They are organized convergently in genetically diverse subtypes of AML and are at least in part associated with inappropriate transcription factor co-expression. Peptidomimetic remodeling of oncogenic MYB complexes is accompanied by specific proteolysis and dynamic redistribution of CBP/P300 with alternative transcription factors such as RUNX1 to induce myeloid differentiation and apoptosis. Thus, aberrant assembly and sequestration of MYB:CBP/P300 complexes provide a unifying mechanism of oncogenic gene expression in AML. This work establishes a compelling strategy for their pharmacologic reprogramming and therapeutic targeting for diverse leukemias and possibly other human cancers caused by dysregulated gene control.

A Molecular Network Comprising MicroRNA-223, E2F1 and C/Ebpα in Granulopoiesis and in Acute Myeloid Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1803-1803
Author(s):  
John Anto Pulikkan ◽  
Viola Dengler ◽  
Abdul Peerzada ◽  
Stefan Bohlander ◽  
Daniel G. Tenen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Transcription Factor ◽  
Protein Level ◽  
Myeloid Leukemia ◽  
Promoter Activity ◽  
Cell Cycle Progression ◽  
Luciferase Assay ◽  
Cycle Progression ◽  
Acute Myeloid

Abstract MicroRNAs play crucial roles in gene expression programmes and have been demonstrated to have major influence in various biological processes. Recent findings suggest aberrant regulation of microRNAs is a hall mark of many cancers including leukemia. MicroRNA-223 (miR-223) is regulated by the transcription factor CCAAT enhancer binding protein α (C/EBPα) and is upregulated during granulopoiesis. miR-223 mutant mice display defects in granulopoiesis pointing out the importance of miR-223 during granulopoiesis. Recent studies suggest that loss of function or expression of C/ EBPα is a major step in the development of acute myeloid leukemia (AML). Using an inducible cell line model, we show that C/EBPα upregulates microRNA-223 expression during granulopoiesis. Based on these findings, we hypothesized that miR-223 could be downregulated in human AML. Here we report that miR-223 is downregulated in different subtypes of AML as analysed by quantitative Real-Time RT-PCR. We investigated what are the critical targets of miR-223 during granulopoiesis. Computational analysis suggests that E2F1, the transcription factor that promotes cell cycle progression which is inhibited by C/EBPα during granulopoiesis, could be a putative target of miR-223. By luciferase assay using 3’UTR of E2F1, we show that E2F1 is a potential target of miR-223. miR-223 downregulates E2F1 by translational repression as revealed by reduction in E2F1 protein level. Silencing of miR-223 leads to upregulation of E2F1 protein level as analyzed by Western blot analysis. Proliferation assays as well as cell cycle analysis demonstrate that miR-223 blocks cell cycle progression in myeloid cells. Interestingly, sequence analysis of miR-223 promoter revealed putative E2F1 binding sites. We demonstrate that E2F1 inhibits the microRNA-223 promoter activity through its transactivation domain as shown by promoter assay. Furthermore, overexpression of E2F1 down regulates the expression of miR-223, suggesting E2F1 acting as a transcriptional repressor of the miR-223 gene. Meanwhile, C/EBPα transactivates miR-223 promoter activity. We also report that E2F1 is able to block granulocytic differentiation. Recent studies demonstrate that disruption of E2F1 inhibition by C/EBPα leads to leukemia, pointing out the significance of E2F1 inhibition in the development of AML. Our data support a circuitry comprising miR-223, C/EBPα and E2F1 as major components of the granulocyte differentiation programme, which is deregulated in AML. Manipulation of miR-223 could be therapeutically relevant in AML subtypes in which E2F1 inhibition is deregulated.

ABR, a Novel Inducer Of Transcription Factor C/EBPα, Is Necessary For Myeloid Differentiation and a Prognostic Factor In Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3814-3814
Author(s):  
Carolina Yaeko Namasu ◽  
Dennis Gerloff ◽  
Alexander Arthur Wurm ◽  
Daniela Braeuer-Hartmann ◽  
Jens-Uwe Hartmann ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Transcription Factor ◽  
Prognostic Factor ◽  
Myeloid Leukemia ◽  
Myeloid Differentiation ◽  
U937 Cells ◽  
Acute Myeloid

Abstract ABR (Active BCR-related) is the only protein in humans and mice closely homologous to BCR. BCR acts as a tumor suppressor in different cancers, such as chronic myeloid leukemia and meningiomas. A putative anti-oncogenic role of ABR has been shown in tumors of the central nervous system, such as medulloblastoma and astrocytomas, in which deletion of ABR was found. However, the role of ABR in hematopoiesis or leukemia remains unclear. We hypothesized that ABR might be important for myelopoiesis via increasing the expression of C/EBPα, a transcription factor known to be pivotal for myeloid differentiation and functionally impaired in acute myeloid leukemia (AML). In fact, we found that ABR expression is dramatically down-regulated (Figure 1, p=0.01) in bone marrow from AML patients (pts; n=63) compared to bone marrow (BM) mononuclear cells from healthy donors (n=3). In agreement with this finding, Abr is significantly increased during M-CSF and G-CSF-stimulated differentiation of primary wild type mouse BM cells (p<0.05). Additionally, we observe that ABR is necessary for monopoiesis induced by PMA (phorbol 12-myristate 13-acetate), since ABR knockdown in leukemic U937 cells results in a significant reduction of about 50% in the number of CD11b+ cells 48h after PMA treatment (p<0.05). Enforced ABR expression induces C/EBPα and its targets M-CSFR, G-CSFR and microRNA (miR)-223 in U937 cells (p<0.01). Moreover, we prove that ABR knockdown prevents induction of CEBPA, M-CSFR and G-CSFR during PMA-mediated differentiation (p<0.05). ABR overexpression blocks cell-cycle progression and down-regulates the known C/EBPα inhibitor E2F1 (p<0.01) in U937 cells, indicating the functional role of ABR as tumor suppressor. Those data suggest that ABR might induce CEBPA expression via inhibition of cell cycle activator E2F1. Finally, we are the first to identify ABR as a good prognostic factor in AML: patients with high ABR expression (median cut) survive significantly longer after allogeneic hematopoietic stem cell transplantation (Figure 2, p=0.04, log-rank test). Furthermore, high ABR expression associates with a low percentage of blasts in the peripheral blood (p=0.006) and high levels of antileukemic miR-181a (p<0.001). In conclusion, these data indicate that ABR, a novel inducer of C/EBPα, is necessary for myelopoiesis and a prognostic factor in AML. Raising ABR levels might be a goal for future therapeutics in AML. Disclosures: No relevant conflicts of interest to declare.

Minimal Reduction of PU.1 Is Sufficient to Induce a Preleukemic State and Promote Development of Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 305-305
Author(s):  
Britta Will ◽  
Thomas O. Vogler ◽  
Swathi-rao Narayanagari ◽  
Boris Bartholdy ◽  
Tihomira I. Todorova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Transcription Factor ◽  
Malignant Transformation ◽  
Myeloid Leukemia ◽  
Complete Loss ◽  
Hematopoietic Stem ◽  
Minimal Reduction ◽  
Acute Myeloid

Abstract Genomic studies have shown that human cancer is rarely associated with a complete loss of transcripts; instead, acquired DNA alterations often occur within the non-coding part of the genome, are enriched in gene-regulatory regions, and cause only moderate transcriptional changes. It is currently not well understood how such moderate gene expression changes impact normal tissue function and how they contribute to malignant transformation. Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) develop through a multi-step transformation process originating in hematopoietic stem cells (HSCs) and mainly present in the elderly (median age of >65 years at diagnosis). Although loss or near-complete loss of the hematopoietic transcription factor PU.1 induces AML in mice, a similar degree of PU.1 impairment is exceedingly rare in human AML; yet moderate PU.1 inhibition is common in AML patients. At the example of the Ets-family transcription factor PU.1, which is indispensable for HSC function and the differentiation of cells within the myeloid as well as lymphoid lineages, we tested the hypothesis that even moderate gene expression alterations of key regulators can drive malignant transformation. We assessed the effects of minimal PU.1 inhibition on hematopoiesis in a novel mouse model that co-models the genomic context found in aging human individuals and patients with MDS/AML. Mice lacking Msh2, the key component of the MutSα and MutSβ complexes mediating DNA mismatch repair, accumulate elevated numbers of point mutations, in particular C/G>T/A transitions and small insertions/deletions resembling the mutation spectrum acquired in HSCs in aging human individuals and patients with MDS and AML. We crossed Msh2-/- mice with animals carrying a heterozygous deletion of an upstream regulatory element of PU.1 (UREΔ/+). UREΔ/+Msh2-/- mice exhibited a significant, but very modest reduction of PU.1 expression on average by 26-37% in fractionated hematopoietic multipotent stem and myeloid progenitor cells. Strikingly, this minimal reduction of PU.1 led to the emergence of an aggressive, transplantable AML in more than two thirds of UREΔ/+Msh2-/- mice which was never observed in URE+/+Msh2-/- mice. Overt leukemia was preceded by a preleukemic phase hallmarked by an expanded population of multipotent murine hematopoietic stem cell enriched cells (HSPCs) that was myeloid-biased and less quiescent than their wild type counterpart. Longitudinal monitoring of preleukemic UREΔ/+Msh2-/- mice revealed a progressive increase in immature myeloid cells along with a gradual decrease in mature myeloid cells, as well as expansion of phenotypic HSPC compartments and multi-lineage dysplasia resembling human MDS. AML progression was accompanied by additional inhibition of a PU.1-cooperating factor, interferon responsive factor 8 (Irf8). Irf8 expression restoration rescued impaired expression of genes harboring PU.1/IRF consensus binding sites, led to the loss of aberrant self-renewal, promoted myeloid differentiation, and induced apoptosis in leukemic UREhetMsh2-/- cells demonstrating that Irf8 impairment functionally cooperates with minimally reduced PU.1 expression in our model. We also found evidence of disease-relevant joint PU.1/IRF8 inhibition in human myeloid leukemogenesis: (1) patients with MDS with a higher risk for the progression to AML had lower IRF8 levels; (2) lower IRF8 expression was detected specifically in AML patients with reduced PU.1 levels; (3) restoration of IRF8 expression induced differentiation in IRF8 low expressing AML cells, and (4) a positive correlation of PU.1 and IRF8 expression was found in human leukemia stem cells, but not in healthy HSCs. Strikingly, comparative pathway analysis revealed a genome-wide molecular resemblance of preleukemic and leukemic UREΔ/+Msh2-/- mice with gene expression profiles from human MDS and AML patients, respectively. Our study demonstrates that minimal reduction of a key lineage-specific transcription factor that commonly occurs in human disease is sufficient to initiate cancer development and provides mechanistic insight into the formation and progression of preleukemic stem cells in MDS and AML. Disclosures No relevant conflicts of interest to declare.

Gene Expression Analyses At Time Of Diagnosis Indicate Biomarkers Predictive Of Therapeutic Response In Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2619-2619 ◽  
Author(s):  
Marita Lagergren Lindberg ◽  
Petra Hååg ◽  
Ali Moshfegh ◽  
Lena Kanter ◽  
Magnus Bjorkholm ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Transcription Factor ◽  
Clinical Outcome ◽  
Myeloid Leukemia ◽  
Differentially Expressed ◽  
Training Cohort ◽  
Gene Expression Analyses ◽  
Diagnostic Samples ◽  
Acute Myeloid

Abstract In this study we aimed to identify biomarkers predictive of clinical response in acute myeloid leukemia (AML). For this purpose mRNA was isolated from diagnostic samples from 42 AML patients (“training cohort”) and subjected to Affymetrix® gene expression analysis. All patients entered complete remission (CR) after high-dose induction chemotherapy, reaching a median CR duration of 161 (range 12-3701) days. Samples from patients with “short CR duration” (<6 months, n=24) and “long CR duration” (>6 months n=18), respectively, were pooled and compared. Gene expression analyses revealed 383 genes to be up-regulated and 610 genes down-regulated more than two fold in samples from patients with short, as compared to those with long CR duration. Ten genes were found to be up-regulated >30 times, with the runt-related transcription factor 1; translocated to 1 (cyclin D-related) (RUNX1T1) gene showing the highest differential expression (116-fold), while annexin 1 (ANXA1) was the most down-regulated gene (58-fold). Significantly higher transcript levels of RUNX1T1 were confirmed in the poor outcome group when performing quantitative real time polymerase chain reaction (qRT-PCR) on individual samples (n=20, p<0.002). Subjecting our data to pathway analysis (Ingenuity®) comparing the same groups, we focused on RUNX1T1 and created a network of RUNX1T1 interacting molecules. Utilizing the IPA database to create a network over interacting molecules of RUNX1T1, we identified a majority of these to be transcriptional regulators and among them the transcription factor 3 (TCF3) to be up-regulated 5-fold in patients with short CR duration. Our training cohort data were validated in silico extracting information from an independent study by Metzeler et al, publicly available from Oncomine® (www.oncomine.org) and encompassing diagnostic samples from 162 AML patients. Among genes differentially and similarly regulated in poor responders in both the training and validation cohorts we observed TCF3, chemokine (C-X-C motif) ligand 3 (CXCL3), Zinc finger, MIZ-type containing 1 (ZMIZ1) (up-regulated) and Peroxiredoxin 2 (PRDX2) (down-regulated). Analyses of clinical outcome revealed that AML patients with a high ZMIZ1 expression had a significantly decreased overall survival (OS) as compared to that of patients with a low ZMIZ1 expression (p <0.03). ZMIZ1 has been reported to be involved in tumor growth in general and suggested to interact with activated NOTCH in inducing leukemia, but its more precise role in AML is still unclear. In conclusion, we report clear differences in gene expression in diagnostic samples from AML patients with subsequent poor vs. better long-term clinical outcome to therapy, thus to constitute possible novel predictive biomarkers for response. In our training set RUNX1T1 was the most differentially expressed gene, while ZMIZ1 was upregulated in both the training and validation sets and significantly associated with survival. Further, functional studies of differentially expressed genes in clinical subsets of AML patients appear warranted. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Inhibition of c-Myc By Apto-253 As an Innovative Therapeutic Approach to Induce Cell Cycle Arrest and Apoptosis in Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1716-1716 ◽  
Author(s):  
Hongying Zhang ◽  
Andrea Local ◽  
Khalid Benbatoul ◽  
Peter Folger ◽  
Susan Sheng ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Transcription Factor ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Time Dependent ◽  
Cycle Arrest ◽  
Dose Dependent ◽  
Acute Myeloid

Abstract The c-Myc multifunctional transcription factor protein, a product on the c-myc proto-oncogene, contributes to the pathogenesis of many types of human cancers through mechanisms of proliferation, apoptosis, cell cycle progression and cellular senescence. c-Myc is frequently overexpressed in acute myeloid leukemia, yet strategies to effectively modulate c-Myc function do not exist. We evaluated inhibition of c-myc gene expression by APTO-253, a small molecule anticancer agent that is being developed clinically for the treatment of acute myelogenous (myeloid) leukemia (AML) and high risk myelodysplastic syndromes (MDS). We first confirmed that c-Myc mRNA level were significantly higher in AML cell lines as compared to peripheral blood mononuclear cells (PBMCs) isolated from healthy human donors. However, the c-Myc expression in AML cells was inhibited by APTO-253 in dose-dependent and time-dependent manners at both the mRNA and protein levels. Likewise, APTO-253 was found to induce AML cell apoptosis in dose-dependent and time-dependent manners as demonstrated by positive Annexin-V staining and increases in cleaved poly (ADP-ribose) polymerase (c-PARP). APTO-253 induced AML cells arrest at G1/G0 phase of cell cycle by increasing p21 expression and decreasing expression of cyclin D3 and cyclin-dependent kinases 4/6 (CDK4/6). For the p53 positive cell lines MV4-11 and EOL-1, p53 was also increased by APTO-253 at early time points (less than 6-hour treatment), suggesting that p53-dependent cell cycle arrest and apoptosis is mechanistically operative as a consequence of treatment with APTO-253. Importantly, we demonstrated that APTO-253 selectively targeted tumor cells but not normal healthy cells, with MV4-11 AML cells and normal PBMCs having IC50s of 0.25±0.03µM and more than 100µM, respectively. Our previous studies (56th ASH abstract #4813) showed that APTO-253 induces the Krüppel-like Factor 4 (KLF4) transcription factor and was effective and well tolerated as a single agent in multiple AML xenograft models without causing bone marrow suppression. Taken together, our results suggested that APTO-253 may serve as an effective and safe agent for AML chemotherapy, and that APTO-253 mechanistically inhibits c-Myc expression in AML cells and subsequently induces cell cycle arrest and apoptosis. Disclosures No relevant conflicts of interest to declare.

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