scholarly journals Paradoxical Enhancement of Leukemogenesis in Acute Myeloid Leukemia Cells with Moderately Attenuated RUNX1 Expressions

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2710-2710
Author(s):  
Kensho Suzuki ◽  
Ken Morita ◽  
Shintaro Maeda ◽  
Hiroki Kiyose ◽  
Souichi Adachi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Free Radicals ◽  
Protein Level ◽  
Myeloid Leukemia ◽  
Cell Cycle Progression ◽  
Leukemia Cells ◽  
Cycle Progression ◽  
Runx1 Expression ◽  
Acute Myeloid

Abstract Although Runt-related transcription factor 1 (RUNX1), a member of RUNX transcription family, is known for its oncogenic role in the development of acute myeloid leukemia (AML), evidence from other groups support the oncosuppressive property of RUNX1 in leukemia cells, casting a question over the bidirectional function of RUNX1 and it is currently highly controversial. Here we report that the dual function of RUNX1 possibly arise from the total level of RUNX family expressions. To examine the precise mechanism of RUNX1 expression in leukemogenesis, we first prepared several tetracycline-inducible short hairpin RNAs (shRNAs) which could attenuate the expressions of RUNX1 at different levels in AML cells (MV4-11 and MOLM-13 cells). Intriguingly, while AML cells transduced with shRNAs which could down-regulate RUNX1 expression below 10% at protein level (sh_Rx1_profound) deteriorated the proliferation speed of AML cells, AML cells transduced with shRNAs which could moderately down-regulate RUNX1 expression to 70% at protein level (sh_Rx1_moderate) paradoxically promoted the cell cycle progression and doubled the growth rate of AML cells. Besides, RUNX1-moderately expressing AML patient cohort exhibited the worse outcome compared to RUNX1-high or RUNX1-low expressing cohorts (n = 187), indicating an underlying mechanism that confer growth advantage to AML cells with moderately inhibited RUNX1 expressions. To further investigate the correspondent gene in this paradoxical enhancement of oncogenesis in sh_Rx1_moderate-transduced AML cells, we performed comprehensive gene expression array and extracted genes that are highly up-regulated in RUNX1 moderate inhibition and down-regulated in AML cells transduced with sh_Rx1_profound. We hereafter focused on the top-listed gene glutathione S-transferase alpha 2 (GSTA2) and addressed the interaction of RUNX1 and GSTA2 and their functions in AML cells. Real time quantitative PCR (RT-qPCR) and immunoblotting revealed that the expression of GSTA2 was actually up-regulated in sh_Rx1_moderate-transduced AML cells and down-regulated in AML cells transduced with sh_Rx1_profound. Interestingly, equivalent level of compensatory up-regulation of RUNX2 and RUNX3 were observed in sh_Rx1_moderate- and sh_Rx1_profound-transduced AML cells, creating an absolute gap in the expression of total amount of RUNX (RUNX1 + RUNX2 + RUNX3), which was confirmed by RT-qPCR (total amount of RUNX expressions were estimated by primers amplifying the specific sequence common to all RUNX family members). Luciferase reporter assay of GSTA2 promoter and chromatin immunoprecipitation (ChIP) assay in the proximal promoter region of GSTA2 gene proved the association of RUNX family members with this genomic region. These results indicated that total amount of RUNX family expressions modulate the expression of GSTA2 in AML cells, which might results in a paradoxical outbursts of RUNX1 moderately-inhibited AML cells. Since GSTA2 catabolizes and scavenges free radicals such as hydrogen peroxide (H2O2), and decreased intracellular free radicals promote acceleration of cell cycle progression, we next measured the intracellular accumulation of H2O2 in RUNX1 inhibited AML cells. As we have expected, intracellular amount of H2O2 was decreased in sh_Rx1_moderate-transduced AML cells and increased in AML cells transduced with sh_Rx1_profound. Additive transduction of sh_RNAs targeting GSTA2 to AML cells with sh_Rx1_moderate reverted the proliferation speed to the control level, underpinning that growth advantage of moderate RUNX1 inhibition could be attributed to the GSTA2 overexpressions. Taken together, these findings indicate that moderately attenuated RUNX1 expressions paradoxically enhance leukemogenesis in AML cells through intracellular environmental change via GSTA2, which could be a novel therapeutic target in anti-leukemia strategy. Disclosures No relevant conflicts of interest to declare.

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.

ATF3 coordinates serine and nucleotide metabolism to drive cell cycle progression in acute myeloid leukemia

2021 ◽  
Author(s):  
Daniela Di Marcantonio ◽  
Esteban Martinez ◽  
Joice S. Kanefsky ◽  
Jacklyn M. Huhn ◽  
Rashid Gabbasov ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Cell Cycle Progression ◽  
Nucleotide Metabolism ◽  
Cycle Progression ◽  
Acute Myeloid

scholarly journals Loss of ISWI ATPase SMARCA5 (SNF2H) in Acute Myeloid Leukemia Cells Inhibits Proliferation and Chromatid Cohesion

2020 ◽  
Vol 21 (6) ◽  
pp. 2073
Author(s):  
Tomas Zikmund ◽  
Helena Paszekova ◽  
Juraj Kokavec ◽  
Paul Kerbs ◽  
Shefali Thakur ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Cell Cycle Progression ◽  
Mitotic Division ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Chromatid Cohesion ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

ISWI chromatin remodeling ATPase SMARCA5 (SNF2H) is a well-known factor for its role in regulation of DNA access via nucleosome sliding and assembly. SMARCA5 transcriptionally inhibits the myeloid master regulator PU.1. Upregulation of SMARCA5 was previously observed in CD34+ hematopoietic progenitors of acute myeloid leukemia (AML) patients. Since high levels of SMARCA5 are necessary for intensive cell proliferation and cell cycle progression of developing hematopoietic stem and progenitor cells in mice, we reasoned that removal of SMARCA5 enzymatic activity could affect the cycling or undifferentiated state of leukemic progenitor-like clones. Indeed, we observed that CRISPR/cas9-mediated SMARCA5 knockout in AML cell lines (S5KO) inhibited the cell cycle progression. We also observed that the SMARCA5 deletion induced karyorrhexis and nuclear budding as well as increased the ploidy, indicating its role in mitotic division of AML cells. The cytogenetic analysis of S5KO cells revealed the premature chromatid separation. We conclude that deleting SMARCA5 in AML blocks leukemic proliferation and chromatid cohesion.

scholarly journals CREB Regulates Cell Cycle Progression through RFC3-PCNA Axis in Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 881-881
Author(s):  
Hee-Don Chae ◽  
Bryan Mitton ◽  
Kathleen Sakamoto
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Promoter Region ◽  
Myeloid Leukemia ◽  
Cell Cycle Progression ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Nuclear Antigen ◽  
Cycle Progression ◽  
Acute Myeloid

Abstract CREB (cAMP Response Element Binding protein) is a transcription factor overexpressed in normal and neoplastic myelopoiesis and regulates cell cycle progression, although its oncogenic mechanism has not been well characterized. Replication Factor C3 (RFC3), a 38 kDa subunit of the RFC complex, is required for chromatin loading of proliferating cell nuclear antigen (PCNA) which is a sliding clamp platform for recruiting numerous proteins in DNA replication and repair processes. CREB1 expression was coupled with RFC3 expression during the G1/S progression in the KG-1 acute myeloid leukemia (AML) cell line, suggesting that RFC3 and CREB1 might be target genes of E2F, a key transcriptional regulator of the G1/S progression. Though there were two potential E2F binding sites in the RFC3 promoter region, chromatin immunoprecipitation assays provided no evidence for E2F1 binding to the RFC3 promoter, whereas E2F1 could directly act on the CREB1 expression. Treatment with the cyclin-dependent kinase (CDK) inhibitor AT7519 decreased expression of CREB1 and RFC3 as well as well-known E2F target genes such as CCNE1, CCNA2 and CCNB1 in KG-1 cells. These results indicate that CREB1 overexpression, a potentially important prognostic marker in leukemia patients, may be associated with dysregulated CDK-E2F activity in leukemia. There was also a direct correlation between the expression of RFC3 and CREB1 in human AML cell lines as well as in AML cells from patients. CREB interacted directly with the CRE site in RFC3 promoter region. CREB knockdown primarily inhibited G1/S cell cycle transition, decreasing expression of RFC3 as well as PCNA loading onto chromatin. Exogenous expression of RFC3 was sufficient to rescue the impaired G1/S progression and PCNA chromatin loading [Chromatin-bound PCNA-positive cells (%), control vs. CREB-knockdown vs. CREB-knockdown with RFC3 overexpression, 8h after release from mitotic arrest: 66.87 +/– 0.90 vs. 24.77 +/– 0.99 vs. 79.17 +/– 0.12, n=3, p< 0.01, mean +/– SEM] caused by CREB knockdown. Taken together, our results suggest that RFC3 may play a role in neoplastic myelopoiesis by promoting the G1/S progression, and its expression is regulated by CREB. These data provide new insight into CREB-driven regulation of the cell cycle in AML cells, and may contribute to leukemogenesis associated with CREB overexpression. Disclosures No relevant conflicts of interest to declare.

scholarly journals Cell-cycle regulator E2F1 and microRNA-223 comprise an autoregulatory negative feedback loop in acute myeloid leukemia

Blood ◽  
2010 ◽  
Vol 115 (9) ◽  
pp. 1768-1778 ◽  
Author(s):  
John A. Pulikkan ◽  
Viola Dengler ◽  
Philomina S. Peramangalam ◽  
Abdul A. Peer Zada ◽  
Carsten Müller-Tidow ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Feedback Loop ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Enhancer Binding Protein ◽  
Blast Cells ◽  
Acute Myeloid ◽  
Transcriptional Target

Abstract Transcription factor CCAAT enhancer binding protein α (C/EBPα) is essential for granulopoiesis and its function is deregulated in leukemia. Inhibition of E2F1, the master regulator of cell-cycle progression, by C/EBPα is pivotal for granulopoiesis. Recent studies show microRNA-223 (miR-223), a transcriptional target of C/EBPα, as a critical player during granulopoiesis. In this report, we demonstrate that during granulopoiesis microRNA-223 targets E2F1. E2F1 protein was up-regulated in miR-223 null mice. We show that miR-223 blocks cell-cycle progression in myeloid cells. miR-223 is down-regulated in different subtypes of acute myeloid leukemia (AML). We further show that E2F1 binds to the miR-223 promoter in AML blast cells and inhibits miR-223 transcription, suggesting that E2F1 is a transcriptional repressor of the miR-223 gene in AML. Our study supports a molecular network involving miR-223, C/EBPα, and E2F1 as major components of the granulocyte differentiation program, which is deregulated in AML.

ATF3 Coordinates Serine and Nucleotide Metabolism to Drive Cell Cycle Progression in Acute Myeloid Leukemia

2021 ◽  
Author(s):  
Daniela Di Marcantonio ◽  
Esteban Martinez ◽  
Joice Kanefsky ◽  
Jacklyn Huhn ◽  
Rashid Gabbasov ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Cell Cycle Progression ◽  
Nucleotide Metabolism ◽  
Cycle Progression ◽  
Acute Myeloid

MLN4924, A Novel First in Class Small Molecule Inhibitor of the Nedd8 Activating Enzyme (NAE), Has Potent Activity in Preclinical Models of Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1021-1021
Author(s):  
Ronan T. Swords ◽  
Kevin R. Kelly ◽  
Peter G. Smith ◽  
James J. Gansey ◽  
Devalingam Mahalingam ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Small Molecule ◽  
Myeloid Leukemia ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Molecule Inhibitor ◽  
Acute Myeloid

Abstract Abstract 1021 Poster Board I-43 The coordinated balance between the synthesis and degradation of proteins is an important regulator of cancer cell biology. The ubiquitin-proteasome system (UPS) is responsible for the timed destruction of many proteins including key mediators of fundamental signaling cascades and critical regulators of cell cycle progression and transcription. Within the UPS, the E3 ligases are multi-protein complexes whose specificity is established by their individual components as well as post-translational modifications by various factors including the ubiquitin-like molecule, Nedd8. The Nedd8 activating enzyme (NAE) has been identified as an essential regulator of the Nedd8 conjugation pathway, which controls the activity of the cullin-dependent E3 ubiquitin ligases. The cullins direct the ubiquitination and subsequent degradation of many proteins with important roles in cell cycle progression (p27, cyclin E), DNA damage (Cdt-1), stress response (NRF-2, HIF1α) and signal transduction (IκBα). Acute myeloid leukemia (AML) is a disease of the elderly and prognosis is extremely poor with a median overall survival of just 2 months for untreated patients. As such, novel therapeutic strategies are urgently needed to improve clinical outcomes. Considering that Nedd8-mediated control of protein homeostasis is vitally important for the survival of AML cells, we hypothesized that disrupting this process would inhibit proliferation and induce cell death. We tested this hypothesis by investigating the preclinical anti-leukemic activity of MLN4924, a novel first in class small molecule inhibitor of the Nedd8 activating enzyme. MLN4924 induced DNA damage followed by rapid and selective caspase-dependent cell death in AML cell lines and primary AML cells from patients, but not in peripheral blood mononuclear cells from healthy donors. Transient exposure to MLN4924 impaired colony formation in a dose-dependent manner. Kinetic analysis of drug-induced effects on cell cycle distribution revealed that AML cells treated with MLN4924 initially arrested at the G1 transition prior to their subsequent accumulation in the sub-G1 compartment. Assays conducted using MV-411 cells with and without stable shRNA-mediated knockdown of FLT3 expression demonstrated that MLN4924 is highly effective independent of FLT3 status. Further investigation revealed that the activity of MLN4924 was preserved when cells were co-cultured with bone marrow stromal cells indicating that it has the ability to overcome the effects of stromal-mediated survival signaling that has been established to blunt the efficacy of relevant standard of care agents. MLN4924 induced a dose and time dependant increase in the expression of phospo-IκB, an important target for degradation through the Nedd8 conjugation pathway. The inhibitory effects of MLN4924 on NFκB were confirmed by demonstrating that the transcriptional activity of the NFκB p65 subunit was significantly reduced following drug exposure. Moreover, treatment of immunodeficient mice implanted with HL-60 human leukemia cells with MLN4924 led to an inhibition of neddylated cullins, accumulation of phospho-IκBα and achieved complete and stable disease regression. Our results indicate that MLN4924 is a highly promising novel agent for the treatment of AML and warrants further evaluation in clinical trials. Disclosures: Smith: Millennium Pharmaceuticals: Employment. Gansey:Millennium Pharmaceuticals: Employment.

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