The Novel Orally Active Aurora A Kinase Inhibitor MLN8237 Is Highly Active in Preclinical Models of Acute Myeloid Leukemia and Significantly Increases the Efficacy of Cytarabine.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2087-2087
Author(s):  
Kevin R. Kelly ◽  
Ronan T. Swords ◽  
Devalingam Mahalingam ◽  
Steffan T. Nawrocki ◽  
Ernest Medina ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Critical Role ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Phase Ii Trials ◽  
Immunodeficient Mice ◽  
Acute Myeloid

Abstract Abstract 2087 Poster Board II-64 Acute myeloid leukemia (AML) most frequently affects the elderly, many of whom are unable to tolerate intensive chemotherapy. Therefore, improvements in clinical outcomes for patients with AML largely depend upon the development of novel targeted therapies. Aurora A is a serine/threonine kinase that plays a key role in mitosis by regulating the G2-M transition, centrosome separation, spindle assembly and chromosome segregation. It is overexpressed in AML and has been implicated in genetic instability, disease progression and drug resistance. MLN8237 is a novel orally available Aurora A inhibitor that has entered Phase I and Phase II trials for cancer therapy. Based on the critical role of Aurora A in cell cycle regulation and its intrinsic overexpression in AML cells, we hypothesized that MLN8237 would possess significant antileukemic activity. We tested our hypothesis in established human AML cell lines, primary AML patient specimens, and a mouse model of AML. MLN8237 potently inhibited the in vitro growth and survival of all AML cell lines and primary human AML cells from patients with newly diagnosed AML and patients with relapsed, chemotherapy resistant disease. Mechanistic studies showed that MLN8237 reduced the activity of Aurora A kinase as evidenced by reduced phosphorylation of Aurora A at Thr288. MLN8237 treatment also disrupted normal cell cycle kinetics and induced apoptosis in a dose- and time-dependant manner characterized by the accumulation of G2/M and aneuploid cells prior to the onset of apoptosis. Exposure to MLN8237 led to activation of FOXO3a followed by a dose-dependent increase in the expression of its transcriptional target Bim. These events appear to be important mediators of the pro-apoptotic effects of this agent. We next investigated the ability of MLN8237 to increase the efficacy of cytarabine in AML. Treatment with the combination of MLN8237 and cytarabine resulted in significantly greater apoptosis and more effective inhibition of survival than treatment with either agent alone in AML cell lines and primary patient specimens. MLN8237 and cytarabine cooperated to enhance mitochondrial-mediated apoptosis as evidenced by increased processing of caspases-9 and -3 to active forms. Daily oral administration of MLN8237 to immunodeficient mice bearing AML xenografts was well tolerated, effectively reduced tumor growth and led to the disruption of mitotic spindles and centrosome amplification. Moreover, MLN8237 significantly enhanced the activity of cytarabine to achieve tumor regression. Our data demonstrates that MLN8237 has a multifaceted mechanism of action comprised of pro-apoptotic and growth inhibitory effects. Our collective findings indicate that the combination of MLN8237 and cytarabine represents a novel, very promising therapeutic strategy for AML. A clinical study investigating the safety and efficacy of this combination in patients with refractory AML is planned. Disclosures: Ecsedy: Millennium Pharmaceuticals: Employment.

Antitumor Effect of Pomolic Acid in Acute Myeloid Leukemia Cells Involves Cell Death, Decreased Cell Growth and Topoisomerases Inhibition

2019 ◽  
Vol 18 (10) ◽  
pp. 1457-1468
Author(s):  
Michelle X.G. Pereira ◽  
Amanda S.O. Hammes ◽  
Flavia C. Vasconcelos ◽  
Aline R. Pozzo ◽  
Thaís H. Pereira ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Natural Compound ◽  
Dna Topoisomerases ◽  
Human Dna ◽  
Pomolic Acid ◽  
Acute Myeloid

Background: Acute myeloid leukemia (AML) represents the largest number of annual deaths from hematologic malignancy. In the United States, it was estimated that 21.380 individuals would be diagnosed with AML and 49.5% of patients would die in 2017. Therefore, the search for novel compounds capable of increasing the overall survival rate to the treatment of AML cells is urgent. Objectives: To investigate the cytotoxicity effect of the natural compound pomolic acid (PA) and to explore the mechanism of action of PA in AML cell lines with different phenotypes. Methods: Three different AML cell lines, HL60, U937 and Kasumi-1 cells with different mechanisms of resistance were used to analyze the effect of PA on the cell cycle progression, on DNA intercalation and on human DNA topoisomerases (hTopo I and IIα) in vitro studies. Theoretical experiments of the inhibition of hTopo I and IIα were done to explore the binding modes of PA. Results: PA reduced cell viability, induced cell death, increased sub-G0/G1 accumulation and activated caspases pathway in all cell lines, altered the cell cycle distribution and inhibited the catalytic activity of both human DNA topoisomerases. Conclusion: Finally, this study showed that PA has powerful antitumor activity against AML cells, suggesting that this natural compound might be a potent antineoplastic agent to improve the treatment scheme of this neoplasm.

RNAi-Based Functional Genomics Screening of the Human Kinome Identifies Major Growth Regulating Kinases in Acute Myeloid Leukemia Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2951-2951
Author(s):  
Raoul Tibes ◽  
Ashish Choudhary ◽  
Amanda Henrichs ◽  
Sadia Guled ◽  
Irma Monzon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Acute Myeloid Leukemia ◽  
Gene Silencing ◽  
Functional Genomics ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Large Scale ◽  
Cell Cycle Checkpoint ◽  
Acute Myeloid

Abstract In order to improve treatment strategies for Acute Myeloid Leukemia (AML), we adapted a functional genomics approach using RNAi screening to identify molecular targets that are vital to the growth of AML. Herein we report the first large-scale kinome gene silencing screen in AML. A high throughput RNAi screen was developed for the efficient siRNA transfection of AML cell lines. Eight commercially available cationic lipid-based transfection reagents were tested for their ability to transfect several AML cell lines with siRNA. These extensive transfection optimization experiments identified two AML cells lines TF-1 and ML4 with up to 95–100 and 70–75% transfection efficiency respectively. Two independent replicate kinome screens were performed on both cell lines using a siRNA library targeting 572 kinase genes with 2 siRNA/gene. At 96 hours post transfection, cell proliferation was assessed and the B-score method was used to background correct and analyze the screening data. Several siRNA to specific kinases were identified that significantly inhibit cell proliferation of up to ~40–88%. Hits were defined at two thresholds: siRNA having a B-score of <−2 providing a statistically significance of p<0.05 (confidence of > 95%) and a cutoff B-score of <−1.5 providing greater than 87% confidence for each siRNA hit. Two different kinases (2 siRNA/gene/screen) were identified as major growth regulating kinases in TF1 cells with all 4 siRNA/gene having a B-score <−2. For two additional kinases, 3/4 siRNA for each gene had a Bscore <−2. Expanding the cutoff to a B-score <−1.5 three further kinases were targeted by at least 3/4 siRNA/gene. Similar analysis using the same criteria for ML4 cells identified one kinase targeted by 3/4 siRNA at a B-score <−2, seven kinases with 2/4 siRNA <−2 and two kinases with 3/4 siRNA/gene at a B-score of <−1.5. Common hits for both cell lines with at least 6/8 siRNA per gene from 4 screens performing at a B-score <−2 identified two kinases, one of them PLK1. Applying a B-score threshold of <−1.5, we identified five kinases for which at least 5/8 siRNA/gene from 4 screens met these criteria. Kinases/genes will be presented at the meeting.Confirmation of gene silencing and validation of growth response is currently underway for a subset of genes. Among the strongest hits are siRNA targeting PLK1, as well as siRNA targeting three other kinase-genes involved in regulating cell cycle progression and checkpoints and gene ontology (GO) analysis showed enrichment in cell cycle and cell cycle-checkpoint processes. Inhibitors against PLK1 and other kinase hits identified in the screen are in (pre)-clinical development and if confirmed, our experiments provide a strong rational to test these in AML. The application of RNAi based screening is useful in the identification of genes important in AML proliferation, which could serve as targets for therapeutic intervention and guide AML drug development. Furthermore, results from these types of functional genomics approaches hold promise to be rapidly translated into clinical application.

scholarly journals Phase I study of the aurora A kinase inhibitor alisertib with induction chemotherapy in patients with acute myeloid leukemia

Haematologica ◽  
2016 ◽  
Vol 102 (4) ◽  
pp. 719-727 ◽  
Author(s):  
Amir T. Fathi ◽  
Seth A. Wander ◽  
Traci M. Blonquist ◽  
Andrew M. Brunner ◽  
Philip C. Amrein ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Phase I ◽  
Induction Chemotherapy ◽  
Myeloid Leukemia ◽  
Phase I Study ◽  
Kinase Inhibitor ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Acute Myeloid

A Machine-Learning Analysis Suggests That FLX925, a FLT3/CDK4/6 Kinase Inhibitor, Is Potent Against FLT3-Wild Type Tumors Via Its CDK4/6 Activity

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3520-3520
Author(s):  
Gene Cutler ◽  
Jordan S Fridman
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Rna Seq ◽  
Cell Cycle Genes ◽  
Equity Ownership ◽  
Anti Tumor Activity ◽  
Acute Myeloid

Abstract Fms-like tyrosine kinase 3 (FLT3) is universally expressed in Acute Myeloid Leukemia (AML) blast cells (Zheng R, et al. FLT3 ligand causes autocrine signaling in acute myeloid leukemia cells. Blood. 2004) and activating mutations of FLT3 are amongst the most common genetic lesions in AML (Levis M. FLT3 mutations in acute myeloid leukemia: what is the best approach in 2013? ASH Education Program Book. 2013). FLX925, a small molecule inhibitor of FLT3 would be expected to have antitumor efficacy against FLT3-driven AML tumors. However, FLX925 also inhibits two closely related cyclin-dependent kinases (CDKs), CDK4 and CDK6 (described together as CDK4/6). Since CDK4/6 are important cell cycle genes and CDK4/6 inhibitors have demonstrated anti-tumor activity, the anti-CDK4/6 activity of FLX925 may drive anti-tumor efficacy in additional and potentially overlapping patient populations. To generate hypotheses about which tumors would be sensitive to FLX925 treatment and to understand the relative contributions of FLX925's FLT3- and CDK4/6-inhibitory activities, a panel of XXX tumor-derived cell lines were screened for sensitivity to FLX925. We performed a Gradient Boosting Machine (GBM) analysis, a machine-learning approach, to generate a model that predicts sensitivity to FLX925 based on RNA-Seq expression data. This sensitivity model can predict which cell lines and tumors are likely to be sensitive to FLX925 and also which gene expression patterns contribute to that sensitivity. The GBM analysis was performed on 133 (75%) of the assayed cell lines with a random 45 (25%) of the cell line datasets reserved for model validation. Pearson's correlations of r=0.96 on the test cell lines and r=0.68 on the validation cell lines were observed, indicating good predictive performance with modest overfitting. The two most important predictive genes in the model were Retinoblastoma 1 (RB1) and by Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A). RB1 and CDKN2A are important cell cycle genes immediately downstream and upstream, respectively, of CDK4/6, suggesting that sensitivity to FLX925 in these cell lines is driven by FLX925's CDK4/6 inhibitory activity. To strengthen this conclusion, the modeling was repeated but gene sets covering cell cycle genes or genes in the FLT3/STAT5 signaling pathway were separately excluded from the analysis. While excluding the FLT3/STAT5 pathway genes had no impact on the predictive power of the model, excluding the cell cycle gene set completely abrogated the ability of the GBM to predict sensitivity to FLX925. Thus, we conclude that in this panel of largely FLT3-wild type (FLT3-WT) cell lines, FLX925-sensitivity is driven solely by the compound's CDK4/6 activity. These results suggest that independent, but potentially overlapping, sets of tumors will be responsive to FLX925 due to both its FLT3- and its CDK4/6-inhibitory activities. To predict which tumor types would be most sensitive to FLX925 treatment, we applied our GBM model to the RNA-Seq expression data for 10,537 tumors from The Cancer Genome Atlas (TCGA) consortium. Even though this model is indifferent to FLT3/STAT5-pathway gene expression, it nevertheless identifies AML tumors as having the highest median predicted sensitivity to FLX925. This is true even when all haem/lymph malignancy-derived cell lines are excluded from the training set. Thus, both FLT3-WT and FLT3-activated tumors are expected to have sensitivity to FLX925 treatment, the former due to FLX925's CDK4/6 activity alone and the latter due to the independent FLT3 and CDK4/6 activities of the drug. The anti-tumor activity of FLX925 is currently being investigated in a Ph1/b dose-escalation study in subject with relapsed or refractory AML (NCT02335814). Disclosures Cutler: Amgen, Inc: Equity Ownership; FLX Bio: Employment, Equity Ownership. Fridman:FLX Bio: Employment, Equity Ownership.

MiR-335-3p inhibits cell proliferation, induces cell cycle arrest and apoptosis in acute myeloid leukemia by targeting EIF3E

2021 ◽  
Author(s):  
Ling Zhang ◽  
Xiaozhen Wang ◽  
Jieying Wu ◽  
Ruozhi Xiao ◽  
Jiajun Liu
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
G1 Arrest ◽  
Cycle Arrest ◽  
Promising Therapeutic Target ◽  
Reverse Transcription Quantitative Pcr ◽  
Acute Myeloid

Abstract Here, we aimed to investigate the biological roles and the regulatory mechanisms of miR-335-3p in acute myeloid leukemia (AML). We first found miR-335-3p was significantly down-regulated in blood samples from leukemia patients and cell lines using reverse transcription quantitative PCR. Through CCK-8 assay and flow cytometry, we observed that miR-335-3p overexpression significantly inhibited cell proliferation, induced cell cycle G0/G1 arrest and apoptosis in AML cell lines (THP-1 and U937). Moreover, miR-335-3p directly targets EIF3E and negatively regulated its expression. More importantly, EIF3E overexpression reversed the effects of miR-335-3p on cell proliferation, G1/S transition and apoptosis. Furthermore, miR-335-3p overexpression obviously downregulated the expression of CDK4, Cyclin D1 and Bcl-2, while upregulated the expression of p21 and Bad, which were significantly rescued by the co-transfection of pcDNA3.1-EIF3E. Collectively, our study proposes that miR-335-3p/EIF3E axis could be a promising therapeutic target to mitigate the progression of AML.

Expression and Role of APP Gene in Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4691-4691
Author(s):  
Fanyi Meng ◽  
Wei Wang ◽  
Zoufang Huang ◽  
Ming Huang ◽  
Lixiang Liu
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Western Blot ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Myeloid Leukemia ◽  
App Gene ◽  
Acute Myeloid

Abstract Abstract 4691 Introduction Amyloid precursor protein(APP) gene was increasingly expressed in solid tumors, promoted the proliferation of tumor cells and the overexpression of APP was a bad prognostic factor to oral squamous cell carcinoma. However, little has been known about the clinical significance and role of APP in acute myeloid leukemia(AML). Methods The expressions of APP mRNA in 85 AML patients and 20 nonmalignant hematological diseases that worked as control were measured by real-time PCR and the expressions of APP in AML cell lines were examined by real-time PCR and western blot. Small interfering RNAs(siRNAs) targeting APP gene were synthesized and transfected into HL60 cell by lipofectamine2000, after RNAi 24h, 48h and 72h, cell growth of HL60 was measured by trypan blue dye exclusion method and MTT, differentiation was observed by Wright-Giemsa staining, cell cycle was examined by PI/RNase staining, apoptosis induction was analyzed by Annexin V/PI and Hoechst33342 staining; apoptosis-related proteins NF-κB, bcl-2 and Caspase-3 were detected by Western blot after RNAi 48h; sensitivity of HL60 to adrimycin was measured by MTT. Results The expression of APP mRNA among AML subtypes was significantly different(P=0.019), M2 with t(8;21) was the highest expression subtype and M5b was the lowest. APP expression had no significant effect on AML clinical characteristic excepting AML subtypes. kasumi-1 was the highest expression cell in AML cell lines and U937 was the lowest(P<0.05), and the expression of APP in HL60/ADM was significantly lower than HL60(P<0.05). The APP expressions in AML cell lines was in agreement with its expressions in primary AML subtypes. After RNAi 24h, 48h, and 72h, no significant differences in proliferation, differentiation, apoptosis, cell cycle and sensitivity of HL60 to adriamycin were detected between interfering group and control groups. Conclusions The APP mRNA expression in M2 with t(8;21) was high and M5b was low. Down-regulation of APP expression had no significant effect on biological behaviour of HL60 and APP was not tightly related to pathogenesis of AML. Disclosures: No relevant conflicts of interest to declare.

Anti-Leukemic Activity of PIK-75, a PI3-Kinase p110α Selective Inhibitor, In Acute Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 659-659
Author(s):  
François Vergez ◽  
Jean-Emmanuel Sarry ◽  
Nathalie Gallay ◽  
Camille Fialin ◽  
Sarah Scotland ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Critical Role ◽  
Selective Inhibitor ◽  
Leukemic Cells ◽  
High Dose ◽  
Inhibition Of Proliferation ◽  
Hl60 Cells ◽  
Acute Myeloid

Abstract Abstract 659 The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway plays a critical role in a variety of tumor cells including hematological malignancies. Class IA PI3Ks are heterodimers that consist of a p85 regulatory and a p110 catalytic subunit. There are several isoforms of both the catalytic (p110α, p110β and p110δ) and regulatory subunits. The p110α isoform of the class IA PI3-Ks was recently genetically validated as a promising target for anticancer therapy. To date, only one compound (imidazo[1,2-a]pyridine, PIK-75) has been described as a very potent and selective inhibitor of this isoform (>100-fold selectivity over p110α and p110δ). In acute myeloid leukemia (AML), aberrant PI3-K activation is detectable in most of cases both in leukemic bulk and in the immature compartment of the leukemic clone. This activation contributes to cell growth, proliferation, survival, and drug-resistance. Furthermore, we have previously shown that the level of Akt phosphorylation on threonine 308, the major target of PI3-K, is correlated with poor outcome in AML patients (Gallay et al, Leukemia 2009, 23(6):1029-38). Therefore, effective targeting of this pathway with pharmacological inhibitors could improve therapeutic outcome in AML. Here, we studied the anti-neoplastic activity of several inhibitors of the PI3-K p110 subunits in AML cell lines and primary patient specimens. Treatment with PIK-75 led to a decrease of the proliferation in all cell lines at low dose (MTT assay, IC50: 62 nM, 144 nM, 173 nM in KG1, HL60 and KG1a cell lines, respectively). This inhibition of proliferation was due to massive apoptosis of KG1 and KG1a cells in both liquid culture but also after adhesion of leukemic cells on a fibronectin matrix. By contrast, p110ß (TGX221, 10 μM) and p110γ (AS252424, 10 μM) inhibitors only slightly decreased cell proliferation in KG1 and HL60 cells while p110δ inhibitor (IC87114) has no effect up to 10 μM. PIK-75 inhibited the phosphorylation of Akt on Thr308, and downstream effectors (4-EBP1 and RPS6) in these cells. These results strongly suggest a major role of p110α subunit which is highly express in AML cell lines and 19/19 patients samples. Next, we assessed the PIK-75 efficacy in 1 AML cell lines, 9 AML samples and 1 normal bone marrow CD34+ cells using clonogenic assays. PIK-75 inhibited AML-CFU in both KG1 cell line and all patient samples tested with an IC50 of 214 nM and 72 nM, respectively. Interestingly, PIK-75 has no effect on normal CFU-GM colonies, even at high dose (IC50 not reached at 1 μM), a result consistent with the normal haematopoiesis observed in p110αfnKO mice (Gritsman et al, Blood (ASH Annual Meeting Abstracts) 2009 114: Abstract 3620). Since leukemic subpopulation bearing the CD34+CD38-CD123+ phenotype is thought to be more resistant to chemotherapy than the leukemic bulk, we have assessed the apoptosis of 17 AML primary cells treated with increasing doses of cytarabine (Ara-C). We found significant differences of IC50 with 9.5 μM and 43 μM in bulk and CD34+38-123+ subpopulation, respectively. By contrast, PIK-75 demonstrated potent activity in both leukemic compartments of 42 AML samples with IC50 of 589 nM and 638 nM, respectively. Interestingly, the effect of PIK75 was not altered at relapse neither in bulk (IC50: 513 nM vs 492 nM at diagnosis and at relapse) nor in CD34+38-123+ subpopulation (IC50: 567 nM vs 254 nM at diagnosis and at relapse) in 8 AML samples. In NOD/SCID mice engrafted with HL60 cells, PIK-75 delivered at 1 and 10 mg/kg/d for 4 days induced a significant decrease in tumor burden after apoptosis induction detected ex vivo by annexin V staining. Further in vivo studies using NSG mice engrafted with primary AML specimens are ongoing. These results demonstrate that PIK-75 is the most potent inhibitor of PI3-K in leukemic cells suggesting that the selective inhibition of the p110α subunit could be a critical target in AML. Moreover, PIK-75 targets both leukemic bulk and chemoresistant leukemic subpopulations paving the way for clinical studies assessing the combination of selective p110α inhibitor with conventional chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Requirement For CDK6 In MLL-Rearranged Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3782-3782
Author(s):  
Theresa Placke ◽  
Katrin Faber ◽  
Atsushi Nonami ◽  
Helmut R. Salih ◽  
Stephen M. Sykes ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Small Molecule ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Myeloid Differentiation ◽  
Cell Cycle Distribution ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Abstract Chromosomal rearrangements involving the H3K4 methyltransferase MLL trigger aberrant gene expression programs in hematopoietic stem and progenitor cells and give rise to an aggressive subtype of acute myeloid leukemia (AML) that is associated with intermediate or poor survival. Insights into MLL fusion-mediated leukemogenesis have not yet translated into better therapies in the clinic, in part because mutant MLL is difficult to target directly and it is incompletely understood which of the genes affected by altered epigenetic regulation in MLL-rearranged AML are responsible for malignant transformation. To search for essential signaling pathways in MLL-rearranged AML that might serve as new therapeutic targets, we performed loss-of-function RNA interference (RNAi) screens in 5 AML cell lines (NOMO-1, THP-1, OCI-AML3, HL-60, U937) using a lentiviral short hairpin RNA (shRNA) library, and observed that the cell cycle regulator CDK6, but not its functional homolog CDK4, was preferentially required by MLL-AF9pos NOMO-1 and THP-1 cells. The enhanced CDK6 dependence of MLL-rearranged cells was confirmed in an expanded panel of AML cell lines (MLL-rearranged, n=6; MLL wildtype [WT], n=4) that also included cell lines harboring other MLL fusions (MLL-AF4 and MLL-AF6), and the RNAi-induced phenotype was countered by overexpression of an shRNA-resistant CDK6 cDNA. Stable knockdown of MLL-AF9 in MLL-AF9pos cell lines and overexpression of MLL-AF9 in WT MLL-expressing cell lines, normal human CD34pos cells, or Ba/F3 murine pro-B cells led to concordant changes in CDK6 mRNA and protein levels that resembled those of HOXA9, a known MLL-AF9 target, indicating that CDK6 is rendered essential via transcriptional activation by truncated MLL. Analysis of cell cycle distribution, apoptosis induction, and myeloid differentiation demonstrated that the differential growth-inhibitory effect of CDK6 suppression was mainly attributable to myeloid differentiation, as MLL-AF9pos cell lines upregulated CD11b expression and assumed a more mature, macrophage-like morphology upon CDK6 knockdown, effects not observed in WT MLL-expressing cells. Furthermore, the immature phenotype of NOMO-1 cells could be rescued by overexpression of an shRNA-resistant CDK6 cDNA. Consistent with the observations in AML cell lines, knockdown of Cdk6 also impaired the proliferation and in vitro clonogenic activity of primary murine bone marrow (BM) cells stably transduced with MLL-AF9, whereas cells expressing another leukemogenic fusion gene (MOZ-TIF2) and Ba/F3 cells were largely unaffected. We also expressed MLL-AF9 in unfractionated BM derived from Cdk6 knockout mice and observed that colony numbers were gradually reduced in cultures initiated with Cdk6+/- and Cdk6-/- BM compared to WT BM. Furthermore, most of the colonies obtained were small and displayed loose morphology in contrast to the large, dense, blast-like colonies seen in cultures initiated with transduced WT BM. We are currently investigating whether Cdk6 is also required for AML development and propagation in vivo using a murine BM transplantation model of MLL-AF9-induced leukemia. The context-dependent effects of lowering CDK6 expression could be recapitulated in cell lines and primary human AML specimens using palbociclib (also known as PD-0332991), a small-molecule inhibitor of CDK4 and CDK6 enzymatic activity that is in clinical development as an anticancer agent. We are currently devising strategies to combine this compound with cytotoxic chemotherapy as well as other targeted therapeutics, such as small-molecule bromodomain inhibitors, to maximize killing of MLL-rearranged AML cells. Together, our data (1) identify CDK6 as a critical and potentially “actionable” effector of MLL fusion proteins in leukemogenesis, (2) link the catalytic activity of CDK6 to arrested myeloid differentiation in MLL-rearranged AML, and (3) underscore that cell cycle regulators thought to normally act redundantly may have distinct functions in different genetic contexts. Disclosures: No relevant conflicts of interest to declare.

scholarly journals MicroRNA-582–3p negatively regulates cell proliferation and cell cycle progression in acute myeloid leukemia by targeting cyclin B2

2019 ◽  
Vol 24 (1) ◽  
Author(s):  
Haixia Li ◽  
Xuefei Tian ◽  
Paoqiu Wang ◽  
Mao Huang ◽  
Ronghua Xu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Cell Cycle Progression ◽  
Target Gene ◽  
Cycle Progression ◽  
Blood Samples ◽  
Acute Myeloid

Abstract Background MicroRNAs (miRNAs) function as post-transcriptional gene expression regulators. Some miRNAs, including the recently discovered miR-582–3p, have been implicated in leukemogenesis. This study aimed to reveal the biological function of miR-582–3p in acute myeloid leukemia (AML), which is one of the most frequently diagnosed hematological malignancies. Methods The expression of miR-582–3p was determined using quantitative real-time PCR in blood samples from leukemia patients and in cell lines. Cell proliferation and cell cycle distribution were analyzed using the CCK-8, colony formation and flow cytometry assays. The target gene of miR-582–3p was verified using a dual-luciferase reporter assay. The G2/M phase arrest-related molecule contents were measured using western blotting analysis. Results We found miR-582–3p was significantly downregulated in the blood samples from leukemia patients and in the cell lines. MiR-582–3p overexpression significantly impaired cell proliferation and induced G2/M cell cycle arrest in THP-1 cells. Furthermore, cyclin B2 (CCNB2) was confirmed as a target gene of miR-582–3p and found to be negatively regulated by miR-582–3p overexpression. More importantly, CCNB2 knockdown showed suppressive effects on cell proliferation and cell cycle progression similar to those caused by miR-582–3p overexpression. The inhibitory effects of miR-582–3p overexpression on cell proliferation and cell cycle progression were abrogated by CCNB2 transfection. Conclusion These findings indicate new functions and mechanisms for miR-582–3p in AML development. Further study could clarify if miR-582–3p and CCNB2 are potential therapeutic targets for the treatment of AML.

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