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.

Degradation of TRAF6 by Bortezomib-Induced Autophagy Contributes to Cell Death in Acute Myeloid Leukemia and Myelodysplastic Syndrome

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2452-2452
Author(s):  
Jing Fang ◽  
Lyndsey Bolanos ◽  
Garrett Rhyasen ◽  
Carmen Rigolino ◽  
Agostino Cortelezzi ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Myelodysplastic Syndrome ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
26S Proteasome ◽  
Negative Regulator ◽  
Myeloid Malignancies ◽  
Chromosome 5Q ◽  
Acute Myeloid

Abstract Abstract 2452 Deletion of chromosome 5q in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients results in loss of miR-146a, which is a negative regulator of the innate immune pathway by targeting TNF receptor associated factor-6 (TRAF6). Therefore, MDS and AML patients with reduced miR-146a expression concomitantly exhibit elevated TRAF6 protein. TRAF6 is an E3 ubiquitin ligase that catalyzes K63-linked polyubiquitin chains on substrates that lead to pathway activation, one of which includes NF-kB. Mice lacking miR-146a, or with overexpression of TRAF6, develop AML- and MDS-like features. Bortezomib (Velcade©), which shows promise alone or in combination with chemotherapy in certain groups of MDS and AML patients, is a selective and reversible inhibitor of the 26S proteasome. Studies on the mechanism of action of Bortezomib have shown that pro-apoptotic proteins are stabilized following proteasome inhibition and contribute to the anti-cancer effect. In this report, paradoxically, we find that Bortezomib induces rapid and complete degradation of TRAF6 protein, but not mRNA, in MDS/AML cell lines and human CD34+ cells. A similar finding was observed when AML cells were treated with MG132, another proteasome inhibitor, indicating that degradation of TRAF6 is secondary to proteasomal inhibition. Interestingly, the reduction in TRAF6 protein coincides with Bortezomib-induced autophagy, as indicated by conversion of LC3B-I to LC3B-II and degradation of SQSTM1/p62, and subsequently with apoptosis in MDS/AML cells. Addition of an autophagy inhibitor (3-methyladenine [3-MA]) to Bortezomib-treated AML cells maintained TRAF6 protein expression and enhanced cell viability. Similarly, TRAF6 degradation was blocked by 3-MA when cells were treated with Rapamycin, an mTOR inhibitor and inducer of autophagy. These findings suggest that a mechanism of Bortezomib-induced cell death in myeloid malignancies involves elimination of TRAF6 protein by autophagosomes. Forced expression of TRAF6 in two AML cell lines partially blocked the cytotoxic effect of Bortezomib, suggesting that TRAF6 is an important target of Bortezomib. To determine whether loss of TRAF6 is sufficient to impede growth of MDS and AML, we used a genetic approach to inhibit TRAF6 in MDS/AML cell lines and bone marrow cells from MDS patients with deletion of chromosome 5q. RNAi-mediated depletion of TRAF6 in MDS and AML samples resulted in impaired malignant hematopoietic stem/progenitor function and rapid apoptosis. To uncover the molecular consequences following loss of TRAF6, we applied gene expression profiling and identified genes relevant to the survival of MDS and AML cells. In summary, these findings implicate TRAF6 in Bortezomib-induced cell death and in the maintenance of myeloid malignancies, and reveal a novel mechanism of TRAF6 regulation through autophagic degradation. Disclosures: Oliva: Celgene: Consultancy.

scholarly journals Inhibiting Mitochondrial Complex II Exposes a Novel Metabolic Vulnerability in Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1300-1300
Author(s):  
Alessia Roma ◽  
Matthew Tcheng ◽  
Nawaz Ahmed ◽  
Sarah Walker ◽  
Preethi Jayanth ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Succinate Dehydrogenase ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Tca Cycle ◽  
Cell Fates ◽  
Reductive Carboxylation ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a hematological malignancy, characterized by an increased reliance on mitochondria-related energetic pathways including oxidative phosphorylation (OXPHOS). Consistent with this, the electron transport chain (ETC), a component of OXPHOS has been demonstrated to be a suitable anti-leukemia target, with ETC complex I inhibitors currently in clinical development. Relative to its counterparts, complex II (CII) is unique in that it directly links the ETC to the tricarboxylic acid (TCA) cycle through succinate dehydrogenase (SDH) activity. Moreover, it is the only ETC complex with elevated activity in AML, relative to normal hematopoietic samples, with indirect inhibition selectively targeting AML cells. However, direct CII inhibition in AML has not been previously investigated, nor have the mechanisms underlying the divergent fates of AML and normal cells upon CII inhibition. A genetic approach was first used to assess the effects of CII impairment on AML growth in vitro and in vivo. Using lentiviral mediated shRNA we generated AML cell lines lacking succinate dehydrogenase assembly factor 1 (Sdhaf1). Sdhaf1 knockdown suppressed CII activity, cell proliferation and clonogenic growth across all three cell lines and delayed leukemia growth in vivo. To recapitulate these effects through a pharmacological approach, we aimed to identify a novel CII inhibitor, since currently available inhibitors are only effective at high doses and are neurotoxic. Through an in silico structural screen and molecular docking study, shikonin was identified as a small molecule that selectively binds to CII. Shikonin inhibited CII activity in the AML cells lines and patient-derived samples, and selectively killed AML cells (EC 50: 1.0μM ± 0.04) while sparing normal progenitors. In murine engraftment models, shikonin (2.0-3.0 mg/kg, 3x/week for 5 weeks) significantly reduced engraftment of patient-derived AML cells but had no effect on normal hematopoiesis. To further characterize the mechanisms governing the divergent cell fates of CII inhibition, we performed stable isotope metabolic tracing using 13C 6- glucose and 13C 5, 15N 2-glutamine in patient-derived AML cells and normal mobilized peripheral blood mononuclear cells (MNCs). Both pharmacological and genetic loss of CII resulted in TCA cycle truncation by impairing oxidative metabolism of both glucose and glutamine. In Sdhaf1 knockdown and primary AML cells, this led to a depletion in steady state levels of TCA metabolites proceeding SDH. Inhibition of CII most notably suppressed levels of aspartate, a nucleotide precursor whose levels dictate the proliferative capacity of a cell under ETC dysfunction. Remarkably, MNCs maintained aspartate levels despite inhibition of CII, which was attributed to reductive carboxylation of glutamine, an alternate metabolic pathway that can regenerate TCA intermediates when OXPHOS is impaired. In contrast, while reductive carboxylation was also active in AML cells after CII inhibition, this activity was insufficient to maintain aspartate levels and resulted in metabolite depletion and cell death. Thus, loss of CII activity results in diverse cell fates whereby normal haematopoietic, but not AML cells sufficiently use reductive carboxylation of glutamine to overcome TCA cycle truncation, sustain aspartate levels and avert cell death. This is further evident through modulation of glutamine entry into the TCA cycle, where supplementation of cell-permeable α-ketoglutarate abrogated shikonin-mediated cell death while concomitant treatment with the glutaminase inhibitor CB-839, sensitized cells. Together, these results expose reductive carboxylation to support aspartate biosynthesis, as a novel metabolic vulnerability in AML that can be pharmacologically targeted through CII inhibition for clinical benefit. Disclosures Minden: Astellas: Consultancy. D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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.

ID: 92: CERAMIDE ACCUMULATION IN MITOCHONDRIA: A NOVEL THERAPEUTIC STRATEGY FOR ACUTE MYELOID LEUKEMIA VIA INDUCING LETHAL MITOPHAGY

2016 ◽  
Vol 64 (4) ◽  
pp. 947.1-947
Author(s):  
M Dany ◽  
B Ogretmen
Keyword(s):  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Targeted Therapy ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Flt3 Inhibitors ◽  
Pyridinium Ring ◽  
Ceramide Accumulation ◽  
Acute Myeloid

Mutations in FLT3 receptor tyrosine kinase are common targets in Acute Myeloid Leukemia (AML); however, FLT3 targeted therapy shows limited success due to development of resistance. Ceramide, a bioactive sphingolipid, is synthesized de novo by Ceramide Synthases (CerS) and mediates cancer cell death in response to various chemotherapeutic agents. This study investigates the biological role of ceramide lipid in the response of AML to FLT3 targeted therapy and aims at finding mechanism-based alternative therapeutic strategies to overcome resistance to FLT3 inhibitors. We found that AML cell lines and patient samples expressing FLT3 have suppressed CerS1 expression and lower levels of its product C18-ceramide compared with FLT3 negative AML cells. Silenced FLT3 expression or its pharmacological inhibition increased CerS1 and C18-ceramide levels while FLT3 overexpression suppressed them. The increase in C18-ceramide after FLT3 inhibition is required for cell death as silencing CerS1 expression or inhibiting its enzymatic activity protected from FLT3 inhibitors-induced cell death in vitro and in vivo. Mechanistically, FLT3 inhibition resulted in CerS1 translocation from cytosol to mitochondria resulting in generation and mitochondrial accumulation of C18-ceramide. The mitochondrial C18-ceramide then binds directly to LC3B-II to recruit autophagosomal membranes to mitochondria for the execution of lethal mitophagy and degradation of mitochondria. We also show that this process is regulated upstream by early Drp1 activation and p-Drp1 S637 de-phosphorylation, whereby silencing Drp1 expression or preventing its S637 dephosphorylation blocked the translocation of CerS1 to mitochondria, prevented ceramide mitochondrial accumulation, halted the events of lethal mitophagy, and protected from FLT3 inhibitors-induced cell death. Due to the importance of ceramide accumulation in mitochondria for AML cells to respond to FLT3 inhibition, we proposed a synthetic lipid compound, LCL-461, composed of C18-ceramide conjugated to a pyridinium ring in the sphingosine backbone. Mass spectrometry proved that LCL-461 accumulates selectively in mitochondrial fractions of AML cells due to the positively charged conjugated pyridinium ring. LCL-461 was effective in inducing cell death in several AML cell lines of different FLT3 mutation statuses and resistance profiles as well as in patient samples and in vivo xenografts, with minimal cytotoxicity effects on normal human bone marrow cells. LCL-461 induced cell death via the same LC3B dependent lethal mitophagy mechanism detected following FLT3 inhibition. This highlights the potential of LCL-461 as an agent that can bypass FLT3 signaling by accumulating in mitochondria to induce lethal mitophagy and AML cell death regardless of whether patients are sensitive or resistant to FLT3 targeted therapy.

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.

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.

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.

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.

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