The BH3 Mimetic, ABT-737, Overcomes Stromal-Mediated Pro-Survival Signals and Synergizes with PHA-767491, a Dual Cdc7/CDK9 Inhibitor, In Acute Myeloid Leukaemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1841-1841
Author(s):  
Ruth C Morrell ◽  
Eva Szegezdi ◽  
Anna Halpin-McCormick ◽  
Karen Cawley ◽  
Afshin Samali ◽  
...  
Keyword(s):  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
In Vivo Studies ◽  
Relative Reduction ◽  
Induced Apoptosis ◽  
Cdk9 Inhibitor ◽  
Acute Myeloid

Abstract Abstract 1841 ABT-737 is a small molecule inhibitor of Bcl-2 and Bcl-xL with reported activity in pre-clinical in-vitro and in-vivo studies of acute myeloid leukaemia(AML) but to date no data has been reported on its activity in an AML co-culture model. To address this, we examined the effects of co-culture of AML cell lines (MOLM-13, ML-1, KG-1, OCI-AML2) with HS5 cells, a human stromal cell line, on sensitivity to Ara-C and ABT-737. All cell lines cultured in the presence of HS-5 stroma demonstrated a significant reduction in Ara-C-induced apoptosis (% relative reduction - OCI-AML2:80%; ML1:65%; MOLM-13:53%; KG-1:55%) as compared to cells cultured in suspension in normal complete media, with the effect on expression of Bcl-2 family members being currently under evaluation. In contrast, in the presence of ABT-737, HS-5 co-culture did not provide any protective effect whatsoever to AML cells, with IC50 ranging from 0.1 to 0.3μM in the cell lines noted above, regardless of the presence of stroma. OCI-AML3, an AML cell line known to express high levels of Mcl-1 was resistant to ABT-737 in both normal suspension cultures and co-culture. Indeed Mcl-1, an important pro-survival protein in haematopoietic cells is thought to be a key factor promoting resistance to ABT-737 and it has recently been reported that transcriptional upregulation of Mcl-1 may follow exposure to ABT-737. Thus, the combination of ABT-737 with strategies to deplete Mcl-1 is particularly attractive. Cdk9 inhibition is such a strategy. Since Cdk9 phosphorylates RNA polymerase II affecting the rate of transcription, inhibition leads to a depletion of proteins with short half-lives, such as Mcl-1. Here we report that resistance of OCI-AML3 cells to ABT-737-induced apoptosis can be overcome by combination with PHA-767491, a novel dual Cdc7/CDK9 inhibitor. OCI-AML3 cells were treated with increasing concentrations of ABT-737, PHA-767491 or both. Co-administration resulted in a strong synergistic apoptosis-inducing effect as assessed by AnnexinV staining, with combination indices, as calculated by Chou et Talalay, for a range of doses of both drugs of <1 (range 0.3–0.9). Importantly, the sensitising effect of PHA-767491 was seen only at concentrations (≥ 2μM) that resulted in significant downregulation of Mcl-1 protein expression, implicating Mcl-1 downregulation as a possible cause of synergy. We are currently investigating the precise role of Mcl-1 in this regard. In conclusion, taken together, these studies support that ABT-737, possibly in combination with agents to deplete Mcl-1, represents a promising therapeutic strategy for AML and warrants further evaluation. Disclosures: No relevant conflicts of interest to declare.

A Crispr/Cas9 Drop-out Screen Identifies Genome-Wide Genetic Valnerubilities in Acute Myeloid Leukaemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 554-554
Author(s):  
Konstantinos Tzelepis ◽  
Hiroko Koike-Yusa ◽  
Etienne De Braekeleer ◽  
Yilong Li ◽  
Emmanouil Metzakopian ◽  
...  
Keyword(s):  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Cell Line ◽  
Cell Lines ◽  
Therapeutic Targets ◽  
Essential Genes ◽  
Drop Out ◽  
Genome Wide ◽  
Ht 29 ◽  
Acute Myeloid

Abstract Despite progress in understanding its genomics and molecular pathogenesis, the therapeutic landscape of acute myeloid leukaemia (AML) has changed little in the last 40 years. Whilst our improved molecular understanding of AML permits some optimism that progress may be forthcoming, an alternative approach for the identification of therapeutic targets is the agnostic interrogation of AML genomes for genetic vulnerabilities. In this study we apply a new and technically robust CRISPR-Cas9 platform to perform genome-wide screens for genetic vulnerabilities in human cancers. To do this, we develop and validate a CRISPR-based functional genomics toolkit composed of: i) lentiviral gRNA expression vectors harbouring an improved sgRNA scaffold, ii) Cas9 activity reporters for choosing cell line clones with high Cas9 nuclease activity and iii) an improved human genome-wide CRISPR library composed of 90,709 gRNAs targeting 18,010 genes. We first describe the timescale over which cells lacking individual essential genes are depleted from a pool of isogenic cells, thus providing the first such genome-wide framework for mammalian cells. As well as being of fundamental interest, such a temporal framework can be used to decide the length of time required for performing genetic screens and to select therapeutic targets. We then proceeded to perform drop-out screens with 30-day latencies in 5 AML cell lines (MV4-11, MOLM-13, OCI-AML2, OCI-AML3 and HL-60) and also in the non-AML lines HT-29 (colorectal adenocarcinoma) and HT-1080 (fibrosarcoma). Drop-out genes were identified using the MAGeCK algorithm as those showing significant depletion across their ≥5 cognate sgRNAs. From each cell line, more than 1,000 genes dropped out at FDR <20%, with the exception of MV4-11 where the number was slightly lower. Using these data we identified 881 "pan-essential genes" defined as those displaying significant depletion across ≥5 cell lines including HT-29 and HT-1080. These 881 genes can be used as a standard set of quality-control genes for future screens. Of these, 335 genes were depleted in all 7 cell lines, showing remarkable consistency across different cellular contexts. Next, we looked for genes that are specifically essential to AML cells by extracting genes depleted in at least 1 of the 5 AML cell lines, but not in HT-29 or HT-1080. This analysis identified approximately 150-200 essential genes for each cell line yielding a total of 510 AML-specific genes. Of these, 59 genes including RUNX1, CEBPA, CEBPB, MEN1, DOT1L and SMARCB1 were essential to 3 or more AML cell lines. GO analysis of these 59 genes showed particular enrichment in processes pertaining to chromatin modification and organisation, transcriptional regulation and nucleotide metabolism. We proceed to validate a number of novel drop-out genes using CRISPR-Cas9 with new sgRNAs and where possible with existing clinical/pre-clinical inhibitors. Furthermore, we identify oncogene-specific cell vulnerabilities, even for leukaemias driven by closely related oncogenes such as the MLL-AF4 (MV4-11) and MLL-AF9 (MOLM-13) fusion genes, which differed in their dependency on several genes including KAT2A and SRPK1. To validate these findings in primary cells, we generate a novel Rosa26-Ef1a-Cas9 mouse model and cross this with mice carrying Flt3-ITD. We then transformed Lin- haemopoietic cells from RosaCas9/+/Flt3ITD/+ mice using MLL-AF4- or MLL-AF9 -expressing retroviruses and validate the findings of our screens using sgRNAs against murine Kat2a and Srpk1. Taken together, these data dissecting the individual vulnerabilities of highly similar initiating mutations demonstrate the power of our screen to identify specific vulnerabilities for individual oncogenes and suggest that similar screens may also help to guide programmes of personalised medicine for patients based on the complement of somatic mutations within their cancer, which in some cases could be achieved through re-purposing of existing therapeutics. Disclosures McDermott: 14M Genomics: Other: co-founder, stock-holder and consultant.

scholarly journals Differences between intrinsic and acquired nucleoside analogue resistance in acute myeloid leukaemia cells

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Tamara Rothenburger ◽  
Dominique Thomas ◽  
Yannick Schreiber ◽  
Paul R. Wratil ◽  
Tamara Pflantz ◽  
...  
Keyword(s):  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Cell Lines ◽  
Nucleoside Analogue ◽  
Acquired Resistance ◽  
Nucleoside Analogues ◽  
Cross Resistance ◽  
Enzymatic Assays ◽  
Anti Cancer ◽  
Acute Myeloid

Abstract Background SAMHD1 mediates resistance to anti-cancer nucleoside analogues, including cytarabine, decitabine, and nelarabine that are commonly used for the treatment of leukaemia, through cleavage of their triphosphorylated forms. Hence, SAMHD1 inhibitors are promising candidates for the sensitisation of leukaemia cells to nucleoside analogue-based therapy. Here, we investigated the effects of the cytosine analogue CNDAC, which has been proposed to be a SAMHD1 inhibitor, in the context of SAMHD1. Methods CNDAC was tested in 13 acute myeloid leukaemia (AML) cell lines, in 26 acute lymphoblastic leukaemia (ALL) cell lines, ten AML sublines adapted to various antileukaemic drugs, 24 single cell-derived clonal AML sublines, and primary leukaemic blasts from 24 AML patients. Moreover, 24 CNDAC-resistant sublines of the AML cell lines HL-60 and PL-21 were established. The SAMHD1 gene was disrupted using CRISPR/Cas9 and SAMHD1 depleted using RNAi, and the viral Vpx protein. Forced DCK expression was achieved by lentiviral transduction. SAMHD1 promoter methylation was determined by PCR after treatment of genomic DNA with the methylation-sensitive HpaII endonuclease. Nucleoside (analogue) triphosphate levels were determined by LC-MS/MS. CNDAC interaction with SAMHD1 was analysed by an enzymatic assay and by crystallisation. Results Although the cytosine analogue CNDAC was anticipated to inhibit SAMHD1, SAMHD1 mediated intrinsic CNDAC resistance in leukaemia cells. Accordingly, SAMHD1 depletion increased CNDAC triphosphate (CNDAC-TP) levels and CNDAC toxicity. Enzymatic assays and crystallisation studies confirmed CNDAC-TP to be a SAMHD1 substrate. In 24 CNDAC-adapted acute myeloid leukaemia (AML) sublines, resistance was driven by DCK (catalyses initial nucleoside phosphorylation) loss. CNDAC-adapted sublines displayed cross-resistance only to other DCK substrates (e.g. cytarabine, decitabine). Cell lines adapted to drugs not affected by DCK or SAMHD1 remained CNDAC sensitive. In cytarabine-adapted AML cells, increased SAMHD1 and reduced DCK levels contributed to cytarabine and CNDAC resistance. Conclusion Intrinsic and acquired resistance to CNDAC and related nucleoside analogues are driven by different mechanisms. The lack of cross-resistance between SAMHD1/ DCK substrates and non-substrates provides scope for next-line therapies after treatment failure.

scholarly journals Suppressor of cytokine signalling-1 gene silencing in acute myeloid leukaemia and human haematopoietic cell lines

2004 ◽  
Vol 126 (5) ◽  
pp. 726-735 ◽  
Author(s):  
Dai Watanabe ◽  
Sachiko Ezoe ◽  
Minoru Fujimoto ◽  
Akihiro Kimura ◽  
Yoshiyuki Saito ◽  
...  
Keyword(s):  
Acute Myeloid Leukaemia ◽  
Gene Silencing ◽  
Myeloid Leukaemia ◽  
Cell Lines ◽  
Haematopoietic Cell ◽  
Acute Myeloid

Characteristics of a Cell Line Established from a Child with Familial Acute Myeloid Leukaemia

2008 ◽  
Vol 46 (1) ◽  
pp. 23-31 ◽  
Author(s):  
Patricia Fischer ◽  
A. Karpas ◽  
Elisabeth Nacheva ◽  
O. Haas ◽  
Heide Winterixitner ◽  
...  
Keyword(s):  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Cell Line ◽  
A Cell ◽  
Acute Myeloid

2′–Cyano–2′–Deoxyarabinofuranosylcytosine Is Active in Acute Myeloid Leukaemia and Acts in Synergy with Cytarabine.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4153-4153 ◽  
Author(s):  
Elisabeth J Walsby ◽  
Chiara Ghiggi ◽  
Ruth H Mackay ◽  
Simon R Green ◽  
Steven Knapper ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Cell Lines ◽  
Prolonged Exposure ◽  
Nucleoside Transporters ◽  
Deoxycytidine Kinase ◽  
Dna Breaks ◽  
Cytotoxic Effects ◽  
Acute Myeloid

Abstract Abstract 4153 2′–Cyano–2′–deoxyarabinofuranosylcytosine (CNDAC) is the metabolic product of sapacitabine following hydrolysis of the palmitoyl sidechain from the pyrimidine analog primarily by plasma, gut and liver amidases. CNDAC is in turn phosphorylated into the active triphosphate form (CNDACTP) by deoxycytidine kinase (dCK). CNDACTP is incorporated into DNA resulting in single stranded DNA breaks during replication and inducing cell cycle arrest. Previously the cytotoxic effects of CNDAC have also been associated with intracellular accumulation of CNDAC triphosphate and chain termination. CNDAC and sapacitabine have overlapping cytotoxic effects. Acute myeloid leukaemia (AML) cell lines NB4 and HL-60 had an LD50 of 0.24μM (± 0.24) for CNDAC and 0.23μM (± 0.21) for cytarabine (AraC) following 24 hours treatment. Primary AML blasts isolated from patients at diagnosis (n = 15) had a higher mean LD50 (25.22μM ± 19.41) for CNDAC and AraC (8.09μM ± 8.93). This is thought to be due to the requirement of cells to be actively cycling in order to be susceptible to these agents. CNDAC induces apoptosis in NB4 and HL-60 cell lines with significant increases in the percentage of cells with increased Annexin V/propidium iodide staining at concentrations of 1.0μM and above (P < 0.04) and significant caspase-3 activation at concentrations of 0.1μM and above (P < 0.05). Treatment with CNDAC also results in a significant concentration-dependent accumulation in the G2 phase of the cell cycle after 24 hours in NB4 and HL-60 cells (P = 0.003 and 0.011 respectively). Synergy was observed in the AML cell lines when CNDAC was combined with AraC at a ratio of 2:1 The mean combination index for CNDAC and AraC was 0.67 (± 0.21). The activity of deoxycytidine kinase (dCK) was blocked by the addition of excess deoxycytidine, under these conditions the effects of CNDAC were abrogated (P < 0.05) in NB4 and HL-60 cells suggesting that CNDAC requires phosphorylation by dCK for its activation in the cells. The nucleoside transporters hENT 1 and 2 and hCNT3 transport a range of nucleoside analogues through the cell membrane into cells, the use of hENT inhibitors led to a 2.5 fold increase in the LD50 for CNDAC (P = 0.028) over 48 hours. This prolonged exposure to CNDAC could have resulted in some passive uptake of CNDAC into the cells potentially explaining why the agent retained some cell killing activity. Equivalent results have been obtained with dCK and hENT inhibitors in other cell lines indicating that there is a general requirement for these enzymes for CNDAC activity. Interestingly, when cells are treated with the parent drug sapacitabine in the presence of excess deoxycytidine the cytotoxicity is reduced, but when cells are treated in the presence of hENT inhibitors, sapacitabine's cytotoxicity is improved. This suggests that the presence of the palmitoyl side-chain allows membrane permeability even in the absence of the traditional nucleoside transporters. Disclosures: Green: Cyclacel Ltd: Employment.

Knockdown of MicroRNA-10a in Acute Myeloid Leukaemia Cells Bearing the Nucleophosmin1 Mutation Causes Cell Death and Reduced Clonogenicity

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1367-1367
Author(s):  
Adam J Bryant ◽  
Catalina A Palma ◽  
Mark Lutherborrow ◽  
Vivek Jayaswal ◽  
Yee Hwa Yang ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Cell Line ◽  
Caspase 3 ◽  
Luciferase Reporter ◽  
Unknown Etiology ◽  
Wild Type ◽  
Over Expression ◽  
Acute Myeloid

Abstract Abstract 1367 Acute Myeloid Leukaemia (AML) with a mutation in the Nucleophosmin1 gene (NPM1c+) accounts for one of the largest subtypes of AML, with an unknown etiology. MicroRNA dysregulation has now been implicated in the oncogenesis of many cancers including AML. We sought to investigate the role of microRNAs in the initiation and development of AML with the NPM1c+ mutation. MicroRNA profiling of bone marrow samples from 28 AML patients and confirmation by qRT-PCR demonstrated a unique microRNA signature in AML-NPM1c+ samples dominated by miR-10a over-expression of 19.6-fold compared to Nucleophosmin1 wild type (NPM1) samples. Functional assessments were performed in the human OCI-AML3 cell line, which is the only cell line to harbour NPM1c+. miR-10a repression was induced by transfection with miRCURY LNA microRNA knockdown probes (Exiqon). Cell growth (MTS) assay demonstrated a significant decrease of 19% in miR-10a knockdown cells compared to the Scrambled control. AnnexinV and Caspase 3 assays assessed the effect of miR-10a knockdown on apoptosis. miR-10a knockdown increased the proportion of AnnexinV positive events when compared to control treated cells by 34.9% and 39.3% at 24 and 48 hours respectively, but had no effect on Caspase 3 expression. Proliferation (BrdU uptake) assays did not show a change, however, clonogenic assays demonstrated a 26.1% decrease in colony number in miR-10a knockdown cells compared to the control. Potential mechanisms were elucidated by determining miR-10a mRNA targets in silico and confirmed by luciferase reporter assays. These included ARNT, GTFH1, ID4, KLF4, MAPRE1, NR4A3, RB1CC1 and TFAP2C. In this study, we have demonstrated that miR-10a was highly differentially expressed between AML-NPM1c+ cells compared to leukaemic cells bearing wild type NPM1. Knockdown of miR-10a in OCI-AML3 cells resulted in increased cell death as detected by AnnexinV binding (but not Caspase 3, indicating an effect independent of the classical apoptotic pathways) and reduced clonogenic capacity. These effects are thought to occur through miR-10a mediated modulation of ARNT, GTFH1, ID4, KLF4, MAPRE1, NR4A3, RB1CC1 and TFAP2C, all of which are associated with neoplastic transformation. Taken together, our results suggest that aberrant miR-10a over-expression in AML-NPM1c+ patients promotes cell survival. Disclosures: No relevant conflicts of interest to declare.

The Potential Role of STAT's in Anti-Leukemic Therapy with Different Drugs

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5120-5120
Author(s):  
Hatice Demet Kiper ◽  
Burcin Tezcanli Kaymaz ◽  
Ozlem Purclutepe ◽  
Ceyda Tunakan Dalgic ◽  
Nur Selvi ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Drug Exposure ◽  
Conflicts Of Interest ◽  
Time Dependent ◽  
Blue Dye ◽  
Dependent Manner ◽  
Down Regulation ◽  
Expression Levels ◽  
Induced Apoptosis

Abstract Abstract 5120 STAT pathways play a pivotal role in oncogenesis and leukemogenesis, thus targeting STAT signalling appears to be an effective anticancer treatment strategy. It has been described that constitutive activation of STAT3 and STAT5 plays a pro-oncogenic role both in acute and chronic myeloid neoplasms. In this study, we aimed to clarify the potential relationship between drug-induced apoptosis with different agents and STAT pathway. A third-generation bisphosphonate; zoledronate, an angiotensin-converting enzyme inhibitor (ACE-I); enalapril, a proteasome inhibitor which is used for treatment of multiple myeloma; bortezomib and a second-generation tyrosine kinase inhibitor; dasatinib were examined in this goal. Cell viability and cytotoxicity tests were conducted by using Trypan blue dye exclusion and XTT assays, respectively. Apoptotic analyses were performed by AnnexinV-EGFP staining method and fluorescence microscopy. Expression levels of STAT3, −5A and −5B genes were analysed in myeloid cell lines by qRT-PCR. The results showed that zoledronate; bortezomib and dasatinib decreased viability and proliferation and induced apoptosis in CML cell line K562 in a dose- and time-dependent manner which is associated by prominent decrease of STAT3, STAT5A and STAT5B mRNA expressions. Enalapril was also found to be cytotoxic and induced apoptosis in APL cell line HL60 in a dose- and time-dependent manner and the expression levels of STAT5A gene have significantly reduced in enalapril-treated HL60 cells as compared to untreated controls. Treatments of cell lines with other drugs were also associated with significant apoptosis in certain time points. The results and changes in expression of STAT's in mRNA level at 72nd hours are summarized in table. Taken together all these data showed that targeting STAT pathways by different drugs may be an appropriate approach in anti-leukemic therapy. This finding is important to propose that discovery or identification of novel agents targeted STATs may open new windows to the other hematological and solid malignancies which are associated with aberrant STAT expression. Table: The changes in STAT expressions after drug exposure in time-dependent manner with the dose of IC50. DRUGS CELL LINE IC50 APOPTOSIS (%) STAT3 mRNA Down Regulation (%) STAT5A mRNA Down Regulation (%) STAT5B mRNA Down Regulation (%) ENALAPRIL HL-60 7 μM 20 20* 76 5* ZOLEDRONATE K562 60 μM 34 63 31 57 BORTEZOMIB K562 177 μM 37 98 100 99 DASATINIB K562 3,314 nM 75 NA 33 78 * : Not significant NA: not applied Disclosures: No relevant conflicts of interest to declare.

scholarly journals Metabolomic Profiling of Drug Responses in Acute Myeloid Leukaemia Cell Lines

PLoS ONE ◽  
2009 ◽  
Vol 4 (1) ◽  
pp. e4251 ◽  
Author(s):  
Stefano Tiziani ◽  
Alessia Lodi ◽  
Farhat L. Khanim ◽  
Mark R. Viant ◽  
Christopher M. Bunce ◽  
...  
Keyword(s):  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Cell Lines ◽  
Leukaemia Cell ◽  
Drug Responses ◽  
Metabolomic Profiling ◽  
Acute Myeloid Leukaemia Cell ◽  
Acute Myeloid

Annexin II cell surface and mRNA expression in human acute myeloid leukaemia cell lines

2005 ◽  
Vol 115 (1-2) ◽  
pp. 109-114 ◽  
Author(s):  
Shane A. Olwill ◽  
Hugh McGlynn ◽  
William S. Gilmore ◽  
H. Denis Alexander
Keyword(s):  
Cell Surface ◽  
Acute Myeloid Leukaemia ◽  
Mrna Expression ◽  
Myeloid Leukaemia ◽  
Cell Lines ◽  
Annexin Ii ◽  
Leukaemia Cell ◽  
Acute Myeloid Leukaemia Cell ◽  
Acute Myeloid
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