14-methyl-20-oxa-5-7-14-26-tetraazatetracyclo(19.3.1.1(2-6).1(8-12))heptacosa-1(25)-2(26)-3-5-8(27)-9-11-16-21-23-decaene and Leukemia--Myeloid

Direct co-operation between sensitiser molecules BAD and NOXA in mediating apoptosis suggests that therapeutic agents which sensitise to BAD may complement agents which sensitise to NOXA. Dynamic BH3 profiling is a novel methodology that we have applied to the measurement of complementarity between sensitiser BH3 peptide mimetics and therapeutic agents. Using dynamic BH3 profiling, we show that the agent TG02, which downregulates MCL-1, sensitises to the BCL-2-inhibitory BAD-BH3 peptide, whereas the BCL-2 antagonist ABT-199 sensitises to MCL-1 inhibitory NOXA-BH3 peptide in acute myeloid leukaemia (AML) cells. At the concentrations used, the peptides did not trigger mitochondrial outer membrane permeabilisation in their own right, but primed cells to release Cytochrome C in the presence of an appropriate trigger of a complementary pathway. In KG-1a cells TG02 and ABT-199 synergised to induce apoptosis. In heterogeneous AML patient samples we noted a range of sensitivities to the two agents. Although some individual samples markedly favoured one agent or the other, in the group as a whole the combination of TG02 + ABT-199 was significantly more cytotoxic than either agent individually. We conclude that dynamic NOXA and BAD BH3 profiling is a sensitive methodology for investigating molecular pathways of drug action and complementary mechanisms of chemoresponsiveness.

Topics: Acute Disease; Antineoplastic Agents; Apoptosis; Biomimetic Materials; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cyclin D1; Cytochromes c; Drug Synergism; Gene Expression Regulation, Leukemic; Heterocyclic Compounds, 4 or More Rings; Humans; Leukemia, Myeloid; Myeloid Cell Leukemia Sequence 1 Protein; Peptide Fragments; Proto-Oncogene Proteins; Reverse Transcriptase Polymerase Chain Reaction; Sulfonamides

Dormant cells are characterised by low RNA synthesis. In contrast, cancer cells can be addicted to high RNA synthesis, including synthesis of survival molecules. We hypothesised that dormant cancer cells, already low in RNA, might be sensitive to apoptosis induced by RNA Polymerase II (RP2) inhibitors that further reduce RNA synthesis.. We cultured leukaemia cells continuously in vitro in the presence of an mTOR inhibitor to model dormancy. Apoptosis, damage, RNA content and reducing capacity were evaluated. We treated dormancy-enriched cells for 48 hours with the nucleoside analogues ara-C, 5-azacytidine and clofarabine, the topoisomerase targeting agents daunorubicin, etoposide and irinotecan and three multikinase inhibitors with activity against RP2 - flavopiridol, roscovitine and TG02, and we measured growth inhibition and apoptosis. We describe use of the parameter 2 × IC50 to measure residual cell targeting. RNA synthesis was measured with 5-ethynyl uridine. Drug-induced apoptosis was measured flow cytometrically in primary cells from patients with acute myeloid leukaemia using a CD34/CD71/annexinV gating strategy to identify dormant apoptotic cells.. Culture of the KG1a cell line continuously in the presence of an mTOR inhibitor induced features of dormancy including low RNA content, low metabolism and low basal ROS formation in the absence of a DNA damage response or apoptosis. All agents were more effective against the unmanipulated than the dormancy-enriched cells, emphasising the chemoresistant nature of dormant cells. However, the percentage of cell reduction by RP2 inhibitors at 2 × IC50 was significantly greater than that of other agents. RP2 inhibitors strongly inhibited RNA synthesis compared with other drugs. We also showed that RP2 inhibitors induce apoptosis in proliferating and dormancy-enriched KG1a cells and in the CD71neg CD34pos subset of primary acute myeloid leukaemia cells.. We suggest that RP2 inhibitors may be a useful class of agent for targeting dormant leukaemia cells.

Topics: Acute Disease; Adenine Nucleotides; Antineoplastic Agents; Apoptosis; Arabinonucleosides; Azacitidine; Cell Line, Tumor; Cell Survival; Clofarabine; Cytarabine; Daunorubicin; Dose-Response Relationship, Drug; Enzyme Inhibitors; Etoposide; Flavonoids; Heterocyclic Compounds, 4 or More Rings; Humans; Leukemia, Myeloid; Piperidines; Purines; RNA Polymerase II; RNA, Neoplasm; Roscovitine; Sirolimus; TOR Serine-Threonine Kinases