Abstract
BackgroundDormant 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.MethodsWe 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.ResultsCulture 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.ConclusionWe suggest that RP2 inhibitors may be a useful class of agent for targeting dormant leukaemia cells.
Highlights
Dormant cells are characterised by low RNA synthesis
Results Mammalian target of rapamycin (mTOR) inhibition induces the principal features of dormant cells Given that inhibition of the mTOR pathway is experimentally proven to maintain the in vivo dormancy and transplantability of haematopoietic and leukaemic cells [25,26,27,28], we experimented with the possibility of inhibiting growth in a leukaemic cell line with the mTOR inhibitor rapamycin
We show that continuous culture of KG1a cells in 100 nM rapamycin for up to 11 days induced no detectable apoptosis, whereas serum withdrawal, the common method for inducing cells to exit the cell cycle, induced a statistically significant induction of Annexin V within 48 hours, and most cells were dead within a week (Figure 1A,B)
Summary
Dormant cells are characterised by low RNA synthesis. 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. Relapse in cancer patients after therapy is due to the continued presence of a subset of cells which is likely to have evaded the effects of treatment by lying dormant [1,2]. Dormant cells are characterised by low levels of RNA, consistent with their lack of proliferation and need to conserve energy [3]. A large amount of material is needed to study mechanisms of drug targeting and resistance, but dormant cancer cells are rare. In contrast to primary samples, leukaemia cell lines are plentiful and highly proliferative, so we sought a suitable method of inducing dormancy in these cells
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