Effectiveness of Transcriptional CDK Inhibitors in Targeting Non-Proliferating CD34+CD38- AML Cells,

Abstract 3482

Quiescent cells conserve energy and are characterised by low RNA synthesis. In contrast, cancer cells are thought to be addicted to high RNA synthesis, particularly synthesis of survival molecules. We hypothesised that quiescent cancer cells, already low in RNA, would be sensitive to apoptosis induced by transcriptional cyclin-dependent kinase (CDK) inhibitors that further deplete RNA. We cultured the CD34+CD38- KG1a cell line continuously in the presence of an mTOR inhibitor, which maintained excellent viability and enriched the cells for quiescent stem cell features including low RNA content, low metabolism, low reactive oxygen species production and decreased size. Sensitivity to mitochondrial pore transition was similar in proliferating and quiescence-enriched cells, indicating that the basal mitochondrial apoptotic machinery is neither impaired nor improved. We treated quiescence-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 transcriptional CDKs - flavopiridol, roscovotine and TG02. All of the agents used showed increased kill in the unmanipulated compared to the quiescence-enriched cells, emphasising the chemoresistant nature of quiescent cells. To put a value on the difficulty of eradicating the leukamic clone, we used the parameter 2 X IC₅₀ (which theoretically would kill 100% cells in a totally homogeneous population). In quiescence-enriched cells, the percentage of cells killed at 2 X IC₅₀ was roscovotine - 80%, TG02 – 72%, flavopiridol - 62%, 5-azacydidine - 61%, daunorubicin, clofarabine and irinotecan - 60%, etoposide - 58%, and for araC no IC₅₀ for quiescence-enriched cells was reached at 20 times the IC₅₀ of proliferating cells. The data show conclusively that, in this model, transcriptional CDK inhibitors outperformed the agents conventionally used in AML chemotherapy. We also showed that transcriptional CDK inhibitors induced serine 2 dephosphorylation of RNA polymerase II in equal measure in proliferating and quiescence- enriched cells. We have already shown that TGO2 targets primary CD34+CD38- cells in vitro (ASH 2010, abstract 1823). Here we also show that in four primary AML samples sensitive to roscovotine in vitro, the median percentage kill is greater in the CD34+CD38- subset, (which consists largely of quiescent cells and tends to contain the leukaemic stem cells,) than in the bulk cells (40.5% versus 19.5% kill at 2 micromolar roscovotine). We suggest that transcriptional CDK inhibitors may be a useful class of agent for targetting the occult quiescent CD34+CD38- cells thought to contribute to relapse in AML.