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BackgroundCancer stem cells are a chemotherapy-resistant population capable of self-renewal and of regenerating the bulk tumor, thereby causing relapse and patient death. Ewing's sarcoma, the second most common form of bone tumor in adolescents and young adults, follows a clinical pattern consistent with the Cancer Stem Cell model – remission is easily achieved, even for patients with metastatic disease, but relapse remains frequent and is usually fatal.Methodology/Principal FindingsWe have isolated a subpopulation of Ewing's sarcoma cells, from both human cell lines and human xenografts grown in immune deficient mice, which express high aldehyde dehydrogenase (ALDHhigh) activity and are enriched for clonogenicity, sphere-formation, and tumor initiation. The ALDHhigh cells are resistant to chemotherapy in vitro, but this can be overcome by the ATP binding cassette transport protein inhibitor, verapamil. Importantly, these cells are not resistant to YK-4-279, a small molecule inhibitor of EWS-FLI1 that is selectively toxic to Ewing's sarcoma cells both in vitro and in vivo.Conclusions/SignificanceEwing's sarcoma contains an ALDHhigh stem-like population of chemotherapy-resistant cells that retain sensitivity to EWS-FLI1 inhibition. Inhibiting the EWS-FLI1 oncoprotein may prove to be an effective means of improving patient outcomes by targeting Ewing's sarcoma stem cells that survive standard chemotherapy.

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Abstract. Purine analogues were used in this study to dissect specific steps in the mechanism of action of nerve growth factor (NGF). Protein kinase N (PKN) is an NGF-activated serine protein kinase that is active in the presence of Mn ÷÷. The activity of PKN was inhibited in vitro by purine analogues, the most effective of which was 6-thioguanine (apparent K~ = 6 #M). Several different criteria indicated that 6-thioguanine is not a general inhibitor of protein kinases and that it is relatively specific for PKN. For instance, it did not affect protein kinases A or C and was without effect on the overall level and pattern of protein phosphorylation by either intact or broken PC12 cells. Since purine analogues rapidly and effectively enter cells, they were also assessed for their actions on both transcription-dependent and -independent responses of PC12 cells to NGE NGF-promoted neurite regeneration was reversibly suppressed by the analogues and at concentrations very similar to those that inhibit PKN. Comparable concentrations of the analogues also blocked NGF-stimulated induction of ornithine decarboxylase activity. In contrast to its inhibition of neurite regeneration and ornithine decarboxylase induction, 6-thioguanine did not suppress NGF-dependent induction of c-los mRNA expression. Thus, purine analogues such as 6-thioguanine appear capable of differentially suppressing some, but not other actions of NGE These findings suggest the presence of multiple pathways in the NGF mechanism and that these can be dissected with purine analogues. Moreover, these data are compatible with a role for protein kinase N in certain of these pathways.S INCE the characterization of nerve growth factor (NGF) ~ (27) as a neurotrophic agent necessary for the development and function of certain peripheral and CNS neurons (11,21,26,32), many events in its mode of action have been revealed (cf. reference 25 for review), but we are still far from a complete understanding of the entire process. The biological effects of NGF comprise both transcription-dependent 05, 24) and -independent responses (4, 18). To provide neuronal differentiation and trophic support, these responses must be coordinated in the cell by means that are still unknown. In this regard, many studies have been performed with the PC12 rat pheochromocytoma cell line (13), a model system that has been extensively used to examine the NGF mechanism of action.A parameter that is likely to be causally involved in many of the events controlled by NGF in the target cell is the regulation of protein phosphorylation. It is well-documented that NGF, like many other growth factors, promotes rapid changes in the phosphorylation of specific cellular proteins 1. Abbreviations used in this paper: 2-AP, 2-aminopurine; NGF, nerve growth factor; ODC, ornithine decarboxylase; PKA, cyclic AMP-dependent protein kinase; PKC, Ca++/phospholipid-dependent protein kinase; PKN, protein kinase N; 6-TG, 6-thioguanine. (1,18,19,34), as well as regulation of several different protein kinase activitie...