Effect of Parthenolide on Stem Cells in Childhood Acute Lymphoblastic Leukaemia.

Abstract

Current therapies for the treatment of childhood acute lymphoblastic leukaemia (ALL) have resulted in vastly improved survival rates of around 80% in recent years. Despite these successes, around 15% of patients die of the disease and relapse is the most common cause of treatment failure. Intensification of treatment to prevent or treat relapse may not be a feasible approach due to an increased risk of significant adverse effects. It is possible that ALL may be maintained by a subpopulation of stem cells that are resistant to regimens designed to kill the bulk population and subsequent relapses may arise from these stem cells. Consequently, there is a need to assess the efficacy of therapeutic agents on ALL stem cells. We have previously shown that ALL cells that are capable of initiating and sustaining the disease in serial xenografts have a CD34+/CD19− phenotype. Furthermore, these putative ALL stem cells were resistant to treatment in vitro with dexamethasone and vincristine, two agents routinely used in the treatment of paediatric leukaemia. In this investigation we have examined the effects of the sesquiterpene lactone parthenolide (PTL), a natural compound which induces oxidative stress and inhibits NF-kB. PTL effectively eradicates stem cells in AML and B-CLL in vitro while sparing normal heamopoietic cells. Unsorted leukaemia cells from 11 cases, with mixed prognostic subgroups, were co-cultured with and without PTL at a range of 0–10μM for 18–24 hours. Cell viability and apoptosis were evaluated by flow cytometry using annexin V and propidium iodide staining. Four out of the 11 cases were relatively resistant to treatment with PTL with only small reductions in viability (<5%) and no significant effect on apoptosis, even at the highest dose evaluated. There was no correlation between the prognostic risk group and the response to PTL. Primary cells from the 4 resistant cases were sorted into CD34+/CD19+ and CD34+/CD19− subfractions to assess the effect of PTL on these cells. The effects of PTL on the CD34+/CD19+ population were similar to that observed with the unsorted leukaemia cells. The CD34+/CD19− population was completely resistant to treatment with PTL, with more cells surviving treatment than the unsorted cells (P=0.03). In the 7 responding cases, the viability of the unsorted cells decreased to 28.4±7.1% and 38±12% were apoptotic following treatment. Very similar effects were observed with the CD34+/CD19+ subfraction in these responding cases with viability reduced to 33.4±6% and 35.9±14% were apoptotic. In contrast, the CD34+/CD19− cells from these 7 cases were significantly resistant to PTL with viabilities >75% at all concentrations evaluated (P<0.003). Apoptosis was 2.6-fold lower at 10μM PTL in the CD34+/CD19− subfraction compared to the unsorted cells (P=0.05). FISH analyses were performed on the viable cells at the end of the time-course and confirmed that leukaemia cells were surviving treatment with PTL. CD34+/CD38− cells from normal peripheral blood samples were also found to be resistant to treatment with PTL and survival of these cells at 10μM was not significantly different to that observed in the CD34+/CD19− ALL population in all 11 cases (P=0.38). Studies to assess the functional capacity of PTL-treated ALL cells are ongoing. These data demonstrate that while PTL shows promising effects on the bulk leukaemia population in some ALL cases, it had no significant effect on the putative ALL stem cell population. In each case examined, the CD34+/CD19− cells were resistant to short term exposure to PTL and responded in a similar manner to normal haemopoietic stem cells. These findings highlight the importance of evaluating therapeutic agents in the context of leukaemia stem cell populations and not just on the bulk leukaemia. Future studies are warranted to gain insight into how the drug sensitivity of ALL stem cells may be mediated.