Combining BCL-2 Inhibitors with Parthenolide to Treat Childhood Acute Lymphoblastic Leukaemia
Authors: Diamanti Paraskevi , Barendt William J , Ede Benjamin C , Cox Charlotte V. , Blair Allison 
Published: October 2019

Abstract
Current therapies for the treatment of childhood acute lymphoblastic leukaemia (ALL) have resulted in vastly improved survival rates of around 90% in recent years. Despite these successes, around 15% of patients die of relapse. It is possible that ALL may be maintained by subpopulations of cells, known as leukaemia stem cells (LSC), that are resistant to therapy and subsequent relapses may arise from these cells. Parthenolide (PTL), a naturally occurring sesquiterpene lactone, is an NF-κB inhibitor that kills leukaemia cells by apoptosis and/or increase of reactive oxygen species. PTL has been shown to be remarkably effective against several LSC subpopulations in vivo, with complete ablation of leukaemia. In a minority of cases, leukaemia burden was reduced following PTL treatment but not eliminated. Therefore, it may be necessary to combine PTL with other agents to improve killing of all LSC subpopulations. Another pathway of increasing interest in the treatment of leukaemias is the BCL-2 family. BCL-2 has been shown to be overexpressed in over 66% of B-ALL cases and is associated with tumourigenesis in several cancers. ABT-263 is an inhibitor of BCL-2, BCL-xL and BCL-w, it has been shown to selectively target AML LSC and is in early clinical trials in lymphoid malignancies. ABT-199 is another promising inhibitor that is currently in clinical trials for CLL. ABT-199 is specific for BCL-2 and has minimal risk for thrombocytopenia. In the present study the effects of both ABT-263 and ABT-199 alone or in combination with PTL were assessed in childhood ALL samples to determine whether toxicity to leukaemia cells could be improved in vitro and in vivo. The viability of bulk cells from 11 B cell precursor (BCP) ALL cases and 11 cord blood (CB) samples following drug treatment for 24 hours were assessed using flow cytometry by staining with Annexin V and propidium iodide. Initially, PTL was used at a range of 1 to 10μM, ABT-263 from 0.025 to 1μM and ABT-199 from 0.1 to 10μM. Only PTL and ABT-263 significantly reduced the viability of ALL cells compared to CB with IC50 values of 1.2μM and 0.125μM (P≤0.01 and P≤0.0015), respectively. In vitro drug combination studies demonstrated synergism when combining PTL with ABT-263 in a 9.5:1 ratio using the Chou Talalay model. The viability of ALL cells following combination therapy (1.2μM PTL with 0.125μM ABT-263) was reduced to 38.3±32.5%, while CB viability was unaffected (96.9±29%, P<0.0001). Using this combined dose, toxicity to ALL cells was increased by a further 35% compared to PTL alone and by 25% compared to ABT-263 alone. Even at the highest combined doses tested (9.6μM PTL: 1μM ABT-263) normal CB remained relatively unaffected with 73.3±25% surviving. The effects of these drugs alone and in combination were also assessed in LSC subpopulations in 3 of these cases. Unsorted ALL cells, CD34+/CD19+ and CD34-/CD19+ subpopulations were the most responsive with viabilities ranging from 17.6±4% to 23.9±11% using 1.2μM PTL and 0.125μM ABT-263. The CD34+/CD19- and CD34-/CD19- cells were more resistant with 70.3±40% and 73.3±15% surviving, respectively. However, since we have previously shown that the effects of in vitro drug treatment do not always accurately reflect the response in vivo, it was important to evaluate the effects of these drugs in mice with established leukaemia. NOD/LtSz-scid IL-2Rγc null (NSG) mice were inoculated with 1-1.15x106 unsorted BCP-ALL cells. Once the levels of leukaemia engraftment in murine peripheral blood reached ≥ 0.1%, mice were treated with 100mg/kg ABT-263 or ABT-199 and vehicle by oral gavage for 21 consecutive days and the levels of leukaemia burden were monitored weekly. Results to date demonstrate that leukaemia levels continued to rise in placebo-treated mice, reaching 49.2±7% by day 21, while the levels in ABT-263 and ABT-199 treated mice were significantly lower at 8.6±10% and 23.7±12%, respectively (P<0.0001). The use of ABT-263 and ABT-199 significantly improved the survival of NSG compared to untreated controls (P=0.0001). These data indicate that combining PTL with ABT-263 shows promising results in the killing of bulk and LSC populations in BCP-ALL. Ongoing in vivo studies will assess the potential of using BCL-2 inhibitors in combination with PTL compared to standard chemotherapeutics.

Disclosures
No relevant conflicts of interest to declare.