Abstract
Background: To improve outcomes for pediatric acute myeloid leukemia (AML), identifying and targeting mechanisms of therapy resistance is critical. Recently, the antimicrobial drug atovaquone (AQ) was shown to reduce IL-6/STAT3 signaling in vitro and to improve relapse-free survival in adult AML patients who used AQ for prophylaxis after bone marrow transplant (Xiang et al, Blood, 2016). Clinically, this FDA approved drug is well tolerated, even in patients with organ dysfunction. It has established pediatric dosing, and plasma concentrations at standard doses are typically 40-80µM. We performed in vitro and in vivo experiments to evaluate the efficacy of AQ as a single agent and in combination with standard induction chemotherapy (cytarabine, daunorubicin, and etoposide). In order to ensure clinical relevance, all experiments examined cytotoxicity at clinically achievable doses.
Methods: AML cell lines were incubated with vehicle control or increasing doses of AQ (1µM - 30µM) for 24, 48, 72, or 96 hours, and then apoptosis was quantified by Annexin-V staining. Primary pediatric AML cells were exposed to 30µM AQ for the same time periods. To reduce spontaneous apoptosis of primary AML cells over prolonged culture, samples were cultured in HS5 stromal cell-conditioned media (CM) or co-cultured with HS5 stromal cells. For in vivo studies, female NSG mice were injected with 5x105 THP-1 cells expressing firefly luciferase (THP-1.ffluc). Mice were randomly assigned to receive AQ 200 mg/kg daily (n=6) or vehicle (n=5) by oral gavage, starting on day 1, and disease burden was monitored by bioluminescent imaging 3 times a week. A cohort of male mice receiving VC or AQ starting the day of injection with THP-1.ffluc cells were followed for survival (n=7 for each group). For in vitro drug combination cytotoxicity assays, 103 THP-1.ffluc cells per well were plated in 96 well plates with media only, single drugs at increasing concentrations, and AQ+chemotherapy combinations (AQ 1-15uM, AraC 0.1-3uM, Dauno 0.03-0.6uM, VP-16 0.3-3uM). After 48 hours, viability was determined by luminescence. Background luminescence of media-only wells was subtracted from all values and means of 4 replicates were used for analysis. All values were normalized to the cells + vehicle wells, which were set to 0 to quantify the percent affected. The Calcusyn method was used to calculate the combination index (CI). An AQ+chemotherapy combination was considered synergistic if CI<0.8, additive if CI>0.8 and <1.2, or antagonistic if CI>1.2.
Results: All 7 AML cell lines demonstrated time and dose-dependent induction of apoptosis in response to 1-30µM doses of AQ. IC50s were within the range of typical clinically achievable doses. For AML cell lines, the IC50s (in µM) after 72 hour incubation were: MOLM13 13.7, MV4-11 14.0, THP-1 15.7, NB4 16.9, Kasumi 1 18.6, HL60 33.7, KG1 98.9. AQ was generally less potent against the CML cell line K562 and the precursor B ALL cell line RS4 (72 hour IC50s were 116.6 and 81.4 respectively). All tested primary samples demonstrated AQ induced apoptosis that increased over time. After subtracting spontaneous apoptosis at each timepoint, the apoptosis induced by AQ at 72 hours for 5 patient samples supported by co-culture were 36, 37, 39, 67 and 87%. Annexin-V+% for the same patient samples supported by CM were 19, 13, 51, 47, and 24%. Bioluminescent imaging of female NSG mice xenografted with THP-1.ffluc cells and treated with single agent AQ daily starting on the day of cell injection demonstrated decreased disease burden compared to control mice (for example on day 21, the average radiance of VC treated mice vs AQ treated mice was 2 x 105 vs 6.3 x 104 p/s/cm²/sr; p=.0048 by ANOVA). Single agent AQ prolonged survival of male xenografted mice with all VC mice succumbing to disease at a median of 63 days vs 1/7 AQ treated mice by day 70 (p=.003 by log-rank test). Our drug combination assays found an antagonistic effect in combination with etoposide, and an additive effect with cytarabine and daunorubicin.
Conclusions: Atovaquone induced dramatic levels of apoptosis of AML cells at clinically achievable doses in vitro ; reduced disease burden and improved survival in AML xenografts; and had an additive effect with standard chemotherapy drugs for pediatric AML. Our data support further evaluation of atovaquone for pediatric AML.
No relevant conflicts of interest to declare.
