Synergistic Effects of Adavosertib, WEE‐1 Inhibitor in Combination with Cytarabine in the Jurkat Leukemia Cell Model

Abstract

Current treatment for leukemia focuses on DNA damaging treatments (DNAdt), including chemo (cytarabine, vincristine, doxorubicin, etc.) and/or radiotherapy. However, in both adult and pediatric patients with Acute Lymphoblastic Leukemia (ALL), value of these treatments are limited by development of chemoresistance. Recent studies evaluated inhibition of the G2‐M gatekeeper (Wee‐1 kinase) as a possible alternative treatment for other types of cancer, such as: breast, prostate, pancreatic, lung, leukemia, glioblastoma, among others. Adavosertib (AZD‐1775), a well‐documented Wee‐1 kinase inhibitor, has shown synergistic; chemo‐sensitization effects, and is currently undergoing clinical trials. Therefore, our working hypothesis is that: AZD‐1775 sensitizes Leukemia cell lines to DNA‐damaging drugs like cytarabine, resulting in a drug combination with significant synergistic effects. This study evaluates the cytotoxic and chemo‐sensitization effect of AZD‐1775 on an ALL T‐cell model (Jurkat). A broad range of drug combinations (high throughput 384 well plates format) in cell viability assays (96 hours; resazurin‐based) was used to determine cytotoxic effects of AZD‐1775 and cytarabine. Corresponding IC50 values were calculated using GraphPad Prism and the nature of the interactions (synergy, additivity, antagonism) determined with open access software CompuSyn, Combenefit, and SynergyFinder. The Combenefit freeware permitted fitting and analysis of the data to various synergy models (Loewe‐additivity, Bliss‐independence and Highest‐single Agent) while SynergyFinder allowed for an additional model (Zero Interaction Potency), and the classical model of CompuSyn allowed estimates of drug combination indexes (CI) and dose‐reduction indexes (DRI). The individual agents exhibited the following rank order of inhibitory potencies (IC50±SEM): cytarabine (160nM±6nM) ≥ AZD‐1775 (370nM±9nM). Drug combinations results demonstrate a strong and statistically significant synergistic effect with the highest synergistic interactions obtained with AZD‐1775 concentrations of 58nM to 270nM, and cytarabine concentrations of 8nM to 125nM. These synergistic interactions were observed independent on the model and software used for the analysis. Furthermore, the drug combination found to produce the highest synergistic effects in our resazurin‐based cytotoxic assay (AZD‐1775 = 97nM; Cytarabine = 63nM), was also used in a study using Jurkat, normal PBMCs, and activated T‐cells with cells analyzed for cell proliferation, viability and biochemical markers evaluated by Western Blot. At these low concentrations combination no synergistic effects were observed in normal PBMC cells; yet, our findings strongly demonstrate that AZD‐1775 can act as a chemosensitizer agent in ALL capable of potentiating the effect and susceptibility to cytarabine.