Combined inhibition of ribonucleotide reductase and WEE1 induces synergistic anticancer activity in Ewing's sarcoma cells

Abstract

Background: Ewing's sarcoma is a childhood bone and soft tissue cancer with poor prognosis. Treatment outcomes for Ewing's sarcoma patients have improved only modestly over the past decades, making the development of new treatment strategies paramount. In this study, the combined targeting of ribonucleotide reductase (RNR) and WEE1 was explored for its effectiveness against Ewing's sarcoma cells.

Methods: The RNR inhibitor triapine and the WEE1 inhibitors adavosertib and ZN-c3 were tested in p53 wild-type and p53 mutant Ewing's sarcoma cells. The combination of adavosertib with the PARP inhibitors olaparib and veliparib was tested for comparison. Combinatorial effects were determined by flow cytometric analyses of cell death, loss of mitochondrial membrane potential and DNA fragmentation as well as by caspase 3/7 activity assay, immunoblotting and real-time RT-PCR. The drug interactions were assessed using combination index analysis.

Results: RNR and WEE1 inhibitors were weakly to moderately effective on their own, but highly effective in combination. The combination treatments were similarly effective in p53 wild-type and p53 mutant cells. They synergistically induced cell death and cooperated to elicit mitochondrial membrane potential decay, to activate caspase 3/7 and to trigger DNA fragmentation, evidencing the induction of the apoptotic cell death cascade. They also cooperated to boost CHK1 phosphorylation, indicating augmented replication stress after combination treatment. In comparison, the combination of adavosertib with PARP inhibitors produced weaker synergistic effects.

Conclusion: Our findings show that combined inhibition of RNR and WEE1 was effective against Ewing's sarcoma in vitro. They thus provide a rationale for the evaluation of the potential of combined targeting of RNR and WEE1 in Ewing's sarcoma in vivo.

Keywords: Adavosertib; Ewing’s sarcoma; Olaparib; PARP; Ribonucleotide reductase; Targeted therapy; Triapine; Veliparib; WEE1; ZN-c3.

Fig. 1

RNRi and WEE1i cooperate in inducing cell death in ES cells. Cells were exposed to triapine in combination with (A) adavosertib or (B) ZN-c3 for 48 h. Cell death was determined by flow-cytometric analysis of PI uptake. Means ± SEM of each three independent measurements are shown

Fig. 2

CI values for triapine plus adavosertib or ZN-c3 in ES cells. Based on data from (A) Fig. 1A and (B) Fig. 1B, CI values were calculated with the Chou-Talalay method

Fig. 3

Triapine and adavosertib cooperate in inducing apoptosis in ES cells. Cells were exposed to drugs for (B) 24 h or (A, C, D, E) 48 h. (C, D, E) z-VAD-fmk was applied 1 h before treatment with triapine-adavosertib. (A, D) Loss of Δψ_m was determined by flow-cytometric analysis of DiOC₆(3) staining. (B) Caspase 3/7 activity was determined using the fluorogenic substrate Ac-DEVD-AMC; relative caspase 3/7 activities are the ratio of treated cells to untreated cells. (C) Cell death was determined by flow-cytometric analysis of PI uptake. (E) sub-G1 cells were determined by flow-cytometric analysis of PI-stained ethanol-fixed cells. Means ± SEM of each three independent measurements are shown (*p < 0.05, **p < 0.01, ***p < 0.001; (C, D) black bars vs. grey bars)

Fig. 4

Triapine and ZN-c3 cooperate in inducing apoptosis in ES cells. Cells were exposed to drugs for (B) 24 h or (A, C, D, E) 48 h. (C, D, E) z-VAD-fmk was applied 1 h before treatment with triapine-ZN-c3. (A, D) Loss of Δψ_m was determined by flow-cytometric analysis of DiOC₆(3) staining. (B) Caspase 3/7 activity was determined using the fluorogenic substrate Ac-DEVD-AMC; relative caspase 3/7 activities are the ratio of treated cells to untreated cells. (C) Cell death was determined by flow-cytometric analysis of PI uptake. (E) sub-G1 cells were determined by flow-cytometric analysis of PI-stained ethanol-fixed cells. Means ± SEM of each three independent measurements are shown (*p < 0.05, **p < 0.01, ***p < 0.001; (C, D) black bars vs. grey bars)

Fig. 5

RNRi and WEE1i cooperate in increasing CHK1 phosphorylation and p53 abundance. Cells were exposed to triapine in combination with adavosertib or ZN-c3 for 24 h. (A, B) p-CHK1, CHK1, p53 and GAPDH abundance were determined by immunoblotting; the blots are representative of each three independent experiments

Fig. 6

RNRi and WEE1i cooperate in inducing p53 target gene expression. Cells were exposed to triapine in combination with adavosertib or ZN-c3 for 24 h. CDKN1A and BBC3 expression levels were determined by real-time RT-PCR and normalised to B2M expression levels; relative gene expression levels are the ratio of treated cells to untreated cells. Means ± SEM of each three independent measurements are shown (*p < 0.05, **p < 0.01, ***p < 0.001)