Cooperative treatment effectiveness of ATR and HSP90 inhibition in Ewing\u2019s sarcoma cells

Christian Marx,Martin Westermann,René Thierbach,Kanstantsin Siniuk,Zhao-Qi Wang,Hauke M Schadwinkel,Doerte Hoelzer,Melisa Halilovic,Katrin Buder,James F Beck,Joanna Kirkpatrick,Jürgen Sonnemann,Marc U Schaarschmidt,Yibo Geng,Lisa Marx-Blümel,Sabine Becker,Felix B Meyer

doi:10.1186/s13578-021-00571-y

Abstract

IntroductionEwing's sarcoma is an aggressive childhood malignancy whose outcome has not substantially improved over the last two decades. In this study, combination treatments of the HSP90 inhibitor AUY922 with either the ATR inhibitor VE821 or the ATM inhibitor KU55933 were investigated for their effectiveness in Ewing's sarcoma cells.MethodsEffects were determined in p53 wild-type and p53 null Ewing's sarcoma cell lines by flow cytometric analyses of cell death, mitochondrial depolarization and cell-cycle distribution as well as fluorescence and transmission electron microscopy. They were molecularly characterized by gene and protein expression profiling, and by quantitative whole proteome analysis.ResultsAUY922 alone induced DNA damage, apoptosis and ER stress, while reducing the abundance of DNA repair proteins. The combination of AUY922 with VE821 led to strong apoptosis induction independent of the cellular p53 status, yet based on different molecular mechanisms. p53 wild-type cells activated pro-apoptotic gene transcription and underwent mitochondria-mediated apoptosis, while p53 null cells accumulated higher levels of DNA damage, ER stress and autophagy, eventually leading to apoptosis. Impaired PI3K/AKT/mTOR signaling further contributed to the antineoplastic combination effects of AUY922 and VE821. In contrast, the combination of AUY922 with KU55933 did not produce a cooperative effect.ConclusionOur study reveals that HSP90 and ATR inhibitor combination treatment may be an effective therapeutic approach for Ewing's sarcoma irrespective of the p53 status.

Highlights

Ewing’s sarcoma is an aggressive childhood malignancy whose outcome has not substantially improved over the last two decades
We applied the ATM inhibitor (ATMi) KU55933 to study the role of different DNA damage response (DDR) pathways upon HSP90 inhibitors (HSP90i) treatment
This demonstrated the potential of AUY922VE821 combinations (AUY-VE) as an effective treatment (See figure on page.) Fig. 1 Increased apoptosis after ATR inhibitors (ATRi) and HSP90i combinations. p53 wild-type WE-68 and p53 null A673 cells were treated with the indicated concentrations of AUY922, VE821, KU55933 and their combinations

Summary

Introduction

Ewing’s sarcoma is an aggressive childhood malignancy whose outcome has not substantially improved over the last two decades. Combination treatments of the HSP90 inhibitor AUY922 with either the ATR inhibitor VE821 or the ATM inhibitor KU55933 were investigated for their effectiveness in Ewing’s sarcoma cells. Ewing’s sarcoma (ES) is the second most frequent bone cancer during childhood and adolescence [1, 2]. While EWSR1 participates in the DNA damage response (DDR) by recruiting BRCA1 to resolve DNA:RNA hybrid structures, so-called R-loops, and for homologous recombination (HR) [1, 3, 4], EWS-FLI1 prevents BRCA1-dependent DNA repair. The anti-ES agents that hold promise to increase treatment effectiveness and to overcome resistance include inhibitors of growth factors, epigenetic modifiers, PARP1 inhibitors, p53 activators and PD-L1-based immune therapies [1, 2]. Inhibitors of ATR and ATR-mediated pathways, as well as of HSP90 have been shown to be effective in ES in vitro [8,9,10,11,12]

Methods

Results

Discussion

Conclusion