iScience: RNA helicase DDX3 regulates RAD51 localization and DNA damage repair in Ewing sarcoma (Bishop)

Matthew E. Randolph 1 2, Marwa Afifi 3, Aparna Gorthi 4, Rachel Weil 1, Breelyn A. Wilky 5, Joshua Weinreb 1, Paul Ciero 1 2, Natalie ter Hoeve 7, Paul J. van Diest 7, Venu Raman 5 6 8, Alexander J.R. Bishop 4, David M. Loeb 1 2 5 9

Highlights

•Impaired DDX3 RNA helicase activity induces radiosensitization of Ewing sarcoma in vivo
•DDX3 inhibition attenuates homologous repair and augments radiation-induced cell death
•DDX3 alters RAD51 localization by modulating cytoplasmic RNA:DNA hybrid abundance
•RK-33 inhibits DDX3 and sequesters RAD51 in the cytoplasm, preventing DNA damage repair

Summary
We previously demonstrated that RNA helicase DDX3X (DDX3) can be a therapeutic target in Ewing sarcoma (EWS), but its role in EWS biology remains unclear. The present work demonstrates that DDX3 plays a unique role in DNA damage repair (DDR). We show that DDX3 interacts with several proteins involved in homologous recombination, including RAD51, RECQL1, RPA32, and XRCC2. In particular, DDX3 colocalizes with RAD51 and RNA:DNA hybrid structures in the cytoplasm of EWS cells. Inhibition of DDX3 RNA helicase activity increases cytoplasmic RNA:DNA hybrids, sequestering RAD51 in the cytoplasm, which impairs nuclear translocation of RAD51 to sites of double-stranded DNA breaks, thus increasing sensitivity of EWS to radiation treatment, both in vitro and in vivo. This discovery lays the foundation for exploring new therapeutic approaches directed at manipulating DDR protein localization in solid tumors.

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