RNA helicase DDX3 regulates RAD51 localization and DNA damage repair in Ewing sarcoma.

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.

RNA Helicase DDX3 Regulates RAD51 Localization and DNA Damage Repair in Ewing Sarcoma.

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.

SMARCA4 biology in alveolar rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and phenocopies a muscle precursor that fails to undergo terminal differentiation. The alveolar subtype (ARMS) has the poorest prognosis and represents the greatest unmet medical need for RMS. Emerging evidence supports the role of epigenetic dysregulation in RMS. Here we show that SMARCA4/BRG1, an ATP-dependent chromatin remodeling enzyme of the SWI/SNF complex, is prominently expressed in primary tumors from ARMS patients and cell cultures. Our validation studies for a CRISPR screen of 400 epigenetic targets identified SMARCA4 as a unique factor for long-term (but not short-term) tumor cell survival in ARMS. A SMARCA4/SMARCA2 protein degrader (ACBI-1) demonstrated similar long-term tumor cell dependence in vitro and in vivo. These results credential SMARCA4 as a tumor cell dependency factor and a therapeutic target in ARMS.

EphB4/EphrinB2 therapeutics in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma affecting children and is often diagnosed with concurrent metastases. Unfortunately, few effective therapies have been discovered that improve the long-term survival rate for children with metastatic disease. Here we determined effectiveness of targeting the receptor tyrosine kinase, EphB4, in both alveolar and embryonal RMS either directly through the inhibitory antibody, VasG3, or indirectly by blocking both forward and reverse signaling of EphB4 binding to EphrinB2, cognate ligand of EphB4. Clinically, EphB4 expression in eRMS was correlated with longer survival. Experimentally, inhibition of EphB4 with VasG3 in both aRMS and eRMS orthotopic xenograft and allograft models failed to alter tumor progression. Inhibition of EphB4 forward signaling using soluble EphB4 protein fused with murine serum albumin failed to affect eRMS model tumor progression, but did moderately slow progression in murine aRMS. We conclude that inhibition of EphB4 signaling with these agents is not a viable monotherapy for rhabdomyosarcoma.