title: RNA helicase DDX3: a novel therapeutic target in Ewing sarcoma.
abstract: RNA helicase DDX3 has oncogenic activity in breast and lung cancers and is required for translation of complex mRNA transcripts, including those encoding key cell-cycle regulatory proteins. We sought to determine the expression and function of DDX3 in sarcoma cells, and to investigate the antitumor activity of a novel small molecule DDX3 inhibitor, RK-33. Utilizing various sarcoma cell lines, xenografts and human tissue microarrays, we measured DDX3 expression at the mRNA and protein levels, and evaluated cytotoxicity of RK-33 in sarcoma cell lines. To study the role of DDX3 in Ewing sarcoma, we generated stable DDX3-knockdown Ewing sarcoma cell lines using DDX3-specific small hairpin RNA (shRNA), and assessed oncogenic activity. DDX3-knockdown and RK-33-treated Ewing sarcoma cells were compared with wild-type cells using an isobaric mass-tag quantitative proteomics approach to identify target proteins impacted by DDX3 inhibition. Overall, we found high expression of DDX3 in numerous human sarcoma subtypes compared with non-malignant mesenchymal cells, and knockdown of DDX3 by RNA interference inhibited oncogenic activity in Ewing sarcoma cells. Treatment with RK-33 was preferentially cytotoxic to sarcoma cells, including chemotherapy-resistant Ewing sarcoma stem cells, while sparing non-malignant cells. Sensitivity to RK-33 correlated with DDX3 protein expression. Growth of human Ewing sarcoma xenografts expressing high DDX3 was inhibited by RK-33 treatment in mice, without overt toxicity. DDX3 inhibition altered the Ewing sarcoma cellular proteome, especially proteins involved in DNA replication, mRNA translation and proteasome function. These data support further investigation of the role of DDX3 in sarcomas, advancement of RK-33 to Ewing sarcoma clinical trials and development of RNA helicase inhibition as a novel anti-neoplastic strategy.
authors.affiliation: Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: B A
authors.initials: BA
authors.lastname: Wilky
authors.affiliation: Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: C
authors.initials: C
authors.lastname: Kim
authors.affiliation: Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: G
authors.initials: G
authors.lastname: McCarty
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: E A
authors.initials: EA
authors.lastname: Montgomery
authors.affiliation: Department of Biostatistics, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: K
authors.initials: K
authors.lastname: Kammers
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: L R
authors.initials: LR
authors.lastname: DeVine
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: R N
authors.initials: RN
authors.lastname: Cole
authors.affiliation: Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA.|Department of Radiology, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: V
authors.initials: V
authors.lastname: Raman
authors.affiliation: Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: D M
authors.initials: DM
authors.lastname: Loeb
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001381:Azepines / Q000494:pharmacology; D045744:Cell Line, Tumor; D053487:DEAD-box RNA Helicases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D051379:Mice; D058990:Molecular Targeted Therapy; D012512:Sarcoma, Ewing / Q000188:drug therapy* / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
chemical_list: C000603512:3,7-dihydro-3,7-bis((4-methoxyphenyl)methyl)-2H-diimidazo(4,5-d:4',5'-f)(1,3)diazepin-2-one; D001381:Azepines; D007093:Imidazoles; C119332:DDX3X protein, human; D053487:DEAD-box RNA Helicases