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Liposarcoma is the most commonly occurring soft tissue sarcoma and is frequently characterized by amplification of chromosome region 12q13-15 harboring the oncogenes MDM2 and CDK4. This unique genetic profile makes liposarcoma an attractive candidate for targeted therapeutics. While CDK4/6 inhibitors are currently employed for treatment of several cancers, MDM2 inhibitors have yet to attain clinical approval. Here, we report the molecular characterization of the response of liposarcoma to the MDM2 inhibitor nutlin-3. Treatment with nutlin-3 led to upregulation of two nodes of the proteostasis network: the ribosome and the proteasome. CRISPR/Cas9 was used to perform a genome-wide loss of function screen that identified PSMD9, which encodes a proteasome subunit, as a regulator of response to nutlin-3. Accordingly, pharmacological studies with a panel of proteasome inhibitors revealed strong combinatorial induction of apoptosis with nutlin-3. Mechanistic studies identified activation of the ATF4/CHOP stress response axis as a potential node of interaction between nutlin-3 and the proteasome inhibitor carfilzomib. CRISPR/Cas9 gene editing experiments confirmed that ATF4, CHOP, and the BH3-only protein, NOXA, are all required for nutlin-3 and carfilzomib-induced apoptosis. Furthermore, activation of the unfolded protein response using tunicamycin and thapsigargin was sufficient to activate the ATF4/CHOP stress response axis and sensitize to nutlin-3. Finally, cell line and patient-derived xenograft models demonstrated combinatorial effects of treatment with idasanutlin and carfilzomib on liposarcoma growth in vivo. Together, these data indicate that targeting of the proteasome could improve the efficacy of MDM2 inhibitors in liposarcoma.

Introduction: Ewing sarcoma (ES) is a primary bone sarcoma occurring in adolescents and young adults, accounting for 15% of childhood/adolescent cancer. We previously reported that an RNA helicase, DDX3, regulates the translation of a variety of proteins in ES cells, including those modulating DNA damage repair. Therefore, we hypothesized that inhibiting DDX3, using a small molecule called RK-33, would radio-sensitize ES cells by preventing DNA damage repair. Methods: ES cell lines (MHH-ES and TC71) were transfected with DDX3- targeted shRNA or a control, scramble shRNA. RT-PCR and western blotting confirmed knockdown of DDX3. RK-33 cytotoxicity was evaluated using cell viability assay. DNA double strand breaks (DSB) were quantified by immunofluorescent staining for γH2AX, and subcellular localization of DDX3 was determined by immunofluorescence. ES patient derived xenografts (PDX) were subcutaneously implanted in 4 cohorts of NSG mice. Results: Expression of DDX3 mRNA and protein was decreased in DDX3-knockdown cells compared with controls. To validate that DDX3 is inhibited by RK-33, we compared the cytotoxicity of this agent in control cells and in the DDX3 knockdown cell lines. Parental ES and control cells (MHH-ES, TC71, MSD-10) were more sensitive to RK-33, with IC50 values that were significantly lower than their corresponding DDX3-knockdown lines (M2C7, M1F6; 4 μM, 2.7 μM and 2.6 μM vs 42 μM and 11.6 μM, respectively, p10 γH2AX foci, but after 24 hours, the average number of irradiated cells with >10 foci was significantly higher in RK-33-treated cells than in cells treated with vehicle alone (p Citation Format: Marwa Afifi, Breelyn A. Wilky, Catherine Kim, Venu Raman, David Loeb. The RNA helicase, DDX3, modulates DNA damage repair in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4170.

High grade sarcomas are among the most common solid tumors in childhood. Although intensive chemotherapy has dramatically improved the outcome of children with localized osteosarcoma, Ewing sarcoma, and rhabdomyosarcoma, the prognosis for children with metastatic or recurrent disease remains dismal, with no substantial improvement since the introduction of multi-agent chemotherapy regimens 30 years ago. The identification of novel therapeutic targets, and the development of drugs to exploit these targets, is therefore an urgent need. RNA helicases are ubiquitous enzymes, aberrantly expressed in a variety of solid and hematologic malignancies, that are involved in all aspects of RNA metabolism, including transcription, translation, mRNA splicing, and regulation of other protein-RNA interactions. The RNA helicase DDX3 is essential for translation of key cell cycle regulatory mRNA transcripts including cyclin A, cyclin E1, TGFB1, STAT1 and others. Expression of DDX3 is dramatically upregulated in mammary epithelial cells exposed to BPDE, a toxin found in cigarette smoke, and overexpression of DDX3 in breast cancer cell lines correlates with invasiveness and aggressive activity. MCF10a mammary epithelial cells induced to overexpress DDX3 undergo epithelial-mesenchymal transition (EMT) with increased invasive behavior, motility, and colony-forming activity, consistent with neoplastic transformation. Novel DDX3 mutations have recently been identified in medulloblastoma. These data suggest DDX3 may play a key role in the development and progression of a wide variety of solid tumors. We therefore investigated DDX3 expression in sarcomas. We found that sarcomas, both cell lines and primary samples, commonly express high levels of DDX3. Knock down of DDX3 expression in cell lines impaired proliferation, clonogenic activity in soft agar, sphere formation when grown under nonadherent conditions, and tumorigenicity in immune deficient mice. We have developed a novel DDX3 inhibitor, known as RK-33, that binds to the ATP binding domain and impairs helicase activity in a DNA unwinding assay. We found that RK-33 is selectively cytotoxic to sarcoma cell lines, compared with normal mesenchymal cells, and that sensitivity correlates with DDX3 expression. Importantly, Ewing sarcoma cells with a stem-like phenotype, characterized by high expression of aldehyde dehydrogenase, are as sensitive to RK-33 as the bulk population, despite being resistant to standard cytotoxic agents. Finally, we found that RK-33 suppresses the growth of human sarcomas in a murine xenograft model, and that sensitivity correlates with DDX3 expression. Thus, we have evidence that the RNA helicase DDX3 is an important mediator of sarcoma proliferation and tumorigenesis, and that inhibition of DDX3 with the small molecule RK-33 is a novel way to target chemotherapy-resistant sarcomas. Citation Format: David M. Loeb, Breelyn A. Wilky, Catherine Kim, Elizabeth Montgomery, Venu Raman. RNA helicase DDX3 – A novel therapeutic target in sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A77.

Chordoma is a rare primary bone malignancy that arises in the skull base, spine and sacrum and originates from remnants of the notochord. Therapy primarily consists of surgical resection and radiation. These tumors are typically resistant to conventional chemotherapy, and to date there are no FDA-approved agents for chordoma. The lack of in vivo models of chordoma has impeded the development of new therapies for this tumor. We have established a chordoma xenograft growing in NOD/SCID/IL-2R gamma-null mice. The histology of the xenograft is consistent with a dedifferentiated chordoma. The xenograft expresses the chordoma marker T brachyury, which is aberrantly localized in the cytoplasm. SNP array shows loss of heterozygosity at the p16 locus, and gene expression profiling shows high expression of numerous cancer-related genes, such as ROS1 and CPA4 The NIH Chemical Genomics Center performed high-throughput screening of 2,816 compounds. Two established chordoma cell lines, U-CH1 and UCH2B, were treated and cell viability measured by CellTiter-Glo assay. Cells were incubated for 48 hours with drug concentrations ranging from 0.5nM to 46uM. The screen yielded several compounds that showed activity and two were tested in the xenograft. Compared with mice treated with vehicle alone, both drugs showed a dramatic inhibition of tumor growth. In conclusion, we have established a xenograft model of dedifferentiated chordoma. High-throughput screening of compounds identified compounds that show activity against chordoma. In vivo testing of two identified compounds showed a dramatic reduction of tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-110. doi:1538-7445.AM2012-LB-110