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Purpose Systemic therapy has improved rhabdomyosarcoma event-free and overall survival; however, approximately 40 % of patients will have progressive or recurrent disease which is difficult to cure and remains a considerable challenge. Minimal progress has been made in improving outcomes for metastatic or relapsed RMS due to a lack of effective therapeutic agents. Targeted therapies are likely to be incorporated into regimens which rely on conventional cytotoxic chemotherapy. A system to evaluate novel combinations of interest is needed. Methods In this study, we explored 8 agents, 5 that are routinely used or similar to agents used in the clinical management of RMS and 3 biologically targeted agents with novel mechanisms of action, the Wee1 inhibitor AZD1775, the tyrosine kinase inhibitor cabozantinib, and the proteasome inhibitor bortezomib. All were tested individually at clinically achievable concentrations for activity in 4 RMS cell lines and then for potential synergy in two-drug combinations. Results We found single-agent activity in five of the agents (or their active metabolites) that constitute the standard of care in RMS and for AZD1775 with mean IC50 values of 207 ng/ml, well below clinically achievable levels. In addition, the combination of individual cytotoxic chemotherapeutics currently used for RMS demonstrated largely synergistic activity with higher, but clinically achievable concentrations of AZD1775 in our assays. Conclusions Prioritization of chemotherapeutics in RMS is possible using an in vitro system that can define novel drug combinations worthy of future investigation. AZD1775 exhibits single-agent activity, as well as synergy with conventional cytotoxic chemotherapy, and is a novel targeted agent that warrants further study in RMS. Electronic supplementary material The online version of this article (doi:10.1007/s00280-016-3077-8) contains supplementary material, which is available to authorized users.

Malignant peripheral nerve sheath tumor (MPNST) is an aggressive soft tissue sarcoma. To more fully characterize the genomic landscape of this tumor type, we performed next generation sequencing studies for mutational and copy number analysis. We analyzed whole exome sequencing data from 12 MPNST and SNP arrays for a subset of these. We additionally conducted a literature review of prior next generation sequencing studies in this disease and compared to the current study. We report recurrent mutations in NF1, SUZ12, EED, TP53 and CDKN2A in our study cohort. Combined with prior studies, we calculate the disease specific incidence of mutation in these genes to be: NF1 (56/64 = 87.5%). SUZ12 (69/123 = 56.1%), EED (40/123 = 32.5%), TP53 (29/72 = 40.3%), and CDKN2A (54/72 = 75.0%). Notably, we also identified frequent Ras pathway activating somatic mutations outside of these previously reported recurrently mutated genes. Five of the 12 MPNST in our cohort (42%) contained such a mutation. In conclusion, our study adds to the growing understanding of the genomic complexity of MPNST. We report a previously underappreciated frequency and variety of secondary or tertiary Ras pathway activating mutations, though not highly recurrent in a single gene.

Box H/ACA small nucleolar and Cajal body ribonucleoprotein particles comprise the most complex pseudouridine synthases and are essential for ribosome and spliceosome maturation. The multistep and multicomponent-mediated enzyme mechanism remains only partially understood. Here we report a crystal structure at 2.35 A of a substrate-bound functional archaeal enzyme containing three of the four proteins, Cbf5, Nop10 and L7Ae, and a box H/ACA RNA that reveals detailed information about the protein-only active site. The substrate RNA, containing 5-fluorouridine at the modification position, is fully docked and catalytically rearranged by the enzyme in a manner similar to that seen in two stand-alone pseudouridine synthases. Structural analysis provides a mechanism for plasticity in the diversity of guide RNA sequences used and identifies a substrate-anchoring loop of Cbf5 that also interacts with Gar1 in unliganded structures. Activity analyses of mutated proteins and RNAs support the structural findings and further suggest a role of the Cbf5 loop in regulation of enzyme activity.