Precision medicine focuses on DNA abnormalities, but not all tumors have tractable genomic alterations. The WINTHER trial () navigated patients to therapy on the basis of fresh biopsy-derived DNA sequencing (arm A; 236 gene panel) or RNA expression (arm B; comparing tumor to normal). The clinical management committee (investigators from five countries) recommended therapies, prioritizing genomic matches; physicians determined the therapy given. Matching scores were calculated post-hoc for each patient, according to drugs received: for DNA, the number of alterations matched divided by the total alteration number; for RNA, expression-matched drug ranks. Overall, 303 patients consented; 107 (35%; 69 in arm A and 38 in arm B) were evaluable for therapy. The median number of previous therapies was three. The most common diagnoses were colon, head and neck, and lung cancers. Among the 107 patients, the rate of stable disease ≥6 months and partial or complete response was 26.2% (arm A: 23.2%; arm B: 31.6% (P=0.37)). The patient proportion with WINTHER versus previous therapy progression-free survival ratio of >1.5 was 22.4%, which did not meet the pre-specified primary end point. Fewer previous therapies, better performance status and higher matching score correlated with longer progression-free survival (all P<0.05, multivariate). Our study shows that genomic and transcriptomic profiling are both useful for improving therapy recommendations and patient outcome, and expands personalized cancer treatment.

We report the design and validation of a fast empirical function for scoring RNA-ligand interactions, and describe its implementation within RiboDock, a virtual screening system for automated flexible docking. Building on well-known protein-ligand scoring function foundations, features were added to describe the interactions of common RNA-binding functional groups that were not handled adequately by conventional terms, to disfavour non-complementary polar contacts, and to control non-specific charged interactions. The results of validation experiments against known structures of RNA-ligand complexes compare favourably with previously reported methods. Binding modes were well predicted in most cases and good discrimination was achieved between native and non-native ligands for each binding site, and between native and non-native binding sites for each ligand. Further evidence of the ability of the method to identify true RNA binders is provided by compound selection ('enrichment factor') experiments based around a series of HIV-1 TAR RNA-binding ligands. Significant enrichment in true binders was achieved amongst high scoring docking hits, even when selection was from a library of structurally related, positively charged molecules. Coupled with a semi-automated cavity detection algorithm for identification of putative ligand binding sites, also described here, the method is suitable for the screening of very large databases of molecules against RNA and RNA-protein interfaces, such as those presented by the bacterial ribosome.

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Subdomain IIId from the hepatitis C virus (HCV) internal ribosome entry site (IRES) has been shown to be essential for cap-independent translation. We have conducted a structural study of a 27-nt fragment, identical in sequence to IIId, to explore the structural features of this subdomain. The proposed secondary structure of IIId is comprised of two 3 bp helical regions separated by an internal loop and closed at one end by a 6-nt terminal loop. NMR and molecular modeling were used interactively to formulate a validated model of the three-dimensional structure of IIId. We found that this fragment contains several noncanonical structural motifs and non-Watson-Crick base pairs, some of which are common to other RNAs. In particular, a motif characteristic of the rRNA a-sarcin/ricin loop was located in the internal loop. The terminal loop, 59-UUGGGU, was found to fold to form a trinucleotide loop closed by a trans-wobble U‫ؠ‬ ‫ؠ‬ ‫ؠ‬G base pair. The sixth nucleotide was bulged out to allow stacking of this U‫ؠ‬ ‫ؠ‬ ‫ؠ‬G pair on the adjacent helical region. In vivo mutational analysis in the context of the full IRES confirmed the importance of each structural motif within IIId for IRES function. These findings may provide clues as to host cellular proteins that play a role in IRES-directed translation and, in particular, the mechanism through which host ribosomes are sequestered for viral function.

A cyclin‐dependent kinase (cdk)‐activating kinase (CAK) has been shown previously to catalyze T‐loop phosphorylation of cdks in most eukaryotic cells. This enzyme exists in either of two forms: the major one contains cdk7, cyclin H and an assembly factor called MAT‐1, whilst the minor one lacks MAT‐1. Cdk7 is unusual among cdks because it contains not one but two residues (S170 and T176 in Xenopus cdk7) in its T‐loop that are phosphorylated in vivo. We have investigated the role of S170 and T176 phosphorylation in the assembly and activity of cyclin H–cdk7 dimers. In the absence of MAT‐1, phosphorylation of the T‐loop appears to be required for cdk7 to bind cyclin H. Phosphorylation of both residues does not require cyclin H binding in vitro. Phosphorylation of S170 is sufficient for cdk7 to bind cyclin H with low affinity, but high affinity binding requires T176 phosphorylation. By mutational analysis, we demonstrate that in addition to its role in promotion of cyclin H binding, S170 phosphorylation plays a direct role in the control of CAK activity. Finally, we show that dual phosphorylation of S170 and T176, or substitution of both phosphorylatable residues by aspartic residues, is sufficient to generate CAK activity to one‐third of its maximal value in vitro, even in the absence of cyclin H and MAT‐1, and may thus provide further clues as to how cyclins activate cdk subunits.

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