Protein translation control in cancer - Mechanistic dissection, in vivo quantification and therapeutic implications

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Aberrant protein translation is seen in many cancers and contributes to aggressiveness by enhancing the expression of oncogenes (e.g. MYC). Initiation is the rate-limiting step of protein translation and depends on the complex interplay of the small ribosomal subunit with various initiation factors and the mRNA, including 5’cap and RNA structural elements. Novel tools like ribosome footprinting in combination with RNA sequencing now allow for detailed analysis of affected mRNA species and the discovery of regulatory elements. When combined with genome-wide screens, the interplay of mRNA structural motifs with translation initiation factors and associated proteins can be revealed and pharmacologically manipulated to develop novel cancer therapies. An example of such upcoming approach is the small-molecule inhibitor of the RNA helicase eIF4A, silvestrol, that has been shown to selectively inhibit translation of oncogenic transcripts containing structured 5’ untranslated regions (UTRs) like G-quadruplexes. Development and validation of such strategies would highly benefit from longitudinal in vivo quantification of protein translation, but currently no positron emission tomography (PET) tracer has been validated for protein translation, and the uptake of the frequently-used amino acid tracers rather indicates expression of amino acid transporters instead of incorporation into nascent proteins. Therefore this work aims to synthesize PET tracers based on the translation inhibitor puromycin and to develop and validate an imaging protocol for quantification of protein translation in rodent models. In parallel, novel factors and sequences involved in this pathway will be identified in biological assays. In particular, the influence of eIF4E perturbation on translation of individual mRNAs will be analyzed in order to identify structural motifs assisting translation initiation. Mechanistical work with reporter systems containing structured 5’UTRs will then define the mode of action, and genome-wide CRISPR/Cas9 screens will elucidate novel interaction partners and potentially hint at hitherto unknown therapeutic targets. As soon as non-invasive protein translation imaging has been validated, it will be deployed to characterize the time-course and dose-dependency of existing (silvestrol) and newly identified therapies in animal models. This proposal brings together the expertise of the host lab on mRNA translation analysis and the required biochemical assays and strategies, as well as the expertise of the applicant on tracer development and PET imaging to achieve these goals.