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Luciferase reporter assays showing reduced protein synthesis due to blockage of translation elongation upon RocA treatment a Structures of dual-luciferase reporters with poly-purine sequences obtained from upstream CDSs of ERGs or 5′ UTRs of IRGs. HCV-like IRES was used for cap-independent translation initiation. b Firefly luciferase activities in relative to Renilla luciferase after transfections of different constructs, followed by 30 min of RocA treatments at different doses. c Design of the reporter constructs for rescue experiments by replacing the poly-purine sequences with poly-pyrimidine in the CDS regions. d Firefly luciferase activities with the constructs in panel c in response to different doses of RocA. Data are presented as mean values ± SD. “ns”, no significance; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. P-values are all calculated by two-sided Student’s t test without adjustments. n = 3 biologically independent samples. Source data are provided as a Source Data file.
Source publication
The quickly accumulating ribosome profiling data is an insightful resource for studying the critical details of translation regulation under various biological contexts. Rocaglamide A (RocA), an antitumor heterotricyclic natural compound, has been shown to inhibit translation initiation of a large group of mRNA species by clamping eIF4A onto poly-p...
Citations
... Among them, the translation inhibition function of rocaglamide A has elicited much interest. For instance, lots of studies have shown that rocaglamide A can specifically target eukaryotic translation inhibition factor 4A (eIF4A) [129,130]. It has been documented that rocaglamide A triggers pancreatic cancer cell apoptosis by inducing mitochondrial dysfunction and mitophagy, while inhibition of mitophagy sensitizes pancreatic cancer cell death to rocaglamide A [131]. ...
Tumor cell amplification of the MYCN transcription factor is seen in half of patients with high-risk neuroblastoma, where it functions as an oncogenic driver associated with metastatic disease and poor survival. Yet, direct targeting of MYCN has been met with little success, prompting efforts to inhibit its expression at multiple levels. MYCN-amplified neuroblastoma cells have an increased requirement for protein synthesis to meet the overwhelming transcriptional burden imposed by oncogenic MYCN. Here, we take advantage of this vulnerability to interrogate the therapeutic potential of inhibiting the activity of the eukaryotic translation initiation factor 4A1 (eIF4A1), an RNA-helicase responsible for resolving structural barriers such as polypurine preponderance within 5′ untranslated regions (UTRs). We observed that eIF4A1 is a key regulator of transcript-specific mRNA recruitment in MYCN-overexpressing neuroblastomas and MYCN-associated transcripts rank highly in polypurine-rich 5′ UTR sequences, the majority of which have critical roles in cell proliferation. Using CMLD012824, a novel synthetic amidino-rocaglate (ADR) derivative, we demonstrate selectively increased eIF4A1 affinity for polypurine-rich 5′ UTRs, including the MYCN mRNA, leading to translation inhibition and cytotoxicity in human neuroblastoma cell lines and animal models. Through ribosome profiling and PAR-CLIP analysis, we show that ADR-mediated clamping of eIF4A1 onto mRNA spans the full lengths of target transcripts, whereas translational inhibition is mediated selectively through 5′ UTR binding. Both cap-dependent and cap-independent translation of MYCN are disrupted, pointing to the ability of CMLD012824 to disrupt non-canonical translation initiation. Our studies provide insights into the functional role of eIF4A1 in meeting the increased protein synthesis demands of MYCN-amplified neuroblastoma and suggest that its disruption may be therapeutically beneficial in this disease.
