MicroRNAs (miRNAs) play critical roles in development, and dysregulation of miRNA expression has been observed in human malignancies. Recent evidence suggests that the processing of several primary miRNA transcripts (pri-miRNAs) is blocked post-transcriptionally in embryonic stem (ES) cells, embryonal carcinoma (EC) cells, and primary tumors. Here we show that Lin-28, a developmentally regulated RNA-binding protein, selectively blocks the processing of pri-let-7 miRNAs in embryonic cells. Using in vitro and in vivo studies, we demonstrate that Lin-28 is necessary and sufficient for blocking Microprocessor-mediated cleavage of pri-let-7 miRNAs. Our results identify Lin-28 as a negative regulator of miRNA biogenesis and suggest that Lin-28 may play a central role in blocking miRNA-mediated differentiation in stem cells and certain cancers.MicroRNAs (miRNAs) constitute a large family of short, noncoding RNAs that posttranscriptionally repress gene expression in metazoans. Mature miRNAs are produced from primary miRNA transcripts (pri-miRNAs) through sequential cleavages by the Microprocessor (1,2) and Dicer (3,4) enzyme complexes to release pre-miRNA and mature miRNA species, respectively. Post-transcriptional control of miRNA expression has been reported to occur in a tissue-specific (5) and developmentally-regulated fashion (6-8). The processing of several pri-miRNAs is blocked in embryonic tissues, with activation of processing occurring only as development proceeds. In addition, it has been reported that certain pri-miRNAs are highly expressed in human(9) and mouse embryonic stem (ES) cells, mouse embryonal carcinoma (EC) cells, and human primary tumors; however, the corresponding mature species are not detectable(7). This suggests that there may be a posttranscriptional block in miRNA biogenesis, the mechanism of which has remained unknown. In ES and EC cells, the magnitude of the Microprocessor processing block is most dramatic for members of the let-7 family of miRNAs although it has been proposed that the processing of all miRNAs may be regulated at the Microprocessor step (7) We observed that the pri-let-7g transcript is readily detectable in ES cells and remains at relatively constant levels over the course of differentiation into embryoid bodies (Fig. 1a). In contrast, mature let-7g is undetectable in undifferentiated ES cells but is strongly induced after day 10 of differentiation ( Fig. 1b). A post-transcriptional induction of let-7g expression has also been reported during the differentiation of P19 EC cells with retinoic acid(7). We sought to understand the mechanism for the post-transcriptional block in miRNA processing in EC and ES cells. We first compared cell extracts from different cell types for their ability 2 co-corresponding authors: Richard Gregory Phone: (617) to inhibit Microprocessor-mediated cleavage of pri-miRNA substrates to the corresponding pre-miRNAs in vitro (Fig. 1c). Radiolabeled pri-miRNA substrates were preincubated with cell extract and subsequently subjected to proc...

Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness1–4. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood1–6. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a nonapoptotic form of cell death induced by the build-up of toxic lipid peroxides7,8. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes8,9. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death8. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFβ-mediated therapy- resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.