An Argonaute phosphorylation cycle promotes microRNA-mediated silencing.

MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). Here we use clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity in human cells to identify new regulators of the miRNA pathway. By using iterative rounds of screening, we reveal a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824-S834), followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Although genetic and biochemical studies demonstrate that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 reveals a pronounced expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per-target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.

eIF5B drives integrated stress response-dependent translation of PD-L1 in lung cancer.

Cancer cells express high levels of PD-L1, a ligand of the PD-1 receptor on T cells, allowing tumors to suppress T cell activity. Clinical trials utilizing antibodies that disrupt the PD-1/PD-L1 checkpoint have yielded remarkable results, with anti-PD-1 immunotherapy approved as first-line therapy for lung cancer patients. We used CRISPR-based screening to identify regulators of PD-L1 in human lung cancer cells, revealing potent induction of PD-L1 upon disruption of heme biosynthesis. Impairment of heme production activates the integrated stress response (ISR), allowing bypass of inhibitory upstream open reading frames in the PD-L1 5' UTR, resulting in enhanced PD-L1 translation and suppression of anti-tumor immunity. We demonstrated that ISR-dependent PD-L1 translation requires the translation initiation factor eIF5B. eIF5B overexpression, which is frequent in lung adenocarcinomas and associated with poor prognosis, is sufficient to induce PD-L1. These findings illuminate mechanisms of immune checkpoint activation and identify targets for therapeutic intervention.

Spheroid-based 3D Cell Cultures Enable Personalized Therapy Testing and Drug Discovery in Head and Neck Cancer.

BACKGROUND/AIM: Chemo-radiation currently serves as first-line therapy of advanced and recurrent head and neck cancer, while new chemotherapy regimens are emerging. However, response rates to any treatment are difficult to predict and underlie broad variation. This study shows the development of a standardized, high-throughput in vitro assay to assess patients' individual response to therapy regimens as a future tool for personalized tumor therapy. MATERIALS AND METHODS: Viability and proliferation analyses after chemo +/- radiation treatment of single spheroids (low adhesion plates/Hanging Drop (HD)) were generated from head and neck squamous cell carcinoma (HNSCC) cell lines and primary human cells from fresh tumor specimens. RESULTS: All cell lines showed reliable growth in all cell culture methods. The spheroids showed significant delay of growth and/or necrosis compared to control groups when exposed to current standard chemotherapeutic regimens. Single 3D spheroids ready for therapy susceptibility testing could be generated from actual tumor specimens after enzymatic and mechanical separation. CONCLUSION: In its current form, this single spheroid-based in vitro assay was able to test individual therapy susceptibility to current standard therapy regimens or, potentially, for testing new targeted drugs in HNSCC treatment. With recent discoveries regarding tumor heterogeneity and individual mutation status, a reliable assay is a prerequisite for personalized therapy in head and neck cancer.

Erratum: Publisher Correction: Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis through distinct mechanisms in Wilms tumours.

[This corrects the article DOI: 10.1038/ncomms5802.].

Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis through distinct mechanisms in Wilms tumours.

Wilms tumour is the most common childhood kidney cancer. Here we report the whole-exome sequencing of 44 Wilms tumours, identifying missense mutations in the microRNA (miRNA)-processing enzymes DROSHA and DICER1, and novel mutations in MYCN, SMARCA4 and ARID1A. Examination of tumour miRNA expression, in vitro processing assays and genomic editing in human cells demonstrates that DICER1 and DROSHA mutations influence miRNA processing through distinct mechanisms. DICER1 RNase IIIB mutations preferentially impair processing of miRNAs deriving from the 5'-arm of pre-miRNA hairpins, while DROSHA RNase IIIB mutations globally inhibit miRNA biogenesis through a dominant-negative mechanism. Both DROSHA and DICER1 mutations impair expression of tumour-suppressing miRNAs, including the let-7 family, important regulators of MYCN, LIN28 and other Wilms tumour oncogenes. These results provide new insights into the mechanisms through which mutations in miRNA biogenesis components reprogramme miRNA expression in human cancer and suggest that these defects define a distinct subclass of Wilms tumours.