Cleavage and polyadenylation machinery as a novel targetable vulnerability for human cancer.

The role of alternative polyadenylation of mRNA in sustaining aggressive features of tumors is quite well established, as it is responsible for the 3'UTR shortening of oncogenes and subsequent relief from miRNA-mediated repression observed in cancer cells. However, the information regarding the vulnerability of cancer cells to the inhibition of cleavage and polyadenylation (CPA) machinery is very scattered. Only few recent reports show the antitumor activity of pharmacological inhibitors of CPSF3, one among CPA factors. More in general, the fact that deregulated CPA can be seen as a new hallmark of cancer and as a potential reservoir of novel therapeutic targets has never been formalized. Here, to extend our view on the potential of CPA inhibition (CPAi) approaches as anticancer therapies, we systematically tested the fitness of about one thousand cell lines of different cancer types upon depletion of all known CPA factors by interrogating genome-scale CRISPR and RNAi dependency maps of the DepMap project. Our analysis confirmed core and accessory CPA factors as novel vulnerabilities for human cancer, thus highlighting the potential of CPAi as anticancer therapy. Among all, CPSF1 appeared as a promising actionable candidate for drug development, as it showed low dependency scores pancancer and particularly in highly proliferating cells. In a personalized medicine perspective, the observed differential vulnerability of cancer cell lines to selected CPA factors may be used to build up signatures to predict response of individual human tumors to CPAi approaches.

The pancancer overexpressed NFYC Antisense 1 controls cell cycle mitotic progression through in cis and in trans modes of action.

Antisense RNAs (asRNAs) represent an underappreciated yet crucial layer of gene expression regulation. Generally thought to modulate their sense genes in cis through sequence complementarity or their act of transcription, asRNAs can also regulate different molecular targets in trans, in the nucleus or in the cytoplasm. Here, we performed an in-depth molecular characterization of NFYC Antisense 1 (NFYC-AS1), the asRNA transcribed head-to-head to NFYC subunit of the proliferation-associated NF-Y transcription factor. Our results show that NFYC-AS1 is a prevalently nuclear asRNA peaking early in the cell cycle. Comparative genomics suggests a narrow phylogenetic distribution, with a probable origin in the common ancestor of mammalian lineages. NFYC-AS1 is overexpressed pancancer, preferentially in association with RB1 mutations. Knockdown of NFYC-AS1 by antisense oligonucleotides impairs cell growth in lung squamous cell carcinoma and small cell lung cancer cells, a phenotype recapitulated by CRISPR/Cas9-deletion of its transcription start site. Surprisingly, expression of the sense gene is affected only when endogenous transcription of NFYC-AS1 is manipulated. This suggests that regulation of cell proliferation is at least in part independent of the in cis transcription-mediated effect on NFYC and is possibly exerted by RNA-dependent in trans effects converging on the regulation of G2/M cell cycle phase genes. Accordingly, NFYC-AS1-depleted cells are stuck in mitosis, indicating defects in mitotic progression. Overall, NFYC-AS1 emerged as a cell cycle-regulating asRNA with dual action, holding therapeutic potential in different cancer types, including the very aggressive RB1-mutated tumors.

Platelets contribute to amyloid-ß aggregation in cerebral vessels through integrin αIIbß3-induced outside-in signaling and clusterin release.

Cerebral amyloid angiopathy (CAA) is a vascular dysfunction disorder characterized by deposits of amyloid-ß (Aß) in the walls of cerebral vessels. CAA and Aß deposition in the brain parenchyma contribute to dementia and Alzheimer's disease (AD). We investigated the contribution of platelets, which accumulate at vascular Aß deposits, to CAA. We found that synthetic monomeric Aß40 bound through its RHDS (Arg-His-Asp-Ser) sequence to integrin αIIbß3, which is the receptor for the extracellular matrix protein fibrinogen, and stimulated the secretion of adenosine diphosphate (ADP) and the chaperone protein clusterin from platelets. Clusterin promoted the formation of fibrillar Aß aggregates, and ADP acted through its receptors P2Y1 and P2Y12 on platelets to enhance integrin αIIbß3 activation, further increasing the secretion of clusterin and Aß40 binding to platelets. Platelets from patients with Glanzmann's thrombasthenia, a bleeding disorder in which platelets have little or dysfunctional αIIbß3, indicated that the abundance of this integrin dictated Aß-induced clusterin release and platelet-induced Aß aggregation. The antiplatelet agent clopidogrel, which irreversibly inhibits P2Y12, inhibited Aß aggregation in platelet cultures; in transgenic AD model mice, this drug reduced the amount of clusterin in the circulation and the incidence of CAA. Our findings indicate that activated platelets directly contribute to CAA by promoting the formation of Aß aggregates and that Aß, in turn, activates platelets, creating a feed-forward loop. Thus, antiplatelet therapy may alleviate fibril formation in cerebral vessels of AD patients.