TRAFfic signals: High-throughput CAR discovery in NK cells reveals novel TRAF-binding endodomains that drive enhanced persistence and cytotoxicity.

Natural killer (NK) cells are a promising alternative therapeutic platform to CAR T cells given their favorable safety profile and potent killing ability. However, CAR NK cells suffer from limited persistence in vivo , which is, in part, thought to be the consequence of limited cytokine signaling. To address this challenge, we developed an innovative high-throughput screening strategy to identify CAR endodomains that could drive enhanced persistence while maintaining potent cytotoxicity. We uncovered a family of TRAF-binding endodomains that outperform benchmarks in primary NK cells along dimensions of persistence and cytotoxicity, even in low IL-2 conditions. This work highlights the importance of cell-type-specific cell therapy engineering and unlocks a wide range of high-throughput molecular engineering avenues in NK cells.

Inducible control of subcellular RNA localization using a synthetic protein-RNA aptamer interaction.

Evidence is accumulating in support of the functional importance of subcellular RNA localization in diverse biological contexts. In different cell types, distinct RNA localization patterns are frequently observed, and the available data indicate that this is achieved through a series of highly coordinated events. Classically, cis-elements within the RNA to be localized are recognized by RNA-binding proteins (RBPs), which then direct specific localization of a target RNA. Until now, the precise control of the spatiotemporal parameters inherent to regulating RNA localization has not been experimentally possible. Here, we demonstrate the development and use of a chemically-inducible RNA-protein interaction to regulate subcellular RNA localization. Our system is composed primarily of two parts: (i) the Tet Repressor protein (TetR) genetically fused to proteins natively involved in localizing endogenous transcripts; and (ii) a target transcript containing genetically encoded TetR-binding RNA aptamers. TetR-fusion protein binding to the target RNA and subsequent localization of the latter are directly regulated by doxycycline. Using this platform, we demonstrate that enhanced and controlled subcellular localization of engineered transcripts are achievable. We also analyze rules for forward engineering this RNA localization system in an effort to facilitate its straightforward application to studying RNA localization more generally.

Direct and specific chemical control of eukaryotic translation with a synthetic RNA-protein interaction.

Sequence-specific RNA-protein interactions, though commonly used in biological systems to regulate translation, are challenging to selectively modulate. Here, we demonstrate the use of a chemically-inducible RNA-protein interaction to regulate eukaryotic translation. By genetically encoding Tet Repressor protein (TetR)-binding RNA elements into the 5'-untranslated region (5'-UTR) of an mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs. In endogenous and synthetic 5'-UTR contexts, this system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from non-functional RNA-TetR interactions. Using a reverse TetR variant, we illustrate the potential for expanding the regulatory properties of the system through protein engineering strategies.

Engineering a direct and inducible protein-RNA interaction to regulate RNA biology.

The importance and pervasiveness of naturally occurring regulation of RNA function in biology is increasingly being recognized. A common mechanism uses inducible protein-RNA interactions to shape diverse aspects of cellular RNA fate. Recapitulating this regulatory mode in cells using a novel set of protein-RNA interactions is appealing given the potential to subsequently modulate RNA biology in a manner decoupled from endogenous cellular physiology. Achieving this outcome, however, has previously proven challenging. Here, we describe a ligand-responsive protein-RNA interaction module, which can be used to target a specific RNA for subsequent regulation. Using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, RNA aptamers binding to the bacterial Tet Repressor protein (TetR) with low- to subnanomolar affinities were obtained. This interaction is reversibly controlled by tetracycline in a manner analogous to the interaction of TetR with its cognate DNA operator. Aptamer minimization and mutational analyses support a functional role for two conserved sequence motifs in TetR binding. As an initial illustration of using this system to achieve protein-based regulation of RNA function in living cells, insertion of a TetR aptamer into the 5'-UTR of a reporter mRNA confers post-transcriptionally regulated, ligand-inducible protein synthesis in E. coli. Altogether, these results define and validate an inducible protein-RNA interaction module that incorporates desirable aspects of a ubiquitous mechanism for regulating RNA function in Nature and can be used as a foundational interaction for functionally and reversibly controlling the multiple fates of RNA in cells.

SNS-314, a pan-Aurora kinase inhibitor, shows potent anti-tumor activity and dosing flexibility in vivo.

PURPOSE: The Aurora family of serine/threonine kinases (Aurora-A, Aurora-B, and Aurora-C) plays a key role in cells orderly progression through mitosis. Elevated expression levels of Aurora kinases have been detected in a high percentage of melanoma, colon, breast, ovarian, gastric, and pancreatic tumors. We characterized the biological and pharmacological properties of SNS-314, an ATP-competitive, selective, and potent inhibitor of Aurora kinases. METHODS: We studied the biochemical potency and selectivity of SNS-314 to inhibit Aurora kinases A, B, and C. The inhibition of cellular proliferation induced by SNS-314 was evaluated in a broad range of tumor cell lines and correlated to inhibition of histone H3 phosphorylation, inhibition of cell-cycle progression, increase in nuclear content and cell size, loss of viability, and induction of apoptosis. The dose and administration schedule of SNS-314 was optimized for in vivo efficacy in mouse xenograft models of human cancer. RESULTS: In the HCT116 human colon cancer xenograft model, administration of 50 and 100 mg/kg SNS-314 led to dose-dependent inhibition of histone H3 phosphorylation for at least 10 h, indicating effective Aurora-B inhibition in vivo. HCT116 tumors from animals treated with SNS-314 showed potent and sustained responses including reduction of phosphorylated histone H3 levels, increased caspase-3 and appearance of increased nuclear size. The compound showed significant tumor growth inhibition in a dose-dependent manner under a variety of dosing schedules including weekly, bi-weekly, and 5 days on/9 days off. CONCLUSIONS: SNS-314 is a potent small-molecule inhibitor of Aurora kinases developed as a novel anti-cancer therapeutic agent for the treatment of diverse human malignancies.

Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex.

Nuclear pore complexes (NPCs) form aqueous conduits in the nuclear envelope and gate the diffusion of large proteins between the cytoplasm and nucleoplasm. NPC proteins (nucleoporins) that contain phenylalanine-glycine motifs in filamentous, natively unfolded domains (FG domains) line the diffusion conduit of the NPC, but their role in the size-selective barrier is unclear. We show that deletion of individual FG domains in yeast relaxes the NPC permeability barrier. At the molecular level, the FG domains of five nucleoporins anchored at the NPC center form a cohesive meshwork of filaments through hydrophobic interactions, which involve phenylalanines in FG motifs and are dispersed by aliphatic alcohols. In contrast, the FG domains of four peripherally anchored nucleoporins are generally noncohesive. The results support a two-gate model of NPC architecture featuring a central diffusion gate formed by a meshwork of cohesive FG nucleoporin filaments and a peripheral gate formed by repulsive FG nucleoporin filaments.