Intratumoral (IT) STING activation results in tumor regression in preclinical models, yet factors dictating the balance between innate and adaptive anti-tumor immunity are unclear. Here, clinical candidate STING agonist ADU-S100 (S100) is used in an IT dosing regimen optimized for adaptive immunity to uncover requirements for a T cell-driven response compatible with checkpoint inhibitors (CPIs). In contrast to highdose tumor ablative regimens that result in systemic S100 distribution, low-dose immunogenic regimens induce local activation of tumor-specific CD8 + effector T cells that are responsible for durable anti-tumor immunity and can be enhanced with CPIs. Both hematopoietic cell STING expression and signaling through IFNAR are required for tumor-specific T cell activation, and in the context of optimized T cell responses, TNFa is dispensable for tumor control. In a poorly immunogenic model, S100 combined with CPIs generates a survival benefit and durable protection. These results provide fundamental mechanistic insights into STING-induced anti-tumor immunity.

The replication-dependent histone mRNAs, the only eukaryotic mRNAs that do not have poly(A) tails, are present only in S-phase cells. Coordinate posttranscriptional regulation of histone mRNAs is mediated by the stem-loop at the 3' end of histone mRNAs. The protein that binds the 3' end of histone mRNA, stem-loop binding protein (SLBP), is required for histone pre-mRNA processing and is involved in multiple aspects of histone mRNA metabolism. SLBP is also regulated during the cell cycle, accumulating as cells enter S phase and being rapidly degraded as cells exit S phase. Mutation of any residues in a TTP sequence (amino acids 60 to 62) or mutation of a consensus cyclin binding site (amino acids 99 to 104) stabilizes SLBP in G2 and mitosis. These two threonines are phosphorylated in late S phase, as determined by mass spectrometry (MS) of purified SLBP from late S-phase cells, triggering SLBP degradation. Cells that express a stable SLBP still degrade histone mRNA at the end of S phase, demonstrating that degradation of SLBP is not required for histone mRNA degradation. Nuclear extracts from G1 and G2 cells are deficient in histone pre-mRNA processing, which is restored by addition of recombinant SLBP, indicating that SLBP is the only cell cycle-regulated factor required for histone pre-mRNA processing.

To facilitate the construction of large genomewide libraries of small interfering RNAs (siRNAs), we have developed a dual promoter system (pDual) in which a synthetic DNA encoding a genespecific siRNA sequence is inserted between two different opposing polymerase III promoters, the mouse U6 and human H1 promoters. Upon transfection into mammalian cells, the sense and antisense strands of the duplex are transcribed by these two opposing promoters from the same template, resulting in a siRNA duplex with a uridine overhang on each 3 terminus. A single-step PCR protocol has been developed by using this dual promoter system that allows the production of siRNA expression cassettes in a high-throughput manner. We have shown that siRNAs transcribed by either the dual promoter vector or siRNA expression cassettes can induce strong and gene-specific suppression of both endogenous genes and ectopically expressed genes in mammalian cells. Furthermore, we have constructed an arrayed siRNA expression cassette library that targets >8,000 genes with two siRNA sequences per gene. A high-throughput screen of this library has revealed both known and unique genes involved in the NF-B signaling pathway. RNA interference (RNAi) is an evolutionarily conserved phenomenon in which gene expression is suppressed by the introduction of homologous double-stranded RNAs (dsRNAs). After dsRNA molecules are delivered to the cytoplasm of a cell, they are cleaved by the RNase III-like enzyme, Dicer, to 21-to 23-nt small interfering RNAs (siRNAs) (1). These siRNAs are then incorporated into a protein complex, the RNA-induced silence complex (RISC). The antisense strand of the duplex siRNA guides the RISC to the homologous mRNA, where the RISC-associated endoribonuclease cleaves the target mRNA, resulting in silencing of the target gene (2). RNAi has been successfully used to suppress gene expression in a variety of organisms including zebrafish, Caenorhabditis elegans, Drosophila, planaria, mice, and mammalian cells (3,4). In C. elegans and Drosophila, RNAi is typically induced by the introduction of a long dsRNA (up to 1-2 kb) produced by in vitro transcription. This simple approach cannot be used in mammalian cells, where introduction of long dsRNA elicits a strong antiviral response that obscures any gene-specific silencing effect. Much of this response is caused by activation of the dsRNA-dependent protein kinase PKR, which phosphorylates and inactivates the translation initiation factor eIF2a (5, 6). However, introduction of 21-nt siRNAs into mammalian cells does not stimulate the antiviral response in mammalian cells and can effectively target specific mRNAs, resulting in gene silencing (7).Short siRNA molecules can be prepared by chemical synthesis or in vitro transcription. Alternatively, they can be transcribed in vivo by using siRNA expression vectors with a RNA polymerase (pol) III promoter (including U6, human H1, and tRNA promoters) (8, 9), or a pol II promoter with a minimal poly(A) signal sequence (10). Tissue-specific pol II promot...

Replication-dependent histone mRNAs are the only eukaryotic cellular mRNAs that are not polyadenylated, ending instead in a conserved stem-loop. The 3 end of histone mRNA is required for histone mRNA translation, as is the stem-loop binding protein (SLBP), which binds the 3 end of histone mRNA. We have identified five conserved residues in a 15-amino-acid region in the amino-terminal portion of SLBP, each of which is required for translation. Using a yeast two-hybrid screen, we identified a novel protein, SLBP-interacting protein 1 (SLIP1), that specifically interacts with this region. Mutations in any of the residues required for translation reduces SLIP1 binding to SLBP. The expression of SLIP1 in Xenopus oocytes together with human SLBP stimulates translation of a reporter mRNA ending in the stem-loop but not a reporter with a poly(A) tail. The expression of SLIP1 in HeLa cells also stimulates the expression of a green fluorescent protein reporter mRNA ending in a stem-loop. RNA interference-mediated downregulation of endogenous SLIP1 reduces the rate of translation of endogenous histone mRNA and also reduces cell viability. SLIP1 may function by bridging the 3 end of the histone mRNA with the 5 end of the mRNA, similar to the mechanism of translation of polyadenylated mRNAs.