Pan-cancer adaptive immune resistance as defined by the Tumor Inflammation Signature (TIS): results from The Cancer Genome Atlas (TCGA).

The Tumor Inflammation Signature (TIS) is an investigational use only (IUO) 18-gene signature that measures a pre-existing but suppressed adaptive immune response within tumors. The TIS has been shown to enrich for patients who respond to the anti-PD1 agent pembrolizumab. To explore this immune phenotype within and across tumor types, we applied the TIS algorithm to over 9000 tumor gene expression profiles downloaded from The Cancer Genome Atlas (TCGA). As expected based on prior evidence, tumors with known clinical sensitivity to anti-programmed cell death protein 1 (PD-1) blockade had higher average TIS scores. Furthermore, TIS scores were more variable within than between tumor types, and within each tumor type a subset of patients with elevated scores was identifiable although with different prevalence associated with each tumor type, the latter consistent with the observed clinical responsiveness to anti PD-1 blockade. Notably, TIS scores only minimally correlated with mutation load in most tumors and ranking tumors by median TIS score showed differing association to clinical sensitivity to PD-1/PD-1 ligand 1 (PD-L1) blockade than ranking of the same tumors by mutation load. The expression patterns of the TIS algorithm genes were conserved across tumor types yet appeared to be minimally prognostic in most cancers, consistent with the TIS score serving as a pan-cancer measurement of the inflamed tumor phenotype. Characterization of the prevalence and variability of TIS will lead to increased understanding of the immune status of untreated tumors and may lead to improved indication selection for testing immunotherapy agents.

Continuously tunable nucleic acid hybridization probes.

In silico-designed nucleic acid probes and primers often do not achieve favorable specificity and sensitivity tradeoffs on the first try, and iterative empirical sequence-based optimization is needed, particularly in multiplexed assays. We present a novel, on-the-fly method of tuning probe affinity and selectivity by adjusting the stoichiometry of auxiliary species, which allows for independent and decoupled adjustment of the hybridization yield for different probes in multiplexed assays. Using this method, we achieved near-continuous tuning of probe effective free energy. To demonstrate our approach, we enforced uniform capture efficiency of 31 DNA molecules (GC content, 0-100%), maximized the signal difference for 11 pairs of single-nucleotide variants and performed tunable hybrid capture of mRNA from total RNA. Using the Nanostring nCounter platform, we applied stoichiometric tuning to simultaneously adjust yields for a 24-plex assay, and we show multiplexed quantitation of RNA sequences and variants from formalin-fixed, paraffin-embedded samples.

Dual regulation of ETTIN (ARF3) gene expression by AS1-AS2, which maintains the DNA methylation level, is involved in stabilization of leaf adaxial-abaxial partitioning in Arabidopsis.

Leaf primordia are generated at the periphery of the shoot apex, developing into flat symmetric organs with adaxial-abaxial polarity, in which the indeterminate state is repressed. Despite the crucial role of the ASYMMETRIC LEAVES1 (AS1)-AS2 nuclear-protein complex in leaf adaxial-abaxial polarity specification, information on mechanisms controlling their downstream genes has remained elusive. We systematically analyzed transcripts by microarray and chromatin immunoprecipitation assays and performed genetic rescue of as1 and as2 phenotypic abnormalities, which identified a new target gene, ETTIN (ETT)/AUXIN RESPONSE FACTOR3 (ARF3), which encodes an abaxial factor acting downstream of the AS1-AS2 complex. While the AS1-AS2 complex represses ETT by direct binding of AS1 to the ETT promoter, it also indirectly activates miR390- and RDR6-dependent post-transcriptional gene silencing to negatively regulate both ETT and ARF4 activities. Furthermore, AS1-AS2 maintains the status of DNA methylation in the ETT coding region. In agreement, filamentous leaves formed in as1 and as2 plants treated with a DNA methylation inhibitor were rescued by loss of ETT and ARF4 activities. We suggest that negative transcriptional, post-transcriptional and epigenetic regulation of the ARFs by AS1-AS2 is important for stabilizing early leaf partitioning into abaxial and adaxial domains.

Failure of the tomato trans-acting short interfering RNA program to regulate AUXIN RESPONSE FACTOR3 and ARF4 underlies the wiry leaf syndrome.

Interfering with small RNA production is a common strategy of plant viruses. A unique class of small RNAs that require microRNA and short interfering (siRNA) biogenesis for their production is termed trans-acting short interfering RNAs (ta-siRNAs). Tomato (Solanum lycopersicum) wiry mutants represent a class of phenotype that mimics viral infection symptoms, including shoestring leaves that lack leaf blade expansion. Here, we show that four WIRY genes are involved in siRNA biogenesis, and in their corresponding mutants, levels of ta-siRNAs that regulate AUXIN RESPONSE FACTOR3 (ARF3) and ARF4 are reduced, while levels of their target ARFs are elevated. Reducing activity of both ARF3 and ARF4 can rescue the wiry leaf lamina, and increased activity of either can phenocopy wiry leaves. Thus, a failure to negatively regulate these ARFs underlies tomato shoestring leaves. Overexpression of these ARFs in Arabidopsis thaliana, tobacco (Nicotiana tabacum), and potato (Solanum tuberosum) failed to produce wiry leaves, suggesting that the dramatic response in tomato is exceptional. As negative regulation of orthologs of these ARFs by ta-siRNA is common to land plants, we propose that ta-siRNA levels serve as universal sensors for interference with small RNA biogenesis, and changes in their levels direct species-specific responses.

Co-ordinated regulation of flowering time, plant architecture and growth by FASCICULATE: the pepper orthologue of SELF PRUNING.

Wild peppers (Capsicum spp.) are either annual or perennial in their native habitat and their shoot architecture is dictated by their sympodial growth habit. To study shoot architecture in pepper, sympodial development is described in wild type and in the classical recessive fasciculate (fa) mutation. The basic sympodial unit in wild-type pepper comprises two leaves and a single terminal flower. fasciculate plants are characterized by the formation of floral clusters separated by short internodes and miniature leaves and by early flowering. Developmental analysis of these clusters revealed shorter sympodial units and, often, precocious termination prior to sympodial leaf formation. fa was mapped to pepper chromosome 6, in a region corresponding to the tomato SELF-PRUNING (SP) locus, the homologue of TFL1 of Arabidopsis. Sequence comparison between wild-type and fa plants revealed a duplication of the second exon in the mutants' orthologue of SP, leading to the formation of a premature stop codon. Ectopic expression of FASCICULATE complemented the Arabidopsis tfl1 mutant plants and as expected, stimulated late flowering. In agreement with the major effect of FASCICULATE imposed on sympodial development, the gene transcripts were localized to the centre of sympodial shoots but could not be detected in the primary shoot. The wide range of pleiotropic effects on plant architecture mediated by a single 'flowering' gene, suggests that it is used to co-ordinate many developmental events, and thus may underlie some of the widespread variation in the Solanaceae shoot architecture.

The making of a compound inflorescence in tomato and related nightshades.

Variation in the branching of plant inflorescences determines flower number and, consequently, reproductive success and crop yield. Nightshade (Solanaceae) species are models for a widespread, yet poorly understood, program of eudicot growth, where short side branches are initiated upon floral termination. This "sympodial" program produces the few-flowered tomato inflorescence, but the classical mutants compound inflorescence (s) and anantha (an) are highly branched, and s bears hundreds of flowers. Here we show that S and AN, which encode a homeobox transcription factor and an F-box protein, respectively, control inflorescence architecture by promoting successive stages in the progression of an inflorescence meristem to floral specification. S and AN are sequentially expressed during this gradual phase transition, and the loss of either gene delays flower formation, resulting in additional branching. Independently arisen alleles of s account for inflorescence variation among domesticated tomatoes, and an stimulates branching in pepper plants that normally have solitary flowers. Our results suggest that variation of Solanaceae inflorescences is modulated through temporal changes in the acquisition of floral fate, providing a flexible evolutionary mechanism to elaborate sympodial inflorescence shoots.

Regulation of LANCEOLATE by miR319 is required for compound-leaf development in tomato.

Plant leaves show pronounced plasticity of size and form. In the classical, partially dominant mutation Lanceolate (La), the large compound leaves of tomato (Solanum lycopersicum) are converted into small simple ones. We show that LA encodes a transcription factor from the TCP family containing an miR319-binding site. Five independent La isolates are gain-of-function alleles that result from point mutations within the miR319-binding site and confer partial resistance of the La transcripts to microRNA (miRNA)-directed inhibition. The reduced sensitivity to miRNA regulation leads to elevated LA expression in very young La leaf primordia and to precocious differentiation of leaf margins. In contrast, downregulation of several LA-like genes using ectopic expression of miR319 resulted in larger leaflets and continuous growth of leaf margins. Our results imply that regulation of LA by miR319 defines a flexible window of morphogenetic competence along the developing leaf margin that is required for leaf elaboration.

Endogenous and synthetic microRNAs stimulate simultaneous, efficient, and localized regulation of multiple targets in diverse species.

Recent studies demonstrated that pattern formation in plants involves regulation of transcription factor families by microRNAs (miRNAs). To explore the potency, autonomy, target range, and functional conservation of miRNA genes, a systematic comparison between plants ectopically expressing pre-miRNAs and plants with corresponding multiple mutant combinations of target genes was performed. We show that regulated expression of several Arabidopsis thaliana pre-miRNA genes induced a range of phenotypic alterations, the most extreme ones being a phenocopy of combined loss of their predicted target genes. This result indicates quantitative regulation by miRNA as a potential source for diversity in developmental outcomes. Remarkably, custom-made, synthetic miRNAs vectored by endogenous pre-miRNA backbones also produced phenocopies of multiple mutant combinations of genes that are not naturally regulated by miRNA. Arabidopsis-based endogenous and synthetic pre-miRNAs were also processed effectively in tomato (Solanum lycopersicum) and tobacco (Nicotiana tabacum). Synthetic miR-ARF targeting Auxin Response Factors 2, 3, and 4 induced dramatic transformations of abaxial tissues into adaxial ones in all three species, which could not cross graft joints. Likewise, organ-specific expression of miR165b that coregulates the PHABULOSA-like adaxial identity genes induced localized abaxial transformations. Thus, miRNAs provide a flexible, quantitative, and autonomous platform that can be employed for regulated expression of multiple related genes in diverse species.

Auxin response factors mediate Arabidopsis organ asymmetry via modulation of KANADI activity.

Members of the KANADI gene family in Arabidopsis thaliana regulate abaxial identity and laminar growth of lateral organs. Promoter APETALA3-mediated ectopic expression of KANADI restricts petal expansion and was used in a genetic screen for factors involved in KANADI-mediated signaling. Through this screen, mutations in ETTIN (ETT; also known as Auxin Response Factor3 [ARF3]) were isolated as second site suppressors and found to ameliorate ectopic KANADI activity throughout the plant as well. Mutant phenotypes of ett are restricted to flowers; however, double mutants with a closely related gene ARF4 exhibit transformation of abaxial tissues into adaxial ones in all aerial parts, resembling mutations in KANADI. Accordingly, the common RNA expression domain of both ARFs was found to be on the abaxial side of all lateral organs. Truncated, negatively acting gene products of strong ett alleles map to an ARF-specific, N-terminal domain of ETT. Such gene products strongly enhance abaxial tissue loss only when ARF activities are compromised. As KANADI is not required for either ETT or ARF4 transcription, and their overexpression cannot rescue kanadi mutants, cooperative activity is implied. ARF proteins are pivotal in mediating auxin responses; thus, we present a model linking transient local auxin gradients and gradual partitioning of lateral organs along the abaxial/adaxial axis.

Reactive oxygen intermediates and glutathione regulate the expression of cytosolic ascorbate peroxidase during iron-mediated oxidative stress in bean.

Excess of free iron is thought to harm plant cells by enhancing the intracellular production of reactive oxygen intermediates (ROI). Cytosolic ascorbate peroxidase (cAPX) is an iron-containing, ROI-detoxifying enzyme induced in response to iron overload or oxidative stress. We studied the expression of cAPX in leaves of de-rooted bean plants in response to iron overload. cAPX expression, i.e., mRNA and protein, was rapidly induced in response to iron overload. This induction correlated with the increase in iron content in leaves and occurred in the light as well as in the dark. Reduced glutathione (GSH), which plays an important role in activating the ROI signal transduction pathway as well as in ROI detoxification, was found to enhance the induction of APX mRNA by iron. To determine whether cAPX induction during iron overload was due to an increase in the amount of free iron, which serves as a co-factor for cAPX synthesis, or due to iron-mediated increase in ROI production, we tested the expression of APX in leaves under low oxygen pressure. This treatment, which suppresses the formation of ROI, completely abolished the induction of cAPX mRNA during iron overload, without affecting the rate of iron uptake by plants. Taken together, our results suggest that high intracellular levels of free iron in plants lead to the enhanced production of ROI, which in turn induces the expression of cAPX, possibly using GSH as an intermediate signal. We further show, using cAPX-antisense transgenic plants, that cAPX expression is essential to prevent iron-mediated tissue damage in tobacco.