Inducing expression of ICOS-L by oncolytic adenovirus to enhance tumor-specific bi-specific antibody efficacy.

BACKGROUND: Intratumoral injection of oncolytic viruses (OVs) shows promise in immunotherapy: ONCOS-102, a genetically engineered OV that encodes Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) demonstrated efficacy in early clinical trials, enhancing T cell infiltration in tumors. This suggests OVs may boost various forms of immunotherapy, including tumor-specific bi-specific antibodies (BsAbs). METHODS: Our study investigated in vitro, how ONCOS-204, a variant of ONCOS-virus expressing the ligand of inducible T-cell co-stimulator (ICOSL), modulates the process of T cell activation induced by a BsAb. ONCOS-102 was used for comparison. Phenotypic and functional changes induced by combination of different OVs, and BsAb in T cell subsets were assessed by flow cytometry, viability, and proliferation assays. RESULTS: Degranulation and IFNγ and TNF production of T cells, especially CD4 + T cells was the most increased upon target cell exposure to ONCOS-204. Unexpectedly, ONCOS-204 profoundly affected CD8 + T cell proliferation and function through ICOS-L/ICOS interaction. The effect solely depended on cell surface expression of ICOS-L as soluble ICOSL did not induce notable T cell activity. CONCLUSIONS: Together, our data suggests that oncolytic adenoviruses encoding ICOSL may enhance functional activity of tumor-specific BsAbs thereby opening a novel avenue for clinical development in immunotherapeutics.

Tissue-specific transcriptome profiling of the Arabidopsis inflorescence stem reveals local cellular signatures.

Genome-wide gene expression maps with a high spatial resolution have substantially accelerated plant molecular science. However, the number of characterized tissues and growth stages is still small due to the limited accessibility of most tissues for protoplast isolation. Here, we provide gene expression profiles of the mature inflorescence stem of Arabidopsis thaliana covering a comprehensive set of distinct tissues. By combining fluorescence-activated nucleus sorting and laser-capture microdissection with next-generation RNA sequencing, we characterized the transcriptomes of xylem vessels, fibers, the proximal and distal cambium, phloem, phloem cap, pith, starch sheath, and epidermis cells. Our analyses classified more than 15,000 genes as being differentially expressed among different stem tissues and revealed known and novel tissue-specific cellular signatures. By determining overrepresented transcription factor binding regions in the promoters of differentially expressed genes, we identified candidate tissue-specific transcriptional regulators. Our datasets predict the expression profiles of an exceptional number of genes and allow hypotheses to be generated about the spatial organization of physiological processes. Moreover, we demonstrate that information about gene expression in a broad range of mature plant tissues can be established at high spatial resolution by nuclear mRNA profiling. Tissue-specific gene expression values can be accessed online at https://arabidopsis-stem.cos.uni-heidelberg.de/.

Developmental and Housekeeping Genes: Two Types of Genetic Organization in the Drosophila Genome.

We developed a procedure for locating genes on Drosophila melanogaster polytene chromosomes and described three types of chromosome structures (gray bands, black bands, and interbands), which differed markedly in morphological and genetic properties. This was reached through the use of our original methods of molecular and genetic analysis, electron microscopy, and bioinformatics data processing. Analysis of the genome-wide distribution of these properties led us to a bioinformatics model of the Drosophila genome organization, in which the genome was divided into two groups of genes. One was constituted by 65, in which the genome was divided into two groups, 62 genes that are expressed in most cell types during life cycle and perform basic cellular functions (the so-called "housekeeping genes"). The other one was made up of 3162 genes that are expressed only at particular stages of development ("developmental genes"). These two groups of genes are so different that we may state that the genome has two types of genetic organization. Different are the timings of their expression, chromatin packaging levels, the composition of activating and deactivating proteins, the sizes of these genes, the lengths of their introns, the organization of the promoter regions of the genes, the locations of origin recognition complexes (ORCs), and DNA replication timings.

Specification and regulation of vascular tissue identity in the Arabidopsis embryo.

Development of plant vascular tissues involves tissue identity specification, growth, pattern formation and cell-type differentiation. Although later developmental steps are understood in some detail, it is still largely unknown how the tissue is initially specified. We used the early Arabidopsis embryo as a simple model to study this process. Using a large collection of marker genes, we found that vascular identity was specified in the 16-cell embryo. After a transient precursor state, however, there was no persistent uniform tissue identity. Auxin is intimately connected to vascular tissue development. We found that, although an AUXIN RESPONSE FACTOR5/MONOPTEROS (ARF5/MP)-dependent auxin response was required, it was not sufficient for tissue specification. We therefore used a large-scale enhanced yeast one-hybrid assay to identify potential regulators of vascular identity. Network and functional analysis of candidate regulators suggest that vascular identity is under robust, complex control. We found that one candidate regulator, the G-class bZIP transcription factor GBF2, can modulate vascular gene expression by tuning MP output through direct interaction. Our work uncovers components of a gene regulatory network that controls the initial specification of vascular tissue identity.

Architecture of DNA elements mediating ARF transcription factor binding and auxin-responsive gene expression in Arabidopsis.

The hormone auxin controls many aspects of the plant life cycle by regulating the expression of thousands of genes. The transcriptional output of the nuclear auxin signaling pathway is determined by the activity of AUXIN RESPONSE transcription FACTORs (ARFs), through their binding to cis-regulatory elements in auxin-responsive genes. Crystal structures, in vitro, and heterologous studies have fueled a model in which ARF dimers bind with high affinity to distinctly spaced repeats of canonical AuxRE motifs. However, the relevance of this "caliper" model, and the mechanisms underlying the binding affinities in vivo, have remained elusive. Here we biochemically and functionally interrogate modes of ARF-DNA interaction. We show that a single additional hydrogen bond in Arabidopsis ARF1 confers high-affinity binding to individual DNA sites. We demonstrate the importance of AuxRE cooperativity within repeats in the Arabidopsis TMO5 and IAA11 promoters in vivo. Meta-analysis of transcriptomes further reveals strong genome-wide association of auxin response with both inverted (IR) and direct (DR) AuxRE repeats, which we experimentally validated. The association of these elements with auxin-induced up-regulation (DR and IR) or down-regulation (IR) was correlated with differential binding affinities of A-class and B-class ARFs, respectively, suggesting a mechanistic basis for the distinct activity of these repeats. Our results support the relevance of high-affinity binding of ARF transcription factors to uniquely spaced DNA elements in vivo, and suggest that differential binding affinities of ARF subfamilies underlie diversity in cis-element function.

Web-MCOT Server for Motif Co-Occurrence Search in ChIP-Seq Data.

(1) Background: The widespread application of ChIP-seq technology requires annotation of cis-regulatory modules through the search of co-occurred motifs. (2) Methods: We present the web server Motifs Co-Occurrence Tool (Web-MCOT) that for a single ChIP-seq dataset detects the composite elements (CEs) or overrepresented homo- and heterotypic pairs of motifs with spacers and overlaps, with any mutual orientations, uncovering various similarities to recognition models within pairs of motifs. The first (Anchor) motif in CEs respects the target transcription factor of the ChIP-seq experiment, while the second one (Partner) can be defined either by a user or a public library of Partner motifs being processed. (3) Results: Web-MCOT computes the significances of CEs without reference to motif conservation and those with more conserved Partner and Anchor motifs. Graphic results show histograms of CE abundance depending on orientations of motifs, overlap and spacer lengths; logos of the most common CE structural types with an overlap of motifs, and heatmaps depicting the abundance of CEs with one motif possessing higher conservation than another. (4) Conclusions: Novel capacities of Web-MCOT allow retrieving from a single ChIP-seq dataset with maximal information on the co-occurrence of motifs and potentiates planning of next ChIP-seq experiments.

Transcription Factors as Important Regulators of Changes in Behavior through Domestication of Gray Rats: Quantitative Data from RNA Sequencing.

Studies on hereditary fixation of the tame-behavior phenotype during animal domestication remain relevant and important because they are of both basic research and applied significance. In model animals, gray rats Rattus norvegicus bred for either an enhancement or reduction in defensive response to humans, for the first time, we used high-throughput RNA sequencing to investigate differential expression of genes in tissue samples from the tegmental region of the midbrain in 2-month-old rats showing either tame or aggressive behavior. A total of 42 differentially expressed genes (DEGs; adjusted p-value < 0.01 and fold-change > 2) were identified, with 20 upregulated and 22 downregulated genes in the tissue samples from tame rats compared with aggressive rats. Among them, three genes encoding transcription factors (TFs) were detected: Ascl3 was upregulated, whereas Fos and Fosb were downregulated in tissue samples from the brains of tame rats brain. Other DEGs were annotated as associated with extracellular matrix components, transporter proteins, the neurotransmitter system, signaling molecules, and immune system proteins. We believe that these DEGs encode proteins that constitute a multifactorial system determining the behavior for which the rats have been artificially selected. We demonstrated that several structural subtypes of E-box motifs­known as binding sites for many developmental TFs of the bHLH class, including the ASCL subfamily of TFs­are enriched in the set of promoters of the DEGs downregulated in the tissue samples of tame rats'. Because ASCL3 may act as a repressor on target genes of other developmental TFs of the bHLH class, we hypothesize that the expression of TF gene Ascl3 in tame rats indicates longer neurogenesis (as compared to aggressive rats), which is a sign of neoteny and domestication. Thus, our domestication model shows a new function of TF ASCL3: it may play the most important role in behavioral changes in animals.

Faint gray bands in Drosophila melanogaster polytene chromosomes are formed by coding sequences of housekeeping genes.

In Drosophila melanogaster, the chromatin of interphase polytene chromosomes appears as alternating decondensed interbands and dense black or thin gray bands. Recently, we uncovered four principle chromatin states (4НММ model) in the fruit fly, and these were matched to the structures observed in polytene chromosomes. Ruby/malachite chromatin states form black bands containing developmental genes, whereas aquamarine chromatin corresponds to interbands enriched with 5' regions of ubiquitously expressed genes. Lazurite chromatin supposedly forms faint gray bands and encompasses the bodies of housekeeping genes. In this report, we test this idea using the X chromosome as the model and MSL1 as a protein marker of the lazurite chromatin. Our bioinformatic analysis indicates that in the X chromosome, it is only the lazurite chromatin that is simultaneously enriched for the proteins and histone marks associated with exons, transcription elongation, and dosage compensation. As a result of FISH and EM mapping of a dosage compensation complex subunit, MSL1, we for the first time provide direct evidence that lazurite chromatin forms faint gray bands. Our analysis proves that overall most of housekeeping genes typically span from the interbands (5' region of the gene) to the gray band (gene body). More rarely, active lazurite chromatin and inactive malachite/ruby chromatin may be found within a common band, where both the housekeeping and the developmental genes reside together.

A single ChIP-seq dataset is sufficient for comprehensive analysis of motifs co-occurrence with MCOT package.

Recognition of composite elements consisting of two transcription factor binding sites gets behind the studies of tissue-, stage- and condition-specific transcription. Genome-wide data on transcription factor binding generated with ChIP-seq method facilitate an identification of composite elements, but the existing bioinformatics tools either require ChIP-seq datasets for both partner transcription factors, or omit composite elements with motifs overlapping. Here we present an universal Motifs Co-Occurrence Tool (MCOT) that retrieves maximum information about overrepresented composite elements from a single ChIP-seq dataset. This includes homo- and heterotypic composite elements of four mutual orientations of motifs, separated with a spacer or overlapping, even if recognition of motifs within composite element requires various stringencies. Analysis of 52 ChIP-seq datasets for 18 human transcription factors confirmed that for over 60% of analyzed datasets and transcription factors predicted co-occurrence of motifs implied experimentally proven protein-protein interaction of respecting transcription factors. Analysis of 164 ChIP-seq datasets for 57 mammalian transcription factors showed that abundance of predicted composite elements with an overlap of motifs compared to those with a spacer more than doubled; and they had 1.5-fold increase of asymmetrical pairs of motifs with one more conservative 'leading' motif and another one 'guided'.

Asymmetric Conservation within Pairs of Co-Occurred Motifs Mediates Weak Direct Binding of Transcription Factors in ChIP-Seq Data.

(1) Background: Transcription factors (TFs) are main regulators of eukaryotic gene expression. The cooperative binding to genomic DNA of at least two TFs is the widespread mechanism of transcription regulation. Cooperating TFs can be revealed through the analysis of co-occurrence of their motifs. (2) Methods: We applied the motifs co-occurrence tool (MCOT) that predicted pairs of spaced or overlapped motifs (composite elements, CEs) for a single ChIP-seq dataset. We improved MCOT capability for the prediction of asymmetric CEs with one of the participating motifs possessing higher conservation than another does. (3) Results: Analysis of 119 ChIP-seq datasets for 45 human TFs revealed that almost for all families of TFs the co-occurrence with an overlap between motifs of target TFs and more conserved partner motifs was significantly higher than that for less conserved partner motifs. The asymmetry toward partner TFs was the most clear for partner motifs of TFs from the ETS (E26 Transformation Specific) family. (4) Conclusion: Co-occurrence with an overlap of less conserved motif of a target TF and more conserved motifs of partner TFs explained a substantial portion of ChIP-seq data lacking conserved motifs of target TFs. Among other TF families, conservative motifs of TFs from ETS family were the most prone to mediate interaction of target TFs with its weak motifs in ChIP-seq.

Nucleosome Positioning around Transcription Start Site Correlates with Gene Expression Only for Active Chromatin State in Drosophila Interphase Chromosomes.

We analyzed the whole-genome experimental maps of nucleosomes in Drosophila melanogaster and classified genes by the expression level in S2 cells (RPKM value, reads per kilobase million) as well as the number of tissues in which a gene was expressed (breadth of expression, BoE). Chromatin in 5'-regions of genes we classified on four states according to the hidden Markov model (4HMM). Only the Aquamarine chromatin state we considered as Active, while the rest three states we defined as Non-Active. Surprisingly, about 20/40% of genes with 5'-regions mapped to Active/Non-Active chromatin possessed the minimal/at least modest RPKM and BoE. We found that regardless of RPKM/BoE the genes of Active chromatin possessed the regular nucleosome arrangement in 5'-regions, while genes of Non-Active chromatin did not show respective specificity. Only for genes of Active chromatin the RPKM/BoE positively correlates with the number of nucleosome sites upstream/around TSS and negatively with that downstream TSS. We propose that for genes of Active chromatin, regardless of RPKM value and BoE the nucleosome arrangement in 5'-regions potentiates transcription, while for genes of Non-Active chromatin, the transcription machinery does not require the substantial support from nucleosome arrangement to influence gene expression.

Diversity of cis-regulatory elements associated with auxin response in Arabidopsis thaliana.

The phytohormone auxin regulates virtually every developmental process in land plants. This regulation is mediated via de-repression of DNA-binding auxin response factors (ARFs). ARFs bind TGTC-containing auxin response cis-elements (AuxREs), but there is growing evidence that additional cis-elements occur in auxin-responsive regulatory regions. The repertoire of auxin-related cis-elements and their involvement in different modes of auxin response are not yet known. Here we analyze the enrichment of nucleotide hexamers in upstream regions of auxin-responsive genes associated with auxin up- or down-regulation, with early or late response, ARF-binding domains, and with different chromatin states. Intriguingly, hexamers potentially bound by basic helix-loop-helix (bHLH) and basic leucine zipper (bZIP) factors as well as a family of A/T-rich hexamers are more highly enriched in auxin-responsive regions than canonical TGTC-containing AuxREs. We classify and annotate the whole spectrum of enriched hexamers and discuss their patterns of enrichment related to different modes of auxin response.

Polytene Chromosomes - A Portrait of Functional Organization of the Drosophila Genome.

This mini-review is devoted to the problem genetic meaning of main polytene chromosome structures - bands and interbands. Generally, densely packed chromatin forms black bands, moderately condensed regions form grey loose bands, whereas decondensed regions of the genome appear as interbands. Recent progress in the annotation of the Drosophila genome and epigenome has made it possible to compare the banding pattern and the structural organization of genes, as well as their activity. This was greatly aided by our ability to establish the borders of bands and interbands on the physical map, which allowed to perform comprehensive side-by-side comparisons of cytology, genetic and epigenetic maps and to uncover the association between the morphological structures and the functional domains of the genome. These studies largely conclude that interbands 5'-ends of housekeeping genes that are active across all cell types. Interbands are enriched with proteins involved in transcription and nucleosome remodeling, as well as with active histone modifications. Notably, most of the replication origins map to interband regions. As for grey loose bands adjacent to interbands, they typically host the bodies of house-keeping genes. Thus, the bipartite structure composed of an interband and an adjacent grey band functions as a standalone genetic unit. Finally, black bands harbor tissue-specific genes with narrow temporal and tissue expression profiles. Thus, the uniform and permanent activity of interbands combined with the inactivity of genes in bands forms the basis of the universal banding pattern observed in various Drosophila tissues.

Protein and Genetic Composition of Four Chromatin Types in Drosophila melanogaster Cell Lines.

BACKGROUND: Recently, we analyzed genome-wide protein binding data for the Drosophila cell lines S2, Kc, BG3 and Cl.8 (modENCODE Consortium) and identified a set of 12 proteins enriched in the regions corresponding to interbands of salivary gland polytene chromosomes. Using these data, we developed a bioinformatic pipeline that partitioned the Drosophila genome into four chromatin types that we hereby refer to as aquamarine, lazurite, malachite and ruby. RESULTS: Here, we describe the properties of these chromatin types across different cell lines. We show that aquamarine chromatin tends to harbor transcription start sites (TSSs) and 5' untranslated regions (5'UTRs) of the genes, is enriched in diverse "open" chromatin proteins, histone modifications, nucleosome remodeling complexes and transcription factors. It encompasses most of the tRNA genes and shows enrichment for non-coding RNAs and miRNA genes. Lazurite chromatin typically encompasses gene bodies. It is rich in proteins involved in transcription elongation. Frequency of both point mutations and natural deletion breakpoints is elevated within lazurite chromatin. Malachite chromatin shows higher frequency of insertions of natural transposons. Finally, ruby chromatin is enriched for proteins and histone modifications typical for the "closed" chromatin. Ruby chromatin has a relatively low frequency of point mutations and is essentially devoid of miRNA and tRNA genes. Aquamarine and ruby chromatin types are highly stable across cell lines and have contrasting properties. Lazurite and malachite chromatin types also display characteristic protein composition, as well as enrichment for specific genomic features. We found that two types of chromatin, aquamarine and ruby, retain their complementary protein patterns in four Drosophila cell lines.

Computational analysis of auxin responsive elements in the Arabidopsis thaliana L. genome.

Auxin responsive elements (AuxRE) were found in upstream regions of target genes for ARFs (Auxin response factors). While Chip-seq data for most of ARFs are still unavailable, prediction of potential AuxRE is restricted by consensus models that detect too many false positive sites. Using sequence analysis of experimentally proven AuxREs, we revealed both an extended nucleotide context pattern for AuxRE itself and three distinct types of its coupling motifs (Y-patch, AuxRE-like, and ABRE-like), which together with AuxRE may form the composite elements. Computational analysis of the genome-wide distribution of the predicted AuxREs and their impact on auxin responsive gene expression allowed us to conclude that: (1) AuxREs are enriched around the transcription start site with the maximum density in 5'UTR; (2) AuxREs mediate auxin responsive up-regulation, not down-regulation. (3) Directly oriented single AuxREs and reverse multiple AuxREs are mostly associated with auxin responsiveness. In the composite AuxRE elements associated with auxin response, ABRE-like and Y-patch are 5'-flanking or overlapping AuxRE, whereas AuxRE-like motif is 3'-flanking. The specificity in location and orientation of the coupling elements suggests them as potential binding sites for ARFs partners.

Application of experimentally verified transcription factor binding sites models for computational analysis of ChIP-Seq data.

BACKGROUND: ChIP-Seq is widely used to detect genomic segments bound by transcription factors (TF), either directly at DNA binding sites (BSs) or indirectly via other proteins. Currently, there are many software tools implementing different approaches to identify TFBSs within ChIP-Seq peaks. However, their use for the interpretation of ChIP-Seq data is usually complicated by the absence of direct experimental verification, making it difficult both to set a threshold to avoid recognition of too many false-positive BSs, and to compare the actual performance of different models. RESULTS: Using ChIP-Seq data for FoxA2 binding loci in mouse adult liver and human HepG2 cells we compared FoxA binding-site predictions for four computational models of two fundamental classes: pattern matching based on existing training set of experimentally confirmed TFBSs (oPWM and SiteGA) and de novo motif discovery (ChIPMunk and diChIPMunk). To properly select prediction thresholds for the models, we experimentally evaluated affinity of 64 predicted FoxA BSs using EMSA that allows safely distinguishing sequences able to bind TF. As a result we identified thousands of reliable FoxA BSs within ChIP-Seq loci from mouse liver and human HepG2 cells. It was found that the performance of conventional position weight matrix (PWM) models was inferior with the highest false positive rate. On the contrary, the best recognition efficiency was achieved by the combination of SiteGA & diChIPMunk/ChIPMunk models, properly identifying FoxA BSs in up to 90% of loci for both mouse and human ChIP-Seq datasets. CONCLUSIONS: The experimental study of TF binding to oligonucleotides corresponding to predicted sites increases the reliability of computational methods for TFBS-recognition in ChIP-Seq data analysis. Regarding ChIP-Seq data interpretation, basic PWMs have inferior TFBS recognition quality compared to the more sophisticated SiteGA and de novo motif discovery methods. A combination of models from different principles allowed identification of proper TFBSs.

Novel method to characterize immune cells from human prostate tissue.

BACKGROUND: Benign prostatic hyperplasia (BPH) is the most common benign adenoma and prostate cancer is the most frequent malignancy in men over 50 years of age in the Western world, where it remains a significant health problem. Prostate lesions are known to contain immune cells, which may contribute to the immune control of tumor progression. However, due to their low numbers and restricted access to necessary material it is difficult to isolate immune cells from prostate tissue to characterize their immunological features. METHODS: An efficient and robust method was developed to process prostate tissue and isolate immune cells for phenotypic analysis by multicolor flow cytometry as downstream application. Fresh prostate tissue from 11 patients undergoing surgery for bladder outlet obstruction due to BPH was processed to evaluate the number, viability, yield, and frequency of various immune cell types. RESULTS: The presented method does not include enzymatic digestion nor incubation steps at 37 °C, increasing cellular viability and avoiding possible phenotypic modification. Various immune cell populations were detected in all patient samples and the median cellular viability was 90%. The number of detected events of individual cell populations varied between patients. The median frequency of different immune cell populations also varied, being 87% for the CD3- and 15% for the CD3+ cell population. CONCLUSIONS: This novel method will allow the phenotypic characterization of immune cell populations present in tumor tissue of prostate cancer patients and promote development of novel approaches to immunotherapy of the disease.

Pilot Study of ONCOS-102 and Pembrolizumab: Remodeling of the Tumor Microenvironment and Clinical Outcomes in Anti-PD-1-Resistant Advanced Melanoma.

PURPOSE: Intratumoral oncolytic virotherapy may overcome anti-PD(L)-1 resistance by triggering pro-inflammatory remodeling of the tumor microenvironment. This pilot study investigated ONCOS-102 (oncolytic adenovirus expressing GM-CSF) plus anti-programmed cell death protein 1 (PD)-1 therapy in anti-PD-1-resistant melanoma. PATIENTS AND METHODS: Patients with advanced melanoma progressing after prior PD-1 blockade received intratumoral ONCOS-102 either as priming with 3 doses (3 × 1011 viral particles) during Week 1 [Part 1 (sequential treatment)] or as 4-dose priming and 8 booster doses every 3 weeks [Part 2 (combination treatment)]. From Week 3, all patients received pembrolizumab every 3 weeks (≤8 doses). The primary endpoint was safety. Objective response rate (ORR), progression-free survival, and immunologic activation in repeat biopsies were also investigated. RESULTS: In 21 patients (Part 1, n = 9; Part 2, n = 12) ONCOS-102 plus pembrolizumab was well tolerated: most adverse events (AE) were mild/moderate in severity. Pyrexia (43%), chills (43%), and nausea (28%) were the most common ONCOS-102-related AEs. There were no dose-limiting toxicities. ORR was 35% [response evaluation in solid tumors (RECIST) 1.1, irRECIST]. Reduction in size of ≥1 non-injected lesions observed in 53% patients indicated a systemic effect. In injected tumors, persistent immune-related gene expression and T-cell infiltration were associated with clinical benefit. Viral persistence and efficacy in injected and non-injected lesions without additional toxicity supported Part 2 dosing regimen in future studies. CONCLUSIONS: ONCOS-102 plus pembrolizumab was well tolerated and led to objective responses in patients with anti-PD-1-resistant advanced melanoma. ONCOS-102 promoted T-cell infiltration, particularly cytotoxic CD8+ T cells, which persisted at Week 9, driving clinical benefit. Further investigation of ONCOS-102 plus PD-1 blockade is warranted. See related commentary by Levi and Boland, p. 3.

ONCOS-102 plus pemetrexed and platinum chemotherapy in malignant pleural mesothelioma: a randomized phase 2 study investigating clinical outcomes and the tumor microenvironment.

BACKGROUND: ONCOS-102, an oncolytic adenovirus expressing granulocyte-macrophage colony-stimulating factor, can alter the tumor microenvironment to an immunostimulatory state. Combining ONCOS-102 with standard-of-care chemotherapy for malignant pleural mesothelioma (MPM) may improve treatment outcomes. METHODS: In this open-label, randomized study, patients with unresectable MPM received intratumoral ONCOS-102 (3×1011 virus particles on days 1, 4, 8, 36, 78, and 120) and pemetrexed plus cisplatin/carboplatin (from day 22), or pemetrexed plus cisplatin/carboplatin alone. The primary endpoint was safety. Overall survival (OS), progression-free survival, objective response rate, and tumor immunologic activation (baseline and day 36 biopsies) were also assessed. RESULTS: In total, 31 patients (safety lead-in: n=6, randomized: n=25) were enrolled. Anemia (15.0% and 27.3%) and neutropenia (40.0% and 45.5%) were the most frequent grade ≥3 adverse events (AEs) in the ONCOS-102 (n=20) and chemotherapy-alone (n=11) cohorts. No patients discontinued ONCOS-102 due to AEs. No statistically significant difference in efficacy endpoints was observed. There was a numerical improvement in OS (30-month OS rate 34.1% vs 0; median OS 20.3 vs 13.5 months) with ONCOS-102 versus chemotherapy alone in chemotherapy-naïve patients (n=17). By day 36, ONCOS-102 was associated with increased T-cell infiltration and immune-related gene expression that was not observed in the control cohort. Substantial immune activation in the tumor microenvironment was associated with survival at month 18 in the ONCOS-102 cohort. CONCLUSIONS: ONCOS-102 plus pemetrexed and cisplatin/carboplatin was well tolerated by patients with MPM. In injected tumors, ONCOS-102 promoted a proinflammatory environment, including T-cell infiltration, which showed association with survival at month 18.

Syntaxin 11 marks a distinct intracellular compartment recruited to the immunological synapse of NK cells to colocalize with cytotoxic granules.

The syntaxin 11 (STX11) gene is mutated in a proportion of patients with familial haemophagocytic lymphohistiocytosis (FHL) and exocytosis of cytotoxic granules is impaired in STX11-deficient NK cells. However, the subcellular localization, regulation of expression and molecular function of STX11 in NK cells and other cytotoxic lymphocytes remain unknown. Here we demonstrate that STX11 expression is strictly controlled by several mechanisms in a cell-type-specific manner and that the enzymatic activity of the proteasome is required for STX11 expression in NK cells. In resting NKL cells, STX11 was localized in the cation-dependent mannose-6-phosphate receptor (CD-M6PR)-containing compartment, which was clearly distinct from cytotoxic granules or Rab27a-expressing vesicles. These subcellular structures appeared to fuse at the contact area with NK-sensitive target cells as demonstrated by partial colocalization of STX11 with perforin and Rab27a. Although STX11-deficent allo-specific cytotoxic T-lymphocytes efficiently lysed target cells and released cytotoxic granules, they exhibited a significantly lower extent of spontaneous association of perforin with Rab27a as compared with STX11-expressing T cells. Thus, our results suggest that STX11 promotes the fusion of Rab27a-expressing vesicles with cytotoxic granules and reveal an additional level of complexity in the spatial/temporal segregation of subcellular structures participating in the process of granule-mediated cytotoxicity.