Polycomb Group Target Research Articles

Sequencing CURLY LEAF-associated RNAs in Arabidopsis revealed prevalent intergenic RNAs from the nuclear mitochondrial sequence

Polycomb Group (PcG) proteins play key roles in development by repressing thousands of targets through histone modifications. However, how PcG is recruited to specific targets is poorly understood. In Arabidopsis, certain noncoding RNAs are necessary for recruiting the PcG protein CURLY LEAF (CLF) to its target sites. However, RNAs associated with CLF have not been analyzed on a genomic scale, thus it is unknown whether long noncoding RNA (lncRNA)-mediated PcG recruitment is a widespread mechanism. Here, we systematically searched for CLF-associated RNAs by RNA immunoprecipitation followed by deep sequencing. We identified 1299 genic and 138 intergenic regions that produced CLF-associated mRNAs and putative lncRNAs, respectively. The genes producing CLF-associated RNAs are depleted in PcG targets, carry active chromatin marks, and are highly expressed, suggesting that CLF may have a non-specific or promiscuous RNA-binding affinity, similar to animal PcG proteins. Notably, a significant portion of the CLF-associated lncRNAs is derived from the nuclear mitochondrial sequence, which is extensively marked by H3K27me3. These findings indicate that while CLF-RNA interactions are widespread, they may not always correlate with PcG target sites, highlighting the complexity of PcG recruitment mechanisms in Arabidopsis.

Dual roles of histone H3 lysine-4 in antagonizing Polycomb group function and promoting target gene expression.

Tight control over cell identity gene expression is necessary for proper adult form and function. The opposing activities of Polycomb and trithorax complexes determine the ON/OFF state of targets like the Hox genes. Trithorax encodes a methyltransferase specific to histone H3 lysine-4 (H3K4). However, there is no direct evidence that H3K4 regulates Polycomb group target genes in vivo . Here, we demonstrate two key roles for replication-dependent histone H3.2K4 in target control. We find that H3.2K4 antagonizes Polycomb group catalytic activity and that it is required for proper target gene activation. We conclude that H3.2K4 directly regulates expression of Polycomb targets.

Polycomb protein binding and looping in the ON transcriptional state.

Polycomb group (PcG) proteins mediate epigenetic silencing of important developmental genes by modifying histones and compacting chromatin through two major protein complexes, PRC1 and PRC2. These complexes are recruited to DNA by CpG islands (CGIs) in mammals and Polycomb response elements (PREs) in Drosophila. When PcG target genes are turned OFF, PcG proteins bind to PREs or CGIs, and PREs serve as anchors that loop together and stabilize gene silencing. Here, we address which PcG proteins bind to PREs and whether PREs mediate looping when their targets are in the ON transcriptional state. While the binding of most PcG proteins decreases at PREs in the ON state, one PRC1 component, Ph, remains bound. Further, PREs can loop to each other and with presumptive enhancers in the ON state and, like CGIs, may act as tethering elements between promoters and enhancers. Overall, our data suggest that PREs are important looping elements for developmental loci in both the ON and OFF states.

Genome-wide temperature-sensitivity of Polycomb group regulation and reduction thereof in temperate Drosophila melanogaster.

Epigenetic regulation varies with the environment. In the fruit fly Drosophila melanogaster, environmental temperature can affect chromatin-based gene regulation. Genes regulated by the Polycomb group can vary in their transcriptional output in response to changes in temperature, which typically increases with decreasing temperature. Here, we studied temperature-sensitive expression of Polycomb group target genes on a genome-wide scale, as well as temperature-sensitive enrichment of two histone modifications associated with the regulation of Polycomb group target genes, H3K27me3 and H3K4me3. We investigated temperature-sensitivity in adult flies, and possible differences thereof between populations adapted to temperate and tropical climates. Compared to genes not targeted by the Polycomb group, an elevated number of target genes showed higher expression at the lower temperature, as it is typically observed for Polycomb group regulation. Many of the Polycomb group target genes also exhibited temperature-sensitive H3K4me3 enrichment in the same direction, and the H3K4me3 temperature response correlated positively with that of expression. A small set of target sites also showed temperature-sensitive enrichment of H3K27me3, again with a higher proportion corresponding to increased transcriptional activation at the lower temperature. Overall, higher transcriptional activity at lower temperature was less pronounced in males compared to females, and in temperate compared to tropical flies. Possible trans- and cis-acting factors responsible for reduced expression plasticity in temperate flies were identified, including factors belonging to the Trithorax group and insulator binding proteins, respectively.

ASXL1 mutations accelerate bone marrow fibrosis via EGR1-TNFA axis mediated neoplastic fibrocyte generation in myeloproliferative neoplasms.

Apart from the central role of activated JAK/STAT signaling pathway, ASXL1 mutations are the most recurrent additional mutations in myeloproliferative neoplasms (MPNs) and occurred much more common in myelofibrosis (MF) than essential thrombocythemia (ET) and polycythemia vera (PV) patients. However, the mechanism of the association with ASXL1 mutations and bone marrow (BM) fibrosis remains unknown. Here, integrating our own data of MPN patients and hematopoietic-specific Asxl1 deletion/Jak2V617F mouse model, we show that ASXL1 mutations are associated with advanced MPN phenotypes and onset of myelofibrosis. ASXL1 mutations induce skewed monocyte/macrophage and neoplastic monocyte-derived fibrocyte differentiation, consequently enhance inflammation and BM fibrosis. Consistently, the loss of ASXL1 and JAK2V617F mutation in hematopoietic stem and progenitor cells (HSPCs) leads to enhancer activation of polycomb group (PcG) target genes, such as EGR1. The upregulation of EGR1, in turn accounts for increased HSPCs commitment to monocyte/macrophage lineage. Moreover, EGR1 induces the activation of TNFA and thereby further drives the differentiation of monocytes to fibrocytes. Accordingly, combined TNFR antagonist with ruxolitinib significantly reduces fibrocytes production in vitro. Altogether, these findings demonstrate that ASXL1 mutations accelerate fibrocyte production and inflammation in MPNs via EGR1-TNFA axis, providing cellular and molecular basis for BM fibrosis and proof-of-concept for anti-fibrosis treatment.

ASXL1 mutations accelerate bone marrow fibrosis via EGR1-TNFA axis-mediated neoplastic fibrocyte generation in myeloproliferative neoplasms.

Apart from the central role of the activated JAK/STAT signaling pathway, ASXL1 mutations are the most recurrent additional mutations in myeloproliferative neoplasms and occur much more commonly in myelofibrosis than in essential thrombocythemia and polycythemia vera. However, the mechanism of the association with ASXL1 mutations and bone marrow fibrosis remains unknown. Here, integrating our own data from patients with myeloproliferative neoplasms and a hematopoietic-specific Asxl1 deletion/Jak2V617F mouse model, we show that ASXL1 mutations are associated with advanced myeloproliferative neoplasm phenotypes and onset of myelofibrosis. ASXL1 mutations induce skewed monocyte/macrophage and neoplastic monocyte-derived fibrocyte differentiation, consequently they enhance inflammation and bone marrow fibrosis. Consistently, the loss of ASXL1 and JAK2V617F mutations in hematopoietic stem and progenitor cells leads to enhanced activation of polycomb group target genes, such as EGR1. The upregulation of EGR1, in turn, accounts for increased hematopoietic stem and progenitor cell commitment to the monocyte/macrophage lineage. Moreover, EGR1 induces the activation of TNFA and thereby further drives the differentiation of monocytes to fibrocytes. Accordingly, combined treatment with a TNFR antagonist and ruxolitinib significantly reduces fibrocyte production in vitro. Altogether, these findings demonstrate that ASXL1 mutations accelerate fibrocyte production and inflammation in myeloproliferative neoplasms via the EGR1-TNFA axis, explaining the cellular and molecular basis for bone marrow fibrosis and the proof-ofconcept for anti-fibrosis treatment.

A prominent gene activation role for C-terminal binding protein in mediating PcG/trxG proteins through Hox gene regulation.

The evolutionarily conserved C-terminal binding protein (CtBP) has been well characterized as a transcriptional co-repressor. Herein, we report a previously unreported function for CtBP, showing that lowering CtBP dosage genetically suppresses Polycomb group (PcG) loss-of-function phenotypes while enhancing that of trithorax group (trxG) in Drosophila, suggesting that the role of CtBP in gene activation is more pronounced in fly development than previously thought. In fly cells, we show that CtBP is required for the derepression of the most direct PcG target genes, which are highly enriched by homeobox transcription factors, including Hox genes. Using ChIP and co-IP assays, we demonstrate that CtBP is directly required for the molecular switch between H3K27me3 and H3K27ac in the derepressed Hox loci. In addition, CtBP physically interacts with many proteins, such as UTX, CBP, Fs(1)h and RNA Pol II, that have activation roles, potentially assisting in their recruitment to promoters and Polycomb response elements that control Hox gene expression. Therefore, we reveal a prominent activation function for CtBP that confers a major role for the epigenetic program of fly segmentation and development.

Differences in temperature-sensitive expression of PcG-regulated genes among natural populations of Drosophila melanogaster.

Environmental temperature can affect chromatin-based gene regulation, in particular in ectotherms such as insects. Genes regulated by the Polycomb group (PcG) vary in their transcriptional output in response to changes in temperature. Expression of PcG-regulated genes typically increases with decreasing temperatures. Here, we examined variations in temperature-sensitive expression of PcG target genes in natural populations from different climates of Drosophila melanogaster, and differences thereof across different fly stages and tissues. Temperature-induced expression plasticity was found to be stage- and sex-specific with differences in the specificity between the examined PcG target genes. Some tissues and stages, however, showed a higher number of PcG target genes with temperature-sensitive expression than others. Overall, we found higher levels of temperature-induced expression plasticity in African tropical flies from the ancestral species range than in flies from temperate Europe. We also observed differences between temperate flies, however, with more reduction of expression plasticity in warm-temperate than in cold-temperate populations. Although in general, temperature-sensitive expression appeared to be detrimental in temperate climates, there were also cases in which plasticity was increased in temperate flies, as well as no changes in expression plasticity between flies from different climates.

Genomewide decoupling of H2AK119ub1 and H3K27me3 in early mouse development

Polycomb group (PcG) proteins are crucial chromatin regulators during development. H2AK119ub1 (H2Aub) and H3K27me3 are catalyzed by Polycomb-repressive complex 1 and 2 (PRC1/2) respectively, and they largely overlap in the genome due to mutual recruitment of the two complexes. However, it is unclear whether PRC1/H2Aub and PRC2/H3K27me3 can also function independently. By developing an ultra-sensitive carrier-DNA-assisted chromatin immunoprecipitation sequencing method termed CATCH-Seq, we generated allelic H2Aub profiles in mouse gametes and early embryos. Our results revealed an unexpected genomewide decoupling of H2Aub and H3K27me3 in mouse preimplantation embryos, where H2Aub but not H3K27me3 was enriched at PcG targets while only H3K27me3 was deposited in the broad distal domains associated with DNA methylation-independent non-canonical imprinting. These observations suggest that H2Aub represses future bivalent genes during early embryogenesis without H3K27me3, but it is not required for the maintenance of non-canonical imprinting, which is mediated by maternal H3K27me3. Thus, our study reveals the distinct depositions and independent functions of H2Aub and H3K27me3 during early mammalian development.

Defining the Boundaries of Polycomb Domains in Drosophila.

Polycomb group (PcG) proteins are an important group of transcriptional repressors that act by modifying chromatin. PcG target genes are covered by the repressive chromatin mark H3K27me3. Polycomb repressive complex 2 (PRC2) is a multiprotein complex that is responsible for generating H3K27me3. In Drosophila, PRC2 is recruited by Polycomb Response Elements (PREs) and then trimethylates flanking nucleosomes, spreading the H3K27me3 mark over large regions of the genome, the "Polycomb domains." What defines the boundary of a Polycomb domain? There is experimental evidence that insulators, PolII, and active transcription can all form the boundaries of Polycomb domains. Here we divide the boundaries of larval Polycomb domains into six different categories. In one category, genes are transcribed toward the Polycomb domain, where active transcription is thought to stop the spreading of H3K27me3. In agreement with this, we show that introducing a transcriptional terminator into such a transcription unit causes an extension of the Polycomb domain. Additional data suggest that active transcription of a boundary gene may restrict the range of enhancer activity of a Polycomb-regulated gene.

Identification of distinct loci for de novo DNA methylation by DNMT3A and DNMT3B during mammalian development

De novo establishment of DNA methylation is accomplished by DNMT3A and DNMT3B. Here, we analyze de novo DNA methylation in mouse embryonic fibroblasts (2i-MEFs) derived from DNA-hypomethylated 2i/L ES cells with genetic ablation of Dnmt3a or Dnmt3b. We identify 355 and 333 uniquely unmethylated genes in Dnmt3a and Dnmt3b knockout (KO) 2i-MEFs, respectively. We find that Dnmt3a is exclusively required for de novo methylation at both TSS regions and gene bodies of Polycomb group (PcG) target developmental genes, while Dnmt3b has a dominant role on the X chromosome. Consistent with this, tissue-specific DNA methylation at PcG target genes is substantially reduced in Dnmt3a KO embryos. Finally, we find that human patients with DNMT3 mutations exhibit reduced DNA methylation at regions that are hypomethylated in Dnmt3 KO 2i-MEFs. In conclusion, here we report a set of unique de novo DNA methylation target sites for both DNMT3 enzymes during mammalian development that overlap with hypomethylated sites in human patients.

The domesticated transposase ALP2 mediates formation of a novel Polycomb protein complex by direct interaction with MSI1, a core subunit of Polycomb Repressive Complex 2 (PRC2).

A large fraction of plant genomes is composed of transposable elements (TE), which provide a potential source of novel genes through "domestication"-the process whereby the proteins encoded by TE diverge in sequence, lose their ability to catalyse transposition and instead acquire novel functions for their hosts. In Arabidopsis, ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) arose by domestication of the nuclease component of Harbinger class TE and acquired a new function as a component of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a histone H3K27me3 methyltransferase involved in regulation of host genes and in some cases TE. It was not clear how ALP1 associated with PRC2, nor what the functional consequence was. Here, we identify ALP2 genetically as a suppressor of Polycomb-group (PcG) mutant phenotypes and show that it arose from the second, DNA binding component of Harbinger transposases. Molecular analysis of PcG compromised backgrounds reveals that ALP genes oppose silencing and H3K27me3 deposition at key PcG target genes. Proteomic analysis reveals that ALP1 and ALP2 are components of a variant PRC2 complex that contains the four core components but lacks plant-specific accessory components such as the H3K27me3 reader LIKE HETEROCHROMATION PROTEIN 1 (LHP1). We show that the N-terminus of ALP2 interacts directly with ALP1, whereas the C-terminus of ALP2 interacts with MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a core component of PRC2. Proteomic analysis reveals that in alp2 mutant backgrounds ALP1 protein no longer associates with PRC2, consistent with a role for ALP2 in recruitment of ALP1. We suggest that the propensity of Harbinger TE to insert in gene-rich regions of the genome, together with the modular two component nature of their transposases, has predisposed them for domestication and incorporation into chromatin modifying complexes.

A central role for canonical PRC1 in shaping the 3D nuclear landscape.

Polycomb group (PcG) proteins silence gene expression by chemically and physically modifying chromatin. A subset of PcG target loci are compacted and cluster in the nucleus; a conformation that is thought to contribute to gene silencing. However, how these interactions influence gross nuclear organization and their relationship with transcription remains poorly understood. Here we examine the role of Polycomb-repressive complex 1 (PRC1) in shaping 3D genome organization in mouse embryonic stem cells (mESCs). Using a combination of imaging and Hi-C analyses, we show that PRC1-mediated long-range interactions are independent of CTCF and can bridge sites at a megabase scale. Impairment of PRC1 enzymatic activity does not directly disrupt these interactions. We demonstrate that PcG targets coalesce in vivo, and that developmentally induced expression of one of the target loci disrupts this spatial arrangement. Finally, we show that transcriptional activation and the loss of PRC1-mediated interactions are separable events. These findings provide important insights into the function of PRC1, while highlighting the complexity of this regulatory system.

Priming exposures to lipopolysaccharides do not affect the induction of Polycomb target genes upon re-exposure.

The Polycomb group (PcG) proteins are chromatin factors underlying the process of transcriptional memory to preserve developmental decisions and keep cellular identities. However, not only developmental signals need to be memorized and thus maintained during the life of an organism. For host protection against pathogens, also a memory of previous exposures to an immunogenic stimulus is crucial to mount a more protective immune response upon re-exposure. The antigen-specific adaptive immunity in vertebrates is an example of such a memory to previous immunogenic stimulation. Recently, adaptive characteristics were also attributed to innate immunity, which was classically seen to lack memory. However, the mechanistic details of an adaptive innate immune response are yet to be fully understood and chromatin-based epigenetic mechanisms seem to play an important role in this phenomenon. Possibly, PcG proteins can contribute to such an epigenetic innate immune memory. In this study, we analyzed whether the PcG system can mediate a transcriptional memory of exposure to lipopolysaccharides (LPS). To this end, various forms of LPS pre-treatment were applied to reporter cells and expression kinetics of PcG target genes were analyzed after a second LPS exposure. Neither single nor multiple LPS pre-treatment affected the induction of endogenous LPS-responsive transcripts upon re-exposure. Altogether, our extensive analyses did not provide any evidence for a PcG system-mediated memory of LPS stimulation.

Polycomb Protects H3K27me3 from Active Demethylation by Utx

Maintenance of cellular identity is a critical developmental process depending on the proper transmission of epigenetic information over cell generations. H3K27me3, controlled by the Polycomb group (PcG) proteins, represents such an epigenetic mark and is correlated to long-term and heritable gene silencing. However, H3K27me3 is also found at many inducible or cell cycle regulated genes, implicating an involvement in more dynamic processes. The chromodomain of Polycomb (Pc) binds the methyl moiety of H3K27 and anchors the PRC1 complex involved in nucleosome compaction. We adapted an Auxin-based degron system to deplete Pc during Drosophila development. The fast degradation of Pc results in the rapid removal of the H3K27me3 mark by the de-methylase Utx and in consequence in the rapid upregulation of Hox genes. This result shows that the chromodomain actively protects the methyl mark, inhibiting removal by Utx, hence, allowing for a much more dynamic regulation of PcG target genes.

De novo recruitment of Polycomb-group proteins in Drosophila embryos.

Polycomb-group (PcG)-mediated transcriptional repression of target genes can be delineated into two phases. First, following initial repression of target genes by gene-specific transcription factors, PcG proteins recognize the repressed state and assume control of the genes' repression. Second, once the silenced state is established, PcG proteins may maintain repression through an indefinite number of cell cycles. Little is understood about how PcG proteins initially recognize the repressed state of target genes and the steps leading to de novo establishment of PcG-mediated repression. We describe a genetic system in which a Drosophila PcG target gene, giant (gt), is ubiquitously repressed during early embryogenesis by a maternally expressed transcription factor, and show the temporal recruitment of components of three PcG protein complexes: PhoRC, PRC1 and PRC2. We show that de novo PcG recruitment follows a temporal hierarchy in which PhoRC stably localizes at the target gene at least 1 h before stable recruitment of PRC2 and concurrent trimethylation of histone H3 at lysine 27 (H3K27me3). The presence of PRC2 and increased levels of H3K27me3 are found to precede stable binding by PRC1.

CBX2 Inhibits Neurite Development by Regulating Neuron-Specific Genes Expression.

Polycomb group (PcG) proteins regulate the epigenetic status of transcription regulatory states during development. Progression from pluripotency to differentiation requires the sequential activation and repression of different PcG target genes, however, the relationship between early patterning signals, PcG expression, and the development of the central nervous system is still unclear. Using various models of neuronal differentiation, we provide evidence that CBX2 is a negative regulator of neuronal differentiation. Knock-down of CBX2 expression promotes neurite development, while overexpression of CBX2 inhibits neurite development. Further, we found that CBX2 is a direct target gene of miR-124. During neuronal differentiation, CBX2 was decreased while miR-124 was increased. Mechanistically, CBX2 directly interacts with the promoter region of several neuro-associated genes and regulates their expression. We found that the neuron-specific GAP-43 gene could contribute to the stimulating effect on neurite development associated with inhibition of CBX2.

Global changes of H3K27me3 domains and Polycomb group protein distribution in the absence of recruiters Spps or Pho

Polycomb group (PcG) proteins maintain the silenced state of key developmental genes in animals, but how these proteins are recruited to specific regions of the genome is still poorly understood. In Drosophila, PcG proteins are recruited to Polycomb response elements (PREs) that include combinations of sites for sequence specific DNA binding "PcG recruiters," including Pho, Cg, and Spps. To understand their roles in PcG recruitment, we compared Pho-, Cg-, and Spps-binding sites against H3K27me3 and key PcG proteins by ChIP-seq in wild-type and mutant third instar larvae. H3K27me3 in canonical Polycomb domains is decreased after the reduction of any recruiter. Reduction of Spps and Pho, but not Cg, causes the redistribution of H3K27me3 to heterochromatin. Regions with dramatically depleted H3K27me3 after Spps knockout are usually accompanied by decreased Pho binding, suggesting their cooperative binding. PcG recruiters, the PRC2 component E(z), and the PRC1 components Psc and Ph cobind thousands of active genes outside of H3K27me3 domains. This study demonstrates the importance of distinct PcG recruiters for the establishment of unique Polycomb domains. Different PcG recruiters can act both cooperatively and independently at specific PcG target genes, highlighting the complexity and diversity of PcG recruitment mechanisms.

Death by HDAC Inhibition in Synovial Sarcoma Cells.

Conventional cytotoxic therapies for synovial sarcoma provide limited benefit, and no drugs specifically targeting the causative SS18-SSX fusion oncoprotein are currently available. Histone deacetylase (HDAC) inhibition has been shown in previous studies to disrupt the synovial sarcoma oncoprotein complex, resulting in apoptosis. To understand the molecular effects of HDAC inhibition, RNA-seq transcriptome analysis was undertaken in six human synovial sarcoma cell lines. HDAC inhibition induced pathways of cell-cycle arrest, neuronal differentiation, and response to oxygen-containing species, effects also observed in other cancers treated with this class of drugs. More specific to synovial sarcoma, polycomb group targets were reactivated, including tumor suppressor CDKN2A, and proapoptotic transcriptional patterns were induced. Functional analyses revealed that ROS-mediated FOXO activation and proapoptotic factors BIK, BIM, and BMF were important to apoptosis induction following HDAC inhibition in synovial sarcoma. HDAC inhibitor pathway activation results in apoptosis and decreased tumor burden following a 7-day quisinostat treatment in the Ptenfl/fl;hSS2 mouse model of synovial sarcoma. This study provides mechanistic support for a particular susceptibility of synovial sarcoma to HDAC inhibition as a means of clinical treatment. Mol Cancer Ther; 16(12); 2656-67. ©2017 AACR.

Highlights of This Issue

Many solid tumors demonstrate dysregulation of fibroblast growth factor receptors (FGFRs) but currently no selective inhibitors of FGFR are approved for cancer treatment. Here, Venetsanakos et al. describe the characterization of a pan-FGFR inhibitor PRN1371 that forms a covalent bond with a cysteine in the FGFR active site. The authors describe the high selectivity and strong anti-tumor activity of PRN1371 and further highlight that covalent binding enables PRN1371 to sustain inhibition of FGFR even after the compound is no longer present in circulation. PRN1371 is currently undergoing clinical trials to explore the benefits that these attributes provide to patients with solid tumors.Histone deacetylase (HDAC) inhibition in synovial sarcoma has been found to disrupt the driving SS18-SSX protein complex, resulting in apoptosis. To better understand the molecular effects of HDAC inhibitors, Laporte and colleagues used RNA-seq transcriptome analysis to uncover mechanisms of cell death in six human synovial sarcoma cells lines. Specific to SS18-SSX positive cell lines, polycomb-group targets were reactivated resulting in pro-apoptotic transcriptional patterns, which was mediated by ROS activity. Furthermore, HDAC inhibition in a GEM model of synovial sarcoma resulted in significantly decreased tumor burden following treatment with novel HDAC inhibitor quisinostat. This study provides mechanistic support for clinical proposals to use HDAC inhibitors in synovial sarcoma and related diseases.Increased MET activity is linked to poor prognosis of several human cancers. In this study, Grandal, Havrylov and colleagues present Sym015, an antibody mixture composed of two IgG1 antibodies against non-overlapping epitopes on MET. Synergistic inhibitory activity of the two antibodies comprising Sym015 was demonstrated in vitro and in vivo. Sym015 was found to block ligand binding and downstream signaling and to induce MET degradation. Sym015 also potently induces secondary effector functions. The enhanced effect of Sym015 is predicted to render Sym015 superior to single antibodies targeting MET. Clinical activity of Sym015 is currently being evaluated in a phase I trial.TRAIL has been considered as promising anti-cancer therapeutic. Clinical studies with soluble TRAIL, however, were largely disappointing. Here, Hutt and colleagues applied defined oligomerization and tumor targeting to improve the activity of a single-chain version of TRAIL (scTRAIL) and compared three EGFR-targeting as well as two non-targeted hexavalent scTRAIL formats. All EGFR-targeted molecules showed similar and improved activity compared to non-targeted formats in vitro, while in vivo studies highlighted superior efficacy of Fc-comprising molecules. Furthermore, depending on the tumor entity, Fc-scTRAIL may induce equally effective anti-tumor activity as targeted counterparts. This study supports the rational development of potent hexavalent TRAIL-based therapeutics.