Tumor cell-directed STING agonist antibody-drug conjugates induce type III interferons and anti-tumor innate immune responses.

Activating interferon responses with STING agonists (STINGa) is a current cancer immunotherapy strategy, and therapeutic modalities that enable tumor-targeted delivery via systemic administration could be beneficial. Here we demonstrate that tumor cell-directed STING agonist antibody-drug-conjugates (STINGa ADCs) activate STING in tumor cells and myeloid cells and induce anti-tumor innate immune responses in in vitro, in vivo (in female mice), and ex vivo tumor models. We show that the tumor cell-directed STINGa ADCs are internalized into myeloid cells by Fcγ-receptor-I in a tumor antigen-dependent manner. Systemic administration of STINGa ADCs in mice leads to STING activation in tumors, with increased anti-tumor activity and reduced serum cytokine elevations compared to a free STING agonist. Furthermore, STINGa ADCs induce type III interferons, which contribute to the anti-tumor activity by upregulating type I interferon and other key chemokines/cytokines. These findings reveal an important role for type III interferons in the anti-tumor activity elicited by STING agonism and provide rationale for the clinical development of tumor cell-directed STINGa ADCs.

Charophytic Green Algae Encode Ancestral Polymerase IV/Polymerase V Subunits and a CLSY/DRD1 Homolog.

In flowering plants, euchromatic transposons are transcriptionally silenced by RNA-directed DNA Methylation, a small RNA-guided de novo methylation pathway. RNA-directed DNA Methylation requires the activity of the RNA Polymerases IV and V, which produce small RNA precursors and noncoding targets of small RNAs, respectively. These polymerases are distinguished from Polymerase II by multiple plant-specific paralogous subunits. Most RNA-directed DNA Methylation components are present in all land plants, and some have been found in the charophytic green algae, a paraphyletic group that is sister to land plants. However, the evolutionary origin of key RNA-directed DNA Methylation components, including the two largest subunits of Polymerase IV and Polymerase V, remains unclear. Here, we show that multiple lineages of charophytic green algae encode a single-copy precursor of the largest subunits of Polymerase IV and Polymerase V, resolving the two presumed duplications in this gene family. We further demonstrate the presence of a Polymerase V-like C-terminal domain, suggesting that the earliest form of RNA-directed DNA Methylation utilized a single Polymerase V-like polymerase. Finally, we reveal that charophytic green algae encode a single CLSY/DRD1-type chromatin remodeling protein, further supporting the presence of a single specialized polymerase in charophytic green algae.

Mating system is associated with seed phenotypes upon loss of RNA-directed DNA methylation in Brassicaceae.

In plants, de novo DNA methylation is guided by 24-nt short interfering (si)RNAs in a process called RNA-directed DNA methylation (RdDM). Primarily targeted at transposons, RdDM causes transcriptional silencing and can indirectly influence expression of neighboring genes. During reproduction, a small number of siRNA loci are dramatically upregulated in the maternally derived seed coat, suggesting that RdDM might have a special function during reproduction. However, the developmental consequence of RdDM has been difficult to dissect because disruption of RdDM does not result in overt phenotypes in Arabidopsis (Arabidopsis thaliana), where the pathway has been most thoroughly studied. In contrast, Brassica rapa mutants lacking RdDM have a severe seed production defect, which is determined by the maternal sporophytic genotype. To explore the factors that underlie the different phenotypes of these species, we produced RdDM mutations in 3 additional members of the Brassicaceae family: Camelina sativa, Capsella rubella, and Capsella grandiflora. Among these 3 species, only mutations in the obligate outcrosser, C. grandiflora, displayed a seed production defect similar to Brassica rapa mutants, suggesting that mating system is a key determinant for reproductive phenotypes in RdDM mutants.

Mapping a mutation causing pale yellow petals in Brassica rapa.

Petal color is an important trait for both ornamental purposes and also for attracting pollinators. Here, we report a mutation of Brassica rapa R-o-18 with pale yellow petals that we retrieved from an EMS population and named whiter shade of pale ( wsp ). Phenotypic segregation ratio of an F2 mapping population indicates the phenotype is controlled by a single recessive gene. Mapping data from the whole genome sequencing coupled with allele frequency analysis suggests the mutation is located in a ~2 Mbp interval on chromosome 2. The interval contains a putative esterase/lipase/thioesterase protein previously demonstrated to account for floral color in B. rapa . We demonstrate that wsp carries a G to A missense mutation causing an aspartate to asparagine substitution within the putative lysophospholipid acyltransferase domain.

Discovery and Preclinical Characterization of XMT-1660, an Optimized B7-H4-Targeted Antibody-Drug Conjugate for the Treatment of Cancer.

Antibody-drug conjugates (ADC) achieve targeted drug delivery to a tumor and have demonstrated clinical success in many tumor types. The activity and safety profile of an ADC depends on its construction: antibody, payload, linker, and conjugation method, as well as the number of payload drugs per antibody [drug-to-antibody ratio (DAR)]. To allow for ADC optimization for a given target antigen, we developed Dolasynthen (DS), a novel ADC platform based on the payload auristatin hydroxypropylamide, that enables precise DAR-ranging and site-specific conjugation. We used the new platform to optimize an ADC that targets B7-H4 (VTCN1), an immune-suppressive protein that is overexpressed in breast, ovarian, and endometrial cancers. XMT-1660 is a site-specific DS DAR 6 ADC that induced complete tumor regressions in xenograft models of breast and ovarian cancer as well as in a syngeneic breast cancer model that is refractory to PD-1 immune checkpoint inhibition. In a panel of 28 breast cancer PDXs, XMT-1660 demonstrated activity that correlated with B7-H4 expression. XMT-1660 has recently entered clinical development in a phase I study (NCT05377996) in patients with cancer.

Plant polymerase IV sensitizes chromatin through histone modifications to preclude spread of silencing into protein-coding domains.

Across eukaryotes, gene regulation is manifested via chromatin states roughly distinguished as heterochromatin and euchromatin. The establishment, maintenance, and modulation of the chromatin states is mediated using several factors including chromatin modifiers. However, factors that avoid the intrusion of silencing signals into protein-coding genes are poorly understood. Here we show that a plant specific paralog of RNA polymerase (Pol) II, named Pol IV, is involved in avoidance of facultative heterochromatic marks in protein-coding genes, in addition to its well-established functions in silencing repeats and transposons. In its absence, H3K27 trimethylation (me3) mark intruded the protein-coding genes, more profoundly in genes embedded with repeats. In a subset of genes, spurious transcriptional activity resulted in small(s) RNA production, leading to post-transcriptional gene silencing. We show that such effects are significantly pronounced in rice, a plant with a larger genome with distributed heterochromatin compared with Arabidopsis Our results indicate the division of labor among plant-specific polymerases, not just in establishing effective silencing via sRNAs and DNA methylation but also in influencing chromatin boundaries.

Small RNA-mediated DNA methylation during plant reproduction.

Reproductive tissues are a rich source of small RNAs, including several classes of short interfering (si)RNAs that are restricted to this stage of development. In addition to RNA polymerase IV-dependent 24-nt siRNAs that trigger canonical RNA-directed DNA methylation, abundant reproductive-specific siRNAs are produced from companion cells adjacent to the developing germ line or zygote and may move intercellularly before inducing methylation. In some cases, these siRNAs are produced via non-canonical biosynthesis mechanisms or from sequences with little similarity to transposons. While the precise role of these siRNAs and the methylation they trigger is unclear, they have been implicated in specifying a single megaspore mother cell, silencing transposons in the male germ line, mediating parental dosage conflict to ensure proper endosperm development, hypermethylation of mature embryos, and trans-chromosomal methylation in hybrids. In this review, we summarize the current knowledge of reproductive siRNAs, including their biosynthesis, transport, and function.

A novel CLAVATA1 mutation causes multilocularity in Brassica rapa.

Locules are the seed-bearing structure of fruits. Multiple locules are associated with increased fruit size and seed set, and therefore, control of locule number is an important agronomic trait. Locule number is controlled in part by the CLAVATA-WUSCHEL pathway. Disruption of either the CLAVATA1 receptor-like kinase or its ligand CLAVATA3 can cause larger floral meristems and an increased number of locules. In an EMS mutagenized population of Brassica rapa, we identified a mutant allele that raises the number of locules from four to a range of from six to eight. Linkage mapping and genetic analysis support that the mutant phenotype is due to a missense mutation in a CLAVATA 1 (CLV1) homolog. In addition to increased locule number, additional internal gynoecia are formed in brclv1 individuals, suggesting a failure to terminate floral meristem development, which results in decreased seed production.

Ovule siRNAs methylate protein-coding genes in trans.

Twenty-four-nucleotide (nt) small interfering RNAs (siRNAs) maintain asymmetric DNA methylation at thousands of euchromatic transposable elements in plant genomes in a process called RNA-directed DNA methylation (RdDM). RdDM is dispensable for growth and development in Arabidopsis thaliana, but is required for reproduction in other plants, such as Brassica rapa. The 24-nt siRNAs are abundant in maternal reproductive tissue, due largely to overwhelming expression from a few loci in the ovule and developing seed coat, termed siren loci. A recent study showed that 24-nt siRNAs produced in the anther tapetal tissue can methylate male meiocyte genes in trans. Here we show that in B. rapa, a similar process takes place in female tissue. siRNAs are produced from gene fragments embedded in some siren loci, and these siRNAs can trigger methylation in trans at related protein-coding genes. This trans-methylation is associated with silencing of some target genes and may be responsible for seed abortion in RdDM mutants. Furthermore, we demonstrate that a consensus sequence in at least two families of DNA transposons is associated with abundant siren expression, most likely through recruitment of CLASSY3, a putative chromatin remodeler. This research describes a mechanism whereby RdDM influences gene expression and sheds light on the role of RdDM during plant reproduction.

Expansion and contraction of small RNA and methylation machinery throughout plant evolution.

The revolution in sequencing has created a wealth of plant genomes that can be mined to understand the evolution of biological complexity. Complexity is often driven by gene duplication, which allows paralogs to specialize in an activity of the ancestral gene or acquire novel functions. Angiosperms encode a variety of gene silencing pathways that share related machinery for small RNA biosynthesis and function. Recent phylogenetic analysis of these gene families plots the expansion, specialization, and occasional contraction of this core machinery. This analysis reveals the ancient origin of RNA-directed DNA Methylation in early land plants, or possibly their algal ancestors, as well as ongoing duplications that evolve novel small RNA pathways.

A null allele of the pol IV second subunit impacts stature and reproductive development in Oryza sativa.

All eukaryotes possess three DNA-dependent RNA polymerases, Pols I-III, while land plants possess two additional polymerases, Pol IV and Pol V. Derived through duplication of Pol II subunits, Pol IV produces 24-nt short interfering RNAs that interact with Pol V transcripts to target de novo DNA methylation and silence transcription of transposons. Members of the grass family encode additional duplicated subunits of Pol IV and V, raising questions regarding the function of each paralog. In this study, we identify a null allele of the putative Pol IV second subunit, NRPD2, and demonstrate that NRPD2 is the sole subunit functioning with NRPD1 in small RNA production and CHH methylation in leaves. Homozygous nrpd2 mutants have neither gametophytic defects nor embryo lethality, although adult plants are dwarf and sterile.

Quality control and evaluation of plant epigenomics data.

Epigenomics is the study of molecular signatures associated with discrete regions within genomes, many of which are important for a wide range of nuclear processes. The ability to profile the epigenomic landscape associated with genes, repetitive regions, transposons, transcription, differential expression, cis-regulatory elements, and 3D chromatin interactions has vastly improved our understanding of plant genomes. However, many epigenomic and single-cell genomic assays are challenging to perform in plants, leading to a wide range of data quality issues; thus, the data require rigorous evaluation prior to downstream analyses and interpretation. In this commentary, we provide considerations for the evaluation of plant epigenomics and single-cell genomics data quality with the aim of improving the quality and utility of studies using those data across diverse plant species.

Embryo CHH hypermethylation is mediated by RdDM and is autonomously directed in Brassica rapa.

BACKGROUND: RNA-directed DNA methylation (RdDM) initiates cytosine methylation in all contexts and maintains asymmetric CHH methylation. Mature plant embryos show one of the highest levels of CHH methylation, and it has been suggested that RdDM is responsible for this hypermethylation. Because loss of RdDM in Brassica rapa causes seed abortion, embryo methylation might play a role in seed development. RdDM is required in the maternal sporophyte, suggesting that small RNAs from the maternal sporophyte might translocate to the developing embryo, triggering DNA methylation that prevents seed abortion. This raises the question of whether embryo hypermethylation is autonomously regulated by the embryo itself or influenced by the maternal sporophyte. RESULTS: Here, we demonstrate that B. rapa embryos are hypermethylated in both euchromatin and heterochromatin and that this process requires RdDM. Contrary to the current models, B. rapa embryo hypermethylation is not correlated with demethylation of the endosperm. We also show that maternal somatic RdDM is not sufficient for global embryo hypermethylation, and we find no compelling evidence for maternal somatic influence over embryo methylation at any locus. Decoupling of maternal and zygotic RdDM leads to successful seed development despite the loss of embryo CHH hypermethylation. CONCLUSIONS: We conclude that embryo CHH hypermethylation is conserved, autonomously controlled, and not required for embryo development. Furthermore, maternal somatic RdDM, while required for seed development, does not directly influence embryo methylation patterns.

The Dolaflexin-based Antibody-Drug Conjugate XMT-1536 Targets the Solid Tumor Lineage Antigen SLC34A2/NaPi2b.

Target selection for antibody-drug conjugates (ADC) frequently focuses on identifying antigens with differential expression in tumor and normal tissue, to mitigate the risk of on-target toxicity. However, this strategy restricts the possible target space. SLC34A2/NaPi2b is a sodium phosphate transporter expressed in a variety of human tumors including lung and ovarian carcinoma, as well as the normal tissues from which these tumors arise. Previous clinical trials with a NaPi2b targeting MMAE-ADCs have shown objective durable responses. However, the protein-based biomarker assay developed for use in that study was unable to discern a statistically significant relationship between NaPi2b protein expression and the probability of response. XMT-1536 is a NaPi2b targeting ADC comprised of a unique humanized antibody conjugated with 10-15 auristatin F- hydroxypropylamide (AF-HPA) payload molecules via the Dolaflexin platform. AF-HPA is a cell-permeable, antimitotic compound that is slowly metabolized intratumorally to an active, very low-permeable metabolite, auristatin F (AF), resulting in controlled bystander killing. We describe the preclinical in vitro and in vivo antitumor effects of XMT-1536 in models of ovarian and lung adenocarcinoma. Pharmacokinetic analysis showed approximately proportional increases in exposure in rat and monkey. Systemic free AF-HPA and AF concentrations were observed to be low in all animal species. Finally, we describe a unique IHC reagent, generated from a chimeric construct of the therapeutic antibody, that was used to derive a target expression and efficacy relationship in a series of ovarian primary xenograft cancer models.

Abundant expression of maternal siRNAs is a conserved feature of seed development.

Small RNAs are abundant in plant reproductive tissues, especially 24-nucleotide (nt) small interfering RNAs (siRNAs). Most 24-nt siRNAs are dependent on RNA Pol IV and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and establish DNA methylation at thousands of genomic loci in a process called RNA-directed DNA methylation (RdDM). In Brassica rapa, RdDM is required in the maternal sporophyte for successful seed development. Here, we demonstrate that a small number of siRNA loci account for over 90% of siRNA expression during B. rapa seed development. These loci exhibit unique characteristics with regard to their copy number and association with genomic features, but they resemble canonical 24-nt siRNA loci in their dependence on RNA Pol IV/RDR2 and role in RdDM. These loci are expressed in ovules before fertilization and in the seed coat, embryo, and endosperm following fertilization. We observed a similar pattern of 24-nt siRNA expression in diverse angiosperms despite rapid sequence evolution at siren loci. In the endosperm, siren siRNAs show a marked maternal bias, and siren expression in maternal sporophytic tissues is required for siren siRNA accumulation. Together, these results demonstrate that seed development occurs under the influence of abundant maternal siRNAs that might be transported to, and function in, filial tissues.

RNA-directed DNA Methylation and sexual reproduction: expanding beyond the seed.

Two trends are changing our understanding of RNA-directed DNA methylation. In model systems like Arabidopsis, tissue-specific analysis of DNA methylation is uncovering dynamic changes in methylation during sexual reproduction and unraveling the contribution of maternal and paternal epigenomes to the developing embryo. These studies indicate that RNA-directed DNA Methylation might be important for mediating balance between maternal and paternal contributions to the endosperm. At the same time, researchers are moving beyond Arabidopsis to illuminate the ancestral role of RdDM in non-flowering plants that lack an endosperm, suggesting that RdDM might play a broader role in sexual reproduction.

Maternal small RNAs mediate spatial-temporal regulation of gene expression, imprinting, and seed development in Arabidopsis.

Arabidopsis seed development involves maternal small interfering RNAs (siRNAs) that induce RNA-directed DNA methylation (RdDM) through the NRPD1-mediated pathway. To investigate their biological functions, we characterized siRNAs in the endosperm and seed coat that were separated by laser-capture microdissection (LCM) in reciprocal genetic crosses with an nrpd1 mutant. We also monitored the spatial-temporal activity of the NRPD1-mediated pathway on seed development using the AGO4:GFP::AGO4 (promoter:GFP::protein) reporter and promoter:GUS sensors of siRNA-mediated silencing. From these approaches, we identified four distinct groups of siRNA loci dependent on or independent of the maternal NRPD1 allele in the endosperm or seed coat. A group of maternally expressed NRPD1-siRNA loci targets endosperm-preferred genes, including those encoding AGAMOUS-LIKE (AGL) transcription factors. Using translational promoter:AGL::GUS constructs as sensors, we demonstrate that spatial and temporal expression patterns of these genes in the endosperm are regulated by the NRPD1-mediated pathway irrespective of complete silencing (AGL91) or incomplete silencing (AGL40) of these target genes. Moreover, altered expression of these siRNA-targeted genes affects seed size. We propose that the corresponding maternal siRNAs could account for parent-of-origin effects on the endosperm in interploidy and hybrid crosses. These analyses reconcile previous studies on siRNAs and imprinted gene expression during seed development.

Evidence for a Unique DNA-Dependent RNA Polymerase in Cereal Crops.

Gene duplication is an important driver for the evolution of new genes and protein functions. Duplication of DNA-dependent RNA polymerase (Pol) II subunits within plants led to the emergence of RNA Pol IV and V complexes, each of which possess unique functions necessary for RNA-directed DNA Methylation. Comprehensive identification of Pol V subunit orthologs across the monocot radiation revealed a duplication of the largest two subunits within the grasses (Poaceae), including critical cereal crops. These paralogous Pol subunits display sequence conservation within catalytic domains, but their carboxy terminal domains differ in length and character of the Ago-binding platform, suggesting unique functional interactions. Phylogenetic analysis of the catalytic region indicates positive selection on one paralog following duplication, consistent with retention via neofunctionalization. Positive selection on residue pairs that are predicted to interact between subunits suggests that paralogous subunits have evolved specific assembly partners. Additional Pol subunits as well as Pol-interacting proteins also possess grass-specific paralogs, supporting the hypothesis that a novel Pol complex with distinct function has evolved in the grass family, Poaceae.