23ME-00610, a genetically informed, first-in-class antibody targeting CD200R1 to enhance antitumor T cell function.

Immune checkpoint inhibition (ICI) has revolutionized cancer treatment; however, only a subset of patients benefit long term. Therefore, methods for identification of novel checkpoint targets and development of therapeutic interventions against them remain a critical challenge. Analysis of human genetics has the potential to inform more successful drug target discovery. We used genome-wide association studies of the 23andMe genetic and health survey database to identify an immuno-oncology signature in which genetic variants are associated with opposing effects on risk for cancer and immune diseases. This signature identified multiple pathway genes mapping to the immune checkpoint comprising CD200, its receptor CD200R1, and the downstream adapter protein DOK2. We confirmed that CD200R1 is elevated on tumor-infiltrating immune cells isolated from cancer patients compared to the matching peripheral blood mononuclear cells. We developed a humanized, effectorless IgG1 antibody (23ME-00610) that bound human CD200R1 with high affinity (KD <0.1 nM), blocked CD200 binding, and inhibited recruitment of DOK2. 23ME-00610 induced T-cell cytokine production and enhanced T cell-mediated tumor cell killing in vitro. Blockade of the CD200:CD200R1 immune checkpoint inhibited tumor growth and engaged immune activation pathways in an S91 tumor cell model of melanoma in mice.

Distinct resistance mechanisms arise to allosteric vs. ATP-competitive AKT inhibitors.

The AKT kinases have emerged as promising therapeutic targets in oncology and both allosteric and ATP-competitive AKT inhibitors have entered clinical investigation. However, long-term efficacy of such inhibitors will likely be challenged by the development of resistance. We have established prostate cancer models of acquired resistance to the allosteric inhibitor MK-2206 or the ATP-competitive inhibitor ipatasertib following prolonged exposure. While alterations in AKT are associated with acquired resistance to MK-2206, ipatasertib resistance is driven by rewired compensatory activity of parallel signaling pathways. Importantly, MK-2206 resistance can be overcome by treatment with ipatasertib, while ipatasertib resistance can be reversed by co-treatment with inhibitors of pathways including PIM signaling. These findings demonstrate that distinct resistance mechanisms arise to the two classes of AKT inhibitors and that combination approaches may reverse resistance to ATP-competitive inhibition.

Therapeutic resistance and susceptibility is shaped by cooperative multi-compartment tumor adaptation.

Emerging research suggests that multiple tumor compartments can influence treatment responsiveness and relapse, yet the search for therapeutic resistance mechanisms remains largely focused on acquired genomic alterations in cancer cells. Here we show how treatment-induced changes occur in multiple tumor compartments during tumor relapse and can reduce benefit of follow-on therapies. By using serial biopsies, next-generation sequencing, and single-cell transcriptomics, we tracked the evolution of multiple cellular compartments within individual lesions during first-line treatment response, relapse, and second-line therapeutic interventions in an autochthonous model of melanoma. We discovered that although treatment-relapsed tumors remained genetically stable, they converged on a shared resistance phenotype characterized by dramatic changes in tumor cell differentiation state, immune infiltration, and extracellular matrix (ECM) composition. Similar alterations in tumor cell differentiation were also observed in more than half of our treatment-relapsed patient tumors. Tumor cell-state changes were coincident with ECM remodeling and increased tumor stiffness, which alone was sufficient to alter tumor cell fate and reduce treatment responses in melanoma cell lines in vitro. Despite the absence of acquired mutations in the targeted pathway, resistant tumors showed significantly decreased responsiveness to second-line therapy intervention within the same pathway. The ability to preclinically model relapse and refractory settings-while capturing dynamics within and crosstalk between all relevant tumor compartments-provides a unique opportunity to better design and sequence appropriate clinical interventions.

Kras mutant genetically engineered mouse models of human cancers are genomically heterogeneous.

KRAS mutant tumors are largely recalcitrant to targeted therapies. Genetically engineered mouse models (GEMMs) of Kras mutant cancer recapitulate critical aspects of this disease and are widely used for preclinical validation of targets and therapies. Through comprehensive profiling of exomes and matched transcriptomes of >200 KrasG12D-initiated GEMM tumors from one lung and two pancreatic cancer models, we discover that significant intratumoral and intertumoral genomic heterogeneity evolves during tumorigenesis. Known oncogenes and tumor suppressor genes, beyond those engineered, are mutated, amplified, and deleted. Unlike human tumors, the GEMM genomic landscapes are dominated by copy number alterations, while protein-altering mutations are rare. However, interspecies comparative analyses of the genomic landscapes demonstrate fidelity between genes altered in KRAS mutant human and murine tumors. Genes that are spontaneously altered during murine tumorigenesis are also among the most prevalent found in human indications. Using targeted therapies, we also demonstrate that this inherent tumor heterogeneity can be exploited preclinically to discover cancer-specific and genotype-specific therapeutic vulnerabilities. Focusing on Kras allelic imbalance, a feature shared by all three models, we discover that MAPK pathway inhibition impinges uniquely on this event, indicating distinct susceptibility and fitness advantage of Kras-mutant cells. These data reveal previously unknown genomic diversity among KrasG12D-initiated GEMM tumors, places them in context of human patients, and demonstrates how to exploit this inherent tumor heterogeneity to discover therapeutic vulnerabilities.

Cupid: simultaneous reconstruction of microRNA-target and ceRNA networks.

We introduce a method for simultaneous prediction of microRNA-target interactions and their mediated competitive endogenous RNA (ceRNA) interactions. Using high-throughput validation assays in breast cancer cell lines, we show that our integrative approach significantly improves on microRNA-target prediction accuracy as assessed by both mRNA and protein level measurements. Our biochemical assays support nearly 500 microRNA-target interactions with evidence for regulation in breast cancer tumors. Moreover, these assays constitute the most extensive validation platform for computationally inferred networks of microRNA-target interactions in breast cancer tumors, providing a useful benchmark to ascertain future improvements.

Proteomics characterization of cytoplasmic and lipid-associated membrane proteins of human pathogen Mycoplasma fermentans M64.

Mycoplasma fermentans is a potent human pathogen which has been implicated in several diseases. Notably, its lipid-associated membrane proteins (LAMPs) play a role in immunomodulation and development of infection-associated inflammatory diseases. However, the systematic protein identification of pathogenic M. fermentans has not been reported. From our recent sequencing results of M. fermentans M64 isolated from human respiratory tract, its genome is around 1.1 Mb and encodes 1050 predicted protein-coding genes. In the present study, soluble proteome of M. fermentans was resolved and analyzed using two-dimensional gel electrophoresis. In addition, Triton X-114 extraction was carried out to enrich amphiphilic proteins including putative lipoproteins and membrane proteins. Subsequent mass spectrometric analyses of these proteins had identified a total of 181 M. fermentans ORFs. Further bioinformatics analysis of these ORFs encoding proteins with known or so far unknown orthologues among bacteria revealed that a total of 131 proteins are homologous to known proteins, 11 proteins are conserved hypothetical proteins, and the remaining 39 proteins are likely M. fermentans-specific proteins. Moreover, Triton X-114-enriched fraction was shown to activate NF-kB activity of raw264.7 macrophage and a total of 21 lipoproteins with predicted signal peptide were identified therefrom. Together, our work provides the first proteome reference map of M. fermentans as well as several putative virulence-associated proteins as diagnostic markers or vaccine candidates for further functional study of this human pathogen.

An extensive microRNA-mediated network of RNA-RNA interactions regulates established oncogenic pathways in glioblastoma.

By analyzing gene expression data in glioblastoma in combination with matched microRNA profiles, we have uncovered a posttranscriptional regulation layer of surprising magnitude, comprising more than 248,000 microRNA (miR)-mediated interactions. These include ∼7,000 genes whose transcripts act as miR "sponges" and 148 genes that act through alternative, nonsponge interactions. Biochemical analyses in cell lines confirmed that this network regulates established drivers of tumor initiation and subtype implementation, including PTEN, PDGFRA, RB1, VEGFA, STAT3, and RUNX1, suggesting that these interactions mediate crosstalk between canonical oncogenic pathways. siRNA silencing of 13 miR-mediated PTEN regulators, whose locus deletions are predictive of PTEN expression variability, was sufficient to downregulate PTEN in a 3'UTR-dependent manner and to increase tumor cell growth rates. Thus, miR-mediated interactions provide a mechanistic, experimentally validated rationale for the loss of PTEN expression in a large number of glioma samples with an intact PTEN locus.

Computational and experimental identification of mirtrons in Drosophila melanogaster and Caenorhabditis elegans.

Mirtrons are intronic hairpin substrates of the dicing machinery that generate functional microRNAs. In this study, we describe experimental assays that defined the essential requirements for entry of introns into the mirtron pathway. These data informed a bioinformatic screen that effectively identified functional mirtrons from the Drosophila melanogaster transcriptome. These included 17 known and six confident novel mirtrons among the top 51 candidates, and additional candidates had limited read evidence in available small RNA data. Our computational model also proved effective on Caenorhabditis elegans, for which the identification of 14 cloned mirtrons among the top 22 candidates more than tripled the number of validated mirtrons in this species. A few low-scoring introns generated mirtron-like read patterns from atypical RNA structures, but their paucity suggests that relatively few such loci were not captured by our model. Unexpectedly, we uncovered examples of clustered mirtrons in both fly and worm genomes, including a <8-kb region in C. elegans harboring eight distinct mirtrons. Altogether, we demonstrate that discovery of functional mirtrons, unlike canonical miRNAs, is amenable to computational methods independent of evolutionary constraint.

MicroRNA biogenesis via splicing and exosome-mediated trimming in Drosophila.

microRNAs (miRNAs) are approximately 22 nucleotide regulatory RNAs derived from hairpins generated either by Drosha cleavage (canonical substrates) or by splicing and debranching of short introns (mirtrons). The 5' end of the highly conserved Drosophila mirtron-like locus mir-1017 is coincident with the splice donor, but a substantial "tail" separates its hairpin from the 3'splice acceptor. Genetic and biochemical studies define a biogenesis pathway involving splicing, lariat debranching, and RNA exosome-mediated "trimming," followed by conventional dicing and loading into AGO1 to yield a miRNA that can repress seed-matched targets. Analysis of cloned small RNAs yielded six additional candidate 3' tailed mirtrons in D. melanogaster. Altogether, these data reveal an unexpected role for the exosome in the biogenesis of miRNAs from hybrid mirtron substrates.

Abundant primary piRNAs, endo-siRNAs, and microRNAs in a Drosophila ovary cell line.

Piwi proteins, a subclass of Argonaute-family proteins, carry approximately 24-30-nt Piwi-interacting RNAs (piRNAs) that mediate gonadal defense against transposable elements (TEs). We analyzed the Drosophila ovary somatic sheet (OSS) cell line and found that it expresses miRNAs, endogenous small interfering RNAs (endo-siRNAs), and piRNAs in abundance. In contrast to intact gonads, which contain mixtures of germline and somatic cell types that express different Piwi-class proteins, OSS cells are a homogenous somatic cell population that expresses only PIWI and primary piRNAs. Detailed examination of its TE-derived piRNAs and endo-siRNAs revealed aspects of TE defense that do not rely upon ping-pong amplification. In particular, we provide evidence that a subset of piRNA master clusters, including flamenco, are specifically expressed in OSS and ovarian follicle cells. These data indicate that the restriction of certain TEs in somatic gonadal cells is largely mediated by a primary piRNA pathway.

The long and short of inverted repeat genes in animals: microRNAs, mirtrons and hairpin RNAs.

MicroRNAs (miRNAs) are endogenous transcripts that contain intramolecular double stranded RNA (dsRNA) and are processed by Dicer. Their mature products are approximately 21-24 nucleotides in length, and they collectively regulate a broad network of endogenous transcripts. A subset of animal miRNAs are produced from mirtrons, short hairpin introns whose splicing bypasses the normal nuclear processing of canonical miRNAs. Recent studies revealed novel, extended intramolecular dsRNA produced by defined transcription units in flies and mammals, termed hairpin RNAs (hpRNAs). Detailed biogenesis studies in Drosophila showed that hpRNAs are not merely "long" miRNAs, but are actually processed by a distinct biogenesis pathway that is related to the canonical RNA interference pathway. We compare and contrast the miRNA and hpRNA pathways in this review, and describe some of the key questions that the recognition of this novel pathway raises.

The evolution and functional diversification of animal microRNA genes.

microRNAs (miRNAs) are an abundant class of approximately 22 nucleotide (nt) regulatory RNAs that are pervasive in higher eukaryotic genomes. In order to fully understand their prominence in genomes, it is necessary to elucidate the molecular mechanisms that can diversify miRNA activities. In this review, we describe some of the many strategies that allow novel miRNA functions to emerge, with particular emphasis on how miRNA genes evolve in animals. These mechanisms include changes in their sequence, processing, or expression pattern; acquisition of miRNA* functionality or antisense processing; and de novo gene birth. The facility and versatility of miRNAs to evolve and change likely underlies how they have become dominant constituents of higher genomes.

Endogenous RNA interference provides a somatic defense against Drosophila transposons.

BACKGROUND: Because of the mutagenic consequences of mobile genetic elements, elaborate defenses have evolved to restrict their activity. A major system that controls the activity of transposable elements (TEs) in flies and vertebrates is mediated by Piwi-interacting RNAs (piRNAs), which are approximately 24-30 nucleotide RNAs that are bound by Piwi-class effectors. The piRNA system is thought to provide primarily a germline defense against TE activity. RESULTS: Here, we describe a second system that represses Drosophila TEs by using endogenous small interfering RNAs (siRNAs), which are 21 nucleotide, 3'-end-modified RNAs that are dependent on Dicer-2 and Argonaute-2. In contrast to piRNAs, we find that the TE-siRNA system is active in somatic tissues, and particularly so in various immortalized cell lines. Analysis of the patterns and properties of TE-derived small RNAs reveals further distinctions between TE regions and genomic loci that are converted into piRNAs and siRNAs, respectively. Finally, functional tests show that many transposon transcripts accumulate to higher levels in cells and animal tissues that are deficient for Dicer-2 or Argonaute-2. CONCLUSIONS: Drosophila utilizes two small-RNA systems to restrict transposon activity in the germline (mostly via piRNAs) and in the soma (mostly via siRNAs).

The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs.

In contrast to microRNAs and Piwi-associated RNAs, short interfering RNAs (siRNAs) are seemingly dispensable for host-directed gene regulation in Drosophila. This notion is based on the fact that mutants lacking the core siRNA-generating enzyme Dicer-2 or the predominant siRNA effector Argonaute 2 are viable, fertile and of relatively normal morphology. Moreover, endogenous Drosophila siRNAs have not yet been identified. Here we report that siRNAs derived from long hairpin RNA genes (hpRNAs) programme Slicer complexes that can repress endogenous target transcripts. The Drosophila hpRNA pathway is a hybrid mechanism that combines canonical RNA interference factors (Dicer-2, Hen1 (known as CG12367) and Argonaute 2) with a canonical microRNA factor (Loquacious) to generate approximately 21-nucleotide siRNAs. These novel regulatory RNAs reveal unexpected complexity in the sorting of small RNAs, and open a window onto the biological usage of endogenous RNA interference in Drosophila.

Functionally distinct regulatory RNAs generated by bidirectional transcription and processing of microRNA loci.

Many microRNA (miRNA) loci exhibit compelling hairpin structures on both sense and antisense strands; however, the possibility that a miRNA gene might produce functional species from its antisense strand has not been examined. We report here that antisense transcription of the Hox miRNA locus mir-iab-4 generates the novel pre-miRNA hairpin mir-iab-8, which is then processed into endogenous mature miRNAs. Sense and antisense iab-4/iab-8 miRNAs are functionally distinguished by their distinct domains of expression and targeting capabilities. We find that miR-iab-8-5p, like miR-iab-4-5p, is also relevant to Hox gene regulation. Ectopic mir-iab-8 can strongly repress the Hox genes Ultrabithorax and abdominal-A via extensive arrays of conserved target sites, and can induce a dramatic homeotic transformation of halteres into wings. We generalize the antisense miRNA principle by showing that several other loci in both invertebrates and vertebrates are endogenously processed on their antisense strands into mature miRNAs with distinct seeds. These findings demonstrate that antisense transcription and processing contributes to the functional diversification of miRNA genes.

Mammalian mirtron genes.

Mirtrons are alternative precursors for microRNA biogenesis that were recently described in invertebrates. These short hairpin introns use splicing to bypass Drosha cleavage, which is otherwise essential for the generation of canonical animal microRNAs. Using computational and experimental strategies, we now establish that mammals have mirtrons as well. We identified 3 mirtrons that are well conserved and expressed in diverse mammals, 16 primate-specific mirtrons, and 46 candidates supported by limited cloning evidence in primates. As with some fly and worm mirtrons, the existence of well-conserved mammalian mirtrons indicates their relatively ancient incorporation into endogenous regulatory pathways. However, as worms, flies, and mammals each have different sets of mirtrons, we hypothesize that different animals may have independently evolved the capacity for this hybrid small RNA pathway. This notion is supported by our observation of several clade-specific features of mammalian and invertebrate mirtrons.

Qualitative and comparative proteomic analysis of Xanthomonas campestris pv. campestris 17.

The bacterium Xanthomonas campestris pathovar campestris (XCC) 17 is a local isolate that causes crucifer black rot disease in Taiwan. In this study, its proteome was separated using 2-DE and the well-resolved proteins were excised, trypsin digested, and analyzed by MS. Over 400 protein spots were analyzed and 281 proteins were identified by searching the MS or MS/MS spectra against the proteome database of the closely related XCC ATCC 33913. Functional categorization of the identified proteins matched 141 (50%) proteins to 81 metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In addition, we performed a comparative proteome analysis of the pathogenic strain 17 and an avirulent strain 11A to reveal the virulence-related proteins. We detected 22 up-regulated proteins in strain 17 including the degrading enzymes EngXCA, HtrA, and PepA, which had been shown to have a role in pathogenesis in other bacteria, and an anti-host defense protein, Ohr. Thus, further functional studies of these up-regulated proteins with respect to their roles in XCC pathogenicity are suggested.