mRNA structure regulates protein expression through changes in functional half-life.

Messenger RNAs (mRNAs) encode information in both their primary sequence and their higher order structure. The independent contributions of factors like codon usage and secondary structure to regulating protein expression are difficult to establish as they are often highly correlated in endogenous sequences. Here, we used 2 approaches, global inclusion of modified nucleotides and rational sequence design of exogenously delivered constructs, to understand the role of mRNA secondary structure independent from codon usage. Unexpectedly, highly expressed mRNAs contained a highly structured coding sequence (CDS). Modified nucleotides that stabilize mRNA secondary structure enabled high expression across a wide variety of primary sequences. Using a set of eGFP mRNAs with independently altered codon usage and CDS structure, we find that the structure of the CDS regulates protein expression through changes in functional mRNA half-life (i.e., mRNA being actively translated). This work highlights an underappreciated role of mRNA secondary structure in the regulation of mRNA stability.

MM-131, a bispecific anti-Met/EpCAM mAb, inhibits HGF-dependent and HGF-independent Met signaling through concurrent binding to EpCAM.

Activation of the Met receptor tyrosine kinase, either by its ligand, hepatocyte growth factor (HGF), or via ligand-independent mechanisms, such as MET amplification or receptor overexpression, has been implicated in driving tumor proliferation, metastasis, and resistance to therapy. Clinical development of Met-targeted antibodies has been challenging, however, as bivalent antibodies exhibit agonistic properties, whereas monovalent antibodies lack potency and the capacity to down-regulate Met. Through computational modeling, we found that the potency of a monovalent antibody targeting Met could be dramatically improved by introducing a second binding site that recognizes an unrelated, highly expressed antigen on the tumor cell surface. Guided by this prediction, we engineered MM-131, a bispecific antibody that is monovalent for both Met and epithelial cell adhesion molecule (EpCAM). MM-131 is a purely antagonistic antibody that blocks ligand-dependent and ligand-independent Met signaling by inhibiting HGF binding to Met and inducing receptor down-regulation. Together, these mechanisms lead to inhibition of proliferation in Met-driven cancer cells, inhibition of HGF-mediated cancer cell migration, and inhibition of tumor growth in HGF-dependent and -independent mouse xenograft models. Consistent with its design, MM-131 is more potent in EpCAM-high cells than in EpCAM-low cells, and its potency decreases when EpCAM levels are reduced by RNAi. Evaluation of Met, EpCAM, and HGF levels in human tumor samples reveals that EpCAM is expressed at high levels in a wide range of Met-positive tumor types, suggesting a broad opportunity for clinical development of MM-131.

Novel circulating tumor cell-based blood test for the assessment of PD-L1 protein expression in treatment-naïve, newly diagnosed patients with non-small cell lung cancer.

We evaluated the analytical and clinical performance of a novel circulating tumor cell (CTC)-based blood test for determination of programmed death ligand 1 (PD-L1) protein expression status in real time in treatment-naïve non-small cell lung cancer (NSCLC) patients. CTCs were detected in 86% of patients with NSCLC (I-IV) at the time of diagnosis, with a 67% PD-L1 positivity rate (≥ 1 PDL + CTC). Among 33 NSCLC patients with PD-L1 results available via both tissue immunohistochemistry (IHC) and CTC assays, 78.9% were positive according to both methods. The CTC test identified an additional ten cases that were positive for PD-L1 expression but that tested negative via IHC analysis. Detection of higher PD-L1 expression on CTCs compared to that in the corresponding tissue was concordant with data obtained using other platforms in previously treated patients. The concordance in PD-L1 expression between tissue and CTCs was approximately 57%, which is higher than that reported by others. In summary, evaluation of PD-L1 protein expression status on CTCs isolated from NSCLC patients is feasible. PD-L1 expression status on CTCs can be determined serially during the disease course, thus overcoming the myriad challenges associated with tissue analysis.

Mouse population-guided resequencing reveals that variants in CD44 contribute to acetaminophen-induced liver injury in humans.

Interindividual variability in response to chemicals and drugs is a common regulatory concern. It is assumed that xenobiotic-induced adverse reactions have a strong genetic basis, but many mechanism-based investigations have not been successful in identifying susceptible individuals. While recent advances in pharmacogenetics of adverse drug reactions show promise, the small size of the populations susceptible to important adverse events limits the utility of whole-genome association studies conducted entirely in humans. We present a strategy to identify genetic polymorphisms that may underlie susceptibility to adverse drug reactions. First, in a cohort of healthy adults who received the maximum recommended dose of acetaminophen (4 g/d x 7 d), we confirm that about one third of subjects develop elevations in serum alanine aminotransferase, indicative of liver injury. To identify the genetic basis for this susceptibility, a panel of 36 inbred mouse strains was used to model genetic diversity. Mice were treated with 300 mg/kg or a range of additional acetaminophen doses, and the extent of liver injury was quantified. We then employed whole-genome association analysis and targeted sequencing to determine that polymorphisms in Ly86, Cd44, Cd59a, and Capn8 correlate strongly with liver injury and demonstrated that dose-curves vary with background. Finally, we demonstrated that variation in the orthologous human gene, CD44, is associated with susceptibility to acetaminophen in two independent cohorts. Our results indicate a role for CD44 in modulation of susceptibility to acetaminophen hepatotoxicity. These studies demonstrate that a diverse mouse population can be used to understand and predict adverse toxicity in heterogeneous human populations through guided resequencing.

3,4'-Linked bis(piperidines) related to the haliclonacyclamine class of marine alkaloids: synthesis using crossed-aldol chemistry and preliminary biological evaluations.

Compounds 2-5, incorporating various elements of the 3,4'-bis(piperidine) core associated with the sponge-derived alkaloid haliclonacyclamine A (HA, 1), have been prepared through, inter alia, aldol-type reactions of N-substituted piperidin-4-ones and certain derivatives. Screening of these compounds in various assays, including an ecological one, reveals that compound 5 exhibits allelochemical properties similar to those associated with HA itself.

Gene set enrichment in eQTL data identifies novel annotations and pathway regulators.

Genome-wide gene expression profiling has been extensively used to generate biological hypotheses based on differential expression. Recently, many studies have used microarrays to measure gene expression levels across genetic mapping populations. These gene expression phenotypes have been used for genome-wide association analyses, an analysis referred to as expression QTL (eQTL) mapping. Here, eQTL analysis was performed in adipose tissue from 28 inbred strains of mice. We focused our analysis on "trans-eQTL bands", defined as instances in which the expression patterns of many genes were all associated to a common genetic locus. Genes comprising trans-eQTL bands were screened for enrichments in functional gene sets representing known biological pathways, and genes located at associated trans-eQTL band loci were considered candidate transcriptional modulators. We demonstrate that these patterns were enriched for previously characterized relationships between known upstream transcriptional regulators and their downstream target genes. Moreover, we used this strategy to identify both novel regulators and novel members of known pathways. Finally, based on a putative regulatory relationship identified in our analysis, we identified and validated a previously uncharacterized role for cyclin H in the regulation of oxidative phosphorylation. We believe that the specific molecular hypotheses generated in this study will reveal many additional pathway members and regulators, and that the analysis approaches described herein will be broadly applicable to other eQTL data sets.

CRISPR-Cas12a System With Synergistic Phage Recombination Proteins for Multiplex Precision Editing in Human Cells.

The development of CRISPR-based gene-editing technologies has brought an unprecedented revolution in the field of genome engineering. Cas12a, a member of the Class 2 Type V CRISPR-associated endonuclease family distinct from Cas9, has been repurposed and developed into versatile gene-editing tools with distinct PAM recognition sites and multiplexed gene targeting capability. However, with current CRISPR/Cas12a technologies, it remains a challenge to perform efficient and precise genome editing of long sequences in mammalian cells. To address this limitation, we utilized phage recombination enzymes and developed an efficient CRISPR/Cas12a tool for multiplexed precision editing in mammalian cells. Through protein engineering, we were able to recruit phage recombination proteins to Cas12a to enhance its homology-directed repair efficiencies. Our phage-recombination-assisted Cas12a system achieved up to 3-fold improvements for kilobase-scale knock-ins in human cells without compromising the specificity of the enzyme. The performance of this system compares favorably against Cas9 references, the commonly used enzyme for gene-editing tasks, with improved specificity. Additionally, we demonstrated multi-target editing with similar improved activities thanks to the RNA-processing activity of the Cas12a system. This compact, multi-target editing tool has the potential to assist in understanding multi-gene interactions. In particular, it paves the way for a gene therapy method for human diseases that complements existing tools and is suitable for polygenic disorders and diseases requiring long-sequence corrections.

Colony-stimulating factor-1 (CSF-1) delivers a proatherogenic signal to human macrophages.

M-CSF/CSF-1 supports the proliferation and differentiation of monocytes and macrophages. In mice, CSF-1 also promotes proinflammatory responses in vivo by regulating mature macrophage functions, but little is known about the acute effects of this growth factor on mature human macrophages. Here, we show that in contrast to its effects on mouse bone marrow-derived macrophages, CSF-1 did not induce expression of urokinase plasminogen activator mRNA, repress expression of apolipoprotein E mRNA, or prime LPS-induced TNF and IL-6 secretion in human monocyte-derived macrophages (HMDM) from several independent donors. Instead, we show by expression profiling that CSF-1 modulates the HMDM transcriptome to favor a proatherogenic environment. CSF-1 induced expression of the proatherogenic chemokines CXCL10/IFN-inducible protein 10, CCL2, and CCL7 but repressed expression of the antiatherogenic chemokine receptor CXCR4. CSF-1 also up-regulated genes encoding enzymes of the cholesterol biosynthetic pathway (HMGCR, MVD, IDI1, FDPS, SQLE, CYP51A1, EBP, NSDHL, DHCR7, and DHCR24), and expression of ABCG1, encoding a cholesterol efflux transporter, was repressed. Consistent with these effects, CSF-1 increased levels of free cholesterol in HMDM, and the selective CSF-1R kinase inhibitor GW2580 ablated this response. These data demonstrate that CSF-1 represents a further link between inflammation and cardiovascular disease and suggest two distinct mechanisms by which CSF-1, which is known to be present in atherosclerotic lesions, may contribute to plaque progression.

Impact of mRNA chemistry and manufacturing process on innate immune activation.

Messenger RNA (mRNA) represents an attractive therapeutic modality for potentially a wide range of clinical indications but requires uridine chemistry modification and/or tuning of the production process to prevent activation of cellular innate immune sensors and a concomitant reduction in protein expression. To decipher the relative contributions of these factors on immune activation, here, we compared, in multiple cell and in vivo models, mRNA that encodes human erythropoietin incorporating either canonical uridine or N1-methyl-pseudouridine (1mΨ), synthesized by either a standard process shown to have double-stranded RNA (dsRNA) impurities or a modified process that yields a highly purified mRNA preparation. Our data demonstrate that the lowest stimulation of immune endpoints was with 1mΨ made by the modified process, while mRNA containing canonical uridine was immunostimulatory regardless of process. These findings confirm that uridine modification and the reduction of dsRNA impurities are both necessary and sufficient at controlling the immune-activating profile of therapeutic mRNA.

Discovery of CYT997: a structurally novel orally active microtubule targeting agent.

CYT997 was discovered as a potent tubulin polymerization inhibitor possessing potent cytotoxic activity against a range of cancer cells. Details of SAR studies, pharmacokinetic investigations and synthesis of compounds leading to the discovery of CYT997 are reported.

Discovery of 2-(alpha-methylbenzylamino) pyrazines as potent Type II inhibitors of FMS.

A series of 2-(alpha-methylbenzylamino) pyrazines have shown to be potent inhibitors of the FMS tyrosine receptor kinase. Details of SAR studies, modeling and synthesis of compounds within this series are reported.

Phenylaminopyrimidines as inhibitors of Janus kinases (JAKs).

A series of phenylaminopyrimidines has been identified as inhibitors of Janus kinases (JAKs). Development of this initial series led to the potent JAK2/JAK1 inhibitor CYT387 (N-(cyanomethyl)-4-[2-[[4-(4-morpholinyl)phenyl]amino]-4-pyrimidinyl]-benzamide). Details of synthesis and SAR studies of these compounds are reported.

Durable anticancer immunity from intratumoral administration of IL-23, IL-36γ, and OX40L mRNAs.

Many solid cancers contain dysfunctional immune microenvironments. Immune system modulators that initiate responses to foreign pathogens could be promising candidates for reigniting productive responses toward tumors. Interleukin-1 (IL-1) and IL-12 cytokine family members cooperate at barrier tissues after microbial invasion, in human inflammatory diseases, and in antitumoral immunity. IL-36γ, in classic alarmin fashion, acts in damaged tissues, whereas IL-23 centrally coordinates immune responses to danger signals. In this study, direct intratumoral delivery of messenger RNAs (mRNAs) encoding these cytokines produced robust anticancer responses in a broad range of tumor microenvironments. The addition of mRNA encoding the T cell costimulator OX40L increased complete response rates in treated and untreated distal tumors compared to the cytokine mRNAs alone. Mice exhibiting complete responses were subsequently protected from tumor rechallenge. Treatments with these mRNA mixtures induced downstream cytokine and chemokine expression, and also activated multiple dendritic cell (DC) and T cell types. Consistent with this, efficacy was dependent on Batf3-dependent cross-presenting DCs and cytotoxic CD8+ T cells. IL-23/IL-36γ/OX40L triplet mRNA mixture triggered substantial immune cell recruitment into tumors, enabling effective tumor destruction irrespective of previous tumoral immune infiltrates. Last, combining triplet mRNA with checkpoint blockade led to efficacy in models otherwise resistant to systemic immune checkpoint inhibition. Human cell studies showed similar cytokine responses to the individual components of this mRNA mixture, suggesting translatability of immunomodulatory activity to human patients.

Two key residues in ephrinB3 are critical for its use as an alternative receptor for Nipah virus.

EphrinB2 was recently discovered as a functional receptor for Nipah virus (NiV), a lethal emerging paramyxovirus. Ephrins constitute a class of homologous ligands for the Eph class of receptor tyrosine kinases and exhibit overlapping expression patterns. Thus, we examined whether other ephrins might serve as alternative receptors for NiV. Here, we show that of all known ephrins (ephrinA1-A5 and ephrinB1-B3), only the soluble Fc-fusion proteins of ephrinB3, in addition to ephrinB2, bound to soluble NiV attachment protein G (NiV-G). Soluble NiV-G bound to cell surface ephrinB3 and B2 with subnanomolar affinities (Kd = 0.58 nM and 0.06 nM for ephrinB3 and B2, respectively). Surface plasmon resonance analysis indicated that the relatively lower affinity of NiV-G for ephrinB3 was largely due to a faster off-rate (K(off) = 1.94 x 10(-3) s(-1) versus 1.06 x 10(-4) s(-1) for ephrinB3 and B2, respectively). EphrinB3 was sufficient to allow for viral entry of both pseudotype and live NiV. Soluble ephrinB2 and B3 were able to compete for NiV-envelope-mediated viral entry on both ephrinB2- and B3-expressing cells, suggesting that NiV-G interacts with both ephrinB2 and B3 via an overlapping site. Mutational analysis indicated that the Leu-Trp residues in the solvent exposed G-H loop of ephrinB2 and B3 were critical determinants of NiV binding and entry. Indeed, replacement of the Tyr-Met residues in the homologous positions in ephrinB1 with Leu-Trp conferred NiV receptor activity to ephrinB1. Thus, ephrinB3 is a bona fide alternate receptor for NiV entry, and two residues in the G-H loop of the ephrin B-class ligands are critical determinants of NiV receptor activity.

Pyrido-pyrimidine derivative CYC10424 inhibits glycosaminoglycan changes on vascular smooth muscle-derived proteoglycans and reduces lipoprotein binding.

Cardiovascular disease is a major cause of mortality with the underlying process being atherosclerosis. Modified proteoglycans bind low-density lipoproteins (LDL), a critical initial step in the atherosclerotic cascade, representing a potential therapeutic target. Platelet-derived growth factor (PDGF) stimulates proteoglycan synthesis and is strongly implicated in atherogenesis. In human vascular smooth muscle cells (VSMCs), CYC10424 (Cytopia Research Ltd), a pyrido-pyrimidine derivative, dose-dependently decreased PDGF-mediated radiolabel incorporation into proteoglycans associated with an increase in electrophoretic mobility by SDS-PAGE. PDGF stimulated increases in both chemically-cleaved and xyloside-associated glycosaminoglycan (GAG) chain size, which were inhibited in the presence of CYC10424 by size exclusion chromatography (Sepharose CL-6B). CYC10424 treatment inhibited the PDGF effect to increase the 6:4 position sulfation ratio of monosulfated disaccharides by fluorophore-assisted carbohydrate electrophoresis. Proteoglycans derived from cells treated with CYC10424 had a decreased binding affinity and capacity to human LDL by gel mobility shift assay. CYC10424 and related compounds are possible candidates as therapeutic agents for the prevention of lipid deposition as characteristic of diseases such as atherosclerosis.

A CSF-1 receptor kinase inhibitor targets effector functions and inhibits pro-inflammatory cytokine production from murine macrophage populations.

CSF-1 regulates macrophage differentiation, survival, and function, and is an attractive therapeutic target for chronic inflammation and malignant diseases. Here we describe the effects of a potent and selective inhibitor of CSF-1R-CYC10268-on CSF-1R-dependent signaling. In in vitro kinase assays, CYC10268 was active in the low nanomolar range and showed selectivity over other kinases such as Abl and Kit. CYC10268 blocked survival mediated by CSF-1R in primary murine bone marrow-derived macrophages (BMM) and in the factor-dependent cell line Ba/F3, in which the CSF-1R was ectopically expressed. CYC10268 also inhibited CSF-1 regulated signaling (Akt, ERK-1/2), gene expression (urokinase plasminogen activator, toll-like receptor 9, and apolipoprotein E), and priming of LPS-inducible cytokine production in BMM. In thioglycollate-elicited peritoneal macrophages (TEPM), which survive in the absence of exogenous CSF-1, CYC10268 impaired LPS-induced cytokine production and regulated expression of known CSF-1 target genes. These observations support the conclusion that TEPM are CSF-1 autocrine and that CSF-1 plays a central role in macrophage effector functions during inflammation. CSF-1R inhibitors such as CYC10268 provide a powerful tool to dissect the role of the CSF-1/CSF-1R signaling system in a range of biological systems and have potential for a number of therapeutic applications.

Sugar and spice: viral envelope-DC-SIGN interactions in HIV pathogenesis.

DC-SIGN is a calcium dependent lectin that binds to HIV envelope, gp120, with high affinity. Its expression on dendritic cells, coupled with its ability to facilitate the binding and subsequent transfer of virions to permissive T-cells, has led to the hypothesis that DC-SIGN may serve as a conduit the transfer of HIV from the peripheral mucosa to secondary lymphoid organs. Studies have shown that DC-SIGN bound virions can maintain their infectivity for prolonged periods of time despite evidence that DC-SIGN itself may serve as an antigen receptor. How HIV subverts the normal function of DC-SIGN to establish a primary infection in the host is unclear. Therefore, understanding the structural and immunological basis for DC-SIGN's function will help us realize the role that DC-SIGN may play in viral transmission and pathogenesis. Importantly, DC-SIGN/envelope interactions may represent a new target for microbicide and vaccine development efforts. Here, we review recent studies on DC-SIGN's structure and function in an effort to present testable models of DC-SIGN's role in HIV pathogenesis.

Optimizing properties of antireceptor antibodies using kinetic computational models and experiments.

Monoclonal antibodies are valuable as anticancer therapeutics because of their ability to selectively bind tumor-associated target proteins like receptor tyrosine kinases. Kinetic computational models that capture protein-protein interactions using mass action kinetics are a valuable tool for understanding the binding properties of monoclonal antibodies to their targets. Insights from the models can be used to explore different formats, to set antibody design specifications such as affinity and valence, and to predict potency. Antibody binding to target is driven by both intrinsic monovalent affinity and bivalent avidity. In this chapter, we describe a combined experimental and computational method of assessing the relative importance of these effects on observed drug potency. The method, which we call virtual flow cytometry (VFC), merges experimental measurements of monovalent antibody binding kinetics and affinity curves of antibody-antigen binding into a kinetic computational model of antibody-antigen interaction. The VFC method introduces a parameter χ, the avidity factor, which characterizes the ability of an antibody to cross-link its target through bivalent binding. This simple parameterization of antibody cross-linking allows the model to successfully describe and predict antibody binding curves across a wide variety of experimental conditions, including variations in target expression level and incubation time of antibody with target. We further demonstrate how computational models of antibody binding to cells can be used to predict target inhibition potency. Importantly, we demonstrate computationally that antibodies with high ability to cross-link antigen have significant potency advantages. We also present data suggesting that the parameter χ is a physical, epitope-dependent property of an antibody, and as a result propose that determination of antibody cross-linking and avidity should be incorporated into the screening of antibody panels for therapeutic development. Overall, our results suggest that antibody cross-linking, in addition to monovalent binding affinity, is a key design parameter of antibody performance.

CYT997: a novel orally active tubulin polymerization inhibitor with potent cytotoxic and vascular disrupting activity in vitro and in vivo.

CYT997 is a wholly synthetic compound that possesses highly potent cytotoxic activity in vitro through inhibition of microtubule polymerization. CYT997 blocks the cell cycle at the G(2)-M boundary, and Western blot analysis indicates an increase in phosphorylated Bcl-2, along with increased expression of cyclin B1. Caspase-3 activation is also observed in cells treated with CYT997 along with the generation of poly(ADP-ribose) polymerase. The compound possesses favorable pharmacokinetic properties, is orally bioavailable, and is efficacious per os in a range of in vivo cancer models, including some refractory to paclitaxel treatment. CYT997 exhibits vascular disrupting activity as measured in vitro by effects on the permeability of human umbilical vein endothelial cell monolayers, and in vivo by effects on tumor blood flow. CYT997 possesses a useful combination of pharmacologic and pharmacokinetic properties and has considerable potential as a novel anticancer agent.