Inhibition of Aurora Kinase Induces Endogenous Retroelements to Induce a Type I/III IFN Response via RIG-I.

Type I IFN signaling is a crucial component of antiviral immunity that has been linked to promoting the efficacy of some chemotherapeutic drugs. We developed a reporter system in HCT116 cells that detects activation of the endogenous IFI27 locus, an IFN target gene. We screened a library of annotated compounds in these cells and discovered Aurora kinase inhibitors (AURKi) as strong hits. Type I IFN signaling was found to be the most enriched gene signature after AURKi treatment in HCT116, and this signature was also strongly enriched in other colorectal cancer cell lines. The ability of AURKi to activate IFN in HCT116 was dependent on MAVS and RIG-I, but independent of STING, whose signaling is deficient in these cells. MAVS dependence was recapitulated in other colorectal cancer lines with STING pathway deficiency, whereas in cells with intact STING signaling, the STING pathway was required for IFN induction by AURKi. AURKis were found to induce expression of endogenous retroviruses (ERV). These ERVs were distinct from those induced by the DNA methyltransferase inhibitors (DNMTi), which can induce IFN signaling via ERV induction, suggesting a novel mechanism of action. The antitumor effect of alisertib in mice was accompanied by an induction of IFN expression in HCT116 or CT26 tumors. CT26 tumor growth inhibition by alisertib was absent in NSG mice versus wildtype (WT) mice, and tumors from WT mice with alisertib treatment showed increased in CD8+ T-cell infiltration, suggesting that antitumor efficacy of AURKi depends, at least in part, on an intact immune response. SIGNIFICANCE: Some cancers deactivate STING signaling to avoid consequences of DNA damage from aberrant cell division. The surprising activation of MAVS/RIG-I signaling by AURKi might represent a vulnerability in STING signaling deficient cancers.

Design and Synthesis of Novel Cereblon Binders for Use in Targeted Protein Degradation.

Modulating the chemical composition of cereblon (CRBN) binders is a critical step in the optimization process of protein degraders that seek to hijack the function of this E3 ligase. Small structural changes can have profound impacts on the overall profile of these compounds, including depth of on-target degradation, neosubstrate degradation selectivity, as well as other drug-like properties. Herein, we report the design and synthesis of a series of novel CRBN binding moieties. These CRBN binders were evaluated for CRBN binding and degradation of common neosubstrates Aiolos and GSPT1. A selection of these binders was employed for an exploratory matrix of heterobifunctional molecules, targeting CRBN-mediated degradation of the androgen receptor.

SETD2 safeguards the genome against isochromosome formation.

Isochromosomes are mirror-imaged chromosomes with simultaneous duplication and deletion of genetic material which may contain two centromeres to create isodicentric chromosomes. Although isochromosomes commonly occur in cancer and developmental disorders and promote genome instability, mechanisms that prevent isochromosomes are not well understood. We show here that the tumor suppressor and methyltransferase SETD2 is essential to prevent these errors. Using cellular and cytogenetic approaches, we demonstrate that loss of SETD2 or its epigenetic mark, histone H3 lysine 36 trimethylation (H3K36me3), results in the formation of isochromosomes as well as isodicentric and acentric chromosomes. These defects arise during DNA replication and are likely due to faulty homologous recombination by RAD52. These data provide a mechanism for isochromosome generation and demonstrate that SETD2 and H3K36me3 are essential to prevent the formation of this common mutable chromatin structure known to initiate a cascade of genomic instability in cancer.

Discovery of the c-Jun N-Terminal Kinase Inhibitor CC-90001.

As a result of emerging biological data suggesting that within the c-Jun N-terminal kinase (JNK) family, JNK1 and not JNK2 or JNK3 may be primarily responsible for fibrosis pathology, we sought to identify JNK inhibitors with an increased JNK1 bias relative to our previous clinical compound tanzisertib (CC-930). This manuscript reports the synthesis and structure-activity relationship (SAR) studies for a novel series of JNK inhibitors demonstrating an increased JNK1 bias. SAR optimization on a series of 2,4-dialkylamino-pyrimidine-5-carboxamides resulted in the identification of compounds possessing low nanomolar JNK inhibitory potency, overall kinome selectivity, and the ability to inhibit cellular phosphorylation of the direct JNK substrate c-Jun. Optimization of physicochemical properties in this series resulted in compounds that demonstrated excellent systemic exposure following oral dosing, enabling in vivo efficacy studies and the selection of a candidate for clinical development, CC-90001, which is currently in clinical trials (Phase II) in patients with idiopathic pulmonary fibrosis (NCT03142191).

Discovery of the Selective Protein Kinase C-θ Kinase Inhibitor, CC-90005.

The PKC-θ isoform of protein kinase C is selectively expressed in T lymphocytes and plays an important role in the T cell antigen receptor (TCR)-triggered activation of mature T cells, T cell proliferation, and the subsequent release of cytokines such as interleukin-2 (IL-2). Herein, we report the synthesis and structure-activity relationship (SAR) of a novel series of PKC-θ inhibitors. Through a combination of structure-guided design and exploratory SAR, suitable replacements for the basic C4 amine of the original lead (3) were identified. Property-guided design enabled the identification of appropriately substituted C2 groups to afford potent analogs with metabolic stability and permeability to support in vivo testing. With exquisite general kinase selectivity, cellular inhibition of T cell activation as assessed by IL-2 expression, a favorable safety profile, and demonstrated in vivo efficacy in models of acute and chronic T cell activation with oral dosing, CC-90005 (57) was selected for clinical development.

Microtubule minus-end stability is dictated by the tubulin off-rate.

Dynamic organization of microtubule minus ends is vital for the formation and maintenance of acentrosomal microtubule arrays. In vitro, both microtubule ends switch between phases of assembly and disassembly, a behavior called dynamic instability. Although minus ends grow slower, their lifetimes are similar to those of plus ends. The mechanisms underlying these distinct dynamics remain unknown. Here, we use an in vitro reconstitution approach to investigate minus-end dynamics. We find that minus-end lifetimes are not defined by the mean size of the protective GTP-tubulin cap. Rather, we conclude that the distinct tubulin off-rate is the primary determinant of the difference between plus- and minus-end dynamics. Further, our results show that the minus-end-directed kinesin-14 HSET/KIFC1 suppresses tubulin off-rate to specifically suppress minus-end catastrophe. HSET maintains its protective minus-end activity even when challenged by a known microtubule depolymerase, kinesin-13 MCAK. Our results provide novel insight into the mechanisms of minus-end dynamics, essential for our understanding of microtubule minus-end regulation in cells.

Motor Dynamics Underlying Cargo Transport by Pairs of Kinesin-1 and Kinesin-3 Motors.

Intracellular cargo transport by kinesin family motor proteins is crucial for many cellular processes, particularly vesicle transport in axons and dendrites. In a number of cases, the transport of specific cargo is carried out by two classes of kinesins that move at different speeds and thus compete during transport. Despite advances in single-molecule characterization and modeling approaches, many questions remain regarding the effect of intermotor tension on motor attachment/reattachment rates during cooperative multimotor transport. To understand the motor dynamics underlying multimotor transport, we analyzed the complexes of kinesin-1 and kinesin-3 motors attached through protein scaffolds moving on immobilized microtubules in vitro. To interpret the observed behavior, simulations were carried out using a model that incorporated motor stepping, attachment/detachment rates, and intermotor force generation. In single-molecule experiments, isolated kinesin-3 motors moved twofold faster and had threefold higher landing rates than kinesin-1. When the positively charged loop 12 of kinesin-3 was swapped with that of kinesin-1, the landing rates reversed, indicating that this "K-loop" is a key determinant of the motor reattachment rate. In contrast, swapping loop 12 had negligible effects on motor velocities. Two-motor complexes containing one kinesin-1 and one kinesin-3 moved at different speeds depending on the identity of their loop 12, indicating the importance of the motor reattachment rate on the cotransport speed. Simulations of these loop-swapped motors using experimentally derived motor parameters were able to reproduce the experimental results and identify best fit parameters for the motor reattachment rates for this geometry. Simulation results also supported previous work, suggesting that kinesin-3 microtubule detachment is very sensitive to load. Overall, the simulations demonstrate that the transport behavior of cargo carried by pairs of kinesin-1 and -3 motors are determined by three properties that differ between these two families: the unloaded velocity, the load dependence of detachment, and the motor reattachment rate.

Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters.

Higher-order structures of the microtubule (MT) cytoskeleton are comprised of two architectures: bundles and asters. Although both architectures are critical for cellular function, the molecular pathways that drive aster formation are poorly understood. Here, we study aster formation by human minus-end-directed kinesin-14 (HSET/KIFC1). We show that HSET is incapable of forming asters from preformed, nongrowing MTs, but rapidly forms MT asters in the presence of soluble (non-MT) tubulin. HSET binds soluble (non-MT) tubulin via its N-terminal tail domain to form heterogeneous HSET-tubulin clusters containing multiple motors. Cluster formation induces motor processivity and rescues the formation of asters from nongrowing MTs. We then show that excess soluble (non-MT) tubulin stimulates aster formation in HeLa cells overexpressing HSET during mitosis. We propose a model where HSET can toggle between MT bundle and aster formation in a manner governed by the availability of soluble (non-MT) tubulin.

Processive Kinesin-14 HSET Exhibits Directional Flexibility Depending on Motor Traffic.

A common mitotic defect observed in cancer cells that possess supernumerary (more than two) centrosomes is multipolar spindle formation [1, 2]. Such structures are resolved into a bipolar geometry by minus-end-directed motor proteins, such as cytoplasmic dynein and the kinesin-14 HSET [3-8]. HSET is also thought to antagonize plus-end-directed kinesin-5 Eg5 to balance spindle forces [4, 5, 7, 9]. However, the biomechanics of this force opposition are unclear, as HSET has previously been defined as a non-processive motor [10-16]. Here, we use optical trapping to elucidate the mechanism of force generation by HSET. We show that a single HSET motor has a processive nature with the ability to complete multiple steps while trapped along a microtubule and when unloaded can move in both directions for microns. Compared to other kinesins, HSET has a relatively weak stall force of 1.1 pN [17, 18]. Moreover, HSET's tail domain and its interaction with the E-hook of tubulin are necessary for long-range motility. In vitro polarity-marked bundle assays revealed that HSET selectively generates force in anti-parallel bundles on the order of its stall force. When combined with varied ratios of Eg5, HSET adopts Eg5's directionality while acting as an antagonizing force brake, requiring at least a 10-fold higher Eg5 concentration to surpass HSET's sliding force. These results reveal HSET's ability to change roles within the spindle from acting as an adjustable microtubule slider and force regulator to a processive motor that aids in minus end focusing.

Aquatic Pseudomonads Inhibit Oomycete Plant Pathogens of Glycine max.

Seedling root rot of soybeans caused by the host-specific pathogen Phytophthora sojae, and a large number of Pythium species, is an economically important disease across the Midwest United States that negatively impacts soybean yields. Research on biocontrol strategies for crop pathogens has focused on compounds produced by microbes from soil, however, recent studies suggest that aquatic bacteria express distinct compounds that efficiently inhibit a wide range of pathogens. Based on these observations, we hypothesized that freshwater strains of pseudomonads might be producing novel antagonistic compounds that inhibit the growth of oomycetes. To test this prediction, we utilized a collection of 330 Pseudomonas strains isolated from soil and freshwater habitats, and determined their activity against a panel of five oomycetes: Phytophthora sojae, Pythium heterothalicum, Pythium irregulare, Pythium sylvaticum, and Pythium ultimum, all of which are pathogenic on soybeans. Among the bacterial strains, 118 exhibited antagonistic activity against at least one oomycete species, and 16 strains were inhibitory to all pathogens. Antagonistic activity toward oomycetes was significantly more common for aquatic isolates than for soil isolates. One water-derived strain, 06C 126, was predicted to express a siderophore and exhibited diverse antagonistic profiles when tested on nutrient rich and iron depleted media suggesting that more than one compound was produced that effectively inhibited oomycetes. These results support the concept that aquatic strains are an efficient source of compounds that inhibit pathogens. We outline a strategy to identify other strains that express unique compounds that may be useful biocontrol agents.

Human CLASP2 specifically regulates microtubule catastrophe and rescue.

Cytoplasmic linker-associated proteins (CLASPs) are microtubule-associated proteins essential for microtubule regulation in many cellular processes. However, the molecular mechanisms underlying CLASP activity are not understood. Here, we use purified protein components and total internal reflection fluorescence microscopy to investigate the effects of human CLASP2 on microtubule dynamics in vitro. We demonstrate that CLASP2 suppresses microtubule catastrophe and promotes rescue without affecting the rates of microtubule growth or shrinkage. Strikingly, when CLASP2 is combined with EB1, a known binding partner, the effects on microtubule dynamics are strongly enhanced. We show that synergy between CLASP2 and EB1 is dependent on a direct interaction, since a truncated EB1 protein that lacks the CLASP2-binding domain does not enhance CLASP2 activity. Further, we find that EB1 targets CLASP2 to microtubules and increases the dwell time of CLASP2 at microtubule tips. Although the temporally averaged microtubule growth rates are unaffected by CLASP2, we find that microtubules grown with CLASP2 display greater variability in growth rates. Our results provide insight into the regulation of microtubule dynamics by CLASP proteins and highlight the importance of the functional interplay between regulatory proteins at dynamic microtubule ends.

Two mechanisms coordinate the recruitment of the chromosomal passenger complex to the plane of cell division.

During cytokinesis, the chromosomal passenger complex (CPC) promotes midzone organization, specifies the cleavage plane, and regulates furrow contractility. The localizations of the CPC are coupled to its cytokinetic functions. At the metaphase-to-anaphase transition, the CPC dissociates from centromeres and localizes to midzone microtubules and the equatorial cortex. CPC relocalization to the cell middle is thought to depend on MKlp2-driven, plus end-directed transport. In support of this idea, MKlp2 depletion impairs cytokinesis; however, cytokinesis failure stems from furrow regression rather than failed initiation of furrowing. This suggests that an alternative mechanism(s) may concentrate the CPC at the division plane. We show here that direct actin binding, via the inner centromere protein (INCENP), enhances CPC enrichment at the equatorial cortex, thus acting in tandem with MKlp2. INCENP overexpression rescues furrowing in MKlp2-depleted cells in an INCENP-actin binding-dependent manner. Using live-cell imaging, we also find that MKlp2-dependent targeting of the CPC is biphasic. MKlp2 targets the CPC to the anti-parallel microtubule overlap of the midzone, after which the MKlp2-CPC complex moves in a nondirected manner. Collectively, our work suggests that both actin binding and MKlp2-dependent midzone targeting cooperate to precisely position the CPC during mitotic exit, and that these pathways converge to ensure successful cleavage furrow ingression.

Environmental vibrios represent a source of antagonistic compounds that inhibit pathogenic Vibrio cholerae and Vibrio parahaemolyticus strains.

With the overuse of antibiotics, many pathogens including Vibrio cholerae and Vibrio parahaemolyticus have evolved multidrug resistance making treatment more difficult. While understanding the mechanisms that underlie pathogenesis is crucial, knowledge of bacterial interactions of V. cholerae and V. parahaemolyticus could provide insight to their susceptibility outside of the human host. Based on previous work showing competition among environmental strains, we predict that marine-derived bacteria should inhibit Vibrio pathogens and may be a source of unique antibiotic compounds. We tested a collection of 3,456 environmental Vibrio isolates from diverse habitats against a panel of V. cholerae and V. parahaemolyticus, and identified 102 strains that inhibited the growth of these pathogens. Phylogenetic analysis revealed that 40 pathogen-inhibiting strains were unique at the hsp60 gene sequence while 62 of the isolates were identical suggesting clonal groups. Genomic comparisons of ten strains revealed diversity even between clonal isolates and were identified as being closely related to known Vibrio crassostreae, Vibrio splendidus, and Vibrio tasmaniensis strains. Further analysis revealed multiple biosynthetic gene clusters within all sequenced genomes that encoded secondary metabolites with potential antagonistic activity. Thus, environmental vibrios represent a source of compounds that inhibit Vibrio pathogens.

Cell Division: Centrosomes Have Separation Anxiety.

Prior to mitosis, duplicated centrosomes are tethered together, which is thought to prevent mitotic defects. A new study establishes the role of tetrameric Kif25, a microtubule minus-end-directed kinesin-14 motor, in preventing premature centrosome separation through a microtubule-dependent pathway.

Evaluation of the immunogenicity of the dabigatran reversal agent idarucizumab during Phase I studies.

AIMS: Idarucizumab, a humanized monoclonal anti-dabigatran antibody fragment, is effective in emergency reversal of dabigatran anticoagulation. Pre-existing and treatment-emergent anti-idarucizumab antibodies (antidrug antibodies; ADA) may affect the safety and efficacy of idarucizumab. This analysis characterized the pre-existing and treatment-emergent ADA and assessed their impact on the pharmacokinetics and pharmacodynamics (PK/PD) of idarucizumab. METHODS: Data were pooled from three Phase I, randomized, double-blind idarucizumab studies in healthy Caucasian subjects; elderly, renally impaired subjects; and healthy Japanese subjects. In plasma sampled before and after idarucizumab dosing, ADA were detected and titrated using a validated electrochemiluminescence method. ADA epitope specificities were examined using idarucizumab and two structurally related molecules. Idarucizumab PK/PD data were compared for subjects with and without pre-existing ADA. RESULTS: Pre-existing ADA were found in 33 out of 283 individuals (11.7%), seven of whom had intermittent ADA. Titres of pre-existing and treatment-emergent ADA were low, estimated equivalent to <0.3% of circulating idarucizumab after a 5 g dose. Pre-existing ADA had no impact on dose-normalized idarucizumab maximum plasma levels and exposure and, although data were limited, no impact on the reversal of dabigatran-induced anticoagulation by idarucizumab. Treatment-emergent ADA were detected in 20 individuals (19 out of 224 treated [8.5%]; 1 out of 59 received placebo [1.7%]) and were transient in ten. The majority had specificity primarily toward the C-terminus of idarucizumab. There were no adverse events indicative of immunogenic reactions. CONCLUSION: Pre-existing and treatment-emergent ADA were present at extremely low levels relative to the idarucizumab dosage under evaluation. The PK/PD of idarucizumab appeared to be unaffected by the presence of pre-existing ADA.

Effect of Age and Renal Function on Idarucizumab Pharmacokinetics and Idarucizumab-Mediated Reversal of Dabigatran Anticoagulant Activity in a Randomized, Double-Blind, Crossover Phase Ib Study.

BACKGROUND AND OBJECTIVES: Idarucizumab is an antibody fragment that specifically reverses dabigatran-mediated anticoagulation. Safety, pharmacokinetics and pharmacodynamics of idarucizumab were investigated in dabigatran-treated, middle-aged, elderly and renally impaired volunteers with characteristics similar to patients receiving anticoagulant therapy. METHODS: In this randomized, double-blind, crossover study, 46 subjects (12 middle-aged, 45-64 years; 16 elderly, 65-80 years; and 18 with mild or moderate renal impairment) received dabigatran etexilate (DE; 220 or 150 mg twice daily) for 4 days. Idarucizumab doses of 1, 2.5 and 5 g or 2 × 2.5 g 1 h apart, or placebo, were administered as a rapid (5 min) infusion ~2 h after DE at steady state. RESULTS: Dabigatran-prolonged diluted thrombin time, ecarin clotting time and activated partial thromboplastin time were reversed to baseline immediately after idarucizumab infusion in all groups. Reversal was sustained with doses ≥2.5 g. Idarucizumab was well tolerated under all conditions. No impact of age on idarucizumab pharmacokinetics was observed; however, subjects with mild or moderate renal impairment demonstrated increased exposure (up to 84 %), decreased clearance and prolonged (by up to 49 %) initial half-life of idarucizumab compared with healthy middle-aged subjects. CONCLUSIONS: Impaired renal function was associated with increased exposure and decreased clearance of idarucizumab. Idarucizumab resulted in immediate, complete and sustained reversal of dabigatran anticoagulant activity, and was safe and well tolerated in middle-aged, elderly and renally impaired volunteers. The results support the clinical use of a 5 g dose of idarucizumab. CLINICAL TRIAL REGISTRATION: http://www.clinicaltrials.gov . Unique identifier: NCT01955720.

Safety, pharmacokinetics and pharmacodynamics of idarucizumab, a specific dabigatran reversal agent in healthy Japanese volunteers: a randomized study.

BACKGROUND: Idarucizumab is a humanized monoclonal antibody fragment that specifically binds with high affinity to dabigatran. OBJECTIVES: This study investigated the safety, tolerability and pharmacokinetics of idarucizumab alone and with dabigatran at steady state, and the effects of idarucizumab on dabigatran-induced anticoagulation. PATIENTS/METHODS: This was a two-part, phase I, randomized, placebo-controlled, double-blind, rising-dose trial in healthy Japanese males. Part 1: 32 subjects (males) received single idarucizumab doses (1, 2, 4 or 8 g [n=6/dose group]) or placebo (n=2/dose group). Part 2: 48 males received dabigatran (220 mg bid) followed by idarucizumab (n=9/dose group) 1, 2, 4 or 5 g (2×2.5 g), or placebo (n=3/dose group). Anti-idarucizumab antibodies (ADAs) and idarucizumab effect on anticoagulation parameters (diluted thrombin time [dTT], ecarin clotting time [ECT], activated partial thromboplastin time [aPTT] and thrombin time [TT]) were assessed. RESULTS: No adverse events were reported in subjects receiving idarucizumab. After single doses of idarucizumab (alone or at steady state of dabigatran), maximum plasma concentration was achieved around the end of each infusion. Mean all anticoagulation parameters fell below the upper limit of normal immediately after idarucizumab infusion in all dose groups; the effect was sustained at 4 and 2×2.5 g over the entire measurement period until 72 h. At 1- and 2-g doses, partial return of the anticoagulant effect occurred. Idarucizumab alone had no effect on coagulation parameters. Treatment-emergent ADAs occurred in 6/60 males receiving idarucizumab. CONCLUSIONS: Idarucizumab infusion achieved immediate, complete and sustained reversal of dabigatran-induced anticoagulation in Japanese volunteers. Idarucizumab was well tolerated with no procoagulant effects. Trial registration number: ClinicalTrials.gov NCT02028780 (completed).

Workplace Violence in Mental Health: A Victorian Mental Health Workforce Survey.

The international literature suggests workplace violence in mental health settings is a significant issue, yet little is known about the frequency, nature, severity and health consequences of staff exposure to violence in Australian mental health services. To address this gap, we examined these aspects of workplace violence as reported by mental health services employees in Victoria, Australia. The project used a cross-sectional, exploratory descriptive design. A random sample of 1600 Health and Community Services Union members were invited to complete a survey investigating exposure to violence in the workplace, and related psychological health outcomes. Participants comprised employees from multiple disciplines including nursing, social work, occupational therapy, psychology and administration staff. A total of 411 members responded to the survey (26% response rate). Of the total sample, 83% reported exposure to at least one form of violence in the previous 12 months. The most frequently reported form of violence was verbal abuse (80%) followed by physical violence (34%) and then bullying/mobbing (30%). Almost one in three victims of violence (33%) rated themselves as being in psychological distress, 54% of whom reported being in severe psychological distress. The more forms of violence to which victims were exposed, the greater the frequency of reports of psychological distress. Workplace violence is prevalent in mental health facilities in Victoria. The nature, severity and health impact of this violence represents a serious safety concern for mental health employees. Strategies must be considered and implemented by healthcare management and policy makers to reduce and prevent violence.