Oncogenic mutations in KRAS account for similar to 25% of all cancers, which has made it a much-researched target for therapeutic intervention [1]. Direct inhibition of KRAS mutants has attracted significant interest in recent years, and the development of small molecule KRAS inhibitors has thus far not proved to be trivial. To date, there is only one KRAS mutant allele-specific covalent inhibitor (Sotarasib/LUMAKRAS (R)) approved by the US FDA for the treatment of non-small cell lung cancer [2]. With the infamy of KRAS as a hitherto `undruggable' target, alternative strategies to indirectly target the KRAS pathway are also being investigated [3]. One strategy is to exploit the vulnerability of KRAS-mutant cancers by targeting synthetic lethal partners of mutant KRAS. The synthetic lethality of two proteins is where the cell remains viable on perturbation of either one alone, but perturbation of both results in cell death. Targeting a gene that is synthetically lethal to a cancer-relevant mutation such as KRAS should theoretically result in cancer cell death, sparing normal cells that do not harbor this mutation [4].

Inhibitors of Chk1 kinase, a key effector of the DNA damage response pathway, are currently undergoing Phase 1 and 2 clinical trials as single agents and in combination with cytotoxic chemotherapy. Understanding the biological effects of Chk1 inhibitors on cancer cells is critical for their continued clinical development. Treatment of adherent HT29 or HCC1937 cancer cells or suspension Jurkat or THP1 cells with a Chk1 inhibitor increased γH2AX in these cells. Chk1i pre-treated HCC1937 or HT29 cells resulted in γH2AX induction in cocultured Jurkat or THP1 cells despite these cells never being treated with a Chk1i. Pre-treatment of HT29 cells with camptothecin or gemcitabine followed by a Chk1i increased the DNA damage bystander effect in naïve cocultured THP1 cells compared to camptothecin or gemcitabine alone. This bystander effect appeared to occur through soluble factors via ATR, ATM, and DNA-PKcs activation in the bystander cells. Chk1 silencing by siRNA in HCC1937 or HT29 cells induced a DNA damage bystander effect in cocultured THP1 cells. However, this bystander effect induced by siRNA appeared mechanistically different to that induced by the Chk1 inhibitor. This work suggests that a Chk1 inhibitor-induced bystander effect may increase the clinical effectiveness of Chk1 inhibitors by inducing additional DNA damage or replication stress in cancer cells not directly exposed to the inhibitor. Conversely, it may also contribute to Chk1 inhibitor toxicity by increasing DNA damage in non-tumour cells.

Utilising Checkpoint Kinase 1 (Chk1) inhibitors to increase cytoplasmic DNA may be a potential strategy to increase the sensitivity of tumours to immune checkpoint modulators. The appearance of DNA in the cytoplasm can drive Cyclic GMP-AMP Synthase-2′,3′-Cyclic Guanosine Monophosphate–Adenosine Monophosphate-Stimulator of Interferon Genes (cGAS-cGAMP-STING) inflammatory, anti-tumour T-cell activity via a type I interferon (IFN) and nuclear factor-κB response. In the THP1-Dual reporter cell line, the STING agonist cGAMP activated both reporters, and increased phosphorylation of the innate immune pathway signallers Tank Binding Kinase 1 (TBK1) and Interferon Regulatory Factor (IRF) 3. Inhibition of Chk1 increased TBK1 but not IRF3 phosphorylation and did not induce IRF or NF-κB reporter activation. cGAMP induced a Type I IFN response in THP1 cells whereas inhibition of Chk1 did not. HT29 or HCC1937 cell treatment with a Chk1 inhibitor increased cytoplasmic dsDNA in treated HCC1937 but not HT29 cells and increased IRF reporter activation in cocultured THP1-Dual cells. HT29 cells pre-treated with gemcitabine or camptothecin had elevated cytoplasmic dsDNA and IRF reporter activation in cocultured THP1-Dual cells. Camptothecin or gemcitabine plus a Chk1 inhibitor increased cytoplasmic dsDNA but Chk1 inhibition suppressed IRF reporter activation in cocultured THP1 cells. In THP1-Dual cells treated with cGAMP, Chk1 inhibition suppressed the activation of the IRF reporter compared to cGAMP alone. These results suggest that, in some cellular models, there is little evidence to support the combination of Chk1 inhibitors with immune checkpoint modulators and, in some combination regimes, may even prove deleterious.

Activating stimulator of interferon genes to turn immunologically refractive cold tumor hot is an exciting therapeutic approach to increase the clinical responsiveness of some human cancers to immune checkpoint inhibitors. DNA damaging drugs and PARP inhibitors are two types of agents that have demonstrated this potential. Inhibitors of Chk1 or Wee1 induce DNA damage in cancer cells in predominantly the S‐phase population. Increased cytoplasmic single‐stranded and double‐stranded DNA (dsDNA) from this DNA damage resulted in increased tank‐binding kinase 1 (TBK1) phosphorylation in a range of cancer cell lines. However, despite robust increases in pTBK1, no downstream consequences of TBK1 phosphorylation were observed (namely no increase in pIRF3/7, interferon regulatory factor (IRF)‐dependent gene expression or a type I IFN response). In combination with cytotoxic chemotherapy such as gemcitabine or camptothecin (CPT), Chk1 inhibition increased cytoplasmic dsDNA compared with the cytotoxic alone but attenuated the cytotoxic chemotherapy‐induced increase in IRF1 protein and STAT1 phosphorylation through inhibition of nuclear RelB translocation. Despite increased cytoplasmic DNA and TBK1 activation, inhibition of Chk1, ataxia telangiectasia and Rad3‐related protein, or Wee1 failed to activate a type I IFN response. We discuss the potential underlying mechanisms for this lack of IRF‐dependent gene response and how this might influence the clinical strategies of combining Chk1 or Wee1 inhibitors with immune checkpoint inhibitors.

Abstract On sustaining damage to their DNA, cells employ a sophisticated mechanism of detection and repair, termed the DNA damage response (DDR). As a critical component of the DDR and G2/M checkpoint, Chk1 kinase represents an attractive target for cancer therapy. We have utilized a structure-based drug design approach to identify and develop VER-250840, a novel, orally active inhibitor of the checkpoint kinase, Chk1. VER-250840 exhibited sub-nM potency against Chk1 kinase with exquisite selectivity over an extensive and diverse panel of kinases. In vitro, VER-250840 inhibited Chk1 autophosphorylation with an IC50 of 1.0 nM and increased the number of S-phase tumor cells staining positive for pan-nuclear γH2AX with an EC50 of 7 - 27 nM. Accumulated genomic DNA damage by Chk1 inhibition led to irreversible cell cycle arrest, inhibition of tumor cell proliferation, increased replication stress, and cell death in both 2D culture and multicellular tumor spheroids. In an in vivo A2058 tumor xenograft PD study, VER-250840 demonstrated rapid and sustained inhibition of Chk1 auto-phosphorylation within 30 minutes of oral administration. Doses of 10 mg/kg and higher PO resulted in greater than 90% inhibition of tumor pChk1 (S296) over 24 hours. In SKOV3 in vivo models, VER-250840 inhibited Chk1 auto-phosphorylation, modulated other biomarkers of replication stress and DNA damage, and exhibited moderate antitumor activity with minimal toxicity when administered orally on a 21-day once-daily schedule. Work is ongoing to further optimize in vivo efficacy. In conclusion, VER-250840 demonstrates potent and selective activity as a monotherapy both in vitro and in vivo. From these findings, further evaluation and optimization of this novel kinase inhibitor is justly merited. Citation Format: Joanne Wayne, Stephen Stokes, Nicolas Foloppe, Helen Browne, Teresa Brooks, Karen Benwell, Lisa Baker, Zoe Daniels, Andrea Fiumana, Christopher Graham, Alba Macias, Daniel Maddox, Sean McKenna, Christopher Northfield, Stuart Ray, Heather Simmonite, Emma Stefaniak, Paul Webb, Mike Wood, Andrew Massey. Identification and preclinical characterisation of VER-250840, a potent, selective Chk1 inhibitor with in vivo oral single-agent antitumor activity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B163.

Chk1 kinase is a critical component of the DNA damage response checkpoint and Chk1 inhibitors are currently under clinical investigation. Chk1 suppresses oncogene-induced replication stress with Chk1 inhibitors demonstrating activity as a monotherapy in numerous cancer types. Understanding the mechanism by which Chk1 inhibitors induce DNA damage and cancer cell death is essential for their future clinical development. Here we characterize the mechanism by which the novel Chk1 inhibitor (V158411) increased DNA damage and cell death in models of human cancer. V158411 induced a time- and concentration-dependent increase in γH2AX-positive nuclei that was restricted to cells actively undergoing DNA synthesis. γH2AX induction was an early event and correlated with activation of the ATR/ATM/DNA-PKcs DNA damage response pathways. The appearance of γH2AX positive nuclei preceded ssDNA appearance and RPA exhaustion. Complete and sustained inhibition of Chk1 kinase was necessary to activate a robust γH2AX induction and growth inhibition. Chk1 inhibitor cytotoxicity correlated with induction of DNA damage with cells undergoing apoptosis, mitotic slippage and DNA damage-induced permanent cell cycle arrest. We identified two distinct classes of Chk1 inhibitors: those that induced a strong increase in γH2AX, pChk1 (S317) and pRPA32 (S4/S8) (including V158411, LY2603618 and ARRY-1A) and those that did not (including MK-8776 and GNE-900). Tumor cell death, induced through increased DNA damage, coupled with abrogation of cell cycle checkpoints makes selective inhibitors of Chk1 a potentially useful therapeutic treatment for multiple human cancers.

Transient treatment with small molecule CDK inhibitors is toxic to cancer cells and leads to depletion of anti-apoptotic proteins and Chk1, coupled with DNA damage and induction of apoptosis. Here we have examined, which of these phenomena are necessary for CDK inhibitors to have an anti-proliferative effect. We find that 24 hours treatment with either a primarily CDK2-specific, or a primarily CDK7/9-specific, antagonist eliminates proliferative potential even if apoptosis is blocked and the tendency of CDK inhibition to result in DNA damage is overcome by expression of recombinant Chk1. Loss of proliferative potential is correlated with irreversible suppression of biomarkers of cell cycle progression. CDK inhibitors dramatically reduced levels of the anti-apoptotic proteins, Mcl-1 and XIAP, but siRNA-mediated suppression of Mcl-1 and XIAP did not induce cell death in the osteosarcoma cells used in this study. Finally, we found that many literature CDK inhibitors do not effectively suppress the CDK/cyclin complexes responsible for cell-cycle progression at the minimum doses required to block proliferation: some are only effective after a substantial delay and may act via inhibition of CDK7.

Understanding how individuals perceive an emerging technology can have a profound impact on how successfully the technology is adopted. We conjectured that the cognitive style of an individual would have a significant bearing on the way the person perceives the technology. We conducted a laboratory experiment to investigate whether cognitive styles affect the perception of RFID, one of the emerging technologies. The findings suggest that a person’ s cognitive style does indeed matter how he/she perceives the usefulness of the technology. Individuals of thinking and judging cognitive style are more likely to perceive higher value with RFID than those of feeling and perceiving cognitive style. We also provide discussion on the managerial and theoretical implications of our findings.

Novel piperazinyl, morpholino and piperidyl derivatives of the pyrazole-based Hsp90 inhibitor CCT018159 are described. Structure-activity relationships have been elucidated by X-ray co-crystal analysis of the new compounds bound to the N-terminal domain of human Hsp90. Key features of the binding mode are essentially identical to the recently reported potent analogue VER-49009. The most potent of the new compounds has a methylsulfonylbenzyl substituent appended to the piperazine nitrogen, possesses an IC50 of less than 600 nM binding against the enzyme and demonstrates low micromolar inhibition of tumour cell proliferation.

Hsp90 encodes a ubiquitous molecular chaperone protein conserved among species which acts on multiple substrates, many of which are important cell-signaling proteins. Inhibition of Hsp90 function has been promoted as a mechanism to degrade client proteins involved in tumorigenesis and disease progression. Several assays to monitor inhibition of Hsp90 function currently exist but are limited in their use for a drug discovery campaign. Using data from the crystal structure of an initial hit compound, we have developed a fluorescence polarization assay to monitor binding of compounds to the ATP-binding site of Hsp90. This assay is very robust (Z' > 0.9) and can detect affinity of compounds with IC50s to 40 nM. We have used this assay in conjunction with cocrystal structures of small molecules to drive a structure-based design program aimed at the discovery and optimization of a novel class of potent Hsp90 inhibitors.

Australian Veterinary JournalVolume 77, Issue 2 p. 98-99 Hypertrophic osteopathy and pneumonia in a macropod J WAYNE, J WAYNE Department of Veterinary Pathology and Anatomy, School of Veterinary Science and Animal Production, The University of Queensland, Queensland 4072Search for more papers by this authorV NICHOLSON, V NICHOLSON Lone Pine Koala Sanctuary, Fig Tree Pocket, Queensland 4069Search for more papers by this author J WAYNE, J WAYNE Department of Veterinary Pathology and Anatomy, School of Veterinary Science and Animal Production, The University of Queensland, Queensland 4072Search for more papers by this authorV NICHOLSON, V NICHOLSON Lone Pine Koala Sanctuary, Fig Tree Pocket, Queensland 4069Search for more papers by this author First published: 10 March 2008 https://doi.org/10.1111/j.1751-0813.1999.tb11676.xCitations: 7Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume77, Issue2February 1999Pages 98-99 RelatedInformation

Resistance of the anterior chamber (AC) of mouse eyes to expression of cell-mediated immunity can be overcome by pre-treating the eye with a dose of recombinant rat gamma-interferon (7IFN) that is of itself noninflammatory. To study the mechanism of this form of intraocular inflammation, cells of the tissues surrounding the AC (iris, ciliary body, cornea) were studied in vivo for alterations in phenotype and in vitro regarding their effects on antigen-driven T cell activation. The results indicate that 7IFN: (1) induced class II major histocompatibility complex (MHC) expression on resident bone marrow-de- rived cells of iris and ciliary body (I/CB), but not the cornea; (2) led to recruitment of bone marrow-de- rived cells into the I/CB stroma; and (3) failed to induce class II MHC expression on ocular epithelial cells. Cell suspensions prepared from 7lFN-treated I/CB superficially resembled normal I/CB cells in that neither were able to activate allogeneic T cells and both were able to suppress antigen-driven T cell activation in vitro. However, unlike cells from normal eyes, I/CB cells from 7lFN-treated eyes sup- pressed T cell activation primarily through the secretion of prostaglandins. These results indicate that the ability of 7IFN-treated eyes to display immunogenic inflammation probably does not result merely from the restoration of conventional antigen presenting cells to this environment, but appears to corre- late with a critical change in the molecular mediators of immunosuppression. The findings are dis- cussed in terms of the possibility that the eye may be able to respond to abrogation of its primary immunosuppressive microenvironment by erecting a secondary microenvironment that also is capable of suppressing immunogenic inflammation with a different set of antiinflammatory mediators. Invest Ophthalmol Vis Sci 33:2304-2315, 1992 The anterior chamber (AC) of the eye has proven to be a rather inhospitable environment for the expres- sion of cell-mediated immunity of the delayed hyper- sensitivity type. This has been especially well demon- strated in mice, where injection of antigen (prepared from Mycobacterium tuberculosa) into the AC of tu- berculin-primed mice failed to elicit a delayed-in- time intraocular inflammatory response. 1 Similarly, parental strain lymphocytes injected into the AC of suitable F, hybrid mice failed to induce a local graft- versus-host response. At least two unusual features of the anterior segment of the eye are thought to be in- volved in limiting or preventing immunogenic intra-