Chk1 inhibition induces a DNA damage bystander effect in cocultured tumour cells.

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.

Targeting DNA damage response pathways to activate the STING innate immune signaling pathway in human cancer cells.

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.

Checkpoint Kinase 1 (Chk1) inhibition fails to activate the Stimulator of Interferon Genes (STING) innate immune signalling in a human coculture cancer system.

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.

Inhibition of Chk1 with the small molecule inhibitor V158411 induces DNA damage and cell death in an unperturbed S-phase.

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 CDK inhibitors eliminates proliferative potential even when their abilities to evoke apoptosis and DNA damage are blocked.

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.

4-Amino derivatives of the Hsp90 inhibitor CCT018159.

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.