Intra-S phase checkpoint kinase Chk1 dissociates replication proteins Treslin and TopBP1 through multiple mechanisms during replication stress.

Replication stress impedes DNA polymerase progression causing activation of the ataxia telangiectasia and Rad3-related signaling pathway, which promotes the intra-S phase checkpoint activity through phosphorylation of checkpoint kinase 1 (Chk1). Chk1 suppresses replication origin firing, in part, by disrupting the interaction between the preinitiation complex components Treslin and TopBP1, an interaction that is mediated by TopBP1 BRCT domain-binding to two cyclin-dependent kinase (CDK) phosphorylation sites, T968 and S1000, in Treslin. Two nonexclusive models for how Chk1 regulates the Treslin-TopBP1 interaction have been proposed in the literature: in one model, these proteins dissociate due to a Chk1-induced decrease in CDK activity that reduces phosphorylation of the Treslin sites that bind TopBP1 and in the second model, Chk1 directly phosphorylates Treslin, resulting in dissociation of TopBP1. However, these models have not been formally examined. We show here that Treslin T968 phosphorylation was decreased in a Chk1-dependent manner, while Treslin S1000 phosphorylation was unchanged, demonstrating that T968 and S1000 are differentially regulated. However, CDK2-mediated phosphorylation alone did not fully account for Chk1 regulation of the Treslin-TopBP1 interaction. We also identified additional Chk1 phosphorylation sites on Treslin that contributed to disruption of the Treslin-TopBP1 interaction, including S1114. Finally, we showed that both of the proposed mechanisms regulate origin firing in cancer cell line models undergoing replication stress, with the relative roles of each mechanism varying among cell lines. This study demonstrates that Chk1 regulates Treslin through multiple mechanisms to promote efficient dissociation of Treslin and TopBP1 and furthers our understanding of Treslin regulation during the intra-S phase checkpoint.

Downregulation of c-Myc expression confers sensitivity to CHK1 inhibitors in hematologic malignancies.

Checkpoint kinase 1 inhibitors (CHK1i) have shown impressive single-agent efficacy in treatment of certain tumors, as monotherapy or potentiators of chemotherapy in clinical trials, but the sensitive tumor types and downstream effectors to dictate the therapeutic responses to CHK1i remains unclear. In this study we first analyzed GDSC (Genomics of Drug Sensitivity in Cancer) and DepMap database and disclosed that hematologic malignancies (HMs) were relatively sensitive to CHK1i or CHK1 knockdown. This notion was confirmed by examining PY34, a new and potent in-house selective CHK1i, which exhibited potent anti-HM effect in vitro and in vivo, as single agent. We demonstrated that the downregulation of c-Myc and its signaling pathway was the common transcriptomic profiling response of sensitive HM cell lines to PY34, whereas overexpressing c-Myc could partially rescue the anticancer effect of PY34. Strikingly, we revealed the significant correlations between downregulation of c-Myc and cell sensitivity to PY34 in 17 HM cell lines and 39 patient-derived cell (PDC) samples. Thus, our results demonstrate that HMs are more sensitive to CHK1i than solid tumors, and c-Myc downregulation could represent the CHK1i efficacy in HMs.

Promyelocytic Leukemia Proteins Regulate Fanconi Anemia Gene Expression.

Promyelocytic leukemia (PML) protein is the core component of subnuclear structures called PML nuclear bodies that are known to play important roles in cell survival, DNA damage responses, and DNA repair. Fanconi anemia (FA) proteins are required for repairing interstrand DNA crosslinks (ICLs). Here we report a novel role of PML proteins, regulating the ICL repair pathway. We found that depletion of the PML protein led to the significant reduction of damage-induced FANCD2 mono-ubiquitination and FANCD2 foci formation. Consistently, the cells treated with siRNA against PML showed enhanced sensitivity to a crosslinking agent, mitomycin C. Further studies showed that depletion of PML reduced the protein expression of FANCA, FANCG, and FANCD2 via reduced transcriptional activity. Interestingly, we observed that damage-induced CHK1 phosphorylation was severely impaired in cells with depleted PML, and we demonstrated that CHK1 regulates FANCA, FANCG, and FANCD2 transcription. Finally, we showed that inhibition of CHK1 phosphorylation further sensitized cancer cells to mitomycin C. Taken together, these findings suggest that the PML is critical for damage-induced CHK1 phosphorylation, which is important for FA gene expression and for repairing ICLs.

Checkpoint Kinase 1 Pharmacological Inhibition Synergizes with DNA-Damaging Agents and Overcomes Platinum Resistance in Basal-Like Breast Cancer.

Basal-like breast cancer is an incurable disease with limited therapeutic options, mainly due to the frequent development of anti-cancer drug resistance. Therefore, identification of druggable targets to improve current therapies and overcome these resistances is a major goal. Targeting DNA repair mechanisms has reached the clinical setting and several strategies, like the inhibition of the CHK1 kinase, are currently in clinical development. Here, using a panel of basal-like cancer cell lines, we explored the synergistic interactions of CHK1 inhibitors (rabusertib and SAR020106) with approved therapies in breast cancer and evaluated their potential to overcome resistance. We identified a synergistic action of these inhibitors with agents that produce DNA damage, like platinum compounds, gemcitabine, and the PARP inhibitor olaparib. Our results demonstrated that the combination of rabusertib with these chemotherapies also has a synergistic impact on tumor initiation, invasion capabilities, and apoptosis in vitro. We also revealed a biochemical effect on DNA damage and caspase-dependent apoptosis pathways through the phosphorylation of H2AX, the degradation of full-length PARP, and the increase of caspases 3 and 8 activity. This agent also demonstrated synergistic activity in a platinum-resistant cell line, inducing an increase in cell death in response to cisplatin only when combined with rabusertib, while no toxic effect was found on non-tumorigenic breast tissue-derived cell lines. Lastly, the combination of CHK1 inhibitor with cisplatin and gemcitabine resulted in more activity than single or double combinations, leading to a higher apoptotic effect. In conclusion, in our study we identify therapeutic options for the clinical development of CHK1 inhibitors, and confirm that the inhibition of this kinase can overcome acquired resistance to cisplatin.

Aven-mediated checkpoint kinase control regulates proliferation and resistance to chemotherapy in conventional osteosarcoma.

Conventional high-grade osteosarcoma is the most common primary bone sarcoma, with relatively high incidence in young people. In this study we found that expression of Aven correlates inversely with metastasis-free survival in osteosarcoma patients and is increased in metastases compared to primary tumours. Aven is an adaptor protein that has been implicated in anti-apoptotic signalling and serves as an oncoprotein in acute lymphoblastic leukaemia. In osteosarcoma cells, silencing Aven triggered G2 cell-cycle arrest; Chk1 protein levels were attenuated and ATR-Chk1 DNA damage response signalling in response to chemotherapy was abolished in Aven-depleted osteosarcoma cells, while ATM, Chk2 and p53 activation remained intact. Osteosarcoma is notoriously difficult to treat with standard chemotherapy, and we examined whether pharmacological inhibition of the Aven-controlled ATR-Chk1 response could sensitize osteosarcoma cells to genotoxic compounds. Indeed, pharmacological inhibitors targeting Chk1/Chk2 or those selective for Chk1 synergized with standard chemotherapy in 2D cultures. Likewise, in 3D extracellular matrix-embedded cultures, Chk1 inhibition led to effective sensitization to chemotherapy. Together, these findings implicate Aven in ATR-Chk1 signalling and point towards Chk1 inhibition as a strategy to sensitize human osteosarcomas to chemotherapy.

Design of granulatimide and isogranulatimide analogues as potential Chk1 inhibitors: Study of amino-platforms for their synthesis.

The two marine alkaloids granulatimide and isogranulatimide have been shown to inhibit the checkpoint kinase 1 (Chk1), a promising target for cancer treatment. A molecular docking study allowing the design of new potential Chk1 inhibitors based on the natural products skeleton and the synthetic work to an amino-target platform to prepare them are described.

Design, Synthesis, and Biological Evaluation of 2-Aminothiazole Derivatives as Novel Checkpoint Kinase 1 (CHK1) Inhibitors.

A series of 2-aminothiazole derivatives were designed, synthesized on the basis of bioisosterism strategy and evaluated for their CHK1 inhibitory activity. Most of them exhibited potent CHK1 inhibition, and excellent antiproliferative activity against MV-4-11 and Z-138 cell lines. Systematic structure-activity relationship (SAR) efforts led to the discovery of a promising compound 8 n, which showed potent CHK1 inhibitory activity with IC50 value of 4.25±0.10 nM, excellent antiproliferative activity against MV-4-11 and Z-138 cells with IC50 value of 42.10±5.77 nM and 24.16±6.67 nM, respectively, as well as moderate oral exposure (AUC(0-t) =1076.25 h ⋅ ng/mL) in mice. Additionally, treatment of MV-4-11 cells with compound 8 n for 2 h led to robust inhibition of CHK1 autophosphorylation on serine 296. Furthermore, kinase selectivity assay revealed that 8 n displayed acceptable selectivity toward 15 kinases. These results demonstrated that compound 8 n may be a promising potential anticancer agent for further development.

Chronic treatment with ATR and CHK1 inhibitors does not substantially increase the mutational burden of human cells.

DNA replication stress (RS) entails the frequent slow down and arrest of replication forks by a variety of conditions that hinder accurate and processive genome duplication. Elevated RS leads to genome instability, replication catastrophe and eventually cell death. RS is particularly prevalent in cancer cells and its exacerbation to unsustainable levels by chemotherapeutic agents remains a cornerstone of cancer treatments. The adverse consequences of RS are normally prevented by the ATR and CHK1 checkpoint kinases that stabilize stressed forks, suppress origin firing and promote cell cycle arrest when replication is perturbed. Specific inhibitors of these kinases have been developed and shown to potentiate RS and cell death in multiple in vitro cancer settings. Ongoing clinical trials are now probing their efficacy against various cancer types, either as single agents or in combination with mainstay chemotherapeutics. Despite their promise as valuable additions to the anti-cancer pharmacopoeia, we still lack a genome-wide view of the potential mutagenicity of these new drugs. To investigate this question, we performed chronic long-term treatments of TP53-depleted human cancer cells with ATR and CHK1 inhibitors (ATRi, AZD6738/ceralasertib and CHK1i, MK8776/SCH-900776). ATR or CHK1 inhibition did not significantly increase the mutational burden of cells, nor generate specific mutational signatures. Indeed, no notable changes in the numbers of base substitutions, short insertions/deletions and larger scale rearrangements were observed despite induction of replication-associated DNA breaks during treatments. Interestingly, ATR inhibition did induce a slight increase in closely-spaced mutations, a feature previously attributed to translesion synthesis DNA polymerases. The results suggest that ATRi and CHK1i do not have substantial mutagenic effects in vitro when used as standalone agents.

Everything Comes with a Price: The Toxicity Profile of DNA-Damage Response Targeting Agents.

Targeting the inherent vulnerability of cancer cells with an impaired DNA Damage Repair (DDR) machinery, Poly-ADP-Ribose-Polymerase (PARP) inhibitors have yielded significant results in several tumor types, eventually entering clinical practice for the treatment of ovarian, breast, pancreatic and prostate cancer. More recently, inhibitors of other key components of DNA repair, such as ATR, CHK1 and WEE1, have been developed and are currently under investigation in clinical trials. The inhibition of DDR inevitably induces on-target and off-target adverse events. Hematological and gastrointestinal toxicities as well as fatigue are common with all DDR-targeting agents, while other adverse events are drug specific, such as hypertension with niraparib and transaminase elevation with rucaparib. Cases of pneumonitis and secondary hematological malignancies have been reported with PARP inhibitors and, despite being overly rare, they deserve particular attention due to their severity. Safety also represents a crucial issue for the development of combination regimens incorporating DDR-targeting agents with other treatments, such as chemotherapy, anti-angiogenics or immunotherapy. As such, overlapping and cumulative toxicities should be considered, especially when more than two classes of drugs are combined. Here, we review the safety profile of DDR-targeting agents when used as single agents or in combination and we provide principles of toxicity management.

In Silico Exploration of Aryl Halides Analogues as Checkpoint Kinase 1 Inhibitors by Using 3D QSAR, Molecular Docking Study, and ADMET Screening.

Purpose: In this review, a set of aryl halides analogs were identified as potent checkpoint kinase 1 (Chk1) inhibitors through a series of computer-aided drug design processes, to develop models with good predictive ability, highlight the important interactions between the ligand and the Chk1 receptor protein and determine properties of the new proposed drugs as Chk1 inhibitors agents. Methods: Three-dimensional quantitative structure-activity relationship (3D-QSAR) modeling, molecular docking and absorption, distribution, metabolism, excretion and toxicity (ADMET) approaches are used to determine structure activity relationship and confirm the stable conformation on the receptor pocket. Results: The statistical analysis results of comparative -molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models that employed for a training set of 24 compounds gives reliable values of Q2 (0.70 and 0.94, respectively) and R2 (0.68 and 0.96, respectively). Conclusion: Computer-aided drug design tools used to develop models that possess good predictive ability, and to determine the stability of the observed and predicted molecules in the receptor pocket, also in silico of pharmacokinetic (ADMET) results shows good properties and bioavailability for these new proposed Chk1 inhibitors agents.

Synthesis and biological evaluation of 2,6-disubstituted-9H-purine, 2,4-disubstitued-thieno[3,2-d]pyrimidine and -7H-pyrrolo[2,3-d]pyrimidine analogues as novel CHK1 inhibitors.

Checkpoint kinase 1 (CHK1) inhibitors can potentiate the effectiveness of deoxyribonucleic acid (DNA) damaging agents in the treatment of cancer. A novel series of 2,6-disubstituted-9H-purine (3a-p, 5a and 5b), 2,4-disubstituted-thieno[3,2-d]pyrimidine (8a-c) and 2,4-disbustituted-7H-pyrrolo[2,3-d]pyrimidine (11a-c) analogues were designed and synthesized as potent CHK1 inhibitors. Compounds (3a, 3d, 3f and 3j-l) with 9H-purine core displayed more potent inhibition against CHK1. The most potent compound (3l) also exhibited low anti-proliferative effects towards HT29 and Hek293 cell lines. In addition, 3l showed strong potentiating effect (7-fold) on the anti-proliferative activity of gemcitabine towards HT29 cells. The results of cell cycle assay indicated that 3l could strikingly affect the cell cycle distribution of the gemcitabine-treated HT29 cells and induce a significant S phase accumulation. The kinase selectivity profile of 3l displayed acceptable selectivity against other kinases. These results rendered 3l a potent lead compound of CHK1 inhibitor for further investigation.

Identification of novel aminothiazole and aminothiadiazole conjugated cyanopyridines as selective CHK1 inhibitors.

Inhibitors of checkpoint kinase 1 (CHK1) are of current interest as potential anti-tumor agents. Novel series of cyanopyridyl-aminothiadiazoles (synthesized from reaction of 1-(3-cyano-4,6-diphenylpyridin-2-yl)-3-phenylthiourea (14) with hydrazonoyl halides) and cyanopyridyl-aminothiazolyl-thiadiazoles (synthesized from treatment of 14 with ethyl chloroacetate followed by reaction of the obtained cyanopyridyl-aminothiazole with hydrazonoyl halides) were synthesized and evaluated for their CHK1 inhibitory potential using a cell-based assay cascade. The tested compounds exhibited a potent and selective CHK1 inhibitory activity at nanomolar levels that reflected their ability to abrogate cell cycle arrest and potentiate the cytotoxic effect of the genotoxic drug gemcitabine in colon cancer cells. Molecular modeling simulation revealed that, the most active compound 28a docked well into the enzyme active site and their complex is stabilized by a key H-bonding with the backbone amide of Cys-87 as well as multiple favorable hydrophobic interactions with different hydrophobic binding regions of the enzyme.