A phase IA dose-escalation study of PHI-101, a new checkpoint kinase 2 inhibitor, for platinum-resistant recurrent ovarian cancer

Background PHI-101 is an orally available, selective checkpoint kinase 2 (Chk2) inhibitor. PHI-101 has shown anti-tumour activity in ovarian cancer cell lines and impaired DNA repair pathways in preclinical experiments. Furthermore, the in vivo study suggests the synergistic effect of PHI-101 through combination with PARP inhibitors for ovarian cancer treatment. The primary objective of this study is to evaluate the safety and tolerability of PHI-101 in platinum-resistant recurrent ovarian cancer. Methods Chk2 inhibitor for Recurrent EpitheliAl periToneal, fallopIan, or oVarian cancEr (CREATIVE) trial is a prospective, multi-centre, phase IA dose-escalation study. Six cohorts of dose levels are planned, and six to 36 patients are expected to be enrolled in this trial. Major inclusion criteria include ≥ 19 years with histologically confirmed epithelial ovarian cancer, fallopian tube carcinoma, or primary peritoneal cancer. Also, patients who showed disease progression during platinum-based chemotherapy or disease progression within 24 weeks from completion of platinum-based chemotherapy will be included, and prior chemotherapy lines of more than five will be excluded. The primary endpoint of this study is to determine the dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of PHI-101. Discussion PHI-101 is the first orally available Chk2 inhibitor, expected to show effectiveness in treating recurrent ovarian cancer. Through this CREATIVE trial, DLT and MTD of this new targeted therapy can be confirmed to find the recommended dose for the phase II clinical trial. This study may contribute to developing a new combination regimen for the treatment of ovarian cancer. Trial registration ClinicalTrials.gov Identifier: NCT04678102.

CHEK2 SIGNALING IS THE KEY REGULATOR OF OOCYTE SURVIVAL AFTER CHEMOTHERAPY

Radiation and chemotherapy can damage the primordial follicle reserve in female cancer patients leading to ovarian failure and infertility. Preservation of ovarian function requires treatment strategies that prevent loss of immature oocytes in primordial follicles during cancer therapy. Checkpoint kinase 2 (CHEK2) inhibition prevents loss of primordial oocytes caused by DNA damage and thus is a promising target for ovoprotective treatment against genotoxic agents. To determine which cancer treatments could benefit from ovoprotective activity of CHEK2 inhibition we investigated oocyte survival in Chek2-/- mice exposed to different chemotherapy drugs. Here, we show that loss of CHEK2 function prevents elimination of primordial oocytes damaged by cisplatin, cyclophosphamide, mafosfamide, doxorubicin, and etoposide, suggesting it could be used to reduce ovarian damage caused by wide range of drugs. Using genetic knockouts we reveal a critical role for TRP53 in oocyte response to chemotherapy drugs and show that both targets of CHEK2, TAp63 and TRP53, are activated by cisplatin and cyclophosphamide. Furthermore, we show that checkpoint kinase inhibitor and radiation- and chemotherapy sensitizer AZD7762 reduces oocyte elimination after radiation and chemotherapy treatments, despite its cytotoxic effect on ovarian somatic cells. Altogether, these findings demonstrate the role for CHEK2 as the master regulator of primordial oocyte survival or death and credential its targeting for ovoprotective treatments.

Mechanisms of loading and release of the 9-1-1 checkpoint clamp

5' single-stranded/double-stranded DNA serve as loading sites for the checkpoint clamp, 9-1-1, which mediates activation of the apical checkpoint kinase, ATRMec1. However, the basis for 9-1-1's recruitment to 5' junctions is unclear. Here, we present structures of the yeast checkpoint clamp loader, Rad24-RFC, in complex with 9-1-1 and a 5' junction and in a post-ATP-hydrolysis state. Unexpectedly, 9-1-1 adopts both closed and planar open states in the presence of Rad24-RFC and DNA. Moreover, Rad24-RFC associates with the DNA junction in the opposite orientation of processivity clamp loaders with Rad24 exclusively coordinating the double-stranded region. ATP hydrolysis stimulates conformational changes in Rad24-RFC, leading to disengagement of DNA-loaded 9-1-1. Together, these structures explain 9-1-1's recruitment to 5' junctions and reveal new principles of sliding clamp loading.

Fms-like tyrosine kinase 3-internal tandem duplications epigenetically activates checkpoint kinase 1 in acute myeloid leukemia cells

It is not clear how Fms-like tyrosine kinase 3-internal tandem duplications (FLT3-ITD) regulates checkpoint kinase 1 (CHK1) in acute myeloid leukemia (AML). In this study, we investigated the regulatory effect of FLT3-ITD on CHK1. Our results showed that CHK1 was highly expressed in FLT3-ITD positive AML. The overall survival rate and disease-free survival rate of AML patients with high CHK1 level were lower than those of patients with low CHK1 level. Mechanistically, FLT3-ITD recruited p300 to the CHK1 promoter and subsequently acetylated H3K27, thereby enhancing the transcription of CHK1. Interfering with the expression of CHK1 significantly inhibited the cell proliferation and induced cell apoptosis in FLT3-ITD positive MV4-11 cells. In addition, CHK1 knockdown promoted the sensitivity of MV4-11 cells to the epigenetic inhibitors JQ1 and C646. This study discovers a new therapeutic target for FLT3-ITD + AML and provided evidence for the combination of epigenetic inhibitors for AML treatment.

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.

Prevalent and Dynamic Binding of the Cell Cycle Checkpoint Kinase Rad53 to Gene Promoters

Replication of the genome must be coordinated with gene transcription and cellular metabolism. These processes are controlled in part by the Rad53 (CHEK2 in mammals) checkpoint kinase and the Mrc1 replisome component, especially following replication stress in the presence of limiting deoxyribonucleotides. We examined cell cycle regulated, genome-wide binding of Rad53 to chromatin. The kinase bound to sites of active DNA replication initiation and fork progression, but unexpectedly to the promoters of numerous genes (>20% of all genes) involved in many cellular functions. At some genes, Rad53 promoter binding correlated with changes in gene expression. Rad53 promoter binding to certain genes is influenced by sequence-specific transcription factors and less by checkpoint signaling. In checkpoint mutants, untimely activation of late-replicating origins reduces the transcription of nearby genes, with concomitant localization of Rad53 to their gene bodies. We suggest that the Rad53 checkpoint kinase coordinates genome-wide replication and transcription under stress conditions.

New Natural Compound Inhibitors of Checkpoint Kinase-1(CHK1) Based on Computational Study

As a malignant tumor of the ovary, the general treatment principle of ovarian cancer is surgical treatment, supplemented by chemotherapy, and some patients can use targeted drugs. Its treatment effect is relatively poor, so the prognosis is poor, the mortality rate is high. To contribute to drug design and refinement, ideal lead compounds with potential inhibitory effects on ATP-competitive CHK1 (Checkpoint kinase-1) inhibitors were downloaded from the drug library (ZINC15 database) and screened afterwards. The ATP-competitive CHK1 inhibitors were identified by using computer-aided virtual screening technology. We first calculated the LibDock score through the docking of proteins and molecules, and then analyzed the pharmacological and toxicological properties. Then, we performed precise docking of the small molecules selected in the above steps with CHK1 protein to analyze their docking mechanism and affinity. Next, we used molecular dynamics simulation to make a assessment if the ligand-CHK1 complex were stable in natural environment. As the result shown, ZINC000008214547 and ZINC000072103632 were proved to bind with CHK1 with a higher binding affinity and stability. Additionally, their toxicological analysis shows that they are less toxic and will not inhibit the activity of cytochrome P-450 2D6. In the simulation of molecular dynamics, we also found that ZINC000008214547-CHK1 and ZINC000072103632-CHK1 complexes’ potential energy were more favorable compared with reference ligand, Prexasertib. Not only that, the two complexes also showed better stability in the natural environment. So, all results elucidated that ZINC000008214547 and ZINC000072103632 were favorable lead inhibitors of CHK1 protein. ZINC000008214547 and ZINC000072103632 were safe and had the potential to inhibit CHK1 protein. They may contribute a solid foundation for the development of CHK1 target drug.