Elucidating The Mechanism Of CHK1 Inhibitor-Induced Activation Of MtDNA-STING Stress Axis To Enhance Anti-Tumor Immunity In Ovarian Cancer

Objective: Ovarian cancer has an insidious and poor prognosis,and new therapeutic strategies are urgently needed.In recent years,multiple studies have found that targeted therapy can activate the anti-tumor immune microenvironment,and the combination of targeted therapy and immunotherapy provides new hope for the treatment of ovarian cancer.CHK1 kinase is a key molecule that regulates the G2/M checkpoint in the cell cycle.Recent data suggest that CHK1 may be a promising target for enhancing the efficacy of immunotherapy,but the molecular mechanism of its action is unclear.Therefore,this study aims to explore the effect of CHK1 inhibitors on the immune response and its mechanism,evaluate the effectiveness of combined immunotherapy,and explore its potential value in clinical practice.Methods: In this study,the effect of CHK1 inhibitor Prexasertib on the interferon pathway and immune response was initially confirmed based on clinical trial data and cellular model validation.Subsequently,by comparing the efficacy of Prexasertib in tumor-bearing mice with normal immune function and deficient mice,it was further determined that the antitumor effect of Prexasertib is dependent on the immune environment.To explore the specific regulatory mechanism of Prexasertib activated immune response,RNA-seq,CRISPY-Cas9,ρ0 cells(without mt DNA),cytoplasmic DNA extraction,EGS cross-linking,COIP,Phostag,Western Blot,q RT-PCR,immunofluorescence confocal,flow cytometry and immunohistochemical staining were employed.Finally,the specific mechanism of Prexasertib in immune regulation was verified in vivo through the construction of CT26tumor-bearing mouse model,and the efficacy of low-dose Prexasertib combined with PDL1 in vivo was explored to provide effective data reference for the real application of Prexasertib in clinical treatment.Results: We have observed that the CHK1 inhibitor Prexasertib activates the interferon pathway and immune response and exerts anti-tumour effects through immunomodulation.We found that Prexasertib promoted the accumulation of intracellular ds DNA,activated the c GAS/STING pathway,induced IFN pathway activation and increased the release of CXCL10 and CCL5.We further demonstrated that ds DNA is of mitochondrial origin and that it is mt DNA.In terms of mechanism,we found that Prexasertib disrupts the dynamic equilibrium between the mitochondria and the nucleus,causing NEK1 to dissociate from VDAC1,and VDAC1 dephosphorylation leads to its conformational change to form oligomers increasing the permeability of the outer mitochondrial membrane leading to mt DNA leakage.Using multicolour flow assays on tumour cells from CT26 transplanted tumours in mice,we found that Prexasertib increased tumour killing T cell recruitment and enhanced lymphocyte infiltration into the tumour microenvironment;meanwhile,a low dose CHK1 inhibitor combined with PD-L1 showed significant anti-tumour effects in the CT26 transplanted tumour model.Conclusions: Our findings reveal a link between the nucleus and mitochondria following DNA damage,providing new insights into the biological mechanisms by which Prexasertib counteracts tumour immune responses,and this new understanding should help in developing strategies to predict prognosis.Furthermore,it emphasizes the importance of combining CHK1 inhibitors and immunotherapy,offering theoretical guidance for the precise use of Prexasertib in clinical practice.