PARP Inhibitor Accumulates B7-H3 On Fibroblasts Via Blocking Autophagic Flux To Potentiate Stroma-associated Immune Evasion In Ovarian Cancer

Objective:Poly(ADP-ribose)polymerase(PARP)inhibitors have become standard treatment for maintenance in ovarian cancer,bringing light for patients exhibiting homologous recombination deficiency or patients with complete or partial response to platinum-based chemotherapy.Recently,emerging researches revealed that PARP inhibitors could promote the infiltration of CD8~+T cells and exerted anti-tumor efficiency besides killing tumor cells via canonical synthetic lethality,rendering PARP inhibitors to be promising combination agents with immune checkpoint blockade therapies.Nevertheless,the reponse rates reported are lower than expected in ovarian cancer treated with Niraparib and Pembrolizumab,indicating more complex immune evasion pathways in ovarian cancer microenvironment exposed to PARP inhibitors.This study is aimed to explore the mechanism of immune escape of ovarian tumors under exposure to PARP inhibitors and attempt to develop new combination strategies for treating ovarian cancer.Methods:Immunofluorescence was used to detect expression changes of immune checkpoint ligands in ovarian cancer tissue derived from patients before and after receiving Niraparib monotherapy in a prospective clinical trial(NANT:NCT04507841).Flow cytometry and western blotting were used to determine changes of immune checkpoint ligands in cell lines before and after stimulation of PARP inhibitors.Target gene wild type and knockout mice as well as mouse embryonic fibroblasts were used to construct mouse ovairan tumor models and the role of immune checkpoint ligands on fibroblasts in the tumor immune microenvironment were explored in vivo.Target gene wild type and knockout mouse embryonic fibroblasts were used to construct co-culture system with T cells and the effect of immune checkpoint ligands expressed in fibroblasts on T cells were explored in vitro.RNA-sequencing data analysis and quantitive real-time PCR were used to explore the mechanism of PARP inhibitors induced changes of immune checkpoint ligands on fibroblasts.LC3 and SQSTM1/p62 were detected using immunofluorescence and western blotting to monitor the autophagy in fibroblasts.The anti-tumor activity of PARP inhibitors combined with immune checkpoint blockade was evaluated in syngenic mouse ovarian tumor models.Results:It was found that B7-H3 significantly increased in ovarian cancer tissue after exposure to Niraparib and B7-H3 expression colocalized with cancer associated fibroblasts(CAFs).Moreover,B7-H3 on CAFs induced by PARP inhibitors weakened T cell functions characterized by compromised expression of IFN-γand Granzyme B both in vivo and in vitro.PARP inhibitors blocked the autophagic flux in CAFs and impaired the degradation of B7-H3 via autophagy,leading to accumulation of B7-H3 on CAFs.The autophagic flux blockade induced by PARP inhibitors in CAFs was mediated by the downregulation of PIP4K2A,a critical gene involved in the fusion of autophagosome and lysosome.Finally,PARP inhibitors combined with B7-H3 antibodies achieved synergistic anti-tumor efficacy in syngeneic mouse ovarian cancer models.Conclusion:1.PARP inhibitors induced the accumulation of immune checkpoint ligand B7-H3 on CAFs and mediated the immune escape of ovarian cancer cells;2.PARP inhibitors blocked the autophagic flux in CAFs through downregulating the critical gene involved in the fusion of autophagosome and lysosome PIP4K2A,resulting in impaired degradation of B7-H3 via autophagy;3.PARP inhibitors in combination with anti-B7-H3antibodies achieved synergistic anti-tumor efficacy.Thus,this study innovatively reveals a tumor immune evasion pathway induced by PARP inhibitors and mediated by B7-H3 on CAFs,and provides promising preclinical evidence for the combination of PARP inhibitors and anti-B7-H3 antibodies to treat ovarian cancer.