Study On The Role And Molecular Mechanism Of PDGFRB/EGR1/PD-L1 In Melanoma Immunotherapy

Background: Immune checkpoint inhibitors(ICIs)have gained great interest as therapeutic methods that can activate immune cells in the body and improve the ability to kill tumor cells by strengthen patients’ own immunity.In clinical studies,ICIs have good clinical application potential.However,there is still a large application bottleneck of ICIs in tumor treatment.Not all oncology patients benefit from ICIs treatment.Therefore,improving the efficacy of tumor immunotherapy is an important breakthrough point in basic and clinical research,and developing new pathways or combining other drugs to increase the effect of immunotherapy is an important research direction of immune checkpoint therapy.PD-L1 level in tumor is a meaningful biomarker to evaluate the clinical response of anti-PD-1/PD-L1 therapy,and elucidating the regulatory mechanism of tumor PD-L1 and improving the clinical efficacy of ICBs are the main research directions for the transformation of basic research into clinical application.Platelet-derived growth factor receptor(PDGFR)has been proved to promote tumor proliferation and metastasis,and it has become a hot spot in tumor treatment that inhibiting tumor growth through the PDGFR pathway.Early growth response factor 1(EGR1)is an improtant transcription factor with zinc finger structure.However,no studies have been found that EGR1 was involved in regulating the stability of PD-L1 proteins.Objectives: To investigate PDGFR suppression can inhibit tumor and affect the tumor microenvironment;To study the molecular mechanism of PDGFR inhibition and regulation of PD-L1,that PDGFRB/EGR1/PD-L1 signal axis;To verify the clinical significance of PDGFRB/EGR1/PD-L1 signaling axis in the diagnosis of melanoma and immunotherapy;To evaluate the efficacy of PDGFR inhibitors in combination with PD-1 or CD73 monoclonal antibody in the treatment of tumors and to provide molecular targets and new strategies for diagnosis and immune treatment.Methods:Mouse source Melanoma cells B16F10 were used to construct a subcutaneous tumorigenic model in immune competent mice,and investigated anti-tumor effect of PDGFR inhibitor CP-673451.The regulatory effect of PDGFR inhibitors on tumor microenvironment and PDL1 expression in tumor cells was explored by flow cytometry.Western blotting,flow cytometry and q RT-PCR were used to detect the effect of CP-673451 on PD-L1 expression in tumor cells;Melanoma PDGFRA and PDGFRB knock-out cell lines were constructed and the regulatory effect of PDGFRB on the expression of PD-L1 in tumor cells was investigated through Western blotting and q RT-PCR.Through T cell co-culture in vitro,the effect of PDGFRB on T cell tumor killing was clarified.A mouse subcutaneous tumorigenesis model was constructed by PDGFRB knockdown cell line,and the effect of PDGFRB on tumor suppression and immunomodulation was verified by flow cytometry and immunofluorescence technology.Bioinformation analysis and q RT-PCR were used to screen the candidate transcription factors which can mediate the expression of PD-L1.Promoter analysis combined with Ch IP was used to confirm the mechanism of PDGFRB regulating PD-L1 protein level and transcription factor binding sites in the PD-L1 promoter region were validated by diluciferase reporter.Mouse melanoma cell B16F10 was used to construct an immunocompetent mouse subcutaneous tumorigenic model to detect the therapeutic effect of CP-673451 and CD73 monoclonal antibody.Results: The study found that PDGFR expression was low in the effective group of patients treated with PD-1,while high in the ineffective group.In immune competent mouse models,PDGFR inhibitor CP-673451 is significantly to inhibit melanoma growth,increase the number and activity of CD8+ T cells in vivo,and reduce tumor PD-L1 expression.CP-673451 is concentration-dependent down-regulation of tumor cell PD-L1 expression at both transcriptional and protein levels.Further exploration found that PDGFR subtype PDGFRB down-regulated the expression of PDL1 in tumor cells.At the same time,transplantation of melanoma cell lines that knocked down PDGFRB in immunosound mouse models showed that tumor growth was significantly slowed down and the number and activity of CD8+ T cells in vivo were enhanced.In vitro functional experiments further confirmed that PDGFRB can enhance the killing ability of T cells to tumor cells.Through cell sequencing and bioinformation analysis,the transcription factor EGR1,which mediates PDGFRB’s regulation of PD-L1,was screened out.Through a series of experiments,such as q RT-PCR,Ch IP experiment,and diluciferase reporter,EGR1 was found to regulate PD-L1 protein expression by transcriptional binding to the PD-L1 promoter region.CP-673451 in combination with CD73 monoclonal antibody can enhance the antitumor effect.Conclusions: 1.PDGFR inhibitor CP-673451 activate anti-tumor immunity and inhibit tumor growth.2.Inhibition of PDGFR can reduce the expression of PD-L1 in melanoma cells.3.The PDGFR subtype PDGFRB regulates tumor PD-L1 through EGR1 transcription and activates CD8+ T cells to inhibit tumor growth.4.Patients who do not respond to clinical PD-1 monoclonal antibody therapy PDGFR expression is high and the expression of PDGFRB/EGR1/PD-L1 signaling axis can be used as a predictor of immunotherapy efficacy.5.PDGFR inhibitor CP-673451 in combination with CD73 monoclonal antibody can enhance the antitumor effect.