Study Of An Anti-Tumor Mechanism Of Spleen Tyrosine Kinase Inhibitor Fostamatinib In Melanoma

BackgroundMelanoma arising from melanocytes,is one of the most malignant and aggressive of skin carcinoma,which causes 80% of deaths from all skin cancer.The incidence of melanoma has risen sharply in the past few decades,leading to heavy economic and health burdens on a global scale.Depending on the stages of the tumor,different treatment options are available.In the early stage of disease,if melanoma can be diagnosed in time,the early localized melanoma can obtain good prognosis through surgery,and the 10-year survival rate can reach 93%.For metastatic melanoma patients,alternative treatment options include conventional chemotherapy(dacarbazine and temozolomide),targeting therapies(BRAF,MEK,and c-KIT),systemic immunotherapies(anti-CTLA4 and anti-PD1)and/or radiation therapy.The mechanism of immune checkpoint inhibition is complex,involving three stages: antigen presentation,antigen recognition,and immune killing effect.Neoantigen is caused by genetic mutations and epigenetic changes in somatic cells,that can be identified by the immune system.After a complex process,the neo-antigen is presented on the surface of an antigen-presenting cell(APC)as the form of peptideMHC complex,and the combined with T-cell receptor(TCR),which on the surface of T-cell.T cell complete activation also requires the participation of co-stimulators signals,to promote T-cell proliferation.On the other hand,a series of co-repressors signals,which delivered by immune checkpoint receptors,are activated to maintain the balance of the immune system and avoid excessive immune damage.The PD-L1 is one of the co-repressors ligand mainly on the surface of tumor-cells,and by interacting with programmed cell death protein-1(PD-1)on T-cell,inhibit the immune function of T cells.At present,the widely used immune checkpoint inhibitors are cytotoxic T-lymphocyte-associated protein-4(CTLA-4)and programmed cell death protein-1(PD-1).The immune checkpoint inhibitor PD-1 has made a unparalleled success as regards the treatment of melanoma and lung cancer,but only a subset of patients can respond effectively.There is no effective drug control disease progression once the patient appears to be resistant to PD-1 or relapsed.Therefore,we need to search for new drugs to be used alone or in combination with PD-1 to solve the drug resistance,alleviate the disease progression,improve the quality of surviving and lengthen patient’s life cycle.ObjectiveThe main purpose of this study was to find a new drug to reduce the expression of PD-L1 in human and mouse melanoma.And then explore the role and mechanism of drug fostamatinib(R788),which can reduce the expression of PD-L1 in melanoma,in the JAK-STAT pathway.Furthermore to determine the antitumor effect of fostamatinib(R788),as the form of signal or combined with PD-1 in vivo.Methods1.In vitro,flow cytometry was used to explore the new drugs,which can reduce the expression of PD-L1 in human and mouse melanoma.Through this experiment,we tested over 115 drugs and found that spleen tyrosine kinase(Syk)inhibitor fostamatinib(R788)can effectively reduce the expression of PD-L1 in melanoma cells.2.MTT assay,clone formation assay and transwell assay were performed to measure the effect of spleen tyrosine kinase(Syk)inhibitor fostamatinib(R788)on the proliferation and migration of melanoma cells.3.In vitro,Quantiative Realtime PCR(Q-PCR),Western Blot(WB)were used to detect the effect of spleen tyrosine kinase(Syk)inhibitor fostamatinib(R788)on the JAK-STAT pathway in melanoma cells.4.In vitro,T cell killing co-culture experiment and the flow cytometry were used to detect the spleen tyrosine kinase(Syk)inhibitor fostamatinib(R788)on the cytotoxic T cells killing effect among different treatment groups.5.In vivo,flow cytometry were used to detect the proportion of various immune cells in tumor tissues among different treatment groups.Results1.we found that expression of PD-L1 is significantly elevated in melanoma cells,after add spleen tyrosine kinase(Syk)inhibitor fostamatinib(R788)in melanoma cells to culture for 24 hours.2.Functional studies demonstrated that spleen tyrosine kinase(Syk)inhibitor fostamatinib(R788)inhibit proliferation and migration of melanoma cells.3.The spleen tyrosine kinase(Syk)inhibitor fostamatinib(R788)reduce the expression of PD-L1 in melanoma may through JAK-STAT pathway.4.In vitro,the spleen tyrosine kinase(Syk)inhibitor fostamatinib(R788)promote the killing effect of cytotoxic T cell after dealed with INF-γ.In vivo,spleen tyrosine kinase(Syk)inhibitor fostamatinib(R788)and anti-PD-1 antibody could inhitit the growth of mouse tumor.ConclusionThe spleen tyrosine kinase(Syk)inhibitor fostamatinib(R788)can reduce the expression of PD-L1 through JAK-STAT signaling in melanoma cells.That indicates that the spleen tyrosine kinase(Syk)inhibitor fostamatinib(R788)may be explored to treatment melanoma.