The Role And Mechanism Of BET Inhibitor Combined With BCG In Melanoma

Background:Cutaneous melanoma is the most malignant skin tumor with a high mortality rate,and the incidence of melanoma has been increasing in recent years.With the emergence of targeted therapy and immunotherapy,the survival rate of patients has been improved,but there are still some patients who cannot benefit from these therapies.Therefore,the treatment of melanoma is still a major clinical research trend,and targeting new therapeutic targets or combination therapeutic strategies is urgently needed.The bromodomain and extraterminal domain(BET)family belongs to the bromodomain protein family,which is overexpressed in a variety of tumors.NHWD-870 is a next-generation BET inhibitor,and is currently undergoing clinical phase I study.For the treatment of melanoma,NHWD-870 has better anti-tumor activity and drug tolerability compared to the conventional BET inhibitor JQ-1.We demonstrated that NHWD-870 also has important modulatory effects on the tumor immune microenvironment(TIME),suggesting that NHWD-870 in combination with some immunomodulators may significantly improve the anti-tumor effect.Mycobacteriμm bovis Bacillus Calmette Guerin(BCG),a live attenuated vaccine,has been used as an immune adjuvant for immunotherapy of bladder cancer and melanoma.We aimed to investigate the effect of this strategy on melanoma development through the combination of NHWD-870 and BCG,and elucidate the mechanism involved,thus providing a new combination treatment strategy for the clinical treatment of advanced melanoma.Objective:(1)To explore the therapeutic effect of the combined strategy of NHWD-870 and BCG on melanoma;(2)To clarify the molecular mechanism of NHWD-870 combined with BCG on melanoma;(3)To provide a theoretical basis for the clinical treatment of skin melanoma.Methods:(1)In C57BL/6 and NSG(or Balb/c nude)mice,subcutaneous tumor-bearing models of YUMM1.7 and B16F10 were constructed to study the effect of NHWD-870 or BCG intervention on the occurrence and development of melanoma;(2)Flow cytometry,immunohistochemistry/immunofluorescence were used to detect the proportion of immune cells in tumor,spleen,blood and lymph node of tumor-bearing mice,and the changes of proliferation,activation or depletion marker;(3)The changes of immune cells in tumor tissues of tumor-bearing mice were analyzed by single-cell sequencing;(4)CD4~+and CD8~+T cells were separated by flow cytometry from tumor tissues of tumor-bearing mice,and the change of Mt1 and chemokine receptor transcription levels were detected by real-time quantitative PCR;(5)Jurkat cells were treated with NHWD-870 to analyze the change of Mt1 expression level;(6)Ch IP-seq analysis of the interaction between BRD4 and MT1DNA;(7)The subcutaneous tumor bearing model of C57BL/6 mice was established to study whether tumor recurrence and memory immune response existed after melanoma suppression under different intervention measures.Results:(1)In the subcutaneous melanoma model of C57BL/6 mice,compared with the control group,the tumor in the BCG group did not change significantly,the tumor in the NHWD-870 group was reduced,and the tumor in the combination of the two drugs was reduced more significantly,and there was a statistical difference between the groups.In NSG mice and Balb/c nude mice,compared with the control group,BCG group did not significantly change the tumor,the tumor was shrinkaged in the NHWD-870 group and combined treatment group,but there was no significant difference between the two groups.(2)By flow cytometry analysis of the changes of immune cells in melanoma tissues,we found that compared with the control group,the proportion of Treg or exhausted CD8~+T cells(FOXP3~+CD4~+T cells,PD-1~+CD8~+T cells)in BCG group was decreased,while effector T cells did not change significantly(GZMB~+CD8~+T cells).In NHWD-870 group,the proportion of Treg or exhausted CD8~+T cells decreased and the proportion of effector T cells increased.In the combined treatment group,the proportion of Treg or exhausted CD8~+T cells decreased,and the proportion of effector T cells increased,and the range of change was more obvious than that of NHWD-870 group.(3)CD4~+T and CD8~+T cell proliferation correlation analysis in single cell sequencing showed that in the control group,NHWD-870group and combined group,the proportion of cells in G1 phase gradually decreased,while the proportion of cells in G2M phase gradually increased.Compared with the control group,proliferation related genes(Mki67,Top2a,Prr11)were up-regulated in NHWD-870 group and the combined treatment group,and the up-regulation was more obvious in the combined treatment group.CD4~+T and CD8~+T cell function analysis showed that compared with the control group,the expression of effect-related genes(Ifng,Gzmb)was up-regulated in the NHWD-870group and the combined treatment group,and the expression of inhibitory genes(Foxp3,Pdcd1)was down-regulated,and the change of gene expression in the combined treatment group was more obvious.Meanwhile,KEGG and GO analysis showed that immune-related signaling pathways(chemokine signaling pathway,cytokine receptor signaling pathway,TNF signaling pathway and IL-17 signaling pathway)were significantly activated after combined treatment compared with the control group.(4)Differential gene results showed that NHWD-870 down-regulated Mt1 expression on CD4~+T and CD8~+T cells;In addition,compared with the NHWD-870 group,the interaction between T cells and tumor cells was significantly increased in the combined group.(5)The expression of Mt1 in CD4~+T and CD8~+T cells was down-regulated after NHWD-870 treatment by flow cytometry and real-time quantitative PCR.(6)Jurkat cells were treated with NHWD-870(6n M)in vitro,and real-time quantitative PCR results showed that NHWD-870 could down-regulate Mt1 expression in Jurkat cells.(7)Ch IP-seq results showed that BRD4 could directly bind to Mt1promoter region,and the binding of BRD4 to Mt1 promoter region was weakened after NHWD-870 treatment or si BRD4 interference.(8)By establishing a melanoma rechallenge model in C57BL/6J mice,the results showed that there was no new tumor formation in the combined treatment group after the second tumor growth.Conclusion:(1)The combination of NHWD-870 and BCG can synergistically promote the proliferation of CD4~+and CD8~+T cells,and enhance the effector function of CTL,and reduce the proportion of Treg and depletion of CD8~+T cells,thus inhibiting the occurrence and development of melanoma.(2)BRD4 can directly bind to the MT1 promoter region of T cells.NHWD-870 inhibited dysfunctional T cells by inhibiting BRD4,thereby down-regulating MT1.BCG also increased the cross-talk between T cells and tumor cells.(3)NHWD-870 combined with BCG can enhance the memory immune response in tumor and reduce the probability of tumor recurrence;Figures 16;Tables 14;References 61...