The Efficacy And Mechanistic Study Of A Novel Phosphoramide Compound,DCZ0847,in Inhibiting The Proliferation Of Diffuse Large B Cell Lymphoma

Background:Cellular DNA damage response（DDR）has long been studied and proven to play a complex role in cancer development and prevention.Diffuse large B-cell lymphoma（DLBCL）is a tumor with aberrant DDR activation,thus prompting the development of novel agents that target DDR factors,as a promising approach for DLBCL therapy.In cooperation with Chinese Academy of Sciences Shanghai Institute of Materia Medica,our group has focused on exploring DDR inhibitors and found that DCZ0847,a novel phosphoramide compound,induces DNA damage in multiple myeloma;however,the effectiveness of DCZ0847 against DLBCL remains unknown.Objective:In this study,we aimed to explore the efficacy and safety of DCZ0847 in killing DLBCL in vivo and in vitro,and to reveal the underlying mechanism of DCZ0847-mediated DDR,prompting novel and promising therapies for DLBCL patients.Methods:The anti-lymphoma effect of DCZ0847 was first analyzed by using the CCK-8 assay and the Ed U assay,which indicates the cell viability and proliferation of DLBCL cells.Furtherly,Annexin V-FITC/PI staining and TUNEL fluorescence staining were used to evaluate the effect of DCZ0847 on apoptosis of DLBCL cells.Western blot was performed to determine the effects of DCZ0847 on the apoptosis-related proteins such as Caspase proteins,Bax and Bcl-2.In addition,Z-VAD-FMK,a broad-spectrum caspase inhibitor,was used to investigate the association of DCZ0847-induced apoptosis with caspase activation.The effect of DCZ0847 on cell cycle was measured by flow cytometry with PI staining,and cell cycle-related proteins was determined by Western blot.RNA-seq analysis was performed on DCZ0847 and vehicle-treated OCI-LY8 cells to explore the mechanisms responsible for DCZ0847-induced cell proliferation suppression.Moreover,the formation of comet tail andγ-H2A.X foci in DLBCL cells,which indicate the DNA damage,were observed by neutral comet assay and immunofluorescence assay.The co-localization ofγ-H2A.X and RAD51 foci and homologous recombination（HR）/non-homologous end joining（NHEJ）reporter assay were conducted to assess the functional role of DCZ0847 in DNA repair.Dual-luciferase reporter assay was performed to discover the potential transcription factors modulated by DCZ0847.Further q RT-PCR and Western blot analysis were used to validate the expression level of significantly differentially expressed genes detected by RNA-seq.Additionally,xenograft mouse model of DLBCL was generated to evaluate the in vivo anti-lymphoma efficacy of DCZ0847 alone or in combination with doxorubicin.The daily weight change in mice and H&E staining of liver and kidney from euthanized mice were used to assess the safety of DCZ0847.The tumor volume was measured daily to assess the efficacy of DCZ0847 and the synergistic effect with doxorubicin.TUNEL staining and immunohistochemical staining of Ki-67 andγ-H2A.X was conducted with tumor tissues to detect the potential mechanism of the in vivo anti-lymphoma efficacy of DCZ0847.What’s more,the expression of DDR-related proteins in cells treated with DCZ0847 and/or doxorubicin were examined by Western blot to verify whether DCZ0847 plays a tumor suppressor role in DLBCL via the DDR pathway.Results:DCZ0847 exhibited a generalized proliferation suppression among GCB-and ABC-DLBCL cell lines in a time-and concentration-dependent manner.Results from RNA-seq analysis identified apoptosis,cell cycle,NF-κB signaling,and DNA-repair pathways were the most influenced pathways by DCZ0847administration.Further flow cytometry analysis combination with Western blot analysis confirmed that DCZ0847 induced a Caspase-dependent cell apoptosis and blocking the cell cycle in the G₀/G₁ phase.Mechanistic studies revealed a dual role of DCZ0847 in activating DNA damage by upregulating levels of phosphorylated ATM and simultaneously inhibiting HR repair via blockage of the recruitment of RAD51 to double-strand break sites.Further results of dual luciferase reporter assay suggested that the dual role of DCZ0847 in the DDR was dependent on the regulation of transcription factor NF-κB2,which modulated the expression of ATM and RAD51.Besides,there was no difference in average body weight between DCZ0847 treated mice and the vehicle treated mice.No significant pathological changes were observed in major organs（liver and kidney）of DCZ0847 treated nude mice,suggesting that it was safe.The calculated combination index values were less than 1 in DLBCL cells treated with DCZ0847 and doxorubicin;The tumor volume in DCZ0847 and doxorubicin combination treated mice were smaller than the tumor volume in DCZ0847 or doxorubicin alone treated mice,indicating that DCZ0847 and doxorubicin had synergistic effects both in vivo and in vitro.Western blot results revealed the mechanism of a synergistic effect between DCZ0847 and doxorubicin was due to the dual role of DCZ0847 in DDR.Conclusion:DCZ0847 exhibits potent inhibitory activity in DLBCL both in vivo and in vitro.Furthermore,the results demonstrated that DCZ0847 played dual roles in the DDR（i.e.,promotion of DNA double strand breaks and suppression of DNA repair）.This characteristic of DCZ0847 allows appropriate synergy with doxorubicin for the treatment of DLBCL.DCZ0847 displayed potent anti-lymphoma activity as a monotherapy and in combination with doxorubicin in the treatment of DLBCL.Collectively,these findings provide a new promising therapeutic strategy for pharmacologic inhibition of DDR through DCZ0847 or in combination with other DNA-damaging drugs for subsets of DLBCL with activated DDR pathways.