Co-administration Of JQ1 And Anti-CD47 Antibody Or Combretastatin A4 Nanoparticles In The Therapy Of Lymphoma And The Associated Mechanism

Background and objective:Non-Hodgkin's lymphoma has always been noted as one of the top 10 in the ranking of the incidence and mortality of all malignancies,seriously threatening public health.Aggressive B-cell lymphoma is the major part of NHL,which is also the focus of the research.Although the introduction of novel agents such as rituximab,bortezomib and lenalidomide has significantly improved the prognosis of patients,chemotherapy still plays an indispensable role.The side effects of chemotherapeutic drugs and the ultimate relapse or progression of lymphoma have always been perplexing the clinicians and patients.The exploration of novel agents and the modification of traditional chemotherapeutic drugs are two important ways to solve this problem,which have become the focus of lymphoma study.In this study,we explored the effect of bromodomain and extra-terminal domain inhibitor JQ1 on lymphoma cells and the function of macrophages.We also loaded combretastatin A4(CA4)with poly(Lglutamic acid)-g-methoxy poly(ethylene glycol).Finally,we explored the effect,safety and associated mechanism of JQ1 and anti-CD47 antibody combination therapy on human Burkitt lymphoma and the effect of JQ1 and CA4 nanoparticles on murine Bcell lymphoma,thus providing new choices for the therapy of lymphoma.Methods:1.Cell experiments: We explored the effect of JQ1 on the viability of Raji,Pfeiffer,BJAB and A20 cells using cck-8 assays.We explored the effect of JQ1 on the apoptosis of Raji,Pfeiffer and A20 cells using Annexin-FITC/PI double staining.Flow cytometry was used to explore the effect of JQ1 on the cell cycle distribution of Raji,Pfeiffer and A20 cells.The effect of JQ1 on the expression level of CD47 and PD-L1 on the surface of lymphoma cells was also detected.RT-PCR was used to explore the effect of JQ1 on the transcription level of C-MYC,CD47 and PD-L1 genes in Raji and A20 cells.2.To explore the effect of JQ1 on the polarization of macrophages,macrophages were induced to M2 by subsequent induction of PMA,IL-4 or IL-13,followed by treatment of JQ1.RT-PCR was used to explore the change on the transcription of M1-and M2-associated genes.Flow cytometry was used to explore the effect of JQ1 on the expression of M1-and M2-associated surface biomarkers.Enzyme linked immunosorbent assay(Elisa)was used to detect the secretion of M1-and M2-associated functional cytokines.3.The synthesis and characterization of CA4-NPs: Poly(L-glutamic acid)-gmethoxy poly(ethylene glycol)was used to load combretastatin A4.The drug loading content,hydrodynamic diameter and release data were detected.4.Animal experiments: We explored the effect,safety and associated mechanism of JQ1 and anti-CD47 antibody combination therapy on human Burkitt lymphoma and the effect of JQ1 and CA4 nanoparticles combination therapy on murine B-cell lymphoma.Results:1.The results of CCK-8 assay,apoptosis assay and cell cycle test: JQ1 could dosedependently inhibit the proliferation of Raji,Pfeiffer,BJAB and A20 cells.JQ1 could induce the apoptosis of Raji,Pfeiffer and A20 cells.JQ1 could block Raji and Pfeiffer cells in the G0/G1 phase,which was not seen in A20 cells.2.The expression level of C-MYC,CD47 and PD-L1 genes: In the Raji,Pfeiffer and A20 cells,JQ1 could inhibit the expression of CD47 on the cell surface.JQ1 could inhibit the expression of PD-L1 on the surface of A20 cells.JQ1 could inhibit the transcription of C-MYC,CD47 genes in the Raji and A20 cells,as well as PD-L1 genes in the A20 cells.3.The effect of JQ1 on the polarization of macrophages: In human macrophages,JQ1 could increase the transcription level of M1-associated genes,CCL3 and TNF-?,while JQ1 reduced the transcription level of M2-associated genes,CCL17 and CCL22.JQ1 could also induce the secretion of M1-associated functional cytokine TNF-? and reduce secretion of M2-associated functional cytokine IL-10 in human macrophages.In murine macrophages,JQ1 could increase the transcription level of M1-associated genes,NOS2 and CXCL10,and reduce the transcription level of M2-associated genes,Arg-1 and Mrc-1.JQ1 could also induce the secretion of M1-associated functional cytokine TNF-? and reduce the expression of CD206 on the surface of murine macrophages.4.The effect of JQ1 and anti-CD47 antibody on the phagocytic index of human macrophages: Compared with PBS,both JQ1 and anti-CD47 antibody could increase the phagocytic index of human macrophages while anti-CD47 antibody was more effective.The combination of these two drugs could further increase the phagocytic index.5.The effect of JQ1 and anti-CD47 antibody on the viability of Raji and BJAB cells: No matter anti-CD47 antibody monotherapy or in combination with JQ1,the viability of Raji and BJAB cells decreased with the increase of anti-CD47 antibody concentration.JQ1 further inhibited the proliferation of lymphoma cells.6.The characterization of CA4-NPs: The loading content and hydrodynamic diameter of CA4-NPs was 11.5% and 83.2 nm,respectively.The release of CA4 was easier in p H7.4 PBS compared with that in pH5.5 PBS.7.Animal experiments: In human Burkitt lymphoma model constructed on NOD/SCID mice,both JQ1 and anti-CD47 antibody could repress the growth of lymphoma while the effect of JQ1 was more obvious.The combination of JQ1 and antiCD47 antibody could further slow the growth but never inhibited it,with no obvious side effect.In murine B-cell lymphoma model constructed on BALB/C mice,both JQ1 and CA4-NPs could repress the growth of lymphoma while no difference between the two groups.The combination of JQ1 and CA4-NPs could further slow the growth and the tumor almost disappeared,with no obvious side effect.Through the immunohistochemistry of A20 tissue,JQ1 and CA4-NPs were found to reduce the expression of Ki-67 and increase the expression of caspase-3,while the combination therapy could further enhance the effect.JQ1 could reduce the expression of C-MYC while CA4-NPs could increase the expression of HIF-1?.JQ1 could reduce the expression of CD47 and PD-L1,while CA4-NPs could reduce the expression of CD47 but increase the expression of PD-L1.The combination therapy ultimately reduced the expression of CD47 and PD-L1.Conclusions:1.JQ1 could repress the growth of lymphoma and the combination therapy with anti-CD47 antibody or CA4-NPs could further stronger the effects,with no obvious side effect.2.The anti-lymphoma effect of JQ1 was associated with following mechanisms:(1)JQ1 could dose-dependently inhibit the proliferation,induce the apoptosis and block the cell cycle of lymphoma cells.(2)JQ1 could reduce the transcription level of C-MYC,CD47 and PD-L1 genes,while reducing the expression of CD47 and PD-L1 on the surface of lymphoma cells.(3)JQ1 could induce the polarization of macrophages from M2 to M1,increase the phagocytic index of macrophages and reduce the viability of lymphoma cells.3.CA4-NPs could increase the hypoxia in lymphoma tissue,leading to antilymphoma effect.