| Background:Diffuse large B cell lymphoma(DLBCL)is associated with aggressive clinical cases with poor prognosis despite recent advances in the treatment of the disease.In activated Bcell-like(ABC)-DLBCL,the most severely damaged signaling pathways converge to aberrantly activate the Toll-like receptor(TLR)7/9/MYD88 pathways,leading to the avoidance of cell death and resistance to chemotherapy.A gain of function mutation in MYD88(MYD88 L265P)enhanced the NF-κB and JAK-STAT signaling pathways,which was associated with dysregulation of TLR signaling in the pathogenesis of ABC-DLBCL.Therefore,the inhibition of the TLR signaling network may be pivotal to improve clinical outcomes.In this study,we designed a de novo synthesized oligodeoxynucleotide-based antagonist of TLR7 and TLR9,referred to as HJ901,which competitively binds to TLR7/9.We profiled HJ901 inhibition in various DLBCL cell lines and verified tumor suppression in a xenograft mouse model.We found that HJ901 treatment significantly reduced TLR7-and TLR9-mediated cell proliferation and cytokine production in a time-and dose-dependent manner in various DLBCL cell lines expressing the MYD88 L265 P mutation.Moreover,HJ901 inhibited tumor growth and downregulated the NF-κB and JAK2-STAT3 signaling pathways in a DLBCL xenograft mouse model with the MYD88 L265 P mutation.These results indicate that the anti-tumor effects of the synthesized oligodeoxynucleotide-based antagonist,HJ901,which competitively binds to TLR7/9,may be associated with the downregulation of the NF-κB and JAK2-STAT3 signaling pathways,which may provide rationale for treating ABC-DLBCL patients with the MYD88 L265 P mutation.Objective:To analyze the effect of HJ901 on MYD88 L265 P mutation and MYD88 wild-type in the cell lines of the ABC-and GCB-DLBCL subtypes in vivo and in vitro,and to further clarify the mechanism of HJ901 inhibiting the proliferation of MYD88 L265 P mutated ABCDLBCL cells.Methods:1.The inhibitory effect of HJ901 on NF-κB activity in the HEK-Blue-h TLR7/TLR9 cells by IMQ or Cp G 685 was determined by the SEAP assay.2.The inhibitory effect of HJ901 on the proliferation and cytokines secretion from h PBMCs was selected by CFSE assay and CBA.3.The inhibitory effect of HJ901 on the proliferation and cytokines secretion from DLBCL cell lines harboring MYD88 L265 P mutation or MYD88 wild-type was detected by WST1 and CBA assay.4.The effect of HJ901 on survival time and tumor suppression was observed in a xenograft mouse model with TMD8 cells harboring MYD88 L265 P mutation or U2932 cells with wild-type MYD88.5.The effect of HJ901 on the tumor histomorphology and expression of Ki-67 was evaluated in the tumor tissues of xenograft mouse model with TMD8 or U2932 cells by histopathological and immune-histochemical analysis.6.The effect of HJ901 on the cell cycle and apoptosis was detected in DLBCL cell lines harboring MYD88 L265 P mutation or MYD88 wild-type by flow cytometer.7.To further elucidate the underlying mechanism of HJ901,the effect of HJ901 on NF-κB and JAK2-STAT3 signal pathway in DLBCL cell lines harboring MYD88 L265 P mutation or MYD88 wild-type was detected by Western blot in vitro and in vivio.Results:1.HJ901 selectively inhibited the SEAP activation of HEK-Blue-h TLR7 or HEK-Blueh TLR9 cells in a time-and dose-dependent manner.2.HJ901 specifically suppressed TLR7-and TLR9-mediated cell proliferation and reduced cytokine production in PBMCs in a time-and dose-dependent manner.3.HJ901 selectively inhibited the cell proliferation and cytokines secretion in OCILy3.3,OCI-Ly10,and TMD8 cells harboring MYD88 L265 P mutation,rather than in U2932,SUDHL-2,and OCI-Ly19 cells with MYD88 wild-type.4.HJ901 treatment decreased tumor growth in a DLBCL xenograft mouse model.We also observed that HJ901(10 or 25 mg/kg)treatment suppressed lymphoma growth in a mouse DLBCL xenogeneic tumor model with TMD8 cells harboring the MYD88 L265 P mutation,rather than U2932 cells with wild-type MYD88.5.Survival analysis showed that mice in all groups did not die under natural circumstances.However,if the tumor reached 2000 mm3 at the time of death,25 mg/kg HJ901 has a delayed effect on the survival time of tumor in TMD8 mice groups,rather than in U2932 mice groups.6.The histopathological and immune-histochemical analysis revealed that HJ901 treatment increased apoptotic and necrotic regions,and decreased the expression of Ki-67 in the tumor tissues of mice with TMD8 cells compared to the control group.However,HJ901 treatment had no effect on tumor tissue in mice injected with U2932 cells.7.HJ901 treatment significantly increased the number of cells in G1 and reduced those in G2/M phase in OCI-Ly3.3,OCI-Ly10,and TMD8 cells but not in SUDHL-2,U2932,and OCI-Ly19 cells compared to the control treatment groups.We observed no change in S phase in any of the cell lines tested.8.HJ901 treatment inhibited NF-κB and JAK2-STAT3 pathway activation in vitro.Our results revealed induction of NF-κB(P65),IκBα,JAK2,and STAT3 phosphorylation in OCI-Ly3.3 and TMD8 cells expressing the My D88 L265 P mutation.9.HJ901 treatment inhibited NF-κB and JAK2-STAT3 pathway activation in vivo.Parallel studies were performed in a mouse xenograft model with TMD8 or U2932 ABCDLBCL cell lines.Western blot analysis revealed that HJ901 treatment inhibited the induction of NF-κB(P65),IκBα,JAK2,and STAT3 phosphorylation in the mouse xenograft model with TMD8.However,these effects of HJ901 treatments were not observed in DBLCL cells expressing wild-type My D88 in vitro and in vivo.Conclusions:1.HJ901,which is an antagonist of TLR7 and TLR9,not only specifically and effectively inhibited TLR7-and TLR9-mediated cell proliferation,but also inhibited the secretion of IL-6 and IL-10 in vitro.2.Moreover,HJ901 treatment significantly reduced tumor growth in a DLBCL xenograft mouse model with the MYD88 L265 P mutation.Our results further indicate that these mechanisms are involved in the downregulation of NF-κB and JAK2-STAT3 signaling pathways in vivo and in vitro.3.Accordingly,the suppressive ODN HJ901 may target the TLR7/9-mediated signaling pathway,which may be used as a novel strategy for treating patients with ABC-DLBCL expressing the MYD88 L265 P mutation.Innovation and significance:We designed a de novo synthesized oligodeoxynucleotide-based antagonist of TLR7 and TLR9,referred to as HJ901,which competitively binds to TLR7/9.HJ901 is diversed with the previously IMO reported,which with the number of repeats of CCT and degree of phosphorothioated unmethylated Cp G ODN.We profiled HJ901 inhibition in various DLBCL cell lines harboring MYD88 L265 P mutation or MYD88 wild-type and verified tumor suppression in a xenograft mouse model using various molecular immunology technologies.This study has broad potential for clinical application using inhibitory oligodeoxynucleotides,and the anti-tumor effect of HJ901 is thought to be an alternative and promising drug for DLBCL protocol therapy. |
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