Disrupting Myddosome Assembly In Diffuse Large B-cell Lymphoma Cells Using The MYD88 Dimerization Inhibitor ST2825

Background: Diffuse large B-cell lymphoma(DLBCL),the most common type of non-Hodgkin lymphoma(NHL),is classified into germinal center B cell(GCB)and activated B cell(ABC)subtype.ABC subtype has a poor prognosis,and the continuous activation of NF-? B signaling pathway plays an important role in the survival of ABC-DLBCL.Toll-like receptor pathway is an important upstream pathway that mediates NF-? B activation,and MYD88 molecule is the most critical adaptor protein in this signaling pathway.Normally,MYD88 is recruited to activated receptors after external stimuli,thereby heterodimerizing with the receptor through TIR-TIR interactions,and homodimerizing with another MYD88 molecule at the same time,these MYD88 oligomers can further recruit IRAK and promote the formation of Myddosome(this complex includes MYD88,IRAK4,IRAK2 and IRAK1).After subsequent phosphorylation and ubiquitination,Myddosome triggers the activation of downstream NF-?B and JAK-STAT3 signaling pathways.MYD88 L265 P mutation is the most prevalent oncogenic mutation in the patients with ABC DLBCL,which indicates inferior outcomes.Contrast to natural MYD88,MYD88 oligomers containing L265 P can trigger Mydsomesome assembly and constitutive activation of NF-? B signals without external stimulation,and play an important role in the survival of ABC DLBCL cells.Therefore,MYD88 can serve as a therapeutic target in DLBCL.The synthetic peptidomimetic compound ST2825 can inhibit the dimerization of MYD88,and may disrupt the assembly of Myddosome in L265 P DLBCL cells,thereby inhibiting tumor cell growth.In this project,DLBCL lymphoma cell lines(OCI-LY10 and TMD8)harboring L265 P mutation were selected as the research model,aiming to detect whether the growth of tumor cells can be inhibited by disrupting the assembly of Myddosome,and trying to illuminate its potential mechanism.Methods: The impact of ST2825 on MYD88 oligomerization was measured in situ in cells by confocal microscopy and HMW fraction experiments.The L265 P harboring ABC DLBCL cell lines were treated with ST2825,cell viability was evaluated by WST-1 reagent,apoptosis was detected by DAPI staining and flow cytometry,and NF-?B activity was detected by WB experiment and NF-?B reporter assays.The association between BTK and MYD88 was studied by coimmunoprecipitation experiment.Drug combination of ST2825 and other inhibitors was evaluated through synergism analysis using Calcu Syn software.Results: To determine whether oligomerization of the MYD88TIR-L265 P mutants could be blocked by ST2825,MYD88 TIR WT or mutant linked to the fluorescent protein mCitrine were expressed in HEK293 T cells.The mCitrine-TIR mutants were strongly aggregated,whilst this was not the case for the WT mCitrine TIR.Furthermore,the aggregation of mCitrine-TIR mutants was markedly inhibited following treatment with ST2825.In addition,MYD88 aggregation was detected in the HMW fraction in DLBCL cell lines with the L265 P mutation(OCI-LY10 and TMD8).The data illustrates a significantly lower pellet:lysate ratio in the ST2825-treated cells compared with the DMSO treated cells,thus indicating that the degree of MYD88 aggregation was significantly decreased following ST2825 treatment.According to these data,it may be that augmented MYD88 oligomerization is blocked by ST2825 in ABC DLBCL cells,resulting in the disruption of Myddosome assembly.The cell viability of OCI-LY10 cells treated with ST2825 for 72 h was decreased in a concentration-dependent manner,with ?50% inhibition at 5?M ST2825,?60% at 10 ?M and ?70% at 20 ?M(P<0.01).Similar results were observed in TMD8 cells(P<0.01).However,the growth inhibition effects of ST2825 on SU-DHL-4 cells(WT MYD88)were only ?20-25%at 10 ?M or 20 ?M after 72 h of treatment,though these results were still significant.Flow cytometry revealed that the percentage of apoptotic cells was significantly increased in OCI-LY10 and TMD8 cells 48 h after treatment with ST2825(P<0.01),while no significant difference was observed in SU-DHL-4 cells.Consistent with these results,the characteristic apoptotic morphological changes(condensed and fragmented nuclear)were observed by DAPI staining in OCI-LY10 and TMD8 cells treated with ST2825,while there were no obvious morphological changes in SU-DHL-4 cells.These data shows that disruption of Myddosome assembly inhibits cell survival and promotes apoptosis in ABC DLBCL cells with the L265 P mutation.After treatment with ST2825,the ratio of phosphorylation/total I?B was reduced,and the transcription of the NF-? B-dependent luciferase reporter decreased in OCI-LY10 and TMD8 cells.Using ELISA,we detected that the secretion of IL-10 and IFN-? in the cell line was significantly reduced after ST2825 treatment(P<0.01).Further immunoprecipitation experiments showed that MYD88 binds to BTK,and the binding can be reduced by ST2825.The combination of ST2825 and the BTK inhibitor ibrutinib promoted OCI-LY10 and TMD8 cell death.Analysis of the CI indicated that ST2825 and ibrutinib were synergistic at most doses,though greater synergistic effects were observed at higher doses of ST2825(? 1.3 ?M)for both OCI-LY10 and TMD8 cells.The enhanced cytotoxicity was associated with stronger inhibition of NF-? B activity in tumor cells treated by dual inhibition,compared with that of each drug alone.In addition,combination treatment with ST2825 and the BCL-2 inhibitor ABT-199 enhanced OCI-LY10 and TMD8 cell death.Analysis of the CI indicated that ST2825 and ABT-199 were synergistic at higher doses of ST2825(?1.3 ?M)and ABT-199(?0.5 ?M)in OCI-LY10 cells.Similar results were revealed in TMD8 cells.This combination treatment also consistently promoted apoptosis,as indicated by the increased number of apoptotic cells detected by flow cytometry,compared with treatment with the individual drugs alone.Conclusions: ST2825 can disrupt the assembly of Myddosome driven by L265 P,thereby mediating the inhibition of proliferation and increased apoptosis of L265 P DLBCL cells.Potential mechanisms may include inhibition of NF-?B activity,reduction of IL10 and IFN-? production,and destruction of the binding between MYD88 and BTK.It was subsequently revealed that combined ST2825,either with the BTK inhibitor ibrutinib,or the BCL-2 inhibitor ABT-199,resulted in synergistic cell death in ABC DLBCL cells.The synergistic mechanism may be related to a stronger inhibition of NF-? B activity or a stronger pro-apoptotic effect.These findings suggest that targeting Myddosome assembly may be a promising therapeutic strategy for MYD88-mutated ABC DLBCL.Patients such as L265 P DLBCL and other B-cell tumors driven by activated MYD88 signals may benefit from it.