Study On The Mechanism Of IAP Family Inhibitor AZD5582 Promoting Apoptosis Of MDS Cells

Background: Myelodysplastic syndrome(MDS)comprises a series of clonal bone marrow disorders characterized by peripheral blood cytopenia and dysplasia due to ineffective hematopoiesis and a variable risk of developing acute myeloid leukemia(AML).MDS patients in the high-risk group have a poor prognosis and are easy to transform into AML,requiring high-intensity treatment,namely chemotherapy and hematopoietic stem cell transplantation.However,high-intensity therapy has high treatmentrelated complications and mortality,and is not suitable for all high-risk MDS patients.For high-risk MDS patients,there is an urgent need to develop and utilize new drugs to improve this situation.AZD5582,a novel inhibitor of small IAP,effectively binds to the BIR3 regions of cIAP1,cIAP2,and XIAP.Aim: Explore the effect and molecular mechanism of AZD5582 on MDS cells;explore the potential application of AZD5582 in MDS.Method: By using the MDS gene expression profile(GEP)dataset GSE19429,GSE18366,GSE58831 and GSE43399 available on the Internet,and collecting clinical samples for q PCR,we verified the difference in the expression of some IAP family molecules between normal persons and MDS patients.The effect of AZD5582 on MDS cell viability was detected by CCK-8 method.The effect of AZD5582 and decitabine combined treatment on the cell viability of MDS cell lines was detected by CCK-8 method,and the synergistic index(CI)of AZD5582 and decitabine was calculated by Compu Syn.The long-term inhibitory effect of AZD5582 on the proliferation of MDS cell lines was detected by soft AGAR clone formation method.Flow cytometry was used to detect the apoptosis induced by AZD5582 in MDS cell lines,RNA sequencing analysis showed that apoptosis pathway was an important pathway for AZD5582 to inhibit MDS cells.Western blot was used to detect the expression changes of related proteins in AZD5582 treated cells.The tumogenesis experiment of nude mice was conducted to detect the inhibitory effect of AZD5582 on the proliferation of MDS cell lines and tumor formation in vivo,and the expression of related proteins in tumor tissues was detected by immunohistochemistry,and the toxicity and side effects of AZD5582 on mice were investigated by HE staining of mouse liver and spleen tissues.Results: The study found that the IAP family members(BIRC2,BIRC3,XIAP)were higher in MDS patients than in normal subjects,and the high expression of these molecules was associated with poor prognosis.The results showed that AZD5582 reduced the cell viability and inhibited the proliferation of MDS cell lines in vitro by CCK-8 method and soft AGAR.Flow cytometry showed that AZD5582 could induce apoptosis of MDS cell lines.RNA sequencing analysis also confirmed that AZD5582 promoted the apoptosis of MDS cell lines.Meanwhile,RNA sequencing analysis showed that AZD5582 was closely related to lysosomal induced apoptosis pathway in MDS cell lines.Western Blot analysis of proteins in NC group and AZD5582 group showed that AZD5582 down-regulated the protein expression of IAP family molecules(cIAP1,cIAP2).Tumor formation experiment in nude mice showed that AZD5582 significantly inhibited the growth of tumor formation in MDS cell lines in vivo.Immunofluorescence(Tunel)and immunohistochemistry(Ki67,Caspase3)were performed on tumor tissues of NC group and AZD5582 group,which showed that AZD5582 promoted apoptosis and inhibited tumor proliferation.The comparison of HE staining sections of liver and kidney tissues of NC group and AZD5582 group showed that AZD5582 had no obvious toxic and side effects on mice in vivo.Conclusion: IAP family members(BIRC3,BIRC2,XIAP)are highly expressed in MDS and are associated with poor prognosis.The inhibitor AZD5582,a member of the IAP family,inhibited the proliferation of MDS cells in vitro,and the combination with decitabine synergistically inhibited the proliferation of MDS cells.AZD5582 promoted the apoptosis of MDS cells,and RNA sequencing analysis showed that apoptosis pathway was an important pathway for AZD5582 to inhibit MDS cells.AZD5582 downregulates protein expression of IAP family molecules.AZD5582 inhibited MDS cell proliferation and promoted MDS cell apoptosis in nude xenograft tumor model.