Effects And Mechanism Of Novel BTK Inhibitor AC0010 Treatment On Mantle Cell Lymphoma

Part I Effects of novel BTK inhibitor AC0010 treatment on Mantle cell lymphoma.Purpose:To research into the effects of AC0010,a novel BTK inhibitor,in Mantle cell lymphoma(MCL)and compare its efficacy and safety with another BTK inhibitor Ibrutinib.Methods:(1)MTS assay was used to detect the growth inhibition caused by AC0010 and Ibrutinib respectively in MCL cell lines(Jeko-1 and JVM-2),primary MCL cells and normal peripheral cells;(2)Apoptosis and cell cycle arrest of Jeko-1 and JVM-2 after exposure into AC0010 and Ibrutinib was conducted by flow cytometry;(3)MCL xenograft was developed by tail vein injection of JVM-2 to testify the efficacy and safety of AC0010 in vivo.Results(1)Both of AC0010 and Ibrutinib could inhibit the growth of MCL cell lines Jeko-1,JVM-2 in a concentration-and time-dependent manner.However,the inhibition of AC0010 on cell growth was excellent than that of Ibrutinib;(2)Both of AC0010 and Ibrutinib could inhibit the growth of primary MCL cells.However,the inhibition on cell growth of AC0010 was excellent than that of Ibrutinib;(3)Neither AC0010 nor Ibrutinib showed damaging effect on normal peripheral cells;(4)With the increase of the concentration of AC0010,cells showed significant apoptosis,while the same concentration of Ibutinib barely induced apoptosis;(5)AC0010 could block JVM-2 cells at G0/G1 phase,but had no obvious cyclic arrest effect on Jeko-1 cells.Ibrutinib didn't exhibit an effect on blocking cell cycle in Jeko-1 and JVM-2 cells as well.(6)AC0010 at 300mg/kg significantly(p<0.001)prolonged the survival rate in MCL xenograft with no adverse effects,while it couldn't prolong the survival rate at 30mg/kg or 100mg/kg.Conclusion(1)AC0010 is surpassing Ibrutinib in inhibiting MCL cell lines and primary MCL cells proliferation rather than normal cells;(2)The pro-apoptotic effect of AC0010 is dramatically better than that of Ibrutinib;(3)AC0010 blocks the cell cycle in JVM-2 cells;(4)AC0010 can prolong the survival rate of animals in JVM-2 survival model.Part ? Mechanism of novel BTK inhibitor AC0010 treatment on mantle cell lymphomaPurpose:(1)To explore the molecular mechanism of novel BTK inhibitor AC0010 on mantle cell lymphoma;(2)To develop a preclinical study for the future therapy of MCL.Methods:(1)We utilized a q-PCR-based approach to profile the gene expression of BTK after extracting total mRNA from Jeko-1 and JVM-2 exposed to AC0010 for 24h;(2)BCR trigger experiment was conducted to detect whether the impact on phosphorylation of BTK made by AC0010 was irreversible;(3)The expression of BTK and p-BTK proteins were tested by western-blot after Jeko-1 and JVM-2 were treated with AC0010 for 24h;(4)Expression of apoptosis related proteins as well as proteins in BCR-BTK and PI3K/AKT signaling pathways were checked by Western-blot.Results(1)Expression of BTK mRNA showed no change in Jeko-1 and JVM-2cells;(2)The phosphorylation of BTK was inhibited obviously after BCR was stimulated by anti-IgG F(ab')2;(3)There was nearly no downregulation in BTK protein but a distinct downregulation in p-BTK protein;(4)In MCL cell lines,a visibly increase in cleavage of caspase-3 and cleavage of PARP was induced by AC0010,and downregulation of Bcl-2 and bcl-xl as well as upregulation of Bax and Bim were observed.(5)The downstream proteins of BTK like p-PLC?2,p-IKK,p-NF-?B were reduced in Jeko-1 and JVM-2,and there was an obvious downregulation of PI3K,p-AKT,p-GSK3? which related to PI3K/AKT signaling pathway.Conclusion(1)AC0010 can irreversibly inhibit the phosphorylation of BTK,but has no effect on its translation and transcription;(2)AC0010 induces apoptosis in MCL cell lines through the casepase family and Bcl family;(3)AC0010 plays an anti-MCL effect by blocking BCR-BTK and PI3K/AKT signaling pathway.