The Inhibition Of Autophagy Enhances The In Vitro And In Vivo Anti-myeloma Effects Of Ibrutinib

Backgrounds and Aims: Bruton’s tyrosine kinase(BTK)participates in the normal development of B cells and the maintenance of the phenotype of malignant cells.BTK is wildly expressed both in B lymphoid cells and many other non-T hematopoietic cells.Therefore,targeting BTK has a wide range of clinical application value.Ibrutinib is the first generation of BTK inhibitor,which has showed obvious clinical efficacy in several B lymphoid malignancies.Multiple myeloma(MM),a common hematologic malignancy,is still incurable up to now.Further exploration of the resistance mechanisms of myeloma,searching for new targeted agents and more effective combination strategies are in urgent need.Ibrutinib is promising in myeloma therapy.Preclinical and clinical studies have also confirmed the effectiveness of ibrutinib in myeloma,but some other studies suggest that the efficacy is limited.Further exploration of the role of ibrutinib in myeloma and the potential mechanisms of drug resistance may contribute to the application of ibrutinib in myeloma.Autophagy is closely related to MM and always acts as a protective mechanism,but it can also initiate autophagic cell death.Autophagy,which can be affected by chemotherapeutic drug pressure,DNA damage and other intracellular or extracellular factors,is a multi-step process and strictly regulated by multi-molecules.The relationship between anti-myeloma activity of ibrutinib and autophagy has not been reported yet.How ibrutinib regulates autophagy in myeloma cells and the influence of autophagy on the anti-myeloma activity of ibrutinib needs to be further studied.In this study,we focused on the effects of ibrutinib on autophagy in myeloma cells,the potential mechanisms of autophagy regulation and the role of autophagy in the anti-myeloma activity of ibrutinib,aiming to explore new combination strategies for improving the efficacy of ibrutinib in MM.Methods: Part 1: 1.MTT assay was used to detect the effects of ibrutinib on viability of myeloma cell lines RPMI8226 and H929;2.Flow cytometry and western blot were performed to examine the effects of ibrutinib on the cell cycle distribution and apoptosis of MM cells;3.The proteins related to DNA damage of MM cells after treatment of ibrutinib were determined by western blot;4.The effect of ibrutinib on the autophagy of MM cells was detected by western blot,PCR and transmission electron microscope;5.The potential mechanism that how ibrutinib initiated autophagy was also studied.First,the effects of DNA damage response(DDR)were explored by the means of interfering DDR initiated by ibrutinib using KU-55933,and western blot was used to detect the LC3 B protein expression;the expression of histone H4 lysine 16 acetylation(H4K16ac)as well as its regulators SIRT1 and h MOF were detected by western blot and PCR,and chromatin immunoprecipitation(Ch IP)combined with q PCR was used to observe the changes of h4k16 ac recruitment in LC3 B promoter region;Part2: 1.Western blot was used to examine the effect of ibrutinib combined with low-dose autophagy inhibitors including 3-methlyadenine(3-MA),hydroxychloroquine(HCQ)and Bafilomycin A1(BAF)on the expression of LC3 B in myeloma cells,while the corresponding changes of autophagy vesicles in myeloma cells were observed by transmission electron microscope;2.The effects of ibrutinib alone or combined with autophagy inhibitors 3-MA,HCQ and BAF on the viability of RPMI8226 and H929 cells were examined by MTT assay;3.Flowcytometry was used to detect the effects of ibrutinib alone or combined with autophagy inhibitors 3-MA,HCQ and BAF on percentages of S phase of MM cells;4.The effects on colony formation of MM cells of ibrutinib alone or in combination with autophagy inhibitors 3-MA,HCQ and BAF were observed by the soft agar colonyformation assay;5.The effects of ibrutinib alone or in combination with autophagy inhibitors 3-MA,HCQ and BAF on DNA damage of MM cells were examined by western blot;6.Flowcytometry and western blot were used to observe the apoptosis induced by ibrutinib alone or in conjunction with autophagy inhibitors 3-MA,HCQ and BAF;7.To prove the effect of co-treatments with ibrutinib and HCQ on MM cells in vivo,we established the subcutaneous plasmacytoma xenograft model of H929 cells in nude mice.HE staining,immunochemistry and TUNEL staining were used to detect the proliferation and apoptosis of tumor cells;8.The cell viability of primary MM cells treated by ibrutinib alone or combined with HCQ and 3-MA were detected by MTT.Results: Part 1:The potential mechanisms of autophagy activation of myeloma cells induced by ibrutinib 1.Ibrutinib inhibited the proliferation of myeloma cells,induced DNA damage as well as apoptosis of MM cells and led to cell cycle arrest in G0/G1 phase which was regulated by p21 and cyclin D1;2.Ibrutinib induced autophagy activation of MM cells,manifestating as the increase of autophagy molecules ATG5,ATG7 and LC3 B at both protein and m RNA levels,while decrease of p62 protein levels;autophagosomes were increased after ibrutinib treatment detected by transmission electron microscope;3.KU-55933 inhibited the autophagy flux induced by ibrutinib revealed by the downregulation of the LC3 B expression;4.Ibrutinib suppressed the transcription and expression of SIRT1,leading to an increase in global levels of H4K16 ac.Concomitant up-regulation of H4K16 ac in the promoter regions of LC3 B was proved by Ch IP and q PCR,which was suspected to promote the transcription of LC3 B gene;Part 2: Inhibiting autophagy response augmented the anti-myeloma activity of ibrutinib 1.3-MA can partly inhibit the expression of LC3B-Ⅱand formation of autophagic vacuoles induced by ibrutinib;HCQ and BAF can further increase the accumulation of LC3B-Ⅱ and aggregation of autophagic vacuoles in MM cells induced by ibrutinib;2.Autophagy inhibitors 3-MA,HCQ and BAF enhanced the cytotoxic effects on MM cells of ibrutinib;3.Autophagy inhibitors 3-MA,HCQ and BAF further decreased the percentage of S phase of MM cells when combined with ibrutinib compared with the treatment of ibrutinib alone;4.Co-treatments with ibrutinib and autophagy inhibitors 3-MA,HCQ and BAF obviously reduced the colony formation revealed by soft agar colony-formation assay;5.DNA damage induced by ibrutinib was increased when combined with autophagy inhibitors 3-MA,HCQ and BAF;6.The apoptotic rates of MM cells induced by ibrutinib were increased when combined with autophagy inhibitors 3-MA,HCQ and BAF;7.In-vivo experiment further implicated that co-treatments with ibrutinib and HCQ resulted in an obvious reduction in tumor growth relative to untreated mice.In the co-treatment group,Ki67-positive proliferating cells were decreased and cleaved caspase-3 positive cells were increased,correspondingly,TUNEL staining showed increased apoptotic cells;8.The combination of ibrutinib and HCQ or 3-MA further inhibited the cell viability of primary myeloma cells.Conclusions: 1.Ibrutinib inhibited the proliferation,induced DNA damage,G0/G1 cell cycle arrest and apoptosis of MM cells;2.Ibrutinib activated autophagy pathways in MM cells,and the mechanisms of autophagy activation may involve histone H4K16 ac epigenetic regulation;3.The autophagy induced by ibrutinib is cytoprotective,and targeting autophagy increased the in vitro and in vivo anti-myeloma activity of ibrutinib.