Identification Of C96as A Novel Inhibitor Of Phosphatidylinositol3-kinase That Displays Potent Preclinical Activity In Multiple Myeloma

Background and Purpose: The PI3K/AKT signaling pathway is constitutiveactivated and plays a critical regulatory role in multiple myeloma pathophysiology,including survival, proliferation, migration, angiogenesis in multiple myeloma, amalignancy of plasma cells. Therefore, PI3K has emerged as a key therapeutic targetin multiple myeloma. In the present study, C96was identified as a novel PI3Kinhibitor by a virtual screen combined with a series of experimental studies. Thepurpose of this study is to determine whether C96can induce MM cell death and tofind out the underlying mechanisms.Methods:(1) MTT assays were used to detect whether C96inhibited proliferation of MMcells.(2) Flow cytometry was used to measure the effects of C96on cell apoptosis inMM cells.(3) Western blotting was performed to examine the expression of AKT,Immunofluorescence assay was used to determine the cell expression and cellularlocalization of p-AKT in OPM2cells, and enzymatic reaction assays was performedto measure the effects of C96on PI3K.(4) Western blotting was performed to examine the expression of protein thatinvolved in cell apoptosis and PI3K/AKT signaling pathway.(5) A myeloma xenograft experiment was used to evaluate the effect of C96onthe growth of multiple myeloma in vivo. Results:(1) C96inhibited proliferation of multiple myeloma cells in a concentrationdependent-manner in seven MM cell lines.(2) C96induced multiple myeloma cells apoptosis.(3) C96inhibited the activation of AKT, not only the constitutive AKT, but alsoIGF-1--triggered AKT signals. C96also precluded AKT phosphorylation in the innerside of the plasma membrane as evidenced by an immunofluorescence analysis, andthe enzymatic reaction assays showed that C96inhibited PI3Ks.(4) The results with Western blotting showed that C96cleaved Caspase3andPARP, down-regulated the expression of anti-apoptotic Bcl-2and up-regulatedapoptotic p53, C96inhibited the PI3K/AKT signaling pathway, it could inhibitedphosphorylation of mTOR, P70S6K, and4E-BP1.(5) C96delayed tumor growth in an MM xenograft model. The results showedthat C96markedly decreased tumor growth without significant lose of body weightwithin the16-day treatment. Further studies showed that C96inhibited thephosphorylation level of both AKT and mTOR from the tumor tissues excised frommice received C96.Conclusion:C96was demonstrated to be a novel PI3K inhibitor, which suppresses thePI3K/AKT signaling and displays potent anti-myeloma activity in both in vitro and invivo. C96could be developed as a drug candidate for the treatment of multiplemyeloma.