Study On The Effects And Molecular Mechanisms Underlying The Induced Differertiation Therapy Of Acute Myeloid Leukemia With A Natural Product Vibsanin A Combined With Tyrosine Kinase Targeted Drugs

Background & Objective Acute myeloid leukemia(AML)is one of the major hematologic neoplasms,and is characterized by defective differentiation and aberrant proliferation and survival of clonal precursor myeloid cells.In the majority of de novo AML patients who achieve a complete remission(CR),leukemia will recur,which is associated with poor outcomes.All-trans retinoic acid(ATRA)is an AML differentiation agent that has introduced a paradigm for success of cell differentiation therapy for acute promyelocytic leukemia(APL),a unique subtype of AML.Unfortunately,ATRA has not been found to be clinically useful for the other AML subtypes,its application was limited to retinoic acid syndrome and retinoic acid resistance.Thus,it is imperative to develop novel differentiation therapies to attempt to improve the prognosis of AML patients.Emerging evidence has implicated aberrant activation of protein tyrosine kinases(PTK)plays an important role in pathogenesis of AML.Many preclinical and clinical studies have provided evidence that tyrosine kinase inhibitor(TKI)s exert mild differentiation-inducing effect of AML cell lines due to their ‘‘off-target’’ effects,and enhancing differentiation induced by ATRA.Vibsanin A,isolated from Viburnum plants,is a natural vibsane-type diterpene,which constitutes more than 80 compounds that possess piscicidal,plant growth regulatory,cytotoxic,and anti-inflammatory activities.We recently reported that vibsanin A is a novel differentiation-inducing agent with potent antileukemic activity.Moreover,we determined that vibsanin A is a protein kinase C(PKC)activator,and that it induces AML cell differentiation through PKC activation.It was also showed that PTK-targeted anticancer drugs could enhance the AML cell differentiation induced by vibsanin A.Based on these results,this study will be designed to screen the optimizing combination,which includes PTK-targeted anticancer drugs and vibsanin A,for synergistic induction of AML cell differentiation.It will be thoroughly investigated that PKC and PTK with their mediating signaling pathways.The molecular targets for the synergistic induction of AML cell differentiation will further be explored by using the advanced proteomic methods.Total these findings will reveal the molecular mechanisms underlying the synergistic induction of AML cell differentiation by PTK-targeted anticancer drugs combined with vibsanin A.Novel therapeutic schedule(s)and drug target(s)will eventually be provided for the differentiation therapy of AML.Methods1.To identify TKIs that may induce or favor the differentiation of AML cells,several FDA-approved and clinical TKIs used at nontoxic concentrations were evaluated for their ability to trigger the differentiation of AML cells when used in combination with vibsanin A.2.The synergistic effect of vibsanin A and TKIs in combination to induce AML cell differentiation was confirmed by cell morphology,analysis of cell differentiation and cell cycle distribution3.A classic PKC activator,PMA,and nonspecific inhibitors of PKC were used to determine the functional role of PKC in the differentiation response to vibsanin A combined with TKIs.4.Western blot analysis,RT-PCR and immunoprecipitations were used to investigate the potential contribution of SFKs to the differentiation effects of the combination of vibsanin A and TKIs.5.Gene silencing or ectopic gene expression was used to signal pathway study and molecular mechanism study.Results1.Vibsanin A sensitizes AML cells to TKI-mediated myeloid differentiation.Several FDA-approved and clinical TKIs used at nontoxic concentrations were evaluated for their ability to trigger the differentiation of HL-60 cells by the detection of the myeloid differentiation marker,CD11 b.Results showed that vibsanin A potentiated the differentiation-inducing action of various TKIs,among which imatinib and saracatinib showed the most significant synergistic activity.2.Vibsanin A enhances TKI-induced growth inhibition and cell cycle arrest.In addition,vibsanin A augmented the ability of TKIs to induce growth inhibition and G1 cell cycle arrest of AML cells,the cell cycle arrest associated with combination-induced differentiation was shown to involve a marked increase in p21 and p27 levels and decrease of CDK4 and CDK6 with resultant dephosphorylation of Rb.3.Activation of PKC is involved in cell differentiation induced by the combination of vibsanin A and TKIs.A similar capacity of PMA to enhance cell differentiation induced by other TKIs was also observed,and the pharmacological inhibition of PKC led to partial reduction in the differentiation induced by the combination treatment or the TKIs alone as measured using both morphological assessment and CD11 b induction.4.Vibsanin A enhances TKI-induced Lyn expression that contributes to AML cell differentiation.Using a pan-SFK antibody that recognizes active site phosphorylation(Y416)in all family members,we found that treatment of HL-60 cells with vibsanin A induced an apparent increase in SFK phosphorylation,which was blocked by co-treatment with TKIs,but TKIs increased total Lyn protein expression,this increase was further enhanced by co-treatment with vibsanin A.Evaluation of immunoprecipitated Lyn suggesting that SFK kinase activity is not necessary for differentiation Lyn activity.Lyn-knockdown significantly diminished the differentiation induced by vibsanin A combined with imatinib,saracatinib or other effective TKIs.Both pharmacological inhibition of PKC and targeting Lyn interference almost completely blocked the differentiation of AML cells.5.Raf/MEK/ERK cascade act as downstream effectors of PKC and Lyn during the combination-induced differentiation.TKIs induced phosphorylation of ERK1/2,MEK,and Raf-1 at S338 and S289/296/301,and combination treatment with vibsanin A plus either inhibitor further enhanced phosphorylation,Activation of Raf-1 was further demonstrated by the decrease in S259 phosphorylation because dephosphorylation of S259 initiated the activation of Raf-1.Consistently,pharmacological blockade of Raf-1 or ERK1/2activation resulted in significant inhibition of cell differentiation induced by the combination treatment.PKC inhibitor pretreatment clearly suppressed the phosphorylation of Raf-1,MEK,and ERK1/2 induced by vibsanin A combined with imatinib or saracatinib,sh RNA-targeting Lyn resulted in a similar decrease in MAPK activation with the same treatment.6.Downregulation of c-Myc expression mediates the differentiation-inducing effects of the combination of vibsanin A and TKIs.Downregulation of c-Myc protein was detectable within 1 hour of treatment and with corresponding decrease in c-Myc m RNA levels.Furthermore,pretreatment of HL-60 cells with sorafenib or U0126 reversed combination-induced downregulation of c-Myc expression that could also be effectively antagonized when the PKC inhibitor GFX was present or Lyn expression was knocked down.Ectopic c-Myc expression significantly abrogated the differentiation induced by vibsanin A combined with TKIs.Conclusion1.Vibsanin A sensitizes AML cells to tyrosine kinase inhibitor(TKI)-induced differentiation.2.Both PKC activation and Lyn induction contribute to combination-induced AML cell differentiation.3.The combination treatment synergistically induces differentiation by activation of PKC and upregulation of Lyn followed by MAPK-mediated c-Myc suppression.4.Vibsanin A coupled with TKIs may lead to new applications of differentiation-based approaches for AML.