Ponatinib elicits caspase 3 dependent cleavage of Mcl 1 Because of the critical

While this manuscript was under review, Lierman et al. reported that ponatinib was active against imatinib resistant FIP1L1 PDGFR ARQ 197 msds mutants. Our results corroborated and extended their findings by pro viding a mechanism for the induction of apoptosis and evidence for in vivo efficacy. Conclusions In conclusion, ponatinib is a potent inhibitor against both WT and T674I FIP1L1 PDGFR. Caspase 3 dependent Mcl 1 cleavage may be a positive feedback mechanism to enhance apoptosis in ponatinib treated cells. Inhibition of PDGFR activity by ponatinib leads to decreased tyrosine phosphorylation of B catenin, decreased protein stability and protein level of B catenin, decreased transcription of TCFLEF regulated genes, and enhanced cytotoxicity. Therefore, regulation of B catenin by PDGFR plays a role in the antineoplastic mechanism of ponatinib.<br><br> Given the FDA approval of oral ponatinib in patients with refractory CML and Ph ALL resistant AZD0530 価格 to the first and second generation of TKIs, our findings warrant a clinical trial of ponatinib in imatinib resistant CEL and other ma lignant disorders harboring T674I PDGFR. Materials and methods Reagents Ponatinib was synthesized in our lab. Imatinib and sorafenib were purchased from Alexis Biochemicals. 4. 6 diamidino 2 phenylindole was from Invitrogen. Cyclohexi mide and propidium iodide were from Sigma Aldrich. TOPflashFOPflash system consisting of optimized TCF binding sites or mutated sites controlling the expression of a luciferase reporter gene was from Upstate Biotechnology. pCMV5 flag human Mcl 1 and pcDNA3 B catenin were kindly provided by Dr.<br><br> Mien Chie Hung. ON TARGETplus SMARTpool small interfering RNA duplexes against human Mcl 1 or PDGFR, and Non Targeting Pool siRNA control were from Dhar macon RNA Tech. Cell culture and cell growth measurement The EOL 1 cell line harboring the FIP1L1 PDGFR fusion oncogene was purchased from DMSZ. BaF3 cells expressing WT or T674I FIP1L1 PDGFR were cultured as described AMN-107 bcr-Abl 阻害剤 previously. Cell viability was assessed by MTS assay. Clonogenicity assay was performed as described. In brief, 2 105ml cells were treated with drugs or dilu ent for 24 h, then washed with PBS and seeded in methylcellulose medium. After incubation for 7 days at 37 C and 5% CO2, colonies with 50 cells were counted. Preparation of whole cell lysates and cytosolic fraction Most experiments of immunoblotting involved whole ly sates prepared with RIPA buffer unless otherwise stated.<br><br> To measure the levels of AIF and cytochrome c in the cytosol, the cytosolic extract was prepared with digitonin extraction buffer. Preparation of cytoplasmic and nuclear fractions Distribution of B catenin was determined in the cyto plasmic and nuclear fractions as we previously de scribed. Immunoblotting Immunoblotting involved use of whole cell lysates pre pared in RIPA buffer. Antibodies and their sources were as followsantibodies against apoptosis inducing factor, Mcl 1, Bax and Bcl XL. antibodies against poly poly merase, X linked inhibitor of apoptosis, caspase 3, active caspase 3, cytochrome c, survivin, and C terminal B catenin. phospho B catenin. antibodies against phospho PDGFR, phospho Erk12 and Erk1 2.