2004;279:20858C20865. JNJ-26854165, used either alone or in combination with TKIs, represents a promising novel targeted approach to overcome TKI resistance and improve patient outcome in CML. studies have shown that induction of p53 through MDM2 inhibition by the small-molecules such as Nutlins and MI219 effectively induces p53-mediated apoptosis in most blast crisis CML cells, with or without mutations including T315I variant [18, 19]. JNJ-26854165 (JNJ-165) is usually a novel small molecule that was initially thought to act as an antagonist to MDM2. [20, 21]. In a phase I trial performed in patients with refractory solid tumors, JNJ-165 displayed a modest anticancer activity and enabled p53 activation . However, recent pre-clinical studies have exhibited antiproliferative activity in various p53 wt and mutant cancer models [20, 23, 24], implying p53-impartial activities. Thus, these two properties provide an advantage to prevent the selection of p53 mutant subclone in cancer during treatment of JNJ-165. The aims of this study were to evaluate the efficacy of JNJ-165 in CML cells with or without p53 mutation and as a single agent and in combination with TKI and to confirm the mechanism of action of this potentially important drug in CML cells. RESULTS Antiproliferative and apoptotic effects of JNJ-165 in models of Imatinib-sensitive and-resistant CML We first examined the antiproliferation effect of JNJ-165 on primary cells from 24 newly diagnosed patients with CML, 9 patients with CML-AP/BC, and 13 cases with CML-CP treated with Imatinib or dasatinib, in whom expression of BCR/ABL mRNA determined by real time RT-PCR was very low or undetectable. The characteristics of the 46 CML patients analyzed in this study are detailed in Supplementary Table S1. CML primary cells were exposed to 2 M JNJ-165 for 72 hours, the viability of cells from the CML-CP patients with BCR/ABL positive and CML AP/BP patients was reduced by 32.9% and 23.4%, respectively, compared with cells from the patients with very low or undetectable BCR/ABL (Determine ?(Figure1A).1A). We next evaluate the cytotoxicity EG00229 of JNJ-165 to normal hematopoietic progenitor cells EG00229 by colony formation assays. The results presented in Supplementary Physique S1 revealed that the number of hematopoietic colonies were not affected by JNJ-165. To investigate the effect of JNJ-165 Igf2r on growth of CML cell lines, K562 and K562/G, an Imatinib-resistant cell line were incubated for 72 hours with escalating concentrations of JNJ-165. Cell viability of both cell types was inhibited with IC50 values of 1 1.54 and 1.67 M, respectively (Determine ?(Physique1B),1B), suggesting comparable sensitivity of these two cell lines to JNJ-165. Next, we treated a pair of murine 32D leukemic EG00229 cell lines stably expressing wt or T315I mutant BCR/ABL (32D-BCR/ABL and 32D- BCR/ABL- T315I) with JNJ-165 and observed their growth remarkably inhibited, with IC50 values of 0.46 and 0.5 M, respectively (Determine ?(Figure1B).1B). These data indicate that JNJ-165 is usually a potential agent to kill Imatinib-sensitive and resistant CML cells including cells with the T315I mutation. EG00229 Open in a separate window Physique 1 JNJ-165 inhibits proliferation and induces death in CML cell lines and primary cells (Imatinib-sensitive and -resistant) via caspase-independent pathway(A) The primary cells were obtained from CML patients, and were cultured with or without 2 M JNJ-165 for 72 h, the viability of cells was assessed by an MTT assay. (B) CML cell lines K562, K562/G, 32D-BCR/ABL, and 32D-BCR/ABL-T315I, were cultured with or without different concentrations of JNJ-165 for 72 h. Growth inhibition by JNJ-165 was assessed by an MTT assay. Data were represented mean SD of three impartial experiments. (C) CML Cell lines K562 and K562/G were harvested at 48 h after treatment with 2 M JNJ-165. Cells were stained by an annexin V/PI-staining method and analyzed by flow cytometry. (D) Cell lines K562 and 32D-BCR/ABL-T315I were incubated for 6 h with 20 M z-VAD-fmk, then exposed to 1 M JNJ-165 for 72 h, the viability of cells was assessed by an MTT assay. Results EG00229 are representative of three impartial experiments and expressed as the mean SD. To further clarify whether the antiproliferative activity of JNJ-165 was related to induction of apoptosis, Annexin V-FITC and PI double staining was performed. Treatment of K562 and K562/G cells with 2 M JNJ-165 for 48 hours resulted in 15.6% and 22.9% apoptotic (annexin V+ and PI+) cells, respectively (Determine ?(Physique1C).1C). This is consistent with a previous report showing that JNJ-165 induced delayed apoptosis in p53 mutant cells including K562 cells . However, JNJ-165 cytotoxicity against K562 and 32D-BCR/ABL-T315I cells was not significantly reduced by z-VAD-fmk pretreatment (Physique ?(Physique1D),1D), suggesting that cell death was caspase-independent. JNJ-165-induced cell death is p53-impartial Since all.