Abstract 3021: FLT3/ITD acute myeloid leukemia (AML) cells show C-MYC- and PIM-1-mediated CDC25A overexpression despite the presence of intrinsic DNA damage

Abstract

Abstract Activating mutations of FLT3 occur in 30% of patients with AML, and are associated with poor prognosis. It has been reported that FLT3/ITD AML cells are sensitive to MDM2 inhibition-induced apoptosis (Blood 2010;116:71) and indeed, FLT3 inhibition synergizes with MDM2 inhibition in the induction of apoptosis in FLT3/ITD AML (Leukemia 2010;24:33). Considering the rarity of TP53 mutations in FLT3/ITD AML, use of MDM2 inhibitors in AML with mutant FLT3 may offer clinical benefits. We investigated the FLT3/ITD-specific p53 signaling profile that may be associated with high susceptibility of FLT3/ITD cells to MDM2 inhibition. [Methods] The MDM2 inhibitor Nutlin-3a was used. Murine pro-B cells stably transfected with expression vectors that encoded wild-type human FLT3 (Ba/F3-FLT3 cells) or human FLT3 containing an ITD mutation (Ba/F3-ITD cells) were provided by Dr Donald Small and AML samples were obtained from patients after informed consent. Apoptosis was quantified by Annexin binding assay and cells were transfected by the Amaxa Nucleofector I. Statistical analysis was performed using t test or Mann-Whitney U test. [Results] Ba/F3-ITD cells were more susceptible to Nutlin-induced apoptosis but not to AraC or doxorubicin than Ba/F3-FLT3 cells; high Nutlin-3a sensitivity was associated with efficient BAX activation (P < .001). Basal levels of total and phospho p53 were 7 times higher in Ba/F3-ITD than in Ba/F3-FLT3 cells; levels of γH2AX, a DNA damage marker, were 6 times higher in Ba/F3-ITD than in Ba/F3-FLT3 cells, and reactive oxygen species levels were also high in Ba/F3-ITD cells (26.2 ± 3.2% vs 6.1 ± 0.1%; P < .001). Although steady state p53 activation was not sufficient to inhibit cell growth or induce cell death in Ba/F3-ITD cells, Nutlin-3a potently activated p53 signaling and induced apoptosis. Interestingly, CDC25A, which is specifically degraded in response to DNA damage, was 30 times higher in Ba/F3-ITD cells compared to Ba/F3-FLT3 cells, despite the presence of DNA damage. Among patient AML samples (n=37), CDC25A levels were significantly higher in samples with mutant FLT3 (n=16) than those with wild-type FLT3 (n=21) (1.7 ± 1.2 vs 17.1 ± 6.7; P < .001). Knockdown experiments showed that CDC25A over-expression was partially induced by C-MYC and PIM-1. Nutlin-3a reduced CDC25A mRNA levels by 63.9 ± 0.4 % (P < 0.01). The proteasome inhibitor MG132 or the pan-caspase inhibitor Z-VAD-FMK did not rescue the CDC25A reduction. [Conclusion] CDC25A over-expression was closely associated with FLT3/ITD in AML. FLT3/ITD cells showed C-MYC- and PIM-1-mediated CDC25A over-expression despite the presence of intrinsic DNA damage. CDC25A over-expression has been reported in a variety of human cancers, and CDC25A has been considered a therapeutic target. CDC25A down-regulation may be potential mechanism by which MDM2 inhibition induces cell death in FLT3/ITD AML cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3021. doi:1538-7445.AM2012-3021