Flt3 Y591 duplication and Bcl-2 overexpression are detected in acute myeloid leukemia cells with high levels of phosphorylated wild-type p53

Abstract

Loss or mutation of the TP53 tumor suppressor gene is not commonly observed in acute myeloid leukemia (AML), suggesting that there is an alternate route for cell transformation. We investigated the hypothesis that previously observed Bcl-2 family member overexpression suppresses wild-type p53 activity in AML. We demonstrate that wild-type p53 protein is expressed in primary leukemic blasts from patients with de novo AML using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and phospho-specific flow cytometry. We found that p53 was heterogeneously expressed and phosphorylated in AML patient samples and could accumulate following DNA damage. Overexpression of antiapoptosis protein Bcl-2 in AML cells was directly correlated with p53 expression and phosphorylation on serine residues 15, 46, and 392. Within those patients with the highest levels of Bcl-2 expression, we identified a mutation in FLT3 that duplicated phosphorylation site Y591. The presence of this mutation correlated with greater than normal IntroductionWe have previously reported that signaling profiles of acute myeloid leukemia (AML) cells identify patients with a poor response to course one of chemotherapy. 1 In that study we showed that mutation of fms-like tyrosine kinase 3 (Flt3) was associated with increased activity of signal transduction and activator of transcription (STAT) family members Stat5 and Stat3, but we did not explore how this signaling might contribute to the observed therapy resistance. Here we examine the connection between a target of altered Stat5 signaling in AML-the Bcl-2 protein-and the suppression of normal apoptotic responses to DNA damage in leukemia cells. Evasion of apoptosis contributes to the formation and continued survival of cancer cells 2 and insight into signaling mechanisms that suppress apoptosis in AML cells could be used to improve the efficacy of existing therapies. 3 AML is particularly relevant to the study of suppressed apoptotic pathways in cancer because p53, a central driver of apoptosis and guardian of genomic integrity, is not generally lost or mutated in this disease. 4,5 p53 is a sequence-specific transcription factor that can halt progression through the cell cycle or initiate apoptosis. 6 Furthermore, p53 is a key tumor suppressor protein often lost or mutated in human cancers; however, TP53 has been reported to be mutant in only 7% of AML cases. 4 Subsequent research has also shown that downstream p53 effector genes, such as p21, are rarely lost in AML and that wild-type p53 protein is expressed in newly established AML derived cell lines. 7 For this study, we measured several properties of p53 in primary AML cells, including TP53 gene sequence, per-cell abundance of p53 protein, and p53 phosphorylation at 5 residues. 8 Following DNA damage and cell stresses, p53 protein is rapidly phosphorylated at several residues, 9 including those we measured here (serines 15, 20, 37, 46, and 392). Phosphorylation of p53 is thought to regulate p53 localization, conformation, and activity, 10 but t...

Correlation analysis of p53 protein isoforms with NPM1/FLT3 mutations and therapy response in acute myeloid leukemia

“…Flt3-mediated activation of downstream signaling pathways may thus stabilize the p53 full-length protein, and the p53 biosignature therefore reveal a high expression of the p53 full-length protein as observed (Figure 4). This also supports our observation that specific FLT3-ITD corresponds to p53 hyperphosphorylation (Irish et al, 2007). On the basis of these previous results, it appears that FLT3-ITD-driven p53 hyperphosphorylation is accompanied by a downstream block of p53 signaling through anti-apoptotic mechanisms (Irish et al, 2007).…”

“…Genetic changes in AML frequently alter signal transduction either directly or indirectly (Irish et al, 2004), subsequently reflected in posttranslational modifications of protein stress sensors and tumor suppressors like p53 (Irish et al, 2007). Given the complexity of p53 regulation including isoform expression and posttranslational modifications, p53 protein is hypothesized to integrate multiple sources of information about intracellular signaling with relevance for chemoresistance (Fridman and Lowe, 2003;Irish et al, 2007).…”

“…Several signaling pathways converge on p53 (Kojima et al, 2007), and we recently reported p53 hyperphosphorylation in sub-populations of AML patients indicating that p53 function may be affected in AML although patients do not display p53 mutations (Irish et al, 2007). We have previously detected mRNA from p53 isoforms b and g in AML patient cells and the corresponding protein appeared detectable by twodimensional immunoblot .…”

“…Consequently, we asked whether regulation of wild-type p53 protein isoforms in AML could be coupled to patient outcome. We visualized individual AML patient p53 biosignatures through protein isoform separation and detection by two-dimensional gel electrophoresis and immunoblot (2DI) Irish et al, 2007). mRNA of D133 p53b and D133 p53g isoforms were not detected in AML patients examined, while all patients tested expressed mRNA of p53 full-length, p53b and p53g isoforms.…”

Overexpression of Bcl2 protein predicts chemoresistance in acute myeloid leukemia: Its correlation with FLT3

“…The Bcl2 expression in AML has been studied by flow cytometry [5,[14][15][16][17][18][19], western blotting [20] and immnunocytochemistry [13,21,22] by various groups reporting heterogenous expression in the range of 34 to 87%. With clinical and hematological pa- Log Rank=1.00, df=1, P=0.31 *P value ≤ 0.05 is significant; DFS, Disease free survival; OS, Overall survival rameters, no significant correlation could be obtained.…”