Purpose: The antioncogene protein product p53 has not been studied previously in cancer patients during in vivo chemotherapy.This study examined the early p53 protein and gene expression during induction chemotherapy in acute myeloid leukemia (AML). Experimental Design: Leukemic cells were collected from five AML patients during their first 18 hours of induction chemotherapy and examined for p53 protein and gene expression by one-and two-dimensional gel immunoblot and high-density gene expression arrays. Results: Up-regulation of p53 protein expression was detected in AML patients posttreatment in vivo. One-and two-dimensional gel immunoblots showed two main forms of p53, denominated ap53 and Dp53, both recognized by various NH 2 -terminal directed antibodies. As a response to treatment, we detected rapid accumulation of ap53, with significantly altered protein expression levels already after 2 hours.The accumulation of ap53 was accompanied by increased transcription of putative p53 target genes and subsequent cytopenia in the patients. Conclusion: Up-regulation of the p53 protein and target genes seems to be a prominent feature in induction chemotherapy of AML.The rapid shift from a shorter p53 protein form (D) toward the full-length protein (a) underscores the complexity of p53 protein modulation in patients undergoing chemotherapy.

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...

Several new therapeutic strategies are now considered for acute myelogenous leukaemia (AML), including modulation of protein lysine acetylation through inhibition of histone deacetylases (HDACs): a large group of enzymes that alters the acetylation and, thereby, the function of a wide range of nuclear and cytoplasmic proteins. Firstly, HDACs can deacetylate histones as well as transcription factors, and can modulate gene expression through both these mechanisms. Secondly, acetylation is an important post-translational modulation of several proteins involved in the regulation of cell proliferation, differentiation and apoptosis (e.g., p53, tubulin, heat-shock protein 90). The only HDAC inhibitors that have been investigated in clinical studies of AML are butyrate derivatives, valproic acid and depsipeptide. In the first studies, the drugs have usually been used as continuous therapy for several weeks or months, and in most studies the drugs were used alone or in combination with all-trans retinoic acid for treatment of patients with relapsed or primary resistant AML. Neurological toxicity and gastrointestinal side effects seem to be common for all three drugs. Complete haematological remission lasting for several months has been reported for a few patients (< 5% of included patients), whereas increased peripheral blood platelet counts seem more common and have been described both for patients with AML and myelodysplastic syndromes. Taken together, these studies suggest that HDAC inhibition can mediate antileukaemic effects in AML, but for most patients the clinical benefit seems limited and further studies of combination therapy are required.

The wild-type tumor-suppressor gene TP53 encodes several isoforms of the p53 protein. However, while the role of p53 in controlling normal cell cycle progression and tumor suppression is well established, the clinical significance of p53 isoform expression is unknown. A novel bioinformatic analysis of p53 isoform expression in 68 patients with acute myeloid leukemia revealed distinct p53 protein biosignatures correlating with clinical outcome. Furthermore, we show that mutated FLT3, a prognostic marker for short survival in AML, is associated with expression of full-length p53. In contrast, mutated NPM1, a prognostic marker for long-term survival, correlated with p53 isoforms b and c expression. In conclusion, p53 biosignatures contain useful information for cancer evaluation and prognostication.

Our studies demonstrate that primary human AML cells show aberrant cytoplasmic expression of cyclin B1 for a majority of patients and a specific humoral immune response was also detected for a subset of patients with untreated leukemia.