The P53 Pathway Is Inactive in Acute Myeloid Leukemia

Abstract 5122

The pathway controlled by the p53 tumor-suppressor protein is altered in most, if not all, human cancers and the TP53 gene is mutated in half of all human tumors. Such mutations are rare in human hematological malignancies, leading to the assumption that the p53 pathway is inactivated by alternative mechanisms. However, to date, the state of activity of the p53 pathway in hematological malignancies is not well understood. We investigated the functional status of the p53 pathway in Acute Myeloid Leukemia (AML) patients, particularly in patients with cytogenetically normal AML (CN-AML) and patients with Acute Promyelocytic Leukemia (APL).

We performed bioinformatic analysis of p53 pathway-related gene expression. For this purpose, we first assembled a list that, to the best of our knowledge, is the most comprehensive list to date of genes related to the p53 pathway. The list consists of 1153 p53 pathway-related genes: 916 p53-related genes and 582 partially overlapping genes related to important components of the p53 pathway (Mdm2, Mdmx, Puma, Slug and Chk2). The list of p53 pathway-related genes was constructed based on gene and protein web databases and literature search. Only genes with proven biochemical relationships to the p53 pathway were included.

Publically available Affymetrix gene expression array data was analyzed which included 290 CN-AML and 34 APL patients at diagnosis in comparison to 63 normal bone marrow (nBM) samples. Differentially expressed genes (DEGs) were identified out of 1153 p53 pathway-related genes using a linear statistical model that produced gene expression contrasts between leukemic samples and nBM. Study effect differences were also corrected by this model. One hundred forty seven DEGs were identified in CN-AML and 172 in APL (fold change>2. 8, p value<0. 01). We found a significant over-representation of p53 pathway related DEGs above the genomic background in both leukemias. Our analysis demonstrated homogeneity of gene expression in APL patients and discovered that CN-AML patients were further divided into 3 sub-groups by hierarchical clustering analysis. Most of the DEGs were down regulated both in CN-AML (108/147) and in APL (135/172) patients. We analyzed the DEGs and concluded that in both leukemias there was no p53-dependent induction of canonical cell cycle arrest genes, canonical pro-apoptotic genes, p53-related antioxidant defense genes, DNA damage repair genes and anti-glycolysis genes.

We compared our bioinformatic results to gene expression signatures related to p53 activation by various stimuli from the literature. This analysis demonstrated that p53 protein did not exert transcriptional activation of the majority of its target genes in CN-AML and APL, implying that p53 pathway is not activated in these leukemias.

We found downregulation of p300, PCAF and CARM1 genes in patient samples compared to nBM. Deregulation of these genes points to decreased acetylation and methylation of the p53 protein that can result in the inhibition of p53 transcriptional activity.

We examined protein levels of p53 and its main inhibitors Mdmx and Mdm2 by immunohistochemistry in 25 CN-AML and 23 APL patients in comparison to 36 nBM biopsies. We found that the fraction of cells expressing p53, Mdmx and Mdm2 proteins was significantly higher in leukemias (70–90%) compared to nBM (10–30%). However, the intensity of Mdm2 staining was not elevated in leukemic blasts compared to nBM and p53 levels were similarly low in both nBM and leukemias. Importantly, Mdmx protein level was significantly upregulated in leukemia cells, offering an explanation for inhibition of p53 transcriptional activity in leukemia. The increased level of Mdmx protein together with low levels of p53 protein is in agreement with inhibition of p53 transcriptional activity in CN-AML and APL demonstrated by our bioinformatic analysis.

Inactivation of p53 pathway shown here may be one of the important leukomogenic events in AML development. Importantly, gene expression and thus the functional status of p53 pathway is very similar in CN-AML and APL patients compared to nBM, despite the different underlying molecular etiology of these diseases. This finding may have important therapeutic implications in that similar reactivation of the p53 pathway may be a therapeutic modality applicable to these two biologically different types of leukemia.