The Role of Mdm2 In Inactivation of the P53 Pathway In APL

Abstract 5111

The pathway controlled by the p53 tumor-suppressor protein is altered in most, if not all, human cancers and the TP53 gene itself is mutated in half of all human tumors. However, mutations in the TP53 gene are rare in human hematological malignancies. This implies that p53 is inactivated by alternative mechanisms such as over expression of its negative regulators. p53 is negatively regulated by Mdm2 through ubiquitin-dependent degradation and by Mdmx through inhibition of transcriptional function. To date, there is no information on the role of Mdm2 and Mdmx in human Acute Promyeloytic Leukemia (APL). We investigated the involvement of these negative regulators of p53 in APL at the gene and protein expression levels. First, we directly sequenced TP53 in 21 APL samples. In all cases TP53 was found to be wild type as expected. We studied Mdm2 and Mdmx gene expression in bone marrow samples of 30 APL patients at diagnosis in comparison to 35 normal bone marrow samples. Quantitative Real-Time PCR analysis showed no statistically significant differences in expression of Mdm2, Mdmx full length or its splicing variant (Mdmx-S) between APL and control samples. Bioinformatics analysis of gene expression of 39 APL patients at diagnosis, using publicly available arrays, showed homogeneous gene expression pattern between patients. We compared the APL arrays to 5 normal promyelocytes arrays. In agreement with our results, no significant difference was detected between APL and normal promyelocytes in the levels of Mdm2 or Mdmx gene expression. Mdm2 and Mdmx are subjected to complex regulation at the protein level. We therefore investigated the level of these proteins by immunohistochemical staining. Bone marrow biopsies from 23 APL patients at diagnosis were compared to 30 normal biopsies and protein levels were evaluated by semi-quantitative score. We found that Mdmx protein was low in APL samples and not significantly different from normal bone marrow. Thus, in APL, Mdmx does not appear to play a major role in p53 inhibition. Remarkably, APL samples showed a bi-modal expression of Mdm2 protein: 11/23 (48%) APL samples had significantly down-regulated Mdm2 protein (p<0.001). This might imply that Mdm2 is not involved in p53 inactivation in these patients. The other 12/23 (52%) patients showed significant over-expression of the Mdm2 protein (p<0.001). This result suggests that in half of APL patients, the p53 pathway may be inactivated by Mdm2. Interestingly, the subset of patients with high Mdm2 protein expression, was also characterized by high levels of p53 (5/12, p=0.058) and the pro-apoptotic p53 target, Puma (5/12). In summary, TP53 is wild type in APL. We found that the Mdm2 protein, a negative regulator of p53, may be accountable for p53 inactivation in about half of APL patients. In the other cases, a different, yet to be determined, mechanism is involved in inhibiting p53. Thus, APL is not a homogeneous disease regarding the inhibition of p53 pathway.