5046 Mutations in the p53 tumor suppressor gene are rare in human hematological malignancies, suggesting that aberrant p53 function may be due to alterations in its regulatory pathways. p53 is negatively regulated by MDM2 through ubiquitin-dependent degradation and by Mdmx through inhibition of transcriptional function. There is little information on the expression of Mdm2 and Mdmx in most myeloid leukemias. We determined the gene expression and protein levels of Mdm2 and Mdmx in bone marrow samples of leukemia patients. We first performed quantitative evaluation of Mdm2 and Mdmx gene expression in bone marrow samples of 144 leukemic patients at the time of diagnosis: 29 de novo AML patients (AML, M0-M7, M3 excluded), 30 de novo AML M3 patients (APL), 39 therapy-related AML patients (t-AML, M0-M7, M3 included) and 46 CML chronic phase (CML-CP) patients in comparison to 35 normal bone marrow samples from Hodgkin's disease patients. Quantitative Real-Time PCR analysis showed no global statistically significant over expression of Mdm2 or Mdmx in any of the tested leukemias. However, a number of patients in both de novo and therapy-related AML had elevated levels of Mdm2 or Mdmx. Significant down regulation of Mdm2, Mdmx and a splicing variant of Mdmx lacking exon 6 (Mdmx-S) was observed in CML-CP. We next performed IHC staining, evaluated by semi-quantitative score, to examine the levels of Mdm2 and Mdmx protein expression in 151 leukemic patients at the time of diagnosis: 40 AML patients, 23 APL, 47 t-AML and 41 CML-CP patients in comparison to 58 normal bone marrow samples from Hodgkin's disease patients. Protein expression analysis also showed no global statistically significant over expression of Mdm2 in any of the tested leukemias. Nonetheless, a number of patients in both de novo and therapy-related AML had elevated levels of Mdm2 protein. Specifically, APL patients segregated into 2 groups: while half of the patients did not express the Mdm2 protein, the other half over-expressed Mdm2. A significant down-regulation of Mdmx was observed in APL. In summary, while in some leukemic patients Mdm2 and Mdmx might play a role in the regulation of p53, our quantitative analysis indicates that these negative regulators do not seem to provoke the inactivation of the p53 pathway in most myeloid leukemias patients. These findings have implications on the possible use of Mdm2 antagonists like nutlin-3 in myeloid leukemias. To the best of our knowledge this is the largest group of myeloid leukemia patients studied for Mdm2 and Mdmx expression. Disclosures No relevant conflicts of interest to declare.
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. Disclosures: No relevant conflicts of interest to declare.
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