BackgroundMechanisms that inactivate the p53 pathway in Acute Myeloid Leukemia (AML), other than rare mutations, are still not well understood.MethodsWe performed a bioinformatics study of the p53 pathway function at the gene expression level on our collection of 1153 p53-pathway related genes. Publically available Affymetrix data of 607 de-novo AML patients at diagnosis were analyzed according to the patients cytogenetic, FAB and molecular mutations subtypes. We further investigated the functional status of the p53 pathway in cytogenetically normal AML (CN-AML) and Acute Promyelocytic Leukemia (APL) patients using bioinformatics, Real-Time PCR and immunohistochemistry.ResultsWe revealed significant and differential alterations of p53 pathway-related gene expression in most of the AML subtypes. We found that p53 pathway-related gene expression was not correlated with the accepted grouping of AML subtypes such as by cytogenetically-based prognosis, morphological stage or by the type of molecular mutation. Our bioinformatic analysis revealed that p53 is not functional in CN-AML and APL blasts at inducing its most important functional outcomes: cell cycle arrest, apoptosis, DNA repair and oxidative stress defense. We revealed transcriptional downregulation of important p53 acetyltransferases in both CN-AML and APL, accompanied by increased Mdmx protein expression and inadequate Chk2 protein activation.ConclusionsOur bioinformatic analysis demonstrated that p53 pathway is differentially inactivated in different AML subtypes. Focused gene and protein analysis of p53 pathway in CN-AML and APL patients imply that functional inactivation of p53 protein can be attributed to its impaired acetylation. Our analysis indicates the need in further accurate evaluation of p53 pathway functioning and regulation in distinct subtypes of AML.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-017-0249-2) contains supplementary material, which is available to authorized users.

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. Disclosures: No relevant conflicts of interest to declare.

1583 A somatic point mutation in the JAK2 gene results in a constitutive activation of the JAK2 kinase in the erythropoietin (epo) signal transduction pathway. The mutation was found in Polycythemia Vera (PV) patients (>95%) as well as in other myeloproliferative disorders (MPD). In MPD the mutation was shown to occur in pluripotential hematopoietic stem cells and to be present in all blood cell lineages. We analyzed DNA of blood donors for the JAK2V617F mutation by allele specific Real Time PCR. The mutation was detected in 9% of 114 samples with a very low mutation frequency of < 0.002% per sample (mutant/wild-type DNA allelic ratio) in comparison to frequency of 50–100% in PV. We speculated that accelerated erythropoiesis with increased erythroid differentiation will result in higher mutation rate. We therefore studied the presence of V617F mutation in two conditions associated with increased erythropoiesis, thalassemia and smoking. In beta-thalassemia major patients, the mutation was found in 22.2% (12/54), a significant 2.4-fold increase in mutation incidence compared to healthy individuals (p=0.016). The mutation frequency was 0.035% per positive sample. The V617F mutation was studied in a case – control study of hospitalized smokers (n=81) and non-smokers (n=61). Smoking was defined as 10 or more cigarettes per day, everyday of the week, for at least 10 consecutive years. Smoking status was assessed by a questionnaire and validated with measurement of exhaled carbon monoxide. Smokers suffered more chronic lung disease than non-smokers (45.7% vs. 6.6%, respectively, p<0.0001) and had a higher hematocrit, RBC and WBC (47.8% vs. 41.7% p<0.0001, 5.2 vs. 4.4×1012 cells/liter, p<0.0001 and 9.4 vs. 7.6×109 cells/liter, p=0.003, respectively). In all subjects epo was within the normal range or higher. The proportion of subjects with higher than normal epo was similar in smokers and non-smokers. The prevalence of the mutation was significantly higher among the smokers (36%) than among the controls (15%) (p= 0.005) The median V617F mutation frequency in smokers was 10 fold higher than in controls (0.032% and 0.0032% respectively; p=0.027). Very low V617F mutation frequency in DNA of non-MPD blood donors led us to question whether in these non-MPD individuals V617F mutation occurs in stem sells as was shown for PV patients. We found that the mutation was more frequent in peripheral blood mononuclear cells than in CD34+ cells purified from these mononuclear cells. Following 3 weeks culturing of CD34+ cells with epo mutation frequency increased by 9-folds (range 6–14) in hemoglobin-containing normoblasts. We next grew individual erythroid colonies from 8 healthy donors in semi-solid culture in the presence of epo. Out of 201 single colonies analyzed 13% contained the JAK2V617F mutation in a very small proportion of the cells (< 0.02%). These results demonstrate that in non-MPD individuals V617F mutation arises in very low frequency during cell differentiation. We established a cell line model to study the occurrence of JAK2V617F mutation during erythroid differentiation. Mouse erythroleukemia (MEL) cells were transfected with the 1kb fragment of human JAK2 gene containing exon 14 sub-cloned into a mammalian expression vector. MEL cells were stimulated to differentiate by hexamethylene bisacetamide and the frequency of sequence changes in the JAK2 exon 14 was investigated. We found that changes in the JAK2 exon 14 sequence occurred at late stages of differentiation. We hypothesize that maintenance of DNA fidelity is down graded with cell differentiation, rendering the cells susceptible for mutations in general and in JAK2 in particular. In summary, accelerated erythropoiesis results in increased V617F mutation frequency such as in beta-thalassemia major patients and in heavy smokers. We demonstrate that unlike in MPD where the JAK2V617F mutation occurs in pluripotential stem cells and expands extensively, in non-MPD individuals it arises at very low frequency during cell differentiation. These differences determine the lack of clinical significance of the JAK2V617F mutation in non-MPD individuals. Disclosures: No relevant conflicts of interest to declare.

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.

A somatic point mutation in the JAK2 gene results in a constitutive activation of the JAK2 kinase in the erythropoietin (epo) signal transduction pathway. The mutation was found in Polycythemia Vera (PV) patients (&gt;95%) as well as in other myeloproliferative disorders (MPD). In MPD the mutation was shown to occur in pluripotential hematopoietic stem cells and to be present in all blood cell lineages. We analyzed DNA samples from healthy individuals for JAK2V617F mutation by allele specific Real Time PCR. The mutation was detected in 9% of 114 samples with less than 0.001% of JAK2 mutant/wild-type (wt) DNA allelic ratio. The clonality of the mutation was studied by growing blood mononuclear cells from 8 healthy individuals in semi-solid culture in the presence of epo, allowing for erythroid differentiation. Individual erythroid colonies were picked up after 2 weeks. The mutation was detected in all 8 individuals at 5.6 – 24% (a median of 13.4%) of the colonies. The mutant/wt allelic ratio was 0.02% per single colony. This low frequency of positive mutant cells within a colony suggests that the mutation occurred at late stages of colony development concurrently with cell differentiation. In contrast, positive colonies derived from PV patients showed mutant/wt allelic ratio of either 50% (heterozygote) or 100% (homozygote), indicating the presence of the mutation at the early erythroid progenitor stage (BFUe). To further demonstrate that in cells from normal individuals the mutation occurs during erythroid differentiation, we studied 4 samples of CD34+ cells (purity &gt;95%). Cells cultured in the presence of epo differentiated to erythroid precursors within 3 weeks. We found 2–4×10−5% of the JAK2V617F mutation in the CD34+ cells prior to culture, which increased more than 2 folds following cell differentiation. We hypothesized that maintenance of DNA fidelity is down graded with cell differentiation, rendering the cells susceptible for mutations in general and in JAK2 in particular. To prove this hypothesis we used mouse erythroleukemia cells (MEL) as a model for differentiation. MEL cells were transfected with plasmid containing human JAK2 wt or V617F DNA sequences and stimulated for differentiation by hemamethylene bisacetamide. After 5 days in culture human V617F sequence (~ 0.001%) was detected in cells transfected with JAK2 wt-containing plasmid and vice versa, indicating incorrect replication of the introduced plasmid. The changes occurred at 2 folds higher frequency in differentiated cells compared to undifferentiated cells. We speculated that accelerated erythropoiesis with increased erythroid differentiation will result in higher mutation rate. We therefore studied 2 conditions associated with increased hematopoiesis, Thalassemia major and smoking. We analyzed 54 samples from Thalassemic patients and detected the V617F mutation in 12 (22.2%) patients - a 2.4 -fold higher frequency compared to healthy individuals. We also screened peripheral blood of 79 heavy smokers and found the mutation in 20 (25%) individuals – a 2.8 fold higher than in non-smokers. As expected, the mutation was detected at very low frequency ~ 0.0024% of total DNA. Our results demonstrate that unlike in MPD where the JAK2V617F mutation occurs in pluripotential stem cells and expands extensively, in non-MPD individuals it arises in very low mutant/wt DNA allelic ratio during cell differentiation. These differences determine the lack of clinical significance of the JAK2V617F mutation in non-MPD individuals.