Mdm2 Maintains Cholesterol Biosynthesis in Hematopoietic Stem/Progenitor Cells Independent of p53

Background: The Mdm2 protein is an E3 ubiquitin ligase that directly interacts with p53 protein leading to its degradation. The expression of MDM2 is controlled by p53 activity through an autoregulatory feedback loop. In addition, a single nucleotide polymorphism (SNP) in the MDM2 promoter modulates its expression and is associated with the risk of cancer. Emerging evidence emphasizes the metabolic activities of MDM2 to be essential for the maintenance of cellular homeostasis. We hypothesized that MDM2 maintains the metabolic homeostasis of hematopoietic stem cells (HSCs) and its downregulation in TP53-mutant leukemias leads to metabolic vulnerabilities independent of p53. Investigation of the metabolic role of MDM2 in hematopoietic stem cells can provide valuable insight into the pathology of TP53 mutant leukemias. Methods: To understand the function of Mdm2 in HSCs, we generated a conditional mouse model driven by Vav-Cre to genetically label and trace the fate of HSCs after heterozygous deletion of Mdm2 in early development and adult bone marrow. We utilized fluorescence microscopy, flow cytometry, apoptosis assays and RNA-seq to functionally characterize the fate of HSCs after heterozygous deletion of Mdm2. Using Trp53 floxed allele and a new Trp53 mutant allele that switches from wildtype to Trp53R172H mutant, we deleted and/or mutated Trp53 gene concomitantly in Mdm2 haplo-insufficient HSCs. Additionally, MDM2 copy number as well as MDM2 SNP309 status were determined in 95 samples from p53 mutant AML patients and 24 controls. Results: Heterozygous deletion of Mdm2 in hematopoietic stem cells (Vav-Cre;mTmG;Mdm2 fl/+) resulted in massive apoptosis of emerging hematopoietic progenitor cells in the aorta-gonad-mesonephros (AGM) region at E11.5. Strikingly, hematopoietic cells residing in fetal liver displayed minimal apoptosis evident by a few TUNEL positive cells. Colony forming assays revealed a myeloid biased hematopoiesis in Mdm2 haplo-insufficient HSCs. Vav-Cre;Mdm2 fl/+ mice displayed a marked reduction in Lin -/CD150 +/c-Kit +/Sca-1 + HSCs cells and significant decrease in peripheral blood counts. Deletion of Trp53 in these mice (Vav-Cre;Trp53 fl/fl;Mdm2 fl/+) resulted in marked decrease in CD19+ B lymphocytes cells whereas the population of CD11b+ myeloid cells did not change. The population of Lin neg-c-Kit + hematopoietic stem/progenitor cells isolated from the bone marrow of Vav-Cre;Mdm2 fl/+ mice displayed marked downregulation of cholesterol biosynthesis and mevalonate pathway (-log2 pvalue=20). Strikingly, 85% of genes involved in cholesterol biosynthesis (29 genes) were downregulated in Vav-Cre;Mdm2 fl/+ mice. Homozygous deletion of Trp53 in Vav-Cre;Mdm2 fl/+ mice did not rescue the metabolic alterations driven by Mdm2 haplo-insufficiency. In addition, the gene signature of oxidative phosphorylation(oxphos), was remarkably upregulated in Vav-Cre;Mdm2 fl/+ mice independent of p53. We further demonstrate that Cre-mediated induction of a Trp53R172 mutation in Mdm2 haplo-insufficient mice resulted in malignant transformation of HSCs leading to acute myeloid leukemia (AML). Of note, mice with homozygote Trp53 mutation and/or deletion without Mdm2- haplo-insufficiency developed lymphoma and not leukemia. In human, MDM2 loss of heterozygosity (MDM2 LOH) in AML was always concomitant with TP53 missense mutations (log2 odds ratio>3, p<.001), and not TP53 deletions or truncations whereas in lymphomas, MDM2 LOH and TP53 mutations were mutually exclusive. Conclusion: Using a genetic model, we have shown that Mdm2 haplo-insufficiency in HSCs leads to apoptosis and clonal selection towards myeloid biased hematopoiesis. Mechanistically, Mdm2 haplo-insufficiency resulted in a metabolic switch from cholesterol biosynthesis to oxphos in HSCs. Notably, this metabolic reprograming is not rescued by deletion of Trp53. However, mutation of Trp53 in Mdm2 haplo-insufficient hematopoietic stem cells resulted in leukemic transformation of HSCs leading to acute myeloid leukemia. Lastly, we demonstrate that MDM2 SNP309 is associated with TP53 mutation in AML and provide clinical evidence that MDM2 loss of heterozygosity is concomitant with TP53 mutations in AML with lower survival compared to TP53 mutant patients with diploid MDM2. Our findings demonstrate a p53-independent role for Mdm2 in metabolic maintenance of hematopoietic stem/progenitor cells. Figure 1 Figure 1. Disclosures Khoury: Kiromic: Research Funding; Angle: Research Funding; Stemline Therapeutics: Research Funding. Andreeff: Glycomimetics: Consultancy; Medicxi: Consultancy; Karyopharm: Research Funding; ONO Pharmaceuticals: Research Funding; Senti-Bio: Consultancy; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Syndax: Consultancy; Amgen: Research Funding; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Oxford Biomedica UK: Research Funding; AstraZeneca: Research Funding; Aptose: Consultancy.

p53 p.Pro72Arg (rs1042522) and Mouse Double Minute 2 (MDM2) Single-Nucleotide Polymorphism (SNP) 309 Variants and Their Interaction in Chronic Lymphocytic Leukemia(CLL): A Survey in CLL Patients from Western Iran

Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. The MDM2 and p53 are interacting proteins that play crucial roles in cell biology. Genetic variations of p53 and MDM2 have been identified in many cancers including CLL; among which are SNP309 in the promoter of MDM2 and SNP codon72 in p53. Materials and Methods: In this study, we sought to find the impact of two SNPs of p53 and MDM2 in the pathogenesis of CLL. A total of 100 CLL patients and 102 healthy controls were recruited. Genomic DNA was extracted, and genotyping was performed using the PCR-RFLP method. The allele and genotype associations were analyzed using the χ2 test. The gene-gene interaction analysis was studied using GMDR v0.9. Results: Our study found the absence of a significant difference between CLL patients and controls related to the allelic frequencies or genotypic distributions for both MDM2 SNP309 and p53 codon72. A significantly higher frequency of p53 C allele was found in patients with disease duration of more than 36 compared to those less than 36 months. However, GMDR analysis suggests genetic interaction between the genes under study. Conclusion: Our findings indicated each polymorphism of p53 codon72 and MDM2 (SNP309) was not a risk factor for CLL but the p53 C allele could be associated with the disease duration. Besides, the interaction between p53/MDM2 genotypes may confer susceptibility to CLL. Our study could be useful in genetic association studies of CLL and the role of gene-gene interactions in the susceptibility to the disease.

Association between TP53 and MDM2 Gene Polymorphisms and Risk of Hepatocellular Carcinoma in Hepatitis C Virus among Egyptian Populations

Background: The Murine double minute 2 (MDM2) gene is overexpressed in several human tumors. The oncogenic potential of MDM2 is partially explained by inhibition of the activity of the tumor suppressor protein P53 (negative regulator of the P53 tumor suppressor protein). A single nucleotide polymorphism (SNP) in the promoter region of MDM2 gene (T to G exchange at nucleotide 309) and TP53 gene (codon 72 exon 4, rs1042522 encoding either C or G) have been independently associated with increased risk of several cancer types. Few studies have analyzed the role of these polymorphisms in the development of hepatocellular carcinoma among Egyptian patients with chronic hepatitis C virus infection. Methods: The study consisted in the comparison of the genotype distribution of TP53 and MDM2 SNP309 in 100 viral hepatitis C-related hepatocellular carcinomas (HCC) cases and 100 controls without HCC matched for age, gender and ethnicity. PCR-RFLP (restriction fragment length polymorphism) and real time PCR methods were used to determine the genotype at the MDM2 SNP309T>G locus and TP53 rs1042522. Results: Overall, our results indicate that frequencies of TP53 alleles (C and G) were not significant different between HCC cases and healthy controls (p=0.093) (Odds Ratio, OR=1.361,95% Confidence Interval, 95% CI=0.949 – 1.951). A significant increase of MDM2 SNP309 G/G and T/G genotypes were observed among HCC cases (Odds Ratio, OR=4.868, 95% Confidence Interval, 95% CI= 2.873 – 8.251). Conclusions: Our finding suggest that people who have G allele increase the risk by 4.868 folds for developing HCC among Egyptian patients, consequently the MDM2 309T>G polymorphism is an important modulator of hepatocellular carcinoma development in Egyptian patients.