Induction of the 5S RNP-Mdm2-p53 ribosomal stress pathway delays the initiation but fails to eradicate established murine acute myeloid leukemia.

Mutations resulting in constitutive activation of signaling pathways that regulate ribosome biogenesis are among the most common genetic events in acute myeloid leukemia (AML). However, whether ribosome biogenesis presents as a therapeutic target to treat AML remains unexplored. Perturbations in ribosome biogenesis trigger the 5S ribonucleoprotein particle (RNP)-Mdm2-p53 ribosomal stress pathway, and induction of this pathway has been shown to have therapeutic efficacy in Myc-driven lymphoma. In the current study we address the physiological and therapeutic role of the 5S RNP-Mdm2-p53 pathway in AML. By utilizing mice that have defective ribosome biogenesis due to downregulation of ribosomal protein S19 (Rps19), we demonstrate that induction of the 5S RNP-Mdm2-p53 pathway significantly delays the initiation of AML. However, even a severe Rps19 deficiency that normally results in acute bone marrow failure has no consistent efficacy on already established disease. Finally, by using mice that harbor a mutation in the Mdm2 gene disrupting its binding to 5S RNP, we show that loss of the 5S RNP-Mdm2-p53 pathway is dispensable for development of AML. Our study suggests that induction of the 5S RNP-Mdm2-p53 ribosomal stress pathway holds limited potential as a single-agent therapy in the treatment of AML.

Disruption of the 5S RNP-Mdm2 interaction significantly improves the erythroid defect in a mouse model for Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by haploinsufficiency of genes encoding ribosomal proteins (RPs). Perturbed ribosome biogenesis in DBA has been shown to induce a p53-mediated ribosomal stress response. However, the mechanisms of p53 activation and its relevance for the erythroid defect remain elusive. Previous studies have indicated that activation of p53 is caused by the inhibition of mouse double minute 2 (Mdm2), the main negative regulator of p53, by the 5S ribonucleoprotein particle (RNP). Meanwhile, it is not clear whether this mechanism solely mediates the p53-dependent component found in DBA. To approach this question, we crossed our mouse model for RPS19-deficient DBA with Mdm2(C305F) knock-in mice that have a disrupted 5S RNP-Mdm2 interaction. Upon induction of the Rps19 deficiency, Mdm2(C305F) reversed the p53 response and improved expansion of hematopoietic progenitors in vitro, and ameliorated the anemia in vivo. Unexpectedly, disruption of the 5S RNP-Mdm2 interaction also led to selective defect in erythropoiesis. Our findings highlight the sensitivity of erythroid progenitor cells to aberrations in p53 homeostasis mediated by the 5S RNP-Mdm2 interaction. Finally, we provide evidence indicating that physiological activation of the 5S RNP-Mdm2-p53 pathway may contribute to functional decline of the hematopoietic system in a cell-autonomous manner over time.

PROX1 is a predictor of survival for gliomas WHO grade II.

BACKGROUND: The clinical course of World Health Organisation grade II gliomas remains variable and their time point of transformation into a more malignant phenotype is unpredictable. Identification of biological markers that can predict prognosis in individual patients is of great clinical value. PROX1 is a transcription factor that has a critical role in the development of various organs. PROX1 has been ascribed both oncogenic and tumour suppressive functions in human cancers. We have recently shown that PROX1 may act as a diagnostic marker for high-grade gliomas. The aim of this study was to address the prognostic value of PROX1 in grade II gliomas. METHODS: A total of 116 samples were evaluated for the presence of PROX1 protein. The number of immunopositive cells was used as a variable in survival analysis, together with established prognostic factors for this patient group. RESULTS: Higher PROX1 protein was associated with poor outcome. In the multivariate analysis, PROX1 was identified as an independent factor for survival (P=0.024), together with the presence of mutated isocitrate dehydrogenase 1 R132H protein, and with combined losses of chromosomal arms 1p/19q in oligodendrocytic tumours. CONCLUSION: PROX1 is a novel predictor of survival for grade II gliomas.

p14ARF homozygous deletion or MDM2 overexpression in Burkitt lymphoma lines carrying wild type p53.

The hallmark of Burkitt lymphoma (BL) is a constitutively activated c-myc gene that drives tumor cell growth. A majority of BL-derived cell lines also carry mutant p53. In addition, the p16INK4a promoter is hypermethylated in most BL biopsies and BL cell lines, leading to silencing of this gene. Activation of c-myc and/or cell cycle dysregulation can induce ARF expression and p53-dependent apoptosis. We therefore investigated the p14ARF-MDM2-p53 pathway in BL cell lines. p14ARF was expressed and localized to nucleoli in all BL carrying mutant p53. Three out of seven BL carrying wt p53 had a homozygous deletion of the CDKN2A locus that encodes both p14ARF and p16INK4a. Three BL carrying wild type p53 retained the CDKN2A locus and overexpressed MDM2. DNA sequencing revealed a point mutation in CDKN2A exon 2 in one of these BL, Seraphine. However, this point mutation did not affect p14ARF's nucleolar localization or ability to induce p53. The Bmi-1 protein that negatively regulates the p14ARF promoter and co-operates with c-myc in tumorigenesis was expressed at low to moderate levels in all BL analysed. Our results indicate that inactivation of the ARF-MDM2-p53 pathway is an essential step during the development of Burkitt lymphoma, presumably as a mechanism to escape c-myc induced apoptosis.

MdmX binding to ARF affects Mdm2 protein stability and p53 transactivation.

Regulation of p53 involves a complex network of protein interactions. The primary regulator of p53 protein stability is the Mdm2 protein. ARF and MdmX are two proteins that have recently been shown to inhibit Mdm2-mediated degradation of p53 via distinct associations with Mdm2. We demonstrate here that ARF is capable of interacting with MdmX and in a manner similar to its association with Mdm2, sequestering MdmX within the nucleolus. The sequestration of MdmX by ARF results in an increase in p53 transactivation. In addition, the redistribution of MdmX by ARF requires that a nucleolar localization signal be present on MdmX. Although expression of either MdmX or ARF leads to Mdm2 stabilization, coexpression of both MdmX and ARF results in a decrease in Mdm2 protein levels. Similarly, increasing ARF protein levels in the presence of constant MdmX and Mdm2 leads to a dose-dependent decrease in Mdm2 levels. Under these conditions, ARF can synergistically reverse the ability of Mdm2 and MdmX to inhibit p53-dependent transactivation. Finally, the association and redistribution of MdmX by ARF has no effect on the protein stability of either ARF or MdmX. Taken together, these results demonstrate that the interaction between MdmX and ARF represents a novel pathway for regulating Mdm2 protein levels. Additionally, both MdmX and Mdm2, either individually or together, are capable of antagonizing the effects of the ARF tumor suppressor on p53 activity.

Immunolocalization of human p14(ARF) to the granular component of the interphase nucleolus.

The human p14(ARF) protein is encoded by an alternative transcript from the INK4a/ARF locus on chromosome 9p21, a locus frequently afflicted in human tumors. By use of two novel specific antisera against p14(ARF) we show that the protein is localized mainly in nucleoli but also in the nucleoplasm. Transfection of full-length and deletion mutant GFP-p14(ARF) fusion proteins confirmed this subcellular localization and assigned the nucleolar localization signal to the exon 2-encoded C-terminal region. In order to determine p14(ARF) expression in human tumor cells, we examined p14(ARF) in 32 tumor cell lines by immunofluorescence staining. Nucleolar p14(ARF) was detected in 10 lines, all of which lacked functional p53. Double immunostaining with p14(ARF) and B23/nucleophosmin or fibrillarin antibodies using 3D microscopy revealed that p14(ARF) is located mainly in the granular component of the nucleolus. p14(ARF) was also found in distinct granular aggregates scattered throughout the nucleoplasm. RNase digestion or selective inhibition of rRNA transcription by low doses of actinomycin D caused nucleoplasmic translocation of p14(ARF). This indicates that the nucleolar localization of p14(ARF) is dependent on ongoing transcriptional activity in intact functional nucleoli.